Bibliography : leaves 165-178. / xii, 178, [73] leaves, [33] leaves of plates : 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 Biochemistry, 1993?

Typescript (photocopy) / vi, 115 leaves, [25] leaves of plates : 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 Plant Pathology, 1982

Errata pasted onto back page. / Bibliography: p. 133-149. / 149 p., [29] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The RhoGEF activity of PBL is shown to be acting predominantly by the activation of Rho1 and downstream signaling pathways required for contractile ring function during cytokinesis. Genetic evidence suggests this could be through the activation of Diaphanous (an FH protein) to reorganize the actin cytoskeleton, as well as through the activation of Rho-kinase which results in the phosphorylation, and activation of myosin. Highlights a possible role for PBL during contractile ring function at a later stage that previously thought. Genetic interaction screens were employed to identify regulators of PBL activity during cytokinesis. CDK1 was identified genetically as a candidate for regulating PFB activity, but functional studies in vivo showed that this regulation was not by direct phophorylation of the PBK consensus CDK1 suites tested. Further screening has identified other possible components pf PBL signaling pathways, but a role during cytokinesis for these interactors remains to be confirmed. The eye phenotypes described provide ideal systems for the identification of components of PBL signaling pathways in Drosophila. The high level of conservation in the mechanism of cytokinesis from yeast to mammals would also suggest that the identified interactors would most likely represent components of cytokinesis pathways in all eukaryotes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2002?

The current epidemic of obesity poses a substantial threat to public health worldwide. Obesity is associated with many deleterious health conditions, including type 2 diabetes, hypertension, dyslipidaemia, respiratory conditions, arthritis, and some forms of cancer. Moreover, the rising prevalence of obesity has been accompanied by a substantial increase in the cost of treating these conditions. Obesity results from a complex interaction between behavioural, environmental, and genetic factors. While the recent increase in the prevalence of obesity is largely due to behavioural factors (for example, physical inactivity); it has also been observed that genetic factors make a large contribution to individual susceptibility. In fact, studies indicate that as much as 50 - 80% of the variation in measures of obesity can be attributed to the effects of genes. Furthermore, closer examination of this genetic component using segregation analysis has indicated the presence of common genes for obesity, with large effects on the phenotype. However, these putative major genes for obesity have not yet been identified. The aim of this thesis was to investigate the role of three distinct genetic loci in obesity and related cardiovascular factors, including type 2 diabetes and dyslipidaemia. The aim of the first investigation was to test whether a common polymorphism (Pro12Ala) in the gene encoding peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2) was associated with obesity and other cardiovascular risk factors in a large group of Caucasian subjects. PPAR-γ2 is an adipogenic transcription factor, which also regulates insulin sensitivity in adipose tissue. No association was observed between the Pro12Ala polymorphism and obesity in Caucasians, but obese subjects carrying the Ala allele displayed an altered blood lipid profile compared with obese Pro/Pro subjects. As the Pro12Ala polymorphism may exacerbate the risk of cardiovascular disease by modifying blood lipid profile in obesity, this relationship was examined further in a separate population. The aim of the second investigation was to determine whether the Pro12Ala polymorphism was associated with obesity, dyslipidaemia, diabetes and carotid intima-medial wall thickening in a population at high risk of developing cardiovascular disease. Australian Aboriginal people display high rates of mortality from cardiovascular disease, and it is possible that their increased susceptibility is due to genetic factors. However, the results from the Aboriginal population confirmed the results of the first study: there was no intrinsic association between the Pro12Ala variant and obesity. In addition, the Ala allele was not associated with deleterious changes in blood lipid profile, as it was in Caucasians. The aim of the third investigation was to confirm the presence of a quantitative trait locus (QTL) for obesity on chromosome 20q13. Highly polymorphic genetic markers in this region were tested for linkage and association with several measures of obesity in a Caucasian population. None of the measures of obesity were linked to or associated with markers spanning 20q13, suggesting that this chromosomal region does not contain a major locus for obesity in this Caucasian population. In the fourth investigation, the 5' sequence of Agouti Signalling Protein (ASIP) was identified. ASIP is a candidate gene for obesity, as it is expressed at high levels in adipocytes, and may participate in several obesity-related processes. Three new exons and two alternative promoters were identified for the ASIP gene. These results may lead to greater understanding of the role of ASIP in obesity and adipocyte metabolism; and may also be used to direct further research into genetic variation within this candidate gene. In conclusion, extensive study of two established candidate genetic loci revealed no association with measures of obesity. Therefore, it is likely that loci other than these make significant contributions to obesity in humans. Further investigation of novel candidate genes, such as ASIP, may allow the identification of novel genetic polymorphisms and new pathways important for the genetic basis of obesity.

