Postsurgical treatments for human malignant gliomas prove ineffective as there are high rates of tumor reoccurrence due to their elevated DNA-repair mechanisms. There is a group of human gliomas, oligodendrogliomas, that present longer survival periods of patients receiving chemotherapy and/or radiation. Certain genetic aberrations, specifically deletions of chromosome arms 1p and 19q, are associated with oligodendrogliomas that may lead to better responses to postsurgical treatment. My work addresses the hypothesis that this observed trend is due to decreased O6-alkylguanine DNA methyltransferase(MGMT) activity, which may be a result of the 1p and 19q deletions. MGMT is a known repair enzyme of DNA that contributes to tumor reoccurrence following alkylating agent based therapies. The MGMT activity is determined by estimating the number of MGMT molecules present in a homogenate through an assay developed in the Silber lab. The highly-sensitive assay exploits the reaction of an alkyl group irreversibly binding to MGMT. By incubating [3H]-labeled O6-methylguanine contained in double-stranded DNA with tumor homogenate, each MGMT molecule is covalently bound to a methyl group. The radioactivity is then counted by a liquid scintillation spectrometer. Preliminary data collected indicates that oligodendrogliomas with the deletions typically have lower amounts of MGMT activity than oligodendrogliomas without the deletion. When analyzing the clinical outcome information available, patients whose tumors have lower MGMT activity tend to follow the trend of higher responsiveness to treatment and longer periods of survival. By better understanding the DNA repair mechanisms of human malignant gliomas, new therapies can be developed that can improve the prognosis of patients.

Agressive cancers are resistant to apoptosis and stem-like cancer cells may be responsible for re-growth of cancerous tumors. This inspired this search for compounds that could aid in the destruction of cancer cells with stem-like properties. Drosophila germ-line stem cells under radiative or chemotoxic stress are protected by a molecular signal from apoptotic daughter cells via the TIE receptor. Thwarting this protective signal by the introduction of a small molecule may lead to the discovery of compounds that can be effective against stem-like cancer cells. The NCI Diversity Set of 1,596 small molecules was screened by undergraduate research students, in-vivo, to find compounds that can induce apoptosis in germ-line stem cells of Drosophila melanogaster. Four compounds have been found that induce apoptosis in both the male and female germ-line stem cells at a low 5 uM concentration. Future investigations will uncover the mechanism of action and efficacy against stem-like cancer cells.

With increasing age and as a result of mitochondrial-generated reactive oxygen species such as hydrogen peroxide, lung fibroblasts cells undergo DNA damage which is leading to a loss of function and premature ageing (senescence). The objective of this project was to assess whether the presence of the mitochondrial-specific antioxidant enzyme catalase (mCAT) could affect the levels of DNA damage and senescence in fibroblasts collected from the lungs of 24 month old mCAT mice versus control. Immunostaining of lung fibroblast cultures, using specific antibodies against markers of DNA damage/premature ageing showed, a significant decrease in DNA damage and a decrease in cell division in mCAT lung fibroblasts against control.
Aging is the most significant risk factor for developing cancer, and majority of malignant cancers that are treated in clinics, occur in older patients that are suffering from cellular senescence/aging and uncontrollable cell division, leading to cancer. Therefore, understanding the consequences of senescence may help with designing new strategies, such as anti-oxidant therapies that lead to prevention of DNA damage and age-related diseases, in the future.
 

Cell adhesion and motility are important cell processes that are necessary for embryo morphogenesis. Prevous studies have identified p120-catenin as a key player in coordinating cell movements by either stabilizing E-cadherin complexes to strengthen adhesion or activating cytoplasmic CDC42 and Rac1 GTPases to initiate motility. Most of these studies were done in-vitro using cells grown in a 2-dimensional plane. Comparatively, few studies have attempted to describe p120-catenin's function in a 3-D space. In an effort to investigate p120-catenin's function in embryos, we are co-immunoprecipitating p120-catenin from developing zebrafish embryos. We are specifically investigating how the tyrosine 228 (Y228) residue of p120-catenin mediates binding to E-cadherin. The hypothesis is that phosphorylation of Y228 causes p120-catenin to bind to E-cadherin. To test this, we are injecting zebrafish embryos with p120-catenin containing either an Y228F of Y228E mutation. If our hypothesis is correct, the Y228F mutation would prevent phosphorylation of the 228 residue and decrease affinity between E-cadherin. The Y228E mutation mimics the negative charge of phosphorylation and would instead cause p120-catenin to bind to E-cadherin. 

Ammonia-oxidizing archaea and bacteria (AOA, AOB) are important contributors to soil ecosystem function, as they convert nitrogen (N) as ammonium (NH4+) into nitrate (NO3-), a more readily available form for plant uptake. This study examines the abundance of AOA and AOB in forest soils of the Pacific Northwest that have experienced wildfire 31, 7, and 5 years previously. Charcoal, a common residue of wildfire, may play a significant role in stimulating nitrification in soils, possibly by adsorbing monoterpenes that may inhibit mono-oxegynase activity in nitrifying organisms. The goal of this study was twofold. We first hypothesized that charcoal would increase the quantity and quality of AOB and/or AOA in wildfire-affected soils. To test this hypothesis, we extracted and compared DNA between burned and unburned sites using real-time quantitative polymerase chain reaction (RT-qPCR). We expect to find a greater abundance of AOA and AOB nucleotides in the burned sites indicated by qPCR, and a larger array of species indicated by DGGE analysis. Secondly, we will introduce monoterpenes (α-pinene) into soils with charcoal present, and soils void of charcoal to assess the interaction between the terpenes and charcoal surfaces. We hypothesize that the addition of terpene will readily adsorb onto charcoal surfaces. Therefore, burned soils will show reduced nitrification inhibition compared to soils without charcoal present and result in higher nitrification rates. These data will be matched with an assessment of the AOA and AOB from the burned and unburned soils, with and without the addition of charcoal in order to reach a better understanding of wildfire effects on forest soils.

As concern over antibiotic resistance continues to grow, there is a critical need to discover and develop new methods of controlling and preventing bacterial infections. Essential oils derived from the peels of oranges are known to possess antimicrobial properties. Previous research has established that limonene, the principle compound present in orange oil extract, is a particularly effective antimicrobial agent that both destroys bacteria and inhibits bacterial growth. To determine whether different varieties of oranges have varying antimicrobial potency, the efficacies of oil distilled from blood, navel, and valencia oranges were compared. Oils extracted through steam distillation were diluted with methanol to concentrations ranging from 1:20 to 1:1. Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli bacteria were exposed to the oils using the disc diffusion method on TSA plates. The minimum inhibitory concentration (MIC) for each oil was determined using the agar diffusion method. Gas Chromatography Mass Spectrometry (GC-MS) was used to compare the relative concentrations of limonene present in each essential oil. A better understanding of the potency of orange oil as an antimicrobial agent could lead to development of novel drugs and products to be used to combat infection and prevent the spread of bacterial pathogens.

Epileptic encephalopathies (EEs) are devastating childhood epilepsies characterized by multiple seizure types that are usually refractory to medication. Next-generation sequencing has revealed the prominent role of de novo mutations in causing EEs with clinical diagnostic labs reporting a molecular diagnosis in as many as 24-40% of cases. Despite this significant progress, the majority of cases are unexplained. Exome sequencing, a next-generation sequencing technique, allows one to look at all the protein coding regions of an individual’s genome, offering an affordable and unbiased approach to gene discovery. Using exome sequence data for 50 trios (proband and unaffected parents), in which the child had seizure onset in the first 2 years of life, we identified 76 candidate genes, of which 37 are confirmed de novo variants through Sanger sequencing. Notably, 2 affected individuals were identified to have a de novo variant in the guanine nucleotide-binding protein (G-protein) beta1 subunit, GNB1. This was a statistically significant finding (p=0.005), and in collaboration with colleagues we have now identified 13 patients with neurodevelopmental delays with and without seizures. This previously unrecognized genetic condition is characterized by global developmental delays (13/13), hypotonia (11/13), seizures (10/13), ocular manifestations (8/13) and growth delay (6/13). These genetic findings will have immediate impact to clinicians and families when discussing diagnosis and prognosis, and hopefully guide future studies of finding effective therapies for these patients. Future work will screen for genetic variants in the candidate genes identified by exome sequencing using a targeted sequencing approach in a cohort of 600 patients with EE of unknown etiology.

Current synthesis for thermoresponsive magnetic nanoparticles such as iron oxide nanoparticles coated with poly(N-isopropylacrylamide), pNIPAm, usually requires extensive surface functionalization or high temperature procedure, and the yield is very limited. Here we present a simple and reproducible approach for the synthesis of thermosensitive iron oxide nanoparticles, via coprecipitation of Fe2+ and Fe3+ ions from basic aqueous solutions at room temperature in the presence of block copolymer, poly(N-isopropylacrylamide)-block-poly(acrylic acid) with a dodecyl tail following the acrylic acid block. The polymer facilitates iron oxide nanoparticle synthesis by forming micelles via hydrophobic dodecyl tail assembly and coordinating iron oxide formation via complexation with the poly(acrylic acid) segment. The block copolymers were synthesized through reversible addition-fragmentation chain transfer polymerization. Proton NMR characterization shows incorporation of 11 acrylic acid monomeric units to pNIPAm macro chain transfer reagent with 150 degree of polymerization. The new synthesis approach is rapid, taking one hour, and can be easily purified via size exclusion chromatography. The resulting magnetic nanoparticles’ surfaces display pNIPAm, which enables particle aggregation when the solution temperature is above 34°C. The particle aggregates exhibit high magnetophoretic mobility, which results in rapid and efficient magnetic separation, ≥95% in few minutes. The particle’s hydrodynamic diameter is ca. 30 nm, characterized using dynamic light scattering. This simple method overcomes the problem of using high temperatures while maintaining reproducibility, which could make it useful for larger scale synthesis.

Loss of heterozygosity (LOH) frequently occurs in tumor cells and is associated with the expression of deleterious recessive phenotypes. However, relatively little is known of its mechanisms or the various functional/regulatory factors involved. The goal of our research is to characterize the mechanisms of LOH and identify the factors involved. One cause of LOH is homology-directed repair (HDR), which uses the homologous chromosome as a donor to repair DNA damage like DNA nicks or double stranded breaks (DSBs). In HDR, the homologous chromosome functions as a template for repairing the damaged DNA strand. In order to study the mechanisms and factors involved in LOH, we developed a novel flow-based reporter model that utilizes an endogenous gene. This model was developed by making genetic modifications in a human cancer cell line (HT1080) in such a way that would allow the detection of a LOH event. Using our flow-based reporter, we will target DNA nicks and DSBs using CRISPR/Cas9 and quantify the resulting LOH events using our flow-based reporter. We will also use siRNA knockdowns to assay other candidate factors potentially involved in the mechanisms and regulation of LOH. To simulate mutations seen in tumors, we will downregulate the expression of common tumor suppressors (BRCA1, BRCA2 and PTEN) with siRNA and determine how these factors affect LOH. When common tumor suppressors are downregulated in our model, mutations that take place are expected to follow a similar mechanism to mutations seen in tumors. Using our model in this way, we will verify which common tumor suppressors are involved in the mechanisms of LOH often seen in tumors. With a better understanding of the factors involved in LOH, prophylactic therapies can be developed to minimize occurrence of LOH and tumorigenesis.

Calcium is an ubiquitous and critical signaling molecule that mediates a wide range of cellular processes. Cytosolic calcium is maintained at concentrations that are orders of magnitude below both the extracellular space and intracellular organelles. Even small, sustained increases in cytosolic calcium can have harmful effects on the cell. As such, calcium homeostasis mediated in large part by the endoplasmic reticulum (ER) and mitochondria that serve to sequester calcium enable normal functioning of the cell. To examine cellular mechanisms that mediate calcium homeostasis, we are using mechanosensory hair cells of the zebrafish lateral line. Mechanosensory hair cells can be stimulated via a mechanical stimulus or drug-mediated calcium release from intracellular stores. The hair cells' abilities to easily uptake drugs and respond to physical stimuli make them an ideal tool to examine calcium homeostasis. Preliminary studies using transgenic zebrafish in which the calcium indicator GCaMP3 is targeted to the cytosol vs. the mitochondrial matrix demonstrates that stimulation of hair cells causes an immediate increase in cytosolic calcium in contrast to a gradual increase in mitochondria calcium. The question is whether the calcium in the ER is changing in response to the changes in mitochondrial and cytosolic calcium. As a first step to investigating the role of ER in calcium homeostasis, we will use gateway cloning to generate transgenic zebrafish lines in which calcium indicators are targeted to the ER. We predict that stimulation of mechanosensory hair cells will cause changes in ER calcium levels. These studies may provide valuable insight in how cells maintain health by buffering intracellular calcium levels in response to activity. 

High-risk human papillomavirus (HR HPV) can establish a persistent infection in epithelial cells through its ability to evade immune detection. HR HPV type E6 is an oncogenic protein that is known to downregulate the innate immune response in human keratinocytes. Previous studies have shown that NFX1-123, an endogenously expressed epithelial protein, acts as a novel binding a partner for HR HPV type 16 E6 (16E6). We examined whether or not NFX1-123 also plays a role in the repression of the innate immune response in human foreskin keratinocytes (HFKs) transduced with HR HPV type 16E6. By stimulating the immune response with Poly(I:C) or double stranded DNA in HFKs, cells with overexpressed levels of NFX1-123 (FWT), and cells with endogenous levels of NFX1-123 (LXSN), we demonstrated that stimulation occurs both at the RNA and protein levels. We found, however, that the immune response of 16E6 HFKs with overexpressed levels of NFX1-123 were more downregulated than those with endogenous levels, confirming the protein’s association with 16E6 in the repression of the innate immune response. By understanding the roles of 16E6 and NFX1-123, we will further understand how together they cause a persistent infection, which can ultimately lead to cancer.

