Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-04, Section: B, page: 2084. Adviser: David P. Toczyski.

<p> Copepod nauplii are important members of the marine planktonic community, and they can be the most abundant component of the microzooplankton. Despite the importance of copepod early life history stages to food web dynamics and carbon flux in the sea, there is a paucity of information about their ecology due to challenges in identifying nauplii to species, and in sampling them quantitatively. I report here on the development and optimization of a new molecular method that uses quantitative PCR (qPCR) to identify and estimate the abundances of nauplii of a common coastal copepod, <i>Parvocalanus crassirostris,</i> in field samples. The following experiments were performed towards this goal: I surveyed the genetic diversity of copepods in the study region, optimized sample treatment for qPCR, developed a size fractionation protocol to separate life stages of the target species, quantified the mitochondrial cytochrome C oxidase subunit I (mtCOI) gene copies in each <i> P. crassirostris</i> life stage, tested the effect of food levels on mtCOI copy number in nauplii, and compared direct counts to qPCR estimates of the target species to validate the qPCR method. The number of mtCOI gene copies in each developmental stage of this species was found to increase by &sim;1.5 orders of magnitude from early nauplii to adult. Food level experiments suggested that mtCOI copy number may be influenced by feeding environment in late naupliar stages. In validation experiments, qPCR estimates were 68 to 130% of the number estimated from direct counts. Both methods had a coefficient of variation of approximately 16%, indicating similar precision across methods. As a field test of the method, daily samples were collected in southern Kane&lsquo;ohe Bay and used to quantify the density <i>P. crassirostris</i> nauplii over a 13-day period in the summer of 2011. The average density of <i> P. crassirostris</i> nauplii in developmental stages NII - NV was found to be 1.5&times;10³ individuals m^-3 over the 13-day period. The qPCR-based method developed here will enable future studies on naupliar ecology in the field, including investigation of food web, population, and community dynamics.</p>

<p> A complex molecular network is put into place at specific phases of the cell cycle to prevent unscheduled cell division that could result in malignant cell growth. Emerging evidence shows that still uncharacterized proteins play crucial functions at those cell cycle transition points. Nuclear protein Msp58 and EDD E3 ubiquitin ligase have been implicated in different aspects of cell proliferation and reported to be abnormally expressed in numerous types of cancers. The molecular mechanisms underlying Msp58 and EDD functions, however, are not well understood. The work presented here shows that Msp58 and EDD form a stable protein complex that regulates cell viability and proliferation. Interestingly, knockdown of EDD by RNA interference leads to a significant accumulation of Msp58 protein, which suggests that EDD serves as a negative regulator of Msp58. In addition, our in vivo ubiquitination assays and analyses of various cell lines treated with translational and proteasomal inhibitors demonstrate that Msp58 is regulated post-translationally by the ubiquitin-proteasome pathway. These results imply that EDD ligase activity is involved in this regulatory process. Using flow cytometry analyses and biochemical characterization of Msp58 and/or EDD depleted cells, we show that the Msp58-EDD complex plays important roles in cell cycle progression via the control of cyclin gene expression. In particular, silencing Msp58 and/or EDD alters the protein levels of cyclins B, D and E. Taken together, our data suggest that a set of the biological roles attributed to Msp58 and EDD may be executed in the context of the complex that they form, thereby revealing a novel molecular mechanism for these two proteins to accomplish their functions.</p>

<p> Through the use of a novel immunodetection assay, our lab identified a series of mutants that internally accumulate the precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY), a phenotype that suggests a defect at the late e&barbelow;n&barbelow;dosome to v&barbelow;acuole (<i> ENV</i>) interface of the biosynthetic pathway. This study focuses on two of the novel genes identified: <i>ENV10</i> and <i>ENV11 </i> and is the first to establish the cellular localization of Env10p in the endoplasmic reticulum. Assays of vacuole and lipid droplet morphology, as well as growth characterization under various stressors demonstrate Env10p has a role in vacuolar protein trafficking and endomembrane system integrity and may operate in a parallel or compensatory manner to Env9p. This study also confirms that Env11p localizes to the nucleus in a saturable fashion where it may be involved in transcriptional regulation of genes involved in vacuole events in conjunction with Vid22p and Tbf1p.</p>

<p> <i>Ustilago maydis</i> is a basidiomycete fungus known to cause tumor formation in maize. It exhibits two distinct forms: yeast-like and filamentous. Both forms exhibit cell polarity, an inherent process observed in all cell types. In the Ascomycota, Tea1 homologs are involved in cell morphogenesis. Tea1 homologs had not been identified in the Basidiomycota. In this study, I have characterized the homolog of the <i>Schizosaccharomyces pombe tea1</i> gene in <i>U. maydis</i>. I have determined the &Delta;<i> tea1</i> null phenotype through a gene deletion strategy, established the subcellular localization of Tea1 and Tea4 using GFP fusions, and determined a Tea1 interaction with itself using a yeast two-hybrid approach. These studies provide support for a major role of Tea1 in cell morphogenesis, in maintaining an axis of polarized growth, and in cell polarity in <i>U. maydis</i>. </p>