Gaucher disease, the most common lysosomal storage disorder, results from a deficiency in the enzyme glucocerebrosidase. Inherited as an autosomal recessive disorder, Gaucher disease is clinically heterogeneous with both non-neuronopathic (Type 1) and neuronopathic (Types 2 and 3) subtypes. Although over 100 mutations in the glucocerebrosidase (GBA) gene have been identified, there still exists a poor correlation between individual genotypes and observed phenotypes, particularly for the neuronopathic subtypes. Using DNA isolated from archival tissue samples and standard molecular biology techniques, two novel and two rare mutations were identified in three individuals with neuronopathic Gaucher disease. One mutation identified only in aboriginals of Cree descent was further characterized by heterologous expression in baculovirus-infected Sf9 cells and displayed moderate levels of residual enzyme activity, despite the corresponding disease severity observed. Heterologous expression studies were extended to examine systems for high-level glucocerebrosidase expression for biochemical analysis and biotherapeutics. While the methylotrophic yeast Pichia pastoris was found to express minimal amounts of human glucocerebrosidase even when selected for high gene copy number, stable transfected Sf9 cells were found to produce functional glucocerebrosidase at a level of 1.0–1.3mg/L of cell culture. A subsequent analysis of synonymous codon usage bias in Pichia pastoris identified a significant difference in codon choice between the expression host and the GBA gene. Codon optimization studies using a 5′ fragment of the GBA gene fused to a luciferase reporter gene found that alterations in both G+C content and codon bias increased expression levels 7.5 to 10 fold. This suggests that codon optimization of the entire GBA gene could significantly improve production levels of this important enzyme in Pichia pastoris and other expression hosts. / Graduate

The 2.7 kilobase transcript of the puf operon of the photosynthetic bacterium Rhodobacter capsulatus has five open reading frames. The gene products of four of these open reading frames (pufB, A, L,and M) are well characterized as structural polypeptides of the reaction center (pufL and M) and the B870 light-harvesting antenna complex (pufB and A), The role of the pufX gene product has been unknown. By deleting the pufX gene from a plasmid carrying the puf operon and using this plasmid to reconstitute a strain of R. capsulatus which had the puf operon deleted, it was possible to characterize the pufX gene product. It was found that the pufX⁻ mutant was unable to grow photosynthetically until a secondary suppressor mutation had occurred. It appeared that either more than one type of suppressor mutation could occur or that one suppressor mutation could be accompanied by further mutations. To determine the nature of the lesion caused by the deletion of pufX, the structure of the photosynthetic unit and the ability of the subunits of the photosynthetic unit to accomplish energy and electron transfer of the mutant were compared to a pseudo-wild type. Spectrophotometric techniques, including fluorescence detection, reduced minus oxidized spectra, flash-induced absorbance change spectra, and ground state absorption spectra were used for these comparisons as well as biochemical assays. The biochemical assays measured the ability of chromatophores to transfer electrons from a quinone analog to horse-heart cytochrome c and to pump protons in response to light irradiation. The results of these comparisons indicated that the individual components of the photosynthetic unit functioned normally but that electron transfer between these components, specifically between the reaction center and the cytochrome b⁄c₁ complex, was impaired. It thus seemed likely that there was some structural defect in the photosynthetic unit. The structure of the photosynthetic units of pseudo-wild type and mutant strains was probed using sodium dodecyl sulfate-polyacrylamide gels, absorption spectroscopy, and electron microscopy. It was determined that the mutant had chromatophore vesicles that were about 50% larger than those of the pseudo-wild type and contained higher levels of reaction center and B870 light-harvesting antenna polypeptides. The suppressor mutants also had altered levels of polypeptides and showed differences in the way the expression of their B800-850 polypeptides was regulated. It was concluded that the pufX gene product plays a role in the correct assembly of the photosynthetic unit as a structural component of the unit and/or as a regulator of its assembly. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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published_or_final_version / abstract / toc / Obstetrics and Gynaecology / Master / Master of Philosophy