<p> ABSTRACT The nervous system comprises more diverse and intricately specialized cell types than any other tissue in the body. Understanding the developmental mechanisms that generate cellular diversity in the nervous system is a major challenge in neuroscience. The nematode <i>C. elegans</i> offers the opportunity to study neuronal development at the molecular level with extraordinary resolution.</p><p> My dissertation focuses on the elucidation of genetic mechanisms required for the proper development of the chemosensory BAG neurons, which are specialized for detecting the respiratory gas carbon dioxide (CO₂). Analogs of these neurons play diverse roles in animals from different phyla. CO₂-sensing neurons in the mammalian brainstem are critical regulators of the respiratory motor program, and their dysfunction has been linked to fatal apneas such as Sudden Infant Death Syndrome. In nematodes, CO₂-sensing neurons mediate an avoidance behavior, but their ethological function was not known.</p><p> In my initial studies of BAG neuron development, I demonstrated that a conserved ETS-family transcription factor directly regulates genes required for CO₂-sensing, including the receptor-type guanylate cyclase, GCY-9, which likely functions as a CO₂ receptor. To uncover other genes that function together with <i>ets-5,</i> I carried out a large-scale chemical mutagenesis screen for mutants with improper BAG neuron differentiation. From this screen I identified two new genes required for BAG neuron development: the Pax6 homolog <i>vab-3</i> and the p38 Mitogen-Activated Protein (MAP) kinase <i>pmk-3</i>.</p><p> VAB-3 likely acts during embryonic development to pattern the expression of ETS-5 in head neurons of <i>C. elegans</i>. In loss of function <i> vab-3</i> mutants, ETS-5 protein is misexpressed in hypodermal cells and a motor neuron, in addition to its expression in BAG. VAB-3 likely represses transcription of ETS-5 in some lineages, such as those that give rise to hypodermal cells.</p><p> I next demonstrated that the p38 MAPK PMK-3 functions in a Toll-like receptor (TLR) signaling pathway. This discovery revealed an unexpected role for TLR signaling in neuronal differentiation. Because TLR signaling was known to be required for behavioral responses to microbes, I tested whether BAG neurons were required for pathogen avoidance. I found that this was the case and propose that TLR signaling functions in pathogen avoidance by promoting the development and function of chemosensory neurons that surveil the metabolic activity of environmental microbes.</p><p> Because ETS-5, VAB-3 and TOL-1 are members of gene families that are conserved between nematodes and vertebrates, a similar mechanism might act in the specification and differentiation of CO₂-sensing neurons in other phyla.</p>

Two processes influence the success of mitosis, the process by which eukaryotic cells divide their replicated genome into two new daughter cells. First, the cell must build a bipolar array of microtubules called the mitotic spindle, which is accomplished by microtubule sliding by the Kinesin-5 Eg5; second, a subset of those microtubules, called kinetochore-microtubules (K-MTs), must attach correctly to each chromosome. The stability of these K-MT attachments to the chromosome influences whether chromosomes will segregate correctly in anaphase, but an influence of K-MT stability on the ability of cells to build or maintain bipolar spindles had not been recognized. Here we show that human cell lines with high K-MT stability are better able to maintain bipolar spindles when Eg5 is inhibited compared to cell lines with low K-MT stability. Artificially stabilizing K-MTs promoted bipolarity maintenance, while destabilizing K-MTs undermined the maintenance of bipolarity. In addition, inhibition of the mitotic master-regulator cyclin-dependent kinase-1 (CDK-1) in the G2 phase of the cell cycle stabilized K-MTs after CDK-1 inhibition was relieved, which promoted bipolar spindle maintenance without Eg5 but undermined mitotic fidelity. These results indicate that the dynamics of K-MTs play a previously unappreciated role in determining the overall geometry of the mitotic spindle.

Myocardial infarction (MI) causes irreversible tissue damage, leading to heart failure. Our laboratory found canonical Wnt signaling and the Wnt10b ligand are strongly induced in mouse and human hearts after MI. Wnt10b regulates cell fate in various organs, yet its role in the heart is unknown. To investigate the effects of Wnt10b gain-of-function on cardiac repair mechanisms and functional outcomes after injury, we generated αMHC-Wnt10b transgenic (TG) mouse line that overexpresses Wnt10b in adult cardiomyocytes. Following acute myocardial injury, the TG mice displayed improved recovery of cardiac function, accompanied by enhanced neovascularization and attenuated scar fibrosis. Wnt10b stimulated expression of vascular endothelial growth factor receptor 2 in endothelial cells and angiopoietin-1 in vascular smooth muscle cells through nuclear factor-κB activation to promote stabilized blood vessel formation. Wnt10b also reduced the number of myofibroblasts to mitigate fibrosis. My findings may lead to novel strategies to optimize the inherent repair capacity of the heart and prevent the onset of heart failure.

