Pollution from human influences is a growing problem in the Chehalis River and tributaries causing deleterious effects on stream health. Stream microbiomes are composed of diverse collection of microbes that play an important role in keeping the surrounding riparian zones and life within the streams healthy and thriving. Stream water has been facing a decline in quality from the leading cause of pollution from agriculture and manufacturer run-offs. Although there have been many studies relating to run-off pollution affecting streams, there are few in-depth studies done on the taxonomy and functionality of microbes in urban water ecosystems, so it is important for this to be further investigated. With the use of 16s rRNA sequencing, each sample site presents two years of data included to indicate differences both by season (Fall, Summer) and sample site. At the phylum level, every season consists of a primary microbial signature shared amongst the sites. These phyla are Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Verrucomicrobia, and Tenericutes. From the family data, common to nearly all sites are the Flavobacteriaceae, Comamonadaceae, and Oxalobacteraceae. Families prevalent at many sites in various combinations include Flexibacteraceae, Crenotrichaceae, Chitinophagaceae, and Verrucobacteraceae. This project presents a two-year microbiome analysis of Alder and Fry Creek to develop trends for the seasonal variation in urban water ecosystems due to human impacts.

A hatchery’s purpose is to supplement falling populations of wild fish, mitigating human impact. However, the effect operating a stream-based hatchery has on the immediate stream environment is not well understood. Varied hatchery designs make it difficult to find a consistent way of assessing an establishment. This project used the idea that consistency is key to evaluate the impact hatcheries made by examining water of the the influent and effluent flow. Water was sampled from where it entered, transitioned, and exited three different fish-bearing hatcheries. Hatcheries were classified into three general water-system types, gravity-fed, spring-fed, and flow-through. Water status was evaluated by measuring electric current, dissolved oxygen, pH, temperature, and bacterial content. In addition to unique location characteristics, containment mitigation measures and extenuating circumstances were considered when evaluating the data. The results showed the trend of influent to effluent water in all three hatcheries increased in temperature while the levels of dissolved oxygen, electric current, and pH decreased. Similar result trends were also found between alike water-system types, although with the small sample results are inconclusive. This study is not meant to be a stand alone. With the inclusion of more locations the data will begin to form an averaged scale which could be referenced for the effectiveness of certain hatchery designs and contaminant mitigation techniques. Having this data-set would eventually serve as a resource for future hatchery construction projects.

Water temperature is one of the largest factors of hatchery efficiency, and this can vary greatly depending on the source of the water. Warming waterways is thought to be a problem with salmonid hatcheries in the Pacific Northwest. For this study, we compared the effects of water source (Lake Aberdeen vs VanWinckle Creek) as well as temperatures on the mortality rates of Oncorhynchus mykiss. Water temperature data was collected from 2015, 2016, 2017, and 2018 to compare the effects of temperatures and water source on the mortality rates of O. mykiss. Microscopy was also used in an attempt to find and identify Tetracapsuloides bryosalmonae, the parasite known to cause Proliferative Kidney Disease in salmonid fishes. This parasite is believed to be a major contributor to high summer mortality rates. We collected multiple morbid and recently deceased juvenile O. mykiss, and examined these samples using a hematoxylin and eosin staining of the kidneys and gills. Initial findings suggest no significant impact of lake versus river water for this specific disease, however gram staining and biochemical assays indicated a presence of a bacterial fish pathogen: Flavobacterium columnare. Because of this, future directions include broadening the scope to a greater variety of infectious diseases. If factors influencing development of PKD can be identified, preventative actions can be taken to lessen the mortality rates and allow for a larger amount of fish to be successfully raised each year.

Glycans are ubiquitous in biology and play a crucial role in cell to cell signaling, immunity, and disease. The A, B, and O blood groups are differentiated by different glycan structures and linkage patterns presented on either glycoproteins or glycolipids. To date, the exact glycan structures that differentiate blood types remain unknown. A clear understanding of these structures would increase the success of transfusion procedures and reduce the risk of complications. Our lab is currently examining glycans from glycoproteins isolated from red blood cell membranes, however we do not have the enzymes needed to remove glycans from glycolipids. ECGase I is an enzyme that was recently shown to effectively cleave glycans from a wide range of glycolipids. We have cloned the gene for ECGase I into a GST fusion vector for recombinant bacterial expression. With this we have been able to generate high yields of purified ECGase I. The EGCase I produced was more active than commercially available product. The ability to grow EGCase I enables large scale investigation of glycolipids involved in the ABO blood group system.

Gravity remains one of the biggest mysteries in physics. Since Einstein’s Theory of Relativity not many new discoveries have happened. There are several big questions remaining about it such as: Why is it so much weaker than the other forces of nature? Is it mediated by a particle like the other forces? Is there a unifying theory between gravity and quantum mechanics? Does gravity have a place in the standard model? There are many theories proposed today which attempt to answer these questions, such as the presence of extra dimensions in string theory. If the effects of these theories exist, they would be present at small length scales (less than 1 mm). In order to test these theories, our lab uses a torsion balance experiment at sub-millimeter lengths. Our torsion balance experiment consists of an attractor mass on a turn table, and a detector mass hanging from a thin wire. Each has wedges cut out of it in the same pattern. The test is run by operating the turntable and measures the gravitational torque experienced by the detector mass. My project is to use a code that simulates the gravitational torque in order to investigate its dependence on different orientations and geometrical aspects of the experiment, as well as to improve on future runs of the experiment. This experiment will shed light on previously unknown aspects of Gravity and hopefully provide new discoveries to the field of gravitational physics.

