Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting 1-2% of individuals above age 65. The mechanisms underlying the pathogenesis of PD are still poorly understood. The most common genetic risk factor for PD is mutations in the gene Glucosidase, Beta Acid 1 (GBA1). GBA1 encodes the lysosomal enzyme glucocerebrosidase, which hydrolyzes glucosylceramide to glucose and ceramide. To study how GBA1 mutations contribute to PD pathogenesis, our laboratory has developed a Drosophila melanogaster model of GBA1 deficiency by introducing a deletion into a Drosophila homolog of human GBA1. My first project utilizes this Drosophila model of GBA1 deficiency to identify genetic modifiers of GBA1 pathogenicity by screening chromosomal deletions for alteration of the GBA1 deficiency phenotype of impaired locomotion and autophagy. Given that only 30% of human GBA1 mutation carriers develop PD by age 80, it is highly likely that GBA1 pathogenicity is modified by genetic factors. For this project, I am currently conducting crosses to obtain flies with the genotypes necessary for the screen. My second project aims to determine whether GBA1-mediated depletion of the lipid ceramide, a biosynthetic precursor to an important class of lipids in membrane composition called sphingosines, contributes to impairment of autophagy and subsequent neurodegeneration through dietary supplementation of ceramide in the Drosophila model of GBA1 deficiency. We hypothesize that ceramide depletion due to low glucocerebrosidase activity contributes to neurodegeneration by lowering cellular sphingosine levels, resulting in altered membrane composition such that fusion of vesicles to the lysosome is impaired. For this project, I conducted one preliminary trial and will conduct further trials aimed at determining an effective method and concentration for dietary supplementation of ceramide in Drosophila. Because lipid metabolism is highly conserved between flies and humans, we expect that genetic modifiers and metabolic effects of GBA1 deficiency identified using the Drosophila model will be applicable to human GBA1-mediated PD.

Recreating digital logic with cellular components has been a newly charted area of engineering research. This has applications in cellular diagnostics and expands our fundamental knowledge of cellular decision making processes. We tested a variety of digital logic systems of dCas9 repression networks in S. cerevisiae (yeast). dCas9 has be re-purposed to be a nucleus localized protein which function as a modular transcription factor, which when bound to specific guide RNA (gRNA) is directed to a distinct 20 base pair genomic sequence determined by the gRNA’s target sequence. Understanding the activity of the dCas9+gRNA complex, allowed us to use gRNA with a particular sequence to repress the expression of other gRNA. Using this insight, multiple repression cascades have been made with varying complexities to build unique logic pathways, also known as Boolean Expressions.  The expression of all gRNA and dCas9 proteins impact cell fitness because they divert carbon and energy for reproduction. The effects of metabolic load these exogenous components add to the cell is a factor that was investigated through cell growth. Cell growth was determined by fitting a logistic population model to a time-series of experimentally measured cell densities. We compared distinct strains of yeast with stable genomic modifications that explored the impact of increasing the network complexity and varying dCas9 expression levels.  We anticipate the results to concur with this statement and realize that a gRNA+dCas9 complex may further increase the metabolic burden of the cell from off target repression. Realizing how dCas9 repression networks lead to metabolic load will help guide their applied use in cellular applications ranging from developing programs for crop species to gene therapies.

Predator-prey models provide invaluable insight into interspecies relationships and fluctuations in population sizes, but like all models, must rely on simplifying assumptions that reduce complex natural processes to functions with explanatory and predictive utility. Logistic equations for population growth predict future population size as a function of current population size and competition for limited resources; the related Lotka-Volterra equations use similar assumptions but with predator-prey interactions limiting population growth. In North America, the Canada Lynx-Snowshoe Hare cycle is an ideal demonstration of interconnected predator-prey population sizes. When vegetation is plentiful, hares have the resources to bear many litters of offspring and they provide a high level of nourishment to lynx. Lynx and hare populations increase in number until vegetation becomes scarce and predation by lynx reduces the size of the hare population. At this point, hares are too scarce to sustain lynx population growth and lynx numbers decrease while plants repopulate the region. Lotka-Volterra equations capture this cyclical relationship but assume genetic uniformity between individuals and no age structure in the population. Extensions of predator-prey models have incorporated age structure but not genetic variation. My model examines the dynamics of this system when both aging and genetic variation for fitness are incorporated into predator-prey dynamics. Using the statistical programming language R, I present a simulation of predator-prey dynamics with these two additional parameters—variation in relative fitness among individuals and age structure. Analysis of “the hare that never got caught” is an inquiry into the influence of age structure on fitness. More broadly speaking, this work attempts to model the effect of aging on the efficiency of natural selection and provides a basis for future research regarding the role of aging in evolutionary biology.

Myeloid Derived Suppressor Cells (MDSCs) are a heterogeneous population of myeloid progenitor cells with an immune suppressive function. Their frequency increases during states of acute and chronic inflammation such as sepsis, cancer, trauma, transplantation and chronic viral infections. They act in a similar way to the Human Immunodeficiency Virus (HIV). We have demonstrated that blood MDSC frequencies increase during HIV infection and these MDSCs specifically inhibit T cell responses. We would like to examine the of role co-infections, recreational drugs, age and health could have in the already elevated frequencies of MDSC in HIV+ individuals. In our study, we enrolled three cohorts. The first cohort consisted of HIV- individuals (n=58). The second and third cohorts consisted of HIV+ individuals on combinational Antiretroviral Therapy (cART), virally suppressed for at least one year. The second cohort consisted of HIV+ individuals (n=57) excluding individuals who had co-infection, cancer or used drugs – recreationally or medically. The third cohort consisted of HIV+ individuals (n=51) and included those with co-infections, cancer and those using drugs. This was to further understand how chronic infections and other factors influence MDSC frequency. Data was collected on age, CD4 count, cancer history, bacterial acute and chronic infections. I was tasked with processing the blood – which I received in heparin tubes - and determining the MDSC frequency of the HIV+ cohorts. As we had shown before, we observed, an increase in MDSC frequency in the HIV+ group versus the HIV- group. I am currently performing statistical analyses on the HIV+ cohorts to determine the role co-factors - other than HIV - have in MDSC frequency. This will help us better inform people living with chronic infection how certain factors could affect their immune system.

Parkinson’s Disease (PD) is an age-related neurodegenerative disorder that leads to progressive decline in motor control. Tens of thousands of new cases are diagnosed each year. Many cases of PD are linked to chronic exposure to rotenone, a commonly used pesticide. As an inhibitor of complex I in the mitochondrial electron transport chain, rotenone causes deterioration of dopamine-producing neurons in the substantia nigra (part of the midbrain). Studies suggest that there is a genetic component to how likely one is to develop symptoms in response to rotenone exposure. To test this hypothesis, we used the Drosophila Genetic Reference Panel (DGRP), a set of 192 fully sequenced inbred lines of the fruit fly, Drosophila melanogaster. Exposure of Drosophila to rotenone has been shown to cause dopaminergic-specific degeneration, similar to the pathology observed in PD patients. When fed highly concentrated rotenone-infused food, we see significant variation in rates of survival among female flies from different lines of the DGRP, allowing us to capture natural variation in response to rotenone exposure. After most of the DGRP has been tested, we will carry out a genome-wide association study to identify specific genetic variants associated with rotenone resistance. In this way, we hope to uncover genetic factors that could point to specific mechanisms behind PD pathogenesis. With further related research, new genetic pathways could lead to new drug targets.

Even though Breast Cancer type 1 gene (BRCA1) was the first gene to be definitively linked to hereditary breast and ovarian cancer, much is still unknown about how BRCA1 influences cancer risk. What is known is that certain BRCA1 variants confer an increased risk of early onset breast, ovarian, prostate and peritoneal cancer, but there are also many patients with variants of unknown significance that have yet to be characterized. Current methods for variant characterization require arduous genetic manipulations of mammalian cell lines, which could be simplified if a homologous model organism was used. A BRCA1 homolog has been identified in the nematode, Caenorhabditis elegans, indicating that the nematode could be a useful BRCA1 model system. My preliminary results indicate that activity of BRCA1, the gene product of BRCA1, is conserved in the worm homolog as both proteins expressed functionally homologous ubiquitin-ligase activity when studied using purified components. In order to investigate the conserved residues in the C. elegans homolog that lead to the conserved activity, I am comparing the interactions of both BRCA1 homologs with the E2 enzyme (required for ubiquitin-ligase activity). Using site-directed mutagenesis, I am producing mutations at the hypothesized E2 binding site on the worm homolog that I predict will inhibit ubiquitin-ligase activity by preventing E2 binding. The impact of these mutations are being studied through biochemical ubiquitin-ligase assays using purified components. A nematode-based BRCA1 model would allow for hypotheses developed through in vitro or in cellulo studies to be tested in a whole organism, significantly expediting variant characterization. Understanding the importance of the conserved residues between the homologs will identify key regions of BRCA1 where patient variations may be more likely to pose a risk.

Approximately 40% of over-the-counter drugs target G protein-coupled receptors (GPCRs), which are seven-transmembrane proteins that become activated when drugs, hormones, or neurotransmitters bind to the receptor. One class of GPCRs, known as adrenergic receptors (ARs), are highly targeted by the endogenous catecholamines, epinephrine and norepinephrine. ARs are comprised of three unique subtypes: α1ARs, α2ARs, and βARs. Focusing on the α1ARs, the different types are α1A, α1B, and α1D. In cancer biology, most research has focused on βARs since many people take medications that specifically target βARs. In my research, I focus on α1BARs due to a recent publication of The Hague Lab, which recently discovered α1BARs in colorectal cancer cells. Reading this publication led me to search for α1BARs in various cancer cell lines by using the Label Free Dynamic Mass Redistribution (EPIC-DMR) method. After adding specific drugs, the EPIC-DMR tracks any conformational change of individual cells to indicate the cellular response to the drugs. Using this method, I discovered α1ARs are present in HCT116 cells, a colorectal cancer cell line. In the future, I intend to determine the α1AR subtype by performing radio-ligand binding, using Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR), and looking at agonist and antagonist responses. This discovery could potentially play an important role in therapeutic drug development for many cancer patients.

This study was conducted by students at the Ocean Research College Academy (ORCA), a dual-enrollment program for high school juniors and seniors at Everett Community College. The study utilized data from the longitudinal State of Possession Sound survey conducted by ORCA students since 2004. Possession Sound is located in the northern reaches of the regional Puget Sound basin, between Everett and Mukilteo, and has a significant freshwater influence from the Snohomish River on its eastern boundary. This study examines the effect of the El Nino weather pattern on the relationship between surface salinity and pH in the Sound at the Buoy site, which, due to its proximity to the river, is directly affected by temporal variation in river discharge. Within an estuarine system pH levels influence the biological processes of many organisms and are critical to the health of the estuary, by regulating the solubility of toxic chemicals. The pH of the Sound is affected by both the salinity of the water and biological productivity. For this study, measurements of surface salinity, pH, temperature, and chlorophyll were taken during the most recent La Nada and El Nino periods, June 2012 to Sept 2014 and December 2014 to June 2016 respectively. Data were collected with various Yellow Springs Instruments (YSI) probes (specifically the YSI 85, YSI 650, and YSI EXO-2 sonde). It was hypothesized that during La Nada, salinity would directly affect pH, with surface pH increasing during summer due to lower river discharge, and decreasing during spring and fall when precipitation and mountain runoff create higher river discharge. During El Nino, it was hypothesized that warmer temperatures and the related decrease in river discharge would cause pH to be more significantly influenced by biological factors, causing a correlation with temperature and chlorophyll instead of salinity.

The inner ear consists of hair cells which are responsible for hearing and balance. Over time, these hair cells die and get damaged, resulting in hearing loss. Curiously, chickens and other related species are capable of regenerating their lost hair cells via supporting cell differentiation, while mammals can only regenerate hair cells in the balance organ. To better understand the mammal regeneration process we are studying Cre-lox mice that allow us to change the gene expression in supporting cells which can differentiate into hair cells. These mice allow us to track supporting cells during regeneration, which types of hair cells they turn into, how they turn into hair cells, and to delete or misexpress genes in supporting cells, in order to test the role of various genes in hair cell regeneration. My research is examining three lines of Cre-lox mice, id2, glast, and sox9, to assess their usefulness for these studies. I studied the images of the mouse utricle, one balance organ, and determined which types of supporting cells, and how many, can be monitored or manipulated in each line of mice. Through the results of this project, we hope to better understand the specific process of mammalian hair cell regeneration, as well as prove this method as viable for analyzing specific gene expression in other cells.

Rhabdomyosarcoma (RMS) is a soft tissue cancer that most likely develops from skeletal muscle stem cells. Like other cancers, RMS is a disease driven by critical mutation(s) in the patients’ genome, causing otherwise normal cells to divide uncontrollably without differentiating. Current treatments of RMS include chemotherapy, radiation therapy and surgical removal of the tumor. However, these treatments are limited in that they are toxic to both cancer and normal cells and are frequently ineffective in killing cancer cells because they do not directly target the genetic mutations causing cancer. One promising strategy to treat cancer is to develop therapies targeting the essential genetic mutations causing cancer. By utilizing the novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated Protein 9 (Cas9) gene editing tool that is more accurate and reliable than its predecessors, my project aims to target and knock-out the deleterious genes that promote growth and progression of RMS cancer cells. The two major subtypes of RMS are embryonal (ERMS) and alveolar (ARMS) with which they are defined largely by either activation of the RAS pathway (ERMS) or the presence of a fusion oncogene between PAX3 and FOXO1 (ARMS). To demonstrate the viability of the CRISPR-mediated gene therapy, I first targeted critical exons in the oncogenes of both RMS subtypes using inducible or viral CRISPR/Cas9 delivery systems. Targeting these essential genes resulted in significant regression of tumor xenografts. To further demonstrate that tumor-specific CRISPR/Cas9 gene therapy can also be achieved, precise targeting of only the mutation site(s) of the oncogene followed. This strategy disrupts the essential cancer oncogenes with minimal impact to normal cells that do not exhibit the unique cancer mutations. This novel approach to cancer therapy provides a promising alternative for traditional treatments while establishing a foundation for further development of CRISPR/Cas9 gene therapy for cancer treatment.

Posttranslational modification with the small protein ubiquitin (Ub) is an universal and essential process in various regulatory pathways of eukaryotes (from yeast to humans), ranging from protein degradation to DNA repair. Misregulation of ubiquitination is associated with diseases such as cancer and neurodegenerative disorders. Ubiquitination is accomplished by an enzymatic cascade that involves a family of ~40 E2 conjugating enzymes, which form high-energy thioester (R–S–CO–R) intermediates with Ub, called E2~Ub conjugates. For the majority of ubiquitination reactions, it is the E2 that covalently attaches Ub to substrate proteins. While the overwhelming majority of E2s ubiquitinate the ε-amino group of substrates’ lysine residues, important exceptions have recently been discovered. For example, Ube2W has been identified as the only human E2 that attaches Ub exclusively to the ɑ-amino group of substrates’ unstructured N-termini. Knockout of Ube2W in mice has lead to severe developmental abnormalities, suggesting it is important for early postnatal survival. Little is known about the molecular mechanism of Ube2W’s unusual specificity and structural insights to the catalytically-relevant species, the Ube2W~Ub conjugate, are lacking. Through mutational analysis and biochemical assays, I seek to understand the following question: What role does Ube2W’s capability to dimerize and its unusually high number of cysteine residues play in its catalytic mechanism? In addition, I am using a combination of chemical biology and nuclear magnetic resonance (NMR) to study the Ube2W~Ub conjugate to track structural changes at the E2’s active site during catalysis. This investigation will provide insight into biochemically fascinating and biologically highly relevant enzyme.

The USDA defines a food desert as any census tract where at least 20% of the residents earn below the poverty line and at least 33% of the residents live more than one mile (urban locations) or ten miles (rural locations) from a supermarket selling fresh groceries. The presence of a food desert has been correlated with negative health outcomes in obesity, diabetes, and heart disease. The Healthy Food Financing Initiative launched in 2010 hoped to eliminate food deserts by 2016, but this result has yet to be seen in communities across America. However, since 2010 the geographical location and density of food deserts has shifted. It is unknown whether this shift is due to government intervention or demographic changes. Likewise, the census tracts where food deserts have appeared or disappeared have yet to be characterized. This study utilized data from the USDA, US census, and American Community Survey to identify how demographic changes and government spending is related to food desert movement between 2010 and 2015. This data was also used to characterize individual census tracts for poverty, SNAP enrollment, and other factors that may explain food desert movement. A regression analysis produced a more complete explanation of this phenomenon through an empirical and quantitative examination of the relationship between these metrics. Ultimately, this geographical and statistical study will serve as both a policy and a demographic analysis that explains trends in recent food desert movement. Researchers and policymakers can expand upon the conclusions of this study to create more targeted, effective, and efficient programming that improves food access across the United States.

Mycobacterium tuberculosis (Mtb) is a widespread disease affecting about 33% of the human population around the world. Treatment of tuberculosis (TB) is hampered by a need for lengthy, multi-drug regimens and drug resistance requiring development of novel therapeutics. Our laboratory is interested in harnessing the host immune response to enhance Mtb killing within the macrophage. Within the host, Mtb is found within lipid-laden macrophages where lipid droplets are hypothesized to both provide nutrients as well as sequester Mtb from intracellular killing mechanisms. Hydroxycarboxylic acid receptor 2 (HCA2) is a G-protein coupled receptor (GPCR) that maintains lipid droplets after activation by its ligands nicotinic acid (NA) and beta-hydroxybutyrate (BHB). In these studies, we assess NA, BHB, and the structurally related small molecules nicotinamide (NAM), sodium butyrate (NaB) and phenylbutyrate (PBA) modulate HCA2 signaling. We have transiently transfected CHO-K1 cells with plasmids encoding human HCA2 and aequorin, the latter which oxidizes coelenterazine in a reaction that generates a luminescent signal. The effective concentration (EC50) of each small molecule as a ligand for HCA2 can then be calculated from the luminescence curve. Preliminary work in our lab suggests that PBA inhibits Mtb replication within the host macrophage, but several mechanisms are likely involved. We hypothesize that NaB, PBA, and NAM will also modulate HCA2 activity given their similar structures to NA and BHB. Future studies will also interrogate whether this panel of small molecules impacts HCA2 gene expression, lipid droplet formation and Mtb replication in macrophages.

