Medicine (Northwest Academic Centre) - Theses
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ItemAssociations between dietary consumption and cardiometabolic disorders among community-dwelling Australian adults( 2017)Background: Cardiometabolic disorders including atherosclerosis, diabetes, and metabolic syndrome (MetS) are major causes of mortality and morbidity. Nutrition is a lifestyle factor that plays an important role in the development of cardiometabolic disorders. Objective: The research project aimed to examine the association of omega-3 fatty acid intake and dietary quality with abdominal aortic calcification (AAC) among community-dwelling adults from a subset of the Melbourne Collaborative Cohort Study (MCCS) and whether protein intakes from different sources are differently associated with the incident diabetes and MetS in the whole population of the MCCS. Methods: Analyses regarding association of omega-3 fatty acid intakes and dietary quality with AAC were based on a subset (n=312) of the MCCS with diet assessed at baseline (1990-1994) and follow-up (2010-2011) and AAC measured using radiography and dual-energy x-ray absorptiometry (DXA) at follow-up (2010-2011). Analyses regarding protein intakes, diabetes and MetS were conducted using data from the MCCS of 41,514 participants. Dietary intakes, diabetes, and MetS were assessed at both baseline (1990-1994) and follow-up (2003-2007). A meta-analysis of the association between protein intakes and incident diabetes was also conducted. Results: Baseline energy-adjusted alpha linolenic acid (ALA) intake showed inverse associations with AAC severity by both radiography (odds ratio (OR) (95% CI) for tertiles 3 vs 1: 0.49 (0.23, 1.02)) and DXA (0.37 (0.16, 0.83)) in women. Women in the third tertile of total omega-3 fatty acid intake had lower AAC severity by radiography with OR (95% CI) 0.33 (0.16, 0.71) and DXA with OR (95% CI) 0.27 (0.12, 0.62) compared with those in the first tertile. Omega-3 fatty acid intake was not associated with AAC severity in men. Higher baseline dietary quality assessed by Alternate Healthy Eating Index-2010 was associated with lower AAC severity by both radiography (OR (95% CI) for tertiles 3 vs 1: 0.53 (0.29, 0.99)) and DXA (0.38 (0.20, 0.70)). Multivariate-adjusted ORs (95% CIs) of incident MetS for the highest compared with lowest quartile of percentage energy intake from total, animal and plant protein were 1.46 (1.01, 2.10), 1.67 (1.13, 2.48) and 0.60 (0.37, 0.97), respectively. The multivariate-adjusted OR (95% CI) for incident type 2 diabetes in the highest compared with the lowest quintile of animal protein intake as % energy was 1.29 (0.99, 1.67). In the meta-analysis of 11 prospective cohort studies, the pooled relative risks for type 2 diabetes comparing the highest with the lowest category of total, animal, and plant protein intakes were 1.09 (1.06, 1.13), 1.19 (1.11, 1.28), and 0.95 (0.89, 1.02), respectively. Conclusion: Higher intakes of ALA and total omega-3 fatty acids and high quality diets were associated with lower risk of AAC in adults. Higher plant protein and lower animal protein consumption, and substitution of animal protein, were associated with lower incidence of MetS and diabetes. This research provides novel evidence regarding the potential benefits of adherence to dietary guidelines especially high intakes of plant foods and low intakes animal foods in the prevention of cardiometabolic disorders.