Historically, only clinicians and researchers have been involved with decisions about what research topics to pursue, and patients have been left out of the process. This creates a gap between clinical application of research and patients’ experience. By expanding the scope of how researchers and clinicians obtain patient data to include research topic priorities, we explore how to incorporate patient perspectives better. Although there are many studies that aim to analyze patient registries, there are limited studies that attempt to elucidate the degree of the participants’ interest, or willingness to get involved in providing input on research activities. Through a study funded by the Patient-Centered Outcomes Research Institute (PCORI), we developed a two-phase approach to prioritize topics for low back pain research. In phase one, we invited participants from a patient registry of older adults with back pain , and in phase two, invited participants using Amazon Mechanical Turk (MTurk), an online crowd-sourcing platform. Participants completed an initial screening survey comprised of the Roland Morris Disability Questionnaire, a standard tool to identify individuals experiencing back pain. Those scoring 7 or higher on the RMDQ were sent a research topic prioritization survey. 1966 people completed the screening survey, and 480 qualified, of which 353 completed the prioritization survey (completion rate: 74%). Using both recruitment strategies, we had a high response rate, and participants are diverse in terms of pain, gender, and age. Responses show participants’ effort to fully answer the survey and voice their opinion, including providing additional topics that had not been identified previously.This work highlights that recruiting from a patient registry or using MTurk are both effective tools to obtain patient input, and increases the flow of information from the public/patient to the researcher, and inevitably the clinician; we expect incorporating these methods will increase patient voice in research.

Overnutrition and sleep loss are widespread problems in today’s society and major risk factors for metabolic diseases. As with overnutrition, sleep loss impairs glucose regulation, though the mechanisms underlying this effect are unclear. Hypothalamic neuroinflammation disrupts leptin and insulin functions, resulting in the promotion of obesity. Sleep loss also elicits neuroinflammation, though little research has focused on determining whether this occurs in brain regions that regulate glucose metabolism. To examine the role of neuroinflammation in insufficient sleep- and overnutrition-induced deficits in glucose regulation, adult C57BL/6J mice were subjected to one of four treatments: undisturbed sleep and standard chow (control); undisturbed sleep and high fat diet (HFD); 18 hours/day sleep fragmentation (SF) and standard chow; 18 h/d sleep fragmentation and high fat diet. After nine days of manipulation, a glucose tolerance test was conducted, and then brain tissues were obtained and processed for immunohistochemical analyses of microglial and astroglial activation in the hypothalamus and brainstem (n=6/group). Sleep fragmentation or high fat diet reduced glucose tolerance compared to the control, with the greatest deficits observed in mice subjected to both sleep fragmentation and high fat diet. Analyses of numbers of activated microglia and astrocytes will determine whether patterns of glial activation across treatments reflect changes in glucose tolerance. These findings will be important in determining the contributions of neuroinflammation to glucose tolerance within the context of chronic insufficient sleep and poor diet.

O6-methylguanine-DNA methyltransferase (MGMT) is the sole DNA repair activity that removes cytotoxic methyl group adducts from the O6 position of guanine. My overall hypothesis is that MGMT in human glioblastoma (GBM), a malignant brain tumor, limits tumor cell killing by chemotherapeutic methylating agents such as temozolomide. To address this hypothesis I will compare MGMT activity in GBMs with a mutated copy of the IDH1 (isocitrate dehydrogenase) gene with that in wild type tumors. IDH1 mutant GBMs differ from wild type in being much more responsive to methylating agent therapy and having longer survival. Mutant IDH1 converts isocitrate to 2-dihydroglutarate that suppresses the expression of epigenetically regulated genes. As MGMT expression is controlled epigetically by a mechanism sensitive to 2-dihydroglutarate, I hypothesize that the better clinical response of IDH1 mutant GBMs is associated with lower MGMT activity than wild type tumors. To address my secondary hypothesis, I will assay MGMT activity in homogenates of human GBM tissue with known mutation status and a complete history of the course of alkylating agent treatment. Tissue and clinical outcome data will be provided by the Neurosurgery Tumor Bank. When necessary I will determine mutation status of archived GBM tissue by an established PCR assay. Finding significantly lower MGMT activity associated with longer survival in mutant tumors will support my hypotheses. I will also examine the association of MGMT activity with survival following alkylator therapy for wild type and mutant separately to provide additional evidence of the role of MGMT as a mechanism of treatment resistance. An inverse association between tumor MGMT activity and treatment response will suggest (1) that MGMT can be used as a prognostic marker for treatment outcome and (2) that inhibition of MGMT as part of alkylator therapy may improve clinical outcome for GBM patients regardless of tumor IDH1 mutation status.
 

Most of the tissues in our body contain adult stem cells that in theory could combat many aging phenotypes observed in human. However, these stem cells do not regenerate the tissue during aging. To understand, and in the future govern adult stem cell regenerative capacity we need to study the regulators of human adult stem cells. DPSCs are adult mesenchymal stem cells with powerful regenerative capacity that can easily be isolated from waste product, extracted wisdom teeth. Due to low cost and easy accessibility, dental pulp is a more feasible source for mesenchymal stem cells than bone marrow. DPSCs are thought to have the ability to differentiate into multiple cell lineages, and thus hold great promises in tissue regeneration. However, little is known about the molecular markers, cellular regulators, and differentiation capacity of these cells. The goal of my research project is to generate a large diversity set of DPSC to carry on a careful analysis of true common markers and differentiation capacity of these cells. At the moment I have optimized the procedures and isolated DPSCs from over 50 patients in partnership with UW dental clinic. We have now identified the molecular markers of DPSCs, and evaluated their differentiability to better understand the key criteria in the self-renewal and differentiation capacity of these cells. Western blot and flow cytometry analysis shows that most of our DPSCs have a robust expression of CD29 and CD146, both molecular markers of mesenchymal stem cells, but displaying an unexpected diversity on the levels of these markers among individuals. In differentiation tests, we also observe diversity in cartilage and adipocyte differentiation efficiency. With this unprecedented diversity set of easily accessible adult stem cells we will now identify the key components and metabolites that enhance the stemness and regenerative capacity of these adult stem cells.

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

Universal health care has been a topic of controversy, largely due to the question of resource management. As part of an Exploration Seminar through the University of Washington, a team of students conducted interviews with health centers in Chile to investigate universal health care. Each team member was responsible for posing questions relevant to their area of study and reporting on the research after the program ended. The goal of the program was to assess health system disparities in Chile.This presentation focuses on the role of regional epidemiology, which studies the population in a local area and tailors the universal health care plan to that region. Open ended interviews with community health workers were conducted at twenty-two medical sites throughout Santiago and Southern Chile. The sites ranged from the private sector headquarters to rural outposts in the Southern Archipelago of Chiloé. Translations between English and Spanish were performed when necessary. The study found that Chile has an overburdened public health care system, particularly in specialty areas. Eighty percent of treatments are not as effective as standards per guidelines. Regional epidemiology decreases waste and increases productivity, especially in the areas of primary care and prevention. The strengthened primary care system reduces the burden of care on the tertiary health system. Regional epidemiology is currently expanding to cities outside of Castro and Chiloé Island. Regional epidemiology is an effective tool for improving the Chilean universal health care system. More research should be done to predict whether regional epidemiology will be effective in private health care and other universal health care systems.

Helicobacter pylori (H. pylori) are gastric bacteria known to cause chronic gastritis, peptic ulcers, and can eventually lead to gastric adenocarcinoma in the long term. Prevalence of H. pylori among Caucasian, Hispanic and African-American populations has been previously surveyed in past studies. However, prevalence and distribution of H. pylori among Asian communities has yet to be exhaustively profiled. While H. pylori exhibits worldwide circulation, studies have shown that infection is more prevalent in developing third world countries. The Vietnam Health Clinic (VHC) is a mobile clinic that provides free healthcare to underserved populations in Vietnam every year. The data collected from the roughly 3000 patients served from VHC trips between 2012 and 2014 presents an invaluable opportunity to learn more about H. pylori prevalence and distribution within the native Vietnamese community. By delving into the patient’s background, medical history and previous treatment history of H. pylori, we give the dataset more meaningful resolution that can facilitate novel prevention and treatment processes in future VHC trips. Data and statistical analysis with Tableau and Excel is currently in progress and the consequent results will prospectively be available in May for the Symposium presentation. As this is a retrospective and exploratory study, a causation link cannot be drawn. However, by providing insight into a patient’s demographic and connection with H. pylori, our research can help to provide a foundation for causally directed studies in the future.

For the past twenty years, scientists have attempted to learn more about the effects of pollution on coral species. However, identifying coral species in today's scientific community is more of an art than a science. For the last five years, researchers have been attempting to address this problem by comparing the skeletal structures of different coral species. Coral calcification is distinctive because it involves the formation of rapid aragonite super saturation. Aragonite is a very unusual form of calcium carbonate. It is much less thermodynamically stable than the primary form of calcium carbonate--calcite. There are two lines of thought as to why aragonite is preferentially precipitated in coral skeletons. One, the proteins secreted by the polyps within the coral create microenvironments that promote aragonite precipitation. Two, the magnesium from the surrounding ocean water interacts with the proteins in such a way that aragonite becomes the more energetically favorable precipitate. Our research examined the proteins and water involved in these calcification sites and compared them among species of coral collected from the Mo’orea Islands using Thermo Gravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC), Energy Dispersive X-Ray Spectroscopy (EDS) and Fourier Transform Infrared Spectroscopy (FT-IR). In short, we wanted to compare the magnesium, water content, and protein content/functional groups between species in the hope of finding species or site-specific trends. Our results confirmed the correlation between magnesium and protein, and discovered that the way in which corals create their exoskeleton is highly evolutionarily conserved.

Cancers are fueled by mutations which impart new capabilities to incipient cancer cells. Spontaneous mutations are, however, more likely to be deleterious than beneficial based on random chance alone. Therefore, inducing more mutations in an heavily mutated population could lead to an “error catastrophe” once the mutation level rises past a certain threshold. Accumulating too many of these debilitating mutations consequently leads to a reduction in the overall population fitness. Our hypothesis is that mutagenic versions of nucleoside analogs (modified versions of the building blocks of DNA) can induce more mutations to reach this ‘error’ threshold and beyond, and that this can selectively kill cancer cells. To evaluate the effectiveness of this strategy, termed therapeutic mutagenesis, we conducted cell culture experiments on various human colon cancer cell lines: HCT116 (mismatch repair deficient colorectal cancer) cells, SWS620 (non-MMR-deficient colorectal cancer), and a control normal human fibroblast line. We screened a library of candidate nucleoside analogues (e.g. O4-Ethyl-Thymidine) for mutagenicity and viability at physiologically relevant concentrations. I then quantified the short-term viability of the treated cell lines with a clonogenic survival assay. I used the highly accurate Duplex genetic sequencing methodology to monitor the changes in a cell line’s overall mutation load after treatment. After validating the concept of therapeutic mutagenesis in cancer, our next steps will be to determine whether possible animal and clinical trials would be possible in the future. Our approach may open up new therapeutic possibilities for patients in the most advanced stages of cancer, especially in the context of complementing increasingly common immunotherapies.

Intestine is responsible for the biotransformation of many orally-exposed chemicals. The nuclear receptor constitutive androstane receptor (CAR/Nr1i3) can be activated by various drugs and environmental chemicals, and in turn it is known to up-regulate the expression of many drug-processing genes (DPGs) in liver to facilitate xenobiotic biotransformation. However, relatively less is known regarding the effect of CAR on the basal and inducible expression of DPGs in various sections of intestine. To fill this critical knowledge gap, sixty-day-old wild-type and CAR-null mice were administered the CAR-ligand TCPOBOP (3mg/kg, i.p.) or vehicle once daily for 4-days. CAR-null mice had altered basal expression of many DPGs in a section-specific manner in intestine. Consistent with the liver data (Aleksunes and Klaassen, 2012), TCPOBOP up-regulated many DPGs in specific sections of small intestine in a CAR-dependent manner. In contrast, the mRNAs of certain DPGs were previously known to be up-regulated by TCPOBOP in liver but were not altered in intestine. Interestingly, many known CAR-target genes were highest expressed in colon where CAR is minimally expressed, suggesting that additional regulators are involved in regulating their expression. In conclusion, the present study has shown that CAR is critical in both maintaining the basal expression as well as the pharmacological regulation of certain DPGs in a section-specific manner of the intestine, and the CAR activation in liver and intestine produces overlapping but not identical results. Therefore, Identification of the xenobiotic responses to CAR activation in intestine is critical for further understanding the adverse drug-drug interactions for many orally-exposed chemicals.

Meier-Gorlin Syndrome (MGS) is a rare form of primordial dwarfism characterized by a short stature, small external ears, and absent kneecaps. Recently, mutations in the pre-replication complex (pre-RC) were linked to MGS. The pre-RC is composed of multiple proteins that must be present in order to initiate DNA replication. Although a genetic link has been made for MGS, it is unclear how these mutations cause the phenotypes observed in humans. To better understand the mutations’ phenotypes on a cellular and molecular level, our lab is utilizing the budding yeast, Saccharomyces cerevisiae. The MGS mutation in the Origin Recognition Complex (ORC) subunit six (ORC6) of the pre-RC occurs in a highly conserved region of the protein where there is a single amino acid substitution (Tyrosine to a Serine) in the C-terminal helix. Since the mutation is conserved from human to yeast, it is predicted that the function of the mutations is also conserved. I introduced the equivalent mutations into budding yeast (orc6^MGS), to investigate the consequences of harboring the MGS mutation on a molecular and cellular level and to elucidate complex genetic interactions these proteins may be involved in. The residues in ORC that recognize origins are unknown. Investigation into this mutation has the potential to illuminate the mechanism behind ORC in eukaryotic cells. The assays performed in this project have exciting implications for the model by which replication initiates and the consequences of a single mutated protein. This genetic approach will hopefully provide insight to how the conserved orc6^MGS mutation is functioning, which has the potential to be translated to the human condition.

Long intergenic non-coding RNAs (lincRNAs) have been discovered to be widespread in mammalian genomes, with thousands existing in humans alone. lincRNAs exceed 200 nucleotides in length and are capped and polyadenylated. They do not overlap with protein-coding genes, and generally exhibit poor sequence conservation across divergent species. The lincRNA cyrano, discovered in zebrafish, possesses a very highly conserved 300 nucletotide region in at least 80 species. Cyrano has also been demonstrated to be essential in embryonic development in zebrafish. Given the sequence conservation of cyrano, we hypothesized that the secondary structure of the 300 nucleotide region was conserved across divergent species. Using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) we determined the RNA structure of this 300 nucleotide conserved region in vitro in mouse, human, and zebrafish. Four structural motifs were found to be common in all of the species. To validate the integrity of these secondary structural motifs, we aim to probe the structure of the 300 nucleotide conserved region as it exists in the cell by performing SHAPE in vivo. Currently, our findings represent some of the first steps towards understanding the roles of lincRNAs and non-coding RNAs in general. The miniscule amount of information we collectively know about this vast new enigma of the genome emphasizes the need for biochemists to tackle this issue and improve the methods at our disposal. 

