Medicine (St Vincent's) - Theses

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    Quality of endoscopy in the detection and management of gastrointestinal pathology
    Yang, Linda Seung-Won ( 2022)
    Endoscopy is essential in gastrointestinal medicine by allowing direct mucosal visualisation, histologic confirmation of pathology, and therapeutic procedures. Defining, measuring, and maintaining the quality of endoscopy is paramount in optimal patient care and cost-effective resource utilisation. Quality assurance in the field of colonoscopy is well-established, with adenoma detection rate and withdrawal time already implemented as key quality indicators. However, the quality of gastroscopy is under-investigated despite its importance. The studies in this thesis examined the current quality of endoscopic practice in Australia and explored methods of improvement. In addition, the use of artificial intelligence was reviewed to guide future research in the quality of modern endoscopy. This thesis identified that the current quality of gastroscopy was suboptimal based on international society guidelines. However, a simple educational intervention on endoscopists improved quality of gastroscopy which resulted in an increased detection of clinically significant pathology. Provision of up-to-date endoscopic procedures is another important aspect of quality assurance. The uptake of novel endoscopic techniques also requires more widespread awareness and better access. Endoscopy provides synergistic benefit in a multidisciplinary setting by improving accuracy of disease assessment. The role of artificial intelligence in endoscopy is increasingly recognised. This will be most beneficial in specific pathologies and patient cohorts where challenges in endoscopic imaging remain, such as dysplasia surveillance in inflammatory bowel disease. With continued advancement in endoscopic technology, future studies on quality need to adapt to the changes in clinical practice for effective measurement and assurance of quality.
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    The HBV-STOP study, a prospective study of nucleot(s)ide analogue discontinuation in non-cirrhotic Hepatitis B e-antigen negative chronic hepatitis B patients who have achieved long term virological suppression
    Hall, Samuel Anthony Lachlan ( 2022)
    It is estimated that 257 million people have chronic hepatitis B (CHB). HBV infection is endemic throughout Asia, sub-Saharan Africa, Polynesia, as well as in indigenous populations in North America, New Zealand and Australia. Nucleot(s)ide analogues (NA) are standard treatment for HBeAg-negative chronic hepatitis B (CHB). The two first-line NA, entecavir (ETV) and tenofovir disoproxil (TDF), are both potent antiviral agents which effect durable viral suppression, reduce hepatic necro-inflammation, fibrosis progression and reduce risk of cirrhosis, liver failure and hepatocellular carcinoma (HCC). However, HBsAg clearance, or functional cure, is rare, and long-term treatment is recommended. Finite therapy for individuals with HBeAg-negative CHB has been identified as an area of unmet need by expert organisations because of concerns regarding cost, long-term side effects and the risk of the development of drug resistance with indefinite treatment. In the past decade, there has been increasing interest in whether NA therapy can be safely stopped in a subset of patients. In 2012, Hadziyannis and colleagues published a landmark study evaluating clinical outcomes after stopping NA treatment in a small cohort of non-cirrhotic patients with HBeAg-negative CHB who were long-term responders to adefovir monotherapy. All patients experienced early virological relapse. Virological relapse was associated with biochemical relapse in 76% of patients, with antiviral therapy resumed in 15/33 patients. Eighteen patients remained off-treatment through five years of follow-up and all achieved a sustained response, defined by serum HBV DNA level < 2,000 IU/mL and normal serum ALT level. HBsAg loss was observed in 72% (13/18) of patients who maintained a sustained response off treatment by the end of 5 years of follow-up. HBsAg loss was associated with lower HBsAg level at the time adefovir was stopped, and was more common among participants who did not restart treatment. Among those patients who did not restart NA therapy, baseline ALT was higher in those who achieved HBsAg loss, suggesting a role for immune-mediated cytolysis of HBV-infected hepatocytes. Stopping treatment was safe, with no episodes of liver decompensation reported. Since this initial study, there have been a number of reports of clinical outcomes after stopping long-term NA therapy in patients with HBeAg-negative CHB. The studies have been heterogeneous, often retrospective in design, and with considerable variation between protocols for ethnicity of cohort, inclusion / exclusion of people with cirrhosis, duration of follow-up, as well as criteria for re-starting NA therapy. There remain important questions about the rate and outcome of virological and biochemical relapse after stopping NA treatment, the safety of this approach, and the rates of HBsAg loss in prospective follow-up. The answers will inform decisions about patient selection for this strategy in clinical practice. Therefore, as part of my thesis, I have planned to perform a prospective multi-centre study to evaluate clinical outcomes after stopping NA therapy in non-cirrhotic individuals with HBeAg-negative CHB after 96 weeks of follow-up, in addition to a meta-analysis of prior NA cessation studies in HBeAg-negative non-cirrhotic individuals. In addition, NA cessation studies provide a unique opportunity to study the immunology of acute hepatitis flares that occur in the setting of HBV reactivation after stopping NA therapy. ALT flares are preceded by a rise in serum HBV DNA and as HBV is a non-cytopathic virus, it is thought that liver injury is primarily immune-mediated. However, the literature characterizing acute flares of HBV is sparse because flares are difficult to predict and most are asymptomatic. Innate immune pathways may participate in ALT flares either directly or in a bystander fashion. To further investigate the role of innate immunity in the host response to HBV infection in the setting of hepatitis flares, as the final component of my thesis, I have also planned to examine the longitudinal expression and activity of TLRs on peripheral monocytes and markers of peripheral NK cell activity in patients experiencing severe hepatitis flares in the setting of HBV reactivation after NA discontinuation by using samples from patients in our prospective multi-centre study investigating clinical outcomes of stopping NA therapy in non-cirrhotic individuals with HBeAg-negative CHB in order.
