Transforming growth factor-beta (TGF-β) signaling in smooth muscle cells (SMC) contributes to normal vascular development but may also play a significant role in the progression of aortic disease. Accordingly, TGF-β signaling is emerging as a therapeutic target, with strategies to block TGF-β signaling proposed as a means of preventing human aortic disease, including aortic aneurysms and dissections. However, because TGF-β signaling contributes to normal vascular development, blockade of TGF-β signaling could be harmful. We hypothesized that elimination of TGF-β signaling in aortic SMC of adult mice would cause aortic disease. We used inducible Cre-lox recombination technology to knock out Tgfbr2 in adult murine SMC. Tgfbr2 encodes TGF-β receptor II, believed to be essential for all TGF-β signaling. Mice were euthanized 4-14 weeks after SMC Tgfbr2 knockout, and effects of Tgfbr2 knockout (compared to control mice) were assessed by gross observation of aortas as well as sectioning and staining of aortic tissue. We found intramural hematomas, penetrating aortic ulcers, aortic dissection, and aortic dilation as well as significant thickening of the medial wall and adventitia in the knockout mice. Our data strongly suggest that loss of TGF-β signaling in SMC has a negative effect on aortic health and support a cautious approach in pursuing blockade of TGF-β signaling as a therapeutic strategy.

Polymeric micelles are nanoscopic structures constructed with amphiphilic diblock copolymers constructed with hydrophobic and hydrophillic portions that spontaneously form micelles in solution. Small hydrophobic molecules, such as cancer drugs taxol and doxorubicin, can be encapsulated in the micelle, which effectively serves as a nanovehicle for drug delivery. Here, we will develop a diblock copolymer integrating polyurethane-based self-immolative polymers (SIPs). SIPs are polymers that sequentially disassemble into their building blocks once the disassembly process is initiated by a triggering event at the trigger head group. By using SIPs, the polymers that make up the micelle are designed to depolymerize which we hope to increase the efficacy of payload release and remove the ability for micelle reformation. For this project, the micelle and all parts of the system will be synthesized from the bottom-up; optimizing the molecular weight of the diblock for micelle formation, investigating the kinetics of depolymerization and thermal trigger activation, and examining the stability of the micelle.

Cocaine and other drugs of abuse have destroyed the lives of many individuals as those who take such drugs struggle with the powerful vice of addiction. Such drugs have been shown to cause changes in the brain, specifically in the cortex and the striatum, which has been linked to the manifestation and maintenance of addiction. Within the striatum there exists cell populations of neurons, which vary in their outputs to the brain and are regulated by glutamate inputs from the cortex. The involvement of these individual cell populations in addiction, including how they are regulated by cortical glutamate, is not yet fully understood. In order to investigate this issue, I collect data in and run rat behavior experiments with rats that have been treated with viral vectors, which temporarily decrease neuron activity in specific cell populations when activated by specific ligands. After the behavioral data has been collected I process the tissues by slicing the brains and stain them with techniques such as immunohistochemistry. Then I determine if the vectors were actually present in the expected cells due to their fluorescence after immunohistochemistry procedures. In some of the work that I have done with Kerry Kerstetter we have found that decreasing activity in cortical glutamate projections to the striatum resulted in an increase in motivation for cocaine. A better understanding of the intricate pathways involved in drug motivation will allow for better treatment and care for those suffering from addiction. Our hope is to provide more information on such pathways to help progress the development of future treatments for victims of addiction.

 The development of drugs is essential for human health. Approximately 40-60% of all currently prescribed drugs target a specific type of membrane protein called a G-protein coupled receptor (GPCR). In the human body, the binding of a neurotransmitter or hormones to a GPCR transmits signals across the membrane to bring about an intracellular response, which ultimately permits cells to communicate with each other and regulate body physiology. The Hague lab studies the molecular mechanism of drugs targeting GPCRs. Currently, we are investigating an important structural portion of certain GPCRs called the PDZ binding motif, which is a site for protein-protein interactions. My role in the lab focuses on the somatostatin (SST) receptor subfamily, which regulates the endocrine system and neurotransmission by inhibiting the release of secondary hormones produced in the gastrointestinal tract and pituitary gland. A drug that targets SST receptors is octreotide. This drug inhibits growth hormone and insulin release, and is FDA approved for the treatment of growth-hormone producing tumors and GI tract disorders. Interestingly, all five SST GPCRs contain this PDZ-binding motif, and my research examined the importance of this motif using a variety of biochemistry techniques that quantified the expression, localization, signaling, and protein-protein interaction network. I created SST receptor mutants lacking the PDZ-binding motif using polymerase chain reaction and then transfected the SST mutants into human cells. These novel clones were subjected to plasma membrane assays, confocal microscopy, and mass spectrometry analysis. I found that removing the PDZ-binding motif had minor impact on overall receptor function, but more studies are necessary to determine the role of the PDZ-binding motif for SST receptor function. Our ultimate goal is to identify novel PDZ-interacting proteins for GPCRs, and then disrupt or enhance this interaction with novel small molecules to treat disease.

Proteins perform a vast array of functions within all living organisms. These functions are heavily dependent on the protein’s 3-D structure. Cellular stress can result in the loss of protein structure and can lead to a wide variety of problems, one being the formation of insoluble protein aggregates. Aggregates can inhibit proper protein function, disrupt cellular homeostasis, and are implicated in many diseases. There exists a family of proteins, known as the Small Heat Shock Proteins (sHSPs), which are thought to combat aggregate formation. They interact with misfolded and aggregate-prone proteins (clients) to delay the formation of insoluble aggregates. How sHSPs delay aggregation is not well understood. I seek to characterize interactions between sHSPs and their clients. A binding interaction between the sHSP αB crystallin and the model client Δ131Δ (a mutant of staphylococcal nuclease) has previously been characterized by NMR. My goal is to confirm this interaction and look for additional interactions between these two proteins through chemical cross-linking. Chemical cross-linking is a process that links two proteins that interact. One can use this method to detect which surfaces of proteins are involved in a protein-protein interaction. The chemical cross-linker I am using is incorporated as an unnatural amino acid (p-benzoyl-L-phenylalanine, or BpA) into the sHSP αB crystallin. Upon exposure to UV light, BpA can covalently cross-link to another protein if it comes into close contact with it. This approach can allow us to explore how sHSPs interact with the client Δ131Δ. By gaining an understanding of this specific sHSP-client interaction, I hope to provide insight into general mechanisms of how sHSPs interact with clients, a process that plays a critical role in cellular health.

Carboxylic acids are ubiquitous in chemistry and used in a variety of industrial applications, ranging from polymer precursors to food preservatives. Modern routes to synthesize these compounds often involve specialized equipment, harsh conditions, and potentially toxic reagents. An alternative synthetic route is the aldehyde-water shift reaction, in which an aldehyde is oxidized by water to form a carboxylic acid and molecular hydrogen. The use of water as both solvent and reagent could potentially reduce the impact of aldehyde oxidation on the environment, especially when compared to the routes . A series of iridium-, rhodium-, and ruthenium-based complexes have been synthesized and investigated for activity in catalyzing this reaction. Using ¹H NMR (proton nuclear magnetic resonance) and GC-FID (gas chromatography) to analyze reaction products, we have observed many of the catalysts to disproportionate aldehydes to alcohols and carboxylic acids in competition with the desired dehydrogenative oxidation to carboxylic acids. Mechanistic studies and catalyst optimization for dihydrogen production are presented.

Autophagy is the process in which eukaryotic cells selectively degrade proteins and organelles. Autophagy has been well documented in organisms ranging from yeast to mammalian cells, with demonstrated roles in response to starvation, aging, and stress. However, there has not been an effective method for measuring autophagy in C. elegans, despite C. elegans being a popular model organism for the study of disease and aging. We refined an assay that uses double-fluorescent protein tagged Atg-8/Lgg-1 to measure autophagy in both yeast as well as C. elegans. This assay reports an increase in autophagy in response to starvation in both model organisms. We then applied this to the study of nematodes containing a mutation in Rheb, an important protein in the upstream signaling cascade that regulates autophagy. Autophagy is damped by signaling through the TORC1 pathway. A mutagenesis screen revealed two different worm mutants (ENU9 and ENU10) which are predicted to hyper-activate TORC1 signaling. Using the aforementioned assay we found that ENU9 and 10 mutants have dramatically reduced levels of autophagy. This work has implications for further research in Alzheimer’s, Parkinson’s and related diseases associated with ageing and proteostasis.

High Density Lipoprotein (HDL) particles are spherical structures, which play a fundamental role in the reverse cholesterol transport (RCT) pathway, where cholesterol is carried to the liver for excretion. Apolipoprotien (Apo) A-1 is the primary protein component of HDL and plays a critical role in the RCT pathway. As lipid and cholesterol are transported from cells, they are encircled by Apo A-1, forming discoidal pre-ß-HDL particles, which subsequently undergo cholesterol-dependent maturation via Lecithin-Cholesterol Acyl Transferease (LCAT), forming spherical HDL. Man-made HDL or reconstituted HDL (rHDL) have many applications in nanotechnology, including cellular delivery of antigens and small interfering RNA. However, the large size distribution of HDL made from full length Apo A-1 raises difficulties in relation to drug delivery variability. Currently, I am using shorter constructs of Apo A-1 lacking the N-terminal globular domain as a membrane scaffold protein for nanodiscs – which resemble pre-ß-HDL particles, but differ in their more uniform size range (~3%). We hypothesize that treatment of nanodiscs containing cholesterol with LCAT will result in spherical HDL-like particles with a more narrow size window. We have demonstrated that LCAT-mediated maturation of cholesterol-containing nanodiscs yields HDL-like particles. This reveals that the N-terminal region is not necessary for maturation. Next, I aim to optimize experimental conditions for generating HDL-like structures and characterize their polydispersity via analytical ultracentrifugation. The insight gleamed from this study will establish a base line for using matured nanodsics as drug delivery vehicles with a uniform size range.

Tuberculosis is the second-leading cause of death due to a single pathogen, Mycobacterium tuberculosis (Mtb), with an estimated two billion people currently infected. Drug-resistant strains have emerged that are unmanageable even with combination drug therapies. Therefore, the development of novel drugs aimed at previously untargeted biochemical pathways in Mtb is of utmost importance. Among potential candidates is a newly identified proteasomal degradation pathway termed pupylation. In this system, proteins in Mtb damaged by the reactive oxygen and nitrogen species of the host’s immune response are tagged with the small prokaryotic ubiquitin-like protein (Pup) and shuttled to the 20S proteasome for degradation. Pup-mediated degradation allows Mtb to recycle key cellular components and remain virulent. Our interest is to study the two key enzymes required for pupylation: the proteasome accessory factor A (PafA) and the deamidase of Pup (Dop), which is also involved in the reverse pathway known as depupylation where Pup is removed from the substrate. Our interest is to characterize the mechanisms and substrate scopes of both Dop and PafA to facilitate the design of novel inhibitors. We synthesized and employed a model fluorescent probe that mimics the site of pupylation on a target protein to study the rates of the pupylation and depupylation enzymatic reactions. Since the Pup-probe conjugate is a substrate for Dop we also studied depupylation. We anticipated Dop to be the faster enzyme since it had fewer substrates to bind than PafA and our studies have revealed that Dop is much faster. Though the purpose of depupylation is currently unclear, our investigation has determined the relative kinetics of these opposing enzymatic pathways. The quicker kinetics of depupylation indicates pupylation of proteins also serves as a post-translational regulator rather than existing exclusively for degradative purposes.

The prevalence of HIV cases in the African born national population versus the African American or national population in general is notably high. In Washington state, there are approximately four times more HIV cases for the foreign born black male versus the African American black male along with about eleven times more HIV cases for the foreign born black female compared to the African American female counterpart. Stigma can impact medication adherence, quality of treatment and undiagnosed contributing mental disorders that, when treated, can improve one’s HIV status. However, little is known about how HIV stigma impacts the treatment and intervention outcomes for the African-born population. My lab as well as my individual role in this research project aims to analyze the nature of HIV stigma among this population, which will provide fundamentals of an intervention that is most ideal for African born population. We conducted semi-structured qualitative interviews with African born patients receiving treatment at Harborview Medical Center Madison HIV Clinic and those receiving social service at BABES YWCA network, and then I as the primary coder analyzed the data through ATLAS.ti, analytic software. Preliminary results indicate that stress due to community paranoia and disturbances due to family history of HIV are common obstacles faced by this population. These findings have important implications for focusing on treatment and other interventions for this understudied population.

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.

In spite of rapid advances in detection and treatment, cancer remains one of the most deadly diseases, killing over 1500 Americans each day. The ability of cancer cells to metastasize is the driving force behind the high mortality. For this reason, early detection and improved treatment technologies are crucial to enhance the survival rate of patients. The progression of cancer pathology begins with the accumulation of DNA mutation that transforms a normal cell into a proliferating tumor cell. This unregulated cell growth is governed by disruption in reactive oxygen species, iron homeostasis, and up-regulation of ribonucelotide reductase. Studies show chelators have significant antiproliferative properties reducing tumor growth through iron chelation. Unfortunately, they also bind to iron in normal cells, therefore, there is a great need to develop chelators that bind specifically to iron in cancer cells to prevent cell proliferation. Our study focuses on the design of a new class of site-directed iron chelators using gold nanoparticles (AuNPs) as targeted molecular carriers. These AuNPs are capped with thiolate ligands that are modified using EDC/NHS coupling chemistry for attachment of chelators and targeting moieties that recognize cancer cell surfaces. We will present the synthesis strategy and characterization methods used for confirming the presence of chelators on the AuNP surface. These methods include ultraviolet-visible, fluorescence, infrared, and dynamic light scattering spectroscopies. Metal binding studies demonstrating iron uptake will also be presented.

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. 

The lateral habenula (LHb), is an important regulator of midbrain dopaminergic systems that are known to be involved in cocaine reinforcement. The LHb exerts an inhibitory effect on dopaminergic neurons in the ventral tegmental area (VTA), though interestingly LHb-VTA projections are known to be glutamatergic (excitatory). The rostromedial tegmental nucleus (RMTg), a recently identified GABAergic (inhibitory) nucleus, that receives direct excitatory inputs from the LHb, has been proposed to be the source of this inhibition of the VTA. Here, firstly, we injected rats with anterograde TAMRA-Lys-Dextan and retrograde FluoSphere tracers in the LHb and RMTg, respectively and demonstrated that the LHb indeed projects to both the VTA and RMTg. Secondly, since the RMTg has no known anatomical boundaries, we gauged the anatomical location of the RMTg using expression of the transcription factor cFos, a marker of neural activation in response to cocaine. A significant increase in cFos expression was found in rats injected with cocaine (10 mg/kg i.p). Finally, we utilized the Designer Receptor Activated Exclusively by Designer Drug (DREADD) technology, to investigate the role of modulating the activity of the LHb on cocaine-self administration. Rats were implanted with jugular venous catheters and trained to self-administer cocaine (0.75 mg/kg/infusion). We found that DREADD-mediated transient silencing of the LHb through the activation of inhibitory G-protein-coupled signaling pathway with clozapine-N-oxide increases cocaine self-administration. In contrast, activation of the excitatory G-protein-coupled signaling pathway decreases self-administration on a progressive ratio schedule. We further show that inactivation of LHb inputs to VTA neurons have no effect on cocaine self-administration. We now propose to investigate the role of LHb projections to the RMTg using a dual-virus strategy using a combination of Cre-dependent DREADD’s and canine adenovirus expressing Cre (that gets transported retrogradely) to specifically target the LHb to RMTg projections for DREADD-mediated modulation on self-administration.

As we move our energy production and consumption economy towards a cleaner, “green” energy paradigm, it has become clear that research into solar energy harvesting is of real world significance. While fundamentally challenging, the capture and storage of solar energy into chemical bonds, such as hydrogen, can be envisioned as a large scale energy solution. In nature, the champion of solar water splitting for energy production and storage is Photosystem I and II (PS I and II). After a photon absorption event, the manganese oxygen evolving center (OEC) in PS II increases its reductive capacity enough to oxidize water into its oxygen and hydrogen constituents. Following the water oxidation reaction, the manganese oxidation state returns to the original to further oxidize additional water molecules. In practice however, it is incredibly difficult to create a regenerative OEC mimic, as high valent Mn is not usually stable. Therefore, our focus is to develop and explore materials which mimic the PS I and II solar water splitting reaction. α-Fe2O3 has shown great promise for the solar water oxidation half reaction. Upon photo-excitation, electrons are collected at the back contact leaving the surface of α-Fe2O3 in an oxidized state, providing a driving force for water oxidation. Additionally, upon introduction of certain catalysts, we can increase the oxidative capacity of α-Fe2O3, and thus improve the overall solar-to-hydrogen efficiency. So far, we’ve observed a significant enhancement in the oxidative efficiency with the use of a certain cobalt catalysts, and we are investigating the use of other metal centered catalysts including: nickel, iridium, silicon, tin, iron, niobium, and titanium. Ideally, we’d like to see these other metal centered catalysts demonstrate a greater increase in solar-to-hydrogen efficiency than our cobalt catalysts. We hope to, one day, apply this technology towards increasing the efficiency of solar powered fuel cells.

