Zebrafish are a valuable model organism in scientific research, however there are limited genetic tools available for inserting/integrating engineered DNA into precise locations in the zebrafish genome. This project has developed a new method of precisely integrating engineered genes into specific zebrafish genomic locations. The heat shock-70 like (hsp70l) gene in the zebrafish genome is an ideal location for new gene (transgene) integration because the hsp70l promoter enables temporal regulation of the transgene through a heat shock-inducible mechanism. CRISPR/Cas9 genome editing technology was used to insert genetic markers, called attP sequences, flanking the green fluorescent protein (GFP) gene into the hsp70l gene locus in zebrafish. I built a construct containing attB sequences flanking the red fluorescent protein (RFP) transgene. These attP and attB markers are recognized by viral integrase proteins, bxb1 and fC31, which cut and recombine the attP and attB sequences causing directional recombination replacing GFP with RFP. I performed a proof-of-principle experiment by injecting zebrafish embryos with this new attB-RFP construct and the old attP-GFP construct with both the bxb1 and fC31 integrase enzymes to show functional integration in zebrafish. I optimized injection techniques to maximize the recombination frequency of the attB-RFP integration at the hsp70l gene in the zebrafish genome. This system allows for the rapid insertion of any transgene precisely into the endogenous hsp70l locus. I used this system to create heat-shock inducible Cas9 transgenic zebrafish as a tool for characterizing the function of novel genes essential for promoting or suppressing cancer growth in zebrafish tumor models. Inducing Cas9-mediated gene knockout is allowing us to determine the specific role a gene plays in cancer progression which helps us identify viable targets for the development of new cancer therapies.

Current weight loss therapies are largely unsuccessful stressing the urgent need to develop new and effective strategies to treat the growing obesity epidemic. Oxytocin (OT) is a downstream target of the adiposity signal, leptin, and is an attractive therapeutic target to treat obesity in humans because it reduces body weight (BW) in diet-induced obese (DIO) rodents even in the face of an impaired ability of leptin to reduce food intake. Recent findings from our lab indicate that, while reductions in food intake contribute to OT’s ability to elicit prolonged weight loss in DIO rodents and nonhuman primates, mounting evidence implicates a role of increased energy expenditure (EE) through sympathetic nervous system (SNS) outflow to interscapular brown adipose tissue (IBAT) as contributing to weight loss. OT neurons in the parvocellular paraventricular nucleus are positioned to control SNS outflow to IBAT through either direct projections to the spinal cord or indirectly via the hindbrain nucleus of the solitary tract. Thus, these circuits are potential targets for pharmacological therapies to reverse the deleterious metabolic effects of central leptin resistance. We hypothesized that OT-induced stimulation of SNS outflow to IBAT contributes to its ability to elicit weight loss in DIO rodents. To test this, we determined the effect of exogenous fourth ventricular (4V) OT administration on IBAT temperature, BW, and body composition in DIO rats. We found that 4V OT increased IBAT temperature, reduced BW and body adiposity. Acute 4V administration of OT increased IBAT temperature during the test period relative to vehicle-treated controls (45 min measurement: vehicle: 37.2 ± 0.03 vs. OT: 38 ± 0.2°C; P<0.05). Collectively, these studies implicate a role for SNS outflow to IBAT in the mechanism by which OT increases EE and elicits BW loss in DIO rats.

Giardiasis is a disease caused by the protozoan parasite Giardia lamblia which affects approximately 20,000 people per year in the United States alone. To this day, there are few anti-Giardiasis drugs available so new therapeutics to address toxicity and resistance issues are needed. In previous studies, we have examined seven protein kinases in Giardia’s genome and these kinases share an unusual structural feature in their active site, which results from an atypically small gatekeeper residue (amino acid residue). This feature confers sensitivity to a class of compounds called bumped kinase inhibitors (BKIs); BKIs demonstrated minimal toxicity and positive pharmacological properties. We repeated the search for small gatekeeper kinases using an updated multisequence alignment program, which identified four new small gatekeeper kinases. We developed a cloning strategy to 3HA tag each of these kinases. Then, we evaluated the expression and cellular localization of the proteins using Western blot and immunofluorescence (IF) assays. With the tagged kinase lines, we utilized translation blocking morpholinos to test the importance of each kinase using cell growth. This data will be used to prioritize protein purification for screening against a BKI library using thermal shift assays. These results may identify lead compounds toward novel treatment options for giardiasis.

Alzheimer’s Disease (AD), the most common form of dementia, is the sixth leading cause of death in the United States. With a prevalence of over five million cases, the number of AD patients is expected to increase as a new diagnosis of AD is made every sixty-six seconds. Early symptoms include short-term memory loss and gradually progress to devastating deficits in cognition, temperament, and behavior. AD is a neurodegenerative disease linked to the accumulation and aggregation of two proteins, Aβ_1-42 and hyperphosphorylated Tau, which form the characteristic amyloid plaques and neurofibrillary tangles found in the brains of AD patients. Previous studies support the notion that genetic variation underlies the wide array of phenotypes observed among AD patients, and several genes have been discovered to cause early onset AD. In sporadic (late onset) AD patients, it remains unknown what effects natural variation may have on the expression and severity of the AD phenotype. To explore the influence of genes on AD, our study utilizes a Drosophila model of AD that allows us to express both the Aβ_1-42 and Tau peptides in the fly eye. Expression of AD-related proteins in the eye leads to degradation of the hundreds of ommatidia that make up the fly eye. To quantify the extent of ommatidial degradation, we use a computer program, ImageJ, which measures the size and circularity of each ommatidium. To introduce natural variation, the AD fly is crossed with flies from the Drosophila Genetic Reference Panel (DGRP), consisting of 192 fully sequenced isogenic lines. By comparing the Aβ_1-42 and Tau-induced degradation in each background, we can identify naturally occurring modifiers of the AD phenotype. We have already identified promising hits, which could eventually help in the development of novel targeted therapies.

Sickle cell disease (SCD) is a genetic blood disorder characterized by recurrent painful episodes. Having frequent pain is a key predictor of morbidity and mortality. Cognitive-behavioral therapy (CBT) interventions for other chronic pain conditions lead to symptom reduction and improved health-related quality of life, and may also be beneficial for adolescents with SCD. However, most adolescents with SCD do not have access to CBT. Internet-based interventions have been used in other populations for providing access to pain self-management strategies. Thus, the primary goal of this pilot study was to determine the feasibility and acceptability of an internet-based CBT program for pain management (Web-MAP), an 8-session program used effectively to reduce pain-related disability in youth with other chronic pain conditions. Twenty-five adolescents, aged 11-18 years (M= 14.76, SD= 2.05), and their parents were randomly assigned to internet CBT (n = 15) or internet pain education (n = 10). They completed assessments at pre-treatment, post-treatment, and six months follow-up. Eight parent-child dyads randomized to CBT also completed qualitative interviews. Results showed that adolescents and parents were highly engaged with Web-MAP, completing an average of 6 out of 8 modules and logging in on average 16.7 and 17.4 times respectively over 8 weeks. 75% of families interviewed would recommend Web-MAP, although they would prefer a program more tailored to youth with SCD. In conclusion, this pilot study suggests that Web-MAP is a feasible and acceptable treatment for adolescents with SCD and their parents, and may be helpful in improving pain-related disability. Future work will focus on specific tailoring of Web-MAP for SCD to better meet the needs of this population.

Traumatic brain injury (TBI) is an illness that affects people in all walks of life, from everyday citizens to soldiers in combat, and both young and old. Among all cases of TBI, there are some patients who recover quickly and some patients who take a long time to see any improvements, suggesting a range of what we call “TBI resistance”. To see if we could identify a genetic basis to this variation, we used the fruit fly, Drosophila melanogaster, as a model of TBI resistance. Specifically, we used a set of fly strains captured in Raleigh, North Carolina, and commonly known as the Drosophila Genome Reference Panel (DGRP). The DGRP is a sequenced set of inbred flies that researchers around the world experiment on, thus establishing a universal platform to identify genes associated with natural variation for a wide range of fly traits. For our study, we created a spring device to model TBI in flies. The device consists of vial of flies connected to a high resistant spring which propels it towards a hard pad causing damage. We compared the mortality rates between the different lines of flies. We hypothesized that we would see some lines display resistance and some lines display sensitivity. This turned out to be true, and we have already identified candidate genes associated with this variation. As a next step, to study TBI resistance on a cellular and molecular level, we will perform metabolomics with both resistant and sensitive lines to see if there is a significant difference between metabolite expression. The expression of certain metabolites in resistant flies, compared to sensitive flies, could hold the key to understanding how resistance is acquired against TBI and we will be one step closer towards understanding ways to protect people from the effects of TBI.

Pediatric rhabdomyosarcoma (RMS) is a rare and aggressive cancer that arises from skeletal muscle precursors in muscle and connective tissues. The molecular mechanisms underlying RMS progression, relapse and metastasis remain poorly characterized. CD82, a novel metastasis suppressor gene, has been shown to decrease tumor progression in a subset of human cancer types when expressed. Recently, two scientific publications investigated the role of CD82 in cellular differentiation and proliferation in muscle precursor cells. Uezumi et. al (2016) claimed that loss of CD82 results in premature differentiation and a depletion of muscle precursor cells. In contrast, Alexander et. Al (2016) discovered opposing results in which loss of CD82 in fetal muscle cells impairs the capacity of myogenic precursor cells to differentiate into mature muscle cells. In RMS, tumor cells keep on proliferating and have lost the capacity to differentiate into mature muscle cells. The role of CD82 in regulating cell proliferation and differentiation in RMS is unknown. In order to investigate the function of CD82 in differentiation of RMS, I used the CRISPR/Cas9 genome engineering system to target the gene in RMS cell lines. To characterize the knockout phenotype, I performed various cell-based assays to characterize the effects of CD82 gene knockout on the cellular phenotypes in tumor cell differentiation, proliferation, and self-renewal. Investigating the role of CD82 in regulating cellular differentiation and proliferation in RMS would provide important insight into the pathogenesis of cancer.

Plants’ ability to sense ambient light conditions is critical for their fitness. Phytochromes (phy) are photoreceptors that translocate to the nucleus when activated by red light. One consequence of this translocation is rapid phosphorylation and turnover of basic helix-loop-helix (bHLH) transcription factors called Phytochrome-interacting factors (PIF). PhyB has been shown to recruit and inactivate PIF5 in seedlings and inhibit elongation of the stem (hypocotyl). Phytochrome mutants in Arabidopsis thaliana exhibit reduced CO₂ uptake, abnormal accumulation of sugars and metabolites during the day, and shade avoidance-like phenotypes. We aim to compare findings in A. thaliana with phenotypes of light signaling mutants in Brassica rapa, a related crop species. We have isolated phyB, and pif5 mutants and are growing them in ambient and high CO₂ conditions. Measurements of photosynthetic rate as well as levels of chlorophyll, sugars, total protein and starch will provide insights into the role of Phytochrome signaling on metabolic processes. Preliminary results show that phyB mutant seedlings exhibit longer hypocotyls in comparison to wild-type plants at ambient CO₂ but have a reduced growth response to elevated CO₂ compared to wild-type. Adult plants have a reduced photosynthetic rate compared to wild-type. In contrast, pif5 mutant seedlings have short stems and reduced root growth in comparison to wild type. Our results to date are consistent with previous studies in A. thaliana. Examining the role phytochromes play in resource allocation, specifically in response to Carbon availability will aid in identifying key phytochrome-mediated developmental pathways. This research has implications in engineering more efficient crops and in improving understanding of plant metabolism as a whole.

Nitrogen (N) is one of the most important nutrients for plant growth, and fertilizers are heavily used worldwide to provide this nutrient. However, fertilizers are costly to produce and have significant negative impacts on the environment. Microorganisms within plants, termed endophytes, were isolated from wild poplar and willow plants and shown to promote growth in several other plant species as broadly diverse as rice, tomato, and Douglas fir. N-fixation was demonstrated in wild poplar and in hybrid poplar inoculated with a consortium of the endophytes from wild poplar. In order to better understand the genes involved in N-fixation by these endophytes, I created mutant libraries by random transposon mutagenesis. I screened the libraries for inability to grow on N-free medium. The Tn insertion sites were identified by sequencing and I analyzed the results. These results as well as a comparative genomics analysis with nodule-inhabiting rhizobia may provide insight into how endophytes protect nitrogenase from inhibitory oxygen. The project will also provide mutants to be tested on plants for determining the relative importance of N-fixation in the observed growth promoting effects of endophytes. A better understanding of the processes involved in N-fixation could lead to the creation of an optimized consortia of naturally occurring, N-fixing endophytes that could reduce the need for fertilizers in the future.

