http://hdl.handle.net/10125/865 Wed, 19 Jun 2013 20:04:10 GMT 2013-06-19T20:04:10Z http://hdl.handle.net/10125/20434 Thesis (Ph.D.)--University of Hawaii at Manoa, 2008.; Two genes that have established roles in the regulation of cell survival are telomerase reverse transcriptase (Tert) and the nicotinamide adenine dinucleotide-dependent protein deactylase Sirt1. However, the relative importance of these genes to the survival of stem cells, and the regulation of expression of these genes in stem cells, has yet to be thoroughly established.; We now show that telomerase suppression in quiescent male primordial germ cells (PGCs) from murine embryos is accompanied by a decrease in expression of TERT. Telomerase activity was detected in quiescent PGCs from transgenic embryos, that constitutively express TERT, demonstrating that re-activation of TERT expression is sufficient to restore telomerase activity in these cells and implying that TERT expression is an important mechanism of telomerase regulation in PGCs. These results also demonstrate that TERT per se does not affect proliferation or development of PGCs in mammals, Sirt1, a member of the sirtuin family of proteins, orthologous to the yeast Sir2, has important physiological roles in regulating glucose metabolism. We have now performed a detailed analysis of the molecular and functional effects of Sirt1 deficiency in the germ line of Sirt1 knock-out (-/-) mice. We find that Sirt1 deficiency markedly attenuates spermatogenesis, but not oogenesis. Microarray analysis in Sirt1 deficient testis revealed dysregulated expression of 85 genes, which were enriched (P<0.05) for genes involved in spermatogenesis and protein sumoylation. To assess the function of Sirt1 deficient germ cells, we compared the efficiency of generating embryos and offspring in in vitro fertilization (IVF) experiments using gametes from Sirt1 deficient mice. While viable animals were derived in all experiments, the efficiency of producing both 2-cell zygotes and viable offspring was diminished when IVF was performed with Sirt1-/- gametes. Using short hairpin RNAs, we found that inhibition of Sirt1 in immortalized human cells enhanced cell growth under normal and nutrient limiting conditions. Hematopoietic stem cells, which were shown to express Sirt1, showed increased growth capacity and decreased dependency on growth factors when the Sirt1 gene was deleted. These data support an important role for Sirt1 in spermatogenesis, including spermatogenic stem cells, as well as germ cell function, but also demonstrate that in certain cell lineages, Sirt1 can act as a growth suppressor.; Includes bibliographical references (leaves 117-132).; Also available by subscription via World Wide Web; 132 leaves, bound 29 cm Tue, 01 Jan 2008 00:00:00 GMT http://hdl.handle.net/10125/20434 2008-01-01T00:00:00Z Coussens, Matthew J http://hdl.handle.net/10125/12112 Thesis (Ph. D.)--University of Hawaii at Manoa, 2005.; Includes bibliographical references (leaves 162-188).; Also available by subscription via World Wide Web; xvi, 188 leaves, bound ill. (some col.) 29 cm Sat, 01 Jan 2005 00:00:00 GMT http://hdl.handle.net/10125/12112 2005-01-01T00:00:00Z Fogelgren, Benjamin C http://hdl.handle.net/10125/12111 Mode of access: World Wide Web.; Thesis (Ph. D.)--University of Hawaii at Manoa, 2005.; Includes bibliographical references (leaves 116-154).; Electronic reproduction.; Also available by subscription via World Wide Web; xvii, 157 leaves, bound ill. 29 cm Sat, 01 Jan 2005 00:00:00 GMT http://hdl.handle.net/10125/12111 2005-01-01T00:00:00Z Ding, Chaonan http://hdl.handle.net/10125/12110 Thesis (Ph. D.)--University of Hawaii at Manoa, 2004.; Includes bibliographical references (leaves 176-192).; Also available by subscription via World Wide Web; xi, 192 leaves, bound ill. (some col.) 29 cm Thu, 01 Jan 2004 00:00:00 GMT http://hdl.handle.net/10125/12110 2004-01-01T00:00:00Z Sotolongo, Barbara http://hdl.handle.net/10125/12109 Thesis (Ph. D.)--University of Hawaii at Manoa, 2004.; Includes bibliographical references (leaves 198-219).; Also available by subscription via World Wide Web; xvii, 219 leaves, bound ill., some col. 29 cm Thu, 01 Jan 2004 00:00:00 GMT http://hdl.handle.net/10125/12109 2004-01-01T00:00:00Z Urschitz, Johann G. E http://hdl.handle.net/10125/12108 Mode of access: World Wide Web.; Thesis (Ph. D.)