The lab I am involved with investigates the molecular events of neuromodulation, a form of neuronal chemical signaling that determines behavior, emotions, learning and memory. Neuromodulators are carried and released by dense core vesicles, a type of cellular vesicle which little is known about. Dr. Ailion's lab has performed genetic screens for C. elegans mutants that have altered dense core vesicle secretion and has isolated many unique mutants. During my time in Dr. Ailion's lab I focused on genetically mapping two of these mutants which were given the names yak90 and yak91. With yak90, I used a well-established mapping method where my mutant strain was crossed to marker strains which contained alleles causing obvious deformities on each chromosome. Generations later, I examined their offspring and was able to pinpoint one mutant phenotype that was not segregating according to Mendelian ratios, indicating linkage to chromosome IV. When a candidate gene was found near the linkage site, I did a complementation test and confirmed that yak90 contains a mutation in an already characterized gene, unc-31. With yak91, a new method was tested using a marker strain that contains alleles for multiple fluorescent tags instead of body deformities. This was necessary because yak91's mutation causes it to be "egl", or distended with mature eggs, and it was difficult to tell it apart from one of the marker phenotypes. The cross with the new strain was a success and after examining the progeny, linkage was found on chromosome IV. Another complementation test was performed, and yak91 was found to contain a mutation in ric-8. Total loss-of-function mutations in ric-8 are lethal so yak91 must contain a splice site or point mutation that significantly alters the function of the protein product. Both genes I identified play important roles in neuromodulation. 

In liver transplantation, increased overall fat content in donor livers causes poor graft function and lower patient survival post-transplant. Longer waiting lists combined with a shortage of organs has resulted in transplant surgeons considering moderately steatotic livers as a part of extended donor criteria. Clinically, the fatty infiltration is diagnosed by the lipid droplet size. Macrovesicular steatosis refers to large droplets or vesicles that distort the nucleus, while microvesicular steatosis refers to droplets equal to or less than the size of the hepatic cell nucleus. While macrovesicular steatosis negatively impacts graft function, livers with microvesicular steatosis result in patient outcomes that are almost the same as that of nonsteatotic livers. Therefore, a method to quantify the amount and droplet size of hepatic steatosis can be an innovative tool for transplant surgeons. In our previous studies, sound speed and attenuation correlated with the presence of visually observable fat. However, significant variabilities were discovered in the attenuation. The next step in this research involves exploring the effect of lipid droplet size on attenuation, using an attenuation spectroscopy method developed in colloid science research. If the total fat content is known, mixture rules based on a volume average of the fat and non-fat components can be used to estimate the intrinsic attenuation at a given frequency. Beyond this estimate, excess attenuation attributable to interactions between fat and non-fat phases of the mixture can be estimated using a coupled-phase model. Here, we quantitatively evaluate the excess attenuation caused by small lipid droplets by comparing attenuation measurements of well-characterized dairy products with model predictions. Results confirm that sub-micron sized lipid droplets produce a measurable amount of excess attenuation at 1 MHz. These results suggest that analogous measurements may be made in quantifying the amount of microvesicular steatosis present in livers with known overall fat percentage.

Chronic allograft injury has replaced acute rejection as the leading cause of renal graft failure following transplantation. Graft failure is of special importance to pediatric patients since they typically require multiple grafts in their lifetime and therefore will benefit from graft life extension (Smith et al, 2010). A relationship has been demonstrated between subclinical Cytomegalovirus (CMV) and subclinical Epstein Barr Virus (EBV) and chronic allograft injury previously. This relationship could lead to insight into the mechanism of chronic allograft injury. To characterize the relationship between subclinical infection and chronic allograft injury we measured T-cell specific response to subclinical CMV and EBV infections. A panel measuring three aspects of T-cell response: activation, memory, and cytokine response, was proposed and validated. T-cell response was quantified using flow cytometry on stimulated whole blood samples from study participants in an intracellular cytokine staining assay. Analysis of T-cell activation, memory, and cytokine production was done using a Boolean gating strategy on FlowJo software. Twenty-six patients are currently enrolled, with whole blood samples being collected every six months over the course of twenty-four months. We are in the process of banking, processing, and analyzing the samples. After all of the patient samples have been analyzed for T-cell memory, activation, and cytokine production, we will quantify the relationship between T-cell response and subclinical viremia. This data will be compiled in conjunction with the GFR, kidney biopsies, and CMV/EBV PCR data to create a longitudinal history, allowing us to understand how CMV and EBV viremia contributes to chronic allograft injury.