Adenomatoid odontogenic tumor (AOT) is a rare odontogenic tumor which is often misdiagnosed as odontogenic cyst. The differential diagnosis of AOT is crucial in terms of surgical management. The purpose of this study is to present 14 cases of AOT highlighting their clinical, radiographic and histologic characteristics, so that oral surgeons can become better familiar with this type of tumor. Fourteen cases of AOT were retrieved from the archives of the Oral Pathology Biopsy Service at the University of Washington. Clinically, it presents as a unilocular radiolucency associated with impacted teeth in the anterior jaws simulating a dentigerous cyst. It affects patients between 10-20 years of age with female to male ratio close to 2:1 and anterior maxilla almost twice as common as the mandible. Of the 14 cases reviewed, 12 were intraosseous and 2 peripheral (gingiva). The patient mean age was 16 and age range was 11-30; 8 females and 6 males, close to the reported age and M:F ratio. Of the 12 within bone, 6 were in the mandible and 6 in the maxilla with equal jaw distribution contrasting with the literature of close to 2:1. Radiographically, 10 of the 12 within bone cases were associated with impacted teeth and two between teeth. Six of the impacted teeth were canines and the other four were lateral, central, first and second premolars. The two peripheral AOT cases presented as small nodules on the buccal gingiva of the anterior maxillary teeth with no radiographic changes. Histologically, all specimens showed duct-like structures with epithelial spheres and spindles shaped epithelial cells. Eleven of the 14 cases showed delicate to significant calcifications, some with cementum-like globules. These results could better help surgeons identify AOT to prevent misdiagnosis, which could lead to unnecessary surgical tissue removal or related procedures.

Recovery after thoracic surgery can be prolonged and fraught with complications if patients are not active after surgery. Postoperative recovery can strongly be influenced by preoperative baseline activity level and overall fitness. Surgeons have historically relied upon patient report to understand these variables; however, given the ubiquitous nature of activity monitors in today’s society, a new opportunity is presented to objectively evaluate perioperative activity. As part of the Perioperative Activity and Outcomes study at Virginia Mason Medical Center, thoracic surgery patients begin wearing a Fitbit prior to surgery and return the device approximately three to five weeks after surgery. However, there are several patient factors that influence the data, such as simply forgetting to wear the device. The objective of this study is to understand whether or not days missed wearing a Fitbit is associated with objective measures of activity. I hypothesize there is a negative correlation between the number of days a patient does not wear their Fitbit and their average daily steps. I will stratify the analysis by preoperative and outpatient postoperative periods, both of which are times where it is incumbent upon the patient to wear the device. In essence, poor compliance with wearing the device may be a surrogate for low activity. The conclusions I draw from this research will contribute to a risk stratification model for improving patient outcomes. By identifying patients at-risk for slow recovery, tailored interventions can be employed to optimize recovery, prevent complications, and improve overall patient outcomes, satisfaction, and quality of life following thoracic surgery.

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.

Traumatic brain injury (TBI) is a pathological condition marked by chronic daytime sleepiness. This sleepiness is associated with the decrease in hypocretin neurons in the hypothalamus. Hypocretin cells are known to be sensitive to inflammation, so research on this inflammatory response is important for developing therapies to treat TBI. One type of inflammatory response to TBI is microgliosis, i.e., an increase in activated microglia, the brain’s immune cells. Depending on inflammatory cytokines released, microglial activation can be beneficial or detrimental to recovery from TBI and is therefore a potential target for TBI therapies. The overall goal of this project is to examine the time course of microglial activation in the hypothalamus using a mouse model of TBI. Mice underwent TBI or sham surgery and brains were examined 3, 7, or 15 days after surgery. Brains were sectioned coronally and numbers of microglia in the hypothalamus were quantified using immunohistochemistry. We predict that microgliosis will be most intense at either 3 or 7 days, since previous studies have shown that microglia peak around the site of injury at 5 to 7 days and then rapidly decline. Determining the extent of microglial activation across time will help to determine an appropriate time to administer therapies targeting microglia.

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.

Adrenergic receptors, a family of G-protein coupled receptors, are a common target by pharmaceutical drugs for cardiovascular disease. With cardiovascular disease being the leading cause of death in the United States, adrenergic receptor targeting drugs are increasing in use. Phenylephrine has long been known to be an agonist of the specific α-1 adrenergic receptor. Past research shows that cancer cells do not contain α-1 adrenergic receptors. Although, using dynamic mass redistribution (DMR), when we introduced U251 human glioma cells to increasing concentrations of phenylephrine, we found evidence of a conformational change in the U251 cells. Other adrenergic receptor agonists (norepinephrine, isoproterenol, and clonidine) exhibited a similar response. This suggests that U251 cells may have α-1 adrenergic receptors. We hypothesize that U251 glioma cells exhibit a structural change in the presence of phenylephrine because they contain α-adrenergic receptors that recognize the ligand and trigger a cellular response. The effects of phenylephrine on U251 cancer cell viability and cell proliferation is still unknown, allowing for a possible future in generating therapies to combat cancer.

Foraging behavior in mammals requires the integration of various sensory cues to allow the subject to navigate through the environment. When the search becomes predictable and repetitive, memory can be used to enhance the sensory cues. To model the dynamic interactions between sensory and memory, we performed a comparative analysis on rats and humans by designing a two-stage task paradigm: search and return. The subjects were trained to search for rewards in a confined space with limited sensory cues. After the end of each trial, they were trained to return to the start position. The rat task was performed in an automated arena that allowed the rats to follow the reward’s olfactory cues but prevented visual and hearing cues. Similarly, the human task simulated the paradigm using a computer game that allowed the subjects to search and return in a virtual room with limited visual cues. We predict that sensory cues have a more significant impact on the search strategies compared to the return strategies. If the reward locations are fixated in every trial, we also expect the subjects to increasingly rely on memory for both the search and return strategies. Our research will provide insight on the tracking methods and analysis in studying the roles of sensory and memory in complex foraging behavior.

Two human retroviruses cause AIDS: HIV-1 and HIV-2. HIV-2 is less pathogenic than HIV-1, with lower plasma viral loads, decreased transmission and mortality rates, and a slower decline in CD4+ T cells. Despite this, many patients progress to clinical AIDS and may benefit from antiretroviral therapy (ART). HIV-2–infected patients failing first-line ART often harbor drug-resistant strains of the virus that limit second-line treatment. Consequently, there is a need for rapid, inexpensive HIV-2 drug resistance testing that can guide second-line therapy. In this study, we optimized and validated a novel genotypic drug resistance test for HIV-2 using dried blood spots (DBS). Specimens for this project were collected as a part of an ongoing cohort study of ART for HIV-2 infection in Senegal, West Africa. Blood samples from HIV-2–infected patients were spotted onto filter paper and shipped via courier “overnight” to Seattle at ambient temperature. We then extracted viral RNA and DNA from the DBS, amplified genes of interest using nested PCR, and identified mutations at major drug resistance sites after population sequencing. After obtaining genotypes from only three of 42 (7%) of DBS from a previous protocol, we designed a new, optimized protocol and were able to genotype 10 of 22 (45%) DBS, including five of seven (71%) with HIV-2 plasma viral loads over 50 copies/mL. Drug resistance mutations V47A (protease) and M184V (reverse transcriptase; RT) were observed in 63% and 75% of genotypes, respectively – 75% had evidence of multi-class resistance to both protease and RT inhibitors. These results show that DBS genotyping can identify major drug resistance mutations in HIV-2 patient samples, thereby guiding second-line ART for HIV-2 infection. Because DBS-based resistance testing is a low-cost testing approach which provides results within a clinically-actionable timeframe, we expect that this innovation will improve HIV-2 patient care in resource-limited settings.

Human immunodeficiency virus (HIV) infection is a global health issue and consists of two main types, HIV-1 and HIV-2. As the backbone of all first-line treatments of HIV, nucleoside reverse transcriptase inhibitors (NRTI) have a critical role in antiretroviral therapy. EFdA (4´-ethynyl-2-fluoro-2´-deoxyadenosine; MK-8591; Merck & Co.) is an investigational NRTI that blocks HIV-1 replication in culture with 50% effective concentrations (EC₅₀) in the low nanomolar to picomolar range. However, studies evaluating the activity of EFdA against HIV-2 and against NRTI-resistant mutants of HIV-2 are lacking. As a result, HIV-1 and HIV-2 isolates from antiretroviral-naïve individuals were tested against EFdA in single-cycle infections of a human cell line (MAGIC-5A cells). Site-directed mutants of HIV-2 reverse transcriptase were generated in a full-length plasmid clone and were evaluated for EFdA resistance in the single-cycle assay. EFdA inhibited HIV-2 infection of MAGIC-5A cells with mean EC₅₀ values (± SD) of 0.55 ± 0.17 nM for 8 HIV-2 group A isolates and 0.50 ± 0.12 nM for 6 HIV-2 group B isolates (range = 0.25–0.76 nM for all 14 HIV-2 strains tested). In contrast, the mean EC₅₀ for 10 HIV-1 isolates, including group M (subtype A, B, C, D and F) and group O isolates, was 2.56 ± 0.56 nM (range = 1.96–3.78 nM; p<.0001 for HIV-1 versus HIV-2, Welch’s t-test). EFdA is the most potent inhibitor of HIV-2 replication described to date and is more active against HIV-2 than against HIV-1 in culture. EFdA also inhibits multi-NRTI–resistant HIV-2 mutants with single-cycle EC₅₀ values <12 nM. Our data indicate that EFdA should be evaluated in clinical studies involving HIV-2-infected individuals.

Mutations that confer resistance to antiretrovirals (ARVs) for HIV can arise from drug selection pressure, HIV error-prone replication, or transmission of resistant variants. Understanding the source of resistant HIV strains present during early acute infection, including those present at low-frequency of the viral population, is important for developing interventions to reduce the spread of HIV drug resistance. Phylogenetically linked cases of HIV transmission were identified in a prospective study of HIV serodiscordant couples in Kenya and Uganda. Deep sequencing by 454 technology identified 29 seroconverters that had HIV variants with resistance mutations present during acute infection. Here we sought to determine whether similar resistance mutations were present in their linked index partners. Plasma samples collected within 6 months prior to and after the estimated date of HIV transmission were available from 19 index partners, resulting in 36 plasma samples tested for resistance by 454 deep sequencing. Mutations with a resistance score of ≥30 per the Stanford HIV Drug Resistance Database were assessed. Bioinformatic analysis of the deep sequencing data is complete for 15 of the 19 linked partners. Of the 15 linked partners, 6 (40%) had resistance mutations. Four (27%) had the same mutations detected in the corresponding seroconverter. The resistant variant was present at <10% of the viral population in the index partner in 1 of these 4 cases of transmitted resistance. Data to date suggests that transmission is not the primary source of resistance during acute infection in this cohort. In addition to resistance testing of the remaining 4 index partners, we also plan to deep sequence and perform phylogenetic analysis of HIV variants from genital samples from the linked index partners to determine whether the transmitted virus is more closely related to virus in the blood or genital tract.

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.

Meier-Gorlin syndrome (MGS) is a rare form of proportionate dwarfism that has been linked to mutations in proteins that function during the initiation and elongation steps of DNA replication. Although mutations in DNA replication proteins have been shown to be causative for MGS, it is still not clear how defects during S-phase lead to the phenotypes observed in patients with this condition. Therefore, further research is necessary to better understand the molecular and cellular consequences of cells harboring MGS mutations. Tools for studying DNA replication in humans are limited, however, there are several well-established tools for studying this process in the budding yeast S. cerevisiae. Some individuals with MGS have been shown to have mutations in the DNA replication initiation factor Cdc45, which functions during both the initiation and elongation steps of DNA replication. Because a specific MGS mutation in CDC45 is in a residue that is conserved between humans and yeast, we have replaced the wild type yeast gene with an allele that contains the equivalent human mutation. Yeast with the MGS mutation exhibit increased sensitivity to elevated temperatures and a reduced ribosomal DNA (rDNA) copy number. In budding yeast, the rDNA locus contains ~150 copies of a 9.1 kb repeat which encodes the template needed to make ribosomal RNA—the main structural component of ribosomes. Additionally, each repeat contains an origin of replication. We hypothesize that this loss of rDNA repeats is a consequence of impaired replication initiation at the rDNA locus and will test this using 2-D gel electrophoresis. We anticipate to expand our study outside of the rDNA locus and examine how replication might be perturbed at other locations across the genome. Ultimately, we hope studying MGS in yeast will elucidate our understanding of the pathogenesis of this condition in humans.