Fibrolamellar carcinoma (FLC) is a life-threatening variant of liver cancer affecting adolescents and young adults. FLC tumors do not respond to chemotherapy, thus, surgery remains the mainstay of therapy. FLC is characterized by a fusion event resulting in a novel chimeric protein that joins the N-terminal domain of DNAJ with the catalytic subunit of protein kinase A (PKAc). However, the underlying mechanism by which the FLC chimeric kinase drives FLC tumor growth remains unknown. Using CRISPR/Cas9 technology, the Scott Lab (UW) has re-created the human FLC fusion protein in a well-characterized and non-transformed hepatocyte cell line. The resulting cells retain hepatocytic differentiation while over-expressing DNAJ-PKAc compared with wild-type PKAc, akin to the human tumors. Using these model cell lines and a computational approach called, Kinome Regularization (KiR), I plan to uncover molecular mechanisms through which chimeric PKAc transforms hepatocytes in FLC. The concept of KiR is built around the fact that most kinase inhibitors are not specific but instead, affect a range of targets. By exploiting the property of polypharmacology in a set of well-characterized kinase inhibitors, we can make use of elastic net regression to compute and deconvolute the kinases that are responsible for a quantitative trait. To do so, we have identified a set of 30 broadly specific inhibitors that give us >85% coverage of the kinases studied. We can then test the effect of each of these 30 inhibitors on cell growth and use the results to identify the kinase important for cell growth. The KiR-based model will also be used to identify kinase inhibitors which can specifically reduce growth of FLC cells. Overall, these studies have a strong translational potential; a new therapeutic option for FLC could give unprecedented hope to the patients who currently face this disease with limited alternatives.

The waters surrounding the San Juan Islands in the Salish Sea near Washington state, USA, possess a variety of physical and chemical conditions that combine to support a rich multitude of marine organisms. Five constituents observed and analyzed in this project represent the larger food web found within the Salish Sea: nutrients, phytoplankton, forage fish, seabirds, and marine mammals. I tracked variability in these constituents during the fall over the course of twelve years from 2006 to 2018. This time range was selected to cover the years before, during, and after the North Pacific marine heat wave (2014-2016), and the subsequent return to normal temperatures in the Salish Sea since those years. The abundance and composition of these constituents varied over this time range. We found a significant decrease in both nutrient concentrations and fish condition in the fall of 2014, which had returned to previously observed levels by the fall of 2018. In contrast, the phytoplankton and marine bird and mammal observations remained comparably low in 2018, similar to values observed in fall 2014. The introduction of the marine heat wave into the Salish Sea during 2014 appears to have consequences that span across multiple years after its dissipation in the Pacific Ocean. As marine heat waves are anticipated to become more prolonged and common with global climate change, it’s crucial to understand their short- and long-term impacts on local marine populations. 

Research has shown transgender children respond similarly to categorical measures of gender identity as their gender-matched cisgender peers, rather than those who share their sex assigned at birth. However, categorical measures may be limiting responses and not encompassing the diversity of gender identities. We presented a continuous measure to 223 transgender children (socially-transitioned children to live and present as a gender opposite the one assigned at birth), 71 gender nonconforming children (children who have not socially transitioned but show characteristics stereotypically associated with a gender other than the one assigned at birth), 281 cisgender control participants, and 181 cisgender siblings of transgender and gender nonconforming participants. Provided a line, participants were asked to mark their gender identity, with the left-most end indicating feeling completely like a boy, the right-most end indicating feeling completely like a girl, and in between representing varying degrees of a mixture of both. Each participant’s mark was converted to a percentage, with 100% indicating feeling completely like the gender they present as (for transgender/gender nonconforming children, opposite the gender assigned at birth). A univariate ANOVA examining the effect of participant group on identity showed a significant main effect, F(3,752)= 38.72, p < .001. Post-hoc Tukey comparisons showed, consistent with previous research, transgender children (M=0.81, SD= .189) did not differ significantly from the cisgender controls (M=0.87, SD= .181), nor cisgender siblings (M=0.87, SD=.206), in their identification with their current gender. However, gender nonconforming participants (M= 0.59, SD = .309) differed significantly in their scores from each of the other groups (ps < .001). These results are consistent with previous research conducted with categorical measures. Additionally, as none of the groups showed 100% binary identification, this study also demonstrates the importance of continuous measures of gender identity within gender development research.

High purity germanium (HPGe) detectors are an important technology in several leading experimental searches for dark matter and neutrinoless double beta decay. Understanding the interaction of various types of radiation on the different surfaces of HPGe detectors is essential to developing methods to reject unwanted signals from radioactive background sources. I have taken a leading role in the construction and use of the Collimated Alphas, Gammas, and Electrons (CAGE) test stand at the University of Washington, whose goal is to evaluate the response of an HPGe detector to different types of radiation on its various surfaces. CAGE is a vacuum cryostat with an internal system of motors that move a radiation source while keeping the detector active. It requires the operation of a liquid nitrogen cryostat, vacuum pump, temperature sensors, and various radioactive sources, all of which must be integrated into a single data acquisition (DAQ) system. We are currently constructing this system, fabricating and installing parts, and are planning to take initial data with the HPGe detector in the summer. In this talk I will present the current status of the CAGE detector, as well as preliminary data from radiation signals in the detector.