<p> Spermatogenesis is governed by vitamin A, as shown by vitamin A deficient (VAD) testes, which lack advanced germ cells. Vitamin A signaling is mediated by retinoid receptors. There are two families of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), each with alpha, beta and gamma subtypes. Retinoic acid receptor alpha (RARA), plays a significant role in the testis such that <i>Rara</i>-null males are infertile because of severe germ cell loss. </p><p> Striking similarities of the testicular phenotypes are detected between <i> Rara</i>-null and VAD mice: severely degenerated testes, lack of germ cells, sloughing of mature spermatids, and infertility. To discern the molecular function of RARA in germ cells, <i>Rara</i> was conditionally deleted using stimulated by retinoic acid 8 (STRA8)-iCRE. With RARA function disabled in germ cells, morphological abnormalities detected in the testes included lack of germ cell organization, lack of lumen, sloughing cells, and vacuolization. Not surprisingly, germ-cell specific <i>Rara</i> conditional knockout mice (cKO) had a dramatic reduction in epididymal sperm number. Further analysis of cKO testes demonstrated decreased spermatogonial proliferation and differentiation, while meiotic defects such as reduced synapsis, synaptonemal fragmentation, and unrepaired double strand breaks were increased. Furthermore, functional spermatogonial transplantation assays pointed to the possibility that RARA regulates spermatogonial stem cell colonization and proliferation, as shown by the reduction of donor-derived spermatogenesis from the cKO donor germ cells. The lack of RARA in the testes clearly shows quantifiable deficiencies during spermatogonial proliferation, differentiation, and meiosis. </p><p> Microarray gene expression studies of mRNAs from the enriched germ cells from wild type and cKO mice provided molecular evidence that RARA regulates spermatogonial differentiation at postnatal day 4 (P4) and meiosis at P8. Cell differentiation, cell adhesion, cell migration, and other pathways related to the early steps of spermatogonial differentiation were found to be functional categories significant in germ cells from P4. These were very distinct from synapsis, synaptonemal complex formation, and crossover formation related to meiosis, which were functional categories significant in germ cells from P8. In conjunction with phenotypic abnormalities, we provide gene expression evidence that RARA mediates retinoic acid function during spermatogonial proliferation, differentiation, and meiosis.</p>

<p> Remodeling of the mouse mammary gland is a highly coordinated process that occurs after the removal of suckling pups from the mother. Involution, or shrinking of the mammary gland, after removal of the pups has been linked to apoptotic events within the mouse mammary tissue during forced weaning. Several transcription factors are hypothesized to be involved in this process. A transcription factor known as GATA-3, which was first identified in the thymus, is also important for maintenance of various tissue types within the mouse mammary gland; its loss leads to epithelial cell detachment and eventual death. Another transcription factor known as fetal zinc liver finger protein 1, or Fliz1, has been found to regulate GATA-3 in T-cells. This interaction had not been elucidated during involution in mouse mammary tissue. I hypothesized that Fliz1 is expressed at heightened levels during mouse mammary gland involution following forced weaning of pups, and that this expression correlates with a decrease in GATA-3 levels, with increased expression of the pro-apoptotic protein BAD. Using qRT-PCR, immunoblotting and immunohistochemistry I have shown that Fliz1 is indeed expressed in involuting mouse mammary gland tissue as well as several other tissue types. However, levels of Fliz1 remain fairly constant during involution. The findings also show that Cathepsin L, a known apoptotic marker for mammary gland involution, is substantially up-regulated during the process of mammary gland involution in the mouse. The study also revealed that GATA-3 levels as hypothesized decrease substantially during the process of mouse mammary gland involution, indicating that GATA-3 is required for maintenance of the mouse mammary gland.</p>