SUMO (small ubiquitin-like modifier) is a protein post-translational modification that has wide ranging influence on protein function. Among its many roles, SUMO is known to regulate transcription, DNA repair, and maintain cellular homeostasis under environmental stress conditions. Changes in sumoylation patterns under environmental stress have been identified, yet the specifics of these pathways remain uncharacterized. By using mass spectrometry (MS), we have identified three yeast proteins that are sumoylated in response to high levels of ethanol: Top2, Smc5, and Smc6. These proteins play a major role in maintaining DNA stability during replication and are conserved through higher eukaryotes. We have tagged these proteins with epitopes identifiable by western blot by yeast transformation, and subjected cells with these tags to ethanol stress, which allowed us to verify the hits from the MS screen. This will allow us to begin further investigation of the role sumoylation plays on these proteins. Our next goals include generating sumo deficient mutants and observing the changes in protein function within the cell after exposure to ethanol stress. As these proteins are involved in DNA stability and repair, we are notably interested in the interactions with chromatin. Through this work, we seek to gain us a better understanding of the signaling underlying adaptation to environmental stress. This research has implications for human health as it allows us to understand how cells respond to toxic levels of a recreationally encountered substance.

The ability to recapitulate the dynamic presentation of signals in a stem cell’s microenvironment remains a major hurdle in tissue engineering. By controlling cell growth and differentiation in 4 dimensions (i.e., time and 3D space) heterogeneous synthetic tissues could be produced that match the complexity of their native counterparts. Combining strategies in both light-programmable hydrogels and recombinant protein engineering, we control the cellular microenvironment. Previously, we have demonstrated the ability to photopattern gels with fluorescent proteins by introducing a biorthogonal synthetic handle site-specifically modified onto our proteins via the sortase enzyme, and a photocaged reactive group in the hydrogrels. The photocaged group uncages upon UV light exposure, allowing for a reaction to occur and a covalent bond to form between the synthetic handle and the hydrogel. While covalent interactions form strong bonds, they are generally irreversible using mild, cytocompatible chemistries. In addition, this technique uses UV light, which may be harmful to cells. By incorporating LOVTRAP into the photopatterning system, we introduce a greater amount of control by allowing for full reversibility. In the dark, LOV2 and Zdk1 dimerize to form a protein-protein bond with similar stability to an antibody-antigen interaction. Upon exposure to blue light, the dimer dissociates, allowing for fully reversible control over conjugation. The Zdk1 domain can be incorporated into a protein of interest using genetic engineering, while still maintaining its binding affinity for LOV2. LOV2 can then be conjugated throughout a hydrogel to make a light responsive protein binding material. By utilizing a light controlled dimerization event between proteins, the LOVTRAP system is fully reversible using only blue light. This technique could be used for tissue engineering applications that require mild treatment conditions and full dynamic control over the presentation of biochemical cues.

Traditional antibiotics are known to wipe out many species of bacteria, but often leave several resistant microbes to dominate. These resistant bacteria are a huge concern for human health. Medical researchers are on the hunt for new approaches to combat bacterial infections on a large scale. It has recently been discovered that bacteria communicate through Quorum Sensing (QS), which if inhibited, presents a promising strategy to control these pathogenic bacteria. Instead of eliminating the weakest strain of bacteria resulting in a proliferation of antibiotic resistance, inhibition of quorum sensing silences gene expression, virulence and pathogenicity. A variety of marine organisms have demonstrated quorum sensing inhibition (QSI) potential, however there are many marine organisms that have yet to be screened for QSI compounds. In this study, three species of marine algae native to the Pacific Northwest were investigated for quorum sensing inhibition (QSI) capabilities. Ulva Lactuca, Chondracanthus exasperatus, and Iridaea Cordata were subjected to QSI capability testing that involved use of the indicator strain Chromobacterium violaceum Bergonzini (ATC 12472). The indicator bacterium uses the QS signal N-acyl-homoserine lactone (AHL) to produce a purple pigment. Each species of algae was overlaid with agar inoculated with Chromobacterium violaceum and incubated overnight. Absence of pigment in the indicator strain suggested the presence of a QSI compound. Preliminary evidence indicated that two of the three species of algae tested demonstrated QSI activity. The next step of this research involves identification of the specific compounds involved in QSI, thereby contributing to the expanding catalogue of known QSI molecules from marine environments. Ultimately this work aims to make a contribution to the fight against antibiotic resistant pathogens.

Managing cultures is a vital task in synthetic biology, but constantly measuring and adjusting culture conditions is both tedious and labor intensive. Modern assays that accomplish this, such as High Precision Liquid Chromatography, can also be prohibitively expensive. Our project aims to reduce the amount of time and effort needed to maintain cultures through the creation of an affordable image analysis system that autonomously reads visual data to measure the current state of a culture and then determines whether to release inducer chemicals based on user input. Our project utilizes the violacein pathway to simulate other metabolic pathways by producing four colored signals dependant on a two gene switch. These outputs are then measured by an open-sourced Raspberry Pi (a small, standalone computer) setup, which captures visual data via camera, measures the culture’s RGB value, and then directs the gradual release of inducer chemicals to maintain or change the culture’s color over time. Currently, yeast strains capable of coexpressing both a violacein and a non-visible pathway are being cloned. In addition to this, a prototype containing 3D-printed syringe pumps, a chemostat, and image analysis software in development. By combining both biological and hardware systems, we expect our unique design to be able to generate previously unavailable visual data regarding certain biosynthesis processes, such as those involved in antibiotic production and fermentation.

Living organisms are composed of cells; thus, cells are called the basic building blocks of life. In order for an organism to grow, cells undergo mitosis. During mitosis, chromosomal DNA is replicated and segregated into two daughter cells. Errors in segregation can lead to the development of some forms of cancer. Because of this, equal chromosome segregation is a critical mitotic process. Microtubules are fibril-like cytoskeletal structures that are responsible for chromosome separation. They originate from two centrosomes. The growth and extension of microtubules push the two centrosomes apart, creating two poles in a mitotic cell. Kinetochores bind to these microtubules and align at the center of the cell. Disassembly of these microtubules is coupled to the equal segregation of sister chromatids. In Saccharomyces cerevisiae, correct kinetochore-microtubule attachment is mediated by the Ndc80 and Dam1 complexes. The heterotetrameric and rod-like Ndc80 complex is the main microtubule-binding component of the kinetochore. The heterodecameric Dam1 complex forms oligomeric rings around microtubules and enhances the microtubule-binding capacity of the Ndc80 complex. In vitro, the Ndc80 complex bridges two Dam1 complex rings, yielding a specific 33 nm inter-ring distance. To investigate the importance of this distance, we constructed various mutant Ndc80 complexes to further increase the Dam1 complex inter-ring distance. Specifically, each Ndc80 complex mutant contained an 8, 10, or 12 heptad repeat insertion that elongated the length of the coiled-coil domain of the Ndc80 complex by 8, 10, and 12 nm, respectively. We tested if these mutant Ndc80 complexes support cell division by transforming a yeast strain that does not contain wild-type Ndc80 gene and selecting for growth on SD-ura low adenine plates. These produced non-sectoring red colonies; therefore, the constructs did not support cell division. This suggests the specific 33 nm Dam1 complex inter-ring distance is important for cell viability.

Although increasing utilization of outreach clinics has increased accessibility to subspecialty care for pediatric patients, the availability of pediatric urologic imaging in local settings has not been previously described. We undertook this study to describe the availability of common radiographic tests ordered by pediatric urologists, and to determine if availability disparities exist between urban and rural locations. We identified 88 freestanding hospitals in Washington and contacted via phone survey asking whether they offered flat plate abdominal X-rays (AXR), renal-bladder ultrasounds (RBUS), voiding cystourethrograms (VCUG), MAG-3 renal scans, and nuclear cystograms (NC) to pediatric patients.74 institutions (84.1%) completed the survey; 17 (23.0%) were rural (population <2500), and 25 (33.8%) were in urban areas (population >50,000). 73 (98.6%) institutions offered AXRs, 68 (91.9%) offered RBUSs, 44 (59.5%) offered VCUGs, 26 (35.1%) offered MAG-3s, and 15 (20.3%) offered NCs to children. The availability of AXRs (100% versus 96%, p=0.88) and RBUSs (70.6% versus 96%, p=0.15) was similar in rural and urban settings, while VCUGs (11.8% vs 72%, p=0.001), MAG-3s (5.9% vs. 60%, p=0.006), and NCs (0% vs 44%, p=0.017) were more commonly available in urban settings. Rural hospitals were less likely to employ in-person radiologists (35.3% vs 96%, p<0.0001) or offer sedation (6.3% vs 36%, p=0.01) during testing, but were equally likely to offer child life (0% vs 20%, p=0.28) and to have age restrictions on the tests offered (17.6% vs 40%, p=0.50). Fellowship-trained pediatric radiologists were employed exclusively in urban settings (16% vs 0%, p=0.39).Our findings portrayed that rural Washington state hospitals have less access to radiographic tests and are promoted by broad radiographic procedures that are not specialized to the individual pediatric patient. These results bring forth a future set of criteria for each Washington state hospital in how they treat pediatric patients with more inclusive treatment plans.

Social determinants of health are non-biologic factors that can strongly influence individual health status, healthcare access, and disease vulnerability. While this subject is well studied, limited data exists on the influence of these social factors on surgery, and more specifically, recovery after surgery. The burden of surgical disease remains taxing globally and disproportionately affects marginalized populations. Due to the limited access to higher level surgical care, patients often travel from eastern Washington and various other rural areas to Virginia Mason Medical Center for cardiothoracic procedures. Through this study I aim to identify social-geographic factors that contribute to this burden, and determine the discrepancies that generate variances in recovery. I hypothesize that among patients undergoing thoracic surgery at Virginia Mason Medical Center, distance from Seattle will be associated with measures of recovery in the postoperative period. I quantify the postoperative recovery of patients by measuring length of stay, postoperative complications, and readmission. Due to the burden of transport, inconvenience, missed work, increased personal costs, and other socioeconomic issues, I expected to see slower recovery, and possibly greater complications among patients coming from further distances. By first characterizing the problem, I then identify unique challenges that arise among various demographics of patients, creating a tailored perioperative education. This optimizes clarity in communication of postoperative planning and potential complications to create an improved set of guidelines, specified towards particular demographics of patients.

Widespread in tropical and subtropical oceans, the marine Cyanobacteria Prochlorococcus are the smallest and most abundant photosynthetic organisms on Earth, playing an important role in global primary production. As a collective group, Prochlorococcus have a very large pangenome, consisting of a conserved core genome and variant accessory genes. Prochlorococcus forms two ecotypes, the low-light (LL) adapted lineages—which possess large amounts of accessory genes—and the more streamlined high-light (HL) adapted lineages. The gain and loss of accessory genes in Prochlorococcus reflects their genomic and physiological differences and allows ocean niche differentiation. Recently, uncultivated lineages of LL-adapted Prochlorococcus have been identified in oxygen deficient zones (ODZs) that dominate under high-nutrient, low-light, and low-oxygen conditions. In my research, I explored the possibility that LL-adapted ODZ Prochlorococcus possess accessory genes related to low-oxygen stress (e.g. redox-sensitive enzymes and redox-dependent metabolic processes such as photosynthetic electron transport and photorespiration) that are likely lost in HL-adapted Prochlorococcus. To understand the effects of low-oxygen stress on the LL-adapted Cyanobacteria Prochlorococcus, I assessed the genomic potential for low-oxygen genes through computational analysis of a metagenomics dataset capturing native ODZ Prochlorococcus as well as the annotated genomes of the LL-adapted cultured isolates MIT9303 and MIT9313. I am currently measuring the physiological response of LL-adapted culture isolate MIT9313 under low oxygen conditions through growth experiments, during which the presence and scope of metabolites showing changes in redox chemistry due to low-oxygen stress will be analyzed using liquid chromatography mass spectrometry. My results will show if LL-IV strains contain low-oxygen accessory genes, and if they combat the negative physiological effects of low O2 concentrations. As ODZs grow the global rising temperatures, knowing how LL-adapted Prochlorococcus responds to low-oxygen stress could give insight on how primary production will be affected by a changing ocean.

Alkanes are the major constituents of natural gas and petroleum. The development of catalysts for the efficient and cost-effective catalytic conversion of alkanes to functionalized organics is highly desirable. Previously, Nishiyama and coworkers found that the Ir^III complex (^dmPhebox)Ir(OAc)₂(OH₂) (Phebox = 3,5-dimethylphenyl-2,6-bis(oxazolinyl)) could activate the C-H bonds of n-octane at high temperatures under nitrogen. Goldberg and coworkers found upon increasing the temperature, β-H elimination could be promoted, giving isomers of n-octene. Reactions of similar Ir complexes with alcohols, ketones and other organic substrates will be presented.

Stimuli-responsive hydrogels represent a promising class of inks for use in 3D-bioprinting. These materials respond to external stimuli such as heat, light, pH, and pressure, and researchers have utilized these different stimuli responses to develop inks for direct-write 3D printing. The objective of our work is to understand how the composition of poly(alkyl glycidyl ethers)--more specifically, allyl-, ethyl-, isopropyl, n-propyl-, and methyl-glycidyl ether homopolymers--affects their thermal response in aqueous solutions. UV-Vis Spectroscopy was used to determine the lower critical solution temperature (LCST) of each homopolymer, varying the molecular weight, concentration and polymer composition to demonstrate poly(alkyl glycidyl ethers) versatility as a platform for synthesis of stimuli-responsive hydrogels. We determined that the identity of the alkyl group significantly affected the temperature-response of the respective hydrogel. These studies will guide our work toward developing temperature and shear-responsive inks for 3D-bioprinting. In particular, these materials can be used to develop printed hydrogel lattices for whole-cell catalysis to produce medicinal compounds.

Diatoms are responsible for roughly 45% of all oceanic primary production and play a unique role in carbon cycling. This crucial role makes understanding their behavior and interactions vital. In the environment, diatom samples are typically xenic (marine bacteria present around and on the diatoms). These diatom-bacterial interactions have been shown to have striking effects on diatom growth rates due to bacterial release phytohormones. These plant-based signaling molecules are well characterized in terrestrial organisms but their marine impact is not fully understood. One such phytohormone, indole-3-actetic acid (IAA), is responsible for bacteria-plant mutualistic behavior on land. Previous work in our lab has shown that addition of IAA to axenic diatoms has no effect at moderate levels and is lethal at high levels. However, when diatoms are co-cultured with IAA-producing bacteria, their growth rate increased. Thus, IAA alone is not sufficient to induce increased growth. We used this information, and the known bacterial IAA biosynthetic pathway to select IAA derivatives and precursors (methyl-IAA, Tryptophol, Tryptamine, Indole-3-acetamide, indole-3-carboxylic acid, and Tryptophan) to test their endogenous expression in marine bacteria found on diatoms in the environment. Of the bacterial species tested, none expressed every compound and most expressed novel combinations. This project tests the effects of various IAA derivatives as well as a cytokinin (phytohormone),6-(γ,γ-Dimethylallylamino)purine (2ip), on diatom growth. We investigate suites of compounds, based on bacterial expression data, as well as individual compounds. We also seek to use phylogenetic tree building to identify the bacterial producers of these compounds using IAA-producing, terrestrial bacteria as a reference. Impacts of bacteria on diatom growth, blooms, behavior and the metabolites left behind from these interactions would aid in analyzing environmental transcriptomic data and ultimately result in a fuller understanding of diatoms and their response to chemical stimuli.

Understanding the mechanisms of plant biotic defense is imperative to help decrease agricultural crop damage by pathogens. Pathogens can potentially enhance infections by inducing DNA damage within the host. One example is DNA double-strand breaks caused by Pseudomonas syringae, a common bacterial pathogen in plants. Plants encode various mechanisms to repair double-strand breaks, including a pathway called homologous recombination repair that is regulated by the protein complex RPA (Replication Protein A) in response to abiotic stress. We further hypothesize that RPA plays a central role in regulating repair of pathogen-induced double-strand breaks. To test this, we will quantify hyper-susceptibility of mutant lines of Arabidopsis thaliana in response to pathogen infection (P. Syringae), employing a plant infiltration assay. This assay involves whole plant infiltration, followed by measurement of CFU counts of persisting bacteria within the leaf tissue. Of particular interest in this study is the role of one subunit of RPA, termed RPA1E, since mutants of the encoding gene display strong hypersensitivity to agents that induce double-strand breaks. Our current results suggest other RPA subunits (RPA1C) display hyper-susceptibility to pathogen infection. These results will ultimately provide initial insight in how agricultural crops defend themselves from microbial invasion by determining whether RPA1E responds to a pathogen infection and if it is required in the defense response of A. thaliana.

Juvenile Idiopathic Arthritis (JIA) is the most common rheumatologic disorder found in children, affecting millions worldwide. It has no known cause, prevention, or cure, and can lead to debilitating destruction of the joints. In adult Rheumatoid Arthritis (RA), previous research suggests a connection between RA and oral bacteria that cause inflammatory diseases. Specifically, Porphyromonas gingivalis, found in patients with periodontitis, expresses an enzyme that modifies the amino acid arginine to citrulline, which the immune system recognizes as foreign, reacting to produce anti--citrullinated protein antibodies (ACPAs), which attack otherwise healthy proteins in the body. Therefore, we hypothesize that in JIA, the unique bacterial colonies of the mouth induce a chronic ACPA production that leads to arthritis. We use enzyme-linked immunosorbent assays to assay patient plasma for ACPAs with 6 peptides. We have tested plasma from 79 patients with JIA, 56 children recruited from dental clinic, and 29 healthy children for antibodies to citrullinated filaggrin, a known autoantigen in RA. Compared to healthy controls, JIA patients had a significant increase in anti-citrullinated filaggrin antibodies (relative levels 0.88 vs. 0.45, p=0.001 by t-test). Anti-filaggrin antibodies were elevated in patients with oligoarticular JIA, (mean 0.62, p=0.0012) and extended-oligoarticular JIA, (mean 0.88, p=0.007), but not polyarticular JIA (mean 0.49, p=0.47). In addition, there is a higher prevalence of bleeding on probing (BOP, an indicator of gingival inflammation) in JIA, with an average BOP score of more than two times that of children from dental clinic (p=0.008). Our next step is to see if there are any specific microbes associated with higher BOP scores and ACPA levels. If we find that oral pathogens are associated with inflammatory disease, prevention can be designed to eliminate plaque-associated pathogens through various dental hygiene practices.

