http://hdl.handle.net/10125/20029 Wed, 22 May 2013 08:26:39 GMT 2013-05-22T08:26:39Z http://hdl.handle.net/10125/20464 Thesis (Ph.D.)--University of Hawaii at Manoa, 2008.; Over the past thirty years, coral reefs have experienced extensive degradation worldwide. One etiology for this global degradation is a syndrome known as coral bleaching, a process whereby corals lose their algal symbionts or the symbiont's photosynthetic pigments degrade. Coral bleaching can be induced by diverse factors including high temperature, intense light or high-energy light, cold temperature, salinity changes and bacterial infection. There are very few models of coral bleaching that incorporate cellular and molecular mechanisms that have been proposed or tested. The overall objective of my dissertation is to elucidate the major cellular aspects of bleaching in coral as a result of high temperature, high light, the compound exposure of both heat stress and light stress, prolonged darkness, and hypo-salinity. The primary thesis of my dissertation is that heat stress and light stress initiate two distinct cellular mechanisms that result in coral bleaching, and that regardless of the initiating mechanisms, these two processes ultimately culminate into a single coordinated pathway whereby the coral host (animal component of the symbiosis) digests the zooxanthellae through an autophagic-related process that I term symbiophagy. Heat stress induces symbiophagy through a non-oxidative-stress regulatory switch. Light stress, a combined heat/light stress, or any stress that imposes an oxidative stress on the coral coral host (e.g., hypo-salinity exposure), probably induces symbiophagy through an oxidation-mediated signal cascade. Bleaching from prolonged darkness is a mechanism distinct from the processes observed from a light-based stress. A second thesis of my dissertation is that there is a defined sequence of events for all the processes of bleaching. Light stress induces significant accumulation of oxidative damage products, dispersion of thylakoid membrane structure, and a collapse of integrity of the chloroplast membrane. Heat stress induces a phase separation and condensation of the thylakoid membrane through a thermodynamic process, and not an oxidative stress process. Heat and light stress are not the only environmentally-relevant stressors known to induce population-level bleaching: hypo-salinity has been associated with large bleaching events. My work demonstrates that a brief hypo-salinity exposure induces an osmotic shock and subsequent oxidative stress, to both the host and the zooxanthellae.; Includes bibliographical references.; Also available by subscription via World Wide Web; 117 leaves, bound 29 cm Tue, 01 Jan 2008 00:00:00 GMT http://hdl.handle.net/10125/20464 2008-01-01T00:00:00Z Downs, Craig A http://hdl.handle.net/10125/20463 Thesis (Ph.D.)--University of Hawaii at Manoa, 2008.; Transport proteins provide essential cellular functions for archaea, bacteria and eukaryotes. Of the over 550 classified transporter families the ATP-Binding Cassette (ABC) Superfamily is one of the largest. The ABC Transporter Superfamily is divided into eight subfamilies designated A thru H. Members of this superfamily are involved in the transport of a variety of physiologically important substrates including ions, sugars, amino acids, vitamins, peptides, lipids and hormones. ABC transporters are also involved in diverse cellular processes such as lipid trafficking, antigen processing, nutrient uptake, and xenobiotic detoxification. Mutations in several ABC transporters result in various genetic diseases, while overexpression of certain key members of the ABCB/Multidrug Resistance (MDR) and ABCC/Multidrug Resistance-associated Protein (MRP) subfamilies can lead to the development of cellular multixenobiotic resistance. Phylogenetic analysis of the Drosophila genome has identified the Drosophila MRP (dMRP) gene as orthologous to three human genes capable of conferring xenobiotic resistance, the human ABCC1/MRP1, ABCC2/MRP2 and ABCC3/MRP3 genes. In vivo and in vitro experiments have shown that the dMRP gene is ubiquitously expressed throughout Drosophila development and established the dMRP gene as a functional ABC transporter. In vivo and in vitro pesticide assays indicated that dMRP is a pesticide-inducible xenobiotic transporter involved in pesticide metabolism.; Includes bibliographical references (leaves 209-240).; Also available by subscription via World Wide Web; 240 leaves, bound 29 cm Tue, 01 Jan 2008 00:00:00 GMT http://hdl.handle.net/10125/20463 2008-01-01T00:00:00Z Cogbill, Jolene Noelani Tarnay http://hdl.handle.net/10125/20462 Thesis (Ph.D.)