The purpose of this research is the development of a precisely electroplated set of vacuum capacitor electrodes for inclusion in the MAJORANA Demonstrator (MJD) - the first stage of the MAJORANA project. Funded by the Department of Energy the MAJORANA program is part of an international effort to study neutrinos, dark matter, and axions led by the UW Center for Experimental Nuclear Physics and Astrophysics (CENPA). The project focus is on making the capacitors with a physical dimension tolerance (<2 microns) that is far less than what can be obtained via machining or other comparable methods of manufacturing. The desired capacitors are made using high-aspect ratio patterns in silicon as an electroplating mold. Silicon wafers are coated with an electroplating seed layer and then bonded to each other using thermcompression wafer bonding. Thermocompression bonding is a technique used to bond wafers by applying heat and force simultaneously. Following bonding, the wafers are patterned, using a photolithography process and Deep Reactive Ion Etching (DRIE), to form the mold cavaties. Photolithography is a multiple step process that includes application and patterning of a light-sensitive material called photoresist. Next, bottom-up copper electroplating is performed until excess deposit of copper called over-plating is achieved. The over-plated structures are polished back to establish dimensional uniformity. The final step is releasing the electroplated structures via a blanket DRIE process. Successful completion of this research project will allow assembly of MJD which in turn will be used to search for neutrinoless double-beta decay – one of the rarest forms of radioactive deacy. This project will also be a primer for more industry-focused capabilities for the Washington Nanofabrication Facility (WNF) with eventual development of through-silicon vias (TSVs) for research applications and a potential foundry process for precision part creation without the limitations of machining.

Barth Syndrome is a rare X-linked genetic disorder that can cause cardiac myopathy, muscle weakness, fatigue, immune dysfunction, and childhood mortality. Like many such diseases, Barth Syndrome is the product of a mutation encoding for a single protein, Tafazzin, a phospholipid-lysophospholipidtransacylase found primarily in cardiac and skeletal muscle, where it plays an integral role in modifying cardiolipin, a component of the mitochondria, or energy production organelles of the body. When Tafazzin, and consequently the modified form of cardiolipin are absent, the function of the mitochondrialelectron transport chain, the primary pathway by which the body converts food into useable energy, is reduced. We hope to rescue defective cardiolipin modification through Tafazzin protein therapy and learn more about tafazzin structure and function. To this end we used X-Ray Crystallography to obtain a three-dimensional electron density map corresponding to the diffraction pattern of the protein, based on the distance and angle between each atom. The determination of structural properties illuminated binding sites and chemical interactions in both functional and mutant Tafazzin. This was supplemented by the synthesis of enzymatically active and stable human and mouse Tafazzin using E. coli (BL21(DE3)pLysS). Overexpression of Tafazzin results in relatively pure inclusion bodies which we we isolated through protein metal affinity chromatography. We produced several protein coding variants of Tafazzin in order to study the enzymatic activity of different mutants. Understanding of structure-activity relationships in Tafazzin will direct future work in Barth Syndrome treatment by facilitating  in vivo studies of Tafazzin protein delivery.

 This project presents the development of a new process flow in the Washington Nanofabrication Facility to create a magnetically neutral Nuclear Magnetic Resonance (NMR) test device for use at the Department of Energy (DOE) Pacific Northwest National Labs (PNNL). At PNNL, within the Environmental Molecular Sciences Laboratory, the devices are used to characterize environmental contaminants, like radioactive isotopes, using NMR spectroscopy. The devices being developed are to be superior in uniformity and layering a stack of thick copper sandwich with a precisely deposited aluminum layer. The project includes computer-aided design (CAD) and fabrication of a precision mask set for fine resolution that limits irregularities in the test structure that could adversely impact the NMR measurement. The fabrication process starts with an electroplating seed, photolithography, and thick electroplating of copper. Following plating, chemical mechanical polishing is performed to yield an atomically smooth surface. After this, a precision thickness aluminum layer, that cannot be conventionally electroplated, is deposited and patterned via a lift-off process. This project will also be a primer for more industry-focused capabilities for the WNF with eventual development of through-silicon vias (TSVs) for research applications.

