American Crow (Corvus brachyrhynchos) calls vary considerably in their acoustic structure and timing, but the function of this variation is still not fully understood. Learning how crows use their ‘caw’ vocalizations to relay information will enrich our ability to more fully comprehend complex communication across species. We examined differences between crow vocalizations made in two contexts with frequent calling. We used a parabolic microphone to record calls both passively at pre-roost aggregations and actively by placing a model owl in a tree while playing back a 75 second recording of crow distress calls to induce predator mobbing responses. We analyzed 75 seconds of audio in each recording for differences in the duration of silence between multisyllabic calls, the duration of silence between caw syllables, the number of caws, the average fundamental frequency of the caws, and the harshness of the caws. We measured harshness as the difference between the atonal, non-harmonic signal existing within caws and random background noise. Preliminary evidence suggests that calls given during mobbing are harsher than those given during pre-roosting. We are currently developing and refining our analyses methods in order to verify our results, which may be useful in future research of avian vocal communication.

A challenge faced by investigators who use Adeno Associated Virus (AAV) vectors lies in verifying successful assembly of the genetic construct developed for a given experimental manipulation. Failure at this step can introduce mutations that produce non-functional proteins and failure to obtain the desired expression. For these reasons, quality control measures support generation of valuable data and prevention of resource loss. Using molecular techniques such as PCR and Sanger Sequencing, we developed a quality control pipeline to amplify and identify viral sequences. Viruses were selected from stock for an identity assay based on relevance to planned optogenetics procedures, and PCR amplicons were designed to contain functional regions of each viral construct. Amplification was carried out using universal primers targeting 5'ITR and WPRE regions, which were common to all assembled viruses. Sanger sequencing was used to confirm that the amplicon sequence matched the viral template. Using the outlined procedure, we can now amplify, purify, and sequence the vector genome to gain nucleotide level confirmation of coding gene sequences prior to use in experiments.

In the United States of America, the nonprofit sector has grown substantially over the last five decades. Despite this, social issues like houselessness appear to be as pervasive as ever. This research seeks to analyze the relationship between the growth of nonprofits focused on the housing crisis and the unhoused population. I hypothesize that the growth of housing-focused (HF) nonprofit organizations will not result in lower numbers of homeless individuals. My underlying theory is that nonprofits, constricted by the structure of the sector, are frequently unable to address structural causes of houselessness. The reliance nonprofits have on wealthy donors is a primary factor. This power dynamic is part of a greater system often referred to as the nonprofit industrial-complex. The growth of HF nonprofits is collected using Candid and measured using nonprofit revenue data from the Internal Revenue Service. Houselessness is measured using data from the United States Department of Housing and Urban Development. To test this hypothesis, I run a multivariate regression analysis between my two variables across two cities: Seattle, Washington and New York City, New York from 2012 to 2020 (a nine-year period). I control government funding, housing costs, poverty and unemployment. I expect to find increasing numbers of unhoused individuals despite HF nonprofit growth. This research does not advance a casual relationship, but contributes to the theoretical discussion as to whether nonprofits create transformative social change and provides rigorous empirical evidence supporting this theory.

During development, cardiomyocytes (CMs) undergo a hypertrophic growth phase to generate load for the heart to pump sufficient blood. However, in the early stages of pathological hypertrophy, stress-induced signal transduction promotes the addition of new contractile units through poorly understood mechanisms to maintain tensional homeostasis. Microtubules provide mechanical resistance in CMs. Our previous data shows that inhibition of contraction by expression of D65A cTnC (a point mutation on the calcium binding site of troponin C) results in complete myofibrillar disarray, with muscle stress fibers emerging in the cellular periphery. Surprisingly, when given topological cues, these cells show aligned myofibrils in the absence of contraction. Thus, the mechanism behind the maintenance of myofibril and passive tension in non-contractile CMs is not explained. My goal was to determine the role of microtubules in maintaining tensional homeostasis in response to change in internal tension in CMs. Here, wild-type (WT) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) were transduced to express cardiac troponin C (cTnC) with point mutations L48Q (hyper-contractile), I61Q (hypo-contractile), and D65A (non-contraction) to study the effect of varying levels of contractility or internal load on microtubules. The percent coverage of microtubules (staining within the CMs for cTnC L48Q, WT, I61Q, D65A) was 42.32%, 50.20%, 64.09%, 75.70% respectively (n=300). Complementary proteomics data have indicated that protein levels related to microtubule proliferation (MAP4) and assembly (Tubb3, Tubb4b, Tuba1a) were elevated in CMs expressing cTnC D65A. This new data gives us insight into how the microtubule remodeling in non-contractile and dysfunctional cardiomyocytes maintains tension in early stages and its possible role in myofibril formation.

 The hippocampus (HPC) and the medial prefrontal cortex (mPFC) are two structures crucial for effective decision-making, and it has been shown that these two structure communicate when spatial working memory (WM) recall is necessary in rats. The hippocampal system is critical for episodic memory, and is especially important in the acquisition and retrieval of spatial information, while the mPFC’s major function is to form and store representations of events and contexts, so that in the future, the most appropriate behavioral response is used in a given context. We will be investigating the correlation between a rat’s behavior and neural activity in the mPFC and the HPC simultaneously to better understand the detailed interaction between these two structures. Rats are trained on a task where they must switch between two types of spatial learning strategies while on the maze to obtain a food reward. While the rat performs the task, we will monitor neural activity in the HPC and mPFC as well as the rat’s behavior. Past research has shown that identification of a certain deliberative behavior can be determined by looking at hippocampal oscillatory data. We predict there will be a correlation between this deliberative behavior made by the rat and an identifiable pattern in hippocampal-prefrontal engagement. We also expect to observe the two structures align their activity as the rat learns to switch from one strategy to the other, giving insight into the involvement of the hippocampal-prefrontal network in learning and decision-making. Understanding the neural mechanism employed in memory-guided decision-making has tremendous implications for the field of medicine, as disadvantageous decision-making and impaired volitional control are characteristic of many mental health disorders, such as addiction, eating disorders, depression, and anxiety disorders.

Brain stimulation has emerged as a novel treatment for stroke, a prevalent cause of death and disability worldwide. Studies in rodent models have shown that post-stroke electrical stimulation results in plasticity and neuroprotective benefits. However, techniques that were effective in rodents have rarely translated into clinically viable therapies in humans due to the significant differences in rodent and human neurophysiology and anatomy. Therefore, the goal of our study is to obtain clinically relevant outcomes that describe the mechanisms of stimulation induced plasticity in non-human primates. We combined electrophysiology and immunohistochemistry to investigate the degree of stimulation-induced plasticity and network dynamics after photothrombotic stroke in 4 macaques. We quantified the expression of two biomarkers, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43) in cells within ~10mm from the lesion penumbra. Since PSD-95 is important for the maturation of excitatory synapses and GAP-43 is involved in axonal branching and elongation, evaluating the expression of these two proteins around the lesion core allowed us to compare post-stroke synaptic and axonal plasticity in 2 control and 2 stimulated monkeys. Based on wide-field epifluorescence imaging, we identified the distance from the lesion penumbra at which there was a distinct difference in biomarker immunoreactivity in control and stimulated animals, and performed high-magnification confocal imaging to further investigate the structure of biomarker expression. Furthermore, analysis of the electrocorticography signal showed a largescale downregulation of neural activity following electrical stimulation, while Nissl staining revealed that stimulated monkeys had smaller lesion volumes than controls. These results indicate that stimulation elicits changes at both neurophysiological and cellular level, and may exert a neuroprotective effect on the post-stroke network by reducing metabolic energy consumption.. Therefore, this study investigates the effects of electrical stimulation on neuroplasticity and protection following injury, which may have a profound impact on future therapeutic interventions for stroke.

The transient receptor potential vanilloid-1 (TRPV1) ion channel is a polymodal receptor expressed in sensory neurons that contributes to inflammatory pain sensation. Interestingly, the receptor channel is modulated by specific phosphoinositide lipid molecules in the plasma membrane. During inflammation, phosphoinositide 3-kinase (PI3K) generates the signaling lipid phosphatidylinositol (3,4,5)-triphosphate (PIP3). This increase in PIP3 levels leads to trafficking and fusion of vesicles containing TRPV1 with the plasma membrane. Previous research from the Gordon Lab revealed that increased PI3K activity is potentiated by TRPV1 during inflammation. However, the mechanism behind potentiation is undetermined. We hypothesize that stabilized association of PI3K with the plasma membrane by TRPV1 results in increased PI3K activity. To visualize the spatial localization of PI3K and its interaction with TRPV1, we employed a special optical technique called total internal reflection fluorescence microscopy (TIRFM). In TIRFM, selective excitation of fluorophores adjacent to the plasma membrane allows us to study translocation of proteins from the cytosol to the plasma membrane, or vice versa. We utilize TIRFM in conjunction with an opto-PI3K system to better understand PI3K signaling events during inflammation. We transiently transfected F-11 cells, model cells for dorsal root ganglion neurons that sense pain, with fluorescently labeled opto-PI3K system components and TRPV1 ion channels. Our fluorescence measurements show that, upon stimulation with 650 nm light, PI3K is retained at the plasma membrane through interaction with TRPV1. TRPV1 retention of PI3K is direct evidence for the hypothesized mechanism of potentiation through membrane localization. Studying PI3K activity in the prescence of TRPV1 will elucidate key properties of potentiation, such as affinity of the interaction, effects on PI3K activity, and substrate selectivity. These findings will aid in the development of inflammatory pain treatments that disrupt the interaction between PI3K and TRPV1.

It is established that stress can promote addictive drug use and relapse in humans with substance use disorders, thus understanding the stress mechanisms responsible is likely important in developing effective treatments for drug addiction. One effect of stress is the stress-induced release of endogenous dynorphin neuropeptide systems in the brain which activate kappa opioid receptors, that in turn stimulate p38 mitogen-activated protein kinase pathway (MAPK). P38 MAPK activation results in the dysphoria experienced during a stress response. We want to learn more about how p38 affects mood and develop efficient CRISPR techniques to manipulate p38 activation. Other techniques of silencing p38 have been used but come with disadvantages. With a CRISPR approach, virus expressing excision sequences for gene editing can be injected into transgenic mice encoding Cre recombinase in a cell-type specific manner. In this study, I tested a CRISPR/Cas9 virus (AAV1-Flex-SaCas9-sqMapk14), designed to excise the p38 gene. As expected, wild type male mice developed significant conditioned place aversion to the KOR agonist, U50,488. In contrast, mice injected with virus bilaterally in the VTA did not acquire aversion. This finding suggested that successful excision of p38 in VTA had occurred. To confirm with immunohistochemistry, I stained CRISPR injected and U50,488 activated brain slices with p38 and phospho-p38 selective antibodies. We expect slices from CRISPR treated mice to show fewer positive cells in the VTA as compared to controls. My characterization of these antibodies is on-going, but preliminary results suggest differences in CRISPR injected and wild type mice. With the development of a technology allowing for efficient manipulation of p38 MAPK within cell site and brain regional specificity, we hope to provide further insight to the stress response and to better understand its role in addiction.

Alcohol withdrawal exists as a significant barrier to care for those who struggle with alcohol use disorder. Furthermore, there is evidence of worsening withdrawal symptoms after repetitive cycles of alcohol use and abstinence. Our preliminary research points to neuroinflammation primarily regulated by microglia, the immune cells in the brain, as a potential causal component in the escalation of recurring alcohol withdrawal symptoms. Previous work in our lab has shown that mice subjected to chronic intermittent exposure (CIE) to ethanol vapor showed elevated markers of neuroinflammation, as evidenced by transcriptome analysis in microglia. We found that the “unfolded protein response (UPR)” was activated; it plays a critical role in the development of neuroinflammation, and the UPR is mediated significantly by the C/EBP homologous protein (CHOP) an apoptotic transcription factor. Therefore, we hypothesize that CHOP in microglia is necessary for promoting alcohol withdrawal symptoms. We expect to observe measurable reductions in the withdrawal symptoms experienced by transgenic male and female mice with and without CHOP following CIE exposure. In our pilot study, transgenic male and female mice with and without CHOP were subjected to five weeks of CIE to ethanol vapor and then tested for withdrawal phenotypes. These tests include body temperature, locomotion, marble-burying behavior, novelty suppressed feeding, and sucrose preference. We have also characterized microglia morphology and CHOP expression in pilot mice brains. Having measured changes in these mice's neuroinflammatory mechanisms and subsequent behaviors, our results will determine the efficacy of decreasing CHOP gene expression to ameliorate the kindling effect in alcoholics. Therefore, our work provides a promising direction in tackling the inescapable cycle of worsening alcohol withdrawal that limits the road to recovery for alcoholics.

Located 100 km north of Oahu and over four kilometers of water, the North Arch Volcanic Field covers an area of 25,000 km² and includes the longest known underwater lava flow. A transect was mapped within the southernmost part of the field using a 30 kHz EM302 multibeam echosounder and 3.5 kHz Knudsen echosounders aboard the R/V Thomas G. Thompson. These surveys yielded a high-resolution bathymetric map and sub-bottom profiles that image shallow structures beneath the ship’s track. This research is investigating the extent and characteristics of these lava flows, as well as ability of Knudsen data to determine the location of flows. The seafloor is 300 meters shallower within the northern parts of the study area, as the transect moved onto the Hawaiian Arch. The lava flows here are more extensive and presumably closer to the eruptive source. Through the analysis of the Knudsen data, 29 lava flow crossings were found that vary in width and height. The lava flows have complex flow morphology, including different boundary structure, sediment pockets, complex flow paths, and elevation changes. There are two main areas of flows within the southern part of the study site with differing characteristics that may be related to the lava viscosity. The lava field is more variable than expected, which suggests that even in the small region sampled, it was formed by several effusive events over a period that had lava with different chemical properties. This work has shown that Knudsen data is extremely useful in classifying marine lava flows, and the further classification of the flow morphology of these lava flows can contribute to the understanding of the history of volcanism within the area, as well as marine effusive lava flows.

I study the evolution of galaxies using cosmological hydrodynamic simulations. These galaxy simulations allow us to explore theoretical explanations for the mechanisms that govern galaxy growth and evolution. I use specialized python packages to investigate the gas and stars that make up these galaxies. My research focuses on understanding the circumgalactic medium (CGM), the halo of gas that surrounds a galaxy. Oxygen is an important element to study within the CGM of Milky Way-mass galaxies. The abundance of different oxygen ions in the CGM can reflect the impact of processes that drive galactic evolution such as: supermassive black hole (SMBH) activity, star formation history, and galaxy growth. By understanding how oxygen plays a role in our simulations, we can better understand the histories driving observations of galaxies in the universe. An initial result of my current work involves the total oxygen mass abundance in the CGM of two different galaxy simulations. Each of my galaxies have significantly different amounts of oxygen in their CGM — by over an order of magnitude. Additionally, the galaxy with less total oxygen mass sees an increase in abundance at larger radii. To determine the evolutionary process that is driving the discrepancy in oxygen mass between these two galaxies, I will investigate the activity of their SMBHs, measure the amounts of different oxygen ions in their CGM, and determine where these galaxies fall on the mass-metallicity relation.

Submarine volcanoes make up 80% of volcanic activity on Earth, but are relatively understudied due to the lack of real-time observations of eruptions and their inaccessibility, often several kilometers below sea level. Underwater volcanoes behave differently than their land counterparts due to the cold sea water surrounding them. Some submarine volcanism is violent as water pours into active vents, rapidly cooling lava and shattering it into fragments. Other eruptions can produce lava flows that extend tens of several kilometers before solidifying. The majority of submarine volcanoes occur in areas of tectonic activity such as mid-ocean ridges and hotspot island chains. One interesting site of submarine volcanic activity is the Submarine North Arch Volcanic Field 100 kilometers north of Oahu, Hawaii, hosting a few of the longest flows on Earth and covering over 25,000 kilometers². The field is associated with the Hawaii hotspots but the source of such extensive volcanism is poorly understood. Past studies have mapped the volcanic field flow but only at low resolution. The southeast portion of the field comprises of a series of lengthy flows, hypothesized to emanate from a 75 kilometer long fissure in the seafloor. However, the resolution of the maps was too low to test this theory. On the R.V. Thomas G. Thompson, our team used the high-resolution Kongsberg EM302 30-kHz multibeam echosounder to map the eastern boundary of the southeastern lava flow where the fissure would likely be located. Although area covered was limited by bad weather, we were able to map a 6 kilometer wide swath track extending 63 kilometers along the inferred fissure. We identified the presence of two features that were potentially part of the fissures. Each extends about 1-2 kilometers and comprise a 5 meter deep and 200-400 meters wide trough with a levee on either sides. 

Exoplanets are any planet that’s outside of our solar system. There’s a myriad of ways to detect these planets, one of which is transit-timing variation. A planet is transiting when it passes in front of its star. If there is more than one planet in the system, there will be variations in the times at which it transits. This is due to gravitational perturbations from the planets on each other. This project focuses on the TRAPPIST-1 system, and more specifically Planet h of this system. We have the transit-timing variation (ttv) data for 7 planets in this system (named Planet b-h). We use an N-Body model to simulate the transit times, and compare this to the data to find the physical parameters of the system. An N-body model of 6 planets, and the data of 6 planets (excluding the Planet h’s data), is used to find the parameters. Then an N-body model of 7 planets is used with the data of the 6 planets. In comparing these models, we predict that the 7 body model will fit better. Although the 7th planet’s data is excluded, the signal from this planet will still be present in the ttvs of the other planets, due to the gravitational perturbations. If the model does fit better using 7 bodies in the N-body simulation but only data from 6 planets, we can see that the transit timing variations are strong enough to tell us that there is a 7th planet. This can be translated to other systems that have similar transit timing variation to see if adding another planet will create a better fit.

Peroxisomes play an integral role in human health. Dysfunction in peroxisome biogenesis can compromise overall peroxisome and cellular function and can lead to severe neuropathologies and metabolic disorders. While a spatiotemporal model of peroxisome biogenesis has been characterized in yeast cells, the extent of the conservation of this model in humans is unknown. Here we perform Kinase Regression Analysis (KiR) using single-cell features extracted from microscopy datasets to deconvolve kinase networks involved in peroxisome biogenesis. Huh7 cells with a KiR panel were fixed and stained for Pex3, Pmp70 and Sec61 to capture Pex3 colocalization at both early and late stages of peroxisome biogenesis. Primary pediatric hepatocyte cells were also stained for Pex3, Pmp70, and actin. CLARITY, a python-based image analysis pipeline, was used for single-cell feature extraction from the image datasets. Images were stacked into multi-channel 3D arrays and restored via deconvolution. The Allen Institute’s deep learning based cell segmenter and structure segmenter were employed to binarize and identify single cells and peroxisomes in the images. Finally, colocalization and peroxisome morphology features were extracted from the images and inputted into the KiR algorithm to predict kinases involved in peroxisome biogenesis. Morphometric analysis of peroxisomes show high variation in peroxisome volume, intensity and number per cell, demonstrating multiple states of peroxisome biogenesis present in a single treatment. Population distributions of Pearson’s Correlation Coefficients demonstrate varying distributions of colocalization between Pex3 and Pmp70 as well as Pex3 and Sec61 in response to different kinase inhibitors. Colocalization metrics were binned and inputted into the KiR algorithm. For each bin, 35-45 kinases were identified and hypothesized to alter Pex3 colocalization. The pairing of single-cell image data extraction with KiR analysis demonstrates a novel method of modeling kinase networks. Future work includes performing validation experiments to verify the predictions of kinases involved in peroxisome biogenesis.

Ocean-bottom seismometers (OBSs) are known to record puzzling signals termed short-duration events (SDEs) at frequencies of 4-18 Hz. SDEs are harmonic and usually have a duration of < 1 s, lack the P and S waves of local earthquakes, and individual events are never recorded on multiple OBSs. These signals have been attributed to either biological activity from animals bumping the seismic sensors or gas bubbles moving beneath the sea floor. In this study, we assess these explanations for SDEs using data from the Cascadia Initiative, a major OBS experiment along the continental margin of the Pacific Northwest that lasted from 2011-2015. After manually calibrating the acceptable trigger amplitude for each station, I ran a recursive short term/long term detector over the year they were deployed. Some shallow stations show a clear diurnal signal with more SDEs during the day, which is consistent with a biological origin, while others show no such pattern. SDEs are also observed on some of seismic stations on the Ocean Observatories Initiative Regional Cabled Array where the sensors are buried and protected from the biological bumps. I categorized the SDEs on representative stations based on parameters including the average frequency, bandwidth, number of peaks, and duration. This data will be used to determine whether the characteristics of the events show a pattern consistent with the biological or gas bubble hypotheses.

Zinc pyrithione (ZPT) is a zinc conjugate that has been used as a preservative in industrial manufacturing supplies for preventing microorganismal growth on materials such as latex and plastics, as well as in shampoos and cosmetics. Little is known about the antibacterial effects of ZPT on common bacterial pathogens, especially when in the form of a solid surface barrier. Here we analyzed the inhibitory effects of ZPT in the form of a solid surface coating, provided by Cao Inc., on the bacterial strains E. coli and E. faecalis. Noufa Khan and I spread plated these bacteria on agar plates that contained a coat of 0%, 2.5%, or 5% ZPT underneath the agar. This indirect ZPT contact did not inhibit E. faecalis by any of the three ZPT materials, while E. coli was inhibited by 5% ZPT only. Noufa and I next tested the effects of direct contact of ZPT on bacterial growth by adding a designated amount of E. coli or E. faecalis directly to 3x3 cm squares of films containing 0%, 2.5% or 5% ZPT, three separate times, each in triplicate. After incubating for 24 hours, we removed the bacteria from these squares by vortexing and sonication into tubes containing liquid media, and plated them after serial dilution onto trypticase-soy agar plates. We counted the colonies to determine the extent of inhibition, if any. With direct ZPT contact, 2.5% and 5% ZPT inhibited E. coli and E. faecalis up to 95-99.9% relative to growth on the 0% ZPT control, with a higher concentration of ZPT showing a higher inhibition rate. These results demonstrate ZPT inhibits growth of common bacterial strains upon direct contact. An application to this research would be to use ZPT as an antibacterial plastic coating on door handles and other commonly touched surfaces.

The purpose of this study is to measure the uptake of arsenic from isolated arsenic resistant bacteria and mixed cultures derived from periphyton that were collected from a local lake with high arsenic concentrations. Periphyton is a complex community of microorganisms, plants, algae, and debris that is found attached to sediments, plants and other surfaces of most aquatic environments. Our colleagues discovered that periphyton collected from Lake Killarney captures arsenic present in the contaminated lake waters. Studying arsenic resistant organisms can help researchers understand their interaction with arsenic and their possible uses for bioremediation, since arsenic contamination in aquatic environments is a worldwide problem. We hypothesized that bacterial members of the periphyton collected from Lake Killarney are responsible for arsenic uptake. Therefore, our study aimed to isolate arsenic resistant bacteria from the periphyton collected and test their arsenic uptake capacity from growth medium. After isolating and growing these arsenic resistant bacteria and mixed cultures with different nutrient conditions, growth periods, and arsenic concentrations, results showed low arsenic concentrations in all the samples. This might indicate that these microorganisms are not responsible for arsenic uptake, but may only be able to tolerate high levels of arsenic. These findings suggest that the isolated bacteria and mixed cultures we tested might not be the organisms that are responsible for arsenic uptake in contaminated water. Therefore, our future research will focus on expanding the taxonomic diversity of periphyton bacteria cultured and tested for arsenic uptake. For example, we can culture Cyanobacteria, a group of photosynthetic bacteria that are abundant in periphyton and were not included in our previous studies. We anticipate different results from Cyanobacteria because of their unique energy requirements and may ultimately be responsible for the heavy arsenic uptake discovered in periphyton.

Gram-negative bacteria are known to secrete outer membrane vesicles (OMVs) which serve as important virulence factors that modulate biofilm formation. In Porphyromonas gingivalis, an oral anaerobic bacteria that is a leading contributor to periodontal disease produces OMVs which play a crucial role in evading host immune responses and the destruction of host tissues. Although their importance in pathogenesis is well characterized, OMV biogenesis is not yet fully understood. Through OMV quantification of various lipid A mutant strains we have shown that lipid A structure is important for OMV biogenesis. We have identified the phosphorylation of C4’ as crucial to inhibiting OMV biogenesis. Our double mutant, PG1587/PG1773 which has C1’ and C4’ phosphatases deleted, showed similar OMV biomass as our WT strain. This led us to hypothesize that OMV cargo composition can be modulated by the structure of lipid A. To test this hypothesis, we added equal amounts of OMVs isolated from either WT or PG1587/PG1773 double mutant cultures to S. gordonii biofilms and measured biofilm dispersal. We also observed dispersal of a fully formed S. gordonii biofilm after the addition of either WT OMVs or WT whole cell cultures. We found that the PG1587/PG1773 double mutant OMVs were significantly diminished in their ability to disperse S. gordonii biofilms, suggesting that gingipain activity per unit of OMV biomass was reduced when lipid A is bis-phosphorylated. We are currently working on quantifying relative amounts of gingipains for each lipid A mutant strain to determine if OMV levels correlate with relative levels of gingipains. Our findings highlight the capacity of P. gingivalis OMVs to shape bacterial communities and suggest that lipid A structure effects the packaging of OMV cargo, a novel finding related to OMV biogenesis.

CRISPR-mediated gene activation (CRISPRa) is an emerging tool for simultaneous regulation of multiple genes in genetic circuit designs. In CRISPRa, a nuclease-deficient dCas9 protein is used to deliver a transcriptional activator domain (MCP-SoxS) upstream of genes of interest. A complementary guide RNA (gRNA) directs dCas9 to bind the DNA target at a designated position. Such circuits are vital in regulating metabolic engineering, providing a more eco-friendly, industrial biosynthesis process and applications to medical therapeutics. Mathematical modeling of guide RNA (gRNA) competition for dCas9 predicted that CRISPRa efficiency will be diminished as the number of gRNAs increases up to 15. To experimentally investigate the limitations and rules for multiplexed CRISPR systems, we designed CRISPRa circuits with an increasing number of gRNAs in P. putida. The circuits were encoded in plasmids constructed with a scalable gRNA insertion technique based on Golden Gate Assembly. Multi-gRNA circuit performance was evaluated by simultaneously regulating multiple fluorescent proteins. Our preliminary results indicated that an increasing number of gRNAs lead to decreasing CRISPRa expression levels on our fluorescent genes. A clear drop in activation ratio was evident when introducing the 4th gRNA to our circuit, falling below the theoretical limit and establishing the limit to our current CRISPRa circuit. We hypothesize that the decrease in CRISPRa efficiency is due to limitations of dCas9 or activator protein. Therefore, by incorporating gRNAs lacking the protein recruitment motif, it is possible to identify whether the limiting factor is dCas9 or activator to provide a better understanding for CRISPR circuit designs. Optimizing multi-gRNA CRISPR circuits pose a potential for transferring to other microorganisms, expanding metabolic engineering capabilities and chemical productions beneficial in a wide range of biosynthetic applications. 

Ischemic preconditioning (IPC) is a phenomenon in which a brief ischemic event confers protection against subsequent prolonged ischemia, such as stroke. Understanding the mechanisms underlying this endogenous neuroprotective response could lead to advances in stroke therapeutics. Most experimental rodent models of stroke focus on injury to grey matter. However, injury to the white matter (WM) is a significant component of ischemic stroke in human patients and is poorly understood. Our laboratory has shown that microglial innate immune signaling is required for WM IPC-induced protection. The nodes of Ranvier (NoR) are critical WM structures that control conductance and action potential propagation along axons. Recent studies have shown elongation of the NoR in the context of WM injury and disease. The mouse optic nerve (MON) is a fully myelinated central nervous system WM tract. We expose MONs to a brief in vivo ischemic event to induce IPC. Seventy-two hours later, we isolate MONs and expose them to ex vivo oxygen-glucose deprivation (OGD) to mimic stroke. Using MONs from mice that underwent pharmacologic depletion of microglia followed by in vivo IPC and then ex vivo OGD, I performed immunohistochemistry with Nav1.6 and Caspr antibodies to label the NoR and image them with a confocal microscope. NoR size, diameter, and length are quantified with ImageJ in conjunction with an in-house MATLAB script. I hypothesize that IPC will protect and conserve structural parameters of the NoR from OGD-induced injury and that this protection will be eliminated by microglial depletion. Our preliminary data show feasibility of this project (i.e., all the methods to carry out this work are in place) and we can generate precise and reproducible measurements of the NoR in MONs. Findings from this project will provide a better understanding of the mechanisms by which microglia mediate IPC-induced axonal protection in WM.
 

The Xpert Xpress SARS-CoV-2 assay is a rapid RT-PCR test developed by Cepheid during the early stages of the COVID-19 pandemic. However, with the emergence of novel variants, concerns have been raised about possible diminished sensitivity of RT-PCR assays that were developed using earlier strains of SARS-CoV-2. We sought to address these concerns by testing known positive Alpha, Beta, Delta, Gamma, and Omicron SARS-CoV-2 samples which, at the time of testing, were classified by the CDC as variants of concern (VOCs). Since these samples were stored in TE buffer, a medium not formally evaluated for use in the Cepheid Xpert Xpress SARS-CoV-2 assay, we also tested positive controls using both TE buffer and the standard viral transport medium (VTM). All samples were run using the Xpert Xpress SARS-CoV-2 assay per Cepheid's provided instructions. Testing of known VOC positives revealed no substantial reduction of PCR sensitivity when compared to a clinically certified RT-qPCR assay. Comparison of TE and VTM samples also revealed no reduction in performance when using TE buffer, validating the use of TE buffer to store SARS-CoV-2 samples. Our findings show that the mutations present in the current circulating VOCs do not lead to false negative patient results, indicating the Xpert Xpress assay may still be used for diagnostic testing.

Nursing is recognized as a high stress career that is linked to depression, anxiety, and burnout. Today's healthcare environment is even more challenging due to distrust of medicine, racial tensions, and understaffing related to the seemingly endless COVID-19 pandemic. The prevalence of burnout, depression, and suicide among healthcare professionals is high. Nursing students are experiencing the chronic stress of COVID-19 as they train in trauma-filled environments. Students need foundational clinical knowledge and skills learned during nursing school; however, they also need resiliency skills to manage stressors throughout their training and into their careers. Resiliency skills have the potential to mitigate poor mental health outcomes and burnout, which ultimately contribute to retaining nurses in the profession. To build resilience, we need to understand the full picture of students' baseline resilience, mental health, and stressors, including adverse childhood events (ACE). The purpose of this study, therefore, is to explore the contributions of ACE scores, university stressors, self-efficacy, and perceived stress on self-reported resilience and mental health of nursing students at University of Washington (UW). We (Vasquez, Santoyo), with our mentor Amy Walker, participated in all aspects of developing and implementing this study. We performed statistical and thematic analysis. We expect to find high levels of perceived stress, numerous university stressors, and low levels of resiliency in undergraduate and graduate nursing students. The findings will be used to identify targets for future interventions to improve the resilience of nursing students during their education and their transition to practice.

In order to fully engage in reproductive justice, it is imperative that we address the ongoing erasure and exclusion of QTBIPoC (Queer, Transgender, Black, Indigenous, People of Color) families and birthing people, and other underserved communities within family building spaces. Perinatal research fails to include the experiences and voices of folks from these communities. This purposeful exclusion was created to continue centering white-cis-hetero bodies and to further oppress folks with identities that deviate from this “standard.” This causes real harm and because of this discrimination, QTBIPoC folks face worse health outcomes and maltreatment within healthcare. This is why we need to center the experiences of QTPBIPoC birthing people and their families, as they are at the highest risk of poor perinatal outcomes. In the Birth Includes Us study pilot my partner and I hope to employ a non-traditional social media recruitment strategy to effectively capture the experiences of QTBIPoC families to address disparities and highlight the communities’ strengths. Current methods fail to capture a large and diverse sample of participants, as they don’t properly reach or build trust within the community. Afterward, my partner and I will be evaluating how effective this recruitment strategy is in engaging a large and diverse sample of QTBIPoC participants. If the recruitment plan is successful, we intend to use this recrutiment strategy as a model to connect with "hard to reach" populations such as the QTBIPoC community and build this model so that others can replicate and better engage the community. In addition if the recruitment strategy is successful, we want to 1) increase our knowledge and inclusion in research, 2) build evidence-based inclusive and affirming practices and interventions to improve care/experiences, and 3) address/eliminate disparities within sexual and reproductive care. 

Cortisol, the primary stress hormone, has important effects on human sleep patterns and sleep quality. An increased presence of cortisol elicited from stress disrupts biological rhythms, affecting both mood and behavior. Since publications in large journals have historically focused on measuring cortisol levels in the context of alcohol abuse, there is not much data on how cortisol levels may be affected by “social drinking.” We aimed to study if social alcohol consumption bidirectionally influenced the measured cortisol levels in a cohort of 11 students enrolled in BIO A 454/455. Students self-collected saliva three times per day, providing two samples in morning to evaluate the cortisol awakening response, and one sample at night; this procedure was paired with a number of surveys where participants recorded daily mood, behaviors, and sleep. Cortisol concentrations were determined in the BIO A 454/455 laboratory at the University of Washington using an enzyme immunoassay protocol designed for use with saliva samples. Students of BIO A 454/455 evaluated their own saliva samples using the salivary cortisol EIA protocol. We aggregated collection averages across subjects, performing t-tests between groups of individuals who self-reported drinking at least one alcoholic beverage within each 24-hour period, and those who did not. Our results demonstrated a general variation of measurable cortisol levels in each individual, but analysis did not produce statistically significant figures between cohorts. A lack of literature on the connections between alcohol consumption and cortisol response still persists. Therefore, while our data proved to be too narrow to extrapolate conclusive results, we hope the format of this study can be used as a framework to explore the topic further.

In response to various forms of stress such as heat shock and oxidative stress, cells produce aggregates of mRNA and proteins called stress granules. These granules sequester mRNA and signaling proteins to promote cell survival. Stress granules are beneficial in the short term, but the chronic presence of stress granules can be cytotoxic and cause hyperaggregation of misfolded proteins. After a heart attack, the heart experiences a lack of oxygen, which creates free radicals and metabolic stress. Whether the stress response is involved in this process is unknown, as most research on stress granules and their role in disease comes from work in neuronal and cancer cells. To test whether the stress granule response is conserved across cell types, I cultured cancer cells, pluripotent stem cells, and stem cell-derived cardiomyocytes (heart muscle cells) and subjected these cells to various stresses, including sodium arsenate poisoning, heat shock, and oxidative stress. I imaged each treatment using immunofluorescence and quantified the number of stress granules per cell. The sodium arsenate treatment induced stress granule formation in all three cell types, but surprisingly, the heat shock and oxidative stress treatments had cell type-specific stress granule responses. It is widely believed the stress response is conserved across a range of cell types, but these results indicate some stress pathways differ between cardiomyocytes, cancer cells, and stem cells. To test how stress granules impact cardiomyocyte function, I generated stem cells with knockouts of the two genes required for stress granule assembly: G3BP1 and G3BP2. In future studies, I will differentiate these cells into cardiomyocytes and test whether the inability to form stress granules affects their ability to survive in response to stress. This work is important in understanding the impact stress granules have on the regeneration of heart cells in damaged heart tissue.

Rejection sensitivity (RS) is a core component of social anxiety that may impact symptom severity and treatment outcomes. There may be substantial differences in both neural and behavioral responses to fear as a function of trait RS in socially anxious individuals, as reflected in altered regional brain activation patterns underlying fear and self-referential processing. Individuals with the highest levels of RS may struggle to extinguish learned social fears but it is unclear if they learn safety differently by watching another person doing so, a process called vicarious extinction learning. Recent work has highlighted the advantage of vicarious extinction learning in fear regulation over traditional extinction learning, but this has not yet been tested in socially anxious populations. Given the relevance of social learning processes in social anxiety, vicarious extinction learning may enhance the effectiveness of exposure-based therapy in this population. In this study, individuals will undergo a social fear task in which they learn to pair images of angry faces with a mild electric shock. During Phase II, participants will watch a video of another person undergoing the same task and learning safety from one of the photos previously associated with a shock. The final phase will test if participants can apply the model's safety learning to themselves. I hypothesize that high trait RS may moderate vicarious extinction learning, as seen by differential activation in the ventromedial prefrontal cortex (vmPFC). It is possible that those with higher RS might show increased vmPFC activation during extinction and might benefit more from vicarious safety learning because social signals are more salient in this population and they have more to gain from learning safety, but it is also possible that they may not benefit, or even worsen, if they are unable to empathize with the learning model and remain vigilant to cues of social rejection, as evident by decreased vmPFC activation during extinction. Regardless of the direction of the effect, trait RS may be an important variable to consider when treating socially anxious patients using extinction-based principles and may influence the development of novel interventions to target this population. 

Platelets aggregate at the site of injury to stop bleeding, but disruptions to hemostasis can cause bleeding or thrombosis. Studying platelet-plug area and contractile force can predict whether bleeding or thrombosis is likely to occur. Microfluidic devices, composed of polydimethylsiloxane (PDMS), are used to study these biomechanics by inducing aggregation through shear flow. As blood flows through the device, the platelets pass over a rigid block in the channel which causes platelets to activate, deflecting a flexible post within the channel. This deflection is used to calculate the platelet forces based on the material properties of the PDMS. These microfluidic devices are single use and require a fabrication process that spans multiple days. Additionally, creating duplicate silicon master molds is a laborious and expensive process that necessitates cleanroom training. I am engineering and implementing a more efficient process for the fabrication of these devices, while limiting the use of the master mold that undergoes long-term wear from repeated uses. My focus is on improving the efficiency of the initial negative mold creation process by using a different material, urethane resin, to replicate the master, which allows us to make more negatives simultaneously without needing to fabricate another silicon master. To compare the devices produced using the onyx master with the silicon master, I am running three blood experiments, each with varying levels of antibodies, on two devices fabricated by the onyx and silicon masters. The aggregation sizes and forces are being observed between each of the experiments. I expect the results to be similar within a degree of certainty, proving the onyx master is equivalent to the silicon master and can be used to increase microfluidic device production and increase the availability of platelet biomechanics studies.

Unlike mammals, western clawed frog (Xenopus tropicalis) tadpoles are able to completely regenerate their spinal cord after tail amputation. This complete spinal cord regeneration is due to the ability of their neural progenitor cells (NPCs) to differentiate into neurons successfully. Our research focuses on two transcription factors—Meis1 and Pbx3– that are upregulated by regenerating neurons and are necessary for successful regeneration. We aim to elucidate how these two proteins are working together to guide successful spinal cord regeneration in X. tropicalis tadpoles. I am specifically investigating Meis1 and Pbx3 splice variant expression during neural regeneration. Previous work in mice found that different known splice variants of Pbx3 have different expression patterns. While X. tropicalis has two predicted splice variants each of Meis1 and Pbx3, nothing is known about their individual expression or function. I sought to fill in this gap by looking at Meis1 and Pbx3 splice variant expression in different tissues and over regenerative time. Based on previous research in mice, I hypothesize that both splice variants of Meis1 and Pbx3 have different gene expression patterns in different cell types over regenerative time. I aimed to investigate this hypothesis by doing two experiments. My first experiment was to study the expression of each splice variant over regenerative time by performing qPCRs in order to look at the presence of splice variant mRNA in uninjured, 24, and 72 hours post-amputation (hpa). For my second experiment, I am making in situ hybridization probes specific for each splice variant to identify their tissue-specific expression patterns. The experiment is being performed over regenerative time to observe how expression in tissues changes at the same time points hpa used in the first experiment.

