| Advisor : | RON BURTON | ||
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| Abstract Title : | Genetic structure of leopard shark populations along the Pacific Coast of North America | ||
| Abstract : | The leopard shark (Triakis semifasciata) is a common nearshore benthic elasmobranch endemic to the Pacific coast of North America, from Washington, USA to Mazatlan, Mexico. Leopard sharks aggregate at specific coastal locations in the spring and summer, but little is known about leopard shark movement patterns once aggregations disperse. As a result, the extent of potential gene flow remains to be fully elucidated. While the leopard shark is not currently a threatened species, understanding gene flow throughout the species' range may provide insight into the population structure of similar species. Five microsatellite markers were used to analyze the genetic population structure of T. semifasciata throughout much of its range. Fin clips were collected from five locations in California and one location in Mexico (total N= 339). Our data show significant structuring among several locations. Evidence of gene flow between Santa Catalina Island and mainland populations is consistent with acoustic tracking data showing that leopard sharks occasionally cross the deep-water channel between Santa Catalina Island and the mainland, a minimum distance of 32 km. This provides an interesting contrast to leopard sharks' generally benthic lifestyle. We conclude that T. semifasciata does not form one panmictic population and significant population structure is present. | ||
| Advisor : | JENNIFER SMITH | ||
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| Abstract Title : | Are herbivores picky eaters? An assessment of functional diversity of Acanthurids in Maui, Hawaii | ||
| Abstract : | Herbivores on coral reefs are instrumental in mitigating the competitive interactions between reef-building corals and fleshy algae; however, not all herbivores provide the same services. While there has been research on functional diversity of herbivores across families, there is limited information on the functional diversity of species within the family Acanthuridae, containing a majority of herbivorous fish species. My study aims to identify functional diversity within this family through observations of foraging behavior and analysis of stomach contents for three species (Acanthurus nigrofuscus, Acanthurus olivaceus, and Ctenochaetus strigosus) at three sites on the leeward side of Maui. | ||
| Advisor : | DR. JOAN HELLER BROWN | ||
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| Abstract Title : | Role of hexokinase II dissociation in the induction of mitophagy in cardiac myocytes | ||
| Abstract : | Damaged mitochondria selectively undergo autophagy in order to cease deleterious signaling and preserve the integrity of surrounding mitochondria. This process is induced by the dissociation of hexokinase II (HKII) from the mitochondria of cardiomyocytes. By overexpressing a dissociation peptide to eliminate mitochondrial HKII binding, we observed a significant decrease in mitochondrial proteins voltage dependent anion channel (VDAC) and cytochrome c oxidase IV (COX-IV). The cytosolic ubiquitin ligase Parkin also translocates to the mitochondria upon dissociation of HKII, and a drastic increase in mitochondrial ubiquitination is observed. The decrease in VDAC and COX-IV is also seen in an ischemia-reperfusion injury model, which has been previously shown to dissociate HKII from mitochondria. Our results indicate that HKII binding at the mitochondria plays a large role in maintaining mitochondrial integrity in the heart and by extension, overall cardiac functioning. | ||
| Advisor : | JENNIFER SMITH | ||
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| Abstract Title : | GLOBAL ASSESSMENT OF THE STATUS CORAL REEF HERBIVOROUS FISHES: Evidence for fishing effects | ||
| Abstract : | ABSTRACT Coral reef herbivores provide important ecological services by regulating competitive interactions between reef building corals and fleshy algae yet little is known about their global status specifically, how fishing may alter community structure. We conduct a global synthesis of coral reef herbivorous fish by compiling data from peer-reviewed sources and scientific monitoring programs. Our results show that herbivorous fish biomass is more than two-times higher at sites not accessible to fisheries than at accessible sites and is independent of regional effects. Analysis of important feeding sub-groups further identifies that fishing disproportionately reduces the abundance of the larger-bodied Scraper/Excavator and Browser groups relative others. Loss of larger bodied groups likely alters the overall effectiveness of the herbivore community to regulate algal abundance on reefs. This is the first global assessment of the variability in and effects of fishing on coral reef herbivore populations and includes many remote locations that may be useful for developing management targets globally. | ||
| Advisor : | WILLIAM GERWICK | ||
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| Abstract Title : | Manipulation of lipase activity to increase lipid yields in Thalassiosira pseudonana | ||