Our research group performs one of the highest-precision tests of Einstein’s equivalence principle, perhaps the most fundamental property of gravitation, using a sensitive rotating torsion balance. Among the leading experimental challenges are temporal and spatial temperature variation. Notably, horizontal temperature gradients across the apparatus, if not properly characterized, can emulate an equivalence-principle violating signal. We have implemented thermal shielding and run tests to measure the thermal effects on our measurement. Past tests have shown a need for both absolute and differential temperature sensors with higher sensitivity. Hence, my research project focuses on investigating the effect of temperature gradient on our experiments by constructing a thermal monitoring system. I have designed, laid out, constructed, and tested sensitive bridge thermistor circuits that can function as both absolute and differential temperature sensors. Current tests of our prototypes have shown that temperature sensitivities reaching 10 micro-Kelvin in one second (10^-5K/Hz^0.5). We are scaling-up these sensors and plan to deploy them in this academic year. Successful completion of this project will yield improved understanding of the temperature gradients within our experimental apparatus, allowing us to test the equivalence principle with yet higher precision.

Chimeric antigen receptor (CAR) T-cell therapy is the latest treatment option available for those suffering from certain forms of cancer such as lymphoma and leukemia. These engineered cells are able to recognize specific proteins found in tumors, and subsequently induce CAR-T cell proliferation, cytokine secretion, and lysis of the cancerous cells. Despite its promise, a percentage of patients who receive this treatment develop a range of neurotoxic symptoms. My research project tests the hypothesis that endothelial activation of vascular tissue in the brain, which would allow for increased permeability of immune cells through the blood-brain barrier, is contributing to the development of these clinical symptoms. Using a technique called immunohistochemistry, I used the antibodies claudin-5 and cd31 to fluorescently label tight-junction proteins and adhesion molecules of endothelial cells from brain tissue harvested from a developed mouse model. This mouse model received CAR-T cell injections and underwent behavioral testing to confirm the presence of neurotoxicity symptoms. I then used microscopy skills to visualize the labeling of the endothelial cells and proteins. If my hypothesis is correct, I expect to see a quantifiable decrease in the number of cerebral tight-junction proteins connecting endothelial cells along the blood-brain barrier, as compared to negative control tissue that received no CAR-T cell injections. In order to make these comparisons, I will use a software program such as Image-Pro Premier software (Media Cybernetics) to help me quantify the positive fluorescence labeling of endothelial cell proteins and adhesion molecules in both the control and experimental tissue. Tissue with less tight-junction proteins and adhesion molecules would permit the influx of foreign particles into the CNS. Understanding the cause of CAR T-cell related neurotoxicity will be first step in promoting prevention and increasing the effectiveness of this new cancer immunotherapy.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that results in progressive degeneration of upper and lower motor neurons, leading to muscle weakness, paralysis and death. Currently, ALS affects 15,000 Americans in the U.S., who have an average life expectancy of three to five years at the time of diagnosis. There are only two FDA-approved medications for ALS treatment, which minimally slow but do not reverse the progression of the disease. Understanding the genetic causes of ALS can help us to identify more effective treatments, therefore we seek to identify pathogenic variants and investigate how they contribute to the death of motor neurons. We previously showed that a tandem repeat in an intron of the gene WD-repeat-containing protein 7 (WDR7) may be involved in ALS. To better characterize this repeat, we PCR-amplified the repeat region and quantified repeat size in 500 ALS samples, including those from sporadic cases and those with known pathogenic variants. These repeat sizes were compared to approximately 500 Parkinson disease samples, 100 Primary Lateral Sclerosis samples, and 500 control samples from the Coriell Cell Repository, to verify whether expansion of this repeat was specific to ALS. Furthermore, to determine if repeat blocks were enhanced in certain subsets of patients, the lengths of the intronic repeats in genomes of ALS patients were profiled and compared with de-identified phenotypic information. To resolve the exact sequence of repeat in ALS samples, a subset of patient DNA samples and controls were sequenced using single molecule real-time (SMRT) sequencing. Together, these findings help us gain insight into the disease and guide us to develop a better treatment.

Epithelial-mesenchymal transition (EMT) refers to a biologic process that allows a polarized epithelial cell, which normally functions in the basement membrane of a cell, to undergo biochemical changes that makes it express as a mesenchymal cell phenotype. This mesenchymal phenotype allows the cell to have enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and increased production of extracellular matrix (ECM) elements. The process of EMT is considered completed once the underlying basement membrane breaks down, and the mesenchymal cell becomes migratory. Another component that proves EMT is the loss of e-cadherin. E-cadherin refers to cell-to-cell adhesion and the degradation of e-cadherin levels are a hallmark of EMT happening. There are three distinct types of EMTs; I will be focusing on type II EMT. Type II EMTs are associated with inflammation/wound repair but usually stops once inflammation subsides. However, in the context of organ fibrosis, type II EMTs can continue to over-respond to a persisting inflammation and can lead to organ death. In my experiment, I hypothesize that in HEK 293 human cells, SNAP -Δ1-91 alpha-1D adrenergic receptors undergo type II EMT. SNAP -Δ1-91 alpha-1D adrenergic receptors are a truncation of the extracellular portion of the receptor. Certain receptors undergo this truncation to increase its expression. It is shown that in SNAP – Full Length alpha-1D adrenergic receptors  (wild type receptors) do not undergo EMT. I will be able to observe the process of type II EMT through imaging the breakdown of the cell membrane in SNAP- Δ1-91 alpha-1D adrenergic receptors and the measuring of e-cadherin levels. The purpose of this research would be to potentially influence future therapeutic interventions that target wild type receptors to induce would repair. 

