"December 2001" / Bibliography: leaves 227-246. / iv, 246 leaves : ill. ; 30 cm / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Environmental Biology, 2002

Bibliography: leaves 97-112. / vii, 112 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 2003

Using 2 - D NMR spectroscopy, the structure of the sex pheromone from Litoria splendida has been determined, in order to elucidate its mode of transport through the aquatic environment. The peptide was found form an α - helical structure, with a central flexible hinge region. The mode of transport through the aquatic environment has been discussed in relation to the structure. Previous work indicated that the Australian amphibian host defence skin peptides that inhibit neuronal nitric oxide synthase ( nNOS ) were likely to act indirectly on the enzyme, by binding to the co - enzyme of nNOS, calmodulin. [superscript 15] N labelled calmodulin was expressed and purified via a bacterial protein expression system and a series of 2 - D NMR [superscript 15] N - HSQC titrations was performed with Australian amphibian host defence skin peptides. in order to determine whether these peptides bind to calmodulin. The three peptides tested were found to bind, and with differing strengths of interaction. One of these was selected for further study. [superscript 15] N and [superscript 13] C doubly labelled calmodulin was then prepared in order to study the complex between this protein and the selected peptide, caerin 1.8, an Australian amphibian skin peptide isolated from Litoria chloris. A series of 3 - D NMR spectra has been recorded on this complex. The backbone atom resonances have been assigned for free calmodulin and for the calmodulin - peptide complex, using a combination of main chain directed and sequential assignment strategies. By analysing the changes in chemical shift that occur upon binding the peptide, it was determined that the mode of binding involves a stronger interaction with the C - terminal domain than the N - terminal domain. / Thesis (Ph.D.)--School of Chemistry and Physics, 2006.

Data from nuclear ribosomal internal transcribed spacer regions (nrITS) and chloroplast DNA (cpDNA) have failed to resolve phylogenetic relationships in Pinus. To provide greater interspecific resolution, five low-copy nuclear genes were developed from mapped conifer anchor loci. Four genes were sequenced from species representing all Pinus subsections. Individual loci do not uniformly support the nrITS or cpDNA hypotheses. Combined analysis of low-copy nuclear loci produces a well-supported subsectional topology. The phylogenetic positions of P. nelsonii and P. krempfii are of systematic interest. Results strongly support P. nelsonii as sister to sect. Parrya, and suggest a moderately well-supported position of P. krempfii as sister to the remaining sect. Quinquefoliae. The most informative locus, a Late Embryogenesis Abundant-like gene, was used to explore phylogenetic relationships among closely related species in subg. Strobus. Thirty-nine species were sequenced, with two or more alleles representing 33 species. Nineteen of 33 species exhibited allelic nonmonophyly in the strict consensus tree. Nucleotide diversity was strongly associated (P<0.0001) with the degree of species monophyly. While species nonmonophyly complicates phylogenetic interpretations, this locus offers greater topological support than cpDNA or nrITS. Lacking evidence for hybridization, recombination, or imperfect taxonomy, incomplete lineage sorting remains the best explanation for trans-species polymorphisms. The absence of allelic coalescence is a severe constraint in the application of phylogenetic methods in Pinus, and taxa sharing similar life history traits may show analogous patterns. While lack of coalescence may limit their utility in traditional phylogenetics, nuclear genes remain highly informative in describing speciation events. Pinus chiapensis is a threatened species originally described as a variety of P. strobus. Prior morphological work suggests P. chiapensis is a distinct species, but that taxonomy is not universally accepted. Multiple accessions of three probable progenitors were sequenced at three nuclear loci. No interspecific allele sharing occurs with P. chiapensis, and its alleles are monophyletic at two loci. Results demonstrate that P. chiapensis is a distinct species. However, determination of the sister species is complicated by lack of species monophyly and interlocus variability. Pinus ayacahuite is the least likely progenitor, but the relationship of P. chiapensis to P. monticola or P. strobus is unclear. / Graduation date: 2006

