Recent advances in genome sequencing have allowed for the study of metagenomes, genetic material recovered directly from environmental samples. This has produced an enormous number of enzyme sequences without efficient methods for characterization. Since it is unknown whether a characterized enzyme is the best catalyst for a specific reaction, uncharacterized, natural enzymes in the metagenome may be better candidates for catalyzing the specific reaction. Thus, I am developing methods to search for and computationally characterize metagenomic enzymes with the same function as enzymes of therapeutic and commercial interest. By utilizing the Rosetta Software Suite to produce highly accurate models of enzyme structures, this method will evaluate an enzyme sequence’s potential for catalysis directly from a gene sequence. In order to validate my computational techniques, I will experimentally determine catalytic rates of enzyme candidates. As the method for an efficient metagenome search is still unknown, I am working to determine proper parameters for a homology search by using visual and statistical analysis of enzyme structure models in order to produce a diverse set of genes for experimental characterization.

Escherichia coli can contaminate food, such as beef and spinach, causing illnesses ranging from recurring diarrhea to death in susceptible individuals. They may produce a toxin typically associated with another harmful bacterium known as Shigella spp. This toxin is referred to as “Shiga-toxin”, and E. coli that produce this toxin are known as STECs. While E. coli O157 remains the most common STEC in the United States there are over 100 STECs that can be just as harmful. My project, conducted in collaboration with individuals at CSUEB and the USFDA, involves identifying chemical indicators that would help detect different strains of non-O157 E. coli in contaminated foods, and determining if these STECS are vulnerable to a coliphage and a Bdellovibrio sp. invasion. Specifically, nine different STECs were chosen to be screened for their ability to produce catecholate siderophores (phenolates), such as enterobactin, chelating compounds important for some pathogenic bacteria in the acquisition of iron. The same STECs were screened for the presence of acyl homoserine lactones, chemicals involved in bacterial communication, also known as quorum sensing, and expression of virulence factors. Assays were conducted to determine if a coliphage and a Bdellovibrio sp. can infect these STECs. Detection and identification of non-O157 STECS is not easy, which hinders determination of their origin and, subsequently, how to eliminate them. If new tools, such as the use of predators or chemical indicators were developed to facilitate detection of these bacteria, then determination of the source of contamination (e.g., packing plants and fields) may occur, and quicken measures that prevent or, at least, decrease further infection, disease and/or death.

Opiates have long been a useful tool in medicine due to their potent analgesic properties. Although there has been a significant increase in prescribed use of opioids in recent years there has also been a large increase in deaths related to prescription opioids that may involve abnormal control of ventilation. Opiate effects on ventilation have largely been considered to be due to depression of respiratory chemosensitivity (sensitivity to carbon dioxide and/or acid-base balance), but another effect may include decreased oxygen consumption and therefore decreased production of carbon dioxide. In this study, we are using a whole body plethysmograph and a respirometer to measure the rate of oxygen consumption levels in juvenile rats 18-23 days) during sleep - before and after administration of morphine at varying dosages (5mg/kg to 45mg/kg). Our initial findings show no decrease in oxygen consumption and surprisingly instead demonstrate increases up to 50% following opioid administration. These unexpected findings illustrate the need for additional investigation on possible causes of prescription opiate associated deaths. Increased oxygen consumption (and therefore increased carbon dioxide production) in the presence of drug induced decreased sensitivity to carbon dioxide may interact to lead to dangerous compromises in the control of ventilation in patients taking a broad range of opiates doses in the community.

Comic books have long been used as an educational tool to improve and protect public health in the U.S. In 2008, the Native Comic Book Project (NCBP) was launched as a youth-focused community education project of Native People for Cancer Control (NPCC); a National Cancer Institute funded Community Network Program Center, based at the University of Washington. The availability of cancer screening tests, high quality health care, encouragement for health promoting behaviors, and access to cancer clinical trials, are often not available to tribal communities. As a result, American Indians and Alaska Natives (AI/ANs) experience the worst cancer related disparities and have the poorest survival rate among all other racial/ethnic groups. The purpose of the NCBP is to reduce cancer risk by involving AI/AN youth in creating comic books on health topic such as health living, tobacco use, cancer risk, disease prevention, and physical fitness. The NCBP is modeled after Dr. Michael Bitz’s The Comic Book Project, but is adapted for AI/AN youth and incorporates Native storytelling and traditional ways of living. We are currently conducting a formal evaluation of the NCBP using pre- and post-intervention assessments to measure knowledge, habits, and decision-making regarding tobacco use, healthy eating, exercise habits, and human papilloma virus (HPV).

Chronic inflammation is a silent killer, contributing to death in patients with chronic kidney disease (CKD) and other chronic disorders, such as diabetes and obesity. Although the specific molecular pathways linking inflammation contribute to CKD pathogenesis still remain unclear, interleukin-6 (IL-6), the prototypic inflammatory biomarker and a potential mediator, is believed to be able to help us understand the mechanism that leads to CKD. We have been investigating the role of IL-6 family of genes using different animal models, such as the Unilateral Ureteral Obstruction (UUO), to induce interstitial fibrosis. The results from our studies have demonstrated that IL-6 and gp130 are profibrotic molecules. Therefore, our goal was to understand the mechanisms of IL-6 in myofibroblasts, the major contributors of collagen during fibrosis. For these studies, we used wild-type mice, IL6 (+/+), IL6 (-/-) and αSMA-Cre-IL-6(fl/fl), in the UUO model of interstitial fibrosis. We hypothesized that IL-6 regulates the crosslinking of collagen by regulating the lysyl-oxidase gene. Therefore, we assessed the Extracellular matrix expansion and collagen accumulation by measuring the changes in gene expression of Col-1, 3, and 4, IL-6, Lysyl-oxidase, Fli-1 and Hells at different time points during renal fibrogenesis. We consider that the change in collagen crosslinking is an important factor in the turnover rate of collagen and it changes its stability to be degraded/accumulated. The examination of the role of IL-6 could potentially provide a new therapeutic breakthrough for treatment and better detection/assessment of renal fibrosis.

The Brome Mosaic Virus (BMV) is an RNA virus that infects granary plants. It consists of three distinct viral particles which consists of each of three distinct BMV RNA molecules wrapped by many copies of the Coat Protein (CP). CP's regulatory roles in the replication processes of BMV are conducted through controlling the efficiency of both RNA replication and protein translation. Previous studies have shown that a 22-residue peptide (CPNT) derived from the N terminal region of CP can penetrate the host cell membrane and deliver the BMV genomic RNA into the host cell. The long-term goal of this project is to investigate the conformational characteristics of CPNT and to explore its binding characteristics on the different parts of BMV genomic RNA. As a first step, a 29 residue-long peptide mimicking the N-terminal region of CP (CPNT-1) was chemically synthesized and its overall conformation was investigated using Circular Dichroism (CD) and nuclear magnetic resonance (NMR). The CD spectra showed that it has only 12% of a-helix structure and 50% of random coil, implicating high conformational flexibility. Given that these Cys residues are not a part of the functionally crucial region of CPNT, we designed a new CPNT construct without any Cys residues (CPNT-2). Our preliminary results showed that the 1 dimensional NMR spectra showed that CPNT forms appreciable amount of secondary and/or tertiary structures in native sequence (27 amino acid residues on N’ end: CPNT-2). When the end of CPNT was replaced by multiple Cys residues (CPNT-1), it is either aggregated or becomes a random, flexible conformation. CD as well as nuclear magnetic resonance analyses have been performed on CPNT-2 and the preliminary data showed that the peptide forms secondary structure. Currently we are investigating the detailed structural characteristics of CPNT-2 using CD and nuclear magnetic resonance analysis.

X-linked severe combined immunodeficiency (X-SCID) is caused by a mutation in the interleukin 2 receptor gamma (IL2Rγ) gene, which is required for lymphocyte development. Affected patients lack functional lymphocytes and die unless cured through bone marrow transplant or gene therapy. However, gene therapy treatment was stopped due to risk of leukemia resulting from insertional mutagenesis, and this prompted the pursuit of alternate treatments, such as gene repair. Gene repair requires the use of two essential components, a homing endonuclease and a repair template, that harness homology directed repair mechanism, which repairs double stranded DNA breaks in cells. Homing endonucleases are enzymes used to target and cut DNA near a mutation in the genome, while a repair template is the DNA sequence used to correct the mutation. My project involved performing experiments to optimize specific components of gene repair using the Sce-SCID mouse model that contained a recognition sequence for the homing endonuclease, I-SceI proximal to the IL2Rγ mutation. I used adeno-associated viral vectors (AAV) to deliver I-SceI and a repair template to hematopoietic stem cells (HSC) isolated from Sce-SCID mice. I tested the hypothesis that delivery of AAV encoding I-SceI homing endonuclease to Sce-SCID HSC would result in recognition and cutting of the Sce locus. Cutting of the Sce locus is essential to gene repair because it initiates the repair of DNA double stranded breaks through homology directed repair with the repair template. Previous experiments in the Rawlings lab demonstrated cutting of the Sce locus with stable I-SceI expression, but my experiments involved transient AAV delivery. Transient delivery is critical for the safety profile of gene repair. Once I-SceI cutting was established with transient delivery, I performed experiments that invovled both I-SceI and repair template in an attempt to correct the SCID phenotype of the Sce-SCID mouse.

Mast cells regulate both inflammatory responses and tissue repair in human diseases but there are conflicting reports on the role of these cells in the pathogenesis of various kidney diseases. Interleukin-6 (IL-6) is an interleukin that acts as both a pro-inflammatory and anti-inflammatory cytokine. From our preliminary studies we have demonstrated that IL-6 and its receptor gp130 are pro-fibrotic in renal fibrogenesis. Here we measure mast cell function in unilateral urethral obstruction, a well-characterized model of renal fibrosis, using mice in which Cre recombinase is expressed under the control of a segment of the carboxypeptidase A3 (Cpa3) promoter bred to IL-6 flox/flox mice. Mast cells from the C57BL/6 Cpa3-IL-6 (fl/fl) mice do not express IL-6. We have groups of 10 for each of the following strains: control1; Cpa3-Cre, control2; IL-6fl/fl and experimental mice; Cpa3-IL-6. Mice under general anesthesia had the ureter ligated at 2 sites distally from the kidney and sacrificed after 14d. We measured the level of fibrosis by measuring hydroxyproline colorimetrically and by measuring area of Picro Sirius red in stained kidney sections, and quantified by pixel analysis. Our preliminary results indicate that the absence of IL-6 in mast cells is important in renal fibrosis. When IL-6 was ablated from mast cells the level of fibrosis measured by collagen and the expansion of extracellular matrix was reduced. These preliminary results suggest that mast cell-derived IL-6 promotes renal fibrosis after unilateral urethral obstruction.

Recovery after severe skeletal muscle crush injury is a major research focus, especially for the armed forces and after major natural disasters. Evaluation of statistics in major American wars displays astonishing numbers of severely injured soldiers, many of them undergoing amputations of major limbs. Much of the research has focused on recovery from exercise-induced muscle injury but little is known about crush injury-induced recovery pattern. Cytokines such as interleukins and tumor-necrosis factors recruit immune cells to the site of injury to clear cell debris, repair cell injury, and stimulate the growth of satellite cells. The time course for the expression of proinflammatory cytokines IL-1beta, IL-6 and TNF-alpha is not well characterized in injured skeletal muscle of mice. The purpose of our investigation is to develop a crush injury animal model to study the expression pattern of these proinflammatory cytokines in the recovery process following crush injury. In our experiments, RNA was extracted and reverse transcribed into cDNA from the right gastrocnemius of injured mice for qPCR to determine the expression levels of IL-1beta, IL-6 and TNF-alpha at 0, 4, 8, 24, 48 and 72 hours post injury. From preliminary data, we see a linear increase in TNF-alpha expression from 0 to 24 hours, and a rapid increase in IL-1beta and IL-6 with a linear decrease over the following 4-24 hour time points. Analysis of later time points is ongoing. This work provides a baseline in preparation of treatment trials to study the healing process and related cytokine production following injury. By understanding the time course and functions of these cytokines and how they interact, we can contribute to the scientific foundation for evaluating the effectiveness of therapies used in managing wounded soldiers.