| Abstract : | The accumulation of fuel precursor-containing lipid droplets in microalgae provides a sustainable alternative to conventional fossil fuels. As large-scale development is pursued, strains that efficiently accumulate triacylglycerol (TAG) without compromised growth rates are of significant interest. We pursued increasing neutral lipid droplet yields in microalgae by inhibiting lipase activity in the diatom Thalassiosira pseudonana. Transcriptomics data identified enzymes likely involved in lipid catabolism, Thaps3_264297 and Thaps3_269487, which were downregulated during lipid accumulation (Smith et al. in preparation). Lipase, phospholipase and acyltransferase activity were detected in Thaps3_264297, a homolog of Cgi-58 (Trentacoste et al., in review), while this study found lipase activity in Thaps3_269487. To inhibit the activity of this specific lipase in T. pseudonana, antisense constructs were transformed into wild-type to obtain single knockdown transformants of Thaps3_269487, and double knockdown transformants of Thaps3_269487 and Thaps3_264297. Both single and double knockdown transformants showed comparable growth rates in replete media and two single knockdown transformants showed twice the BODIPY fluorescence of wild-type, indicating higher lipid accumulation in stationary phase. The results indicate that downregulation of lipase activity can result in increased lipid content without affecting growth, and that strains expressing the knockdown could be useful for improved biofuel production. | ||
| Advisor : | YIMIN ZOU | ||
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| Abstract Title : | Wnt Inhibition combined with pre-conditioning promotes plasticity and functional recovery | ||
| Abstract : | Our lab developed a new method of peripheral conditioning that leaves peripheral circuitry intact and permits functional testing after SCI. After showing that Wnt expression limits axonal plasticity of lesioned neurons, we found that decreasing Wnt signaling promotes axonal plasticity with functional recovery after a high cervical lesion. | ||
| Advisor : | DR. JOHN GUATELLI | ||
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| Abstract Title : | Vpu Activity of Transmitted/Founder and Chronic Clade B HIV-1 | ||
| Abstract : | Acute HIV-1 infection is characterized by a robust type I interferon response, resulting in the induction of several host restriction factors. HIV-1 has evolved to counteract these restriction factors, and one such adaptation, the ability of Vpu to counteract BST2/tetherin, is associated with the evolution of SIVcpz into the pandemic group M HIV-1. Vpu also downmodulates the expression of CD4, counteracting a negative effect on the infectivity of virions that could in principle inhibit viral transmission. During transmission between individuals, very few or even a single virus, the "transmitted/founder (T/F) virus," gives rise to the new infection, but in the new host the selective pressure of the immune response yields the diverse "quasispecies" of chronic infection. In this study we examine the functional characteristics of Vpu proteins encoded by T/F viruses compared to those from acute and chronic viruses from longitudinally sampled subjects. T/F Vpu proteins showed a trend towards optimized CD4 downregulation compared to chronic Vpu proteins but did not differ substantially in their ability to downregulate BST2 or enhance virion release, although individual clones from each group were impaired in these activities. Analysis of the functionally impaired clones identified a C-terminal residue, W76, as important specifically for Vpu enhancement of virion release. Vpu clones with a W76G polymorphism, or site-directed mutants, were impaired in their ability to enhance virion release, but they were not defective for BST2 surface-downregulation, challenging the notion that Vpu enhances virion release exclusively by decreasing the total amount of BST2 at the cell surface. | ||
| Advisor : | DR. JAMES HAGOOD | ||
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| Abstract Title : | Mechanisms of Thy-1 and its effects on signaling pathways regulating myofibroblast differentiation in pulmonary fibrosis | ||
| Abstract : | There has been significant evidence that harnessing the anti-myofibroblastic effects of Thy-1 may allow for a therapeutic 'phenotype switch' in lung myofibroblasts, and thus either halt the progression or speed the resolution of lung fibrosis. Thy-1 expression affects myofibroblast phenotype broadly, i.e. expression of muscle and ECM proteins, myogenic transcription factors, and mechanotransduction of TGFβ activation. Global targeting of myofibroblast phenotype offers the best hope for effective idiopathic pulmonary fibrosis (IPF) therapy as it is characterized by excessive myofibroblast differentiation and accumulation of extracellular matrix. Thy-1 is a fibrosis suppressor that modulates certain aspects of fibrogenic phenotype (i.e. proliferation, cytokine and growth factor expression and responsiveness, migration, myofibroblastic differentiation and cell survival). Thy-1 inhibits lipid raft-associated signaling via the Src-family kinase (SFK) and focal adhesion kinase (FAK) pathways, promoting fibroblast adhesion and limiting migration. Recent studies have displayed Thy-1 interaction with αv integrins and syndecan-4 at the cell surface, modulating cell-cell and cell-matrix interactions and mechanical coupling, to inhibit TGF-β activation and myofibroblast differentiation. It is anticipated that Thy-1 will suppress myofibroblastic differentiation via RLD amino acid sequence and Syn4 interaction will lead to prevented activation of SFK, FAK, and PI3K signaling cascades. Together, these effects will lead to a better understanding of the mechanisms of Thy-1 anti-myofibroblastic effects and eventually to the contribution of innovative therapeutic treatments. | ||
| Advisor : | DR. JENNIFER SMITH | ||
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| Abstract Title : | Response of calcified and noncalcified southern California macroalgae to increased CO2 and temperature | ||
