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.

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.

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.

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.

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.

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.

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. 

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.

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.

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.

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.

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.

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. 

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.

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.

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.

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. 

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.
 

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.

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.

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.

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.

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. 

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

 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.

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.

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. 

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.

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.

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.

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.

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.

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.

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.

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.

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. 

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.

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.

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. 

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.

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.

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.

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.

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.

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.

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. 

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.

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.

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.

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.

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.

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. 

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.
 

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. 

 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.