Medicine & Health

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  • (2020) Lau, Derrick
    Thesis
    The HIV-1 capsid is a protein shell utilised by the virus as a binding platform to hijack host machineries to promote infection. Characterisation of novel cofactors binding to capsid demands sensitive and quantitative assays to better understand their underlying binding mechanisms. Here, I have developed three complementary fluorescence microscopy-based platforms for quantitative characterisation of capsid binding analytes using tubular, spherical or conical lattices self-assembled from recombinant capsid protein (CA) with engineered cysteine residues for cross-linking as surrogates for the authentic capsid. The first approach focused on the development of a TIRFM biosensor using CA tubes immobilised on a glass surface as the biorecognition substrate for sensitive single-molecule binding studies. The biosensor was assessed in its ability to measure binding affinity, stoichiometry and kinetics using known capsid-binding cofactors as part of its validation process. Biosensor measurements confirmed that binding of the host cell protein cyclophilin A (CypA) to the tubular lattice is substoichiometric due to steric hindrance between CypA molecules. Binding analysis of cleavage and polyadenylation specificity factor subunit 6 (CPSF6) expressed as a full-length protein in a cell-free expression system suggested a role of oligomerisation to enhance binding via avidity. In the second approach, the biosensor surface was modified with cross-linked CA spheres as an alternative substrate containing pentameric defects and highly curved regions that are lacking in CA tubes. Binding of CypA to spheres revealed a significantly higher CypA to CA binding ratio at saturation suggesting a role of curvature in accommodating specific host cofactors. The third approach utilises confocal microscopy-based single molecule spectroscopy and was developed as a medium-throughput capsid interactor screening platform that uses conical CA self-assemblies as the substrate. Fluorescence-tagged analytes are identified as capsid binders when they accumulate on the surface of the CA cones, which can be detected in the fluorescence intensity traces as the appearance of spikes in the analyte channel or as an increase in the coincidence between the analyte signal and CA signal. The assay can be adapted for competition studies by using antagonistic molecules or different CA mutants to dissect binding interfaces on the capsid. The three assays developed herein are complementary methods to accelerate characterisation of novel capsid binders.

  • (2021) Cheng, Lap
    Thesis
    Chronic kidney disease (CKD) is a major public health issue affecting 10% of the global population and resulting in 1.2 million deaths. The risk of all-cause and cardiovascular mortality is inversely proportional to declining eGFR such that individuals with CKD are more likely to die, primarily due to a cardiovascular cause, than survive to the point of requiring dialysis. These poor outcomes are related to a myriad of factors including multimorbidity, medial vascular calcification and underlying haemostatic dysfunction. Polypharmacy is a key consequence of the disease burden experienced by CKD patients. It is linked with poor adherence, adverse drug reactions, falls and increased hospitalisations. A post-hoc analysis of the CKD-FIX study was conducted to assess the prevalence and predictors of polypharmacy in CKD patients. It found that polypharmacy, defined as ≥5 medications, and hyperpolypharmacy, defined as ≥10 medications, were found in 77.5% and 34.3% of patients respectively. Age ≥65 yrs, diabetes, cardiovascular disease and hyperlipidemia were independently associated with polypharmacy. However, limiting polypharmacy via appropriate prescribing is hampered by the lack of data in patients with advanced CKD. Despite the disproportionate cardiovascular disease burden in CKD, the benefits and risks of dual antiplatelet therapy are not known. Therefore a systematic review of randomised controlled trials on the effectiveness of dual antiplatelet therapy in CKD was conducted. Nineteen trials with 27,308 participants were analysed with all but 3 trials excluding participants with dialysis-dependent kidney failure. Compared with aspirin monotherapy or no study medication, P2Y12 inhibitor-based dual antiplatelet therapy significantly reduced the risks of major adverse cardiovascular events, myocardial infarction and stroke; but increased the risk of major bleeding. There was insufficient evidence to conclude whether patients with advanced stages of CKD and dialysis-dependent kidney failure derived benefit. In conclusion, CKD patients represent an expanding population that is at high risk of adverse outcomes. Polypharmacy is common in patients with CKD and is related to age and multimorbidity. Further research on medication appropriateness and deprescribing are needed. In particular, adequately powered randomized trials are required to evaluate the effectiveness of dual antiplatelet therapy in patients with advanced stages of CKD and cardiovascular disease.