Protein homeostasis encompasses the creation, maintenance, and degradation of proteins, and it is essential for organisms to function properly. Yet, environmental stressors can disrupt protein homeostasis, which can lead to protein aggregation. Protein aggregation has been linked to serious aging diseases such as Huntington’s, Parkinson’s, and Alzheimer’s disease. In order to gain a better understanding of the pathways important for maintaining and disrupting protein homeostasis, we subject Caenorhabditis elegans to various environmental stressors that disrupt protein homeostasis. We have found that specific concentrations of hypoxia, or low oxygen conditions, induce an increased amount of protein aggregates in C. elegans compared to animals in room air, suggesting defects in protein homeostasis. In addition, the AMP-activated protein kinase (AMPK) seems to be crucial for allowing the formation of aggregates in hypoxia. AMPK mutants that are placed in hypoxia show a decrease in protein aggregation compared to wild-type animals. Furthermore, another environmental stressor, heat shock, also affects protein homeostasis. Heat shocking C. elegans prior to the hypoxic exposure, decreases the amount of aggregates compared to worms that did not experience heat shock. However, unlike hypoxia-induced protein aggregation, heat shock does not seem to require the presence of functional AMPK in order to influence protein aggregation.

Von Willebrand factor (VWF) is a multimeric blood glycoprotein involved in the process blood clotting. Its primary function is to be an adhesive proteins that platelets can bind to a wound site. In the Sniadecki lab, VWF is used as a matrix protein for capturing platelets under shear flow in a microfluidic device for blood coagulation studies. Currently, VWF is purchased from a company and coated on the surface of the microchannels. High molecular weight VWF has a higher ability to form a clot, while low molecular weight VWF has a lower ability. Since the VWF comes from a commercial source, the purity of its molecular weight weight is unknown. The goal of my project is to improve the VWF coating by purifying VWF from Enzyme Research. We hypothesized that low molecular weight VWF does not support clot formation and that high molecular weight VWF will show more clot formation than the control vWF in the microfluidic device. My research efforts have been to purify VWF using the fractionation gel filtration technique developed by Professor Dominic Chung’s lab at the Puget Sound Blood Center. The gel filtration technique can isolate the vWF according to size. Gel filtration was done using a 10x10 Sephacryl 500 Column. After the filtration process, vWF of different molecular weights are then applied primarily to the surface of PDMS so that platelets can bind to them under fast flow, in order to measure forces of platelet clotting. We found that there is a link between the molecular weight of the vWF and the rate of clot formation. Low molecular weight vWF does not support clot formation and that high molecular weight vWF will show more clot formation than the control vWF.

For the first time we are able to non-invasively measure levels of a molecule that indicates the ageing and disease state of an individual. This molecule called nictotinamide adenine dinucleotide (NAD) and its other forms (NAD, NAD+, NADH) are related to aging and muscle function. High levels of the form NADH is found in individuals with disease and old age while high levels of NAD+ is found in active and younger individuals. Through exercise on elderly subjects we show that ageing effects on muscle can be reversed and levels of NAD+ and NADH can be restored to those found in adults. The form NAD+ is an index of mitochondrial capacity for making energy. With aging the oxidative capacity of the mitochondria declines, the amount of mitochondria declines and the levels of NAD+ declines. Through exercise training this effect can be reversed to yield higher muscle capacity in elderly. Our measurements were made on a 1.5 Tesla Magnet. We acquired Phosphorus spectra on the FDI (First Dorsal Interosseous) muscle and analyzed the data using software called jMRUI. From the Phosphorous spectra we are able to extract and analyze the NAD+ and NADH peaks.

Gas chromatography - mass spectrometry (GC-MS) is a widely used analytical technique for separating and identifying volatile and semi-volatile compounds in complex mixtures. It is beneficial to research ways of increasing sample throughput (i.e. decreasing analysis time) while not losing any chemical information. This study investigates the change in volatile chemicals (odors) that are emitted from banana peels in varying stages of ripeness. The headspace of banana peels was first analyzed using Solid Phase Micro-Extraction (SPME) with a standard gas chromatograph and a quadrupole mass spectrometer (qMS). For demonstrating a viable platform for increased sample throughput, the samples were then analyzed on a LTM (Low Thermal Mass GC) coupled to a Time-of-Flight mass spectrometer. The banana peel analyses on the LTM-TOFMS decreases the run time by tenfold when compared with the traditional GC-qMS. A new data analysis program developed by the Synovec Lab is used to visualize the data and identify compounds.

Age-related cardiac pathologies are currently endemic in most developed nations and account for a large number of premature deaths and huge medical expenditures. Although lifestyle-associated risk factors have frequently been correlated with risk of cardiac impairment, it is also clear that aging impairs heart function even in the absence of these risk factors. Caloric restriction (CR) is known to extend lifespan and offset age-related impairment in diastolic function in murine models. Rapamycin is a CR mimetic that also extends lifespan (when given throughout life) and is protective in models of cardiac hypertrophy and failure. However, the potential role of Rapamycin for treatment of impaired heart function in aging has not been established. To determine the effect of short-term Rapamycin treatment on cardiac aging, we monitored cardiac function in 25 months old C57BL6/CR mice fed for 10 weeks with a control diet or a diet containing 14ppm encapsulated Rapamycin. Analysis by tissue dropper imaging showed improvement in diastolic function in the treatment group as early as 4 weeks. Normalized heart weights were also lower in the treatment group compared to old control mice, suggesting that short-term sub-acute Rapamycin treatment can reverse cardiac aging and regress age-related cardiac hypertrophy. Proteomic analysis showed increased stability (half-life) of the cardiac global proteome, increased abundance of mitochondrial proteins, and decreased oxidative damage of proteins in the treatment group. Further analysis revealed metabolic remodeling in the glycolytic and TCA pathways that improve energy metabolism and cardiac function in 10 weeks Rapamycin treated mice. Data from murine models offer potent approaches that may be replicable in humans and may prove effective in management of cardiac and perhaps other age-related ailments. In this project, we both assist in preparing the control and treatment diets, mice handling, imaging preparation, tissues harvesting, data analysis, and experimental design for future experiments.

Mitochondria are essential organelles in eukaryotic cells and are most commonly known for their prominent role in energy production. In addition, mitochondria are involved in other important cellular processes such as lipid biosynthesis and apoptotic cell death. Mitochondria also have dynamic properties including fusion, division, and movement, which are all essential to maintain mitochondrial function. Given the plethora of essential cellular activities that require mitochondrial function, it is not surprising that defects in mitochondrial function are implicated in many different human diseases and disorders including diabetes and myopathies. Mitochondrial movement is important for the distribution of mitochondria, which is important in all cells, but especially important for asymmetric cells such as neurons. Transport is also required for mitochondrial fusion – the two organelles must move close enough to each other to fuse. Mitochondrial fusion in particular is required to maintain mitochondrial DNA and therefore, mitochondrial function. We are interested in the mechanism of mitochondrial movement and the connection between mitochondrial fusion and transport. Mitochondria move on microtubules using a complex of proteins. Miro 1 and Miro 2 are anchored to the mitochondrial outer membrane and interact with two adaptor proteins, TRAK1 and TRAK2. The TRAK proteins also interact with Kinesin, which binds to the microtubules and mediates microtubule-directed movement. Understanding the assembly and regulation of this complex and the stoichiometry of its components will provide insights into the mechanism of microtubule-directed mitochondrial movement. Our goal is to clone TRAK1 and TRAK2 complementary DNA (cDNA) into expression vectors for protein production in Escherichia coli cells and then to develop a purification protocol for TRAK1 and TRAK2. This will facilitate biochemical analysis of the mitochondrial motility complex.

Müller glia are the predominant glial cell types in the retina that play a critical role in development and neuronal support. In adult mammalian retinas Müller glia are mitotically quiescent. However in other organisms, such as zebrafish, the Müller glia re-enter the cell cycle, proliferate and differentiate into new neurons upon retinal damage to replace lost ones. The goal of this study was to promote Müller glial proliferation and regenerate neurons in the adult mammalian retina. p27 is a tumor suppressor and plays a role in controlling cell proliferation. To test if the loss of p27 promotes Müller glial proliferation in adult retinas, we have generated Müller glia-specific p27 knockout mouse. Using tamoxifen-inducible, CreER/lox system to control timing of p27 deletion in Müller glia our Western blot data shows that daily injections of tamoxifen for four days efficiently eliminated p27 expression in adult Müller glia. When p27 expression is lost, adult Müller glia appeared to proliferate. Our data suggest that p27 plays a role in keeping adult Müller glia from re-entering the cell cycle. The controlled re-differentiation of Müller glia and proliferation into new neurons could be used to cure diseases involving retinal damage or degeneration like macular degeneration.

Rumination has been linked to the onset and development of depression and anxiety. Rumination is a process of repetitive negative feelings and thoughts associated with an upsetting event or situation. In this study, I investigate whether rumination explains the relationship between different types of environmental adversity and symptoms of depression and anxiety in teens. Specifically, does rumination explain the association of trauma exposure, poverty, and race/ethnicity related with the onset of depression or anxiety? Data come from a sample of 168 adolescents recruited from the community in Boston and Cambridge, MA. To examine mediation I examined four sets of models testing the following relationships: 1) the association between each measure of environmental adversity and depression/anxiety, 2) the association between each measure of environmental adversity and rumination, 3) the association between rumination and depression/anxiety, and 4) a final model that included the adversity variables as well as rumination to see the attenuation in associations of adversity with depression and anxiety once rumination was added to the model. The findings suggest that, 1) child abuse was associated with elevations in depression and anxiety symptoms, and community violence was associated with depressive symptoms and marginally with anxiety symptoms; 2) child abuse was associated with higher engagement in rumination; higher income-to-needs ratio was also related to greater rumination; 3) higher degrees of rumination predicted both depression and anxiety symptoms; and 4) the association between child abuse and depressive symptoms was reduced by 24.5% after accounting for rumination, and the association between child abuse and anxiety symptoms was reduced by 39.8% after accounting for rumination. These findings fill the void in the literature regarding mechanisms linking different types of environmental stressors to the onset of depression and anxiety. This is important for development of interventions for preventing mental illness.

Every plant needs Nitrogen to grow. Nitrogen is usually given through synthetic fertilizer which is expensive and causes a lot of environmental problems. Synthetic fertilizer is bad for the envieronment not only it need a lot of fossil fuel to make the high temperature and pressure condition for Nitrogen reaction, but also excess Fertilizer will bring blooming algae to the surround ponds, which will make the organism in the water die out because lack of Oxygen since the sunshine cannot get through algae and help underwater plants to photosynthesis. Since previous research in the lab showed that endophytes isolated from Willow and Poplar trees can promote growth of crops such as pepper, tomatoes, and sweet corn under low nitrogen conditions, we hope that Endophyte can reduce or completely substitute synthetic fertilizer of other commercial plants, such as corn. Endophytes (microbes living inside plants) can produce phytohormones and provide fixed nitrogen for plants. My research focuses on corn, a bioenergy crop species typically grown with large inputs of fertilizer. I screened ten commercial varieties of corn and compared the growth of endophyte inoculated plants with the uninoculated controls, in low nitrogen soil. In one variety of corn there was increased biomass in the inoculated plants compared to the controls. Currently I am testing which isolate is responsible for the plant growth promotion. These findings will have a great impact on commercial production of corn in a sustainable way.

Pollination syndrome refers to suites of floral traits that influence biotic or abiotic vectors transporting pollen from one flower to another. These traits typically include flower color, position, shape, size, scent and reward. Scent is a major component of pollination syndrome, as the volatile organic compounds (VOCs) that compose “scent” often attract pollinators or deter predators. The genus Thalictrum in the Ranunculaceae (buttercup family) includes both insect- and wind-pollinated species, enabling comparisons of the role of scent in floral diversification among recently diverged taxa with distinct pollination syndromes. To characterize floral scent, we identified and compared the bouquet of VOCs emitted by insect- and wind-pollination species, as well as by floral vs. vegetative tissue. Scent was collected from tissue samples by traps in a filtered air system, then eluted and analyzed with gas chromatography mass spectrometry. Quantitation and emission rate were calculated to generate floral bouquets. Electroantennograms were performed on selected compounds to examine pollinator response. Focusing on eleven species representative of the two pollination syndromes and spanning the Thalictrum phylogeny, we identified ten key compounds of which five are deterrents against herbivory, two are pollinator attractants, and three have unknown functions. Given that these scent profiles consist primarily of defensive rather than attractive compounds, we hypothesize that repeated transitions from insect- to wind-pollination in Thalictrum may be due to the breakdown of an ancestrally weak insect pollination syndrome. Moreover, scent bouquets appear to follow phylogenetic patterns more so than pollination mode. The variation in distribution of VOCs across species in a phylogenetic framework allows us to test evolutionary patterns of compound co-option during evolution.

The retina contains a dense web of photoreceptors, interneurons, and retinal ganglion cells. Although much work has been done on interactions between homologous photoreceptors, little has been done to characterize interactions between the two different types of photoreceptor cells: rods and cones. Electrophysiological recordings done in the Rieke lab found that selective activation of rod cells inhibited the response of long wavelength cones within a subsequent time window. The goal of my work was to probe whether this intercellular retinal phenomenon is robust enough so as to actually be perceptible by human observers in psychophysical trials. Using a dual monitor haploscope setup, I was able to present rod or cone isolating visual stimuli with different time offsets to subjects, who then performed a “brightness” matching task. Indeed, my results identified a perceptual suppression of cone mediated signals with a strength and latency comparable to that observed in electrophysiological recordings. This work contributes to our understanding of the neural circuitry underlying vision, particularly under mesopic or moderately dark light levels.

There is currently an estimated 350 million people worldwide chronically infected with the Hepatitis B virus (HBV). These individuals, over the course of their life, are at an increased risk of developing liver cancer. While there are currently antiviral drugs that can suppress HBV replication, latent HBV so far has proven difficult to eradicate. Homing endonucleases, enzymes that recognize long DNA sequences and induce DNA double strand breaks, could be a means by which latent HBV is targeted and inactivated. In particular, we aim to modify the recognition site of the wild type I-GzeII homing endonuclease (HE) into a HBV specific DNA targeting enzyme that will recognize specific sequences in the HBV genome and introduce double-strand DNA breaks. By exploiting the error prone nature of non-homologous end joining, the process cells undergo to repair double-strand breaks, these modified HEs will have a mutagenic effect on target sequences in the HBV genome. Repeated HE activity will eventually cripple the replicative ability of the latent virus. To selectively modify the structure of homing endonucleases, the amino acid residues that interact with the DNA substrate are identified and randomized. A library of variant forms of I-GzeII is generated. Variants that encoded for active forms with the appropriate HBV specific recognition site are selected for by in vitro compartmentalization. After several rounds of selection, bacterial selection assays and in vitro cleavage assays using a reporter cell line will be utilized to select for structurally stable variants that functions in vitro. The final step is to test the HE product on an in vitro HBV cell line. Through our research, we hope to produce a proof of concept that latent viruses can be targeted and inactivated through mutagenesis by homing endonucleases and that specificity and target of the enzyme can be modified and assessed through this protocol.

Compound I is the intermediate responsible for substrate oxidation in the catalytic cycles of a variety of heme-containing metalloenzymes, such as peroxidases, oxygenases, and importantly, cytochrome P450s. Cytochrome P450s, through compound I, oxidize approximately 75% of foreign substances that enter the body, such as pharmaceuticals and chemicals from the environment, so there is an interest in the mechanism by which this reaction occurs. The goal of this project is to probe the potential reversibility of Compound I formation, a step traditionally thought to be irreversible. Due to its stability and availability, horseradish peroxidase (HRP) was used as the model enzyme. In the case of HRP, hydrogen peroxide is reduced to water at the heme cofactor, while oxidizing a variety of substrates. In the presence of an oxidant and the absence of a substrate to oxidize, hydrogen peroxide coordinates to the iron of the heme, the O-O bond is cleaved, which forms water and compound I. A key experiment reacts HRP with unlabeled H₂O₂ in ¹⁸O-labeled water, and determines whether mixed-labled H₂O₂ is formed. The presence of mixed-labeled H₂O₂ indicates that there is reversibility of the O-O bond cleavage that forms compound I. The existence of mixed-labeled H₂O₂ is ascertained by first removing HRP by filtration, leaving the H₂O₂ in the filtrate. This is followed by the addition of a water soluble phosphine. In this case, the phosphine cannot be oxidized by Compound I, but can readily react with free hydrogen peroxide in solution to give the corresponding phosphine oxide. Mass spectroscopy is used to determine whether the phosphine oxide has incorporated the ¹⁸O label. Results show that when the experiment is performed in the presence of ¹⁸OH₂, there was no observable increase in the intensity of a peak corresponding to ¹⁸O-phosphine oxide, so reversibility of Compound I formation was not confirmed.