This study compares narrative abilities of 11 adults with autism spectrum disorders (ASD) and 11 adults with fetal alcohol syndrome disorder (FASD) versus their 23 age- and sex-matched controls. The study aims to investigate the differences between ASD and FASD groups in the use of local measures (mental states and temporal relations) and the use of global measures (mean length of utterance, numbers of different words, and narrative scoring scheme) during a story-telling task using the children’s book Frog, Where are You?. We predict no significant differences in global narrative abilities between ASD and FASD to their comparison groups, but local narrative deficits in people with ASD and FASD. We predict they use fewer temporal references and mental state words. Comparing ASD and FASD groups, we predict that the ASD group will perform worse on both levels compared to the FASD group. The findings of this research will provide further evidence for differences in linguistic impairments between ASD and FASD and how social competence - the ability to understand a listener’s states of knowledge - influences linguistic abilities.

Sindbis Virus (SINV) is an alphavirus that is transmitted between birds via mosquitos and causes disease in humans after spillover events. While many alphaviruses have potential to cause severe disease such as Venezuelan Equine Encephalitis Virus (VEEV), SINV is known to cause less life-threatening but still severe and debilitating chronic illness predominantly associated with fever, arthralgia, and myalgia. Given its lower morbidity, SINV often serves as a model system for infectivity and pathogenesis studies of alphavirus. Comparison of the genomes of virulent strains (AR86) and avirulent strains (Girdwood) of SINV help us to define elements in the viral genome of SINV that contribute to pathogenesis. Previous studies have identified key pathogenesis determinants within the E2 glycoprotein and 5’ noncoding regions. Additionally, SINV contains elements in the 3’ UTR that are capable of repressing deadenylation contributing to evasion of host cell mRNA decay machinery. Our recent work with VEEV has identified additional novel RNA structures in the E1 segment that contribute to replication in macrophages and serves as a basis for further exploration of the role of RNA structure in pathogenesis and immune evasion which has been largely unexplored. By exchanging a 4000bp nucleotide segment of the 3’ end in SINV strains Girdwood and AR86, we hope that important conclusions can be made about the importance of differing genomic code and underlying RNA structure between these two strains after observing virus production, replication, and pathogenesis of constructed chimeras. This in turn can be used to further discern the genomic mutations and RNA structure requirements to shift from avirulent to virulent strains and could play an important role in predicting the mutational requirements necessary for SINV and other alphaviruses to make the jump from endemic bird/mosquito hosts to the human population as well as any mutations that contribute to greater pathogenicity.

In some traditional societies, a strong bimodal pattern of postpartum amenorrhea (PPA) duration has been observed in breastfeeding women. We have recently shown that early breastfeeding behavior is predicative of short-duration (~3 months) or long-duration (~15 months) amenorrhea, but the physiological mechanisms leading to this pattern are unclear. We investigate the relationship between hormone levels and duration of PPA in a sample of rural Bangladeshi women. Data came from an 11-month prospective study in which 261 breastfeeding women were interviewed and provided a urine specimen twice-weekly. It is known that breastfeeding disrupts ovarian cycles by inhibiting release of luteinizing hormone (LH) which in turn inhibits growth of ovarian follicles and release of ovarian steroids (estradiol and progesterone). During this time, women are infecundable. We assay hormone levels of estradiol metabolites, progesterone metabolites, LH, follicle-stimulating hormone (FSH), and cortisol across stages of PPA until eventual return to menses. We examine how these hormones correlate with the probability of resumption of menses and self-reports of breastfeeding behavior using survival analysis. We expect to see an earlier rise in LH and FSH spikes and increased levels of estradiol and progesterone metabolites in women who experienced a shorter duration PPA. We also expect that cortisol remains unaffected by the length of PPA. These results are used to explore the unknown mechanism that leads to bimodality in PPA.

Adeno-Associated Virus (AAV) has been used as a vector to deliver DNA to cells for transgene expression. When used to target cells in the brain, these injections are typically made directly into that tissue. Several factors determine whether injection of AAV causes expression of the transgene. A neutralizing antibody response may impede transgene expression through the binding of antibodies to viral vectors, which would prevent gene delivery. Different AAV serotypes have different epitopes, or binding regions for antibodies. However, epitopes may be shared between closely-related AAV serotypes. AAV-PHP.eB is a modification of AAV9 with improved ability to cross the blood brain barrier. This serotype could be injected intravenously to transfect cells in the brain, allowing for less invasive gene delivery as compared to brain injection. It is unclear whether a neutralizing antibody response to AAV9 would also prevent AAV-PHP.eB from infecting neurons. To test this, I will infect human embryonic kidney (HEK293) cells with AAV-PHP.eB in the presence and absence of blood serum taken from macaques before and after injection with AAV9, AAV-PHP.eB and other AAV serotypes. I hope to determine whether blood serum from macaques that have been injected with either AAV9 or AAV-PHP.eB, but not both, contains neutralizing antibodies that reduce the efficacy of the AAV serotype that was not injected. While blood serum from some macaques indicates the presence of antibodies for AAV serotypes other than the ones they were injected with, further analysis must be done before drawing conclusions. This study has implications for researchers conducting gene therapy research both in non-human primates and in human clinical trials, as the choice of serotype may determine whether or not the gene product is effectively delivered.

In 2020 marijuana use among college students was at an all-time high with 44% reporting using in the past year. Research demonstrates individuals with ADHD are at risk for increased marijuana use later in life. Additionally, some studies demonstrate self-reported symptoms of ADHD among undiagnosed college students prospectively relate to increased marijuana use, which was mediated by changes in perceptions of typical peer marijuana use (i.e., descriptive norms). However, this is complicated by frequent marijuana use being linked to issues with attention, impulsivity, and memory; all captured by self-reported ADHD measures. This study will evaluate bidirectional relations between ADHD-related symptoms, marijuana descriptive norms, and marijuana use among college students. Data collection is in progress and will be completed in March 2022 as part of a larger study. Students completed assessments at baseline, 6 months, and 12 months. A cross-lagged panel model will be conducted in R using the lavaan package to evaluate the bidirectional associations over a 1-year period, as well as test whether marijuana norms at 6 months mediate the longitudinal associations between ADHD-related symptoms and marijuana use from baseline to 12 months.  We hypothesize those reporting more ADHD-related symptoms will use marijuana more frequently 6 months later. Additionally, those who use marijuana more frequently will report more ADHD-related symptoms after 6 months. We also hypothesize that those experiencing more ADHD-related symptoms at baseline will have increased marijuana norms at 6 months and subsequent greater marijuana use at 12 months. This study attempts to disentangle the bidirectional associations between ADHD-related symptoms, marijuana norms, and marijuana use over a one-year period. Conclusions and future directions for research will be presented. Additionally, this study may have implications for norms-based interventions to better address memory, impulsivity, and other ADHD-related symptoms in association with marijuana use and perceptions.

Disruption of ion channel function is increasingly associated with neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD). ASD has complex etiology and symptoms, but recent studies suggest that individuals with ASD have disrupted mesostriatal network activity associated with social interaction deficits. Although mutated ion channels play a key role in NDDs, the degree to which they disrupt mesostriatal network activity is not well understood. The Kv7 family of voltage-gated potassium channels encoded by KCNQ1-5 genes are increasingly linked to NDDs including ASD. These channels are broadly expressed, including within the ventral tegmental area (VTA), a nucleus of the mesostriatal pathway that plays an important role in social behavior. Here we assessed the impact of the ASD-associated variant KCNQ3(R2C) on VTA dopamine neuron excitability and social behavior in mice. We used a viral-based strategy to conditionally inactivate endogenous Kv7.3 with CRISPR/SaCas9 and re-express human wildtype (hKv7.3/WT) or mutant (hKv7.3/R2C) KCNQ3. Whole-cell patch clamp electrophysiology in brain slices revealed that hKv7.3/R2C significantly decreased excitability of dopamine neurons. I also observed a loss of social novelty preference using the three-chamber social interaction task in mice expressing hKv7.3/R2C but not in controls expressing hKv7.3/WT. These results suggest that hKv7.3/R2C and other similar Kv7 mutant variants may disrupt mesostriatal network activity and social interaction by decreasing dopamine neuron excitability.

Ceramic petrography involves the examination of the mineralogical and microstructural composition of ceramic artifacts known as sherds. Traditionally, this examination to generate quantitative data from the ceramic material is often done by the hand, particularly the examination of ceramic inclusions in both their size, amounts, shapes, and density. This process is time-consuming, error-prone, and leads to limited datasets which are difficult to compare. By leveraging Python imaging analysis capabilities, a standardized and rapid method to generate repeatable datasets can be achieved on a greater scale. From work previously performed by my mentor, Alec Iacobucci, Alec created a robust set of internally consistent functions to collect multivariate data from ceramic sherd scans including inclusion size and count. This was done through Scikit-Image, an open-source image processing library for Python. However, Scikit-Image has its limitations, specifically in its computational efficiency as processing times are long and extensive. My goal for this project is to move the current material we have in Scikit-Image and translate it into OpenCV which is another library for image processing, but OpenCV is better optimized and specializes in computer vision. I will match similar functions and capabilities between Scikit-Image and OpenCV, perform data analysis on the ceramic inclusions to see the degree in which OpenCV outperforms Scikit-Image, and expand on previous determined variables such as average color of inclusions, orientation, and shape. The broader implications of this research is to reduce processing time of inclusion counting in the field which not only allows for larger and standardized datasets that can be compared and analyzed but to automate the ceramic petrographic process beginning at the dig site all the way to data analysis. This will be through the creation of an on-site scanner that implements our algorithms that is both portable and efficient where researchers can carry with them. 

Despite advances in treatment regimens for people living with HIV (PLWH), structural factors like food insecurity and housing inaccessibility continue to impact the populations’ health outcomes. As effective treatments, such as antiretroviral therapy (ART), improve health outcomes of PLWH, a new demographic of aging PLWH emerges. Older PLWH balance typical aging conditions like fragility and inflammation buildup with ART polypharmacy adherence within a complex system of social determinants of health (SDOH). SDOH are societal-driven conditions that impact prospective health outcomes such as race, socioeconomic status, access to adequate, nutritious food and shelter. Food security or the consistent access to sufficient, affordable, and healthy food is an upstream factor that greatly influences inflammation. Specific diet patterns such as high-carb and fat diets can also increase inflammation, in turn affecting health outcomes for aging PLWH. Inflammation induces negative side effects for aging PLWH on ART. Using the data collected from the four-year prospective PROSPER-HIV study, we assess the impact of food insecurity and inflammation on PLWH. We evaluate food insecurity using self-reported questions regarding food access scaled on a 5-point Likert scale. In addition, we assess diet-associated inflammation through a dietitian-led food recall. We hypothesize a strong correlation between food insecurity and aging. We expect the results to show how systemic factors significantly impact the health outcomes of PLWH. The findings have implications for policy development and resource needs for marginalized communities facing food insecurity. Uplifting marginalized communities occurs by identifying research gaps and implementing system-wide policies that address those shortcomings to diminish health disparities and promote health equity.

Despite the well-known role of the neurotransmitter dopamine in reinforcement learning, generating the brain’s patterns of dopamine release for conditioned response learning remains unresolved. The Zweifel laboratory discovered that two ion channel subunits, Kv4.3 and BKCa1.1, expressed in ventral tegmental area (VTA) dopamine-producing neurons control the pattern of dopamine neuron firing at the cell body and dopamine release in the nucleus accumbens (NAc) on different time scales to regulate separate phases of reinforced behavior in awake and behaving mice (in vivo). However, whether the impact on dopamine release is dependent on cell body loss of function (LOF) or possible effects of local terminal regulation remains unknown. DLight—a fluorescent, genetically modified dopamine receptor—was injected into the NAc. Chrimson, a photoactivatable ion channel, and CRISPR/Cas9 viruses that knocked out either of the two ion channel subunits were injected into the VTA for selective expression in dopamine neurons. Three conditions were imposed: control (no subunit knockout), Kv4.3 LOF, or BKCa1.1 LOF. After at least six weeks, NAc brain slices were prepared for two-photon imaging of dLight following photoactivation of VTA dopamine terminals. I measured changes in dLight fluorescence, indicating dopamine release, using ImageJ and analyzed them using ImageJ, Excel, and GraphPad Prism. VTA dopamine terminals in the dLight-expressing NAc of control and Kv4.3 LOF mice were photostimulated; the BKCa1.1 LOF mice are in progress. I found that loss of Kv4.3 in VTA dopamine neurons does not impact dopamine release ex vivo. Observed differences in dopamine release in vivo in Kv4.3 LOF mice and lack of differences in ex vivo suggest that the ion channel effects at the cell body impact dopamine release downstream, highlighting that regulators of dopamine neuron cell body activity patterns impact dopamine release and affect dopamine-mediated behaviors like learning and motivation.

The ongoing opioid epidemic has made apparent the consequences of repeat opioid use and the opioid withdrawal that follows. Avoidance of opioid withdrawal and its associated symptoms has led to a need to understand signaling within neural circuits involved. The parabrachial nucleus (PBN) is a region in the brainstem that relays sensory information throughout critical forebrain regions, informing specific behavioral actions and learning mechanisms. A genetically defined cell population with the PBN expressing the calcitonin gene related peptide (CGRP, gene name Calca) has been demonstrated to relay aversive sensory and visceral information when activated. I hypothesized that PBN-CGRP neurons may be a neuron population that is recruited during opioid withdrawal. To investigate whether the aversive experience of opioid withdrawal recruits PBN-CGRP neuron activity, I induced morphine-dependence in mice expressing a yellow fluorescent protein in PBN-CGRP neurons. After establishing morphine dependence, precipitated opioid withdrawal was induced with an injection of naloxone. Ninety minutes after induction of precipitated opioid withdrawal, mice were perfused to fix brain tissue. I then performed immunohistochemistry for cFos, an immediate early gene and a marker of increased neural activity, in brain sections with the PBN. I am quantifying the expression of cFos, a marker of increased neural activity, in PBN neurons that contain the neuropeptide CGRP following naloxone precipitated withdrawal. I expect to see increased cFos expression indicating the recruitment of PBN-CGRP neurons in mice that are suffering from opiod withdrawal compared to the control mice. By understanding whether the PBN is recruited during opioid withdrawal, I can begin to explore options to alter neuronal activity in the PBN to lessen the severity of symptoms felt during opioid withdrawal, a key contributor to the prevalence of relapse in those overusing opiates.

Planetary Nebulae, named for their sometimes circular ‘planet-like’ appearance, are gas clouds that are ejected by almost all ordinary stars as they approach their final phase of evolution. When stars with up to approximately 8 times the mass of the Sun (representing the vast majority of stars in the sky) run out of fuel, they undergo a series of rapid expansion events that eventually eject their outer layers. These ejections enrich interstellar gas with the carbon that forms pre-biotic molecules. We are investigating the poorly understood mass ejection process using Hubble Space Telescope images separated by 10 years to trace the changes in form and motions of sharp-edged features within the two “wings” of the extraordinarily active “Butterfly Nebula” NGC 6302. The ejection ages of the features are estimated by dividing their measured 10-year displacements from the star at the center by their measured 10-year expansion rate. The key outcome and highly unexpected result is that the principal features within the butterfly wings of NGC 6302 have a wide range of ages. This multiple ejection pattern is unique among planetary nebulae observed so far and will provide fresh insight into the stellar ejection process.

With age, proteins have an increased tendency to misfold, and the cellular processes that are meant to repair these aggregates begin to lose function. Alzheimer’s disease (AD) is partially characterized by the presence of neurofibrillary tangles and beta-amyloid senile plaques. These aggregates have been linked to neurotoxicity and the successive degeneration of neurons. We used a model organism, C. elegans, a nematode that has functional counterparts in humans, a fast life cycle, and transparent body plan. The GMC101 strain of C.elegans expresses the full-length human amyloid-beta protein, and this expression leads to quick and progressive paralysis in animals when they are temperature upshifted to 25°C at the fourth larval stage (pre-adulthood). The goal of this project is to test whether we can extend this model by inducing the paralysis phenotype, and by proxy, beta-amyloid accumulation, when animals are upshifted to a restrictive temperature in mid and late adulthood rather than pre-adulthood. Since AD is primarily a disease of old age in humans, recapitulating the beginning of symptoms in older age animals would make this model more relevant to late-onset AD. Based on preliminary results, we hypothesized that when they are upshifted to a restrictive temperature in mid or late adulthood, animals would paralyze as quickly as they do at pre-adulthood. If true, this would suggest that we can use the GMC101 model as a screening tool for late-onset AD. To test our hypothesis, I shifted animals of the GMC101 and control strain to the restrictive temperature at various adult ages and screened for paralysis using the WormBot system developed in our laboratory. I will describe results from this set of experiments and discuss preclinical screening strategies we are currently pursuing using this model.

Irritable Bowel Syndrome (IBS) is a functional condition of the bowels which affects 10% - 15% of the global population. IBS is characterized by abdominal pain and changes in bowel movement frequency and consistency. While the cause of IBS is unknown, evidence suggests that developmental and psychological factors play a significant role. Patients who score high in Adverse Childhood Experience (ACE) and Early Trauma Inventory Self Report-Short Form (ETI-SR) assessments have an increased risk of developing IBS. The purpose of this study is to compare heart rate variability (HRV) and IBS subtypes between individuals with IBS with and without a history of sexual and physical abuse. We recruited women with IBS and healthy controls using community advertisements. IBS subtypes were recorded. Participants completed measures of traumatic experience during adolescence (Childhood Trauma Questionnaire) and adulthood (Lifetime Sexual and Physical Abuse Questionnaire). HRV was measured using 12 hours of Holter ECG recordings. Participants were categorized via ROME-III diagnostic criteria for IBS. ROME-III Criteria defines IBS by recurrent abdominal pain or discomfort for 3 days per month within a 12 week period, along with stool consistency patterns, the latter of which is used to define a patient's IBS subtype:  IBS-Constipation (IBS-C), IBS-Diarrhea (IBS-D),  IBS-Mixed (IBS-M), or IBS-Unclassified (IBS-U). We hypothesize that individuals with a history of abuse will have lower night heart night-time variability, as well as specific IBS subtypes. We expect that the findings from this study to broaden our understanding of how traumatic experience in childhood impacts IBS symptoms and related physiology in adulthood, as well as the causes of IBS. Findings have implications for developing trauma-informed care practices for patients with IBS, as well as trauma-exposed children at risk of developing IBS.

Irritable bowel syndrome (IBS) is a gastrointestinal disorder that affects more than 11.2% of the global population. Anxiety, a common comorbidity, has been shown to exacerbate the progression of IBS. This relationship between anxiety and IBS is hypothesized to be connected by the gut microbiome through the gut-brain axis. The gut-brain axis is a two-way connection, in which disruption in the gut causes an imbalance in the brain and vice versa. Therefore, understanding how bacterial populations may shift among IBS patients with anxiety compared to those without anxiety could provide insight into IBS development. We have data from a cohort of patients with IBS that includes profiled bacterial populations through 16S sequencing, demographics, and Hospital Anxiety Depression Scale (HADS) scores for anxiety. We are identifying IBS patients with and without anxiety based on their HADS score for anxiety. We are comparing the ratio of two bacterial phyla (Firmicutes and Bacteroidetes) in the gut of patients with IBS without anxiety, IBS and anxiety, and healthy controls with neither IBS nor anxiety. Additionally, we are examining other factors such as race, age, and sex. We hypothesize that individuals with IBS and anxiety will have a higher ratio of Firmicutes to Bacteroidetes compared to individuals with IBS without anxiety and healthy controls. By further understanding the relationship of the gut-brain axis, we hope to introduce new research directions to improve therapeutic options for people with IBS and anxiety.

The comorbidities (e.g., depression and anxiety) often shared by Alzheimer’s disease (AD) and epilepsy patients can increase the burden of disease. Antiseizure drugs (ASDs) can affect anxiety-like behaviors of laboratory rodents; however, there is a need to better quantify the effects of therapeutically relevant doses of ASDs across age and sex. This project characterized the pharmacological sensitivity and feasibility of a rodent light-dark (LD) box assay. We hypothesized that there were age- and sex-related differences in anxiety-like behaviors and that these behaviors could be acutely modified with ASDs. Each mouse was first challenged in an open field (OF) behavioral assay to establish baseline anxiety-like behavioral phenotype. Spending more time in the center of the OF is indicative of generally reduced anxiety-like behavior. Twenty-four hours later, I randomized female and male wild-type mice aged 1-2 or 6-7 months across four intraperitoneal (IP) treatment groups: lamotrigine, levetiracetam, FG-7142, or vehicle (VEH). I selected FG-7142 as a positive control. I administered each agent before assessing anxiety-like behavior in the LD box assay. Spending more time in the dark compartment is indicative of generally heightened anxiety-like behavior. Preliminary findings indicate that female mice aged 6-months-old ambulated significantly more in the OF prior to the drug administration session (t=2.894, p=0.055). Similarly, males spent a greater percentage of their total vertical activity in the center of the OF (t=3.458, p=0.0011), suggesting generally reduced anxiety-like behaviors. LD box findings suggest that the percent of total distance traveled in the light was markedly affected by acute ASD treatment (F(3,45)=3.386, p=0.0261). Post-hoc Dunnett’s test indicated that levetiracetam administration to 6-month-old female mice reduced time spent in the light, suggesting increased anxiety-like behavior with this treatment (p=0.0374). Overall, this study established a sensitive assay to subsequently assess anxiety-like behavioral comorbidities in rodent models of neurological disease, including AD and epilepsy.

Sleep deficiency is a common challenge during pregnancy. Lack of sleep puts pregnant individuals and their fetuses at risk for poorer health outcomes. Pregnancy symptoms are known to be one of the main obstacles that prevent pregnant individuals from getting quality sleep. Mindfulness is a therapeutic practice where individuals focus on their feelings, thoughts, and bodily sensations in the present moment. Mindfulness meditation targets emotional and cognitive reactivity, which is a common pathway for developing depression and insomnia symptoms. Mindfulness-based interventions are known to help with sleep disorders, but little research has been conducted in regards to the possible relationship between mindfulness and sleep during pregnancy. A cross-sectional study was done to explore the associations amongst sleep hygiene, mood, and mindfulness. The aim of this study is to evaluate the relationship between pregnancy symptoms and morning restfulness in pregnant individuals with insomnia prior to the mindfulness intervention. We are examining the relationships among daily sleep habits, pregnancy symptoms, and quality of sleep in sleep diaries of pregnant individuals with insomnia. Preliminary findings show stronger relations between symptoms of worry and sickness to poorer sleep quality. These preliminary results will better inform mindfulness-intervention design specific to pregnant individuals with insomnia. 

Late Onset Alzheimer’s Disease (LOAD) is a common neurodegenerative disorder that affects ~5 million Americans. A primary risk factor in sporadic LOAD is the apolipoprotein E (APOE) gene, with carriers of the ɛ4 allele (ɛ3/ɛ4 heterozygotes or ɛ4/ɛ4 homozygotes) being at highest risk compared to ɛ3/ɛ3 homozygotes. LOAD is strongly sex-biased, with increased frequency in women (XX), but also increased severity in men (XY). The impact of genetic sex differences combined with APOE genotypes has not been studied. Our goal is to build models with various sex chromosome complements and APOE genotypes to understand interactions of these genetic factors in sex differences in LOAD pathology. The Disteche lab has derived isogenic pairs of human induced pluripotent stem cell (iPSC) lines with different numbers of sex chromosomes, e.g. XY/XXY, X0/XX, or X0/XXX. These pairs are genetically identical, save for their sex chromosomes. My project is to use CRISPR/Cas9 editing to generate different APOE genotypes in these paired lines. Starting from an XXY/XY isogenic pair from a heterozygote ɛ4/ɛ3, I am generating ɛ3/ɛ3 clones with either the XXY or XY genotype. To accomplish this, I transfect iPSCs with Cas9 and APOE-specific guide RNAs in the presence of a single-stranded DNA donor that contains the ɛ3 allele. After DNA cleavage and replacement, edited ɛ3/ɛ3 single-cell clones and control ɛ4/ɛ3 clones are identified using PCR and DNA sequencing, followed by karyotyping and verifying the absence of off-target effects and epigenetic instability. The new isogenic iPSC lines with different APOE genotypes and sex chromosome complements will be differentiated to LOAD-relevant cell types, including neurons, microglia, and cortical organoids, for transcriptomic and functional analyses to better understand how APOE genotypes and genetic sex interact to modulate risk in LOAD pathology.

With advancements in antiretroviral therapy (ART), the prevalence of older people living with HIV (PLWH) continues to rise. Accompanying this increase in life expectancy among PLWH, there has also been subsequent evolving symptomatology. These include an increased burden of age-related comorbidities such as increased fatigue, frailty (i.e., muscle shrinking, weakness, slowness, poor endurance), and a lower sense of perceived well-being (e.g., poor mental health, depression, self-image). Research has demonstrated that physical activity (PA) can reduce some of these symptomatologies, especially those related to physical health. However, the implications of using PA to prevent the evolving symptomatologies of older PLWH are still yet to be explored. This study examines the variability of exercise training responses between men and women with HIV. The study considers multiple scalable data and measurements (e.g., Short Physical Performance Battery, fried frailty criteria, patient health questionnaire-9). Using descriptive analysis, this study provides further insight into the impacts of exercise response for PLWH based on Sex. Sex is likely to influence the physical functioning of PLWH. Data was obtained from the High-Intensity Exercise Study to Attenuate Limitations and Train Habits in Older Adults with HIV (HEALTH). This ongoing study incorporates exercise and biobehavioral coaching interventions to determine the efficacy of high-intensity interval training (HIIT) in older PLWH. The HEALTH study involves a randomized trial of 100 older PLWH (≥ 50 years of age) who self-report fatigue and a sedentary lifestyle. Due to the current limitation of sample sizes, the study has been considered with respect to sex-based analyses in the general population. Results of the study is expected to indicate differences in the physical and mental benefits of exercise comparing sex.

We can use human pluripotent stem cells to derive cardiomyocytes (hPSC-CMs) in vitro, with the goal of transplanting them into hearts of heart attack survivors to restore contractile function. Studies using animal models have shown that transplanted hPSC-CMs can improve heart function, however they result in arrhythmias, which may not be well-tolerated. These arrhythmias are likely caused by the cells' immature electrical properties (pacemaker-like activity) and their lack of resemblance to adult cardiomyocytes. We aim to mature hPSC-CMs in vitro by controlling gene expression to engineer adult-like cells. To do this, I am studying whether DNA methylation (modification associated with transcriptional repression) plays a role in regulating hPSC-CM maturation. To investigate this, I have cultured hPSC-CMs with the methylation-inhibiting drug 5-azacytidine and used quantitative real-time PCR to measure gene expression. 5-azacytidine treatment increased expression for ATP2A2, MYL2, and SCN5A. Next, I treated hPSC-CM DNA with bisulfite reagent and used PCR to determine whether cardiac gene promoters are methylated. I found evidence of methylation at the MYL2 promoter, a maturation gene that encodes for regulatory myosin light chain and triggers contraction. To understand how methylation regulates MYL2 expression, I used a stem cell line where MYL2 is tagged with GFP. After 5-azacytidine treatment, MYL2 RNA expression increased four-fold compared to control hPSC-CMs. Next, I tested whether MYL2 expression changes after treatment due to the number of hPSC-CMs expressing MYL2 and/or the intensity of MYL2 expression in individual cells, using flow cytometry to measure GFP intensity. My data suggests the latter, and supports a role for DNA methylation in restricting hPSC-CM maturation. By regulating DNA methylation, we can engineer cells in vitro to match expression patterns measured in adult cardiomyocytes, leading to enhanced contractile function. Ultimately, this will contribute to improved hPSC-CM therapies to restore cardiac function after a heart attack.

Vestibular dysfunction is prevalent among all demographics and is commonly caused by the death of sensory hair cells within the inner ear. Vestibular dysfunction can significantly disrupt the quality of life of an individual and can have large impacts on emotional health. The primary function of hair cells is to respond to auditory and vestibular stimuli to facilitate perception of sound, head movement, and gravity.  In mammals, hair cell loss leads to permanent loss of auditory and vestibular function  due to the inability of mammals to regenerate these cells.  In contrast to mammals, zebrafish are highly regenerative and can robustly regenerate hair cells in the inner ear.  The zebrafish inner ear can serve as a functional model vestibular system to explore hearing loss, hair cell function, and to investigate genetic models of inner ear damage because of its homology with the mammalian inner ear on genetic and structural levels. Before studying regeneration in the zebrafish inner ear, we must quantify hair cell addition under homeostatic conditions. We will approach this by using a transgenic fish line(myo6b:NLS-Eos) that expresses photoconvertible protein Eos in the hair cell nuclei. By photoconverting nuclei at specific time points we can assess where new (unconverted) cells are added and determine whether old (converted) hair cells are lost.  Here, we describe efforts to optimize a method for dissection and imaging of the larval zebrafish utricle, a vestibular organ that contains hair cells, and quantify hair cell growth and turnover at early and late larval stages.  Using the zebrafish model system allows us to use a genetically encoded method of hair cell ablation to explore hair cell regeneration, and may ultimately lead to the development of therapeutics that promote hair cell regeneration in mammals.

The liver is a vital organ that secrets waste, metabolizes drugs, and breaks down blood to provide nutrients to the rest of the body. The liver is also the sole organ in the human body capable of regenerating after transplant surgery or treatment of cirrhosis, unfortunately the human body is not always efficient in the regenerative process, especially in the case of severe damage to the tissue. My research is centered around the use of 3D bio-printers to replicate the intricate biometric structures within the liver for artificial tissue transplantation. I focus on using CAD design through the SolidWorks program to design such intricate structures while simultaneously testing the resolution of the printer and its ability to print microscale channels within the structures. Additionally, I have focused on a heat producing circuit to promote biocompatibility and the efficiency of the printer during the printing process. Heat is a natural stimulus for gene expression and regulated temperature is vital for living cells. Implementing a component such as an induction heater and controlling its applied energy may be able to provide the necessary electrical current to heat cells at livable temperatures during the printing process and promote gene expression, without denaturing the cell. If 3D printers can effectively and efficiently be used to replicate microscale structures, scientist will be able to print artifical livers at low costs and fast rates for patients who are suffering from liver diseases. 

Microphysiological systems (MPS) or Organs-on-a-chip (OOC) are novel in vitro models being used to understand human disease and support drug development. Using a Kidney MPS, we are investigating potential causal factors, Ochratoxin A (OTA) and heat stress, for a disease state termed Chronic Kidney Disease of Unknown Etiology (CKDu). Thus far, we have conducted experiments treating human primary proximal tubule cells (PTECs) with OTA +/- transient heat stress (39 degrees for 24 hours) in conventional 2D cultures and MPS to understand the synergistic nephrotoxic effects of the two treatments. We found that gene expression of CDKN1A is increased in cells treated with OTA +/- heat. CDKN1A codes for the protein p21, which inhibits cyclin dependent kinases responsible for regulating the cell cycle. Chronic cell cycle arrest in G1/S and G2/M checkpoints induced by high levels of p21 can lead to injury and subsequent development of CKD. To further probe this finding, we are developing a protocol to increase the efficiency of obtaining sections of the kidney tubule from our MPS to be used for the purpose of immunocytochemistry (ICC) staining. A reagent called Optimal Cutting Temperature (OCT) solidifies the tissue into a block so that it can be cut using a cryostat. Then, we can stain OTA and heat-treated kidney tubule cross sections for Ki67, a marker for cell proliferation, and p21 using ICC. The expected results should demonstrate a higher expression of p21 in the cytoplasm and less staining of Ki67 in OTA +/- heat treated tissue in the nucleus. We have optimized antibody concentrations for staining and observed appropriate localization of Ki67 in the nucleus and p21 in the cytoplasm in 2D cell culture samples. By understanding OTA’s mechanism of toxicity, preventative interventions can be implemented to mitigate CDKu risk in impacted regions around the world.

Human adenoviruses (HAdVs) are known to infect and cause diseases in multiple organ systems. HAdV serotypes are associated with particular diseases, giving us reason to believe there is a genetic link to tissue tropism. To understand this relationship, we are using mouse adenoviruses (MAdVs). Like HAdVs, MAdV serotypes are associated with distinct tissue tropisms: MAdV-1 infects macrophages whereas MAdV-2 infects intestinal epithelial cells. By swapping genes between MAdV-1 and MAdV-2, we hope to uncover the genetic basis of tissue tropism in MAdVs. These studies are aided by the availability of a cell culture system that recapitulates the cellularity of the intestinal epithelium and supports MAdV-2 replication but not MAdV-1 replication. Phylogenic analysis indicates that MAdVs are the oldest lineage of Mastadenoviridae, the genus that includes AdVs of mammals. Thus, functions of viral genes that are conserved among viruses within the genus and important for MAdV tissue tropism are likely to apply to HAdVs. A major determinant of viral cellular tropism is receptor usage, which may also play a primary role in tissue tropism. For both MAdV species, the trimeric fiber protein that extends from the icosahedral capsid is thought to be the viral attachment protein that binds to a host receptor. MAdV-1 has been shown to use specific cell adhesion proteins called integrins as a receptor for viral entry. Although the receptor for MAdV-2 is unknown, it differs from MAdV-1. To gain insight into tissue tropism, I have designed a chimeric MAdV-1-M2f virus, where the fiber gene of MAdV-2 has been inserted in place of the native fiber gene in the MAdV-1 genome. A prior student in the Smith lab created the inverse chimera. Using these viruses, we can infect intestinal epithelial cells to measure replication. Doing so will help uncover whether the fiber/receptor interaction plays a central role in determining tissue tropism.

Rotaviruses (RVs) are non-enveloped, fecal-oral pathogens of the Reoviridae family that cause acute severe gastroenteritis. RVs infect the small intestine, where they encounter diverse host immune defenses. One intestinal innate immune mechanism is the abundant secretion of antimicrobial peptides, including alpha-defensins, which can interact with RVs and modulate their infection. Defensins are small, amphipathic, cationic peptides with broad antimicrobial activity. Our lab has recently shown that some RV strains are neutralized by alpha-defensins, while infection of other strains is enhanced or resistant to alpha-defensins. For instance, mouse rotavirus (EDIM) infection is enhanced by human enteric alpha-defensin (HD5) and rhesus macaque myeloid alpha-defensin (RMAD1), while rhesus rotavirus (RRV) is neutralized by these defensins. Using viral genetics, we identified viral protein 4 (VP4), the spike protein, as the determinant for defensin-mediated neutralization of RRV. In the intestine, VP4 is cleaved by trypsin into two subdomains, VP5* and VP8*. This cleavage makes the virus infectious. VP5* and VP8* have distinct functions, membrane penetration and cell binding, respectively. The goal of my project is to uncover the crucial subdomain of VP4 that determines defensin-mediated neutralization. To accomplish this, I designed and created plasmids encoding chimeric VP4 proteins consisting of combinations of VP5* and VP8* from defensin-enhanced RVs (EDIM) and defensin neutralized RVs (RRV and SA11). Currently, I am in the process of using a newly described RV reverse genetics system to generate infectious RVs containing these chimeric VP4 proteins. Once I have rescued my chimeric viruses, I will do infectivity assays in the presence of defensins to assess how defensins modulate the chimeric viruses compared to the wildtype RVs. By identifying the defensin determinant, we will gain important insight into the mechanism of defensin-mediated neutralization.

We analyze the International Study of Asthma and Allergies in Childhood (ISAAC) data for Seattle and apply statistical clustering methods to identify asthma phenotypes. This study focuses on Phase One of ISAAC, conducted between 1994 and 1995, in which approximately 3,000 adolescent asthma patients and their parents filled out detailed questionnaires about asthma, rhinitis and eczema symptoms. Asthma is a heterogeneous disease, meaning it has a highly variable clinical presentation, so in order to identify distinct phenotypes, we apply a hierarchical bottom-up clustering method on categorical variables from the questionnaire and verified cluster stability using various linkage methods. The clusters we obtained were distinguished primarily by differences in the severity of respiratory symptoms and the presence of eczema symptoms. After assessing the accuracy of several alternative clustering methods, we conclude by comparing the clusters identified by this analysis to clinically recognized asthma phenotypes. Accurate characterization of asthma phenotypes is important for informing management and treatment strategies for urban adolescents with asthma. Techniques that identify severe phenotypes in population data sets can help target treatment to those who may benefit from high-intensity treatment regimens, careful attention to potential exposures to environmental allergens, and specialist level care.

 As a protein’s sequence directly impacts its function, being able to understand and map protein sequence to function is integral to developing improved proteins with applications in various biotech fields such as medicine, agriculture, and energy. Machine learning algorithms can use protein mutation datasets to learn the relationship between sequence and function. The purpose of our project is to investigate the hypothesis that information encoded by convolutional neural networks (CNNs), a structure-based machine learning model, trained on protein sequences predominantly learn on secondary structural features such as alpha helices and beta sheets rather than on other regions of the protein that lack defined secondary structures. In order to test this hypothesis, we trained a CNN using the mutation data of several different proteins with the goal of finding a correlation between secondary structure fraction and model accuracy. Furthermore, in the future we will train on a protein that the CNN traditionally performed poorly on using data limited to protein mutations inside secondary structural features. If our hypothesis is upheld, the accuracy of the model will improve although fewer data are used to train the model overall. Moreover, an upheld hypothesis will help answer a frequently raised question in the literature as to why graphical CNNs (GCNs), or CNNs that can consider additional information about the tertiary structure of a protein, do not typically perform better than sequence-only CNNs: if the model does primarily learn on secondary structures, information on tertiary structure would not be expected to aid performance. The results of this project will enhance our understanding of how models learn sequence-function mapping and help produce improved models to better design new proteins with more desirable properties.

Vascular malformations (VM) are congenital malformations of veins, arteries, capillaries, and/or lymphatic vessels. They are primarily caused by post-zygotic activating mutations within oncogenes of the PI3K-MTOR-RAS-MAPK pathways. The major types of VM include capillary malformations (CM), lymphatic malformations (LM), venous malformation (VeM), and arteriovenous malformations (AVM). Identifying the molecular etiology of VM is important, as there are increasingly targeted medical therapies. However, current molecular diagnosis requires surgically excised tissues, as the causative mutations are somatic and not present in blood cells. Cell-free DNA (cfDNA) is an emerging diagnostic analyte that is already in wide use in cancer diagnosis and prenatal genetic screening.  Given the role of mutant endothelial cells in the development of VM and their proximity to blood and lymphatic fluid, we hypothesized that mutant cfDNA will be detectable in plasma or cyst fluid from individuals with VMs. This project aims to develop a non-invasive methods to identify genetic causes of VM without surgery to reduce the costs and risks relating to surgical biopsy. Utilizing digital droplet polymerase chain reaction (ddPCR) technology, we are able to amplify cfDNA fragments to detect specific well known “hotspot” DNA mutations in PIK3CA, BRAF, TEK, and others. We continue to determine the sensitivity of allele-specific cfDNA analysis in individuals with known, tissue-based mutations from a retrospective cohort, and test prospective cfDNA from VM patients with unknown mutations using both ddPCR screening and/or a high sensitivity gene sequencing panel. Ultimately, our hope is to bring these techniques into the clinical arena to increase the possibility of medical therapies for individuals with vascular malformations.