Cardiovascular disease (CVD) continues as the leading cause of death worldwide. The underlying cause of CVD is atherosclerosis, characterized by fatty plaques in the inner walls of the artery, and exacerbated by oxidative stress, inflammation and immune cells. The high-density lipoprotein (HDL)-associated enzyme paraoxonase-1 (PON1) plays a significant role in protecting against CVD. However, the mechanism of PON1’s protection is not well understood. Our laboratory and others have reported a progressive decrease of PON1 activity in several diseases. Our goal is to understand how PON1 prevents oxidative stress and its potential measurement as an early biomarker of risk of disease. Our hypothesis is that oxidative stress inhibits PON1 enzymatic activity, leading to progression of the atherosclerotic process in CVD. The objective of this research is to generate a mouse model to allow us to study the human enzyme PON1 in induced atherosclerosis in vivo.  Starting from two strains of knockout (KO) mice, we have generated the Pon1/apolipoprotein E (apoE) double KO mouse, known to be susceptible to atherosclerosis. We are now crossing this double KO mouse with Pon1 KO mice that express human PON1 (transgenic human PON1 mice, tgHuPON1). We collect ear punches for DNA extraction and blood from the saphenous vein for activity assays. We use polymerase chain reaction (PCR) genotyping methodology, which amplifies DNA, to identify mice that are KO for mouse Pon1 and apoE and that express human PON1. We run enzymatic assays to determine activity levels of the human PON1 in mice. Once we have created the tgHuPON1/apoE KO mouse, we will feed mice with an atherogenic diet, which will induce subclinical atherosclerosis. This knowledge will contribute to understanding the relationship between PON1 and CVD, and will likely generate a useful biomarker for risk of disease.      

For many high schoolers having a mentor can significantly alter their life course. For marginalized youth, research has shown that quality mentoring increases the likelihood for high school graduation, addresses socio-emotional needs, and improves mentee’s behavioral skills. Mentoring is one of many interventions schools might use to address students with multiple risk factors such as poverty, housing instability, food insecurity, and discrimination. These many risk factors, in turn, lead to a disproportionately negative high school experience and higher rates of socio-emotional and behavioral issues than students with fewer risk factors. However, increasingly schools have turned to more punitive carceral approaches to manage these negative behaviors by increasing the presence of police officers, security guards, or school resource officers into public school settings. One Portland program works to address this issue by implementing a community care based mentoring program. This study seeks to understand the experiences of both policing and community care mentoring for freshman and sophomore students at three urban public high schools. This study will take on a mixed-methods approach. A survey will be composed to collect quantitative self-report data, as well as semi-structured interview guide to collect qualitative data in the form of open ended questions. The findings from this study will inform the power of community care in high school in relation to mentoring students of color vulnerable to the school-to-prison pipeline, which can be used to inform the argument to remove police from schools and for the continuation of mentoring programs and the allocation of increased federal funding to provide further support for this program and ones similar.

With the increased development of space programs globally, more space probes are being funded for sample-return missions. Versatility and power limitations are some of the critical issues that these extraterrestrial sampling rovers are seeking to overcome in order to reliably deploy autonomously. Exploring the rough and unpredictable surfaces of other celestial bodies requires more adaptable and energy efficient robotics, which is what makes bio-inspired origami structures so appealing. Leaf-like origami is reconfigurable, which means that it can walk, jump, grasp, and actuate other useful motions all within the same device. Typical robots have redundant actuators and structural systems, but origami devices can build up potential energy before converting it into a mechanical motion. Due to this, rigid origami design approaches have the potential to be more compact, versatile, and energy efficient than conventional devices. Studying reconfigurable origami-based robotics can lead to devices that can be transformed into multiple configurations for various tasks. To design a versatile origami-based structure, I followed up on the research that Professor Jinkyu Yang and the members of his research group had on leaf-like origami, specifically leaf-out origami. This origami structure shows multi-transformable features. This means that the structure can be configured in a stable-stored or stable-deployed shape without having an external power supply maintaining its configuration. By finalizing the design, fabrication method, and discovering the optimized folding patterns for jumping and grasping motions, we will be able to start implementing the structure into more complex systems. Designing more structurally efficient systems may be the most practical solution we currently have to combat power and versatility limitations for autonomous space probes.