<p> In the presence of arachidonic acid (AA), <i>Saccharomyces cerevisiae </i> produces prostaglandin E₂ (PGE₂). <i> S. cerevisiae</i> and its metabolites may be consumed in products manufactured using the yeast (e.g. beer). Neutrophils are immune cells present in the gastrointestinal (GI) tract during inflammation. As a lipid-signaling molecule, PGE₂ can potentially modify neutrophil functions and exacerbate pre-existing inflammation. As neutrophil migration is a hallmark of inflammation, we investigated the impact of PGE₂ on neutrophil chemotaxis. Chemotaxis assays were performed on neutrophils isolated from human whole blood using the chemotactic agents f-Met-Leu-Phe (fMLP) or interleukin-8 (IL-8). Neutrophil chemotaxis was concentration dependent as it was enhanced 3.5-fold at low concentrations of PGE₂ (0.1 nM-10 nM) and reduced 3.0-fold at higher concentrations of PGE₂ (100 nM).</p><p> The biochemical pathway utilized by <i>S. cerevisiae</i> to produce PGE₂ is unknown. Identifying enzymes that metabolize AA may direct approaches to reduce the impact that yeast PGE₂ may have on neutrophils. <i>S. cerevisiae</i> does not have genes homologous to those involved in mammalian AA metabolism. We employed RNAseq transcriptome sequencing to study the lipid biosynthetic pathway in <i>S. cerevisiae </i> and observed 1248 genes upregulated in yeast that were cultured in the presence of AA relative to yeast that were cultured without AA. Notably, genes that mediate beta-oxidation of fatty acids (<i>Pot1, Pox1, Faa1 and Faa2</i>) were upregulated up to 2.3-fold.</p><p> The results demonstrate that low concentrations of PGE₂ enhance neutrophil chemotaxis that is mediated by fMLP or IL-8, suggesting that PGE₂ may aid in recruiting neutrophils from regions that are distant to a site of inflammation. Once a higher concentration of PGE₂ is encountered by neutrophils, neutrophils may halt their migration and engage effector functions such as phagocytosis and superoxide production. Increased expression of genes involved with fatty acid metabolism points to enzymes that may utilize AA to produce PGE₂ in <i>S. cerevisiae</i>. Experiments testing PGE₂ levels in knock-out strains of yeast will identify genes involved in PGE₂ production. Results of this study have implications to reduce potential off-target effects caused by yeast PGE₂ in consumables.</p>

<p> Small non-coding RNAs (sRNAs) are being increasingly recognized as critical regulators of a wide variety of processes in bacteria. To investigate the contribution of unknown sRNAs to virulence gene regulation in <i>Vibrio cholerae,</i> we undertook a screen to identify previously uncharacterized sRNAs under the control of the major virulence gene activator in <i> V. cholerae,</i> ToxT. Using a combination of direct sRNA cloning and sequencing together with a genome-wide ToxT <i>in vitro</i> binding assay, we identified 18 putative ToxT-regulated sRNAs. Two of these ToxT regulated sRNAs were located within the <i>Vibrio</i> Pathogenicity Island-1 (VPI-1), the genetic element that encodes ToxT and the Toxin Co-regulated Pilus (TCP). We verified regulation of these sRNAs by ToxT and showed that deletion of one of them, now designated <i>tarB</i>, caused a variable colonization phenotype when competed against the parental strain in an infant mouse model of <i>V. cholerae</i> infection. Infections progressing for 18 hours or less showed the &Delta;<i>tarB</i> strain was out-competed by the wild type strain, while those carried out longer, showed &Delta;<i> tarB</i> out-competing the wild type. Additionally, if inoculated from a resource poor environment the &Delta;<i>tarB</i> strain also showed decreased colonization relative to wild type. Using a bioinformatic approach, we identified that <i>tarB</i>-mediated regulation of the gene <i> tcpF</i> was primarily responsible for the <i>tarB</i> mutant's <i> in vivo</i> colonization phenotype. Further investigation of genes regulated by <i>tarB</i> using genome-wide transcriptional profiling of a <i> tarB</i> over-expressing strain revealed that <i>tarB</i> also directly regulates genes involved in iron and amino acid uptake. We determined that <i>tarB</i> has a repressive effect on many genes within the VPI-1, but has an activating effect on <i>tcpP/tcpH,</i> encoding regulators upstream of ToxT. Taken together, the data suggest that <i> tarB</i> plays an important role in regulating virulence and metabolic genes early after <i>V. cholerae</i> infection, but that this repressive effect on virulence genes later in infection may lead to reduced replication <i> in vivo.</i></p>

<p> Blast injury-induced neurotrauma (BINT) is steadily increasing in prevalence due to escalated terror activity and constitutes the signature injury associated with current military conflicts. BINT produces significant neurological deficiencies and there is a growing concern that the injury may produce long-term consequences that affect the resilience and the performance of soldiers. One of the potential consequences is an increased susceptibility to Parkinson's disease (PD). A vital goal aimed at curtailing the post-deployment long-term consequences of blast injury-induced neurotrauma is to further our knowledge of pathogenic mechanisms responsible for the escalation of post injury diseases. <i> The purpose of this project is to investigate the molecular mechanism underlying the susceptibility of PD in post-blast rats.</i> We have identified acrolein, a highly reactive aldehyde that persists days to weeks following brain-injury and perpetuates oxidative insult, as a potential therapeutic target to curtail chemically mediated damage, a common feature of BINT and PD. <b>Our hypothesis is that acrolein is a key pathological factor linking BINT and the development of PD in our rat model.</b></p>