Medicine (Austin & Northern Health) - Theses
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ItemTargeting malnutrition to improve patient and clinical outcomes in liver tranpslantation( 2023-12)Background: Malnutrition and sarcopenia are highly prevalent in decompensated cirrhosis and are independently associated with increased morbidity and mortality before and after liver transplantation. Broadly defined, malnutrition in liver disease is characterised by dry weight loss, muscle wasting, fatigue, and weakness; occurring when the diet does not provide sufficient calories and protein to maintain nutritional status, or the body is unable to fully absorb or utilise food consumed secondary to liver disease. Sarcopenia encompasses the complete spectrum of reduced muscle mass, muscle strength and muscle function. Despite the established interplay of malnutrition, sarcopenia and poor patient outcome, effective therapies to improve nutritional status and muscle function in the pre-transplant period have mostly remained elusive, and any subsequent impact on post-transplant outcomes have not been described. The ability to accurately define and subsequently achieve energy requirements in cirrhosis is poorly described in the current literature and may contribute to the outcomes described thus far, which are conflicting and fail to demonstrate consistent improvement in muscle and nutrition parameters. Aims: This research aims to discover nutritional interventions that prevent nutritional decline and improve functional status in patients awaiting liver transplantation, and to accurately characterise energy requirements in cirrhosis. We hypothesise that targeted enteral feeding in the pre-transplant period will be superior to standard high-energy, high-protein oral diet in delivering improved nutritional, functional and clinical outcomes in patients before and after liver transplantation. Methods: A retrospective analysis of 373 patients consecutively transplanted at a single centre was conducted to assess the impact of pre-transplant nutritional status and muscle function on post-transplant clinical outcomes and healthcare costs. A prospective observational study then followed 110 candidates under assessment for liver transplant and compared their measured energy expenditure (via indirect calorimetry) with estimated energy requirements (via predictive equations). Patient and clinical factors predictive of measured energy expenditure were also explored. An additional observational study of patients treated with continuous terlipressin infusion for management of portal hypertension, evaluated the impact of terlipressin therapy on dietary intake and muscle strength in a small cohort of patients awaiting liver transplant. Finally, a prospective randomised controlled trial comparing pre-transplant enteral feeding with standard high-energy high-protein diet in 50 malnourished and sarcopenic patients, evaluated the effect of enteral feeding on patients’ nutrition, muscle and immune function both before and after transplant. Results: In patients undergoing liver transplant surgery, severe malnutrition and low grip strength were independent predictors of adverse post-transplant outcomes including ICU length of stay, hospital length of stay, and post-transplant infection (p all < 0.05). Additionally, hospital costs were 30% higher in severely malnourished compared to well-nourished recipients (p = 0.012). When prospectively analysing predicted versus measured energy requirements in cirrhotic patients, there was poor correlation between the two methods, and hypermetabolism was common. Treatment of portal hypertension with continuous terlipressin infusion was associated with significantly increased dietary energy and protein intake, by 54% and 56%, respectively (both p < 0.001); which translated to a 3.13 kg (SD 3.55), or 12% increase in HGS (p < 0.001). The randomised controlled trial demonstrated that nasogastric feeding for a median 84.5 days (IQR 40.25 - 130.5) resulted in a median increase in HGS of 3.90kg (2.02 - 5.05), compared to a decrease of 0.35kg (-3.02 to 0.63) in controls (p < 0.001). Dry body weight, mid upper-arm circumference, triceps skinfold and immune function all increased significantly for the nasogastric feed group compared to no change in controls. Post-transplant clinical outcomes were similar between groups. Conclusion: Malnutrition and reduced muscle strength are highly prevalent in cirrhotic patients and are independent predictors of poor outcomes after LT which significantly increases healthcare costs. Nutritional interventions in this cohort should involve measurement of individual energy requirements, as relying on predictive equations in this patient population may result in significant under or over feeding. Effective treatment of portal hypertension with terlipressin infusion appears to improve nutritional and muscle parameters not previously described and warrants further investigation. Finally, targeted enteral feeding before liver transplant improves grip strength, anthropometric markers, and immune function in severely malnourished patients. Larger-scale studies are required to assess the effect of enteral feeding on clinically meaningful endpoints such as infection prevalence, post-transplant length of stay and survival.