Diabetic Retinopathy (DR) is a leading cause of blindness worldwide, and its prevalence is growing. Current therapies for DR address only the later stages of the disease, are invasive and are of limited effectiveness. Retinal pericyte death is an early pathologic
feature of DR. Though it has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown. A novel pro-apoptotic pathway initiated by the interaction between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the E3 ubiquitin ligase, seven in absentia homolog 1 (Siah1), was identified to play a significant role in human retinal pericyte apoptosis. Inhibition of the GAPDH/Siah1 pro-apoptotic complex blocks diabetes-induced pericyte apoptosis, widely considered a hallmark feature of DR.

The Wnt signaling pathway plays essential roles in a wide variety of biological processes including early animal development, cell fate determination, cell proliferation, organogenesis, and stem-cell renewal. Deregulation of the Wnt pathway can lead to human disease (e.g. developmental defects and cancers). Our lab had previously demonstrated that X-linked Inhibitor of Apoptosis (XIAP) is required for Wnt signaling via a mechanism that involves XIAP-facilitated dissociation of Gro/TLE from TCF/Lef. We had shown that XIAP is recruited onto TCF/Lef complexes upon Wnt pathway activation and ubiquitinates Gro/TLE-bound TCF/Lef. Ubiquitinated Gro/TLE has decreased affinity for TCF/Lef, allowing beta-catenin to bind. Inhibition of GSK3 by lithium causes XIAP to be recruited to TCF/Lef, although the precise mechanism was unknown. We found that XIAP is phosphorylated by GSK3, a known component of the Wnt pathway. Using mass spectrometry analysis with purified proteins, we identified two GSK3 phosphorylation sites on XIAP that are strongly phosphorylated. Mutational analysis of these two sites indicate that they are required by XIAP to fully activate Wnt signaling, as assessed by reporter assays in cultured mammalian cells and axis duplication assays in Xenopus embryos. Using purified proteins, we found that the XIAP phosphomutants have similar ubiquitination activity as wild-type XIAP. In cultured mammalian cells, however, the XIAP phosphomutants have a markedly decreased capacity to ubiquitinate Gro/TLE. We also showed that the phosphomutants also have a decreased affinity for Gro/TLE. We propose a model in which phosphorylation is necessary for the interaction between XIAP and Gro/TLE to activate Wnt signaling. GSK3 phosphorylates XIAP at T180 and S239 to facilitate its binding to Gro/TLE. XIAP can then ubiquitinate and remove Gro/TLE for subsequent binding of beta-catenin to form the transcriptionally active complex.

<p> Polycystic Ovarian Syndrome (PCOS) is a metabolic and endocrine disorder that is the most common cause of infertility. PCOS can manifest itself as a long and short term disability and is characterized by insulin resistance (IR), hyperandrogenism, anovulation, hyperinsulinaemia and polycystic ovaries. Our lack of understanding of this disorder and its long term effects has complicated the treatment of the disorder; yet, it is clear that PCOS involves the intricate interaction between genetics, environments and behaviors. To study this disease, scientists have used various animal models. Since the <i> Drosophila</i> model for PCOS has only been postulated,in this work, we determined whether starvation along with the addition of steroid hormones would induce a PCOS-like disorder in <i>D. melanogaster</i> after 24 hour exposure. </p><p> In women with PCOS, testosterone levels and the expression of the androgen receptor are elevated. In fruit flies, ecdysone (E) and its &ldquo;active&rdquo; form, 20-hydroxyecdysone (20E), are homologous to the human testosterone and 20-hydroxytestosterone, respectively. This hormone is required for circadian cycles, molting, and maturation in insects. More specifically, this hormone is also located in ovarian tissue and aids in follicular development. The receptor for ecdysone is the ecdysone receptor (EcR). In this work, we examined the expression of the ecdysone receptor (EcR) upon starvation for up to 24 hours by immunofluorescence microcopy. Using qRT-PCR, we determined the levels of expression of genes usually associated with inflammation. Ovarian dysfunction was examined by measuring the fecundity of the females. Starvation increases the expression of the EcR and pro-inflammatory gene expression and decreases fecundity, suggesting that <i>Drosophila melanogaster</i> is a potentially useful model organism in the study of PCOS.</p>

The spliceosome is a dynamic macromolecular machine composed of five different small nuclear ribonucleoproteins (snRNPs), the Nineteen Complex (NTC), RNA helicases, and other proteins that catalyze the removal of introns from pre-mRNA. Although the spliceosome has been extensively studied, there are still many unanswered questions regarding the molecular mechanisms of pre-mRNA splicing. In this work we used the Schizosaccharomyces pombe model system to characterize the structure and function of the spliceosome. By examining the in vitro RNA ability of S. pombe Cdc5, a core component of the NTC, we were able to propose a mechanism where protein components of the NTC can bind and stabilize RNA within the active site of the spliceosome. We also used an RNA helicase mutant, S. pombe prp16-1, to isolate a second step splicing complex. This work has provided insight into the general mechanisms on how the spliceosome functions to process pre-mRNA.

No description available.

No description available.

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