Solar technology is a potential way to help meet the growing demand for clean, renewable energy. Hybrid composites of inorganic quantum dots, with organic semiconducting polymers, offer a potential means of producing low-cost, solution-processable photovoltaics. Photovoltaics can convert sunlight directly into electricity to capture solar energy. The synthesis of quantum dots typically involves the use of large surfactant molecules to facilitate particle growth and solubility. These native ligands act as electrical insulators that impede charge transport in photovoltaic devices. Therefore, it is necessary to exchange these large ligands with small molecules to achieve efficient charge carrier photogeneration and transport. In this research, we examine bulk heterojunction blends of low band gap PbS quantum dots with the conjugated polymer poly((4,8-bis(octyloxy)benzo(1,2-b:4,5-b')dithiophene-2,6-diyl)(2-((dodecyloxy)carbonyl)thieno(3,4-b)thiophenediyl)) (PTB1) treated with different ligands including halide ions and organic crosslinkers. By using photoinduced absorption and transient photovoltage techniques, we study the long-lived charge generation dynamics and recombination kinetics of our devices. We find that the ligand treatments exhibiting higher device performance correlate to longer carrier recombination lifetimes in both spectroscopic and photovoltaic device measurements.

Composites of conjugated polymers with semiconducting nanocrystals (NCs) are of interest for low-cost, solution- processable photovoltaics. However, compared to typical polymer/fullerene composites, relatively little is known about factors influencing charge generation. Without understanding these factors, design principles for improving solar cell applications remain elusive. Previous studies of blends of the polymer poly(3-hexylthiophene-2,5-diyl) (P3HT) with PbS nanocrystal quantum dots showed the excited NC undergoing charge transfer by hole transfer from the NCs to the polymer. To further investigate this hole transfer mechanism, quasi-steady-state PhotoInduced Absorption (PIA) was performed on blends of P3HT with PbS nanocrystal quantum dots as a function of excitation energy. As the energy exciting the PbS quantum dots was increased, the yield, per photon absorbed, of positive charge carriers on the organic host polymer increased. This occurred even at excitation wavelengths that only the NCs could absorb at. Our result provide direct evidence that holes generated on the NC retain some of the additional energy from excitation, e.g. hot holes. Further understanding of these results could provide even more insight to the mechanisms of organic/inorganic photovoltaics.

Physicochemical DNA research contributes to cutting edge medical applications such as single nucleotide polymorphism (SNP) detection and drug delivery. DNA can be modified with azobenzene moieties to form azobenzene DNA. I used UV light to induce a trans to cis isomerization reaction of the azobenzene moiety which causes double stranded azobenzene DNA (dsDNA) to split into a single stranded DNA (ssDNA). We study the effects of UV light on the azobenzene photoisomerization of perfectly matched and mismatched DNA. For the experiments, a glass substrate is functionalized with amino-silane molecules and single stranded azobenzene DNA. I hybridized the azobenzene DNA with either a perfectly matched or a mismatched strand to form double stranded DNA. I destabilize the DNA duplex by adding UV light to the hybridized DNA sample. Since the mismatched strand has a single base mismatch, it is less intrinsically stable than a perfectly matched strand due to less contributing intermolecular forces. Consequently, the melting temperature is lower for the mismatched strand. The mismatched DNA strand returns a less intense fluorescent signal in my detection. Experimental parameters such as hybridization time, UV exposure time and temperature applied to the experimental set-up are varied in order to study changes in ssDNA vs. dsDNA fluorescence. Consistent reproduction of these results is vital for a reliable DNA chip assay that can detect single nucleotide polymorphisms (SNP) in fragments of DNA.

We describe an exo-selective cyclization of enantioenriched allenols to form cyclic ethers containing an α-tetrasubstituted stereocenter. Cyclic ethers bearing α-tetrasubstituted stereocenter are present in acetogenins, macrodiolides, ionophores, and lignans. These natural products have been found to have anti-fugal, anti-tumor, and/or antibiotic properties. However, an asymmetric catalytic synthesis of α-tertrasubstituted cyclic ethers remains a major synthetic challenge. Our method works with as little as 0.02 mol % of a gold catalyst for the exo-selective cyclization of enantioenriched allenols, and proceeds with excellent chirality transfer, and high functional group compatibility.

Sleep disordered breathing (SDB) in children is a serious health concern associated with significant adverse health outcomes and high use of health care resources. It is characterized by habitual snoring and repeated episodes of partial or complete upper airway obstruction during sleep. In a recent study from our laboratory, 40% of a sample of school aged children with Juvenile Idiopathic Arthritis (JIA) showed signs of SDB. JIA is an inflammatory disease with no definite known cause and one of the most common rheumatologic chronic conditions in children. The purpose of this study is to examine SDB and neurobehavioral performance in 81 children (n=40 JIA) compared to a group of age, sex matched school age children. Children underwent one night of polysomnography in the UW School of Nursing Sleep Research Laboratory and completed neurocognitive tests (WASI, CANTAB). Parents completed the Behavior Rating Inventory of Executive Function (BRIEF), an 86-item measure that assesses executive function behaviors at school and home. Data was analyzed using SPSS, and analyses were blocked into conceptual categories and then analyzed for group differences. Each category was considered a separate analysis with significance set at p <.05 (2-sided). The first set of analyses addressed group differences in all study variables between JIA and control children (using a Chi-square test). Second, we compared the proportion of children with SDB (apnea/hypopnea index >1.5/hr) as measured by polysomnography to those without SDB. Third, we examined relations among disease-related variables, sleep disordered breathing, and neurocognition (WASI, CANTAB, BRIEF), with a series of bivariate correlations controlling for age, medications, and disease condition. Finally, a linear regression analysis was performed to identify potential predictors of neurocognition (WASI, BRIEF, reaction time). We explored how much of the variance in neurocognition was explained by sleep disordered breathing, controlling for age, medications, and disease condition.

 Using mice as an animal model, we are investigating the possibility that fetal ultrasound exposure in utero has contributed to the observed dramatic increase in the number of cases of autism. A leading hypothesis suggests that autism may arise through a combination of genetic predisposition and exposure to certain environmental factors during a vulnerable period of neuro-development in utero. To test this hypothesis we exposed mice to diagnostic ultrasound in utero during a specific developmental time window to check for possible post-birth induction of autistic-like behavior and neuro-anatomy. Specifically, we exposed anesthetized pregnant mice to ultrasound for 30 minutes on embryonic day 12. When mice pups were three weeks of age, we used a variety of behavioral tests to assay for autistic-like characteristics. Additionally, we performed histology on the mouse brains, analyzing them for neuroanatomical characteristics of autism. Our published results show significant social behavioral differences between ultrasound-exposed and sham-exposed mice. Also, preliminary analysis of the brains found decreased minicolumnar width and variability in the ultrasound-exposed group relative to the sham-exposed group. Both the behavioral and neuroanatomical results are consistent with the hypothesis that diagnostic ultrasound exposure in utero represents a potential risk factor for autism. Future research will target study of possible bio-physical mechanisms that may underlie our observations. Specifically, we intend to tag newborn cortical neurons via injection of BrdU in an attempt to quantify ultrasound’s effect on the migration of these neurons, anticipating a relationship between dose of ultrasound and extent of disruption of the cortical layers within the brains of ultrasound-exposed mice.

An efficient and environmentally benign means of creating semiconducting polymers is necessary for the commercialization of organic electronics. Furthermore, it is crucial that as much control as possible be exerted over the polymerization process. To these ends, two polymerization methods are useful – C-H activation and living polymerization. First, C-H activation is a collection of synthetic techniques where C-H bonds undergo direct catalytic alteration to yield new homo- or heteroatom linkages. Whereas organometallic reagents were traditionally used to achieve these results, C-H activation allows for shorter syntheses utilizing less-hazardous reagents and broader functional group compatibility. Living polymerization is a form of addition polymerization and in our case, the number of chains is constant during the reaction and dependent on the catalyst-to-monomer ratio. This allows for much narrower mass distributions of the desired polymer and more consistent growth. To achieve this, benzoxazole-based monomers containing a C-O electrophile are to be reacted in the presence of a zero-valent nickel species with electron-rich phosphine ligands. By increasing the electron density on the metal center, we aim to facilitate a “ring-walking” mechanism at higher temperatures than previously thought possible. Monomer synthesis and polymerization results are detailed.

Thaumarchaeota are widespread in marine and terrestrial environments. Many of them are thought to carry genes coding for a putative ammonia monooxygenase (AMO), suggesting a capacity for ammonia oxidation. However, strong evidence from isotopic studies and molecular surveys, as well as direct physiological characterization shows some populations of ammonia-oxidizing archaea (AOA) have a capacity to use alternative fixed carbon for biosynthesis, and suggests their mixotrophic lifestyle. In this study, we investigate the influence of organic substrates on growth and activity of the only two available isolated representatives of marine AOA, Nitrosopumilus maritimus strain SCM1 and strain HCA1. The certain intermediates of the TCA cycle are supplemented into AOA pure cultures, and the growth response of each strain to different organic substrates are monitored by measuring nitrite production and cell counts. The calculated specific growth rate, doubling time and specific cell yields are compared between organic carbon amended cultures and organic carbon-free controls. The SCM1 and HCA1 growth rate and cell yield were greater in cultures supplemented with alpha ketoglutaric acid (from 93.4 to 112.6 x 1012 cells mol-1 NH4+ for SCM1, and no growth to 80.8 x 1012 cells mol-1 NH4+ for HCA1), which suggest that marine AOA may prefer a mixotrophic lifestyle. This data expands our knowledge of thaumarchaeal metabolic capabilities and can provide information on the competition between AOA and other microbial groups to improve our understanding of microbial ecology.

Binary chromophore systems contribute to more favorable intermolecular interactions and can significantly enhance electro-optic (EO) activity of EO materials, which have potential applications for telecommunications. Optimization of EO activity of binary chromophore systems consisting of dendrimer host (i.e. PSLD-33) and guest chromophore (i.e. FTC) will be achieved by determining the ideal host to guest concentration ratio. Synthetic schemes for known dendritic systems were proposed and synthesis is currently underway. Linear optical properties, including max absorbance (λmax) and molar absorptivity (ε), of materials in solutions with chloroform will be characterized using UV-vis spectroscopy. These properties have implications in chromophore packing density, which up to a thresh hold can improve EO activity. Additional steps include making films of the materials. EO activity of the materials will be characterized using the Teng-Man method, which has proven to be a convenient way to determine the EO coefficient (r33) in the Dalton lab over the past ten years.

Pericytes are mesenchymal cells that are attached to peritubular capillaries. The activation and differentiation of progenitor pericytes to myofibroblasts leads to matrix deposition, renal scarring, and is a major contributing factor leading to chronic kidney disease after injury. Connective tissue growth factor (CTGF) plays an important role in fibrogenesis by promoting the production and secretion of extracellular matrix components. The goal of this project is to dissect the contribution of CTGF/Wnt signaling pathways in pericytes during the development of kidney fibrosis and to examine the effect of using the canonical Wnt extracellular regulator Dickkopf related protein-1 (DKK-1) on CTGF/Wnt signaling. In-vitro signaling studies on purified kidney pericytes, migration assays, and immunological staining were used to characterize the roles of DKK-1 and CTGF in the regulation of renal fibrosis. Results show that CTGF induces Wnt/β-catenin signaling in pericytes. DKK-1 inhibits CTGF induced fibrotic gene activation, changes the morphology, and the capability of migration of kidney pericytes through JNK MAP kinase, and canonical WNT independent pathway.

Currently there is extensive interest in the health of honeybee colonies, both in the United States, and worldwide. A healthy and functioning agricultural system requires honeybees, and bees in turn, require nutritious, diverse forage that includes both nectar and pollen. This study involved analyzing the pollen collected by honeybee colonies maintained on the campus of Edmonds Community College, and matching that with pollen collected directly from local plants. We isolated pollen from honey collected and stored by the bees and also collected pollen directly from cells in the hive. In order to identify the different pollen species, the pollen was chemically extracted and acetolyzed to remove sugars and waxes. The treated pollen was then stained, slide-mounted and viewed by light microscopy. These pollen samples were compared to samples of pollen that were collected directly from local plants as well as to existing pollen databases. Preliminary analysis revealed the presence of pollen from brassica, salvia, and trifolium. We were unable to identify some of the extracted pollen species. During the coming foraging season we will expand our database of locally-collected pollen and will generate a local pollen identification key. This will allow us to conclusively identify all of the pollen found in the hives. Results of this study will help us track what the bees are eating and will inform decisions about what best to plant to insure healthy colonies.

Vascular calcification, the accumulation of calcium-phosphate salts in blood vessels, is a common complication associated with atherosclerosis and type II diabetes mellitus (T2DM). We have shown that reprogramming of vascular smooth muscle cells (SMCs) into osteochondrogenic cells is important in T2DM vascular calcification, and endothelin-1 (ET1) and its type A receptor (ETA) were found in the reprogrammed SMCs. Along with the in vitro study that showed a substantial inhibition of the high glucose-induced SMC calcification upon ETA antagonism, we hypothesize that signaling through ET1 and ETA may play an important role in ectopic calcification of atherosclerotic blood vessels of T2DM. To test this hypothesis, we fed low-density-lipoprotein receptor knockout (LDLr-/-) mice with a customized diet for 18-24 weeks to induce T2DM, atherosclerosis, and vascular calcification. Upon 5 weeks diet fed, LDLr-/- mice developed hyperglycemia with no detectable atherosclerosis and vascular cartilaginous metaplasia and calcification. Half of these mice were then treated with an ETA antagonist, atrasentan, to inhibit ET1-ETA signaling. Mice were terminated at 18 weeks under the diet for histological analysis and 24 weeks under the diet for quantification of vascular calcification. We found that atrasentan significantly reduced fasted blood glucose levels of the T2DM mice but not affecting their body weight. Surprisingly, quantitative analysis showed no difference of calcium amounts in aortic arches between the treated and untreated groups. We are currently in the process of analyzing the lesion sizes of atherosclerosis, cartilaginous metaplasia and calcification of 18-week diet-fed mice via morphometric analysis to determine whether atrasentan affects vascular calcification at an earlier time point and whether vascular calcification is correlated with atherosclerotic lesion size. Finally, our finding of ETA antagonism in the control of hyperglycemia provides a new therapeutic role of atrasentan in improving T2DM, which may be beneficial in alleviating the hyperglycemia-related complication.

Diatoms are an important taxon of phytoplankton that contribute to both freshwater and marine ecosystem function. For example, marine diatoms are responsible for about 14% of global primary production. My research is focused on determining the ability of these algae to regulate the production of chlorophyll a- the core pigment component of photosynthetic complexes. The enzyme light-dependent NADPH protochlorophyllide oxidoreductase (POR) is a key regulator of chlorophyll synthesis. Whereas most organisms have just one POR enzyme, diatoms have two (POR1 and POR2). The goals of this project are to understand how these enzymes are metabolically different from one another in: (a.) their enzymology and (b.) their regulation at the transcriptional and posttranslational levels. To this end, plasmids were constructed encoding the cDNAs of the por genes, adding N-terminal histidine tags to assist in affinity purification. E. coli, transformed with these plasmids, was induced to express the proteins; harvested, and lysed. Recovered POR1 and POR2 proteins were purified from the lysate by affinity chromatography. Antibodies raised against these proteins were used in Western blot protein identification and quantitation of the targeted proteins. Preliminary experiments demonstrate good affinity of generated antibodies for POR proteins, and indicate that POR 2 may be post-transcriptionally truncated. Developing an understanding of chlorophyll metabolism in diatoms has not only ecological implication, but also could prove important in commercial aspects of biofuel development- increasingly important issues driven by changing ocean conditions and the critical need for generating alternative fuel sources. Determining the properties of these enzymes and their regulation is just one step towards understanding the successful productivity of diatoms. Further studies may be required to determine if there are additional regulatory pathways and enzymes that are critical in photosynthetic production.