Gluten intolerance disorders such as Celiac sprue elicit a painful immunological response in the intestines of patients who consume gluten. Gluten is a polypeptide found in wheat, barley, and rye, and contains repeating Pro-Gln-Leu-Pro (PQLP) motifs. Currently the only treatment for gluten intolerance is elimination of gluten from the diet. Our goal is to design an enzyme therapeutic that can be taken as a drug therapeutic in pill form. The engineered enzyme will break down the immunogenic PQLP motifs before gluten reaches the small intestine and can elicit an inflammatory response. Kumamolisin-As, a protein isolated from Alicyclobacillus sendaiensis, demonstrates promising proteolysis on PQ residues, and works in the low pH of the stomach. Using the protein-engineering program “FoldIt,” we designed novel mutations to Kumamolisin to improve its activity on PQLP. Our best version of the enzyme, dubbed KumaMax, degrades gluten over 700 times faster at pH 4 than SC-PEP, an enzyme currently in clinical trials. After further protein characterization, we hope to start clinical trials on KumaMax with the future goal of creating an effective treatment for gluten intolerance.

Chronic social-stress is known to lead to a number of aberrant behaviors, including anxiety and depression. Using a mouse model of social-defeat stress, this project aims to better understand the relationship between the kappa-opioid receptor (KOR), a G-protein coupled receptor (GPCR) previously shown to be activated by multiple forms of stress, and its role in generating anxiety-like and aversion behaviors in mice. Different forms of stress lead to the release of the endogenous KOR agonist, dynorphin, which activates an intracellular signal transduction cascade resulting in anxiety-like and aversion behaviors. Prior studies have demonstrated that the long-acting KOR antagonist, norbinaltorphimine (norBNI), is able to reduce social-defeat induced social avoidance behaviors when administered prior to social defeat stress exposure. Although these studies have implicated the KOR system in social-defeat induced behaviors, pretreatment of pharmacotherapies prior to a stress event is limited in therapeutic benefit.  In this research, the ability of norBNI to reverse repeated social-defeat induced changes in social behaviors was tested by administering norBNI following exposure to the social-defeat stress. The results have so far shown that post treatment with norBNI is able to effectively counter the altered social interaction behaviors caused by a repeated social-defeat stress; moreover, the reversal of the stress-induced social interaction deficits also correlates with a reduction in anxiety-like behaviors. These data suggest that social interaction deficits are due to increased anxiety in previously benign social settings, and norBNI has the therapeutic potential to reverse these social-stress induced behavior alterations.

RNA Polymerase V is a DNA-dependent RNA polymerase involved in RNA-directed DNA methylation (RdDM) (Matzke M et al 2007). RdDM is required for transcriptional silencing of intergenic regions, repetitive DNA, and transposons genome-wide. Arabidopsis polV mutants show increased developmental instability and aberrant root hairs. We hypothesize these phenotypes are the result of alterations in transcriptional silencing and we predict changes in transcriptional silencing are due to altered chromatin accessibility. To asses chromatin accessibility in polV mutant plants we will use a technique called DNaseI-seq that chops up chromatin with a restriction enzyme. This technique allows us to measure which regions of the genome are highly accessible to transcriptional machinery and which are not. Currently, I am developing transgenic plant strains as well as quality control assays to improve DNaseI-seq feasibility in polV mutant plants.  Thus the knowledge will enhance our understanding of developmental instabilities in Arabidopsis and plants in general.
 

What the media has termed as Mad Cow disease is a form of Transmissible Spongiform Encephalopathy (TSE). It is also known as a prion disease and occurs when the normal prion protein in the brain misfolds. This is thought to induce other prion proteins around it to misfold, leading to brain degeneration. Scrapie is a form of TSE in sheep; primarily infecting the central nervous and lymphoreticular systems. There is currently little explanation for why the process occurs, although, studies show that cytokines may be involved. Scrapie will not readily cause disease in normal mice. However, transgenic mice can be infected. These transgenic mice have a prion gene knockout with an insertion of the sheep version of the prion gene, which makes them susceptible when directly infected with scrapie prions. To further our understanding of prion diseases, we aimed to learn more about its source material, transmission, host genetic markers, and immune response with the help of a transgenic mouse model. We challenged transgenic mice with scrapie via the intracerebral route and observed them for signs of disease. Certain tissues were collected and analyzed at multiple points of infection for infectivity, potential co-localized proteins, cytokine dynamics, and growth factors. Various cytokine levels could increase, decrease, or stay the same throughout the infection period. This will enable a better characterization of immune response to prion disease and potentially provide a measure of disease progression using blood cytokine levels.

A key factor involved in the exploration of Mars is the possibility that Mars may have harbored life early in its history. As the presence of liquid water is essential for life, NASA is pursuing the identification of aqueous salt minerals on the Martian surface. These minerals include magnesium sulfate of various hydration states, Magnesium Chloride, Calcium Sulfate, Magnesium Perchlorate, Potassium Perchlorate, and Sodium Perchlorate.  Recently, two NASA spacecrafts have returned imagery of mineral salt deposits on the Martian surface and it is hypothesized that these crystalline solids were formed when a salt solution cooled below its so-called eutectic temperature – the point at which everything becomes solidified. These cold salt deposit are known as “cryogenites”. These cryogenites can be crystallographically distinguished from “evaporates”, which are crystals left behind after water has evaporated. It is possible to create laboratory simulations of these aqueous salt deposits that are believed to be in abundance on Mars to compare the morphologies and compositions of the different cryogenites. The temperature and concentration of the solution affect the number of water molecules attached to the resulting crystals and therefore the morphologies of the different crystals. We used multiple cooling methods including walk-in cold rooms, chest freezers, and standard refrigerators to cool our salt solutions below their respective eutectic temperatures. After cooling, some eutectic solids contained ice that needed to be sublimated which further required the use of hydrophilic chemicals such as CaCl2 to pull off the moisture and isolate the salt crystals. Another method we used was to desiccate the solid by freeze drying. After photographing the cryogenite crystals under a microscope, crystal morphologies will be compared with that from the lander imagery. We will examine the lander images in the context of our laboratory images, noting both similarities and differences.

Quorum sensing is cell density-dependent communication that regulates many functions in bacteria. Signal synthases (I) produce acyl-homoserine lactones, which diffuse into the environment and activate signal receptors (R) when they reach a critical concentration. Synthases and receptors often form cognate pairs. The soil bacterium Burkholderia thailandensis encodes three I-R pairs. The I-R1 system controls cell-cell aggregation and activates many putative aggregation factors including the bce genes that likely encode an exopolysaccharide. The I-R2 system controls antibiotic production. The antibiotics are members of the bactobolin family and are effective against both Gram-positive and Gram-negative bacteria. Both aggregation and antibiotics are believed to promote competitiveness in the diverse soil communities where B. thailandensis is found. We have developed genetic screens to learn more about quorum-controlled antibiotics and aggregation in B. thailandensis. The first screen identified bactobolin-resistant isolates of the soil bacterium Bacillis subtilis, which is normally sensitive to bactobolin. We submitted these mutants for whole genome sequencing to investigate the mutations that confer resistance, and with our results we hope to gain insight into how bactobolin acts on target cells. The second screen was designed to isolate variants that overproduce aggregation factors. Quorum sensing mutants do not wrinkle but variants arise that produce aggregation factors by other means. We are isolating such variants and identifying the underlying mutations to understand how they compensate for the loss of quorum sensing regulation. This may reveal the elements involved in regulating aggregation. These variants will also be useful to learn about the advantages of aggregation in different contexts. Through our two screens we hope to better understand quorum-controlled aggregation and antibiotic phenotypes. Our findings will be useful in further studies of the importance of each of these in competition.

Most adults have some degree of atherosclerosis. In a subset of these individuals, atherosclerosis progresses more rapidly. Increased activity of proteases (enzymes that digest proteins) in atherosclerotic plaques is a plausible cause both of atherosclerosis progression and of atherosclerotic plaque rupture. Plaque rupture is often a terminal event that results in blockage of arteries, heart attacks and strokes. Urokinase plasminogen activator (uPA, a protease that is present in plaques) functions by converting plasminogen (a zymogen, or enzyme precursor) into its active form, plasmin. The uPA/plasminogen pathway facilitates macrophage migration, enhances the release of inflammatory mediators, and activates other proteolytic enzymes, all of which may contribute to atherosclerosis progression and plaque rupture. We hypothesize that inhibition of uPA will decrease atherosclerosis progression and prevent plaque rupture. To begin to test our hypothesis, we use a mouse model of uPA-accelerated atherosclerosis (mice that are genetically predisposed to develop atherosclerosis and express high levels of uPA). We inject an antibody (mU1) that binds to and inhibits uPA, or a control antibody, and we measure atherosclerosis in aortic roots and pinned aortas. We anticipate finding less atherosclerosis in mice treated with mU1. We also expect to find fewer macrophages in atherosclerotic plaques of mice treated with mU1. If we find that inhibition of uPA decreases atherosclerosis progression and that it is also well tolerated by mice, we will later set up an experiment in older mice to determine whether anti-uPA treatment with mU1 will prevent plaque rupture. If uPA activity contributes to atherosclerosis progression, and if inhibiting uPA decreases atherosclerosis progression and prevents plaque rupture, drugs could be developed that inhibit uPA. These drugs could be used to treat humans who are at high risk for atherosclerosis progression and plaque rupture.

Many occurrences of glomerulonephritis present recurrent acute inflammation episodes. With time these conditions often lead to progressive fibrosis and chronic renal failure. My project studies the role of Interleukin-6 (IL-6), one of the most proinflammatory cytokines, using the nephrotoxic serum (NTS) nephritis mouse model. The ubiquitous expression of IL-6 makes it a challenge to study this interleukin. We have genetically engineered mice with ablated IL-6 in the F4/80+ macrophages and the Ytgax (CD11c+) dendritic cells. We hypothesize a greater level of glomerular damage and disease in the wild type mice compared to the IL-6 depleted mice. IL-6 +/+ (IL6WT) and -/- mice (IL6KO) were injected with NTS in the tail vein and placed in metabolic cages for seven days. Urine was collected to measure renal failure and progression of the disease. Mice were sacrificed at 7 days after the injection and both kidneys harvested and sectioned. Extraction, quantification and quality analysis (RNA integrity number) from the IL6WT and IL6KO frozen kidney samples were conducted and later cDNA synthesized. Quantitative PCR (qPCR) reactions were carried out to analyze gene expression of IL-6 family genes and pathological analysis of frozen or paraffin embedded tissue was done to assess glomerular and interstitial damage. Preliminary results from histopathology analysis and qPCR suggest a significant role played by IL-6 in glomerular damage. IL6WT diseased mice show upregulated expression of IL-6 and greater degree of disease relative to the IL6KO. We plan to continue gene expression quantifications and perform immunostaining to compile more data and validate our results. These findings will help to establish the efficacy of IL-6 neutralization as a therapeutic option for kidney disease.

Transcription, the first step in gene regulation in which RNA is created from DNA, is a process that occurs in the nucleus. When messenger RNA is transcribed, its function is to code for a protein product. This message is transported from the nucleus to the cytoplasm, where it can serve its purpose, and over time gets degraded. Transcription and degradation were always thought to be uncoupled, but recent evidence suggests otherwise.  Coordination of synthesis and decay rates of an mRNA transcript could be beneficial to the cell. It has been shown that copies of the exact same message encoded by different promoters can have different rates of degradation in the cytoplasm depending on the promoter used to activate transcription. I hypothesize that an RNA-binding motif on a protein could affect the lifespan of a specific message, and plan to test this hypothesis using the awesome power of yeast genetics and molecular biology.  By culturing wild type and deletion strains of yeast, and de-repressing the activity of gluconeogenic enzymes by starving the cells of glucose, I can test my prediction. SNF1 is a protein kinase that is active only during gluconeogenesis, and I have shown that when it is inhibited, the rate of mRNA degradation is more rapid than under normal conditions. I will test my prediction by deleting genes encoding putative RNA binding proteins that are phosphorylated by SNF1 and measuring mRNA levels by Reverse Transcriptase-quantitative polymerase chain reaction.  The idea of mRNA degradation being linked to the way that mRNA was transcribed is new, and I am interested to see what I will find.  I am hoping to identify both positive and negative effectors of mRNA stability using this approach.