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ItemGenetic study of thyroid eye disease( 2016)Thyroid-associated orbitopathy (TO) is an autoimmune-mediated orbital inflammation that affects 25% of patients with Graves’ disease. The close temporal relationship between onset of Graves’ disease and TO suggests they share a common aetiology. While the complex inheritance of Graves’ disease is better characterized, little is known of the genetic susceptibility in TO. Multiple environmental factors such as smoking, male, older age are known risk factors for development of TO, however the extent of gene-environmental interaction remains largely unknown. The molecular mechanisms driving the development of TO is incompletely understood, hence targeted treatment options for TO remained limited. This thesis is undertaken to test the hypothesis that there is genetic susceptibility that predispose to development of TO. The research project initially examined exogenous risk factors associated with TO in a large Australian cohort with Graves’ disease, in order to identify environmental factors important for subsequent covariates adjustment when analyzing genetic findings. The risk factors association study found smoking, older age and longer duration of Graves’ disease correlated positively with TO, and secondarily there was relative selenium deficiency in TO cases compared to Graves’ disease patients without eye involvement. A genome-wide association study using deoxyribonucleic acid pooling approach and high-throughout array platform were used to discover gene variants associated with thyroid-associated orbitopathy in a case-control study design. The genetic findings were followed by a second stage individual genotyping targeting fewer markers to validate the genetics variants identified through genome-wide association study in the discovery cohort and independent replication study cohort. MACROD2, a novel gene that encodes an eraser of mono-ADP-ribosylation, possibly has a role in nuclear factor κβ signaling, showed evidence of association with TO in genome-wide association study and also in validation genotyping. A secondary aim of the thesis is to determine differentially expressed genes a priori in active TO orbital adipose tissue using microarray to explore molecular mechanisms of TO and to correlate gene expression findings with the genetic study. The study found TIMD4, DEFA1, DEFA1B, and DEFA 3 were over-expressed in active TO compared with inactive TO suggesting a pathogenic role of the innate immune response in TO. Active TO was marked by up-regulation of multiple genes involved in cell-mediated, innate and inflammatory responses with concurrent enhancement of orbital adipogenesis. For the first time, epigenetic factors was implicated in the pathogenesis of TO. However MACROD2 were not differentially expressed in active TO when compared with either inactive TO or normal control. Overall the findings from this thesis further our understanding on the genetic and environmental risk factors involved in thyroid-associated orbitopathy and give new insights into the underlying complex molecular mechanisms. The novel insights into candidate molecules and pathways can be explored to develop alternative treatment strategies for TO.
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ItemThe role of placental heparan sulphate proteoglycans in the pathogenesis of pre-eclampsia( 2015)Introduction: Uncomplicated pregnancies represent a hypercoaguable state. However, placental thrombosis is rare in these pregnancies which suggests that thrombin generation must be tightly regulated. In contrast, pregnancy disorders such as pre-eclampsia not only demonstrate an exaggerated increase in procoagulant activity which may contribute to the thrombotic lesions observed in the uteroplacental circulation of these pregnancies, but there is evidence of substantial cell growth, differentiation and angiogenic functional defects. Proteoglycans are abundantly expressed within the placenta compared to other human tissues. Particularly of interest are heparan sulphate proteoglycans which have important anticoagulant, anti-inflammatory and angiogenic properties. Hypothesis: Altered abundance, structure or function of placental heparan sulphate proteoglycans contributes to the development of pre-eclampsia by: increasing placental thrombin generation, interfering with cellular growth, differentiation, disrupting normal angiogenesis and altering growth factor interactions. Aims : 1) To determine the mRNA expression, protein abundance and cellular localisation of placental heparan sulphate proteoglycans from pregnancies complicated by pre-eclampsia and compare these to gestation matched controls. 2) To investigate the functional consequence of reduced HSPGs in placental cells 3) To determine the differences in the abundance and structure of heparan sulphate proteoglycans and heparan sulphate GAGs from placentae obtained from pregnancies complicated by pre-eclampsia and compare these to gestation matched controls. Methods: 1) The mRNA, protein abundance and cellular localisation of placental heparan sulphate proteoglycans was determined using real-time PCR, western immunoblotting and immunofluorescence, respectively. 2) To investigate the functions of reduced heparan sulphate proteoglycans, a cell culture model using short interference RNA was used. Cellular growth will be assessed using the xCELLigence system, thrombin generation using the calibrated automated thrombogram system and angiogenesis will be determined using a matrigel based assay. Cellular differentiation and apoptosis will be determined using real-time PCR. Growth factor signalling will be determined using a real-time PCR growth factor array. 3) To isolate proteoglycans from placenta, anion exchange chromatography will be utilised. Enzymatic digestion will be used to isolate the glycosaminoglycans, and enzyme-linked immunosorbant assays will be undertaken to determine the abundance of PGs and GAGs. Results: The mRNA expression of heparan sulphate proteoglycans, Syndecan 1, Syndecan 2, Glypican 1 and Glypican 3 are significantly reduced in the placentae of women whose pregnancies are complicated by pre-eclampsia. A cell culture model was utilised to determine the functions of heparan sulphate proteoglycans in the placenta. Successful downregulation of heparan sulphate proteoglycans was achieved in the cell lines using short interference RNA treatment, and a number of functions were significantly altered as a result. The downregulation of Syndecan 1, Glypican 1 and Glypican 3 resulted in significant alterations in the downstream growth factor targets. Reduced Syndecan 2 expression resulted in a significant reduction in the thrombin generation potential of endothelial cells. Investigation into the abundance and structure of glycosaminoglycans within the placenta, demonstrated heparan sulphate glycosaminoglycans to be significantly reduced in pregnancies complicated with pre-eclampsia compared to gestation matched controls. Conclusion: The reduction in heparan sulphate proteoglycans expression observed in pregnancies complicated with pre-eclampsia may be responsible for the altered growth factor interaction commonly observed in preeclamptic pregnancies. This study has provided us with a greater understanding of the biological role of heparan sulphate proteoglycans within the human placenta and its potential implications in the pathogenesis of pre-eclampsia.