Genetic diseases affecting the skeletal system present with a wide range of symptoms and phenotypes that make diagnosis and treatment difficult. One such disease, Bruck Syndrome, is a rare form of osteogenesis imperfecta, or brittle bone disease, and has been associated with the PLOD2 gene in human genome-wide association studies. Due to advances in 3D phenotyping and CRISPR gene editing, zebrafish have become a good model system for assessing the genetic contributions to skeletal disease. While the phenotype of the homologous zebrafish gene (plod2) has been elucidated in the axial skeleton, the effects on the craniofacial skeleton have not yet been quantified. The objective of this study was to quantify how plod2 crispant zebrafish skulls differ from their wildtype siblings. We used the open-source 3DSlicer software to place 21 traditional landmarks and 372 pseudolandmarks on micro-CT scans of 22 zebrafish (11 crispant and 11 wildtype), and used geometric morphometric methods implemented in R to analyze the complex shape differences between the two groups. Preliminary results show that plod2 crispants have skulls that are wider and shorter than their wildtype siblings. The plod2 crispant fish as a group exhibit more variation than wildtype fish across the morphospace, which may be consistent with the broad array of phenotypes associated with PLOD2 mutations and Bruck Syndrome in clinical observations. Using a zebrafish model and 3D data pipeline could enable rapid screening to investigate the causes of other human skeletal diseases. In addition to these clinical implications, this project contributes to an understanding of the genotype to phenotype pipeline, one of the most active areas of biological inquiry.

In the midst of the pandemic, our team prototyped a volatile organic compound (VOC) sensor that seeks to detect COVID-19 using mechanisms from our noses: olfactory proteins. In taking the concept from design to testing, a massive amount of data was compiled and produced. Protein sequences were gathered from hundreds of publications on odorant binding proteins (OBPs) and cross-referenced against protein structures in the Protein Data Bank (PDB), sensor molecules were simulated in molecular dynamics, and candidates were screened using multiple experiment methods. I built and deployed the database that tied together signature disease VOCs, protein binding affinities, and protein and peptide sequences, along with molecular dynamics experimental results. In addtion, I had also pulled seqeunces from PDB and had contributed to the literature search. We demonstrate how intelligent data management enabled and accelerated a project to tackle rapid detection of COVID-19.

Opioid abuse is an epidemic problem in the United States, with more than 40,000 overdoses each year and a substantial increase in the past few years. Moreover, opioid withdrawal is difficult to treat because its severe withdrawal causes users to develop hypersensitivity to emotional stress and relapse, continuing the cycle of addiction. In our project, we are studying whether the Lateral Habenula (LHb), a brain region which processes stressful events, is activated by fentanyl withdrawal and communicates to the Ventral Tegmental Area (VTA), a brain region known to motivate behaviors. Our hypothesis is that the LHb-->VTA pathway is active during withdrawal or withdrawal avoidance. This hypothesis was tested with pathway-specific fiber photometry, a technique for measuring activity in an animal’s brain, specifically via a fluorescent calcium indicator (gCamp7). gCamp7 can be stimulated to emit green light that is correlated with the amount of activity in brain cells. Fiber photometry recordings were used to indicate if the LHb is activated by fentanyl withdrawal or by rats’ avoidance of a withdrawal paired chamber in a conditioned place aversion task (a psychological behavioral experiment). Independently, I sliced brain tissue, performed IP injections, recorded the LHb-->VTA pathway, ran behavioral experiments, and aided with fiber placement surgeries. I also used DeepSqueak, a software system that detects rat vocalizations to measure animals affect during behavior. ANOVA, a statistical test, was used to analyze the behavioral results of the project. By studying this significant pathway in the rat brain, we will determine if the LHb-->VTA pathway helps motivate avoidance of opioid withdrawal, and possibly find a new neural target for opioid withdrawal therapies. 

Tuberculosis is still one of the leading causes of death worldwide, primarily due to multidrug-resistant tuberculosis and lengthy drug regimens. Efforts are being made to understand the mechanisms of resistance and to develop novel antimycobacterial drugs. Drug resistance has been found in the Esx-3 type VII secretion system of Mycobacterium tuberculosis, which is involved in metal ion homeostasis. Rv0474 is a transcriptional copper-responsive regulator in M. tuberculosis with a copper binding motif. Under toxic copper conditions, Rv0474 binds to the rpoB promoter, which blocks the transcription of the RNA polymerase beta subunit leading to growth arrest. Rv0474 has also been found to be involved in mediating resistance to the aminothiazole (AmT) and 8-hydroxyquinoline (8HQ) drug series. I am studying the Rv0474 homolog (MSMEG_0918) in Mycobacterium smegmatis, a fast-growing species being used as a model organism. MSMEG_0918 lacks the copper binding motif, permitting determination of whether copper binding activity is required to confer resistance. We propose that if copper binding activity is involved in AmT/8HQ resistance, overexpression, or downregulation of MSMEG_0918 would have no effect on compound activity, because it lacks the copper binding motif. To test the effect of changes in MSMEG_0918 expression on AmT and 8HQ activity, I have constructed and sequenced recombinant plasmids containing the CRISPRi backbone and sites essential for target gene knockdown. Using CRISPRi knockdown technique, I have generated strains with reduced expression of MSMEG_0918, along with overexpression strains. Using growth curve measurements and PCR analysis, I have characterized these strains. Knockdown and overexpression strains will enable me to determine growth, AmT/8HQ sensitivity and copper sensitivity. This work will determine whether MSMEG_0918 plays an equivalent role to Rv0474 in conferring resistance to AmT and 8HQ compounds. This research will provide knowledge in understanding the mechanism of resistance to AmT and 8HQ compounds in M. tuberculosis.

Embryonic development is a process of regulated growth by which all cells are initially established. Previous studies suggest that there may be differential metabolic regulation in developing embryonic tissues. However, little research has been conducted to determine the specific metabolic factors that are differentially active during development and the key regulatory elements behind these metabolic processes, such as glycolysis. Hypoxy-inducible factor 1-alpha, hif1α, is a stress-induced transcription factor that is known to regulate glycolysis under hypoxic conditions. This project aims to investigate the role of hif1α in regulating the expression of glycolytic genes in the developing axial tissues of the Xenopus tropicalis embryo. X. tropicalis is a unique model for studying embryonic development. Due to their complete early-stage embryo cleavages, inhibitors can be restricted to one side of the embryo allowing for direct comparison with an internal control on the other side of the embryo. Preliminary data from in situ hybridization suggests that the inhibition of hif1α using translation-blocking morpholinos reduces glycolytic gene expression in early-stage X. tropicalis embryos. Based on these results, we plan to further test the regionalization of glycolysis, by determining what causes hif1α to activate a glycolytic gradient in certain tissues in the X. tropicalis embryo. This research implicates hif1α as a potentially important transcriptional regulator of glycolytic gene expression during embryonic development, and can lead to discovering new ways in which differential metabolic regulation can contribute to the form and function of embryos.

While humans cannot regrow a limb after amputation, animals like the Xenopus tropicalis tadpoles can fully regenerate their limbs. How metabolic pathways are reprogrammed to support the demand for anabolic building blocks needed for regeneration is still not understood. Our group has found that while regeneration increases glucose intake, tadpole tail regeneration does not depend on glycolysis for precursors to support proliferation. This result suggests a redirection of glucose flux to pathways other than glycolysis, specifically the pentose phosphate pathway (PPP). Previously, I inhibited glucose-6-phosphate dehydrogenase (G6PD), a key enzyme in the PPP, with the pharmacological antagonists dehydroepiandrosterone (DHEA) and G6PDi and showed that inhibitor-treated tails are much shorter compared to dimethyl sulfoxide (DMSO) controls. From these results, I predicted that the PPP is required throughout regeneration to support increased cell proliferation rates. To investigate if the PPP is required for increased cell proliferation during regeneration, I performed phosphohistone-H3 (pH3) immunohistochemistry to label mitotic cells. Histone-H3 is phosphorylated at the start of mitosis, making pH3 a marker for actively dividing cells. The results showed that amputated tadpoles with inhibited PPP have reduced cell proliferation rates compared to controls, confirming that the PPP is required to support rapid cell proliferation during regeneration. To determine if the PPP has a shorter critical activation window during regeneration, I treated amputated tadpoles with PPP inhibitors while varying the length and start-time of treatments. PPP-inhibited tadpoles have significantly shorter tails as treatment length increases, regardless of the start time for PPP inhibition. This result suggests that PPP activity must be sustained throughout regeneration to fully regrow the tail. My work has therefore identified the PPP as a previously unknown but critical metabolic pathway promoting tadpole tail regeneration. This insight advances our understanding of how metabolic reprogramming provides the carbon building blocks for regeneration.

Sub-Saharan Africa (SSA) accounts for the majority of global preterm births. Preterm birth can occur for various reasons but often the etiology is unknown. One potential cause is the impact of stress on the hypothalamic-pituitary adrenal axis which contributes to inflammatory processes. Chronic stressors may exacerbate during pregnancy and lead to adverse birth outcomes. Few studies have examined biomarkers for stress among pregnant populations in SSA who have unique considerations. To date, there is no specific biomarker for identifying individuals with high stress and an increased risk of preterm birth. In this study, we evaluate hair cortisol concentrations (HCC) as a biomarker for perinatal stress. In a cohort study in Kenya, 4500 cisgender women were enrolled during pregnancy and parent-infant pairs were followed through 9 months postpartum. HCC levels were extracted using ELISA methods from hair specimens collected from participants at 6 weeks postpartum. We evaluated the association between HCC, maternal stressors (e.g., intimate partner violence, behavioral risk for HIV), and preterm births using linear regression models. HIV risk behaviors were assessed using an empirical risk score validated to predict risk of HIV acquisition among pregnant cisgender women (high risk = scores >6). Among 153 participants included in this study, those with a partner known to be living with HIV or a unknown status had mean HCC levels that were 42.0% (95% confidence interval [CI]:4.9%- 92.2%) greater than those with HIV-negative partners (p=0.023). Individuals with high HIV acquisition risk had 43.1% (95% CI:6.1%-93.1%) higher mean HCC compared to those with lower HIV risk (p=0.019). Mean HCC levels did not differ between participants who had preterm vs. term births (mean difference 8.3%, 95% CI:-30.6%-68.8%, p=0.725). In summary, HCC levels were associated with some stressors during the perinatal period and could have implications for using HCC as a biomarker for antenatal stress. 

Peripherally restricted drugs are extremely appealing to drug developers because they remain effective while minimizing adverse side effects such as addiction. This is possible because the drugs stay out of the central nervous system, which allows them to have an impact on the body without affecting the brain. I examined Klight 1.2A transfected Neuro 2a cells under a fluorescent microscope when treated with three different drugs that are thought to be peripherally restricted. Klight is a Kappa opioid receptor agonist sensor that makes the cells fluoresce when they are activated. Neuro 2a cells are a neuroblastoma mouse cell line that is used to study signaling pathways. When I imaged these cells under different drug treatments I quantified the fluorescence. I then compared that quantity to the fluorescence of the cells when treated with U50, a full agonist, at maximal activation. I repeated this procedure for both a high and low drug concentration for each drug. The first two drugs this study examined are Difelikefalin and TP-2021 which are anti-itch drugs from Titan Pharmaceuticals. The third is Asimadonline which plays a role in hot flashes during menopause. My results of these experiments showed significant dose-dependent activation of KLight for all three drugs. These in vitro experiments on plated cells will provide a baseline which can then be compared to the in vivo fluorescence in the brain. In future experiments, when we compare the in vitro and in vivo levels of fluorescence, we can determine if the drugs are peripherally restricted or not.

 Often, after a night of observation, the images that a telescope collects will have pairs of point sources so close together that the observer will not be able to resolve the distance between them. The observer may incorrectly conclude there is one source with the combined flux of the two real sources. This source confusion is the issue that we wanted to undertake. Accurate estimations of the flux of stars are very important: astronomers use these measurements for standard candles to measure distances to nearby celestial bodies, to discover the presence of binary systems or exoplanets, and generally in time-domain astronomy. We created computer models of two-star stellar images with varying fluxes, distances between sources, and Gaussian and Poisson distributions of noise. We then wrote maximum likelihood fits to find the threshold in which you can determine the existence of two stars. Through this method, we began to determine trends in this threshold as a function of source flux ratio and distance between sources, and the noise level. We then selected a fitting function that best modeled our simulated data, which can be used to predict the distance in which a user can tell two stars apart. In the future, we plan to test our method against existing catalogs of blended stars.

The escalating epidemics of obesity and Type 2 Diabetes (T2D) are among the most impactful and costly biomedical challenges confronting modern society. Despite intensive research efforts, few non-surgical treatments are available that have shown sustained efficacy. However, growing evidence points to hypothalamic neurocircuits involved in energy homeostasis as potential therapeutic targets. Previous studies have identified fibroblast growth factor 1 (FGF1) to be an attractive candidate for the treatment of obesity and T2D as exogenous intracerebroventricular administration of FGF1 elicits potent weight-loss and anti-diabetic effects. FGF1 is also endogenously expressed in the hypothalamus, but the potential contribution of endogenous hypothalamic FGF1 signaling in brain control of energy and glucose homeostasis is unknown. We report that in both rats and mice, FGF1 is expressed by tanycytes and neurons in hypothalamic nuclei that are well-recognized to participate in energy and glucose homeostasis including the arcuate nucleus, ventromedial nucleus, dorsomedial hypothalamus, and lateral hypothalamus. However, the extent to which further cell types express FGF1 awaits further investigation. We aim to further these findings by determining endogenous FGF1 expression by hypothalamic nuclei and cell type using double-labeled immunohistochemistry to colocalize FGF1 with cell markers for neurons, astrocytes, and microglia. Next, we found that hypothalamic FGF1 mRNA expression is significantly decreased in fasted mice deprived of food for 24 h. Similarly, hypothalamic FGF1 protein levels were decreased by fasting, but rapidly increase 2 h after refeeding. Surprisingly, we found that in contrast to white adipose tissue in which FGF1 expression increases during high fat diet (HFD) feeding, hypothalamic FGF1 mRNA and protein levels are potently reduced seven days after being switched onto a HFD. These findings demonstrate that endogenous hypothalamic FGF1 mRNA and protein expression are dynamically regulated by acute changes in metabolic state, and that HFD may disrupt endogenous hypothalamic FGF1 signaling. 

Graffiti surrounds us everywhere we go. It can be found throughout Seattle, ranging from small tags or stickers that are just about anywhere to large pieces on walls and the sides of buildings. Even though it is so widespread, the locational aspects of graffiti remain unexplored. The data used in this project was collected by students using a mobile phone app as part of the fall 2021 course Archaeology 235: Exploring Graffiti: Combining Landscape Archaeology and Data Science. This study attempts to determine whether different types of locations emerge when attending to various locational factors (e.g., viewing potential and accessibility). To this end, I use various multivariate techniques (e.g., clustering, multiple correspondence analysis) utilizing various Python packages (Pandas, Scikit-Learn). These techniques reveal natural clusters within the data by grouping data points that are more similar to each other than they are to data points in other groups. Each technique does this differently. The goal of this research is to explore how locational factors of graffiti interact with each other and to investigate which clustering techniques are best at uncovering these interactions. The findings of this research show how different aspects of graffiti are associated with one another, allowing me to understand how various factors influence graffiti production. With this understanding, we can study Seattle’s graffiti scene on a larger scale.
 

Graffiti is everywhere - from the low-lying corners of a university bathroom to the soaring heights of a highway overpass. No matter where it is found, the interplay of colors, techniques, style, and wordplay act as a vortex - guiding eyes into a world of abstract creation and interpretation. Based on the sheer magnitude of graffiti present throughout Seattle, it is evident that this community has survived and continues to be quite active. From this, one may ask: What are key patterns exhibited by graffiti artists that are displayed through data analysis? Are there locations writers choose over other spots? Is the type of graffiti hindered by a quality-vs-quantity relationship? Many of these are explored to better understand writers' behavior and intent. By using landscape archaeology and data-science techniques, the observed patterns of three writers have been analyzed and further studied to understand canvas location, graffiti type, color use, visual composition, and other related components. By using data collected from the ARCHY 235: Explore Graffiti course, I have been able to culminate and process individual entries through the computing platform Jupyter Notebooks. Through Python coding, I have been able to derive maps, various descriptive statistics, and charts to help gain insight into each artist's work. Furthermore, by focusing on three specific and well-documented writers, commonalities and distinctive traits have been uncovered. An example of these findings includes the use of high-contrast black and white markers. These are used because of their possible reflective capabilities. Additionally, stickers are popular because artists can mass produce their tag and dramatically reduce the time it takes to leave their mark while significantly decreasing the chance of being caught. Thus, as a result, the study has shed light on this sociological phenomenon by focusing on examining and investigating individual practices exhibited by these artists through their graffiti.

Despite recent advancements in cancer treatment, the overall 5-year survival rate for glioblastoma, a very aggressive brain cancer, is only 7.4%. The greatest challenge in brain cancer treatment is the blood-brain barrier (BBB), a physical barrier that protects the central nervous system (CNS) from circulating solutes in the blood but prevents therapeutics from entering the brain space. While surgery is the gold-standard treatment, this procedure is high-risk. Thus, research into nanoscale injectable therapies that can cross the BBB to treat brain tumors is critical as they are non-invasive and can be targeted to specific cells. The Pun Lab is developing nanoparticles to cross the BBB via receptor-mediated transcytosis (RMT) via the transferrin receptor. To further this research, I developed a cellular model of the BBB to assess the ability of different nanoparticle formulations to cross the BBB in vitro. Specifically, I developed a Transwell culture of brain endothelial cells, which are the main regulators of the BBB due to tight junction formation. I investigated additional targeting ligands through analysis of a polymer panel to improve transport through the BBB. Finally, I will validate endosomal escape through the barrier with confocal microscopy. Through this project, I (i) developed a representative in vitro model of the BBB, (ii) explored alternative receptor-binding ligands and (iii) validated the mechanism through which the nanoparticles travel to enhance nanoparticle transport through the BBB. Ultimately, these three aims enable better direction of nanoparticle behavior in vivo and across the BBB. Non-invasive nano-therapeutics are critical to the future treatment of glioblastoma as current treatment options are limited, extremely risky, and lack long term efficacy.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by deficiency of the protein dystrophin and affects approximately 1/3500 boys. Basic/translational studies of exercise effects in DMD have been conducted for decades, but with often contradictory results concerning benefit vs. detriment especially for cardiac muscle. However, these studies have been exclusively performed in a mouse model which does not fully recapitulate the DMD cardiac phenotype. Here we present the first known characterization of exercise and physical function in a new DMD mdx rat model that better mimics human DMD. DMD mdx rats (n=6) and healthy, sibling wild types (WT, n=9) were compared in this study. I performed a battery of functional and exercise tests  when the rats were at 14-15 weeks of age, including max treadmill endurance, volitional wheel running distance, forelimb grip strength, and in-vivo hindlimb force testing. Total work performed in treadmill endurance testing was lower for DMD mdx (2.6±0.3 Kpm) vs. WT (29.3±2.2 Kpm), p<0.001. 24-hr wheel running distance was lower for DMD mdx (702±65m) vs. WT (1038±66m), p<0.001. Forelimb grip strength was lower for DMD mdx (211±16g) vs. WT (318±13g), p<0.001. Finally, in electrically-stimulated in-vivo hindlimb force testing, both muscular endurance (% of initial force at 200-s) and peak torque (at 120Hz) were less in DMDmdx (55±2%, 0.33±0.02nM*m/g) vs. WT (69±2%, 0.45±0.030nM*m/g) p<0.01. Capacity for volitional and forced exercise is severely impaired in the novel DMD rat, congruent with limitations observed in functional muscular strength and endurance. Ongoing work is examining exercise adaptations and cardiac responses in this model which may better represent training responses in DMD patients and be useful for establishing exercise guidelines in this population.

Osteoporosis is an orthopedic disease in which old bone begins to reabsorb but is not replaced by new bone. WNT16 is a critical gene influencing genetic risk for osteoporosis. The WNT16 locus harbors genetic variants associated with bone mineral density (BMD). Mice with global or conditional null mutations in Wnt16 exhibit reduced cortical bone mass and strength. Recently, our lab has shown that zebrafish with null mutations in wnt16 exhibit reduced vertebral bone length with otherwise normal bone morphology. My objective was to generate somatic mutant zebrafish with protein-truncating mutations in wnt16 and assess the effects of these mutations on adult zebrafish spine morphology and mineralization. My hypothesis was that protein-truncating mutations in wnt16 induce severe alterations in adult vertebral bone mass and morphology that are not observed in wnt16 null mutants. CRISPR-based gene editing was used to generate somatic mutants, wnt16^trunc, by targeting the terminal exon of wnt16, resulting in a prematurely truncated gene product. FishCuT was used to assess measures of bone morphology and mineralization in the spine; this included the volume, thickness, and tissue mineral density of the centrum, and neural and haemal arches. FishCuT analysis revealed wnt16^trunc mutants had significantly reduced centrum volume and length. Our results indicate that zebrafish somatic mutants with wnt16 protein-truncating mutants exhibit severe phenotypic changes and morphological abnormalities not apparent in wnt16 null mutants. This could reveal potential targets with regards to the development of therapeutic agents for manipulating WNT16 signaling in humans. These results also allow for interpretation of the functional consequences of human genetic variants in WNT16 that are predicted to result in truncated protein products. Future studies are focused on generating germline mutants with wnt16 protein-truncating mutations and determining the molecular mechanism by which such mutations give rise to severe phenotypes not present in wnt16 knockout animals.

Cone photoreceptors are specialized cells within the eye responsible for sensing light and starting the process of seeing. Using adaptive optics, a tool borrowed from astronomy, we can image these cells in a living human eye. Being able to track these individual cells over time in different images of the retina and from different imaging modalities is helpful for diagnosing, understanding, and finding ways to treat retinal diseases. But it is time consuming for even an expert to match cells between images from the same instrument and even more challenging between different modalities. And solving this problem is not as simple as performing an image match, because missing cells, image warping due to eye movement, and different lighting present challenges. This research investigates ways to algorithmically match cells between two images of the retina while overcoming these obstacles. We started with a basic algorithm to match an image of cells to itself, and then we made iterative improvements and additions to the algorithm as we ran it on images with more differences in warping and missing cells, improving its robustness. The result was a specialized algorithm to match cells based only on the local patterns of neighboring cells, which solved the problem of global warping and different lighting because local cell locations are unaffected by these factors. This algorithm helps automate the process of matching cells across multiple images taken at different times and with different instruments, and decreases the time to do so from minutes or hours to just seconds. In the future, we hope to make this algorithm more resilient to missing cells by interpolating matches in regions between areas where we have confident matches. By automating this matching task, we can aid and speed up research into these cells that are progressively affected by retinal diseases.

The eyecup consists of retinal pigment epithelium (RPE), choroid, and sclera. Eyecup tissue is used as a proxy for RPE. RPE metabolism is assumed to be dominant in eyecup preparations, but this has yet to be proven rigorously. In this study we probe the contribution of retinal pigment epithelial cells to eyecup metabolism. We approach this question by determining metabolic flux in eyecup tissue from control and mice injected with 50 mg/kg of the selective RPE cell toxin sodium iodate (NaIO₃). Seven days later we sacrificed mice and dissected eyecup tissue into physiological buffer, then quantified extracellular flux of glucose and lactate using spectrophotometric assays or intracellular flux using gas chromatography-mass spectrometry. We compared glucose flux in eyecups from saline-injected control mice to eyecups from NaIO₃-injected mice. NaIO₃ treated eyecups released 42% less lactate from media than controls (p<0.05), despite negligible glucose consumption. Surprisingly, most glycolytic and tricarboxylic acid (TCA) cycle metabolite levels were unchanged by NaIO₃ injection. NaIO₃ treatment did however significantly decrease levels of lactate and the TCA cycle metabolites malate and fumarate. Flux from glucose to lactate and malate was also decreased by NaIO₃ treatment. Our results suggest that lactate export in the eyecup is partly due to retinal pigment epithelium metabolism. However, metabolite levels and flux were partly maintained, implying that RPE metabolism may not be dominant in the eyecup. Remaining glucose metabolism may be from endothelial cells, or microglial cells recruited to the eyecup after NaIO₃ treatment. These findings may give insight into diseases affected by RPE metabolism, including some forms of retinitis pigmentosa and age-related macular degeneration. However, further analysis is still required to fully understand the role of RPE metabolism in the eyecup.

Osteoporosis is a condition in which bones become weak and brittle which leads to increased risk of bone fractures. Approximately 60-80% of bone mineral density is determined by genetics. One gene in humans that is known to contribute to bone resorption is TNFSF11. In normal conditions, TNFSF11 encodes for receptor activator of nuclear factor kappa-B ligand (RANKL). When this ligand binds its receptor RANK, this stimulates osteoclast activity and bone resorption occurs. Zebrafish are an emerging model organism for bone biomedical research. My question is: are zebrafish an appropriate model organism to study human bone biology regarding TNFSF11 and other genes that it interacts with? If so, we can use zebrafish to study this gene and eventually apply our findings to humans. My hypothesis was that zebrafish with loss-of-function mutations in the TNFSF11 gene would exhibit high bone mass, due to a decreased level of RANKL released resulting in decreased osteoclast activity. To test this, CRISPR technology was used to mutate the TNFSF11 gene rendering it functionless. 12 wildtype and 12 mutant fish that were scanned via microcomputed tomography. I generated maximum intensity projections using a program called FIJI and then analyzed the fish projections using a computer program called FishCuT (developed by Dr. Kwon and his colleagues) which analyzes bone mineral density and measurements related to bone microarchitecture. When analyzing the data using R, I was able to find statistically significant difference between bone measurements in the wildtype and mutant fish. The mutant fish had bones that were more mineralized in comparison to the wildtype fish. These data support that TNFSF11 in humans and fish serves the same function because hyper-mineralized bones are expected from decreased bone resorption. Future studies will be directed at investigating the effects of these mutations in germline mutant fish.

Syphilis is a sexually transmitted infection (STI) caused by the bacterial spirochete Treponema pallidum subsp. pallidum. Syphilis is a multistage infection with the stages aptly named primary, secondary and tertiary syphilis and can also manifest itself latently and congenitally (LaFond & Lukehart, 2006). The exact immunology of syphilis has not been clearly defined. The purpose of this research was to elucidate antigens and immunogenic epitopes of interest to help characterize the immune response against T. pallidum. In this experiment, peripheral blood mononuclear cells were enriched and T. pallidum specific CD4+ T cells were isolated and expanded from a convalescent patient blood sample. I created recombinant proteins from select orfeomes Tp870 and Tp684 of the T. pallidum genome, which had previously been predicted to stimulated CD4+ T cells. Synthetic peptides comprising these two proteins were purchased and assayed with an ELISA. By measuring the reactivity of the CD4+ T cell reactivity to each, I was able to preliminarily characterize T. pallidum epitopes. This workflow is useful for epitope discovery and progressing the understanding of syphilis immunology.

Though there are roughly 2 million people of Korean descent in the U.S., there is limited understanding of bilingual Korean-English infants’ and toddlers’ language development. Assessment of bilingual language development can be more challenging due to the lack of language-specific research. Caregiver reports of language abilities may be one way to approach measuring language development in bilingual infants and toddlers. This remote study investigates how caregiver-reported measures of receptive and expressive language differs in English versus Korean for typically developing Korean-English bilingual infants and toddlers. Participants were recruited through both online flyers and physical flyers in the greater Seattle area. To obtain caregiver-reported measures of language skills, the “Understands”, “Understands and Says” of the Korean and English MacArthur-Bates Communicative Development Inventories (CDI) were administered. The “Listening and Understanding” and “Talking” subscales of the English Vineland Adaptive Behavior Scales – Third Edition (Vineland-3) were also administered to each bilingual caregiver. A thorough background questionnaire defining each infant’s and toddler’s language exposure and Hollingshead Four Factor Index of Socioeconomic Status score were collected to facilitate interpretation of our findings. Scores from the Korean CDI, English CDI, and English Vineland-3 were age-matched and compared. With greater exposure to Korean at home, we expect higher Korean receptive and expressive language scores compared to English. Findings from this work may offer important insight into expected differences between a Korean-English bilingual child’s Korean and English language skills in typical bilingual language acquisition and may potentially shed light on early signs of speech and language delays in bilingual children. I participated in the study design, participant recruitment, and data presentation for this study.

Although the best psychological treatments, such as cognitive behavioral therapy, for anxiety disorders already rely on fear extinction mechanisms, these treatments underutilize principles of vicarious extinction learning. Vicarious extinction learning is a form of social safety learning that involves learning safety from the experience of others and can potentially enhance treatment potency. Social anxiety disorder (SAD), which is characterized by persistent social avoidance due to fear of negative evaluation by others, may uniquely benefit from vicarious extinction learning. Broader social contexts, like cultural norms, can also have influence on social learning and the efficacy in treatments of social anxiety. Although culture is a broad and nuanced construct, interdependent and independent self-construals have been shown to capture two of the many facets of culture. In this study, we propose that cultural factors, such as interdependent and independent self-construals, may impact vicarious extinction learning in people with high levels of social anxiety symptoms. Twenty socially anxious adults and twenty healthy controls will complete the Self-Construal Scale (SCS) and undergo a vicarious extinction task while undergoing fMRI. We expect that those with interdependent self-construals will show enhanced safety learning due to their value for seeing themselves as similar to others, as compared to those with independent self-construals. This will be evidenced by lower skin conductance responses (SCR) and greater ventromedial prefrontal cortex activation during the reinstatement phase of the task. Our results will highlight a potentially important cultural moderator of social safety learning and advance our understanding of the boundaries and clinical implications of learning safety from others.

Ocean acidification, the persistent lowering of pH in marine environments, is influenced by water chemistry, biological aspects, seasonal changes, and human activity. Low pH (acidic conditions) can contribute to hypoxia, coral bleaching, and other dangerous conditions for the environment. Estuarine environments contain all these influences. In this study, changes in pH in Possession Sound, WA were examined over a five-year period, with the context of changes in river discharge. This was accomplished using a YSI EXO Sonde periodically deployed nearshore in Mukilteo, Washington, USA used in partnership with Ocean Research College Academy. This site, being within the Snohomish River Estuary, is affected by both oceanic factors and the Snohomish River, including any runoff that comes through those waters. These measurements create a depiction of changes in pH mostly due to seasonal factors, like river discharge and upwelling. Early results from these data demonstrate a clear seasonal pattern without significant annual trends toward lower pH. As climate change progresses, consistent monitoring of ocean pH will be essential to understanding the effects of ocean acidification and the ways we might combat them in the future. While this study was limited by its short timeframe, these results provide an important baseline for continued collection and analysis of these data.

Combined sewer outflow (CSO) overflows are a threat to water quality, particularly in such urbanized areas as the Puget Sound. CSOs contribute to spills of untreated sewage mixed with stormwater that wash into water systems during heavy rain events. Possession Sound, fed by the Snohomish River, has 13 CSO outfalls in Everett, Washington, some of which experience CSO events fairly regularly. Contaminants in these CSO overflows can release pathogens, solids, nutrients, toxins, and oxygen-consuming pollutants into the water. These variables can in turn affect DO mg/L (dissolved oxygen) and turbidity NTU (measure of water clarity) – two important measures of water quality. Past research has found that wastewater spills cause major decreases in DO and increases in turbidity. DO and turbidity data were collected using a CTD in the Everett Marina throughout 2019. This data, in addition to the combined volume of water discharged from the CSOs during overflows, the duration of these spills, and the depth of precipitation in inches during the overflow, were analyzed using the Principal Component Analysis (PCA) method to find which components were most effecting the change in DO and turbidity. Using the components that covered over 90% of the variability in the data from the PCA, a Principal Component Regression (PCR) was made to be the foundation for two predictive models, one for projected DO, the other for turbidity. It is expected that a regression based on these components will make a model that covers the majority of change in DO and turbidity with a statistically significant R2 value. These models may make analysis of the effects of CSO overflows on Possession Sound a much simpler process and provide important insights into the impacts of these overflows on water quality.

Possession Sound, a fjord-type estuary system in the Salish Sea, is home not only to incredible biodiversity but also to some unique clines. A cline, such as a thermocline or a halocline, is a portion of the water column where a physical property changes significantly with depth. The haloclines of the Possession Sound fjord system vary periodically in stratification, particularly at river mouths. One such area can be found at the mouth of the Snohomish River, where cold freshwater flows into warmer seawater, creating a stratified but unstable water column. This study aims to draw connections and seek out patterns between current speeds and temperatures. Temperature data was collected from the Ocean Research College Academy's Conductivity, Temperature and Depth (CTD) sensor mooring at the Everett Marina, and current speed data was collected from their Acoustic Doppler Current Profiler (ADCP) in the same location. Prior research suggests that during most parts of the tidal cycle, temperature readings at the surface correlate strongly with tidal stages: the surface becomes colder when river water flows out to sea at low tide and warmer as the seawater pushes the river water back at high tide. However, when the tide cycle becomes less intense and current speeds decrease, this correlation becomes muddied. It is hypothesized that this pattern represents a decrease in thermocline stratification during periods of slower current speeds. Prior research done on this correlation at the mouth of the Snohomish River lacked in scope and statistical support. By expanding the scope by several months and incorporating statistical support, this study has reinforced previous findings, supporting the hypothesis posed previously: a linear correlation between the variables was supported with a p-value of 2.89E-25, and the inverse linear correlation between temperature and tide height was stronger during periods of greater average current speed.

Ocean acidification is a global crisis that is mainly caused by too much carbon dioxide in the atmosphere being absorbed by bodies of water, altering the water chemistry. Ocean acidification has large visual consequences, such as the bleaching of coral reefs, but less obvious, small scale influences are also found in the Salish Sea. A major indication of global warming’s effects on local water systems is pH, or the measure of how acidic or basic a solution is. Previous global studies have shown that pH has been decreasing (becoming more acidic) over the years, while the overall temperatures have been rising. The goal of this study is to observe how changes in lowernig pH are related to temperature changes in the watercolumn of Possession Sound, a salt wedge estuary, near Everett, Washington. We utilized pH and temperature vertical profiles collected from a YSI EXO Sonde, a deployment device that utilizes sensors and precise calibrations to monitor water quality, over six years to assess the degree of ocean acidification locally. Trends were analyzed according to depth and season. Preliminary studies of this particular site have shown minor changes compared to the extreme trends recorded in other ocean environments. Given the potential for negative impacts on the estuary, it is worth expanding the study by investigating a longer time frame. Local estuary data regarding depth and season will allow people to better understand how these variables change in our environment and gain a greater understanding of climate change’s influences on ocean acidification locally.

Harbor seals fill a critical role in the balance of the Salish Sea. Prey availability is known to be a strong indicator of seal presence; however, there are many more subtle environmental influences on harbor seal presence as well. This study hones in on the harbor seals of the Snohomish River Estuary and how their haul-out habits may be influenced by the unique water circulation of the area. This study analyzed data compiled by the Ocean Research College Academy at multiple log boom haul-out sites in the Snohomish River from 2015-2022. I analyzed seal data through the lens of the tide's movement of water in this estuary and compiled tide data from the National Oceanic and Atmospheric Administration (NOAA). I expected that there would be an increase in seals hauled-out at flood tide as well as in the beginning of the ebbing tide due to the colder temperatures experienced during high tide. Early results suggest no direct or strong correlations between tidal height and overall seal presence at sampling sites. This study seeks to better understand the presence and behavior of harbor seals at the mouth of the Snohomish River. 

Nutrient levels in marine environments can vary widely due to local geography, the placement of various manmade input sources, seasonal factors, and tidal patterns. They are important in understanding the overall health of an ecosystem, as they can be an indicator of potential pollution. They also have a significant impact on plankton populations and, as a result, primary production. Unnaturally high nutrient levels can affect other water chemistry variables, contributing to events such as harmful algal blooms, hypoxia, and ocean acidification. In this study, I analyze 13 years of nutrient data from ten Possession Sound sampling sites, at varying distances from the mouth of the Snohomish River. Nitrate and phosphate levels were analyzed temporally, and tidal, weather, and river discharge data was overlaid to analyze the relationship between nutrients and other facets of the surrounding environment. My early analysis indicates that seasons play a large role in nutrient levels, likely due to the weather of the Pacific Northwest and runoff from the Snohomish River. Figures also support the relative similarity of values between sites, showing that nutrient levels in the Snohomish River estuary are collectively affected by nutrient flow rather than having site specific characteristics. Studies of this type can provide insight about specific characteristics of our local nutrient pathways and can provide context for changes in our ecosystem. For further research, oceanic parameters such as dissolved oxygen, pH levels, and plankton densities should be analyzed in comparison to nutrients in order to gain a better understanding of the actual relative impact of nutrients in this local marine system.

Pugettia producta, native to the Salish Sea, alter their diet seasonally based on availability, shifting from algae in spring and summer to mussels in fall and winter. Our hypothesis was that when presented with both algae and mussels simultaneously, the kelp crabs would prioritize one food over another. Additionally, the environmental conditions of a tank may also have an effect on the amount of food consumed. We also hypothesized that by mimicking the seafloor, the consumption rate would be higher. Crabs were placed in two tanks, one with sediment and one without, and were accompanied by a measured amount of bull kelp and mussels. Tanks were covered nightly at sunset to mimic outdoor lighting conditions, and salinity, pH, water flow, and temperature were regulated evenly for both tanks. Measurements were taken daily of the remaining food supply, and each trial was run for 48 hours. We found that the tank with the sediment had less food remaining, and in both tanks, more algae was consumed than mussels. Algae may have been preferred because it takes less energy to consume, which may mean that kelp crabs spend more energy eating in the winter, and likely suffer a tradeoff with less energy to spend on other factors, such as growth and reproduction. Imitating the sea floor may have made a difference in alleviating stress levels in the crabs, which led to more consumption. Understanding dietary preferences is important when considering the potential future effects of anthropogenic climate change and ocean acidification on marine ecosystems.  Additionally, mimicking field conditions in a laboratory in conjunction with manipulating variables of interest for any experiment likely provides more accurate results than an empty tank, and may be a viable option wherever possible to obtain results representative of a natural environment when field experiments are not possible.  

In any marine ecosystem, water currents are an important factor in both the biological aspects and the physical movements of a body of water. The focus of this study, the Possession Sound estuary in the Salish Sea, lies in an interesting area that is affected both by discharge from the Snohomish River and surrounding streams, and incoming ocean currents from the Pacific Ocean. In Possession Sound the currents can affect everything from the regular boat traffic through the area to the transportation of debris and other natural or harmful substances in the water. I used an Acoustic Doppler Current Profiler (ADCP), moored in the Everett marina at the mouth of the Snohomish River, to collect data on current velocity for 10–30-minute intervals over the period of seven months in 2020, and almost two months in 2021. The collated data I then used to analyze the current directions and speeds of the water to determine potential local trends in the currents. Preliminary analysis shows that the currents flowing near the moored ADCP tend to flow north and south with fewer currents going to the east or west. However, the currents going east and west are often faster than the north and south streams. These two trends are likely caused by the north moving ocean currents and the south moving river currents, but more research utilizing related data such as river discharge is necessary. Because of the estuary’s diverse current sources, the analysis of these data allows for a greater understanding of the movements of the water column, and insight into the transportation of important substances within it such as nutrients and heavy metals.