Exposure to ionizing radiation is the longest-established environmental cause of human breast cancer in both women and men. Evidence indicates that HER2-positive breast cancers are more likely to spread and are more resistant to anticancer-treatments. The goal of our study is to investigate genetic variations that are introduced during breast carcinogenesis. As a progressive stages of breast carcinogenesis cell model, normal human breast stem cells were sequentially transformed with SV40 large T antigen, x-ray radiation, and HER2 (ERBB2) overexpression to generate non-tumorigenic/immortalized cells, weakly tumorigenic cells, and highly tumorigenic cells, respectively. We sequenced 82 genes that are frequently mutated in breast cancer using next generation sequencing. We identified 26 radiation-specific mutations in exonic or intronic splicing regions that are not present in immortalized cells, but are present only in weakly tumorigenic cells. Among the 26 mutations, a single nucleotide deletion in RECQL gene, which is involved in DNA repair, is highly mutated (43%). Mutations induced by the overexpression of HER2 were identified by analyzing mutations present in highly tumorigenic cells but not in weakly tumorigenic cells. Two HER2 overexpression-specific mutations PRSS1 p.K225R and RET p.F683Y were identified with the mutation RET p.F683Y present at a high prevalence of 54%. The RECQL deletion induced by radiation and the two nonsynonymous mutations PRSS1 p.K225R and RET p.F683Y induced by HER2 overexpression have not been previously reported by others. Studies are underway to further characterize genetic variations that are induced by SV40-T Ag, radiation, and HER2 overexpression in breast carcinogenesis. The identified mutations that are specific to these oncogenic treatments aid in characterizing molecular subtypes of breast cancers and provide a foundation of identifying potential target mutations that can serve as biomarkers for the sequential stages of breast carcinogenesis.

Aggressive cancers are resistant to apoptosis, and stem-like cancer cells might be responsible for re-growth of cancerous tumors. This inspires the search for compounds that could aid in the destruction of cancer cells with stem-like properties. Drosophila germ line stem cells under radiative or chemo-toxic stress are protected by a molecular signal from apoptotic daughter cells via the TIE receptor. Thwarting this protective signal by the introduction of a small molecule may lead to the discovery of compounds that can be effective against stem-like cancer cells. The National Cancer Institute (NCI) diversity set of 1,596 small molecules is screened, in vivo, to find compounds that can induce apoptosis in the germ-line stem cells of Drosophila melanogaster. We tested several compounds from the NCI diversity set on 0-5 day-old fruit flies to get an understanding of the effects of the compounds on stem cells. Multiple compounds, including Camptothecin and Maytansinol, have been found that can induce apoptosis in both the male and female germ-line stem cells. Future investigations will uncover the mechanism of action and efficacy against stem-like cancer cells. After determining which compounds work effectively to target stem cells, this knowledge will be applied to understanding the mechanism responsible for resistance to cell death by stem cells and potentially some cancer cells, which could lead to more effective cancer-eradicating drugs.

The epithelial-mesenchymal transition (EMT) is a reversible process in which epithelial cells adopt mesenchymal properties by altering their morphology, adhesion, and migratory capacity. During tumor progression, the EMT underlies a process that allows benign tumor cells to infiltrate surrounding tissue and metastasize to distant sites. Our lab has identified a non-canonical Wnt signaling that involves ligands (Wnt 5a/b) and Frizzled2 receptor (Fzd2) in regulating EMT as well as tumor metastasis. Inhibition of Fzd2 signaling suppresses EMT and the progression of metastatic cancer cells both in vitro and in vivo. Recently, we have discovered a non-receptor proline-rich tyrosine kinase 2 (Pyk2) that plays a critical role in Fzd2-mediated cell migration. However, details of Fzd2-mediated activation of Pyk2 and mechanistic understanding of Pyk2 interactions in the pathway still need to be elucidated. We hypothesized that phosphorylation of Fzd2 induces activation of Pyk2, a possible contributor in Fzd2-mediated EMT pathway and tumor metastasis and aimed to uncover 1) the role Pyk2 in Fzd2-mediated EMT induction and cancer metastasis 2) the new mechanistic understanding of how Pyk2 interacts with proteins in the Fzd2 complex. Our preliminary data showed that inhibiting kinase activity of Pyk2 downregulated mesenchymal markers such as vimentin and upregulated epithelial markers such as E-cadherin. Then, the study focused on perturbing Pyk2 expression by RNAi and assessing EMT markers, and cell motility as well as gaining a mechanistic understanding of how Pyk2 interacts with other proteins in the complex. A better understanding of Fzd2-Pyk2 signaling would not only help in connecting critical nodes in Wnt pathway but may open up potential treatment options for metastatic cancers.

Youth with T1D, although encouraged to exercise as part of their treatment plan, are not implementing recommendations to adjust insulin around exercise. Little is known as to whether insulin delivery method impacts these behaviors. Our aim was to compare behaviors surrounding exercise between youth on insulin pump therapy and those on insulin injections, specifically focusing on evening behaviors after exercise. We recruited 50 youth aged 10-18 years with T1D who completed the Type 1 Diabetes Report of Exercise Practices Survey (T1D-REPS), a questionnaire designed to assess specific practices around insulin management, nutrient intake, and glucose monitoring. 30 were on insulin pump therapy and 20 on injection therapy. We measured frequency of glucose checks and hemoglobin A1C (HbA1c). HbA1c measures average blood glucose level over the preceding 3 months. Higher HbA1c indicates worse blood glucose levels. Pump users were more likely to be male (73%), had lower HbA1c (8.5% ±1.3 v. 9.8% ±2.7, p<.05), and reported more glucose checks per day (5.1±3.0 v. 3.2±1.9, p<.05). Pump users were less likely to check their blood glucose prior to exercise (37% of pump users reported often or always checking v. 60% of non-pump users; p=.07). Pump users were also less likely to adjust insulin for bedtime snacks following exercise (8% of pump users v. 31% of non-pump users, p<.05).  Overall, pump users reported less glucose monitoring before exercise and were less likely to implement interventions to prevent overnight hypoglycemia, though they displayed more frequent monitoring around exercise. Understanding populations at risk for glycemic excursions around exercise will allow for the development of intervention strategies to improve adherence to these recommendations.

Cancer stem cells (CSCs) are small populations found within tumors and appear to be more resistant to anti-cancer treatments. Although evidence supports that CSCs play a major role in initiation and in the clinical outcome of cancers, the roles of breast CSCs in metastasis and invasiveness are not fully understood. Moreover, the association of the invasiveness of breast CSCs with genomic variations is unclear. The overall goal of this study is to investigate the migration abilities and genomic expression changes in breast CSCs. Human breast CSCs and immortalized/non-tumorigenic cells were derived from the same parental breast normal stem cells. To recapitulate tumor microenvironment, the cells were cultured on nanotopography-defined extracellular matrix (ECM)-mimetic platforms. We have employed a live cell imaging microscopy to measure migration at the single cell level as an indicator of invasiveness in breast tumorigenic and immortalized cells. Our preliminary results indicate that breast CSCs migrate significantly faster than immortalized cells on ECM mimics. Interestingly, we only find this difference under ECM-mimetic culture conditions, but not under flat control surfaces lacking ECM structures. This suggests that the invasive abilities of breast CSCs are lost when they are cultured in vitro on flat control surfaces without ECM structures. We will perform RNA sequencing to identify differences between the transcriptomes of breast CSCs and immortalized cells, and between cells cultured on ECM-mimics and substrates without ECM-mimics. The current study will characterize transcriptome profiling in breast CSCs and identify the genes that are differentially expressed between breast CSCs and immortalized cells. This will have implications in classifying molecular subtypes of breast cancers and in identifying potential therapeutic targets to regulate specific gene expressions for the inhibition of breast CSC invasiveness.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation in either of the genes PKD1 or PKD2. This disease has been the focus of ongoing research due to its high prevalence and lack of curative treatments. How does ADPKD develop? To understand cyst formation in ADPKD, we are utilizing the murine model PKD1 p.R3277C (RC). Human dominant expression of the APKD phenotype is not similarly found in mice; null mutations of PKD1 or PKD2 are embryonic lethal, and the cystic phenotype of mice is observed only in conditional deletion mutants. However, the RC murine model recapitulates the late onset cystic growth of human ADPKD. The morphology of the pre-cystic kidney was characterized by formalin-fixed paraffin embedded (FFPE) sectioning in combination with H & E staining performed on RC homozygous mutant and control kidneys at post-natal time points of p20 (days), p40, p60, and p80. In the future, RNA from RC mutant and wild-type kidneys at p20 will be collected and sequenced. RNA sequencing will be utilized to determine pre-cystic expression changes between affected and unaffected phenotypes. Although less advanced, cyst formation has been shown to occur as early as p20. Because of this, the earlier time point of p10 was also assessed. By investigating these time-points we aim to identify the earliest differences that we hypothesize are more likely to be causal for cyst formation, versus changes that are a secondary consequence of PKD pathology.

DNA replication is the process by which genetic information is duplicated and passed from cell to cell. The process of DNA replication occurs with the assistance of various enzymes, including DNA polymerases, which synthesize new DNA molecules using template DNA and deoxyribonucleotides (dNTPs). Faithful DNA replication is vital for population survival and protected by exonuclease domains on the major DNA polymerases, which proofread replication errors. A secondary error-correction pathway known as mismatch repair (MMR) also scans the genome for replication errors. Defects in proofreading and MMR lead to "mutator phenotypes" marked by elevated mutation rates and increased cellular mutation burden.  In microbial populations, mutators spontaneously emerge and can increase adaptation to new environments by fueling genetic diversity. However, at extreme mutation rates, the accumulation of harmful mutations can also drive extinction of mutators.  Some mutator cells survive this "error-induced extinction" (EEX) by acquiring antimutator mutations that suppress the mutator phenotype. We have identified Saccharomyces cerevisiae strains that suppress elevated mutation rates through a screen for mutants that survive EEX. Using these eex mutant strains, we are currently mapping the genetic changes responsible for the antimutator phenotypes. Changes in mutation rates in haploid spores derived from these eex suppressor strains are being tracked to identify antimutator segregants, which are then being subjected to whole genome sequencing. As mutator phenotypes are important drivers in cancer, our analyses may reveal novel pathways that influence cancer evolution.

Alzheimer’s disease (AD) is the most prominent cause of dementia worldwide, affecting up to 5.3 million elderly Americans. AD pathology includes decreased neuronal count and synaptic connections, intracellular pathology and increased affinity for neurofibrillary tangles resulting in cognitive deterioration. The hippocampus is known to be particularly vulnerable and is an early structural biomarker of AD. Our aim is to investigate the longitudinal relationships between hippocampal volume and memory performance using neuroimaging data from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database. The ADNI database contains biomarker, neuroimaging, cognitive, and behavioral data from hundreds of elderly individuals, divided into groups including normal/normal aging (CN), Alzheimer’s Disease (AD), Early Mild Cognitive Impairment (EMCI), and Late Mild Cognitive Impairment (LMCI). Cerebrospinal fluid biomarkers are a “gold standard” for detecting the presence of amyloid and tau pathology. Neuroimaging methods include amyloid PET tracers (i.e. F-18 florbetapir), which detects amyloid beta plaques and FDG-PET imaging which measures metabolic activity within the brain. Both techniques reflect neurodegeneration and synaptic dysfunction. Structural MRI imaging is used to assess neuroanatomy as a measure of brain atrophy and changes in neuronal density. Cognitive assessment data quantifies the extent of memory loss and general cognitive decline. Using the R programming language, we will conduct longitudinal analyses of neuroimaging analyses to examine the relationship between cognitive, structural and metabolic neuroimaging variables across different diagnostic groups over time. This initial investigation is preparation for learning longitudinal methods that will later be applied to physiological measures within the entire brain. Our hypothesis is that a decrease in hippocampal volume will be less correlated to decline in memory in CN than in other groups. If our hypothesis is correct, it suggests that compensatory mechanisms preserve cognition in early stages of AD, which we can explore using subsequent extended analyses of functional connectivity.

Serotonin is a key player in the regulation of anxiety, fear, and stress. While acute stress is associated with increased activity in serotonergic neurons, chronic stress is characterized by the transition to decreased serotonergic neuron activity, the desensitization of serotonin autoreceptors, and the development of maladaptive emotional behaviors. We hypothesize that chronic stress causes dysregulation of serotonergic neuron excitability, and that these adaptations affect emotional responses to future exposure to stressful and hedonic stimuli. The goal of this study is to determine if alterations to serotonergic neuron excitability can alter the effects of stress on subsequent emotional behavior. We predict that reducing or intensifying the excitability of serotoninergic neurons will decrease and increase maladaptive behavioral responses to chronic stress, respectively. We expose experimental groups of wild type and transgenic mice expressing either excitatory or inhibitory DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) to chronic stress. The mice undergo 5 consecutive days of social defeat stress, during which they receive injections of saline or clozapine-N-oxide (the ligand for DREADDs). Following chronic exposure to stress, behavioral responses are measured through sucrose preference, social interaction, and immobility during forced swim tests. Mice expressing inhibitory DREADDs are anticipated to exhibit greater hedonic responses (increased sucrose preference), improved sociability, and decreased immobility during forced swim, compared to wild-type mice. Mice expressing excitatory DREADDS are anticipated to show decreased sucrose preference, decreased sociability, and increased mobility during forced swim. By altering the activity of serotonergic neurons, we can potentially modify the emotional and behavioral effects of stress. This study could provide new insight into the role of the serotonergic system in behavioral responses to chronic stress, and yield new therapeutic targets for conditions such as depression and post-traumatic stress disorder.

Sorsby Fundus Dystrophy (SFD) is a rare autosomal-dominant macular dystrophy that causes central vision loss in early adulthood due to mutations in the TIMP3 gene, yet its pathogenesis remains unclear. SFD is clinically similar to age-related macular degeneration (AMD), the leading cause of vision loss in older persons in the United States. Preliminary data revealed structural and functional differences between retinal pigment epithelial (RPE) cells differentiated from SFD patient-derived induced pluripotent stem cells (iPSC) and control iPSC-derived RPE cells. In contrast to control iPSC-derived RPE, SFD RPE does not accumulate any detectable extracellular matrix (ECM) and demonstrates a 40-fold increase in the level of intracellular hydroxyproline, a collagen degradation product whose increased expression adversely impacts SFD RPE metabolism and increases its susceptibility to oxidative damage. We wanted to determine whether differential protein expression between SFD and control RPE cells could elucidate the mechanisms behind these changes. A shotgun proteomics approach using isobaric Tag for Relative and Absolute Quantification (iTRAQ) identified peptide sequences present in these cells, and proteins were identified based on these sequences. Proteins with a twofold increase or decrease between cell types were noted, which included glutathione-s-transferases, gamma-glutamyl transferase 5, and chitinase-3-like 1. I am confirming these results through Western Blot and will analyze them by protein network analysis to determine their relevance to the disease model based on previously obtained metabolic data. The results from this experiment will help reveal the mechanisms behind SFD pathogenesis and potentially identify targets for disease treatment.

Cytosolic pH is sustained by the movement of protons out of the cytosol. The vacuolar ATPase (V-ATPase) is a conserved protein complex that localizes to the membrane of organelles and transfers protons from the cytosol into the organelle lumen. The V-ATPase couples the energy of ATP hydrolysis to transport protons. Proper pH homeostasis is important for many organelles such as endosomes, lysosomes, and secretory vesicles. In Saccharomyces cerevisiae, the Pma1 protein localizes to the plasma membrane and pumps protons out of the cell, helping to maintain cellular pH homeostasis. In Saccharomyces cerevisiae, we have found that loss of activity of the V-ATPase results in a growth arrest at low temperature. We generated 66 yeast V-ATPase mutant strains that are able to suppress the low temperature induced growth arrest. In complementation tests of these mutants, our results suggest that all of the mutants belong in the same complementation group. We have performed whole genome sequencing on one strain and analysis of the whole genome sequencing data has identified a single putative mutation. We are validating that the identified mutation is causative for the phenotypic suppression, and performing targeted sequencing of the candidate gene in the other suppressor strains.

Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is the etiological agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor predominantly made up of cells of endothelial origin. KSHV establishes a predominantly latent infection in endothelial cells in culture and in the KS tumor. KSHV latent infection alters cellular metabolism to improve the survival of the infected cells. These metabolic changes resemble a common alteration in cancer cells, termed the Warburg effect. This refers to an increase in glycolysis in the presence of oxygen and a decrease in oxidative phosphorylation. The mechanism of KSHV induction of the Warburg effect is currently unknown. Hypoxia-induced factors (HIFs) are likely candidates for KSHV induction of the Warburg effect as both HIF1α and 2α are induced by KSHV latent infection. To determine if HIF1α or 2α is required for the survival of KSHV infected endothelial cells, I constructed HIF1α or 2α knockouts using the CRISPR/Cas9 gene editing technique in a lentivirus vector. I expressed the endonuclease Cas9 and guide RNAs that lead Cas9 to cut at the HIF1α or 2α genomic sequences leading to genomic mutations in HIF1α and 2α respectively. To determine if HIF1α or 2α is required for the survival of latently infected endothelial cells, I infected the HIF1α and 2α knockout cells with KSHV and looked for cell death at 48 hours after infection. To evaluate if HIFs are required for KSHV induction of the Warburg effect, I will determine if one or both of the knockout cells produce less lactic acid and increase oxygen consumption at 48 hours post KSHV infection as compared to infection of wild type cells indicating the Warburg effect is not being induced by KSHV in the knockout cells. These results will aid in the future efforts to develop antiviral drugs by inhibiting viral latency.

Cells can be thought of as aqueous solutions of proteins enclosed within a lipid membrane. A surprising and exciting discovery in the past decade is that several proteins inside of cells aggregate to form separate liquid droplets. This discovery has potential medical applications because the types of proteins that undergo aggregation are associated with prion diseases. In the lab, researchers use a specific type of alcohol (1,6-hexanediol) to disrupt droplets in protein solutions. However, it is unknown whether hexanediol disrupts lipid membranes at the same concentration that it is used to disrupt liquid droplets. If so, then hexanediol cannot be used in cell systems. By probing the effects of 1,6 hexanediol on model lipid membrane physical properties, this study will determine the suitability of 1,6 hexanediol for in vivo studies of liquid droplets.