Glioblastomas (GBMs) are malignant brain tumors associated with lethal cancers that have a great tendency to rapidly invade healthy brain tissue, leaving patients with short survival times and decreased quality of life. The median survival time for patients treated with the chemotherapeutic, temozolomide (TMZ), and radiation therapy is around 12 months, and many patients develop resistance to TMZ due to high activity of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repairing protein. However, the inclusion of an MGMT inhibitor, such as O6-benzylguanosine (BGS), could work to combat this problem. Currently, the Zhang lab is working on creating nanoparticles (NP) with drug-delivery capabilities, which can cross the blood-brain barrier and deliver chemotherapeutics to targeted glioma cells. This highly drug loaded NP model (IOPH-pBGS) efficiently inhibits MGMT and sensitizes GBM cells to TMZ. However, we wanted to know to what extent a targeting agent would improve this model, and how much efficacy can be increased through optimization of the targeting agent conjugation process. We hypothesized that we can significantly increase drug delivery to tumors by avoiding nonspecific uptake of NPs in healthy cells. My research project was to optimize chlorotoxin (CTX) conjugation to the NPs for use against GBMs. CTX is a small amino-acid peptide sequence that has the ability to target GBM cells. We optimized: the point in the NP synthesis process to add CTX, the length of polyethylene glycol linker to attach CTX to the NP surface, and the reaction ratio of CTX to NP in order to optimize both drug loading and targeting efficacy. In order to evaluate the effectiveness of the various CTX conjugated NPs, SDS-PAGE peptide quantitation assays, cell targeting assays, MGMT quantitation assays and clonogenic cell survival assays were utilized.

Helicobacter pylori is a gastric bacterial pathogen that infects 50% of the world’s population. H. pylori’s helical shape is presumed to help it drill through the mucus lining of the stomach and arrive at the surface layer of gastric epithelial cells, its preferred niche. The bacteria then use a type IV secretion system to deliver toxins to the gastric epithelial cells. Once infected, the gastric epithelial cells initiate an immune response via numerous receptors resulting in a signaling cascade that leads to activation of pro-inflammatory genes including a cytokine called interleukin 8 (IL-8). Gastric epithelial cells secrete IL-8 in an attempt to recruit immune cells to fight the bacterial infection. However, this immune response seldom results in clearance of the infection. This is because H. pylori controls the host’s immune response through a set of virulence factors that are responsible for invoking and maintaining a pro-inflammatory response. Inducing inflammation allows H. pylori to thrive, however, too much inflammation will result in a clearance of the infection. My goal is to understand which H. pylori genes are responsible for modulating the immune response in gastric epithelial cells. To find these genes, I am screening a mutant library that targets virtually all non-essential genes (approximately 1000 out of 1500) in the H. pylori genome. I co-culture individual H. pylori mutants with gastric epithelial cells and assay their response to the bacterial infection. Using enzyme-linked immunosorbent assay (ELISA), I can quantify the amount of IL-8 gastric epithelial cells secreted into the supernatant. By determining which mutant bacteria in our library induce more or less IL-8 secretion in gastric epithelial cells, I can identify bacterial pathways that are important for modulating the host immune response. This approach may potentially uncover bacterial factors that can be targeted therapeutically.

Parkinson’s disease (PD) is a progressive, neurodegenerative disease with a global and national prevalence of four million people and one million people affected respectively. Though PD can be clinically characterized by dyskinesia, resting tremors and rigidity, it remains largely idiopathic biochemically. PD patients can be pathologically characterized post-mortem however, by the presence of Lewy bodies in the Substantia Nigra pars compacta (SNpc) of the ventral midbrain. While the major protein constituent of Lewy bodies is known to be α-Synuclein (α-Syn), the pathogenesis of α-Syn fibrillation in the SNpc remains elusive. One mitochondrial chaperone protein, mortalin, is thought to play a part in PD pathogenesis for its anti-apoptotic effects, role in stress responses, and through its regulation of transcription factor p53. We previously found mortalin to be down-regulated in the corpus striatum of manganese (Mn) miners, as well as primary murine glial cells. In the study at hand, we evaluated the connection between mortalin and p53 and the resultant effect on α-Syn oligomerization in astroglia with Mn-induced neurotoxicity. Using primary astrocyte cultures, western blot analysis, immunofluorescence and confocal microscopy, we evaluated this possible connection and its implications for α-Synucleinopathy. Within Mn-induced neurotoxic conditions, we saw decreases in total p53 of cortical and striatal mortalin knock-down astrocytes and increases in the density of cytosolic oligomeric α-Syn of these cells. Notably, we found that overexpressing mortalin under the same conditions decreased the density of oligomeric α-Syn dose-dependently. Taken together, these data suggest that the interaction of mortalin with p53 could have a role in α-Syn up-regulation, resulting in oligomerization within Mn-induced neurotoxicity. Further study is necessary to elucidate mortalin’s role in α-Synucleinopathy and Lewy Body pathology more precisely, but this work illuminates an important protein-protein interaction and provides implications for understanding PD pathogenesis and for developing therapies against α-Syn fibrillation in Parkinsonian diseases.
 

Approximately 300,000 cases of appendicitis are diagnosed in the United States on a yearly basis. Appendicitis is the condition in which the appendix, located in the right lower quadrant (RLQ) of the abdomen, becomes inflamed usually due to a bacterial infection. A common symptom of appendicitis is when RLQ pain is sustained over time. We, the Surgical Outcomes Research Center (SORCE), hypothesize that antibiotics for intra-abdominal infections recommended by the Infectious Diseases Society of America and American Surgical Association are just as effective as appendectomies for treatment. This project will illustrate to both clinicians and patients that they will have more options when treating appendicitis. The Comparing Outcomes of Drugs and Appendectomy (CODA) clinical trial aims to investigate appendicitis treatment options and understand if antibiotics are just as good as appendectomies (the current standard of care) from the patients' and clinicians' perspectives. Previous studies in Europe suggest the usage of antibiotics is a viable option when treating appendicitis. Patients consenting to the CODA trial are randomized to determine which treatment they receive, either the surgical appendectomy or the antibiotic medication. If the patient wishes to not be randomized, they can opt-into an observational or electronic medical record cohort in which they choose their treatment option. We are currently still in the data collection stage as we continue to monitor and assess patient reported and clinical outcomes for two-years following initial presentation. This research is important because patient reported outcomes gained from the trial will educate patients about their treatment options, therefore patients can decide the treatment best suited for them. In addition, the same results may demonstrate to current healthcare professionals that appendicitis may be treated though other methods, namely antibiotics, which if successful, will revolutionize current perspectives on how appendicitis is treated in the United States.

Many inherited diseases are attributed to monogenic mutations; however, some human disorders and conditions are genetically complex, requiring mutations at multiple loci. One of the research goals at the Waterston Lab is to identify genetic interactions that could affect developmental pathways related to human disease. Using the small nematode, Caenorhabditis elegans, as a model organism, we have developed a high-throughput approach to look for interactions. RNAi bacteria were grown separately for knockdown of forty different transcription factor (TF) genes on pooled populations composed of 38 mutated and fully sequenced strains of C. elegans from the Million Mutation Project (MMP). After growth for several generations, the abundance of strains in each condition was compared by targeting strain-specific single nucleotide variants with Molecular Inversion Probes and sequencing on an Illumina MiSeq system. From ~115,000 possible screened genetic interactions, we identified a number of candidate interactions and focused our efforts on a positive interaction between the strain VC20424 and the TF gene B0261.1. While most strains had poor growth on the B0261.1 RNAi, VC20424 had a much higher abundance. We confirmed this candidate interaction by re-testing VC20424 alone and against a control strain on B0261.1 RNAi. We then genetically mapped the VC20424 interaction to a region on chromosome X. In this region we identified a deletion in C25A11.2 which shows similar expression patterns as B0261.1, suggesting a good interaction candidate. We will test additional MMP strains that share the same allele of C25A11.2 on B0261.1 RNAi to look for a similar interaction. We also plan to use CRISPR/Cas-9 to induce the mutation into a control strain and look for conferred resistance to B0261.1, which would further validate our results. Broadly, studying the nature of genetic interactions and potential roles of transcription factors may contribute to the elucidation of human development and complex disease.

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.

Spontaneous electrical activity in the mouse neocortex has been extensively studied. Two main types of activity are observed: synchronous waves and asynchronous single cell activity. This synchronous activity has been believed to be unique to the developing brain. This research project investigated whether the phenomenon is as much a property of sleep as it is development. Synchronous waves exhibit remarkably similar dynamics to the up-down oscillations of slow wave sleep. Conversely, asynchronous single cell activity is very similar in form to the "persistent up" or wakeful brain state. Thus, the two types of spontaneous activity generally observed in the mouse neocortex may actually be a sign of overall brain state, with asynchronous single cell activity dominating during states of wakefulness and synchronous waves taking over during sleep states. Calcium imaging of slices of neonatal mouse cortex has revealed that application of curare, an acetylcholine antagonist, decreases asynchronous single cell activity and induces synchronous waves. As blocking acetylcholine puts the brain in a state associated with sleep, we hypothesize that the neurotransmitter may be involved in keeping the brain awake. Further experiments were conducted on the effect of curare on synchronous waves. According to our hypothesis, it should increase their frequency and/or propagation. The same experiments were performed with other acetylcholine antagonists, such as mecamylamine, to ensure the observed effect is not unique to curare as a drug. We also conducted experiments with acetylcholine agonists as well to see if they have the opposite effect. These synchronous waves are known to be involved in several developmental neural processes, and this research project may reveal they are a property of sleep, and could serve as precursors to adult slow-wave sleep.

DNA stores genetic instructions that are required to program cell activities. Therefore, it is important for DNA to replicate in order to provide a copy of genetic material to daughter cells during mitosis or meiosis. Origin recognition complex (ORC) is a six-subunit assemblage that promotes the initiation of DNA replication. ORC proteins bind in all eukaryotes to a specific sequence found within an origin of replication to assemble and bring together other key initiation factors. Although the ORC proteins themselves are highly conserved throughout evolution, dramatic changes in the origin DNA sequence motif have been observed across species. These changes are most likely accompanied by changes in one or more subunits within the ORC complex. The aim of this project is to understand how changes in the ORC’s binding preferences across species are determined by changes in ORC protein sequences, specifically within the yeast phylogeny. Using genetic engineering approaches, I will introduce foreign ORC alleles into knockout strains of Sacchoromyces cerevisiae which we have generated. Previous data generated by members of our lab and others show how ARS (autonomous replication sequence) Consensus Sequences (ACS, the binding site for ORC) differ across the yeast species. The data indicate that Lachancea kluyveri and Lachancea waltii have intermediate levels of ACS differentiation from Sacchoromyces cerevisiae, so I am introducing alleles from these species as a starting point. Differences in the cell growth rate, size, and fitness will be observed within haploid strains of Sacchoromyces cerevisiae containing the foreign ORC protein in order to determine which ORC subunit(s) is/are responsible for binding preferences for different ACS sequences within the yeast phylogeny. This research can provide a fundamental understanding of evolution of the genome sequence and the proteins that bind it.

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.

Accurate quantification of DNA replication errors is difficult to measure since mutations are exceedingly rare. Recent attempts have relied on Next Generation Sequencing however, they are limited by its high error rate, with 5 x 10^-5 as their lowest detected mutation rate. Duplex Sequencing is an error correction method that reduces sequencing error through barcoding strands with unique identifiers and comparing the complementary strands, to achieve error rates as low as 10^-7. Our assay starts with a cloned single-stranded m13mp18 phage DNA with a base accuracy of 10^-7 as a template for replication. After annealing a forward primer we have our test polymerase extend the complementary strand and then we excise and isolate an 850 base pair region to undergo Duplex Sequencing. Through the improved baseline error rate of Duplex Sequencing, we hope to assay high fidelity polymerases, such as human polymerase delta. Accurately measuring the in vitro mutation rate and mutational profile of human polymerases is important in establishing the role of DNA polymerase generated misincorporations in somatic mutagenesis and human cancers. Furthermore, we have the potential to define a mutator signature that indicates the misincorporation of nucleotides in cancer by a specific DNA polymerase.

The patterning of skin appendages, such as hair, feathers, teeth, and scales, is crucial for a vertebrate’s ability to survive in their environment. For example, an increased density of eccrine sweat glands in humans has a large impact on our capability to maintain our body temperature. While some key developmental genes have been identified, little is known about the molecular mechanisms involved in skin appendage patterning. All skin appendages evolved from a single ancestor, and although they have different morphologies they share many developmental mechanisms. Zebrafish scales are simple skin appendages made of dentin-like extracellular matrix that form a partially overlapping pattern resembling roof tiles. While mouse and chick-models of skin appendage development require examination of fixed specimens, we can conduct conditional genetic manipulations while imaging the entire skin through scale patterning in live animals. Therefore, zebrafish scales are an ideal system to gain insight into mechanisms underlying skin appendage patterning. We manipulated conserved cell-cell signaling pathways and assayed scale patterning. The zebrafish genome harbors multiple Fgf receptors that are partially redundant, and removing one of them results in fewer, larger scales, while inhibition of all Fgf receptors completely abolishes scale development. This suggests that Fgf signaling, likely through multiple Fgf receptors, plays a role in scale initiation and patterning. Experimentally increasing Fgf signaling results in smaller and more numerous scales, likely due to delayed squamation, indicating that both Fgf and the timing of scale development ultimately determines density. This also implies that a specific level of Fgf signaling may be necessary for scales to form correctly. Inhibition of the Wnt signaling pathway also results in failed scale development. In summary, these results suggest that skin appendage density is dependent upon the timing of their development, and is controlled by a mechanism involving multiple signaling pathways.