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    Defining mechanisms by which EphrinB2 in osteocytes controls bone strength and material quality
    Blank, Martha Alexandra ( 2022)
    Bone strength is determined by bone mass, geometry, and material quality. Mechanisms determining bone mass have been well-studied but what maintains material quality remains poorly understood. We have previously provided evidence that EphrinB2 in osteocytes may mediate one pathway supporting bone material quality. Knockdown of EphrinB2 in osteocytes led to lower bone strength without any difference in bone mass or geometry. The bone strength defect originated from greater levels of collagen and mineral in cortical bone suggesting that EphrinB2 in osteocytes limits their accrual. In this thesis, I investigate the cellular and molecular downstream effects of EphrinB2-deletion in osteocytes in vitro and assess differences in the bone material quality due to absence of EphrinB2 in osteocytes in vivo and in vitro. Since our initial data suggested that greater autophagy in osteocytes may cause the greater mineral content I sought to determine the role of EphrinB2 in autophagy. I showed that EphrinB2-Fc treatment limits autophagosome numbers and that stimulation of autophagy can increase mineralisation in osteocytes in vitro. However, further investigation showed that EphrinB2-deficiency in osteocytes leads to lower lysosome content and cis-Golgi apparatus fragmentation compared to control cells. This suggests that autophagosomes accumulate in EphrinB2-deficient osteocytes due to impaired degradation by lysosomes. Thus, I suggest that EprhinB2 deletion in osteocytes limits lysosome formation. To address the low lysosome content in EphrinB2-deficient osteocytes, I next investigated the potential dysregulated pathways supporting lysosome formation in these cells, including the transcription factor EB (TFEB) and mTOR activity specifically mTORC1. I found that EphrinB2-deficiency in osteocytes resulted in lower Tfeb mRNA and TFEB protein levels, possibly caused by greater mTORC1 activity, an inhibitor of TFEB activity. Proteomics analysis additionally showed lower levels of bone material-degrading enzymes such as cathepsins and matrix metalloproteinases, which are direct downstream targets of TFEB. However, Tfeb knockout in osteocytes did not mimic the greater mineral levels observed when EphrinB2 was absent in osteocytes suggesting that the downregulation of TFEB is not the primary mechanism by which EphrinB2 deletion leads to mineral accrual. Since the bones of mice with EphrinB2 deficient osteocytes exhibit lower bone strength due to a defective material, and my data showed lower levels of bone matrix-degrading enzymes, I next sought to determine the effects of EphrinB2-deficiency in osteocytes on the bone matrix. I found that, when cultured in a collagenous matrix, absence of EphrinB2 in osteocytes led to less contraction of their collagenous environment compared to control cells. This could explain additional observations I made in vivo, where bones with EphrinB2-deficiency in osteocytes exhibited thicker and less parallel-oriented collagen fibre bundles. When I assessed crystal length in cortical bone in vivo I observed no differences between mice with EphrinB2-deficient osteocytes and controls. This suggests that the origin of the lower bone strength caused by the deficiency of EphrinB2 in osteocytes may be due to a gradual formation of a defective collagenous matrix which provides more binding space for mineral crystals to nucleate and this leads to brittle bones. Having initially shown that stimulating autophagy using rapamycin can increase mineral deposition in vitro I wanted to investigate whether the stimulation of autophagy can be used as a therapeutical tool to improve bone material quality. For this I used a mouse of osteogenesis imperfecta (OI) and tested whether the removal of toxic collagen I aggregates in osteoblasts could improve the severity of the OI phenotype using the FDA-approved autophagy-inducing drug carbamazepine (CBZ). Neither short- (3 weeks) nor long-term treatment (6 weeks) improved bone size, strength, or material quality in growing OI mice. Additionally, long-term treatment with CBZ had no effect on bone material quality, but led to lower bone strength in control mice caused by lower cortical and trabecular bone mass. These data suggest that CBZ-induced autophagy does not improve the OI phenotype but inhibits osteoblast function and bone growth in young individuals. In summary, I showed that EphrinB2 deficient osteocytes have a lower lysosome content and fragmented cis-Golgi apparatus and produce lower levels of bone matrix-degrading proteins compared to control cells in vitro. Additionally, EphrinB2-deficiency in osteocytes led to less collagen arrangement in vitro and thicker and less-parallel oriented collagen fibre bundles in bones in vivo without any difference in mineral crystal length compared to control bone. This suggests that EprhinB2 in osteocytes is essential for lysosome formation and supports lysosomal enzyme production and thereby maintains collagen fibre integrity. This reveals a novel physiological role for osteocytes to gradually limit mineral and collagen accrual in bone tissue to maintain bone strength.