Xenobiotic biotransformation is a vital process in the body that detoxifies various drugs, dietary factors, and environmental toxicants. This process is mediated by various drug-metabolizing enzymes in Phase-I (oxidation, reduction, and hydrolysis) and Phase-II, as well as transporters. The hepatic nuclear factor 4alpha (HNF4α) is a master regulator that transcriptionally up-regulates a wide spectrum of drug-metabolizing enzymes and transporters in liver. HNF4α also plays an important role in liver development and nutrient homeostasis. Previously, it has been shown that HNF4α can trans-activate the constitutive androstane receptor (CAR), which is a major ligand-activated xenobiotic-sensing nuclear receptor. However, very little is known about the effect of CAR activation by xenobiotics on the regulation of HNF4α in liver. In the present study, male mice at either 5-day-old neonatal age or 60-day-old adult age in wild-type, CAR-null, or humanized CAR-transgenic (hCAR-TG) genotypes were treated with vehicle (corn oil, which served as the control), or a species-appropriate CAR ligand (TCPOBOP for mouse CAR and CITCO for hCAR), once daily for 4 days. A novel truncated coding exon 9 was observed for the HNF4α transcript variant in mouse liver (RNA-Seq). Isoform-specific RT-PCR and gel electrophoresis confirmed the presence of the novel HNF4α isoform. During development, both the known and the novel isoform had increased expression with age. The mouse CAR ligand, TCPOBOP, down-regulated HNF4α in a mouse CAR-dependent manner only at mice at age 60, whereas hCAR did not appear to regulate the expression of HNF4α at any ages. In conclusion, the present study has identified a novel HNF4α transcript variant in the mouse liver, and discovered that both the known and novel isoforms were regulated by CAR in a species and age-specific manner.

This project will inform a systems biology platform for identifying adverse responses to environmental compounds on testicular development in mice, a commonly used model system in the field of Toxicology. In order to validate the organotypic culture system, we have investigated the adverse effects of the known testicular toxicant, cadmium. To initiate the culture model, we isolate and plate testicular cells from immature mice on postnatal day (PND) 9 in order to capture a developmental window of susceptibility. Based on a literature search derived developmental timeline, we chose to treat the culture on days in vitro (DIV) 2, 5 and 15 with cadmium and quantify effects 24 hours later. We hypothesized that dosing with cadmium during these time points would correspond to impacts on steroid regulation, proliferation, and spermatogenesis processes. In order to evaluate a dose response relationship in our testicular culture, we treated the testis cells with 2.5, 5.0 and 10 uM concentrations of cadmium. To quantify the effects of cadmium in our cell culture model we evaluated; total protein, testosterone production, cell type specific proteins with western blots, LDH cytotoxicity, 3 color assay for live and dead cells and immunofluorescence to capture morphology for DIV 3,7 and 16, 24 hours post treatment for all three doses. Our initial studies have observed that cadmium treatment for all three doses impacted testosterone production, germ cell morphology and proliferation on DIV 7 and 16.  

Food security is the measure of an individual’s ability to access and utilize sufficient food to ensure a healthy and productive lifestyle. Previous research has highlighted this issue in various populations in the Seattle area. However, the effectiveness and coverage of the available nutritional programming has yet to be determined. The goals of this project were to 1) better understand which areas of Seattle are at the greatest risk of hunger and food insecurity as measured by income, and 2) to measure which areas are most underserved by nutrition programming. A visual representation of Seattle was created to identify specific census tracts with a population that is at-risk of food insecurity by incorporating economic, social, and demographic data from the 2013 American Community Survey [ACS] and the 2010 Decennial Census. From this map highlighting at-risk populations and data regarding numerous social programs run by nonprofits, governmental agencies, and businesses, a geographical analysis was performed to determine the coverage and the ability of these programs to fulfill the studied population’s nutritional needs. The results of this study will enable nutritional programs and future researchers to better serve at-risk populations by focusing their efforts on underserved areas and allow more efficient allocation of resources to ensure food equity in Seattle.

Acute myeloid leukemia (AML) is characterized by uncontrolled proliferation of abnormal immature bone marrow progenitor cells (“blasts”). AML blasts reside in the bone marrow microenvironment via adhesion molecules such as CXCR-4 and VLA-4, which confer protection from chemotherapy in AML. L-selectin is a surface receptor utilized by leukocytes (white blood cells) for rolling on the endothelial lining of blood vessels and adhesion to sites of inflammation. CXCR-4 is a chemokine receptor for stromal derived factor (SDF-1, CXCL12) produced by bone marrow stromal cells. VLA4 is an α4β1 integrin that mediates adhesion to alternatively spliced fibronectin and cellular vascular cell adhesion molecule 1 (VCAM1). Previous studies in our laboratory and others suggested that expression of CXCR-4 and VLA-4 is associated with prognosis in AML, and L-selectin expression increases with older age in AML patients. The hypothesis of my project is that L-selectin expression level is associated with chemotherapy resistance in AML. We performed experiments on blasts with high vs. low L-selectin surface expression as measured by flow cytometry assessment of mean fluorescence intensity (MFI). We allowed the AML blasts to be attached to plates coated with PSGL-1, an L-selectin ligand, or bovine serum albumin (BSA), a control protein. Then blasts were treated with cytarabine (Ara-C), one of the most active chemotherapy drugs in AML, for 72 hours. Data were collected by manual counting of viable cells, Cell-Titer Glo luminescent viability assay, and flow cytometry for Annexin V, as a measure of apoptosis. Our data showed that PSGL-1 binding protected AML blasts from chemotherapy in 4/15 (27%) of the samples, which had higher L-selectin (p = 0.05), lower VLA-4 expression (p = 0.10) and higher CXCR-4 (p=0.11) surface expression. Therefore, these results suggest that differences in expression and function of L-selectin, CXCR-4 and VLA-4 are associated with chemotherapy sensitivity in AML blasts.

Hydrogen sulfide (H₂S) is an important signaling molecule in humans, however the mechanisms behind H₂S signaling and its toxicity, which is thought to be similar to hydrogen cyanide (HCN), are poorly understood. Caenorhabditis elegans (C. elegans) is a convenient model to study exposure to H₂S and HCN as the proteins that mediate the organismal responses are highly conserved. Our previous work has shown that low levels of H₂S can increase thermotolerance and lifespans in C. elegans, but at high levels H₂S has toxic effects. The disparate effects of signaling and toxicity of H₂S on C. elegans led us to ask how the response to HCN compares to that of H₂S. To investigate this, we exposed different C. elegans strains to gaseous H₂S and HCN in sealed chambers and scored the survival rates between the two gases. We see that while both H₂S and HCN are toxic to C. elegans, HCN leads to death at lower concentrations, suggesting different modes of toxicity. We confirmed that HIF-1, a key transcription factor in the response to both H₂S and HCN, is necessary for survival in both H₂S and HCN. To determine if there are mechanistic similarities between the responses to these gases we exposed mutants that suppress hif-1 lethality in H₂S to both H₂S and HCN, and found that mutants that increased survival in H₂S did not necessarily increase resistance to HCN. This allows us to identify proteins that play a role in the response to H₂S. Understanding the proteins which differentially mediate toxicity in H₂S versus HCN will help us better understand the signaling roles of H₂S.

Sec1/Munc18 related proteins (SM) are essential cofactors of SNARE–mediated membrane fusion. They function in conjunction with SNARE proteins, a family of zipper-like proteins that drive the two opposing membranes toward fusion, and other SNARE cofactors. Membrane fusion is one of the most fundamental cellular processes. However, universal mechanisms of SM function and regulation have been elusive to membrane biologists for a long time. Sly1 is an SM protein that functions at the Golgi. Here, we present genetic and biochemical results, which show how a SNARE–mediated tethering mechanism of Sly1 is regulated, and which suggest that key aspects of SM function are evolutionarily conserved across the SM family. Unlike other SMs, Sly1p contains a loop that covers a portion of domain 3a on the protein. Previous studies show that mutations at the tip of the loop can suppress requirements for the Rab GTPase Ypt1 and tethering protein Uso1. It was hypothesized that the loop serves an auto–inhibitory function. Moreover, the recent crystal structure of another SM, Vps33, shows that domain 3a serves as a binding site for the cognate R–SNARE. We performed a screen for Sly1 mutants that suppress the loss of the Golgi tether, Uso1. The new mutants, as well as limited proteolysis experiments, suggest that the entire loop swings away to reveal a SNARE binding site on domain 3a, thereby activating the tethering and SNARE assembly functions of Sly1. These experiments reveal new features of Sly1 regulation, and in addition to the results from Vps33, suggest a universal mechanism of SM–SNARE interaction during vesicle tethering.

Systemic immune activation (IA) is a proven key factor in human immunodeficiency virus type 1 (HIV) infection pathogenesis and progression to acquired immunodeficiency syndrome. The reproductive hormone progesterone plays a role in regulating the immune system, and most cells contain progesterone receptors. Previous studies have linked high-progestogen states in women, such as pregnancy and progestin-based contraception use, to increased infectivity in HIV-seropositive women and increased HIV acquisition in seronegative women. Progestin-based contraceptives such as depot-medroxyprogesterone acetate (DMPA) can create high plasma levels of progesterone analogs. Therefore, studying DMPA use by HIV-seropositive and seronegative women may clarify DMPA’s role in HIV-related IA and genital shedding, and immune changes related to HIV acquisition. We hypothesize that DMPA increases systemic IA. Increased IA could be clinically significant by contributing to increased HIV acquisition in high-risk HIV-seronegative women, and by increasing genital shedding and infectivity among HIV-seropositive women. To answer this question, peripheral blood mononuclear cells (PBMC) were collected at three postpartum time points over six months from 120 Kenyan women with both HIV-seropositive and seronegative status, including a sub-population in each category that elected to receive DMPA for contraception. Two immunophenotyping panels were developed to measure T-cell IA and cellular subsets of interest using flow cytometry (CD38, HLA-DR, Ki-67, Bcl-2 for T-cell activation; FOXP3, CD25, CD127 for regulatory T-cells; CCR7, CD45RA, CD27, CD28, CD57 for memory subsets; CCR5 for the HIV co-receptor). With preliminary analysis completed for most samples, specific antibody populations have been gated with FlowJo software. After completion, we will compare different population percentages in the four arms of the study, observing how these change with DMPA administration. These calculations will help elucidate expected differences in inflammatory and regulatory changes in both HIV-seropositive and seronegative women with respect to the relationships between DMPA, immune activation, and HIV susceptibility and infectivity.

In order to successfully invade and survive within a host cell, pathogens such as the bacteria Shigella flexneri have to suppress the host cell’s immune response. To do this, Shigella flexneri inject bacterial effector proteins into the host cell that hamper the host cell’s ability to detect and respond to invasion. One such protein is IpaH4.5, a bacterial E3 ubiquitin ligase, which hijacks the host cell’s ubiquitylation machinery to transfer ubiquitin, a small signaling protein, to a substrate. Ubiquitin signaling impacts all aspects of eukaryotic cell biology. Often modification of a protein by ubiquitin serves to mark protein for destruction. IpaH4.5 targets proteins involved in the host cell’s immune response, dampening the host cell’s ability to respond to invasion by Shigella flexneri. I am investigating IpaH4.5 and its interactions with both host substrates and the host ubiquitin machinery. After sub cloning out the various domains of TbK1, a known substrate of IpaH4.5, I will perform ubiquitilation assays in order to determine which domain of TBK1 is polyubiquitilated, indicating that IpaH4.5 interacts with this domain. Then, this domain can be used for structural and mechanical analysis of IpaH4.5, such as determining how mutations in IpaH4.5 affect ubiquitilation through biochemical assays. Understanding how IpaH4.5 binds to and interacts with its substrates will increase our understanding of how Shigella flexneri dampens host immune response as well as help us better understand the mechanisms of bacterial E3 ubiquitin ligases. Since IpaH4.5 functions differently than eukaryotic E3 ubiquitin ligases, increasing our understanding of its mechanisms could allow us to use it as a target for future drug development. Finding new targets for drug development is becoming increasingly important with the spread of antibiotic resistant bacteria.

Disorders and diseases of chronic pain have placed a tremendous burden on modern society, imposing myriad consequences on many individuals and populations. Despite extensive investment to date, therapies to treat chronic and acute pain remain limited; the most-widely used, researched, and prescribed drugs often act generally upon the nervous system. This characteristic of most existing therapies carries with it many unintended consequences and unwanted effects for pain patients. The limitations of these therapies are often traceable to our limited understanding of the neuronal circuitry responsible for the sensation and transduction of noxious or other pain-inducing stimuli. We utilize zebrafish in our forward genetic screen to identify mutations that disrupt the ability to perceive pain. Behavioral tracking of larval zebrafish, specifically the progeny of mutated genetic lines, allows us to screen for a large number of potential phenotypic mutations. By identifying mutant lineages in this screen, we hope to further elucidate and characterize the neuron cell populations responsible for nociception. Our genetic screen has identified several lineage of fish that lacked the behavioral “escape” response typical of embryonic zebrafish when immersed in a noxious chemical environment, in this case, 10µM allyl isothiocyanate (AITC), the pungent compound in mustard oil. Subsequent complementation to known mutants suggested that one of these families carried a mutation in a previously identified nociceptive ion channel, TRPA1, which is specifically expressed in somatosensory neurons, and has been shown to be required for the pain response induced by noxious chemicals, including AITC. We have crossed mutants into well-characterized genetic backgrounds and used RNA-seq based mapping to narrow these lesions to a region of Linkage Group 9. Following fine-mapping, we will subsequently study the role these gene(s) play in the sensation of pain. Our findings suggest that our screening strategy is likely to be successful and is feasible for identifying genes involved in nociception.