Albinism is a genetic mutation that strips those affected of the pigment in their skin, hair, and eyes. This alters the appearance of the affected and at times, subjects them to ostracism when interacting with their ethnic group(s). Albinism affects less than 1% of the human population and it is for this reason that understanding their attitudes and opinions on their racial-ethnic identity is important. The goal of this study is not only to investigate participants’ attitudes on and experiences with ethnicity and race, but to spread awareness of participants’ experiences. Participants attitudes and experiences were assessed using a survey and interviews which elaborated on the survey responses. Data for this study are in the preliminary stages of analysis. However, once completed this study they will prove to be one of the few psychological studies on Albinism. This study’s findings also aim to provide information to families of those with Albinism to better care for individuals with this mutation.

In this research project, a method was developed to quantify Gordonia and Microthrix parvicella filamentous bacteria in primary, secondary and digester sludge from three different wastewater treatment facilities in King County. The goal of developing this method was to determine the concentrations of filamentous bacteria for the purpose of relating those values to the foaming process in anaerobic digesters. The method utilized gram staining to identify Gordonia and M. parvicella by microscopy. After visual identification, the method required counting of the filamentous bacteria. These counts were then related to the measurements of biomass to calculate the overall concentrations of Gordonia and Microthrix parvicella in sludge samples. The method was validated by quantitative polymerase chain reaction (qPCR), which used DNA to quantify the filamentous bacteria. This method was successful in determining the concentration of these filamentous bacteria in sludge samples manipulated in the lab. However, it did not succeed with sludge samples directly from wastewater treatment plants. To ensure this method determines the concentration of Gordonia and Microthrix parvicella in samples directly from treatment plants, suggestions for future improvement of the method was made. Suggested improvements include different drying methods, different dilutions or additional preparation to homogenize sludge samples.
 

The identification and characterization of natural and artificial protein-protein interactions are fundamental to science and engineering. Although many technologies have been established to expedite research in this area, all can hardly meet the need to analyze vastly diverse protein-interactome networks, dynamics, evolution, and druggability. To develop a high-throughput quantitative interaction profiling technology, multimeric protein interaction sequencing (mPI-seq), we performed a 20×20-plex binding assay in a single aqueous solution using a set of de novo designed heterodimeric pairs. First, we barcoded the proteins with DNAs by ribosome and mRNA display. Then, barcoded proteins were assayed en masse in aqueous solution and immobilized into an ultrathin polyacrylamide gel layer attached to glass surface for in situ sequencing. DNA barcodes were amplified into discrete DNA clusters (polymerase colonies or polonies) and then sequenced using sequencing by synthesis. Finally, the protein-protein interactions were measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Through this project, we aim to establish a robust quantitative method to measure protein binding affinity massively in parallel and to use the large-scale functional data set to guide computational protein design.

Chemically induced dimerization (CID), in which two proteins dimerize only in the presence of a small molecule, has been widely used to control cell signaling, regulatory, and metabolic pathways, and used as logic gates for biological computation in living mammalian cells. However, few naturally occuring CID systems and their derivatives are currently available. Creating a CID system with desired affinity and specificity for any given small molecule remains an unsolved problem for computational design and other protein engineering approaches. To address this challenge, we have used a novel strategy to select CID binders from a vastly diverse combinatorial nanobody library. We have created new CID systems that can sense cholecalciferol and artemisinin. We are validating CID biosensors by a yeast three-hybrid system and built structural models to understand the small molecule-induced dimerization. Our work is a proof-of-concept that can be generalized to create CID systems for many applications.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor composed of cells of endothelial origin. KSHV, while possessing both lytic and latent replication programs, predominantly exists in the latent form during infection. While KSHV infects both blood (BECs) and lymphatic (LECs) endothelial cells, LECs are more susceptible to infection and express fewer antiviral genes during infection compared to BECs. Recent experiments have shown that LECs, but not BECs, have a defect in STING, a critical signaling protein that is activated during herpesvirus infections and results in the production of antiviral signaling molecules such as IFN-β. It remains unknown whether the defect in STING plays a direct role in increasing susceptibility to KSHV infection and if the defect impacts the ability of STING to suppress lytic reactivation in LECs. Accordingly, I propose to construct a constitutively active (CA)-STING and express it in LECs. Because CA-STING results in the continuous induction of IFN-β, I hypothesize that CA-STING-LECS will show decreased susceptibility to the establishment of latency by KSHV and have increased ability to suppress lytic reactivation compared to empty vector-expressing (EV)-LECs. First, I will infect EV-LECs and CA-STING-LECs with KSHV and measure the infection rates 48 hours post infection (hpi). I expect the number of infected cells in the CA-STING-LECs to be decreased relative to the EV-LECs. Next, I will infect EV-LECs and CA-STING-LECs with KSHV, and at 48 hpi, I will induce lytic reactivation in the two cell types and quantify the virus produced. If CA-STING suppresses lytic reactivation in LECs, I expect less virion production from CA-STING-LECs than from EV-LECs. The results from these experiments will further elucidate how KSHV exploits defects in innate-immunity to infect and transform host cells.

