American Foulbrood (AFB), a disease of honeybee larvae caused by the bacterium Paenibacillus larvae, has global economic significance. AFB spreads through colonies killing the next generation of bees. AFB has traditionally been treated with antibiotics or by burning infected hives and exposed materials. The former method is complicated by antibiotic resistance and bacterial sporulation, while the latter method carries severe financial liability for beekeepers. One alternative treatment is the use of bacteriophages, viruses that infect and destroy bacteria. Bacteriophages are host-specific and co-evolve with their host, unlike antibiotics, thus making resistance and collateral damage a non-issue. Characterizing these phages will be key to future treatments of infected hives. The P. larvae phage Willow, isolated in our lab, is both lysogenic (allow the infected bacterium to survive) and lytic (kill the infected bacterium). Currently, we have confirmed Willow lysogeny in P. larvae strain 3650. This relationship presents a model system for investigation. A comprehensive understanding of the host-pathogen interactions between Willow and P. larvae will aid in the effective targeting of P. larvae in the field. Our laboratory also seeks to determine the relatedness of four P. larvae phages to Willow to further characterize the genes responsible for lysogenic/lytic decision making. In sensitivity assays, lysogens (bacteria with integrated phage) are plated to form a lawn and dilutions of a different phage are spotted onto the plate. An identical wild-type plate is made, and comparison yields plating efficiencies. Plating efficiency determines what degree of protection is offered by the integrated phage. I hypothesize that genetic sequencing will show measurable genetic differences in the putative C1/Cro region of the phages under investigation, and that all genome sequence similarity will correlate directly with established plating efficiencies. Furthermore, I used sequence alignment, product alignment, and dot-plots to illustrate the degree of sequence similarity between phages.

Heterotopia are organized structures consisting of mixed elements of neurons, glia and neuronal progenitors arranged in an architectural pattern inappropriate to the considered tissue. Heterotopia are observed in pathological preparations of the cerebral cortex and the cerebellum as a feature of many neurological disorders yet the mechanisms driving their development remain unclear. We have analyzed 28 Dandy Walker Malformation (DWM) human fetal cerebella and found that more than 40% of cases contain heterotopia. DWM is the most common structural birth defect of the human cerebellum and is characterized by hypoplasia and foliation defects. A subset of cases are caused by loss of FOXC1, a transcription factor expressed in the mesenchyme adjacent to the developing cerebellum. We previously reported that in mice, loss of FOXC1 causes loss of the mesenchyme-secreted factor SDF1-alpha. Further, loss of SDF1-alpha is sufficient to cause cerebellar heterotopia, highlighting the importance of mesenchymal signaling in the maintenance of the highly stereotypical laminar architecture of the mature cerebellum. We hypothesized that granule neuronal progenitors (GCPs) adjacent to the mesenchyme are the primary cellular target of SDF1-alpha mesenchymal signaling and thus, the primary cell type driving heterotopia formation. To test our hypothesis, we specifically deleted the SDF1-alpha receptor from just GCPs using a Cre-loxP genetic scheme in mice. We determined that loss of SDF1-alpha in just this one cell type caused their premature migration into the developing cerebellar anlage and secondarily caused other cerebellar cell types to form structured heterotopia. We also observed defects in cerebellar foliation in the treatment group. Our data further highlights the importance of SDF1-alpha dependent mesenchymal signaling in cerebellar development and identifies heterotopia as a new phenotype in DWM.

This abstract is no longer available. Please contact urp@uw.edu if you have any questions.

Alzheimer’s Disease (AD) is a progressive brain disorder that results in middle to late life dementia. AD, the most common form of dementia, is the sixth leading cause of death in the United States, and the third leading cause of death for older people over 65 years of age. AD is pathologically characterized by the accumulation and aggregation of an alternatively spliced amyloid-beta (Aβ) and hyperphosphorylated tau, which lead to the formation of neuritic plaques and neurofibrillary tangles (NFT’s) in the brain and ultimately to decreased neural function and cell death. Previous studies indicate that the extent to which an individual is affected by the interaction of these proteins and the severity of AD is largely influenced by genetic variation. However, the specific genes involved, their regulation and expression remain largely unknown. Our ongoing project explores the genetic variation underlying Drosophila susceptibility to severe eye cell degeneration caused by human Aβ and tau. We express Aβ and tau in the fly eye through the UAS-GAL4 system to create our AD model Drosophila, and cross them with a panel of flies (the Drosophila Genome Reference Panel, DGRP) that allows us to map natural genetic variation for resistance to Aβ and tau. Our preliminary results show significant variation in neuronal eye cell degeneration among DGRP lines, implying that neurodegeneration would also differ between DGRP lines if Aβ and tau were expressed in the nervous system. Based on current progress, this new project will continue exploring variation in DGRP lines, but this time we will express them in the fly brain. I will measure how different genotypes shape the effect of brain-specific Aβ and tau on lifespan and protein aggregation, and will use Genome Wide Association Studies to identify single nucleotide polymorphisms associated with variation in expression of Aβ and tau.