Possession Sound, located in Everett, Washington, includes the second largest source of freshwater in Puget Sound from the Snohomish River. This salt-wedge estuary serves as a home to a wide selection of phytoplankton, which provide energy to a variety of organisms farther up the food chain. Water chemistry often determines where phytoplankton accumulate prior to their recycling as nutrients. Ocean Research College Academy students utilize water chemistry data (temperature, salinity, dissolved oxygen, pH, turbidity and chlorophyll concentration) from two sensors deployed in Possession Sound: One in the river and one two miles away at Mukilteo. While plankton samples are collected in the Sound, rarely are plankton collected in the river and compared to chlorophyll concentrations. This study will look at abundance and diversity of phytoplankton collected in the river at various tide stages and compare these to Mukilteo samples. I hypothesize that flood tide samples will be similar, while ebb tide phytoplankton and chlorophyll levels will decrease. The preliminary data revealed that chlorophyll and temperature levels did not vary significantly between the two sites despite the widely differing salinity levels. The next steps of the study are to determine the plankton density of phytoplankton species across the two most recent years of data. Results will enable us to explore further into plankton presence in relation to chemical variance in water systems. 

Insects have superlative capabilities over contemporary robots: increased mobility, redundancy, coverage area, and can utilize different sensors. Perhaps most importantly, having reduced mass, launch costs can be exponentially lower. The goal of my research project was to create a simulation to compute the pathway/logistics of an insect robot landing on Mars, code a simulation, linearize data and arrays, and learn more about insect robots and space to investigate how to land insect-sized flying robots on Mars. I first reviewed existing information on spacecraft transiting from Earth to Mars, including the need to protect insect-sized robots from space and radiation while in transit. Of great interest would be potentially different de-orbiting and landing scenarios given a lower mass spacecraft, which is still traveling a hundred times faster than a bullet during initial deorbiting. My research speculated that simpler landing strategies like Spirit and Opportunity vs Endurance might be employed for the carrier spacecraft, and once on the surface, a carrier could deploy a small rover to act as a home base supplying power and communications to the flying insect-sized robots, greatly extending the range of the science data collection. My research captured the general characteristics of insect robotics and using a Python program I created, simulated reentry paths and maximum heating rates, which were still high as expected. My next steps would be to test different ballistic coefficients to see if a small payload direct from deorbit landing is possible. The broader implication is the potential for delivering many tiny distributed sensors on Mars to dramatically improve our understanding of the planet and at a lower cost.

Rotary encoders are present in many electronic devices, and are used to measure changes in rotation. The common photo type encoder has remained largely unchanged in its fundamental operation, and faces limitations in resolution at small sizes. This research explores an encoder design utilizing polarizers, that can achieve higher resolutions than similarly sized photo encoders. Current photo encoders achieve their rotational measurements through a rotating disk, with perforations located along the circumference. As the disk rotates, a photodiode receives pulses of light through the perforations from a light source. The resulting signal(s) consists of a multitude of digital pulses that require compiling and processing, before position can be determined. To achieve higher resolutions, more perforations are required, and more photodiodes are needed. High-resolution encoders can therefore become very expensive as the perforations increase, and the perforations, and therefore resolution, is limited at smaller sizes. However, there is a way to inexpensively achieve indefinite resolution and absolute position, at sizes that are unfeasible with current encoder technologies. Such an encoder utilizes an initially polarized light source, followed by an analyzer polarizer, and then a photodiode. As the analyzer is rotated, the photodiode receives a gradually increasing, and then decreasing, change in brightness (translating into current). Rotating the analyzer continuously, produces a sine graph of current change, with each point corresponding to a position of the analyzer. In this research, this configuration has been adapted to create a small scale, and high-resolution rotary encoder. This polarizer encoder measures only ten millimeters in diameter, and achieves greater resolution than current similarly sized rotary encoders.

Our lab has recently implemented a virtual reality (VR) experimental platform for research with human patients undergoing invasive, intracranial neural monitoring for seizure localization. The goal of our project is to design a software interface enabling high-dimensional kinematic tracking during commercial VR gameplay. This will allow us to collect a large dataset of synchronized neural signals and continuous kinematic variables such as hand location and angular velocity during the dynamic/naturalistic human movements evoked by VR gameplay, that can be used to support neural decoding of 3D human movement for brain computer interface (BCI) development in labs at UW. It can also be published as a benchmark dataset for human movement decoding to the larger community of computational neuroscientists. Currently, only 2D datasets of human movement tracking during concurrent intracranial neural recording have been provided to the larger computational community. The distribution of a 3D movement dataset with intracranial neural recording is thus vital and would facilitate many avenues of clinical, computational, and neural network (ANN) modeling research. Specifically, our project is to create a software interface, called a “layer”, that can record VR controller/headset positions and other tracked variables while a subject plays a commercial VR game that utilizes the industry-standard OpenXR framework. Our layer works by intercepting different functions in the OpenXR framework, and uses this to track movement data. Specifically, our layer will include a manifest file which holds our layer’s identification, and intercepts commands such as xrNegotiateLoaderApiLayerInterface, xrNegotiateLoaderInfo, xrCreateActionSpace, and more to specifically track the movement. The positions and data collected will be outputted to a .json file which will then be used in the clinical research detailed above.

As Internet of Things (IoT) devices--networked computing technologies embedded directly in objects that interact with or sense the environment in some way--are increasingly incorporated into the built environment, the amount of data collected on users simultaneously increases. From this arises a quantitative concern of managing data points and the security of devices, as well as a societal concern of ensuring privacy. Thus, it is imperative to address not only the benefits of IoT devices, but also the tangible and potential harms, including privacy harms. From this baseline, I have researched the following question: How does the data collected by connected devices--both aggregate and personally-identifiable--affect the appropriate flow of information and reshape conceptions of privacy? In studying this question, I have centered my methodology on analyzing the privacy risks of devices in both public and private spaces by conducting a literature review of privacy theory and policy. In doing so, preliminary results have yielded that, while privacy policies may be targeted to one generic user, privacy harm does not affect populations congruently. My research has visualized how subjective and objective privacy harms in the built environment have varying effects on individuals, as people's conceptions of privacy are not congruent to start with and are reshaped differentially by connected devices. To curb these vulnerabilities and differing effects, scholars have proposed a social contract theory of privacy. Instead of placing the burden of privacy on a single actor, privacy may instead be seen as a social contract, in which individuals and groups obtain the agency to determine how their information is communicated. In this way, the differential vulnerabilities of populations can be preemptively accounted for, reducing the risk of privacy harm in a proactive manner.

The resistive random-access memory (RRAM) is a promising candidate for next-generation nonvolatile memory (NVM). This technology is considered one of the most standout emerging memory technologies due to its potential high storage density, fast access speed, low power consumption, and low cost. If the technology is well-understood, it could significantly change the memory industry and trigger more advancement in computing and, thus, the scientific field. One of the most widely discussed applications of resistive memories is neuromorphic computing. Neuromorphic computing uses very large-scale integrated circuits to mimic neurological architectures. The human brain can compute with extremely low energy consumption since neurons and synapses combine storage and computation. However, the scalability of current semiconductor technology could be problematic as such synapses density in the neurological system is hard to achieve by conventional means. Resistive random-access memories could ease the problem by providing high scalability and computing capacity to mimic neuron and synaptic functions and thus push important technological advancements in autonomous systems, which learn and interact with the environment in real-time and open the door to so many possibilities. The research project’s main focus would be the theoretical modeling and simulations of the properties and behaviors of the conductive filament in the resistive random-access memories using high-performance computing (HPC) resources at the University of Washington. More simulations will be done to understand further the physical principles behind two configurations: unipolar RRAM and bipolar RRAM. In unipolar RRAM, the voltage to reset and set the memory cell has the same polarity, while in bipolar RRAM, the voltage to reset and set has reverse polarity. While some of the theoretical models are still not complete, these simulations might help understand the phenomenon and shed some light on future modeling.

The thermal optimization of computer systems is a study dating back to their creation. Because powerful computers typically generate additional heat, the more heat one can remove from a computer, the more powerful they can make it. Over the years, it has become a popular solution for many companies to build computers using standardized, modular hardware. Our research questions the cooling efficiency of this standardized hardware. Initially, we are creating a 3D simulation of a desktop computer that will allow us to quickly test various internal component layouts for thermal efficiency. Using a custom-programmed microcontroller for data collection, our real-world testing includes the building and monitoring of both a computer built in this standardized fashion and one reconfigured to other custom layouts, which, from our simulations, we anticipate will improve the cooling capabilities of the computer. By using this data in conjunction with observations from our digital 3D simulations, we hope to test potential improvements to the layout of computer components to enhance the performance of both high-end computers and everyday desktops.

Computer vision is a branch of artificial intelligence that involves applications of mathematical methods and computers for machine learning from digital images and videos. Here, we apply computer vision-based methods for optical character recognition (OCR) and image compression. OCR has important applications such as process automation like check clearing, digitizing text and image records for online databases, automated analysis of surveillance camera videos for security, automated reading of text from car license plates in a parking lot, etc. But how can we feed visual information to a computer in a form that it can understand and operate on? To this end, we digitized images into vectorized arrays and analyzed data using vector and scalar projections. Further, we applied algorithms with foundations in linear algebra and wrote programs using Python scientific libraries for optical character recognition and image compression. Using IPython, we characterized color and grayscale images as arrays and implemented singular value decompositions (SVD) and principal component analysis (PCA) for grayscale and color image compression studies and OCR. These studies illustrate how mathematical transformations and data reduction methods can be used for optical character recognition, image compression, identification and encryption. This project elucidates the key role of mathematical modeling for computer vision applications.

Motion algorithms are foundational for effective autonomous robot movement. For surface robotics, one particularly useful algorithm is known as pure pursuit, where a robot follows a point along a path that is a constant distance away from the robot. This work hopes to improve the pure pursuit motion algorithm to account for differences in the robot's features by implementing closed loop full state feedback (FSF) control. In addition, this project aims to provide more abilities to the pure pursuit algorithm, such as specification of angle at each point, allow for moving points, and ensure fast and efficient movements. The algorithm additions are made by modifying the calculations or control loop, and using simulations to verify effectiveness. So far, this work has shown promise by enabling intricate movements while being effective. As a whole, the role of this research is to make pure pursuit more useful and effective for any robot operating on a surface.

According to the World Bank, Somalia has a mere gross domestic product (GDP) per capita of $309 USD, as compared to the $63,500 of the United States. The negative impacts of terrorists, government corruption, and poverty/famine have left Somalia a war-torn country battling a 30-year civil war. Due to this, foreign countries have become reluctant to provide aid. Fortunately, there has been some viable potential for economic growth through partnership of Non-government Organizations (NGOs) and promoting civic engagement. Recently, returnees and Internally Displaced Persons (IDPs), who were forced to flee the country due to war and economic instability, have returned and partnered with government officials to take an initiative to improve the country's well-being. The question this report seeks to answer is what methods can be applied to solve Somalia's economic dilemma? Economists know the most important factors that determine a country’s well-being are education, infrastructure, human capital, and literacy rate to name a few. To gain better insight into each of these categories, I compared case studies of the economic driving factors of now industrially advanced countries. Countries like South Korea and Singapore who once experienced economic depravity, I then compared those economic factors against Somalia’s economy. I have found that from an economic standpoint, Somalia should focus on free or low-cost education for the masses, enabling international trade through specialization, and improving productivity per citizen through supplying more jobs.

The student movement of the late 1960s roiled postwar West Germany with its demands for sweeping social and political change, leaving a controversial legacy. The purpose of this research is to analyze the concrete policy impact of the student movement on West German politics in the areas of education, democratization, and select foreign policy issues. The presentation consists of two parts: First, I describe key demands of the student movement as addressed in student-produced flyers from the 1960s, during interviews with student leader Rudi Dutschke, and by German scholars and journalists. Second, I present the results of a comparative analysis of the election programs of West Germany’s center-right Christian Democratic Union (CDU) and center-left Social Democratic Party (SPD) in the years 1965 and 1969 and discuss how these parties responded to the student movement. The results show that the student movement succeeded in influencing both parties, with the CDU and SPD addressing many of the students’ core concerns for the first time in 1969. While the parties yielded to student demands in certain areas, including education reform and parliamentary reform, they opposed students’ demands on other issues, including internal security and international military alliances. By examining the impact of the student movement on the two parties’ election programs, my research provides insight into institutional responses to popular movements, illustrating both the potential of these movements to shape the political agenda and the limits of their influence.

Washington state is rapidly increasing renewable energy infrastructure, such as utility solar, to meet the state’s zero emission goals by 2045. However, struggles over land have raised concerns about the impacts of utility solar on largely rural, energy producing communities. Those impacts are spread unevenly across class, race, and indigeneity. In Klickitat County, residents of Goldendale have mobilized to create CEASE – Citizens Educated Against Solar Energy. Some Yakama Nation members have worked with the group to oppose solar infrastructure. I am investigating how solar energy impacts energy producing communities. I am conducting ethnographic interviews with relevant parties to solar siting in Klickitat, including: CEASE, members of the Yakama Nation, political representatives, developers, and agricultural organizations. This research expands on the growing field of energy justice literature that investigates how racial capitalism functions within energy systems. Is the use of rural and tribal land for energy extraction evidence of perpetual dispossession and colonization? Solar corporations in Goldendale are using state apparatuses to privitize and profit from rural and indigenous land, placing the burdens of energy production on rural residents and indigenous communities. Research on land use and solar energy helps us better understand how the clean energy transition interacts with power inequities, class, and indigeneity.

There is a long and unfortunate history of violence in communities of color at the hands of police. I aim to examine the role of police in the oppression of vulnerable communities, specifically communities of color, and how community alternatives bring equity to the justice system. Community alternatives are programs that divest from or abolish police departments. Divestment is a practice by which funds are redirected away from police towards community-focused departments and programs. Abolition is a practice that involves the full-on breakdown of a police department to replace it with community policing or mental health services. I would advocate for divesting first with abolition as a secondary measure should divestment fail. Some preliminary studies in Denver and Oakland, show a savings of $34 million dollars by removing police from schools. Austin and San Francisco have reallocated funding from police departments into programs like housing and healthcare. These early results are evidence of the efficacy of moving to a reallocation or abolitionist model. To explore this I have examined the history of policing in America, as well as current reports and investigations by civil rights groups and federal agencies to analyze historical and continued oppression by police departments. I have also reviewed abolitionist literature in order to paint the most accurate picture of the more equitable justice system. This research has revealed data from investigations into police departments from cities like Ferguson and Minneapolis that display gross abuses of power against people of color. Abolitionist literature discusses solutions such as the debate surrounding including social workers in the law enforcement process. The majority of the research and literature surrounding abolition is fairly young, so going forward information relevant to the topic will be monitored. Ultimately, the goal of reimagining policing is to ensure safety, security, and equity for all.

This project explores gender and identity during the Siege of Leningrad (September 8 1941 - January 27 1944) as articulated by child diarists Elena Mukhina and Iura Riabinkin. Iura is a disabled young man, who struggles to reestablish a sense of masculinity and purpose after being rejected from the army; he slowly loses his physical strength, a pillar of his male identity, due to extreme starvation and grapples with feelings of worthlessness. Lena is a young woman who grounds her femininity, and thus her self-worth, in her work as a hospital orderly; once she is required to return to school, she attempts to commit herself to the genderless “Soviet student” ideal but finds difficulty in relinquishing her feminine identity. During the German encirclement of the city during WWII, Leningraders suffered tremendously, fighting to survive without ample food, electricity, or water, all while isolated from the rest of the USSR. This deeply traumatic experience redefined Leningrad’s cultural landscape, specifically raising questions about Soviet gender culture. In their diaries, these two 16-year-old students demonstrate the changing norms through their perceptions of traditional gender roles, self-worth, and their own masculinity and femininity. This project focuses on the diarists’ use of gendered language and how it changes over the course of the war, offering historical context to suggest explanations for these changes. In the academic discourse surrounding the Siege, scholars have approached the event as a psychological phenomenon or a primarily female experience, but rarely as a child’s experience. Iura’s diary is the only published and translated diary by a male child, even then only available in excerpts, while Lena is one of very few female child diarists published in full. Through this project, I aim to illuminate this neglected aspect of Siege study and illustrate the nuanced gender discussion explored by these extraordinary children.

My research questions for this paper are: What are the effects of the Extra Score Policy on Indigenous student enrollment in colleges and universities in Taiwan and is the Aboriginal Language Proficiency Certificate, in the context of the Extra Score Policy, an effective method of language revitalization? My hypotheses are that this policy has increased Indigenous student enrollment in colleges in Taiwan and the Aboriginal Language Proficiency Certificate is an effective method of language revitalization. Indigenous students have a higher chance at attending colleges with a boost to their university entrance exam score and with the addition of the Aboriginal Language Proficiency Certificate, students can receive an even greater boost in their score, so it should incentivize students to learn their Indigenous language to receive a higher score. Studying the enrollment and university population trends among Indigenous students, as well as the Aboriginal Language Proficiency Exam results, I examine the impacts of the Extra Score Policy. Through my study, I found that the Extra Score Policy increased Indigenous student enrollment in colleges and universities in Taiwan. However, I also found that there is no significant data which prove that the Aboriginal Language Proficiency Exam is an effective method of language revitalization. While this policy enables greater Indigenous student enrollment in college, Indigenous students are not attaining a college education at an equitable rate in comparison to non-Indigenous students. Furthermore, students do not have the proper support in these colleges to graduate from college and receive an equitable education. In turn, the implications of my research suggest that one policy cannot undo a system of inequity. Rather it is necessary to use this policy as a starting point to understand the impacts of it and highlight areas of improvements ranging from financial support for Indigenous students to culturally competent curricula.

The goal of my project is twofold: firstly, to analyze the philosopher Iris Marion Young’s (1949-2006) application of a “social connection model” to the problem of responsibility for historical injustice in her book Responsibility for Justice (2011) and pose a critique to this model in addressing harms against Black Americans. I do this by putting her chapter on historical injustice in conversation with Roy Brooks’ Atonement and Forgiveness (2004), a work of legal scholarship on the similarly constructed “atonement model” of reparations in the case of anti-Black racial injustice in the United States. Secondly, I hope to explore new avenues of philosophical research that are conducted in partnership with reparations organizations and individuals with a clear connection to specific past injustices. I do this through analyzing three group interviews of 3-4 activists each (Black, white, and integrated) and making use of three reparationist conference recordings in collaboration with the African American Redress Network (AARN), a coalition of national, state, and local reparations organizations. Overall, I argue that neither Young’s social connection model nor Brooks’ atonement model offer a satisfying solution to the problem of conceptualizing the Black redress movement in light of testimonies by and interviews with reparations activists. Instead, I propose a relational approach based on the principles of relational egalitarianism and the concept of “moral repair” in Margaret Urban Walker’s 2006 book of the same name. In this way, I hope to not only bring more on-the-ground perspectives into the philosophical debate on Black redress (and historical redress more generally), but also reflect on ways that philosophy can be reconceptualized as a tool to achieve racial justice goals.

This research will address climate change and its impact on migration to the US, specifically how it impacts Central America. Migration from this region is at an all-time high. Climate change, especially droughts and the decrease in precipitation, as well as extreme weather events, have agitated migration patterns in Central America and will compound in the near future with the increase of climate refugees from the region, putting additional stress on the already failing US immigration system. Before this problem gets out of hand, research needs to be conducted to understand the severity of the situation and steps towards a solution. This paper aims to uncover hidden data that often goes unseen that demonstrates the hardships and impossible situations that inhabitants of Central America are faced with. These hardships, compounded with new and emerging climate crises, are forcing many to migrate to more hospitable environments, namely, the United States. This study will first begin with an overview of the current situation and explain the problem at hand. It will then delve into the personal experiences of the participants of this study, those that are immigrants themselves, and those that have various and unique perspectives on this climate migration. This research will study how they have been impacted by climate-related issues such as extreme weather events, variability of precipitation, crop failure, etc. Finally, it will discuss the future of this issue and propose solutions and methods of adaptation for the coming decades.

Preferred methods to engage with music vary among d/Deaf and Hard of Hearing people (d/DHH), depending on a number of social, environmental, and technological factors. Previous research on music for the d/DHH population has pursued specific methods such as dance, sound vibrations, and captioning. Other literature has focused on applying a hearing standard of music engagement and fluency to the d/DHH community and is outdated with regards to technological advances that improve music access. In this generative research, our goals are to understand how various social and technical environments affect music experiences and discover design opportunities to improve music accessibility for d/DHH people. We are conducting semi-structured interviews with d/DHH adults ages 18-49. These interviews undergo design research activities consisting of word exercise, image exercise, and cognitive mapping to uncover the background, current, and ideal music experiences of participants. We are developing a design prototype based on research findings that addresses the accessibility shortcomings of music experiences and adapts to numerous music scenarios and environments. This prototype can be used as a tool to inform future design solutions for existing music platforms to accommodate the needs and wants of the d/DHH community.

I, along with advisor Dr. Simpkins and student peer Katie Ward conducted research on integrating disability studies in their classrooms, by surveying faculty and students who self-describe as having a disability across an extensive tri-campus state school system. With an emphasis on intersectionality, interdisciplinarity, and interrogating diversity, the team researchers, including myself, will offer tools for bringing disability studies into conversation with existing curricula. In spending the past two quarters on this project, I have extensively read about disability, intersectionality, and interdisciplinarity. I have since utilized these readings to correctly write the surveys the team used for collecting data, being a significant contributor to our paper, and being a significant contributor to the term reference collection and academic resource collection. At the symposium, I will outlay the progress our team has made thus far, including writing the survey, sending them out to all three University of Washington campuses, and receiving the data back to analyze it and make conclusive understandings. I will also present the disability term book and resource collection that we compiled for faculty, staff, and students to access utilizing disability curriculum content. The rest of the team and I will then address our future hopes for implementing a disability curriculum and the concerns of both groups with integrating disability studies in classrooms across the curriculum. I will also describe their use of Pressbooks to publish an open-source resource that collects their findings; this resource offers suggested materials, teaching strategies, and research guides to support instructors who are less familiar with disability culture and disability studies to bring disability-related content into their teaching. 

The COVID-19 pandemic necessitated a shift to online learning options which brought significant challenges to courses with major hands-on components. The Remote Hub Lab (RHLab) of the University of Washington's Electrical and Computer Engineering department was established in response to the pivot to online instruction. The RHLab provided students with remote access to industry-grade hardware, an initiative that eliminated the need for shipping lab kits during the pandemic as well as the maintenance and purchase of new components after the return to in-person instruction. The lab has been used successfully since Autumn 2020 in delivering the Electrical Engineering (EE) 371 course. A recent study showed that students who use the RHLab for assignments have similar learning outcomes to those in traditional offerings with physical lab kits. This research builds on that study, exploring educational improvements to engineering laboratories and seeking to increase scalability and advance accessibility for wider audiences and underrepresented communities. By discovering how engineering laboratories can be more accessible for learners in a post-pandemic lens, this research identifies an optimal combination of remote methods for engineering laboratories through three data-gathering approaches. First, I conducted a literature review about equitable access in engineering education and remote engineering labs in a post-pandemic context. Then, I conducted a survey among winter 2022 EE 371 students, qualifying and quantifying their definition of equitable and scalable access. Finally, I developed an introductory digital logic lab curriculum which leads to Hardware Description Language (HDL) proficiency. These primer projects which leverage the RHLab allow for self-guided exploration of digital logic, and the curriculum will be used in future RHLab documentation and advancements. Expected findings include the surveyed students’ suggestions for improving the accessibility and scalability of remote labs and engineering education, results which will be implemented in the RHLab’s future work.

Child-to-Parent Violence (CPV) is defined as youth's actions to gain power and control over a parent or caregiver with the deliberate and pervasive use of physical, psychological, and/or financial means. Research shows that 7-22% of the population experiences CPV, also known as APV (Adolescent-to-Parent Abuse). CPV is one of the most rapidly growing, underreported, and under-researched societal problems. This literature review tracks the evolution of CPV in definition and scope. It categorizes abusers and abuses including underlying psychopathology while defining victim and perpetrator indicators and risk factors. It highlights barriers in seeking, obtaining, and completing rehabilitation which include cultural contexts and stigma to familial role reversals and inadequate resources. The complexity of CPV is reflected in the lack of acceptance, array of responses, and inadequate interventions shown to exaggerate the recidivism and drop-out rate of intervention systems that are not robust or adequately designed specifically for CPV. The review shows that without intervention, the cycles of multi-generational family abuse including CPV will continue to rise and gain intensity. I reviewed peer-reviewed scholarly articles and government documents to evaluate multiple aspects of CPV responses and interventions categorized as crisis response, therapeutic interventions, judicial interventions, and hybrid models in their availability and effectiveness. I found that interventions which offer full-family/community, wrap-around, multi-faceted methods are the most effective to reduce stigma, improve overall family well-being, while reducing negative outcomes. Therefore, these systematic CPV intervention methods should be modeled everywhere with encouragement for more robust research in the phenomenon of Child-to-Parent Violence resulting in healthy societies.

Previous studies show that the high prevalence of body image concern and disordered eating among Western women precipitates from the promotion of an unrealistically thin body ideal. Exposure to thin-idealized images on social media has been found to increase body dissatisfaction and perpetuate internalization of anti-fat attitudes. The present research examined whether implicit anti-fat attitudes can be internalized without ever being visually exposed to body shapes. Preliminary results from a sample of blind women (N = 18) in Israel revealed that implicit anti-fat attitudes in blind females are comparable to those of sighted controls. The purpose of this study was to expand on these original findings by administering a novel auditory weight-bias Implicit Association Test (IAT) to larger samples of blind women (N = 30) and sighted controls (N = 30) across the United States. Participants completed an auditory IAT in which they paired words describing thin bodies and words describing overweight bodies with either negative or positive words in congruent and incongruent blocks. Afterwards, they answered self-report questionnaires to evaluate body dissatisfaction, pursuit of the thin ideal, and perceived pressure from family, friends, significant others, and the media to attain a thin body. In our analysis, I helped compare reaction times of blind and sighted female participants. Implicit anti-fat bias was reflected as slower reaction times when words describing overweight bodies were paired with positive words compared to when they were paired with negative words. If results of the present study replicate the preliminary results, we will develop stronger evidence towards the existence of non-visual influences that contribute to thin-ideal internalization. Additionally, these results would demonstrate that internalization of the thin-ideal can occur without any visual exposure to body shapes.These results may shed light on sociocultural factors that should be considered in evaluating disordered eating risk among blind women.

Tent encampments are an increasingly common form of dwelling for the homeless population in the West Coast. Recently, the Ninth US Circuit Court of Appeals has stated “[a]s long as there is no option of sleeping indoors, the government cannot criminalize indigent, homeless people for sleeping outdoors, on public property, on the false premise they had a choice in the matter.” However, many city residents worry about issues such as local impact on the environment and criminal activity. Oftentimes city officials are pressured into action by homeowners for encampments not sanctioned by the local government. Removing (or sweeping) encampments not sanctioned by the city eliminates their impact on the surrounding community, but also disrupts the lives of the already-vulnerable people living in them. Debates among the public, activists, and the government on how to address encampments in public spaces are salient, polarized, and evolving. However, knowledge of the number and locations of encampments is still limited. Using data from Google Street View, combined with reports from The Seattle Times and the City of Seattle reports on removals, my study will chart the spatial growth of tent encampments and measure changes in responses to the City of Seattle’s interventions from 2008 to 2021. By linking the dataset of tent encampments removals with neighborhood level US Census American Community Survey data, I will use event history analysis and spatial statistics to measure the impact of encampment removal activities on local neighborhood demographic change, gentrification, and crime rate. This study expands the scope of homelessness encampment displacement research over the last decade in Seattle. Understanding encampment sweeps in Seattle is important for policy researchers and social scientists studying West Coast urban environments.

 Improper hand hygiene can lead to the transmission of various diseases including gastrointestinal infections, such as salmonellosis, and respiratory infections, such as influenza, colds and coronavirus. The area under the fingernails (subungual area) is a source of bacteria accumulation that is often overlooked during hand washing. This research evaluates the effect of fingernail length on the effectiveness of a 20 second hand wash on reducing subungual bacteria counts. We performed a randomized controlled trial to determine total bacteria counts of the subungual area right before and after a 20 second hand wash with antibacterial soap. Bacteria counts were determined on standard media after a 72 hour incubation. Subjects were grouped by fingernail lengths of 0mm, 0-1mm, 1-2mm, >2mm, with a control group of 0mm. The results suggest that regularly trimming fingernails and paying attention to the subungual area during hand washing may be an important tool in preventing infections and diseases caused by pathogenic bacteria and viruses. This is especially important for people with impaired immune systems or young children who are more susceptible to infections, as well as for people who can transmit diseases to others, like healthcare personnel and childcare providers.

Mental state terms refer to the narrator’s interpretation of a character’s cognitive state such as what the character says, wants, intends, or thinks (Altman et al., 2015). However, previous research on mental state terms has documented conflicting results regarding the frequency of mental state terms used across languages (Altman et al., 2015; Fichman et al., 2021). The current research examines children’s use of mental state terms by elementary school students (1st, 3rd, 5th, and 6th grade) enrolled in a Mandarin-English Dual Language Immersion (DLI) program in California. Children completed a story-telling task, and their narratives were based on the wordless picture book “Frog where are you?” in English, and “Frog goes to dinner” in Mandarin. Speech samples were collected and recorded over Zoom and then transcribed on Computerized Language Analysis (CLAN) software. Transcripts were coded for instances in which individuals of each grade level used mental state terms. Data collection is complete and analysis is in progress. In this study we will answer: (1) How often are children using mental state terms during a narrative production task in Mandarin and English? (2) What kind of emotional language use differences are seen across Mandarin-English and grade levels? We hypothesize that the frequency of mental state terms will be higher in English because English-speaking cultures place greater emphasis on conveying motivations or feelings involved (Sah & Torng, 2016). Across grade levels, we expect older children to use mental state terms at a higher frequency than their younger peers. Investigating how children make use of these devices in narrative production as they acquire two languages can provide insight into cultural priorities around storytelling.

Disability Services (DS) are the most common strategy for ensuring that students with disabilities have access to higher education. These offices provide accommodations for students that can apply to both physical (e.g., building) and digital (e.g., course content) accessibility. Prior work has shown that the access needs of disabled students are not always successfully provided, and if they are, they are not always accomplished in a timely manner. Consequently, there may be dissonance between when professors believe an accommodation has been fulfilled (e.g., when a PDF they posted has been made accessible by DS) and when it is actually completed. While prior work has documented the accessibility challenges faced on university campuses and with disability services, work has not yet focused on how to better support DS, student, and faculty interactions and communication. This study aims to do so by investigating how communication currently flows, the issues that arise within interactions and implementations, and whether technical solutions can better support interaction for the accommodation fulfillment process.

The Internet of Things, also known as IoT, refers to the network of physical devices that can send and receive data through the Internet. These devices are often embedded with sensors and process data which is then communicated to humans, giving us greater control over the spaces that we inhabit and introduces ways to interact with technology that goes beyond screen interfaces and into the physical world. Internet of Light (IoL) is a Human Centered Design & Engineering (HCDE) research group where we leverage IoT technologies for creative expression, data visualization, and human connection. Students in the group design and develop an interactive lighting installation located in Sieg Hall on the University of Washington campus, utilizing the canvas of Philips Hue Smart Lighting. However, students, both new and returning, are tasked to learn how to program the lights, design an installation idea, and build it all within 10 weeks. Too much time is spent on learning the technicalities of programming the installation, leaving little time for students to dive deeper into the creative process, experiment with different possible interactive modes, and execute their ideas. This project is developing a framework for designing interactive spatial experiences that aims to address recurring issues and maximize learning efficiency, which would allow future runs of the research group to be more successful. I've designed and created a set of learning materials including slide decks, video tutorials, and organizational tools aimed at cutting down learning time from a previous average of 3-5 weeks to 1-2 weeks. Establishing this framework gives future students the necessary tools and additional time to experiment, design, build, and evaluate their installations. I anticipate that future iterations of the IoL research group will offer students a more complete installation designing experience and produce higher quality installation experiences as a result.

Autism Spectrum Disorder (ASD) is a developmental disorder that can impair social and communication skills. Many individuals diagnosed with ASD experience other mental health disorders such as anxiety and depression. Research has estimated that between 63-70% of people with ASD suffer from depression while 50-58% experience anxiety. Anxiety and depression both have been found to have a negative correlation with self-compassion for ASD children and typically developing (TD) children. Self-compassion is the concept of being caring towards oneself and is characterized by three facets: (1) self-kindness (2) common humanity, and (3) mindfulness. The present study seeks to explore the relationship between self-compassion and anxiety/depression and how this may vary for children with ASD compared to TD children. To examine this, 55 children (ASD=21), ages 7-17 years from the longitudinal ACE2 study, a five-site NIH-funded study on gender differences in autism were included in the analysis. ASD diagnosis was confirmed via standardized measures. Self-compassion was measured using the Self-Compassion Inventory for Youth, a self-report tool that generates scores on two scales: self-compassionate coping scale and self-punitive coping scale. Anxiety and depression were measured using the Youth Self Report (YSR), a self-questionnaire assessing symptoms of anxiety and depression. We expect that children with ASD will experience a stronger negative correlation between self-compassion and anxiety/depression than TD children. This relationship is critical to understand as individuals with ASD can experience difficulty practicing self-compassion. Cognizance of the relationship between these two constructs may help inform beneficial interventions and treatments for children with ASD to alleviate symptoms of anxiety and depression.

The COVID-19 pandemic exacerbated the impact of social determinants of health factors which consequently led to a disproportionate effect of the virus on the health of immigrant communities. This study focuses on African communities in King County, WA and explores how socio-economic factors such as lack of adequate healthcare, low-income status, and lack of social safety nets were made worsened by the COVID-19 pandemic. The research team recruited African immigrants from a local community health center, Health Point Community Health Center in Kent, Washington. The respondents (n=8) were interviewed via zoom or in-person and were asked 10 open-ended questions which addressed the experience of social, economic, and psychological challenges during the global pandemic. Thematic analyses were conducted of transcripted responses. Several themes emerged: (1) Stress and distress attributed to the inability to provide for respondents’ basic familial needs; (2) Experience of anxiety attributed to housing insecurity; (3) Emotional trauma associated with inadequate access to healthcare resources. These findings demonstrate that African communities experienced substantial social and economic disadvantages, however, these factors were substantially worsened by the global pandemic and placed this community at heightened risk. It is recommended that members of racial and ethnic minoritized groups, such as the African community, must be supported with resources enabling access to basic social, psychological, and healthcare needs. Future public health strategies should focus on empowering the community by engaging its members in policy-making processes that can help assure that their needs can be met.

Drug addiction and abuse exacerbate many health issues globally. The nucleus accumbens (NAc), a region within the brain, contains dopamine receptor D1 and D2 medium spiny neurons (MSNs) with relevance to addiction-related behaviors. I investigated the effect of D2 MSNs on behavioral economics of cocaine-seeking through chemogenetic activation of Designer Receptors Exclusively Activated by Designer Drugs (DREADD). I used a transgenic line of rats specifically expressing the Cre-recombinase enzyme in D2 MSNs. This enzyme is responsible for selective expression of otherwise-inert viral vectors by reversing its sequence, allowing for neuron-specific genetic manipulation. I bilaterally injected either a Cre-dependent AAV containing the DREADD hM3Dq or a fluorescent marker as control into the NAc of male rats; additionally, I catheterized the rats to allow for cocaine self-administration. Activation of hM3Dq required binding to clozapine-N-oxide (CNO)—the “designer drug”—and because only D2 MSNs expressed this DREADD, they were the only neurons to activate upon administration of CNO. After recovery, rats were trained to press a lever to receive cocaine infusions; and once this self-administration task was learned, the animals were introduced to reinforcement schedules with blocks of trials with differing unit doses of cocaine. Once this phase was learned, the rats were tested in sessions that were preceded either with an injection of saline or CNO, to test how chemogenetic activation of D2 MSNs affects the cocaine demand curve. Based on studies discussing the roles of D2 MSNs, I predict that chemogenetic activation of these neurons will correlate with decreased demand for cocaine concentrations compared to those injected with vehicle or green fluorescent protein alone. This study can produce valuable insight into the role this pathway plays in the development of drug-seeking behavior, ultimately leading to a deeper understanding of the system and possible avenues for treating addiction.

Spinocerebellar ataxias (SCAs) are a group of hereditary diseases that are characterized by slowly progressive incoordination in gait, hand and eye movement, and cerebellum degeneration. Most forms of SCAs are caused by an expansion of short tandem repeats; however, SCA 14 is an autosomal dominant form of the disease associated with mutations in the Protein Kinase C gamma (PRKCG) gene, which encodes for the PKCγ protein, a serine/threonine kinase that plays a role in signaling and regulating cerebellar Purkinje cells' development. Our lab first reported this genetic cause in SCA 14 patients. To understand the pathogenesis of SCA 14, our lab generated mouse models of SCA 14 mutant (H101Y, F643L) and wild type (WT) PKCγ transgenic (Tg) mice using modified human-BAC constructs. By 3 months of age, both mutant Tg lines demonstrated impaired rotarod performance as compared to WT-Tg mice and showed PKCγ aggregates. Here I further examined the pathological changes by quantitatively analyzing the morphology and fluorescent intensity changes of Purkinje cells in mice cerebellum across age groups of 2-months, 6-months, and 12-months. I observed the dendritic arborization abnormality at an early age, and the abnormality is more severe in the Tg-PKCγ-F643L mice. PKCγ intensity in the cell body decreased in 2-month H101Y and F643L mice. These results revealed pathological changes and provided evidence the missense mutations caused an early development of SCA 14 cerebellar disease. It will be interesting to compare and verify these findings from the Tg mice in SCA 14 patients’ autopsy brain tissues as it could provide novel measures to characterize SCA 14 pathology.

Morphine and other mu-opioid receptor (MOR) ligands are commonly used treatments for pain management. Long-term administration of opiates results in receptor desensitization and tolerance, inhibiting treatment efficacy and contributing to the current opioid epidemic. Following opioid administration, peroxiredoxin 6 (PRDX6) is recruited to the opioid receptor complex through JNK (cJun N-terminal Kinase)-dependent signaling. PRDX6 activation generates reactive oxygen species (ROS), resulting in opioid receptor desensitization and one form of opioid tolerance. Recently, cannabidiol (CBD) has been implicated in decreasing acute morphine tolerance. The present study will establish a connection between morphine and CBD cotreatment with the hypothesis that cotreatment will decrease ROS production. To measure ROS, I will employ the genetically encoded ROS sensor HRM63, which fluoresces proportionally to ROS production. Because ROS is a product of JNK/PRDX6 signaling, measuring the strength of ROS-dependent fluorescence is a simple way to visualize my hypothesis using in vitro cell models. In each experiment, treatments of morphine, CBD, or combined morphine and CBD will be delivered to plated HEK293 cells stably expressing both MOR and HRM63. I will then image the cells using fluorescence microscopy to quantify ROS response to treatment. I predict that cotreatment of CBD with morphine will result in lower ROS production compared to morphine treatment alone. These results will be crucial in the ongoing characterization of CBD's role in opioid tolerance.

