Gastric dilatation volvulus (GDV) is a life-threatening condition that occurs in large breed dogs. Previous research investigating three candidate genes of major histocompatibility complex (MHC), DLA88, DRB1 and the innate immunity gene, TLR5 found diagnostic markers that could be used to identify Great Danes at risk for GDV. The Endoplasmic Reticulum Aminopeptidase 1 gene (ERAP1), is now known to be an important up-stream component of the antigen-presentation system of the MHC, and human polymorphisms in this gene have been linked to the same inflammatory disorders. I hypothesize that Great Danes with certain ERAP1 single nucleotide polymorphism (SNP) variants on exons 3,7,12,13,16 associate with the risk for GDV in Great Danes. We conduct the experiment on two groups of Great Danes; eighty dogs are recruited for this study. Half comprise a GDV group consisting of dogs that have undergone emergency surgery for GDV, and a second age-matched gender-matched control group is dogs with no history of gastric problems. We perform polymerase chain reaction (PCR) and sequencing on subjects from both groups in the variable regions of the ERAP1, evaluating alleles from the gene for association with GDV while controlling for dog family, ages, and diet intake. The goal of this work is to establish a list of specific “risk”or “protective" alleles of ERAP1 that are associated with GDV in Great Danes. If the dominant genotype that predisposes dogs to GDV can be determined, this canine GDV model marker will provide a means to detect at-risk animals, especially in Great Danes. Early genetic testing, probiotic, drug-based approaches and gastropexy (a surgery to prevent GDV) could reduce the incidence of GDV.

Multiple studies have demonstrated an association between a long-term exposure to air pollutants and health outcomes in area of cardiovascular and respiratory diseases. However, there is suggestive, but insufficient, evidence linking the association between long-term exposure to air pollution and mental health outcomes, such as depression. In South Korea, a nationwide population-based, cross-sectional study investigated the relationship between long-term exposure to ambient air pollutants and mental health status. The study of 124,205 participants found a positive associations between Particulate Matter (PM10 and PM2.5 ), Nitrogen Dioxide (NO2), and Carbon Monoxide (CO) exposures and mental health status. The goal of this study is to determine whether the air pollution-depression relationship holds true in the ACT (Adult Changes in Thought) cohort by conducting a similar study to the cross-sectional study conducted in South Korea. In this study, I will consider air pollutants (PM2.5 and PM10) as exposure and depression as a measure of health outcome. The central working hypothesis of this study is that higher levels of PM2.5 and PM10 will be associated with an increase in CES-D (Center for Epidemiologic Studies Depression) score. Participants from the ACT cohort (members of the Kaiser Permanente in Seattle, Washington) who had an ACT study visit between 11/1/2000-4/1/2003 will be the study population. The primary analysis aims to evaluate the association between PM (PM2.5 and PM10) and CES-D scores using linear regression model after adjusting for age, sex, smoking, income, education, cardiovascular disease, and cerebrovascular disease. The secondary analysis aims to evaluate the effect modification of age, alcohol history and early vs. late onset of depression has on this association. It is hoped that the results from this study will expand our understanding of the relationship between air pollution and depression.

Photoresist is a viscous light sensitive organic solution which is used to coat the surface of silicon substrates in nanofabrication processes. An essential step in nanofabrication is photolithography. During this procedure, photoresist is deposited in a uniform, thin film over the wafer surface using a tool called the Spin Coater.  The purpose of our experiment is to develop spin curves that will examine the correlation between the different speeds or RPM value that the wafer is spun at and the uniformity of the photoresist coated wafer. The thickness and uniformity are critical to determine the correct UV exposure dose and plasma etch times. We will analyze the results for both positive and negative photoresist. In the case of a positive photoresist, the regions that are exposed to light are dissolved in developer. On the other hand, with negative photoresist, regions that were not exposed to light are dissolved. After the wafers are spun, the uniformities will be measured by an automated thickness mapping system called Filmetrics. Filmetrics is a machine that allows for the surface to be measured at the nano-meter level. The spin curves experiment will allow researchers to obtain wafers using these photoresists to achieve photolithography with their desired thickness.

Blood transfusion is a cornerstone of modern medicine, with a transfusion performed every 2 seconds in the United States. It is critical to accurately determine both patient and donor blood type prior to transfusion, as mixing non-complementary blood types can trigger life threatening reactions. While the ABO antigen system was first described over a century ago by Nobel Laureate Karl Landsteiner, to this day safe transfusion remains burdened by the nuance of blood type. Many of the current blood typing tests over simplify classification and often disregard ABO subgroups, despite widespread recognition of their significance. Moreover, our understanding of the ABO blood types (A, B, AB, and O) is still incomplete, as the full structure and microheterogeneity of these ubiquitous blood group antigens is not yet fully described. This project characterizes the biochemistry of ABO blood types through an interdisciplinary collaboration between UW Bioengineering, Medicinal Chemistry, and Bloodworks Northwest (the regional blood center). Our study employs exhaustive isolation of red blood cell (RBC) membranes from genotyped donors for comprehensive biochemical and biophysical analysis. The RBC membranes are treated with cocktails of enzymes - namely PNGase F, EGCase and Neuraminidase - to cleave glycan structures at specific locations. Reactivity to different antibodies and lectins provides insight into the structure of the glycan antigen. Results have shown that the clinical anti-A antibody binds disproportionally to N-linked associated antigens. These findings inform ongoing mass spectrometric and biosensing work to further elaborate ABO structure and bioactivity, with implications for transfusion and transplant medicine.