Chronic epilepsy is attributed to dysfunctional ion channels, including hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which are regulated by a signal cascade mediated by p38 mitogen activated protein kinase (p38 MAPK). Previous researchers in the Poolos Lab manipulated this cascade and discovered an increase in activation of c-Jun N-terminal kinase (JNK), seen by increased phosphorylated JNK in animals experiencing seizures. This is a novel connection and my project explores the timeline of JNK activity during the development of epilepsy. I hypothesize that JNK activity precedes epilepsy onset and may be causal of chronic epilepsy. To study epilepsy, we administer research-bred rats a pilocarpine injection that induces status epilepticus (SE), a state of continuous seizures. After one hour, the rat receives a phenobarbital injection to halt SE. Seizures begin around one week post-SE and achieve steady-state frequency around six weeks. I analyze brain tissue taken after one hour, one day, and one week post-SE using Western blotting to observe JNK differences from pilocarpine-treated rats to control rats. There are three isoforms of JNK; JNK 1 and 3 run on a 46 kDa band and JNK 2 runs on a 54 kDa band. JNK 3 is thought to have most significance in modulating brain neuronal activity. I have discovered a significant increase of the 54 kDa band at one hour post-SE (130 ± 10%, p<0.05, n=8) and one day post-SE (129 ± 11%, p<0.05, n=11); however, the 46 kDa band was insignificantly changed at both one hour post-SE (104 ± 7%, p>0.5, n=9) and one day post-SE (120 ± 12%, p=0.11, n=11). Gathering data at one week post-SE will show us if JNK activation is potentially causative of epilepsy. By understanding JNK activation, we can explore ways to prevent epilepsy development or lessen its severity after brain injury, through antiepileptic drugs and other therapeutic options.

Newborns and children are more vulnerable to xenobiotics (foreign chemical substances) partly because little is known regarding the developmental regulation of drug-processing genes (DPGs) by drugs and environmental chemicals. The xenobiotic-sensing nuclear receptors, namely the pregnane X receptor (PXR) and constitutive androstane receptor (CAR), are well-known to up-regulate many DPGs in liver. However, relatively less is known regarding the effect of developmental exposure to PXR- or CAR-activators on the expression of DPGs in intestine, where the biotransformation of orally-exposed xenobiotics takes place. Therefore, the goal of this study was to determine both the acute and the potential long-term effects of neonatal exposure to PXR- and CAR-activators on the regulation of DPGs in neonatal and adult intestine. Wild-type mice were pharmacologically administered the PXR-ligand PCN (the activator of PXR in mice), the CAR-ligand TCPOBOP (the activator of CAR in mice), or vehicle, at 3-day-old neonatal age. Intestines were collected 24h post-dose (whole intestine) or at 60-day-old adult age (duodenum, jejunum, ileum, and colon). Neonatal exposure to TCPOBOP and PCN up-regulated many DPGs 24h postdose. Interestingly, at 60-days adult age, neonatal exposure to TCPOBOP persistently up-regulated many DPGs especially in small intestine, but down-regulated particularly in colon (large intestine). Neonatal exposure to PCN persistently up-regulated 6 DPGs but down-regulated 11 DPGs in specific sections of intestine. In conclusion, this study has demonstrated that neonatal exposure to xenobiotics that activate PXR and CAR not only produce acute induction effect on the DPG expression in newborns, but also lead to persistent alterations on the expression of certain DPGs in adult intestine, and this may potentially alter the pharmacokinetics of orally-exposed chemicals in adults.

Stille coupling, Suzuki coupling, and Kumada coupling are some of the most commonly used reactions in the synthesis of polymers for organic solar cells and organic electrical devices. They are highly effective in creating new carbon-carbon (C-C) bonds. However, when synthesizing a conjugated polymer for organic solar cell applications through these conventional reactions, the process can be challenging and lead to many synthetic steps. In addition, some of the materials used are expensive and extremely toxic to both human health and the environment. In this project, I work individually to synthesize a new benzobisoxazole-based compound which can potentially serves as the monomer for conjugated polymer of organic polymer. The goal of the project is to create a compound in which the inert carbon-hydrogen (C-H) bonds are strongly weakened and easily broken. Potentially, organic solar cells production via this new compound can be more efficient, and as a result, reduces the cost and makes it more environmentally friendly. Recently, the lab’s collaborators at Emory University synthesized a benzobisthiazole-based monomer with two solubilizing alkyl thiophene groups. However, the monomer has limited solubility in organic solvents as well as C-H activation ability. To improve the monomer, I attached a further 2-ethyl hexyl chain to the 2-position on each thiophene solubilizing group and also replaced thiazole with the more electron deficient oxazole unit. Right now, I have successfully synthesized the desired compound and been in the process of activating it using a Gold-based catalyst. If C-H activation is possible for this compound, oligomers of at least three monomers are expected to form.

Chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is the gold standard for assaying the binding sites of chromatin-binding proteins and their modifications, such as histone tail modifications. One major limititation for this method is that it requires large amounts of starting material, therefore limiting applications in those cases where only a limited number of cells can be recovered, e.g. rare cell populations obtained directly from patients. In the last few years, great efforts have been made to develop new ChIP-seq protocols to overcome this limitation. In my lab, they have developed a method called carrier ChIP-seq (cChIP-seq). The method is based on the use of a DNA-free carrier to enhance recovery of DNA after IP. Using cChIP-seq they have been able to perform ChIP-seq on 10,000 cells for several histone tail modifications. I tested a series of modifications on the cChIP-seq protocol. The goal of my project was to develop a protocol that can be performed in one day. Currently the cChIP-seq protocol takes 3 days to be complete. This is mainly due to two overnight incubations. The first one is the immunoprecipitation, so I performed multiple trials to identity the minimum time sufficient for immunoprecitating the chromatin. The second overnight incubation is to reverse crosslink immuneprecipitated chromatin. I tested a version of the cChIP-seq protocol for native chromatin, meaning the DNA is not crosslinked. By optimizing and combining these two modifications I was able to shorten the cChIP-seq to a single day. Through my project I contributed to a larger project with the goal of scaling down the number of cells used for ChIP-seq.

Due to the complex nature of the steroidogenic pathway, which involves enzymes with multiple substrates as well as multiple enzymes acting upon the same substrates, rigorous methods for molecularly dissecting this pathway are required for understanding it’s role in AR signaling and prostate cancer. Over the course of current androgen deprivation treatments, many patients develop castration resistant prostate cancer (CRPC). When the disease reaches this stage it has been known that cancer utilizes multiple pathways, known as the backdoor or bypass pathways, in order to continue synthesizing the androgens which ultimately drive cancer progression. To learn more about these alternate pathways, isoform-specific CRISPR/Cas9-mediated knockouts of these highly similar genes can be used to determine the specific contributions of each of these factors to de novo androgen synthesis and androgen scavenging in prostate cancer tumors. These CRISPR constructs will be used to generate gene specific knockout prostate cancer cell lines. Validation of each line will be done using a T7 endonuclease assay followed by western blot to confirm knockout of the protein. Further analyses using these specific knockout cell lines will provide insight on which genes are involved in the new pathway when the classical pathway is shunted.

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

 Diabetes affects about 10% of the US population and is the 7th leading cause of death. Diabetes results from either a lack of insulin production (Type 1) or resistance to it (Type 2). The active form of insulin is a 51 amino acid monomer which regulates homeostasis of blood-glucose levels. It is crucial for insulin to be structurally sound and not degraded, especially for people using insulin pumps. However, insulin has a shelf life of about 28 days unopened and only 14 days after opening the container. Efforts to improve the stability of insulin may facilitate the development of implantable pump technologies for insulin delivery and allow for longer storage of insulin. Based on a review of the literature, I hypothesize that solvation in an Ionic Liquid solution will decrease the degradation and increase the shelf-life of insulin. To better understand the stability of the insulin protein, I compared its activity at an air-water interface to an air-ionic liquid interface. The approach consists of modeling the structure of insulin using the simulation software GROMACS and PLUMED. 10 nanosecond trials in different air-liquid interfaces were then analyzed to optimize the best insulin backbone stability. The goal is to extrapolate an ammonium- or imidazolium-based ionic liquid solution that will decrease the aggregation of the insulin monomers. Compared to the bulk insulin form, we found that the ionic liquid-protein solution demonstrates a lower radius of gyration and a root-mean-square deviation value that levels out towards 1 Angstrom representing a stable protein. These results are also supported by a decreased hydrodynamic radius and an increased free energy of unfolding the insulin protein. These findings suggest that insulin in an ionic liquid solution will last longer in vitro than in a water-based solution.

Our project is to characterize the Caenorhabditis elegans (C. elegans) ortholog of PICK1 and we will henceforth refer to this C. elegans gene as pick-1. In other systems, PICK1 is involved with dense-core vesicle (DCV) biology and has been shown to affect the trafficking of AMPA glutamate receptors to the cell membrane, which are believed to be trafficked in a non-DCV process. DCVs are a specialized organelle in neuron and neuroendocrine cells that are loaded with, and release, several important neuromodulators. These DCV-release neuromodulators regulate a broad spectrum of biological processes such as blood glucose homeostasis, the plasticity of the nervous system, and even appetite. AMPA trafficking is crucial to signaling in the brain. Glutamate is the major transmitter of excitatory signals in the brain. Our project aims to figure out if, and how, pick-1 is affecting these processes in C. elegans. To investigate pick-1's involvement in DCV biology we tested its interaction with RIC-19 (ICA69 in mammalian systems), a protein that is known to interact with PICK1 systems. To test pick-1's interaction with AMPA receptors, we tested how PICK1 affects the localization of GLR-1 (an AMPA receptor). If PICK1 does not affect the localization of GLR-1, then we would expect to see GLR-1 not properly trafficked to the synapse. If PICK1 is involved in DCV biology, we would expect a deletion of pick-1 would create lower levels of RIC-19 in the worms. To this point, our raw data has suggested that PICK1 does not play a role in DCV biology or AMPA receptor trafficking in C. elegans.

Gastrulation is a stage in embryogenesis in which most progenitor cells commit to a muscle or neural fate. However, a population of bipotential progentitor cels does not differentiate during gastrulation and allows the embryos to dynamically allocate neural and muscle tissue in the correct proportion as the body axis is formed. The regulation of this process is crucial for proper neuromuscular development. The maintenance of these bipotential progenitor cells requires the repression of tbx16, a transcription factor that progenitors express when they commit to a muscle fate. Here, we examine two genes of interest termed eve1 and vent, and investigate their roles in the regulation of tbx16. While previous studies relied on Morpholino anti-sense oloigonucleotides (MOs), the credibility of MOs has recently become seriously questioned because they can cause off-target effects, generating misleading effects that don't reflect genetic mutants. Revolutionary new gene-editing techonology known as CRISPR, is hailed as the new standard for gene analysis because it allows the production of true genetic mutants in specific genes. CRISPR accurately and efficiently creates small deletions in target genes, which can result in complete loss of function. Using the CRISPR method, we will generate F1 lines of zebrafish with eve1 and vent mutations. Through high-resolution melt anaylsis (HRM) and DNA sequencing, we have thus far identified two eve1 F0 founder fish. We will then test how the absence of eve1 and vent affects the expression of tbx16 during zebrafish embryogenesis using in-situ hybridizations. By analyzing how embryogenesis in normal zebrafish embryos differs from that of eve1 or vent mutant embryos, we can identify the role that these genes play during development. Ultimately, our goal is to gain a better understanding of how serious birth defects and genetic disorders arise from complications in development. 

Current chemotherapies exploit the larger pore size of tumor vasculature to reach cancer cells. However, this rudimentary form of targeting offers low selectivity and carries the risk of severe systemic toxicity. In addition, chemotherapy distribution to tumors is limited by the large distance between blood vessels in tumors, increased interstitial fluid pressure, and physiological resistance by the extracellular matrix (ECM). With the goal of addressing these limitations, researchers have developed drug-bearing polymers of various sizes and architectures that selectively target tumor cells. The Pun lab previously produced a novel drug delivery polymer, the sunflower polymer, that can be utilized for anticancer drug-delivery. The sunflower polymer is a macrocyclic brush polymer that can be functionalized with a folic acid targeting ligand and an anticancer drug, doxorubicin, that is released in the acidic tumor microenvironment. In order to maximize the potential of anti-cancer drug-delivery polymers such as this, the impact of polymer size and architecture on tumor penetration must be better understood. This is because the efficacy of any anticancer drug, regardless of its mechanism of action, is limited by its ability to navigate the tumor microenvironment and reach all viable cells in a tumor. This project seeks to discover how polymer size and architecture impacts tumor penetration by using a 3D in vitro perfusion chip model of the tumor microenvironment. The model consists of microfluidic channels seeded with KB carcinoma cells and ECM, and has been optimized using model polymers of polyethylene glycol (PEG) and dextran. Tumor penetration is studied by perfusing fluorescently labelled polymers of various morphologies through the seeded microfluidic channels. The information obtained from this project could potentially be used to optimize the size and architecture of drug-delivery polymers for tumor penetration. In turn, these optimizations could present a significant contribution to the development of anticancer drug-delivery polymers.

Amyloid diseases like Alzheimer’s disease, type 2 diabetes, and Parkinson’s disease represent some of the most pressing concerns facing our healthcare system today. Human consequences aside, these disorders place an enormous economic burden on society; Alzheimer’s disease care in the United States alone costs upwards of $226 billion annually, and these expenses will continue to rise as the population ages. As such, substantial effort has been invested in the development of novel compounds that would halt or reverse the effects of these diseases. Our group’s previous research suggests that amyloid diseases, though caused by the selective dysfunction of different and unrelated proteins, share a common mechanism of pathogenicity, wherein misfolded proteins aggregate into toxic oligomers typified by novel α-sheet secondary structure. Synthetic peptides designed to adopt a structure complementary to this motif inhibit in vitro aggregation of several different amyloid systems. In this study, we show that the inhibitory effects of these molecules also extend into the realm of simple biological models, in this case inhibition of toxicity in neuroblastoma cells. We co-incubated IMR-32 cells with Aβ42 peptide (whose aggregation is associated with Alzheimer’s disease) and our synthetic α-sheet compounds for periods of twenty-four to forty-eight hours, then assessed cytotoxicity by a MTT cell viability assay. When compared with neat Aβ42, the designs were able to rescue cells from necrosis, significantly lessening detectable toxicity. While preliminary, these results suggest that our peptides may have promise as potential therapeutic agents for treating a broad spectrum of amyloid diseases.