The world’s population is rapidly aging, and we now increasingly face challenges related to the prevalence of age-related diseases. Studies from Geroscience have identified hallmarks of aging, such as the cellular senescence and mechanistic target of rapamycin (mTOR) pathways, that can be disrupted to improve health and lifespan in model organisms. Previous studies have shown that these pathways can be targeted pharmacologically by the mTOR inhibitor rapamycin or the senolytic drug combination Dasatinib + Quercetin (DQ). Our laboratory’s published data shows that rapamycin restored oral health in aged mice and reversed periodontal disease. While attempting to uncover the mechanism behind this reversal, our laboratory discovered that the age-related increase in cellular senescence was attenuated by rapamycin treatment. Thus, we hypothesized that targeting cellular senescence may provide an alternative therapeutic strategy to phenocopy the impact of rapamycin on aging oral tissues. I compared the effects of control (n=5), rapamycin (n=5), and the senolytic drug combination, DQ (n=5), in aged male mice (20-21 months old). Western Blot was performed on the mandible and salivary gland tissues to analyze the senescence marker p16INK4a. Histological assessment of H&E and lipofuscin staining was completed on the salivary glands to compare the effects of age, rapamycin, and DQ treatment. In male salivary glands, I discovered the age-related increase in p16 expression was decreased after rapamycin treatment, but not with DQ treatment. There were no significant changes in levels of p16 in the mandible with age or with the administration of rapamycin or DQ. Rapamycin treatment attenuated cellular senescence in male salivary glands, while the senolytic cocktail DQ had no impact on the aging male salivary glands. Future studies could be performed on female mice, with the addition of alternative senolytics which may target other markers of cellular senescence beyond p16. 

In the retina, loss of neurons results in blindness, because the mammalian retina cannot regenerate. Although mammals cannot regenerate neurons, species such as fish and amphibians can make fully functional neurons after injury and restore their vision. Muller Glia (MG) cells in fish and amphibians are the source of this regeneration. These cells respond to injury by dedifferentiating into progenitor cells that then become neurons, replacing the dead neurons and in turn restoring sight. At the Reh Lab, we have found a way to stimulate functional regeneration in mammals through these MG cells by expressing a gene called Ascl1. This technique has limitations however, as it only causes 25% of MG to undergo neurogenesis. One of the variables potentially limiting regeneration is inflammation, as inflammation has been known to be detrimental to neurogenesis. Monocytes invade the retina after injury and potentially cause inflammation that limits retinal regeneration. To determine monocyte impact on retinal regeneration we employed a transgenic technique to ablate monocytes. I then performed our retinal regeneration paradigm and determined whether regeneration is enhanced in the absence of monocyte invasion. Using immunohistochemistry and confocal microscopy I found more regenerated neurons in retinas that lacked monocytes. These data further confirm that inflammation limits the regeneration capacity of the retina, and provides future topics to improve neural repair through modifying the immune response.

One of the great outstanding challenges in neuroscience is to understand how the human brain represents distinct objects. Studies have shown that a significant number of neurons in the visual cortex of non-human primates responded differentially to the sides of figures in 2D images even when the figures were outside of their classical receptive fields. This selectivity, termed border ownership, is believed to be responsible for the Gestalt idea of figure-ground organization, a process that allows specific regions of an image to be grouped together and given "thing-like" qualities. Many computational models have been developed to reproduce the experimental results of border ownership studies. At the same time, convolutional neural networks (CNNs), especially those specialized in image segmentation, are able to learn to solve the problem of figure-ground organization through supervised learning, all without the need for explicitly defined computational rules. We hypothesize that there is knowledge to be gained from CNNs, for they are excellent computational models for visual selectivities. Our novel 'artiphysiology' technique allows us to study the border-ownership phenomena in CNNs at a single-unit level in the same way an electrophysiologist studies the brain. The technique takes advantage of the accessibility and lack of noise of CNNs to enable high-throughput identification and analysis of circuit mechanisms. Starting from border ownership, the research aims to elucidate the mechanism by which figure-ground organization occurs at different layer depths and in different CNNs, using natural and artificial visual stimuli. The research has several applications, including improving CNN efficiency and interpretability as well as allowing for a better understanding of object recognition.

Mammalian retinas function in a more hypoxic environment than most tissues. The retina thus does not exclusively use oxygen as a terminal electron acceptor in the electron transport chain (ETC). As a result, expression of proteins involved in energy metabolism may be affected. We determined the levels of metabolic proteins of various tissues, and found that compared to eyecup (consisting of retinal pigment epithelium and choroid vasculature), kidney, and cerebellum tissue, the retina expresses higher levels of Hexokinase I. This suggests that glycolysis may occur faster in the retina than in other tissues. We also observed higher levels of the ETC proteins cytochrome c and subunit 4 of cytochrome c oxidase levels compared to both the eyecup and cerebellum. This implies an increased capacity for electron transport in the retina, despite a lower O2 tension. We also investigated post-translational protein modifications that could be affected by a hypoxic tissue microenvironment. Lysine succinylation is one such modification, and is controlled by regulators of energy metabolism such as SIRT5. I observed that succinyl-lysine intensities were higher in the retina than in the cerebellum, kidney, liver, and eyecup. Further investigation will be necessary to determine the role that lysine succinylation plays in the retina. Through these experiments we show that retina tissue is well-suited for rapid energy metabolism in spite of its hypoxic environment.

Enzymes have recently been incorporated into multiple high-value industrial syntheses, demonstrating the utility of enzymes as highly selective catalysts for practical industrial processes. However, the current scope of non-biological enzymatic reactions is narrow and new reactions and reaction pathways need to be engineered. The goal of our work in the Zalatan lab is engineering enzymes as catalysts in carbon-hydrogen bond functionalization reactions, a transformation critical for practical industrial synthesis where selective catalysis is still a major challenge. Importantly, we are interested in exploring more efficient and informed engineering approaches by establishing structure-function relationships with the enzymes that we work with. Our model system for this work is the non-heme iron(II) 2-oxoglutarate dependent oxygenase superfamily (Fe(II)-2OGs). We are using a high-throughput microfluidics based kinetic assay to determine key sites that we can target for mutagenesis and directed evolution in a candidate Fe(II)-2OG found to catalyze a new reaction. Overall, we expect that this work will enable new directions and principles for engineering Fe(II)-2OGs, and that lessons learned here can then be extended to additional industrially-relevant enzyme families.

Small heat shock proteins (sHSPs) fall into a class of proteins known as protein chaperones, molecular tools that help prevent aggregation, an often-unhealthy phenomenon in which proteins clump together. This protection against aggregation plays a critical role in helping to prevent diseases such as cataracts, and may play a role in preventing neurological diseases associated with protein aggregation such as Alzheimer's disease or Parkinson's disease. Yet little is known about the mechanism by which sHSPs prevent misfolded proteins from aggregating. The study of this mechanism is significantly complicated by the dynamic nature of sHSPs; rather than having just one structure, these protein chaperones fluctuate between several different stable structures. The purpose of this research project was to see what insights into sHSP structure and function could be gleaned by simplifying this dynamic structure. We focused on the central region of a small heat shock protein, known as the alpha-crystallin domain (ACD). Individual ACDs associate to form dimers in an antiparallel fashion, and in doing so form a central fold known as the dimer-interface groove. In a typical wild type small heat shock protein, these ACDs would continuously slide against each other along this groove. We created three distinct cysteine mutations in the ACD of the small heat shock protein HSPB5, producing the R116C, E117C, and F118C mutants. We expected each of these mutant dimers to form a disulfide bond tethering the two ACD subunits of the dimer together at the dimer interface groove, preventing them from sliding against each other, and therefore locking the dimer's central region into one of three distinct conformations. Additionally, we expected the affinities of other proteins for the mutants' dimer-interface grooves to differ between mutants, giving us insight into how the conformational state of the ACD affects its ability to interact with other proteins.

Individuals with Developmental Language Disorder (DLD; previously known as specific language impairment) have difficulty acquiring their native language with no apparent underlying cause. While this disorder is common (7% of the population, or 2 in every classroom), there is little research investigating structural differences in the brain. My mentor Dr. Cler and colleagues previously found differences related to myelination of the caudate nucleus in children with DLD. They used a specialized form of MRI, multiparameter mapping (MPM), which reveals specific aspects about the microstructure of the neural tissue. The purpose of this current research is to confirm and expand upon these findings by obtaining MPMs on adults with DLD. Twenty participants between the ages of 18 and 45 are recruited via Facebook flier, ten typically-developing and ten with DLD. Because of the lack of instruments available to assess DLD in adults, my role has been to compile a battery of tests that can accurately differentiate individuals with DLD from TD as well as from other communication disorders. The selected battery evaluates participant’s IQ, motor skills,  ability to define words, spell, and follow complex directions. Scanning is conducted at the Center for Human Neuroscience using a 3T Siemens Prisma. MPM quantitative imaging provides maps of myelin and iron content in the brain. We hypothesize that we will find reduced myelination in the caudate nucleus, a part of the basal ganglia implicated in implicit learning. The findings of the study will contribute to current neural models of DLD, helping us to identify the brain processes that are disrupted by the disorder. This information can inform future clinical interventions to help individuals with DLD in their speech and language development.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder that affects people who have mutations in the PKD1 or PKD2 genes. The prominent feature of this disease is the formation of cysts in the kidneys. Despite ADPKD being genetically heterozygous, cystic tissue from patients with ADPKD also contains cells with homozygous mutations. This causes speculation within the PKD community about whether heterozygous mutations with one mutated copy of the PKD1 or PKD2 gene are sufficient to induce cystogenesis or if two mutated copies are needed. This concept is referred to as the two-hit hypothesis, a somatic mutation (second hit) occurs during the lifetime of the kidney and gives rise to clonal populations of cells that give rise to cysts. The goal of my project is to investigate the two-hit hypothesis and determine if there is cyst formation in the heterozygous organoids. To do so, I use patient-derived hSPC lines modified using CRISPR to express PKD1 or PKD2 mutations. I follow a 21 day differentiation process to produce PKD organoids. I then pick individual organoids to place in suspension for 14 days and track cyst growth by area and amount. If cysts form in heterozygous PKD organoid models at the same rate as the control, it would suggest that heterozygous mutants are unlikely to cause cyst formation, indicating the two-hit hypothesis applies to PKD. Current data collected on mutations in PKD2 have shown heterozygous organoids do not form cysts, however more data is needed to test the PKD1 site. Future applications involve inducing cyst formation in heterozygous mutants using forskolin, an agonist of adenylyl cyclase shown to induce cyst growth. Establishing if heterozygous mutant organoids form cysts has significant implications in ADPKD research by potentially creating a better representation of the disease process, which has been difficult to study in other models.

Protein kinase R (PKR) is a host antiviral factor that shuts off translation in response to double-stranded RNA that accumulates during viral infection. Many viruses, including cytomegaloviruses (CMVs), encode proteins that inhibit PKR and rescue viral protein synthesis in infected cells. In the case of CMV, the “terminal repeat short 1” (TRS1) protein antagonizes PKR. Analyses have shown that PKR has been evolving under positive selection among primates, likely in order to evade TRS1 and other viral antagonists. Consequently, the ability of TRS1 to block PKR is quite species-specific. For example, the TRS1 homolog in human and Old-World monkeys (OWMs) CMVs only inhibit PKR from great apes and OWMs, respectively. Thus, it is surprising that TRS1 encoded by a New World monkey (NWM) virus, squirrel monkey CMV, antagonizes hominoid and OWM PKRs. I hypothesize that squirrel monkey CMV TRS1 has broad activity against diverse PKRs because it interacts with a conserved domain of PKR that is different from the region bound by TRS1 encoded by human CMV or OWM CMVs. To test this hypothesis, I studied how TRS1 and PKR compete to control protein synthesis in infected cells by using assays of protein synthesis and of replication of wild-type and mutant viruses. Because there has been no study of squirrel monkey PKR (smPKR), I first tested its function using a reporter gene transfection assay and demonstrated that it is active in inhibiting translation, as expected. I cloned smPKR into a doxycycline-inducible lentiviral vector and tested the susceptibility of it and other NWM PKRs to alternative TRS1 genes. By understanding the evolution and molecular mechanisms that have diverged in the ongoing “arms race” between NWMs and their viruses we can reveal insights into the risks and barriers to cross-species transmission of viruses.

Chronic nonbacterial osteomyelitis (CNO) is an autoimmune condition characterized by excessive activity of osteoclasts predominantly in children. It remains unclear what roles the precursor cells present in peripheral blood mononucleocyte cells (PBMCs) and their environment (serum) play in the disease pathogenesis. We hypothesize that the serum from children with active CNO have increased stimulating effects on osteoclastogenesis derived from PBMCs compared to the serum from patients with inactive CNO, inactive juvenile idiopathic arthritis (JIA), or healthy controls. With IRB approval, patient serum is utilized in varying concentrations of 0.1%, 1%, and 10% across different patient groups. PBMCs were ordered from ZenBio. In-vitro cultures of patient serum mixed with a donor PBMC sample were conducted for 7 days and supplemented with receptor activator of nuclear factor kappa-Β ligand (RANKL) and macrophage colony stimulating factor (M-CSF) to simulate physiologic conditions. During the incubation period, media exchange was carried out twice. After 7 days, cells were stained with tartrate-resistant alkaline phosphatase (TRAP) and DAPI. Cells with 3 or more nuclei and stained positive for TRAP were counted as an osteoclast using ImageJ. Osteoclast counts from all groups were normalized to the counts of the serum-free well. The findings from this study are expected to inform on whether serum from patients with active CNO contribute to the process of osteoclastogenesis. Currently, the results suggest that as serum concentration increases there is a correlated increase in osteoclast counts, with the exception of patient samples from the inactive CNO group. As the study continues, we expect to gain a more comprehensive understanding of the association between serum concentration and cell types. Future studies can explore inflammatory markers in CNO samples that promote osteoclastogenesis as potential drug targets.

Irritable bowel syndrome (IBS) is a disease that affects many people worldwide. Clinical trials using probiotic bacteria to treat IBS show promising outcomes, although the molecular mechanisms of probiotics and their effect on gut health is not completely understood. Multiple studies comparing different strains of probiotics show Lactobacillus plantarum and Bifidobacterium longum as the most effective strains in inhibiting the release of proinflammatory factors in the gut and promoting proliferation of healthy gut bacteria. The purpose of this study is to evaluate the protein profile of probiotic bacteria Lactobacillus plantarum and Bifidobacterium longum in both healthy gut and IBS gut conditions. To observe how Lactobacillus plantarum and Bifidobacterium longum respond to different simulated gut conditions, samples of the bacteria will be isolated from probiotic supplements. These samples will be grown in aliquots representing different simulated anaerobic gut conditions for 12-16 hours at 37°C. Lactobacillus plantarum and Bifidobacterium longum will be grown separately and grown combined within these conditions. To enrich secreted proteins, the supernatant from each aliquot sample will be clarified using centrifugation. Secreted proteins will be solubilized and denatured with sodium deoxycholate. The denatured proteins will be reduced with dithiothreitol and alkylated with iodoacetamide. The sample will be diluted using ammonium bicarbonate and digested by trypsin. Tryptic peptides will be analyzed using liquid chromatography and mass spectrometry. Tryptic peptides will be identified using the SEQUEST search engine with a reference database. I anticipate the probiotic strains will demonstrate differential proteomics when isolated and combined in the healthy gut and IBS gut conditions. The secreted protein analysis completed in this study will further the understanding of the probiotic molecular mechanism of promoting gut health.

Blindness caused by retinal degeneration is untreatable, and is a condition currently suffered by millions. The field of retinal reprogramming aims to establish a way to treat this type of vision loss, by finding a way for the eye to essentially produce new retinal neurons after they have been lost to degeneration. Often, this is done through directing cell fate by expressing or repressing various transcription factors such as Ascl1, a potent pro-neural transcription factor involved in retinal reprogramming. Ascl1 expression in mouse Muller glia, a support cell in the retina, can stimulate the regeneration of certain subtypes of retinal neurons, but the variety of retinal cells produced through this strategy is still limited. The class of transcription factors known as ‘Krüppel-like factors,’ or KLFs, regulates important cell processes, such as cell proliferation and development, with several KLFs functioning in the development of neurons. Due to the inhibitory function of KLFs during neuron development, we propose that the loss of inhibitory KLF genes in Muller glia may allow for activation of neurogenesis. Therefore, coupling the knockout of KLF genes with Ascl1 expression in Muller glia may be a key to enhancing reprogramming capabilities of Ascl1. For this project, I knocked out KLF genes in young mouse Muller glia in culture using the CRISPR/Cas9 system, and induced Ascl1 expression to stimulate reprogramming of the Muller glia. We used scRNA sequencing to determine if the knockout reprogrammed cells were molecularly similar to various retinal cell types that are typically lost with degenerative blindness. Preliminary sequencing results revealed that knockout of one candidate, KLF15, appears to promote neurogenesis. Findings from this experiment will allow us to increase our understanding of the role of KLF genes in retinal cell development as we work towards a future treatment for degenerative vision loss.

Platelet transfusions are used to treat patients with acute hemorrhage, low platelet count, and dysfunctioning platelets, but the ideal storage conditions for platelets prior to transfusion is poorly understood. Storage at room temperature is the current gold standard for platelets, but there is a limited shelf life of 5-7 days due to storage lesions and risk of bacterial growth. As a result, there is a need for alternative approaches that can increase the shelf life of platelets and increase their availability in remote locations or during major catastrophes. Here, we examined whether cold storage of platelets, e.g. 4º C, which can extend their shelf life to 14 days, has an impact on their hemostatic function. Specifically, the function of cold-stored and room temperature-stored platelets were investigated by utilizing a microfluidic shear assay that measures platelet aggregation and contractile force during the formation of a thrombus. Blood was perfused through the microfluidic channels, and platelet aggregation and production of contractile forces were calculated using custom MATLAB code. The previous code required user input at several steps to complete the analysis, so improved code was written and tested that quantified area and force with significantly less user input. Results from experiments and subsequent analysis found that cold-stored platelets formed larger aggregates and similar contractile forces as room temperature-stored platelets, indicating that their hemostatic function was not impaired. This investigation highlights the need for further investigations on cold-stored platelets as a means to increase shelf life and availability for transfusions.

The purpose of this prevalence study is to determine if MAGEA1 specific cytotoxic T cells are good therapy for head and neck squamous cell carcinoma (HNSCC) and hepatocellular carcinoma (HCC). MAGEA1 gene is a member of the MAGEA gene family and MAGE-A antigens are expressed in a variety of cancers. Previous studies have shown a correlation between the MAGEA1 gene with HNSCC and HCC but further studies were not performed to validate it. We performed a prevalence study by immunohistochemistry staining (IHC) using tissue microarray (TMA) slides. We stained a total of 9 representative formalin-fixed paraffin embedded cores of HNSCC (HN804 and HN602) and 124 cores of HCC (LV809b and LV631) tumor tissue using mouse monoclonal antibody clone MA454 at a concentration of 8 ug/ml. We performed antigen retrieval using pH 9.0 Tris-EDTA buffer and achieved visualization via deposition of DAB chromogen by horseradish peroxidase reaction. Then, we use IHC to test and validate this protein marker and evaluate whether it is a good biological target in treatments for certain population groups. Two pathologists defined positive staining tumors as those demonstrating an intensity of 1+ or greater on nuclear or cytoplasmic compartments of the cell. Staining seen on stroma, artifactual folds, or necrotic tissue was not counted. By analyzing the percentage that is positive for this biomarker, we can determine if the MAGEA1 gene is a useful target for these cancer patients. After examining 129 cases of HNSCC, 10 positive cases indicated a prevalence rate of 7.75%; of the 124 HCC (liver cancer) cases examined, 14 positive cases showed a prevalence rate of 11.29%. In conclusion, about 8% of HNSCC cases and about 11% of HCC cases can be targeted with the TCR treatment so it can be potentially beneficial as a targeted therapy worthwhile to pursue.

Renal Cell Carcinoma (RCC) kills more than 14,800 people annually in the United States alone. While immune and targeted therapies have shown some promise, late stage RCC is an aggressive and lethal disease, and so, new therapies that can treat RCC are urgently needed. TRAIL (Tumor necrosis factor (TNF)-related apoptosis-inducing ligand) has been shown previously to induce apoptosis in cancer cells by binding to TRAIL receptors DR4 and DR5. Unfortunately, DR5 antibodies have failed to improve patient survival in phase II clinical trials due, in part, to the inability to cluster enough membrane receptors and kill RCC cells in the body. This project is investigating the effects that computationally designed protein nanocages bound to DR5 antibodies have on kidney cancer cells in culture. In collaboration with the Institute for Protein Design, we have recently shown that these nanoparticles induce significant activation of caspase 8 and caspase 3 in renal carcinoma TRAIL resistant cells, but not in healthy primary kidney tubular cells. To further investigate the nanocage-induced apoptosis specificity to DR5 and the involvement of other TRAIL receptors, we used CRISPR/Cas9 to generate knockouts of TRAIL receptors in RCC. We are also investigating the effects of these nanocages on other downstream pathways including the NFkB and cFLIP pathways. This project advances the knowledge of mechanisms involved in TRAIL receptor-mediated apoptosis, and our results suggest a potential new treatment for RCC.

Altered cellular metabolism is intimately linked to cancer, both by supporting the increased metabolic demands of cell proliferation and through changes in metabolism that initiate tumorigenesis. For example, loss of function mutations to the metabolic enzymes Succinate Dehydrogenase (SDH) or Fumarase (FH) of the tricarboxylic acid cycle are sufficient to promote renal cell carcinomas (RCC). Normally, these enzymes fulfill metabolic roles, where SDH converts succinate to fumarate, which is then converted to malate by FH. Inactivating mutations to SDH and FH cause the accumulation of their substrates succinate and fumarate, respectively, which can drive cancer relevant signaling changes. Recently, studies have shown that perturbations to SDH cause resistance to ferroptosis, an iron-dependent form of nonapoptotic cell death. Inversely, another study has shown that FH-inactivation causes ferroptosis sensitivity. Considering SDH and FH are metabolically adjacent and have similar oncogenic consequences, it is unknown why they have opposing effects on ferroptosis induction. Cell death by ferroptosis occurs in response to the accumulation of oxidized polyunsaturated fatty acid-containing membrane lipids. I hypothesize that FH and SDH mutations have different effects on RCC cell lipid metabolism that cause divergent responses to ferroptosis induction. To test how SDH and FH alterations affect ferroptosis induction, I am performing a series of dose response assays with pro-ferroptosis treatments and measuring the effect on proliferation and lipid peroxidation in wild type, SDH-impaired, or FH-impaired cells. To inhibit SDH and FH, I will use two different patients derived RCC cell lines, UOK269 and UOK262, which have endogenous SDH and FH mutations, respectively. Each cell line will be compared to their respective SDH or FH addbacks. Ferroptosis induction is a potential opportunity for cancer treatment. Thus, it is essential to understand how different metabolic alterations affect ferroptosis sensitivity to identify conditions that may be most amenable to ferroptosis induction therapy.

Type 1 diabetes (T1D) is an autoimmune disease in which the pancreas is unable to produce enough insulin to effectively regulate sugar into the body’s cells. Recent literature suggests that T1D has a prevalence and incidence of 9.5% and 15 per 100,000 people respectively. T1D risk is multifactorial but is heavily dependent on genetics and the environment. Twin studies have shown that although disease risk for the general population is 0.4%, children of diabetic patients are 2 to 9% at risk and identical twins are up to 70% at risk of developing T1D. Genome-wide association studies have shown that many genetic variants associated with T1D risk are found in gene regulatory regions such as enhancer elements. As T1D is an autoimmune disease with a strong genetic component particularly in non-coding, regulatory regions, it follows that thorough genomic profiling of immune cells such as T cells can help identify functional non-coding genetic variants that alter gene regulation for target genes associated with disease risk. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) allows for unbiased identification of cis-regulatory elements (CREs). By utilizing ATAC-seq on various T cell types of both healthy control donors and T1D patients, chromatin accessibility can be collected, and the underlying sequence data can be used to determine allelic differences in transcription factor binding due to T1D-associated genetic variants. I am currently applying ATAC-seq to three T cell subtypes isolated from both donor cohorts. Through these data functional links will be made between non-coding genetic variants and associated target genes to better understand how they impact disease risk in T1D.

Early-stage melanoma, squamous and basal cell carcinomas have local control rates of >90% with wide excision after pathologically clear margins and >95% with Mohs micrographic surgery. Local control rates for these approaches are not well defined in Merkel cell carcinoma (MCC). Herein, we analyzed data from 80 patients (pts) in a Seattle-based IRB-approved registry who had local MCC and underwent surgical excision with pathologically clear margins. Patients who had local radiation therapy after surgery were excluded as radiation affects recurrence rate independent of surgery. We also performed meta-analysis of 13 published studies (846 pts) based on a random-effects model. For the 80 pt cohort, local recurrence rate (LRR; ≤2 cm from the primary tumor) was 10%. In-transit recurrence rate (ITR; >2cm from primary) was 1%. Regional nodal recurrence rate (RRR) was 5%. This cohort had low-risk characteristics with small primary tumors (74% were ≤1 cm, 23% were 1-2, and 4% were >2 cm). No residual tumor was found in 60% of re-excisions while 29% had closest pathologic margins <1 cm. Meta-analysis of 9 published studies (745 pts) who underwent excision with clear pathological margins yielded 16.4% LRR [95% CI 8.3-26.5], 9.5% ITR [95% CI 5.4-14.6], and 32.1% RRR [95% CI 19.1-46.7]. Data from 4 studies (101 pts) who underwent Mohs yielded 3.6% LRR [95% CI 0-16.3], 12.8% ITR [95% CI 6.4-21.1], and 20.7% RRR [95% CI 13.8-38.3]. In each cohort, LRR/IRR following surgical excision with pathologically clear margins was >10%. This suggests MCC is more likely to recur near the excision site than other skin cancers and may reflect biological difference in the MCC local extension pattern (discontinuous spread beyond pathologically clear margins). Even for pathologically negative excisions, higher risk tumors may benefit from adjuvant radiotherapy.

Microtubules play an essential role in neuronal health as they contribute to the regulation of neuronal morphology, transport and polarity. Our post-translational modification of interest, polyglutamylation, involves the elongation of glutamate chains of microtubules by a set of enzymes called glutamylases. Polyglutamylation is involved in the regulation of microtubule functions, however, the specific role of tubulin glutamylation in its impact on neurodegeneration is not well understood. While there are five glutamylases encoded in the C. elegans genome, there are only two carboxypeptidase enzymes, ccpp-1 and ccpp-6, which catalyze the shortening of polyglutamate chains. A deletion of these carboxypeptidases can cause hyperglutamylation since the shortening mechanism is absent. The purpose of this study is to utilize the loss-of-function mutation in carboxypeptidases to investigate the connection between hyperglutamylation and neurodegeneration using C. elegans as a model organism. We will utilize an Alzheimer’s model strain, GMC101, in which an overexpression of amyloid-beta protein in the body-muscle tissue causes a paralysis phenotype.Using GMC101 helps us explore the neurodegenerative effects of hyperglutamylation in the ccpp-1 and ccpp-6 mutants. We will establish baseline data of ccpp-1, ccpp-6 and GMC101 mutant strains using dye filling and paralysis assays to characterize each strain individually. Next, we will develop a double mutant ccpp-1/GMC101 and ccpp-6/GMC101, to investigate whether these mutations improve or worsens the neuronal morphology and the paralysis phenotype. Based on the literature, we expect the double mutant strains to have a worsened paralysis phenotype as well as a worsened neuronal morphology determined through dye filling assays. Investigating the specific contributions of hyperglutamylation to modulating microtubule properties in neurodegeneration is essential since there is a link between an increase in hyperglutamylation enzymatic activity in neurodegenerative regions. Further understanding of this mechanism could contribute toward developing therapeutics involved in polyglutamylation for individuals with neurodegenerative diseases.

Antibiotics are medicines that inhibit the growth of or kill microorganisms. The discovery and production of new antibiotics has greatly decreased in the past 30 years, while the number of antibiotic-resistant microorganisms has increased. Microorganisms growing in soil are a potential source to discover new antibiotics. The goal of our study was to identify and characterize soil microbes that are antibiotic producers. A soil sample was diluted in water and plated onto complex media. Of the 300-400 colonies that grew, 26 soil isolates were screened for the ability to produce antibiotics and inhibit the growth of the five ESKAPE-safe relatives (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and several species of Enterobacter). Of the 26 soil isolates screen, 7.7% were found to produce antibiotics. The identity of the antibiotic producers was determined by PCR of the 16S rRNA gene and DNA sequence analysis. One of the antibiotic producers was of the genus Flavobacterium and the other is Streptomyces. Our plan is to perform future biochemical analysis of the antibiotic producers. In conclusion, our study supports the hypotheses that soil is an excellent source for potential antibiotic discovery.

Gastrointestinal helminth infections are among the most common infections worldwide affecting about 1.5 billion people. Previous work shows that during infection with Trichuris muris, a mouse model of human whipworm in the large intestine, basophils, rare innate immune cells, accumulate at the site of infection and upregulate the expression of Notch receptors, part of a signaling pathway that regulates gene expression. Due to this discovery, we are investigating the importance of Notch receptor expression in basophils for the anti-helminth immune response and parasite clearance during infection. With single-cell RNA sequencing (scRNAseq) we are able to analyze the expression of individual cells to better understand immune cell response to infection. To investigate how Notch signaling in basophils impacts CD4 T cell function during infection, we performed scRNAseq on CD4 T cells from the intestine of naive and T. muris infected wild-type mice and mice without Notch signaling specifically in basophils. Using this approach, for the first time, we have characterized the gene expression profile of CD4 T cells at the site of infection, allowing us to identify different CD4 T cell subpopulations and their differential gene expression. This will provide insight into how T. muris infection impacts CD4 T cell gene expression and function, and the importance of Notch signaling in basophils for promoting T cell function during infection. Ongoing work is addressing how CD4 T cells and basophils interact in the large intestine, how the highly regulated environment impacts the development of the CD4 T cell response and the T cell receptor repertoire in T. muris infection, and the importance of basophil Notch signaling for this process. A better understanding of the response to helminth infection and manipulation of Notch signaling-dependent cellular responses can lead to the discovery of novel treatments for helminth infection and allergic diseases.

Antimicrobial peptide (AMPS) hold promise as novel antibiotics for treatment of human infections. However, in general, they fail to reach clinical application because of issues with human cell toxicity or other factors. The AMP colistin (CST), is a rare AMP in clinical use and despite its nephrotoxic effects, has seen increasing use as a last resort to antibiotics. There’s now widespread CST resistance among bacterial pathogens. Our chemist colleagues recently synthesized a new class of AMP mimics, main chain cationic polyimidazoliums (PIMs). Their lead PIMs are potent broad spectrum antibiotics with a novel mode of action. We are interested in developing PIMs for inhalation therapy to treat respiratory infections in people with the genetic disease cystic fibrosis (CF). The bacterium, Pseudomonas aeruginosa, is the major contributor of disease in patients with CF lung infection. Unlike CST, PIM-resistant P. aeruginosa do not emerge in laboratory evolution experiments. We wanted to assess a particularly potent PIM called PIM1D as a potential CF therapeutic. Many antibiotics are inhibited by the secretions in CF lung and we expected this might be true of PIM1D. We used a special in vitro culture medium designed to mimic CF sputum, synthetic CF medium (SCFM). The medium contains the nutrient composition of CF sputum (SCFM-1) along with mucin and DNA (SCFM-2), polymers present at high concentrations in CF lungs. These polymers are known to inhibit many antibiotics’ activity. PIM1D retains high activity in SCFM-1 but activity is reduced by 90% in SCFM-2 compared to SCFM-1 and standard media. The inhibition might be due to either mucin, DNA, or both together. We aim to answer which of these molecules is responsible for inhibition of PIM1D activity. In collaboration with chemists who synthesized PIMs, they will provide PIM1D derivatives and PIMs packaged in nano-carriers to test for better activity in SCFM-2. 

The mechanistic target of rapamycin (mTOR) pathway is a central nutrient signaling pathway involved in regulating cell proliferation and metabolism. Targeting this pathway has promising implications for treating a variety of diseases, especially cancer and age-related diseases. Rapamycin is an allosteric inhibitor of mTOR that has been shown to extend longevity in the fruit fly Drosophila melanogaster. Despite this, rapamycin can interfere with healthy mTOR pathways necessary for survival, which is why further research on rapamycin’s mechanism is necessary to improve clinical usage. Previous research has shown that larval size is significantly decreased in fly larvae following inhibition of the mTOR pathway. However, this effect has not been studied across fly strains with differing levels of rapamycin sensitivity. In the Promislow Lab, we have found that fly strains vary in sensitivity to the effect of rapamycin on developmental timing. We are now comparing variation in sensitivity of development time with variation in rapamycin’s effect on larval size. Using D. melanogaster, we are able to selectively expose larvae to rapamycin from embryogenesis to model the effects of rapamycin. We are collecting larvae at various time points throughout their development, comparing individuals exposed to rapamycin or control conditions. We use ImageJ software to quantify larval size. Because of mTOR’s involvement in growth pathways, we hypothesized that larvae with greater rapamycin sensitivity would have significantly smaller sizes at the same time points compared with larvae with lesser rapamycin sensitivity following rapamycin exposure. These experimental results will provide insight into how rapamycin sensitivity is linked to the phenotypic effects of rapamycin on larval size. They will also help us investigate what contributes to the sensitivity differences seen between genotypes. The spectrum of rapamycin resistance in humans is unknown and so our work could help us identify targets for treatments and therapies of age-related diseases.

With the impending implications of climate change and global warming, space colonization in the near future will be necessary for the long-term survival of humans. We are examining how exposure to space affects biological aspects of humans by studying a model organism using a CubeSat. A CubeSat is a miniaturized satellite used in space research, built to a set of standardized measurements (10 cm per side), allowing small research projects to be launched simultaneously. We asked the questions: what would the experimental design look like investigating microgravity and UV radiation’s impact on the model organism C. elegans and what is a feasible experiment to design on a CubeSat? In this project we developed this research question to design a molecular biosciences project on a CubeSat. As part of this project, we conducted a literature review to determine 1) how small-scale satellites can be used for this kind of research, 2) what model organism is best suited for our project, and 3) how we can build upon the existing body of knowledge. The literature review, done in consultation with experts in the field, focused on the effects of microgravity and UV radiation on living organisms in the space environment. We didn’t limit our model organism research to C. elegans in the literature review. We used the constraints of the CubeSat to determine our data sampling methods and developed a research question. We anticipate research from our literature review will help us determine the next steps to take in designing our project. We hope to continue this research in preparation for implementing our work in collaboration with UW CubeSat. Research projects and experiments done in CubeSats like this one can advance the research and help address challenges our Earth faces with a rapidly expanding human population and ongoing climate change.

Mitochondrial dysfunction is characterized by loss of structural integrity and decreased efficiency of the Electron Transport Chain. These changes are linked to cellular senescence, an irreversible end to the cell-cycle that contributes to aging phenotypes and is marked by a cellular senescence-associated secretory phenotype. Understanding this impact of late age mitochondrial dysfunction interventions will contribute to the field of, ‘senolytics’, anti-senescent drugs dedicated to improving health span. Previously, systematic treatment of old mice with tetrapeptide, Elamipretide (ELAM), which interacts with the inner mitochondrial membrane to improve cristae structure and function, reduced mitochondrial dysfunction and senescence in kidney and heart. We hypothesized that additional intervention with Nicotinamide Mononucleotide (NMN), a NAD+ precursor, which contributes to adenosine triphosphate generation, would supplement this finding by enhancing mitochondrial energetic capability and decreasing mitochondrial dysfunction and senescence in aged kidneys. Liver was selected to clarify intervention effects as global vs. kidney-specific mechanisms. Old mice were treated at 24 months-old (mo) for 8 weeks with: ELAM through osmotic pump (3mg/kg), NMN via drinking water (300mg/kg), or both simultaneously. Untreated control mice were 4 mo (young) and 26 mo (old). Contrary to our hypothesis, NMN treatment proved detrimental in kidney by increasing mRNA expression of IL-1b, an inflammatory cytokine, relative to untreated aged mice. Further exploration of IL-1b downstream targets showed consistent upregulation of CCL2, an inflammatory chemokine and KIM-1, a marker of proximal tubule injury in NMN treated mice. ELAM, however, provided rescue in the coupled treatment group, by significantly reducing mRNA expression of IL-1b and CCL2 relative to NMN alone. These data suggest that ELAM lessens senescent burden by reducing renal inflammation; conversely NMN exacerbates existing inflammation pathways in aged kidneys. This result was not observed in liver, demonstrating tissue specificity. Further work will investigate the cellular source of inflammation through RNA in situ hybridization.

Skin is a very important organ that facilitates our sense of touch. The touch system is complex and versatile. For example, specialized cells detect a variety of tactile stimuli, including temperature, pain, and textures. Merkel cells are specialized skin cells responsible for detecting light touch and textures in most vertebrates. Despite being discovered over a hundred years ago, their development remains poorly understood. Zebrafish are a good model organism for studying Merkel cells because the fish skin is transparent and easy to image. In wildtype zebrafish, Merkel cells are distributed in clusters, corresponding to the location of scales. The ectodysplasin (Eda) signaling pathway regulates the formation of many types of skin appendages, including mammalian hair follicles and zebrafish scales. In eda mutants lacking scales, Merkel cells appear at a lower density and are uniformly distributed across the trunk. This suggests that blocking Eda-dependent scale formation inhibited Merkel cell development, but what would be the effect of altering scale shape and size? To address this question, we examined the skin of hagoromo mutant fish. The hagoromo mutation is a viral insertion that causes an overexpression of fgf8a, another important signal pathway for scale formation. By imaging transgenic zebrafish with marked Merkel cells and osteoblasts, we used ImageJ to calculate Merkel cell density, scale area, scale Feret’s diameter, and scale aspect ratio. We found that juvenile zebrafish with the hagoromo mutation had highly variable scale shapes and sizes. Interestingly, the clusters of Merkel cells expanded or shrank to match the new scale shapes in the mutants. However, there seemed to be no significant difference in Merkel cell density. We expect to see similar results in adult zebrafish. Our research will help us understand the development of Merkel cells, therefore helping us understand skin development in vertebrates better. 