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    Creating a state-wide registry to drive new epidemiological, forensic and clinical insights into sudden cardiac arrest in young Australians
    Paratz, Elizabeth Davida ( 2022)
    Sudden cardiac arrest (SCA) is a leading cause of death globally, accounting for approximately half of all deaths from cardiovascular disease. As demonstrated in this thesis, in young Australians it accounts for one-quarter of all deaths and is associated with approximately 90% mortality. Despite the condition’s high mortality rate and prevalence, systematic and adjudicated data collection remains lacking. As indicated in Chapter 1, only a few registries worldwide provide comprehensive multi-source surveillance of all SCAs, and many important questions in the field of SCA remain unanswered. The substantial economic cost imposed by young SCA in Australia is calculated for the first time in this thesis and provides an economic as well as medical imperative to better understand SCA. In Chapter 2, the aims of this PhD project are established as being to design a multi-source SCA registry with comprehensive case capture and adjudication. Such a registry would enable the secondary aims of identifying epidemiological and forensic insights, as well as creating an opportunity to highlight the experiences of selected patient groups. Chapter 3 of this thesis describes in detail the process of establishing the End Unexplained Cardiac Death (EndUCD) registry including its hierarchical consent process, data flow, data quality control and governance processes. From the establishment of the EndUCD registry, large-scale epidemiological assessments have been undertaken (Chapter 4). These include delineating overarching features of the entire patient group such as causes of arrest, the role of obesity and the role of rural residence. We have also identified variations in administrative data coding that will create under-appreciation of the community burden of SCA. The registry also enabled several projects to be undertaken in close collaboration with the Victorian Institute of Forensic Medicine (Chapter 5), yielding novel forensic information in the investigation of young sudden cardiac death (SCD). These projects include identifying rates of referral and potential under-referral of young SCD for forensic investigation, investigating a novel approach of performing post-mortem coronary artery calcium scores to assist cause of death determination, and examining the diagnostic yield of post-mortem interrogation of implanted cardiac devices such as pacemakers. The EndUCD registry’s unfortunately substantial number of patients created an opportunity to examine multiple relevant clinical questions (Chapter 6). These include more targeted clinical scenarios such as the outcomes of pregnant women experiencing SCA, those with coronary artery anomalies and unusual cases such as IgG4 coronary arteritis causing SCA. Two registry participants also graciously waived their anonymity and shared their experiences in being affected by SCA. In conclusion, the EndUCD registry has been established in line with best practice globally, and successfully commenced operations. Data from this registry has enabled diverse important projects providing new insights into the management of SCA on epidemiological, forensic and clinical levels.