Becoming a mother can be an incredibly beautiful process, but it can also become overwhelming and extremely stressful, especially when lacking support and resources. The chronic and significant stress that low-income, pregnant women experience put them at greater risk for adverse health outcomes and their infant at risk for poor developmental outcomes. At the University of Washington’s Center for Child and Family Well-Being, our research team has developed mindfulness-based interventions to help new moms adjust to their new life. Our goal is to examine the effects of stress on the mother-infant dyad and to evaluate whether these programs are beneficial for moms and their babies. Expecting mothers are randomly assigned to attend one mindfulness-based program that focuses on preparing for childbirth, reducing postpartum stress, or developing parenting skills. Administration of extensive questionnaires to the mother and recordings of the mother and baby completing various tasks, before and after the program, measure the pair’s socioemotional development. We also collect cortisol, a stress hormone, and measure heart rate and breathing during a stress-reactivity paradigm to inform us of the physiological effects of stress. We hypothesize that mothers who exhibit high levels of mindfulness are more likely to engage in consistent, warm, and responsive parenting skills. As a result, these mothers' infants will display better self-regulation and focus. We also predict that mothers and babies who participate in these mindfulness interventions are more likely to show a decrease in cortisol production and have controlled cardiovascular reactivity. Recommendations for future research include establishing a systematic way of identifying mothers at risk so that we may prevent further harms caused by stress. Implications of the findings can be used to advocate for equitable, accessible mental health programs and implementation of public health policy centered around protecting and empowering vulnerable women and children in our community.

2D cell models have traditionally been used in labs to test the effects of new drugs on certain cell types due to the ease and convenience of use. While 2D methods are great, they often simplify the cell-to-cell interactions and may not accurately represent cell systems in humans. 3D methods show the complex cell communication systems and better simulate actual organ systems. Research comparing these two methods can inform scientists on the benefits of 3D models which can help efficiency in creating new drugs. Our lab looked into various 3D models to determine their effectiveness and reliability and looked into the differences in perceived cell mechanics and functionality between 2D and 3D methods. We tried Corning Matrigel and Corning 3D Spheroid microplates for 3D cell modeling using HEK293 cells, which are human embryonic kidney cells that were grown in lab. They are known for being easy to grow and transfect. We used SNAP-Gels, which are protein assays that show the protein levels in the cells, to ensure that the protein levels were similar between the 2D and 3D systems. We then did florescent imaging to determine cell localization and EPIC dynamic mass redistribution (DMR) to determine cell functionality. We found Matrigel to have inconsistent results, so we focused on using the spheroid microplates. Based on our initial results, we saw increased functionality and expression levels for full-length protein cells compared to cells with a truncated N-terminal protein in the 3D method. This increase in functionality and expression levels was not seen in the 2D method. Our results show that 3D modeling methods can be reliable, and do show results that differ from 2D models. This is important for future studies that require cell modeling because 3D models can provide a more accurate and reliable modeling system to create novel therapeutics.

Many psychiatric disorders, including substance abuse, have been linked to risky decision-making, but the mechanisms underlying these pathologies remain unclear. Cortical intratelencephalic and pyramidal tract (PT) neurons have distinct projections, morphology, and firing properties, but their role in behavioral regulation remains unknown. PT neurons have been identified in the orbitofrontal cortex (OFC), which is known to be heavily involved in the cognitive process of decision making. Based on previous studies in our lab demonstrating that inactivation of PT neurons increases reward preference, we hypothesized that PT neuron inhibition would increase risky decision-making. To test this hypothesis, rats were trained on a risky-decision task (RDT) in which they were trained to lever press for a food pellet reward. In order to target PT neurons in the OFC, CAV2-CRE virus was bilaterally injected into the pontine reticular nucleus (PnC), and an inhibitory Designer Receptor Exclusively-Activated by Designer Drugs (DREADD; DIO-hM4Di) was injected bilaterally into the OFC; this strategy allows selective expression of DREADDs in PT neurons. In the RDT, two options were presented: a “risky” choice associated with descending probability of administration (100%, 50%, 25%, and 12.5%) for delivery of four food pellets, or a “safe” lever that always delivers one food pellet. To evaluate response flexibility, rats underwent a reversal task whereby five consecutive responses on the active lever switched the “active” lever to the previously inactive one. In both tasks, rats underwent two sessions: in the test session, animals received an injection clozapine-N-oxide (CNO) to activate DREADDs, and in the control session, animals received an injection of vehicle (DMSO). Interestingly, we found that inhibiting PT neurons did not significantly alter decision-making or reversal-learning. Future studies will monitor activity of PT neurons using in vivo calcium imaging to determine the contribution of this cell population to decision-making tasks.

G-protein coupled receptors (GPCRs) - characterized by seven transmembrane alpha helical domains - are the largest family of membrane proteins, constituting ~1% of the human genome. The α1D-adrenergic receptor (A1DAR) is a GPCR that regulates function of the cardiovascular, urinary, and central nervous systems. Dysfunction of this receptor can lead to various diseases including schizophrenia, benign prostate hypertrophy, hypertension, and PTSD. Prazosin, a non-specific α1-antagonist is the first line treatment for PTSD, however, chronic use has deleterious side effects including orthostatic hypotension and potentially fatal reflex tachycardia due to interactions with off-target related receptors. Thus, understanding how A1DARs are regulated will allow for the development of targeted therapeutics. To this end, the Hague Lab has previously discovered that A1DAR undergoes an endogenous cleavage of its extracellular N-terminal domain, affecting its membrane localization and response to agonist stimulation. Located within the N-terminal domain of A1DAR are two glycosylation sites at amino acids 65 and 82. Currently, how glycosylation of these sites regulates the cleavage event remains unknown. To characterize this phenomena, I used molecular cloning to mutate the glycosylation sites of A1DAR in the pSNAP vector for expression in Human Embryonic Kidney 293 (HEK293) cells. Near Infrared PAGE analysis revealed that glycosylation of both amino acids is required for cleavage and proper expression of A1DAR. Sucrose density gradient and dynamic mass redistribution further showed that glycosylation controls function and trafficking of A1DAR to the membrane. These results allow for the development of targeted medications specific to the N-terminal glycosylation sites of A1DAR, further reducing the potential side effects experienced by patients.