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ItemThe therapeutics of Diabetes Mellitus : developmental and regulatory aspects(University of Melbourne, 2006)
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ItemThe expression and functional roles of angiotensin II receptors in prostate cancer(University of Melbourne, 2006)
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ItemClinical studies and interventions for sarcopenia in cirrhosis( 2023-09)Sarcopenia, defined as reduced muscle mass and impaired muscle function, is associated with higher morbidity and mortality in patients with advanced liver disease. Despite its recognised prognostic importance, radiological tools recommended for the measurement of muscle mass are either not widely validated in cirrhotic cohorts or poorly accessible amongst clinicians. As such, uptake of sarcopenia assessment in the clinical setting is greatly limited. The mechanisms underlying sarcopenia associated with cirrhosis are complex. The pathophysiology of muscle loss in this population differs from sarcopenia associated with aging and other chronic diseases as it is driven by different metabolic, hormonal and physiological changes that are specific to advanced liver disease. In particular, the development of portal hypertension and its sequelae such as ascites may contribute to reduced nutritional intake, impaired nutrient absorption, increased heat loss and systemic inflammation. Branched-chain amino acids (BCAAs) are an integral part of muscle homeostasis and promote muscle protein synthesis and anabolism. BCAA levels are reduced in cirrhosis due to protein malnutrition and utilisation in extra-hepatic ammonia clearance. These mechanisms provide several targets for interventional clinical trials. Yet there is a lack of prospective studies examining therapies to prevent or ameliorate sarcopenia in cirrhosis. It is also uncertain whether addressing the drivers of muscle loss in cirrhosis will improve outcomes. This thesis sought to address current deficiencies in the cirrhotic literature surrounding diagnostic tools and interventions for sarcopenia. The first study validated the use of an open access web-based program for the measurement of muscle mass using CT scans amongst clinicians in a cohort of cirrhotic patients. This was compared to a widely used dedicated software program. The new program showed excellent inter-rater and between-program agreement for the measurement of muscle mass and provided an accurate and accessible platform for clinicians to assess sarcopenia in patients with cirrhosis undergoing CT scans. The second diagnostic study aimed to assess the use of dual-energy x-ray absorptiometry (DEXA) in predicting post-transplant outcomes in patients with cirrhosis undergoing liver transplantation. We identified reduced upper limb lean mass as a novel predictor of adverse post-transplant outcomes including sepsis and length and stay in men undergoing liver transplantation. Two prospective studies were conducted aiming to evaluate different interventions on sarcopenia, frailty and physical performance in patients with cirrhosis. The first was a cohort study of 12 patients who were referred for transjugular-intrahepatic portosystemic shunt (TIPS) insertion. This is a radiological procedure used to treat complications of portal hypertension, a major driver of sarcopenia in cirrhosis. Compared to baseline, at 6-months post TIPS insertion, patients demonstrated a significant increase in muscle mass and subcutaneous fat mass. There however was no improvement in muscle quality or function or physical frailty. TIPS insertion was associated with improvements in immune function as measured by a novel global immune function assay. Finally, we conducted a double-blinded randomised controlled trial of 150 patients to assess the effect of oral BCAAs supplementation on sarcopenia and clinical outcomes. Compared to a standard protein control, BCAAs did not improve measures of muscle strength, mass, performance or quality of life.
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ItemImmunopathogenic role of T cells in diabetic kidney disease (DKD)( 2023-05)Diabetic kidney disease (DKD) is a highly prevalent complication of diabetes mellitus (DM) and is the leading cause of end-stage kidney disease (ESKD). Approximately one-third of people with diabetes ultimately develop DKD, and the majority of them fail to receive kidney replacement therapy before dying from cardiovascular diseases. While components of the innate immune system have been shown to be active in the early stages of diabetes over time with disease’s progression in experimental models of DKD, the initiating factor of the increased activity of the adaptive immune system has not been fully understood. As for the role of the adaptive immune system, studies have demonstrated a high number of intrarenal CD4+, CD8+ and CD20+ cells, and found a positive association with the degree of albuminuria in people with type 2 diabetes mellitus (T2DM). In this thesis, we aimed to investigate the quantitative change in T cells from the adaptive immune system in the pathogenesis of DKD, and the sequence of infiltration of the adaptive immune system components in the kidneys of people with diabetes over the development of DKD. The overall hypothesis is that hyperglycemia leads to the quantitative and phenotypic change in T cells proportion, which promotes the proinflammatory milieu and kidney injury. Multiple experimental methodologies were undertaken in this study. First, our work utilised a pre-clinical diabetic model, db/db mouse model, with leptin receptor defect that mimics human T2DM and DKD. We also collected human kidney samples from individuals with T2DM, and we localized the distribution of T cells in the kidneys of people with different degrees of albuminuria with histomorphology approaches. In this histomorphology study, we found the influx of inflammatory cells in the kidney specimen of people with DKD, as presented by the presence of CD3+ T cells in the interstitial compartment of the kidneys. We then analysed the surface phenotypic characteristics of gamma delta T cells, monocytes/macrophages, regulatory T cells (Tregs), memory T cells and TRM. As a result, we identified a reduced level of a T cells subset not circulating in the blood, called tissue-resident memory T cells (TRM), in the cortex compartment of people with DKD compared to controls. In the db/db mouse model, we showed similar results exhibiting the infiltration of CD3+ T cells but a decreased fraction of TRM in the kidneys of diabetic mice compared to control, which may be due to the loss of the ligand for the binding of CD103 (one of the prime markers for TRM) in the kidney locally, termed E-cadherin. Next, we explored the novel miRNAs, mRNAs and proteins to explore the possibility of new biomarkers, comparing between traditional and machine learning methods using a panel of human kidney biopsies. Machine learning provides a useful tool for identifying a novel panel of miRNAs, mRNAs and proteins. Additionally, we found that a selected panel of important miRNAs, mRNAs and proteins could be utilized to predict eGFR efficiently. Therefore, machine learning was considered as a complementary approach in identifying novel targets in miRNAs, mRNAs and proteins during the development of DKD.