Regulation of metabolic pathways is often implicated in many normal as well as disease processes in organisms. Our previous work discovered changes in the metabolome associated with longevity in eleven different species of fruit flies. The metabolic pathways associated with longevity included gluconeogenesis, the Krebs Cycle, and amino acid degradation. However, it is still unknown how basic nutrient parameters influence metabolic pathways in flies. We used mass spectrometry coupled with liquid chromatography to quantify the small molecules present in whole fly tissue. After finding metabolites associated with age, we completed nutrition assays to determine if there were associations between nutrient parameters and metabolomic profiles. The measurement of basic nutrient parameters consisted of colorimetric assays for total glucose and protein levels. Data analysis revealed correlations between the levels of each nutrient found in whole fly tissue and corresponding metabolomic profiles. In our future work, we plan to manipulate metabolic pathways or nutrient parameters found to be significantly associated with age and observe whether there are changes in longevity.

Treatment resistant bacterial infections are a major public health concern. Staphylococcus aureus, in particular, is responsible for thousands of deaths and hospitalizations every year in the U. S. alone. S. aureus has many virulent characteristics, including its ability to subvert the immune system via neutrophil lysis and biofilm secretion. Biofilm secretion is a common bacterial defense mechanism in which a mixture of specific proteins and cellular debris are assembled into a protective matrix. This matrix serves to shield bacteria from antibiotic treatment and mechanical disruption. Interestingly, phenol soluble modulins (PSMs) – small, soluble, amphipathic proteins which are the main constituent of biofilms – also modulate neutrophil lysis. Neutrophils are one of the body’s primary defense mechanisms against infection. Unfortunately, S. aureus have evolved to evade the normal function of these cells by lysing them from the inside. PSMs’ membrane-lytic behavior largely contributes to this pathology. Despite their importance in the promotion of persistent S. aureus infection, little is known about the biophysical mechanism of action of PSMs. In this project, I look into these two interconnected pathways using a series of high-resolution spectroscopic techniques, including aggregation fluorescence assays, fluorescence correlation spectroscopy, high performance liquid chromatography, and electron microscopy. Greater understanding of these pathways will help lead to better treatments to combat the global burden of resistant bacterial infections caused by S. aureus.

X-Link Myotubular Myopathy (XLMTM) is a progressive congenital pediatric disease that affects one in every 50,000 male births. XLMTM is caused by a mutation of the MTM1 gene. MTM1 codes for myotubularin, an enzyme that is involved in the excitation-contraction coupling of muscle cells. XLMTM is characterized by a severe phenotype marked with muscle weakness and hypotonia that can result in many life-threatening complications, including diaphragmatic weakness leading to respiratory failure. Adeno-associated virus (AAV)-mediated gene therapy has reversed muscle pathology in murine and canine models. Treatment of the canine models with an AAV8-MTM1 vector indicated improved muscle strength as well as prolonged life. The field of muscle disease research would benefit greatly if an in vitro patient-specific induced pluripotent stem cell (iPSC) model were utilized to generate XLMTM-null muscle myotubes. It is necessary to develop an in vitro model of the disease using specific patient-derived cells because the patient-specific disease model has the predictive utility to determine if there are aspects of the disease not mitigated by gene therapy. This will help pinpoint the areas the disease affects in order to come up with a cure. To do this, MTM1-mutant iPSCs were differentiated stepwise to myogenic precursors and then to multinucleated myotubes using a small molecule approach that has been shown to mimic embryonic muscle specification. Extent of myotube differentiation was qualified with immunochemistry, using muscle-specific markers such as Myogenin, MyoD, and MF20. Experiments are currently underway to identify the disease phenotype in vitro and correlate with known pathology observed in the patients with XLMTM.

The diffusion of ions into a nano-scale ZnO lattice has gained attention in the past decade as a possible solution to the rapid fracturing of ZnO electrodes in bulk batteries and as an alternative to conventional LiCoO₂ and LiFePO₄ materials which are expensive to manufacture and environmentally hazardous. Experiments have shown that utilizing bulk (ie. not nano-structured) ZnO electrodes fracture in discrete segments (a leapfrog effect) after only a few charge/discharge cycles, diminishing the effective charging/discharging capacity. Current efforts are aimed at using nano-scale lattices which fall within the size range of the cracking separations and thus are not susceptible to leapfrog fracturing. Using an optimized quantum dot structure and conventional ab initio electron structure theory methods, the effects of lithium ion intercalation into a Zn₃₃O₃₃ quantum dot lattice were investigated. The main research questions were: (1) where are the potential energy valleys in which lithium ions can settle; (2) What effect does lattice expansion have on the energy of a lithium-ion charged quantum dot; and (3) how does doping the dot with iron affect the energy? Using a rigid and a relaxed scan of a lithium entering and exiting through the C3v axis of the quantum dot, the potential energy landscape was mapped, illustrating the presence of multiple potential valleys in which ions could settle. We used single point calculations to determine the optimal expansion factor after observing expansion in previous molecular dynamics simulations. Currently preliminary results indicate a possible trend regarding iron positioning within the lattice and we hope to improve our data by introducing a larger Zn₈₄O₈₄ dot. Hopefully our findings will demonstrate that nano-scale iron doped ZnO lattices are not subject to leapfrog fracturing and may therefore be cheap and viable options as battery electrodes.

Alkanes, or saturated hydrocarbons, are the major constituents of natural gas and petroleum. Conversion of the chemically inert bonds of alkanes to more useful functional groups (like alcohols, aldehydes, and carboxylic acids) requires a great deal of energy and is often expensive. For this reason, functionality is often added using olefin precursors, or unsaturated hydrocarbons. Thus, more efficient and cost-effective conversions of alkanes to olefins, or alkane dehydrogenation, are highly desirable. Prior work shows alkane dehydrogenation may be achieved with iridium and rhodium pincer complexes. While significant success in this type of alkane functionalization has been achieved with Ir^I systems, these C–H bond-activating systems are inhibited by nitrogen and water, and can be self-inhibited by olefins. In contrast, Ir^III complexes have been shown to be tolerant to these inhibitors and are able to activate the C–H bonds of alkanes. Our experimental and computational results show usage of Brønsted acids to generate new Ir^III systems, in particular (^dmPhebox)Ir(OAc)(X) complexes, leads to an increase in the rate of C-H activation over the previously reported (^dmPhebox)Ir(OAc)₂(OH₂) system. The product of this activation and dehydrogenation reaction is olefin and (^dmPhebox)Ir(OAc)(H). Optimized conditions for this alkane dehydrogenation system and mechanistic studies will be presented.

All biological systems are dependent on properly folded protein molecules to function correctly. To enable major developments in biotechnology and medicine, an understanding of the protein folding process is needed. Advances in this field have been made, however most of the knowledge gained has been concentrated on structured beta-sheets and alpha-helices. Experimental studies of beta-strand association and the effects of conformational dynamics in unstructured loop regions remain scarce, in part because the timescales of the dynamics leave few spectroscopic methods available for measuring kinetics. Using a model system consisting of two beta-strands connected by a loop of variable length and composition, measurements of beta-strand association dynamics on microsecond to millisecond timescales have been made by employing Relaxation Dispersion Nuclear Magnetic Resonance (NMR) techniques. In addition, using multidimensional NMR and Circular Dichroism spectroscopy, sequence-specific thermodynamic effects in the folding of these peptides have been examined. Loop closure was originally thought to be so fast that it would be negligible in the protein folding process, however our measurements show that loop closure rates can vary significantly depending on size and composition. Peptides containing all-glycine loops of length 3 – 16 have been shown to have folding rate constants (1/kf) of 12.3 – 109 microseconds with unfolding rate constants (1/ku) of 636 – 191 microseconds at 300 Kelvin. Insertion of structured beta-turns into the loop region were shown to increase the rate of folding, from 231 microseconds for an all glycine 10-loop to 25.0 microseconds for a length-10 loop containing an organized turn. These measurements have shown that the interplay of conformational entropy and residual segmental motion are crucial to understanding the stability and dynamics of beta-sheets. Current and future work on this project seeks to understand how tuning the dynamics of beta-sheets will affect thermodynamics and kinetics of ligand binding in biologically relevant systems.

Transient receptor potential vanilloid receptor 1(TRPV1) is a nonselective cation channel whose activity can be regulated by different kinds of factors including vanilloids such as capsaicin. Nerve growth factor (NGF) has been shown to cause the rapid sensitization of nociceptors neurons expressing TRPV1 under inflammatory hyperalgesia. In other words, TRPV1 is involved the sensation of pain caused by inflammation. It’s been suggested that tropomyosin receptor kinase A (trkA) and its downstream effectors including phosphoinositide 3-kinase (PI3K) play a key role in TRPV1 potentiation and we want to further examine the molecular signaling mechanism. For the purpose of our study, we utilize a system based on a light-sensitive molecule phytochrome B (PhyB) to mimic inflammatory responses in cells. Under 650nm light, PhyB undergoes conformational change and binds to phytochrome interaction factor (PIF). By fusing proteins of interest to PIF, PhyB-PIF interaction controlled by light can be used to manipulate signaling pathways including PI3K pathway. To study the potentiating effect of PI3K on TRPV1, we examine NIH3T3 cells transfected with proteins of interest using calcium imaging. We compare cell responses to capsaicin before and after PI3K translocation activated by 650 nm light. Our experimental data suggest a significant increase of calcium signalling after translocating PI3K to the plasma membrane. In the future, we are hoping to learn more about how the downstream effectors can affect TRPV1 trafficking. We will perform similar procedures as described using dominant-negative Akt and Rac1 cells and investigate the specific mechanisms. Eventually, we hope to utilize what we know about TRPV1 in pain treatment development.

Multiple strains of rats and mice are used to evaluate differences in response after exposures to various toxicants at the molecular or cellular levels. However, neuronal toxicity in different strains of species has received much less attention. Therefore, the objective of our research was to compare and characterize fetal mouse micromass culture system using two different strains of mouse—C57BL/6 and A/J—which may be used to screen and prioritize variety of neurodevelopmental toxicants. Our mouse midbrain micromass culture system, a high-density in vitro cultures of primary midbrain cells, is an alternative method to in vivo neurodevelopmental testing because of the ability of cells within the cultures to differentiate in vitro without additional stimulants. Differentiation is purely driven by high cell density. For our 3D in vitro micromass culture system, we isolated the primary midbrain cells from fetus at gestational day 11 (for C57BL/6 mice) or 12 (for A/J mice). The embryos were surgically removed, and midbrain cells of fetal mice were digested into single neuronal cells. These primary in vitro neuronal cells were plated onto plates and cultured up to 22 days in vitro. When morphology was examined throughout 22 days in culture with a 400x phase microscope, cell proliferation, differentiation, and migration were observed for both strains of mice. Proliferation (PCNA, Nestin) and differentiation (B-tubulin, EN1, MAP-2) related protein expression levels were examined using western blot for both C57BL/6 and A/J mouse strains. Dynamics of protein expression levels in western blot indicated that proliferation may be at its highest point between DIV 4-6 while differentiation is between DIV 8-14. If midbrain cells from multiple mouse strains can be harvested at similar neurodevelopmental stages, differences in response due to genetic variability may be studied using our 3D fetal mouse micromass model.

Carnivorous plants inhabit low nitrogen soils and must consume insects for nutrition. They achieve this by using modified leaves, called traps, which can catch prey. Thus, the ability of carnivorous plants to attract prey is key for their survival. In this study, we tested the hypothesis that carnivorous plants use scents to lure insect prey. We considered two possible cases: 1) prey are lured by only trap scents, so they will only detect trap scents or 2) prey animals are lured by both trap and flower scents, so they will detect scents from both organs. We focused on Sarracenia pitcher plants, a genus that is indigenous to the southeastern United States, and their interactions with calliphorid flies (commonly known as blow flies), one of their major prey. We used electroanntenograms (EAGs) to determine whether flies were responding to specific smells. We did this by hooking up a whole fly prep to the EAG machine, pulsing specific scent compounds over the antennae, and recording the electrical responses produced by the fly. In our tests so far, we have found that flies respond most strongly to pulses of DMT, a compound which is found in the scents of traps that have captured prey. Flies also respond to beta-ionone, a compound that is found in pitcher plant flowers. Our preliminary results indicate that flies are able to detect both trap and flower scents, so this supports the hypothesis that pitcher plants use a combination of trap and flower scents to attract their prey. We plan on testing a wider range of trap and flower scents using both EAG pulses and gas chromatography - electroantennography (GC-EAG).

 Protein aggregation is implicated in many human diseases. For example, Alzheimer’s disease is characterized by the misfolding of the amyloid beta (abeta) protein that disrupts cellular function by accumulating into fibrils with a characteristic cross-beta sheet structure. A wide variety of computational, sequence based tools are available to predict amyloidogenic “hot spots”; the regions in proteins that cause the formation of these toxic aggregates. Recently we have performed a comprehensive mutational analysis of abeta using a high throughput method termed Deep Mutational Scanning (DMS). Four hundred and fifty abeta variants were simultaneously assayed for solubility. An aggregation score that reflects the effect on the aggregation propensity of abeta was calculated for each variant. Using these data as a gold standard, we benchmarked aggregation prediction tools and assessed the ability of each algorithm to accurately predict aggregation. In particular, we were interested in identifying methodological weaknesses in these tools and then determining how to improve aggregation propensity prediction. I measured the correlation between several tools’ aggregation predictions for abeta single mutants with the measured aggregation scores. My initial analysis revealed that these tools are often insensitive and over-predict amyloidogenic regions, indicating an opportunity to improve these methods. Having comprehensively measured the relationship between abeta sequence and aggregation propensity, we are now in a position to assess and improve current aggregation prediction tools. Improving our ability to predict this critical process will enhance our understanding of diseases of protein aggregation.

The drug rapamycin is a specific inhibitor of the mechanistic target of rapamycin (mTOR) and has been previously shown to increase lifespan in yeast, worms, flies, and mice. Rapamycin inhibits mTOR by binding the Fpr1 protein (FKBP12 in mammals); the rapamycin-Fpr1 complex then dissociates the mTOR complex I. In order to identify new, natural product inhibitors of mTOR that function similarly to rapamycin we have developed a high resolution assay for growth inhibition in yeast using a Bioscreen C MBR machine. We are currently testing twenty-two natural product compounds, and their mixes in this assay to determine if they inhibit the TOR pathway, change mitochondrial function, or have any effect on replicative lifespan in yeast. Each compound and mixture is being assessed for its ability to alter the growth kinetics of four yeast strains: wild-type, TOR1 deletion, FPR1 deletion, and SIT4 deletion. Tor1p is a kinase in the TOR complex, and deletion of TOR1 leads to rapamycin hypersensitivity. The fpr1Δ mutant is resistant to rapamycin. The sit4Δ deletion is sensitive to multiple drugs. Compounds were dissolved in the solvent DMSO, or in some cases other solvents, and diluted to 1 µg/ml, 10 µg/ml, and 100 µg/ml in culture medium. All tests were performed in yeast extract peptone dextrose (YEPD) solution. We used a Bioscreen C MBR analyzer to measure the optical density of growing yeast in the YEPD with the compounds. The bioscreen analyzer generates an optical density curve over time that can be further analyzed by using the web tool, Yeast Outgrowth Data Analyzer (YODA), to selectively examine and compute the doubling time of the cultured yeast cells. The doubling time data was used to evaluate the effect of supplements on growth rate compared to rapamycin, in order to identify compounds of interest for further study.

Genetic robustness is the ability of an organism to gain mutations without changes to its fitness or functions. Evolutionary conditions that might select for genetic robustness are unclear, but some studies suggest that proteins that are more thermostable (able to fold properly at high temperatures) are also genetically robust to mutations. We are examining this relationship in phi-6, a virus that infects bacteria. We have evolved phi-6 for thermostability by subjecting it to high temperatures and then sequenced the viruses for mutations. We used homology-modeling programs to visualize the structures of mutated viral proteins and made predictions about the molecular basis of thermostability and genetic robustness. In order to evaluate whether the thermostable strains are genetically robust, we ran random mutagenesis on both thermostable mutants and the wild type virus. Our work shows that evolution for thermostability may be a viable mechanism to evolve genetic robustness.

The model yeast, Saccharomyces cerevisiae, can evolve the ability to clump, a trait called flocculation that protects the yeast cells from stress factors including antimicrobial treatments. Understanding the genetics behind this trait could give us insight into treatment options for biofilm-related complications in hospitals as well as targets for microbe engineering in the case of industrial production. In this project, we are interested in identifying the mutations that occur when yeast evolve flocculation. We evolved S. cerevisiae from a non-flocculent ancestor strain for 300 generations in miniature chemostats, multiplexed culture devices that maintain a constant environment. Strains that flocculate have a selective advantage in these devices, and 35% of our final populations evolved this trait. In backcrossing these flocculent strains with their ancestor and dissecting the tetrads into four spores, 20 out of 22 strains segregated the flocculation trait with a 2:2 ratio, indicative of a mutation in a single gene causing the trait. We identified causal alleles using bulk segregant analysis, a pooling and sequencing approach, including a causal transposable element insertion in the promoter of FLO1, a gene known to control flocculation. We hypothesize that this mutation is responsible for changing how FLO1 is expressed and have now identified this mutation in 13 of the clones, some of which exhibit varying phenotypes. We believe that additional mutations may be responsible for this phenotypic variation and are using microscopy to target our sequencing for the gene(s) of interest. We also used these findings to engineer a new strain with FLO1 deleted, which we hypothesized would take longer to flocculate. We report the results of a new evolution experiment comparing flocculation time between the deletion strain and the wild-type strain and demonstrate that our engineering effectively decreases flocculation ability. This is a promising result for both medical and industrial applications.

Phenotypes of mutant alleles in a given environment can be used to determine which processes depend on the normal function of a gene. Although genes involved in specific biosynthesis pathways are well characterized in the model organism Saccharomyces cerevisiae, there are genes involved in these processes that still have uncharacterized function. The interaction between gene function and the environmental condition is complex, and requires a comparative approach across yeast species to identify the function of uncharacterized genes in a variety of conditions. We have used a Tn7 transposon insertion mutagenesis system in Saccharomyces uvarum to randomly mutate genes across the genome and pooled all resulting insertional mutants together. We used deep sequencing to identify the insertion sites, and previously predicted 809 genes to be essential for growth in standard laboratory conditions in S. uvarum. Using these same methods, we will apply different selective pressures to libraries of mutant yeast, and aim to understand which genes are critical for growth under specific conditions, such as glucose, phosphate, and sulfate limiting media. We expect that it will be more common to find genes that were non-essential in normal growth media to be essential in more limiting conditions, whereas most genes that were essential in normal growth media should also be essential in limiting conditions. Studying yeast in conditions where they are naturally found (such as grape-must) can provide insight into new, critical genes. This information can be used to make commercial processes such as fermentation more efficient by optimizing metabolic pathways. Additionally, this data can be used to improve gene annotations and increase our knowledge of the biochemical pathways that underlie these essential processes. Thus, acquiring phenotypic information of mutant genes across species and conditions can even help discover genes involved in previously well-studied pathways.

The plasma protein von Willebrand Factor (VWF) is secreted by vascular endothelial cells. VWF’s primary function is to recruit platelets to the site of blood vessel injury. The interaction between VWF and platelets happens through binding between the VWF A1 domain and the platelet surface receptor, glycoprotein Ibα (GpIbα). VWF requires shear to be activated. Recently, it has been shown that oxidizing agents increase the platelet-binding activity of VWF, although the exact molecular mechanism is not understood. To explore whether neighboring domains of A1 play a role in the oxidant-induced activation of VWF, we used a flow chamber microscopy assay. Two experimental constructs of VWF, one containing the A1 domain and the neighboring A2 and A3 domains, the second containing A1 and the neighboring D`D3 domain were expressed and bound to separate plates. One control construct containing only the A1 domain was also expressed and bound to plates. Then GpIbα coated beads were washed at different shears over plates that had either been pre-oxidized or not. The movement of beads was recorded. Subsequently, rolling velocities and total number of beads were measured and analyzed using MATLAB. The results showed that, at high shear, both the A1A2A3 and the D`D3A1 constructs were more active in binding to beads when oxidized compared to the unoxidized controls. The control construct did not show a difference in binding to beads when oxidized compared to unoxidized controls. These findings suggest that the A2, A3 and D`D3 domains could have an inhibitory function on the A1 domain and that oxidation removes this inhibition resulting in an increased binding activity of VWF to platelets. Understanding how oxidation alters the function of VWF domains is necessary to explain the increased risk of thrombosis in patients suffering of chronic or acute inflammation.

Previous studies have shown the importance of bitewing radiographs, xrays taken to detect caries (also known as cavities) in between teeth. Some dental schools have adopted individualized decision-making protocol in order to monitor the decay that lead to performing restorations. However, it is not clear if selective radiographic examinations based on caries risk have been applied routinely. The goals of this project are (1) to examine the frequency of dental radiographic examinations in both children and adult populations, and (2) to analyze the relationship between the frequency of dental examinations, bitewing radiograph prescriptions, and risk of dental caries. Factors that may influence how often radiographic examinations were prescribed, including patients’ age, gender, distance from clinics, and status of dental insurance, were investigated. Patients’ dental records and radiographic images at the University of Washington School of Dentistry from 2007 to 2015 were reviewed. Patients who have been examined for more than 3 visits over a span more than 2 years were selected. Data was collected from two clinics, the adult (n=109) and children's (n=172) clinics. Patients lacking posterior teeth, fewer than 3 exams, or no radiograph prescriptions were excluded from the study. The caries risk was calculated as the number of decay, missing, and filled teeth for the adult clinic and recorded as decay and filled teeth for the children's clinic. Missing teeth were not considered part of the caries risk due to the mixed dentition nature of most children. Our results showed no significant correlation between the clinical examination and radiograph prescription frequencies and the caries risk, age, gender, and distance from clinics. There are significant differences in both radiograph prescription frequencies between clinics and how soon patients return for their second visit among insurance statuses. Our data suggests that dental bitewing radiograph prescriptions were determined by clinic/providers, not caries risk factors.

In the aging community, our ultimate goal is to lifespan and healthspan, the period of time that an individual lives healthily. Previous studies have found that aging results in decline of heart functions and increases prevalence of cardiovascular disease. Characteristics of cardiac aging include enlargement of the left ventricle, impaired diastolic functions, and reduced myocardial performance. Oxidative stress has been implicated as one of the mechanisms underlying cardiac aging. SS-31 peptide, a tetrapeptide that targets the inner membrane of the mitochondria, has been shown to interact with cardiolipin and enhance the electron carrying function of cytochrome c, an essential component of the electron transport chain. SS-31 peptide also limits oxidative damage to cardiolipin, a phospholipid that is almost exclusively located in the inner mitochondrial membrane, by reducing peroxidase activity of cytochrome C. SS-31 treatment has been shown to protect mitochondria in pressure-overload induced heart failure and oxidative injury in other tissues. In this study, we performed echocardiography and treadmill running and showed that 8 weeks of the SS-31 treatment improved diastolic function, myocardial performance, and enhanced exercise endurance in old mice. Using quantitative real-time PCR, I am assessing the gene expression changes regulated by the SS-31 treatment. Understanding the mechanism of how SS-31 improves cardiac aging will provide insight on potential therapies to improve cardiac health in the elderly populations.

The psychology literature delves into understanding the role of men's self perception of masculinity in help-seeking behavior as well as help-seeking behavior in men with cancer. Help-seeking behavior is behavior that explains the utilization of healthcare of a population or group. What is the role of self perceptions of masculinity in help-seeking behavior of men with prostate cancer? This literature review focuses on understanding this role in men specifically with prostate cancer. The resources used for the review are MedLine, PsycINFO, PsycArticles, PubMed, and references within articles. The majority of the literature used were qualitative studies due to the availability in the literature. The literature suggests that men believe that prostate cancer affects their masculinity in both physical and emotional ways such as their physical aspects like strength, ability to provide for and protect their families, reaction to physical pain through peer pressure and social norms, and being viewed as unmanly. There are men that efficiently seek help from healthcare professionals for this disease to preserve their masculinity. Men also inefficiently seek help due to the potential embarrassment of those qualities being diminished by the disease. The limitations to writing the review included small sample sizes, support group participants, and participants that were already diagnosed. The review is also limited because there was no research on men that focused on how masculinity may have contributed to their help-seeking in the initial stages. Healthcare services, such as interventions or programs, should focus on the following indicators when promoting help-seeking behavior to men with prostate cancer: stoicism, privacy, family providing and protection, and being viewed as unmanly due to the physical effects on a man's body. If the healthcare system can overcome these barriers, it will be able to reach men more efficiently and prevent further advancement of diseases like prostate cancer. 

 Cyclic diadenosine monophosphate (c-di-AMP) is an essential nucleotide for bacterial virulence. C-di-AMP has been extensively studied in Listeria monocytogenes (Lm), but is also produced by other pathogens including E. coli, C. trachomatis, and M. tuberculosis. Although c-di-AMP is a signaling molecule in bacteria, host cells detect it as a pathogen-associated molecular pattern. During infection, the host receptor STING senses c-di-AMP and stimulates expression of interferon-beta, an anti-viral molecule. To date, STING is the only known sensor of c-di-AMP, and its promotion of an anti-viral versus anti-bacterial response has been somewhat perplexing. However, our lab has recently discovered another host c-di-AMP sensor, the murine aldo-keto reductase1C13 (AKR1C13) or “RECON”, that induces an alternative, anti-bacterial cellular response to infection. Based on the importance of bacterial clearance and the ubiquity of AKRs across species, we began to investigate whether other murine and human AKRs respond similarly to c-di-AMP. Sequence alignment of AKR genes and data on tissue-specific expression of these genes returned six candidates: the murine AKR1C12 as a potential paralogue, and human AKRs 1C1, 1C2, 1C3, and 1C4 as potential homologues. Cloning techniques allowed me to insert these genes into an E. coli expression vector that I used to purify recombinant protein via affinity chromatography. The binding affinity with c-di-AMP of each protein was tested with radio-ligand binding assays to determine dissociation constants (K_d) and we additionally used enzyme activity assays to quantify inhibition of protein activity. AKRs that bind c-di-AMP will be further characterized in vivo in mammalian cell lines in which expression has been knocked down via lentiviral transduction. Further work in cell lines may elucidate novel pathways that are activated downstream of c-di-AMP detection. Understanding the pathways that naturally clear infections is important to potential medical advances in bacterial infection management and prevention.

Aging and severe mitochondrial disease are disparate, complex processes whose causes have yet to be fully understood. One protein involved in both is the mechanistic target of rapamycin (mTOR), which regulates cell growth and metabolism. mTOR protein is a component of two complexes, the mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Inhibiting mTORC1 with the drug rapamycin increases lifespan in model organisms and suppresses severe mitochondrial disease and shortened lifespan in the Ndufs4 knockout (KO) mouse model of Leigh Syndrome. In mouse models, KO of the S6K1 protein, a substrate of mTORC1, also increases lifespan. Therefore, we hypothesized that KO of S6K1 in the whole body and specific tissues (brain, liver, fat) would rescue the short lifespan of Ndufs4 KO mice. A breeding strategy utilizing Cre-recombinase mice, S6K1^Flox/Flox mice, and Ndufs4 +/- mice was employed to ultimately produce tissue-specific Cre +/- Ndufs4 -/- S6K1^Flox/Flox mice. PCR and Western blot analysis confirmed the whole body and tissue-specific deletion of S6K1 gene and depletion of the protein in each mouse. Our findings indicate that whole body and liver-specific S6K1 KO modestly increased lifespan and delayed the onset of neurological symptoms originating from the mitochondrial disease. However, S6K1 KO is still less effective in rescuing mitochondrial dysfunction than the optimal dose of rapamycin. We speculate that S6K1 partially contributes to the mitochondrial disease of Ndufs4 KO mice, and the liver may play an important role in regulating lifespan. In the long run, these findings may enable us to potentially produce drug treatments to extend the lifespan of healthy humans as well as Leigh Syndrome patients.

