Peripheral insulin resistance is characterised by reduced insulin-stimulated glucose uptake in skeletal muscle and adipose tissue, and the condition represents one of the earliest hallmarks in the development of type 2 diabetes (T2D). In patients with T2D, protein expression of the insulin-stimulated glucose transporter, GLUT4, is reduced in adipose tissue, but preserved in skeletal muscle. Transgenic studies in rodents provide evidence that overexpression of GLUT4 selectively in either skeletal muscle or adipose tissue enhances whole-body insulin action. Since skeletal muscle accounts for the majority of insulin-stimulated glucose disposal, the effect of adipose tissue GLUT4 on insulin sensitivity is thought to be secondary to an altered secretion of adipokines which affect insulin action in muscle, in the context of a ‘metabolic crosstalk’ between insulin sensitive tissues. Increasing GLUT4 expression in skeletal muscle and adipose tissue could be an effective therapy in the treatment of insulin resistance and T2D. Exercise training increases GLUT4 protein expression in skeletal muscle of patients with T2D. This adaptation occurs in the face of enhanced insulin sensitivity, and results from the cumulative and transient increase in GLUT4 mRNA following each acute exercise bout. Less is known regarding the regulation of skeletal muscle GLUT4 expression by a single bout of exercise in patients with T2D, or the effect of exercise training on GLUT4 expression in adipose tissue. / The primary aim of the studies undertaken for this thesis was to enhance understanding of exercise-mediated GLUT4 expression in skeletal muscle and adipose tissue of patients with T2D. The first investigation determined the effect of a single bout of exercise on skeletal muscle GLUT4 mRNA, and the signalling pathways which regulate GLUT4 expression, in patients with T2D and healthy control volunteers, matched for age and BMI. Increased (p<0.05) expression of GLUT4 and PGC-1α mRNA, together with increased (p<0.05) phosphorylation of AMPK and p38 MAPK was observed following exercise in patients with T2D, to a similar extent as in age- and BMI-matched control subjects. These findings lead to the conclusion that exercise-mediated regulation of GLUT4 expression is normal in patients with T2D. The second investigation of this thesis sought to identify the effect of a 4 week exercise training program on skeletal muscle and adipose tissue GLUT4 expression in patients with T2D. It was found that exercise training increased (p<0.05) GLUT4 protein expression by ~36% and ~20% in adipose tissue and skeletal muscle, respectively. These adaptations occurred in the absence of changes in insulin sensitivity or plasma levels of adipokines, adiponectin and resistin. Accordingly, the third study of this thesis sought to identify novel adipokines that regulate peripheral glucose metabolism in an adipocyte model of GLUT4 overexpression. Amyloid precursor protein (APP) was reduced (p<0.05) in culture media of GLUT4 overexpressing adipocytes, and the APP cleavage product, amyloid-beta (Aβ), reduced (p<0.05) insulin-stimulated Akt phosphorylation in L6 myocytes in vitro. / These observations lead to the conclusion that increased adipose tissue GLUT4 expression may influence whole body glucose metabolism through reduced levels of Aβ. The primary aim of the final study undertaken was to identify novel changes in the abundance of proteins in skeletal muscle following exercise training in patients with T2D, including proteins of glucose metabolism, which may regulate of GLUT4 expression. Exercise training altered the abundance of several proteins involved in energy metabolism, as well as some novel proteins which may play a role in cytoskeleton interactions with mitochondria. In summary, this thesis demonstrated that skeletal muscle from patients with T2D responds normally to an acute exercise bout in terms of increased GLUT4 mRNA expression. In addition, it was shown that exercise training increased GLUT4 protein expression, not only in skeletal muscle, but also in adipose tissue of patients with T2D. This is significant because adipose tissue GLUT4 overexpression enhances insulin sensitivity. Data from this thesis suggest that improvements in insulin sensitivity may be secondary to altered secretion of Aβ from adipose tissue. Collectively, the findings provide a number of therapeutic targets for the treatment of insulin resistance and T2D.