Arthropods represent 29 percent of the world’s animal biomass and comprise over 500 million years of evolutionary diversity. Arthropods such as spiders and moths constitute a perfect milieu for the discovery of multitudes of highly diverse RNA viruses of unknown pathogenic potential. Sequencing and characterizing these viruses is of immense value for understanding many basic science questions about viruses. The sequences obtained and described can also provide great insight into future zoonotic outbreaks. We sequenced RNA extracted from 10 moths and 6 spiders collected from western Washington. We developed a custom bioinformatics pipeline for automatic discovery and characterization of completely new viruses. The custom software also automates the process of depositing viral sequences into the National Center for Biotechnology Information’s Genbank archives, the main database in which DNA and RNA sequences are shared among scientists and researchers. A total of 12 novel viral genomes were discovered in these 16 samples. These new viruses belong to 4 highly different viral families, all of which contain species known to infect humans. We also present what is likely a completely new viral clade within Picornavirales, the viral family that contains polio and many other diseases afflicting humans. This new viral clade has a never seen before genome organization and contains a highly divergent RNA dependent RNA polymerase. New and highly divergent strains such as the ones outlined here provide extremely valuable insight into the inner workings of viral biology and genetics.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor made up of cells of endothelial origin. KSHV establishes a predominantly latent infection in endothelial cells in culture and in the KS tumor. A previous study has shown that KSHV induction of the Warburg effect is required for the survival of latently infected endothelial cells. The Warburg effect, a common metabolic alteration in cancer cells, refers to an increase in glycolysis and a decrease in oxidative phosphorylation. The mechanism of Warburg induction by KSHV is currently unknown. I proposed to evaluate the role of endothelial cell specific hypoxia-induced factors (HIFs) on KSHV Warburg induction since HIFs have been implicated in Warburg induction in other types of cancer. I hypothesized that HIF2α mediates KSHV Warburg induction through expression of glycolytic genes. To test this, I constructed HIF2α knock-out cells using CRISPR/Cas9 gene editing. I then used RT-qPCR to measure glycolytic gene expression during KSHV infection of wild-type and HIF2α knock-out cells. I found that the transcript levels of certain glycolytic genes remained constant in KSHV-infected HIF2α knock-out cells as compared to KSHV-infected wild-type cells, showing that HIF2α is not responsible for glycolytic gene expression during KSHV infection. I am following up on the role of the KSHV latent gene KapA on the induction of glycolytic gene expression. KapA was previously found to interact with components of the Ras pathway. As the Ras pathway activates glycolytic genes through HIF1α, I hypothesize that exogenous expression of KapA will lead to increased glycolytic gene expression through increased expression of HIF1α. I will construct an endothelial cell line that overexpresses KapA using CRISPR/Cas9 system and then use RT-qPCR to evaluate glycolytic gene expression. These results will aid in the future efforts to develop antiviral drugs by targeting cellular metabolism.

Antibiotic resistance is a major public health problem. For example, in people with the genetic disease cystic fibrosis, Pseudomonas aeruginosa causes chronic lung infections that resist treatment to all existing antibiotics. Gallium is a promising new antimicrobial agent that works through a novel mechanism of action. Bacterial uptake systems are unable to distinguish gallium from iron due to their nearly identical ionic radii. Once inside, bacterial gallium is thought to disrupt iron-dependent processes because Ga³⁺ cannot be reduced in physiological conditions, and iron's biological functions depend on its capacity for redox cycling. Here I sought to better understand gallium’s mechanism of action by identifying gene mutations that decrease P. aeruginosa gallium sensitivity. To accomplish this, I used transposon (Tn) mutagenesis, which transfers a genetic element to random locations on the bacterial chromosome thereby inactivating genes. Of the 295,000 mutants generated, I selected for mutants capable of growing in the presence of increased gallium compared to the parental strain. Using a procedure known as rescue cloning, a restriction digest was performed on the chromosomal DNA of these mutants to produce individual fragments. One of these DNA fragments will contain the Tn insert and chromosomal DNA flanking that insert. The Tn insert encodes resistance to gentamycin and holds the capacity to replicate as a plasmid in E. coli. I ligated the isolated fragments and transformed E. coli, selecting for gentamycin resistance. I isolated this plasmid DNA from two mutants and their Tn insertion sites (and the genes inactivated by the Tn) were identified by sequencing the DNA surrounding the Tn inserts. These genes were found to be involved in iron transport. By completing these experiments, I hope to suggest hypotheses for the roles these genes play in gallium sensitivity and to provide data useful toward guiding its development as an antibacterial agent.