Physical resilience is a measure of healthy aging, but current assessment methods are underdeveloped, relying on a frailty panel of subjective tests. A quantitative, reliable test for resilience using endpoints aligning with healthy aging is needed to provide a standardized measurement of resistance to age-associated decline. The chemotherapeutic drug cyclophosphamide (CYP) is an immunosuppressant used in clinical medicine for certain types of cancer and autoimmune diseases. Precursor neutrophils are especially sensitive to this chemical stressor, such that a single low-level dose stops cell cycling with a decrease in mature neutrophils, but neutrophils rebound as the drug is eliminated from the body. This study sought to characterize the neutrophil rebound response in aging mice as a biological sensor for how an organism might respond to physical stress with increasing age. One intraperitoneal dose of CYP at 100 mg/kg was given to groups of mice at various ages. White blood cell differential and total counts were enumerated to monitor cellular response over a 25-day period. A robust and predictable drop in neutrophils occurred across age groups five days after injection of CYP, followed by a robust increase in neutrophils beyond baseline measurements at day seven in an age-dependent manner. Strength of the neutrophil rebound had a moderate correlation with strength and stamina as measured by rod hanging time, a test that corresponds to hand grip assessment in humans as an indication of frailty. This observation suggests that the neutrophil rebound response to the chemical stressor CYP in young adult mice can predict resilience to aging in older adult mice. The clinical implications are that an in vitro bench test could be developed using CYP-induced rebound response of human precursor neutrophils to predict resistance to frailty and produce a more accurate assessment of resilient healthy aging in older adults.

Aging is characterized by the loss of physiological and cellular functionality, however the mechanisms that underlie this deterioration are still unclear. Emerging evidence indicates that aging is associated with increased cell-to-cell variation in gene expression within tissues: homologous cells within tissues start expressing the same gene at varying levels. The causes of this age-related variation of gene expression are not known. Here, we aim to investigate this age-related dissimilarity in gene expression using C. elegans as a model system. We hypothesize that increase of gene expression variation is an early event during aging that may therefore underlie subsequent deterioration of tissues functionality. By characterizing aging in C. elegans, we hope to provide further insight into the molecular characteristics of aging in humans, and possible points of intervention. Previously, we have found that young C. elegans animals exhibit nearly identical stoichiometry of independent genes expression with very little difference between individual animals of the same genetic background. Our initial results support the idea of increased cell-to-cell and animal-to-animal variation of gene expression with age in C. elegans. Here, I use quantitative microscopy to measure animal-to-animal and cell-to-cell variation of genes expression in middle aged C. elegans using fluorescently-tagged proteins and quantitative microscopy. In addition, I use the methodology of molecular cloning and transgenics developed by the Mendenhall lab to create new transgenic strains of C. elegans. These transgenic strains will be also be used for quantitative microscopic analysis. By examining existing strains and developing new ones, I will determine if increase of gene expression variation represents early event in the aging of C. elegans.

Recent studies have shown just how important microbiomes are for individual health, population health, and environmental health. Unfortunately, these studies are often limited by the costs of meta-Genomics. Furthermore, meta-Genomic data itself is limiting by only providing information on population characteristics, but not the functional contributions of members within the population. Single cell transcriptomic sequencing aims to lessen the latter issue by providing information on the gene expression of each individual cell within a sample. Even so, current single cell sequencing technologies are costly and require specialized equipment. SPLiT-seq is a single cell transcriptomic technology developed by the Seelig lab at the University of Washington that uses split-pool ligation to create uniquely barcoded cDNA for each cell using every-day laboratory bench tools and techniques and costs only one cent per cell. Currently, SPLiT-seq is well-optimized for mammalian cells. However, using this method on bacteria requires its own set of optimized procedures given the morphological and biochemical differences between eukaryotes and prokaryotes. The aims of this project are to deal with these biological differences to increase the information obtained from messenger RNA and decrease the amount received from ribosomal RNA, as well as reduce the amount of cells that get the same cDNA barcodes. By optimizing this single-cell transcriptomic technique for bacteria, future studies involving microbial communities will be able to obtain more robust information on the individuals within those populations.

Herpes simplex virus (HSV) infects mucosal epithelial cells and can cause recurrent, painful ulcers. HSV is not curable and establishes lifelong latency in host neuronal ganglia. Herpes affects more than 400 million people globally, with an increased risk of genital ulcer disease, HIV acquisition, and transmission of HSV-2 to partners or neonates. Persons with altered T cell immunity have been reported to have prolonged and more frequent lesions, suggesting that the adaptive immune responses are associated with HSV clearance. Additionally, there is evidence of innate cell and T lymphocyte recruitment to the infection site to help clear the lesion. While there is extensive evidence on the role of T cells in these lesion infiltrates, little is known about B cell activation and antibody response during HSV reactivation. The study of B cells and antibodies can be used in developing a HSV vaccine since all licensed vaccines induce robust antibody responses. Antibodies block virus entry and mediate antibody-dependent cell-mediated cytotoxicity (ADCC). That is why a better understanding of local antibody responses against HSV-2 is needed to develop a successful vaccine. My project examines whether HSV antibody-producing B cells are found in areas where HSV-2 reactivates and the potential role that these antibody-producing cells play in host clearance of the virus. To examine this question, ddPCR using B-cell-specific probes were used to identify different B-cell subtypes responding to the HSV infection. Results to date have shown high variability with few emergent patterns. In the future, additional samples will be examined using new protocols for better cDNA synthesis, and more positive and negative controls to increase the accuracy of the data.