Antibiotic resistance is increasing worldwide, which is threatening the effectiveness of even the most potent and recent antibiotics. The successful treatment of disease is hampered due to the multidrug resistant (MDR) phenotype exhibited by the bacterial pathogens. Therefore, the aims of this thesis were to investigate MDR through several different approaches. Integrons are important contributors to the MDR profile of nosocomial isolates within Australia, therefore the incidence of integrons was assessed in a collection of 72 conjugative clinical plasmids isolated from E. coli, a cohort of 30 urinary tract infection (UTI) isolates and a cohort of four bacteria producing metallo-beta-lactamases (MBLs). Integrons were found in 63% (45/72) of the conjugative plasmids by polymerase chain reaction (PCR). Sequencing of gene cassette arrays revealed that cassettes of the dfr and aadA families were most common. Within the cohort of UTI bacteria, 37% (11/30) were positive for class 1 integrons, and the dfrA17-aadA5 gene cassette array was most common. The four MBL-producers contained the gene cassette blaIMP-4 found within a class 1 integron which was responsible for the MBL phenotype. An assay based on real-time PCR was also developed to measure the recombination activity of the integron integrase (IntI) enzymes. The existing method of IntI measurement, the in vivo conduction assay, was used as a basis for the development of the real-time PCR assay. Five 59-be from the gene cassettes aadB, orfA, sat2, dfrA1 and aacA4 were cloned as recognition sites used in the real-time PCR assay. IntI1 was the most active integrase and showed an activity of 2.31 ?? 10??-1 when recombining the aadB and orfA 59-be. The highest level of class 2 integrase activity was 2.00 ?? 10-1?? during recombination of the sat2 and dfrA1 59-be, while IntI3 showed its highest recombination frequency of 2.29 ?? 10-1?? when the aadB and orfA 59-be were used. Additionally, the real-time PCR assay was used assess the levels of IntI activity over time. Using this method, the level of recombination as time progressed remained stable at a level of 4.10 ?? 10-2????. MDR was also analysed in 37 Acinetobacter baumannii isolates which were collected from four hospitals in Sydney. Minimum inhibitory concentration (MIC) analysis to 25 antibiotics revealed that all isolates showed a reduced susceptibility to between five and 24 antibiotics. PCR was performed to detect the presence of resistance determinants. Class 1 integrons encoding resistance to aminoglycosides, antiseptics and disinfectants were found in 35 % (13/37) of the isolates. Aminoglycoside resistance genes including aphA1 (12/37), strA (1/37) and strB (22/39) were also found. Resistance to beta-lactams was also observed in all isolates, which correlated with the presence of the ampC and blaOXA-51-like genes. The insertion sequence ISAba1 which provides an alternative promoter leading to increased gene expression was found upstream of the ampC gene in 29 isolates; the same isolates also contained the identical insertion sequence upstream of the carbapenemase resistance gene blaOXA-23. These 29 isolates also possessed the tetracycline resistance gene tetB. All but one of these 29 isolates also contained the gene blaTEM-1. Resistance to quinolones and fluoroquinolones was attributed to the presence of a Ser83-Leu83 gyrA mutation present in 36 resistant isolates. Furthermore, a putative dihydrofolate resistance gene, folA, was found in all isolates. Repetitive extragenic palindromic PCR revealed the presence of seven clonal groups. Overall, this study demonstrated the widespread impact and dissemination of MDR within nosocomial settings in Australia. The use of new assays, such as the real-time PCR assay developed in this thesis, is essential to the understanding of dissemination of antibiotic resistance.