APOBEC3G (A3G) is a host protein that acts as a cytidine deaminase to inhibit HIV replication as part of the innate immune system. A3G has been shown to cause Guanine to Adenosine hypermutation. This host protein cannot deaminate the proviral HIV DNA because of the HIV accessory protein Vif. The Vif protein interrupts the antiviral activity of A3G by inducing ubiquitination and degradation via the host proteasome. In this project, we investigated the deamination ability of A3G and deamination inhibition ability of Vif in vivo. While much of the published work on A3G has involved the investigation of Vif-induced A3G degradation, bacterial models have also revealed a degradation-independent mechanism of A3G inactivation by Vif. We looked for direct inhibition of A3G deamination ability by Vif in vivo. We found that the deamination of the proviral genome was correlated with the concentration of A3G in both NL4-3 and NL4-3 (vif-) strains. Unexpectedly, we observed deamination in the viral genome, in NL4-3 at higher concentration. In comparison, packaging of A3G into NL4-3(vif-) virions caused much larger amount of deamination than in Vif-expressing NL4-3 virions. In the case of wild-type NL4-3, the virus remained infectious despite significant levels of deamination. This represents a novel phenomenon that suggests that the Vif protein has an additional function within the viral replication cycle. Our results, although preliminary, set the stage for a more extensive analysis that may provide novel insights into the biological function of Vif. By studying A3G and Vif interactions in vivo, we are getting closer to understanding the viral replication cycle as it occurs in the human body. By understanding the complete range of function of Vif, new antiviral therapies could be developed to target Vif.

Klebsiella pneumoniae is a gram-negative bacterium that asymptomatically colonizes the intestinal and upper respiratory tract of healthy humans. It is also an opportunistic pathogen that causes bacteremia, pneumonia, urinary tract infections, and other severe infections in individuals with significant comorbidities. Klebsiella infections are often healthcare-associated and antibiotic-resistance is a major problem for treatment. A K. pneumoniae lineage known as multilocus sequence type 258 (ST258) is resistant to virtually all beta-lactam antibiotics, including the carbapenems, and many clinical isolates are also resistant to fluoroquinolones and aminoglycosides. Additionally, the capsule of ST258 confers resistance to phagocytosis by neutrophils and killing by the complement system. However, it is readily killed if phagocytosed. One possible therapeutic approach is to develop an antibody-based immunotherapy that targets the capsular polysaccharide (CPS) of ST258. As a step toward developing the tools needed for such an approach, we developed an enzyme-linked immunosorbent assay (ELISA) that can be used to detect CPS and titer CPS-specific IgG antibodies. Polystyrene ELISA plates were coated with purified ST258 CPS and then incubated with rabbit antiserum containing antibodies specific for CPS. Anti-CPS IgG was then detected with goat anti-rabbit IgG antibodies and a detection reagent. We anticipate the ELISA will be an important quantitative tool in our efforts to develop an immunotherapy that targets the CPS of K. pneumoniae.

Human immunodeficiency virus (HIV) infections are often paired with microbial translocation within the gastrointestinal tract (GI). In-vivo studies show that HIV-associated microbial translocation results from multiple immunopathological events occurring at the GI mucosa: early, detrimental, severe CD4(+) depletion in mucosal regions, mucosal immune hyperactivation and inflammation, and intestinal epithelium damage. Previous human studies have shown anti-inflammatory effects of cannabis, along with slowed GI tract disease progression. This project aims to further understand HIV infection and its response to patients’ cannabis use. We hypothesize the anti-inflammatory effects of cannabis should decrease the microbial translocation within HIV+ individuals. Using stool from HIV+ and HIV- patients, DNA and short chain fatty acid (SCFA) extractions were performed. The DNA was extracted using Power Fecal DNA extraction kits. In order to understand microbial community and how it relates to SCFA production, the extracted genomic DNA was PCR-amplified and will be sequenced with an Illumina MISEQ. SCFA extraction was done with ethyl acetate. We analyzed the samples using gas chromatography-mass spectrometry, and it showed HIV+ patients had a decrease in acetic (p=0.0057), propionic (p=0.0325), butyric (p=0.0007), iso-butyric (p=0.0039), and valeric acid (p=0.0133). When compared with HIV- patients, there’s an overall decrease in total SCFA (p=0.0012). These results demonstrate the detrimental effects HIV infection has on SCFA production, which can lead to a decrease in available energy for colon cells and disrupt gut microbial community health. However, amongst HIV+ with cannabis use, we saw an increase in acetic (p=0.0173), propionic (p=0.0087), butyric (p=0.0173), iso-butyric (p=0.0043), and total SCFA (p=0.0057). The HIV+ patients that used cannabis saw improvement in microbial community health, which is a promising sign that cannabis assists with the recovery of antiretroviral treatment.

Conventional diagnostics for tuberculosis use the patient’s sputum, mucus from the lower airways, as the primary sample type. Collecting sputum can be difficult and hazardous and for some patients, especially pediatric cases, sputum must be induced through an invasive procedure. An alternative to sputum samples is oral swabs, which have been used to detect the presence of Mycobacterium tuberculosis DNA in clinical trials. However, the current threshold of oral swabs is not yet sufficient for widespread implementation. In a new phase of assessing the viability of oral swab samples, nine different swab types were compared to improve DNA yield and subsequent qPCR readouts. The comparison was based on qPCR Cq values after spiking the swabs with mTB DNA, and analysis of each swab’s suitability for culturing. Earlier results suggest that oral swabs are a feasible alternative to sputum samples when using swabs that are heavily “flocked”, meaning that they have many small fiber particles deposited across a surface. Moreover, samples performed with dissolvable swabs may be better suited for further molecular and microbiological analysis.