| Abstract : | Declining oceanic pH associated with Ocean Acidification (OA) is expected to negatively affect calcified macroalgae, but it is unclear how noncalcified macroalgae will respond. Global warming projects increased oceanic temperatures which could further help or harm macroalgae. We determined how CO2 enrichment and temperature increase affected seven species of common southern California macroalgae growth and chlorophyll fluorescence. One noncalcified alga increased growth rates and one calcified alga decreased growth rates under CO2 enrichment; the others were not affected. Another noncalcified alga showed decreased growth rates under increased temperature treatment while calcified alga was not affected. Neither treatment affected chlorophyll fluorescence. | ||
| Advisor : | DR. MILTON SAIER | ||
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| Abstract Title : | 1) Characterization of the The Tetraspanning Junctional Complex (4-JC) Superfamily and 2) Establishing Homology Between Mitochondrial Calcium Uniporters and Prokaryotic Magnesium Transporters | ||
| Abstract : | 1) Connexins and innexins form gap junctions, while claudins and occludins form tight junctions. In this study, convincing statistical data, derived using novel software, indicate that these four junctional protein families and six other families, including the Calcium Homeostasis Modulator Ca2+ Channel (CALHM-c), Ecm7p of SUR7 (SUR7), Intracellular Chloride Channels (ICC), Hair Cell Mechanotransduction Channels (HCMC), Ca2+ Channel Auxiliary Subunits γ1-γ8 (CCAγ), and the Low Affinity Ca2+ Channels (LACC), are related by common descent. These proteins all share the same 4 TMS topology; evidence is presented that they arose via an intragenic duplication event, whereby a 2 TMS-encoding genetic element duplicated to give 4 TMS proteins. Comparison scores in excess of values required to establish homology were obtained. A phylogenetic tree including members of all ten families demonstrated that the HCMC, SUR7, LACC and CCAγ families are most similar to the claudin family, while the ICC family is more similar to the innexin and connexin families. The occludins and the CALHM-c family clustered separately between the two major groups. Comparison scores were in general agreement with the phylogenetic results. These observations provide insight into the evolutionary origins and subfunctions of these proteins as well as guides concerning their structural and functional relationships. 2) Mitochondrial calcium uniporters, MCUs, are oligomeric channel proteins found in the mitochondrial inner membrane. MCUs have a highly conserved linker between their two predicted transmembrane helices (TMSs), which is also found in bacterial MCUs. Through topological analysis, a motif encompassing these conserved linkers was found in prokaryotic Mg2+ transporters, AtpI and AtpZ. Comparison scores of up to 14 S.D. were obtained, indicating homology between the MCUs and the Mg2+ transporters. A phylogenetic tree containing these proteins, in addition to eukaryotic AtpI homologues, showed that the AtpI and AtpZ proteins cluster separately from each other and the MCUs, while the eukaryotic AtpIs cluster in between these two major groups. The MCUs and AtpZs share the same 2 TMS topology, but the AtpIs have 4 TMSs. Binary alignments and comparison scores showed that TMSs 1 and 2 align with TMSs 3 and 4, suggesting that the 4 TMS AtpI proteins arose via an intragenic duplication event. These findings establish an evolutionary link interconnecting eukaryotic and prokaryotic Ca2+ and Mg2+ transporters and reveal their structural relationships. | ||
| Advisor : | DR. VIRGIL WOODS JR / DR. PALMER TAYLOR | ||
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| Abstract Title : | Elucidating Dynamic Interactions of Neuroligin4-Alpha/Beta-Neurexin1 Binding via Deuterium Exchange Mass Spectrometry | ||
| Abstract : | Neuroligin-Neurexin cellular adhesion molecular binding complications have long been correlated with issues surrounding Autism Spectrum Disease. Yet why two distinct α- and β- neurexin protein isoforms emerged in the synapse is still not well understood. Neurexins are composed of distinct α- forms containing six distinct Laminin-Neurexin-Sex (LNS 1-6) hormone binding globulin domains and three epidermal growth factor like domains (EGF 1-3), and β- forms which are solely composed of the sixth LNS domain. Both serve as presynaptic cell adhesion molecules and bind to postsynaptic neuroligins which are part of the α-β hydrolase fold family. It is hypothesized that though binding of neuroligin to neurexins is strongest at the sixth LNS domain, or β-neurexin, it may not have to occur there exclusively due to observed variance in protein function and such contributes to synaptic function diversity. Hydrogen/Deuterium Exchange Mass Spectrometry (DXMS) was utilized to elucidate the structural components of neuroligin4 and α- and β-neurexin1 and probe for changes induced by binding interactions. This revealed in neuroligin a novel point of interaction with α-neurexin through reductions in deuterium exchange rates absent when bound to beta-neurexin1. α-neurexin1 exchange profiles also affirmed physical occlusion at LNS 4 and 5 yet neuroligin?s binding site remained specific to LNS 6. Furthermore β-neurexin1 displayed binding trends highly similar to α-neurexin1's LNS 6 and confirmed that both bind neuroligin4 in the same fashion and that neuroligin interaction with LNS 4 and 5 exert no noticeable effects on LNS 6 binding. | ||
| Advisor : | JASON K. SICKLICK | ||
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| Abstract Title : | Hh Signaling Regulates KIT Expression in Gastrointestinal Stromal Tumors | ||