A national organization named 2-1-1 provides assistance to populations with basic needs, mainly informing and answering questions, and researching resources covering 50 states. Not only is the demand for information growing, but also the complexity of calls is increasing. For example, a person may need information on shelters, medical issues, legal assistance, debt management and other services. Consequently, calls are taking longer, and with limited resources, wait times are increasing and even critical calls are sometimes abandoned. Assigning more staff to a call center is not realistic under limited budgets. Hence, an efficient call center system must be designed. Our research seeks to address the efficiency of the WA 2-1-1 call center system, in Washington State. We hypothesize that the application of call prioritization and skill-based routing can reduce the holding-call time, and eliminate call abandonment for those with urgent needs. We will identify classifications of calls, and evaluate how automation can be used to navigate or direct callers to a correct resource and expedite the whole process. Appropriate classification of calls and prioritization may not only decrease the waiting time but also help training operators with an appropriate skill set. We will apply the concept of operations research to forecast seasonal demand using available data within Washington State, and will estimate the number and allocation of resources the organization should provide. The anticipated result of the research is a call center design that will decrease the number of abandoned calls, provide an immediate response to an urgent call, decrease waiting time, and assign calls to available operators to exploit the limited resources. The WA 2-1-1 call center is important to provide assistance to efficiently serve populations in need. Increasing the efficiency of call centers with limited resources will enable WA 2-1-1 to reach more people in need.

Indoleacetic acid(IAA) is a common and well understood auxin class phytohormone that promotes plant growth by increasing cell division and elongation. IAA has also been shown to increase infectious adhesion and filamentation in certain strains of Saccharomyces cerevisiae. Increased IAA production by the plant’s microbiome has been demonstrated to stimulate host and symbiont growth. The IAA production in multiple strains of S. cerevisiae from the USDA ARS library was quantified using the Salkowski colorimetric technique, then the highest IAA producing strains were treated onto corn to examine the effect on biomass growth. Preliminary results have shown greatly increased root mass and moderately increased shoot mass in treated corn. This symbiotic yeast treatment could have agricultural applications, increasing crop yields without increased application of fertilizer, pesticides, or other products that could have a negative ecologial impact or detrimental effects on the crop.

G protein-coupled receptors (GPCRs) contain seven transmembrane domains and are the largest family of cell surface receptors, making up ~1% of the human genome. GPCRs can interact with a variety of ligands, such as odors, pheromones, hormones, and neurotransmitters. At least 30 human GPCRs contain a C-terminal Type-I PDZ ligand that allows for interactions with adapter proteins which can regulate receptor trafficking, stability, and signaling. The Hague Lab has previously found that the α1D-adrenergic receptor, which contains a C-terminal Type-I PDZ ligand, also undergoes an endogenous N-terminal cleavage event, which enhances receptor function and may play a role in which PDZ domain containing proteins interact with this receptor. We propose that this unique observation of the α1D-adrenergic receptor may be prototypical of a new class of GPCRs which contain a Type-I PDZ ligand and undergo an N-terminal cleavage. CysLT2, MAS1, and NPFFR2 are understudied GPCRs and potential members of this family with distinct PDZ ligands, though it remains unknown if their extracellular N-terminal domains regulates receptor function. Thus, I have cloned wildtype and N-terminal truncation mutants of these three GPCRs into the pSNAP vector to create fusion proteins with N-terminal SNAP tags. These constructs were transfected into HEK293 cells and subjected to near infrared PAGE analysis to elucidate the presence of N-terminal processing. Furthermore, dynamic mass redistribution revealed how the N-termini modulate receptor signaling. The combination of biochemical and pharmacological techniques allows me to determine if these receptors belong to this new subfamily of GPCRs. These results increase our understanding of how GPCRs are regulated within the cell, which can lead to the development of more efficient drugs.

As of February 2019, 3,916 extrasolar planets (planets orbiting another star) have been confirmed. A limited number of these planets have published spectral data of their atmospheres. This data comes from transmission spectroscopy taken during the transit of the extrasolar planet: as the exoplanet passes between the earth and its host star, light from the host star goes through the exoplanets’ atmosphere, which shows different absorption patterns based on its composition. The goal of this research is to gather published transmission spectroscopy data on atmospheric compositions of extrasolar planets, and to look for possible trends. In particular, we are searching for trends in relation to the different physical properties of the extrasolar planets: radius, mass, orbital period, temperature, etc. As a first step, our research has focused on hot Jupiters with periods from one to three Earth days, and radii between one and two Jupiter radii. From the 78 planets that fit in this parameter space, seventeen had published spectral data, and out of those only eight had spectra that covered an overlapping large wavelength region (480 to 900 nanometers). Out of these planets, unfortunately only one showed absorption in their atmospheric data, which did not allow us to look for any potential trends. We will present the results of an expansion of this work to a larger parameter space of radius versus orbital period to search for trends between the absorption characteristics of the extrasolar planet’s atmosphere and their physical properties.