Ion Mobility Mass Spectrometry (IMMS) has become an important tool for separating small molecules such as peptides, glycans, and petroleum molecules. Ion Mobility is most often performed in He, N₂ or air. There is a great interest in using alternative drift gases to increase selectivity of ion mobility separations, specifically for complex mixtures like petroleum. In this study we measured arrival time distributions (ATD) of a set of double bond equivalents (DBE) in four different gases (He, N_2, CO₂, Ar) which span a range of masses and polarizabilities. Quinoline, isoquinoline, and their DBE were chosen because they are aromatic compounds that are easily protonated due to the presence of nitrogen and are similar to molecules found in petroleum. The measured ATDs were plotted as a function of reciprocal drift voltage; the resulting slope is inversely proportional to mobility. Using the mobility and the Mason-Schamp equation, we determined the collision cross section of each molecule with each drift gas. We also investigated the relationship between the dipole moment of an ion and its collision cross section. In general, collision cross sections increase with drift gas size and polarizability. Additionally some of the DBE were better separated with larger, more polarizable drift gases (N₂ and CO₂). We have shown that IMMS can be used with different gases to improve separation between ions of similar size and shape. This method can be used in future investigations of petroleum mixtures to potentially identify a wider range of molecules.

Aging is a process which causes the deterioration of cellular function in organisms. In order to understand the mechanisms of aging and to begin to identify ways to extend healthy life expectancy, research in the Kaeberlein lab uses Saccharomyces cerevisiae, a species of yeast. The technique that our lab uses to quantitate lifespan in yeast is known as the replicative lifespan (RLS) assay. When a yeast cell divides, the original “mother” cell and the newly produced “daughter” cell can be differentiated based on size. Daughter cells are microdissected away from mother cells, the cell division is recorded, and this cycle repeats until the mother is no longer able to produce daughters. We have tested the effects on lifespan resulting from the manipulation of thousands of genes and numerous environmental conditions. This data can be analyzed to understand the process of aging in yeast cells. By studying the mechanisms of aging in unicellular yeast cells, we can transfer these findings to identify conserved factors in the aging process that are also present in multicellular organisms. The study of the basic biology of aging in yeast can yield insights into the aging process that may contribute to our understanding of aging in humans and help facilitate the development of therapies aimed at decreasing age-related diseases.

The axons in the nervous system are collectively referred to as white matter. White matter forms intricate fiber connections among the brain’s grey matter (neurons). These connections change with development, learning, and disease, and therefore are of interest to several medical researchers. Tract-Based Spatial Statistics (TBSS) is an analysis technique which is used to analyze white matter integrity. Currently, this is the best existing method to align and analyze white matter. However, we are creating and evaluating an improved method to more accurately measure changes to integrity. Diffusion Tensor Imaging (DTI) Voxel Based Morphometry (DTIVBM) is a technique that we are developing which allows us to compare white matter statistics by using nonlinear registration to align the DTI image to a structural image and then a standard template. We are comparing several variations of DTIVBM with the established method of TBSS. We are using four different datasets, three of which contain adult brains ranging from ages 18-35, and one containing preteen/teen brains ranging from ages 11-15. One of the datasets also contains a mixture of normal brains and brains affected by neurodegenerative disease. Each subject has two separate brain images taken within a month’s gap. By running both the DTIVBM scripts and TBSS scripts on these brain images, we will be able to determine which method is more reliable, as measured using an Intra-Class Correlation (ICC) statistic. We are currently in the process of gathering the results, but we expect to see that DTIVBM will have higher reliability on most datasets than TBSS over a wider range of white matter. If this is true, we would have demonstrated a potentially better method to analyze white matter changes through aging and development, which will require fewer people and allow us to see changes in more of the brain.

Long non-coding RNAs (lncRNAs) are defined as non-protein-coding transcripts longer than 200 nucleotides. Recent studies have recognized lncRNAs as crucial regulators of transcription, mRNA processing and protein activity. However, little is known regarding the pharmacological regulation of lncRNAs in liver, which is an important organ for xenobiotic biotransformation. The goal of this study was to investigate the correlation between lncRNAs and the constitutive androstane receptor (CAR/Nr1i3), which is a liver-enriched xenobiotic-sensing nuclear receptor that plays an important role in xenobiotic biotransformation and intermediary metabolism. Three genotypes of mice, namely wild-type (WT), CAR-null and humanized CAR-transgenic (hCAR-TG) mice at 5- or 60-days of age were administered a species-appropriate CAR ligand (TCPOBOP for mCAR, and CITCO for hCAR) or vehicle once daily for 4-days, and livers were removed on the 5th day. The lncRNA gene expression was determined using RNA-Seq (n=3 per group, Cuffdiff, FDR-BH<0.05). At Day 5 neonatal age, mCAR activation altered more lncRNAs than hCAR activation (877 vs. 234). Interestingly, only 87 lncRNAs were co-regulated by both mCAR and hCAR, whereas 790 were only regulated by mCAR, and 147 were only regulated by hCAR. At Day 60, mCAR activation also altered more lncRNAs than hCAR activation. Among the differentially regulated lncRNAs, 126 lncRNAs were co-regulated, 1110 and 87 were uniquely altered by mCAR and hCAR respectively. The results also showed the significance of age in the expression levels of lnRNAs. For example, mCAR activation produced more changes in lncRNA expression at Day 60 than at Day 5; however, his pattern was not observed in hCAR-TG mice. This study highlights the age and the species differences of the lncRNA regulation in liver following pharmacological activation of CAR. The differentially regulated lncRNAs may serve as novel biomarkers or therapeutic targets for human diseases such as metabolic syndrome and drug-induced liver injuries.

Plants respond to abiotic factors in natural settings in order to regulate physical responses at the most appropriate timing. The timing of flowering affects seed and/or fruit production in many agriculturally relevant plants, so understanding its mechanism is essential for addressing agricultural needs as climate changes. To address this aim, we have used Arabidopsis thaliana as a model plant to understand the molecular mechanism of seasonal flowering. It has been shown that the expression of FLOWERING LOCUS T (FT), which encodes florigen, peaks in the evening under long-day lab conditions (16 hours light, 8 hours dark, 22°C) and promotes flowering in a day-length specific manner. However, our lab found that FT also peaks in the morning under outside long-day conditions over the summer solstice in Seattle (16 hours light, 8 hours dark, highest average temperature 21°C). This double-peak of FT can be re-created by changing the light quality and temperature settings of long-day lab conditions. In addition, we found that a mutant of PHYTOCHROME A (PHYA), which encodes a red/far-red light photoreceptor in plants, lacks FT expression in a morning-specific manner, suggesting that PHYA is involved in regulating morning expression of FT. In order to understand the molecular mechanism of PHYA-mediated FT regulation, we are in the process of identifying transcription factors and other molecules that work with PHYA to induce FT expression. We have successfully cloned various transcription factors that are reported to act in the flowering regulatory pathway and are in the process of testing protein-protein interaction against PHYA by yeast-two-hybrid methods. Identifying the components that act in the PHYA-FT pathway will allow us to further understand how light affects flowering regulation.

 Minority actors continue to be under-represented in major Hollywood films, something brought to national light last year through the return of the “OscarsSoWhite” hashtag on Twitter. Though there have been some efforts to increase racial diversity in blockbuster roles, there has been serious backlash from minority actors and viewers who have reacted vehemently to the “white-washing” of minority roles, including characters in Doctor Strange, The Great Wall, and Aloha. This study aims to gauge young (ages 18 - 25), self-identified Asian American adults’ responses to this issue of unequal media representation using an interpretive perspective. Data will be gathered through initial survey “screeners” and in-person semistructured interviews. First and foremost, I will ask Asian Americans if they interact with and/or view forms of media. If so, do they feel (accurately) represented in the media? Do they perceive differences in minority roles in major films versus television? Key terms such as “representation” and “white-washing” will be clarified during the interview, as needed. This research will lay important groundwork for assessing Asian Amerian consumers’ views regarding media representations, especially regarding the intersection of identity and mental health. With this work, we can establish need for future quantitative studies and hopefully address issues associated with unequal representation.

The protein Transient Receptor Potential Ankyrin type 1 (TRPA1) is an ion channel found in nociceptive (pain-sensing) sensory neurons. TRPA1 is activated by several noxious compounds, including those found in mustard plants, garlic, smoke, and tear gas, among others. It is responsible for the sensation of irritation and pain that these compounds cause, as well as some related chronic pain disorders. A better understanding of TRPA1 could lead to novel therapeutics against chronic pain. For this reason, the mechanism by which TRPA1 activates is an active area of research. TRPA1 contains a coiled-coil domain at its C-terminal end and several ankyrin repeat domains (ARDs) at its N-terminal end. These are both very common repeating protein motifs; ARDs in particular often modulate protein-protein interactions. Many channels in the Transient Receptor Potential (TRP) family, which includes TRPA1, contain these domains, but their role in channel activation is not fully understood. However, a recently solved atomic structure of TRPA1 provided some key insights. The structure showed that TRPA1’s coiled-coil domain is tightly enveloped by its ARDs, and they appear to interact with each other. No other TRP channel with a known structure exhibits this unique structural arrangement. My project aims to better characterize the interaction between the ARDs and coiled-coil. I am studying a mutation in the ARDs of the human TRPA1 channel, which is at its interface with the coiled-coil. The mutation, K591E, changes a key lysine amino acid residue, which is positively charged, to a negatively charged glutamate. It is found in rattlesnake TRPA1, which unlike the human protein is activated by temperatures above 27 °C. Current results show that the K591E mutant is active at room temperature, even without any other compounds. A stronger understanding of TRPA1’s activation mechanism will be vital to the development of effective next-generation pain therapies.

The blood-brain barrier is a very selective membrane that works to protect the brain from most pathogens; however, this means that it is a difficult therapeutic target for drug targeting. Cutting-edge nanotechnology research is being performed by Dr. Elizabeth Nance on targeted drug delivery mechanisms to combat brain tumors such as glioblastomas, neuromas etc. Nance’s group has found and supported good drug release results with poly-ethylene glycol coating (PEG) for which they hope to perform controlled and targeted release. Currently, there is a lack of molecular level understanding of the drug-polymer interactions that dictate controlled release. Better computational models are needed to understand release kinetics and facilitate rational design of biomaterials to control the release or retention of a particular drug or enzyme. In the present work, molecular dynamics (MD) simulations were used with GROMACS software to study glutamate diffusion in water to validate our method and set a baseline for more advanced systems. Diffusion constants calculated with the Einstein equation based on glutamate diffusion in MD were consistent with experimental values of ∼0.75 μm2/msec. We are currently extending this approach to probe glutamate diffusion in the presence of biocompatible polymers such as PLGA (poly(lactic-co-glycolic acid)) and PEG. Then, we compared and contrasted the diffusion constants to make sure they were on the same order of magnitude as glutamate. We also studied the system kinetics to discover the polymeric matrix that allows the system to get closest to a diffusion-limited rate. We will relay this to Dr. Nance to modify her experiments to lead to improved drug delivery systems that help fight off brain cancers.

Heart failure (HF) is a serious medical epidemic, and the number of those suffering from end-stage, advanced HF continues to rise along with the use of long-term left ventricular assist devices (LVADs) as treatment. Despite improvements in LVAD technology and clinical management, patient response remains significantly heterogeneous with variable predicted outcomes based on measurement of traditional biomarkers. Soluble suppression of tumorigenicity-2 (sST2) is upregulated in HF due to myocyte stretching and fibrosis during disease progression; this protein, as a novel biomarker, may provide insight into pathophysiologic changes that occur following LVAD implantation and potentially help predict patient response. More comprehensive patient selection would reduce the use of unfavorable and expensive surgery, saving money, and lives of those suffering from HF. Thus, we aim to quantify changing levels of plasma sST2 in adult patients who have undergone LVAD implantation. Plasma samples were collected from 100 individuals prior to surgical implantation of a LVAD and at 1-, 3-, and 6-months post-operatively. Levels of sST2 will be measured in all available plasma samples using commercially-available quantitative sandwich monoclonal ELISA kits (Critical Diagnostics, San Diego, CA). Data will be analyzed using repeated measures ANOVA and modeled using growth modeling approaches, as appropriate. We anticipate that plasma sST2 levels will significantly decrease from pre-LVAD to 6-months post-LVAD in this cohort of patients. Moreover, we anticipate discovering differing trajectories of sST2 change that will help differentiate who will do better or worse following LVAD implantation. Our findings may support use of sST2 as a clinically relevant biomarker in HF patients following LVAD implantation. Incorporating sST2 into the model of response to LVAD implantation could lead to improved outcome prediction to LVAD implantation.

High-resolution structures of membrane proteins are necessary for guiding the rational design of novel therapeutics. Membrane proteins make up 30% of our proteome and roughly 60% of all drug targets, yet they only represent ~4% of the structures in the Protein Data Bank. This is because membrane proteins have proven to be extremely difficult to crystallize for traditional X-ray crystallographic studies. Recent advances in single-particle electron cryo-microscopy (CryoEM) technology are beginning to revolutionize the field of membrane protein structural biology by providing near-atomic resolution structural information without the need for protein crystallization. Despite these advances, CryoEM is limited in its ability to characterize small membrane proteins (less than ~200 kDa in size) and dynamic protein-protein interactions. Several important classes of membrane proteins are “too small for CryoEM”, such as the G-Protein Coupled Receptors and the Major Facilitator Superfamily transporters. Moreover, our ability to capture membrane proteins in complex with cytosolic signaling proteins is commonly limited by their inherently short-lived low-affinity interactions. Our research group aims to overcome these hurdles to characterize the regulatory complex formed between the ~100 kDA small membrane water channel, Aquaporin-0 (AQP0) and the calcium signaling protein, Calmodulin (CaM). We intend to enhance the small size of the AQP0 water channel by applying Nanodisc technology, which offers the added benefit of a near native lipid bilayer environment to study the membrane protein. While the system requires further optimization, a preliminary Nanodisc reconstitution has an enhanced mass of ~250 kDa, which is sufficient for high-resolution CryoEM analysis. We are also exploring the use of engineered avidity as a novel approach to enhance the native multi-valent interaction between CaM and AQP0 tetramers. We believe these strategies represent general approaches that may be applied to thousands of other small membrane proteins and transient protein complexes to facilitate their structural characterization. "

As cancer remains a looming threat to the public, understanding the genetic and biochemical basis of how cancers operate is necessary in order to develop new medicines and treatments. A topic of critical importance when considering the biochemistry of cancers is how specific changes in gene expression control the growth of tumors. I explore this topic by using Drosophila genetics in order to investigate the crosstalk amongst three key growth regulators, RAC-alpha serine/threonine-protein kinase (Akt1), Translationally Controlled Tumor Protein (TCTP), and Yorkie (Yki). The overexpression of Yki in intestinal cells is known to cause intestinal tumors, and our preliminary results indicate that Akt1, TCTP, and Yki interact to control intestinal stem cell proliferation. Nevertheless, signaling crosstalk amongst these three genes is still ambiguous. By using the advantageous genetic toolbox of Drosophila, I created experimental fly lines which allow me to manipulate the expression of each gene or a combination of these genes in Drosophila intestinal stem cells. My goal in this project is to anatomize this signaling crosstalk through monitoring how manipulation of these genes in combinations influence the proliferation of intestinal stem cells using confocal microscopy. Ultimately, we hope to attain a greater understanding of growth in relation to genetic networks that control it.

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

During development, the repression of transcription factor tbx16 allows embryos to dynamically allocate neural and muscle tissue in the correction proportion as the body axis is formed. Understanding how tbx16 is regulated is crucial for identifying the underlying mechanisms of neuromuscular developmental defects. Currently, we are investigating how two genes of interest termed eve1 and vent interact to regulate the expression of tbx16 in zebrafish embryogenesis. Previous studies have relied on Morpholino anti-sense oligonucleotides (MOs), but MOs often create off-target effects and misleading phenotypes that don't reflect genetic mutants. Using the CRISPR-Cas9 system in zebrafish, we have created true genetic knockouts for eve1 and vent. By analyzing the difference in phenotypes between eve1 mutants, vent mutants, and eve 1/vent double mutants, we can determine if eve1 and vent act redundantly to repress tbx16. Our goal is to uncover the mechanisms behind these mutant phenotypes in order to identify the role that eve1 and vent play during embryogenesis. Our research aims to further our understanding about how serious birth deficits arise from gene deficiencies and complications in development.

Mammalian cardiac myocytes (CMs) decrease proliferation soon after birth. This is caused by silencing of cell cycle gene expression. Gene activity is epigenetically regulated by physically changing chromatin structure. Generally, gene silencing is mediated by heterochromatin because of its tightly packed structure, which limits accessibility for transcription factors to bind to the gene promoters. Silenced chromatin is believed to form when heterochromatin protein 1 (HP1) binds to histone H3 lysine 9 tri-methylation (H3K9me3) to condense chromatin into heterochromatin. We previously found that demethylation of H3K9me3 inhibited cell cycle gene silencing in CMs and resulted in hyperplastic heart growth. This suggests that H3K9me3/HP1 pathway is an important regulator for cell cycle gene silencing. There are three isoforms of HP1 (alpha, beta and gamma) expressed in mammals, but we found that HP1 gamma was most highly expressed in CMs. Therefore, we created a cardiac specific HP1 gamma knockout (KO) mouse and tested our hypothesis that loss of HP1 gamma would reactivate cell cycle gene expression and induces cardiac growth. Using RT-PCR we confirmed deletion of critical exons of HP1 gamma mRNA. Western blot demonstrated significant reduction of HP1 gamma protein in KO CMs. Immunofluorescence staining showed HP1 gamma expression was reduced specifically in CMs. HP1 gamma KO mice showed normal heart growth and no significant difference in heart mass was observed compared to control hearts. Consistent with this result, there were no significant differences in cell cycle or cardiac gene expression between HP1 gamma KO and control CMs. Interestingly we found an increase in protein level of HP1 beta, but not HP1 alpha, in HP1 gamma KO CMs. These data demonstrate that HP1 gamma’s function is redundant in cell cycle gene silencing and cardiac growth, which could be due to HP1 beta compensating for the loss of HP1 gamma in CMs.