| Abstract : | Background: Gastrointestinal Stromal Tumors (GISTs), the most common sarcoma, arise within the gut due to overexpression of mutated KIT genes within the Interstitial Cells of Cajal (ICC). Earlier work demonstrated that gastrointestinal mesenchymal development depends upon Hedgehog (Hh) signaling, while Gli3, a transcription factor in the Hh pathway, regulates KIT mRNA expression in ureteral ICC-like cells. Therefore, we hypothesized that Gli transcription factors (Gli1/2/3) within the Hh pathway may regulate KIT expression in human GISTs, and therefore serve as novel targets for treating these KIT-driven tumors. Methods: Using genetic (siRNA knockdown and gene overexpression) and pharmacologic (Gli inhibitor-GANT61) studies, we investigated the effects of regulating the activating (Gli1 and Gli2) and the repressing (Gli3) members of the Gli transcription factor family in 2 human GIST lines (GIST-T1 and GIST882). We employed quantitative RT-PCR and immunoblot analyses, as well as cell viability and apoptosis assays to characterize the effects of Gli regulation within the lines. Results: In two GIST cell lines, KIT mRNA expression is inverse to Gli3 mRNA expression (KIT/Gli3 ratio: T1=0.38; 882=1.98). In the KIThiGli3lo line (882), Gli3 siRNA induces a 62%-fold increase in KIT mRNA, while Gli3 overexpression induces a 270%-fold decrease in KIT mRNA. The latter results in a 24% reduction in KIT protein levels. Inversely, Gli1 and Gli2 siRNA knockdowns result in 55% and 19% decreases in KIT mRNA expression, respectively. Treatment with GANT61, a Gli1/2 inhibitor, decreases KIT mRNA by 50%. Given that KIT is the target of anti-GIST therapies, we found that increasing KIT (i.e., Gli3 siRNA) slightly increases cell viability (5%) while decreasing KIT modestly decreases cell viability (Gli2 siRNA: 10%) and increases apoptosis (Gli3 overexpression: 20%). Finally, GANT61 treatment decreases the cell viability in a dose-dependent manner in both lines (IC50: T1=3.0 uM; 882=15.4 uM). Conclusion: Herein, we provide the first evidence that Hh signaling (namely Gli1/2/3) transcriptionally regulates mRNA expression of the KIT oncogene. Gli1 and Gli2 activate KIT expression while Gli3 represses KIT expression. Taken together, these transcription factors may serve as novel targets for treating GIST. Further investigations are underway in order to define the role of Hh regulation upon other GIST-related genes. | ||
| Advisor : | DR. ROGER TSIEN & DR. QUYEN NGUYEN | ||
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| Abstract Title : | Viral Transduction of Human Cancer Cell Lines with an Optimized Triple Modality Reporter for Quantifiable Tumor Imaging and Therapy Evaluation In Vivo | ||
| Abstract : | Development of noninvasive imaging technologies is leading to significant advancements in the field of molecular imaging. This research presents optimization of a triple modality reporter combining genes for a far-red fluorescent protein (E2-Crimson), luciferase enzyme (Luc2), and thymidine kinase (truncated herpes simplex virus I thymidine kinase). This schematic allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. This triple reporter improves on previous designs by replacing the shorter wavelength GFP or mRFP with E2-Crimson to increase penetration of fluorescence signal through mammalian tissues. Additionally, self-cleaving viral 2A sequences separate each component, ensuring equal stoichiometry of the reporter genes without requiring protein fusion. Cleavage between each protein gene product allows for proper protein folding, trafficking, and full activity of each modality. This triple reporter construct was cloned into a lentiviral plasmid from which a lentivirus was produced. Two human cancer cell lines were virally transduced with this construct, and the therapeutic response of the breast cancer cell line MDA231 to chemotherapeutic agents was successfully quantified in vivo utilizing this optimized triple modality reporter. | ||
| Advisor : | JILL LEUTGEB | ||
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| Abstract Title : | Interneurons robustly and consistently increase their firing rates during the minutes preceding behavioral seizures in the chronic model of temporal lobe epilepsy | ||
| Abstract : | Seizures reflect abnormal synchronized activity of a neuronal network, however, the activity dynamics preceding seizure onset are still poorly understood and algorithms for seizure prediction typically rely on local field potential recordings. Recent research has asked whether single-unit recordings might improve the predictability of seizures. Neuronal activity was found to change inconsistently before behavioral seizure onset such that an increase in variability was observed in the minutes before the onset. GABAergic interneurons integrate excitatory inputs from local and afferent networks. Such convergence of input may allow interneurons to be more sensitive to widespread neural synchrony that leads to seizures compared to principal cells. We asked whether interneurons might be more reliable predictors of seizures than principal cells. To record activity patterns of interneurons and principal cells before behavioral seizures, we used a kainate-acid model to induce male Wistar rats with temporal lobe epilepsy. We implanted animals that developed epilepsy (2 spontaneous seizures) with tetrode arrays and video monitored the rats (n=3) while local field potentials and single unit activity were recorded from CA1 and CA3. We identified a total of 20 behavioral seizures (rat 1: 4 seizures across 4 days, rat 2: 5 seizures across 3 days, rat 3: 11 seizures across 5 days). We assessed the activity patterns of hippocampal pyramidal cells (CA1, n=79 cells; CA3, n=34 cells) and interneurons (CA1, n=13 cells; CA3/DG, n=18 cells) during the 5 minutes preceding the behavioral seizure onset. First, we characterized baseline firing rates during 100 second epochs. We found that only ~10.8% of principal cells