  • (2021) McKenzie, Briar
    Thesis
    A quarter of adult deaths are attributable to suboptimal diets globally, with cardiovascular (CVD) and metabolic diseases being the leading cause of diet-related deaths. The aim of this thesis was to investigate the relationship of sex and gender with diet and cardiometabolic diseases via a range of geographically diverse studies, through a mixed methods approach. Three quantitative studies focusing on sex differences were conducted: one looking at biases in relation to self-reported energy intake by a systematic review and meta-analysis; one cross-sectional analysis of dietary behaviours and associations with cardiometabolic risk factors in seven low- and middle-income countries (LMICs); and a prospective analysis of cohort data from the UK looking at dietary intake and associated risks of CVD and premature mortality. Questions arising from these studies were explored through qualitative studies in Fiji: a policy landscape analysis and focus group discussions to understand gender differences in diet knowledge, attitudes and behaviours and gender considerations in policies. No sex bias in the accuracy of dietary assessment was identified, with similar levels of energy underestimation by women and men. Across the seven LMICs in the cross-sectional analysis, both women and men had poor dietary behaviours, however, women who reported positive (good) salt use behaviour were less likely to have undiagnosed hypertension (not evident for men). Diets of women and men were also poor in the UK cohort, yet an association between specific combinations of macronutrients and a reduced risk of mortality was identified for women and men, and a reduced risk of CVD for men. The policy analysis conducted in Fiji revealed a conflation between “gender” and “reproductive health”, and that gender differences in diet-related diseases were not viewed as policy issues. Finally, the focus group discussions identified gender constructs around food, however, upstream determinants of poor diets such as climate change and socioeconomic factors were identified as crucial influences on diet, by women and men. Collectively, findings identified poor diets for both sexes, with some modest sex differences in associations between diet and disease, which are unlikely due to differences in reporting. Results from the qualitative studies highlight the importance of considering gender in view of other equity factors. These findings will be important in the development of equitable food policy globally.

  • (2020) Dereli, Ayse
    Thesis
    Chronic respiratory diseases including chronic obstructive pulmonary disease, obesity hypoventilation syndrome and obstructive sleep apnoea involve living in a chronic hypercapnic state and are associated with high mortality rate, poor prognosis and low quality of life. Affected individuals exhibit a reduction in CO2-stimulated ventilatory responses, indicating plasticity in ventilatory behaviour. This thesis investigates whether neurons mediating the central respiratory chemoreflex are involved in this plasticity. The retrotrapezoid nucleus (RTN) contains chemoreceptor neurons that modulate ventilation via glutamatergic innervation of the ventral respiratory column (VRC), the central pattern generator. Recently, RTN neurons were found to express inducible neuropeptides including the inhibitory neuropeptide galanin. Microinjections of galanin into the VRC blunts CO2-mediated chemoreflex responses, however the function of galanin in the respiratory chemoreflex circuit is not clear. Hence, this thesis hypothesised that galaninergic RTN-VRC circuitry contributes to centrally mediated adaptation to long-term hypercapnia (LH) and is involved in the pathophysiology of associated respiratory disorders. The first aim explored neuropeptide gene expression in mouse RTN following room air, short-term hypercapnia (SH, 6 or 8 hrs, 5 or 8% CO2) or LH (10 days, 8% CO2). The results revealed a biphasic pattern of neuropeptide mRNA expression, with a decrease following SH and increase following LH. The second aim investigated CO2-responsiveness of chemoreceptor populations following LH (c-Fos immunoreactivity). LH blunted responsiveness of all regions to AH (1 hr, 10% CO2) except for the RTN which retained its CO2-sensitivity. The last aim used retrograde tracing to identify all galaninergic neurons projecting to the VRC, and whether VRC neurons express galanin receptors (GalR), using fluorescent in situ hybridisation. It was revealed that 29 brain regions project to the VRC, amongst which 5 were galaninergic and only RTN neurons were CO2-responsive (increased c-Fos). Finally, VRC neurons contained ~850 GalR1+ neurons with 27% co-labelled with glycine transporter 2. In conclusion, this thesis characterises galaninergic VRC circuitry and suggests that galaninergic neurotransmission from the RTN contributes to diminished hyperventilatory response observed during adaptation to LH.