The somatic mutation theory of aging states that aging is the result of accumulated DNA mutations in somatic cells, which lead to loss of physiological integrity through tissue and organ level dysfunction. In mouse model systems, decreases in lifespan through high mutation rates have been documented by creating mutator phenotypes in these organisms. Mutator phenotypes are marked by increased cellular mutation rates, and are known drivers of tumor development in human cancers. Mutator-driven adaptations occur in natural microbial populations. Mutators represent a unique opportunity to test the somatic mutation accumulation theory of aging due to their sustained high mutation rates. Nutritional status, particularly carbohydrate metabolism, influences lifespan in numerous organisms. In yeast, which preferentially ferments glucose to ethanol before switching to respiratory metabolism upon glucose depletion, the induction of respiratory metabolism is associated with increased lifespan. This study examines the effects of high mutation rate on yeast replicative lifespan and whether induction of respiratory metabolism is sufficient to change lifespan in organisms with increased mutation load. To do this, yeast defective for DNA replication proofreading, mismatch repair, and base-excision repair were grown under dietary restriction (reduced glucose) and in the presence of non-fermentable carbon sources. Then, mutation rates and replicative lifespan were assayed. Our results show that increased mutation rates correlate with decreased lifespan in strong mutators and that this decrease in lifespan can be partially suppressed through growth on limited glucose and non-fermentable media. These findings suggest that the metabolic state of the cell modulates the severity of mutator phenotypes, which has important implications for the role that mutators play in carcinogenesis.

Micro RNAs (miRNAs) control translation of certain proteins through degradation of the messenger RNA which code for these proteins. These non coding RNAs are often up-regulated in many forms of cancer and can inhibit the expression of tumor suppressor proteins. The goal of this project is to engineer a protein which can bind to and inhibit the biogenesis of a miRNA-21, which is overexpressed in many cancers. Previous reports have shown that the RNA recognition domain (RRM) of human splicing factor Fox-1 protein has high affinity and specificity for the UGCAUG sequence. We are engineering this RRM to target the stem-loop sequence of precursor miRNA-21. Amino acid site changes were introduced into Fox-1 to generate a protein which will inhibit production of the oncogenic miRNA-21 in cancer cells. This project involves the application of various methods in biochemistry, including PCR site-directed mutagenesis, cell transfection, DNA cloning, protein expression and purification, RNA transcription and purification as well as gel electrophoresis assays and 3-Dimensional structural analyses of protein and RNA through nuclear magnetic resonance studies. Currently, a stable protein has been expressed and the precursor species of miRNA-21 is being prepared to characterize its interaction with the engineered protein biochemically, biophysically and structurally.

FLOWERING LOCUS T (FT) is a highly conserved protein in plants which promotes flowering by influencing the differentiation of shoot apex meristem cells from vegetative to floral identity. CONSTANS (CO) is a protein involved in sensing photoperiodic cues and promoting FT expression. Arabidopsis thaliana express a single evening peak of FT when placed under long-day and constant temperature conditions. Under natural conditions, temperature fluctuates throughout the course of a day. In order to determine if experimental conditions of constant temperature accurately describe flowering time mechanisms in nature, wild type Arabidopsis thaliana plants were grown outside during the long days around the summer solstice of 2013. Real Time qPCR analysis of FT mRNA expression levels in 14-day-old plants over the course of 3 days revealed a morning peak of FT absent under long-day constant temperature conditions – in addition to the evening peak of FT present under long-day, constant temperature conditions. To determine what factors might induce this morning peak, Arabidopsis thaliana were grown in two sets of environmental chambers. Both sets simulated average outdoor temperature conditions over the week of original tissue collection. One set simulated average outdoor light intensity conditions over the same period, while the other used constant light intensity. Real Time qPCR analysis of plants grown under these conditions revealed that the simulation of outdoor temperature conditions is sufficient in producing the morning peak of FT. Different mutants of genes associated with flowering time were then grown under simulated temperature conditions to determine what genes might be involved in the induction of the morning peak of FT. By looking at the different expression profiles of FT and CO, we seek to determine what genes are involved in the induction of this morning FT peak.

For the past year, we have researched the contributions made by Guy Shearman Peterkin, an early pioneer urologist living in Seattle in the early 20th century. Our research focused on the life of Peterkin himself as well as his numerous scientific publications to understand his place in not only Seattle history, but the history of urology as a specialty field of medicine. We spent two quarters scouring the archives of the King County Medical located in the First Hill neighborhood to find references and publications relating to Peterkin. We also spent time in Special Collections here at UW collecting additional primary resources. We also utilized UW’s microfilm collections to search through old articles from the Seattle Times and other newspapers. The Department of Bioethics and Humanities owns a large collection of antique medical instruments. One of those exhibits is a presentation prepared by Peterkin circa 1920, “Mechanics in Developing the Cystoscope” that inspired this research to begin with. Peterkin was a pillar in Seattle society for his involvement in the King County Medical Society, the Washington State Medical Association, the American Urological Association and other professional groups. He wrote extensively on the relationship between urology and psychoanalysis of venereal diseases (i.e syphilis) with special emphasis on scientific and ethical standards. Peterkin’s legacy is overshadowed by his dramatic death in which he shot and killed his son and himself in a case of what he believed to be a mercy killing.

 Stomata play a crucial role in global carbon and water cycles, and disruption of stomatal function could impact our life and sustenance. In order for stomata to function correctly, proper density and distribution of stomata need to be tightly regulated. Several family members of cysteine rich secreted peptides called Epidermal Patterning Factors(EPFs) have been discovered by the Torii Lab and other groups as important signaling molecules controlling stomatal patterning and differentiation in Arabidopsis. Within the EPF family, EPF2 is secreted from stomatal precursor cells to suppress neighboring cells from differentiating into stomata by interacting with ERECTA-family receptor kinases (RKs) and a receptor-like protein, TMM. Thus EPF2 acts as a negative signal of stomatal development. In contrast, EPFL9 (Stomagen), was discovered to enhance stomata development, therefore acting as a positive signal of stomatal development. Conversely, suppression of EPFL9 gene expression by RNA interference leads to reduction in stomatal numbers. The question I addressed was whether EPFL9 requires ERECTA-family RKs and how ERECTA-family RKs process negative signals and positive signals simultaneously. I hypothesized that EPF2 and Stomagen activate and inhibit ERECTA-family receptors and that TMM influence the receptor activity. I addressed this hypothesis through characterizing the effects of their loss-of-function and gain-of-function mutant phenotypes and genetic interactions. For loss-of-function approach, I reduced EPFL9 expression in different genetic backgrounds with respect to different combinations of erecta-family RKs and tmm mutants to analyze the stomata density and index . For gain-of-function approach, I overexpressed EPFL9 in different genetic backgrounds to analyze the stomata density and index. Through the data, I am able to delineate the mechanism by which EPFL9 interacts with ERECTA-family RKs. This will further help to understand stomatal signal regulation and interaction by antagonistic peptide hormones in Arabidopsis.

Most bacterial infections are initiated when binding to receptors on the tissue surface via bacterial adhesive proteins. After adhesion, they form biofilms and become more resistant to the immune system and antibiotics. A novel approach to preventing infection is the use of anti-adhesive agents that inhibits bacterial adhesion to host tissues and thus blocks the infectious process. Therefore, understanding bacterial adhesion and physiology under different conditions is essential for the development of anti-adhesive drug therapies. This project involves studying the effects of pulsatile flow on Escherichia coli adhesion and testing the hypothesis that flow pulsatility enhances bacterial adhesion because bacteria are adapted to in vivo pulsatile flows caused by intestinal peristalsis, swallowing saliva, or beating of the heart. I used a programmable syringe pump to produce a pulsatile laminar flow composed of a low shear (e.g. 0.1 Pascal) and high shear (e.g. 1 Pascal) in a parallel plate flow chamber. The frequency and amplitude of this pulsatile flow was in the physiological range. Through a microscope with a digital camera, I recorded the number of E. coli that bound to the bottom plate of the flow chamber coated with the model receptor mannan. The preliminary results demonstrated that E. coli accumulation after 5 minutes under pulsatile flow was not significantly different from that under steady low shear but accumulation was more stationary with pulsatile flow. However, the difference in accumulation became significant when comparing with the steady high shear. These data suggest that E. coli initiate adhesion at low shear and they firmly maintain binding at high shear. Future work will require the adhesion assays of wide range of bacteria under various pulsatile flow conditions. This research will help us understand the bacterial physiology and advance the development of anti-adhesive drug therapies.

Large chromosomal amplicons and deletions or copy number variation (CNV) are found in many different cancers. However, the inheritance, stability, and fixation of these amplicons and deletions within a population are widely debated and poorly understood. In an effort to better understand how the stability of this large chromosomal rearrangement, I analyzed the stability of a large segmental amplification in the yeast S. cerevisiae. This amplification is commonly observed in clones evolved under sulfate-limited conditions and contains the sulfate transporter SUL1. This amplification is an important adaptive strategy used by the cells to improve their ability to extract the limited supply of sulfate available in the media. High copy number of this gene confers a competitive fitness advantage over other clones that have only one copy of the gene. We used a GFP marker integrated next to SUL1 to differentiate green clones, which contain a single copy of the SUL1 gene, from “super green” clones that have multiple copies of the gene. A super green evolved clone was grown in steady state growth vessels for ~30 generations in media that is non-selective for adaptation and our selective media as a control. I monitored the population using flow cytometry and quantitative PCR to find clones that have lost the amplification. These green clones were isolated using flow sorting and then had their genome sequenced to look at the scar that was left behind. This tells us how efficiently the cells can remove amplifications without causing detrimental effects to their fitness.

The three mammalian MAPRE proteins play important roles in the microtubule dynamics of the cell cytoskeleton, and facilitate the binding of multiple +TIP proteins to microtubule plus ends. Work conducted by the 1000 Genomes Project, the Exome Sequencing Project, and many other groups has led to the identification of non-conservative coding sequence allelic variants of the MAPRE proteins in the human population that result in the production of mutant MAPRE proteins. Disruption of MAPRE protein function could decrease cell signaling fidelity (how well a cell receives and transmits information) and microtubule function. The MAPRE proteins are related by descent from a common ancestor to the Bim1 protein in budding yeast. Here I designed and constructed mutant versions of Bim1 that contained mutations corresponding to known MAPRE variants and expressed them in yeast. The yeast pheromone response pathway was utilized as a model signaling system to test the effects of the MAPRE variants on cell signaling. The output of the pheromone response pathway, and therefore amount of signal received and transmitted by the cell, was quantified using fluorescent reporters under the control of a pheromone responsive promoter in yeast strains expressing the mutant versions of Bim1. By comparing the pheromone pathway output via fluorescence intensity of the mutant MAPRE strains to the output of the wildtype MAPRE strains, I was able to discern effects of the Bim1 mutants on cell signaling. Deletion of Bim1 resulted in increased variation in the pheromone pathway output. Also, a mutant MAPRE allele consisting of a C-terminal domain deletion resulted in diminished pathway output and increased output variation. Adverse effects on cell signaling could result in poor decision making and environmental response, potentially leading to the generation of disease. By better understanding the effects of allelic variants in the human population, we will be able to develop individualized health plans and therapies.

Chromosomal DNA synthesis begins at origins of replication, with some origins activating earlier than others during S-phase. Neither the mechanism by which origin activation time is determined nor the biological significance of this temporal program is fully understood. Interestingly centromeres, sites where kinetochore complexes form to correctly separate chromosomes during mitosis, replicate early. In the budding yeast Saccharomyces cerevisiae, centromeres actively promote their own early replication by advancing the activation time of their neighboring origins. We previously showed, in cells lacking the mitotic checkpoint, an artificially delayed, late replicating centromere causes a dramatic increase in chromosome instability. In this study, I am investigating the function of centromere-like regions (CLRs) in S. cerevisiae. These sequences can bind the centromeric histone (Cse4), especially when CSE4 is overexpressed. Under this condition, additional kinetochore proteins can build up and CLRs take on some of the properties of functional centromeres. Because the kinetochore protein Ctf19 recruits a replication initiation factor, I hypothesize that the centromere-mimicking DNA sequences are also capable of recruiting the kinetochore protein associated with early replication to advance the initiation time of nearby origins. To test this possibility, I will examine the effects of the CLR on replication origin activation using a plasmid that contains two identical origins. I will clone a CLR next to one of the origins and determine whether this origin is now the earlier activated origin on the plasmid when CSE4 is overexpressed. The CLR I will use resides in an early replicating part of the yeast genome. In a complementary experiment I will delete the genomic copy of this CLR and measure its effect on firing time of the adjacent origins. The results of this study will provide us deeper insights on how cells orchestrate when and where in their genome to start duplication.

American Indians and Alaska Natives suffer disproportionately from end-stage renal disease. For those with chronic kidney disease, transplantation may be the most effective treatment option. However, Native people do not donate organs at a rate that is commensurate with the need in the population. We developed a semi-structured, 10-question interview guide and used it to facilitate interviews with American Indians on their experiences with community activities to promote awareness of organ and tissue donation. Our nine key informants were project partners at Tribal Colleges and Universities and other tribal leaders. According to our informants, successful strategies to promote donation awareness included community engagement and active listening. Challenges included cultural beliefs and fear of discussing organ donation and death. However, community perceptions of living donation were positive, and key informants felt that living donation should be emphasized in future efforts. More education on organ and tissue donation is needed in Native communities. Information on living donation should be included in future educational efforts as part of the strategy to reduce donor shortages.

In the Hague Lab we study a class of proteins called G-protein coupled receptors (GPCR's). GPCR's are seven-pass transmembrane proteins that are involved in cell signaling. One GPCR we study is called alpha-1D adrenergic receptor. Adrenergic receptors bind the endogenous catecholamines adrenaline and noradrenaline. An important functional role of the alpha-1D GPCR in the body is to regulate blood pressure by affecting blood vessel diameter.  Our lab recently discovered that the alpha-1D adrenergic receptor's relatively long N-terminal domain is cleaved in human cells. We are currently elucidating the specific cleavage site - which we suspect is a GL matrix metalloprotease domain - and the physiological purpose of the cleavage. Over the last few months we have narrowed down the region of cleavage to approximately 15 amino acids. We also have a working theory that the N-terminal cleavage of the protein allows for better cell surface localization. We are currently examining how the cleavage event effects the signaling characteristics of the protein.

It has been shown that the chemical nor-binaltorphimine (norBNI) inactivates the k-opioid receptor (KOR) for days or weeks in vivo, and also results in downstream activation of c-Jun N-terminal kinase (JNK). If JNK is blocked in vivo by SP610025 or by knocking out the JNK1 gene, norBNI inactivates the KOR for just hours. We hypothesize that there is a JNK mediated inactivation of the KOR. To further investigate this, I will create constructs that will allow us to study the interactions of JNK and arrestin, two proteins that are involved in the transduction of KOR signaling. I aim to express a version of each JNK1 splice variant tagged with luciferase and arrestin3 tagged with Venus in human embryonic kidney (HEK293) cells that have been transfected with rat KOR. Venus and luciferase are two bioluminescent molecules that emit different wavelengths of light depending on their distance from one another. After treating these cells with norBNI, I can dtermine the level of their interaction by using a bioluminescence resonance energy transfer (BRET) assay. These experiments will provide useful data in determining the extent of the interactions between JNK1 and arrestin3. This data will help us understand the protein cascade that results in the deactivation of the KOR, and if certain drugs activate certain splice variants. Understanding this pathway has implications in creating analgesics with smaller potential for abuse and addiction, and creating pharmaceuticals that could help with stress related depression and addiction relapse.

Of the many types of devastating spinal cord injuries, incomplete injuries of the cervical spinal cord are the most common among patients. For these individuals, the highest treatment priority is restoration of hand and arm function; much more important than any other symptom of paralysis. The goal of our project is to restore hand and arm function by recording movement intention in the brain and using it in real time to control spinal cord stimulation. We use single unit activity encoding movement intention in the rat motor cortex, and intra-spinal microstimulation (ISMS) for movement induction. ISMS may be superior to direct muscle stimulation (FES), since it causes little fatigue and a more natural recruitment of motor pools. ISMS in the cervical and lumbar regions of the spinal cord can also be utilized to produce a wide array of functional limb movements. By using rats that I have trained to perform a lever-pressing task, I can quantify the functional relevance of movements evoked by varying ISMS parameters. By combining both recorded cortical data and ISMS, we have created a brain-machine spinal interface (BMSI). This BMSI can deliver brain-controlled, functionally useful stimulation directly to the spinal cord and caudal to a contusion injury in order to restore some movements to the animal’s injured forelimb. Our long-term goal is to translate this device to a clinical setting, where it could be used to restore hand and arm function to patients with complete spinal cord injuries.