Extracellular vesicles (ECVs) are small vesicles secreted into the extracellular space to transfer proteins, lipids, and genetic material throughout the organism. Although ECV signaling is thought to be a conserved cell-signaling modality, seen by all cells from bacteria to human, very little is known how this signaling occurs in a living multicellular organism. I used the simple genetic worm model organism C. elegans to conduct genetic screens to understand the mechanisms by which ECVs are formed and secreted in a living animal. In addition, I generated worm lines with fluorescent ECV markers allowing myself and others to monitor ECV signaling in an intact organism. C. elegans uses the ECV machinery to secrete extracellular vesicles that are needed to form alae – two rows of three “racing stripes” that run down the lateral sides of the worm. This clear phenotype allows me to perform a reverse genetics screen using RNAi (RNA interference) to identify the key molecular components for proper signaling. In my first screen, I selected 20 genes that my mass-spectrometry experiments indicated were on ECVs. I found that several of them also prevented proper alae formation, indicating they are key for proper ECV signaling. I then screened all 21 members of the C. elegans tetraspanin family, an ECV-associated transmembrane protein family that is implicated in loading, secretion and cell targeting of the vesicles. Then I generated transgenic lines with artificially introduced DNA that produce fluorescent versions of the genes that are key for ECV secretion. This research is significant because it will uncover new genes necessary for ECV secretion in C. elegans and will find markers that will monitor ECV trafficking in vivo. This results of this will open up the study of ECV signaling in a powerful model system, allowing us to identify physiologically relevant signaling pathways that utilize this communication modality.

Dacarbazine (DTIC) is a chemotherapeutic drug, which targets cells at the time of cell division by damaging the DNA or RNA, thus interrupting cell duplication and division. DTIC is a DNA-methylating agent and is commonly used with adriamycin, bleomycin, and vincristine to fight certain types of cancer such as malignant melanoma and Hodgkin’s Lymphoma. Hormones and enzymes change during pregnancy, which affects the efficacy and toxicity of drugs in the body. With this study, we better understand how Dacarbazine is metabolized during pregnancy as compared to the non-pregnant state. Two pregnant patients with Hodgkin’s Lymphoma were recruited for our study. The first was a 31-year-old who presented with enlarged supraclavicular nodes at 10 weeks gestation and the second subject was a 34-year-old who sought medical attention for enlarged cervical nodes at 15 weeks gestation. Dacarbazine relies on the P450 enzyme CYP1A2 as the primary pathway for DTIC metabolism. It has been reported in the literature that CYP1A2 activity during pregnancy is significantly reduced. As a result, the concentrations of DTIC, an inactive pro-drug, are higher and its active metabolites, MTIC, HMMTIC, and AIC, are lower during pregnancy compared to postpartum. Since DTIC is inactive and the lower enzyme activity leads to lower concentrations of the active metabolites, the lower CYP1A2 activity potentially decreases the efficacy of DTIC during pregnancy. Although the women in this study had lower concentrations of the active metabolites of dacarbazine during pregnancy, both patients still successfully achieved remission of their lymphomas. This research will help us better prescribe chemotherapeutic drugs that interact with P450 enzyme CYP1A2 to pregnant women who have certain types of cancer. Personally, I analyzed the specimen results to determine drug concentrations in the blood and excretion along with other PK parameters and wrote a manuscript describing the results.

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.

We have recently identified a new cytosolic sensor for bacterial cyclic dinucleotides, the aldo-keto reductase RECON. RECON plays a critical role in surveying the host cytosol for intracellular bacterial pathogens, orchestrating the innate immune response to those pathogens and aiding in bacterial clearance. In the host, RECON is most highly expressed in the small intestinal epithelium, enterocytes and M cells. Its high expression at these important mucosal immune sites raises the question as to whether RECON is part of the intestinal immune system. We have recently made a RECON knockout (KO) mouse and found significant alterations in intestinal inflammation and commensal abundance, particularly an increase in segmented filamentous bacteria (SFB). Using quantitative real-time PCR copy number analyses of SFB 16S gRNA, we detected high SFB burden in the RECON KO mice while SFB was undetectable in wild-type mice. SFB have been shown to impact the development of the immune system, particularly T cell populations, in rodents by colonizing and attaching to the follicle-associated epithelium of the small intestines. We predict that the high abundance of SFB in RECON KO mice may either be the result of immunosuppression due to the presence of too many T regulatory cells or will associate with the increased presence of pro-inflammatory T helper type 17 cells. Current work is aimed at establishing whether there are changes in these T helper cell types in the intestine, as well as looking at the broader dysbiosis by 16S deep sequencing. This project will establish which immune axes are dysregulated in the absence of RECON and will direct future mechanistic investigations into how loss of RECON enzymatic activity is driving immune dysregulation.

Studies using primary airway epithelial cells from asthmatic and healthy donors have reported differences in gene expression of a variety of genes between cells from asthmatic and healthy donors. However, few studies have investigated the stability of gene expression by primary cells with increasing cell passage number, or how viral infection might affect the stability of gene expression with increasing cell passage number. This research investigated: 1) whether expression by primary bronchial epithelial cells (BEC) of genes IFIH1, which codes for a viral sensor protein with an important role in the innate immune response, and TGFβ2, a pro-remodeling cytokine, from asthmatic and healthy children, is stable with increasing cell passage number, and 2) if BEC infection with respiratory syncytial virus (RSV) alters the stability of IFIH1 and TGFβ2 expression with increasing cell passage number. To answer these questions, we differentiated passage 1, 2, 3, 4, and 5 BECs from asthmatic and healthy children at an air-liquid interface for 3 weeks. RNA was then harvested from BECs and RT-PCR was performed for TGFβ2 and IFIH1. To assess the expression stability across increasing cell passage number for each gene, we compared expression at passages 2-5 to expression at passage 1. Preliminary data collected shows that expression of TGFβ2 and IFIH1 from asthmatic and healthy children was stable with no significant differences between passages 1, 2 and 3. However, expression at cell passages 4 and 5 was significantly greater and more variable than by passage 1 BECs. We anticipate gene expression to be even more variable for RSV-infected passage 4 and 5 BECs. These observations illustrate the importance of using BECs from passage ≤ 3 when comparing gene expression between asthmatic and healthy primary BECs, and of characterizing the expression pattern across increasing cell passage number for each gene studied.

Mast cells are bone marrow-derived, granule-containing immune cells that are widely distributed in connective and mucosal tissues. Although mast cells are known to store and release histamine and other inflammatory agents in IgE-mediated allergic reactions, the specifics of their extracellular signaling and regulatory mechanisms are not fully understood. Therefore, this study aimed to further characterize the mast cell surface proteome to better understand the signals that promote mast cell activation. Murine bone marrow-derived mast cells were cultured and their membrane proteins were purified using hydrazide chemistry to isolate galactose and sialic-acid moiety-containing glycopeptides; these peptides are involved in post-translational modifications and anchorage of proteins to cell membranes. Post-cell lysis and purification, both liquid chromatography and mass spectrometry were used to classify and quantify detectable proteins. Use of online databases identified characteristics of these proteins, such as the presence of transmembrane domains, glycosylphosphatidylinositol (GPI) linkage, or target sites. As the cell surface proteome of bone marrow-derived macrophages is well characterized, they were subjected to the same treatments as mast cells, serving as a control. By comparing the mast cell and macrophage cell surface proteomes, we distinguished which proteins are mast cell-specific. Results show the presence of 69 transmembrane proteins including proteins known to be expressed by mast cells such as receptors for stem cell factor (c-kit) and interleukin-33 (ST2). Additionally, we detected and confirmed the expression of a potential cell surface histidine transporter, solute carrier family 15A4 (SLC15A4), with quantitative polymerase chain reaction (qPCR). The identification of mast cell-specific proteins can help us further understand their effects on mast cell activation and subsequent degranulation.

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

From blood vessels to the small intestine to the spinal cord, tubes are an essential part of nearly all multi-cellular organisms. Errors in tube formation, called tubulogenesis, cause many of the birth defects that afflict infants, including congenital heart defects and spina bifida, a failure to close the neural tube. Our lab uses the fruit fly Drosophila melanogaster to study tube formation because of the highly conserved nature of this morphological process between our species. My project focuses specifically on a family of genes called Imaginal Disc Growth Factors (IDGFs), which are linked to tubulogenesis in Drosophila and are closely related to a human protein family (CLPs) that have been found to be dysregulated in numerous diseases, including arthritis, and in metastasizing tumor cells. While this homology indicates that IDGFs have a role in cell behaviour, the mechanisms by which these genes act remain unclear. Last year, I used the gene-editing technology CRISPR/Cas9 to investigate the function of one IDGF, the gene Idgf6, by deleting it entirely. Analysis of these knock-out mutants suggests that Idgf6 plays an important role in making and shaping tubes and that removal of Idgf6 results in branched structures instead of discrete tubes. This branching phenotype likely limits the flow of oxygen to the developing embryo, resulting in decreased survival of offspring. My current research explores this genetic pathway further by testing tube morphogenesis under hypoxia and other stress conditions such as crowding, with the aim of further defining the mechanism of tube dysfunction in these mutants.

We demonstrate that results from genetic association studies of telomere length performed in European populations are not generalizable to African Americans. Telomeres are repeated segments of DNA at the ends of chromosomes important for maintaining healthy chromosomes. Telomeres have been associated with a number of diseases and age-related conditions, however this relationship is complex because it is uncertain if an individual’s telomere length increases their risk for disease, or if disease and other environmental exposures are modifying the individual’s telomere length. Recent studies have scanned the entire genome for genetic variants that may be associated with telomere length. Nine highly significant genetic variants associated with telomere length have been identified, though the study populations have been of exclusively European descent. Since it is well known that the genetic architecture varies extensively across racial populations, we examined whether the nine genetic variants were associated with telomere length in 1,442 African American children (age 8-21) from the Study of African Americans, Asthma, Genes and Environments. We examined the genotypes for each variant using multivariable linear regression to test whether the nine genotyped variants were associated with telomere length. We found that the nine variants identified in European populations were not significantly associated with telomere length in African American children. In addition to these nine variants, we examined locations across the entire genome for association with telomere length. We found several genetic loci associated with telomere length that were specific to sex and asthma status. These loci map to chromosome positions 6p22.1, 13q21.1, 20p11.23 and genes INPPB4 and CFTR. To our knowledge, this is the first study demonstrating that the nine variants associated with telomere length in European populations are not generalizable to African Americans. Our study underscores the necessity for including diverse populations in biomedical research.

Properly timed seasonal flowering in plants is necessary for reproductive viability in natural and agricultural settings. We use Arabidopsis thaliana as a model organism to study the molecular mechanisms that underlie the developmental transition to flowering, a process that is mediated by both internally propagated circadian cycles as well as information about day length (photoperiod) detected via photoreception. Transcriptional regulation of the FT (FLOWERING LOCUS T) gene is the primary mechanism of controlling the flowering response, and flowering occurs when requisite levels of FT proteins are transported from the leaves to apical stem cell tissue. FT expression is initiated by CO (CONSTANS), a transcription factor whose own expression is tightly regulated by molecular outputs of the endogenous circadian clock. One such output, CDF1 (CYCLING DOF FACTOR 1) is responsible for repression of CO and FT expression. We established that CDF1 forms a complex with the TPL (TOPLESS) protein, an interaction that is dependent on a conserved peptide sequence at the N-terminal of CDF1. We generated phloem companion tissue-specific mutants of TPL in a variety of flowering time genetic backgrounds and found that mutants of TPL consistently flowered earlier than nonmutants, similar to the phenotype witnessed in cdf mutants. tpl mutation had a similar effect on CO and FT expression across 24-hour timecourse to cdf-1,2,3,5 mutants in both long and short day conditions. We have shown that TPL protein associates with CO and FT promoters during the mornings of long days, and that this recruitment of TPL to the promoters is enhanced by the addition of excess CDF1 protein. We demonstrate that TPL protein is required for the repressive activity of CDF1 and are working to better understand the complex role it plays in regulating flowering time in Arabidopsis.

FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1) is a blue light photoreceptor which induces photoperiodic flowering in Arabidopsis. Depending on blue light, FKF1, along with GIGANTEA, forms a complex to induce flowering by degrading a transcriptional repressor, CYCLING DOF FACTOR 1 (CDF1). FKF1 stays in light status for a long time, allowing plants to continue induce flowering. ZEITLUPE (ZTL), a close homolog of FKF1, is a circadian clock regulator with a faster photocycle LOV domain. A photocycle is the sequence of structural changes that takes place in molecules when exposed to light. ZTL has high affinity with GI in presence of light, but also degrades its targets quickly in the dark. The faster ZTL photocycle allows it to act as a sensor to the night periods in circadian oscillation phases. Therefore, we hypothesize that slower photocycles of FKF1 is necessary for FKF1 to keep light information and induce photoperiodic flowering. To test the effect of photocycle on the FKF1 LOV domain in photoperiodic flowering, we generated mutations on the FKF1 LOV domain based upon sequence information of the ZTL LOV domain, to speed up the photocycle kinetics. Quantitative real time PCR (qPCR) was used to select stable, transgenic lines expressing comparable levels of mutated FKF1. Then we analyzed samples for expressions of downstream genes CONSTANS, FLOWERING LOCUS T, along with flowering times to measure FKF1 functionality. As a control, wild type plants were used to provide regular flowering time data. We gathered samples to establish lines to test the effect of each mutation on FKF1 function. We expect to find that the mutations’ faster conversion to dark status decreases its affinity to GIGANTEA and CDF1 degradation, delaying the flowering time. This suggests the slow conversion of FKF1 LOV domain to dark status is necessary to induce photoperiodic flowering in Arabidopsis.

Cocha Cashu Biological Station in the Amazonian rainforest of Southeastern Peru hosts a myriad of plants and animals, with one study reporting 230 avian species found in 80-100 ha study plots. Such incredible species diversity may be attributed to the adaptation of specialized hunting behavior and morphological traits allowing for habitat partitioning of forest strata. In our study we hypothesized that we would find differences in wing and tarsus morphology in birds living at different forest strata. Over a five-day period, twenty birds were captured by mist nets and used for data collection. From these individuals, wing length to weight and tarsus length to weight ratios were explored to seek a correlation between them and the heights at which different avian species were observed. We predicted birds with lower wing length to weight ratios, and thus more compact morphology, specialized for flight at lower strata. Furthermore, ground-dwelling birds tend to have a longer tarsi for efficient bipedal locomotion on the forest floor, leading us to predict that birds with lower tarsus length to weight ratios would be observed at higher strata. We found that individuals with shorter tarsi and longer wings relative to body mass tended to be found in higher strata. Our results supported our hypothesis, suggesting adaptations in wing and tarsus morphology facilitate partitioning of forest strata. Morphological analysis of avian populations foraging at different heights is critical for developing an understanding of the evolutionary processes underlying these specializations, and for effective conservation management. Although our predictions regarding correlations between morphology and vertical distribution were confirmed in this study, a larger dataset is necessary to better support our findings. Continued research will allow for accurate predictions to be made concerning how avian populations will respond to fluctuating environmental conditions such as altered migration patterns, climate change, and deforestation.

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.

DNA replication is a crucial step in the duplication of all cells, requiring the cooperative function of multiple enzymes. In one of the final steps of chromosomal DNA replication, short pieces of DNA, Okazaki fragments, are joined by the essential enzyme DNA ligase. Mutations in human DNA ligase have been linked to genome instability. To study the consequences of ligase deficiency, my lab uses a temperature-sensitive mutation in the CDC9 gene that encodes DNA ligase in the budding yeast Saccharomyces cerevisiae. When cdc9^ts mutant yeast cells replicate at restrictive temperatures, they lose viability while simultaneously and unexpectedly accumulating loose pieces of single-stranded DNA (ssDNA). The current model of DNA replication cannot explain this accumulation, as newly formed ssDNA fragments should be base-paired to the template strand. We hypothesize that the absence of ligase causes this accumulation of ssDNA, which in turn causes cells to lose viability. I am currently testing this hypothesis in S. cerevisiae cells that have the cdc9^ts mutation as well as a mutation in either EXO1 or RAD27, two genes that code for exonucleases. These exonucleases process Okazaki fragments before ligation, and preliminary evidence suggests that the rad27 mutation decreases viability, while the exo1 mutation improves viability. By growing mutant cells at restrictive temperatures and visualizing ssDNA quantity at multiple time points while determining viability, I am correlating these two phenotypes. If ssDNA causes loss of viability, cdc9^ts rad27 double-mutant cells should have more accumulated ssDNA, correlating to decreased viability, while cdc9^ts exo1 double-mutants should have less accumulated ssDNA. These experiments could provide novel insights into a universal process—DNA replication—and clarify the consequences of defects in that process. Additionally, because ssDNA accumulation has been linked to autoimmune disorders in humans, understanding the causes and consequences of ssDNA accumulation may have implications for human health.

Wound healing is a biological process that tissues use to repair themselves after damage. If wound healing is impaired, the tissue may scar or the wound may be fatal. Our lab uses the fruit fly Drosophila melanogaster as a model organism to study the cellular and molecular mechanisms involved in tissue growth, morphogenesis, and homeostasis. Recently while characterizing a novel family of growth factors, Imaginal Disc Growth Factors (IDGFs), our data suggested that one family member, IDGF3, may be upregulated following damage to imaginal discs, the future adult tissue. To explore the potential involvement of the IDGFs in wound healing, I have quantifed their expression by in situ hybridization and localized the mRNA sequences at wound sites in the Drosophila wing imaginal disc. Next, I plan to determine whether specific molecular pathways such as the canonical Wnt pathway or the beta-Akt pathway, which are known to play a role in innate immunity, interact with IDGFs during wound healing. Subsequently, I will use a yeast transcription factor, Gal4, to express RNAi that interferes with mRNA in the same wing disc to determine the relationship between the different pathways. These studies will contribute to our understanding of the mechanisms that regulate tissue healing during development.

Alzheimer’s Disease (AD) is a debilitating neurodegenerative disease that currently affects over 5 million Americans. We have previously demonstrated genetic and neuropathology-based variation across cognitively-defined AD subgroups. Here we sought to determine whether patterns of vascular risk factors differed across cognitively defined subgroups. We used data from the Seattle-based Adult Changes in Thought (ACT) prospective cohort study of individuals age 65+ and dementia-free at enrollment. ACT follows people at 2-year intervals to identify incident dementia and AD. We used cognitive data at the time of AD diagnosis to determine scores for memory, visuospatial abilities, language, and executive functioning. We used these scores to determine each individual’s average cognition and then domain-specific deficits below that individual average. We used these data to define six groups: no prominent domain, memory-, visuospatial-, language-, or executive functioning-prominent. We evaluated risk factors for these six groups from self-reported medical conditions including diabetes, stroke, and hypertension, and diagnosed atrial fibrillation or coronary artery disease. We used multinomial logistic regression models with the no prominent domain group as the reference. To account for multiple comparisons, we present tests of the null that each risk factor is unrelated to each subgroup, and an omnibus test of the association between each risk factor and any subgroup. Of 825 cases, nearly half had no prominent domain, a few had multiple prominent domains, and a single domain was prominent for the remainder. Memory-prominent domain was more common in people with diabetes and executive functioning-prominent was less common, although the omnibus test did not reach statistical significance (p=0.08). Despite low statistical power due to small group sizes, our results suggest possible differences in risk associated with diabetes. Subsequent work will refine these investigations using medications and evaluating glucose levels over time. These results support additional efforts to further understand cognitively-defined AD subgroups

Sudden unexpected death in epilepsy (SUDEP) is the most common type of death in people with intractable epilepsies, including Dravet syndrome (DS). DS is a treatment-resistant infantile-onset epilepsy syndrome with comorbidities of cognitive impairment and premature death. DS is caused by a heterozygous loss-of-function mutation in SCN1A, the gene encoding the α subunit of the type I voltage-gated sodium channel Na_v1.1. Cardiovascular dysfunctions have been identified as the main causes of SUDEP. Recent studies have indicated that changes in cardiorespiratory coupling can indicate signs of disease and predict disease susceptibility, such as schizophrenia. We used the established mouse model of DS, which carries a global knock out of Scn1a, and conducted an examination of cardiac and respiratory functions. We recorded video recordings, electroencephalogram (EEG), electrocardiogram (ECC), whole body plethysmography, and LabChart Software 8.0 (AD Instruments) in freely moving DS and wild type (WT) control mice. We then identified and characterized the defects in cardiorespiratory coupling strength associated with SUDEP risk in the DS mice. We hypothesize that cardiorespiratory coupling of DS mice, compared to the WT mice, is disturbed and results in increased complexity between the heart rate and respiration. Findings from these studies may indicate that cardiorespiratory coupling parameters can be used as biomarkers of susceptibility to sudden death in intractable epilepsies and in other severe neurological disorders.

Pierre Robin Sequence (PRS) is a human congenital disorder manifesting as a combination of several orofacial anomalies that include a small lower jaw (micrognathia), posterior placement of the tongue (glossoptosis), and high incidence of cleft palate. The resulting airway obstruction in affected infants and young children can be life-threatening. While lower jaw growth is thought to permit tongue descent to allow palatal shelf elevation and fusion, it remains unclear how tongue malposition and cleft palate results from lower jaw malformation in PRS. The aim of this study is to analyze the physical interactions between the tongue and other orofacial structures, such as the palatal shelves and lower jaw, to determine if dysregulation of these interactions plays a role in the pathogenesis of PRS. We used a Wnt1-Cre driver, a method to specifically knockout genes in the craniofacial mesenchyme, to disrupt hyaluronic acid synthase 2 (Has2), an enzyme crucial to the extracellular matrix and overall craniofacial development. This allowed us to produce conditional knockout (CKO) mouse models that developed the entire spectrum of PRS phenotypes including micrognathia, cleft palate, and a high-arched tongue interfering with the nasopharynx. Combining histology with 3D imaging techniques, we analyzed morphological changes of the palatal shelves, tongue and lower jaw in Wnt1-Cre, Has2 CKO mice. Our results indicated that reduced lower jaw growth affects tongue growth and its interaction with the nasomaxillary complex, while reduced palatal shelf growth affects tongue shape changes. This suggests the abnormal shape and positioning of the tongue in PRS may result from abnormal orofacial tissue interactions due to defects in the lower jaw and palatal shelves. The implications of this research will allow us to better understand the regulatory mechanisms of orofacial development and the pathogenesis of PRS in humans, which is essential for diagnosis and treatment of this disorder.

In resource-limited settings, premature and small for gestational age (SGA) births are common and increase risk of infant mortality. During pregnancy, exposure to high pathogen levels and other health risks increases inflammation and may result in premature or SGA infants. Cytokines are small secreted proteins involved in the inflammatory immune response. This experiment examines variations in 25 cytokine levels in sera collected from pregnant women during the second trimester of gestation and again at the time of delivery. Samples selected from women who delivered full term SGA (n=50), preterm SGA (n=50), preterm appropriate for gestational age (AGA) (n=50), and full-term, AGA (n=50) infants were tested using a Luminex 25-Plex Magnetic Assay to compare cytokine level changes in these populations. I plan to conduct and analyze this assay which has not yet been completed, but preliminary data shows we should expect elevated levels of cytokines in those mothers with preterm and SGA infants. Overall this experiment provides a better understanding of cytokine profiles unique to preterm and SGA infants in resource poor conditions, and could provide novel therapeutic targets for the prevention of infant mortality associated with these conditions.

Ocean warming, due to climate change, is currently altering marine ecosystems. In the Salish Sea, temperature anomalies of up to 4°C per year have been reported. Sea urchins hold an important ecological role as community engineers with the ability to alter the ecosystem in which they are found. The green sea urchin (Strongylocentrotus droebachiensis) relies on phytoplankton as a food source for development in the field, but has been shown to consume kelp detritus in the laboratory. Studies have separately shown the effects of different diets and temperatures on larval growth but have not studied the stresses of increasing ocean temperature with changing food sources and abundance. Here we studied the combined effects of food concentration, food type, and temperature on sea urchin larval development. Contrary to prior studies, our results show that at warmer temperatures, larvae can develop under lower food concentrations, but in agreement with past studies we confirm that larvae develop best under a high concentration phytoplankton diet. Interestingly, we saw increased mortality for larvae grown under warm temperatures and fed a high concentration kelp detritus diet, potentially caused by the introduction of microbes that thrive at warmer temperatures. Warming ocean conditions, causing nutrient composition shifts, could be harmful for sea urchin larvae.

Crop production will have to double by 2050 to meet projected global food demands. Insect herbivory currently accounts for nearly 15% of global crop losses; thus, plants better able to repel insect attack should have a significant impact on food security. One successful strategy for engineering plants is the constitutive expression of insecticidal toxins such as Bacillus thuringiensis endotoxin (BT). However, the constant production of these toxins decreases crop yield and increases the development of BT-resistant pests. When plants are damaged by insects, they produce the phytohormone jasmonate. We propose a new plant engineering strategy that links the expression of BT to jasmonate signaling. To create this link we have built a synthetic transcription factor called a Jasmonate Degradable CRISPR Transcription Factor (JDCTF). This transcription factor is easily retargeted to any gene of interest by changing the dcas9-associated guide RNA. The JDCTF responds to jasmonate via a jasmonate sensitive degradation domain. Using the plant model Arabidopsis thaliana, we’ve generated transgenic plant lines expressing a JDCTF that targets a Venus-Luciferase reporter. We have shown the system works using a bioluminescence time course assay on seedlings treated with the jasmonate analog coronatine. After induction with coronatine, we observed an increase in bioluminescence in the treated seedlings and no increase in biolumescence for the control. Currently we are characterizing the dynamic response to both exogenous jasmonate, as well as simulated and real insect herbivory on mature leaves. Future work will focus on regulating BT expression with the JDCTF system. Our system has the potential to increase yields, increase consumer safety, and slow down the development of insecticide resistance in pests.