There are countless times in the past where people have attempted to expose systematic injustices committed by the U.S. government. Despite the fact that Hanford was a critical site in producing plutonium for the atomic bombs dropped during World War II, the contamination and subsequent health disparities remain shrouded in mystery and present dangers to future generations. In this project, we explore how the Hanford facility and the release of radioactive waste impacted the culture and daily activities related to diet, health, and faith of indigenous peoples in Washington State, as well as efforts by the government and native tribes that have been made in regards to cleaning Hanford and protecting tribal lands. By comparing interviews of Native Americans affected by the Manhattan Project, their oral histories, literature, public speeches, and using website discourse analysis of governmental sites, we made several important observations. Through our research, we found that among the devastating results of the Hanford contamination, the most common theme was the upheaval and marginalization of indigenous culture and the resulting inability of Natives to take part in community building activities such as wild game hunting, fishing, spiritual ceremonies, and trade with neighboring tribes. Not only did contamination tarnish relationships that local natives had with each other and the land, but there was and remains a strong correlation between subsequent cancer-related health disparities and bioaccumulation in natural resources. Nuclear radiation resulting from the Manhattan Project is not an issue of the past; its effects are far-reaching and will continue to mutate the physical and cultural conditions of the Native American tribes who lived in the area, relied on its resources, and were biologically affected by the processes involved at Hanford.

Theiler's murine encephalomyelitis virus (TMEV) is a positive-sense RNA Picornavirus used to model epilepsy, poliomyelitis and multiple sclerosis. Previous research has demonstrated a dependence of some Picornaviruses on host glutathione (GSH) in order to produce viable progeny. GSH depletion in host cells has resulted in unstable viral capsids, reducing viral fitness due to a lack of oxidative homeostasis. The objective of this study was to investigate the dependence of TMEV on GSH and determine the consequences of oxidative stress in the host cell due to the absence of GSH. Incubation of TMEV at denaturing temperatures with increasing doses of reduced GSH demonstrated that GSH stabilizes viral particles and 54% viral infectivity was retained with doses as low as 25mM GSH. Using L-Buthionine-sulfoximine (BSO) to deplete GSH in host cells, a 3-fold decrease in viral production was observed when compared with untreated cells. Interestingly, when GSH was restored in BSO treated cells using cell permeable GSH ethyl ester, TMEV viral production was partially restored and viral plaque formation was comparable in yield to an untreated viral infection. Virus production and viability were quantified using plaque assays on host cell monolayers. Next steps include monitoring viral genome production via RT-qPCR and quantifying GSH levels using fluorescence plate assays. RT-qPCR will be further utilized to examine other host stress pathways. This project will use TMEV to look at a host-virus relationship as it relates to an important host antioxidant system interacting with the capsid, assembly, and genome replication of the virus life cycle.

Food contamination outbreaks have become increasingly prevalent in the United States. Food processing companies often do not have sufficient resources to accurately monitor pathogenic bacteria and biofilms on their food preparation surfaces. Unfortunately, consumers assume that the food is safe because it is packaged and ‘screened’ but invisible bacterial contamination may be present. This creates an opportunity to improve methods for detecting pathogenic bacteria in food handling and processing environments. BioVizia provides an alternative solution for detecting pathogenic bacteria on food processing surfaces that is efficient yet effective. We have developed a device that uses an LED to excite a fluorescent dye. Fluorescein is an FDA approved dye that exhibits pH dependent spectral properties. When viable pathogenic bacteria metabolize sugar, they produce acidic byproducts. Therefore an optical system using fluorescein as a pH disclosing agent may provide sufficient sensitivity for detecting bacterial contamination. Our optical system records the fluorescein emission spectra which contains two overlapping, pH dependent spectral features. The fractional amount of the two contributing bands are calculated using a spectral unmixing algorithm. A calibration with standardized pH buffers yields a linear relationship between pH and the fractional amount of each band. This noncontact optical pH measurement method has been successfully employed in an in vivo dental study to measure the pH drop produced by dental plaque following a sucrose rinse. We seek to demonstrate that BioVizia can make the detection of pathogenic bacteria and biofilms on food preparation surfaces fast and quantitative.

The construction of the Hanford Nuclear Site was to aid the production of the atom bomb for the United States in World War II. This led to an era of secrecy, especially for the Hanford workers. A whistleblower is someone that brings about safety concerns about nuclear production to the public, but only with the risk of losing their job. Recent trends have shown that there are major consequences to whistleblowing, as many in the past who whistleblowed lost their jobs. However, whistleblowing is crucial to sustain the safety and health of many workers. Our purpose is to identify the motivations of these workers for either speaking out against the program or staying silent. Through analyzing interviews, literature and discourse, we hypothesize that whistleblowers at Hanford spoke out when they felt their moral obligations outweighed any monetary or social gain. Because the government heavily suppressed the voices of workers, we wish to better understand how society fosters environments where people are comfortable to speak out as opposed to promoting an environment of secrecy.

When bacteria experience high salinity environments, such as the brines of sea ice, they take up compatible solutes to protect against osmotic stress. Examples of compatible solutes include amino acids, betaines, and other organic molecules that accumulate in the cells, balancing the osmotic difference between cytosol and external environment without impacting intracellular functions. I am exploring the mutualism of sea-ice bacteria and diatoms (an algal source of compatible solutes for bacteria), and the effects of salinity shifts on this dynamic. I have determined specific growth rates for five strains of bacteria at different combinations of temperature (–3°C to 1°C) and salinity (17 – 55 ppt) that mimic sea-ice conditions. Four strains derive from a collection of Antarctic bacteria found growing mutualistically in diatom cultures: 1) strain Fc1, most closely related (by16S rRNA gene sequence analysis) to Marinobacter psychrophilus strain i20041; 2) Fc4, closest relative Pseudoalteromonas arctica strain A 37-1-2; 3) Nl1, closest relative Glaciecola pallidula strain DSM 14239; and 4) Tr1, closest relative Colwellia rossensis strain S51-W. The fifth was isolated from Arctic sediments but has since been found in sea ice: Colwelia psychrerythraea strain 34H These strains were tested in a defined medium, composed of glucose, vitamins, and a nitrogen source (GVaN), and a complex medium, Marine Broth 2216. Those subjected to higher salinities could be tested at subzero temperatures due to the lowering of freezing point by the salts. During the incubations optical density and cell counts were determined and used for calculations. After determining permissive growth conditions from calculated growth rates, strains will be selected for experiments using specific compatible solutes and/or diatom exudates.