Touch is an extremely important sense to any organism’s understanding of their environment. The anatomy of the touch system is well-characterized: sensory neurons project axons to the skin where they form complexes with many specialized cells. One such specialized skin cell is the Merkel cell, known for its ability to sense gentle touch and texture. Although Merkel cells have been well studied in rodents during adult stages, little is known about Merkel cells during development. The Rasmussen lab recently identified a novel population of zebrafish skin cells sharing many characteristics of mammalian Merkel cells. In both zebrafish and mammals, Merkel cells arise from basal keratinocytes, a stem cell population in the epidermis, during normal skin development. However, the precise mechanisms involved in this process remain unclear. Stem cells can undergo two types of cell division: asymmetric or symmetric division. I hypothesize that Merkel cells develop from asymmetric division of basal keratinocytes, which would allow the production of new Merkel cells while also maintaining a certain level of stem cells. To test my hypothesis, I examined the Merkel cell lineage during zebrafish scale regeneration (to simulate recovery after injury) which we have established as an experimentally tractable system to study Merkel cell differentiation. Testing of my hypothesis has been conducted through two methods: EdU labeling/antibody staining of Merkel cells and photoconversion of Merkel cells. Preliminary results suggest asymmetric division to be occurring, with no “doublets” of recently divided Merkel cells shown in either method to support the occurrence of symmetric division. My results provide novel insights into the lineage connecting basal keratinocytes to differentiation of specialized sensory cells in the skin. I hope these findings can be used to better our understanding of the restoration of touch systems after injury.

Organelles - membrane bound structures within eukaryotic cells - allow compartmentalization and simultaneous execution of thousands of biochemical reactions within one cell. Rather than serving just one purpose, they are constantly interacting with outside molecules and each other to complete processes. This function is described as being signaling platforms, surfaces that can facilitate interactions between proteins. Mitochondria house signaling proteins that are important for innate immunity and mitochondrial quality control on their surfaces and has been shown to have signaling complexes that associate with the outer mitochondrial membrane to coordinate local protein synthesis. Based on these findings, we hypothesized that mitochondria function as signaling platforms for protein interactions. We chose to focus on two signaling cascades, insulin and calcium, due to their significance in metabolic processes and clinical relevance. To investigate, we used proximity labeling technology, in which a molecular tag is attached to proteins within a certain cellular location. We labeled proteins that alter their mitochondrial association in response to these two stimuli. After cells were treated with stimuli, the lysed and tagged proteins were visualized through Western blots. This process also enables their identification through mass spectrometry. Our goal is to have a detailed characterization of mitochondria associated proteins after insulin and calcium stimulation. Such data will create openings for further research into the mitochondria as a signaling platform and potential for implications in the medical world.

Control of fungal pathogens remains a significant challenge facing agriculture, causing plant death and post-harvest spoilage. Existing methods for fungal control, such as chemical fungicides or transgenic crops, are often non-specific or come with environmental concerns. Prior research indicates that some bacterial symbionts, naturally occurring in plants, can increase resistance to pathogens. In the Sharon L. Doty Plant Microbiology lab, I explored the use of endophyte bacteria to inhibit the growth of three fungal pathogens common among fruit and fruit trees. I investigated fungal growth inhibition through the secretion of volatile chemicals without direct contact. To do this, I grew putative antifungal bacteria together with the fungal pathogens, so that the two cultures were not in direct contact but shared airspace. I identified six strains that were highly successful against multiple fungal pathogens. I cultured these strains to collect any volatiles produced for gas chromatography-mass spectrometry analysis. Several of these strains produce similar volatiles; although the exact structure is not known, the chemical formula is the same among them. In addition, some of these strains produce carbon dioxide, which may be responsible for the inhibitory effect. I also used various bioinformatics pipelines to identify genes responsible for the biosynthesis of these inhibitory volatiles, allowing for deeper understanding of how these strains are able to inhibit fungal growth. These strains have strong potential for use as biocontrol agents. This use would allow for greater pathogen resistance without many of the drawbacks associated with chemical fertilizers or genetically modified organisms.

The geologic record has become an increasingly important source of information for scientists to observe how plant communities of the past have responded to disturbance. Currently, there is a limited ability to recognize disturbance as a primary driver of plant community change, as there is limited evidence of how functional traits – plant traits that relate directly with plant function and ecological strategy – that can be measured in fossil leaves vary across succession. In this study I will measure a functional trait to help better identify disturbance in the fossil record, the carbon stable isotopic composition (δ13C) of bulk organic matter in leaves sampled across a successional gradient following a disturbance. This trait is often preserved during leaf fossilization and is representative of a plant's water use efficiency (WUE), or the amount of carbon dioxide used by the plant during photosynthesis for a given amount of water that is lost during transpiration. It is currently not known the extent to which carbon isotopes measured at the community-scale reflect the successional stage of a plant community. In an effort to develop this tool, I hypothesize that the WUE of plant species within a community will become more conservative in later successional stages. In support of this hypothesis, I predict that the abundance-weighted community average of leaf δ13Cwill increase through succession. In addition, I hypothesize that δ13C as a proxy for WUE will be most confounded in early succession, before a tree canopy forms, due to seedling utilizing water resources more rapidly without having established root systems and thus predict a higher variance of δ13Cvalues in this earliest stage of succession (Cernusak 2020). This research will help develop a method of identifying disturbances within geologic records which can give guidance on management decisions regarding modern ecosystems.

Eelgrass (Zostera marina) is an ecologically important species of flowering marine plant that inhabits sublittoral sediments in the northern hemisphere. Eelgrass beds provide critical habitat for many species of invertebrates, birds, and larval fish. As a true plant with roots, leaves and a vascular system, eelgrass is capable of extracting heavy metals from its environment, potentially making it a sentinel accumulator for heavy metal contamination and sourcing for ecological monitoring. Previous studies of eelgrass beds found that metal concentrations were higher in eelgrass tissues than sediment; however, it is uncertain whether the source of the Pb is the sediment, the water, or both. To determine metal sources and accumulation in eelgrass, we used the natural and anthropogenic variability of lead (Pb) isotopes to fingerprint the source of Pb in bottom sediments and in eelgrass of the Puget Sound. We collected eelgrass and sediment from five sites in Possession Sound, WA between 2018-2019. After purification of Pb from the samples, we analyzed Pb isotope ratios by multi-collector ICP mass spectrometry in the Dept. of Earth & Space Sciences. Significant variation was observed between sites – Hat Island and Whidbey sediments were undifferentiated but Mount Baker Terminal (MBT) sediments have different Pb isotope ratios. Eelgrass from the MBT site also has Pb isotope ratios distinct from other eelgrass and sediment samples, indicating a higher proportion of anthropogenic lead. Site MBT has more pollutant input from the city of Everett, whereas the Hat and Whidbey sites, which are adjacent to uninhabited land, may have more Snohomish River influence. Paired sediment and eelgrass samples from the same sites did not have the same lead isotope ratios, implying that eelgrass accesses multiple sources of Pb during growth. Testing this observation at other sites is important to understanding metal cycling in sublittoral environments.

Trees are sessile organisms and require effective root architecture for water and nutrient uptake. Despite their importance, research in plant root systems is limited in comparison to shoots as roots are difficult to access. Rhizotrons, flat plexiglass pots, enable direct imaging of roots, but are restricted in their capabilities since plant material is started from seed with a limited growth period. These restrictions also limit the ability to model interactions between stress and tree root architecture. Effective experimental systems and accurate biological models are needed to predict how trees will respond to a rapidly changing environment. Here, I demonstrate the ability of a rhizotron using vegetatively propagated material and extended growth times to phenotype the effects of nutrient stress on root architecture in the model tree species, Populus trichocarpa. I then show how Populus traits generated from this system can parameterize a 3D functional-structural plant model (FSPM) for roots called CropRootBox. Populus cuttings were transplanted to rhizotron pots containing high contrast soil media and experienced a nutrient stress treatment. Images collected throughout the growth period were analyzed with RhizoVision and ImageJ to extract root traits including root depth, angle, branching frequency, and lateral root density. These traits advised Populus’ addition to CropRootBox, incorporating the response to nutrient stress. Using this platform, I expected nutrient deficit to decrease primary root elongation and increase lateral root elongation, branching, and density. I also expected these results to improve CropRootBox’s predictions of Populus root responses to nutrient stress. This accessible phenotyping platform accelerated my study on root architecture. CropRootBox showcases modeling’s ability to extend the derived functionality beyond just early growth nutrient stress. In combination, these tools have great potential to aid our understanding of trees and their rhizosphere.

Grasses are a diverse group of plants that play a significant role in many terrestrial ecosystems globally. Despite their importance, very little is known about where and when grasses originated. In particular, it remains unclear in which environment early grasses evolved. Current hypotheses, based primarily on phylogenetic work, suggest that early grasses emerged in closed habitats, such as forests, or in more open habitats along forest margins. However, there is little direct paleobotanical evidence to support either option. To understand the environmental context grasses evolved in, I will be reconstructing canopy openness using phytoliths from Argentinian fossil sites. Phytoliths are microscopic silica bodies deposited in or around plant cells, which can be preserved in the fossil record and used to reconstruct past vegetation. The phytoliths that I am analyzing are from the Las Violetas Formation (57.9-50.6 Ma) of Argentine Patagonia, a rock formation within an area known to hold the oldest records of grass phytoliths in South America. As a proxy for vegetation structure, I am using reconstructed Leaf Area Index (rLAI), which takes the area to perimeter ratio of non-grass phytoliths originated in the leaf epidermis and determines a value that corresponds to how much light passes through a canopy. A canopy with a high leaf coverage correlates to a high rLAI value and vice versa. Based on preliminary rLAI results, I expect to find that ancestral grasses lived in habitats with semi-open canopies, similar to modern shrublands. Investigating the ancestral environment of grasses can help us better understand their evolutionary history and potential environmental drivers that led to their success in terrestrial ecosystems. This information can help us gain insight on the vulnerability of grasses and grass-dominated habitats to environmental changes in the past, present and future.

Trait-based plant ecology can serve as a means to better understand shifts in ecological strategies within plant communities and how that affects greater ecosystem processes, such as productivity, across a period of major climate change. The most recent major global warming event prior to modern anthropogenic influences was the Miocene Climatic Optimum (MCO) ca. 17-14 million years ago. This event was a short aberration to a long-term cooling trend of the last 53 million years, with global temperature averages up to 8°C warmer than preindustrial averages. Changing climate conditions during the MCO may have led to plant community reshuffling, with many ecosystems possibly restabilizing with notably different optimal trait distributions. Functional traits such as leaf shape, size, and toothedness can be indicative of a plant’s ecological strategy. These leaf morphology variables have been shown to closely correlate with climate, highlighting their role in plant function and strategy, and can thus be used to statistically analyze community diversity across the MCO. I hypothesize that the MCO caused an overall increase in functional trait diversity through an increase in favorable environments, allowing plant reshuffling or migration of plants with new ecological strategies into existing communities. We expect to see this through trait distributions in a community diversifying as the MCO progresses. This study uses leaf functional trait data measured digitally from a range of Miocene fossil sites to assess trends and variances from before, during, and after the MCO. Statistical analysis will make use of a previously developed R package to assess functional diversity. These results will be crucial information in understanding the ecological response to today’s far more rapid climate change, as well as humanity’s response to the possible need for human-assisted plant community reshuffling by providing an example of how global warming affected vegetation in Earth’s past.

The deinstitutionalization of massive psychiatric hospitals in the 1970s and 1980s improved the physical and social environment of psychiatric hospitals and aided in the transition away from prison-like hospitals. Still, there remains opportunities for growth if the psychiatry field aims to engage and provide patients with a fully therapeutic setting in psychiatric hospitals. This research explores the relationship between a patient’s social and physical experience in a psychiatric hospital and their engagement with mental health treatment after discharge. I analyze the physical environment of a psychiatric unit and explore mental health professionals’ views on guidelines for maintaining a therapeutic environment in these spaces. Using qualitative methodology, including ethnographic observations of psychiatric hospitals in the Seattle area, photographs, and interviews, I discuss the environmental factors of psychiatric rooms through the evaluation of room design and its effectiveness in creating a therapeutic environment. Preliminary findings point to a possible relationship between consent and a patient's receptivity to post-hospitalization treatment after being exposed to the psychiatric hospitalization environment. Additional results could indicate a negative relationship between a patient’s socio-physical experience in an inpatient psychiatric facility and their engagement in future treatment after discharge. These research findings will enable mental health providers to better understand the relationship of socio-physical aspects in psychiatric hospitals to the continuation of a patient’s treatment after discharge. This information will help providers improve upon these experiences and increase a patient’s receptivity to post-hospitalization treatment.

Culture tends to view psychology as interesting but medicine as important. These two perceptions, while not mutually exclusive, may differentially influence journalistic practices around scientific reporting and create disparities in the fidelity and nuance with which news outlets convey scientific information on these topics to the general public. The capacity for even the most well-intentioned news media to sensationalize, misrepresent, or oversimplify information makes it important to characterize how the popular press talks about research findings, especially those pertaining to mental and physical health. The present study aims to build this understanding. Using a web-scraping algorithm, we collected articles reporting psychological and/or biomedical findings in popular press outlets worldwide. A team of trained raters reviewed these e-articles and systematically evaluated the extent to which each article (1) describes the findings' limitations; (2) identifies the specific samples (of people, methods, and contexts) involved in the cited study and how they potentially constrain the findings' generalizability; and (3) avoids making unsubstantiated claims without providing caveats. We expect that the popular press frequently neglects to call attention to scientific findings' constraints on generality (COGs) and other limitations, and frequently makes unsubstantiated claims without caveats when reporting on such findings. In particular, we hypothesize that these problems are more prevalent when findings are relevant to psychology and/or mental health compared to when they are relevant to biology and/or physical health. This investigation provides important information to researchers, journalists, healthcare providers, and consumers about the current state of scientific reporting, how to constructively navigate it, and how to improve it.

Soldiers who are transitioning to civilian life have alarmingly high rates of suicide, alcohol abuse, and mental illness. There are multiple facets that may influence these poor outcomes for veterans. Organizational dissatisfaction is a risk factor for poor mental health and lack of access to mental health support for veterans, but this has yet to be studied in-depth. The aim of this project is to assess how demographic variables and organizational dynamics impact mental health during the transition process. First, we will use preliminary data from The Network Study, one of the first longitudinal studies to follow the mental health, alcohol use, and socioeconomic stressors of service members as they transition out of the Army. Multiple regression will be used to analyze the influence of interpersonal support and conflict within the Army on depressive and anxiety symptoms. Mental health symptoms were measured by the PHQ-9 and GAD-7, two common screening questionnaires used to briefly assess depressive and anxiety symptoms, respectively. We will additionally assess various demographic variables as moderators. Next, findings will be used to develop questions for qualitative interviews to be conducted with recent veterans. We predict there will be a positive association between organizational support and positive mental health outcomes, and qualitative interviews will show lower work-related stress and increased access to mental health supports as major themes for those with positive work relationships. In line with previous research, we predict these associations and themes will be strongest for women, racial and ethnic minorities, and those of lower rank. Support and intraunit conflict are potentially modifiable factors, so our research demonstrates a potential pathway for prevention of negative mental health outcomes for veterans.

Homelessness and environmental disasters are two issues that are becoming more widespread and concerning in the wake of increasing housing prices and climate change. People experiencing homelessness are at a greater risk of harm from environmental disasters, however there has been very little empirical evidence gathered on the way these two problems intersect, with most of the existing research relying on a more theoretical framework. In this project, I collect and analyze data on the effects of environmental disasters on people experiencing homelessness in both Seattle and New Orleans in order to provide more quantifiable research on this subject. I collect the cause of death records from recent years for people who were experiencing homelessness to observe how many of these causes were related to environmental disasters, such as extreme heat or cold. This data provides evidence of how much of an impact these disasters can have on the health and safety of people experiencing homelessness. I also compare maps of the areas of these cities most at risk of environmental disaster with the areas where most of the homeless population lives to analyze how much overlap there is between the two. These maps show how much more vulnerable people experiencing homelessness are to environmental disasters from a spatial perspective. I compare the data from these two methods between the two cities in order to see how a city like Seattle, where environmental disasters are less frequent, relates to a city like New Orleans, where they generally occur more often and with greater severity. I use the results of this project to either back up, or refute, the more theoretical claims regarding the vulnerability of people experiencing homelessness to environmental disasters, and explore potential reasons for why these results occurred.

Engagement barriers are barriers that prevent individuals from participating in Attention Deficit Hyperactivity Disorder (ADHD) therapy. Kazdin et al. (1997) found that having fewer engagement barriers serves as a protective factor to prevent treatment dropout. Despite knowledge behind the importance of having few engagement barriers, who experiences what barriers is largely unexplored. By understanding what engagement barriers can arise during behavioral therapy, and who experiences them, therapists can generate tools to overcome these barriers, thus improving the treatment of adolescent ADHD. Our purpose of this study was to ascertain and understand any relationships between engagement barriers in behavioral therapy and clinical characteristics/sociodemographic factors. Participants were 121 adolescents with ADHD (which came from an ethnically diverse sample) and their parents. Twenty-five different barriers were sorted across three subtypes (cognitive/attitudinal, behavioral, and logistical), three sources (parent, teen, or dyadic) and three treatment phases (engagement, skills, and planning). Minority status, gender, ADHD medication status, family adversity, Oppositional Defiant Disorder (ODD) comorbidity, and parental ADHD served as independent variables. I used multiple regression models to analyze relationships between each independent variable and barrier subtype, source, and treatment phase. ODD comorbidity and parental ADHD each significantly predicted a primary outcome of an overall increase in barrier frequency. Significant secondary outcomes included ODD comorbidity and parental ADHD each significantly predicting greater cognitive barriers, teen barriers, engagement phase, and skills phase barriers frequencies. ODD also predicted significant increases in planning phase barriers frequencies, while minority status significantly predicted lower planning phase barriers frequencies. These results demonstrate that populations of adolescents diagnosed with ADHD and comorbid ODD or who have parents that are also diagnosed with ADHD are at risk to experiencing more engagement barriers. Additional research is required to develop therapeutic tools. These tools will help therapists better serve adolescents with comorbid ADHD/ODD, and parents with ADHD.

In the United States, caregivers carry a higher level of physical and emotional stress than non-caregivers. According to the National Alliance for Caregiving, over 60% of caregivers consider their caregiving situations to be moderate to highly stressful. In response to the multifaceted needs of family caregivers, a team of health researchers and engineers initiated a research project called COCO ("Care for Caregivers Online"). COCO’s goal is to design and implement innovative methods to support caregivers emotionally. We have implemented a system with two platforms connecting family caregivers with healthcare providers: a web app with a conversational agent (“chatbot”) for caregivers and a provider platform using artificially intelligent (AI) technologies for healthcare providers to provide effective and efficient support through the mobile app. We conducted a pre-post study with forty caregivers who chatted with COCO through the system. The initial result shows that COCO significantly reduces caregivers’ negative feelings including guilt, sadness, and fatigue. We are expecting more positive results after deploying the mobile application with the chatbot embedded. Additionally, I, along with other reasearchers, conducted research on chatbot’s ability of emotion detection. We collected caregivers’ emotional state data through a two-week diary study and I personally preprocessed and analyzed the data.  We then used these data to develop a natural language understanding (NLU) model. In simplicity, we incorporated the ability of commonsense into the chatbot, achieving better results in emotional state inference and grounding response recommendation. I am currently leading a digital human project, creating a virtual human profile for COCO to ahieve better user experience. The integrated care approach COCO takes with AI-enhanced empathetic and therapeutic dialogs has the potential to address the critical needs of millions of family caregivers in an accessible, scalable, and cost-effective way.

This research aims to understand why such a reliance exists within the disability parent community in Washington State in terms of navigating healthcare, and how the healthcare system can do a better job of supporting families who have children with developmental disabilities. There is an incredible disconnect and lack of support at the time of diagnosis for these families, and often it is not the healthcare providers that support these families and assist in their navigation of the health system. Rather, the experience of other parents that have gone through the process of navigating the health system for their child with a developmental disability are the best resources for these newly diagnosed families. My major research question is: How can the disability community and the healthcare system work more in unison to ensure that families are able to maneuver through the health system successfully from the start of their journey with a child with a disability? I have conducted ethnographic research through interviews with disability parents, healthcare providers in developmental medicine, patient navigators, and various in-person and Facebook disability parent support groups. The results of this work are presented as a written thesis, highlighting the challenges these families face, the nature of the reliance on community to navigate care, and proposed suggestions for the healthcare community to assist families in accessing parent support networks. This research highlights the strength, reliance, and efficacy of the disability parent community in helping one another navigate a health system that is lacking adequate support for newly diagnosed families. This research is being utilized to bridge a gap between the healthcare system and the disability parent community, so that they can work more in unison to serve families with children with developmental disabilities, and most importantly, improve the lives of these children.

Currently, it is not well understood how young people in Low and Middle-Income Countries (LMICs) access health services such as primary care and mental health services, which are easier to access in other parts of the world. Since adolescents are a significant, unique population in these countries, there is evidence to suggest that health services need distinct features and considerations to be adolescent-friendly and responsive. However, there is no universal, comprehensive definition of adolescent-friendly health services. To fill this gap in the body of knowledge, we are conducting a systematic review to collate, synthesize and identify the barriers and facilitators of adolescent-responsive health services. We worked closely with a research librarian to perform a robust search through a range of databases (CAB Direct, CINAHL, Cochrane, Embase, Global health Medicus, PsychINFO, and PubMed) which resulted in 4751 unique abstracts. During the title and abstract screening, two members of the team reviewed each article. Currently, 171 articles have been selected for full-text screening to confirm they are appropriate for inclusion and for analysis. Next, we will create a narrative synthesis of the features of service, keywords, factors, and outcomes identified. The research will provide a clear, comprehensive definition of adolescent-responsive health care and services in LMICs. This information will be particularly useful to international organizations, like the UN, healthcare providers, and policymakers as it will inform new programs, initiatives, or guidelines to improve the quality of adolescent health services in LMICs.

Laser-induced graphene’s (LIG) simple and rapid fabrication has led to the development of flexible sensors with various applications in wearable electronics. LIG is produced in ambient air through CO₂ laser scribing on a polyimide film. Although LIG has been incorporated into flexible chemical and strain sensors, its sensitivity to resistance changes under deformation and instability prevents it from being fully utilized as a flexible conductor. This work presents a versatile technique to increase the electrical conductivity of LIG and enhance its structural stability so that it can be used as flexible conductors in printed electronics. This is achieved by the deposition and activation of functionalized liquid metal (LM) nanoparticles on LIG traces. To overcome the repulsion of LM on LIG’s surface, the CO₂ laser’s settings are adjusted to create LIG traces with a superhydrophilic inside and superhydrophobic border. Additionally, the adhesion between the LIG and LM was improved through surface functionalization of the liquid metal droplets. Our results show the resistance of LM-LIG traces to be 3 orders of magnitude smaller than that of LIG traces. Electromechanical characterization of the LM-LIG traces demonstrate low resistance changes under large bending deformations. The combination of the liquid-phase conductor and 3D structure of graphene enables the fabrication of customizable, solder-free, flexible circuits with high mechanical stability. We demonstrate this technique with the fabrication of flexible light-dependent resistor circuits that serve as a basis for further flexible sensor and biosensor exploration.

Development and testing of sensors and power methods for insect-based robots is a difficult task. Due to the high cost of manufacture with regards to both training time and funds, finding a sustainable and easy-to-produce method to test sensors and power options is essential. Previously, the only option for testing new sensors and power options was using one of the robotic insects, which is risky considering their high costs. Drawing from prior results, we believe a lightweight quadcopter would be faster, easier to produce, more robust, and able to serve as a suitable replacement in sensor testing and development. My goal will be to create the world's lightest and smallest quad-rotor helicopter, “TinyQuad,” with a target mass of 1–2 g. The new helicopter I have designed will enable the testing of new sensors such as cameras and power options such as radio frequency-based charging. This design allows for testing a variety of sensors and electronics configurations very quickly, with the potential to rapidly speed up the prototyping process. I will demonstrate flight capabilities through utilizing wireless charging, and sensor-based feedback control to improve flight stability and duration. I completed calculations and design of this hardware, and anticipate seeing that the collected flight data supports the utilization of a lightweight quadcopter in insect robotics development. This will allow us to rapidly develop and refine sensors for use onboard the RoboFly robotic insect platform. Creating a working quadcopter would result in accelerated prototyping that allows for more unusual sensor and payload designs, and for further research in developing new sensors and power methods for insect robotics, smaller quadcopters, and improved design of micro aerial vehicles.

This work proposes a novel generative design tool for passive grippers—robot end effectors that have no additional actuation and instead leverage the existing degrees of freedom in a robotic arm to perform grasping tasks. Passive grippers offer interesting trade-offs between cost and capabilities. However, existing designs are limited in the types of shapes that can be grasped. This work proposes to use rapid-manufacturing and design optimization to expand the space of shapes that can be passively grasped. Our novel generative design algorithm takes in an object and its orientation with respect to a robotic arm and generates a 3D printable passive gripper that can stably pick the object up. To achieve this, we address the key challenge by jointly optimizing the gripper shape and the insert trajectory to enlarge the set of objects that can be pasively grasped. We evaluate our method on a testing suite of 23 objects, all of which were evaluated with physical experiments to bridge the virtual-to-real gap. Inspired by the true cost of repurposing infrastructures in assembly lines following the recent changes in demand early in the COVID-19 crisis, our work allows a cost effective solution to rapidly generate, fabricate, and deploy custom passive grippers on the existing robot arms for the new products in demand.

Traditional foams are fabricated via stochastic chemical processes that yield homogeneous material properties. Foams can exhibit a wide range of material properties by varying process controls allowing them to be used in many industrial and commercial applications. Previously, additive manufacturing could only produce foam approximations in the form of traditional lattice infill. My work employs viscous thread printing (VTP) of thermoplastic polyurethane (TPU) on a fused filament fabrication (FFF) printer, exploiting the semi-viscous nature of extruded filament to coil producing a new type of printed foam. Specimens were tested under compression to determine uniformity along principal axes and behavior under strain when compared to infill patterns, such as grid and cubic. My work establishes that VTP, using elastic materials, can be used to manufacture programmable stiffness foams as a function of density, suited to a variety of needs and should be considered as an alternative to traditional foams and other printed lattice geometries.

Social robots have been used to support mental health, because they are capable of forming meaningful interactions with humans due to their social intelligence, which boost user engagement with social robots. Thus, social robots can better foster emotional support with mental health data visualization compared to other technologies in collecting and displaying relevant data from humans. In this Human-Robot Interaction (HRI) research work, I explored their potential as community-based tools rendering data visualizations that represent a community’s mood patterns. Collecting and visualizing the mood patterns of a community might help the community members raise awareness about the mental health of the community, and whether there are significant factors negatively impacting the mental health of community members. In a two-day study, twelve participants recruited from a university community engaged with a robot displaying mood data. I created a data visualization template to show random mood data and placed it in a social robot. The design choices (e.g., spectrum of data varieties, data representation) of the template were made hypothetically in a random manner with no evidence basis from any particular community’s moods. During the study, participants were asked questions verbally and through written prompts on paper to express their feelings about robot-rendered visualizations’ usefulness in their lives. Given the feedback from participants, I modified the data visualization on the robot screen with the aim of increasing accessibility, universality, and usefulness of such visualizations. Currently I am implementing a real-time data collection and visualization system in the robot’s end-user-programming tool. In the near future, I plan on conducting studies to assess how interacting with the robot’s automated data collection and visualization software affects users and if it produces an improved experience for them. Eventually, I hope to evaluate how data visualization enhances a sense of belonging and support among community members.

Wrist and hand motion is continuously present and necessary in daily life, making it important that we understand the biomechanics of the joints along with the effects of injury. The use of cadaveric studies is crucial in understanding the biomechanics of the wrist and hand. Currently, most cadaveric studies on wrists are performed statically, which can fail to represent the complexity of dynamic wrist motion. To address the inaccuracy of static testing, we designed, constructed, and tested a system capable of dynamic testing of the wrist under prescribed loads. The dynamic cadaveric test bed utilizes a robotic pulley system to load the tendons that actuate the wrist and hand along with a fuzzy logic control system to recreate physiologically accurate wrist motions. Biomechanical data, such as tendon forces and joint kinematics, are collected simultaneously during motion to provide wholistic insight on the biomechanics of the wrist and hand. To validate the cadaveric test bed, a 3D printed wrist model was used to ensure accurate control of the control system and robust data collection. The wrist model consisted of a model forearm, palm, and wrist joint that simulated wrist motion using string in lieu of tendons. After successful trials using the hand model, the test bed is now being utilized for biomechanical wrist and hand cadaveric studies, including studies regarding surgery effectiveness and injury analysis. The dynamic cadaveric test bed’s ability to more accurately study wrist and hand motion could lead improved treatment practices for wrist injuries.

The conventional signalized diamond interchange provides numerous essential freeway-to-arterial connections. However, it tends to become operationally inefficient when high traffic demands exist on the arterial or off-ramps. To address this problem, diverging diamond interchanges (DDIs) have been implemented at many sites to replace conventional diamond interchanges (CDIs). While DDIs have been shown to outperform CDIs under high left-turn demands, their operational performance diminishes when through demands on the arterial become heavy. In this work, I proposed a new service interchange design named the “one-sided diverging diamond interchange” (one-sided DDI) as a replacement for congested CDIs and DDIs. Through a comprehensive series of microscopic simulation tests with the software PTV Vissim, I analyzed and compared the operational attributes of the one-sided DDI to those of the CDI and DDI over a range of traffic demands. Overall, the results from the simulation tests indicate that the one-sided DDI significantly outperforms both the CDI and DDI in vehicle travel time and throughput when through demands on the arterial are dominant. On the other hand, the one-sided DDI tends to modestly outperform the traditional DDI in travel time when handling moderate to high proportions of left-turning traffic.

In many languages, nouns have what is called "grammatical gender," which is a seemingly arbitrary gender that is assigned to each noun. A common example would be Spanish, where nouns with the article la are feminine and those with el are masculine. In German, these noun genders are notoriously difficult to learn. This is, in part, due to the fact that German has three grammatical genders (masculine, feminine, and neuter) compared to the more common two (masculine and feminine). In addition, there are often no markers at the ends of nouns to indicate whether the noun is masculine, feminine, or neuter, which means learners cannot tell the gender of the word simply by looking at it. For example, in Spanish, feminine nouns often end with -a and masculine nouns with -o, which make the gender relatively easy to identify. This is not the case in German. Learners must, for the most part, learn noun genders through rote memorization. In this study, I test three different methods of helping native English speakers acquire the grammatical gender of nouns in German. I investigate whether learners can effectively acquire the gender and meaning of twelve German nouns with three mnemonic devices: color association, images, and videos with gendered actors. Participants are split into four groups, one control and one for each mnemonic device, where they are exposed to the nouns. The short-term effects of these visual aids are then revealed through two post-tests following the session. Based on previous research, I believe that the video condition will be the most effective at helping learners acquire noun gender. Given that memorization of grammatical gender is crucial to effectively learning a language, it is worth exploring innovative ways of helping students acquire this feature, especially for speakers whose first language does not have gendered nouns.

Inequities in healthcare have led to disparities for many populations, including autistic individuals. One structural factor associated with health inequities for autistic individuals are the tools used to screen for autism, which do not work equitably within minority populations. There is, therefore, a great need for culturally sensitive screening tools. My study assesses the cultural sensitivity of “My Toddler’s Social Communication” (MTSC), a tool developed by UW’s ReadiLab to screen toddlers at autism risk. Unlike existing screening tools, MTSC employs photographs of toddlers engaging in social communication behaviors, along with brief behavioral descriptions. I used a sequential explanatory mixed-methods design to investigate whether caregivers of toddlers find MTSC to be feasible for use with culturally and linguistically diverse (CLD) families; and find the photos and behaviors culturally relevant. For the first phase, 10 caregivers of toddlers completed MTSC and answered questions regarding its acceptability and feasibility for CLD families. For the second phase, I interviewed 15 CLD caregivers. I then analyzed survey data using descriptive statistics and the interviews using thematic analysis. Survey results showed that 80% of caregivers found the photos representative of a broad range of cultures. However, only 30% of respondents agreed that MTSC was easy to complete for caregivers from diverse backgrounds. Thematic analysis revealed five themes: visual representation deficits, cultural relevance differences, accessibility to non-native English speakers, and accuracy challenges. Overall, results suggest that MTSC has fair visual representation, but can become more feasible and accessible by accommodating caregivers who utilize daycare services, and including photos with a greater range of races, cultures, and caregiver genders. This study is one step of many toward creating measures that can assess the cultural sensitivity of tools to make cultural sensitivity accessible for researchers developing instruments and improve the lack of diversity and equity in Clinical Psychology.

Episodic memory is a crucial element of the human condition. It allows us to remember what happened, where it happened, when it happened, and then piece these components together to form comprehensive memories. However, the ability to remember when an event happened lags years behind the ability to remember what happened and where it took place. At the root of this phenomenon is the concept that temporal reasoning ability takes a more gradual path to maturation as well. In several cultures, the challenge of reasoning with time is alleviated using a spatiotemporal model called the mental timeline—a representation of time based on a left-to-right linear frame. With the proposed research I aim to prime 32 6-year-old children with a left-to-right spatiotemporal model, and 32 additional 6-year-old children with a nonlinear model of events before examining each of their spatial and temporal memory binding abilities. The first phase of this study—in which children are primed to think about time in a spatial format—entails a short animation of a character visiting nine places around the world. Icons corresponding to each event are then placed either in order from left to right, or in a nonlinear format. After completing the priming task, children participate in an unrelated memory game to assess the effects of spatiotemporal priming on their temporal memory abilities. They watch three animations, each consisting of a character completing nine activities in nine different locations. After each animation, they answer three corresponding location-related and order-related questions each. I expect children primed with a left-to-right model to outperform children in the nonlinear priming group on temporal-order probing questions. We hope that the anticipated results can be used in academic settings to strengthen memory abilities in young children and shift the nature of how we communicate with children about time.

Despite trends in hearing technology, there is little understanding of how listeners judge quality of speech. The lack of understanding of these perceptual mechanisms prevents development of efficient approaches to improving speech quality technologies. The proposed research addresses this knowledge gap and is among the first studies to elucidate the differences in listening strategies between speech understanding and speech quality judgments. It is well established that the frequencies in speech contributing to speech understanding varies--certain frequencies are more essential than others. The relative importance of different frequencies, known as spectral weights, represents listeners' strategies when performing speech recognition tasks (i.e., repeating back the presented speech). We hypothesize the spectral weightings of listeners judging speech quality compared to speech understanding will differ--our preliminary results have proved our hypothesis true as frequencies are proving to contribute disparately for understanding versus recognition. Previous research demonstrates clarity of pitch being a key contributor to perceived speech quality, while speech understanding may be maintained when voice pitch is heavily distorted. Therefore, the frequencies carrying information in pitch may become more important in speech quality judgment. As research continues, I will test this hypothesis by analyzing the differences in the participants' spectral weightings of a speech recognition task and a speech quality judgment task. To discern the spectral weighting, a correlational analysis is done for each of the two tasks using averages in the responses. This correlational analysis averages data from the participants' responses to find how each frequency band of interest contributed to understanding versus recognition. This project provides one of the first estimates on the contribution of frequencies in perceptual judgments of speech quality, which is valuable for future hearing technology aiming to improve perceived speech quality in that it pinpoints the frequency regions requiring attention in audiological research to come.

Noisy environments pose difficulty for efficiency of communication and understanding of conversation. However, some individuals are able to hear better in these environments than others, with age and hearing ability controlled. A contributing factor may be the natural rhythm of speech. All of speech contains rhythm, we speak at different paces with words timed at certain paces. When this rhythm is altered, it is shown to reduce speech understanding. This study investigates whether people are the same in terms of how much they rely on speech rhythm to decipher speech in background noise. The purpose of this study is to use these results to create more effective and customizable hearing aids catered to each individual's hearing differences. We looked at two promising factors in this study that may predict increased ability to use the natural rhythms of speech in order to better understand it: working memory and non-speech rhythmic abilities. We created remote tasks on an online platform, Gorilla, to measure these factors in normal hearing individuals. More specifically, these tasks included a working memory task, Reading Span; a rhythmic abilities task, Beat-Based Advantage; and a rhythmic speech in noise task. We used stepwise linear regression to analyze whether these two factors impact one’s reliance on rhythm in speech. The findings of this study support that better working memory skills and better rhythmic abilities improve speech understanding in noisy environments, as these participants utilized speech rhythm to do so. We can use this knowledge on future research with participants with hearing loss. We still live in a “one-size fits all” model for hearing aids. However, by understanding the factors that improve speech comprehension in noise, we can create individually-tailored hearing aids that adapt to people’s abilities and thus improve communication in all types of environments.

There is often a stigma associated with bilingualism due to the mistaken belief that bilingual children develop language skills at a significantly slower rate than monolingual children. To combat this misconception, it is necessary to conduct research showing that bilingualism either helps or does not harm language acquisition when compared to monolingualism. This study attempts to meet that general need by determining how bilingual and monolingual children compare in metaphor comprehension. I decided to focus on metaphors because they can be challenging for children to interpret correctly, and I hypothesized that bilingual children would perform better on metaphor comprehension tasks than monolingual children because of their regular use of second labels. I investigated how 127 monolingual and bilingual children ages 2.5-4.5 years old responded to a set of metaphors by using a game-like format to gather data via Qualtrics. Specifically, I gave children a series of 10 metaphor questions in which they needed to choose which of two pictures best fit a provided metaphorical phrase. Children were also given 10 vocabulary questions derived from the metaphor questions to ensure that children knew the literal meaning of the words. Results showed that bilingual children performed slightly but significantly better than monolingual children on metaphor comprehension questions. Currently, I am also collecting data for a follow-up study to replicate and extend my findings. These results are important because they give us insight into how child language development differs between monolingual and bilingual children and counter negative stereotypes about bilingualism.

Early childhood language capability is closely tied to future success. Thus, it is vital to research how best to support the language acquisition process of young children. Extant literature emphasizes the role of children's parents but largely ignores the impact of other family members such as siblings. The primary goal of my research is to address this absence by investigating the effects of older sibling presence upon the language development outcomes of infants. I use a longitudinal dataset of audio recordings taken by Language ENvironment Analysis (LENA) software. This software uses a small device worn by a child over an entire day to record their naturalistic linguistic environment for up to 16 hours. I focus on two main indicators of language input quality: parentese and conversational turns (CTs), both of which have been shown to promote language acquisition. Commonly referred to as “baby talk,” parentese is the acoustically exaggerated speech style typically used while addressing infants. CTs are alternations between speakers in one-on-one conversation, and often include nonverbal aspects of communication upon which infants rely for learning. My present findings demonstrate that infants with older siblings experience lower levels of these variables compared to infants without older siblings. However, the differences between language outcomes of the two groups were not as pronounced as expected based on this difference. Thus, with the help of fellow undergraduate coders, I am now coding and transcribing segments of LENA recordings of infants with older siblings that have the highest amount of speech from other children. I hope to uncover sibling input variables that may help infants with siblings to "catch up" to those without siblings regarding language outcomes. My results will inform those involved in early childhood development regarding how to facilitate the language acquisition process of young children with older siblings most effectively.