exhibited firing rates that deviated by more than 3 standard deviations from the baseline in the minute before the seizure onset, whereas ~50% of interneurons deviated from baseline up to 2 minutes before seizure onset. The average increase in firing rate of all interneurons was +60% compared to baseline during the 2 minutes before the seizure onset. Interneurons are thus a much better predictor of seizures during the minutes before the seizure onset. Because of the large effect size, even small numbers of recorded interneurons can reliably predict a seizure without false positive detection. Of the cells that were tracked across 2 seizures (n=14 principal cells and 8 interneurons in two rats), 31 of 32 showed a consistent change in firing rate preceding seizure onset. Together, these data suggest that knowing the change in firing from a previous seizure improves prediction, but that the gains from using interneurons for detection algorithms would be much more robust. | ||
| Advisor : | MARK H. TUSZYNSKI | ||
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| Abstract Title : | Utilizing automated screening methods to identify regeneration associated genes | ||
| Abstract : | Damage to the spinal cord results in debilitating effects, including loss of normal motor and sensory function; thus patients require lifelong support. Unlike the peripheral nervous system (PNS), the adult central nervous system (CNS) is characterized by its inability to regenerate after injury; however, in dorsal root ganglia neurons (DRGs) if a PNS injury precedes a CNS injury of the same neuron, the expression of regeneration associated genes can be utilized for both PNS and CNS axonal regeneration. Gene array analysis revealed hundreds of differentially expressed genes upon regeneration. Our hypothesis is that genes activated for PNS regeneration are also utilized for CNS regeneration of DRG neurons. In order to determine which genes are responsible for regeneration, an in vitro screening process is required to narrow down possible candidates. Through transfection of gene candidates (with a GFP reporter) into DRG neurons, we are investigating the effects of these candidates on neurite outgrowth; however, there are many caveats in measuring neurite outgrowth in response to ectopic gene expression. Ideally, upon transfection, the reporter and transgene would be fully expressed; unfortunately this is not the case. One solution to this problem would be inhibiting neurite outgrowth, which allows the transgene and reporter enough time to be expressed and be able to impact neurite outgrowth. In this study, we investigate the effects of nutrients deprivation and inhibitory substrates such as, myelin-associated glycoprotein expressing CHO cells and myelin extract coatings. Myelin extract coating produces over a 70% reduction in neurite outgrowth, making it an ideal inhibitory candidate for the in vitro screening. By identifying genes responsible for regeneration we can hopefully develop gene therapies to treat patients with spinal cord injury. | ||
| Advisor : | DONG-ER ZHANG | ||
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| Abstract Title : | Role of splicing factor SC35 in the whole blood system and in the development of MDS. | ||
| Abstract : | Acute myeloid leukemia (AML) is one of the deadliest forms of leukemia, a prominent cancer in the United States, with a median survival rate of three months. Myelodysplastic syndrome (MDS) is a common precursor to AML. Recently, it was found that mutations in the splicing factor SC35 is present in over 20% of MDS patients. However, current knowledge on the role of SC35 in the blood system is incomplete. We used conditional knock-out (KO) mice of SC35 to elucidate the role of SC35 in steady-state and stress hematopoiesis. Blood-specific knock-out (vav-cre(+) SC35f/f) was embryonic lethal between embryonic day 16 and 18. Although KO fetal liver cells showed increased apoptosis and G1 cell cycle arrest, adult Mx-cre(+) SC35f/f mice are unexpectedly alive after poly-IC injection. There is no phenotype in hetero mice in steady state. To evaluate the effect of haploinsufficiency under stress condition, mice heterozygous for SC35 in their blood system were injected weekly with the chemotherapeutic drug 5-flurouracil or underwent single sublethal irradiation (5 Gy). SC35 hetero mice survived significantly longer than wild type (WT) mice (median survival rate 20 days vs 39 days, p= 0.0243), while white cell blood count recovery was slower in hetero mice than WT mice. To get insights into the mechanisms, bone marrow transplantation (BMT) of SC35 overexpressing cells, BMT of Mx-cre(+) SC35 f/f or w/f cells followed by poly-IC injection, and RNA sequencing of lineage-depleted bone marrow cells overexpressing wild-type and mutant SC35 are underway. Through these in vitro and in vivo experiments we hope to discover more about SC35?s role in the blood system and in the development of MDS. | ||
| Advisor : | WILLIS LI | ||
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| Abstract Title : | Drug Screen for Heterochromatin Promoting Drugs in Drosophila Melanogaster | ||
| Abstract : | It was previously found that the JAK/STAT pathway directly plays a role in heterochromatin formation, a form of chromatin that is tightly compact and is essential for gene silencing, chromosome organization and preservation of genome integrity. Antagonizing heterochromatin formation induces tumor genesis in Drosophila melanogaster hematopoietic tumor model. Observing this, we intend to screen a small set of molecule compounds for their ability to promote heterochromatin formation in hopes of discovering a potential anti cancer drug. | ||
| Advisor : | PAUL A. INSEL | ||