  • (2021) Thittamranahalli Kariyappa, Jyothi
    Thesis
    This thesis focuses on variation in cortical structure across diprotodontid marsupials and aims to elucidate the archetypal features of the mammalian cortex and those transformations that are of evolutionary importance. The study analysed these features both qualitatively, quantitatively and also compared MRI and DTI derived connectome reconstructions in representatives of the major families of diprotodontids with a representative eutherian (the laboratory mouse). Overall findings suggest that a number of features of the cerebral cortex are retained in all the species examined and have been conserved throughout mammalian evolution and likely represent key aspects of neocortical organization. The differnces observed may be solutions to environmental challenges. For example, a different scaling relationship of hippocampal volume against brain volume has been found amongst the diprotodontids and eutherians. Very small-brained diprotodontids have quite small hippocampal formation volume for their brain size, but the hippocampal formation size increases more steeply with brain size amongst diprotodontids compared to eutherians. The role of the hippocampus in spatial cognition is of direct significance to home range and in species where social behaviour plays an important role, the involvement of the hippocampus in social information processing is also of ecological significance.

  • (2021) Peng, Wang
    Thesis
    The HIV-1 capsid interacts with mutiple host factors during its early life cycle to achieve succesfful infection. In this thesis we aim to expand our understanding of how interactions between the capsid and host cell factors regulate viral activities, with chapter 4 and 5 specifically focusing on the cytoplasmic transport process. Based on cellular and biochemical evidence, recent studies suggest that HIV-1 capsid hijacks both dynein and kinesin-1, via the adaptor proteins BICD2 and FEZ1 respectively, for active transport along microtubule network. We will present our work to reconstitute these complexes and demonstrate their motilities along microtubles in vitro, providing more direct evidence to support these models. To investigate the interactions between HIV-1 capsid and the motor adaptors, we applied a fluorescence fluctuation spectroscopy based binding assay and a total internal reflection microscopy (TIRFm) based binding assay. These approaches allowed us to generate quantitative descriptions of the interactions between HIV-1 capsid and the adaptor proteins. Subsequently, we reconstituted the motor-adaptor-cargo complexes (DDBC and KFC) using recombinant proteins as well as components isolated from native tissues. We have successfully demonstrated both the dynein- and kinesin-dependent transport of HIV-1 capsid along microtubules in vitro using a TIRFm based single molecule motility assay. We also further charaterized the motile behaviors and properties of the KFC complex. Our work validated the proposed models for the cytoplasmic transport of HIV-1 capsid and demonstrated the minimum requirement for this process. In general, this work has made solid contributions towards the understanding both HIV viology and motor-driven cargo transport, as well as opened oppotutnities to a range of exciting research questions in both fields.