The Ser/Thr kinase Akt plays an important role in many of insulin's actions including GLUT4 translocation to the plasma membrane (PM). However, there are several features of Akt's regulation of GLUT4 translocation that remain unclear. The goal of my thesis was to resolve some of the following questions: Is activation of Akt sufficient to stimulate GLUT4 translocation? What is the quantitative relationship in signal transmission between individual components within the Akt cascade? What is the role of Akt in insulin resistance? To determine if activation of Akt is sufficient to mediate GLUT4 translocation, I developed a drug-inducible heterodimerisation strategy to activate Akt2 independently of other potential insulin signalling pathways. These studies revealed that activation of Akt2 resulted in rapid stimulation of GLUT4 translocation to a similar extent with maximum insulin, indicating that Akt2 is sufficient for this event. It was previously observed that maximum effect of insulin on GLUT4 translocation was obtained with minimum activation of Akt. To resolve this discrepancy, the relationship between Akt signalling components was examined using a quantitative kinetic and dose response approach combined with hierarchical cluster analysis. Most notably I observed a strong relationship between Akt at the PM, but not Akt in the whole cell lysate, with its substrate phosphorylation. Active pools of phospho-Akt and -AS160, a major substrate involved in GLUT4 translocation, were found in the lipid raft, highlighting the importance of subcellular partitioning of key signalling components for achieving biological specificity. The involvement of Akt in insulin resistance was investigated using the heterodimerisation strategy. These studies revealed that insulin itself initiates a pathway that causes insulin resistance by converging on target(s) downstream of Akt. This inhibitory pathway emanates from PI3-kinase and is likely induced by a range of insults including chronic insulin and dexamethasone. In conclusion, Akt is a crucial element in the insulin action pathway that exhibits precise spatial regulation. While the role of this nanoregulation of Akt in disease remains to be evaluated, my studies suggest that the major defect contributing to insulin resistance occurs downstream of Akt. The elucidation of this target will have major implications for metabolic diseases.

As anti-cancer agents, retinoid induce cell growth arrest and differentiation, while HDACIs cause cell differentiation, growth inhibition, death and inhibit angiogenesis in many cancer types. However, a proportion of patients respond poorly to these therapies. My studies, presented here, aimed to improve the anti-cancer effects of these agents by identifying key factors which mediate cancer cell sensitivity or resistance to their action. In this study I have found that the clinically used retinoid, 13-cis RA, exerts its anti-cancer signal in a manner similar to atRA, by modulating the transcriptional response of retinoid-regulated genes. HDACI-induced cytotoxicity is significantly enhanced when combined with IFNα in 8 out of 9 cancer cell lines from various organ origins. Sensitivity to the combination treatment correlated with an absence of basal p21 protein expression, and cell cycle arrest. Knocking-down p21 gene expression further sensitized cancer cells to the combination therapy. Moreover, IFNα and HDACI co-operatively inhibited pro-angiogenic gene expression in cancer cells, and the combination therapy decreased endothelial cell migration, invasion, and capillary tubule formation. Further experiments on p21 as a resistance factor to anti-cancer treatment demonstrated that conditioned media from breast cancer MCF-7 cells transfected with p21 siRNA, induced significantly less endothelial cell migration, invasion and vascular sprouting, compared with media from cells transfected with scrambled siRNA. LC/MS analysis of the conditioned media revealed that Trx secretion was significantly reduced after p21 knockdown. The reduction in Trx secretion following p21 knockdown was due to a direct effect of p21 siRNA on the expression of intracellular TBP2 in neuroblastoma, prostate and lung cancer cells. Consistent with this result, media from MCF7 cells transfected with TBP2-specific siRNA alone, promoted endothelial cell invasion and vascular sprouting, Trx knockdown resulted in opposite effects, and the anti-angiogenic effect of p21 siRNA was offset by simultaneous TBP2 siRNA transfection. ChIP assay revealed that p21 directly bound to an E2F1-bindng site in the TBP2 gene promoter. These data indicate that p21 promoted tumour-driven angiogenesis through transcriptional repression of TBP2. Collectively, my experiments indicate several potential treatment targets directed toward enhancing the effectiveness of HDACIs and retinoids.