The evolutionarily conserved Hippo cell signaling pathway is important in tissue and organ development through its regulation of cell death, proliferation, and differentiation; as such, the Hippo pathway is often dysregulated in cancer. The phosphotransferases serine/threonine-protein kinase 3 and 4 (STK3 and STK4), are core components of the Hippo pathway. When active, these kinases negatively regulate the transcription factors yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). The activities of STK3 and 4 are regulated by phosphorylation on multiple serine, threonine and tyrosine residues. Recently, we discovered that the central cellular signaling regulator cAMP-dependent protein kinase (PKA) can phosphorylate STK3 on serine 15 (S15). Since it has been shown before that PKA can suppress the transcriptional activity of YAP/TAZ, we hypothesized that this phosphorylation event activates STK3. To investigate the functional consequences of STK3-S15 phosphorylation, I used molecular cloning to construct active and kinase-dead (KD) FLAG-tagged STK3 mutants in which S15 was replaced by alanine (S15A, non-phosphorylatable) or aspartate (S15D, phosphomimic), respectively. I transfected plasmid DNA encoding these mutants into human embryonic kidney cells (HEK293T) to investigate if overexpression of each STK3 variant differentially affects activation of the Hippo pathway. Western blot analyses of transfected cells showed that active STK3 S15A and S15D but not the kinase-dead versions were able to phosphorylate known STK3 substrates. Further, I used anti-FLAG antibodies to enrich FLAG-tagged STK3 variants and co-precipitating protein complexes for mass spectrometric (MS) analysis to examine the effect of STK3-S15 phosphorylation on protein-protein interactions. MS analysis revealed that the STK3 S15D mutant preferentially bound to STK4, suggesting pathway activation. Insights into the mechanism of Hippo pathway regulation by PKA will have important implications for cancer research since targeted therapeutics modulating the cAMP - PKA signaling axis may be directly applicable in tumors with aberrant YAP/TAZ activity.

Previous research on JNK-mediated stress signals demonstrated that stem cells in the posterior midgut of Drosophila Melanogaster only undergo compensatory proliferation or apoptosis. However, our group discovered that the stem cells can also undergo the process of basal extrusion and dissemination, resulting in the cells being eliminated from the tissue into the hemocoel, the blood containing intertissue body cavity. The JNK signal promotes the cells to exit the epithelium of the gut, move through the muscle layer, and be released to the hemocoel, which resembles the process of metastasis in human cancer. In order to understand the mechanism of this extrusion process, we carried out an RNA Interference (RNAi) screen and sought to find genes that are necessary for the stem cell extrusion in the JNK activator, HepCA, expressed flies. We used the ESG-GAL4, UAS-GFP, TUB-GAL80TS(EGT) genetic system to study the knockdown effect from the RNAi of each gene. We selected 215 lines of kinases and phosphatases of flies to test if the knocked-down gene results in suppression of cell elimination by the JNK stress signal. After 4 days of inducement, which is the sufficient time for the JNK signal can promote complete extrusion of the intestinal stem cells, the intestines were dissected, fixed, mounted, and examined with a fluorescent microscope for any presence of stem cells. Through this screen, we could find 33 lines that had strong suppression of cell elimination, 39 lines that had a moderate effect. Our results suggest that the knocked down genes with strong suppression of cell elimination are involved in the mechanism of basal cell extrusion, and future research dictates an investigation into the molecular function of each of these genes.

X-ray diffraction gratings are an array of equally spaced structures with defined width designed to diffract optical waves. These waves create a spectrum that allows for the characterization of the wavelength or angles created by each diffraction. X-ray diffraction is commonly used in x-ray crystallography, where x-rays are diffracted off crystal lattices and give information about its three-dimensional structure. The goal of this project is to develop a microfabrication process for high aspect ratio x-ray diffraction gratings. These devices will expand the x-ray diffraction capabilities of the University of Washington and the National Nanotechnology Coordinated Infrastructure (NNCI) at large. In developing this process, our main objectives are to attain smooth vertical sidewalls for the grating features, minimize gold waste in the plating process and to be highly repeatable. For our research, each fabrication step will be tested, troubleshot, and documented for future use. The microfabrication steps required in our process include photolithography, etching, grinding and polishing, wafer bonding, and electrochemistry metal plating. The main challenges of this project are producing a photoresist pattern thick enough to withstand deep etching without affecting the sidewall profile, attaining precise grinding thicknesses and overcoming wafer bonding complications. The work towards maturing each process through inspection and troubleshooting for fabricating high aspect ratio x-ray gratings will be discussed.