With the discovery of radioactive materials, many institutions have conducted numerous radiation experiments in order to further understand the effects of radiation and radioactive contamination on human bodies and their environments. While doing so, there has been a great amount of unequal health disparities among the different communities involved. Institutions, like the University of Washington, while held in high regard, have participated in experiments that have affected the livelihood of many people. To this day, common knowledge of the University of Washington’s participation is still obscured. There are many institutions in the United States that are like UW and are known for their extensive and groundbreaking research; with the funding and power they hold, people often don’t question their research or their ethical approach to conducting it. From our research, we obtained a greater understanding of what occurred throughout the experiments - thus our goal is to bring attention to the actions of the University of Washington researchers’, C. Alvin Paulsen’s and Lauren R. Donaldson’s, unethical research practices in the Marshall Islands and UW Walla Walla Prison Radiation Experiments. Our methods include analyzation of interviews with Tom Carpenter - the Executive Director of Hanford Challenge, discourse analysis, reviews of relevant literature and interpretations of visual elements. Because the research of C. Alvin Paulsen and Lauren R. Donaldson is locked away in UW’s Special Collections Library, we were unable to analyze their research directly. We anticipate that this project will ensue more curiosity and lead to the general public learning more about the University of Washington’s participation in human radiation experiments.

The sensory mechanisms by which organisms orient towards potential food prior to initiating movement are well-researched. However, behavioral mechanisms in stimulus-poor environments which determine foraging through seemingly stochastic movement variability or random-walk models remain poorly understood. Existing literature describe analyses of a variety of single-organism emergent movement patterns but do not appear to offer comparative analysis between single- and multi-individual environments nor within degrees of resource availability. We introduce a new analysis of observed fluctuations in spontaneous movement by Caenorhabditis elegans exposed to diverse conditions of competition and resource abundance. We describe a sequence of experiments which quantify the movement patterns of C. elegans through video imaging pattern recognition codified by run-length-time and turn-angle-time in comparison to recursively updating algorithmic position estimation. We expect to observe movement patterned on optimized explore-exploit strategies—such as simple random walk or Lévy flight—with frequency of implementation influenced by both population and resource density.

In the last decade, a large amount of studies have been done using virtual environment (VE) because of the increased control over the sensory stimuli used and the detailed observation of the associated behavioral and sometimes neuronal responses. In this study, we placed honeybee in a VE and exposed them to visual stimulus in closed-loop. We showed that honeybees are able to fixate the stimulus regardless of the level of feedback fed to the sensors(e.g., the gain between the animal motion and the stimulus motion on the VE). Our next step is to investigate how different neuromodulators such as the octopamine and dopamine, may be important for fixation and adaptation to the different level of sensor feedback. Octopamine (OA) is key in modulates physiological process in invertebrates including honeybees. Recent studies have identified OA neurons that are critical for visual behaviors and that increase their activity during active behavioral state. To identify the effects on octopamine and dopamine (DA) on honeybee’s ability to fixate, we are injecting bees with OA, DA or their respective antagonists in a visual processing brain area. Potential results of this study will impact the fields of neuroethology, providing insights on the modularity of neural processes and the feedback between sensory and motor pathways.

Gut Microbiome is increasingly recognized as a pivotal player in toxicological responses, thus dysbiosis or microbial imbalance may worsen chemical-induced adverse outcomes such as inflammation, metabolic syndrome, and cancer. Early life exposure to environmental contaminants may produce long term toxicities in adulthood, and little is known to what extent early life exposure to environmental contaminants modulate the gut microbiome beyond adulthood. Therefore, this study tested the effects of perinatal exposure to 3 human health relevant environmental contaminants (BDE-47, TBBPA, and BPS), on the composition and functions of the gut microbiome of perinatally exposed adult male mice. CD-1 mouse dams were orally exposed to vehicle (corn-oil, 10ml/kg), BDE-47 (0.2mg/kg), TBBPA (0.2mg/kg), and BPS (0.2mg/kg) once daily from gestational day 8 to the end of lactation (postnatal day 21). Feces from male pups were collected at 12-weeks of age (n=14-23/group). Microbial DNA was isolated, subjected to 16rDNA sequencing, and analyzed using QIIME. Microbial biomarkers for each chemical exposure were predicted using LefSe. Microbial functions and key taxa that drive functional changes were predicted using PICRUSt and FishTaco, respectively. None of the 3 chemicals markedly altered the overall richness of the gut microbiome in adult male pups. However, principle coordinate analysis showed a distinct separation among different exposure groups, and especially between BPS and vehicle exposure groups. A total of 73 taxa were persistently altered by at least 1 chemical exposure, among which 12 taxa were commonly regulated by all 3 chemicals. The most representative microbial biomarkers for each exposure condition were Clostridiales for vehicle, S24-7 for BDE-47, Rikenellaceae for TBBPA, and Lactobacillus for BPS. Together, these observations suggest early life exposure to these human health relevant environmental contaminants produce persistent gut dysbiosis in adult male offspring, leading to functional shifts that may play important roles in regulating certain diseases of the host.