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| Abstract Title : | G protein-coupled receptor expression and function in Pulmonary Artery Smooth Muscle Cells: Novel Targets in Pulmonary Arterial Hypertension | ||
| Abstract : | Pulmonary arterial hypertension (PAH) is characterized by increased proliferation of pulmonary artery smooth muscle cells (PASMC). The second messenger cAMP decreases PASMC proliferation, thus G protein-coupled receptors (GPCRs) that couple to Gαs/Gαi are targets for PAH. Using a TaqMan® GPCR array we found PASMC express >135 GPCRs, >50 of which regulate cAMP. GPCR expression correlated with function (Gαs-coupled GPCRs increased cAMP and inhibited PASMC proliferation). PAH-PASMC were associated with an increase in >41 GPCRs compared to control, the greatest of which was an orphan. We uncovered GPCRs that contribute to the physiology of PASMC that could be targets for PAH. | ||
| Advisor : | DR. ELSA CLELAND | ||
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| Abstract Title : | Does soil organic matter content promote invasive grasses in competition with native plants in the Anza-Borrego desert? | ||
| Abstract : | Exotic annual grasses are an increasing problem across western North American ecosystems. These invasive grasses often deter native plants from establishing and decrease species diversity specifically in arid and semi-arid regions. It has been shown that higher soil moisture favors exotics over native species, either through increased rainfall or an increase in soil organic matter content. Exotic grasses lay a thick layer of roots when they establish and this can increase the soil moisture creating a positive feedback for further grass invasion. We tested these ideas with a greenhouse experiment consisting of four native and four exotic desert species grown individually and in combination under varying watering frequency and root additions (high versus low). After a short growing season the exotic grasses outperformed natives under all conditions. However, when plants were grown together exotics favored low levels of root biomass while natives were favorable to high root levels. This suggests further soil dynamics could be involved where organic matter addition stimulates microbial activity, therefore altering the competitive dynamics between exotic and native plants. | ||
| Advisor : | DR. STEVEN BRIGGS | ||
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| Abstract Title : | Conservation of plant stress hormone responses in the unicellular alga, Scenedesmus dimorphus | ||
| Abstract : | Scenedesmus dimorphus is a microalga with potential use as a biofuel crop. Outdoor ponds are the most practical means to cultivate algae for biofuel production. S. dimorphus grown in ponds is susceptible to infection and disease caused by primitive fungi including Cryptomycota. Infection can kill virtually all of the algal cells in a pond within days. Disease phenotypes include flocculation, loss of chlorophyll, and cell death. Infection can be observed in the laboratory using three different pathogens isolated from naturally-infected algal cultivation ponds in New Mexico; they are designated FD01, FD61, and FD95; only FD01 has been identified at the species level (Amoeboaphelidium protococcarum). Infection in vitro results in culture death 3-4 days post-infection. Green algae gave rise to higher plants, thus we hypothesize that S. dimorphus possesses the evolutionary origins of the higher plant immune system. If this turns out to be true then the extensive knowledge of plant immunity can be used to guide breeding efforts to develop disease-resistant algal strains. We are testing whether S. dimorphus responds predictably to stress hormones that are known to modulate immunity in higher plants but which are not known to occur in algae. Plant receptors for pathogen-produced molecules activate biosynthesis of the stress hormones salicylic acid (SA) and jasmonic acid (JA) and consequent signaling triggered by the hormones; this induces defense against biotrophic pathogens and necrotrophic pathogens, respectively. To investigate the existence of these hormone response pathways in S. dimorphus we treated cultures with BTH (an SA analog) or me-JA and measured culture appearance, growth, and time to death following infection. Changes in algal-pathogen interactions caused by the hormones was similar to their effects on plants indicating conservation of the hormone response pathways and strongly implying that algae have an immune system related to that of higher plants. | ||
| Advisor : | DR. TRACY JOHNSON | ||
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| Abstract Title : | Investigating the Regulation of GCR1 during Glucose Starvation in the Yeast Saccharomyces Cerevisiae. | ||
| Abstract : | In a dynamic nutrient environment, cells commit to metabolic pathways to utilize the nutrient that is presently available. This commitment is determined at the level of gene regulation, thus studying cells in response to nutrient flux can lend insight into gene regulation mechanisms. Specifically, cells of the yeast Saccharomyces cerevisiae will undergo a metabolic shift from Glycolysis to Respiration in the absence of the fermentable carbon source glucose. This phenomenon is known as a diauxic shift and is conserved among all eukaryotic and aerobic prokaryote species. A gene tightly regulated during this process, GCR1, encodes a transcription factor also known to regulate about 75% of all Pol II transcripts in the cell. For these reasons information about GCR1 regulation will not only expand our understanding in the field of RNA biology, but will also contribute to fields including cancer biology, wherein dynamic gene regulation is eminent. Recent work by Claggett et. al. demonstrates that Gcr1 protein levels are regulated in a manner that is glucose-dependent. Unexpectedly, GCR1 generates multiple protein isoforms when cells are grown in a glucose-rich environment. Remarkably, exclusive expression of each isoform causes overlapping and distinct effects on genome-wide RNA expression, suggesting that changing the ratio of Gcr1 isoforms provides a means by which the cell can elicit highly specific changes in gene expression. Consistent with this, Gcr1 isoforms are differentially regulated in response to glucose depletion, supporting a model whereby the cell alters the Gcr1 isoform ratio to enable robust metabolic adjustment when exposed to a fluctuating glucose environment. | ||