--University of Hawaii at Manoa, 2004.; Electronic reproduction.; Also available by subscription via World Wide Web; xii, 142 leaves, bound ill. (some col.) 29 cm Thu, 01 Jan 2004 00:00:00 GMT http://hdl.handle.net/10125/12108 2004-01-01T00:00:00Z Vine, Alex L http://hdl.handle.net/10125/1260 Quorum sensing is a cell density-dependent bacterial gene regulatory mechanism used for the expression of colonization-related genes. The symbiotic relationship between the luminescent bacterium Vibrio fischeri and the Hawaiian bobtail squid Euprymna scolopes serves as a model system to study the molecular processes underlying bacterial colonization. This system is especially well-suited for the investigation of the impact of quorum sensing on colonization because (i) it is an easily accessible, natural, two-species colonization model, and (ii) quorum sensing regulates luminescence expression in V. fischeri, which allows the non-invasive detection of quorum-sensing activity both in culture and in symbiosis. While the impact of one of V. fischeri's quorum-sensing systems, lux, on luminescence expression and symbiotic competence has been extensively studied, little was known about other putative systems. The results of this study demonstrate that the V. fischeri ain system is essential for both maximal luminescence expression and symbiotic competence. The ain system predominantly induces luminescence expression at intermediate cell densities, which occur in culture, while the lux system is responsible for luminescence expression at the high cell densities found in symbiosis, suggesting the sequential induction of luminescence gene expression by these two systems. Furthermore, the ain quorum sensing system is important for the processes underlying colonization initiation, while the impact of the lux system is apparent only in later stages of the symbiosis, indicating distinct functions of these two systems during the colonization process. A global transcriptome. analysis of quorum-sensing mutants revealed that ain quorum sensing represses motility gene expression, providing a likely explanation for the initiation defect. Although it has been known that many bacterial species possess multiple quorum-sensing systems, this is the first study demonstrating that two quorum-sensing systems are employed to specifically regulate functions important at distinct cell densities occurring during the colonization process. Mon, 01 Dec 2003 00:00:00 GMT http://hdl.handle.net/10125/1260 2003-12-01T00:00:00Z Lupp, Claudia http://hdl.handle.net/10125/1259 All animals exist in lifelong relations with a complement of bacteria. Because of the ubiquity of these symbioses as well as the derived biomedical applications, the study of both beneficial and pathogenic host-microbe associations has long been established. The monospecific light organ association between the Hawaiian sepiolid squid Euprymnascolopes and the marine luminous bacterium Vibrio fischeri has been used as a experimental model for the study of the most common type of animal-bacterial interaction, i.e., the association of coevolved Gram-negative bacteria with the extracellular apical surfaces of polarized epithelia. A fundamental step for understanding the mechanisms of host-symbiont associations lies in defining the genetic components involved; specifically defining changes in host gene expression. The studies presented in this dissertation identify and characterize V. fischeri-induced changes in host gene expression at both the transcript and protein level. Mon, 01 Dec 2003 00:00:00 GMT http://hdl.handle.net/10125/1259 2003-12-01T00:00:00Z Kimbell, Jennifer Loraine http://hdl.handle.net/10125/1258 Several members of the lysyl oxidase family of copper-dependent amine oxidases have been implicated in tumor development. The Iysyl oxidase (LOX) and LOX-like 2 (LOXL2) genes have been mapped to chromosomal regions affected by loss of heterozygosity (LOH) in several cancers, including those of the colon and esophagus. Indeed, there have been numerous reports of reduced LOX and a few reports of reduced LOXL2 expression in various cancers. Identification of microsatellite markers within the LOX locus and the LOXL2 gene allowed for evaluation ofthe status of these gene alleles in colon and esophageal tumors. There was significant LOH of the LOX locus in colon tumors that was accompanied by reduced mRNA expression and a spectrum of alterations and mutations affecting the LOX gene. This study demonstrated, for the first time, that genetic events, namely LOH, deletions and mutations ofthe LOX gene, were responsible, at least partly, for the reduction of LOX gene expression. There was also significant LOH of the LOXL2 gene in both colon and esophageal tumors. However, instead of a reduction of LOXL2 expression, there was increased expression that correlated with less differentiated tumors and absent elastosis, both indicators of poor