The fungi Sclerotinia minor and S. sclerotiorum are the causal agents of two similar diseases of peanut (Arachis hypogaea L.). Both diseases cause significant losses in the Australian peanut industry. Chemical and cultural control methods will not provide complete control. Development of cultivars with resistance to Sclerotinia will be an important component of integrated control. The capacity to breed and select for such resistance efficiently must be established before serious investment of resources is made to develop Sclerotinia resistant cultivars. The aims of this project are: (1) to generate information that will assist in the improvement of Sclerotinia resistance in peanut; (2) to develop screening techniques; (3) to identify Sclerotinia resistant peanut germplasm; (4) to understand the inheritance and estimate heritability of resistance to S. minor; (5) to assess response to selection for resistance; and (6) to test the effectiveness of identified sources of resistance against both S. minor and S. sclerotiorum. Experiments were conducted to develop screening techniques applicable for this project and a full scale breeding program. A previously unpublished technique for evaluating physiological resistance was described and modified. The artificial inoculation technique using colonised bean pod segments was found to be more robust than using colonised agar disks: discriminating among the physiological resistance of peanut genotypes to Sclerotinia whether used in controlled environment cabinets or the simple tent structures, and working well with both whole plants and detached stems. Cultivars and lines with the shortest lesion length in this test have demonstrated resistance to S. minor in field experiments in both Australia and in the USA. The use of a tent structure in place of a controlled environment cabinet (CEC) to create a high humidity environment allowed larger numbers of individuals to be tested at one time. With this technique, glasshouse space and labour costs, rather than the size of the available CECs, are the limits to the number of individuals that can be tested at one time. This will allow mass screening of segregating populations or replicated testing of progenies in a breeding program context that would not be possible with limited CEC facilities. Calculating a Moderated Lesion Length (MLL) by eliminating the failed or very small lesions was found to improve precision in measuring resistance responses of genotypes. Compared to average lesion length, the MLL had a stronger association with foci count (FC), a measure of resistance in the field. In cases where the small lesions do not occur independently of peanut genotype, a small lesion count (SLC) can be used to describe that variation. The detached stem technique examined in this thesis was another useful tool for screening, particularly in situations where seed production by the plants is deemed critical, or where the seed quantity available would not provide sufficient replication for the pot-based glasshouse test. This study has clearly established that material which shows resistance to S. minor in the USA, is resistant to S. minor and quite likely to be resistant to S. sclerotiorum in Australia. The high level of resistance to both S. minor and S. sclerotiorum in germplasm from Texas, particularly TxAG-4, was confirmed. The component lines of Virginia 93 Bunch showed good resistance in the field, which is primarily architectural resistance. Physiological resistance to S. minor was also identified in a cultivar and a landrace from Indonesia and three rust resistant breeding lines from Queensland. All germplasm found to have high physiological resistance to S. minor belonged to the Spanish type. Inheritance of physiological resistance to S. minor was studied using a Generation Means Analysis (GMA) of the cross TxAG-4/VA 861102 and its reciprocal. The broad-sense heritability of physiological resistance on a single plant basis was estimated at 47%, much higher than earlier estimates obtained in field studies. The average gene action of Sclerotinia resistance genes from TxAG-4 was found to be additive. No dominance effects were detected in the GMA. A small but significant reciprocal effect between TxAG-4 and VA 861102 indicated that VA 861102 passed on some physiological resistance maternally. Selection of single F2 and F3 plants successfully achieved an improvement in physiological resistance as measured by MLL. Selection was based on both MLL and seed production and further work should be conducted to quantify the comparative contribution of these two criteria. To estimate a realised heritability of physiological resistance in early generation selection it may be necessary to use a detached stem technique so that seed production occurs independently of expression of disease resistance. Successful selection of highly resistant genotypes from small F2 and F3 populations was taken as a possible indication of oligogenic control of resistance. An experiment was conducted to confirm the value of resistance against both S. minor and S. sclerotiorum. TxAG-4 was found to have physiological resistance to both S. minor and S. sclerotiorum. This resistance was expressed against both Sclerotinia species by progeny that were selected for resistance to S. minor. On the basis of the information obtained, the comparative advantages of three strategies for Sclerotinia resistant cultivar development are described: (1) introduction of germplasm; (2) recurrent backcrossing with screening and crossing in the BCnF1 generation; and (3) pedigree selection. At present introduction and backcrossing are recommended as the preferred strategies. Pedigree selection for Sclerotinia resistance is expected to become increasingly valuable after the development, by introduction and backcrossing, of Sclerotinia resistant cultivars with other desirable

Thesis (M.S.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references (ℓ. 65-73)

Thesis (Th. M.)--Calvin Theological Seminary, 1988. / Bibliography: leaves 165-173.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--University of Adelaide, 1993. / Includes bibliographical references (leaves 203-220).

Thesis (Ph. D.)--University of Adelaide, Dept. of Crop Protection, 1996? / Includes bibliographical references (leaves 159-184).