Aging is associated with a decline in functionality and cellular organization. These changes are reflected in the morphology of cellular organelles, such as the cytoskeleton, which plays an integral role in cell movement and signaling. Age-related changes in the cytoskeletal system make it a great point of interest in age-related studies. However, its roles in aging and longevity are still largely unknown. We hypothesized that alteration of the actin cytoskeleton results in an accumulation of age-related cellular pathologies. To test this hypothesis, we examined cellular markers of aging in C. elegans worms with deactivated actin proteins. The genes for the cytoskeletal system are well conserved between humans and Caenorhabditis elegans, making it a great and popular model system for examining the role of the cytoskeleton in aging. We used RNA interference (RNAi) to knock down the expression of cytoskeletal actins in the worms. Afterwards, we used fluorescent microscopy to examine markers of cellular aging: pH, nucleoskeleton integrity morphology, and mitochondrial network morphology. We have found that perturbation of actin cytoskeleton partially mimics age-related cellular changes. We expect the results to be of help in the ongoing study of the role of the cellular cytoskeletal complex in the human aging process.

Chemically induced dimerization (CID) systems, which are systems that involve the dimerization of two proteins only in the presence of a specific molecule, have shown a great potential in studying and controlling cellular signaling pathways. However, current methods for identifying CID systems for any given target based on small molecule-protein interactions remains a challenge in the area of protein design. Our research team proposes a general approach for identifying new CID systems for which may have clinical and therapeutic applications. To do this, we aim to create nanobody-based CID binders. Using a technique known as “Trinucleotide-Directed Mutagenesis,” our team has been able to create a diverse nanobody library of over 109. Phage display allows us to screen this library for specific nanobodies that can bind with high specificity and affinity to a specific molecule. Currently, we are using cannabidiol (CBD) as a target molecule. After performing several rounds of selection, in order to validate our system’s specificity and selectivity, we use ELISA assay tests as well as Bio-Layer Interferometry to measure the binding affinity and binding rate of our system. In order to ensure the specificity of our CID system we tested it against, tetrahydrocannabinol (THC), a structural homolog. To date, we have successfully found three different nanobodies that can bind with high affinity and specificity to our small molecule of choice. As we move into phase two of our project, we are currently working on identifying the second nanobody in order to form a complete CID system. Designed CID systems will not only provide personalized choices for patients showing different drug responses, but also serve as orthogonal chemical control of different cell subpopulations and cell behaviors to improve therapeutic efficacy and safety.

Store-and-forward teldermatology (SFT) has grown in use nationwide to increase accessibility of dermatological care especially towards underserved populations. It has demonstrated its cost-effective services by minimizing avoidable referrals to dermatologists and granting quicker overall access to dermatologists for initial interventions. However, due to transportation and other logistics, there are concerns that SFT may have significantly longer delays and lengths of treatment of malignant melanoma than face-to-face (FTF) dermatology. Melanoma is the most lethal type of skin cancer which makes it imperative to seek intervention as soon as possible. We conducted a descriptive comparison of melanoma discovery and timeline for treatment among veterans whose melanomas were discovered and treated through face-to-face and store-and-forward teledermatology. We tested the hypothesis that SFTD is non-inferior to FTF dermatology in discovery and treatment of primary cutaneous melanoma. We ascertained chart data for Veterans diagnosed with melanoma between January 1, 2006 through December 31, 2013 from VA corporate data warehouse (CDW), Veterans Integrated Service Network 20 (VISN 20) and and Computerized Patient Record System (CPRS). To determine possible opportunities to discover a melanoma, and lengths of each episode of care, we retrospectively analyzed 300 cases of melanomas 365 days prior to the date of their biopsy to the date of its definitive procedure. The mean days between the earliest opportunity to discover and discovery, discovery to biopsy, and biopsy to definitive procedure was 90, 39, and 41 days respectively for melanoma cases seen by FTF dermatology and 44, 50, 40 days respectively for melanoma cases seen by SFTD. With no biological significance between the timelines of care, SFTD is non-inferior to FTF dermatology in timeliness to treat melanoma. 

Although syphilis is often labeled as a disease of the past, this infection is still a significant public health concern in both developing countries, where most cases occur, and developed ones, including the United States, where syphilis has been resurgent for over a decade. Public health initiatives aimed at syphilis control would greatly benefit from the availability of a vaccine that could confer complete immunological protection against Treponema pallidum (T. pallidum), the syphilis agent. Modern pre-clinical vaccination/challenge trials focused on the use of recombinant T. pallidum antigens and although were effective in conferring partial protection against syphilis, complete protection was never achieved. A possible explanation for this result is that these immunizations were performed with antigens lacking their natural three-dimensional conformation. This, in turn, might have abrogated production of antibodies to conformational epitopes of these proteins and diminished their protectivity. To evaluate whether administration of antigens in their proper conformation could improve current vaccine formulations, we expressed T. pallidum antigens in a bacterial surrogate system that utilizes a lab-derived strain of Borrelia burgdorferi (B. burgdorferi), a spirochete genetically and structurally related to T. pallidum but engineered to be non-pathogenic. We used B. burgdorferi to express selected T. pallidum antigens that have been shown in the past to be partially protective and then injected these B. burgdorferi strains into New Zealand white rabbits to induce humoral and cellular immunity against the T. pallidum proteins. Following immunization, rabbits were challenged with infectious T. pallidum to evaluate the efficacy of this new vaccine design. Immunized animals developed a specific antibody response to the T. pallidum antigens, but were only partially protected after challenge. We showed that our innovative vaccine design is very effective in inducing an immune response to T. pallidum antigens and will be a useful tool to test additional vaccine candidates.