Chokepoints are strategic, narrow passages that connect two large areas to one another. When it comes to maritime trade, these are typically straits or canals that see high volumes of traffic because of their optimal location. In March 2021, due to a strong wind, a large container ship called the Ever Given blocked the Suez Canal, a narrow trading waterway (a maritime chokepoint) that essentially connects Europe and Asia, while also handling approximately 12% of global trade. The blockage resulted in 9.6 billion dollars of goods and services to be delayed. Furthermore, it worsened the existing supply chain constraints that were caused by the COVID-19 pandemic. Nonetheless, this event displayed how reliant or dependent international trade is on a chokepoint and showed how easily the whole process can be disrupted because of an incident that could be considered by many as insignificant. This paper is an event study of the Ever Given blockage, which aims to see how it has impacted volume of trade between countries that are situated on either side of the Suez Canal. Using a gravity model of trade, I examine and compare the effect that the event had on the volume of trade of various countries bordering the Mediterranean Sea, with Saudi Arabia on the Asian side of the canal. The broader ambition of this paper is to stress the economic reliance of international trade, in terms of volume of trade, on major chokepoints in order to motivate us to find, create and develop alternative trading waterways that could be used at the event of future disruptions at strategic chokepoints.

The Washington Creative Districts program, managed by ArtsWA, is a place-based investment strategy that offers state certification to geographically defined areas focused on cultural and creative enterprises. ArtsWA provides technical and professional assistance, marketing, and funding to certified Creative Districts to encourage localized economic development through tourism and hubs of economic activity. My research examines Washington Creative Districts policy to determine what unintended consequences may be felt by communities pursuing state certification. I identify potential unintended consequences by reviewing enacted legislation in the Revised Code of Washington, Creative Districts guidelines, academic literature, and recent Washington state employment and housing data. I find the state’s interpretation of the creative economy includes professions with high social capital, such as writers and photographers, and professions with high economic capital, such as software developers and computer programmers of the knowledge economy. This broad framing of the creative economy could result in Creative Districts using social capital of traditional artistic professions for branding while primarily courting the mobile, economic capital of the knowledge economy. I expect to find that communities pursuing state certification, some of which are in economically distressed regions, may accelerate cycles of gentrification in urban areas or introduce gentrification to rural ones, resulting in long-term resident displacement. Additional research is needed to document market, demographic, and population change trends present in Washington Creative Districts and determine if their relationship to state certification is causal or a correlation. Additional work is also needed to identify local and state policy changes that could better equip Creative Districts communities to mitigate any unintended consequences and focus state support and investment in areas of the local creative economy where it is most needed.

While many fields of study, such as psychology and sociology, have long offered various investigations and explanations of crime, starting in the latter half of the Twentieth Century, economists have begun attempting to understand crime through an economics lens. Our research has furthered this inquiry by looking at how crime rates are driven by changes in compensation for legal work as affected by changes in state income taxes. Specifically, we examined how a four-year long increase in Illinois’s flat income tax that began in 2011 affected crime rates in the state in comparison to crime rates in similar states and nationally as well. We used a difference-in-difference analysis to understand how the change in state income tax rates led to a change in crime rates in our treatment state, Illinois, when compared to those states that represent our control group. Using data on tax rates levied in each state, crime data aggregated by the federal government, and other publicly available data to inform our analysis, we have an idea of the relationship between changes in state-level income taxation and changes in crime rates as observed in Illinois. We expect that an increase in taxation will put upward pressure on crime rates in Illinois as the returns to legal work will be comparatively decreased. The hope for such work is not necessarily to inform the conversation surrounding appropriate tax rates but to contribute to an economic model of crime. Such models empower policymakers to combat crime not as a function of some base flaw in a criminal’s humanity or cognition, but as the outcome of some malleable analysis of personal costs and benefits.

Between 1973 and 1990, Chile was ruled by Augusto Pinochet, a military dictator known for his persecution of leftists and political opponents. Although Chile transitioned to democracy, ratified the American Convention on Human Rights and joined the Inter-American System for the protection of human rights (IAHRS) in 1990, Pinochet's legacy of human rights abuse lingers. This is exemplified by the Chilean military justice system's ongoing jurisdiction over cases in which military personnel harmed civilians, which violates the Convention's guarantee of access to competent courts and fair trials. This research fills a gap in the existing literature on this violation by exploring the following question: how do the combined structural weaknesses of the Chilean judiciary and IAHRS limit the Chilean military justice system's compliance with the Convention? To address this question, I qualitatively analyzed the foundational documents of the Chilean judiciary and IAHRS with a focus on how vague or inconsistent language in the articles that are procedurally connected in the interactions between the two institutions overlap and create loopholes that allow for Chile's ongoing Convention violation. This thesis argues that ambiguous and contradictory language in the articles of the Chilean and IAHRS documents involved in the referral of human rights petitions to the Inter-American Court limits compliance by jeopardizing the legitimacy of petitions against Chile for inappropriate military jurisdiction and by creating significant deterrents for those considering petitioning against Chile. As many Latin American countries were ruled by military dictatorships and have since ratified the Convention, this research could illuminate tendencies central to understanding and rooting out dictatorial legacies in the region and inform more effective collaboration between the IAHRS and its member states in addressing impunity for human rights abuse. 

In the last two decades, Chinese state lenders have been responsible for $153 billion in loans to African countries. Acknowledging assertions in the literature that this lending has had tangible negative effects on debt sustainability in several of those African countries, my research project analyzes the effectiveness of this lending in improving welfare through the financing of energy projects. This represents a first step in answering the broader question of whether official Chinese lending to African nations, in its current state, is worth the accompanying risks.

Combining data from Horn, Reinhart, and Trebesch (2021), William & Mary's AidData Initiative, and other macro-level datasets on national electrification, I build a comprehensive picture of Chinese lending to African energy generation projects, as well as the long-term success of those projects and their effects on national energy outcomes. My analysis will be based in part on descriptive statistics generated from my dataset and in part on an econometric analysis run on the dataset that factors in costs, energy output, and national debt and labor metrics.

This research is expected to yield the result that while Chinese investment is largely effective and prioritizes sustainable energy generation projects, opportunities exist for more accommodative lending practices and greater domestic labor involvement in project implementations.

In 1979, the administration of Iran was overthrown and in its place, the Islamic Republic of Iran was proclaimed. The event challenged understanding of what Islam is in the modern world and questioned the dominance of secular society. How did Islamic influence in Iran evolve to champion the cause of a revolution? What was the context which gave it its influence? This thesis examines the effects of Iran's economic conditions prior to the revolution. It connects large scale modernization projects and the economic and religious histories of Iran. The comparison reveals a distinct adaptation of religious rhetoric which champions the economic adversities of the common citizen and galvanized support against economic exploitation in the country. To accurately analyze these themes, I utilize a quantitative and qualitative approach. The paper collects economic data such as growth and income inequality from international and Iranian sources. By comparing this data with the primary accounts of prominent Iranian Islamic academics, the paper bridges a connection between economic condition and religious rhetoric. It reveals how Islam becomes a means for economic justice and a defense against the disenfranchisements of modernization. The paper implicates the importance of financial well-being on the ideological outlook of a society and provides a comparison to future ideological change within a nation.

This research compares the COVID-19 control policies and the use of Contact Tracing Apps (CTAs) in Shenzhen, China, and Seattle (King County, WA). The research examines the differences between local government's policies of pandemic control, contact tracing response, and the discrepancies between CTAs design, mechanism, and the ways of defining close contacts. This first part provides background for a better understanding of the differences in government policies and CTAs for the two metropolises. The second part of the research analyzes data available to show the effectiveness of contact tracing apps and the results of differences in government pandemic control policies and response time. Visualizations are created using existing data and construct timelines of the pandemic and usage of CTAs, demonstrating the effects of CTAs on certain indicators such as numbers of hospitalizations, people in quarantine, cases, and deaths. The timeline will be divided based on different COVID-19 mutations that were dominant in the areas, as well as the version updates on the CTAs. After analyzing the effectiveness of CTAs on pandemic control, the research explores the overall impacts of CTA, COVID-19, and government’s pandemic responses on society as a whole, including the inequalities between races, different income groups, and migrations. This research may generate insights on the possible policies and ways of utilizing CTAs that are beneficial to the pandemic control as well as the economy and residents' daily life. Besides, as there are few studies on CTAs’ effectiveness on COVID-19 control, this study can also suggest possible directions for other researchers. In the end, the research generates visualizations that tell a story from the beginning of how the government deals with the pandemic and how their responses and CTAs had an effect on the pandemic control and the society, especially on migration and inequalities.

In biomimetics, nudibranch sea slugs serve as a model for suction technology suited for rough and uneven surfaces. This study quantified and compared the attachment strengths of several nudibranch species [Dirona pellucid (n=12), Janolus fuscus (n=23), Hermissenda crassicornis (n=20), Aeolidia papillosa (n=13)] from the rocky intertidal of the San Juan Islands (SJI), WA on 3 different substrates – rough rock, smooth rock, and a plank of bull kelp. In individual trials, the substrate and each sea slug were placed in a mini flume tank (0.02 m³ volume) with a rotor engine generating a known current velocity to simulate wave exposure. Water velocity was increased in a stepwise manner (increments of 0.025 m/s to a maximum of 0.25 m/s) until the nudibranch fell from the substrate (referred to as the Critical Point Force, CPF) or until the full trial period (10 mins) elapsed. The hydrodynamic force on the nudibranch was calculated assuming negligible body mass and used as a proxy for nudibranch attachment force, standardized according to nudibranch body length. Attachment strengths were cross compared to calculated average hydrodynamic force in the SJI according to ocean current velocities obtained from NOAA. D. pellucid was the only species to have significantly greater CPF than the other 3 species. Few H. crassicornis individuals fell (n=3), so their CPF could not be accurately quantified. Estimated average hydrodynamic force for SJI currents on a model nudibranch was 56.4 mN while average experimental nudibranch CPF was 5.06 mN. This difference may be due to higher water velocities in the SJI channel than at the dock where the nudibranchs were collected. Across all trials, 54% of nudibranchs fell, so further research should incorporate experimental water velocities exceeding 0.25 m/sec. No significant difference in each species’ CPF across the varied substrates was detected, highlighting nudibranchs’ potential versatility in biomimetics.

Prochlorococcus is a cyanobacterium smaller than 1 μm that accounts for much of the primary production in nutrient-poor areas such as the North Pacific Subtropical Gyre (NPSG). In the transition from the NPSG to more productive coastal regions, there are fronts that have sharp changes in chemical, physical, and biological properties. In more coastal, nutrient-rich conditions, larger phytoplankton are more abundant, including Synechococcus and picoeukaryotes. Data previously collected on cruises going north from the NPSG (the Gradients cruises) were compared to data I collected on the TN398 cruise going east from the NPSG to the California coast. A SeaFlow flow cytometer measured small phytoplankton, including Prochlorococcus, Synechococcus, and picoeukaryotes. Prochlorococcus was most abundant in nutrient-poor conditions in the NPSG, reaching a concentration of 300 cells/μL, and larger phytoplankton, including Synechococcus and picoeukaryotes, were most abundant in the coastal ocean and subpolar region. The diameters of Prochlorococcus, Synechococcus, and picoeukaryotes varied on a diel cycle that was most strongly observed in the gyre. The average diameter of Prochlorococcus and Synechococcus increased by about 0.2 μm outside the NPSG, while the diameter of picoeukaryotes observed by SeaFlow decreased by about 0.4 μm. Prochlorococcus abundance was negatively correlated with nitrate and nitrite. In the future, these variables could be compared seasonally, annually, and across ocean basins to better understand how these populations are responding to climate change.

Pacific sand lance (PSL) are a pelagic plankton feeding forage fish that also exhibit benthic associated behavior, related to burial in sediment on the ocean floor. They represent a key trophic link between plankton and upper-level predators. Despite their ecological importance, these fish are relatively under researched. This study provides new insights related to sediment preference and the effects of light availability on foraging behavior, and quantifies movement patterns related to various response behaviors. We found that PSL may prefer sediments made up of fine and coarse grain sand, as well as require a light cue to leave sediment habitats and forage. Additionally, we quantified a multitude of behaviors exhibited in PSL and found statistically significant relationships therein. We observed positive correlations between fish length and velocity in schooling fish and in fish movement related to burial in sediment. We also observed a negative correlation between fish length and both burial depth and steepness of body position within sediments (burial angle). In short, longer fish were generally faster and buried deeper within sediment at a flatter angle. Schooling distances were compared through two methodologies: stereo camera and video imaging analyses. A significant difference was found between the two, likely due to human error. Further research should be conducted on sediment preference as it relates to spatial habitat, as well as other behavior motion and schooling analysis.

Rorqual whales are some of the largest animals on the planet, yet feed on some of the smallest animals in the ocean. When feeding, rorquals will lunge toward dense masses of food, engulfing both prey and water. Their upper jaws are lined with baleen – a filtering fringe composed of keratin plates and bristle-like hairs. Larger keratin plates (major) are situated labially with smaller plates (minor) lingual to them. Rorquals can feed on plankton, like krill, and fish. We hypothesize that baleen morphology varies with species and diet. We investigated the hierarchical anatomy of baleen in five whale species: humpback, fin, gray, blue and minke – which span a great range in body and prey size. We used computed tomography (CT) and scanning electron microscopy (SEM) to image, quantify, and understand how variability in baleen morphology may affect filtration. CT revealed cylindrical keratin hairs embedded in the baleen plates that grow out of the gum. These hairs extend out of the ventral side creating a fringe. The size of the hairs, the keratin sheath surrounding each hair, and the keratin matrix of the baleen plates vary by species. SEM revealed that the keratin plates are worn away at the ventral side exposing the bristles by peeling away the exterior layers. Hair diameter differs within an individual plate for each of the species. Hair diameter varies with species, but does not correlate with whale body size i.e. bigger whales do not have thicker hairs.

Microplastics (plastics < 5mm) are increasingly prevalent in marine systems and of growing concern, as a source of marine pollution and in potential biomagnification in food webs. This has consequences for high trophic level consumers, including humans. Despite being an emerging area of study with many unknowns, there is increased interest in understanding the abundance and consequences of marine plastics, given evidence that links plastics to degraded ecosystem and organismal health. Microplastics contain and can absorb harmful chemicals, which may serve as endocrine disruptors. This may have implications for growth, reproductive health and longevity. To expand current knowledge on Salish Sea microplastics, I processed and examined 233 Pacific sandlance, sculpin, Pacific salmon, and Arctic cod stomachs for microplastics. 28% of all fish stomachs contained at least one microplastic particle. There was variation of prevalence between sites, ranging from 3% to 49%. Differences between prevalence of plastics differed between beach sites for both sandlance and sculpin and also differed from sandlance sampled in offshore benthic habitats. All forage and benthic samples indicated potential biomagnification to salmon based off of biomagnification factor values (BMF). Comparisons between plastic concentrations and stomach fullness was compared with plastic concentration. Further investigation is needed to develop a more thorough understanding of the microplastic issue in the San Juan Archipelago

Scripps’s murrelets (Synthliboramphus scrippsi; hereafter murrelets) are an endangered seabird with one of the largest breeding colonies in the U.S. at Anacapa Island within Channel Islands National Park. Recent monitoring on Anacapa Island has revealed predation of adult murrelets by common ravens (Corvus corax) that target incubating individuals at their nests. Murrelets are long-lived seabirds, which suggests that adult survival is an important demographic parameter for the viability of this population. Our study will determine the efficacy of remote monitoring tools to detect predation events, predator visitation, and murrelet activity. We deployed camera traps and autonomous recording units (ARUs) at five known nesting caves during the 2021 breeding season. We will compare predator visitation and predation events captured on camera traps with acoustic data from the ARUs to determine the extent of predator vocalization; the same will be done to capture murrelet activity. ARU and camera trap detections will be compared to traditional monitoring methods where humans perform tri-weekly nest checks for predation evidence such as murrelet carcasses or feather piles. We anticipate camera traps will perform better than ARUs at detecting predator visitation and predation events, but groups of two or greater ravens will have higher vocalization rates. In contrast, we anticipate that ARUs will perform better than camera traps at detecting murrelet activity due to their small stature making them less likely to trigger the cameras. We hypothesize the combined power of both remote monitoring tools may be comparable to traditional monitoring methods. Our comparative analysis of monitoring methods will allow park management to make informed decisions for future monitoring protocols for murrelets. In addition, our results will provide the park with a better understanding of the impact of raven predation on murrelets as well as management options to mitigate this impact.

Pacific herring (Clupea pallasii) play a critical ecological role in the Puget Sound food web and are experiencing declines throughout the Sound. Presently, no single cause has been identified, and consequently, no policy action has been implemented to reverse the declines. Here we evaluate the hypothesis that terrestrial landscape features govern the trends in local stocks (defined by aggregations during reproduction) through run-off, noise, light, and other indirect effects. We predicted that status of local stocks would show associations with terrestrial landscape features such as land use and human density. To test these predictions, we gathered a time series of spawning biomass estimates for sub-stocks throughout Puget Sound and used multiple metrics to evaluate the population status of each subpopulation. We then related these metrics with information on local land-use and other localized anthropogenic disturbances. Across all metrics, a majority of the 22 stocks showed impaired status over the past 15 years. However, we found no association between local status and landscape metrics. This result indicates that landscape level effects are not solely responsible for the impaired status of Pacific herring, but we caution that this may be because there are multiple, interacting causes for declines that need to be addressed to recover this valuable species.

Charged particles in the heliosphere can be continuously accelerated by interplanetary shocks and eventually escape from these shocks without returning to it. Acceleration and escape are highly intertwined and both contribute to the shaping of the particles’ momentum spectrum at the shock. The simplest model which describes this phenomenon is called Diffusive Shock Acceleration (DSA). DSA has been very successful in describing several observations. However, DSA does not include an energy-dependent escape from the foreshock region. We expand upon DSA by presenting a model for interplanetary shock acceleration which includes this energy-dependent particle escape. We analytically solve a one-dimensional transport equation with a diffusion coefficient and an escape time that describes both the turbulence self-generated by the shock and the far upstream pre-existing turbulence. We consider the case where a shock encounters a population of pre-existing charged particles with a power law energy distribution as measured by spacecraft. We find that at lower energies our solution is concave, whereas at higher energies it asymptotically approaches a power law whose slope depends on the original energy spectrum’s power law index and shock parameters. We fitted the solution obtained from this transport equation to shock data measured from multiple shock events collected by ACE/EPAM (the Electron, Proton, and Alpha Monitor aboard the Advanced Composition Explorer spacecraft). We find that for the shock events considered, our model’s best fit parameters match very well with the predicted values, obtained by using the measured shock parameters. From this model, we can better understand the mechanism of interplanetary shock acceleration and how this phenomenon energizes charged particles near the Sun and around other objects (for example, blazars and supernova remnants).

In order to compare observations of massive stars with theoretical predictions, stars must be classified accurately. Classification traditionally relies upon expensive new telescope observations. As the field of astronomy enters a new era of Big Data, Modern computational techniques may be used in place of these methods; however the results still require rigorous validation in order to be trusted. Recently, Dorn-Wallenstein et al. (2021) utilized a novel machine learning technique to classify a large sample of massive stars. This resulted in putative classifications for ~2550 stars. Our project serves as a follow-up to validate the results of Dorn-Wallenstein et al. and identify stars with rare evolutionary phases that are most useful for probing stellar evolution. We test the hypothesis that these classifications are reliable by obtaining new observations with the ARCES instrument mounted on the Apache Point Observatory 3.5-meter telescope. Using these data, we assigned classifications to the stars in our sample, focusing on identifying rare objects and evolved supergiants. To this end, we have developed custom software designed to navigate through the key features in our observations and allow for easy identification of an object's spectral type (i.e., its evolutionary stage). We find that our observations support the classifications made by Dorn-Wallenstein et al. Our future work will focus on expanding our sample with further observations in the Southern Hemisphere. This work is critical in order to prepare for the age of Big Data in astronomy.

Take a circular sheet of paper. Pick two random points on the edge of the circle and draw a line segment between them. Repeat this many times. Then you take scissors and cut the circle along those line segments. How many pieces of paper do you get? Such questions are a part of a subject called geometric probability. Examples include the famous 18th century Buffon's needle problem that lets one estimate the value of pi by running a random experiment on the computer. In modern times geometric probability is used to estimate the coverage of cell phone towers and communicating drones. I review historical research in geometric probability and graph theory dating between the 18th and 20th centuries to find the expectation and the variance of the number of pieces in the circular sheet. Then I prove a Central Limit-type behavior (converges to normal distribution) of the number of pieces that is conjectured from experiments. The proof is from a modern result of probability theory, called Stein's method for the Central Limit Theorem. Several open questions remain to be answered, including how likely one is to get a piece with a very large or small area. The results of this research enrich the content of geometric probability with possible applications in geology, image processing, transportation, and so on.

Mentored by Kirill Golubnichiy, this research project aims to apply mathematical finance and machine learning (ML) to forecast stock option prices. We create and evaluate new empirical mathematical models for the Black-Scholes equation to analyze data for 177,000 companies. For each company, we have 13 elements including stock and options’ daily prices, volatility, minimizer, etc. Because the market is so complicated that there exists no perfect model, we apply ML to train algorithms to make the best prediction. We first analyze several existing stock and option prediction models: Quasi-Reversibility Method (QRM), Binary Classification, and Regression Neural Network (RNN) ML. QRM is an analytical and analytical approach to find the minimizer by solving the Black-Scholes equation as an ill-posed problem; whereas the latter two combine QRM with ML. The current stage of research attempts to combine QRM with Convolutional Neural Networks (CNN), which learns information across a large number of data points simultaneously. Our current focus is to apply CNN to generate new results by programming, implementing, testing, and validating sample data. We will compare our CNN model with previous models to see if it is possible to achieve a higher profitable rate. If our CNN model successfully forecasts prices for a majority of stock options, it might be possible to deploy the model in the real world and help investors make better investment decisions.

When conducting surveys containing sensitive questions, Social Desirability Bias (SDB), people’s tendency to provide socially acceptable answers rather than truthful ones, often leads to low response rate or worse, untruthful responding. Randomized Response Techniques (RRT) combat SDB by allowing respondents to provide scrambled responses. However, if a respondent does not trust the RRT model, data accuracy will be compromised. Lack of trust in binary RRT models has been shown to lead to untruthfulness, and thus unreliable data and unreliable estimates of the sensitive trait. Yet, no quantitative RRT model currently accounts for respondent lack of trust. We propose an Optional Enhanced Trust (OET) RRT model that extends Warner’s Additive Model, a quantitative RRT model with additive noise, by allowing additional multiplicative noise if the respondent does not trust the Warner Additive Model. Using the Unified Measure, a combined metric of respondent privacy and model efficiency, we demonstrate both theoretically and empirically that the proposed OET model is superior to existing models in terms of respondent privacy and estimator accuracy.

An invertible matrix is called a Perron similarity if one of its columns and the corresponding row of its inverse are both nonnegative or both nonpositive, a definition introduced in a previous work by Johnson and Paparella (2016). For a given matrix S, the spectracone is a polyhedral cone formed by the vectors that produce nonnegative matrices when multiplied on the left by S and the right by S-inverse. The spectratope is a set defined similarly, with the added condition that the included vectors have an infinity norm of one. This work identifies previously unknown relationships between Perron similarities and their Kronecker products. First, if two matrices are Perron similarities, their Kronecker product is also a Perron similarity. Second, the Kronecker product of spectracones (spectratopes) is a subset of the spectracone (spectratope) of the Kronecker product. In addition, if two matrices are Perron similarities with dimensions greater than 1, then the Kronecker product of the spectracones (spectratopes) is properly contained in the spectracone (spectratope) of the Kronecker product. Another significant result is if S is a Perron similarity, then the ray through the all-ones vector is properly contained in the spectracone of S, but the converse is not true. One reason this work is relevant is that Perron similarities were previously defined to have a necessary and sufficient condition that the ray through the all-ones vector is properly contained in the spectracone. With the publication of this article, the condition was found to only be necessary, and the definition was corrected. Perron similarities are important when examining the nonnegative inverse eigenvalue problem, or NIEP, which is concerned with the spectrum of nonnegative matrices. This research expands on previous work related to the NIEP by examining relationships between Perron similarities and their Kronecker products.

The circumgalactic medium (CGM) is the extended gaseous halo that typically surrounds the visible parts of a galaxy - the beautiful spiraling disks that one may recognize from an image captured by the Hubble Space Telescope. Although the CGM harbors no stars, making it only faintly illuminated by the interior galaxy and background sources, it is believed to contain far more mass than the galaxy itself. Understanding the CGM is thus key to understanding galaxy formation and address questions such as: Why are some galaxies red and some blue? Why are we only observing ~20% of the baryons (regular matter, not dark matter) we should observe in galaxies? How do galaxies like our own sustain star formation, enabling the particle diversity we see everywhere around us? The way we currently observe the CGM’s properties is through absorption spectroscopy by using a bright background light source (quasar) and observing how the light is absorbed as it travels through the CGM before reaching our telescopes. However, this limits our observations to only a “pinhole” view of the CGM’s properties. With the advent of instruments sensitive enough to observe the light emitted from the CGM, we will be able to create so-called “emission-maps” of the full state of the gas - including temperature, density, and element abundances. Our project has identified what wavelengths (ionic lines) are most observable with more sensitive telescopes, some of which are already underway. I have run computer simulations in the Cloudy program to identify these emission lines and developed models estimating their intensity. The culmination of our work shows that many of these CGM emission lines are detectable with feasible instruments in the near future, laying the groundwork to justify future missions targeting these specific lines in order to investigate the most pressing questions of galaxy evolution.

During a microbial infection, cytokine and interferon communication downstream of JAK-STAT signaling molecules are critical for the activation of pro- and anti-inflammatory gene programs. The prevailing model relies on JAKs (Janus Kinases) activation upon cytokine or interferon docking with its receptor at the membrane, which phosphorylates STATs (signal transducers and activators of transcription proteins) and leads to their translocation into the nucleus to either activate or repress the transcription of genes. In this study, we have identified the role of an endocytic pathway responsible for JAK-STAT activation upon interactions between the IL-10 family proteins and the cytokine receptor. Using skin epithelial cell lines, I have performed experiments to show that clathrin-mediated endocytosis is required for the signaling of cytokines that use the IL-10R2 receptor. According to my preliminary results, the inhibition of clathrin-mediated endocytosis using chemical inhibitor dynasore, prior to treatment with IL-26, results in complete shutdown of pSTAT3, pSTAT1, and pErk1/2. These downstream molecules are required for activation of inflammatory gene programming. We now propose to investigate the roles of other inhibitors within the endocytic pathway including chloroquine, bafilomycin A1, and a Rab7a inhibitor on subsequent JAK-STAT signaling transduction. In order to visualize these results, confocal microscopy studies will be used to investigate trafficking in these vacuoles, followed by colocalization studies of JAK proteins with receptors in early endosomes. Our studies suggest that a change in receptor mobilization controlled through the endosomal trafficking mechanism modulates JAK-STAT signaling and downstream gene expression. This proposal is a paradigm shift which challenges the prevailing dogma that IL-10R2 dependent cytokines can activate JAK-STATs at the cell membrane to induce inflammatory responses. Furthermore, this new signaling model has the potential to impact pharmaceutical interventions as the IL-10 family plays a critical role in inflammatory bowel diseases as well as fungal and bacterial infections.

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that is estimated to infect around 23 million people per year. Despite high global prevalence, there are not any approved vaccines that are therapeutic or preventative. Most vaccines that we use today rely on injecting antigens intramuscularly in order to elicit an adaptive immune response. However, given that most pathogens gain entry to the host across barrier surfaces, a focus on eliciting mucosal immunity may enhance protection; vaccine-induced local immunity at the site of first pathogen exposure may have the best chance at preventing the spread of infection beyond the pathogen portal of entry. We hypothesized that a mucosal immunization would prime memory T cells to reside in vaginal tissues and provide better protection against vaginal HSV-2 exposure than other routes of immunization. Our initial data suggests that intranasal immunization is effective in protecting mice from vaginal HSV-2 infection. Ongoing work focuses on characterizing the role of vaccine-elicited mucosal CD8+ T cells in preventing infection to provide insight into the mechanisms of protection induced by mucosal immunization for HSV-2. These findings contribute to the efforts to generate an effective vaccine to prevent HSV-2 infection and disease.

Cystic fibrosis (CF) is a genetic disease characterized by defects in lung host defenses against infection. Chronic lung infections by the bacterium Pseudomonas aeruginosa (Pa) are a major cause of death in CF, and recent work shows that infecting Pa strains can evolve extensive genetic diversity in vivo during infection. A new class of drugs called modulators markedly improves the CF host defense defect and improves symptoms but does not frequently produce infection eradication. We hypothesize that the extent of phenotypic diversity within Pa populations infecting individual subjects will negatively affect Pa clearance after modulator treatment. This hypothesis stems from observations in environmental systems showing that diversity generally increases the stress resistance of populations. To test this hypothesis, we sampled the lungs of 9 subjects with bronchoscopy before and 18 months after they started the most effective modulator currently available and collected ~ 100 Pa isolates from each of the five different lung regions. The collected isolates are being tested for variation in resistance to different classes of antibiotics and their nutritional requirements, motility, capacity to secret virulence factors, and attach to and aggregate on surfaces. These measurements will enable us to compare the phenotypic diversity of Pa populations infecting subjects that do and do not eradicate infection. In my preliminary work examining Pa populations from two subjects, I found much lower levels of diversity in resistance to three antibiotic classes in the subject that cleared infection as compared to the subject that did not (mean variance in resistance of 0.22 vs 75.3, respectively), which is consistent with my hypothesis. Understanding the consequences of Pa diversity in CF infections could shed light on factors causing different infection outcomes and suggest new approaches to treatment.

Hemagglutinin (HA) is a glycoprotein found on the surface of the influenza virus. HA is responsible for binding to sialic acid receptors on host cells and mediating membrane fusion. Because it is involved in these essential functions, HA is targeted by neutralizing antibodies (nAb) against the influenza virus. When the antigen-binding fragment (Fab) domain of a nAb binds to HA, it can neutralize infectivity either by blocking receptor binding of HA, by inhibiting conformational changes required for membrane fusion, or by disrupting HA structure. All of these mechanisms can prohibit the virus from entering the cell. In a hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiment, we attempted to map the epitope-- the site on the antigen where the antibody binds-- of the 5E10 neutralizing antibody on the HA from the 2011 Victoria H3 influenza strain. A deuterium exchange time course was performed and HDX-MS was used to determine the rate at which deuterium is exchanged for hydrogen on specific peptide segments of the HA protein backbone. We hypothesized that the epitope would be an area that becomes more protected, meaning that it would take up less deuterium, due to local structural ordering from the bound Fab fragment. Through our research, we have identified two potential epitope sites. Identifying the 5E10 epitope on H3 HA will tell us about sites of vulnerability that can be targeted by the immune system. Furthermore, it will help us understand the way the antibody can neutralize the virus as well as predict if the antibody would be able to neutralize other strains of flu based on comparisons of amino acid conservation at the epitope. This information can be used by other scientists to design vaccines that direct an immune response to a specific epitope.

Since the beginning of the Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) pandemic, a large number of COVID-19 patients have suffered a variety of cardiovascular complications. In a previous study from the Murry Lab, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) were infected with live SARS-CoV-2 alpha variant, resulting in increased cell death and functional abnormalities. Once infected with SARS-CoV-2 delta variant, hPSC-CMs showed reduced cell death, compared with the ones infected with alpha. The SARS-CoV-2 genome has at least 14 open reading frames (ORFs), which is the transcribed part of a gene. A difference in expressed ORFs can create new variants. The delta variant has 60 amino acid deletions in the ORF7a sequence, resulting in the absence of the ORF7a protein. Thus, I hypothesized that this protein might play a role in cardiomyocytes’ cell death. To demonstrate that this protein has a direct effect on hPSC-CMs cell death, I have created a lentivirus plasmid construct that expresses only ORF7a protein. To assemble the virus, I transfected HEK-293 cells with the ORF7a plasmid with two other plasmids encoding for the packaging and the replication of the lentivirus. Once the virus is ready, I will then infect hPSC-CMs with the lentivirus. If the ORF7a protein is responsible for cell death, we expect to see increased cell death in the hPSC-CMs infected with the ORF7a lentivirus. Showing support of the ORF7a protein inducing harmful effects on cardiomyocytes has the potential to provide valuable insight for scientists to target this particular protein for drug development to combat cardiovascular complications in COVID-19 patients.

Nucleic-acid based vaccines, including RNA and DNA, provide protective immunity by eliciting antibody (Ab), and cytotoxic T lymphocyte (CTL) responses. FDA approval of mRNA vaccines against SARS-CoV-2 (COVID-19) provide ample evidence that mRNA vaccines are a viable vaccine platform. Once a mRNA vaccine enters a cells cytoplasm, mRNA encoded antigens are produced rapidly inducing an immune response. The production of mRNA encoded antigens will wane over time as mRNA degrades and transfected cells die. Alpha viruses, a RNA virus, have a unique replication process whereby this virus amplifies its RNA genome upon entering a cell. We, as well as others, have developed RNA vaccines that, like alpha viruses, self-amplify once inside of a cell to create more copies of mRNA than entered cell. This self-replicating RNA vaccine is called a replicon RNA vaccine (repRNA). RepRNA induces robust and sustained antigen production and immune responses. Currently, mRNA vaccines are administered as a homologous prime/boost vaccine regimen where the same mRNA vaccine is given as a priming vaccine and boosting vaccine, but antibody titers wane over time allowing for potential infections. This project aims to evaluate If utilizing a heterologous prime/boost regimen with DNA and repRNA vaccines confers more robust and longer lasting antibody and CTL responses than homologous regimens with DNA or repRNA. Mice will be vaccinated with DNA as a priming vaccine and repRNA as a boosting vaccine that encode SARS-CoV-2 spike protein. Anti-SARS-CoV-2 spike IgG antibody titers will be measured at 3-4 different timepoints, and CTL responses will be evaluated using an interferon gamma (IFN-γ) enzyme-linked immune absorbent spot (ELISpot) assay. The insights gained from this project will help to inform future DNA vaccine formulations and regimens as more DNA vaccines and repRNA vaccines enter clinical trials.

Cystic fibrosis (CF) is a genetic disease characterized by polymicrobial lung infections. Staphylococcus aureus, a Gram-positive pathogen, is commonly cultured from the secretions of people with CF (PwCF). Treatment for S. aureus infections requires antibiotics, such as the bactericidal antibiotic trimethoprim-sulfamethoxazole (SXT). SXT prohibits growth by targeting folate biosynthesis, a pathway important for DNA replication and maintenance and production of cell metabolites. In many PwCF, S. aureus generally persists despite antibiotic treatment. Our data shows that S. aureus can survive SXT treatment through the accumulation of adaptive mutations. In this project, we examined these adaptive mutations in S. aureus in vitro to better understand the mechanisms of resistance. We grew several S. aureus isolates with adaptive mutations in Luria Bertani (LB) broth. We sampled the culture tubes at several times in a 24-hour period, measuring viable bacterial counts on chocolate agar. We found that isolates with mutations in the sugar transport gene, ptsI, persisted better under SXT selection, relative to wild-type S. aureus. Other S. aureus isolates with mutations in pathways for aerobic respiration, including menaquinone (menB, thiN) and hemin synthesis (hemB), also better survived SXT compared to wild-type. Many of these mutations were also identified in S. aureus infecting PwCF. These results indicate that adaptive mutations in pathways associated with important metabolic processes may allow survival with folate inhibition. We hypothesize that limiting aerobic respiration may assist in S. aureus surviving SXT. As S. aureus can survive without oxygen, we are currently studying whether limiting oxygen and, consequently, aerobic respiration in wild-type S. aureus will improve survival with SXT. This work will help us understand the mechanisms of SXT action and S. aureus’ response, in an effort to improve treatment for S. aureus infections.

In this study we developed a nonhuman primate model of simian immunodeficiency virus (SIV)-Zika virus (ZIKV) co-infection to understand how HIV infection impacts ZIKV pathogenesis and test our hypothesis that ZIKV pathogenesis is enhanced in people living with untreated HIV. Previously, we have found delayed viral clearance, as well as delayed and dampened expansion of whole blood monocytes, the ZIKV cellular targets, during SIV infection. Here, we sought to further characterize the innate immune responses of SIV-ZIKV co-infection, by assessing cytokine and chemokine release. Pigtail macaques (n=7) were infected with SIVmac239M and co-infected with ZIKV at 9 weeks post-SIV infection. Co-infected animals were compared to control animals (n=7) infected with ZIKV only. Longitudinal plasma and cerebral spinal fluid (CSF) were collected at timepoints pre- and post-infection and assayed using a multiplex immunoassay to quantify 24 different cytokine and chemokines ex vivo. SIV and ZIKV both induced pro-inflammatory responses, characterized by transient increases in interleukin-17 (IL-17A) and monocyte chemoattractant protein-1 (MCP-1), with no major differences between experimental groups. Plasma MCP-1 concentrations were also found to be consistent with dampened and delayed whole blood monocyte frequencies. Pro-inflammatory interleukin-8 (IL-8), a chemokine needed for recruitment of neutrophils, increased in the plasma during SIV infection but not following ZIKV infection, a result that is in contrast to our previous findings. Transient increases in IL-8 were detected in a few animals in the CSF after ZIKV infection, which may be evidence for neuroinflammation. Overall, no significant differences between SIV+ vs SIV- groups were found for any analytes detected in plasma or CSF during ZIKV infection. Collectively, our results demonstrate that both SIV and ZIKV infections induce a pro-inflammatory response, that is not enhanced by SIV-ZIKV co-infection. This suggests SIV induced immunosuppression does not impair pro-inflammatory cytokine responses during ZIKV infection.

Adoptive cell immunotherapy (ACT) with engineered T cells has shown impressive efficacy against some cancers, particularly CD19+ leukemias. However, ACT efficacy in solid tumors can be limited by restrictive tumor microenvironments (TMEs), with increased inhibitory signals, reduced T cell infiltration/accumulation, and inadequate metabolic substrates. We engineer T cells with novel switch receptors that combine an inhibitory ectodomain with a costimulatory signaling endodomain, to “replace a break with an accelerator”. FasL is a death receptor ligand that is overexpressed in the majority of human TMEs and can protect tumor cells from immunity by giving T cells inhibitory signals upon binding. We engineered a Fas-4-1BB switch receptor to convert the Fas death signal to a costimulatory 4-1BB signal and demonstrated enhanced Fas switch-receptor-T cell in vivo persistence and therapeutic efficacy in a murine pancreatic cancer model (KPC). We developed new switch receptor candidates by combining CD200R, an inhibitory signaling receptor, ectodomain with various co-stimulatory endodomains. Our aim is to screen for CD200R switch receptors that exhibit the best expression on T cells, best ability in tumor-lysis and proliferation with the least exhaustion in TMEs, and test the best candidates in KPC models. We are able to transduce mouse T cells to express our CD200R constructs, which is verified by flow cytometry, and ready to start in vitro killing assay to assess their ability in tumor-lysis. This T cell engineering strategy may help overcome the restrictive TMEs, catalyze an endogenous immune response, and greatly improve T cell anticancer efficacy.