Drosophila alpha-tubulin acetylase (dTat) is an enzyme that covalently modifies microtubules by addition of acetyl groups to the microtubule lumen. Prior studies demonstrated dTat is essential for mechanosensation in Drosophila. Mutation of alpha-tubulin lysine 40, which is covalently modified by dTat, similarly compromises mechanosensation, but our preliminary results suggest that alpha-tubulin 40 mutation and dTat mutation differentially affect some forms of somatosensation. Consistent with this observation, recent studies suggest that dTat may additionally regulate microtubule stability independent of its enzymatic activity. Whether this non-enzymatic function of dTat influences neuronal function remains to be determined. In my studies, I aim to determine the relative contributions of enzymatic and non-enzymatic functions of dTat to somatosensory function in Drosophila larvae. To this end, I have assayed requirements for dTat function in a variety of larval mechanosensory and thermal responses, which are governed by defined classes of somatosensory neurons. For each of these behaviors, I used transgenes to resupply dTat function to dTat mutant flies, comparing the ability of wild type and enzyme-dead versions of dTat to support neuronal function. Results from these studies provide an entry point to understanding enzyme-independent function of dTat.

For many years, concerted efforts to combat malaria through the use of antimalarial drugs, bed nets, and other public health measures led to marked reductions in morbidity and mortality. Unfortunately, progress has stalled. Reductions in malaria have leveled off and even reversed in certain areas (WHO, 2017). As of 2016, there were 216 million cases and 445,000 deaths annually due to Plasmodium infections (WHO, 2017). To regain momentum and accelerate malaria eradication efforts, an effective and durable vaccine is needed. The Murphy Laboratory focuses on developing novel pre-erythrocytic (PE) malaria vaccines that can effectively stop the Plasmodium sporozoite (spz) before the clinically symptomatic blood stage begins. Identification and inclusion of multiple different protective Plasmodium antigens is thought to be crucial to developing a broad immune response and durable protection against this intracellular parasite. To test and define protective antigens, the Laboratory developed an “Acute Challenge” (AC) model in order to sensitively measure T-cell responses that are completely or partially protective. In this model, DNA vaccines encoding Plasmodium yoelii proteins are delivered by gene gun to induce CD8+ T-cell responses in BALB/c mice. At the peak of the immune response, we challenge the mice with luciferase-expressing P. yoelli sporozoites and measure the parasite burden and protection using IVIS imaging. A known protective epitope derived from P. yoelii circumsporozoite protein (CSP) induces a potent and protective response in this system. My project is to utilize the AC model to assess P. yoelli candidate antigens, of unknown protective potential, that are putatively exported or secreted from the parasite-containing vacuole into the host cell cytoplasm. Confirmed protective antigens will then be assessed for their localization and defined T-cell epitopes. The results will be used to create vaccines designed to maximize such responses and target the responding T-cells to the liver.

Mouse adenoviruses (MAdVs) are non-enveloped double stranded DNA viruses. There are two different types of MAdVs with different tropisms or ability to infect particular cells or tissues. MAdV-1 infects macrophages and endothelial cells and causes encephalitis. MAdV-2 infects intestinal cells but causes no disease. Although, the MAdV fiber capsid proteins are important for attachment of the virus to host cells, it is not known if they are the major determinant of tissue tropism in the mouse. To address this question, I use recombination-mediated genetic engineering to make chimeric MAdVs, wherein I keep most of the genome of one strain but replace the fiber protein with that of the other strain. I then use transfection to introduce the DNA of the chimeric virus into a mouse cell line to allow the virus to replicate. I am currently designing and testing the proper chimeric fiber construct that will result in a replication-competent virus. Ultimately, I compare infection of the chimeric virus to that of the parent viruses in intestinal organoids, a tissue culture model that allows us to faithfully test tropism without the need for mouse studies. These experiments may reveal general principles of AdV tropism that will allow us to understand why different human AdVs cause disease in specific organs.

Mouse adenoviruses (MAdV), like human adenoviruses (HAdVs), have specific tissue tropisms. MAdV-1 infects macrophages and vascular endothelial cells, which can result in encephalitis, while MAdV-2 infects epithelial cells of the intestine but does not cause overt disease. The viral protein that determines MAdV tropism is unknown; however, for many viral families it is the viral attachment protein that is critical. For MAdVs, fiber is the viral attachment protein, and the receptors used by MAdV-1 and MAdV-2, although unknown, are distinct. To test whether MAdV receptor usage dictates tissue tropism, I constructed a MAdV-2 chimeric virus, replacing its fiber protein with that of MAdV-1 using a gene-editing recombination system. The chimera was used to infect a 3D culture model of the intestinal epithelium called “enteroids.” As expected, MAdV-1 does not replicate in enteroids and MAdV-2 does, consistent with their in vivo tropisms. Remarkably, the chimera replicated efficiently, indicating that the fiber protein is not the sole determinant of MAdV-2 intestinal tropism. Although fiber is not the main contributor to tropism, its interactions with host factors are still likely important for productive infection. A recent study identified N-acetylglucosamine (GlcNAc) as a specific ligand for MAdV-2 fiber. We have shown that GlcNAc is not the primary receptor for MAdV-2; however, binding to GlcNAc may aid in adhesion of MAdV-2 and penetration through the mouse intestinal mucus layer. To test this hypothesis, I mutated the GlcNAc interacting residues in MAdV-2 fiber to prevent GlcNAc binding. I am currently comparing the infectivity of this mutant virus to wild type MAdV-2 in both epithelial tumor cells and enteroids. Unlike tumor cell cultures, enteroids contain mucus-secreting goblet cells which will recreate the in vivo context more accurately. Together, these studies of MAdV may help us to understand why different HAdVs infect specific tissues.