The Occupy Movement represented the American contingent of the populist uprisings of Spain and Egypt. It adeptly deployed popular, personalized political involvement with mobilization of networking tools and crowd tactics to create the ubiquitous meme “We Are The 99%”. The emerging, novel form of protest embodied by Occupy suggests the possible trajectory of contemporary social movements but it has been criticized for failing to assert demands and institute changes. The divergence with traditional social movements who make claims on the state extends to the realm of rights. This raises the question of whether the absence of rights-talk reflects a broader ‘exhaustion of rights.’ To investigate this claim I drew on activists’ accounts as well as academics studying the mechanisms behind movement formation and development. I sought to establish a framework to understand activist’s choices to reject demands and whether this logic was consciously extended to rights. I also incorporated academic works to understand and situate the movement in the neoliberal state to which the movement was, in part, a response. I found that activists were limited by the movement’s internal structure created out of a desire to instantiate horizontality, the absence of leadership, and inclusivity, broad participation, that were crucial to its legitimacy. More importantly, the perceived dominance of neoliberalism made activists wary of institutionalizing or making demands on a system that disproportionately favored the 1%. Consequences of the neoliberal project have intensified the critique of rights, leaving rights-talk perilous or powerless for activists. This view of rights delimits rights to state-claims and ignores their unintended and potentially transformative capacities. Consequently, although rights existed within the movement, they were not extant in broader discussions, missing an opportunity to seize upon the platform created by the movement to contextualize novel rights claims giving them political salience.

Pseudo-nitzschia is a cosmopolitan genus of marine diatoms known for its production of the neurotoxin domoic acid (DA) and resultant harmful algal blooms. The production of DA varies across species; of approximately 37 defined Pseudo-nitzschia species, at least 14 have produced DA in laboratory studies. Previous work based on ribosomal RNA sequences suggested the genus Pseudo-nitzschia could be divided into two major clades, or groups made up of an ancestor and its descendants. With the discovery of new species and identification of cryptic species complexes, the structure of these clades has eroded. We sought to integrate phylogeny, morphology and physiology of Pseudo-nitzschia, by re-examining their phylogeny, using both ribosomal LSU sequences and the rbcL gene, which encodes the large subunit of the carbon fixation enzyme, ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO). Trees based on rbcL sequences provided higher resolution to resolve the branching orders of Pseudo-nitzschia species. By mapping physiological characteristics such as cell size, frustule morphology, pigment content and DA production onto the tree we can interpret the evolutionary history of these features and species distributions in the context of our presumed clades.

The cosmopolitan pennate diatom Pseudo-nitzschia is notable for production of the neurotoxin domoic acid. Recently, a virus was isolated on Pseudo-nitzschia multiseries that has a broad host range, infecting many genera of diatoms. This is contrary to previous reports of very specific host ranges amongst diatom viruses, and suggests a nested infection network whereby specialist viruses infect only the most permissive hosts, while generalist viruses can also infect hosts that are well-defended. To explore the infection patterns of Pseudo-nitzschia and its viruses, new Pseudo-nitzschia strains were isolated monthly at a coastal and estuarine location in the Pacific Northwest for one year. Over 100 new strains of Pseudo-nitzschia were identified by sequencing the ITS region. These new strains are then challenged with environmental viral communities that were sampled at each location concurrently. To date, 28 of 47 strains tested are susceptible to infection by at least one viral community. Three strains are lysed by the majority of viral communities, suggesting we have identified permissive hosts. This analysis will continue until all currently isolated strains are challenged. Seasonally, May and July contained viruses that infected the widest range of strains, suggesting either high concentration of generalist viruses in those months, or high concentration of both specialist and generalists together. Understanding the seasonal structure of these infection networks will inform future virus isolation attempts and provide insight into a key component of these diatom-virus interactions.

From 1951 to 1981, the Canadian weathership program collected nearly continuous wind and wave observations at Station Papa, a common reference site in the North Pacific. The length of this time series makes it a valuable tool in determining long-term trends and statistics of North Pacific wind and waves. Due to the uncertain quality and poor documentation of the data, extensive cross-referencing and quality control of disparate data sources was performed, leading to the development of a single, high-quality dataset. To verify the accuracy and consistency of the weathership measurements, histograms of wind speed, wave height, and wave period were compared with those from modern wave buoy measurements at Station Papa, taken since 2010. While the wind measurements aligned well with the newer data, the wave observations showed notable biases. These biases are likely due to the outdated method of visually observing waves from the weatherships. A time-series model with an annual oscillation and autoregressive component was fit to the wind measurements. Such a model accurately represents both the seasonal weather cycle and short-term storm pattern at Station Papa. In addition, it was found that the wind and wave statistics were significantly correlated on interannual timescales with the El Niño Southern Oscillation (ENSO) and Pacific Decadal Oscillation (PDO). Quantifying the impacts of these trends has practical implications for predicting future changes in wind and wave climate.

Muscle contraction results from interactions between thick and thin-filament proteins within the sarcomere of a muscle cell. The movement of tropomyosin (Tm) over actin on the thin filament exposes myosin binding sites and is a fundamental step in the regulation of muscle contraction. Single amino acid substitutions in Tm and regulatory mechanisms such as Tm phosphorylation can greatly impact muscle function. Tm monomers link end to end over actin, so shifting of one Tm monomer to expose myosin binding sites may cause strain in neighboring monomers via end-to-end interactions, resulting in cooperative behavior. The flexibility of Tm may play a role in modulating the strain induced in neighboring monomers, thus we chose to assess the flexibility of phosphorylation-mimicking and clinically relevant Tm mutants that are known to affect contractile function. We produced and purified recombinant Tm in E. Coli and imaged it using rotary-shadowed electron microscopy. The flexibility of single molecules was determined by skeletonizing Tm to determine its persistence length, a quantity from statistical mechanics that describes the curvature of rod-like objects. Skeletonization and analysis were performed manually and with a novel automated image analysis program. Molecular dynamics simulations were also performed with the clinically relevant Tm mutant to determine structural mechanisms that could lead to the altered contractile function observed in muscle containing mutant Tm. Preliminary results show decreased flexibility near the region of phosphorylation in phosphomimetic Tm compared to wildtype Tm. This result is expected because Tm phosphorylation has been shown to increase cooperative behavior and the phosphorylation site is located in the middle of the overlapping region of each Tm monomer.

Distal Arthrogryposis (DA) is an umbrella term for non progressive congenital skeletal muscle defect characterized by abnormal contractures of the limbs and the face. It occurs in 1 in every 3,000 live births and is inherited in an autosomal dominant manner. Currently, there are no treatments for the disease, and the patients usually go through regular physical therapy throughout their lifetime. Severe cases require surgical intervention and amputation. One of the more common syndromes is DA 2B, or Sheldon Hall syndrome, which is caused by mutations in two genes, TNNI2 and TNNT3. I am particularly interested in studying the R63H mutation that impacts the fast skeletal troponin T (TNNT 3) subunit. The troponin subunits are critical in regulating the interaction between actin and myosin myofilaments. Mutations in the TnT can alter the way the troponin subunit interacts with the tropomyosin and have large impacts on overall muscle mechanics. I hypothesize that this mutation increases the contractility and tension in mutated proteins. To verify the hypothesis, I will compare the wildtype to the mutated version of TNNT 3 by putting a fluorescent tag on the proteins and observe the differences in calcium sensitivity, ATPase, and maximum filamented actin velocity in a sliding filament assay known as the in vitro motility assay (IVM) to piece together information about how the mutation impacts the overall muscle mechanics. My project seeks to gather information about the mechanics of the mutated muscles. Putting all of the data together will enable me to identify causes in the mechanics behind abnormal contractions. Also, it will allow me to make connections between the genotype and the phenotype of the disease to better understand it on a molecular level, and thus open up doors for therapies.

Hypertrophic cardiomyopathy (HCM) is a heritable disease in which the heart undergoes pathological remodeling and thickening, leading to sudden cardiac death, especially in young adults. I am investigating how modifications to myofilament proteins, which are responsible for muscle contractions, contribute to HCM functional characteristics. My goal is to determine how the P82S mutation of cardiac troponin I (cTnI) alters the dynamic properties of cardiac troponin (cTn), such as the Ca²⁺ binding affinity to cTn and interactions between cardiac troponin C and cTnI (C-I interaction), as well as the overall effect of P82S on muscle activation and relaxation. I am testing the hypothesis that the C-I interaction serves as a critical point in contractile regulation, regulating systolic performance at basal activity and particularly during β-adrenergic stimulation. During β-adrenergic stimulation, the cardiac-specific N-terminus of cTnI is phosphorylated by protein kinase A (PKA), and the shifting protein conformation results in a cascade of conformational changes in regulatory proteins that promote myosin unbinding from actin, accelerating relaxation rates in cardiac muscle. This work combines state-of-the-art protein biochemistry, myofibril & cellular mechanics/kinetics and multi-scale computational modeling to study the behavior of P82S-cTnI with wild type (WT) cTnI. For each, recombinant troponin complexes containing WT-cTnI, P82S-cTnI, non-phosphorylatable cTnI (S23/24A), and phosphomimetic cTnI (S23/24D) are studied and compared. Initial results from the WT-cTnI variants show that with respect to the WT-cTnI, the S23/24A and S23/24D mutated troponin have decreased Ca²⁺ sensitivity to cTn, as well as the C-I affinity. Accompanying experiments are currently being done on the P82S variants. A better understanding of how protein structure and function is altered by mutations will bring the HAMM Lab closer to developing approaches to mitigate the impact of HCM-related mutations.