Climate change is threatening global food security. There is a pressing need for better crops that can prevent severe losses. We hope to apply our current understanding of plant developmental mechanisms that are involved in responses to harsh external stimuli to engineer more resistant crop varieties. The plant hormone auxin plays an important role in organogenesis, the process of new organ formation. Auxin controls organogenesis by accumulating in specific tissues at specific developmental times. These accumulations are due to the polar flow of auxin through the plant. These flows are in turn reinforced by auxin through a feedback mechanism called canalization. PIN1 is a an auxin efflux transporter that is required for polar auxin transport. Loss of PIN1 results in several developmental defects. These phenotypes and other analyses have led to mathematical models of organogenesis. We used these models to predict plant phenotype in response to increased canalization. We have engineered transgenic plants with a library of synthetic promoters with varying auxin sensitivity driving expression of PIN1. We are characterizing the shoot architectures of these plants, specifically the number of branches and the pattern of lateral organ initiation. We are also studying how the increased canalization affects the distribution of auxin in our plant lines using fluorescent reporters. We hope that our results will demonstrate how plant synthetic biology and predictive models can be used to rationally design plant developmental phenotypes.

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.

The Amazon tidal river consists of the lowermost 800 km of the Amazon River that is affected by tides.  The effect of tides on surface suspended sediment concentration (SSC) in the Amazon has not yet been characterized, and may impact estimates of surface SSC as monitored through remote sensing.  To assess potential tidal influence on surface SSC, water samples were collected and filtered in field; published remote sensing maps were also collected and compared to field measurements.  Thus far, this project has identified discrepancies between in situ and remotely-sensed estimates of surface SSC, and constrained the influence of tides on the observed signals.  In situ surface SSCs more accurately matched with remote sensing maps on sites located directly in the Amazon mainstem, while tributaries showed exaggerated differences in surface SSC.  Field surface SSCs in the Amazon mainstem also did not vary significantly with tidal  seasons, suggesting that ocean tides play a smaller role in surface sediment transport than originally hypothesized.  Further conclusions will look into the roll that tides play on total bedload, especially at sites close to the river mouth where tidal influence is strongest.  Explanations for surface SSC discrepancies will also be included in the paper's conclusions; since many of the discrepancies occur in freshwater tribuataries, the seasonality of phytoplankton blooms will be cross-linked to satellite SSCs.  Finally, suggestions for future usage of remote sensing in strong tidal areas of the Amazon River will be given and the success rate/percentage of correlation of satellite surface SSC with field SSCs will be assessed.  With a better understanding of the effects of tides on surface SSC and how well remote sensing captures variability, we can better monitor sediment discharge into the tropical Atlantic Ocean as well as sediment delivery to the floodplain of the Amazon River during seasonal floods.

There are many age-related cardiac diseases that relate to impairment of muscle function, including heart failure and arrhythmia. Previous studies show that increased mitochondrial function results in the enhancement of cardiac and skeletal muscle function as mice age. In these studies, the SS-31 tetrapeptide was used, an agent that serves as an antioxidant by binding to cardiolipin in the inner mitochondrial membrane, enhancing electron transport and increasing ATP generation, and protecting against damage from Reactive Oxygen Species (ROS) by reducing their generation. However prior literature has suggested that ultrastructural morphology does not change in relation to age. The goal of this study is to quantitatively assess interfibrillar mitochondrial damage caused by these ROS', and to see if there is structural improvement after treatment with the SS-31 peptide. Eight 24 month-old mice were split into two treatment groups, four treated with SS-31 for 8 weeks and four untreated. Four 8 month-old untreated mice were used as controls for youthful, “ideal” mitochondrial structure. The mice were euthanized, and cardiac tissue was harvested, sectioned, and examined by transmission electron microscopy (TEM). Quantitative analysis using NIH ImageJ software allowed the comparison of the SS-31 treated and untreated old hearts to young hearts. This revealed stark differences when comparing the young and old ultrastructure. Untreated old mice exhibited higher amounts of vacuolation, greater lack of mitochondrial axial organization, and lower density of mitochondrial cristae than the younger untreated mice. Older mice treated with SS-31 appear to exhibit improvement in ultrastructural definition compared to untreated older mice, but additional quantitation is necessary to validate this observation. In further testing, we are examining the persistence of the SS-31 tetrapeptide's positive effects after its administration period ends, and how ultrastructure changes during this refractory period.

Tyrosine kinases are critical drug targets in oncology due to their role in tumorigenesis. Accordingly, the treatment of specific cancers has been revolutionized by the development of tyrosine kinase inhibitors (TKIs). However, the long-term effectiveness of TKIs is undermined by the emergence of drug resistance. This resistance is often caused by mutations that prevent drug binding. Understanding how a given mutation affects drug sensitivity can enable physicians to personalize their treatment based on a patient's specific tumor genotype. However, current methods to identify resistance mutations are laborious and can only isolate a small subset of possible mutations. Previously, our lab developed a technique to simultaneously measure the functional effect of all single mutations within a protein of interest. We leveraged this method in order to generate a novel system for the rapid and comprehensive characterization of resistance mutations in oncogenic tyrosine kinases, thus overcoming previous technical limitations. Key to this system was choosing a functional assay that could accurately discriminate between the mutations of interest (in our case, drug resistance mutations). Our functional assay was built around Ba/F3 cells; a mammalian cell line whose growth is uniquely dependent on oncogenic kinase signaling. The first step in this project was to validate this growth phenotype by using three clinically relevant oncogenes: FGFR2, ALK, and JAK2. To do this, I cloned each oncogene into a mammalian expression vector, transfected the cloned vectors into Ba/F3 cells, and measured the growth rates of each transfected cell line. As I anticipated, only cells harboring an oncogenic tyrosine kinase grew, thus validating the cell line for further development. These results represent the first step toward the generation of a system for widespread identification of drug resistance mutations in cancer relevant kinases.

Investigation of the phenotypic features of a putatively unaffected individual, in a family diagnosed with a multiple congenital contracture disorder called Distal Arthrogryposis Type 2B (DA2B), identified joint hypermobility, a phenotypic trait not commonly associated with DA2B. Review of the family history revealed joint hypermobility in multiple other family members extending back four generations. Analysis of exome data from the family, identified two candidate variants, one in exon 1 of the gene SRL and another in exon 8 of NPR3, which may explain the joint hypermobility observed in this family. Recently, we used Sanger sequencing to genotype five family members with hypermobility and four unaffected family members for the two candidate variants to see if either variant tracks with the hypermobility phenotype. Briefly, we amplified exons via polymerase chain reaction and verified product sizes using gel electrophoresis.  We then sequenced the amplicons using di-deoxy chain termination sequencing and generated electropherograms using an automated DNA sequencer. Using the computer program CodonCode, we aligned and examined the sequences. Analysis of the data is underway. Through pinpointing mutations that cause hypermobility, characterization of Distal Arthrogryposis syndromes will become more accurate which may lead to better patient outcomes for individuals with additional features like severe hypermobility.

Hair cells are mechanosensory receptors of the auditory and vestibular systems in vertebrates. Loss of hair cells or hair cell dysfunction, leads to defects in hearing and balance. There are several mitochondrial gene mutations that lead to hearing loss, however, whether these mutations affect hair cells directly remains unknown. Two mitochondrial proteins known to be involved in sensorineural hearing loss, CLPP protease, and Timm8a translocate, were chosen as the focus of this study. To investigate the role of these proteins in hair cell function and susceptibility, I used the zebrafish lateral line as a model. Lateral line hair cells are structurally and functionally similar to those in the mammalian inner ear, but instead detect changes in water flow. CRIPSPR/Cas9 was used to introduce dsDNA breaks in CLPP and Timm8a in zebrafish embryos. Non-homologous end repair following dsDNA breaks is error prone, and can introduce small insertions or deletions (indels) in the protein coding sequence which often leads to a loss of function in the protein. In order to quantify hair cell susceptibility, I treated embryos 5 days post-fertilization with varying concentrations of neomycin, a well characterized ototoxin, and counted the hair cells remaining after treatment. Future characterization of these mutants will involve examining mitochondrial activity and oxidation levels. These studies will provide insight into how mutations in CLPP and Timm8a affect hair cell function and susceptibility to damage. Additionally, they will more broadly inform our understanding of how changes in mitochondrial function can cause deafness.

An organism’s ability to sense, interpret, and respond to its environment is an essential requirement of life itself. At the cellular level, this process of connecting signals to behaviors is governed by a diverse set of mechanisms such as mitogen activated protein (MAP) kinase cascades, which are broadly conserved across animals, plants and fungi. Alterations to these connections that arise from evolution or disease can have significant phenotypic effects. MAP kinases transmit signals by colocalizing with a target protein and altering its structure through phosphorylation and thereby producing downstream behavioral changes in the cell. Engineering novel phosphorylation triggered interaction domains will allow MAP kinase signaling networks to be rewired to correct pathological dysfunction or to generate novel responses to environmental signals. We intend to use an engineered protein scaffold with a phosphorylatable motif and an occluded binding motif, which was designed by collaborators in the Baker lab, to create a phosphorylation triggered interaction motif in Saccharomyces cerevisiae. By rewiring the S. cerevisiae mating MAP kinase cascade to phosphorylate this scaffold and incorporating a yeast three hybrid output, we hope to demonstrate the function of this new class of interaction domains in vivo. We hypothesize that upon phosphorylation, the scaffold will undergo a conformational change revealing the previously occluded binding motif, allowing another protein to bind and produce downstream effects. Such novel interaction domains will serve as a “toolbox” to allow the rewiring of MAP kinase cascades in any context in which they naturally occur. This approach could be used to fix pathological miswiring of MAP kinase cascades that contribute to anomalies of development or diseases such as cancer. Additionally, the creation of new connections using these interaction domains could be utilized to engineer organisms or cells with useful functions such as plants with enhanced defensive traits or cancer targeting T-cells.

Necroptosis is a lytic and immunogenic form of programmed cell death (PCD), characterized by pro-inflammatory cytokine production and the release of intracellular molecules, including cell-associated antigens. In the context of tumor immunology, tumor cell death is a necessary step in causing the release of tumor antigens, allowing for antigen-specific tumor recognition by the immune system. However, how distinct forms of PCD within tumor cells could differentially instruct immune recognition of tumor-associated antigens remains poorly understood. Our research question addresses how the immune system responds to cancer cell death, and will help determine whether necroptosis can promote tumor immunity. We propose that induction of necroptosis within tumor cells could promote anti-tumor immune responses by releasing tumor antigens accompanied by pro-inflammatory signals, resulting in potent immune recognition of cancerous cells. To study the immune responses to cell death in tumors, we have modeled the effects of various cancer cell death programs using flank tumors implanted in B6/J mice. Using flank tumors engineered to express versions of various pro-death signaling proteins that can be activated, we show that induction of necroptosis within the tumor microenvironment leads to robust tumor control. Measuring serum cytokine levels, in addition to analyzing the identity and activation status of immune cells isolated from the tumor tissue following treatment, will yield critical insights towards the immune cell subsets and signaling axes responsible for the observed response to necroptosis. These experiments provide us with a comprehensive understanding of whether cell death in a solid tumor has a therapeutic benefit as well as the underlying mechanism of the immune response to dying cells within a solid tumor. Considering the pro-inflammatory nature of necroptosis, it represents a promising therapeutic target in the context of cancer immunology.

Mycobacterium tuberculosis (M.tb) is the causative agent of tuberculosis, an infectious disease responsible for nearly two million deaths around the world every year. Mycobacteria express a number of cell wall lipids that activate our immune system. For example, T cells are activated by lipids when bound to highly conserved CD1 molecules on the surface of antigen-presenting cells. T cells express a unique T cell receptor (TCR) that is the result of genetic recombination and mutation. Thus, the DNA sequence of a TCR is specific for the antigen recognized by a T cell. Virulent M.tb strains contain sulfoglycolipids (SGLs), but the diversity of SGL-specific T cells has not been determined. This information could be helpful in distinguishing infection from exposure or vaccination. The objective of this work is to determine the diversity of TCRs expressed by SGL-specific T cells. We isolated mRNA from SGL-specific T-cells that were derived from a patient with a latent M.tb infection and used template-switched PCR to amplify the TCR sequences. We will present data summarizing the diversity and conservation of sequence motifs required for recognizing SGLs. The identification of conserved sequences could be utilized to develop novel TCR sequence based diagnostics for M.tb.