The dynamic nature of chromatin, the complex of DNA and histones, is not fully understood and continues to be studied. During infection, viruses utilize host cell machinery, including chromatin, to promote viral proliferation. Adenovirus protein VII is a small histone-like core protein that tightly binds DNA and disrupts host chromatin in the infected cell . By studying protein VII, we aim to uncover how chromatin manipulation affects the health of the cell. To this end, our lab is using the model eukaryote, Saccharomyces cerevisiae or budding yeast, to understand the interaction between protein VII and chromatin. Yeast is an excellent model for studying chromatin due to the high conservation of histone proteins across all eukaryotes. By inducibly expressing protein VII in yeast, we tested how the expression of protein VII affects yeast growth and viability. Our preliminary findings show that protein VII expression is toxic to yeast—cell growth is slowed resulting in the formation of fewer and smaller colonies. This result suggests that protein VII perturbation of host chromatin is sufficient to inhibit growth in our model organism. Currently, we are generating yeast mutants and expressing different viral protein variants to understand the mechanism of these phenotypes . Through studying the chromatin perturbation caused by adenovirus protein VII, we will further our understanding of fundamental chromatin structure.

Crops are constantly exposed to pathogens and pests, but being sessile, they cannot physically move away from danger. In order to defend themselves, plants have mechanisms to recognize and respond to threats. One way that plants do this is through the Jasmonic acid pathway, which is specifically activated in response to the wounding of plant tissue. Surprisingly, there are many functionally similar Jasmonate-ZIM domain (JAZ) proteins involved in this response, and the reason for this redundancy is unknown. Studying this pathway in depth presents a challenge when using low-throughput methods. In order to better understand this complex system, we built a workflow, which will be integrated into an automated system using the Aquarium Laboratory Operating System, designed by the Klavins Lab. This will help us track data from high throughput experiments. By developing automated methods to track maintenance of cell cultures, production of protoplasts, and delivery of dCAS9 transcription regulators, we hope to produce sufficient data to better understand the JA-signaling pathway. We anticipate that these experiments will illustrate a relationship between the manipulated activity of JAZ gene regulators and subsequent immune response activity, and have potential implications in the agricultural and food industries, where pests and stress conditions are a significant concern.

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

During HIV infection, CD8+T-cells are crucial for the control of viral replication. Increased CD8+T-cell polyfunctionality, which is the ability for one cell to perform more than one function within the immune response, was associated with improved clinical outcomes, is now the focus of experimental curative therapies for HIV. One therapy that showed promise is a Conserved Elements DNA (CE) vaccine, which is designed to prime the immune system with essential regions of the virus, thereby eliciting polyfunctionality and circumventing escape mutants. A novel immunotherapeutic combinatorial approach was investigated for the ability to reduce or eliminate viral burden in Simian Human Immunodeficiency Virus (SHIV) infected macaques receiving antiretroviral drug therapy. We evaluated effects of this regimen on CD8+T-cell polyfunctionality and its correlation to viral control after analytical antiretroviral treatment interruption (ATI). Our combinatorial immunotherapy regimen uses the CE vaccine to target highly conserved viral sequences, latency reversal agent GS986, exhaustion reversal agent anti-PD-1, and CCR5 gene editing to delete the co-receptor for viral entry in CD4+T-cells. Analysis of CD8+T-cell polyfunctionality was performed using intracellular cytokine staining and flow cytometry to measure the frequency of CD8+T-cells secreting one or more of the following effector functions: TNFα, IFNγ, IL-2, and both CD107a and Granzyme B. Polyfunctionality induced by the combinatorial regimen was algorithmically quantified and compared to a control group receiving no interventions and a group receiving CE vaccine and CCR5 gene editing alone. Our analysis showed neither significant differences in polyfunctionality between treated and control groups, nor any changes in polyfunctionality within the subset of CD8 targeting conserved regions of the virus. However, we observed diverse viral control post-ATI among all animals, and additional experiments are in progress to determine if CD8 polyfunctionality played a role in improved viral control post-ATI.

Telomeres represent the structural ends of genomic chromosome molecules and are composed of hexamer repeats of the form [TTAGGG] which play a protective role against DNA damage and genetic information loss. The telomere length of normal diploid cells decreases over time and can be correlated with issues related to cancer and the aging process. Our preliminary work utilizes methods developed initially by Cawthon and O’Callaghan and is applied to DNA samples obtained from research studies involving the elderly as well as those with aneurisms. In contrast to standard measurements which use real-time PCR in order to calculate relative telomere lengths, our method uses absolute quantification through the use of standard curves generated with a synthetic 84-mer telomere (control gene) and another synthetic oligomer (36B4 ‘housekeeping gene’). Through the use of both synthetic standards in a typical RT-PCR experiment utilizing SybrGreen and specific primers for either telomere sequence or housekeeping gene, the true length of a diploid telomere can be calculated by extrapolation from standard curves. Telomere lengths obtained through this method have been compared to results derived from commercially available test kits which used relative RT-PCR as a quantification measure. Results obtained through the use of this technique can be incorporated in research studies encompassing cell apoptosis, processes involved with aging. This technique can also be used to ascertain the effects of environmental impacts involving DNA damage on plants, bacteria, and animal cells.