Malaria, due to parasitic infection by Plasmodium protozoa, is a severe global disease with 300 million annual cases leading to 1-2 million deaths. While a few anti-malarial treatments continue to work, the continual emergence of new drug resistant parasites is an urgent public health concern. Previous studies in the Rathod laboratory suggest that chemical tools can be used to distinguish variations in evolutionary capabilities of different malaria parasite strains. Some strains such as Dd2 display the Accelerated Resistance to Multiple Drugs (ARMD) phenotype, whereas the HB3 strain displays the non-ARMD phenotype. In previous studies, Dd2 strain acquires resistance to several antimalarials at a significantly higher frequency than the HB3 strain. In this experiment, a cyclopropyl carboxamide (GSK2645947), a novel class of antimalarial agent, was used to select for resistant parasites from Dd2 and HB3 with the aim of comparing their relative frequencies of resistance to GSK2645947. A series of selection experiments were carried out utilizing different concentrations of GSK2645947 and different numbers of starting parasites. The parasites acquired resistance to GSK2645947 at similar frequencies in Dd2 and HB3 cells. The extent of resistance to GSK2645947, measured using EC50 assays, was similar in Dd2 and HB3 resistant clones; resistant parasites from neither lines showed cross-resistance to other antimalarial drugs. The present results point to a novel mechanism for rapidly acquiring resistance to GSK2645947, a mechanism that is equally prevalent in Dd2 and HB3 strains of P. falciparum parasites.

Rotavirus is the main cause of viral diarrhea among young children. There exist licensed vaccines, but rotavirus still kills nearly half a million children yearly. Live, attenuated vaccines quickly lose potency at room temperature. My host lab is assisting a Seattle NGO callednPATH and the Bill and Melinda Gates Foundation with developing temperature-stabilized, inexpensive vaccines. The current industry standard assay for measuring vaccine potency is time-consuming, subjective, and low throughput. My goal is to develop an objective, inexpensive, automatable, and high-throughput assay of the potency of candidate, next-generation, temperature-stable rotavirus vaccines. To develop this assay, susceptible cells are infected with candidate vaccine-strain viruses manufactured in India and undergoing development at PATH. After cell membrane modification to allow antibody access to cytoplasmic viral proteins, an anti-rotavirus antibody and a fluorescent secondary antibody are used to detect viral protein within infected cells. Experimental samples undergo single-cell flow cytometry analysis of individual cells to differentiate infected and uninfected cells. The vaccine industry-standard potency assay is performed in parallel to directly compare assays. Comparison and titration of polyclonal and monoclonal rotavirus-specific antibodies identified the optimal antibody and concentration for specific intracellular recognition of rotavirus protein. Testing of secondary antibodies and fluorochromes resulted in cell fluorescence characteristics yielding a favorable signal-to-noise ratio without causing false-positive results. A direct relationship between the amount of live rotavirus input and the percentage of infected cells with good linearity and acceptable coefficients of variation was observed. Future work is focused on reducing material costs, comparing the flow cytometry assay to the industry standard, and adapting the assay to additional rotavirus vaccine strains. Based on the above results, flow cytometry is a promising candidate assay for quantifying rotavirus vaccine potency to assist the development of novel live rotavirus vaccine formulations and storage formats.

Pre-erythrocytic malaria vaccines may need to target numerous sporozoite (spz) and/or liver-stage proteins to be effective. If the protective antigens could be definitively identified, multi-component subunit vaccines could be produced. Using a novel high-throughput T cell screening system, we recently identified a CD8 response against the Plasmodium yoelii L3 ribosomal protein in sporozoite-immunized BALB/c mice. Unlike the CD8 T cell response against the circumsporozoite protein (CSP) that increases with each parasite exposure in our system, the L3-specific response is not boosted by repeated exposures to attenuated sporozoites. We have shown that L3 -specific cells have no cell intrinsic defects that counteract their re-expansion or function but rather that broad anti-sporozoite immune responses in secondary or later exposures eliminate expression of L3, thereby preventing any opportunity for activation of memory L3-specific CD8+ T cells. This T cell outcome following immunization may be emblematic of other T cells with liver-stage targets as well. Here, we tested the L3-specific T cell response in another species (P. berghei) and also employed heterologous (cross-species) prime-boost approaches. We explore the difference in parasite load and L3-specific CD8+ T cell responses between homologous and heterologous immunization regimens. We screened mice against 37 liver-stage proteins and found that cross-species immunization boosted more responses than homologous immunization. The identities of the specific targets are under investigation. Cross-species boosting may be useful as we work to identify antigens that promote an L3-like CD8+ T cell response for inclusion in a novel multivalent malaria vaccine.

Trypanosoma brucei (T. brucei) is an ideal model system for the study of a diverse range of biological processes, a number of which are unique to the kinetoplastid pathogens. The ability to genetically manipulate these parasites allows us to investigate the molecular machinery and mechanisms underlying these major processes. We have focused specifically on two areas of study: first, the identification and functions of essential T. brucei kinases; second, the functions of a number of uncharacterized proteins that we predict are essential for precise RNA editing in the mitochondrion of T. brucei. We created conditional null cell lines in which both endogenous alleles of a gene of interest are eliminated and a tetracycline-regulatable copy is inserted. Through the presence or absence of tetracycline, we regulated gene expression and assessed the phenotypic effects. Using this method, we were able to identify a set of protein kinases essential for cell viability, potentially functioning in cell cycle regulation and in connection with other cellular processes. RNA editing is an essential process unique to kinetoplastids, in which multi-protein complexes called editosomes insert or delete uridine nucleotides to create functional mitochondrial mRNAs. The major components of editosomes have been identified, though our understanding of the functional and structural organization of these proteins is limited. This is especially true for a number of proteins with limited detectable homology to other known proteins or domains. By generating and analyzing conditional null cell lines, we will assess the functional and structural roles these proteins play within editosomes for precise RNA editing. We anticipate that these proteins are key to understanding why specific sites in mRNAs are edited by insertion, whilst others are edited by deletion of uridine residues, which is a major outstanding question in the field.

Malaria is an infectious disease caused by various species of the Plasmodium parasite which affects an estimated 207 million individuals, resulting in approximately 627,000 deaths per year. While this disease is primarily prevalent in developing nations, over half of the world’s population is at risk for malaria. Among existing malaria drugs, primaquine is uniquely effective against late-stage Plasmodium gametocytes and liver hypnozoites; however, it is dangerous to patients who are G6PD deficient. G6PD deficiency is caused primarily by point mutations in the Glucose-6-Phosphate Dehydrogenase (G6PD) protein—an enzyme that catalyzes the formation of NAPDH (a reducing agent) in the Pentose Phosphate Pathway. This condition affects around 400,000 individuals worldwide, most of whom live in areas at risk for malaria. Screening for G6PD deficiency is not readily available in many of these at risk areas and thus complicates treatment of malaria with primaquine. Therefore, screening for compounds that stabilize mutant forms of G6PD may yield potential candidates that can be used in combination with primaquine for a new therapeutic regimen. In a screen of ~100,000 compounds, 71 compounds were found to have stabilizing effects on mutant G6PD R454H (Andalus). Screening was done with a standard biochemical assay that measured enzyme activity through the detection of reaction product, NADPH (detected by fluorescence). Additional biochemical assays and studies on these 71 hits will follow to assess the mechanism and extent to which these compounds increase mutant G6PD Andalus activity. Candidate compounds that make it through these follow up experiments will then be tested with other G6PD mutants in search of a stabilizer of multiple G6PD mutants.

Trypanosoma cruzi is a protozoan parasite prevalent in South and Central America that causes life-long infection in humans. Approximately 30% of infected individuals develop a condition called Chagas disease which usually manifests as life-threatening cardiomyopathy or pathologies in the gastrointestinal system. Over 8 million people are believed to be infected. Unfortunately, current drugs for treating Chagas disease have low antiparasitic activity, are expensive, and are known to cause harmful side effects. As a result, research needs to focus on discovering new drug targets in Trypanosoma cruzi to help lead to improved drugs. Most eukaryotic organisms synthesize sterols such as cholesterol and ergosterol for essential biological functions that can lead to cell death if these sterols were made incorrectly. This research is investigating the effects of blocking the synthesis of ergosterol by deactivating an enzyme called sterol 14-demethylase. This enzyme catalyzes one of the intermediate of 20 steps of ergosterol synthesis. Inhibitors of sterol 14-demethylase, known as azoles, have been shown to be extremely active on T.cruzi in vitro and are now the center of research regarding anti T.cruzi drug discovery. Further evidence shows that blocking sterol 14-demethylase can lead to an accumulation of sterol intermediates that are converted into toxic agents in the presence of another enzyme, ERG3 enzyme, which is normally active in a later step in the biosynthesis of ergosterol. The objective of the research project I am presenting is to analyze the ERG3 homologs of T.cruzi in the context of the ergosterol synthesis. The putative homologs have been cloned and sequenced. The research now is focused on engineering genetic knockouts of the ERG3 genes to test the hypothesis that the knockout parasites may be resistant to treatment with azole drugs.

Tumor necrosis factor (TNF) is a key host factor in tuberculosis (TB). In the zebrafish model for TB, excess TNF triggers necrosis of infected macrophages which leads to exuberant extracellular bacterial growth and host susceptibility. In infected macrophages, excess TNF induces production of reactive oxygen species (ROS) which activate two death pathways one of which involves the mitochondrial transition pore, and the other leads to ceramide production and lysosomal permeabilization. In exploring the events downstream of ceramide production we found that the pro-apoptotic factor Bax is required for macrophage necrosis in excess TNF conditions. Bax has been well-characterized for its role during apoptosis, but this result suggested that Bax might also play a role in necrosis. To determine if this was the case, we engineered Bax mutants that lack its different functional domains. We studied the ability of each mutant protein to regulate apoptosis and necrosis in the zebrafish and found the BH3 domain, which is required for Bax oligomerization during apoptosis, not to be required for necrosis under excess TNF conditions. Rather, an C-terminal transmembrane helix was found to be required for necrosis. These findings demonstrate that the pro-apoptotic factor Bax also regulates necrosis but through a distinct functional domain. Since excess TNF-mediated necrosis has been implicated in human TB pathogenesis, this understanding of how Bax regulates necrosis may guide host-derived therapies.

Tyrosinemia Type II is a genetic disorder caused by a mutation in the enzyme tyrosine aminotransferase (TAT), which leads to an elevated level of tyrosine in the blood. If untreated, individuals with tyrosinemia develop life-threatening clinical symptoms that include an increased tendency to bleed, damage to the nervous system, damage to the eyes, and organ failure. Unexpectedly, mutation of TAT in the nematode Caenorhabditis elegans results in animals that are long lived in comparison with wild-type (WT) worms. This study aims to elucidate the cellular mechanism and workings of TAT in the mitochondria, its role in the pathology of the disease, and its possible links with aging in C. elegans. Thus far, we have developed a simple method to measure the CO2 output of the worms and shown that the TAT mutant’s metabolism is about 50% compared to WT. This significant decrease in CO2 output was matched comparable to a strain deficient for mitochondrial Cytochrome C Oxidase, which served as a positive control. Cytochrome C Oxidase, or Electron Transport Chain Complex IV, is one of the three complexes that create the proton gradient inside the mitochondrial matrix upon accepting electrons. Notably, mutation of the gene acting downstream to TAT in the tyrosine degradation pathway, which causes Tyrosinemia Type III in people, or any other mutation in tyrosine metabolism that we have tested, causes no significant change in CO2 output of C. elegans. Furthermore, through our preliminary metabolomics assay we have identified three metabolites that are accumulated to extremely high levels in comparison to positive and negative controls. The future steps will include determining whether these metabolites play a direct role in the effects of TAT mutation on mitochondrial function and aging.

Aging research depends on the use of model organisms to test hypotheses about the biological mechanisms common to many organisms. One commonly used model organism is the roundworm Caenorhabditis elegans. Unlike homeothermic mammals, C. elegans are poikilotherms, and as a result of a variable internal temperature, they also exhibit variable longevity based on the ambient temperature. Under standard laboratory conditions, C. elegans are commonly grown in incubators ranging between 15°C and 25°C, and lifespan varies inversely with temperature; worms live longer at colder temperatures. Our recent data show that the relationship between temperature and aging has a genetic component. By measuring the lifespan of worm strains with mutations in genes found in established aging pathways, we have identified mutations that have opposing effects on lifespan at different temperatures. For example, utilizing lifespan assays I have conducted, we have found worm strains that live longer than controls at high or low temperature, but not both, as well as strains that are short-lived at specific temperatures. These experiments suggest there may be overlapping but distinct mechanisms driving aging at different temperatures, and we are developing hypotheses to explain the temperature-dependent mechanisms of aging. Thus far, our data are consistent with the hypothesis that genes that contribute to protein homeostasis promote longevity at higher temperatures, whereas genes that limit effects of toxic compounds (both internally and externally produced) promote longevity at low temperatures. Understanding the temperature-dependent mechanisms of aging in C. elegans will advance the aging research field by extending knowledge on a widely used model organism and by testing how the mechanisms of aging and disease may be conserved in homeotherms. We hope our results will suggest how and why many genetic longevity pathways are conserved from worms to mammals despite drastically different life histories.

It is well known that tumors can be “seen” by the immune system, but immune responses differ depending on the nature and context of the target antigen: tumor antigens that are self-proteins are generally weakly immunogenic due to pre-existing self-tolerance, whereas tumor antigens that are truly tumor-specific (viral and mutated proteins) are potentially highly immunogenic because the immune system has not been previously exposed to these antigens. Therefore, tumor-specific T cells that recognize these antigens as non-self should be able to eliminate cancer cells expressing such tumor-specific neo-antigens. We developed a spontaneous Tamoxifen-inducible cancer mouse model to investigate the fate of naïve tumor-specific T cells encountering a tumor-specific neo-antigen during the pre-malignant phase of tumor development. We found, contrary to our expectation, that tumor-specific T cells were rendered tolerant and unresponsive to the cancer cell as early as 8 days post-tumor initiation and displayed very similar phenotypic and functional characteristics compared to self-tolerant T cells. Interestingly, this unresponsiveness was not due to global tumor-induced immune suppression in the microenvironment but instead the result of continuous antigen encounter, as tumor-infiltrating control T cells specific for an antigen not expressed by the cancer cell remained functional in the tumor site. Transcriptional profiling uncovered the molecular program underlying this early cancer antigen-induced T cell dysfunction. Thus, an antigenic stimulus in a non-inflammatory context, whether by a self-antigen or tumor-specific antigen, induces a distinct state of “tolerance-specific” functional unresponsiveness. While many immunotherapies aim to reverse the immune suppression mediated by the tumor microenvironment, we demonstrate that there is another level of antigen-specific, cell-intrinsic dysfunction that must also be overcome.

Colorectal cancer (CRC) is the third most prevalent (136,830 new cases estimated for 2014) and fatal cancers (50,310 deaths estimated) in the world, according to the American Cancer Society. Despite an expanding knowledge of its molecular pathogenesis during the past two decades, robust biomarkers to enable surveillance, screening, and primary prevention of CRC are still lacking. In the present study, we propose a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolic profiling approach for highly sensitive and specific colorectal cancer (CRC) detection using human serum samples. 158 metabolites from 25 metabolic pathways of potential significance were monitored. 234 serum samples from three groups of patients (66 CRC patients, 76 polyp patients, and 92 healthy controls) were analyzed. We detected 113 metabolites out of the 158 monitored, with 42 of them showing statistical significance between CRC cancer and healthy controls, 48 showing statistical difference between CRC cancer and polyp patients, and 8 between healthy controls and polyp patients. Partial least squares-discriminate analysis (PLS-DA) models established using 13 or 14 metabolites proved to be powerful for distinguishing CRC patients from either healthy controls or polyp patients, respectively. Receiver operator characteristic (ROC) curves generated based on these PLS-DA models gave high sensitivities, good specificities, low false discovery rates, and excellent areas under the curve (0.93 and 0.95 respectively, for differentiating CRC patients from healthy controls or polyp patients). Monte Carlo cross validation (MCCV) was also applied, demonstrating the robust diagnostic power of this metabolic profiling approach. To the best of our knowledge, this is the first time that an LC-MS/MS targeted serum metabolic profiling approach has been applied for comparing CRC patients to healthy controls and polyp patients, and our results demonstrate that a panel of serum metabolites enhanced by clinical factors (age, gender, smoking and alcohol status), can potentially serve as novel disease biomarkers for CRC diagnosis.

Thirty-five million people worldwide currently live with HIV. Despite the availability of highly-active antiretroviral therapy (HAART) which may prolong the lifespan of HIV patients, HAART does not target the latent viral reservoir in infected cells, enabling viral rebound in the absence of treatment. Introducing genetically-engineered DNA cleavage enzymes (endonucleases) into HIV-infected cells may disrupt the integrated provirus. The HIV-directed site-specific DNA cutting activity of these endonucleases operating in conjunction with cellular error-prone DNA repair mechanisms can introduce targeted mutations in the integrated HIV genome that prevent viral reactivation. We are investigating three classes of engineered endonucleases: homing endonucleases (HEs), zinc finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENS). While the DNA binding domains of these endonucleases allow for customization to a desired target sequence, delivering these enzymes to HIV-infected cells poses a challenge. TALENS in particular require a DNA coding sequence beyond the capacity of many viral vectors. Thus, a method of co-transduction must be pursued, whereby two halves of the endonuclease coding sequence are delivered by separate viral vectors and coordinately expressed in the target cell. My project aims to determine the optimal capsid serotype, promoter, and multiplicity of infection (MOI) for co-transducing HIV-infected cells using adeno-associated virus (AAV) vectors. I first determined AAV-1 to be the optimal serotype of AAV for infecting SupT1 cells, a cell line used as a model for HIV-infected T-cells. I then constructed two sets of AAV-1 viral vectors to express either Green Fluorescent Protein (GFP) or a red fluorescent protein (mCherry) as surrogates for endonuclease genes and infected SupT1 cells with both sets of vectors at variable MOIs, using flow cytometry to determine the rate of co-transduction. Through this method, I have successfully co-transduced cells with two AAV vectors. These results will have applications in future HIV research and clinical therapy.