| Advisor : | DR. AMY KIGER | ||
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| Abstract Title : | Sbf complex members regulate the toll-signaling pathway in Drosophila Melanogaster | ||
| Abstract : | In Drosophila Melanogaster (fruit fly) the toll-signaling pathway is well characterized. The toll receptor is activated by a gram positive and fungal (Drosophila toll) infection. Initially the Spätzle ligand binds the toll receptor, and subsequently the toll receptor is endocytosed. Both the binding and the endocytosis are required for an appropriate innate immune response. Thus, understanding the signaling pathway and the trafficking of toll is important for understanding immunity. In the fruit fly, Sbf is catalytically inactive and is thereby classified as a MTM pseudo-phosphatase. Sbf interacts with the class-2-kinase Pi3K68D. This interaction allows for the recruitment of mtm. This promotes turnover of a Pi3P pool that is essential for endosomal trafficking. Rab21 is associated with activation of the Pi3P endosomes. While Sbf is a guanine exchange factor (GEF) for Rab21, Rab21 is a small GTPase. Sbf coordinates Pi3P and Rab21 regulation, along with the endosomal pathway. While studying Sbf, Rab21, and Pi3K68D interactions, our lab found that loss of function of Rab21 causes the formation of melanotic masses. Along with Rab 21 loss of function, over-expression of the toll-signaling pathway through Toll10B:GFP, a constitutively active mutant, causes melanotic masses. Given the similar phenotype between Rab21 LoF, the trafficking of the toll receptor and transduction of the signal were examined in the fruit fly. Preliminary results suggested that the toll pathway could be improperly regulated in larvae depleted of Sbf complex members. Thus, I hypothesized whether over-expression or loss of function of Pi3K68D, Sbf, or Rab21, while co-expressed with a hyper-activated toll-signaling pathway, would attenuate the signaling pathway and impact the production of melanotic masses. Here I will present evidence that indeed, Sbf complex members regulate toll. | ||
| Advisor : | DR. MILTON H SAIER | ||
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| Abstract Title : | Identifying Novel Transport Proteins Promoting E. coli Pathogenesis | ||
| Abstract : | Eight prominent types of E. coli pathovars have been identified based on varying mechanisms of infection. These strains cause urinary tract infections, gastroenteritis, pyelonephritis, diarrhoea, haemorrhagic colitis and neonatal meningitis. The general paradigm of the pathogenic mechanism associated with E. coli virulence involves (1) adhesion, (2) protein injection into host cells, (3) subversion of signaling mechanisms and (4) colonization leading to impaired immune responses, membrane potential disruption, and cytoskeletal manipulation. In my study, seven well-characterized pathovars were examined with respect to their transport protein compositions and compared with the non-pathogenic E. coli strain K12 to identify novel transport proteins related to pathogenesis. Pathovar 55989, an enteroaggregative bacterium, possesses a complete and newly characterized type VI protein secretion system for injecting effector proteins into host cells. Other protein secretion systems characterized the different pathovars. Autotransporters, siderophore receptors and transporters, and other outer membrane proteins lacking in K12 were distinctively identified in the various pathogens. These results provide clues as to transport systems relevant to various types of E. coli pathogenesis that can be exploited in future studies. | ||
| Advisor : | JENS LYKKE-ANDERSEN | ||
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| Abstract Title : | Identification of 3'UTR Elements that Inhibit Nonsense-Mediated Decay | ||
| Abstract : | Nonsense-Mediated Decay (NMD) pathway is a quality control mechanism that targets mRNA containing premature termination codons (PTCs). This surveillance pathway rids cells of mRNAs that if otherwise translated would produce potentially deleterious truncated proteins. The size of the 3? Untranslated Region (UTR) constitutes an important NMD feature. Indeed, long 3?UTRs place the PolyA Binding Protein (PABP) away from terminating ribosomes and therefore favor the assembly of the NMD complex containing UPF1, UPF2, and UPF3 which then targets mRNA for degradation. Intriguingly, microarray analyses demonstrate that numerous mRNAs containing long 3?UTRs, which will be sufficient to trigger NMD, actually evade the NMD pathway. The question we are addressing is how those mRNAs with long 3? UTR evade the NMD pathway. Our hypothesis is that mRNAs with long 3?UTR contain cis elements that allow them to evade NMD. The focus of our investigation is to identify those cis elements. To begin, we selected candidate mRNAs that have a long 3?UTR and that evade NMD. We confirmed the 3? UTR of those genes was actually resistant to NMD by performing decay assay using β-Globin WT reporter mRNA containing our candidates 3?UTR. Preliminary evidence suggests that the first 200 nucleotides of the 3?UTR might contain those cis elements responsible for NMD resistance. Currently, we are testing whether the first 200 nucleotides of other candidate 3?UTRs contain such elements that allow them to evade NMD. We have cloned the first 200 nucleotides of each of the long 3?UTRs into the NMD reporter β-Globin WT GFP and we are currently testing those constructs in decay assay. This study could uncover a common mechanism by which mRNAs with long 3?UTR have evolved to evade NMD. Further studies will be necessary to describe the mechanism by which those cis elements inhibit NMD and to identify potential trans factors that bind those cis elements. Understanding how mRNA with long 3?UTR evade the NMD pathway is crucial to better understand the role of the NMD pathway in gene regulation. | ||