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ItemIdentification and characterisation of novel calcitonin receptor isoforms expressed in stressed cell lines derived from glioblastoma and its potential therapeutic advantages( 2023-08)Glioblastoma (GBM) is a deadly brain tumour without effective treatment resulting in only 50% patient survival 8 months post diagnosis and shows high resistance to conventional therapeutic treatments. It infiltrates the neuropil in a diffuse manner making its complete removal unachievable during surgery. The current treatment methods combining chemotherapy with radiotherapy are also inefficient in stopping relapse of the tumour. Therefore, there is a need to identify novel targets for effective therapeutic interventions to treat GBM. Previous studies by our group have implicated expression of calcitonin receptor (CT Receptor) by malignant tumour cells in 78% to 88% of GBM patient biopsies using in-house developed human anti-CT Receptor antibodies. CT Receptor was also found to be pharmacologically inactive in a majority of high-grade glioma (HGG) cell lines representing glioma stem cells pointing towards a novel role of CT Receptor. Further, there are two major splice variants of CT Receptor – highly characterised CTa Receptor and a longer isoform, CTb Receptor, with an additional 48 nucleotides in intracellular loop 1. Unlike CTa Receptor, CTb Receptor is poorly trafficked to the plasma membrane and its physiological roles have not been described. Chapter 1 is a comprehensive literature review elaborating characteristics of CT Receptor, glioblastoma and their relationship. Chapter 2 describes the vast expression of CT Receptor in mammals, developmental stages, diseases and conditions such as stress and quiescence; and the significance of CTb Receptor and its potential roles. Chapter 3 consists of a novel methodology using cDNA long-range nanopore sequencing to detect changes in CT Receptor expression and mRNA transcript profile in GBM cells induced with stress and presents anti-CT Receptor-fluorophore conjugates as potent indicators of programmed cell death. The study in chapter 4 explores the underlying mutations and/or splice variants of CT Receptor in GBM cell line U-87 MG using the cDNA nanopore sequencing strategy developed in chapter 3. We observed that the CTb Receptor isoform which is normally expressed by CT Receptor mRNA transcript variant 1 was actually a product of mRNA transcript variant 2 that gives rise to CTa Receptor isoform. This suggests that alternate splicing is evident in U-87 MG cells, a mechanism heavily exploited by cancer cells for survival. Other identified novel isoforms were found to be stochastically expressed and lacked exon(s) forming important protein domains which could be the reason behind abrogation of downstream signalling post ligand stimulation. Based on prior knowledge of CT Receptor distribution in Cos-7 transfected cells and U-87 MG data from chapter 4, four high-grade glioma cell lines were investigated for novel CT receptor isoforms in chapter 5. These cell lines (WK1, JK2, SB2b and PB1) were derived from GBM stem cell populations from patients and express CT Receptor at mRNA as well as protein level. CT Receptor in GBM cell lines is known to traffic to the membrane as well as localise in the cytoplasmic domain, predominantly near the nucleus. So, it was hypothesised that CT Receptor is associated with intracellular trafficking compartments and involved in efflux of chemotherapeutic drugs possibly via exosome pathway, attributing to an oncogenic survival function. Immunofluorescence observations in HGG cells lines using in-house as well as commercial antibodies and high-resolution confocal microscopy reveal association of both CTa Receptor and CTb Receptor with Rab11 (recycling vesicle), Rab27a (secretory vesicle) and LC3B (autophagosome) supporting our hypothesis. This led to our exploration of CT Receptor as a potential therapeutic target for treating GBM. Our group had conjugated plant toxins (dianthin-30 or gelonin, ribosome-inactivating protein (RIP)) and mAb2C4 which binds to an extracellular epitope of CT Receptor (detects both isoforms) and developed a potent immunotoxin with EC50 to be 10-20 pM in HGG cell lines. The last chapter of this thesis describes a method of immunotoxin development using click-chemistry to improve synthesis as well as penetration into solid tumours and eventually test this immunotoxin for uptake and toxicity in the HGG cell lines. The immunotoxin was successfully developed, however, the unprecedented developments due to Covid-19 pandemic caused a substantial impact on timelines and access to research facility. As a result, the fluorophore tagged toxin-antibody conjugate (Dianthin mAb2C4-Cy5.5) could not be characterised in HGG cell lines. Overall, this thesis examines CT Receptor from an oncoprotein lens in glioblastoma cell lines, aims to decipher CT Receptor mRNA isoforms expressed during stress and shed some light on CTb Receptor functionality.