Tube formation in the fruit fly (Drosophila melanogaster) egg chamber is an excellent model for vertebrate neural tube formation, as they closely resemble each other. During egg chamber development, a subset of cells, the roof cells and the floor cells, form two tubes that make eggshell structures called dorsal appendages (DAs). Roof cells, which express the protein Broad, constrict their apices (inner surfaces) to curve up and out of the epithelium. The floor cells elongate, dive under the roof cells, and zipper together to seal the floor; they are distinguished by expression of the protein Rhomboid. Defects in this process, especially in floor closure, create open tubes, similar to spina bifida. To observe cell movement in the developing egg chamber, we want to outline the shapes of cells with a fluorescent molecule. mCherry is a red fluorescent protein that has good fluorescence and a short maturation time (15 minutes), so we can observe the rapid process of tube formation. If one links the CD2 gene, which encodes a cell membrane adhesion protein, to the mCherry gene, red fluorescence will appear all around the cell surfaces. Such a fusion protein lets us observe changes in cell shape and cell movements during tube formation. When the CD2-mCherry segment is connected to the regulatory region for the rhomboid gene, fluorescence occurs in just the floor cells. I have created a rhomboid-CD2-mCherry construct that I will introduce into flies through injection with a recombinase that will integrate the construct into the fly genome. With this construct I can visualize DA tube development live by observing fluorescence in the floor cells. Analysis of these cells can give insight into conserved tube-forming processes.

There are currently very few effective treatments to repair damage resulting from spinal cord injury (SCI). The project I have been working on in the Moritz Lab involves the exploration of Intraspinal Microstimulation (ISMS) and its ability to promote functional recovery after SCI in a rodent animal model. Our group employs a unilateral incomplete spinal cord contusion injury using the Ohio State University contusion device at the level of the fourth lamina in the cervical spine, or C4. We then place stimulating wires in the spinal cord, specifically in the ventral gray matter caudal to the injury at C6-C8, in order to deliver ISMS to the motor pools innervating the triceps and other forelimb muscles. My independent project in the lab is a histological analysis of triceps and forelimb muscles taken from the SCI models to determine whether the use of ISMS preferentially recruits fatigue resistant (FR) muscle fibers in the affected forelimb of these rats. After paresis resulting from SCI, muscles atrophy and undergo a "slow-to-fast" transition in the myosin heavy chain (MHC) isoform content. My goal is to examine whether ISMS can slow or even reverse this slow-to-fast conversion after SCI. I am using immunohistochemistry to differentiate muscle fiber types in the triceps muscle. I stain for the unique MHC isoform that is expressed by the various slow and fast type fibers. My hypothesis is that there will be a higher population of type I and type IIA FR fibers in the stimulated muscles of ISMS group than in the control group, which will mostly consist of fast fatigable types IIB and type IID. The long-term goal of my project is to determine the benefits of using ISMS as a neural prosthesis and regenerative therapy for individuals who have suffered from spinal cord contusion injuries.

 The effect of viral evolution on the duration of latency in Human Immunodeficiency Virus Type 1 (HIV-1) infection has been studied extensively in men. The asymptomatic latency period, after HIV-1 infection and before the onset of Acquired Immune Deficiency Syndrome (AIDS), is characterized by high viral variability. In many cases, a viral phenotype that utilizes CXCR4 and/or CCR5 receptors on target host cells to advance HIV-1 infection progress also emerges during this period. Despite known sex-dependent variation in the progression of HIV-1, the effect of viral evolution and CXCR4/CCR5 utilizing virus (X4 virus) prevalence on HIV-1 latency has not yet been studied in women. I am investigating trends in viral evolution in 9 women from seroconversion until initiation of antiretroviral therapy. I will then compare my results to previously studied male viral evolutionary trends. Ultimately, a total of 72 blood plasma samples, an average of 8 samples from each woman collected progressively during the course of infection, will be used to generate 25-30 HIV-1 sequences per time point. These sequences will then be evaluated through 1) viral divergence from a founder strain 2) viral population diversity and 3) emergence and prevalence of X4 viruses. I will then analyze the relationship between viral evolutionary trends and X4 virus prevalence, and the duration of HIV-1 infection before the onset of AIDS. Ultimately, a comparison between the sexes will be made. Furthering the scientific communities’ knowledge of sex-related HIV-1 infection variation could lead to more effective sex-specific treatment and vaccine development. The important biological question of whether sex can influence HIV-1’s evolutionary path will be addressed. This project is currently in the preliminary stage of sequence generation. Trend analysis and conclusions will take place in Fall 2012.

Fear conditioning is a form of classical conditioning where an initially neutral conditioned stimulus (CS) is paired with an inherently aversive unconditioned stimulus (US). After multiple CS-US pairings, CS alone produces conditioned fear responses. The amygdala has long been considered the key brain structure where fear learning-related plasticity primarily occurs. However, it has not been well studied how the US directs CS-US association in the amygdala. We recently found that stimulation of the dorsal periaqueductal gray (dPAG) could serve as a US in tone fear conditioning, indicating the dPAG may be a key structure in relaying US information to the amygdala during fear conditioning. In this study, we investigated the role of the amygdala in auditory fear conditioning where dPAG stimulation acted as the US. Male Sprague-Dawley rats were bilaterally implanted with stimulating electrodes in their dPAG and with guide cannulae in their amygdala. Prior to training, muscimol (0.3 µg/0.3 µL per side) was infused into the amygdala using infusion cannula and a microsyringe pump. For training, a tone (2.9 kHz, 85 dB, 20 sec) was used as a CS, and electrical stimulation (0.1-ms width, 100 Hz, 2 sec) of the dPAG as a US which co-terminated with the CS. Next day, an 8-min tone was presented continuously in a novel chamber as a tone testing, and the following day, 8-min exposure to the training chamber was given to the rats as a context testing. While the muscimol-injected animals displayed comparable level of freezing to the control animals during training, their freezing to the tone and context was disrupted during the testing, indicating fear conditioning was blocked by the amygdalar inactivation. These results suggest that the amygdala is critical for dPAG stimulation fear conditioning and may be the center of dPAG-directed US and tone CS convergence and plasticity.

Neurons contain two highly specialized compartments: the dendrite, which receives synaptic or sensory inputs, and the axon, which relays these signals to other cells. Different types of neurons often have unique, type-specific dendrite morphologies and proper dendrite patterning is important for normal neuronal function. Underscoring the importance of dendrite morphology to neuronal function, defects in dendrite patterning likely contribute to numerous neurological disease states and the cognitive decline that accompanies normal aging. However, the many genes that contribute to the complex dendrite patterns remain largely uncharacterized. To identify genes involved in this process, we conducted a genetic screen for mutants that alter the patterning of sensory neuron dendrites that innervate the body wall of fruit fly larvae. Briefly, we mutagenized fruit flies with EMS, a chemical mutagen, and screened for late-larval stage lethal mutations that perturb dendrite patterning using a fluorescent protein that allowed us to visualize a single functional class of sensory neurons in living larvae. Here, we report our analysis of a novel mutant, messi. Using live cell imaging and quantitative analysis, we found that messi leads to exuberant dendrite branching and intermingling of dendrites in sensory neurons. This latter defect is of particular interest since “self-avoidance” may represent a general mechanism for organizing sensory dendrites and little is known about the genetic basis for this phenomenon. Using time-lapse microscopy, we found that messi causes progressive dendrite defects: the dendrite patterning of messi mutants is indistinguishable from wild type early in development, but becomes abnormal later in development. Similarly, dendrite defects in many neurological disorders are progressive, therefore further analysis of messi may contribute to our understanding of how dendrite maintenance is deregulated in diseased states. Currently, we are working towards identifying the gene affected in messi mutants and determining whether messi affects dendrite patterning in other functional classes of sensory neurons.

Alzheimer's disease (AD) is the leading cause of dementia, and its precise cause is still unknown. Mutations in the two Presenilin (PS) genes, Presenilin 1 and Presenilin 2 (PS2), cause AD. Understanding how AD mutations impact PS function may help us understand AD pathogenesis. One function of PS involves calcium (Ca2+) transfer between the endoplasmic reticulum (ER) and mitochondria. Presenilin 2 (PS2) and mutant PS2 (mtPS2) in particular have been shown to increase this transfer, possibly causing mitochondrial Ca2+ overload. This could lead to metabolic dysfunction, which has been suggested as a key pathogenic mechanism in AD. Establishing a link between PS mutations and mitochondrial dysfunction would support this hypothesis. One symptom of mitochondrial dysfunction is changes in mitochondrial fragmentation and localization. "Fragmented," rather than interconnected, "branchlike" mitochondrial networks are associated with increased ER-mitochondria Ca2+-transfer and apoptosis. Furthermore, while healthy mitochondria move towards energy-demanding neuronal processes, mitochondria with reduced ATP production stay near the nucleus. To determine the effects of PS2 and mtPS2 on mitochondrial fragmentation and localization, wild-type (wt) and mtPS2 (N141I) transgenic mouse cortical neurons were infected with a lentiviral vector expressing mitochondrial targeted red fluorescent protein (DsRed). Fixed cultures were imaged using confocal microscopy and analyzed using Huygens software. Currently, we have plated, infected, and analyzed sample preparations for mitochondrial length, sphericity, and other characteristics associated with fragmentation, as well as mitochondrial volume as a function of distance from the soma. Using this paradigm, we will also challenge cultured wt and mtPS2 neurons with camptothecin and staurosporine, inducers of mitochondrial fission and fusion, respectively. If mtPS2 induces mitochondrial dysfunction, mtPS2-expressing neurons should exhibit more mitochondrial fragmentation and mitochondria localized nearer to the soma. This would provide evidence for mitochondrial dysfunction in AD, supporting further research into mitochondrial changes in AD and a better understanding of AD’s etiology.

The effects of amphetamine on learning and memory are important areas of research due to the widespread use of amphetamine among people, ranging from prescription to recreational. It has been shown in animals that repeated treatment of amphetamine will result in sensitization to the drug. Sensitization is a process through which treated subjects develop an elevated response to a drug. Long term potentiation (LTP) is a physical change strengthening the connection between synapses that is thought to underlie learning and memory. Enhancement of LTP in the striatum has been shown to occur as a result of amphetamine use. The striatum is a brain region important in mediating a large number of behaviors including motor control and learning. For this reason it is possible that sensitization to amphetamine may affect learning in mice. To investigate this possibility, wild-type mice sensitized to amphetamine were tested in behavioral paradigms designed to measure learning ability. It was found that they do not show enhanced performance in motor learning, appetitive learning, or spatial learning, but they may show an improvement over controls when they are required to alter their strategy to escape from a water-maze. In order to explore the neurological pathway through which sensitization to amphetamine may affect learning, we also tested knockout mice that lack N-methyl-D–aspartate-type glutamate receptors (NMDARs) in medium spiny neurons, the primary neuron type in the striatum. In many brain regions, NMDARs are important for various forms of learning and memory, and it has been shown that these knockout mice display impaired motor and appetitive learning and yet are still able to sensitize to amphetamine. Using the same learning paradigms as with our wild-type mice, we found that knockout mice did not show any enhancement in motor or appetitive learning as a result of amphetamine sensitization.

Bacterial conjugation is a form of horizontal gene transfer that serves as an important source of genetic plasticity and diversity in both Gram positive and Gram negative bacteria. It has also been proved to be advantageous for dissemination of antibiotic resistance. Conjugation is a plasmid-mediated mechanism that allows single stranded DNA to be transferred across cell membranes from a donor to a recipient cell. In Escherichia coli, the conjugative F (fertility) plasmid encodes all necessary proteins for assembling the transfer apparatus thus allowing transfer and propagation of DNA into a recipient bacterial cell. Previous conflicting studies of bacterial conjugation in E. coli have shown DNA transfer to occur between donor and recipient pairs in intimate cell-cell contact or between distant donor and recipient pairs, connected only by the pilus, a filamentous protein extended from the donor cell. Using various fluorescent protein constructs to differentially mark donor and recipient cells and F plasmid DNA itself, we visualize conjugation in real time with single-cell resolution by fluorescent microscopy. Here we show that conjugation occurs only when donor and recipient cells are in intimate and close physical contact with each other. In addition, there appears to be no preference for cellular orientation for transfer. Furthermore, we propose a simple rate equation to model DNA transfer between bacteria and measure the rate of DNA transfer per unit time per cell contact. This quantitative model is a tool to elucidate conjugation at a single-cell scale and will provide a deeper understanding into the mechanism of bacterial conjugation.