Congenital Heart Disease (CHD) is the most common inborn defect in babies and is responsible for 1/3 of all major congenital anomalies. With its continuous prevalence in the human population, the genetic causes remain poorly understood. Previous findings have shown that Pbx transcription factors control gene expression during development. Studies in zebrafish shows that pbx4 has significant requirements for heart muscle differentiation. Our hypothesis states that mutations in human Pbx genes can lead to heart defects. The purpose of this study is to acquire further insight about the role of Pbx genes in the context of CHD and analyze cardiac dysmorphic patterns in pbx gene mutants. By using the genomic editing system CRISPR/Cas9 (Clustered regulatory interspaced short palindromic repeats), we have been able to make exact genetic changes in Pbx genes in zebrafish (Danio rerio) animal models. In particular, we have engineered a Pbx gene mutation found in humans with heart defects, known as Pbx4 SNP (single nucleotide polymorphism). For our experiments, samples of zebrafish DNA are obtained and genotyped with various assays for mutations. We use microscopy and transgenic markers to analyze heart defects in mutant embryos. Pbx4 mutant zebrafish display distinct phenotypessuch as precardial edema and bulged ventricles, but it is not clear at what stages these morphogenesis defects occur. To further investigate the dysmorphic phenotypes, our next step is to conduct cardiomyocyte counting analysis in Pbx4 SNP and Pbx2/Pbx4 double mutant fish and gather data at 24 and 48 hours of development. This experiment will assist in better understanding the heart defects previously seen in the single Pbx4 mutant samples. Understanding the mechanisms and discovering the genes of CHD, as well as cardiac morphogenesis patterns, will contribute to finding therapies and improving genetic screenings for patients with lifelong heart defects.

Cerebral malaria (CM) is a multifactorial disease with lethal neurological complications plus increased risks of cognitive and neurological impairments making CM a leading cause of childhood neuro-disability. The malaria parasite, Plasmodium falciparum, accounts for most mortality from malaria, and primarily kills children under five in sub-Saharan Africa. The deadly CM complication develops when red blood cells infected with parasites (iRBCs) bind to the endothelial cell wall in brain microvasculature. This process of parasite sequestration via cytoadhesion is mediated by the parasite ligand, P. falciparum erythrocyte membrane protein 1 (PfEMP1) encoded by a family of 60 var genes. An individual parasite, however, strategically undergoes allelic exclusion to express only a single distinct PfEMP1 variant. PfEMP1 proteins possess the ability to bind relatively few host receptors despite seemingly limitless sequence diversity. Requirements for maintaining binding surfaces conserved in structure and binding potential despite significant sequence diversity remain a mystery. Understanding these requirements will be critical for developing therapeutics to mitigate disease severity. A subset of PfEMP1 proteins associated with severe/cerebral malaria bind highly conserved regions of the host receptor, endothelial protein C receptor (EPCR), and inhibit binding of its natural ligand, activated protein C (APC). There are six PfEMP1 subdomains that bind EPCR (CIDRα1.1 and CIDRα1.4–1.8 domains), which share limited sequence identity among PfEMP1 variants. The data so far suggests the presence or otherwise absence of a coiled coil domain within CIDRα1 determines binding to EPCR. Our study uses computational and molecular biology tools to investigate the molecular and structural determinants for malaria parasite adhesion to EPCR. 
 

Chronic epilepsy is thought to develop due to dysfunctions in various ion channels. Previous researchers in the Poolos lab manipulated a signaling cascade regulating these channels and discovered an increase in activation of c-Jun N-terminal kinase (JNK) in animals experiencing seizures. My project aims to understand this connection and the timeline of JNK activity during the development of epilepsy, hypothesizing that JNK activity will precede epilepsy onset and may be a cause of chronic epilepsy. To study epilepsy, we administer research-bred rats a pilocarpine injection. Shortly after, the rat is in status epilepticus (SE), a state of continuous seizures. After one hour, a phenobarbital injection is administered to halt SE. Seizures begin around one week post-SE and achieve steady-state frequency around four weeks. I analyze brain tissue samples taken after one hour, one day, and one week post-SE using Western-blotting to obtain a relationship between phosphorylated-JNK (pJNK) levels from pilocarpine-treated rats to control rats. There are three JNK isoforms, separated into two bands: 54kDa and 46kDa. JNK 1 is primarily in the 46kDa band, JNK 2 is present in both bands, and JNK 3 is predominately in the 54kDa band. For the 54kDa band, I have discovered a significant increase of pJNK one hour post-SE (124±8.1%, p<0.05, n=7), one day post-SE (132±9.2%, p<0.05, n=14), an insignificant change at one week post-SE (100±9.6%, p>0.05, n=10) and a significant increase in chronic epilepsy (126±7.6%, p<0.05, n=12). For the 46kDa band, I have discovered an insignificant change of pJNK at one hour post-SE (101±7.1%, p>0.05, n=8), a significant increase one day post-SE (123±12.8%, p<0.05, n=12), one week post-SE (122±7.6%, p<0.05, n=10) and in chronic epilepsy (116±5.9%, p<0.05, n=12). We hope that, by understanding JNK activation, we can explore avenues for medicine and treatments for epilepsy, through antiepileptic drugs and other therapeutic options.

Proteins serve very specific and powerful functions in the cell by maintaining a specific shape through chemical interactions in the protein sequence. Under stress conditions however, these chemical interactions holding the protein together may break apart, leading to the protein’s loss of function and exposure of hydrophobic patches that can interact with other proteins to form toxic aggregates. Heat shock proteins (sHSPs) act as safeguards under conditions of stress and protein misfolding as molecular chaperones that interact with exposed hydrophobic patches to stabilize unfolded proteins. The importance of sHSPs in the cell is exemplified through various disease associated mutations of sHSPs. sHSPs are a large and diverse family of proteins distinctive in their ability to form multimers of various sizes, which correlates with varying ability to prevent aggregation. However, little is known about how different structural elements of sHSPs influence function. The Klevit lab aims to better understand the molecular interactions of human sHSPs with themselves, how these sHSPs are activated in cellular conditions, and how they recognize and bind unfolded protein. My work focuses on the characterization of the structure and function of the human sHSP, HSPB8. Specifically, I am studying the effects of HSPB8 binding to zinc, the cellular levels of which vary in response to stress and regulate the function of many other proteins. By comparing zinc bound and non-zinc bound protein samples using various biophysical techniques such as NMR, circular dichroism and fluorescence, I aim to elucidate the effects of zinc binding on structural and functional elements of HSPB8.

With the rise of antibiotic resistant bacteria, understanding how pathogenic bacteria invade and survive within our bodies is becoming more important as we search for novel ways to combat bacterial infection. Some pathogens, such as Salmonella typhimurium, inject host cells with proteins that suppress the host cell’s ability to respond to bacterial invasion. One such protein, SspH1, is an E3 ubiquitin ligase. E3 ubiquitin ligases attach ubiquitin, an important regulatory protein used to mark other proteins for degradation, to a substrate. Ubiquitin, the biochemical machinery that activates ubiquitin, and the pathways that utilize ubiquitin as a signaling molecule, are not found in bacteria, only in eukaryotes. However, bacteria have evolved proteins, such as SspH1, that hijack a host cell’s biochemical machinery in order to target host proteins for degradation. SspH1 consists of two domains: a catalytic E3 domain that transfers ubiquitin to substrate, and a substrate recognizing Leucine-Rich-Repeat (LRR) domain. I have shown through biochemical assays that the LRR domain inhibits the catalytic activity of the E3 domain. I have also shown that this inhibition is relieved in the presence of substrate. Through the use of 2D NMR and further biochemical assays, I hope to characterize the binding between the LRR and E3 domain in order to further the understanding of how SspH1 is inhibited. Understanding the mechanism of SspH1 auto-inhibition can increase our knowledge of the mechanisms and activities of related bacterial effector proteins, furthering our understanding of how pathogenic bacteria evade our immune system, and potentially lead to the development of novel treatments that can combat bacterial infection.

Model yeast Saccharomyces cerevisiae normally grows in the lab as isolated cells but can evolve the ability to clump, protecting the cells from stress factors including antimicrobial treatments. Clumping can occur either due to a failure of cell separation during division or through physical cell adhesion (“flocculation”). Understanding the genetics behind these traits could give us insight into how microbes develop antimicrobial resistance. Previously, we evolved S. cerevisiae from a non-clumping ancestor strain for 300 generations in miniature chemostats, multiplexed culture devices that maintain a constant environment. Using a combination of whole genome sequencing and bulk segregant analysis, a pooling and sequencing approach, we identified causal mutations in 23 independent, clumping clones from the evolution experiments. We identified two strains with cell separation defects which had mutations in both budding gene BEM2 and cell separation gene ACE2. In the remaining clones, the majority of the causal mutations affected the regulation of known flocculation gene FLO1. Despite this shared mutation, a number of these clones exhibited varying flocculation phenotypes. We hypothesized that other “modifier” mutations might also be contributing to the complexity of the phenotype. For one strain with unusual cell morphology, targeted sequencing of candidate modifier genes revealed that the modifier mutation is either in the IRA1 or HSL7 gene, which are genetically linked. The goal of our current experiments is to determine the contributions of BEM2 and ACE2 to the separation defect phenotype and of HSL7 and IRA1 to the flocculation phenotype. We are using a method called complementation testing, in which the wild-type gene is added to the mutated strain using molecular cloning techniques to see if the mutant phenotype is repaired. This will enable us to identify which genes are contributing to the complexity of these traits and facilitate a better understanding of their underlying mechanisms.

Successful genome replication is essential for cell division and has been correlated with longevity in the model organism Saccharomyces cerevisiae. DNA replication is initiated at specific sites on chromosomes called origins and requires the recruitment of several low abundance replication factors. The highly repetitive ribosomal DNA (rDNA) locus, known to encode the RNA components of ribosomes in both yeast and humans, has recently been suggested to be influential in genome replication and stability. For instance, the genome of the budding yeast S. cerevisiae contains 450 origins, 150 of which reside at the rDNA locus and could therefore create competition with origins located in the rest of the genome. We hypothesize that the preponderance of rDNA origins compete for essential replication factors and that reduction of rDNA origin competition, achieved by reducing rDNA copy number or compromising the rDNA origin sequence, would promote non-rDNA genome replication. We wanted to examine the role of rDNA competition by using four yeast strains that have either high or low rDNA copy number and either a normal or compromised rDNA origin sequence. We measured the ability of these four strains to maintain a non-essential plasmid containing a non-rDNA origin of replication as an indicator of genome replication. We expect that if rDNA origins compete with other origins, including those on the plasmid, DNA replication will be unable to initiate at the plasmid origin. Therefore, we predict that strains with high rDNA copy number arrays and strong rDNA origins will lose the plasmid at a greater rate than strains with low rDNA copy number and weak rDNA origins. Cells with such intense competition for replication factors could consequently struggle to complete chromosome replication, potentially leading to decreased cellular health and lifespan in yeast, which could be extended to our understanding of human cellular health.

In March of 2011 Dr. Bonita Brewer published a paper proposing a model to explain how a particular error in replication can lead to the amplification of chromosomal segments in an inverted orientation, such as those seen in classes of chromosomal rearrangements that arise in human developmental disorders and cancers. She hypothesized that when a replication fork encounters a short inverted repeat, the leading strands could erroneously become ligated to the lagging strand and produce an extrachromosomal palindromic DNA intermediate. Through my research I am investigating if this model can explain the amplification of the ADH4 locus in yeast, what features of the short inverted repeats lead to the inverted junctions, and what environmental or genetic factors influence this mode of gene amplification. Yeast cells that lack a functional copy of ADH1 (a gene that encodes the major form of alcohol dehydrogenase) become sensitive to the drug Antimycin-A. However, these cells can regain Antimycin-A resistance by amplification of ADH4 (a gene that encodes a minor form of alcohol dehydrogenase). Previously it has been found that the extra copies of ADH4 reside on an extrachromosomal palindromic DNA molecule. Through my research I aim to determine if the extrachromosomal amplification of ADH4 is occurring via this new model. To do so, I have created my own adh1∆:KanMX strain, confirmed that high copy number of ADH4 is sufficient to confer resistance to Antimycin-A, and will isolate the extrachromosomal fragment via pulse-field gel electrophoresis and characterize its structure in detail. In an extension of this work I will test whether genetic and environmental factors that stress the replication process influence the frequency of this form of gene amplification, possibly providing insights into amplification events that contribute to human developmental disorders and cancers.

Previous research has emphasized that the basal forebrain partially controls high-order neural processes such as memory. For this reason, it has often been studied in the context of Alzheimer’s and Parkinson’s disease, which both correspond to impaired cognitive function. In spite of the importance of this research, however, there stands much to learn and even revise about our understanding of the neurons which populate the basal forebrain. Neurons expressing the gene T-Box Brain 1 (TBR1) found in the Horizontal Limb Diagonal Band (HDB) and the Substantia Innominata (SI)—both regions within the basal forebrain—have been identified in the past as cholinergic neurons, meaning that they employ acetylcholine as their neurotransmitter. However, immunohistochemical stains of mouse brain sections in the Hevner lab have revealed counterevidence to this claim. We have hypothesized instead that the neurons which populate the HDB and SI are actually glutamatergic neurons, not cholinergic. In order to test this claim, we harvested mouse brains from different ages, ranging from embryonic, postnatal, to adult mice. These brains were embedded in Optimal Cutting Temperature Compound then sectioned these into 12 μm thick slices. These slices were then mounted on slides and stained via immunohistochemical techniques or via endogenous expression. High-magnification images were then collected using AxioVision software to confirm morphology and colocalization of neurons. Employing the above techniques, we have concluded that Tbr1-expressing neurons in the HBD and SI are not cholingeric. Rather, they are glutamatergic neurons. This distinction will be important as research into Alzheimer’s and Parkinson’s diseases develop. An understanding of the precise physical bases for complex, intangible processes is the necessary prerequisite to developing effective therapies.

Systemic Lupus Erythematosus (SLE) is one of the complicated chronic autoimmune diseases that can involve many different organs including liver, kidney and skin. There is evidence that proliferation and apoptosis (cell death) of autoreactive T lymphocytes are defective in SLE, sustaining the inflammatory response. One specific protein that may contribute to T cell dysregulation is CD30. The main goal of my project is to study the expression of CD30 on T cells from SLE patients, specifically Lupus Nephritis (LN) patients. CD30 is expressed on activated memory T cells and it regulates communication between T and B cells along with initiation of apoptosis. Previous studies suggest that there is elevated soluble CD30 in LN patients. There is an FDA approved drug that causes apoptosis by targeting CD30 expressing cells, which is used to treat lymphoma patients. The drug Brentuximab Vedotin could potentially replace chemotherapy for those who cannot tolerate its toxicity and used as a primary treatment. Results of this study provide principle data to support the approach of therapeutically targeting chronic T cell activation in SLE via CD30. I hypothesize that the LN patients with more severe disease will have higher numbers of CD30 expressing cells depending on their diagnosed classes. Patient samples are stained and evaluated by immunofluorescence. The expression of CD30 was correlated to disease status at the time biopsies or urine samples collection. We expect that the higher classes such as III and IV are more likely to have higher number of CD30 positive cells compared to classes I and II as well as non LN patients. Therefore, I compared the frequency of CD30 expressing cells in SLE patients to those without SLE and those with different classes of LN. This research has great implications for SLE patients and could replace previously expensive and highly toxic treatments.

Stressed cells may be able to avoid elimination but continue to respond to compensatory growth signals and accumulate, resulting in tumor formation. This ability to avoid cell elimination processes, such as apoptosis, is one of the hallmarks of cancer. The Jun N-terminal Kinase (JNK) mediates cellular stress responses by inducing apoptosis of stressed cells and promoting compensatory cell proliferation. Using immunohistochemistry and the GAL4-UAS targeted gene expression system, we are able to distinguish intestinal stem cells (ISCs) from enteroblasts, enterocytes, and other intestinal cells through a confocal microscope. We found that activation of JNK in fruit fly intestinal stem cells (ISCs) induced temporary over-proliferation. On the other hand, persistent activation of JNK resulted in elimination of the ISCs. Strikingly, we discovered that ISCs with constitutive activation of JNK 'extrude' out from the epithelial layer of fly intestines. Our findings suggest that cell extrusion may be a mechanism for JNK-mediated tumor suppression, preventing the accumulation of cancerous cells. Alternatively, cell extrusion may be involved in metastasis, enabling cancerous cells to spread to other parts of the body. We aim to further investigate the molecular mechanisms and gene expression underlying cell extrusion in various genotypic backgrounds, which may be helpful to understand how cancerous cells evade cell elimination and undergo metastasis. 

Aging is a phenomenon that brings about the onset of disease and eventually death. One of the hallmarks of aging is the decline in proteome maintenance that can lead to the accumulation of misfolded proteins. In extreme cases, protein misfolding can induce such devastating neurodegenerative diseases such as Huntington’s disease and Alzheimer’s for which very limited treatment is currently available. It has been observed previously that dietary restriction is a method that increases lifespan and delays the loss of function caused by age-associated pathologies. Specifically, complete food removal leads to robust increase in longevity in the roundworm Caenhorbabditis elegans, a popular model organism for aging research. Remarkably, when pre-reproductive adult worms are subjected to starvation, they enter a condition known as adult reproductive diapause (ARD). Upon reintroduction to food after prolonged starvation, ARD worms experience a pronounced morphological recovery and have normal lifespan. To understand how diapaused animals preserve this rejuvenation potential and normal lifespan we aim to analyze how proteome quality is maintained during the diapause. We hypothesize that proteome quality is either well maintained during the diapause or is restored back to youthful state upon exit from the diapause. To discriminate between these possibilities, we use C. elegans models of proteotoxicity wherein toxic protein species produced in muscle cells induce paralysis with age. By analyzing the accumulation of insoluble protein aggregates and motility during and after the diapause we will gain a better understanding of the physiological aspects of the ARD state. These insights will be useful in developing novel approaches to counter age-associated pathologies.

Mutations in the gene Presenilin 2 (PSEN2) cause familial Alzheimer’s disease (fAD). FAD shares clinical and pathological features of sporadic, late onset AD thus fAD cell models can be useful to study mechanisms relevant to all forms of AD which is critical to developing effective AD therapeutics. Presenilin 2 protein (PS2) forms the catalytic subunit of the γ-secretase complex, which cleaves amyloid precursor protein and releases Aβ1-42, considered a pathogenic contributor to Alzheimer’s disease (AD). Our laboratory is interested in a fAD associated PSEN2 mutation, a frameshift two base-pair deletion (PSEN2 K115Fx) . The PSEN2 K115Fx is predicted to either lead to a truncated protein suggesting that the mutation may create a shortened peptide that interferes with normal cellular function (dominant negative or toxic gain of function) or result in degradation of the RNA transcript and subsequent loss of normal amount of PS2 protein (loss of function). To better understand how PSEN2 mutations cause disease, we have two objectives. The first aim of this project is to determine if the PSEN2 K115Fx does indeed result in a truncated protein or influence levels of wildtype PS2. I will be collecting human cultured fibroblasts isolated from AD patients with the PSEN2 mutations or controls and prepare cell lysate for analysis by Western blot. My colleague will also be analyzing mRNA levels from those same samples to determine the stability of the PSEN2 mutant and wildtype transcripts in all cases. The second aim is to determine the impact of the mutation on PS2 enzymatic activity. I will culture the cells described above, then infect with a luciferase based enzyme reporter assay to compare the impact of PSEN2 mutations on γ-secretase mediated cleavage of APP. My work will help identify the candidate mechanisms by which the PSEN2 K115Fx mutation causes AD.