  • (2021) Santos, Joseph Alvin
    Thesis
    Background: The totality of evidence shows that high salt intake increases cardiovascular risk, and a number of studies have suggested its link with diabetes mellitus. Many studies also indicate that reducing salt intake will reduce these risks; however, most of the evidence is from high income countries, and little is known about how this applies to LMICs. Methods: This PhD combined systematic reviews of existing evidence and Bayesian modelling to contribute evidence on the effectiveness of salt reduction interventions, the relationship between salt intake and cardiometabolic outcomes, and factors (individual, household, national-level) affecting salt intake, with a view to better informing future interventions in LMICs. The research comprises three components: (1) a review of national salt reduction initiatives around the world; (2) an in-depth examination of evaluated salt reduction interventions at all levels of implementation in LMICs, and; (3) secondary analysis of data from LMICs using Bayesian methods. Results: The number of national salt reduction initiatives globally increased from 75 in 2014 to 96 in 2019, with a 43% and 18% increase in the number of initiatives in upper-middle- and lower-middle-income countries, respectively. Evaluations of salt reduction interventions in LMICs, implemented at all levels (i.e. not just national), showed that they were successful in producing positive outcomes such as decreased salt intake, lower sodium levels in foods, improved knowledge, attitudes and behaviours towards salt, or decreased blood pressure. The secondary analysis found limited data on cardiometabolic outcomes, apart from systolic blood pressure, and most datasets only contained individual-level factors. The Bayesian analysis showed that salt intake was positively associated with systolic blood pressure (β 0.15, 95% credible interval 0.07 to 0.22) and the factors that influenced salt intake in LMICs were sex, age, BMI, education level, history of diabetes, and salt-related attitudes. Conclusion: While there was little data on other cardiometabolic outcomes, this research reaffirms the relationship between high salt intake and blood pressure, and shows the factors that influence salt intake in LMICs. Combined with existing global evidence on the effectiveness of salt reduction interventions, the findings from this PhD produce a strong case for urgent action to reduce salt intake in LMICs.

  • (2021) Gupta, Medhavi
    Thesis
    Globally, drowning is the second largest cause of death by injury in children aged 1-14 years old. Risk factors for child drowning include poor supervision, lower socioeconomic status, poor swimming and rescue skills, and the proximity of open water near homes. These are more prevalent in low-and middle-income countries(LMICs). The WHO has developed recommended interventions for drowning prevention in rural LMIC contexts, such as the provision of supervised childcare to prevent access to nearby water bodies. This thesis explores the process of developing and evaluating drowning prevention programs in two high-risk LMIC regions: the Sundarbans in India and the Barishal Division in Bangladesh. As no previous research on drowning burden and prevention has been conducted in India, the main aims were to: (1) Identify the burden of child drowning in the Sundarbans, and (2) identify implementation strategies for drowning prevention programs. Conversely, drowning prevention programs have been implemented in Bangladesh, but evaluation of their implementation remains. The Anchal program provides supervised childcare to younger children, while SwimSafe provides swim training to older children. The main aims in Bangladesh were to: (1) Understand implementation implications and best practices, and (2) understand the impact of gender norms on implementation. The findings from the Sundarbans mortality survey showed a significant burden of drowning, with a rate of 243.8/100 000 for 1-4-year-old children, and 38.8/100 000 for 5-9-year-old children. Common circumstances were the lack of effective adult supervision, no physical barriers against water, and proximity of open water to homes. Findings from the analysis of relevant government policy and interviews with community-based stakeholders identified three existing government programs that could be leveraged for the implementation of drowning interventions. In Bangladesh, the mixed-methods process evaluation of the Anchal program showed that while the program was acceptable in the community, geographical barriers to access, cultural beliefs and inadequate resources reduced attendance, limiting effectiveness. The gender analyses of both Anchal and SwimSafe programs revealed opportunities to ensure equitability. Fewer older girls enrolled in SwimSafe classes compared to boys due to cultural concerns. Female community-based staff found that employment in the programs improved social status, physical mobility and access to resources.