MicroRNAs (miRNAs) are small noncoding RNA molecules that post-transcriptionally regulate gene expression. MiRNA biogenesis involves two key ribonucleases, Drosha and Dicer. I am investigating the Drosha enzyme. Previous research from the Ruohola-Baker lab reveals that reducing levels of Drosha in stem cells significantly reduces levels of some miRNAs, while only slightly reducing levels of another group of miRNAs. This surprising result led us to conduct a bioinformatics analysis to identify characteristics that differentiate these two groups of miRNAs. We discovered structural differences, specifically that bulges in the central region of miRNAs were more frequent in heavily reduced miRNAs than in miRNAs that were not affected. My mentor, Henrik Sperber and I propose a model explaining why miRNAs with mismatches (bulges) in the central region of the sequence are more sensitive to reduced levels of Drosha than rigid miRNAs with no mismatches in that region. To test whether a bulge causes differential miRNA processing when Drosha is the limiting factor, I am adding and removing bulges to primary miRNA transcripts, overexpressing them in cells with low Drosha levels, and comparing their mature levels using qPCR. In addition, I am testing whether in nature cells vary Drosha levels as a mode of regulation. A previous study shows that T cells at specific stages of differentiation reduce Drosha expression by as much as 10 fold. My own bioinformatics analysis reveals that certain tissues in mice have an 8-fold reduction of Drosha. To see whether reduction of Drosha levels correlates with differential microRNA processing, I am using microarray data to find subsets of microRNAs that have dramatically reduced levels when Drosha expression is significantly low. With my research, I hope to discover a potentially novel mechanism of regulation in which cells alter Drosha levels to selectively process a subgroup of miRNAs.

Ubiquitin is a small post-translational signaling molecule. Its roles in repairing damaged DNA, apoptosis and protein degradation make it critical for tumor suppression. Genetic mutations affecting proteins in the ubiquitination pathway have been implicated in multiple inherited human cancers. The ubiquitination pathway attaches the protein ubiquitin (Ub) onto a substrate molecule with the help of three enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). Transfer of Ub onto the substrate requires the formation of a thioester bond between Ub and E2, denoted as E2~Ub. A catalytically active E3 ligase which can bind E2~Ub, but never directly binds Ub, is also necessary. E3’s catalytic mechanism is not fully understood. One function of E3 is to bring E2~Ub and substrate in close enough proximity to facilitate a reaction. However, E3 enhances the release of Ub from E2~Ub in the absence of substrate molecules. This suggests E3 plays a role in priming E2~Ub for transfer of Ub. Using site directed mutagenesis and protein-protein interaction assays visualized through western blotting, I have investigated what electrostatic properties of Ub are important for E3’s catalytic activity. Informative Ub mutations don’t stop formation of E2~Ub, but disrupt the catalytic activity of E3. I propose that polar/charged Ub residues in the contact region between Ub and E2 affect E2~Ub binding modes, promoting inhabitance of E2~Ub conformations favorable to Ub transfer upon binding of E3.

Embryonic stem cells are isolated from the early developing embryo and are capable of forming the different cell types found in the body. Understanding how these cells develop and maintain their specialized state is critical to understanding how they function. We hypothesize that embryonic stem cells acquire a unique metabolic state that aids them in maintaining their specialized state. Using two distinct mouse pluripotent states, embryonic stem cells (ESC’s) and epiblast stem cells (EpiSC’s) representing cells isolated from the inner cell mass of the blastocyst and the epiblast, a later stage in embryonic development, we found that EpiSC’s are much more glycolytic in terms of their energy usage compared to ESC’s as measured by oxygen consumption and extracellular acidification rates. Furthermore, using quantitative polymerase chain reaction experiments, we found that genes key to regulating and maintaining high rates of glycolysis are significantly upregulated in EpiSC’s compared to ESC’s. We showed that the overexpression of HIF1α, a known key regulator of these glycolytic genes, in ESC’s is able to shift the ESC’s towards a more glycolytic metabolic state and cellular morphology reminiscent of EpiSC’s. In addition, transition of ESC’s to EpiSC’s using the known chemical inducers Activin and Fibroblast Growth Factor 2 revealed that changes in the expression of metabolic genes occur prior to changes in expression of cell fate markers, which suggest that a metabolic transition in the ESC’s may be key in its transition to an EpiSC-like state.

Understanding stem cell maintenance is crucial for delineating conditions such as tissue loss, aging, and cancer. Using the Drosophila female germ-line stem cell (GSC) model to observe stem cells in vivo, several extrinsic and intrinsic factors have been demonstrated to regulate maintenance of Drosophila GSCs; they have also been found to be conserved in mammalian systems. The search is on for novel regulatory factors of stem cell maintenance with the hope of extending our understanding to stem cells in mammalian systems. My project involves the characterization of the molecular function of Pineapple eye gene (Pie), a novel regulator of GSC maintenance. Through a loss-of-function screen in GSCs, it has been demonstrated that mutations of pie result in GSC maintenance failure. The amino acid sequence of pie revealed that it contains ring-domains suggesting possible E3 ubiquitin ligase function. Such proteins are key components of the ubiquitination pathway, which are involved in protein tagging and degradation. To test pie’s function as an E3 ligase, I am using a novel assay that employs the fact that bacteria do not have endogenous ubiquitination pathway and therefore utilizes Escheria coli as an in vivo system to express the components of the ubiquitination pathway and detect the formation of ubiquitin chains, a key indicator of E3 Ligase activity. I have demonstrated that the expression of pie’s ring-domains without a substrate is not sufficient for the detection of ubiquitin chains. I will now test whether the full length pie is necessary for proper ubiquitination. Further, I will also test whether pie’s functional domain needs a substrate in order to function effectively. Successful characterization of pie can reveal new pathways to target for conditions involving stem cell maintenance.

Plants use hormone-signaling pathways to regulate growth and development. Brassinosteroids (BRs) are critical plant hormones that regulate growth such as the elongation of the embryonic stem. We study the specific mechanism of this process by testing the hormone-induced growth responses of both wild type and mutant Arabidopsis seedlings. We are testing whether growth repressor SPATULA is involved in BR growth response. We hypothesized that the growth repressor SPATULA is downstream of the BR pathway. To test this hypothesis, we acquired mutants that lack SPATULA function. After confirming the genotype of the mutants by PCR, we used a high-resolution camera to observe growth throughout early development in response to hormone treatments. Data is recorded by the camera over the course of 132 hours. The images are then analyzed and growth rates are calculated. Using these data, we determined if SPATULA is involved in the BR growth response.

The reduction of CO₂ has become a global priority as the concerns of climate change and the adverse effects of atmospheric carbon dioxide on the environment continue to rise. One method of reducing CO₂ emissions is through conversion and utilization as a chemical feedstock. CO₂ is cheap, nontoxic, and can be recycled to produce valuable new products, making it an attractive feedstock. The increasing importance of CO₂ utilization processes requires a better understanding of the underlying mechanisms of its conversion. The industrial conversion of CO₂ is typically achieved using a heterogeneous catalyst, usually consisting of small transition metal particles supported on a high surface area metal oxide. Our project focuses on elucidating the mechanism of the reverse water-gas shift (RWGS) reaction on platinum, an extensively studied catalyst with a highly active surface. Our goals are (1) to quantitatively measure the RWGS rate under conditions of low H coverage and (2) to compare the measured rate to the rate of CO₂ dissociation (the rate-limiting step according to the surface redox mechanism) and to the theoretical rate calculated using a micro-kinetic model based on the associative mechanism. The information we gather from the investigation will clarify whether the RWGS reaction proceeds on platinum via the surface redox mechanism or the associative mechanism.

Significantly reducing caloric intake without malnutrition (calorie restriction) has been shown to extend lifespan in many organisms including yeast, nematodes, and mammals. Since calorie restriction (CR) can also result in the extension of healthspan, CR or CR mimetics may be useful in the prevention of aging related disorders such as cancer, neurodegenerative disease, and cardiovascular disease. However, before long-term CR or CR mimetics can be considered safe for humans, more information is needed on the molecular mechanisms responsible for the effects of CR. In addition, since long-term voluntary CR may prove difficult to endure in humans, the consequences of reinstating a normal or ad libitum (AL) diet after prolonged CR must be investigated. We hypothesized that switching back to an AL diet after prolonged CR would reactivate pro-aging molecular pathways, such as the target of rapamycin (TOR) pathway. To test this, we obtained kidney tissue from aged F344 rats that had been subjected to long-term CR. Some of the long-term CR treated rats had been gradually switched to an AL diet and the rest had been maintained on the CR diet. Proteins were extracted from the kidney tissue and then analyzed by Western blot. Switching to an AL diet after long-term CR resulted in a significant increase in the phosphorylation of downstream signaling proteins in the TOR pathway in kidney tissue. In addition, after switching to an AL diet, there were significant changes in autophagy, which is regulated by the TOR pathway, as indicated by increases in the autophagy markers LC3 and p62. These results indicate that reinstating an AL diet after prolonged CR results in an increase in the activation of the pro-aging TOR pathway. This suggests that the TOR pathway may regulate some of the physiological effects of reinstating a normal or ad libitum (AL) diet after prolonged CR.

Multiple Sclerosis is a disease characterized by inflammation and demyelination in the central nervous system. Experimental Autoimmune Encephalomyelitis (EAE) is a mouse model used to study MS. Previous studies have shown that EAE can be induced by helper T cells of both the Th17 (IL-17 producing) and Th1 (IFNg-producing) lineages. In classic EAE induced by Th1 cells, inflammation is observed predominantly in the spinal cord, however in our model of atypical EAE, induced by transfer of Th17-skewed cells, inflammation is observed in both the brain and spinal cord. This atypical model is closer to the disease course of MS in humans, because only rarely do MS patients have spinal cord inflammation without accompanying brain inflammation. Using mice that lack IL-17RA, a protein required for IL-17 signal transduction, we can study the mechanism of IL-17-induced inflammation in the brain versus the spinal cord. In our model we have shown that IL-17 promotes neutrophil recruitment to the brain, but is not required for neutrophil recruitment to the spinal cord. The presence of neutrophils in the spinal cord in IL-17 receptor knockout mice suggests some other mechanism must be used to recruit neutrophils in the spinal cord. Using RT-PCR we have been looking at the role of other factors (IFN-g, CXCL5, CXCL2, CXCL1, GMCFS, and IL-1b) in recruiting neutrophils in spinal cord independent of IL-17.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases (GOLD, 2011). There are two classes of medications to treat COPD, bronchodilators and corticosteroids. Some patients are prescribed multiple inhalers to maximize treatment effectiveness. Correct inhaler technique is important to ensure proper deposition of medications in the airways; however, administration techniques vary across devices. This raises the question as to if there is a relationship between the number of prescribed inhalers with patients’ confidence in their inhaler technique and medication behavior. We hypothesized that patients using multiple inhalers would have decreased confidence in their inhaler technique and adherence to inhaler use may be lower. To answer this question, we used data collected from an ongoing longitudinal observational study looking at biological causes and functional consequences of depression in COPD patients. Our analyses showed that patients who were on 3 or more inhalers had the same level of confidence in taking their inhalers correctly as patients who were on 2 or fewer inhalers. They also did not differ in forgetting to take or being careless about their medications compared to patients who took fewer inhalers. In addition, they were no less likely to take their inhalers when there were changes in their daily routine. These results refute our hypothesis, and instead, demonstrate that there is no relationship between the number of prescribed inhalers and inhaler technique confidence and medication behavior.

The complexity of the factors underlying the aging process and difficulty in categorizing definitive aging phenotypes complicates efforts to elucidate the pathology of aging. One approach toward understanding human biology is to look to model organisms as simpler starting points. Specifically, we can ask which aging genes have counterparts in more complex organisms and, of those, which also influence longevity. The Kaeberlein lab is in the process of completing a genome-wide screen of the budding yeast Saccharomyces cerevisiae for genes that alter its lifespan. The more difficult challenge is to piece together the pathways connecting the myriad products of these genes into a cohesive model for their effect on the aging process. A pathway of particular interest is the mitochondrial unfolded protein response (mtUPR). This stress response pathway is activated by accumulation of unfolded or misfolded proteins in the mitochondrion. After stress, signaling from the mitochondria to the nucleus can increase mitochondrial chaperone presence to promote proper protein folding. This pathway might play a role in some of the lifespan extending effects of the genes identified through the genomic screen, and there is evidence that some mechanisms of longevity in other organisms also involve the mtUPR. Our goal is to develop a GFP reporter system for the yeast mtUPR and investigate mtUPR activation in the lifespan mutants already identified by the lab. By creating a yeast GFP-promoter construct, we will be able to conduct experiments using fluorescence microscopy and flow cytometry to quantitatively determine mtUPR activation for a variety of genotypes and environmental conditions.