Mutations are imbedded in every individual’s DNA. A key challenge in human genetics is to determine the functional phenotype of specific mutations. The goal of my research in the Monnat Lab is to develop an efficient, quantitative, cell-based assay to study the phenotype of mutations in a gene in parallel. Our target disease is Fanconi Anemia (FA), a recessive human genetic disease associated with cancer, bone marrow failure, and sensitivity to crosslinking agents such as mitomycin-C. Among of the 21 “FANC” genes that could result FA, we focused on FANCA gene as it is mutated in 2/3 of FA patients. A set of nearly 500 FANCA variants was then used to build a plasmid library encoding each protein variant. In order to achieve this, each variant, linked to a unique DNA molecular 'barcode', will be inserted at a unique chromosomal location in FANCA-mutant human cells in order to determine their cellular phenotype. We will assess cell survival as a function of mitomycin-C dose and the expressed transgene. Preliminary results have already been generated to confirm the feasibility of this approach. My effort thus far has been focused on generating a library of ~500 single nucleotide variants of FANCA. In addition to capturing each variant, I developed an optimized workflow to generate barcoded plasmids with only desired variant without additional mutations in the FANCA. In the coming quarter, I will work with other members to develop a corresponding cell library in which each of these FANCA variants is expressed in a common, FANCA-deficient background for functional phenotyping. Results of my research will help identify new and existing deleterious as well as innocuous FANCA variants, and thus will enable a more accurate molecular diagnosis of FA.

The Possession Sound is formed where freshwater from the Snohomish River and the salt water from Puget Sound creates an estuarine habitat that supports biodiversity within this ecosystem. Decreases in pH make it difficult for calcifiers to produce their calcium-carbonate shells, thus decreasing their survival rate in a higher acidity environment. Ocean Research College Academy (ORCA) has been sampling water chemistry at multiple sites in Possession Sound for 11 years. This study utilized ORCA data to compare two sites, one near shore and a second in a deep, central location. The purpose of this investigation was to study the pH and salinity trends from 2008 to 2016, considering influence of surface runoff, temperature, river discharge, and climate variations. It was hypothesized that the pH would decrease, becoming more acidic over the eight year period, and the salinity would remain within normal limits. Salinity and pH data were collected in 1-meter increments using a YSI 650 instrument. Initial data demonstrates an inverse relationship between salinity and pH. In 2013, salinity ranged with depth from 22.2-29.5 ppt near shore and 19.2-30.2 ppt at the deep site, correlating with higher surface pH that decreased with depth of 6.8-7.3 near shore and 6.5-7.1 at the deep site. This relationship was consistently demonstrated over the eight-year period, with the exceptions of 2011 and 2015. Further investigations include exploring the impact of climate patterns on the Snohomish River discharge and its influence on pH and salinity trends, as well as looking for seasonal trends in primary productivity of phytoplankton and their impact on temporal and spatial trends.

Brassinosteroids and auxin are plant hormones that are crucial in plant growth development. Although it is known that brassinosteroids and auxin signaling interact, it is not known exactly how. We are focusing our attention on the question of how auxin and brassinosteroids interact during the formation of lateral roots. Lateral roots are roots that extend horizontally from the primary root and the developmental tradeoff between the primary and lateral root formation helps the plant optimize its water and nutrient uptake. Previous studies have demonstrated that auxin promotes specification of founder cells, the first step in making a new root. To test if brassinosteroids are also involved in this process, I am quantifying the density of founder cells in seedlings treated with brassinosteroids alone, auxin alone or both hormones together. In this study, I will be able to distinguish whether the hormones act additively or synergistically (a much greater effect than a simply addition of the effect of each hormone individually). Our study will deepen our understanding of root formation which will have agricultural significance as strong roots lead to effective nutrient and water intake and stability for the plant under varying weather conditions.

The meeting of the fresh water from the Snohomish River and the salt water from Possession Sound creates a unique habitat that is important for many species that make up the biodiversity within this ecosystem. The purpose of this investigation was to observe seasonal variation trends from the pH, chlorophyll, dissolved oxygen (DO) and temperature data at the Deep site in the Possession Sound in Everett, Washington. The study was conducted using a Yellow Springs Instrument 85 and Exo Sonde to collect temperature, dissolved oxygen, pH, and chlorophyll data between 0-3 meters in comparison to 7-10 meters in the the water column. It was hypothesized that during the spring and summer months, there will be a higher chlorophyll concentration due to photosynthesis causing an increase in pH and DO throughout the water column. We also predicted during fall and winter months, there will be a decrease in photosynthesis causing a decrease in chlorophyll, pH, and DO. The hypothesis was not supported. There was an inverse relationship between temperature and dissolved oxygen; as temperature was increasing during the summer months, dissolved oxygen was decreasing. When the temperature was decreasing during the Fall and Winter months, there was a increase in dissolved oxygen.

Photovoltaic devices based on polycrystalline films of hybrid organic-inorganic lead perovskite semiconductors have demonstrated impressive power conversion efficiencies of over 22% while still retaining the advantage of low-cost solution processability. However, a fundamental understanding of many of the photophysical and charge transport properties of these materials must be further developed. In this project, we synthesize a series of n-type doped single crystals of organic-inorganic methylammonium lead bromide (CH₃NH₃Pb_1-xBi_xBr₃) and all-inorganic cesium lead bromide (CsPb_1-xBi_xBr₃) by varying the amounts of Bi3+ cation and use optical spectroscopy and x-ray diffraction to characterize their optical and structural properties. In collaboration with the Chu group in the UW Department of Physics, we use these crystals to compare the charge transport properties of organic-inorganic to all-inorganic perovskite as a function of temperature and doping concentration, in order to understand the mechanisms that determine the charge carrier mobility in these materials. Insight gained from studies on these crystals can be applied to improve device fabrication procedures, ultimately improving perovskite device performance and stability and thereby hastening the introduction of efficient perovskite-based materials into the emerging solar energy market.

Small cell lung tumor (SCLC) is a type of lung tumor that accounts for 10-15% of new lung cancer cases. It is an aggressive cancer that is often not detected before metastasis occurs. Even with chemotherapy and radiotherapy, SCLC has a very high mortality rate and patients tend to develop chemoresistance quickly. Unlike other types of lung cancers, SCLC has no available gene-targeted therapies. This project examines the development of chemoresistance in SCLC genetic mouse models . This approach deletes tumor suppressor genes, such as Rb1 and Tp53, and/or activates oncogenes like lmyc, using a Cre-expressing virus in mouse lung tissue, which leads to the development of SCLC. My role included verifying tumor burden following magnetic resonance imaging (MRI) and quantification of tumor volume in the lungs using MRI data. Once MRI confirmed tumor burden, mice are treated with chemotherapy and tumor volume measured over three weeks of drug treatment. The tumor response to chemotherapy will be compared in different genetic mouse models. At the end of treatment, lung tumors are collected and I use immunohistochemistry to measure the response of the tumors collected from the genetic mouse models. I examine markers of cell proliferation and cell death to assess drug effectiveness. These results could be relevant clinically if certain genetic mutations are found to be associated with chemoresistance.

Autism spectrum disorder (ASD) is characterized by deficits in social communication, repetitive patterns of behavior, and sensory sensitivities. Notably, studies have found that greater ASD severity is associated with lower levels of anxiety and depression. This finding raises the question as to which of these defining ASD characteristics influences internalizing behaviors. We explored the influence of social communication and interaction (SCI) on internalizing behavior symptoms between children with ASD and two comparison groups: typically developing (TD) children and children with sensory processing challenges (SPC). It was hypothesized that children with ASD would show greater levels of internalizing behavior symptoms than both the TD and SPC groups, and that social dysfunction would have a greater influence on internalizing behavior in children with ASD than in our comparison groups. Analyses of variance (ANOVA) were conducted to test for group differences in the relationship between internalizing behavior scores and social subscales from the Vineland and Social Responsiveness Scale surveys. An overall group difference in internalizing behavior symptoms F(2, 42)=21.46, p < 0.001 was found. However, a follow-up t-test did not yield differences between symptoms in children with ASD and SPC t(25)=1.03, p=0.793. We then ran a multiple regression to test for group differences in the effect of SCI on internalizing behaviors. We found a significant group difference in the relationship between SCI on internalizing behaviors, significantly explained by the slope of the ASD group, R2 =.495, t(39)=2.988 p =.005. These results suggest that while children with ASD and SPC show similar rates of social deficits and internalizing behaviors, social dysfunction contributes to internalizing behaviors significantly more in children with ASD than children with SPC. This finding indicates that in children with ASD, social dysfunction may be a contributing factor to internalizing behaviors and consequently an important target for intervention.

Cystic Fibrosis (CF) patients are highly susceptible to colonization and infection by Pseudomonas aeruginosa, a bacterium that is a major contributor to CF-associated morbidity and mortality. Inflammation is a key part of the immune response to bacterial infection. However, inflammation can damage the lung and in the case of cystic fibrosis, lead to permanent structural damage. Respiratory failure is the leading cause of death for CF patients. The inflammasome is a protein complex that, upon activation in macrophages, leads to release of signaling molecules, IL-1b, and pyroptotic cell death. P. aeruginosa interacts with the host cell to trigger a protein, NLRC4, that mediates inflammasome activation. We recently found that a common variant of the NLRC4 gene (rs212704) is associated with lower mRNA levels in monocytes and is a genetic marker of NLRC4 deficiency. The cellular and clinical consequences of human NLRC4 deficiency in response to bacterial pathogens are largely unknown. We hypothesize that NLRC4 deficiency regulates host macrophage responses to NLRC4 ligands and infection by Pseudomonas. To test this hypothesis, we stimulated macrophages with ligands known to induce NLRC4 mediated inflammasome activation as well as with P. aeruginosa. We established an optimal dose and exposure time to monitor IL-1b secretion and pyroptosis before applying this assay to monocytes collected from people with the rs212704 variant. We expect less IL-1b production and pyroptosis in macrophages with the variant compared to wild-type macrophages due to an altered inflammasome response in these cells. The common variant allows us to study the mechanisms by which inflammasome activation affects CF-like outcomes. Additionally, we aim to knockout the NLRC4 gene using CRISPR technology and study the impact that it will have on the inflammatory response. The results derived from the experiment will ultimately help in the identification of novel therapeutic targets for patients with cystic fibrosis.

NASA confronts a forward contamination problem with the launch of spacecraft and probes into extraterrestrial environments with potential for detection of life. Earth-origin microbes risk threatening planetary systems, compromising the data obtained, and require implementing microbial control for planetary protection from cross contamination. While generally effective, currently approved decontamination methods are costly, involve high heat and chemical treatments, and risk damaging thermally sensitive spacecraft materials and components. Alternative methods, including atmospheric pressure plasma jets (APPJs), show promise as effective technologies for microbial control. Researchers at Edmonds Community College partnered with Eagle Harbor Technologies (EHT) of Seattle, WA to test the capabilities of an APPJ developed by EHT, performing characterization of killing efficacy. Bacillus atrophaeus endospores were spread on tryptic soy agar plates to quantify killing efficacy of over 900 combinations of APPJ parameters including pulse width, frequency, voltage, distance, time, electrode placement, jet configuration, gas composition, and flow rate. Unprecedented independent control of APPJ pulse width, frequency, and voltage allowed researchers to identify combinations of these parameters resulting in greater than 4-log reduction of endospores. A decimal reduction time (D-value) was additionally determined at an average of 160 seconds on aluminum. High humidity and low concentrations of O₂ were identified as conditions that enhanced killing of endospores. Further characterization of higher voltage, frequency, and humidity, as well as variable pulse width and low O₂ should lead to improved APPJ killing efficacy with decreased exposure time and allow for development of an APPJ optimized for microbial reduction for large-area spacecraft.

 In today’s children, the increased use of electronic devices during early stages of development is providing more audio and visual stimulation than ever before. At the same time, attentional deficits later in life are becoming significantly more common. This has lead us to hypothesize that excessive sensory stimulation (ESS), from media sources like TV, Smartphones, video games, etc., during development increases the risk of neurological disorders, most notably attention deficit hyperactive disorder (ADHD). Previous studies in our lab have found that mice subjected to a model of excessive sensory stimulation have damaged cognitive abilities, are less risk-averse, and display hyperactive behavior compared to controls. Our current study aims to build on this data and investigate the impacts of ESS on attention and impulsivity by using the Five Choice Serial Reaction Time Task (5CSRTT), a behavioral test in which mice nose poke a temporarily illuminated target in order to receive a food reward. CD-1 mice were subjected to ESS in the form of flashing lights and sounds for six hours per day for a total of 42 days and subsequently trained and tested on the 5CSRTT. While there was no difference in accuracy between groups, ESS mice displayed significantly increased premature responses in comparison to the controls indicating higher levels of impulsive behavior. These findings suggest a causal link between excessive sensory stimulation and impulsivity, and further illustrate the possible ramifications of the increased use of technology during development.

Bacteria are widely used in metabolic engineering because they can synthesize chemical products from cheap, renewable carbon sources. A major current problem, however, is that toxic intermediates can build up when bacteria express heterologous biosynthetic pathways. In principle, we can control metabolic gene expression levels so that downstream enzymes quickly consume toxic intermediates. To achieve this goal, we use a programmable gene regulatory tool called the CRISPR-Cas system. To optimize gene activation activity of CRISPR-Cas in bacteria, we sought to identify modular transcriptional activation domains that could be recruited to specific genes to activate transcription. This strategy has been previously used successfully in eukaryotic cells, but with only limited success in bacteria. We used CRISPR-Cas to recruit a number of potential synthetic transcriptional activators to specific loci in E. coli, and identified candidates that can activate a fluorescent reporter gene. We also used protein-engineering approaches to optimize the gene activation activity of our CRISPR-Cas system. The transcription factor screening and protein engineering we did gave an 80-100-fold increase in activation compared to basal transcription levels. To make this new tool useful for practical metabolic engineering, we plan to use this system to dynamically respond to metabolic stresses. This tool could potentially be used to optimize biosynthesis of useful polymer precursors such as lactate and p-aminostyrene. Production of these types of cheap, biosynthetic precursors will help us move towards a greener industrial future.

The obligate human pathogen, Mycobacterium tuberculosis (Mtb), is the primary causative agent of the infectious disease tuberculosis (TB) and ranks as one of the world’s deadliest pathogens; infecting about 1.5 billion people worldwide. Mtb evades the host immune response via intracellular persistence in alveolar macrophages, where it faces chemical stresses such as a drop in pH, various hydrolytic enzymes, increased toxic ion concentration, and highly reactive oxygen (ROI) and nitrogen intermediates (RNI). Modification of mycobacterial proteins by the prokaryotic ubiquitin-like protein (Pup) and their subsequent proteasomal degradation plays a critical role in Mtb survival in this harsh environment. Thus enzymes associated with protein pupylation may be valuable targets in anti-tubercular therapy. This knowledge is exploited in a rational approach to drug design wherein known structural information about the sole Pup ligase, PafA, is used to develop specifically targeted inhibitors. These inhibitors take the form of stabilized short α-helical peptides, which mimic the hydrophobic and electrostatic interactions in Pup essential for binding to PafA. Production of these compounds was achieved using both automated and manual solid-phase peptide synthesis, as well as 'Click' chemistry to modify cyclized variants. Quantitation of inhibitor performance was accomplished by densitometric analysis of SDS-PAGE results from in vitro pupylation assays with purified proteins. Here I report my work on the successful design, synthesis, and testing of helical peptides which display significant activity as PafA inhibitors.

Fundamental understanding of how effector T-cells are transformed into quiescent memory T-cells is vital for developing immunotherapeutic treatments for autoimmune diseases and cancer, and for the development of vaccines against diseases such as AIDS, tuberculosis and malaria. Previous research on conversion of memory precursor effector cells (MPECs) into memory T cells has found evidence of Treg cell involvement in the process. Treg cells are regulatory helper T cells which selectively inhibit effector T cells to prevent and suppress overly strong immune responses and autoimmune responses. Research has also shown that MPECs produce their own IL-2, a cytokine involved in immune tolerance and activation. Based on these data, we hypothesize that direct interaction between MPECs and Treg cells, possibly via MPEC-produced IL-2, is required for conversion of MPECs into memory T-cells. We also addressed colocalization of Treg cells with short-lived effector cells (SLECs). SLECs are IL-2 non-producing effector cell counterparts of MPECs and do not form memory. Potential MPEC or SLEC interaction with Treg cells was visualized through immunofluorescence microscopy of the secondary lymphoid organs (spleen and lymph nodes) of experimental mice injected with lymphocytic choriomeningitis virus (LCMV). To bypass the low precursor physiological frequencies of MPECs we employed the P14 LCMV GP33-specific TCR transgenic mouse system, which is a well-regarded model system for studying memory differentiation. To test the role of IL-2 in cell-cell interactions, we used IL-2 agonist and antagonist antibodies. A detailed understanding of how Treg cells are involved in the process of MPEC to memory T cell conversion is key to developing novel immunotherapeutic treatments.

Cell movement during development is crucial as the location of a cell determines what that cell will ultimately become. Cells require both adhesion and motility for movement. Adhesion is cells holding on to each other via proteins called cadherins. Motility is movement through projections called fillopodia and lamelliopodia. We are interested in the protein p120-catenin, which determines how cells move. We purpose that phosphorylation, or the addition of negative PO4- groups at specific amino acid residues changes p120 catenin’s function. Specifically we are interested in tyrosine (Y) residues 228 and 335 which likely effect p120 antagonistically, by mediating adhesion and motility, respectively. In order to mimic the process of phosphorylation we mutated each residue to either glutamate (E) or phenylalanine (F). Glutamate, which is charged, mimics phosphorylation, while phenylalanine cannot be phosphorylated, but is otherwise similar to tyrosine. To examine which pathway is activated, we quantified whether proteins associated with motility or adhesion were interacting with p120 catenin for each mutation. The primary technique we used to explore this is coimmunoprecipitation. Coimmunoprecipitation works by using antibodies to link p120 catenin to magnetic beads, as well as any proteins interacting with it. The beads can then be extracted and we can determine the amount of each protein interacting with p120 catenin through a process called western blot. So far we have verified that our mutations for Y228 and Y335 produce proteins, which guarantees that any later results are due to phosphorylation. Also preliminary coimmunoprecipitations suggest Y228 does mediate adhesion. Additionally injection of Y228E in embryos, results in embryos with shortened tails and poor yolk constriction, which are associated with excessive adhesion. Taken together these results strongly support our hypothesis for Y228 mediating adhesion. Based on these results, in the future we plan on examining Y335 and verifying our Y228 results.