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ItemVitamin D and HIV: exploring determinants of 25-hydroxyvitamin D and altered vitamin D metabolism in HIV-infected adults( 2015)Background: Vitamin D deficiency is a widespread global problem of particular importance for people with HIV. Vitamin D is an important hormone for endocrine and extra-endocrine functions, including modulating the immune system. Vitamin D deficiency is associated with reduced bone mineral density and infectious diseases, both common in people with HIV. Maintaining optimal vitamin D levels may help to reduce the risk of developing these diseases. The overall research questions for this thesis are: Firstly, which factors influence vitamin D metabolites, and secondly, what effect does vitamin D status have on bone and immunological outcomes, in people living with HIV infection? Method: To answer these questions, five studies were performed: 1. Cross-sectional comparison of 25(OH)D levels in HIV-infected and uninfected individuals in southern Australia. 2. Cross-sectional analysis of the determinants of 25(OH)D in people with HIV in Queensland and Melbourne, Australia. 3. Cross-sectional analysis of parathyroid hormone levels in antiretroviral-treated individuals. 4. Cross-sectional analysis of the effect of 25(OH)D on bone mineral density in antiretroviral-treated individuals. 5. Longitudinal analysis of CD4 cell count trajectory in antiretroviral-untreated individuals. Results: 1. People with HIV were more likely to be vitamin D deficient when compared with people without HIV in southern Australia. 2. Determinants of 25(OH)D levels: a. UV index and location are important determinants of 25(OH)D levels in people with HIV in Australia. b. Antiretroviral therapy impacts 25(OH)D levels: i. Efavirenz is associated with lower 25(OH)D levels, particularly in those with dyslipidaemia. ii. Protease inhibitors are associated with higher 25(OH)D levels. 3. The effect of tenofovir on parathyroid hormone levels depends on sex and ethnicity; parathyroid hormone levels were higher in non-white males using tenofovir. 4. Influence of ART and 25(OH)D on 1,25(OH)2D levels and bone mineral density: a. People using tenofovir have higher 1,25(OH)2D levels and people using protease inhibitors have lower 1,25(OH)2D levels. b. There is an interaction between tenofovir and 25(OH)D status on 1,25(OH)2D levels and bone mineral density. Vitamin D deficiency increases the odds of low bone mineral density only in those not using tenofovir. 5. Vitamin D deficiency may reduce the time to CD4 decline to <350 cells/μL in people living with HIV and untreated with antiretroviral therapy. Conclusions: Traditional risk factors for vitamin D deficiency and bone mineral density appear to be more important than HIV-related factors in people with HIV. In spite of this, antiretroviral therapy clearly alters vitamin D and bone metabolism. Understanding the mechanisms behind these alterations may assist in modifying the negative effects of vitamin D deficiency.
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ItemCross‐priming of TCD8+ specific for cell‐associated antigens( 2014)TCD8+ of the adaptive immune system play critical roles in the host defence against viruses and other pathogens through elimination of infected host cells. After infection, TCD8+ proliferate and adopt effector functions following recognition of specific antigenic‐peptides in the groove of a MHC class I molecule expressed by antigen presenting cells (APC). Professional APCs such as dendritic cells (DCs) are specialized for the priming and activation of naïve TCD8+. DCs are the key cell type responsible for T cell priming. They can either be directly infected and present viral antigens (direct‐priming) or phagocytose infected cell debris and process and present phagocytosed antigens to the specific TCD8+ (cross‐priming). Little is known about the actual contribution of direct versus cross‐priming during an immune response against viral infections. Understanding how DCs regulate TCD8+ responses is central for our ability to favourably manipulate the immune system as well as develop effective targeting strategies for optimal anti‐viral and antitumoral vaccination. To determine the contributions of direct versus cross‐priming to the clearance of an in vivo viral infection, we generated a Cre‐indicator transgenic mouse model utilising cutaneous HSV‐Cre infection to conditionally trigger model antigen expression in infected cells. This was expected to enable us to discriminate between virally infected and hence direct‐presenting DCs from uninfected and cross‐presenting ones in vivo for the first time. Unexpectedly, all generated transgenic mouse lines showed various levels of tolerance towards our model antigen due to undesired protein leakiness at steady state. However, TCD8+ in these neo‐transgene expresser transgenic mice possessed antigen ignorant properties and were non‐specifically activated following acute viral infection and the introduction of other innate immune cell ligands. Therefore, although these generated transgenic mice could not be used for their initial study purpose, their varying expression levels of model antigen make them an excellent model for studying peripheral tolerance induction and its maintenance. Cross‐priming is especially important for anti‐tumour immunity as tumour cells, although carrying tumour associated antigens, do not activate naïve TCD8+ efficiently due to an absence of co‐stimulatory molecules. Remarkably, our group has recently shown that influenza A virus (IAV) infection of allogeneic cells led to tumour protection due to enhanced cross‐priming of TCD8+ specific to cellular antigen. We have previously demonstrated that this enhancement was partially mediated through TLR7 sensing and entirely dependent on MyD88 and IFN signalling pathways, yet independent of the IL‐1β‐inflammasome. To further increase our understanding of cross‐priming enhancement mechanisms found in our system, we have additionally investigated the involvement of other immunological mechanisms in this thesis. Here, we show that IAV enhanced crosspriming is independent from the IL‐18‐inflammasome signalling pathway but that TCD4+ helper play a surprisingly important role for optimal enhancement. Also, through investigations using Batf3‐/‐ mice, we not only confirm the specificity of CD8α+ and CD103+ DC subsets for cross‐presenting, but also demonstrated that there are two types of cross‐priming outcomes: a baseline cross‐priming that is innate signalling independent and an innate immune signal enhanced crosspriming pathway. Interestingly, both types of cross‐priming events were abolished in Batf3‐/‐ mice. This knowledge will be useful to aid future efforts to develop more robust cancer vaccines. Finally, efficient antigen processing and presentation of the TCD8+ epitope is a central factor that determines the extent of specific T‐cell responses. Our group has identified the most immunodominant T‐cell response in the Balb/c mice after IAV infection. Interestingly, this novel epitope is encoded by the intronic region of the non‐structural protein mRNA. To dissect the mechanism of such epitope translation, as being either through spliced mRNA translation or generation using an alternative open reading frame (AltORF), we disabled splicing mechanisms and discovered that generation of the novel epitope occurred through AltORF translation. We will focus on the identification of the exact mechanism(s) underlying the efficient generation of this novel epitope as such mechanisms may provide a great opportunity for developing more efficient IAV T‐cell based vaccine strategies.
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ItemLong term effects of dietary calcium intake on fractures, mortality, cardiovascular diseases and abdominal aortic calcification: a prospective cohort study( 2014)High calcium intake can prevent age-related bone loss; however, its efficacy in fracture prevention is not clear. Some recent studies have associated calcium supplementation with adverse cardiovascular events (CVE). Scientific evidence on the long term effects of dietary calcium intake (DCI) and fracture or CVD risk lacks consistency.