The study of chronological and replicative aging in Saccharomyces cerevisiae has provided both post-mitotic and mitotic aging models in this single-celled organism. In chronological aging, lifespan is determined by the length of time a yeast cell can survive in a non-dividing state, whereas in replicative aging, the lifespan is determined by the number of daughter cells produced by a mother cell prior to irreversible cell cycle arrest. The primary goals of this study were to observe how the chronological aging of yeast cells influences replicative lifespan (RLS) and to determine whether changes in the extracellular environment that influence chronological lifespan (CLS) would also influence subsequent RLS. By buffering the pH of the medium to 6.0 or reducing the concentration of available glucose (dietary restriction), we show that these manipulations were able to suppress the reduction in RLS associated with chronological aging, when compared with cells aged in synthetic complete medium with 2% glucose. The production of ethanol and acetic acid due to yeast fermentation may be responsible for cellular damage leading to reduced RLS, and it can be inferred that by buffering the pH to 6.0 or dietary restricting the cells, this damage is attenuated and replicative capacity is increased following chronological aging.

The nuclear transport pathway performs the fundamental function of moving cargo between the cytoplasm and nucleus in all eukaryotes. Nuclear transport is carried out through a highly conserved mechanism across all eukaryotes. Yet, in Drosophila, several components of the nuclear transport apparatus evolve rapidly under positive selection. Genetic conflict with selfish elements has been suggested as a possible cause for this pattern of rapid evolution. Here, we present a comprehensive phylogenomic analysis of importin gene evolution in Drosophila. Importins are adapter molecules that directly mediate the transport of cargo into the nucleus. Our analysis reveals a recurrent pattern of gain and loss of importin paralogs across independent lineages of Drosophila. Interestingly, we discovered that almost all new copies of importins have acquired a testes-specific expression pattern since their birth through gene duplication. This pattern of repeated gains of testes-specific copies of importins and signatures of episodic lineage-specific positive selection suggests a function in suppressing genetic conlifcts such as segregation distortion in the male germline.

HIV has infected over 30 million people worldwide and has resulted in the loss of millions in healthcare costs. HIV vaccines have the potential to prevent infection and drastically reduce infection rates. A potential strategy for vaccine development is to elicit a mucosal immune response, since sexual transmission of the virus is the leading cause of new infections. A successful functioning vaccine would deliver an HIV antigen, such as the highly conserved regions of the HIV membrane surface protein gp41, and an adjuvant, such as the synthetic organic molecule R848, through mucosal and cellular barriers. Here, a delivery vehicle for a complex HIV vaccine is synthesized by using a single emulsion method to formulate a polymeric nanoparticle, consisting of a biodegradable polymer core surrounded by a lipid layer. Previous research has shown that a lipid shell increases serum stability and immunocompatibility in vivo. First, design parameters, such as solvent and surfactant properties, were modified to yield consistently monodisperse populations of nanoparticles with controlled size and surface chemistries. Different methods for encapsulating the adjuvant into the polymer core were also explored. The encapsulation efficiency and drug loading of the adjuvant, as measured using liquid chromatography, was less than 1%. We delivered an HIV antigen by intercalating it within the lipid layer surrounding the polymeric core. Encapsulation efficiency of the antigen was measured with a proof-of-concept peptide attached to a fluorophore. We used ELISA and cell culture studies to test for the controlled release and immunogenicity of the immunogens. Finally, a mouse model will be constructed to test the particles in vivo. This project demonstrates a design for creating nanoparticles with controlled characteristics for eliciting a comprehensive immune response against HIV.

Ulnar-Mammary Syndrome (UMS) is a congenital syndrome that is characterized by ulnar abnormalities and breast hypoplasia. The Bamshad Lab discovered that UMS is caused by mutations in the gene, TBX3. The goal of our project is to find novel TBX3 mutations that cause UMS, and expand the knowledge of how different mutations correlate to the severity of the disease. We collected DNA samples from three UMS families, and sequenced the exons of TBX3 via Sanger sequencing. Sequence analysis software allowed us to search the sequence for mutations and their locations within the gene. In the family K31928, previous screening showed that the proband and her sister, have a mutation called 1294_1301dup8. We screened the parents for mutations in TBX3, and we found that the father had the insertion mutation as well. The father’s clinical findings were very mild, but this screening confirmed that he passed the mutation causing UMS to his daughters. No mutations were found in members of the other two UMS families. The next step of this project is to investigate, in the families without TBX3 mutations, other candidate genes, as well as regulatory regions of TBX3, for mutations that could cause UMS.

The Community Action Research and Empowerment Project (CARE), launched in September of 2010, is a participatory action research project where elders and youth work together to assess the strengths and needs of the Vietnamese community in Seattle, and develop a long-term plan to address those needs and advance the community. Data from 16 focus groups, 85 interviews, and 316 surveys were collected. We learned that the Vietnamese community members have a broad range of concerns, such as youth and education, housing, jobs, health and social services. During Phase 2 (2011-2012), a strategic action plan will be created with extensive input from community members to address the issues identified—thereby creating a shared vision for the Vietnamese community.

Evolutionary conflicts involving mimicry are found throughout nature ranging in a multitude of levels from ecosystems to molecules. Moreover, structural mimicry is increasingly being recognized at the interface between viruses and hosts. The goal of my project is to provide structural and mechanistic insight into a newly discovered evolutionary ‘arms race’ between a viral protein and a host immunity protein. We have found that a poxviral protein IBP1 (IFIT Binding Protein 1) binds a host immunity protein, IFIT2, which represses viral mRNA translation. I hypothesize that IBP1 has convergently evolved to structurally mimic IFIT2’s binding partner, eIF3c, as a strategy to restore viral protein production. Because IBP1 has no known amino acid similarity to any other viral or host protein, the best way to test my hypothesis is to determine the molecular structure of IBP1 and compare it to eIF3c. I will biochemically engineer constructs to optimize the expression and amount of soluble IBP1 in E. coli and then utilize affinity columns to isolate IBP1, which will then be subjected to a myriad of crystallization conditions. Once optimized crystallization conditions have been found I will use X-ray diffraction techniques to solve the structure. Regardless of whether my hypothesis is correct, my work will provide atomic resolution detail for IBP1, which is a protein that is important for the fitness of the virus. If I find that IBP1 is indeed using molecular mimicry it will not only reveal the mechanism of IBP1 action, but it will also provide a case of molecular mimicry marked by the signature of convergent evolution. Furthermore, my work will make progress towards further understanding the interactions between hosts and viruses, which determine the results of potentially deadly infections in so many people.

Helicobacter pylori is a bacterium that infects the stomach of about half the world’s population. While usually asymptomatic, H. pylori infection is the main cause of peptic ulceration, gastric lymphoma, and gastric adenocarcinoma. This suggests variability within populations of H. pylori in their ability to cause disease. I am researching the effect of a mutation in the gene cagY on the ability of H. pylori to cause disease. This gene produces CagY, a structural protein of the bacteria’s Type IV Secretion System, which is involved in causing disease. H. pylori transports different materials across the Type IV Secretion System, including the protein CagA. Host cells receiving this protein elongate and narrow (a change called the hummingbird phenotype) and produce interleukin-8, a chemical messenger of the immune system. In host cells, the protein CagA is phosphorylated (a process by which a phosphate group is added to a molecule). To test the effects of the cagY mutation on the bacteria’s Type IV Secretion System, I will infect human stomach cancer cells with the cagY mutant. To detect changes in these cells caused by the infection, I will use two techniques that detect specific proteins, Western Blot and ELISA. The Western Blot will detect the presence of phosphorylated CagA, while the ELISA will detect interleukin-8. The length of infected cells will be measured to detect the hummingbird phenotype. These tests will measure the function of the Type IV Secretion System in the cagY mutant. The absence of a functioning Type IV Secretion System in the cagY mutant will suggest a change in bacteria and host interaction and a potentially decreased ability for the bacterium to cause disease.

Colloidal semiconductor nanocrystals (NCs), often referred to as quantum dots, are a class of materials that exhibit size dependent photo physical properties. This class of materials has great potential for application in a variety of fields such as photovoltaics, light emitting diodes and spintronics. Doping NCs with transition metals provides additional tunability of the optical and physical properties has been synthesized. By tuning the energy gap of these NCs within thermal reach of the Mn2+ 4T1 excited state a highly temperature sensitive dual emission is acheived. The synthetic route to these dual emitting nanocrystals required the synthesis of a Mn2+:ZnSe core followed by the sequential growth of three shell layers. Doping Mn2+ into a Zn1-xCdxSe allow however, could prove to be a more efficient route to dual emitting nanocrystals with higher stability. Successful incorporation of Mn2+ into Zn1-xCdxSe alloys has resulted in dual emitting NCs.

Dr. Gerald Pollack and lab associates have discovered that when a polymer or gel is placed in a chamber with an aqueous suspension of polystyrene microspheres, the microspheres migrate away from the polymer or gel surface to about ~200-500 micrometers, leaving a microsphere free zone. This region is known as an “exclusion zone” (EZ) and is characterized by crystalline water. We tested the effects of changing the solution temperature on the size of the exclusion zone. A critical aspect of this experiment is what role thermal energy plays, if any, on the exclusion zone. In this experiment, we placed the polymer (Nafion) in an aluminum chamber, which was then filled with microsphere solution (500:1 deionized water to polystyrene microspheres). We then placed this chamber into a larger chamber filled with water. This larger chamber was connected to a water circulator with which we could control the temperature of the water surrounding the aluminum chamber. In our experiments, we varied the temperature of the circulating water at 15 randomly chosen temperatures between 5˚C and 75˚C, and measured the size of the EZ. We found that EZ size increases as temperature diminishes. This finding has fundamental implications for understanding basic properties of water, and particularly the EZ phase of water.

Surface plasmons of metal nanoparticles have been the source of much scientific study over the last decade due to their immediate and significant applications in biosensing, surface-enhanced Raman Specroscopy (SERS), and light absorption engineering in semiconductor nanodevices. Surface plasmons can be generated by high-energy electrons coupling with natural plasmonic resonances of a nanometal’s electron cloud. An abundant source of such relativistic electrons can be found in common electron energy-loss spectroscopy (EELS) experiment, in which surface plasmons generated by the experiment can further couple with plasmons of other nearby nanoparticles, giving rise to “hot spots” with very strong electric fields. Often these hot spots can magnify an incident electric field over 100-fold, giving rise to many of the applications mentioned previously. This work aims to produce a computational model that computes plasmon excitation due to a fast electron by using an existing Discrete Dipole Approximation (DDA) framework, and to compare theoretical results from metal nano-rods and metal nano-cubes with experimental data. Unlike experimental data, computational models are not limited by detector resolution; meaning that such a model will give significant insight into details of the EEL experiment. In this work we explore results that are higher-resolution than what is currently obtainable by modern techniques. The existence of such a model will allow researchers to obtain preliminary data before running time-consuming and expensive experiments, will allow enormous insight into the deeper physics of surface plasmons, and will give experimental results a theoretical foothold while also improving on their accuracy.

It is well known that mammals contain several types of phospholipase A2. The cytosolic phospholipases A2 (cPLA₂s) are one type, and are composed of six enzymes: cPLA₂alpha, cPLA₂beta, cPLA₂gamma, cPLA₂delta, cPLA₂epsilon, and cPLA₂zeta. There has been significant interest in the cPLA₂alpha isoform because of the enzyme's 10 fold preference for the hydrolysis at the sn-2 position of the glycerol backbone in phospholipids, resulting in the liberation of arachidonic acid. Arachidonic acid serves as a precursor for several highly regulated inflammatory mediators that play an important role in asthma, atherosclerosis, and arthritis. To address these problems, inhibitors that target cPLA₂alpha have been developed to serve as anti-inflammatory therapeutics. Wyeth pharmaceuticals has released a class of indole inhibitors that inhibit cPLA₂zeta at <10 nM IC₅₀. However, a recent study has shown that in cPLA₂alpha^-/- stimulated lung fibroblasts arachidonic acid production is lessened but still pressent. cPLA₂zeta has been identified as the other enzyme involved in the release of arachidonic acid. Consequently, my research project is to develop a selective and potent inhibitor of the cPLA₂zeta isoform. Our synthetic strategy is to modify the scaffold of Wyeth's cPLA₂alpha inhibitor. Analysis of the structure-activity relationships and 3D molecular modeling has revealed 4 different places on the scaffold to introduce substitutions. To date, we have generated and assayed over 20 inhibitors, with many more in progress. These inhibitors display low nanomolar potency, but are not selective for cPLA₂zeta. Our main focus is to increase the selectivity of our inhibitors towards cPLA₂zeta in order to further regulate and understand cPLA₂zeta's role in arachidonic acid production. 