Magnetic microbeads are a common tool used in biological assay purification, however, they don’t diffuse quickly nor do they have a high surface area, which results in low binding efficiency. Magnetic nanoparticles have more total surface area and can diffuse quickly, but cannot be separated magnetically as individual particles. The research I do uses magnetic nanoparticles (MNPs) coupled with pNIPAAm (p(N-isopropylacrylamide)) as a temperature-responsive reagent system to purify solutions for diagnostic tests in order to increase the sensitivity and specificity. This works by combining MNPs coated with pNIPAAm and free pNIPAAM-antibody conjugates. The antigen binds to the antibody and when heated to the cloud point temperature (around 37°C for pNIPAAm), the polymers aggregate, allowing the MNPs and the antibody conjugates to be magnetically separated together. The conjugates are synthesized using NHS chemistry and purified through HPLC. The separation efficiency of these conjugates is determined though ELISA and can reach near 100% and the total aggregation and capture time for this system is less than 4 minutes. This is useful because it can rapidly concentrate and purify samples to reduce false positives and allow for a lower limit of detection. This can be incorporated into a multitude of point-of-care tests to deliver earlier, faster, and more accurate results.

 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.

Affecting millions of people across the globe, the WHO deems myocardial infarctions (heart attacks) as the largest cause of death worldwide. The heart’s weak regenerative capacity leads to scarring of the dead tissue and a reduction in cardiac output that presents numerous health consequences. Tissue engineering is a promising outlook in restoring the structural integrity and contractile force of the damaged area. Recent findings have demonstrated that engineered heart tissues grown from stem cells can display significantly improved characteristics when subjected to mechanical stimuli. Inspired by these results, this project seeks to improve tissue performance even further by investigating a novel concept where stem cell-derived cardiac tissues are treated with the same mechanical profile experienced by native heart tissue. In order to simulate and implement these forces, tissues are grown and then anchored onto two posts that can vary in distance and stiffness. Upon contracting, post stiffness is increased providing a resistance to each beat. This resistance simulates the afterload stress that the ventricular wall contracts against to eject blood into the aorta. Next, the tissues are stretched by increasing the distance between each post. The stress produced by elongating the tissues simulates the preload stress exhibited by the ventricular wall as blood is pumped into the chamber prior to the next contraction. Finally, combining the stiffness (afterload) and stretching (preload) schemes allows the tissues to experience the entire force profile exhibited by the native heart. Simulating the cardiac mechanical environment gives the engineered tissues the stimulus to yield contractile forces and a cellular structure at par with their native counterparts. This investigation thus aims to advance regenerative medicine by bringing stem-cell derived cardiac tissues one step closer towards curing myocardial infarctions.

Polymerases epsilon and delta are responsible for replicating the leading and lagging strands of DNA, respectively. Accurate DNA replication occurs through Watson-Crick base pairing catalyzed by the enzymes’ polymerase domains, and also through the action of the polymerases’ exonuclease (proofreading) domains. The exonuclease domain of polymerase epsilon (POLE) has recently been reported to be mutated in ~4% of sporadic colorectal cancers and is associated with a high frequency of clonal base substitutions throughout the tumor genome. We hypothesize that the extent of mutational heterogeneity within a tumor will be predictive of the likelihood of resistance to any given chemotherapeutic agent and that the mutational heterogeneity due to mutations in POLE may also allow us to clinically stratify patents.The ultimate goal of the project is to identify POLE mutations in a tumor tissue, if applicable, and to find the quantity of mutations to compare with normal tissue and tumor tissue without POLE. I will utilize the newly developed Duplex Sequencing methodology to identify mutations that are present in a small number of cells within a tumor. By independently tagging and sequencing each of the two strands of individual DNA duplexes, this approach greatly reduces the number of errors. As the two strands of DNA are complementary, true mutations should be found at the same position in both strands. I will first identify tumors carrying somatic POLE mutations and use Duplex Sequencing to quantify the subclonal mutational load. My research team and I expect that the extent of mutational heterogeneity will be greatly increased in tumors bearing POLE mutations due to its impairment of proofreading capacity. For the symposium, we hope to present the quantity of sequencing errors in each of the tissues listed above or at least explain the process if we fail to collect the results in time.

Our perception of the color of an object is strongly affected by the other nearby stimuli, i.e., the object’s context. A yellow stimulus changes to brown when surrounded by bright stimuli; this change comes with a shift of red-green balance where more red light is needed to produce balanced brown and more green light is needed for balanced yellow. However, the perceptual or physiological nature of these effects and their relation to other brightness and color context effects remain poorly understood. Our project explores aspects of the spatial and temporal dependence of both the induction of brown and the accompanying red-green shift. Our study measures: (a) the threshold of brown by adjusting the luminance of a test stimulus to find the light level that appeared pure brown and (b) the red-green balance of brown or yellow stimuli by varying the red and green lights creating the stimulus. For both measures, the test stimulus is a foveally-fixated 2° diameter disk; subjects are presented with a flashing (30 or 1000 ms) stimulus, surrounded by full-screen black or single concentric wide white ring that is either contiguous with the test or has a gap ranging from 0.25° to 3.0° in width between the stimulus and the ring. We investigate spatial influence by varying gap width and temporal influence by varying stimulus duration. Based on studies of brightness perception, we predict that brown will be stronger when bright context features are near the test stimulus and the test duration is shorter. In any case, the results will help us to understand the perceptual and physiological mechanisms underlying the induction of brown and the accompanying shift of red-green balance.

The Rieske iron-sulfur protein (Rip1) is a nuclear encoded protein that assembles into the cytochrome bc1 complex of the electron transport chain in budding yeast, Saccharomyces cerevisiae. The iron-sulfur cluster bound by this protein facilitates the flow of electrons during cellular respiration in the inner membrane of the mitochondrial matrix. In the nematode worm, Caenorhabditis elegans, a mutation in a conserved residue of the Rieske iron-sulfur protein has been found that delays development and extends the lifespan of this organism. Mutagenesis experiments from our laboratory have identified suppressors of this C. elegans developmental phenotype that are due to secondary mutations in conserved residues within a flexible hinge region of the Rieske iron-sulfur protein. The primary mutation and secondary suppressing mutations were introduced to the yeast Rip1 gene through site directed PCR mutagenesis. Transgenic yeast have been created expressing the mutant forms of Rip1 homologous to those identified in C. elegans. The next stages of my project will entail the characterization of these mutations in yeast. I plan to assay the replicative lifespan, growth rate, respiratory efficiency, as well as sensitivity to variance in temperature, reactive oxygen species and carbon source in an effort to understand how this mutant’s lifespan extension fits into the larger picture of known aging pathways. The extrapolation of the mechanisms found in these model organisms serve as the basis for understanding our own complex aging process.

Parkinson’s Disease (PD) is the second most common neurodegenerative disorder in the world, affecting nearly 7 million people globally and more than 1 million people in the U.S. Symptoms of PD typically include tremors, rigidity, postural instability and slowness of movement. These motor control deficiencies can be attributed to a decrease in the expression of the neurotransmitter dopamine and the loss of many dopaminergic neurons in the substantia nigra of the brain. To study PD a mouse model is used in which C57BL/6 mice are administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is a neurotoxin that inhibits complex I activity in the mitochondrial electron transport chain. Mice treated with MPTP exhibit PD-like symptoms, including the loss of dopamine producing neurons and physical motor control deficiencies analogous to PD. Specifically, we are interested in utilizing the MPTP mouse model to understand a mechanism by which the drug rapamycin might prevent or treat symptoms of PD. Rapamycin inhibits signaling of the mechanistic target of rapamycin (mTOR) pathway, which is a key regulator of many functions, including cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. Previous research by our lab has shown that rapamycin dramatically attenuates disease progression in a mouse model of mitochondrial complex I deficiency. Because PD patients also exhibit defects in complex I, we believe that rapamycin may alleviate Parkinson’s disease progression by a mechanism similar to the effects we have seen. Currently we are administering high doses of encapsulated rapamycin in mouse diet during the three weeks prior to MPTP exposure. The expression of PD-like biomarkers in rapamycin fed mice will be measured through immunohistochemistry and western blot assays and compared to a control fed group. We hypothesize that pre-treatment with high doses of rapamycin in the diet will have robustly beneficial effects in MPTP mice.

Neurological outcomes for premature infants with cerebellar hemorrhages are poor, but the mechanisms of injury are not understood. We hypothesize that the addition of blood to cerebellar slices would decrease external granule layer (EGL) proliferation and impair migration of cells leading to developmental disruption. Our objective is to develop a slice culture model for understanding the effects of cerebellar hemorrhage on cerebellum development. Postnatal day six mice were decapitated and trunk blood obtained using a heparinized syringe. Whole brains were removed and 200 µm thick sagittal cerebellar slices were obtained using a Vibratome. Slices were placed on a cell culture insert with serum containing media. Trunk blood (4µL) was applied directly to treatment slices. Slices are incubated for 48 hours. Media samples were collected for lactate dehydrogenase (LDH) assays. BrdU was added 24 or 2 hours before fixation. Slices were fixed in 4% paraformaldehyde overnight. Immunohistochemistry for calbindin, BrdU, GFAP, PCNA was performed. Slices were processed with LacZ and Nissl staining. Images were taken on a confocal microscope and analyzed for EGL cell counts and thickness. LDH, a marker of cell death, increased in the media of blood-exposed slices than control slices for 4, 24, and 48 hours. Blood-exposed slices have thinner EGL (control 141 versus 120 µm blood-exposed). Bergmann glia were more disorganized after blood exposure and lacked the radial appearance of control slices. Purkinje cell arborization was decreased after blood exposure. Cell proliferation was lower in blood-exposed slices than in control slices (control 77 versus 26 blood-exposed for PCNA; control 80 versus 55 blood-exposed for BrdU). In conclusion, the addition of blood to neonatal mouse cerebellum slice cultures decreased proliferation of cells in the EGL after 48 hours. Additional experiments are underway to better characterize the mechanism of action.

Previous literature has illustrated that Asian culture emphasizes interdependence, family assistance, and fulfillment of social roles, while mainstream American culture emphasizes independence, individual choice, and autonomy. Do bicultural Asian-Americans face conflict when their interdependent and independent value systems directly collide? The present study examined the psychological consequences of simultaneously holding both interdependent (family values) and independent (autonomy) cultural considerations for bicultural Asian-Americans, as compared to monocultural European-Americans. Participants (N=121 European Americans, N=70 Asian-Americans) were presented with vignettes that required them to choose between family values and the self as a way of measuring conflict between incongruent cultural values. As predicted, Asian-Americans were more distressed when making the decision to go against the family as compared to European-Americans, suggesting that they do identify with Asian values. However, Asian-Americans felt equally distressed as European-Americans when deciding to go against the self for the family, suggesting that they have also adopted American values. The differences in how bad they felt were partially explained by interdependent and independent self-construals, and attitudes about respecting family. Our results suggest one source of distress and conflict, which Asian-American youth are particularly susceptible to when it comes to decision-making, as well as some of the factors that explain that discomfort and psychological distress.

The results of Na-cyclamate (Na-c) experience generalize for a variety of sweeteners: taste sensitivities for the monosaccharides glucose (pyranose) and fructose (furanose), and the disaccharide maltose (pyranose), are significantly increased, while increased sensitivity for the disaccharide sucrose (furanose) approaches significance. Sensitivities for the structurally different sweeteners Na-c and sucralose also are increased, but D-tryptophan (D-tryp) and monosodium glutamate sensitivities are decreased (Kennedy et al., 2013). Human psychophysics and animal neurophysiology data suggest that the changes involve peripheral mechanisms, in or before the receptor cells. (Faurion et al., 2002; Hassan et al., 2006; Gonzalez et al, 2009). Here we tested both the general hypothesis that the receptor subunit hT1R3 plays a modulatory role in experience-induced changes of taste sensitivity and a sub-hypothesis that Na-c experience affects responses to amino acids and peptides differently than responses to sugars and other sweeteners. Subjects rinsed their tongues with 4mM Na-cyclamate or distilled water for 10 sec once a day for 10 days. On day 11 or 12, they tasted a concentration series of aspartame, each concentration paired with water, and indicated which of each pair was “the sweetener.” Subjects treated with Na-c made fewer correct identifications than those treated with water (p= 0.03, Logistic Regression), and thus showed decreased aspartame sensitivity. Together with the overall generalization results, these data support the hypothesis that Na-c interaction with hT1R3 leads to modulation of the whole receptor response and thus to experience-induced changes in perceptual sensitivity. These data also support a difference in the mechanism for experience-induced changes of sensitivities for amino acids and peptides.

The mammalian Target of Rapamycin (mTOR) is a protein kinase that integrates signaling from growth factors and nutrient availability to regulate metabolism and cell growth. Reducing mTOR signaling with rapamycin extends lifespan in several model organisms such as yeast, flies, and mice, and rescues virtually all symptoms in a mouse model of Leigh syndrome, a neurodegenerative disorder that causes loss of motor function and early death. Previous observations from our laboratory suggest that rapamycin induces changes in the metabolism of mice modeling Leigh syndrome, favoring fatty and amino acid catabolism over glycolysis. We believe this shift in metabolism is, at least in part, responsible for the beneficial effects of rapamycin in normative aging. In order to test this hypothesis, we are examining the effects of daily rapamycin injections in 4, 12, and 22 months old mice. We performed baseline measurements of serum metabolites, forelimb strength, voluntary running, gait and balance, body composition, blood glucose, and hemoglobin A1c, and are currently administering 8mg/kg of rapamycin daily via intra-peritoneal injections for 60 days. During this period we are monitoring body weight and food intake daily, as well as blood glucose every 15 days. At the end of this period we will measure body composition, gait and balance, forelimb strength, and voluntary running again, while maintaining the mice under treatment with rapamycin for an additional 30 days. Finally, we will sacrifice the mice and analyze serum, liver, brain, adipose, and other tissues for the activation of metabolic pathways, metabolite composition, and signs of pathology. We expect rapamycin to improve most parameters studied and profoundly alter the metabolic profile of mice at all three ages. These results will ultimately allow us to investigate how modulating metabolic pathways can increase healthspan in mice and provide insight on how to promote healthy aging in humans.

Clonal evolution is a key feature of cancer progression and relapse. As cancers progress they accumulate large numbers of mutations. Some of these mutations are present in all of cells (clonal mutations) and drive the malignant progress. We will ask if there are other mutations that are present in only a subset of cells within tumors; as the environment changes during therapy these could become new driver mutations and could be the source of resistance to therapy. The subclones with the best evolutionary fitness will, in time, come to dominate the cancer cell population. Studying the number and identity of subclonal mutations may help us to predict whether or not a tumor has potential resistance to different drugs. Our hypothesis is that the large amount of subclonal mutations within a cancer will increase the overall fitness of the tumor, by providing a greater diversity from which new phenotypes can arise. We will modify the newly developed sequencing methodology, termed Duplex Sequencing, to identify specific subclonal mutations present in colorectal cancers. By adding a barcoded, partially non-complementary adapters to both ends of an individual DNA molecules which allow mutations in both strands of a DNA duplex to be identified, this approach greatly reduces sequencing errors. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be identified as technical error. We believe that most colorectal cancers will have a complex subclonal structure and may show clusters of subclonal variants, including subclonal driver mutations. Furthermore, presence of a subclonal driver mutation may be an independent risk factor for disease progression. Our study will thus uncover patterns of mutational evolution in colorectal cancer, providing insights into its stepwise clonal evolution.

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.