The growing use of opioids in the United States has become a dangerous epidemic. The people most susceptible to overdosing on opioids are those who relapse after extended drug abstinence, whether forced or by choice. This study investigates the effects of heroin addiction on KCNQ channels in the basal ganglia, which plays an integral role in addiction.  More specifically, our research is focused on relapse after a prolonged period of no drug use. If the population of KCNQ channels are affected by heroin use and addiction, then these channels are a possible site for treatment to target. The drug retigabine, a KCNQ channel opener, has shown promise in reducing self-administration after abstinence by affecting dopamine levels in the striatum, a neurological center often involved in addiction. Given that dopamine release is critical in establishing and affecting addiction, retigabine is a likely candidate to diminish relapse behaviors and, therefore, reduce the occurrence of relapse-related overdoses. This study measures the effect of a global administration of retigabine. Catheters were surgically into the animals’ jugular vein to allow for self-administration of heroin with limited researcher interference. Over the course of three weeks, we ran our animals inoperant boxes for periods of self-administration following various drug availability periods and ending with forced abstinence. After two weeks of forced abstinence, the animals with high and low responding on self-administration were separated into three different treatment groups: Retigabine, XE991 — a  drug that has the opposite effects of retigabine — and a control solution (all administered intraperitoneally). Our animals then underwent one hour of continuous access self-administration where they could relapse. Behaviors such as drug intake, drug seeking, drug taking, and motivation to administer were measured across all six groups.The results from this study will provide evidence on the efficacy of retigabine in reducing relapse behavior after abstinence.

Risk taking is strongly associated with many disordered behaviors. Many studies implicate the orbitofrontal cortex (OFC) in such behaviors, though its modulation yields inconsistent results. This could be due to possible contrasting effects of intratelencephalic (IT) and pyramidal tract (PT) neurons, which have distinct morphologies and projections. Despite this, specific functionality differences are unknown. Given that 5-HT2A and D1 receptors have predominant expression in IT neurons over PT neurons, and antagonism of these receptors decreases risk-seeking, it was hypothesized that inhibition of all IT neuron activity in the OFC would also result in risk aversion. IT neurons in rats were inhibited during a risky-decision making task (RDT). To modulate IT neurons, an inhibitory designer receptor exclusively activated by designer drugs (DREADD) was injected into lateral OFC bilaterally, conjugated to CRE and FLP drivers injected into contralateral nucleus accumbens. In the full RDT, animals were presented with two levers: The “safe” lever delivered one pellet reward per press, while the “risky” lever delivered four pellets, with descending probability of reward administration (100%, 50%, 25%, 12.5%). Two test sessions were conducted whereby animals received intraperitoneal injections of clozapine-N-oxide (CNO) or a vehicle (DMSO) 30 minutes prior to task onset. Animals receiving CNO the first time received vehicle the second time. To determine if IT neurons were involved in perseverative responding, animals underwent a reversal task. They were presented with an active lever -which administered a food pellet per press -, and an inactive lever. Five consecutive responses on the active lever switched the inactive lever to the “active” lever. Results showed no significant changes in decision-making or reversal learning upon receiving CNO or vehicle. IT neuron modulation may need to be more temporally specific; future studies will focus on monitoring IT activity during decision-making paradigms to further elucidate their contributions within OFC.

All-trans-retinoic acid (RA) serves an important role in maintaining tissue health, either deficient or excessive levels can lead to health issues. Aldehyde dehydrogenase 1A1 (ALDH1A1), is generally believed to be the main enzyme responsible for the conversion of retinaldehyde (RAL) to RA in the liver, requiring the cofactor nicotinamide adenine dinucleotide (NAD+). However, previous studies indicated that after WIN18,446, a potent inhibitor of ALDH1A enzymes, was administered to mice; liver RA concentrations were not significantly altered, and in vitro the RA formation in mouse liver was only inhibited by about 50% suggesting other enzymes except ALDH1A1 synthesis RA in mouse liver. Mouse aldehyde oxidase has previously been proposed to synthesize RA. Hence, in the current study we tested the hypothesis that aldehyde oxidase (AOX) also contributes to the formation of RA in human liver. Our data shows that purified human recombinant AOX catalyzes the oxidation of RAL to RA. The K_m (indicating substrate binding affinity to the enzyme) and k_cat (indicating maximum enzyme velocity) values were determined by enzyme kinetic assays as 1.4 μM and 3.5 min-1. In the absence of NAD+(AOX mediated activity), RA formation was observed in human liver S9 fractions (HLS9) and the RA formation rate was, on average, 60% lower than that measured in the presence of NAD+(n=4). In addition, hydralazine, a selective AOX inhibitor, inhibited about 55% of RA formation in HLS9 in the presence of NAD+ while combining hydralazine and WIN18,446, more than 85% of RA formation in HLS9 was inhibited when compared to the control. In conclusion, this data shows that AOX and ALDH1A1 each contribute about 50% of RA biosynthesis in human liver. This research helps us better understand the regulation of retinoid homeostasis in humans.