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ItemOptimizing Cardiovascular Outcomes in Oncology Patients( 2023-06)Heart disease and cancer are the two leading causes of death in the developed world. Public health campaigns targeting these conditions have led to significant improvement in population awareness and prevention of disease, however, there remains an inadequate acknowledgement of their coexistence. Due to advancements in modern cancer therapy, we are seeing higher rates of cure and the conversion of a terminal illness into a chronic disease. As a result, cardiovascular disease now competes with cancer as the leading cause of death in survivors of certain tumour streams. Attention to reducing the risk of cardiovascular disease should thus be a priority in the long-term management of oncology patients. In other areas of cardiology, interventional and medical therapies are tested through rigorous large scale randomized controlled trials. These data form the backbone of national and international guidelines. In contrast, the cardiac management of comorbid cardio-oncology patients remains subject to significant physician bias as until 2022 there was a lack of evidence-based guidelines relevant to cancer patients, who have been traditionally excluded from cardiovascular trials. Using a combination of single and multi-site, prospective research, and the meta-analysis of available trials in evolving areas of cardio-oncology, this thesis focuses on the risk stratification and prevention of cardiotoxicity and cardiovascular management of patients with a past or present diagnosis of cancer. The topics addressed range from risk prediction of major adverse cardiovascular events and the echocardiographic and biomarker surveillance of breast cancer patients to the efficacy and safety of common cardiovascular interventions in patients with active cancer and cancer survivors. Finally, through a multi-centre randomized controlled trial of a smartphone-based cardiovascular risk reduction program in breast cancer patients, this thesis underscores the value of an innovative model of care to optimize physical activity and cardiovascular risk factor management in breast cancer patients.
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ItemApplication of High Resolution Peripheral Quantitative Computed Tomography to the Study of Bone Microstructure and Matrix Mineral Density in Health and Disease( 2023-06)Longevity increases the proportion of elderly persons in the community. Advancing age is accompanied by bone loss which compromises the volume, microarchitecture, and strength of both cortical and trabecular bone, particularly in women after menopause. Increasing fragility fracture risk is likely to be due to trabeculae thinning, perforation of trabeculae or reduction of trabecular numbers, thinning of the cortex and increased cortical porosity. Bone also becomes fragile due to changes in the material composition such as increased matrix mineral density, glycation of collagen and accumulation of unrepaired microdamage. These changes impact the mechanical properties of bone and make bones less able to resist loading, leading to an increased risk of fragility fractures. The diagnosis of bone fragility is currently based on evaluation of clinical risk factor and with measurement of areal bone mineral density (aBMD) of the lumbar spine, and proximal hip using dual-energy X-ray absorptiometry (DXA). BMD is normally distributed. Over 10% of people in the community have ‘so-called’ normal BMD (T-score more than - 1.0 SD), around 60% of persons have osteopenia (T-score - 1.0 to -2.5 SD) and around 30% have a BMD T-score less than - 2.5 SD below the young normal mean, the diagnostic threshold for ‘osteoporosis’. Because of the population distribution of BMD, most patients with fragility fractures in the community and most patients with diseases affecting the skeleton such as chronic kidney disease who suffer fragility fractures have osteopenia or normal BMD, not osteoporosis, so the use of aBMD threshold of less than – 2.5 SD has led to underestimation of the burden of bone fragility in the community. High-Resolution peripheral Quantitative Computed Tomography (HR-pQCT) with 82 or 61-micron isotropic voxel size is an in-vivo imaging modality. HR-pQCT enables quantifying bone microarchitecture, distinguishing trabecular and cortical compartments, and measuring cortical and trabecular microarchitecture with minimal radiation exposure (5 micro-Sv, equivalent to one day of background radiation) has made it an important modality to identify bone fragility in persons independent of their BMD level. HR-pQCT is used in musculoskeletal research to quantify bone microstructure of appendicular regions of interest, both in normal and pathological conditions. However, there are limitations in the application of this imaging method and in the interpretation of findings, that need to be recognised such as accurate segmentation of cortical from