Emulsions of oil droplets decorated with gold nanoparticles can be used to enhance contrast in ultrasound and photoacoustic imaging. Plasmon resonance in the gold nanoparticles enables absorption of near infra-red (NIR) light, which can penetrate two to three centimeters below the skin in the human body. Upon irradiation with a NIR laser, oil droplets with a shell of gold nanoparticles will heat and vaporize as the gold absorbs the light. The vaporization of the oil creates a microbubble, providing contrast enhancement in imaging as well as a mechanical pressure wave which could be used to deliver mechanical forces inside the body. If the gold nanoparticles are functionalized with ligands that are designed to target specific areas or threats inside the body, the process could also be used to deliver targeted therapy. Emulsions are thermodynamically unstable and will coalesce over time without external stabilization. We utilize gold nanoparticle surfactants to stabilize the oil-water interface. We investigated a charge based stabilization of perfluorohexane emulsions with cationic gold nanoparticles. Size is characterized by Dynamic Light Scattering (DLS). Successful adsorption of the cationic gold nanoparticles to the oil-water interface is represented by zeta potential, which approximates surface charge. Emulsions of oil have a negative surface charge and the presence of cationic gold at the interface will result in a positive charge. Ultra-Violet Visible Spectroscopy (UV-Vis) is used to measure the NIR absorbance of the emulsions, which should be maximized to enable rapid heating of the gold nanoparticles and subsequent oil vaporization. Stability over time is determined by changes in the DLS and UV-Vis curves. The results of the investigation are analyzed in terms of size, stability, and NIR absorbance in order to determine the potential effectiveness of a charge based stabilization mechanism in creating plasmonic emulsions for contrast agents and therapeutics.

The pancreatic islet β-cell secretes insulin in response to nutrients such as glucose. In type 2 diabetes, insulin secretion by β-cells is impaired, causing hyperglycemia. Our group investigates this impairment, with the goal of identifying and understanding the underlying mechanisms. Pancreatic islets contain an extensive capillary network, including endothelial cells, which produce soluble factors that may modulate β-cell function. Specifically, our group has shown that media from islet endothelial cells cultured under control conditions (5.5 mM glucose) and subsequently incubated with islets enhances insulin secretion from those islets. In contrast, media from islet endothelial cells cultured in diabetic conditions (high glucose; 25 mM) shows no effect on insulin secretion. Thus, we hypothesize that islet endothelial cells normally secrete paracrine factor(s) – signaling molecules produced by one cell that cause particular reactions in nearby cells – that enhance glucose-stimulated insulin release but that in diabetes, production of these paracrine factor(s) is reduced. The goal of this project is to identify one or more of these paracrine factors. We cultured islet endothelial cells under control and diabetic conditions. mRNA was extracted and sent for whole transcriptome analysis (RNA-Seq), creating an expression profile showing the expression amplitude for each gene. Analysis of RNA-Seq data revealed patterns of differential gene expression between control and diabetic samples. Changes in expression for genes of interest identified by RNA-Seq are being verified by quantitative real time PCR (qPCR). Subsequent protein analysis via ELISA or western blot will confirm the RNA-Seq and qPCR data. Viral vectors or siRNA will be used to either overexpress or silence respectively the gene(s) of interest in islet endothelial cells. Media will be collected from these cells and used on islets to determine its effect on glucose-stimulated insulin release. This project should reveal novel mechanisms by which islet endothelial cells influence insulin release.

Cyanide-bridged transition metal complexes serve as model systems to study photoinduced charge transfer reactions. These complexes are important to understand because they play a major role in processes such as photosynthesis and the conversion of light to electronic energy in solar cells. Using RuCl₃•XH₂O salt, 2,2':6',2"-terpyridine (tpy), 2,2'-bipyridine (bpy) and KCN we synthesized [Ru(tpy)(bpy)CN]²⁺, [(tpy)(bpy)Ru-{CNRu(tpy)(bpy)]³⁺ and [(bpy)₂Ru-{CNRu(tpy)(bpy)]⁴⁺. These multistep syntheses were monitored using FTIR, UV-vis and ¹H NMR in order to verify that the compounds of interest were made. These compounds are interesting because of their metal-to-ligand charge transfer and metal-to-metal charge transfer processes when excited with visible light.  In the future these complexes will be studied using nonlinear spectroscopies in the visible and infrared regions of the spectrum in order to investigate their charge transfer capabilities.