Secondary nucleotide messengers are used by all domains of life to sense and respond to the changes in their environment. In bacteria these secondary nucleotide messengers play a role in regulating several signaling pathways such as cell wall homeostasis, motility, and the expression of virulence genes. The nucleotide cyclic di- 3, 5’ adenosine monophosphate (c-di-AMP) was recently added to the list of secondary nucleotides. C-di-AMP is found in many bacteria such as S. aureus, S. pneumoniae, B. subtilis, and L. monocytogenes (Lm). C-di-AMP has been best characterized in Lm, a well-studied intracellular pathogen. Lm has adapted to survive and replicate in the host cell cytosol by evading host cell defenses through use of key virulence factors. In Lm, synthesis of c-di-AMP is catalyzed by the diadenylate cyclase dacA and degradation is coordinated by the phosphodiesterases, pdeA and pgpH. Studies using Lm mutants that lack both pdeA and pgpH contain abnormal c-di-AMP levels that cause growth and virulence defects of about four logs compared to wild type Lm. This highlights the importance of c-di-AMP regulation for bacterial virulence and growth, but we still know very little about c-di-AMP regulation and toxicity. Our goal is to further understand the toxicity of high levels of c-di-AMP during bacterial infection. We aim to create a transposon library in the double phosphodiesterase KO (ΔΔ Pde) background to identify suppressor mutations. Previous approaches to analyzing suppressor mutations in the ΔΔ Pde strain has not been thorough or cannot be utilized in vivo. Therefore, we have created an amenable phosphodiesterase mutant that knocks out the phosphodiesterases in Lm (pdeA and pgpH) to grow in vivo successfully to investigate c-di-AMP regulation. Understanding the regulation of c-di-AMP could result in targets for novel treatments against Lm and allow for ways to investigate regulation methods of c-di-AMP in other organisms.

Single-cell gel electrophoresis, or a Comet Assay, can be used to assess DNA damage in cells as it liberates single-strand DNA breaks (SSB), alkali labile sites (ALS), and DNA cross-links, then uses microscopy to determine levels of chromosome damage. Various methods have been used to detect DNA damage; advantages of this assay include its capacity to detect low levels of DNA damage, the small number of cells required, low cost, and the ability to utilize various cell types. Our lab used lymphocytes withdrawn from elderly patients as we targeted quantifying the effects on an individual’s DNA due to aging, stress, and drugs. Lymphocytes were suspended in low-melting point agarose and placed on a slide pre-coated with regular agarose. Once cells were fixed on the slide, they were placed in lysis solution to remove membranes and liberate DNA. Lymphocytes then went through an alkaline treatment to unwind DNA super-coils. The assay has been tried under neutral conditions however, we used the alkaline method because it is known to increase reproducibility and specificity. The cells were then placed in an electrophoresis chamber at 5 V/cm for 30 minutes, the SSB’s, ALS’s, and DNA cross-link fragments traveled towards the anode, forming a comet like appearance behind the cell. The cells were then neutralized, stained with ethidium bromide, and the comets were visualized. Data can be quantified using software provided by Instem Technologies. A comparison of Comet values derived from varying cell densities as well as untreated control samples are also presented, confirming good reliability using this technique.

The HIV Vaccine Trials Network (HVTN) has flow cytometry labs at both the Fred Hutchinson Cancer Research Center (FHCRC) and the Cape Town HVTN Immunology Lab (CHIL) which both perform testing on HIV vaccine clinical trial samples. There can be a significant level of variability in the performance of flow cytometers, potentially leading to inconsistent data. The large degree of intra- and inter-instrument variability necessitates a quality assurance (QA) protocol be followed to standardize new flow cytometers to the instruments currently in use in both HVTN laboratories to ensure accurate, consistent results between instruments. Using a modified version of Perfetto et al.’s protocol which utilized sets of standardized calibration beads as well as singly stained CD4 T-cells, we successfully optimized, calibrated, standardized, and qualified a new BD FACSymphonyTM (Becton-Dickinson, San Jose, CA) flow cytometer for future use at CHIL. The successful completion of this QA protocol ensures that this instrument will be able to collect accurate and reproducible data for HIV vaccine clinical trials. It is iimportant to guarantee the quality of the data collected during these clinical trials so that the effectiveness of candidiate HIV vaccines can be properly assessed.

Interferons are signal proteins released by cells in response to infections which induce neighboring cells to synthesize anti-viral genes like Mx2, which encodes the MxB protein. MxB was thought to only restrict lentiviruses but recent studies have shown that MxB also restricts herpesviruses. It is hypothesized that herpesvirus and MxB have co-evolved in an ‘arms race’ to compete against each other. This suggests that regions of MxB that are under positive selection may have interfaces for its anti-herpesvirus activity. In order to test this hypothesis, I plan to determine whether different homologs of MxB can restrict human herpesviruses. I created MxB inducible cell lines using MxB homologs from three different primates - humans, rhesus monkeys and owl monkeys. Subsequently, I infected these cells with the following GFP labeled viruses: herpes simplex virus 1 (HSV-1), Kaposi’s sarcoma-associated herpesvirus (KSHV), and human cytomegalovirus (HCMV). I assessed the effect of these MxB alleles on viral replication by measuring viral titer and viral protein production (including GFP). I expect that the MxB restriction of herpesviruses will be species-specific, with only human MxB showing meaningful reduction in human herpesvirus replication. These results will guide future research aiming to understand the physical interactions and mechanisms of suppression between MxB and herpesviruses.