Olfaction is an important method of communication for organisms such as mice. However, metabolism is significantly influenced by the integrity of sleep and the amount of dietary fat. Here I examined whether environmental factors such as sleep and diet could affect volatile profiles, because these may in turn affect the biological signals that are relayed between conspecifics. We hypothesized that the volatile profiles in mice would change following sleep disruption and/or increased fat levels intake. A group of one strain of mice was divided into two groups: one control group with undisturbed sleep and one experimental group that underwent sleep disturbance. Each group was divided again into two groups: one received regular chow diet and the other received high-fat diet. All mice were individually housed during the 4-week treatment period, and fecal samples were taken 2 weeks after cessation of sleep and diet manipulations through solid-phase microextraction (SPME). Following gas chromatography and mass spectroscopy (GC-MS) of these samples, I analyzed the data and found differences in abundance of volatile compounds among the experimental groups. Namely, production of pentanoic acid and benzaldehyde decreased considerably as a result of sleep disruption. Several other compounds were also found to undergo change in abundance as a result of fat level in diet or from a combination of both factors. My work in this project could potentially be a starting point in investigating the metabolic pathways associated with sleep disruption and diet that are linked to the above changes in volatile production.

 Eukaryotic cells contain membrane-bound organelles that serve to compartmentalize certain cellular functions. Different organelles contain optimal pH levels necessary for the organelle function. The vacuolar ATPase (V-ATPase) is a highly evolutionarily conserved proton pump that is responsible for acidification of many organelles. This proton pump consists of various protein subunits and loss of any these subunits results in disruption of the entire protein complex, which in return is not compatible with life for most organisms. Within certain conditions, S.cerevisiae (yeast) is able to survive in the absence of a functional V-ATPase, which makes it an ideal model organism to study V-ATPase function. In yeast, the proteins that comprise the V-ATPase are encoded for by vacuolar membrane ATPase (VMA) genes. Loss of any individual VMA gene results in pleiotropic phenotypes including an inability to grow on nonfermentable carbon sources and shortened replicative lifespan. In order to understand more about how the V-ATPase functions, we have performed multiple screens to identify genes that when overexpressed or mutated can suppress the respiratory phenotype of VMA mutants. Our results implicate iron homeostasis as a process that is crucially disturbed when the V-ATPase is disrupted.

The human Tumor Suppressing Subtransferable Candidate 1 (TSSC1) gene was first discovered in 1997 as a gene whose likely location was within a chromosomal region that suppressed tumor growth. A recent study reported that decreased TSSC1 gene expression results in increased metastasis of breast cancer cells to bone. However, the underlying mechanism by which the TSSC1 protein acts in cancer cells remains unknown. Interestingly, TSSC1 is a highly conserved protein among eukaryotes. We are using Drosophila melanogaster as a model organism to define the normal function of TSSC1. Previously, our lab showed that the Drosophila protein, dTSSC1, localizes near Trans-Golgi Network (TGN), the region of the Golgi that sorts and packages membrane and secreted proteins into vesicles for transport to post-Golgi destinations. We have also isolated two mutations in dTSSC1 and observed that they cause defective formation of the acrosome, a Golgi-derived organelle that is localized to the apical tip of mature sperm and is required for fertilization. Based on these observations, we propose that dTSSC1 acts in spermatids to regulate acrosomal protein and vesicular trafficking. We are expressing tagged membrane proteins to ask whether dTSSC1 is required specifically for trafficking of acrosomal proteins or if it has a more general impact on membrane protein trafficking. These studies use expression in the male germ line. Notably, dTSSC1 is expressed in many tissue types, similar to its counterpart in humans. Therefore, we are also investigating whether dTSSC1 mutations have effects on protein trafficking in somatic cells. Our results should define the normal cellular role of dTSSC1 in Drosophila and may also provide insight into the role of the human protein in normal and cancer cells.

Exposure to manganese (Mn) causes a neurodegenerative syndrome with clinical manifestations of Parkinsonism (PS) similar to Parkinson’s disease (PD). Although both PD and Mn-induced PS feature movement dysfunction resulting from decreased striatal dopamine, pathologically, Mn-induced PS is thought to be related to striatal degeneration, while in PD, dopaminergic neuron bodies residing in the substantia nigra pars compacta are largely affected. Recent evidence has suggested that astrocytes, glial cells that play a vital role in maintaining homeostasis such as uptake and release of important molecules like precursors for neurotransmitters, may play a vital role in neuronal survival in PD and PS. In order to determine whether the regional differences in sensitivity to Mn are conferred by inherent differences in astrocytes from these brain regions I examined the role of such astrocytes in neuron survival. Contact co-cultures of two neuron-like immortalized cell lines, MES and SH-SY5Y cells, were made with astrocytes from the ventral mesencephalon (vm), striatum (str), and cortex (ctx). These were treated with 10μM and 100μM of Mn and stained for the markers MAP2, TH, and GFAP, imaged, and neurons were counted. Based on previous results, MES cells grown with str astrocytes are expected to have a lower survival rate when treated with Mn, versus those plated with vm and ctx astrocytes. If this trend is observed in the separate SH-SY5Y cell line, this would suggest other neuron-like cell lines and dopaminergic neurons respond similarly to Mn. By examining if Mn affects astrocytes, and in turn neuron survival, this could not only elucidate the pathogenesis of Mn-induced PS, but also elucidate the region specific astrocyte-neuron relationships in the brain.

HIV remains a major global health problem due to high transmission levels and emergence of drug resistant strains. According to WHO, in 2014, sub-Saharan Africa had 25 million people living with HIV and 70% of new HIV infections globally. A high number of circulating drug resistant HIV remains undetected due to lack of affordable HIV genotyping methods. The lab based oligonucleotide ligation assay (OLA) is a highly sensitive method for detecting HIV drug resistance as an alternative to the expensive and complex complete-genome sequencing, but its reliance on moderately expensive lab equipment prevents its universal access.  Our lab, in collaboration with the Frenkel Lab (Seattle Children’s Research Institute) and the Lai Lab (UW BioE), focuses on developing a simplified OLA-based HIV drug resistance test. The ligation step of the OLA relies on the specificity of DNA ligase to target single nucleotide polymorphisms (SNP’s) that can confer HIV drug resistance. However, ligation currently requires a thermal cycler to cycle between 95 °C to de-hybridize target DNA and 37 °C for annealing of oligonucleotide probes to target DNA. Eliminating the temperature cycling component of ligation would be a key advantage because thermal cyclers are not widely available in low resource settings. We measured ligation at intervals from 25 °C to 70 °C and were able to pinpoint an optimal temperature range for isothermal ligation. We plan to further increase ligation yield, and therefore detection signal, by optimizing the concentrations of salts and cofactors, adjusting probe lengths, and modifying probe sequences.  With the development of isothermal ligation, the OLA will be further simplified for potential widespread use in low resource settings. This will allow HIV infected individuals to be accurately diagnosed and receive the proper line of HIV drugs, controlling transmission rates and reducing the evolution of drug resistant HIV within entire populations. 

The Ndufs4 gene knockout (Ndufs4-/-) mice show the phenotype of severe neurodegenerative mitochondrial disease analogous to human Leigh syndrome. Phosphoproteomics is a large-scale study of phosphorylated proteins, which are post-translational modifications that can turn on and off biological signaling pathways. Phosphoproteomics and proteomics experiments using mass spectrometry can allow us to quantify protein levels in mouse tissues. The broad goal of my project is to define the underlying mechanism of mitochondrial dysfunction using a phosphoproteomics approach in the Ndufs4-/- mouse model and translate the findings to improve the health of patients with severe mitochondrial disorders. Our lab previously showed that rapamycin, a drug that inhibits mTOR kinase, partially rescued the disease phenotype of Ndufs4-/- mice. However, the downstream biological pathways affected by rapamycin in this setting are not understood yet. Those may be discovered by analyzing differences in phosphorylated proteins among brain tissues of wildtype, Ndufs4-/-, and rapamycin treated Ndufs4-/- mice. For the initial analysis, protein data sets with quantitative measurements of more than 4,000 proteins were analyzed. Proteins that may be important and most affected by mitochondrial disease and rescued by rapamycin were pinpointed. After the analysis of the data was complete, the proteins that passed the filtering thresholds were quantified with Western blots, a biochemical technique to quantify protein level individually. We have already identified two signaling pathways from the phosphoproteomics and will test the effect of inhibitors of these pathways in the mice. It is our hope that a better mechanistic understanding of disease progression in this mitochondrial disease mouse model will yield new therapies to treat mitochondrial disease in patients for whom there is not currently any effective treatment.
 

Acute myeloid leukemia (AML) is a generally aggressive blood cancer with poor prognosis for which the fundamental treatment approach has not changed substantially. The need for new therapies is thus unquestioned. One therapy currently under phase I/II investigation at UW/SCCA for patients with newly diagnosed AML and those with relapsed or refractory disease, is G-CLAM (Granulocyte-colony stimulating factor, Cladribine, cytArabine, and Mitoxantrone). The phase I portion of the clinical trial involved evaluating the regimen for safety and escalating the dosage of mitoxantrone to determine the maximally tolerated dose. The main purpose of phase II, which is now ongoing, is to evaluate the effectiveness of the regimen, measured by the rates of complete remission and overall survival. Patients are accepted into the trial after having their medical history, laboratory, pathology and radiology results thoroughly reviewed to ensure all of the eligibility criteria have been met. As in all clinical trials the eligibility criteria are designed to help minimize risks to patients and homogenize the study population. However, the use of a set of criteria to define trial eligibility introduces a potential bias in the study and raises the question as to the degree to which findings can ultimately be extrapolated to the AML population at large. Studying the latter question is facilitated by the fact that a larger number of patients have received the same treatment off-protocol, e.g. because they were too sick to be eligible or did not have insurance coverage for study participation. We are collecting data on these off-study patients to compare their characteristics and treatment outcomes with those treated on-study. Findings are anticipated to answer whether the promising treatment results we have seen in the on-protocol patients carries over to those treated off-protocol as well and thus whether our results can be validly applied to the general AML population. 

Students from the Ocean Research College Academy (ORCA), a program through Everett Community College for high school students that focuses on marine science research, have collected monthly water quality data from the Possession Sound (PS) over the past twelve years. This longitudinal data set is combined with collections of current water quality by this research group. Nutrients play an important role in the health of the PS ecosystem. We evaluated the variations in the levels of phosphates and nitrates in relation to season in the north Whidbey Basin of Puget Sound from 2012-2015. Data were collected monthly from four locations in PS and we averaged these values to analyze the resulting trends in nutrient amounts. Surface water temperature was used as a proxy for the seasonal changes in direct sunlight. Nutrients and sunlight are required by phytoplankton for photosynthesis, so as phytoplankton populations increase nutrients become less abundant as they are utilized. Nitrates and phosphates were hypothesized to decrease in spring months because there was more photosynthesis taking place during the spring with increased sunlight, while the nutrients would peak in the winter. The resulting trends supported this hypothesis with the highest average levels of nitrates in October, at 15.54 μmol, and phosphates peaking in February at 2.15 μmol. The lowest average nitrate and phosphate levels occurred in June, at 3.08 μmol and 0.43 μmol, respectively. Our study gives insight into the health of the PS ecosystem by analyzing nutrients that limit the primary production of phytoplankton. Future parameters to be studied in correlation with this research include silicate and chlorophyll levels.
 

Aging is characterized by time-dependent deterioration of cellular and physiological functions. Due to the numerous challenges and limitations of studying aging in humans, model organisms are used to further extend our understanding of basic mechanisms of aging. The Kaeberlein laboratory uses the single celled budding yeast Saccharomyces cerevisiae as one such model. Advantages of this model organism include the ability to measure the lifespan of great numbers of organisms under various environmental conditions. One method of measuring a yeast cell’s longevity is by assaying its replicative lifespan (RLS). The RLS model refers to the number of daughter cells that can be produced by a mother cell before senescence. The number of budded daughters can be determined by micro-dissection with fiber optic needles from mother cells after each round of incubation. This process is repeated until mother cells no longer generate daughters. Approximately 600 mother cells are assessed weekly and incubation cycles last one month. Yeast lifespan can be manipulated through genetic changes, altering media content, addition of pharmacological agents, and change of temperature. Data collected is compared to control groups and to past and expected results. These results clarify how senescence is regulated. RLS is thought to be a better representation of senescence in mitotically active cells, like certain tissues in multicellular organisms. By analyzing thousands of cells, a variety of genes and environmental conditions that impact aging have been identified. These genes and interventions can be further studied and applied to other organisms. Studying budding yeast enhances our understanding of the molecular basis of aging.​

One of the leading causes of depression is stress, and yet not enough is known about the exact mechanism by which stress alters depression. Of the four opioid receptor systems (mu, delta, kappa, and opioid receptor like-1), the kappa opioid receptor (KOR) is what my research focuses on. KOR is activated by its endogenous ligand, dynorphin. This is responsible for promoting analgesia, and depression-like phenotypes such as swim-stress immobility. The mammalian target of rapamycin (mTOR) is a protein kinase involved in regulation of functions such as cell growth, and metabolism. When dysregulated, it may lead to obesity, depression or cancer. Both mTOR and KOR have been shown to independently regulate stress-induced depression. However, there are no studies which tie these two ideas together. One possible link could be the stimulation of p38 MAPK kinase. Stimulation of KOR causes G-protein coupled receptor kinase (GRK)-dependent activation of the p38. Additionally, it is known that p38 causes the inhibition of TSC2, promoting phosphorylation of mTOR. Based on our results, activation of KOR with U50,488 causes an increase in phosphorylation in mTOR in vitro. However, this is also present in negative control cells not expressing KOR, which could indicate that U50,488 also has an off-target effect on a non-KOR mediated pathway. A similar treatment on mice decreases phosphorylation of mTOR after 28 hours in the brain, indicating that after a stressful event, mTOR activation is transient and acute. Furthermore, decreased mTOR phosphorylation after 28 hours in the brain was found to be blocked in GRK3 knockout mice. This implies that decreased mTOR phosphorylation after 28 hours with U50,488, requires the GRK gene. Further experiments, including behavioral testing, will be needed to investigate a deeper relationship between KOR and mTOR. With further research, these studies may lead to developing novel anti-depressants, revolutionizing depression treatment.