Mitochondria, the energy producing organelles in eukaryotic cells, play a critical role in cell metabolism and regulation. Interestingly, mitochondrial dysfunction promotes lifespan extension in Caenorhabditis elegans. One proposed mechanism is that the induction of mitochondrial unfolded protein response (UPR^mt) increases the expression of mitochondrial chaperones, nuclear-encoded enzymes that assist in folding of misfolded or aggregated proteins, to maintain mitochondrial protein homeostasis. In an effort to elucidate genes involved in the UPR^mt pathway, we performed a genome-wide RNAi screen in transgenic animals expressing mitochondrial chaperone GFP reporter hsp-6_pr::gfp. Unexpectedly, we found that knockdown of genes involved in the pentose phosphate pathway (PPP) activates hsp-6_pr::gfp expression. The PPP is a metabolic shunt off of glycolysis that produces NADPH, a reducing equivalent used for glutathione reduction and lipid metabolism, and ribose 5-phosphate for nucleotide synthesis. In addition, knockdown of transaldolase, a gene in the reductive phase of the PPP, increases the lifespan of C. elegans. Through lifespan epistasis analysis, we found that transaldolase deficiency promotes longevity by a mechanism that is genetically distinct from known longevity factors such as the insulin-like/IGF-1 target (DAF-16), the hypoxia inducible factor-1 (HIF-1), AMP kinase (AAK-2), the UPR^mt transcription factor (ATFS-1), or the germline signaling (GLP-1). This suggests that the PPP inhibition promotes longevity through an uncharacterized pathway that may respond to mitochondrial dysfunction. In addition, we found that transaldolase knockdown alters expression of fatty acid desaturase, an enzyme that oxidizes fatty acid chains. We hypothesize that dysregulation of lipid metabolism by inhibition of the PPP may cause a defect in mitochondrial lipid composition, which may increase mitochondrial proteotoxic stress, inducing the UPR^mt. Understanding the genetics behind the UPR^mt and the PPP’s role in aging has enormous benefits to human health as both the PPP and mitochondrial dysfunction are highly correlated with many age-related diseases including cancer and neurodegenerative diseases.

CT and MR imaging are widely used for the staging of a newly diagnosed H&N cancer, where treatment decision highly depends on imaging findings. Currently, there is no data regarding whether or not the TNM staging is routinely incorporated into radiology reports. We conducted a national survey to determine whether radiologists routinely address the staging, in particular regards to T (primary), and N (nodal). In addition, we explored the barriers to and perceived values of the reporting incorporating TNM staging among radiologists. A total 199 responses were submitted within a week (25.4% response rate). Over 60% of the responders were at an academic practice, having more than 8 years of experience. 73% of the responders were subspecialized in H&N radiology. Only 26% stated that they routinely assigned TNM staging in radiology reports. The most common barriers were a) unable to remember the staging classification (58%), b) afraid of inaccuracy (57%), followed by c) time consuming (46%), and d) not required (37%). All responders who assign TNM staging indicated that the reasons for such practice are a) believe in added values (31%) and b) help treatment decision (26%). 75% indicated they measure a primary tumor in three dimensions. Approximately half (49%) of the responders thought that incorporating TNM staging is important, 33% were neutral, and 17% thought it was not important.

Leigh Syndrome (LS) is a common pediatric mitochondrial disease comprised of many biochemically distinct but symptomatically similar disorders, and affects approximately 1 in 40,000 children. We are currently studying what is considered classical Leigh Syndrome by using a mouse model with a homozygous loss of the Ndufs4 gene, which encodes an essential subunit of Complex I in the mitochondrial electron transport chain, and thus results in energetic defects. Animals lacking Ndufs4 (Ndufs4KO mice) display progressive neurodegeneration, ataxia, and respiratory failure that leads to death. Selective inactivation of Ndufs4 in neurons and glia leads to manifestation of a disease phenotype indistinguishable from Ndufs4KO mice. Accordingly, lesions in the brainstem and cerebellum are common in human LS patients. However, the neuronal cell types susceptible to the disease are unknown. Thus, to determine the role of certain neuronal types in disease progression, we used the Cre-Lox system to inactivate Ndufs4 in glutamatergic, cholinergic, and GABAergic neurons. Glutamate, acetylcholine, and GABA are three primary neurotransmitters which define specific subsets of neurons widely present in the brain. So far, it appears that inactivation of Ndufs4 in cholinergic and GABAergic neurons does not yield a disease phenotype comparable to that of a full-body Ndufs4 knockout, as they are indistinguishable from wild-type mice. Inactivation of Ndufs4 in glutamatergic neurons, however, appears to yield a mouse with a significantly shortened lifespan that is statistically indistinguishable from the full-body Ndufs4KO mouse.  

Due to rapid mutation of the Hemagglutinin (HA) antigen, the current influenza vaccine only induces strain specific protection, requiring people to get vaccinated yearly. Hemagglutinin is the main glycoprotein on the surface of the influenza virus and consists of two main components, the variable head and the conserved stem. Previous studies have shown that vaccination with the conserved stem region of the HA antigen induces the production of broadly neutralizing antibodies, which are able to neutralize a broad spectrum of influenza sub-types. Therefore we designed a vaccine regimen in order to increase the effectiveness of our current DNA vaccination delivered by a gene gun to induce antibodies against the stem. We hypothesized that priming mice with the conserved stem region of the HA antigen followed by a boost with the full-length HA would lead to an increase in antibodies against the stem and the production of broadly neutralizing antibodies. Groups of mice were primed with either the conserved stem or the full-length HA and were boosted 6 weeks later with either full-length HA or the conserved stem. Two weeks after the boost, the mice were challenged with a lethal dose of a homologous or heterologous strain of influenza. Weight loss was monitored and blood was collected throughout the experiment. Neutralization and binding antibody assays were performed in order to compare the relative ability of each immunization regimen to induce antibodies against the HA head vs. stem.

 Sleep is essential for survival, yet its function remains unknown. In our society, sleep deprivation and disruption of sleep architecture, namely the temporal structure of rapid eye movement (REM) and non-REM sleep stages—are common. Sleep deprivation impairs memory consolidation dependent on the hippocampus region of the brain. We hypothesize that disruption of sleep architecture in the absence of sleep deprivation impairs hippocampus-dependent memory. Our laboratory developed a rat model in which sleep architecture is predictably disrupted without the confounding effects of sleep deprivation, and without genetic, pharmacological or surgical manipulations. Exposure to an artificially short light-dark (LD) cycle (22 hours) produces two circadian rhythms within the same animal. In this “forced desynchrony” protocol, animals cycle between “aligned” days, during which the two oscillations are in phase and sleep architecture is similar to that of 24-hour LD cycle control animals, and “misaligned” days, during which oscillations are out of phase and sleep architecture is disrupted. Previous results indicated that rats trained in a contextual fear-conditioning hippocampus-dependent memory task during misaligned days show impaired memory consolidation. Because memory consolidation was measured as “freezing”, the animal’s immobility in the fearful context, we asked whether memory consolidation would also be impaired in a hippocampus-dependent task requiring active spatial navigation (the Morris water maze or MWM). We trained aligned, misaligned and control animals to swim to a hidden escape platform within a pool containing visual cues. After removing the platform, we tested memory consolidation by measuring latency to reach the original platform location along with other parameters. Our analysis showed that while circadian forced desynchrony associated with sleep-stage architecture disruption may impact memory acquisition in the MWM task, it does not appear to impair long-term memory consolidation. We conclude that normal sleep architecture may be critical for some hippocampus-dependent memory tasks but not others.

Positive outward remodeling of arteries and formation of arteries from arterioles in response to arterial injury is highly dependent on the proliferation and migration of smooth muscle cells (SMC). To form new arteries, SMCs also have to degrade the extracellular matrix (ECM) around the existing vessel to serve as a starting point for arteriogenesis. The p27 protein is a cyclin-dependent kinase inhibitor that stalls the cell cycle. We have previously demonstrated that mice lacking the p27 protein have improved arterial enlargement in response to arterial injury. We hypothesize that decreased expression of p27 improves SMC proliferation and migration, as well as ECM degradation. To test this hypothesis, we extracted aortic SMC isolated from p27 knockout and wild type mice and performed several in vitro assays to assess proliferation and migration. We used a growth assay to measure proliferation. We used a wound assay and a modified Boyden Chamber assay to measure migration in the absence (wound assay) and presence of a chemoattractant, platelet-derived growth factor-BB (Boyden Chamber assay). The wound assay is done under growth arrest to study migration independently of proliferation. The Boyden Chamber assay is performed with and without matrix metalloproteinase inhibitors (MMPIs) to assess migration in the presence and absence of ECM degradation. We performed these assays under varying conditions such as nutrient starvation and hypoxia to better model recovery from ischemia or under wound conditions. Preliminary wound assay data has shown that certain isolates of p27 KO SMCs exhibit a higher degree of migration. A better understanding of p27’s role in arterial remodeling may lead to improved therapies to stimulate arterial growth to treat ischemia resulting from arterial injury or to inhibit arterial growth to treat cancer.

Measurement of tumor proliferation following cancer therapy can provide information on the effectiveness of that therapeutic treatment. One method for measuring tumor proliferation in vivo is Positron Emission Tomography (PET). In PET, a patient is injected with a radiolabeled compound that is selectively taken up by tumors. The radioisotope used decays with the ejection of a positron, which subsequently interacts with an electron to produce two 0.51 MeV gamma rays traveling 180 degrees from each other. The two gamma rays then strike a detector surrounding the patient. The in vivo location of the positron-electron interaction can be determined by comparing the energies of the two coincident photons. The PET tracers used to measure proliferation are either thymidine or thymidine analogs. Recent studies on one of these tracers, 4’-[methyl]-thiothymidine (4DST), found that the kinetics of 4DST uptake in tumor cells was much slower than thymidine (TdR) or 3’-deoxy-3’-fluorothymidine (FLT). Differences in the kinetics of uptake could be due to variations in transport kinetics, different rates of phosphorylation, or different rates of nucleotide incorporation into DNA. Understanding the factors that underlie different tracer uptake kinetics improves our ability to interpret PET information and thereby develop more effective treatment options. In the present study we examined whether the different rates of uptake observed for 4DST were due to differences in the first step in nucleoside metabolism, phosphorylation by thymidine kinase-1 (TK1). The kinetics of phosphorylation for 4DST was determined using purified TK1 in an in vitro assay. Reactions were stopped at various time points and phosphorylated products were collected by ion-exchange filtration. Results were compared to assays of TdR and FLT phosphorylation. Preliminary results suggested that 4DST phosphorylation was slower than either TdR or FLT phosphorylation. Slower phosphorylation by TK1 could explain the slow kinetics of 4DST uptake in cells.

The toll-like receptor (TLR) proteins are expressed on cells of the innate immune system and recognize structural motifs present on microbial pathogens. Recognition of pathogens by TLRs activates an intracellular signaling cascade and activation of NF-kB, a transcription factor that is critical for expression of inflammatory mediators. Previous studies from our laboratory have shown that single nucleotide variations present in human TLR1 lead to altered expression of TLR1 and are associated with increased risk of death in patients with sepsis. We tested the hypothesis that variations of mouse Tlr1 which correspond to common human variants would result in comparable changes in TLR1 activity. We used site-directed mutagenesis to introduce coding variations in the Tlr1 cDNA. After transfecting the variants into cultured human cells, we stimulated them with Pam3CSK4, a triacylated lipoprotein known to be recognized by the TLR1/TLR2 complex. We then used a bioluminescent luciferase reporter system to measure the levels of NF-kB transcriptional activity. Tlr1 with an Ile allele at position 605 showed increased activity relative to a Leu allele, and the Tlr1 with a Ser allele at position 605 showed increased activity compared to Leu, but to a lesser degree than the Ile allele. Tlr1 with a Ser allele at position 251 showed increased activity compared to Asn, but elucidating its specific effect will require further investigation. These results indicate that variations in the transmembrane domain of mouse TLR1 result in alterations in function comparable to that of human variants in the same region. These findings suggest that mouse models may be used to study TLR1 variants’ effect on the immune response in humans. This could lead to a better understanding of the mechanisms leading to poor clinical outcomes for patients with sepsis and other inflammatory diseases.

Aging is associated with increasing degrees of drifts in gene expression, thought to be epigenetic in origin. The resulting variegated gene expression would have the potential to lead to a loss of proliferative homeostasis, resulting in multifocal tissue atrophies and hyperplasias. Because of enhanced rates of DNA replication, the latter could lead to neoplasms via mutations in oncogenes and tumor suppressor genes. As an initial test of this hypothesis, I immunostained leiomyomas (a benign uterine tumor)of six patients for nuclear proteins, including p53, sirt 1, Cdk4 (Cyclin-dependent kinase 4) and p16/Ink4a (CDKN2A), and examined the degrees of variegated gene expression at the three sites: cells within benign leiomyomas of the uterus and of smooth muscle immediately contiguous to and distal to these lesions. The preliminary results showed that the majority of specimens gave results consistent with the hypothesis of clonal expansions derived from precursor populations of cells with more extensive variegation. There were several exceptional results, however, perhaps related to technical issues. These pilot studies encourage additional studies, including the use of alternative methodologies for the detection of neutral mutations associated with the predicted clonal expansions. In addition, these studies may provide new prospectives for diagnostic approaches to tumor formation in uterine.

Disruption of the nervous system has been widely associated with aberrant bone function. The capacity for zebrafish to undergo rapid bone regeneration following fin amputation confers unique opportunities to examine neuroskeletal signaling within a highly tractable experimental system. In this study, we developed a model of cholinergic disruption in the regenerating tail fin using intramuscular injections of Botuliunum toxin B (BTxB). The tail fin is muscularized only at the base. By administering BTxB into the base, we were able to examine remote effects of BTxB at the distal tip while minimizing the influence of impaired muscle near the regenerate. BTxB resulted in multi-faceted effects on bone regeneration in adult zebrafish. For example, BTxB fish exhibited a significant decrease in the % length of bone regrowth compared to saline controls (sal: 74.8±8.1%, btx: 35.2±4.6%, p<0.001 for treatment, n=5). The degree of inhibition was significantly more pronounced in the middle of the regenerate compared to the dorsal/ventral regions (p<0.05 for region: treatment), suggesting that the trophic influence of fin base nerves can be spatially focused to distinct fin regions. Finally, when fish were calcein stained, we found that BTxB dramatically decreased osteoblast activity in regenerating bone. Interestingly, the effects of BTxB appeared to be spatially restricted within individual rays, as evidenced by marked differences in osteoblast activity between adjacent rays. This suggests a potential role for intra- and/or inter-ray nerves in mediating the effects of nerve disruption in the fin base on osteoblast activity in the distal tip. Collectively, these studies demonstrate the capacity for focal cholinergic disruption to remotely alter bone regeneration in a multi-faceted manner. Given the genetic and optical tools for dissecting neural pathways in zebrafish, this model provides a powerful platform for identifying novel mechanisms underlying neural control of bone growth.

Cardiac cell death has been implicated in the pathogenesis of various forms of cardiomyopathy and end-stage heart failure. Here, a novel signaling molecule, TAK1 (TGFβ-activated kinase 1, gene name Map3k7), is identified as a nodal regulator of apoptotic and necroptotic myocyte death, myocardial remodeling, and heart failure propensity. I found that genetic or pharmacological inactivation of TAK1 in cardiac myocytes markedly induced programmed necrosis and apoptosis in response to TNFα (tumor necrosis factor alpha), an inflammatory cytokine. Conversely, overexpression of the constitutively active mutant TAK1ΔN (N-terminal truncation), or TAK1 plus its activator TAB1 protected cardiac myocytes from TNFα-induced cell death. Mechanistically, inactivation of TAK1 promoted formation of the necroptotic cell death complex consisting of RIP1 (Receptor-interacting protein 1), RIP3 (Receptor-interacting protein 3), caspase 8 and FADD (Fas-Associated protein with Death Domain). In addition, my data showed that genetic ablation of cytoplasmic cell death signaling molecules such as RIP1, RIP3, caspase 8, or FADD largely blocked TNFα-induced cell death in TAK1-deficient cells, whereas deletion of mitochondrial cell death proteins, Bax/Bak or cyclophilin D, showed no effects. Further, the transcription factor NFκB-mediated cell survival signaling was greatly impaired in TAK-1 deficient cardiac myocytes. Taken together, the data indicate that TAK1 functions as a critical “molecular switch” in TNFα-induced programmed necrosis in cardiomyocytes by interacting with the RIP1/3-caspase 8-FADD cell death pathway as well as the NFκB cell survival pathway. These results also reveal a new paradigm where TAK1 functions as a necessary survival factor in the heart by directly antagonizing necroptosis.

 DNA sequencing is essential to life sciences and personalized medicine. Nanopore sequencing is a single molecule sequencing technique that promises both an increase in sequencing speed and a reduction in cost compared to current sequencing technologies. In nanopore sequencing, DNA is drawn through a nanometer-wide pore while we record any changes in ionic current. These current changes depending on the nucleotide sequence. Our lab uses the nanopore Mycobacterium smegmatis porin A (MspA), which was engineered to examine DNA with single nucleotide resolution. MspA is sensitive to the four standard nucleotides (A, T, C, G) as well as small chemical changes to those nucleotides. Uracil is a nucleotide that differs from thymine by a single methyl group which we now show we can detect with MspA. Using a process called bisulfite treatment, which converts cytosine to uracil, we obtain information that helps us to determine the input DNA sequence.