Organisms have evolved specific mechanisms to respond to molecular and environmental stress. These stress-response pathways are evolutionarily conserved and can modify lifespan in model organisms, making them popular targets of aging research. Using the nematode Caenorhabditis elegans (C. elegans), we have found the loss of important genes in stress-response pathways can cause a phenomenon called a vulval integrity defect (Vid). Worms with Vid show a small protrusion near their vulva that can eventually lead to rupture of the gonad and premature death. While many labs have noted this phenotype, it is unknown if Vid is a natural part of C. elegans aging or if other genetic and environmental factors induce this phenotype, leading to confusion as to whether worms with Vid should be censored in aging experiments. The goal of our project is to determine the mechanism of Vid and whether it is part of normal worm aging. To further define the mechanism of Vid, we are using three experimental approaches: (1) using mutant worm strains, we are testing the genetic pathways important for causing and preventing Vid, (2) because Vid is temperature dependent, with frequency increasing inversely with temperature, we are testing at what point in their lifespan worms can still be rescued from Vid, (3) beginning with the gonad and vulval muscle, we are looking for cellular death and/or structural weaknesses that could be the direct mechanism behind Vid. Our initial work has identified several genes and genetic pathways important for Vid, including the hypoxic response pathway, the oxidative stress response pathway, and the protein assembly pathway. Our current work is close to identifying when worms are most susceptible to Vid and has begun to describe the physical mechanism of Vid. We intend to combine the results from these experiments to develop a model for Vid.

Aging research seeks to determine the processes that cause degeneration and disease in aged organisms, and to discover ways to prevent and/or slow their progression. Previous studies have shown that aging can be slowed in several model organisms through various means; however much is still not understood. In the model organism Caenorhabditis elegans, previous work shows that decreasing oxygen levels (hypoxia) significantly increases lifespan. The hypoxia-induced lifespan increase is observed when worms are exposed to hypoxia either from a late larval stage for life or if they are deprived of oxygen for just one day at the same larval stage. The low oxygen environment activates the hypoxic response pathway, mediated by the hypoxia-inducible factor (HIF-1). Previous data show that an increase of the HIF-1 protein, via genetic mutation or alterations in atmospheric oxygen, is sufficient to extend lifespan. However, much about this pathway remains unknown, including the mechanism for increased longevity, the difference between genetic and environmental manipulations of HIF-1, and the downstream proteins involved. My project examines the genetic components responsible for extending lifespan in hypoxia. To test this, we measured whether hypoxia increases the lifespans of worm strains with deletions or transgenic overexpression of known aging genes. Our results suggest that disruption of most of the non-HIF-1-related pathways have no effect on hypoxia-induced lifespan extension. However, the data show that the forkhead transcription factor, DAF-16, is required for hypoxia to increase lifespan, and subsequent work has shown that hypoxia causes DAF-16 to enter the nucleus. Interestingly, results indicate that hypoxia decreases lifespan when the oxidative stress response factor, SKN-1, is overexpressed, suggesting that SKN-1 is somehow toxic to worms in low oxygen. Collectively, the results show that hypoxic treatment increases lifespan in worms, but that the hypoxic pathway is intertwined with other stress responsive pathways.

The regulation of organismal life cycles by genetic components is a research area that has shown great promise in recent years. The process of aging is becoming more important to understand, not only due to the increasing incidence of age-related diseases, but also in order to determine what factors contribute to prolonged health and longevity. A variety of physiological pathways can regulate lifespan in C. elegans nematodes. Among these, one pathway of great interest involves dietary restriction. It is known that “stresses” on the worm, such as reduced food intake, can extend its lifespan. A mutation in the eat-2 gene affects the pharyngeal pumping mechanism whereby the worms ingest bacteria, thus creating a genetic model for dietary restriction. This research investigates the role of steroid signaling in dietary restriction-induced lifespan extension and improved stress resistance, using eat-2 mutant worms as a background and using a heat shock assay to induce stress at various points during the worms’ life cycle. By studying genetic determinants of longevity in C. elegans, my research will help to uncover more information about what can make nematodes long-lived, thus contributing to our understanding of the genetic basis for longevity regulation. More importantly, the existence of human orthologs for many longevity genes in C. elegans suggests that our findings will be valuable in identifying factors that are crucial for human longevity and health.

Spinocerebellar ataxia 7 (SCA7) is a heritable disease caused by a mutation in the gene for the protein ataxin-7. SCA7 results in progressive neurodegeneration in the cerebellum, brain stem, and retina, which causes patients to lose motor control and vision. The disease disrupts the intricate circuitry of the cerebellum, impacting cells such as Purkinje cell (PC) neurons and the Bergmann glia (BG) that support them. BG and PC function is interdependent such that dysfunction in one cell type causes damage in the other. I set out to characterize how BG function is altered in SCA7 by examining brain tissue from 40 week old mice with or without the ataxin-7 mutation. The tissue was analyzed for various markers of BG function and morphology. I addressed whether mutant ataxin-7 expression in BG leads to changes in their morphology and identity as radial glia using markers for radial glia such as Sox9. I also used the marker for GLAST, a glutamate transporter, to study changes in their ability to support proper PC neurotransmission through glutamate uptake from the synapses. BG distribution in the cerebellum was studied using the markers Calbindin and S100β to mark PC and BG respectively. The APC/β-catenin intracellular signaling pathway in BG was studied using markers for APC and β-catenin, as this pathway may impact BG’s ability to maintain its structure and thus, its function. The results of this study may suggest which BG functions are affected by SCA7. These functional changes may then provide insight into possible mechanisms for cerebellar neurodegeneration in SCA7.

DEER (double electron electron resonance) is a pulsed EPR (electron paramagnetic resonance) spectroscopy technique that is used to measure distances between unpaired electrons. DEER can be applied in structural biology to accurately characterize the structure of spin-labeled proteins, at the scale of several nanometers. EPR is commonly conducted using microwave frequencies at around 10 GHz (X-band), but spectral resolution and sensitivity can be improved by increasing the microwave frequencies to around 35 GHz (Q-band). Microwaves are concentrated on the sample of interest through the use of a resonator. Resonators developed for use with traditional continuous wave (CW) EPR experiments are designed with a high quality factor (i.e. most efficient storage of microwave energy) at a single resonance frequency. However, DEER requires a wide resonance bandwidth, which means that the quality factor of the resonator must be lowered, often at the expense of sensitivity. The goal of this project is to develop a Q-band resonant cavity for use with DEER that combines a bandwidth of 60-70 MHz with high accuracy and sensitivity. The resonator is designed using CST Microwave Studio, an electromagnetics modeling software package, to maximize its bandwidth and power conversion efficiency. A cylindrical cavity geometry is chosen due its ease of construction and simple tuning procedure. We anticipate that, after further development and testing, this initial design will serve as a platform that can be easily modified to accomodate other advanced EPR techniques.

The spinal cord represents a key site of convergence for sensory feedback and cortical commands, but determining how each contributes to movement has not been uncovered.  Understanding how spinal circuits are structured and utilized to generate complex movements will be necessary to understand how the nervous system processes multiple inputs.  One approach to understanding motor circuits is to know how the output effects of neurons change between tasks.  Here we investigate the state of the spinal cord by stimulating at specific sites in the cord and recording electromyographic activity (EMG) in the forelimb muscles of a non-human primate.  By comparing the output effects of stimulation between two behaviors, a flexion/extension wrist task and a reaching task, we hope to understand the effects of cortical and sensory interactions in shaping motor output.  In addition, by creating a spinal map of stimulus triggered EMG activity, we hope to reveal the functional organization of spinal outputs during the behaviors.  In this experiment, we compare the effects of stimulus triggered averages of EMG between these two tasks at various sties in the cord.  Our results show a change in the effects on EMG activity dependent on the behavior performed in roughly 2/3 of sites.  Indicating that the configuration of the circuit that our electrode stimulates is dependent on the task performed.  When the non-human primate performs a wrist task, cortical input and sensory feedback recruit specific spinal pathways to activity certain muscles in order to perform the task.  When the task is altered, different cortical inputs and sensory feedback may inhibit or further excite that circuit, changing EMG activity.  Understanding the circuits responsible for the control of arm movements and how these circuits change with varying tasks, may allow us to utilize and exploit these spinal circuits to treat patients with spinal cord injury.

T lymphocytes protect against the universe of pathogens by generating a diverse repertoire of antigen receptors called T cell receptors (TCRs). Each individual T cell expresses TCRs with a single antigen specificity. The induction of self-tolerance is crucial to the health of the individual because it culls the T cell population of those cells that bear self-antigen reactive receptors. Such autoreactive T cells are the driving force behind autoimmune diseases. One mechanism for inducing self-tolerance is TCR revision, by which peripheral T cells replace self-reactive TCRs with newly generated, non-autoreactive TCRs. This replacement results in cell rescue and involves cleaving DNA, which requires implementation of many controls to protect genome stability. Cells undergoing TCR revision appear to be localized in the spleen and lymph nodes inside a confined microenvironment known as the germinal center (GC), and this constrainment may provide a means for controlling this process. Given the rarity of revising T cells, my project focused on potential means for increasing the frequency of cells undergoing TCR revision using as a model system TCR transgenic mice, in which the majority of T cells express TCRs of the same antigen specificity. We hypothesized that expanding the GC T cell compartment would increase the frequency of cells undergoing TCR revision. I carried out injections of lipopolysaccharide, sheep red blood cells, and anti-CD40 antibody into TCR transgenic mice in an effort to induce GC formation. I then analyzed the accumulation of post-revision and revising T cell populations by flow cytometry. Use of any of these agents to increase TCR revision would facilitate the isolation of post-revision and revising T cells and allow further investigation into the mechanistic details of this tolerance process. Our goal is to understand how the challenging balance between having a diverse T cell repertoire and avoiding autoimmunity is maintained.

Angiogenesis is the development of new blood vessels from pre-existing vasculature and is a hallmark of Kaposi’s sarcoma (KS), a highly vascularized neoplasm prevalent among immunosuppressed individuals. The etiological agent of KS, Kaposi’s sarcoma herpesvirus (KSHV), induces angiogenic phenotypes in endothelial cells during latent infection. Interestingly, KSHV infection leads to down-regulation of transforming growth factor-beta 2 (TGF-beta2), an anti-angiogenic cytokine. In addition to six viral genes, KSHV also encodes 17 recently discovered microRNAs (miRNAs) during latent infection that appear to directly contribute to pathogenesis and angiogenesis by targeting host gene expression.  These endogenously encoded RNAs of 19-23 nucleotides post-transcriptionally regulate gene expression through basepairing interactions with the 3’ untranslated region (UTR) of target messenger RNAs (mRNAs) and either mark them for degradation or prevent ribosomal binding. We have screened the miRNAs of KSHV to determine if any are involved in the downregulation of TGF-beta2 by KSHV. We found that two KSHV miRNAs, miK-K3 and miR-K8, are sufficient to downregulate TGF-beta2. To determine if these miRNAs act directly on the mRNA of TGF-beta2 as opposed to altering genes that affect its expression, I proposed two aims: (1) use quantitative RT-PCR to determine the levels of TGF-beta2 expression during KSHV infection when miR-K3 and miR-K8 are inhibited by antagomirs, small antisense inhibitors of miRNAs, and (2) determine if miK-K3 and miR-K8 directly bind to the 3ʼ UTR of TGF-beta2 mRNA using luciferase reporter constructs. By identifying specific targets of KSHVʼs miRNAs such as TGF-beta2, we can improve our understanding of how KSHV is able to maintain latent infection and disrupt the normal regulation of cell proliferation. These studies are the first steps to better understanding KSHV pathogenesis and to developing targets that disrupt KSHV replication.

Populus trichocarpa, or Black Cottonwood, form a mutualistic relationship with diazotrophic bacteria, in which nitrogen is provided to the host plant in exchange for protection and energy. Understanding this form of symbiosis is essential in determining how biologically available nitrogen is cycled throughout our planet’s biomes. A well-known diazotrophic mutualism is found between leguminous species and Rhizobia or Frankia bacteria, where colonies are established inside root nodules of the host plant. Black Cottonwoods differ from legumes in that they form an endosymbiosis with nitrogen-fixing bacteria recruited from the environment, where the bacteria form colonies throughout the plant tissue. To determine the endophyte source, bacterial samples were obtained from sand, river water, and seed collected at the Three Forks Park in Washington to determine where in the environment Black Cottonwoods are coming into contact with these endophytes, and which species are found at each source. Nitrogen-fixing species were selected for by culturing bacteria using nitrogen free media, and were then subjected to PCR for amplification of the nitrogenase gene for confirmation. Species identification was performed by amplifying the 16sRNA, followed by sequencing and local alignment matching. Of the 29 samples collected from the Three Forks site, 27 were found to carry the nitrogenase gene, and species identification underway. Black Cottonwoods, in addition to other poplar species, have been found to be very useful industrially as biofuels and in the bioremediation of environmental pollutants. Nitrogen-fixation provides a major benefit for using poplars, where the use of fertilizers is reduced. It is thought that there is a common evolutionary link between more plant species and diazotrophic bacteria than is currently known, and understanding which species exist outside of the plant hosts may lead to the reduction of nitrogen-based fertilizer in other industrially useful plants.