Infantile Spasms (IS), classified as an epileptic encephalopathy (EE), is a severe epileptic syndrome that mostly occurs in newborn babies. Because these seizures are hard to treat, IS can lead to other forms of epilepsy and is often associated with intellectual disability. Many IS cases have an underlying genetic cause – specifically, mutations acquired during DNA replication that disrupt key genetic pathways of synaptic function and brain development. However, many IS cases remain without a genetic diagnoses due to the genetic heterogeneity of the disease. Because IS shares many phenotypic traits with other EEs, my research uses a candidate gene approach to identify genetic variants in the coding regions of 51 known EE genes in a cohort of 105 patients with IS of unknown cause. Sequencing these candidate genes from a population of patients with IS will determine if mutations in these EE genes cause IS. I used next-generation DNA sequencing to screen for variants in 51 known EE genes. I filtered the data to identify 1) variants that result in an alternative amino acid sequence, which may lead to the disrupted function of a protein, and 2) variants that are unreported in a healthy population – identifying 16 variants. IS is most commonly the result of de novo mutations, where the patient has a pathogenic genetic mutation that was not inherited from either healthy parent. I performed segregation testing to determine the inheritance of the 16 identified variants. Of the 105 patients with IS, I identified 4 de novo, pathogenic mutations in 3 genes and am currently waiting on parents to determine the mode of inheritance of 4 other variants. My lab has solved 10% of the IS cases in total. These new genetic discoveries will contribute to a better understanding of IS and lead to more accurate diagnoses.

Adolescents without college attending parents and from minority communities have lower post-secondary enrollment rates. Non-profit college preparedness organizations targeting these adolescents can provide support to help increase these enrollment rates. An important aspect of reaching adolescents is outreach through social media, given its prevalence among this population. However, pilot evaluations show infrequent social media use by college preparedness organizations, and it remains unclear which online outreach approaches yield the highest user engagement. The purpose of this study is to understand approaches used by college preparedness organizations in social media outreach, and which factors are associated with user engagement. In this content analysis study, seven college preparedness organizations listed as partner organizations on the Seattle government website were randomly selected. A codebook was developed to evaluate outreach approaches using components derived from the Facebook Influence and non-profit organization models, which will use variables for how organizations communicate information, build communities, create immersion, and facilitate action in their social media posts. The codebook was used to evaluate each organization’s posts from their Facebook and Twitter profiles for a period of a year. Likes, Shares, and Retweet totals were also collected for each post. Associations between social media outreach approaches and user engagement were examined. Organizations may use more community and immersive orientated posts to generate more likes and followers. The results of this study could potentially be used to develop strategies that will allow other college preparedness organizations or organizations in other outreach based fields to increase their user engagement.

Student-graded practice exams (PEs) have been used to facilitate learning in large classrooms. In a large introductory biology class, we investigated the impact of self vs peer-grading of PEs. We hypothesized that students who graded their own PEs would grade more accurately than peer graders because they are more motivated and reflective in their grading. Over time this more accurate grading would lead to increased performance on course exams. One question from each week’s PE was graded by a team of experts. We calculated PE grading accuracy by comparing the expert grade to the grade assigned by the student. We analyzed the correlation of PE grading accuracy with exam performance. For each metric we investigated the impact of treatment, incoming GPA, and student demographics (e.g., gender, socioeconomic status, first generation, race/ethnicity) using general linear models. There was no difference in average expert PE grade between the two treatments. Incoming GPA was the only significant predictor of expert PE grade with GPA positively correlated with expert PE grade. There was a significant interaction between incoming GPA and treatment on grading accuracy. Students with high GPAs who graded peers consistently underestimated their peers’ PE performance. Students with lower GPAs who graded peers either graded similarly to experts or slightly overestimated their peers’ PE performance. Students who graded their own PEs more accurately predicted their PE performance regardless of incoming GPA. There was an interaction between GPA and treatment on course exam performance, as high GPA self-graders earned more course exam points than high GPA peer-graders. Average GPA students earned the same amount of course exam points regardless of treatment. Given these findings and the logistical complications of peer-grading in large classes, instructors can use the more easily administered self-grading method of providing feedback on low-stakes practice exams.

Atherosclerosis is a disease in which cholesterol accumulates in the walls of arteries, limits blood flow, and causes heart attacks and strokes. We aim to prevent and reverse atherosclerosis with a gene therapy that increases levels of apolipoprotein A-I (apoA-I), a protein that removes cholesterol from the artery wall. This therapy increases expression of apoA-I specifically in endothelial cells (EC), cells that line the artery wall. Our initial vectors for expressing apoA-I contained the murine endothelin-1 (mET-1) promoter (an EC-specific promoter) both without (1XETE) and with (4XETE) three additional copies of an endothelin-1 enhancer (ETE). We used two approaches to increase apoA-I expression: 1) literature searches for regulatory sequences that increase EC gene expression in other contexts; 2) novel bioinformatics approaches that identify EC-specific cis-regulatory sequences (CRMs) throughout the genome based on ENCODE data, relative local abundance of EC-related transcription factor binding sites, and interspecies conservation. We ligate regulatory sequences into the apoA-I vectors mentioned above. We transfect these constructs into cultured EC and measure apoA-I expression. Using the first approach, we found that addition of a shear-stress-response element into the 4XETE vector did not alter apoA-I expression. Addition of a single MEF2C enhancer sequence into the 4XETE vector decreased expression; however, after this decline, addition of more copies of the MEF2C enhancer increased apoA-I expression. Lower expression may be due to unfavorable interactions between multiple enhancer elements. Using the second approach, we identified eleven CRMs (55-352 bp) clustered around the promoters of genes highly expressed in EC: CDH5, EFEMP1, THBS1, and VWF. In conclusion, trial-and-error addition of enhancers has not reliably improved EC apoA-I expression. A bioinformatics-based approach identified CRMs associated with high transcriptional activity in EC. We inserted the eleven CRMs into both the 1XETE and 4XETE apoA-I vectors and will test whether they increase EC apoA-I expression.

Impairments in the DNA mismatch repair system give rise to mutations in microsatellite loci, which are short, repetitive stretches of DNA. Also known as microsatellite instability (MSI), these mutations manifest as new alleles of variable length. MSI is an important marker of cancer outcomes, and moreover, can directly drive oncogenesis. Despite their clinical relevance, microsatellite loci are challenging to reliably sequence using existing high throughput sequencing methods. Here, we have performed exploratory analyses using high throughput next-generation sequencing (NGS) in conjunction with single molecule molecular inversion probes (smMIPS) to enable typing of large numbers of cancer-relevant microsatellite loci. smMIPs integrate multiplex targeted sequencing with molecular-tag-based error correction, resulting in a cost-effective method for large-scale, accurate genotyping of microsatellite alleles. smMIPs are 80bp oligonucleotides, each containing a random, unique molecular tag, that hybridize to flanking regions around microsatellite loci. The targeted sequence is copied into the probe through the action of DNA polymerase and ligase. Capture reactions are then exonuclease treated, PCR amplified, and sequenced using the Illumina NGS platform. Error correction is achieved through consensus mapping of probes associated with a unique molecular tag, eliminating analytic errors otherwise inherent to the process. We have developed a panel of probes for detecting microsatellite instability events in 150 cancer-linked loci. We have compared our data to conventional NGS methods and have demonstrated higher accuracy genotyping compared to existing methods. The combination of high-throughput sequencing and robust error correction suggests that further development of this technology will be well suited for clinical laboratories searching for streamlined and accurate approaches to MSI diagnostics. The intent of this project is to develop a smMIP panel that detects microsatellite instability in heterogeneous tumor samples and that offers other advantages over existing testing methods.

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.

Extant mammals derive from three lineages: monotremes, marsupials, and placentals. The divergence of marsupials and placentals is estimated to have occurred 160 million years ago. Marsupials typically have very small, underdeveloped offspring born after a short gestation period, which are then nursed for long periods of time in a pouch. In contrast, placental mammals give birth to large, more developed young, which are nursed for a shorter period of time. Sexual dimorphism, the anatomical differences between males and females, has been demonstrated in the pelves of many species, both marsupial and placental, but no studies to date have compared the degree of sexual dimorphism in the pelves of these two groups. This study compares representative pelves from 30 species, 9 marsupial and 21 placental, across a wide range of taxa. One male and one female pelvis from each species was scanned using either a laser scanner or a microCT scanner to create a 3D surface scan. A set of landmark points were placed on each pelvis and the distances between points were calculated. The pelves were compared to calculate a value representing the amount of sexual dimorphism in each species. These values were then compared between placentals and marsupials using a t-test. Locomotion, adult female size, offspring size, number of offspring per litter, were included in the analysis as possible influencing factors. Placentals are expected to show a greater degree of sexual dimorphism due to the burden on the females for bearing larger, more developed offspring. More specialized modes of locomotion are expected to reduce the amount of sexual dimorphism. This study provides important insight into the evolutionary development of the two dominant mammalian lineages today. To our knowledge, no other study has examined sexual dimorphism of the pelvis in such a broad range of taxa.

We have evaluated the viability of a system for detecting auxin with a yeast culture using the red buildup of P-ribosylformyl glycinamidine as a reporter that can be used to reliably identify auxin in water without the need for advanced equipment. To accomplish this we have fused an IAA17 protein to an ADE2 enzyme with a linker that preserves function, then transformed that into a yeast strain that lacks native ADE2 and has an Fbox AFB2. We plated these on adenine deficient agar with various concentrations of auxin to test for sensitivity. As controls, we plated basic strains w303a and w303alpha and found that the ADE2-lacking w303alpha still needs a little bit of adenine or it will not grow at all. We tested two different types of media, YPD and SC with minimal adenine concentration, both of these have just enough adenine to keep the yeast alive but still induce the red color. We conclude that the method of detecting auxin via the degradation of an IAA17 tagged protein is a viable strategy, but that using ADE2 as our target enzyme presents complications when considering the ultimate goal of our project. The reporter is not simple to use as a very specific concentration of supplemental adenine is required to allow growth but still induce the ADE pathway. To mix this media would require high-precision equipment usually available only in a lab. This problem can be solved by changing the reporter to allow the yeast to be functional in any media, which will be our next project.

The forefront of modern medical research involves decoding the biological pathways that make disease transmission and perpetuation possible. Protein-based site-specific affinity reagents could allow us to antagonize harmful molecular interactions employed by pathogens, disrupt their toxic effects, and develop cutting-edge treatments. Consequently, the ability to express and purify rationally-designed proteins is the key to assessing their structure and function. To increase solubility and stability during recombinant expression, a genetic fusion to a small-ubiquitin-like-modifier (SUMO) is commonly used. The highly-specific SUMO protease is then used to cleave the SUMO fusion, leaving a completely intact and unmodified protein-of-interest. The current protease used originates from Saccharomyces cerevisiae. Known as Ulp1, the instability of this enzyme due to thermosensitivity and solvent-exposed hydrophobicity presents considerable limitations and can result in incomplete digestion of SUMO-tagged constructs. We present two approaches to mitigate these complications. First, we have discovered a homologous protease with a conserved catalytic site in the thermophilic eukaryote Chaetomium thermophilium. Second, using the Baker Lab’s pioneering computational design program, Rosetta, we have exchanged the hydrophobic surface residues of Ulp1 with hydrophilic side chains with the aim of improving stability in aqueous solution. We have characterized the kinetic function of these protease variants by monitoring digest efficacy through gel-based assays. Effectively, the improved proteases can equip the scientific community with a powerful new tool to facilitate protein-based drug design research.

Prolonged activation of cardiac fibroblasts after acute wound healing post myocardial infarction results in excessive extracellular matrix deposition in the myocardium and formation of fibrotic scar at the site of injury. This adverse fibrotic remodeling disrupts physiological function in the heart and leads to increased risk for heart failure which is detrimental for patient outcomes. The reasons why activated fibroblasts, known as myofibroblasts, persist after wound stabilization are poorly understood due to the lack of a myofibroblast reporter system capable of direct longitudinal study. Current reporting modalities are invasive, resource and time intensive, wasteful, and indirect. The present project aims to design and develop an in vivo, repeatable, and real time reporter system for myofibroblast cell fate as a surrogate for cardiac fibrosis. To achieve this, we have designed a chimeric promoter to drive firefly luciferase vector expression specifically in myofibroblasts. We have built 9x tandem repeats of serum response factor (SRF) and nucleated factor of activated T-cells (NFAT) binding sequence since they are key mediators in myofibroblast activation. These repeats were ligated onto smooth muscle alpha actin (ACTA2) and collagen type 1 alpha 2 (COL1A2) minimal promoters, and these constructs were cloned into pGL3 luciferase reporter vectors. The specificity of the promoter was evaluated and optimized through in vitro luciferase assays with NIH 3T3 primary mouse fibroblasts.  With the successful creation of this reporter, wasteful use of animal models will be mitigated and direct mechanisms in the pathobiology of chronic cardiac fibrosis can be elucidated. Furthermore, efficacy of novel therapies can be evaluated in vivo.