prognosis. Further studies indicated that both LOX and LOXL2 are absent in non-invasive tumor cell lines but re-expressed in invasive cell lines, likely as part of the thelial-mesenchymal transition that occurs in the last steps of tumorigenesis to facilitate metastasis. The results presented and research strategy outlined in this dissertation will define the importance of LOXL2 amine oxidase activity and protein interactions in the critical but poorly understood process oftumor cell migration and invasion. Mon, 01 Dec 2003 00:00:00 GMT http://hdl.handle.net/10125/1258 2003-12-01T00:00:00Z Fong, Sheri Fumiko Tsuda http://hdl.handle.net/10125/987 Regulation of N-myc oncogene expression is an important determinant of the biological behavior of neuroblastoma. The N-myc promoter contains several potential binding sites for transcription factors of the Sp1 family. Mutation of a CT-box motif contained within a 26 base pair region required for N-myc downregulation by retinoic acid decreased basal transcriptional activity and altered DNA-protein interactions of the promoter, while mutations flanking this motif did neither. On gel shift this region generated 3 specific DNA-protein complexes that were reliant on wild type sequence of the core CT element within it. Both Spl and Sp3 bound to the wild type probe as distinct complexes in specifically retarded bands, while neither protein was present on mutated sequences. Lysates from Drosophila S2 cells expressing exogenous Sp1 and Sp3 proteins were able to reproduce the gel shift complexes seen with neuroblastoma nuclear extract. Transient transfections of S2 cells showed that individually or together, Sp1 and Sp3 were able to trans-activate a N-myc CT-box-containing luciferase reporter construct in a dose-dependent manner. Conversely, transfection of CT-box oligonucleotide was able to decrease endogenous N-myc expression in neuroblastoma cells. Together these results suggest that the CT-box element serves a critical functional role, and in the basal state allows for N-myc transactivation by Spl and Sp3. Mon, 01 Dec 2003 00:00:00 GMT http://hdl.handle.net/10125/987 2003-12-01T00:00:00Z Tuthill, Matthew Charles http://hdl.handle.net/10125/986 Differential gene expression plays a key role in developmental pathways within an organism. Examples of such pathways include primary sex determination signaling and the formation of secondary sexual characteristics. This dissertation is focused on the use of suppression subtractive hybridization (SSH) to identify genes that are differentially expressed and involved in some aspect of sexual development in the Mediterranean fruit fly (medfly), Ceratitis capitata. In the course ofthis project, a method for sexing individual specimens from pre-adult stages was developed. This method was used to collect sex-specific RNAs at different developmental stages for use in SSH. A total of25 subtraction products were obtained across all the stages examined. Analysis of these products revealed that approximately half were similar to cytoplasmic ribosomal proteins and mitochondrial ribosomal RNA The remaining products represent putative medfly homologs of other previously identified genes or potentially novel genes One ofthe subtraction products, representing a potentially novel gene, was characterized in detail. This gene, named mapotge', represents a novel medfly gene that appears to encode a polypeptide of 299 amino acids. The N-terminus of this polypeptide contains a BTB-POZ domain. This domain functions as a protein-protein interaction motif found in a wide range of organisms from humans to Drosophila that mediates protein dimerization and oligomerization. The temporal expression pattern of mapotge' was determined using RT-PCR and Northern blot analysis. These revealed that the transcript is expressed throughout embryogenesis in both females and males, and in adult females that are > 0.5 days post-eclosion. Minimal expression is observed in female and male third instar larvae, early pupae, and in adult males. Studies were also initiated to characterize the representation of additioual sequences containing a BTB-POZ domain in the medfly genome. This was performed using Southern blot analysis and degenerate primers for the polymerase chain reaction (PCR). These results indicate the presence of at least three sequences in the medfly, in addition to 'mapotge', that contain a BTB-POZ domain. Potential evolutionary relationships ofthe BTB-POZ domain sequences from the medfly and other insect species were also analyzed. Sun, 01 Dec 2002 00:00:00 GMT http://hdl.handle.net/10125/986 2002-12-01T00:00:00Z Untalan, Pia Marie