--University of Hawaii at Manoa, 2008.; Harmful algal blooms (HABs) are associated with several acute and chronic diseases in humans worldwide, which are characterized by gastrointestinal, neurological, and/or cardiovascular disturbances. Ciguatera fish poisoning, the most commonly reported marine toxin disease in the world, is the primary and most important human health manifestation of HAB and results from the consumption of fish containing high levels of ciguatoxins (CTXs), a family of complex, lipid-soluble compounds produced by the benthic marine dinoflagellate, Gambierdiscus toxicus.; Quantitative, reliable methods to detect ciguatoxins in fish tissue are not widely accessible, thus rendering the detection of CTX in fish destined for human consumption a serious public health concern. This dissertation addresses an important food safety issue by developing and validating a sensitive and specific enzyme linked immunoassay (ELISA) capable of detecting picogram quantities of ciguatoxin in fish flesh. Development of the assay represents the first ELISA for detecting ciguatoxin using chicken antibody (IgY) as an immunoanalytic tool, and further establishes a relatively rapid, reliable method for screening large quantities of fish.; The results of testing over 300 fish commonly implicated in ciguatera fish poisoning, specifically two species of amberjacks (kahala) and the blue spotted grouper (roi) using both the ELISA and a secondary bioassay to validate results leads to the following conclusions: (a) the ELISA is a valid screening tool for evaluation of presence or absence of ciguatoxin in fish tissues with reasonable sensitivity and specificity; (b) the fish farmed in open ocean aquaculture cages in Hawai'i are not likely to be susceptible to ciguatoxin bioaccumulation despite the discovery of G. toxicus in or on the cages; (c) CTX is present in three fish species commonly implicated in ciguatera fish poisoning at overall rates lower than previously reported; and (d) the prevalence of ciguatoxin in wild-caught fish cannot reliably be correlated to the size of the fish or the depth at which it was caught. This dissertation also demonstrates that the antibody used in assay development is reactive with purified ciguatoxin and provides evidence that the AB epitope of the CTX molecule is active at the sodium channel in neuroblastoma cells.; Includes bibliographical references (leaves 115-131).; Also available by subscription via World Wide Web; 131 leaves, bound 29 cm Tue, 01 Jan 2008 00:00:00 GMT http://hdl.handle.net/10125/20462 2008-01-01T00:00:00Z Campora, Cara Empey http://hdl.handle.net/10125/20460 Thesis (Ph.D.)--University of Hawaii at Manoa, 2008.; Selenium is an essential micronutrient linked to various aspects of health. Selenium exerts its biological activity through incorporation of the amino acid, selenocysteine (Sec), into a unique class of proteins termed selenoproteins. Sec incorporation occurs cotranslationally at UGA codons in archaea, prokaryotes, and eukaryotes. UGA codons specify Sec coding rather than termination by the presence of secondary structures in mRNAs termed selenocysteine insertion (SECIS) elements, and trans-acting factors that associate with SECIS elements. Selenoprotein mRNAs are potential targets for degradation via nonsense-mediated decay, due to the presence of in-frame UGA codons. When UGA recoding is inefficient, as occurs when selenium is limiting, termination occurs at these positions. Based on predicted exon-intron structure, fourteen of the 25 human selenoprotein mRNAs are predicted to be sensitive to nonsense-mediated decay. Among these, sensitivity varies, resulting in a hierarchy of selenoprotein synthesis. Potential factors dictating this hierarchy are the SECIS binding proteins, SBP2 and nucleolin. To investigate the role of these proteins in the hierarchy of selenoprotein synthesis, we carried out knockdowns of SBP2 expression and assessed the effects on selenoprotein mRNA levels. We also investigated in vivo binding of selenoprotein mRNAs by SBP2 and nucleolin, via immunoprecipitation and quantitation of bound mRNAs. We report that SBP2 exhibits preferential binding to some selenoprotein mRNAs over others, whereas nucleolin exhibits minimal binding differences. Thus SBP2 is a determinant in dictating the hierarchy of selenoprotein synthesis via differential selenoprotein mRNA translation and sensitivity to nonsense-mediated decay.; Selenophosphate synthetase 1 (SPS1) and selenophosphate synthetase 2 (SPS2) have been implicated as essential components in selenoprotein biosynthesis. In vitro studies have demonstrated that SPS2, but not SPS1, generates the active selenium donor for Sec synthesis, monoselenophosphate. To establish the functions of these two proteins in vivo, siRNAs were used to knockdown SPS1 and SPS2 expression, and DNA constructs were designed to overexpress the two proteins in HEK-293 and MSTO-211H cells. Knockdown of SPS1 and SPS2 decreased the protein levels of several selenoproteins whereas the overexpression of SPS1 and SPS2 increased selenoprotein levels. These results reconfirm the role of SPS2 and establish SPS1 as a factor that regulates selenoprotein biosynthesis.; Includes bibliographical references (leaves xxx-xxx).; Also available by subscription via World Wide Web; 83 leaves, bound 29 cm Tue, 01 Jan 2008 00:00:00 GMT http://hdl.handle.net/10125/20460 2008-01-01T00:00:00Z Squires, Jeffrey E