 Impaired systolic function resulting from acute injury, congenital defect, or aging can lead to cardiac complications and heart failure. Current therapies are unable to rescue cardiac function, but merely slow disease progression. We have demonstrated that when myosin uses 2-deoxy-ATP (dATP) as an energy substrate for contraction instead of ATP, cardiac contractility is enhanced by 20% or more. Viral-vector mediated overexpression of R1R2, the enzyme that catalyzes production of deoxy-nucleotides, increases intracellular dATP. In this study we determined the expression pattern of R1R2 in mice following systemic delivery of recombinant adeno-associated viral vectors pseudotyped with the type 6 capsid (rAAV6) carrying R1 and R2 subunits, and a cardiac specific gene regulatory cassette (cTnT455). Cardiac function was assessed in vivo via echocardiography, and ex vivo using Langendorf perfusion to test the hypothesis that elevating myocardial dATP by a gene therapy approach enhances cardiac function. We also performed metabolomics evaluation of both skeletal and cardiac muscle to correlate with changes in cardiac function. Our current data suggest that systemic delivery of this rAAV6 system results in myocardial specific overexpression of R1R2 and improves basal cardiac performance, which supports our previously published data for transgenic animals with long-term overexpression of R1R2. Metabolic analysis suggests that R1R2 overexpression in the heart alone alters the glucose metabolism of both skeletal and cardiac muscle, indicating improved basal cardiac performance can directly impact skeletal muscle metabolism. Preliminary studies in an aging model demonstrate that treatment with this rAAV6 vector increases oxidative metabolism in cardiac tissue and increases glycolysis in fast twitch skeletal muscle. These data indicate that vector-mediated overexpression of R1R2 improves basal cardiac function and impacts skeletal muscle metabolism, demonstrating potential to improve cardiac function of patients with systolic dysfunction. HL11197.

Simian immunodeficiency viruses (SIVs) naturally infect over 40 species of African primates. From these SIV strains, only two have crossed to humans giving rise to HIV-1 and HIV-2 which have now infected over 40 million people worldwide leading to several million AIDS-related deaths per year. The goal of this project is to understand what determines the ability of the SIVs to cross between hosts and to evaluate the risk of future SIV cross-species transmissions to humans. To efficiently transmit between species, an SIV needs to be able to overcome the multiple immunological barriers imposed by a new host or rapidly adapt to gain this ability. Several of these barriers are encoded by the antiviral proteins that limit virus replication and thus constitute an innate immunological response against lentiviral infection. In this study, we studied the evolution and adaptation of SIVs during cross-species transmission by identifying the amino acid mutations that the viral protein, Vif, needs to undergo to antagonize the human antiviral protein APOBEC3G (A3G). Since, A3G has been described as one of the major barriers for cross-species transmission its degradation by the virus might facilitate SIV transmission to humans. In order to identify the adaptive mutations required to gain anti-human A3G activity, a virus encoding for an HIV-1 backbone and a SIV Vif protein was serially passaged in a human CD4+T cell line expressing human A3G. Preliminary data suggests that in short-term infection the virus is not able to overcome human A3G. We will continue to passage the virus to determine whether or not viral replication is rescued which would indicate that Vif acquired resistance to A3G. The acquired resistance then will be mapped to specific amino acid mutations through sequencing. To test the anti-human A3G activity of these mutations, molecular clones of the virus containing the mutations will be generated using site-directed mutagenesis and tested in specific A3G degradation assays.

Glucose and glutamine are primary carbon sources for cells to produce biomolecules and energy. Increased aerobic glycolysis, de novo fatty acid synthesis (FAS), and glutaminolysis are hallmarks of cancer cell metabolism and many tumors are dependent upon these for their survival. Likewise, virally infected cells also have considerably altered metabolic requirements. Our lab studies Kaposi’s Sarcoma Herpes Virus (KSHV), the etiologic agent of Kaposi’s Sarcoma (KS). KS is the most common tumor found in AIDS patients. Previous work from our lab showed that latent KSHV infected endothelial cells also require increased aerobic glycolysis and FAS. Our lab also published a global metabolic analysis of latent KSHV infection, revealing that latent KSHV infection also induces increased glutamine levels. Exogenous glutamine is metabolized through Glutaminolysis; glutamine is converted to glutamate and alpha-ketoglurarate (αKG) through two key enzymes, glutaminase (GLS) and glutamate dehydrogenase (GDH). Further, αKG can enter the tricarboxylic acid cycle (TCA) and support ATP production. Cancer cells utilize glutamine to replenish the TCA cycle when glucose is primarily used to produce lactate and fatty acids. Preliminary data shows that exogenous glutamine is required for latent KSHV infection through starving infected endothelial cells of glutamine. While glutamine starvation does not reduce viability of mock-infected endothelial cells, the absence of glutamine leads to significant cell death in KSHV infected cells. I hypothesize that glutamine is required during latent KSHV infection to replenish the TCA cycle. To study this, I inhibited glutaminolysis using a compound called BPTES, an inhibitor of GLS. Inhibiting glutaminolysis also leads to significant cell death of KSHV infected cells. This data indicates that glutamine is required during latent KSHV infection to replenish the TCA cycle. In the future I will add back TCA cycle intermediates to rescue cell death. Through our metabolomics studies of latent KSHV infection, we are identifying potential targets for KS therapy.