Understanding cold stress response pathways in plants is key to the development of new agricultural techniques, which help prevent crop loss or degeneration due to cold snaps or early frosts. Epigenetic modifications by histone acetyltransferases appear to regulate these pathways. HAC1 is a histone acetyltransferase identified in Arabidopsis thaliana that is involved in cold stress response, and binds with transcription factors to mediate gene expression. CsWRKY46, a transcription factor recently identified in cucumbers, was found to be deeply involved in cold stress response. We identified the WRKY71 gene as the Arabidopsis homolog most similar in sequence to CsWRKY46. The WRKY71 transcription factor has also been identified as part of the response pathway to general abiotic response. We hypothesized that HAC1 regulates cold response in A. thaliana by localizing with WRKY71 and acetylating histones of nearby cold response genes such as COR47. We predicted that in cold temperature, WRKY71 and COR47 transcripts would be up-regulated only if the HAC1 protein is present. We designed an experiment with 6 A. thaliana plants: 3 wild type and 3 Salk_080380 hac1 mutants. Wild type Colombia ecostrain and hac1 A. thaliana were subjected to a 4°C cold temperature treatment of 0, 2, or 6 hours under constant light cycle conditions. RNA was extracted from the leaf tissue of each plant and RT-PCR and gel electrophoresis were performed to measure gene expression of HAC1, WRKY71, COR47, and e1f4-III (control gene). Results from this pilot study of HAC1/WRKY71 interaction with RT-PCR assays were inconclusive due to nonspecific primers. Without RT-PCR results with which to analyze the relative expression levels of eIF4-III, COR47 and WRKY71 throughout cold stress, we can neither support nor refute our hypothesis. We propose repeating the experiment with larger, more specific primers and examining WRKY71-HAC1 protein interaction through a co-immunoprecipitation assay or similar test.

 The percentage of obese children in the United States has tripled since the 1970’s, with one in five school-aged children now categorized as obese. Obesity development and response to intervention has been found to depend on factors such as impulsivity, though its influence as a major risk factor is unknown. This project seeks to determine if impulsivity affects weight reduction outcomes in family-based behavioral treatment. I hypothesize that increased impulsivity will be associated with increased food intake, increased food reward value and subsequently poorer weight reduction outcomes. Forty-nine obese males and females aged nine to eleven years old were evaluated before and after they participated in a 6-month family-based behavioral treatment intervention to determine the relationship between impulsivity and treatment outcomes. Impulsivity was assessed using a Developmental Neuropsychological Assessment. Each subject’s food reward value was measured using interviewer-administered tasks in which participants were questioned on their choice between preferred snack foods vs. an appealing alternative. Calorie intake was measured by providing study participants with a buffet consisting of a wide variety of foods, with which they had access to for thirty minutes. Calorie intake and composition was measured by weighing back uneaten food. Success of obesity treatment was measured in the comparison of adiposity using BMI z-scores, given that a z-score decrease of at least 0.25 improves blood pressure, lipids and insulin sensitivity. Obese children categorized as impulsive are predicted to consume more calories and rate a food reward value compared to non-impulsive obese children, resulting in poorer weight reduction outcomes, indicating that childhood obesity is correlated with impulsivity on the basis of an inability to engage cognitive control of food intake. This research will provide new insights into the neurobiological basis of child obesity, potentially improving the cost-effectively and long-term results of obesity interventions.

Macrophages are specialized cells of the immune system that direct the pro-inflammatory response and its resolution. Matrix Metalloproteinase 28 (MMP28) is an extracellular matrix proteinase expressed in alveolar macrophages during lung inflammation and is involved in macrophage recruitment and polarization. Our aim was to explore the regulation of Mmp28 expression in macrophages. We hypothesized that LPS (lipopolysaccharide, a component of bacterial cell membranes which simulates bacterial infection) stimulation would increase Mmp28 expression via a TLR4-dependent (Toll-Like Receptor 4) pathway involving Type I Interferon (IFN) signaling. We also hypothesized that Polyinosinic-polycytidylic acid (Poly(I:C), a synthetic form of double-stranded RNA which simulates viral infection) would increase Mmp28 by a TLR3-dependent pathway. To test these hypotheses, we generated bone-marrow derived macrophages (BMDM) from wild-type mice. We exposed these cells to purified recombinant IFNα/β (two forms of IFN involved in antiviral responses), LPS, Poly(I:C), or control media; 4-6 h after treatment, we isolated RNA from the BMDM, synthesized cDNA, and used qPCR to measure mRNA levels of several inflammatory genes. We also included BMDMs from knockout mice for several proteins thought to be part of the Mmp28 regulatory pathway. We confirmed that LPS and Poly(I:C) increased Mmp28 expression in BMDM. By comparing knockout mice for two adaptor molecules shared by TLR4, we found that LPS-stimulated Mmp28 expression was retained in MyD88-/- but decreased in TRIF-/- macrophages. Since both poly(I:C) and the TRIF-dependent response to LPS induce a type I IFN response, we tested if Mmp28 expression was dependent on type I IFN by using IFNAR-/- (IFN Receptor) macrophages. We found that both TLR4 and TLR3 ligands require IFNAR for Mmp28 expression. These data link type I IFN responses to regulation of MMP28 and development of experimental COPD and emphysema models of lung disease, and further our understanding of how MMP28 may regulate macrophage responses.

Small Ubiquitin-like Modifier, SUMO for short, is a protein involved in cellular regulation which can also be used as a biochemistry tool to express difficult-to-express proteins in the lab. I am investigating several SUMO proteases in order to learn more about the regulatory pathways it is involved in. We are focused on the traditionally used protease from Saccharomyces cerevisiae, a thermophilic version from Chaetomium thermophilium, as well as several redesigns of a homologous protein from the Saccharomyces SUMO protease to improve solubility by mutating solvent-exposed hydrophobic amino acids to polar amino acids using a computational protein design program called Rosetta. I am using thermal melting techniques to find melting points and gel comparison techniques to analyze the efficiency of each protease. Understanding of the kinetics and properties of these proteases and comparing them to each other will yield a greater understanding of how the system works, which versions are more efficient for in vitro use, and how the system can be manipulated in vivo.The goal of my research is to create a set of biochemical tools associated with SUMO proteins to be used in cancer research, as the SUMO system is known to be dysregulated in cancer. Understanding of the role SUMO plays in cancer will provide context for the development of a new generation of therapeutics, and understanding of the SUMO system as a whole will improve a current biochemical technique.

Sulfate conjugation is an important metabolic pathway which has been shown to play a vital role in modulating the biological activity and disposition of drugs and endogenous compounds. Studies have shown that sulfotransferases (SULTs) have a broad substrate selectivity that includes hormones, neurotransmitters and xenobiotics. SULT proteins are expressed ubiquitously in human tissue, but is largely expressed in the gastrointestinal tract, liver and bone marrow. Numerous SULT isoforms exist in humans and the most predominate of these isoforms in the liver is SULT1A1. In this study, we evaluate the impact of various single nucleotide polymorphisms (SNPs) present in the SULT1A1 gene using samples obtained by St Jude Children’s Research Hospital and the University of Washington. Of particular interest is the effect these SNPs have on the RNA and protein expression of SULT1A1. In total, 17 SNPs within the SULT1A1 gene was identified. A subsequent analysis using RNA-seq data found that 7 out of the 17 SNPs were common variants with an allele frequency greater than 5%. To assess the effects of these SNPs on the protein expression of SULT1A1, samples of human liver cytosol was analyzed using a mass spectrometry based protein quantification method. While this protein analysis continues to be an ongoing effort, we expect results from this analysis to be consistent with genotypic trends observed in our RNA-seq dataset. By evaluating the relationship between various SNPs in the SULT1A1 gene and their effect on SULT1A1 RNA and protein levels, we hope to expand on our knowledge of the impact SNPs have on the disposition of substrates that undergo sulfonation.

During embryogenesis, phosphorylation of serine and threonine residues on p120 catenin causes disassociation from cadherin transmembrane proteins and activation of downstream pathways leading to cell motility. We have mutated a single amino acid on p120 catenin, T310, to determine its involvement in cell migration of the dorsal axis during zebrafish embryogenesis in vivo. T310 is a target of glycogen synthase kinase 3 (GSK3) and phosphorylation of this site facilitates disassociation from membrane-bound N-cadherin in astrocytes, decreasing cell-cell adhesion and activating the cell motility pathway. We created two mutations of p120 catenin: T310E that mimics the negatively charged phosphate and T310A containing a neutral residue that cannot be phosphorylated. Either mutated or wild type mRNA was co-injected with morpholino, which knocks down endogenous p120 catenin, to determine the injected protein’s ability to rescue the short-tailed knockdown phenotype. As expected, co-injection of wild type p120 catenin mRNA and morpholino rescued the damaged phenotype resulting in embryos with normal tail extension. Co-injecting either T310A or E with morpholino resulted in partial rescue, suggesting that phosphorylation at this site may be variable during development or that the effects cannot be measured by phenotypic quantification methods. We are currently studying the effects of phosphorylation at other sites, S268 and S269, which may give insight on the downstream effects p120 catenin in cell motility, including morphological bone abnormalities and metastasizing cancer cells.

Joubert syndrome (JS) is a genetic neurodevelopmental disorder with medical features including a distinctive hindbrain malformation and developmental delay. All 35 JS-associated genes to date encode gene products localizing to the primary cilium but the mechanism behind JS is still unknown. The primary cilium serves as a cellular antenna for signaling, and when dysfunctional, can lead to the above features and more. One JS-associated gene, INPP5E, is an enzyme that controls phosphatidylinositol (PI) subtype distribution in the ciliary membrane. We hypothesize that abnormal distribution of PI subtypes PI(4,5)P₂ and PI(4)P in primary cilia is central to the JS mechanism, and therefore should be present across genetic causes. To test this hypothesis, I used fluorescently-tagged PI biosensors, domains from naturally occurring proteins that bind to different PI subtypes, and a domain from serotonin receptor (5-HT₆) that marks cilia. The PI biosensors were already tagged with GFP (green fluorescent protein) so I replaced GFP for RFP (red fluorescent protein) on the PI(4,5)P₂ biosensor, and inserted BFP (blue fluorescent protein) on the 5-HT₆ domain, allowing me to distinguish all three molecules in the same primary cilium. To do this, I removed the GFP sequence from our PI(4,5)P₂ plasmid using restriction enzymes that cut DNA then inserted RFP (amplified from a template plasmid carrying RFP) using DNA ligase. I confirmed the RFP insert by restriction enzyme digests specific for RFP and by direct DNA sequencing. PI(4,5)P₂-RFP plasmids were transformed into cells with PI(4)P-GFP and our BFP ciliary marker, then live-imaged. These tools will make it possible to determine if abnormal PI subtype distribution in primary cilia is a unifying cellular defect in JS patient cell lines. If so, we can then explore the effects of altered PI distribution and determine whether modulating PI distribution has the potential for mitigating the effects of Joubert syndrome.

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.

Quorum Sensing (QS) is a chemical signal based communication system by which bacteria regulate gene expression according to changes in population density. This communication mechanism is used by the environmental bacterium and opportunistic human pathogen P. aeruginosa, which is a major cause of Cystic Fibrosis (CF) lung infections. P. aeruginosa employs two interconnected QS systems: las and rhl. These systems are regulated by the transcription factors LasR and RhlR, each of which binds a unique chemical signal that further activates transcriptional regulation. LasR and RhlR together control the expression of virulence factors critical for many infection processes. In laboratory strains, the LasR and RhlR QS systems are arranged hierarchically, such that LasR transcriptionally activates RhlR. Although this QS hierarchy is common to laboratory strains of P. aeruginosa, some CF clinical isolates are capable of RhlR activation in the absence of functional LasR. However, the origin of this LasR-independent regulation remains unclear. In this study, we investigate whether the LasR–RhlR hierarchy is conserved in environmental P. aeruginosa. We collected several environmental isolates and genetically engineered them through allelic exchange mutagenesis to disrupt LasR function. We then transformed wild type and LasR-null isolates with reporter constructs that produce GFP (Green Fluorescent Protein) in response to either LasR or RhlR. In these two conditions, we measured protein levels of LasR/RhlR, signal concentrations and the production of several QS-regulated products. In most cases, we observed that RhlR activity was substantially reduced in the absence of functional LasR. The observed decrease in RhlR function is so far consistent with the idea that the QS hierarchy is a conserved feature of P. aeruginosa. However, mechanisms of quorum regulation in environmental strains may differ from laboratory strains and a proper understanding of them can potentially contribute to the development of non-antibiotic therapies.