| Advisor : | DR. JANE C. BURNS | ||
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| Abstract Title : | Whole blood microRNA profiles in adenovirus-infected children | ||
| Abstract : | Adenovirus causes acute self-limited illnesses including respiratory infections, gastroenteritis and conjunctivitis in children. microRNAs (miRs) are small non-coding RNAs that post-transcriptionally regulate genes associated with immune response and inflammation. A miR profile was created to understand the role of miRs in modulating the host response to adenovirus infection. Total RNA extracted from whole blood of 6 acute adenovirus-infected children and 12 healthy controls were analyzed by small RNA sequencing. The top 5 differentially expressed miRs (miRs-3614-5p, -16, -15a, 126* and -126 (nominal p-value < 0.05)) were elevated in adenovirus-infected children. These miRs were then tested by qRT-PCR in the whole blood of an independent cohort of children infected with adenovirus (n=24) and healthy controls (n=24). miR-3614-5p was validated as being significantly differentially expressed and was subjected to pathway analysis to reveal predicted gene targets. The top 5 predicted gene targets were cross referenced with previously analyzed Lymphochip microarray data sets, which revealed POU2F1 to be down-regulated in adenovirus-infected patients. POU2F1, however, was not validated in an independent cohort by qRT-PCR. Sequencing of small RNA allowed discovery of miRs that may participate in host defense of adenovirus infection. miR-3614-5p was shown to be differentially expressed. POU2F1 is a predicted target of miR-3614-5p, which is an important transcription factor that increases inflammation by regulating iNOS, E-selectin and VCAM- 1in the host as a mechanism to limit adenovirus replication. Failure to demonstrate down-regulation of the transcription factor POU2F1 may be due to timing of sample collection from these patients. | ||
| Advisor : | MARTIN KAGNOFF | ||
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| Abstract Title : | Role of epithelial microRNAs during rotavirus infection in humans | ||
| Abstract : | MicroRNAs play a key role in mRNA regulation by RNA silencing within the cell, but several human encoded miRNAs have also been shown to inhibit key proteins involved in viral replication. No studies to date have been published about the role of microRNAS, cellular or viral, during rotavirus infection. Knockdown of Dicer and Drosha, the enzymes responsible for all microRNA processing and solely cellular microRNA processing, respectively, led to an increase in rotavirus replication and IFN-B production. This suggests the existence of human microRNAs that target rotavirus. Understanding the role of miRNAs in rotavirus pathogenicity may aid in the development of therapeutic miRNAs or an improved vaccine construct. | ||
| Advisor : | GERY SCHULTEIS | ||
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| Abstract Title : | Reduction of Brain Reward Threshold Elevation During Acute Opioid Withdrawal by Decreasing Noradrenergic Activity in the Central Amygdala | ||
| Abstract : | Although abuse of drugs like opioids may begin with positive reinforcement (e.g. taking a drug to experience pleasurable/euphoric effects), this can transition to negative reinforcement as neuronal changes result in adaptation to the recurring presence of neuropharmacologic agents, such that individuals may experience drug-opposite effects (e.g. dysphoria) during abstinence/withdrawal from the drug. The central amygdala (CeA) has recently been identified as a region within the limbic system that undergoes rapid neuroadaptation upon acute opioid use, with increased levels of corticotropin releasing factor (CRF) identified as contributing to opioid withdrawal dysphoria. CRF is known to interact with Norepenephrine (NE) throughout the brain, including the CeA, and this study looks at the role of NE in the CeA during withdrawal from acute opioid (morphine) intoxication in a rat model. Using the Intracranial Self-Stimulation (ICSS) technique, rats were trained to turn a wheel in order to receive discrete electrical pulses to their reward pathways. After achieving a stable baseline with ICSS reward thresholds, rats were given an acute injection of morphine, followed by intracranial infusion of one of two antiadrenergic agents 3.75 hours later, and the opioid receptor blocker naloxone given 4 hours post-morphine and right before an ICSS session. The antiadrenergic agents used were the Alpha-1 receptor blocker Prazosin, and the Alpha-2 Agonist UK 14,304 which reduces NE release. The results indicate that both prazosin and UK 14,304 significantly reduced, but did not completely reverse, the dysphoria-like elevations in ICSS thresholds precipitated by naloxone during acute morphine withdrawal. These results indicate that NE, in addition to CRF, in the CeA may be involved with states of dysphoria seen in the withdrawal phase of opioid use. | ||