Human cytomegalovirus (HCMV) is a member of the Herpesvirus family. Most of the US population has been exposed to this virus, but it is only deadly in immunocompromised individuals. When a human cell becomes infected, one of the earliest mechanisms of anti-viral defense is the activation of protein kinase R (PKR). Active PKR phosphorylates the translation initiation factor, eIF2α, shutting down most protein synthesis in the cell. HCMV circumvents cellular shutdown by expressing protein TRS1, which binds to PKR and inhibits it. Another cellular kinase, the heme-regulated inhibitor (HRI), also phosphorylates eIF2α upon activation by reactive oxygen species. Because HRI and PKR are similar in structure, I hypothesize that HRI activation during HCMV infection is inhibited by TRS1. I infected PKR-knockout human fibroblasts with HCMV and then activated HRI expression by inducing production of reactive oxygen species. These cells also have a luciferase gene, expression of which is induced upon eIF2α phosphorylation. I measured the abundance of phosphorylated eIF2α (eIF2α-P) and luciferase activity in the lysate of infected cells in which HRI has been activated. I expect that TRS1 will bind and inhibit HRI, therefore eIF2α-P levels and luciferase activity will be low compared to cells lacking TRS1. These results reveal that HCMV replication is sensitive to HRI activation in reactive oxygen-rich inflammatory tissues and provides insights into the evolutionary history and structural similarities between HRI and PKR.

Living cells must go through several phases as they grow and divide. During the phase known as mitosis, attachment of microtubules to chromosomes through specialized protein structures called kinetochores is indispensable for chromosome segregation. The process is prone to error due to the random nature of chromosome-microtubule attachments. A protein located near kinetochores, Aurora B kinase, phosphorylates erroneous connections, facilitating detachment of the microtubules and allowing another opportunity for correct attachments to form. Classic studies have suggested that a lack of tension on kinetochores is an error indicator for cell division checkpoints, because artificially applying force to chromosomes was sufficient to prevent their reorientation from mal-oriented configurations in insect spermatocytes. Based on this observation, a popular belief is that tension suppresses Aurora B triggered detachments. However, the relationship between chromosome reorientations observed in insect spermatocytes and the activity of Aurora B remains uncertain. To test the involvement of Aurora B, we utilized a small molecular inhibitor, AZD, to specifically reduce the activity of Aurora B. House cricket’s spermatocytes were extracted and soaked in a mixture of Grace’s insect medium and AZD to allow the inhibitor to permeate the cell membrane. After AZD treatment, the cells were spread on a chamber slide and filmed under a phase contrast microscope for approximately 5 hours. Our results show near complete prevention of cell division in the AZD treatment group. In the control group without AZD, transitions from metaphase to anaphase were much more frequent. Thus, Aurora B appears to have a crucial role during insect spermatocyte mitosis. This observation brings us one step closer to a definitive test of whether the tension-suppressed reorientation of chromosomes in insect spermatocytes is dependent on Aurora activity.

In the past few years, tetanus has caused many cases of the disease, according to the World Health Organization, specifically 34019 newborns died in 2015 from Maternal and Neonatal Tetanus (MNT). Many studies have been done on its transmission pathways and its effect on patients. This project explored research in academic journals and other professional publications with keywords such as “maternal and neonatal tetanus” and “transmission.” We explored existing transmission pathways of tetanus, as well as current recommendations for these specific cases, such as proper wound care regarding umbilical cord cutting and vaccination of pregnant mothers. Such recommendations would be to prevent illness and death, as well as diminishing the risk of infant demise. It was found that tetanus is endemic in certain regions. Its contamination can be prevented through proper wound care, vaccination, and proper umbilical cord practices. This review used the disciplinary lenses of microbiology, clinical medicine, epidemiology, and public health. There is overwhelming evidence pointing to the recommendation to vaccinate in the circumstances of a wound or as a form of prevention. This review will suggest implementation in regards to vaccination and prevention options to accompany current healthcare provider efforts to further assist their endeavors in eliminating the specific presentation of MNT in the remaining 15 countries where it remains a public health threat, such as Mali or Pakistan.