 Each year about 33 children 18 years of age or younger are hospitalized and 19 die from firearm injuries in Washington State. Safe firearm storage, including storing firearms locked, unloaded, and separate from locked ammunition is associated with a lower risk of such injuries. The purpose of this study is to evaluate the effectiveness of 2 community-based firearm safety events that included the provision of a free firearm locking device (lock box or trigger lock) among firearm owners in Washington State, particularly among families with young children in the home. At each event, participants received training to operate the safety device and advice to store their firearms safely. Participants were asked to complete voluntary onsite questionnaires that established household firearm storage practices prior to receiving the lock box or trigger lock and consent to follow-up phone surveys approximately 4-6 weeks later. Surveys included questions assessing demographics, motivations for firearm storage practices, level of comfort with potential firearm safety counselors (e.g., physicians, law enforcement officers), and changes in household firearm storage practices since the safety event. Participants were compensated for their time with $5 gift certificates. Of 398 event participants, 195 completed both surveys and were included in the study sample. About one-half were female and 81.4% reported keeping a firearm in the home. The results from this research will help determine the best way to improve gun safety within the firearm community.

The first member of the vanilloid sensing sub-family of transient receptor potential cation channels (TRPV1) is an ion channel located primarily in the nociceptive (pain-sensing) neurons of the peripheral nervous system. It is most strongly activated by high temperatures, protons, and capsaicin, the molecule responsible for the spice of hot chili peppers. From these examples of TRPV1 activators, it has been deduced that this ion channel plays a key role in the sensory transduction of pain that results from thermal burns, acid burns, and ingestion of noxious chemicals. Thus, the TRPV1 ion channel has potential to be a drug target for innovative pain medications and therapies. Before this is feasible, physiologically relevant details of TRPV1 structure and function must be elucidated. Expanding the understanding of how TRPV1 interacts and changes in various environments is also crucial. My research aims to determine if the lipid environment surrounding TRPV1 has significant impacts on its structural conformation. Electron resonance techniques such as EPR and DEER can be used to detect structural information of proteins; however, a number of steps precede this goal. First, I expressed and purifyied TRPV1 in large quantities. Next, I reconstituted the purified channel into vesicles. Vesicles are spheres of phospholipid bilayer and more accurately resemble the plasma membrane environment in which TRPV1 naturally occurs. Then electron resonance techniques were used to compare the structure of detergent-stabilized TRPV1 and TRPV1 reconstituted into vesicles. The comparison of TRPV1 structure in different lipid environments has a lot of implications in the applicability of structural studies conducted outside of physiologically relevant systems and for the future study of TRPV1.

A Mycobacterium bovis-based Bacillus Calmette-Guerin (BCG) vaccine is protective against childhood TB meningitis but insufficient for controlling the disease. The development of efficacious vaccines requires mechanistic understanding of host immune response to MTb and the BCG vaccine. We have previously identified that single nucleotide polymorphisms in the gene HSP90B1 are associated with development of TB disease in South African children. HSP90B1 is a molecular chaperone for integrins and most Toll-like receptors (TLRs). TLRs recognize pathogen-associated molecular patterns (PAMPs), and are critical for the innate immune response to infection. In a mouse model, HSP90B1-deficient macrophages were unresponsive to ligands that target TLR2, 4, 5, 7, and 9. Our lab has generated an HSP90B1 gene knockout using CRISPR/Cas9 technology in a human monocyte cell line, and I have confirmed successful mutagenesis via a restriction fragment length polymorphism analysis. Our preliminary data indicates that exposure to TLR ligands PAM2, PAM3 and LPS, in addition to MTb whole cell lysate is correlated with a reduction in Tumor Necrosis Factor (TNF-α) secretion in the knockout cells compared to controls. We hypothesize that HSP90B1 is a critical modulator of the MTb-induced TLR signaling pathway, which drives the production of inflammatory cytokines and plays a key role in the immune response to MTb. My objective is to characterize the effects of HSP90B1 on the TLR signaling pathway by stimulating cells with TLR ligands such as flagellin for TLR5, imiquimod for TLR7, and performing cytokine quantification via enzyme-linked immunosorbent assays (ELISA). Moreover, I will conduct Griess assays to evaluate effects of HSP90B1 upon nitric oxide production, which is an important defense molecule in enhancing efferocytosis, the process of up taking apoptotic cells. These studies will improve our understanding of host immune responses to MTb and potentially lead to development of more efficacious TB vaccines.

Expansion Microscopy (ExM) is a revolutionary type of fluorescence microscopy that enables super-resolution on conventional microscopes of fixed specimens that have been chemically incorporated into a hydrogel polymer matrix, enzymatically digested, and physically expanded. The original ExM approach utilized special tri-functional antibodies containing a DNA-dye-linker complex to tether the fluorescent dyes to the expandable polymer and yielded an isotropic expansion of ~4x in all directions and a lateral spatial resolution of ~70nm. Recently, we have created a simplified ExM methodology, using conventional fluorescently labeled antibodies or fluorescent proteins, and a variety of commonly available reagents yielding increased fluorescence retention for cultured cells and tissue specimens. Additionally, we have extended this new approach of ExM to Drosophila larvae and brain tissue using an optimized solution of enzymes to digest the robust outer surface of larvae (cuticle) and allow for uniform expansion of the polymer. These simplified methods for expansion microscopy will enable super-resolution microscopy on a wide range of biological samples without the need for specialized reagents or equipment.

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

Voltage-gated calcium channels are vital to electrical signaling in the human body, and their defects are associated with various neurological and cardiovascular disorders. Finding the 3-D molecular structure of these channels allows us to understand the physical basis for their function of selectively allowing calcium ions through the cell membrane. The Catterall lab uses X-ray crystallography, a technique in which a protein is grown into crystals and then diffracted with X-rays, to study the structure of Ca_vAb, a voltage-gated calcium channel from the bacterium Arcobacter butzleri. However, crystals of Ca_vAb are currently weak and give structures with relatively low resolution. This makes it difficult to see details in the protein’s structure and limits these structures' usefulness. To this end, I have introduced a zinc-binding site into Ca_vAb that increases the strength of the protein-protein interactions that cause it to crystallize. I anticipate that crystals of this altered form of Ca_vAb, when grown in the presence of zinc ions, will incorporate zinc into the introduced binding site, strengthening the crystal. Current results show some evidence for the inclusion of this zinc-binding site in crystals of Ca_vAb. The crystals show X-ray absorption and electron density within the binding site that indicate the presence of zinc. While further studies are needed to completely elucidate the effect of this zinc-binding site, current results are promising. In the future, we hope to take advantage of the anticipated crystal quality from this zinc-binding site for further structural studies on Ca_vAb. For instance, greater resolution will allow our lab to fully determine the mechanism of action by which calcium channel blocker drugs bind to a voltage-gated calcium channel. Furthermore, similar introduced metal-binding sites could be applied to the crystallization of other proteins that have so far evaded structure determination by crystallography.

Factors such as temperature, pressure, presence of a catalyst, and reactant concentration are all known to affect the rate of chemical reactions and are well exploited in industry to drive chemical synthesis to favor certain mechanistic pathways. Here we consider the effect of using magnetism to control the rate of reaction for the Briggs Ruascher reaction. This reaction was chosen as a model for magnetic manipulation because it is mediated by both radical and non-radical pathways that compete with oscillatory behavior to produce the same product. Dramatic color changes serve as a visual cue to indicate which path is dominating. Here we put forth magnetic radical arrest hypothesis for the Brigg’s Ruascher reaction. It is known that electrons act like tiny magnets that pair with opposite spins. Because radical species carry a lone unpaired electron, presumably their magnetic fields are not concealed. We proposed that due to the exposed nature of the unpaired radical electrons in the Briggs Ruascher reaction, the progress of the reaction would be affected by the application of a magnetic field. Upon exposure to strong magnets, the radical intermediates of the Briggs Ruascher reaction were expected to aligned their spins with the applied magnetic field affecting their reactivity. Several types of neodymium magnets with various strengths were applied to the reaction in order to determine whether magnetism could prevent or greatly reduce the velocity of the color phase changes for the radical mediated portion of the reaction. We also built an electromagnet to expose the reaction to in order to achieve even greater field strength. The Brigg’s Rouscher reaction was found to be significantly robust and there was insufficient evidence to suggest that the reaction was affected by neodymium fields below a range of 5.8 mT. Results of exposure to the electromagnet are still in progress. The color changing cycles of the Briggs Ruascher reaction were found to follow a decreasing sigmoid mathematical model before going to completion after 9-10 oscillations.

The purpose of this study is to determine whether blood-oxygenation level dependent (BOLD) variability could be a marker of neurodegeneration caused by Parkinson's Disease (PD). BOLD variability is related to higher cognitive performance and it decreases with age. Also, studies have shown that BOLD variability is correlated with lower levels of dopamine, a neurotransmitter that is decreased in PD and contributes to cognitive and motor symptoms. This motivates our hypothesis that BOLD variability may be correlated with cerebrospinal fluid (CSF) levels of alpha synuclein (α-Syn) and amyloid-β1–42 (Aβ42), markers that decrease with advanced PD. We obtained resting state and structural MRI data from 31 de novo PD subjects from the Parkinson’s Progression Markers Initiative (PPMI) cohort, who were scanned within 3 months of lumbar puncture. We first regressed out sources of noise such as scanner instabilities, subject motion, and physiological artifacts (e.g. respiration and cardiac effects). Second, we coregistered (registering one person’s brain to other brain image modality (rest to T1)) each data and conducted spatial smoothing. Then, we calculated the standard deviation of the BOLD signal (SDbold) and registered all the subjects’ brains to standard Montreal Neurological Institute (MNI) template. Finally, we computed the correlation between each subject’s SDbold and CSF markers We observed a significant correlation between SDbold and α-Syn in anterior cingulate gyrus and premotor cortex, areas that are known to be affected by PD. We did not see the same relationship with Aβ42. Our results suggest a specific relationship between physiological disruption related to PD and α-Syn that might be mediated by dopamine.

One limitation of chemotherapy drugs is their impaired ability to penetrate tight junctions between epithelial cancer cells to access the inside of a tumor. Adenoviruses have devised a method for moving between epithelial cells by virtue of binding adenoviral fiber proteins to the human protein desmoglein-2 (DSG-2) found within these junctions. When used in coordination with chemotherapy drugs, a recombinant adenoviral fiber protein, termed JO-1, was shown to improve the efficacy of the cancer drug by opening tight junctions thereby facilitating entry of the drug into the tumor mass. Mutagenic library studies with JO-1 identified point mutations that resulted in increased affinities for DSG-2. An affinity enhanced variant, JO-4, containing a V239D mutation is currently being evaluated for tumor co-therapy. Two additional mutations were identified, one (N293S) lead to an increased association rate, and a second mutation (Y250F) slowed dissociation by stabilizing the complex between the fiber protein and DSG-2. We hypothesized that combining multiple advantageous mutations in a single protein would result in an overall additive effect on affinity and activity. Utilizing more active proteins in cancer therapy can allow for smaller doses and higher potency of chemotherapy drugs in human patients. Two proteins based on JO-4 and containing the Y250F or the N293S mutations were designed in silico, cloned, and produced in E. coli. These proteins were then characterized for functional activities based on the ability to compete with adenovirus infection and the ability to enhance chemotherapy in mouse tumor models.  We expect the proteins with heightened affinity to DSG-2 will block virus infection at lower doses than JO-4 and in conjuction with chemotherapy drugs will stunt the growth of tumors in mice compared to chemotherapy alone.  

Arsenic is a toxic heavy metal to humans that can cause serious health damages. Exposure to arsenic can cause various health conditions such as skin damage, lung, bladder, kidney and liver cancer, as well as nerve and circulatory problems. Arsenic contamination in water and soil can be naturally sourced from local geology or human sourced from pesticide and preservative uses. Studies have shown that rice will uptake arsenic from water and soil. Rice is a very easily digestible grain which makes it very suitable source of nutrition for children, whom are more susceptible to toxins. Therefore, it is important to develop and continue the study of methods that can help to quantitatively detect arsenic in rice. For this experiment, the objective was to look at a previously published method used to detect arsenic and validate its effectiveness on five store-bought rice samples. The broader implication of this experiment is to validate a method and modify it to gain quantitative data in order to assess whether harmful levels can be detected and to determine background levels of arsenic in rice. Rice was powdered and 1.0 g of rice sample was digested in 10mL of 0.28M nitric acid for 90 minutes at 95°C. The samples were analyzed with ICP-MS to detect arsenic based on its mass. A calibration plot with a correlation coefficient of 0.96278 and a slope of 1856.48 was created. The calibration plot was made from concentration of 1ppb, 5 ppb, 10 ppb, and 50ppb in which all had intensities that were detectable. The detection value of the samples falls in between the calibration intensities. The data that is obtained suggests mean concentration of arsenic range from 1ppb to 30 ppb in 10mL solution of HNO3. This indicates that there is a range of 10 ppb to 300ppb per grams of rice. These data are corroborated by other studies analyzing levels of arsenic in rice.

Retinal degeneration is one of the leading causes for blindness in working-age people. It results from the loss of one or more of the seven different cell types in the retina which eventually disrupts the structure and signaling between the cells, ultimately leading to irreversible vision loss. Unfortunately, mammalian retinas cannot regenerate lost neurons, and current therapies only slow down disease progression. Alternatively, cell therapy using self-renewing, pluripotent embryonic stem cells (ESCs) could provide an unlimited source of replacement retinal cells. Many groups, including our own have developed protocols to derive retinal neurons from ESCs. While these can generate photoreceptors well, the generation of complete retinas from ESCs in a consistent and more efficient manner has remained elusive. Our research thus focused on optimizing the existing retinal differentiation protocols for mouse ESCs, with the goal of producing more laminated retinas consisting of all retinal cell types. In the current study, we used the three-dimensional method to produce embryoid bodies (EB) from our ESCs. We tested the effectiveness of Fetal Bovine Serum (FBS) and hyperoxia to enhance retinal differentiation by supplying them to our EBs. The samples were collected every week for five weeks and analyzed by immunohistochemistry. We found that both conditions increased production of inner retinal neurons such as ganglion cells and amacrine cells, and EBs grown under hyperoxia were healthier looking. We then compared the ESC-derived retinas to age-matured mouse retinas and found that both retinas were similar in structure with the different cell types organized in the correct arrangement. These results suggest that FBS and oxygen are additional factors that can be integrated into existing protocols to improve the differentiation of mouse and possibly also human ESCs into retinal neurons.