  • (2021) Ouyang, Menglu
    Thesis
    Introduction: Although clinical guidelines recommend various care processes to improve outcomes of patients with stroke, evidence to support many of them, such as the management of post-stroke infections and the monitoring of abnormal physiological variables, are scarce. While for those care processes with more evidence, very few studies have quantified their variations across regions and what factors influence their implementation in clinical practice. This thesis aims to determine the utilisation of guideline-recommended care processes for patients with acute stroke, and explore various strategies that may improve their implementation. Methods: I conducted secondary analyses of a large clinical trial to explore the associations of care processes and clinical outcomes, using data of 11,093 patients with acute stroke from nine countries. These care processes included dysphagia screening, indwelling urinary catheterisation (IUC), and early detection of low blood pressure (BP) and oxygen saturation (SaO2) levels. To explore variations in the utilisation of care processes, I compared the evidence-based recommendations for stroke unit care across Australia/UK, China, India/Sri Lanka and South America. I also conducted a process evaluation of a ‘quality improvement’ intervention within an ongoing trial involving the management of patients with acute intracerebral haemorrhage in China, to explore what factors could improve the implementation of systems to improve the quality of care. Results: Patients who failed a dysphagia screen, had an IUC, had SBP <120mmHg or SaO2 <93% during the acute phase (up to 7 days after stroke onset) had increased odds of poor outcome. The utilisation of care processes varied across regions, with lower probabilities of reperfusion therapy and allied health care in low- and middle-income countries (LMICs) than high-income countries. Constant training with the clinicians, case reviews, optimisation of workflow within available resources, and having a dedicated team, may facilitate the implementation of evidence-based care. Conclusions: The utilisations of guideline-recommended care processes are associated with patient outcomes and vary across regions. Timely assessment and appropriate management should be provided to those with dysphagia, IUC, low BP, and low SaO2 levels, in an effort to improve their recovery after stroke. Future studies are needed to confirm the causality of these associations and to examine opportunities to promote the delivery of evidence-based stroke care, especially in LMICs.

  • (2021) Kirubakaran, Ranita
    Thesis
    Tacrolimus is the key immunosuppressant used in most solid-organ transplant recipients, including heart transplants, to prevent graft rejection. However, tacrolimus dosing strategies are complicated by the narrow therapeutic window and considerable pharmacokinetic variability. Individualising lifelong tacrolimus therapy to avoid graft rejection and minimise adverse effects is essential for heart transplant recipients. This thesis aimed to investigate the individualisation of tacrolimus therapy in adult heart transplant recipients using the pharmacokinetic modelling approach. In Chapter 1, I present an overview of tacrolimus clinical pharmacology, including clinical factors influencing tacrolimus pharmacokinetics (e.g., concomitant azole antifungal therapy). In Chapter 2, I explore tacrolimus dosing and monitoring practices in heart transplant recipients (n=87) at St. Vincent’s Hospital Sydney, a major heart transplant centre in Australia. Additionally, I assess the ability of a Bayesian dosing software, approved by the Therapeutic Goods Administration to predict tacrolimus concentrations in heart transplant recipients. Tacrolimus dosing and monitoring practices were discordant with the hospital guidelines. The population pharmacokinetic model integrated within the software was suitable in guiding tacrolimus dosing only after 11 days of therapy. This finding necessitated the identification of other model(s) that might be more suitable for use in heart transplant recipients, particularly for the immediate post-transplantation phase. In Chapter 3, I conduct a systematic review summarising published population pharmacokinetic models of tacrolimus (n=69) developed from various organ transplant recipient populations. In Chapter 4, I select relevant tacrolimus models (n=17) from the systematic review and evaluated their predictive performance in heart transplant recipients (n=85). The evaluated models displayed poor predictive performances. This finding complements the work from Chapters 2 and 3 highlighting a tacrolimus model for heart transplant recipients is required. In Chapter 5, I successfully develop a tacrolimus population pharmacokinetic model for heart transplant recipients. The model incorporated the effects of concomitant azole antifungal use, haematocrit, and body weight on tacrolimus pharmacokinetics. Model evaluation in an independent heart transplant recipient cohort displayed good model performance. The model can be implemented in clinical practice to individualise tacrolimus dosing in heart transplant recipients. In Chapter 6, I discuss the clinical implication of this work and recommendations for future research.