trabecular bone using threshold or non- threshold-based image analysis algorithms and challenges in the correct choice of a region of interest (ROI) which is chosen as a fixed distance from the distal joint line. This region differs in persons differing in arm length; it is more distal in taller individuals, more proximal in shorter individuals. For this reason, sex differences, racial differences and growth related differences in microarchitecture may be the result of differences in the positioning of the region of interest. The aim of this thesis was to address several of these technical limitations in methodology by using a non-threshold-based HR-pQCT image analysis and standardising the location of the ROI, so that sex- and race-specific differences in bone morphology could be correctly determined free of positioning errors. This method was applied in various disease states to establish the effects of disease on bone microarchitecture. We investigated distal radius bone morphology of 158 healthy Asians and Caucasian women and men aged from 21 to 53 years using HR-pQCT. Errors in positioning the region of interest misrepresents accurate quantification of bone microarchitecture because it varies point by point (slice-by slice) along the length of a bone. For example, adjusting the scanned ROI resulted in a 0.51 SD higher cortical porosity in Asian women and 0.32 SD higher cortical porosity in Caucasian women. Failure to correct the ROI overestimated porosity by 0.21 SD in Asian men and by 0.39 SD in Caucasian men. Failure to correct the ROI comparing older Caucasian women, 33 postmenopausal women aged from 73 to 95 years, underestimate the age-related increase in porosity by 0.40 SD. We investigated the effects of spinal paralysis on bone microstructure of distal radius and distal tibia and fibula of individuals with spinal cord injury. In this study, 32 men, 12 with tetraplegia and 20 with paraplegia, that were within 0.5 to 18.5 years of paralysis, were scanned by HR-pQCT. We report that following unloading caused by spinal cord injury, weight bearing regions adapted to accommodate greater peak strains, and strain rates changes had more severe microarchitectural deterioration than non-weight bearing regions that are normally adapted to lower peak strains and strain rates prior injury. These observations highlight the site specificity of the strain thresholds regulating the cellular activity of mechano-transduction. The rapidity of the change and irreversible microarchitectural deterioration suggest prompt intervention with antiresorptive, anabolic therapy, or both, warrants consideration. We also studied the effect of chronic kidney disease (CKD) on bone microstructure of 128 patients with CKD and compared them with 275 age- and sex-matched controls. We report that most patients with CKD stages 4-5D and kidney transplant recipients had osteopenia or normal femoral neck BMD, not osteoporosis. Despite these modest deficits in femoral neck BMD, distal tibial and distal radial microarchitecture and estimated failure load were compromised, even in patients with normal BMD. Cortical and trabecular distal tibia and distal radius microarchitecture, not femoral neck BMD, were independently associated with estimated failure load and accounted for over 85% of the variance in failure load estimated at these metaphyseal locations. Resistance to fracture is achieved by modelling and remodelling adding bone to, or removing bone from, its periosteal (external) and endocortical, intracortical, and trabecular components of its endosteal (internal) surfaces. Quantification of macro/micro-structure of 18 radii and 5 femora human post-mortem specimens show that bone mass is constant along the radius, femur, and femoral neck. Along the metaphyseal regions, the constant mass was fashioned with a large void volume and high surface area/matrix volume forming mainly trabecular bone. At the middiaphyseal regions the same mass was fashioned with small intracortical and medullary void volumes and low surface area/matrix volume forming cortical bone. In addition, quantification of distal radius and tibia of 94 women, using HR-pQCT, showed that bigger bones are assemble with relatively less cortical mass of higher porosity and lower matrix mineral density. In conclusion, the aging population is at higher risk of fragility fractures due to age-related bone loss and deteriorated bone microstructure. HR-pQCT provides three-dimensional bone microstructure analysis of human extremities as in-vivo, with minimal radiation exposure and high-resolution images. We offered a methodological solution for standardizing positioning and scanning of ROI and accurately quantifying bone microarchitecture, and in pathological conditions. We investigated the impact of spinal cord injury on bone microstructure and the effects of chronic kidney disease on bone strength, highlighting the significance of cortical and trabecular microarchitecture in assessing bone health.