This project will use historical and contemporary photographs to test the hypothesis that the average size of salmon has noticeably declined over the last two hundred years in the Salish Sea. Native Americans and early settlers described the population of salmon species in this area with metaphors such as, “you could walk across a river on their backs,” but quantitative measurements began long after human impacts had diminished their populations. The sizes and populations that we record today are drastically smaller than they once were. Salmonids that once weighed over one hundred pounds now weigh closer to twenty pounds, on average. It is believed that human impacts are to blame for the decline of these salmon species. Shifting baselines is the idea that the benchmark of fish size has shifted over the years. We are building upon the research of author Loren McClenachan, whose work was published in Conservation Biology (June, 2009) documenting the reduction of large fish in the Florida Keys. We will be using her methods of reviewing and collecting copies of historical photos to document the considerable decline in size of Pacific Northwest salmonids over time. This research is especially significant because it is the first application of McClenachan's methods to the Salish Sea. By acknowledging former baselines of size and population, we can improve our attempts at restoring our water ways and salmonid habitats.

Kaposi’s sarcoma-associated herpesvirus (KSHV), is the infectious agent that causes Kaposi’s sarcoma (KS)—a tumor that is the most prevalent cancer found in untreated HIV-infected individuals worldwide. KSHV establishes predominantly latent infection both in cells in vitro, as well as in KS tumors. KSHV, like all viruses, lacks an independent metabolism, and it has been shown to dramatically alter host cellular metabolism, particularly in carbon utilization pathways. Similar to cancer cells, latent KSHV-infected cells require increases in the most abundant carbon sources, glucose and glutamine for survival. However, it is still unknown how glutamine is utilized during latent KSHV infection. Glutathione (GSH) is the most abundant antioxidant in eukaryotic cells. GSH is essential for cell survival because it functions to stabilize reactive oxygen species (ROS), which are deleterious to the cell. I hypothesized that latent KSHV-infected cells require glutamine to produce GSH and to stabilize ROS in infected cells. KSHV-infected cells have been shown to be under chronic oxidative stress, and increased levels of GSH could explain how latent KSHV-infected cells inactivate excess ROS to avoid cell death. To test my hypothesis, I will determine the abundance of GSH in uninfected samples compared to that of KSHV-infected samples. I will use a fluorescent GSH detection reagent and measure GSH absorbance through a spectrophotometer or flow cytometry. Secondly, to determine if glutamine is the necessary precursor for GSH production, I will compare GSH levels between KSHV-infected samples in the presence of all carbon sources and KSHV-infected samples starved of glutamine. If glutamine is the required carbon source, I expect to see decreased GSH levels in KSHV-infected cells in the absence of glutamine. This finding could reveal important drug targets for future strategies to inhibit and treat latent-KSHV infection and ultimately KS tumors.

Phylogeography is the study of the geographic and spatiotemporal distributions of populations, and it is an important tool for investigating the evolutionary history of species. Determining when and where species originated, merged, or moved is critical in understanding the factors that generate biodiversity. Novel Bayesian phylogeographic methods provide a useful tool for obtaining accurate estimates of these important population dynamic parameters. West Africa is a global biodiversity hotspot with a high number of endemic species, and Agama lizards are a diverse and prominent member of this distinctive ecoregion. To investigate the phylogeography of Agama lizards, I constructed phylogenetic trees from over 300 unique samples of Agama lizards from 20 countries using Bayesian phylogeographic inference. Molecular genetic data from a quickly evolving gene (16S rRNA) provided sufficient variation for estimating the divergences between distinct populations. Visualization of the phylogeographic history for the populations was conducted with Google Earth. This group of species is predicted to have originated in the central part of West Africa (i.e., Cameroon, Nigeria, and Equatorial Guinea). At least five distinctive species formed across the region from that one ancestral population, and one paraphyletic group requires further investigation. Outlining the history of species formation in subtropical West Africa, with an emphasis on when and where populations divided, moved, and reconnected, serves to show the benefits of phylogeographic studies as they can reveal patterns that are otherwise too difficult to detect using other approaches. These studies also serve as tools in providing information for the detection of distinct species.