Immunotherapy has yielded exciting results in the clinic, primarily against blood cancers. Moving toward applying immunotherapy to solid cancers, the field has seen success in treating certain cancers, such as lung cancer, with immunotherapy. However, only a fraction of patients respond to treatment, highlighting a need for continued research in this area. The ‘KP’ model is a genetically engineered mouse model of lung cancer, relying on Cre-recombinase inducible activation of oncogenic Kras and inactivation of the tumor suppressor p53, the two most frequently mutated genes in human lung adenocarcinoma. Tumors are initiated by delivery of Cre into lung alveolar cells by the administration of Cre-expressing viruses. Using this virally-induced model, we can control the timing of tumor induction and tumor burden, and introduce additional genetic modifications into tumors. However, the use of viruses can cause inflammation, potentially impacting the immune responses that we are studying. Improving this model will make the study of lung cancer more physiologically relevant. Therefore, we are developing a system for separating viral infection from tumor initiation, dependent on a tamoxifen-inducible Cre-ER allele. By developing viruses that express Cre-ER, mice can be infected and the induction of tumors can be delayed until a timepoint when virus-related inflammation has subsided. Then, Cre activity can be induced by administration of tamoxifen. Additionally, this system allows us to control the number tumors that form, by titrating the doses of tamoxifen, to better recapitulate human disease. To date, we have cloned DNA fragments and performed Gibson Assembly, and are currently validating clones. We have produced lentiviruses and titered the viruses using GreenGO cells, a cell line carrying a Green Fluorescent Protein reporter of Cre activity. To test Cre-ER functionality and sensitivity, we will use GreenGO cells to assay differences in Cre–ER activity, using different concentrations of tamoxifen in vitro.

Numerous imaging studies have reported brain chemical alterations in children with autism spectrum disorder (ASD). Neurochemical concentrations have been linked to specific cognitive abilities and behaviors in both typical and atypical populations. This preliminary study investigates the relationship between neurochemical profiles in high-functioning children between 8 and 12 years of age with ASD (n = 17) and typically developing (TD) children (n = 21) in relation to IQ as measured by the Wechsler Abbreviated Scale of Intelligence (WASI). All data were collected as part of an ongoing study. Cognitive, behavioral, and diagnostic evaluations were performed by clinical psychologists at the UW Autism Center. Concentrations of N-acetylaspartate, choline, creatine, and glutamate in the left cerebellum and left amygdala were measured using single-voxel proton magnetic resonance spectroscopy (1H-MRS) on a 3T MR system. Significant positive correlations between choline concentration and Performance (p<0.01), Verbal (p<0.05), and Full Scale (p<0.01) IQ were found in the ASD sample in the amygdala, and between choline concentration and Performance IQ (p<0.01) in the cerebellum. No significant correlations were found between brain metabolites and IQ scores in the TD sample, or between other metabolites and WASI IQ scores in the ASD sample. Additionally, there were no significant group differences in metabolite concentrations between the ASD and TD groups. Choline is a precursor to the neurotransmitter acetylcholine, which plays an important role in memory, attention, and cognitive flexibility. These preliminary findings suggest that small differences in brain choline concentrations may have a larger impact on cognition in children with ASD than in TD children.

Targeted therapies designed to inhibit hyperactive oncogenic signaling have demonstrated some encouraging clinical responses. However, many of these responses are not durable as tumors may develop new mutations to “work around” pathway inhibition leading to drug resistance. Technologies that enable more efficient discovery of “suites” of drugs that inhibit multiple pathway nodes are needed. The SABRE (Splice Acceptor Brilliant Reporter) platform was developed to provide an unbiased method to screen large compound libraries and dramatically improve the speed and efficiency for which novel targeted therapeutics can be identified. SABRE is built on the premise that oncogenic changes in gene transcription can be harnessed as powerful reporters of pathway activation status. SABRE utilizes gene trap technology coupled with a drug selection process to isolate cells that generate a robust “off to on” luciferase signal in response to pathway inhibition. Via massively parallel comparative analysis, multiple traps let nature provide the best reporter for further analysis and drug discovery. We employed the SABRE technology to identify insertion sites specific to the MAPK (mitogen-activated protein kinase) pathway, an oncogenic pathway that is implicated in a plethora of cancers. Experiments were performed using the human melanoma cell line A375, containing BRAFV600E mutation. SABRE lentiviral transduced A375 cells were treated with trametinib, an inhibitor of MAPK pathway, and clones were isolated that emitted a positive luciferase signal upon drug treatment. To determine if these reporters were MAPK pathway specific, the platform was used to screen a 6000+ compound library. Results from the screen found that 70% (28/40) of the top drug hits were known to directly modulate the MAPK pathway revealing the power of the SABRE gene trap technology to generate a panel of reporters specific to an unlimited number of cancer signaling pathways. SABRE holds the promise for discovering untapped drug therapies for cancer.

36.7 million people are living with HIV worldwide, with most in low- to middle-income countries. Among many clinical interventions for controlling HIV, Antiretroviral Therapy (ART) has shown success in reducing HIV transmission. However, the effectiveness of these ART is undermined by rising drug-resistant HIV. The lab-based Oligonucleotide Ligation Assay (OLA) can specifically detect HIV drug resistance as an alternative to expensive complete-genome sequencing, but its reliance on moderately expensive lab equipment prevents its universal access. Our lab, in collaboration with the Frenkel Lab (Seattle Children’s Research Institute) and the Lai Lab (UW Bioengineering), is developing a simplified OLA-based HIV drug resistance test. In the OLA, PCR amplification of the HIV Pol region is necessary before subsequent detection of Single Nucleotide Polymorphisms (SNPs) conferring HIV drug resistance. Current amplification relies on a thermal cycler, a >$2.5k instrument not widely available in low resource settings. Isothermal Helicase Dependent Amplification (HDA) eliminates need for a thermal cycler. HDA has been used to amplify the conserved Gag and Env regions of HIV, however it has never been used for amplification of the Pol region, a non-conserved region containing mutations that interfere with DNA annealing in isothermal amplification. We have designed and validated three sets of primers that successfully amplified three regions in the HIV Pol gene covering six common SNPs that confer HIV drug resistance against 1st-line ART. These primer sets each could detect down to 10 copies of HIV Pol DNA per reaction.  We are currently working on a multiplexed HDA assay that simultaneously amplifies three regions in a single tube to reduce cost and consolidate workflow. Implementation of HDA will allow OLA to be further simplified for use in low resource settings by allowing accurate diagnoses of HIV-infected individuals, controlling transmission rates, and reducing the evolution of drug-resistant HIV within entire populations.

The purpose of this research project is to demonstrate the feasibility of incorporating a new photocatalytic Diels-Alder reaction into the curriculum of undergraduate organic chemistry laboratories at University of Washington. Although the traditional Diels-Alder reaction is thermally allowed and does not require UV light catalysis, it fails to succeed when both the dienophile and diene are electron rich due to electrostatic repulsion between the reagents. Recently, an alternative photocatalytic Diels-Alder reaction was reported in the chemical literature involving a ruthenium bipyrazine catalyst, Ru(bpz)₃X, where X denotes the counter anion. This new photocatalytic method is advantageous because it allows the Diel-Alder reaction to work when both the diene and the dienophile are electron rich. Consequently, we believe that the new photocatalytic Diels-Alder reaction would be an attractive addition to our undergraduate laboratory curriculum by increasing the versatility of Diels Alder reagents to accommodate both electron rich substrates. Because the ruthenium catalyst is not commercially available, it must be synthesized from chloropyrazine. Two equivalents of chloropyrazine were coupled together under heated conditions and a Pd(Ph₃)₄ catalyst to yield a solution mixture containing bipyrazine, which was purified via silica flash chromatography. Subsequently, bipyrazine was reacted with RuCl₃xH₂O in ethylene glycol at 170°C to afford a solution mixture containing Ru(bpz)₃Cl₂. This chloride salt was reacted with NaBArF to precipitate the desired catalyst, Ru(bpz)₃X, where X represents the anion BArF-. Finally, the catalyst was purified through alumina flash chromatography. We intend to test the efficacy of this new photocatalytic method using the electron rich substrates trans anethole (dienophile) and isoprene (diene) in the upcoming months. We expect that the two photocatalysts we have synthesized will catalyze the cycloaddition reaction between isoprene and trans anethole with endo stereoselectivity.

Spinal-cord injuries can lead to loss of motor function in affected limbs. Our laboratory has demonstrated enhanced recovery of forelimb motor function after spinal-cord injury using a neuroprosthetic intervention, targeted activity-dependent spinal stimulation (TADSS), to direct plasticity in spared motor pathways. TADSS uses a recurrent neural-computer interface to detect muscle activity in the affected forelimb and deliver (electromyographic) activity-triggered electrical stimulation at a functionally related spinal site below the lesion. To further enhance motor recovery outcomes and make them longer lasting, we are combining TADSS with delivery of plasticity-enhancing neuromodulators, such as brain-derived neurotrophic factor (BDNF) and quipazine, a serotonin-receptor agonist. Rats with cervical contusion injuries have been receiving TADSS in conjunction with delivery of BDNF and quipazine for 5 hours/day through chronically implanted Pt/Ir tubes. Recovery of forelimb movements over a 12-week therapy period is assessed through performance on a trained food pellet reach-grasp retrieval task, administered twice per day during the 5-hour therapy period, which further serves as physical retraining of the injured limb. We expect our combined electrochemical therapeutic strategy to lead to enhanced motor recovery outcomes compared to delivery of TADSS alone. This study may lead to development of novel therapies for targeting spinal-cord injuries.

The family of Transient Receptor Potential (TRP) proteins contains ion channels with a wide array of functions, including invertebrate phototransduction, responding to painful stimuli, responding to temperature changes, and many others. Of particular interest is the polymodal receptor TRPV1, which responds to many stimuli, including capsaicin, heat, pH, etc. TRPV1 is known to play a role in the sensation of both pain and heat; however, the structural dynamics that underlie TRPV1’s ability to transduce these signals are still incompletely understood. Therefore, an understanding of the activation, regulation, and structure of TRPV1 are of clear importance. To address these questions, I have sought to use Xenopus laevis oocytes as an expression platform to perform studies on the structure and function of TRPV1. Since TRPV1 is not naturally expressed in Xenopus oocytes, the genetic information needed for the cell to build the protein was provided for the oocyte via microinjection of RNA. After an incubation period of several days, Western blot techniques were applied to analyze the presence and strength of expression of TRPV1. To better understand specific structural changes made when in the activated conformation, the fluorescent, non-canonical amino acid L-ANAP was integrated into TRPV1 using amber stop codon suppression and an engineered tRNA synthetase. Integrating the non-canonical amino acid ANAP enables structural and functional analysis of the membrane protein via fluorescence microscopy. The results of applying such methods to understand the function of TRPV1 will provide insight into the use of TRPV1 for therapeutic purposes, most prominently the reduction of the sensation of pain.

Aging can be defined as the time-related deterioration of physiological functions that are essential to the fertility and survival of a living organism. In the Kaeberlein Lab, we study how cells change with age and how these changes contribute to age-associated diseases in order to help people live healthier lives. The eukaryote Saccharomyces cerevisiae (yeast) is used as a model for cellular aging. Due to its conservation of cellular functions through evolution, modeling aging using yeast can provide insights into how mammalian cells age. Through replicative lifespan (RLS) analysis, we are able to determine the effects of interventions on aging by measuring the number of divisions that a single yeast cell goes through before it stops dividing. We are testing various nutraceuticals in order to identify novel pharmacological interventions that increase lifespan. In a screen of twenty nutraceuticals, we found that Pterocarpus marsupium (PME) extract significantly extended lifespan. P. marsupium is a deciduous tree native to the Indian regions. It is well-known to Ayurvedic medicine thanks to its purported curative and lenitive properties. The flowers are said to be effective in lowering fever; its heartwood may possess purifying and detoxifying effects; its wood is used to treat chest and body pain as well as indigestion. Additional studies will test whether the RLS-extending effects of PME can be attributed to pterostilbene, a compound found in PME that is structurally related to resveratrol. We tested concentrations of pterostilbene ranging from 1-50 µM to identify its effect on replicative lifespan. These studies will allow for further development of PME and pterostilbene for use as an intervention to promote healthy longevity.

One of the research goals of the Stoll lab is to characterize how the behavior of tryptophan radicals is influenced by their nano-environment. Tryptophan is an aromatic amino acid that facilitates electron transport during reduction or oxidation reactions within a protein. The ability for a protein to fine-tune tryptophan’s reduction potential by modulating its nano-environment is a crucial aspect of the function of enzymes such as cryptochromes, which are involved in regulation of circadian rhythm, and photolyases, which are involved in DNA repair. In order to study the relationship between protein environment and tryptophan oxidation, we have designed and synthesized model peptides to engineer specific environments. The peptides used in this study fold into small cages surrounding a tryptophan residue. Mutations to the peptide change the local environment around tryptophan from charged to hydrophobic with varying levels of solvent exposure. The reduction potential of the tryptophan in these peptides was then determined using differential pulse voltammetry experiments. Each peptide was tested over a pH range from 1-7.5 in order to determine how the pH dependence of tryptophan's reduction potential responds to changes in its nano-environment. We have identified some initial trends such as the tendency for more cationic environments to increase the potential at which tryptophan oxidation occurs. Further analysis will allow us to develop a model which will be able to directly predict tryptophan oxidation behavior as a function of the pKas of surrounding residues and a weighting metric based on solvent exposure. We hope to extend this model to be able to describe the tryptophan redox behavior in full proteins.

Expansion Microscopy (ExM) is a revolutionary fluorescence microscopy technique that enables super-resolution imaging of fixed biological specimens on ordinary microscopes. By chemically incorporating the specimens into a hydrogel polymer matrix, enzymatically digesting the proteins that hold the structures together, and physically expanding the hydrogel complex, this technique yields expansion of fourfold along each x/y/z axis and a spatial resolution of ~70nm. The original ExM approach utilized special tri-functional antibodies containing a DNA-dye-linker complex to attach the fluorescent dyes to the expandable polymer. Previously, our group has developed an improved and simplified ExM protocol, which is compatible with conventional antibodies and fluorescent proteins, by using only commonly available reagents, yielding increased fluorophore retention for cultured cells and tissue specimens. In this project, we extend this new approach of ExM to enable ultrahigh resolution imaging of Drosophila melanogaster tissues which had previously been incompatible with expansion. To do so, we adapted our existing ExM protocol to Drosophila embryos, larval brains, and larval and adult body walls by utilizing an enzyme system that can efficiently digest the tough outer cuticle of the organism. We assessed the uniformity of expansion achieved by our new procedure using correlative pre-expansion and post-expansion imaging and found the distortions to be minimal (<2%). With our new ExM approach, we were able to resolve features of presynaptic active zone structure and study the interactions between the epidermis and dendrites of somatosensory neurons in Drosophila. This work should open up this powerful super-resolution microscopy method to a large community of researchers who utilize Drosophila as an important model system in biology.

On Twitter, there are over 52,000 tweets containing the word “antibiotic”, many of which portray inaccurate information. Young adults are social media’s most avid users and may view such content. It remains unclear if and how college students decipher information in inaccurate tweets. The purpose of this study is to investigate how youth perceive and assess the accuracy of Twitter posts about antibiotics. College students were recruited from the Seattle area through snowball sampling and participated in focus groups. Four focus groups were conducted with a range of 3-5 students in each group. Researchers developed a mock Twitter feed composed of real tweets discussing antibiotics. The tweets chosen conveyed accurate information (e.g., “Antibiotics don’t help viral infections…”), made inaccurate claims (e.g., “Doctor gave me some antibiotics for my flu…but a least I’ll recover”), or embodied disconnect from health assertions (e.g., “Finished antibiotic Tue...”). The facilitator read a vignette about being a college student, sick during finals weeks, who turned to Twitter, and searched “antibiotics”. Students were asked to imagine that they are this person when answering questions throughout the focus group. The mock Twitter feed was presented. Students discussed initial perceptions of the tweets, followed by being asked to group the tweets in any way they saw fit. Then students were asked to categorize the tweets again, but this time into accurate or inaccurate claims about antibiotics. After, a discussion was held about the categorizing processes. The focus groups were recorded, transcribed, and analyzed for reoccurring themes throughout the discussions and categorizing process. This study helps inform health professionals of a need to improve education about antibiotics particularly around evaluating the accuracy of social media content.

We are studying the regulation of p120 catenin in zebrafish embryonic development. P120 catenin plays an essential role in the pathway for cell migration and normal embryonic development. Our research on cell migration and the extension of the dorsal axis can be applied to all vertebrates. These functions are also relevant to many clinical issues such as Spina Bifida in humans, wound healing, and cancer metastasis. The particular protein we're studying (p120 catenin) facilitates the activation of Rac1 GTPase when it is bound to both Vav2 and Rac1 GTPase. Vav2 is a guanine nucleotide exchange factor (GEF) that activates Rac1 by exchanging GTP for GDP. Rac1 GTPase activates the actin polymerization and lamellipodia. There has been research suggesting that the dephosphorylation of certain tyrosine residues in p120 catenin is necessary for this binding and subsequent Rac activation. Rac activation promotes cell migration in zebrafish embryonic development. This research has prompted us to ask how phosphorylation at Y217 and co-phosphorylation at Y228-Y217 of p120 catenin affect gastrulation of zebrafish embryos. Based on previous research, we expect embryonic injection of mRNA containing Y217E and Y228E-Y217E mutations to result in abnormal gastrulation. Abnormal phenotypes exhibit delays with cell migration and yoke constriction. In contrast, mutations Y217F and Y228F-Y217F should result in a rescue phenotype similar to the wild type, since the phenyalannine residues are not negatively charged. In order to test this, we designed primers for each mutation, and inserted them using PCR. We used gel electrophoresis to confirm the effectiveness of the PCR by comparing the size of the PCR product with its expected length. We amplify the DNA in E. coli, followed by DNA purification, and sequencing. With confirmed mutations, the next step is linearization of the DNA followed by production of mRNA. We are now in the process of synthesizing mRNA for the multiple DNA mutations we have created. We will then inject them into zebrafish embryos with knocked down wild type p120 catenin and look for resulting phenotypes.