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. 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-mediated signaling, a critical antiviral pathway that is activated during herpesvirus infections. STING induces expression of the antiviral signaling molecule interferon and pro-inflammatory cytokines through the transcription factors IRF3 and NF-kB, respectively. While STING is unable to signal through IRF3 in LECs, it is unknown if NF-kB activation is similarly impaired. Moreover, it is unknown if the defect contributes to the increased susceptibility to KSHV infection in LECs. Accordingly, I hypothesize that in LECs, NF-kB signaling via STING will be impaired and that the STING defect increases susceptibility to infection relative to BECs. To determine if STING activates NF-kB, I will stimulate STING in BECs and LECs and measure expression of NF-kB-activated genes. I expect NF-kB-activated genes to be expressed in BECs but not in LECs, indicating that STING is unable to signal through NF-kB in LECs. To determine if the STING defect renders LECs more susceptible to KSHV infection, I will use CRISPR-Cas9 to knock out STING in BECs. I will then use KSHV to infect the knock-out BECs as well as wild-type BECs and LECs and compare infection rates. I expect the infection rate in the knockouts to be increased relative to wild-type BECs and similar to the infection rate in LECs. The results from these experiments will further elucidate how KSHV exploits defects in innate-immunity to infect and transform host cells.

As an airborne virus, influenza A is a widespread threat to global economies and a consistent danger to public health. Through high reassortment and evolutionary rates, influenza is even able to infect those who have been previously vaccinated against the virus. The innate immune system serves as a key first line of defense against this pathogen, with the signaling components, called interferons, driving the production of a potent cellular antiviral response. Studies have indicated that viral populations replete in defective virus particles, virions with a deletion in a portion of their genome, are less efficient at blocking the antiviral response, as shown by increased interferon in the host. Our project seeks to explore this phenomenon of RNA deletions leading to increased interferon expression in host cells by testing the hypothesis that deletions in the three polymerase genes of influenza alone are sufficient to cause an increase in the interferon response. In addition, we are currently testing if mutational deactivation of one of the other genome segments, or absence of such segments, is capable of producing a more robust immune response when combined with polymerase gene deletions. In order to support this analysis, I began by creating pure populations of PA defective influenza particles grown on PA expressing host cells. Similar to results observed by my mentor Dr. Alistair Russell with PB1 and PB2 defective populations, it was found that these PA defective influenza particles were sufficient to induce the interferon response. Recently, I have assisted in the creation of multiple influenza protein expressing cell lines and influenza populations with simultaneous modifications to the HA, NS, and polymerase genes. It is hoped that immune stimulation data derived from these custom viruses, in combination with previous findings, will improve current antiviral therapies and models of the human immune response to influenza.

The last clinical case of wild poliovirus type 2 (WPV2) was in 1999, with eradication declared in 2015. However, the type 2 vaccine strain is responsible for nearly 90% circulating vaccine-derived polio virus (cVDPV2) cases. Poliovirus (PV) is spread in areas with low immunization rates, poor sanitation and resource limited settings. Environmental monitoring of PV is a useful complement to ongoing disease-based clinical surveillance. Current PV detection methods, such as plaque assays and real time quantitative polymerase chain reaction (RT-qPCR), are costly, time consuming, and require specialized facilities. Reverse transcription recombinase polymerase amplification (RT-RPA) is an isothermal molecular amplification technique that has been adapted for PV2 detection. The high sensitivity and specificity of this method could provide a tool for rapid PV2 screening of environmental concentrates. To simulate testing of environmental samples, we collected and concentrated raw sewage from a local wastewater treatment plant using ViroCap filters. We performed secondary concentration using one of two methods, skimmed milk flocculation (SMF) and polyethylene glycol (PEG) precipitation. We added known amounts of PV2 to each concentrated sample, and compared detection results between RT-RPA, RT-qPCR and plaque assays to determine any matrix associated inhibition. RT-RPA detects target RNA with a change in fluorescence (mV). A change >200 mV indicates a positive reading for PV2. I conducted experiments with varying initial PV2 concentrations, and this resulted in positive detection by RPA in 46% of SMF and 88% of PEG samples. All RT-qPCR and plaque assay results yielded positive PV2 recovery. These preliminary findings suggest inhibition in SMF samples; further testing is required to determine the cause of inhibition. Future research should include the addition of PV2 directly into wastewater samples prior to concentration, to determine recovery with RPA. This is important for the successful implementation of RT-RPA with environmental samples.

Aging is characterized by the increasing loss of physiological and cellular functionality, however the mechanism that underlies this deterioration is still unclear. Emerging evidence indicates that aging is associated with increased cell-to-cell variation in gene expression within tissues: homologous cells within tissues express the same gene at varying levels. The causes of this age-related variation of gene expression are not known. Here we aim to investigate the mechanisms of increased cell-to-cell variation in gene expression with age using C. elegans as a model system. 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. We hypothesize that stochastic noise of transcription can lead to increased gene expression variation with age. Previously we have found that stochastic noise of gene expression is incredibly restricted in young C. elegans animals. Using quantitative fluorescent microscopy we have analyzed expression of reporter genes in old animals and have dissected the potential contribution of stochastic transcription noise into age-related variation of gene expression.