During early development, spontaneous waves of electrical activity propagate across many different structures in the central nervous system. These waves are a critical feature of neuronal development, regulating neuronal migration, physiological maturation, and synaptic connectivity. In slices of mouse cerebral cortex taken at early stages (embryonic day 18 – postnatal day 1), we observed circular clusters of cells (domains) that were synchronously active during times when the slice was not generating spontaneous waves of activity. The domains consisted of approximately 10 to 50 cells and activity appeared to initiate from a center cell before the synchronous activation of the surrounding cluster of cells. We hypothesize that domains may form the functional basis of activity that propagates across the entire cortex. We have applied a range of drugs to elucidate the mechanism that triggers these small coactive domains. The domains do not appear to be dependent on electrical activity, they are not mediated by the neurotransmitters gamma-aminobutyric acid (GABA) or glutamate, and they are not dependent on calcium channels or the release of internal stores of calcium. Application of the gap junction blocker octanol inhibited the appearance of domains, but questions remain as to the specificity of octanol. Future work will identify the type of cell that triggers the domain and further study the relationship between the presence of domains and development of the neocortex.

The mitotic spindle orchestrates proper alignment of chromosomes during cell division. The spindle is composed of microtubules that attach to kinetochores, multi-protein complexes that assemble at the centromeres of chromosomes. Understanding kinetochore function is essential to understanding the process of cell division. Through mass spectrometry analysis, the Davis Lab has found an abundance of the urmylation component Uba4 at the kinetochore. The urmylation pathway is highly similar to ubiquitination, and involves conjugation of a ubiquitin related modifier (Urm1) to proteins and RNAs. The role of urmylation at the kinetochore is not known. We hypothesize that post-translational modification of kinetochore proteins through urmylation is a regulator of chromosome segregation. To examine the effects of urmylation on the kinetochore I will be using an Ncs2 mutant, a protein involved in the urmylation pathway, By fluorescently labeling the kinetochore protein Spc105 and spindle pole body protein Spc110, I am using fluorescence microscopy and computational analysis to look for abnormalities in spindle appearance. These results will help elucidate the role of the urmylation pathway in the regulation of cell division.

The Institute of Medicine‘s (IOM) report on health disparities, “Unequal Treatment”, identified the medical system’s inability to distinguish minority groups with meaningful specificity as a primary barrier to recognizing critical disparities in health care and outcomes. A recent follow-up report on “Race, Ethnicity, and Language Data” called for more detailed demographic data collection. This study evaluated the validity of self-reported primary language to detect disparities in diabetes care across language groups of first generation, mostly non-English speaking outpatients at an international clinic. The REALL (Race, Ethnicity and Language proficiency Level) study is a cross-sectional pilot study, surveying a convenience sample of immigrant and refugee patients receiving primary care at an urban, county hospital. Participants were eligible for this study if they had diabetes based on medical record data and completed the REALL survey question about how many and what languages they speak, their reading ability in those languages, and the culture they most identify with. Survey data were linked with medical record data that included hemoglobin A1C (HA1C), low density lipoprotein (LDL) and systolic blood pressure (SBP). Diabetes control was evaluated across the 6 largest language groups. 250 patients were eligible for the study. The survey data were 90.84% concordant with registration data for primary language. Mean HA1Cs were lowest among Vietnamese patients, significantly lower than Somali patients and Cambodian patients. While 52% of Vietnamese patients had HA1C<7, only 26% of Somali patients had HA1C<7. There were no statistically significant differences across language groups in mean LDL level or SBP. Results suggest that there is variability in diabetic outcomes across language groups in first generation immigrant communities. Recent refugee communities are potentially more stressed due to lack of acculturation and community establishment. Further inquiry into their duration in the US, the clinical care system, and their dietary practices are advised.

The spindle assembly checkpoint is important for detecting errors in spindle assembly during mitosis. When the checkpoint is activated, it arrests the cell cycle so that errors such as unattached kinetochores can be detected and corrected before progressing through the cell cycle. Problems with kinetochore attachment can be detrimental and lead to cells with an abnormal number of chromosomes which can then cause birth defects. Misregulation of the cell cycle can lead to abnormal cell division and cancer. Spindle pole bodies in yeast are functionally equivalent to human centrosomes, which organize microtubules in the cell cycle. Spindle pole bodies increase in size when the cell cycle is arrested at the checkpoint. Mad1 is an essential component of the checkpoint and interacts with the Nup60 protein in the nuclear pore complex, a channel through which information traffics between the nucleus and cytoplasm. The N-terminus of Mad1 protein is required to localize Mad1 to the nuclear pore complex, but is not required for the checkpoint arrest. We found previously that if Nup60 is deleted, the spindle pole bodies do not grow in cells arrested at the checkpoint. Mad1 mutants containing only the C-terminus of the protein will be constructed. I will test if the ability of Mad1 to bind to Nup60 is required for spindle pole body growth. Spindle pole body size will be measured by fluorescence microscopy after arresting the cells in metaphase. I hypothesize that although Mad1 interaction with Nup60 is not required for the cell cycle arrest, it is required to signal spindle pole body growth. If true, then deletion of the Nup60 binding domain of Mad1 should block spindle pole body growth. Discovering what interactions are vital in these cells will give us a deeper understanding of how regulation of the cell cycle is maintained.

In eukaryotic organisms, ubiquitin is a small protein modifier that is attached to proteins and targets them for proteasomal degradation. In an analogous fashion, in Mycobacterium tuberculosis (Mtb), the prokaryotic ubiquitin-like protein (Pup) is conjugated to lysine side-chains of substrate proteins and targets them for degradation by the Mtb 20S proteasome. This pathway has been shown to be essential for Mtb infection in mice, and is a potential target for theraputic reseach. The enzyme PafA is responsible for attaching Pup to its substrates and has previously been shown to be essential for lethal infections by Mtb in mice. We have developed a fluorescent activity-based probe to monitor PafA activity in vitro by conjugating Fluorescein isothiocyanate (FITC) to free lysine, a known substrate of PafA. FITC-Lys demonstrates a high degree of specificity for PafA, even in the presence of cell lysates. This probe was employed to visualize PafA activity by in-gel fluorescence. It has allowed us to identify a minimal sequence of Pup, the C-terminal 26 amino acids, that is recognized by PafA. Alanine mutants of the 25th and 26th C-terminal residues demonstrate that leucines at these positions are critical for PafA interaction.

Marine bacteria carry out nutrient transformations that maintain the chemistry of seawater. Most marine bacteria are uncultured and little is known about their specific roles in marine biogeochemical cycles. We identified several cultures from the beta-proteobacterial OM43 and gamma proteobacterial sulfur oxidizer (GSO) clades in a culturing experiment from the North Pacific. Members of the OM43 clade use single carbon compounds produced by phytoplankton, suggesting that they contribute to carbon cycling in surface waters. Members of the GSO clade use energy from sulfur oxidation to assimilate organic and inorganic carbon, suggesting that they contribute to carbon and sulfur cycling throughout the water column. We selected four OM43 and three GSO cultures to establish a culture collection. Several OM43 and GSO cultures were revived from freezer stocks and purified. Their identity was confirmed by 16S rRNA gene analyses and they were added to the UW marine bacterial culture collection. OM43 and GSO isolates obtained in this study will be used to further characterize the roles of these important groups of bacteria in marine carbon and sulfur cycles.

The Dunham group studies the costs and benefits of changes in gene and chromosome copy number (aneuploidy) in S. cerevisiae. Gene copy number change is a common evolutionary mechanism, and is often seen in cancers. Previously, the lab carried out a series of experiments to determine the consequences of changing the copy number of every gene in the yeast genome. 4383 yeast strains, each carrying a plasmid containing a yeast gene and a unique barcode sequence, were competed in nutrient-limited continuous-culture environments. The relative success of each strain was found by performing high-throughput sequencing on the barcodes. Our project explored three assumptions of this experiment, using direct chemostat competition assays. First, we confirmed that fitness effects observed in the pooled experiment could be repeated in individual experiments. We performed competition experiments using 22 genes from the experiment that were found to either be beneficial in certain environments or potentially harmful when duplicated. We also found that the plasmid alone had a fitness effect, by competing a strain containing a plasmid without a gene insert against a wild-type strain. Finally we tested the fitness effects of Autonomously Replicating Sequences (ARSs), which are origins of DNA replication found in many of the loci we are investigating. In a competition between a strain transformed with a plasmid with two ARSs and a strain with a plasmid containing a single ARS, a fitness benefit was observed for the presence of an extra ARS on the plasmid. These data are important controls for the pooled experiments previously performed in the Dunham lab and will help to identify the precise molecular causes of fitness changes associated with aneuploidy, and, perhaps in the future, provide some new insights into cancer biology.

Huntington’s disease is a hereditary neurodegenerative disease that is characterized by motor and cognitive dysfunction and currently has no known cure. One of the earliest molecular events in Huntington’s disease pathogenesis is the down-regulation of the cannabinoid 1 receptor (CB₁), occurring prior to symptom onset and loss of any other receptor. Though the exact mechanism is unclear, it has been shown that CB₁ loss occurs almost exclusively in medium spiny neurons (MSNs), cells that play a key role in movement and degenerate later in disease progression. CB₁ knockouts in mouse models also exhibit exacerbated disease phenotype. Previous studies have attempted to rescue CB₁ function in mouse models by administering chronic cannabinoid agonists, but have produced inconclusive results. Our study investigates the effects of CB₁ receptor rescue from a genetic approach, by inserting flox-stop CB₁ (fsCB₁) specifically activated in striatal MSNs of R6/2 mice, the best characterized Huntington’s disease mouse model. We first confirmed the localization and functionality of the knocked-in fsCB₁ in R6/2 and CB₁ null mice (control), and then measured motor phenotype and synaptic integrity over time. Performance of R6/2 mice with and without fsCB₁ rescue was measured using rotarod, open field, novel object recognition, and clasping behavior tests biweekly from weeks 4 to 12 of age. MSN axon integrity was measured by IHC staining for caspase-6 and NPY-Y1R, markers for Wallerian-type degeneration. By observing behavior and axon integrity from pre- through late-symptomatic stages, we hope to establish a relationship between early MSN axonal loss and impaired motor phenotype in R6/2 mice, as well as link CB₁ function to axonal integrity through R6/2 and fsCB₁-R6/2 comparisons. This research will increase the present understanding of molecular mechanisms behind Huntington’s disease and provide insight into CB₁ as a therapeutic target.

Glioblastoma multiforms (GBMs) are malignant brain tumors and are among the most lethal cancers in the United States. Current clinical treatments involve surgical resection followed by chemo radiation therapy. Temozolomide (TMZ) is a DNA methylating agent that has shown promise in post-operative chemo radiation therapy, yet its effects are mediated greatly by the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). The addition of DNA repair inhibitor O6-benzylguanine (BG) as part of a concurrent treatment with TMZ has shown promise, but suffers from unacceptable toxicity due to BGs poor pharmacokinetics. Here we report the development of nanoparticles (NPs) loaded with a BG payload that can efficiently cross the blood brain barrier (BBB) and with the addition of targeting agents may be a more effective method for drug delivery by reducing toxicity in healthy tissue. These NPs are composed of a magnetic core and biocompatible polymer surface coating. The NPs are then conjugated with BG (NP-BG) by attachment to the polymer surface. NP-BG is further modified with chlorotoxin (NP-BG-CTX), a tumor targeting peptide, through a polyethylene glycol linker. NP-BG-CTX had a mean hydrodynamic size of 75nm with a surface charge of about +4mV and have demonstrated specific targeting in vitro of GBM cell line SF767. Finally, we have shown that NP-BG substantially reduces MGMT activity in SF767 cells in vitro. These results suggest that NP-BG-CTX have a higher specificity to tumor cells than normal healthy cells in cancer patients. This would decrease drug toxicity in patients and reduce tumor resistance to TMZ, potentially providing a more effective treatment and improve clinical outcome.