After a myocardial infarction (heart attack), affected areas of the heart have limited function and capacity for regeneration. Injectable therapies with human embryonic stem cell-derived cardiomyocytes (immature heart cells derived from stem cells) can improve the function of damaged tissues, but graft-cell integration with the host is limited. Studying the migratory behavior patterns of these cells may provide insight to understand the basic biology of how the heart develops and may suggest new approaches to improve graft-cell integration. Toward this end, I have tested the hypothesis that a low-cost, high-throughput gap closure assay can be used to identify novel small-molecules that will promote the migratory behavior of human embryonic stem cell-derived cardiomyocytes. In particular, I developed a modified gap-closure assay that can be employed in the University of Washington's small-molecule screening core. Using a bottomless 384-well cell culture plate as the main platform, I used surface-modifying plasma lithography techniques to create a bottom surface that has alternating high- and low-affinity regions. Cells seeded into the multi-well plates with this patterned bottom initially adhered in the high-affinity regions and I used microscopy to monitor their migration into the low-affinity areas during exposure to various small-molecules introduced by robotics. To ensure these results were valid, I tested the assay using previously identified pro-migratory factors as a positive control. This assay can be a customizable and low-cost alternative to commercially available products, which is beneficial for studies requiring mass screening. Although this high-throughput assay was used to study the migratory behavior of stem cell-derived cardiomyocytes, it can be used for a variety of cell types.

Chronic heart failure as a result of damaged myocardium affects millions of individuals around the world. Our laboratory has shown that human embryonic stem cell derived cardiomyocytes (hESC-CMs) are capable of improving contractile function and decreasing arrythmias in a guinea pig model of myocardial infarction (Shiba et al. Nature, 2012). We have also shown that hESC-CMs migrate in response to gradients of the soluble extracellular matrix protein Wnt5a in vitro (Moyes et al. Stem Cells Dev, 2013). We hypothesize that a gradient of Wnt5a, delivered by sustained release from gelatin microspheres, will drive the migration graft hESC-CMs towards healthy host tissue and increase the physical contact between graft and host cardiomyocytes. We fabricated gelatin microspheres by an oil/aqueous phase emulsion and controlled their diameter to range from 4-6 um in order to prevent capillary obstruction. We loaded Wnt5a into the microspheres overnight and characterized the release kinetics over 14 days through an ELISA assay of samples taken throughout that time. We verified the bioactivity of the eluted protein with a cell-based fluorescent response to a sample at a late time point. The migration-inducing effects of the sustained Wnt5a gradient on hESC-CMs can be modeled in future via in vitro, three dimensional collagen gel seeding experiments. The resultant in vitro data will inform future in vivo transplantation studies in which we will test the ability of Wnt-5a loaded microspheres to direct the migration of grafted hESC-CMs to locations where they can couple with host myocardium and more effectively contribute force generating units to a failing heart.

On September 1st, 1991 a large nation state of Soviets died, giving birth to the Republic of Uzbekistan. As a result, thousands of non-Uzbek people became citizens by default in the newly independent republic. They had no chance but to stay in Uzbekistan – now their only homeland. Uzbekistan is a highly understudied region, especially when it comes to the national identities of Russian-speaking population that used to be labeled as the Soviets. The main research question is how linguistically russified non-Russian youth identify their national/ethnic identity in Tashkent, Uzbekistan today? I hypothesize that the majority of this population in Tashkent connect themselves to the Uzbek culture while integrating Russian cultural culture in their everyday lives. Russian speaking population today, though varying in gender, ethnicity and class, has similar experiences thus forming an identity that we can name as “Central Asians.” Methods I have employed are my positionality (I am a Russian-speaking citizen of Uzbekistan), interviews (informal interviews conducted in Uzbekistan with 5 adults), and participant observation (done in the summer of 2013 in Tashkent, Uzbekistan). The research sample are young adults between the ages of 20 to 22, raised after the collapse of the Soviet Union with Russian as their native tongue, but mixed ethnic backgrounds (linguistically russified non-Russians) who live in the capitol of Uzbekistan, Tashkent. The findings provided by this research are a significant step in understanding national formations in understudied Uzbekistan. Theoretically, studying independent formation of national identity of ethnic minorities in the post-Soviet nation-states can help understand identity formation of ostracized groups at the time of the mobilized nation building promoted by the government in the region. Urgent need for such research in post-Soviet territories is highlighted by the recent events in Ukraine and Crimean territory.