The Fu lab, in collaboration with the Yager and Lutz labs, develops low cost disposable diagnostic assays, much like the common pregnancy strip, but capable of performing more complex sample processing. Our paper-based assays are capable of automatically sending multiple reagents over a detection zone in sequence. These assays have been developed to perform the detection of markers for diseases such as dengue fever and malaria. My project is to further expand the capability of these assays by providing methods to passively control the flow rate through the detection zone. By optimizing the flow rate for each reagent delivery step, the time to result of the assay can be reduced without sacrificing signal strength. Devices are designed in AUTOCAD and fabricated from nitrocellulose and cellulose using a CO2 laser system. Flow speed within a device is quantified using a fluid marking system, in conjunction with custom video tracking software composed in MATLAB. Our experiments demonstrate several preliminary designs, in which flow is modulated by modifying the geometry of the assay substrate. In one design, a steady flow rate eventually transitions to a secondary, slower steady flow. A second design performs the opposite, where a steady flow rate eventually transitions to a secondary faster steady flow. The third preliminary design incorporates both these results to have a slow steady flow, which eventually transitions to a faster steady flow, and then finally falls back to a slower steady flow again. These flow profiles are well tailored for common protocols currently in use, where fast flowing rinse steps alternate in between slow flowing reagent binding steps.

Childhood epileptic encephalopathies (CEE) are a subset of rare epilepsies, largely characterized by seizures and mental disabilities. Recent studies, including those by the Mefford lab, have shown that about 30% of CEE is caused by mutations in specific genes.These mutations are ‘de novo’ – they arise during fertilization or the development of the embryo. While many types of epilepsy have a hereditary basis, de novo mutations are increasingly recognized as significant risk factors in some epilepsies. The mutations also change the amino acids that make up proteins and are mostly in exons – parts of the DNA that code for protein. Few studies focus on synonymous mutations that do not change the amino acids of a protein or mutations in introns –pieces of DNA that have regulatory function but do not code for protein. We have previously sequenced 29 known epilepsy genes in a cohort of 800 CEE patients, but focused primarily on exonic mutations that change the protein sequence. My goal is to understand whether intronic or synonymous mutations that we have already found in these patients are inherited or de novo. We hypothesize that these classes of de novo mutations disrupt the typical formation of RNA transcripts, which in turn results in an absent or mutant protein. I will use molecular inversion probe (MIP) capture to determine the inheritance of intronic variants in patients with CEE. A MIP is a piece of DNA that is designed to target specific regions of DNA which are then amplified and analyzed; the technique enables us to capture a large number of variants at once. Discovering de novo variants in intronic regions of genes will deepen our understanding of the cause of CEE. These variants will inform us of affected biological pathways and contribute information for future CEE treatments.

Nitrogen is important for plants because it helps makes chlorophyll and keeps the plants healthy, however use of fertilizer is expensive and also environmentally damaging. Previous studies in our lab have shown that inoculating crop plants with endophytes isolated from poplar and willow trees enhanced biomass and fruit yields of peppers and tomatoes. In this study, we screened commercial varieties of rice in low nitrogen soil in a greenhouse setting and found that one out of the six variety of rice inoculated with an endophyte consortium had increased shoot biomass in comparison to the uninoculated control plants. We are further experimenting with the endophyte inoculation on this particular rice variety to identify which individual isolate has a positive impact on the growth of rice in low nitrogen conditions. With rice being one of the most important crops around the world, the impact of these findings could hugely reduce the environmental impact of chemical fertilizers in rice farms around the world.

Patients with Alzheimer’s disease experience progressive loss of memory and thinking skills, gradual declines in the ability to carry out simple tasks and, in severe cases, full blown dementia. The brains of patients with Alzheimer’s accumulate extracellular plaques of a family of beta amyloid peptides (strings of amino acids cleaved from a larger protein called Amyloid Precursor Protein) in multiple regions of the cerebral cortex resulting in losses of synapses and neurons. Given the fact that beta amyloid peptides have been evolutionarily conserved, our group became interested in the potential normative roles of these peptides. Observations by a member of the Martin lab led to the hypothesis that small concentrations of beta amyloid may have a proliferative effect on cells. My project explores this idea with experiments that compare the proliferative effects of various members of the beta amyloid family and dose-response experiments that determine optimal concentrations for proliferative effects. In my experiments, various concentrations of beta amyloid 1-40 (and, as controls, comparable levels of a scrambled beta amyloid peptide 40-1) are added to a standard culture medium along with 10% embryonic serum (serum without detectible levels of beta amyloid peptides) and established required growth factors. The cells’ division rates within those two week periods are determined for individual clones though crystal violet staining. From these initial experiments, we have concluded that cells are indeed able to divide more rapidly in media that has beta amyloid 1-40 present and that further attempts to determine optimal concentrations should focus within the range of 1-10 nM and above 10 nM to the point at which we anticipate evidence of toxicity. Future experiments will probe changes in gene expression resulting from treatment with optimal concentrations of beta amyloid for the enhancement of cell proliferation.

Roux-en-Y gastric bypass (RYGB) surgery, which promotes major, sustained weight loss, is the most commonly performed bariatric operation in the United States. A ramification of RYGB is the remission of type 2 diabetes mellitus (T2DM) in patients. With 25.8 million people in the U.S. suffering from diabetes and 90-95% of adult diabetes being T2DM, we hope to understand the underlying mechanisms of RYGB on glycemic improvement in T2DM patients. The two leading hypotheses for the impact of RYGB on T2DM resolution are the lower intestinal hypothesis and the upper intestinal hypothesis. The lower intestinal hypothesis postulates that increase in nutrient delivery to the distal intestine stimulates glucagon-like peptide-1 and increased insulin secretion. The upper intestinal hypothesis postulates that elimination of contact between the ingested nutrients and the proximal small intestine exerts direct anti-diabetic effects. To test the two hypotheses, we use a porcine model and perform various bariatric gastrointestinal (GI) operations to assess the effects of each operation on glucose homeostasis. Ossabaw pigs are used for their similarities in gastrointestinal anatomy to humans. We feed them a high-fat diet to induce obesity and insulin-resistance resembling those seen in T2DM patients. The four GI operations - Gastrojejunostomy (GJ), GJ with Duodenal Exclusion, Roux-en-Y Gastric Bypass (RYGB), and RYGB with Vagotomy - rearrange various parts of the stomach and small intestine. To assess the effects of surgery on glucose homeostasis, we perform two types of blood tests: intravenous glucose tolerance test and meal tolerance test. The monitoring of the post-operative changes in insulin and gut peptide hormone levels provides insight into the mechanism of T2DM remission. Although more data is needed to support either hypothesis, the results of this study will help identify new targets for surgical and medical interventions for T2DM patients.

Merkel Cell Carcinoma (MCC) is a rare, yet aggressive skin cancer with a disease-associated mortality of approximately 40%. Although surgery is typically a component of standard care, it is unclear how often patients should also receive radiation to control disease at the primary site. One retrospective study of 1254 patients found that of the patients who received surgery alone, 39% recurred locally compared to only 12% of the patients who received surgery in combination with radiation therapy. In contrast, in a cohort of 243 patients from New York treated with surgery alone, only 3.8% recurred locally. In order to establish a better standard of care for low risk MCC patients and attempt to resolve this discrepancy in the literature, we identified low risk patients within our cohort based on the following inclusion criteria: 1) primary tumor ≤1cm in diameter, 2) microscopically negative margins on the surgical excision, 3) absence of lymphovascular invasion in the primary tumor, 4) absence of profound, chronic immunosuppression, and 5) no microscopic evidence of nodal metastasis in the sentinel lymph node biopsy. Of the 29 patients who met these criteria, 17 were treated with surgery alone, while 12 received adjuvant radiation to the primary site after surgery. We found that 4 patients recurred locally (24%) among the patients treated with surgery monotherapy, while there were no recurrences among patients treated with surgery and adjuvant radiation. Even patients with low-risk MCC still have an appreciable chance of local recurrence if treated with surgery alone. While this risk may be acceptable to some patients, adding radiation therapy can lower the chance of local recurrence.

Duchenne muscular dystrophy (DMD), is a degenerative disease caused by a recessive X-chromosome mutation, resulting in lack of the protein dystrophin. Affected males experience profound muscular weakness in childhood and by adolescence the heart and respiratory muscles are affected. The Golden Retriever muscular dystrophy (GRMD) dog model displays phenotypes analogous to patients and is considered the premier preclinical model. We evaluated changes in limb function by assessing gait in GRMD (n=3), wildtype (n=2), and in GRMD dogs injected with an adeno-associated virus carrying a dystrophin replacement gene in a single limb (n=3). Dogs were walked at a self-selected pace along an instrumented carpet to measure gait parameters. The average velocity over time of non-injected GRMD dogs was 60.39% of wildtype. Treated dogs displayed slightly increased velocities (67.17% of normal). By the final timepoint, velocity in all groups was comparable. The average paw pressure of the non-injected GRMD group increased 8.05%, while the average paw pressure for the wildtype decreased by 12.75%. The ratio of mean paw pressure for untreated GRMD: wildtype increases from 0.819 to 1.014 over time. We therefore determined that paw pressure might be a sensitive indicator of a treatment effect. To estimate the minimum sample size required for future studies, a power of analysis was performed. Results indicate that n=14 is required to detect differences between groups with a confidence level of 5% and power of 80%. Given that human patients suffer multiple symptoms of muscular weakness analogous to the dogs, including reduced limb function, changes in treated GRMD dogs as measured through gait analysis may evaluate the efficacy of the gene therapy as treatment for DMD. Human patients suffer multiple symptoms from muscular weakness (non-ambulatory, cardiac impairment, etc). Future studies hope to transition treatment to human patients to improve ambulation and increase quality of life.

Although patients diagnosed with pulmonary hypertension (PHTN) are at a higher risk for postoperative complications, studies to date have not distinguished whether this applies equally to all PHTN patients, nor have they determined the severity of postoperative complications should they occur. Pre-operative screenings take place in the form of echocardiograms (ECHO), which are accurate but rather expensive. This study, one of the largest pulmonary hypertension studies to date, is headed by Dr. Gail van Norman, director of the Pre-Anesthesia Clinic at the University of Washington Medical Center (UWMC). We look to explore the correlation between the severity of pre-existing pulmonary hypertension and post-operative complications. This study additionally explores the correlation between metabolic equivalences (METS) and PHTN, which could yield a new, more cost-effective method of screening for the disease. As research interns on this project, we abstract data from the medical records of eligible patients at UWMC. We focus on patients who have undergone surgery with moderate to severe PHTN, and collect pre-op and post-op data using hospital affiliated databases. Patients with these specific conditions are believed to have <4 METS and are at an elevated risk for post-operative complications. Should these hypotheses be proven correct, the results of this study will benefit both patients and providers, reducing the cost of screening, as well as improving patient care by allowing doctors to better manage the disease in the peri- and postoperative settings, reducing both complication and mortality rates.

Sepsis is a medical condition characterized by potentially fatal generalized inflammation, typically resulting from infection. Despite previous attempts to identify mechanistic therapies, no targeted intervention has impacted survival rates. Prior hypotheses focused on immunologic mechanisms, but this project investigates metabolic responses in sepsis using a mouse model. The current target of investigation is a nuclear hormone transcription factor called PPARalpha. PPARalpha regulates the expression of proteins and enzymes associated with inflammation, stress, lipid metabolism and is related to both human and mouse survival in sepsis. We hypothesize that PPARalpha is necessary to maintain energy production sufficient to support organ function in sepsis and that deficient PPARalpha activity results in organ system failure. Because cardiac tissues preferentially use fatty acids as an energy source, we hypothesize that it is the organ that will be most affected in sepsis. Sepsis was surgically induced in mice, tissues were harvested at specific time points and gene expression was quantified using real time qPCR. Seven genes involved in fatty acid metabolism were analyzed at time points 0, 24, 48 and 72 hours. PPARalpha expression was higher in wild-type tissues, but it was down-regulated in both wild type and knockout mice. In addition, wild type mice showed greater expression of fatty acid oxidation genes than PPARalpha-knockout mice at every time point though this group of genes was down-regulated in both cohorts. Knock out mice also showed increased cardiac PPARgamma expression at 24 and 48 hours, which is a marker of reduced survival in other models. Our findings indicate that wild type mice are better able to maintain energy production necessary to preserve tissue function. These results support the hypothesis that the major cause of death in septic mice is cardiac failure due to impaired PPARalpha expression and therefore inability to sufficiently metabolize fatty acids.

Stroke is the fourth leading cause of death and the leading cause of long-term disability in the United States. Microglia are the resident immune cells in the central nervous system and play critical roles in stroke pathophysiology. During ischemic stroke, cytokines known as interferons (IFNs) are released from CNS cells and bind to microglial cell surface receptors. This binding initiates a signaling cascade that leads to increased expression of Interferon-Stimulated Genes (ISGs). We hypothesize that increased expression of ISGs contributes to post-stroke neuroprotective pathways. There are three types of IFNs, and Type 1 includes IFNs α and β, which are well-known immunomodulatory factors. Type 2 IFNs include IFNγ, a prototypical pro-inflammatory activator. Type 3 IFNs (IFNλs) are the most recently discovered and least well characterized. IFNλs are effective in treating several viral illnesses and they are better tolerated (fewer side effects) than Type 1 IFNs. We have recently shown that Type 1 IFNs can induce increases in ISG expression in microglia. My project goal was to characterize the ability of IFNλs to induce ISG expression in microglia and compare their pharmacological potency to that of Type 1 IFNs. To do this, I stimulated primary microglia with varying dosages of IFNλ3 and for varying amounts of time. I extracted RNA and prepared protein lysates from the stimulated cells. ISG expression was quantified by qRT-PCR and immune (Western) blotting. I also collected conditioned medium and assayed for the release of specific ISG chemokines, such as CCL5 and CXCL10 using ELISA. I obtained dose response curves and time course data. Our results showed robust up-regulation of multiple ISG mRNA transcripts and increases in release of specific chemokines following stimulation of microglia with IFNλ3. These studies will help us continue to characterize important cellular and molecular targets for pharmacological intervention in stroke.

“Fetal alcohol spectrum disorders” (FASD) are considered to be the leading preventable cause of mental retardation. Among this spectrum Fetal Alcohol Syndrome (FAS) represents the most extreme consequence of maternal alcohol consumption and as such associated facial dysmorphologies and neurological defects have been heavily investigated. In this project, our lab is interested in assessing the extent craniofacial development is affected due to chronic paternal and pre-conception maternal alcohol consumption using a mouse model. For the paternal exposure project, male mice were first bred with normal females as control and then given a 10% ethanol water solution for six weeks, which covers a full cycle of spermatogenesis in mice, and mated with normal females again as experimental compare. For the pre-conception exposure project, female mice are divided into three groups given different solutions respectively, four days a week for ten weeks: 10% ethanol water exposure, calorie-matched sucrose solution, and water. Upon completion of exposure, they were bred with normal males. When litters are at 60 days of age, the craniofacial phenotype of the specimens will be assessed using high-resolution microCT and their shape differences will be statistically assessed using a set of landmark and geometric morphometric methods (GMM). By the time of the Symposium, we expect to complete the image acquisition of the control group, as well as some of the initial GMM analysis. We will also be able to report differences in litter size, birth weight, and growth rate of litters between groups in both projects. As a whole, we expect to see subtle but significant differences in craniofacial development between mice that are born to fathers or mothers consuming alcohol and their controls. From there, the leading mechanism and degrees of alteration of craniofacial development can be related to the effects of chronic alcohol exposure in humans.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer among US men and women, and it disproportionately affects Korean-Americans (KAs). Currently, CRC related deaths are decreasing among all racial/ethnic groups, but increasing among KAs. Prevention and early detection of CRC, through cancer screening reduces disease incidence and death. However, research shows that KAs may not be adhering to the current CRC screening guidelines. The purpose of this study was to investigate whether KAs residing in Washington State are up-to-date with the current CRC screening guidelines and identify demographic predictors of CRC screening up-to-date status. We conducted a cross-sectional survey using convenience sampling among KAs ages 50-75 residing in three WA State counties (-Snohomish, King, and Pierce). Participants were recruited from Korean community organizations including churches, community health centers, and senior centers. The interviewers administered the 15 minutes survey in person. The total sample size was 170 participants. We analyzed data to investigate the number of KAs in the WA State who reported being up-to-date with CRC screening, based on current medical guidelines. We also examined demographic predictors that relate to CRC screening up-to-date status such as insurance status, having a primary care provider, annual household income, English proficiency, and education level. The findings highlighted the CRC screening up-to-date rates among KAs in WA State and demographic predictors that are associated with CRC screening up-to-date status. Identifying these predictors will allow researchers to understand the barriers and facilitators of CRC screening and develop interventions targeted to a particular high-risk group within KAs.