Volatile organic compounds (VOCs) in indoor household air are carcinogens that many people are exposed to everyday. Examples of VOCs in the home are benzene, chloroform, and trichloroethylene. Genetically transformed plants are capable of degrading these VOCs by oxidizing them into less harmful compounds. The Strand lab has previously shown that poplar trees and tobacco, genetically transformed with cytochrome P450 2E1 (a rabbit gene expressed in the liver), successfully phytoremediated VOCs (phytoremediation is the use of plants in environmental treatment). My project at the Strand lab is to transform a houseplant with the same 2E1 gene, so people may raise this plant in their home to degrade indoor VOCs. Epipremnum aureum, a house ivy under the common name of pothos, was chosen for this project because the project follows the protocol for pothos transformation published by Kotsuka and Tada. Additionally, pothos does not flower, so the spread of the 2E1 gene can be kept in check. The project was executed in three steps: transformation, regeneration, and confirmation. The first part was to transform explants (pieces of pothos) by way of Agrobacterium infection, in which the bacteria insert the 2E1 gene into the pothos genome. The second step was to induce growth of calli in the explants, from which a new, genetically identical plant could be regenerated. The last step is to confirm that the new pothos was actually transformed and capable of degrading VOCs. This will be done through such tests as PCR, rtPCR, Western blot, and activity assays. I expect the pothos to be able to degrade VOCs as did the transgenic poplar and tobacco. These VOCs include trichloroethylene, vinyl chloride, chloroform, carbon tetrachloride, and benzene. If this result is not achieved, then future work may be devoted to find why the 2E1 gene is not compatible with pothos.

Gastrulation is a crucial step in embryogenesis, creating differentiation between ectoderm, mesoderm, and endoderm, as well as forming necessary organs. Transmembrane and juxtamembrane proteins located at the adherens junction, such as cadherins and p120 catenin, work together to either promote cell motility or adhesion. Using Danio rerio embryos as a model organism, we hypothesized that when p120 catenin is at the plasma membrane it binds and stabilizes the cadherins, causing cell clustering and increased adhesion. When p120 catenin is in the cytoplasm it activates Rac1 and Cdc42 (two members of the Rho GTPase family), which causes cell motility. By using a “depletion-rescue” experimental approach, p120 catenin levels are depleted by injecting a splice-morpholino, then “rescued” by coinjecting wild-type Cdc42 or Rac1. This indicates that p120 catenin in healthy embryos works to activate Cdc42 and Rac1 during gastrulation, resulting in cellular motility. When dominant-negative and constitutively-active mutants of Cdc42 or Rac1 are injected in combination with morpholino, the embryos do not “rescue” well, if at all. We postulate that wild type GTPases “rescue” well because the can receive a signal and activate migration directionally while the constitutively-active GTPases activate migration in all directions. These studies on the regulation of cell motility can help to elucidate larger signaling pathways, leading to additional effects on cell survival, growth, and invasiveness, all of which may be caused by the upstream p120 catenin.

The mechanism to direct auditory attention consciously is not well understood. For hearing impaired people, it is very hard to direct attention efficiently between conversations with background noise. My project investigated the differences between maintaining and switching attention prompted by visual cues. In the behavioral task, two streams were presented simultaneously, one with high pitch and the other, low. Each stream consisted two triplets of spoken letters and the two triplets were separated by a silent gap that varied in duration. In the beginning of each trial, subjects were prompted by two spoken letters which stream (high or low) to pay attention to initially. Simultaneously, a visual cue directed subjects to either stay or switch between two streams during the silent gap period. Subjects were asked to respond when they heard the second ‘E’ from one of two streams that they attended to as quickly and accurately as possible. Preliminary results suggest that subjects needed more time to respond when they had to switch between streams. As expected, there were no differences between subjects attending to the high or low pitch stream. We also hypothesized that the shorter the gap period, the longer the response time will be—I hope to test this hypothesis through more in-depth data analysis. The result from this project will be analyzed together with neural data obtained using magnetoencephalography where subjects will be performing a similar task. The findings of this project will provide insight into the underlying mechanism of switching of auditory attention—a task that hearing impaired people find difficult to perform in a noisy environment, even with the help of hearing aids or other augmentative devices.

In Acute Myeloid Leukemia (AML) the adhesion interaction between the AML blast cells and the bone marrow microenvironment confers resistance of the leukemia cells to the chemotherapy drugs. One possible receptor that might facilitate this adhesion is the VLA-6 integrin. Our laboratory has been found it to be highly expressed in the AML stem cells, the cells that are believed to give rise to leukemic relapse. To investigate the role of the VLA-6 receptor, I performed assays to observe the effects of blocking this receptor on cell adhesion as well as the chemotherapy resistance. The adhesion assay consists of measuring AML patient cell binding to laminin-coated wells, as laminin is the ligand for the VLA-6 adhesion receptor. Adhesion mediated by VLA-6 is assessed by measuring binding in the presence or absence of the anti-VLA-6 antibody. The chemotherapy resistance assay involved the same conditions as the adhesion assay. We expected that blocking the VLA- 6 receptor would prevent the cells from adhering to the laminin and that this would also lead to lessened drug resistance. However, for the cells from one patient studied so far, there has not been a significant difference between the amount of adhered cells in these conditions. Additional experiments are in progress with more patient samples to elucidate the significance of the VLA-6 receptor in AML physiology and survival.

Many important enzymatic reactions require the transport of electrons to function properly. This transport is commonly achieved by passing an electron to several different residues on the peptide chain which composes the enzyme. Tryptophan, a common amino acid, participates in this electron transfer by way of an oxidized radical tryptophan intermediate. We hypothesize that hydrogen bonding characteristics have significant influence on the electron transfer behavior of tryptophan. Understanding the effects and implications of hydrogen bonding would greatly affect how we engineer enzyme structure for reactions involving this type of mechanism. Using EPR (electron paramagnetic resonance) spectroscopy, we recently discovered that the strength and position of the hydrogen bond correlates directly to shifts in the g-tensor, a proportionality constant used to describe the resonance condition of the radical electron. Using computational chemistry software, we are gathering data to better understand how g-tensors change as the geometry of the hydrogen bond is altered. Our main goal is to determine trends which describe how changing specific structural features alters the corresponding g-tensor values of the radical tryptophan. We hope these trends will allow us to understand if and how hydrogen bonding is occurring at a radical tryptophan.

In this study, chitosan, a biocompatible, biodegradable, and nontoxic polymer obtained from crab shells, was used to produce 3D scaffolds which closely resemble the mechanical and physical properties of human bone tissue. One of the critical stages in tissue engineering for successful regeneration is to design a scaffold that mimics the architecture of actual human tissue. While chitosan has been acknowledged for its potential to be used in human tissue engineering, its weak mechanical strength restrained chitosan usage in bone tissue engineering. In order to cope with the drawbacks, mechanical properties of 3D chitosan scaffolds were examined in relation to different chitosan concentration (4, 6, 8, 12 wt%). Compression test and tensile test were done on each scaffold to measure the mechanical properties and study the relationship between increasing chitosan concentration and mechanical strengths. Then, MG-63 osteoblasts were seeded on each scaffold to examine attachment and proliferation of the cells. Immunoblotting will be performed to detect the levels of osteocalcin expression on walls of the scaffolds, and Scanning Electron Microscope imaging will be used to assess the proliferation pattern of MG-63. Upon completion of the study, we predict to observe direct relations between the concentration of chitosan and mechanical properties of the scaffolds.

Duchenne Muscular Dystrophy (DMD) patients have mutated dystrophin genes and suffer from muscular degeneration, which leads to cardiac, respiratory and general skeletal muscle disabilities. Potential treatment of DMD patients could involve gene therapy in which Adeno-Associated Viruses (AAV) deliver functional dystrophin cDNA ligated to muscle specific regulatory cassettes that express dystrophin exclusively in striated muscle tissues. The Hauschka lab has been designing regulatory cassettes (e.g. MHCK7) that contain control regions from genes that are highly active in skeletal muscles such as the muscle Creatine Kinase (MCK) and α-Myosin Heavy Chain (α-MHC) genes. Due to the limited size of DNA that can fit into AAV capsids, the cassettes need to be as compact as possible. My goal is to test whether adding a small intronic enhancer (SIE) from the MCK gene, that is known to have high activity in skeletal muscle in vitro, will improve transcriptional activity in cardiac and skeletal muscle cultures compared to MHCK7. The SIE was isolated and cloned immediately 5’ of MHCK7. After sequencing to confirm the SIE orientation, we carried out a pilot study in which 5’-SIE-MHCK7 was transiently transfected into skeletal muscle cultures. 5’-SIE-MHCK7 activity, as measured by the reporter gene firefly luciferase, was several fold higher than MHCK7. This suggests that the addition of the SIE to two known muscle specific enhancers increases transcriptional activity in skeletal muscle cells. Future studies will focus on confirming this result, and comparing 5’-SIE-MHCK7 activity to aMHCK7-SIE cassette in which the SIE is located 3’ of the luciferase reporter, so as to mimic its normal location in the MCK gene. The 5’-SIE-MHCK7 cassette will also be tested in cardiomyocyte cultures, and smaller cassette versions will be designed that eliminates additional non-conserved DNA sequences from the SIE and MHCK7 components.

Cancer vaccines offer a promising method of manipulating the immune system to seek and destroy malignant cells. However, there have been many limitations on their ability to enhance stimulation and persistence of CD4+ Type 1 helper (Th1) and CD8+ cytotoxic T lymphocytes (CTL) necessary to promote adequate tumor regression. The long-term goal of this research is to use polymeric carriers to deliver antigens and immunomodulatory siRNA to antigen presenting cells (APCs), in order to promote anti-tumor cellular immunity. We have designed pH-responsive, endosomolytic, “smart” polymers that are capable of being conjugated to antigens and complexed to siRNA. Also, we have demonstrated the ability of these polymers to enhance class I antigen presentation by APCs. In addition, we have shown that polymeric delivery of siRNA promotes knock down of a model gene, glyceraldehyde 3-phosphate dehydrogenase, in dendritic cells. We plan to demonstrate similar results with other immune cells, both immortalized and primary, and genes of interest, as well as extend this technology to relevant tumor-associated antigens. These data point to the modular nature of our polymeric delivery systems, as they provide a promising way of approaching challenges in cancer immunotherapy through multiple delivery modalities.

Blindness is a condition that impacts people across the globe. Some of these people are born blind due to a genetic defect while others become blind through an accident or as the result of disease. Thus, dissecting the underlying molecular causes of blindness is a critical component in improving the lives of many people. To study causes of blindness, we are analyzing disease causing mutations in the model organism Danio Rerio, or zebrafish. The recessive ZVM10 mutation causes early degeneration of the retinal photoreceptors, causing complete blindness. To study this mutation we are determining the identity of the mutated gene. The first step is to determine the position of the mutation within the zebrafish genome (i.e. map the mutation). This is done by mating the ZVM10 AB fish with a polymorphic fish strain. The crossovers between the different DNAs are traced, and used to identify the area homozygous for the AB allele. By continually reducing this region we specify a small group of genes that potentially have mutated. Using this method, I have established a general region on chromosome 20 between Z markers G40149 and G47173 containing the ZVM10 mutation. Identification and study of this mutation will enable scientists to better understand the role of this gene in vision. It will also permit the identification of people who are blind due to this genetic mutation, through genetic screening of the patient. Finally, if this gene is also involved in human blindness, it will become a candidate for rescuing blindness through gene therapy.

Research studies suggest that up to 40% of hepatocellular carcinomas may be caused by liver stem cell carcinogenesis, in which stem cells, due to their high proliferative abilities, become neoplastic and maintain cancer growth and metastasis. MicroRNA are post-transcriptional gene regulators that have been found to be involved in many cellular processes such as differentiation of stem cells. This project focused on the role microRNA play specifically in liver stem cell differentiation. Finding which microRNA are important in liver cell differentiation could provide new methods for diagnosis and treatment of liver cancer originating from stem cells. I used a stem-cell-like liver progenitor cell line capable of differentiating into mature liver tissue from a proliferative state, providing an ideal in vitro model for liver stem cell differentiation. Relative quantification of microRNA by real-time PCR and microarray was performed on samples taken from various time points during differentiation leading to the characterization of two microRNAs strongly upregulated in differentiation. In order to validate the importance of these microRNAs, I will use a viral vector to induce their overexpression and monitor any subsequent changes in the differentiation process. In the end, this project may provide additional insights into neoplastic liver stem cells.