Tuberculosis (TB) remains a major international health concern and one of the top 10 causes of death worldwide, according to the World Health Organization. Previous clinical work in our lab demonstrated that oral swab analysis (OSA) can successfully diagnose tuberculosis by detecting Mycobacterium tuberculosis DNA in the mouths of infected patients. In order to strengthen OSA against traditional but more invasive methods, such as sputum sampling, improvements to DNA extraction and swab type must be investigated. I am comparing different degrees of automation with MolBio’s Trueprep, the AudioLyse, and ThermoFisher’s KingFisher against our previously validated manual Qiagen extraction protocol—with the comparison lying in sensitivity and efficiency. In order to increase the versatility and sensitivity of OSA, I am also investigating boil preparations and dissolvable swabs. Boil preparation of swabs is a relatively simple extraction procedure, and early results have demonstrated its comparability against the Qiagen extraction. Meanwhile, dissolvable swabs have a hypothetical 100% yield of sample material. I have successfully dissolved calcium alginate swabs from Puritan in acidic sodium citrate solutions, and Luna swabs were dissolved in chaotropic agents. DNA yields are compared to non-dissolvable, previously validated swabs.

The influenza virus is known for its rapid evolution, or the ability to fix many mutations over a short period of time. Some of these mutations lead to amino acid substitutions in regions of the virus targeted by the immune system. Such changes are often selected for because they confer a fitness advantage by allowing the virus to "escape" immune response. This pattern of repeated immune escape is a detriment to public health because it necessitates an annual update to the influenza vaccine. Therefore, identifying sites on the influenza virus which are targeted by the immune system could help predict which influenza strain will circulate in the future, inform vaccine design, and help understand basic evolutionary questions. Using molecular phylogenetic techniques, we can identify sites potentially targeted by the immune system by looking for "positive selection", a phenomenon which manifests as a higher than expected rate of evolution. To identify sites evolving faster than expected, we defined a null expectation of the evolutionary rate of influenza in the absence of immune pressure. This null model is defined using empirical measurements from a high-throughput functional assay known as deep mutational scanning. This null model differs from traditional phylogenetic models in that it describes the constraints on influenza on a site-specific basis and, as a result, has been shown to be a more accurate and powerful null model. I have implemented an empirical Bayes approach to identify sites which deviate from the null model by an unexpectedly high evolutionary rate, suggesting positive selection. Preliminary results show that my method outperforms other methods for identifying sites under positive selection. Next, I will apply these methods to the influenza virus surface protein, hemagglutinin, which is a major target of the immune system.

Previous research has established extracellular vesicle (EV) signaling as a ubiquitous cell-signaling modality used by all cells. In this modality RNA, peptide, and protein signals are packaged together in small vesicles and directed to specific downstream cell targets. In addition to being necessary for healthy physiology, EVs can also contribute to pathological processes such as neurodegeneration in Alzheimer’s disease through carrying toxic proteins tau, Aβ and β-synuclein between cell types. Recent research in the Kaeberlein and Mendenhall labs has shown that when human tau is specifically expressed in neurons of C. elegans, there is a significant increase in HSP90 chaperone and proteostatic stress reporter expression in the intestine. I hypothesize that the induced human tau neuron-to intestine stress signal is conveyed by the extracellular vesicles. To determine the signal pathway, extracellular vesicle signaling was disrupted specifically in neurons of a transgenic nematode line with neuron-specific human tau and fluorescent intestinal proteostatic stress reporter (daf-21::GFP). RAL-1 is a small GTPase that has been shown to be necessary for EV signaling in the C. elegans hypodermis and intestine. Therefore, RAL-1 function is disrupted though knocking down neuron-specific expression with either a double-stranded RNA ral-1 construct or a dominant-negative form of RAL-1 The degree of daf-21p::GFP expression was compared between the reporter lines and those with disrupted ral-1 function. Many studies utilizing C. elegans have shown that neurons send cellular stress signals to other tissues which strongly affect lifespan, metabolism, and proteostatic stress. Therefore, the results from my experiments contribute to determining whether EV-signaling carries neuronal stress-signals induced by human tau and establish a methodology for using C. elegans as a model for studying this important signaling pathway.

There is a critical lack of knowledge regarding the effects of human immunodeficiency virus (HIV) and zika virus (ZIKV) coinfection in respect to ZIKV pathogenesis, vertical transmission and current vaccine strategies. ZIKV has received global attention because of its clinical complications including congenital malformations during vertical transmission and Guillain-Barré syndrome, a neurocognitive disorder in adults. Recently, ZIKV outbreaks have occurred in tropical and subtropical regions endemic with HIV, therefore it is imperative to understand the impact HIV-ZIKV coinfection may have when moving forward with ZIKV vaccine design. Furthermore, in humans and non-human primates (NHPs) frequencies of blood monocytes increase during HIV and simian immunodeficiency virus (SIV) infection and monocytes are early targets of ZIKV infection. Therefore, we hypothesize that cells from HIV infected individuals have the capacity to harbor increased ZIKV replication and could lead to enhanced ZIKV viremia and pathogenesis. For this study, we use a NHP model to determine the impact of acute SIV infection on ZIKV pathogenesis. We determine ZIKV cellular targets in the blood and preliminary results show an increase in NHP blood monocyte within the first six weeks of SIV infection. We evaluate whether SIV infection increases the susceptibility of cells to ZIKV infection by isolating cells from SIV- and SIV+ animals, infecting them in vitro with ZIKV, and assessing viral replication by plaque assay. Our preliminary findings suggest that in vitro ZIKV replication may increase in cells from SIV+ NHP blood when compared to SIV- blood. Future studies will look at the impact of SIV infection on in vivo ZIKV replication and whether ZIKV replication is enhanced in cells from HIV-infected individuals.