The work presented in this dissertation examines possible modes of action for growth inhibition by anthropogenic endocrine disrupting chemicals (EDCs) as well as endogenous hormones associated with growth in fish. Using the sheepshead minnow (SHM) (Cyprinodon variegatus) as a model, I developed methods to examine perturbations in the endocrine axis controlling fish growth, and also examined effects of EDCs on the whole fish. I used two relatively new techniques to study the endocrine growth axis, quantitative real-time PCR (TaqMan) and differential display analysis. TaqMan analysis is a highly sensitive method to measure specific sequences from a small amount of total RNA using a fluorescent probe and specific primer pairs. I optimized a TaqMan assay for SHM IGF-I to measure hepatic IGF-I mRNA concentrations. in fish injected with hormones known to influence fish growth (GH, T���, E���, insulin, or a carrier control). IGF-I mRNA levels increased in fish injected with GH, T��� and insulin, peaking at 12 h post-injection. IGF-I mRNA levels decreased significantly at 8 h and 12 h post-injection in fish injected with E���, suggesting that pharmacological levels of E��� may affect the GH/IGF-I axis and could have consequences for fish living in waters polluted by EDCs. Differences in growth were observed in fish exposed for 18 weeks to E��� or chlorpyrifos (an organophsophate). Fish exposed to the highest dose of E��� grew larger than controls only during the last week of the experiment. Fish exposed to the lower dose of E��� were not significantly different from controls. The fish exposed to all doses of chloryprifos grew significantly less than controls in a dose-dependent manner. No significant differences were found in hepatic IGF-I mRNA levels in any treatments. To establish patterns of gene up- or down-regulation, I performed differential display analysis on livers of several fish from the previous two experiments. Several genes were identified as being similar to fish including a microsatellite sequence, a choriogenin (vitelline envelope) protein mRNA sequence, a transferrin mRNA sequence and several ribosomal RNA sequences. This technique to evaluate gene expression will become more useful when more fish genes are added to the data bases. / Graduation date: 2003

The role of viral proteins in the pathogenesis of infectious hematopoietic necrosis virus (IHNV) was studied at the molecular level. The expression of the viral genes at the protein and RNA level, and their cellular localization, were characterized to further our understanding of viral pathogenesis. The pathogenic effect of individual viral proteins was also investigated and a method for detecting viral RNA in infected fish tissues was developed. The polarity of transcription was confirmed in terms of the relative amounts of each viral protein. Also, cells treated with glycosylation inhibitors did not exhibit cytopathic effect, demonstrating that a functioning host glycosylation system is necessary for viral replication. These studies also revealed a previously undescribed non-glycosylated protein, S, which appeared to be virus-encoded. The expression of the nonvirion protein (NV), was also detected in infected kidney tissues. The location of M2 and NV in the cell was found to be the nucleus and cytoplasm. The expression of the NV gene was further analyzed at the level of transcription and the regulation signals for IHNV transcription were investigated. Unique transcriptional initiation and terminational signals for the fish lyssa-like rhabdoviruses were identified. The transcriptional initiation signal, 3'-CGUG-5', was distinctly different from that of the other rhabdoviruses, 3'-UUGU-5'. The role of the M2 and NV proteins in viral pathogenesis was investigated by transient expression of these proteins individually in cultured fish cells. The M2 protein alone resulted in inhibition of host-directed gene expression at the level of transcription and induction of nuclear fragmentation. The NV protein was not involved in the regulation of the host gene expression, but was involved in another type of cytopathic effect characterized as cell rounding. This is the first biological function attributed to the NV protein. A PCR method was developed for detecting IHNV N-specific RNA in formalin-fixed, paraffin-embedded fish tissues. The method is sensitive and specific. The technique is capable of detecting viral RNA in samples that have been remained at room temperature in 10% buffered formalin for over 2 years. / Graduation date: 1997

published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Anatomy / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy