Medicine (Northwest Academic Centre) - Theses
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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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ItemSun exposure and type 2 diabetes mellitus: Can sun exposure lower type 2 diabetes risk?( 2015)Background: Lower serum 25-hydroxyvitamin D (25OHD) levels have been consistently associated with increased type 2 diabetes mellitus (T2DM) prevalence and incidence in systematic reviews and meta-analyses of observational studies, however this association has not consistently been replicated in vitamin D supplementation trials. This disparity may be due to a number of different factors: lack of power in the trials due to small sample size, insufficient duration of dosing, baseline vitamin D or glycaemic status differing between studies, low supplementation compliance or insufficient vitamin D dose. Alternatively, lower 25OHD levels may be a product, rather than a cause of ill-health, or they may share pathology earlier in life or in disease progression so that supplementing with vitamin D in adulthood has no effect on disease outcome. This body of work presents another explanation: given that sun exposure is the most influential contributor to serum 25OHD levels, observational studies may be reporting an effect of sun exposure, rather than vitamin D, on T2DM. Therefore vitamin D supplementation trials may be failing to capture any additional benefits of sun exposure through non-vitamin D pathways. The aim of this body of work was to investigate the possible association between sun exposure and T2DM endpoints reported in scientific literature as well as in an original analysis. A major objective of the original analysis was to determine whether or not any association found between sun exposure and T2DM incidence was through non-vitamin D pathways. Methods: Following a literature review, a systematic review of observational studies reporting on associations between sun exposure variables and T2DM-related endpoints was conducted. The potential of an association between sun exposure- measured using ambient ultraviolet radiation (UVR), and five-year cumulative incidence of T2DM was explored using a prospective, national diabetes cohort (AusDiab). A causal mediation analysis was undertaken to explore whether or not there were effects of ambient UVR on cumulative T2DM incidence, via non-vitamin D pathways. Results: The systematic review revealed that high-level evidence for an association between sun exposure and T2DM-related outcomes was lacking. There was moderate-level evidence for greater sun exposure reducing T2DM incidence. The opposite was found in the original analysis using the AusDiab cohort: ambient UVR was associated with increased T2DM incidence (OR=1.17, 95% CI: 1.01-1.36, p=0.04). This association was independent of an effect of age, sex, body mass index, physical activity, ethnicity, smoking status and serum 25OHD levels, but was likely to be confounded by area-level determinants of health due to the nature of the exposure variable. The major limitations of this work were that the sun exposure measures were suboptimal. Time-of-year measures were the most common sun exposure variables contained in the systematic review, and ambient UVR at the site of participant recruitment was the proxy for sun exposure in the original analysis. Conclusion: There is likely to be a complex relationship between sun exposure and T2DM. The direction of the association between sun exposure and T2DM incidence, as well as delineation of the mechanistic pathways through which this association may exist, are yet to be confirmed. Future studies are encouraged to use person-level sun exposure measurements, and findings from such studies may influence sun protection policy.