Non-Hodgkin's Lymphoma (NHL) is the most common malignancy of the blood and bone marrow, responsible for about 4% of all cancers. The anti-apoptotic MCL-1 and BCL-xL proteins have been linked to the development of various types of NHL. Radiation is an effective treatment, as it is known that lymphomas are generally highly sensitive to radiation, but toxicity can limit the dose. Our goal is to use different treatments in varying combinations to find points of synergy where the effect of the treatments surpasses the expected additive response. We first tested in vitro response of NHL cell lines to the BCL-xL inhibitor A1155463 and the MCL-1 inhibitor S63845. We then evaluated combinations between the two inhibitor drugs with radiation treatments. To date we have tested cytotoxicity of these treatment combinations in six NHL cell lines. All lines tested with the combination of the MCL-1 and BCL-xL inhibitors demonstrated an increase in cell death attributable to a synergistic effect between the two drugs. Furthermore, when treated with radiation prior to the two drug treatments, three out of the six lines demonstrated a synergistic response to the triple treatment combination. The other three lines displayed an additive response to the triple combination. Moving forward we aim to test a total of 14 NHL cell lines, and given the positive results to date, will also expand the work into mouse models. These largely synergist results show promise because they suggest that this triple combination treatment could reduce treatment side effects while also improving the curative response for NHL patients.

Breast cancer is one of the most common types of cancer in women, resulting in ~42,000 deaths annually in the United States. Tumor cells can travel to distant tissues and form new tumors, a process known as metastasis, which ultimately kills the patient. These disseminated tumor cells (DTCs) that stray from the original tumor may undergo a period of dormancy prior to awakening and growing into lethal metastases. Dormancy is when a DTC is in a non-dividing (i.e., quiescent) state, but remains receptive to microenvironmental cues that trigger proliferation. The relationship between DTCs and their surrounding immune microenvironments is not well understood, thus we opted to explore whether dormancy of DTCs is impacted by the immune milieu. We used the mouse lung as an experimental model. Following intravenous injection of D2.0R mammary tumor cells, we stained for MHC-2 to identify myeloid and B cell populations in the lung. Surprisingly, we found that MHC-2+ immune cells border dormant DTCs. We performed a control analysis protocol by placing random dots (representing random cells) in the stained tissue images and found that MHC-2+ cells are not in immediate proximity to our random ‘cells’, suggesting that the association between MHC-2+ cells and DTCs is likely not arbitrary. Our next step in this investigation is to identify precisely what these MHC-2+ cells are, looking specifically at markers for myeloid and B cell subsets including F4/80, CD11c, and CD19. This study will provide us with a better understanding of the relationships between dormant DTCs and immune cells in their immediate lung microenvironment. Our long-term goal is to use what we learn from these studies to aid in the development of immunotherapies to target DTCs and prevent metastasis. 

Cancer is a disease that is characterized by uncontrolled cell growth and proliferation. As these cells spread throughout the body they can invade and damage major organs resulting in death. It has been shown that most cancers arise from genetic changes in the signaling pathways that control the cell growth and proliferation. One major challenge cancer researchers face is to understand the behavior of these pathways. As an example, the behavior of the Endothelial Growth Factor receptor (EGFR) pathway, a commonly mutated pathway in cancer, has a broad range of behaviors. Under different conditions, the pathway can exhibit bistability, amplification and even oscillations in protein levels. Predicting such behaviors can significantly aid our understanding of how cancer arises as well as provide us with insights to therapeutically treat patients. The goal of this project was to develop a high accuracy machine learning model that is capable of predicting the behaviors of signaling pathways. Over the first quarter, I generated over 3000 artificial signaling networks utilizing existing software written by the lab. For each of these networks I then extracted the network architecture, the connections and relationships between species in the pathways, into matrices such as the stoichiometry matrix and scaled Jacobian matrix. These matrices were then treated as pseudo-images and used to train a convolutional neural network to predict oscillatory behavior. We expect this model to have greater than 70 percent accuracy given preliminary models trained on similar smaller datasets. The development of this high accuracy model provides cancer researchers with valuable insights into the behavior of these signaling pathways and how they can best therapeutically treat patients.

Chimeric antigen receptor (CAR) T cell therapy is a highly effective treatment for blood cancers; however, it is correlated with neurotoxicity in about 40% of cases. Neurotoxicity manifests as headaches, delirium, and in more severe cases, seizures, coma, and death. In a mouse model of CD19-directed CAR T cell therapy, we observed that mice developed behavioral changes, as well stalled blood flow in over 10% of brain capillaries. This was caused by circulating leukocytes becoming stuck in the capillaries. My goal for this project was to investigate the underlying mechanism of capillary stalls in the context of CAR T cell therapy. We predicted that capillary stalls are caused by increased expression of adhesion molecules, such as Intercellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1), by the endothelial cells that comprise the capillary walls. These adhesion molecules grip onto molecules on the surfaces of leukocytes, creating plugs and halting blood flow through the vessels. To test this hypothesis, I used immunohistochemistry to compare the expression of ICAM-1 and VCAM-1 between 4 treatment groups: mock, tumor, CAR T, and tumor+CAR T. Mice from each treatment group were perfused and their brains were collected and sectioned. I incubated mouse brain sections in a primary antibody solution targeting ICAM-1 and VCAM-1, and then in a secondary fluorescently conjugated antibody solution that bound to the previous antibodies, enabling specific labeling of the molecules of interest. I then imaged the sections with a confocal microscope and analyzed them using ImageJ software. I quantified the brightness of fluorescence of ICAM-1 and VCAM-1 to determine the relative expression of these molecules between control mice and those receiving CAR T. The findings from this project have contributed to our overall understanding of the mechanisms of neurotoxicity associated with CAR T cell therapy.

Decades of research have demonstrated that RNA molecules can serve as architectural scaffolds, templating the assembly of subcellular compartments in all kingdoms of life. In mammals, architectural RNAs scaffold an array of subnuclear structures that are essential to cellular function including metabolism, DNA repair, and epigenetic programming. Many of these architectural RNAs are causally dysregulated in diseases, including cancer and neurodegenerative disorders. However, the molecular mechanisms of these architectural RNAs remain poorly understood, partially because technologies for identifying the molecules (proteins, DNAs, other RNAs), with which RNAs interact are lacking. To address this challenge, the Shechner Lab has developed a technology termed Oligonucleotide-Directed Biotinylation (ODB), a universal method for elucidating RNA subcellular interactions. ODB applies a powerful method called proximity-biotinylation to individual RNAs. In proximity-biotinylation, a promiscuous biotinylating enzyme (e.g. Horseradish Peroxidase, HRP) is targeted to a subcellular compartment of interest. This enzyme then tags nearby (~10 nm) molecules with biotin, enabling their straightforward isolation and analysis. ODB advances this technology by using RNA-in situ Hybridization (RNA¬–FISH) methods to precisely deploy HRP to individual RNAs. Ongoing work, using a series of model RNA targets, has demonstrated that ODB can reveal the proteins, RNAs, and genomic loci near a target RNA at exceptional depth and precision. My goal is to generalize this ODB protocol, developing methods that can be applied to any RNA target. To examine how RNA abundance influences ODB experimental design, I use pulse-chase, RNA decay experiments to manipulate the expression of the architectural RNA NEAT1, and examine how to adjust parameters of the ODB protocol to compensate for this altered expression. Likewise, I am using imaging-based assays to investigate how ODB's biotinylation radius varies with labeling conditions, increasing the range of ODB’s target radii. Collectively, this establishes general guidelines for adapting ODB to novel RNA targets.

Retinal diseases such as macular degeneration and glaucoma lead to various forms of blindness as neurons in the retina die. Unlike amphibians or fish, the neurons of the mammalian retina cannot regenerate on their own and any damage is permanent. Previous research has shown that we can recover some of the lost regenerative capacity in mammalian retinas by mimicking the regeneration process in other species. This is possible by overexpressing transcription factor Ascl1 in Müller glia (MG), which are the main support cells in the retina. However, expressing Ascl1 alone can only lead to the neurogenesis of one type of neuron in the retina. This limits the therapeutic applicability of this approach because in diseases like macular degeneration or glaucoma, specific neuronal cell types are lost such as photoreceptors and ganglion cells respectively. Therefore, to regenerate these neurons we need to identify the right cocktail of transcription factors for MG reprogramming. Recent single-cell analysis from our lab has identified candidate developmental factors that could push reprogrammed cells to photoreceptors or retinal ganglion cells. This project aims to investigate the role of these factors in influencing cell fate after MG cells have been pushed to a progenitor state with Ascl1. Using lentiviral vectors, I will induce the overexpression of these candidate genes in primary cultures of young adult mouse MG that have been engineered to express Ascl1. In order to identify the nature of resulting neurons from these cultures I will perform immunocytochemistry paired with confocal microscopy, and to better understand changes in functionality of these cells I aim to perform calcium imaging. Since the electrophysiological responses of glia and neurons are distinct, cultures with reprogrammed neurons would record differently. Overall, this analysis evaluates the influence of new transcription factors on mammalian retinal regeneration.

In fish and amphibians there is a specialized zone of retinal stem-cells at the edge of the retina, called the ciliary margin zone (CMZ) which replenishes the retina with new cells if it is damaged in adult animals. However, the presence of these stem-cells has not been observed specifically in the CMZ of the developing human. Here, I investigate the developing human retina to understand if it contains stem-cells that could be harnessed for repair. I first utilized CMZ stem-cell markers found in fish and amphibians to assess in the developing human retina, including BLBP, C-myc, cyclin D3, Six3, SMAD1/5, and Zic1. Following the stainings, I observed expression of C-myc and BLBP. To maintain their long-term proliferation, stem-cells will replicate slowly. Therefore I analyzed cell cycle kinetics in the CMZ. Primary cultures of fetal human retina, called retinospheres, were made by dissecting the fetal retina into small pieces containing a portion of the CMZ and growing them in culture with retinal differentiation media. EdU, a dye that is integrated into DNA only in replicating cells, was then added to the media for different incubation intervals with EdU being 30min, 1hr, 2hrs, 4hrs, 6hrs, 8.5hrs and 25hrs, then retinospheres were fixed in PFA. Retinospheres were IHC stained with antibodies and dyes: Pax6 (a stem-cell marker), EdU (marker of cell division), Ki67 (marker of replicating cells) and DAPI. The total Ki67 positive cells in the CMZ and of EdU and Ki67 positive cells were counted so that the S-phase of mitosis could be measured to discover how fast the cells in the CMZ were dividing. I observed that cells in the CMZ were replicating slower than those further away from the CMZ, consistent with the possibility that there is a population of stem-cells in the CMZ of the developing human retina.

Mutations in the gene glucosidase, beta acid 1 (GBA) are not only the strongest genetic risk factor for Parkinson’s Disease (PD), but also accelerate the progression of PD. We hypothesize that GBA mutations accelerate disease progression by promoting propagation of Lewy pathology from cell to cell via dysregulated extracellular vesicles (EVs). To investigate this, we developed a Drosophila model of GBA deficiency (GBA^del) manifesting neurodegeneration and accelerated protein aggregation. We also developed a human neuronal model by generating human induced pluripotent stem cells (iPSCs) from an individual with PD heterozygous for a null GBA mutation (GBA^IVS PD). Neurons were differentiated from GBA^IVS PD iPSCs, isogenic GBA^WT PD iPSCs, and iPSCs from an age- and sex-matched healthy control. I performed immunocytochemistry and western blots to evaluate protein aggregation within neurons. Additionally, I isolated neuronal EVs by size exclusion chromatography and analyzed them using a ZetaView nanoparticle analyzer. We previously found the expression of wildtype GBA in muscles of GBA^del mutant flies rescued levels of protein aggregation in the brain. This non-cell autonomous rescue was accompanied by normalization of alterations observed in EVs from GBA^del flies. Similar to our fly model, I found human GBA^IVS PD neurons and EVs have increased ubiquitinated proteins when compared to GBA^WT PD or healthy control neurons and EVs collected from these neurons. Our results suggest that GBA deficiency mediates PD pathogenesis by accelerating propagation of pathogenic protein aggregation through the alteration of EV protein cargo. We are now further investigating how GBA influences endolysosomal trafficking and EV biogenesis and I will now test whether GBA^IVS PD EVs can propagate protein aggregation faster in recipient neurons than control EVs. Understanding mechanisms regulating the spread of protein aggregates could reveal novel therapeutic targets to slow the rate of progression of neurodegeneration.

The exact cellular and molecular mechanisms for the progression of amyloidogenic diseases such as Alzheimer’s and Parkinson’s disease are elusive. During amyloidogenesis, soluble protein monomers aggregate to form soluble oligomers, which further aggregate to form insoluble, ꞵ-sheet rich fibrils. The Daggett lab investigates the involvement of a nonstandard secondary structure called ɑ-sheet in the aggregation pathway of amyloidogenic proteins. The ɑ-sheet hypothesis states that ɑ-sheet is formed in the soluble oligomeric species during protein aggregation. This soluble, oligomeric form of the protein with ɑ-sheet secondary structure is implicated to be the toxic species in amyloidogenesis and a driver of disease pathology. One amyloidogenic protein is ɑ-synuclein, the major constituent of Lewy bodies, which are a pathological hallmark of Parkinson's disease. Preliminary data reveal the presence of ɑ-sheet in Parkinson's disease patient samples, showing promise for detection of Parkinson’s disease earlier than previously possible utilizing ɑ-sheet secondary structure. I am working on connecting the ɑ-sheet hypothesis with the amyloidogenesis of ɑ-synuclein by using synthetic ɑ-synuclein to link together data from different types of experiments. Experiments I conducted include the purification of ɑ-synuclein from E. coli, thioflavin T aggregation assays to measure the progression of the protein from monomer to fibril, circular dichroism spectroscopy to measure the predominant secondary structure in a peptide solution, and a soluble oligomer binding assay to detect the amount of ɑ-sheet content in patient or synthetic peptide samples. The purpose of my experiments is to demonstrate that ɑ-sheet is formed during the amyloidogenesis of ɑ-synuclein prior to fibril formation by linking together the above experiments, supporting the idea that detection of ɑ-sheet in patient samples may lead to methods of catching the progression of amyloid diseases earlier than previously possible.

As of 2021, Alzheimer’s Disease (AD) afflicts 6.2 million Americans. This number is expected to climb rapidly in the coming decades as the number of individuals at the age of increased risk rises. AD is characterized by the deposition of insoluble, β-sheet-rich amyloid plaques of the amyloid β (Aβ) peptide. However, the AD-causing toxicity of Aβ is mediated by soluble, oligomeric species of the peptide formed in the process of aggregating into plaques (amyloidogenesis). These oligomers have been shown to adopt a unique secondary structure known as “α-sheet.” The onset of AD is typically sporadic, but some cases, known as familial Alzheimer’s Disease (FAD), are inherited via mutations in Aβ. None of these mutations have been studied for their ability to adopt the pathogenic α-sheet conformation. Therefore, I am investigating the impact of the Arctic (E22G), Iowa (D23N), Flemish (A21G), and Osaka (E22Δ) mutations on the amyloidogenesis and toxicity of Aβ to explain their role in FAD. I accomplish this through a Soluble Oligomer Binding Assay (SOBA) designed in the Daggett Lab to capture and quantify α-sheet conformations. Additionally I use circular dichroism, MTT cell toxicity assays and Thioflavin-T curves to investigate the aggregation pathway of these mutants. I have found that these mutations confer unique aggregation patterns to Aβ. Many of these Aβ mutants appear to adopt a stabilized α-sheet consistent with their tie to FAD. This provides an explanation of how these mutants cause FAD and offers insight into the factors that contribute to the adoption of α-sheet structure and pathogenesis of AD.

To expand my experience with the human brain beyond dissections, I worked in the Intelligent Networks Laboratory (INL) in UW Bothell’s Computing and Software System (CSS) Division to simulate activity in a biological neural network. INL has a computer software system called “Graphitti” that allows users to run simulations of large biological neural networks. The specific simulations I analyzed were of growth and development in cultures of initially dissociated cortical neurons, which produce bursts of activity that propagate as waves across the network. Networks were grown over 28 simulated days on a 100 by 100 neuron grid, producing 500,000 synaptic connections. Two previous student papers suggest that this bursting activity is stable with 10% inhibitory neurons distributed across the simulated culture. With the original raw data stored in Matlab software (www.mathworks.com), I ran other code to pull burst location, speed, and distance from four simulations with different neuron firing rates and percentage of inhibitory neurons. In one simulation, burst origin locations were scattered across the network, indicating bursting behavior throughout the network. Two other simulations had a few, stable origin locations, suggesting a discernible pattern in bursting behavior. The burst propagation speed graphs for each network demonstrated growth and stabilization of activity by the end of the simulations. Working with this neural network has provided me with a fresh and "live" perspective on the connectivity of neurons. By understanding the dynamics of the network under these conditions, the data inspires further research investigating the connectivity effects of neurodegenerative diseases such as Parkinson's Disease.

Community composition and structure of modern benthic foraminiferal assemblages are commonly used as indicators of integrative water quality. The San Juan Archipelago is a tidally mixed estuarine environment located in the Salish Sea. This is an oceanographically complex area due to ocean input from the Strait of Juan de Fuca and estuarine input from the Fraser River. To assess the feasibility of using foraminifera as water quality indicators, foraminiferal assemblages from four oceanographically distinct locations in the San Juan Archipelago were collected and subsequently identified to pair water conditions with associated differences in assemblage. I carried out a survey of foraminiferal tests from Rosario Strait, Strait of Juan de Fuca, and San Juan Channel. Density of the number of tests decreased in high current environments and water-column stratification had no impact on assemblage. Abundance of Lobatula lobatula and Rotalinoides gaimardii were significantly correlated with oxygen, temperature, salinity, and nitrate. Depth and individual water conditions were observed to impact diversity of foraminiferal assemblage more than stratification. Foraminifera are incredibly resilient and present in most sediments of the San Juan Archipelago allowing foraminiferal assemblage to be used as a tool for water quality assessment in the San Juan Archipelago based on the abundance of species like L. lobatula and R. gaimardii.

A novel freshwater methane seep with unique physics and chemistry was discovered off the Oregon coast in 2014. This seep, called Pythia’s Oasis, is located ~20 km east from where the Juan de Fuca plate subducts beneath the North American plate along the Cascadia Margin. Studying microorganisms from deep within subduction zones is difficult and requires deep-sea drilling expeditions that can disrupt the ecosystem. Recent analyses of Pythia’s Oasis fluids suggests that they likely originate from high temperatures deep within the Cascadia Margin, providing a window into the region where deep subduction-zone fluids are formed. The Pythias’s plume has a vent-orifice temperature of 12.08˚C, ~3 times warmer than background seawater and is characterized by low-salinity and methane-saturated fluids. Nothing is currently known about the microorganisms that inhabit this type of system. In August of 2021, the deep-sea remotely operated vehicle Jason collected 5 L of fluid emanating from the Pythia’s Oasis’s orifice. The goal of this project is to extract and sequence community-genomic DNA to identify the dominant taxa present in this system and to elucidate the diversity of functions present. The resulting metagenomic data will be analyzed using established bioinformatic tools that can assess diversity and genetic potential. Results of this research will provide new insights into the chemical and biological conditions within the subsurface of the Cascadia Margin and similar systems in the deep subsurface.

The North Arch Volcanic Field lies north of O’ahu, covering 24,000 km² of seafloor. It is a part of the greater Hawai’ian Arch, a region of uplifted seafloor surrounding the Hawai'ian hotspot. The North Arch is a fairly recent discovery, with little known about it. It is a product of recent basaltic volcanism, the mechanism of which is not understood. The magnetic minerals found in basalts retain the signature of the Earth’s magnetic field at the time they cooled. Thus, basaltic lava flows generally have high remnant magnetization, distinguishing them from the surrounding, weakly-magnetized sediments. My objective was to use a magnetometer to assess the feasibility of using magnetic anomalies to determine the thickness of lava flows in the southern North Arch. On board the R/V Thomas G. Thompson, a sub-bottom profiler and multibeam sonar system were used to resolve lava flow boundaries while I collected data with a towed magnetometer. The ship followed 5 east-west track lines before heading north-west along an inferred rift on the seafloor, then west to exit the field. I obtained magnetic anomalies by correcting the raw magnetic data with the International Geomagnetic Reference Field and a time series from Honolulu’s magnetic observatory. I then used a modeling program to compare observed anomalies with those predicted for lava flows of varying thicknesses. A modeled flow along the northmost track line yielded a flow maximum thickness of 50 m, thinning to 5 km towards the east edge of the field. The results show that the magnetic anomaly produced by these flows was strong enough to be detected and yielded flow thicknesses of tens of meters that are consistent with other observations. This research demonstrates that magnetometry is an effective tool for studying the North Arch Volcanic Field and could be used on other submarine volcanic fields as well.

Ocean conveyor belt circulation is conceptually foundational in oceanographic education. It is the idea that oceans slowly overturn through the movement of deep water toward the equator and surface water toward the poles. Introductory courses often spend the majority of a semester building the skills required to understand the basics of this theory. Ocean circulation is also typically covered through multiple courses to build upon its complexity through different study lenses. The multi-dimensional concept of ocean circulation is commonly conveyed through 2D diagrams and animations rather than presenting ideas in a 3D space that allows students to form connections between theory and physical space. Students were asked to interact with a 3D printed model that recreates circulation diagrams by connecting cross-sections of oceanographic data to specific locations on the model. Furthermore, students were asked to describe how perturbations to surface conditions could change ocean stratification and how water circulates based on its temperature and salinity. Previous 3D printing experience allowed us to expand students’ experiences while struggling to parse these interdisciplinary oceanographic topics. Here we show that using a physical model in teaching thermohaline circulation enhances the speed and depth at which students understand ocean circulation compared to the traditional 2D approach. Surveys were given to assess students’ understanding of the driving factors behind thermohaline circulation prior to and after interacting with the model. Preliminary results show that students can better connect oceanographic concepts taught in lectures to data and locations essential to ocean circulation after completing exercises that ask them to interact with the model. We anticipate students to exhibit further proficiency in concepts related to ocean circulation after interacting with this model. We also expect to find that students will express a desire to see the utilization of similar models in more of their core oceanography courses.

Freshwater wetlands are the largest natural source of methane, a powerful greenhouse gas (GHG), to the atmosphere. However, the mechanisms and magnitude of wetland methane (CH₄) emissions are not well understood. There is significant variability of flux estimates between field sites and a lack of agreement between top-down and bottom-up estimations of methane flux, indicating a high level of uncertainty. This study provides the first estimation of aquatic methane flux in the Union Bay Natural Area (UBNA). This site can be categorized as a freshwater wetland and is an urban site that is adjacent to a former landfill. We hypothesize that the flux will be dominated by ebullition (bubble released) emissions and that the total emissions will decrease as depth increases. To measure the total CH₄ flux, we measured diffusive emissions using a flux chamber and LGR portable greenhouse gas analyzer and ebullition emissions with bubble traps that were left in the field site for 48 hours. In addition to these measurements we collected water parameter measurements using an Exo sonde and dissolved gas samples at each diffusive sampling location. With this data, we were able to calculate the measured CH₄ flux and model the flux so that the two values could be compared. This study indicates the spatial variability and depth-dependent patterns of methane flux in the UBNA. There are multiple possible extensions of the research. If more sampling campaigns were conducted throughout the year, the data could be used to describe temporal variability in flux. Additionally, if gas samples are collected from the ventilation systems on the landfill, an isotopic analysis could reveal the percentage of methane emissions that can be attributed to decomposition in the landfill.

The deformation processes in the brittle-ductile transition region are poorly constrained and critical to understanding both the magnitude of earthquakes within the subduction seismogenic zone, and slow slip and tremor downdip of this region. Here we present field observations and microstructural data in the form of photomicrographs, thin-section scale x-ray maps and electron backscatter diffraction (EBSD) to constrain the rheology of a paleosubduction interface in the Central Alps during deformation and fluid-rock interaction. We studied three samples across a 10 meter transect within the lowest 10 meters of the overring Austroalpine plate at the contact with the Penninic subduction plate shear zone. Our granodiorite samples are exhumed from paleodepths of 30-35 km. Cataclasites, microfractures, and crack-seal quartz ± calcite veins are evidence of brittle deformation while a ductile foliation fabric indicates viscous deformation at conditions of 400-430ºC & 0.80-0.95 GPa. EBSD data shows progressive increase in viscous deformation and mineralogic changes closer to the contact. As the samples progress towards the shear zone the crystallographic preferred orientation and grain misorientation of quartz and albite decreases suggesting the controlling deformation process changes from dislocation creep to diffusion creep due to pinning of the albite and quartz by phengite. This coincides with progressive fluid-rock modification observed in photomicrographs and chemical x-ray maps. We observe a decrease in albite replaced by an increase in quartz and phengite across the three samples. The breakdown of albite, and its replacement with more water rich minerals like phengite, the quartz +/- calcite veins, and the progressive shift from dislocation to fluid-aided diffusive mechanisms demonstrates increased fluid-rich interactions nearer to the plate interface contact. Increased fluid-rock interaction impacts the viscous deformation mechanisms and strength of these rocks. Diffusion creep occurs in low stress environments, suggesting that fluid- rock interactions lead to a low stress environment below the locked seismogenic zone.

Although the dominant forcing for earthquakes is plate tectonics, a wide variety of other processes have also been shown to modulate the tectonic release of elastic strain such as glacial mass changes, reservoir impoundment, and monsoonal loading. Here, we examine the relationship between surface mass loading and seismicity. We examine data from the Gravity Recovery and Climate Experiment (GRACE) and GRACE Follow-On (GRACE-FO) that have been monitoring global gravity changes from 2002 to 2022 and compare this record to the USGS earthquake catalog with magnitudes greater than 5.4 during the same time period. We compare the distribution of the surface mass loading during all time periods to the distribution of the surface mass loading during time periods with an earthquake occurrence. We use Bayes’ theorem to calculate the relative conditional probability of an earthquake occurrence given the surface loading. We use the Kolmogorov-Smirnov and Cramér-von Mises statistical tests to confirm that the surface mass load during periods with an earthquake are significantly different than the background. We conclude that there is a higher relative probability for earthquakes to occur during periods of large surface mass loading and unloading. The existence of surface load induced events suggest novel interactions between cascading hazards, such as earthquakes and landslides. Our analysis provides evidence for the hypothesis suggesting that cascading solid Earth and Earth-surficial hazards exhibit two-way coupling.

We are living in a world of gender, but it’s difficult to grasp its shape because of its fluidity and complexity. And yet, many of us are taught to believe that gender only exist as “either-or”, or that it is merely about our own identities. My research about gender in my gender and sexuality course GWSS 200 and GWSS 390 have offered me the tools to better articulate historic and current ideas and experiences related to gender and place. With this interactive zine project, I invite my audiences to question and interrogate assumptions about gender and our gendered world through words and images. I introduce a series of terms and their definitions accompanied by abstract graphics based on my own understanding and experiences. During the reading process, I encourage my readers to embrace and embody those terms through their deeply personal lenses and experiences, as well as through guided intentional practices, like making art pieces or writing poems with the terms and ideas provided in the zine. in so doing, participants examine their own vulnerabilities around gender in a safe space. Unfamiliar vocabularies are intended to evoke questions if not some confusions, but that’s the point. I hope to deepen our awareness of gender-related topics and ongoing issues through a humane, educational, and playful strategy, and eventually, cultivate more empathy within us. 

Mount St. Helens shook local communities and spewed volcanic ash into the sky for two months before it finally had its major eruption. In those two months newspapers eagerly tracked the activity, crafted a story, and relayed it to their audiences. When the mountain erupted on May 18, 1980, the world caught a glimpse of nature’s power and found a dramatic climax to their two-month story. In the immediate aftermath, accounts of what had happened took various forms, ranging from personal hymns to films, with each of them showing a different response to the eruption. These responses showcase a population reconsidering what it means to live alongside nature. Today, forty years later, Mount St. Helens’ story is still being told through a wide array of sources. In my research, I analyze works approaching the eruption, reacting in the immediate aftermath, and those which have come out in memory. I note the content of these sources as well as their framing to construct an analysis of how changing treatment of the Mount St. Helens story reveals society’s approach to nature before the eruption and how that approach changed in response to the events of May 18. I specifically focus on sources consumed and produced by the broader public, such as films and songs, using private correspondence or scientific conferences only as a source of what does not make widespread narratives. This approach encapsulates how people of various communities make sense of living alongside the natural world, and especially how they conceptualize sudden change events like volcanic eruptions. My research uncovers both flaws in the population’s conceptual relationship to nature as well as their tendencies to remember natural events, specifically Mount St. Helens, in a way that maintains or minimally changes the way the see themselves in the world.

Becoming an adult is a significant life course transition which brings much uncertainty to an individual's life. Previous studies attribute this uncertainty to navigating a growing sense of agency, with more outlets to explore emerging adulthood and personal identities. As young adults increasingly engage with these opportunities, more time is spent away from their families. As a result, they are less susceptible to the parental, familial, and cultural pressures in the home, where parents enforce their expectations for adulthood onto their children. The lifestyle of young adults of Asian immigrant parents who identify as a gender or sexual minority (AAGSM) often conflicts with their parents' ideal of a successful transition into adulthood. Yet, they find themselves poorly represented within gender and sexual minority (GSM) spaces, often downplaying their racial or ethnic identities to feel accepted. Scholars are only beginning to examine the outcomes of this identity conflict with AAGSM young adults, and so far, none have made a clear connection between the transition to adulthood literature and the (in)visibility of Asian-American coming-out narratives. The goal of my study is to understand how parental closeness and expectations for emerging adulthood affect how AAGSM young adults perceive their preparedness for adulthood. This goal will be met by conducting semi-structured interviews, asking participants to reflect on their parents' and their own expectations for adulthood with the consideration of their AAGSM identity and relational closeness. As I am using grounded theory to conduct my analysis, the anticipated results of my study have yet to be determined. Focusing on AAGSM young adulthood is a promising area for future research, as it holds important implications on how to accomodate the intersectional experiences of individuals as culture continues to evolve. 

The location of Bangkok on the Chao Phraya River delta as it enters the north end of the Gulf of Thailand makes the city particularly vulnerable to climate change-induced changes in hydrological systems. Rising sea levels, low-lying land, and rapid development on unstable marshes - which has led to the city sinking at an annual rate of 1 to 2 cm - combine to make it necessary for Bangkok to grapple with how it will survive into the future. In this work, the majority of climate change research takes a quantitative angle, with dominant discourse often surrounding models, numerically determined ‘threshold’ points, and specific environmental impacts. It's hard to overestimate the importance of scientific work on climate change; however, it is also true that 'hard' scientific data has limited influence on opinion and has not prevented the continuing climate crisis. In this project, I turn to fiction and narrative analysis as alternative, qualitative methods for examining problems of climate change. Building on the work of ecocriticism and literary theory, this project explores narratives used in policy and fiction as they depict Bangkok’s future under climate change in order to understand how narratives influence how we imagine - and thus, determine - potential futures. This thesis examines the stories that shape our lives and our role in creating and responding to them. In close readings of Pitchaya Sudbanthad’s novel Bangkok Wakes to Rain (2019) alongside the government-led Bangkok Resilience Strategy produced by the Bangkok Metropolitan Administration and the 100 Resilient Cities initiative, this project examines ecocritical claims to the virtues of climate change fiction in interdisciplinary research and argues for the importance of including qualitative values into humanity's attempts to confront the challenges of climate change.

The Civil Rights Movement of the 1960s in the United States galvanized millions of Americans to fight for a more free and democratic society, banding together to protest racial segregation and other forms of systemic racism such as police violence against minorities. Newspapers covered these actions extensively, spreading the message of civil rights across the US. People eager for change in cities far from the centers of civil rights activism in the South, such as Seattle, responded to this national political fervor by fighting for change locally. In Seattle, activists sought an end to job and housing discrimination, de facto school segregation, and police violence through non-violent direct action. Seattle’s major print newspapers, The Seattle Times and the Seattle Post-Intelligencer covered these issues extensively, spreading news and controversial developments to their readers. In my research, I analyze newspaper coverage on activism and cases of police violence which garnered a strong public demand for justice. With the support of other sources, such as the biographies of Seattle activists, the histories of local civil rights organizations, and studies on media coverage of the police, I construct an analysis of how these newspapers shifted their coverage of civil rights activism and police violence throughout the 1960s as a response to community activism. This critical angle focuses on how the actions of Seattle’s activist community influenced newspaper media, prompting the newspapers to include more activist perspectives in their news coverage. This research, therefore, displays the power that local activists held in influencing print media coverage of their actions, and with that, the influence that activists had to shift the public perspective towards activism in the 1960s in Seattle.

In this project I completed last spring, I examine both reception and textual content of renowned author Matsuura Rieko's 1987 short story collection Natural Woman to explore what makes the work an affirmative portrayal of queer women, focusing on the joy, pain, and growth the women themselves derive from their relationships (rather than painting them in a scandalous or pornographic light). I first review reception from both queer women in Japan and literary critics at the time of the work's release, revealing that the work was embraced by queer women at the time for its true-to-life portrayals but generally overlooked by literary critics for its queer themes. I then explore three main aspects of the work - the protagonist Yōko's position as a first person narrator of the three stories, the nonlinear ordering of the stories, and the use of space in the stories - to depict lesbianism as something that is intrinsic to Yōko's character and allows her to explore her identity and grow. These three aspects show Yōko’s growth as a character, all while her continued attraction towards women is not seen as a barrier to her growth, but rather, a means of growth. Additionally, they make the perspective of Yōko and the emotions of the women she is involved with the focal point of the story, giving queer women voices and depicting pleasure for their own sake. As works of contemporary Japanese literature depicting same-sex relationships between women have largely yet to be examined in depth, my hope is that this research brings attention to the potential for visibility of queer women in contemporary Japanese literature that is gained by examining what makes for portrayals of queer women that are affirmative.

The Philippines is experiencing a new kind of era ever since the onset of Rodrigo Duterte’s presidency. Although the Philippines deems itself as a republic, the president exhibits an authoritarian nature. His tactics, rhetoric, and worldview have garnered attention and controversy, leading to a reevaluation of the country. Despite disagreement and condemnation from human rights groups and other entities, for many people Duterte stands as an effective leader in the Philippines. Even with his brutality, there is a strong presence of approval. In this project, I aim to explore and uncover what makes Duterte such a likable figure among Filipino citizens. A review of the literature reveals many plausible reasons. Duterte veils himself as a ‘common man.’ His efforts to combat the war on drugs are also admirable to many Filipinos; they feel much safer with some of his policies. Initially, my research was supposed to manifest in an academic paper. However, I found that one of my parents shared the same sentiments as other Filipinos. To highlight my findings, I chose to interview my dad and borrowing from the work of Ocean Vuong, I utilized the form of a letter to my father. With this project, I aimed to connect with my dad in hopes of understanding more about my people. Additionally, I was able to hear the genuine thoughts and background of my dad. This brought me closer to my roots, and my dad. This research contributes further to the broader question of what makes dictators and authoritative leaders, such as Duterte, ‘likable’ people to their citizens.  

 My research paper investigates whether the Supreme Court is influenced by public opinion. It is important to be aware of whether the Court is inclined to uphold laws that have been passed by legislative majorities or overturn them. Is the Supreme Court restrained or does it have a tendency for judicial activism? The question that guides my work is: During the period of approximately the last six decades, has the Supreme Court been influenced by public opinion when deciding politically important (i.e., salient) cases? My theory is that the Supreme Court is influenced by public opinion when deciding cases that are salient due, in part, to the Court’s inherent political nature. Justices ascend through a partisan appointment process that is predicated on the sharing of power between the President and the Senate. Consequently, justices and their institution cannot uncouple from the political ecosystem. Specifically, I posit that fear of non-implementation constrains a justice’s decision-making. Non-implementation would occur if the Court’s ruling was not enforced by either a lower court or a non-judicial authority. And the potential consequence of recurring non-implementation is to degrade the Court’s legitimacy. To test my hypothesis systematically, I will use multivariate regression analysis to examine the relationship between the ideological direction of Supreme Court case decisions and prevailing public opinion, while accounting for the influences of other factors. My analysis incorporates data from (approximately) 5,000 individual Supreme Court cases, extending from 1958 to 2018. I predict that public opinion does influence Supreme Court decision-making when rulings will ‘resolve’ the national debate around issues that have significant public policy implications.

This paper uses national data collected through China’s population census to estimate the effect of housing prices on fertility rates between 2000-2020 across 31 provincial-level administrative regions in mainland China. I theorize that higher housing price would lead to a decrease in fertility rates. I take housing price as a proxy for the overall costs of living. As houses become more expensive, younger generations are forced to save money for years to afford buying a house. When individuals’ income remains unchanged, higher housing prices lower people’s purchasing ability. This lowered purchasing ability leaves people less financially capable of raising a child and results in individuals being less willing to have a child. This paper employs a multivariate regression model and controls for other relevant variables. I expect to find that with the continuous increase in housing price, there is a steady decrease in fertility rates in each administrative region. Furthermore, since a more rapid rise in commodity prices is associated with fast economic development especially in Beijing and Shanghai, the fertility rates in these two regions would be the lowest, corresponding with a weak per capita purchasing power of individual households. This analysis of the relationship between housing price and fertility rate is important because it explains why there is a fertility decline despite robust economic growth in a developing country. This paper calls for changes in maternity policy to accommodate possible decline in workforce and productivity in China and its aging society in the next 20 years.

Recent years have seen a great expansion in the public consciousness of human impacts on global environment, as well as wider acceptance of the reality of anthropogenic climate change. Although individual willingness to take pro-environmental action is a crucial component to real progress on the climate dilemma, it is a phenomenon whose antecedents are complex and diffuse. While much of the literature on this topic focuses on the identity characteristics which may account for individual pro-environmental behaviors, in this research project I build on research in environmental psychology and other disciplines to hypothesize that spending time in nature impacts the pro-environmental behaviors of individuals. I argue that, because it facilitates deeper connection and measurable psychological changes toward cooperative behaviors, time in nature is likely an influence on future actions on behalf of the natural world. I further theorize that although time in nature may impact Democrats and Republicans differently, there is an effect regardless of partisanship. To test this hypothesis, I employ a multivariate regression analysis which tests the relationship between time spent in nature and pro-environmental behaviors while controlling for possible confounding variables including income, education, gender, and partisanship. To investigate the effects of partisanship, I also run a model testing whether time in nature influences the behavior of Democrats and Republicans differently. I expect to find in each case that the relationship between time in nature and pro-environmental behaviors is statistically significant. Such findings could serve to emphasize the importance of access to natural spaces for the future of environmental attitude formation and activism.

In 2013, the Supreme Court rendered unenforceable the federal preclearance provision of the Voting Rights Act of 1965 – a decision that many feared would undo decades of progress in eliminating voting discrimination. Chief Justice John Roberts claimed that federal preclearance was an “extraordinary measure” that is no longer warranted. Was he correct? Or have our worst fears been realized? Almost a decade since the decision, I explore these questions by mapping the landscape of election laws across all fifty states and analyzing their effects on minority participation in each election cycle from 2006 through 2020. I identify de facto voter suppression by state legislatures, such as voter I.D. requirements and felon disenfranchisement, and assess whether these have increased since the Shelby County v. Holder decision. I then use multiple regression analysis to assess whether these laws widen the gap in voter registration and turnout rates between white and minority voters. I theorize that, contrary to Chief Justice Roberts’ claim, voting discrimination had been kept at bay by the federal preclearance provision, rather than by previously covered state legislatures’ own accord. In the absence of federal preclearance, I expect to see an increase in voter suppression following the 2013 decision and a negative relationship between voter suppression and minority participation. These findings may inform major voting rights legislation moving through Congress this year.