Research in human cohorts and animal models has established a link between prenatal stress (PS) and alterations in development of the fetal hypothalamic-pituitary-adrenal (HPA) axis and autonomic nervous system (ANS). These alterations are physiological, cognitive and behavioral and reach into adulthood. One of the mechanisms is an increased transfer of stress hormones, glucocorticoids, to the fetus. Therefore, interest lies in identifying biological markers that indicate the onset of future developmental problems caused by PS on HPA and ANS pathways.This project is investigating how heart rate (HR) fluctuations, a proxy for ANS activity, can act as a biological marker. The animal model is chronically instrumented unanesthetized fetal sheep. We measure the impact of PS on maternal and fetal glucose, cortisol, ACTH and HR. PS is modeled by repetitive-intermittent-unpredictable three hour isolations during the last trimester. Sheep and humans have remarkably similar physiological responses to stress. Their endocrine system and gestation patterns mirror humans, causing them to be prone to the same developmental issues and diseases. These similarities and the ability to perform chronic fetal instrumentation and long-term monitoring, make sheep ideal for studying the effects of PS on fetal development. ECG and intra-arterial glucose levels are monitored continuously through the experiment to derive HR and quantify chronic-metabolic effects of elevated maternal stress. Maternal plasma samples are obtained pre- and post-isolation for both control and isolation animals. Postmortem, both maternal and fetal adrenals, fetal liver and fetal brain tissues are analyzed histologically. While the model is now established, the data is currently being analyzed. We expect HR patterns to correlate with hormone levels and tissue outcomes, rendering HR as a non-invasive method of identifying changes in ANS caused by PS. Such HR patterns can be used as a predictor for complications during pregnancy, and neurodevelopmental issues post-partum due to PS in humans.

The California Current region encompasses a gradient between coastal upwelling and oligotrophic offshore zones, where the phytoplankton biomass is dominated by species of large and small celled phytoplankton, respectively. Mesoscale eddies are ubiquitous in the mixed region between the coastal and offshore zones, and they mediate the offshore transport of coastal waters. Mesoscale eddies are known to trap and transport water masses and their associated biogeochemical signatures, acting much like traveling mesocosms. It is unclear to what extent phytoplankton communities are trapped by the eddies, and how much they mingle with the communities found in the surrounding waters. In this study we characterized the phytoplankton community structure found within three mesoscale eddies, two cyclones and one anticyclone, using flow cytometry and 16S and 18S tag sequencing. We tracked the age and region of formation of the eddies from sea surface height. The two cyclonic eddies were formed in the same region, but at different times, C1 was 22 days old and C2 was 214 days old at the time of sampling. The anticyclonic eddy, A1, was formed further to the south and was 19 days old at the time of sampling. We assess to what extent the phytoplankton communities within each of the eddies are distinct from their surrounding waters, differ from each other, and how much the phytoplankton communities vary within each of the eddies with distance and depth. Our results show that the phytoplankton community is structured, to first order, by the physical environment, such that the phytoplankton metacommunities differ between eddies. However, we also see finer scale features with community changes within and across the eddies. These deviations reveal patchiness in the communities likely driven by corresponding local patchiness in nutrient and trace metal concentrations.

Studies have shown disparities in environmental justice as manifest by the variation of air pollution exposure based on race and socioeconomic status (SES) on a national scale. This study focused on determining whether a similar association exists in Washington. I hypothesized that in areas where people of color are the majority residents, exposure to air pollution as measured in PM2.5, will be higher. I also hypothesized that there would be a higher average concentration of PM2.5 in low-income areas. PM2.5 are solid particles with a diameter smaller than 2.5 micrometers. In this cross-sectional study, I used linear regression and analysis of variance to evaluate my hypotheses where I treated income as a continuous variable and majority residency by race as a categorical variable. Overall, I found a negative association between PM2.5 and income, which supports the hypothesis. I estimated that PM2.5 is expected to decrease by 0.03 µg/m3 on average per 10,000 dollars increase in income [95% CI: -0.032, -0.02.8]. I controlled for urbanity and education. When I considered effect modification, I found that the negative association was not present in rural areas. In descriptive analyses, I considered four different racial categories (White, Black, Latinos, and Asian). I observed that for areas where black people are the majority, the PM2.5 exposure is the highest, with a mean of 6.86 µg/m3 [95% CI: 6.82, 6.87], with a maximum of 7.65 µg/m3 and a minimum of 6.06 µg/m3. In comparison, in areas where white people are the majority population, the mean was 6.12 µg/m3 [95% CI: 6.11, 6.13], with a maximum of 8.12 µg/m3 and a minimum of 3.54 µg/m3. I conclude that there is a disparity in air quality based on ethnicity and SES in Washington State. Directly or indirectly, environmental injustice contributes a greater risk of health issues and diseases.