Alzheimer’s disease (AD) and cancer are two of the most devastating public health challenges in modern society costing 361 billion dollars and 685,240 lives each year. As these two classes of pathologies progress they both generate protein stress inside cells. Tumor cells are often characterized by greatly increased protein production while most neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease are classified as a “proteinopathies”; characterized by the accumulation of the misfolded proteins. Some recent reports suggest that the Parkinson’s disease related protein α- synuclein influences cancer growth in mice and cell culture models. The goal of our project is to uncover the cellular pathways that are shared between neurodegeneration and oncogenesis. We will study this using the C. elegans genetic model system. I have generated a transgenic line that ubiquitously expresses a human form of the AD-related protein, Amyloid-βeta, and observed that it develops similar tumors to cancer-model worm lines. We are also generating transgenic worm lines that ubiquitously express the other canonical neurodegenerative proteins α-synuclein and Tau to see if they also form tumors. These transgenic lines will allow me to knock down different cellular pathways using RNA interference (RNAi) which silences or inhibits the gene expression using double stranded RNA. First we will look chaperone functioning because this class of proteins is necessary for the cells to deal with protein stress. Research in our lab has already shown that RNAi of some chaperones decreases the occurrence of tumors in a worm cancer line. Next, we will generate worm lines with fluorescent reporters of the tumor altering genes and reporters of cancer-related cell-signaling pathways. Thus, we will begin to uncover the ways in which AD-related protein stress impacts cell processes that promote cancer. This could lead to new gene targets for future therapies.

Our laboratory has identified a group of neurons that expresses calcitonin gene-related peptide (CGRP) in the parabrachial nucleus (PBN), which are involved in appetite suppression and responses to fear. For example, CGRPPBN neurons mediate the suppression of food intake during a conditioned taste aversion (CTA) and are activated following conditioned fear responses [1,2,3]. Here, we investigate whether CGRPPBN neurons mediate anorexia associated with a diet deficient in essential amino acids (EAA-def anorexia). We inactivated CGRPPBN neurons by injecting an associated adenovirus (AAV) carrying the tetanus toxin light chain (Tet tox) in the PBN of CalcaCre/+ mice (Calca is the CGRP gene) and showed increased food intake and decreased body weight loss compared to control animals on a prolonged diet deficient in EAAs. In a pair fed group of mice injected with the Tet tox virus, there was not a significant difference in body weight loss. These results suggest that CGRPPBN neurons modulate appetite during EAA-def anorexia, but are not responsible for a change in energy homeostasis during EAA-def anorexia.

Since DNA replication and transcription are concurrent in most bacteria, collisions (or conflicts) occur between these two processes. Conflicts have deleterious effects on cells, including double strand breaks and mutagenesis, especially when a gene is transcribed head-on to replication. Because conflicts increase mutagenesis in specifically genes that are oriented head-on to replication, this process may be a conserved strategy for targeted evolution. Consistent with this, a bioinformatics analysis determined that many genes in the head-on orientation are involved in stress response. We previously showed in B. subtilis that the error prone translesion synthesis (TLS) polymerase YqjH is required for the asymmetric mutagenesis of genes in the two orientations and that transcription-coupled nucleotide excision repair (TC-NER) is also involved in this repair pathway. Mutation rate (or reversion assays) and survival assay experiments performed under DNA damaging conditions, however, indicate that a different TLS polymerase, YqjW may be responsible for mutational asymmetry, and that YqjW functions in the same pathway as TC-NER. Thus, YqjW may facilitate targeted evolution under DNA damage conditions. 

Müller glia (MG) are the predominant glia of the neural retina. In fish and birds, retinal injury causes these glial cells to de-differentiate into retinal progenitor cells (RPCs), and these RPCs repair the damaged tissue by generating new neurons. This process does not happen in mammals, and so retinal damage is permanent. microRNAs (miRNAs, miRs) are small RNA molecules that regulate gene expression. Several miRNAs are expressed in the RPCs, but not in the Müller glia, and we call these RPC-miRs. This study focused on if artificially expressing the RPC-miRs would stimulate mammalian MG to regenerate new neurons in postnatal mouse retinal explants. Two leading questions were explored: Will the Müller glia treated with RPC miRNAs regenerate new neurons? What is the best time window to convert Müller glia into retinal progenitors with high efficiency? Retinas from postnatal (P) 4 up to P12 wild type or transgenic reporter mice were dissected from surrounding eye tissue and electroporated to introduce RPC artificial miRNAs (such as miR-15a, miR-17, miR-19, and miR-20a mimics) or control miRNA mimics, along with a reporter mRNA.  Explants were cultured for 3/4 or 6/7 days ex vitro (DEV), fixed, cross sectioned, stained (immunofluorescent labeling) and analyzed using confocal microscopy. The proliferation marker EdU was added to track cell proliferation. Our data showed that RPC-miRs led to an increased proliferative response in the Müller glia, indicating that the cells were adopting an RPC-like state. Moreover, the age of the explants was an important factor; the areas that were successfully electroporated became smaller in older explants and therefore fewer Müller glia can be targeted in older tissue. Our data shows that miRNAs from RPCs can promote Müller glia to adopt some RPC-like characteristics and therefore could be used to stimulate regeneration in the retina.

According to the World Health Organization, an estimated, 780 million people do not have access to clean water sources. Disasters are another potential cause of disruption to the supply of clean water. When the safety of a water source is unknown, a cost effective method to determine water safety is necessary to reduce unsafe water related disease and death. Prior synthetic biology research has been conducted utilizing transgenic yeast as a biosensor for heavy metal detection, such as using plasmids expressing Green Fluorescent Protein (GFP) which are promoted by heavy metal binding transcription factors. While able to detect copper ions in a dose dependent fashion, the relative instability of the plasmid, reporters which are difficult to detect in situ, and limited targeting to copper and silver ions make the efficacy of the designs impractical. Using modern methods of plasmid integration and the modulation of the copper sensitive pCUP1 promoter expression we sought to produce a stable and more sensitive biosensor for the detection of copper ions in water. We successfully integrated the genetic cassette pCUP1-yeGFP into the yeast genome and produced a viable biosensor for the detection of aqueous copper. We tested this yeast strain via a cytometer after exposure to copper of varying concentrations at varied time intervals. The results showed a dose response curve that positively correlated with copper concentration in a semi-linear fashion, which indicated the pCUP1-yeGFP transgene was a viable sensitive reporter for the detection of copper. Currently, variable constitutive promoters are being altered to increase copper detection sensitivity by modulation of the expression of the metal binding transcription factor Ace1. The team is currently exploring the effect of Ace1 upregulation on the sensitivity of copper sensing. After investigating sensitivity modulation, an exploration of practical field detectable reporters, such as violacein, will be undertaken.

Mutator phenotypes due to mutations in genes encoding DNA polymerases or mismatch repair proteins lead to increased error rates during DNA replication that accelerate the evolution of cancer cells and contribute to chemotherapy resistance. Work in the yeast Saccharomyces cerevisae indicates that excessive DNA replication errors can lead to error-induced extinction (EEX), where every cell within the population dies due to a random lethal mutation. Thus, a possible direction for cancer therapy may be to modulate mutation rates of mutator cells – to either increase mutation rates to a lethal level or suppress mutation rates to their baseline levels in order to slow the rate of tumor evolution. Genetic pathways that may be targeted to modulate mutator phenotypes have yet to be fully elucidated. To identify such pathways, we isolated spontaneous haploid yeast eex mutants that escaped from error-induced extinction caused by combining a mismatch repair defect (msh2Δ) with the pol2-L439V allele, which models a human cancer mutation affecting DNA polymerase epsilon. We crossed the eex mutants to wild type yeast, generating diploid strains that were heterozygous for msh2Δ, pol2-L439V, and the eex mutation. Following sporulation, we measured the mutation rates of haploid MMR-proficient pol2-L439V progeny in order to assess the presence or absence of the antimutator mutation. For each mapping experiment, cells showing evidence of suppressed mutation rates were pooled separately from those exhibiting the normal pol2-L439V mutator phenotype. Unique mutations present only in the antimutator pool were identified by Illumina Next Generation Sequencing. Candidate eex mutations were engineered into fresh strains to assess the antimutator phenotype. Our compiled list of antimutator mutations and their functions in yeast may allow us to identify analogous mutations in the human genome and explore new methods of cancer therapy by which specific genes can be targeted to lower mutation rates to normal levels.

Post-surgical endophthalmitis is a serious vision-threatening inflammation following any ocular procedure. Detection of endophthalmitis with culture has been traditionally used; however, endophthalmitis caused by viruses are overlooked. In a 1995 Endophthalmitis Vitrectomy Study by RK Forster, 33% of patient samples were culture negative. This represents a significant portion of patients who could have had endophthalmitis due to viruses. Using alternative techniques, we can characterize viral pathogens found in post-surgical endophthalmitis with qPCR (quantitative polymerase chain reaction) and polymerase chain reaction (PCR). qPCR quantifies the number of viral DNA strands whereas PCR indicates presence or absence of the viral DNA. This allows doctors to treat more patients which would have been previously thought to not have endophthalmitis. Patients diagnosed with endophthalmitis following intraocular procedures were recruited from Wills Eye Hospital and University of Washington from Sept 1st , 2014 to Dec 31st , 2015. All patients underwent vitreous or aqueous tap. The specimens were sent for microbial culture. Following DNA extraction, qPCR for Torque Teno Virus (TTV), Merkel Cell Polyomavirus (MCV), and actin were performed in all samples. qPCR results were verified via PCR and gel electrophoresis. A total of 29 samples were collected (12 culture-positive [41.38%], 17 culture-negative [58.62%]). Overall, 18 (62.07%) samples were positive for TTV and 15 (51.72%) for MCV. The presence of TTV or MCV was not associated with culture positivity by Fisher’s exact test. Viral DNA sequences are unexpectedly common in endophthalmitis. The role of these viruses in this disease process remains unclear.

Frailty is an age-related, clinically recognizable state of heightened vulnerability that is defined by having 3 of the following criteria: muscle weakness, slow walking speed, exhaustion, unintentional weight loss, and low physical activity. Frailty is associated with increased risk of disability, hospitalization, and mortality among older adults and therefore there is a critical need to identify potentially modifiable risk factors. It is conceivable that chronic pain increases the risk of frailty through multiple biopsychosocial mechanisms, including physical deconditioning. Accordingly, we sought to determine whether pain and the number of pain sites increases risk of frailty in a nationally representative, prospective cohort study of community-dwelling older adults. We analyzed data on 7,601 Medicare beneficiaries aged ≥65 years who participated in the National Health and Aging Trends Study. Participants were interviewed annually in their homes from 2011 through 2015. Over the 4-year follow-up period, the cumulative incidence of frailty was 17.6% (1,327 new cases). Incidence increased with advancing age and was higher in women than in men. Importantly, frailty was significantly more likely to develop among older adults with pain (23.0%) than in those without pain (12.5%). When adjusted for demographic and health characteristics, the risk of frailty was 54% higher in those with pain than without pain (Incidence Ratio [IR]=1.54; 95% confidence interval [CI]: 1.32-1.80). Frailty incidence also increased significantly with the total number of pain sites. Compared to those without pain, the adjusted IRs for frailty were 1.14 (95% CI: 0.89-1.47), 1.34 (95% CI: 1.10-1.64), 1.52 (95% CI: 1.23-1.88), and 2.08 (95% CI: 1.66-2.61) for older adults with 1, 2, 3, and ≥4 pain sites, respectively. In summary, pain was independently associated with increased risk of frailty among community-dwelling older adults. This finding suggests that multicomponent intervention programs should integrate pain management to more effectively prevent frailty among older adults.

The hippocampus, a brain area traditionally thought to play a key role in memory consolidation, has been shown to be involved in encoding contextual information during reward-motivated decision-making tasks. Projections to and from the hippocampus connect it with structures known to be involved in decision-making behavior and encoding cost/benefit values, making it well situated to contribute to the circuitry processing choice selection. Inactivation of hippocampal areas in rats have been shown to increase variability of choice preference between large rewards with an associated delay, and small rewards received immediately. To elucidate the role of the hippocampus in encoding economic factors of rewards, we recorded from place cells in a hippocampal subregion known to be necessary for spatial coding of salient objects and events, called CA1, during a delay-discounting maze-based task. The task was designed on an elevated T-maze to have three blocks during each daily recording session. During each block the rat chooses several times between a large reward (4 sugar pellets) with a long delay specific to that block (10, 20, or 40 seconds) and a small reward (1 sugar pellet) with a short delay of 3 seconds across all blocks. The rats were familiarized with the delays at the beginning of each block by undergoing 5 forced trials on each side. Behavior measured over 10 free choice trials then completed the block. As expected, preference for the large reward was maximal during 10-second blocks and declined as the delay increased. Preliminary analysis of place fields shows remapping between different delay conditions indicating spatial encoding of reward costs by hippocampus. Remapping was also observed between free and forced trials. These results imply that the place field representation of maze areas reflects contextual spatial information that includes the expected cost of rewards.

The embryonic development of vertebrates involves the anterior movement of progenitor cells, which form and proliferate at the posterior end of the embryonic body and then move anteriorly to differentiate into neural and muscle cells. Our interest is in understanding how these progenitor cells migrate, and how their movement is regulated, using the zebrafish as a model system because of its outstanding optical qualities. In my project, I am examining the role of specific proteins that are hypothesized to be important in this migration. To alter the expression of these proteins, I injected messenger RNAs encoding these proteins and also some newly developed transgenic lines where the expression of these proteins is regulated by a short period heat shock. For the mRNA injections I examined the phenotype of the whole embryo, whereas with the transgenic lines I microdissected the most posterior end of the growing embryo (called the “tailbud”) and imaged cell movement using a confocal microscope, with image analysis using software such as Slidebook and ImageJ. My results show that a change in the expression of several interesting proteins produced abnormal embryos and yielded abnormal movement of progenitor cells and impaired formation of the tail. These results provide useful information that helps us understand vertebrate development, including human embryogenesis.

Human pluripotent stem cells (hPSCs) are defined by their ability to self-renew, proliferate, and differentiate into the three germ layers. Controversy remains as to the role of canonical Wnt/β-catenin signaling (β-catenin dependent) in hPSC maintenance and mesoderm formation. Wnt/β-catenin signaling has shown to be important in the context of cellular homeostasis and embryonic development, and improper signaling can cause growth of cancerous tumors. By determining the role of this signaling pathway in hPSCs, we aim to improve our overall understanding of the molecular processes behind hPSC directed differentiations and early embryonic development. We assessed the role of Wnt/β-catenin signaling in hPSCs by generating a CRISPR/Cas9 mediated knockout (KO) of the signal transducer β-catenin. Expression levels of protein biomarkers of pluripotency (SSEA4 and OCT4) were evaluated and no difference was noted between wild type (WT) and β-catenin KO hPSCs. Additionally, the relative level of cell proliferation between WT and β-catenin KO cells was analyzed by quantification of EDU incorporation using flow cytometry, and no difference was noted between the two cell types. After confirming β-catenin KO hPSCs can self-renew and proliferate, we assessed the ability of β-catenin KO cells to differentiate to form mesoderm in vitro. In order to further demonstrate the level of pluripotency of the KO cells, we also attempted to differentiate the KO cells towards ectoderm. Analysis of the effectiveness of both the mesoderm and the ectoderm differentiations show that while KO cells cannot effectively form mesoderm, they are still able to form ectoderm. In this study, we demonstrate that hPSCs lacking β-catenin can still proliferate, fail to form mesoderm, but can still form ectoderm. Thus, β-catenin may not be required for maintaining the proliferative characteristics of hPSCs, but is required for formation of the mesoderm germ layer, and is therefore an important component of early embryonic development.

The medial geniculate body (MGB) is part of the auditory thalamus and is critical to the processing of sound. It receives input from the inferior colliculus (IC), projects to the auditory cortex (AC), then receives extensive descending projections from the AC, forming a feedback loop. Some evidence suggests that the MGB may remain plastic longer than other parts of the subcortical auditory pathway, and may play a role in learning and experience-dependent plasticity. To further understand the development of the MGB, we measured the volume of the MGB in the Big Brown Bat (Eptesicus fuscus) relative to the rest of the brainstem from prenatal ages through adulthood and found that its relative size did increase postnatally. To look for causes of volume expansion, we measured neuron size and cell density. We found an increase in neuron size and a decrease in cell density. This suggests that not only are neurons becoming larger, there is also a proliferation of connections as evidenced by greater volume of neuropil. These findings are consistent with the idea that new connections are forming as young bats learn. As the next step in our study, we are currently working on collecting the same data for the IC.

The Food and Drug Administration (FDA) approved drug rapamycin lengthens lifespan and rescues rodents from age-related diseases. Previous studies have shown that a 3-months regimen of rapamycin for old mice of the C57BL/6 strain changes the composition of gut bacteria that includes increased bacterial species such as lactobacillus, bacteroides and segmented filamentous bacteria (SFB). In mouse models with a mitochondrial disease, there was a decreased amount of bacteria and an extremely short lifespan. Injection of rapamycin increased the amount of bacteria and prolonged lifespan, showing an association between the quantity of bacteria and lifespan. We are interested in the effect that this experiment would have on a different strain of mice, Balb/c, which had not been previously tested for the rapamycin effect on lifespan nor the change in the composition of gut bacteria. First, we conducted a 90-day injection study on Balb/c mice, in which the mice were injected with different frequencies of rapamycin via an intraperitoneal (IP) injection. After collecting fecal samples at three different time stages – at the start of the 90-day injection period, immediately after the 90-day period, and 3 months after the 90-day period, we ran a qPCR (quantitative polymerase chain reaction) for each of the fecal samples collected, which quantifies the relative amount of microorganisms in the sample. We also monitored the lifespan of those rapamycin-treated Balb/c mice. This allowed us to see if the lifespan extension and gut microbiome change observed in the original model is reproducible in a different mouse strain. We hope that our findings help to support a future translational research strategy that applies the effect of rapamycin on specific types of bacteria to produce an intervention on gut bacteria to slow aging in pet animals and humans.