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    Addressing knowledge gaps in the assessment of disease status in systemic sclerosis
    Ross, Laura ( 2022)
    Systemic sclerosis (SSc, or scleroderma) is a rare, orphan condition associated with high morbidity and mortality. There remain significant areas of unmet need in terms of the understanding of the underlying pathogenesis of the disease, how disease manifestations are identified and measured in clinical trials, as well as in clinical practice, in addition to a lack of effective, targeted therapeutics that offer improved survival and quality of life to patients. SSc is frequently a fatal disease, with a mortality risk up to seven times that of the general population and it is associated with a large burden of physical and mental ill-health, notable from the time of onset of disease. Our understanding of the disease has progressed significantly over the past 100 years, since Matsui confirmed that SSc was in fact a multi-system disease, not an exclusively dermatological condition. The importance of vascular changes and auto-inflammatory mechanisms in addition to excessive collagen deposition in the development of SSc and subsequent disease progression are now appreciated. Important pathogenic pathways that lead to the dense fibrotic extra-cellular matrix deposition and obliterative vasculopathy that characterise advanced SSc have been identified. However there is an ongoing absence of evidence to link levels and activities of cytokines and other cell mediators to specific pathogenic effects in SSc. The precise pathogenic mechanisms of specific disease manifestations remain incompletely understood and insights are often gained by working backwards through studying those individuals with advanced disease and identifying the clinical and pathogenic mechanisms that set them apart from others with mild disease. Heterogeneity, both of disease presentation and progression, is a clinical hallmark of SSc, and this has impeded efforts to both define the presence of disease and measure important changes in disease status, either in response to novel therapies in a clinical trial or in observational, longitudinal cohort studies. Important disease manifestations such as cardiac disease or gastrointestinal disease remain largely undefined and the lack of fully-validated multi-system measures of disease status have contributed to the failure of multiple randomised controlled therapeutic trials. Treatment options in SSc are limited, and it remains one of the few rheumatic diseases without a targeted treatment that alters the disease course. Optimal clinical trial design remains debated and effective recruitment strategies to identify those individuals most likely to benefit from treatment are controversial. Methodological inconsistencies remain an Achilles heel of SSc clinical trials, meaning accurate, valid measurement of disease status and comparison between study cohorts cannot be performed. In this context, this thesis aims to further our understanding of SSc by studying specific organ effects of SSc and reviewing and developing methodologically sound means of disease assessment. Specifically, this thesis will address the significant knowledge gaps that currently limit our ability to develop a global measure of disease activity in SSc. I will address the face, content and construct validity of items to measure activity within particular organ systems and will present a preliminary multi-system activity index. Additionally, I will explore SSc-associated heart involvement. This organ system was prioritised in my thesis research because of the prognostic importance of cardiac disease in SSc and the lack of understanding of the full spectrum and natural history of heart involvement, and the absence of valid methods of diagnosis. I present results that quantify the prevalence of myocardial fibrosis in SSc, evaluate the utility of commonly available cardiac investigations in the diagnosis of SSc heart involvement and the progress made in the development of classification criteria for the diagnosis of SSc-associated heart involvement.
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    Targeting cytokines involved in T cell proliferation and function in a mouse model of type 1 diabetes
    Ge, Tingting ( 2022)
    Cytokines functioning through the JAK-STAT pathway amplify immune responses in autoimmune and inflammatory diseases and drive some T cell lymphomas. Therefore, JAK inhibitors have shown efficacy in multiple autoimmune diseases as well as T cell malignancies. My PhD work focused on the role of common gamma chain cytokines in T cell proliferation and their contribution to autoimmune diabetes and T cell lymphomas. In chapter 2, I used the JAK1 selective inhibitor ABT 317 to reverse spontaneous diabetes in NOD mice. I confirmed that the JAK1 inhibitor can block IFNgamma activity to prevent MHC class I upregulation on beta cells and weaken the interaction between T cells and beta cells. More importantly, my research revealed that the JAK1 inhibitor reduced common chain cytokines, IL-21, IL-2, IL-15 and IL-7 signalling in T cells and inhibited both CD4+ and CD8+ T cell proliferation and differentiation. In a recent study by our lab, Jhala et al showed that islet autoantigen-specific T cells from NOD mice lacking IFNgamma receptors (Ifngr1 mutant mice) are present at >10-fold in number compared to wild-type NOD mice, and this expansion is due to increased response of Ifngr1 mutant T cells to common gamma chain cytokines. It emphasises reducing T-cell proliferation as an important way that JAK inhibitors preserve beta cells. In chapter 3, I studied checkpoint inhibitor-induced diabetes by injecting NOD mice with anti-PD-L1 antibodies. Diabetes was induced rapidly after the administration of anti-PD-L1 due to activation of already existing islet-specific T cells that can destroy beta cells. There is currently no effective therapy to prevent checkpoint inhibitor-induced diabetes. I used a JAK1/JAK2 inhibitor to block the effect of cytokines on T cells and beta cells. The JAK1/JAK2 inhibitor prevented anti-PD-L1 induced diabetes. Using JAK inhibitors after checkpoint inhibitors in a tumour model did not reverse or abrogate the anti-tumour effects of checkpoint inhibitors. This provides preclinical validation for using a JAK inhibitor to prevent checkpoint inhibitor-induced diabetes, a rare immune-related side effect of this cancer therapy. We observed a high incidence of lymphomas in Ifngr1 mutant mice. Phenotype studies of these lymphomas showed that most lymphomas originated from immature T cells. Lymphoma cells showed increased phosphorylation of STAT3 and STAT5. Ifngr1 mutant mice had increased proliferation of pathogenic beta cell specific CD8+ T cells due to decreased SOCS1 and increased proliferative response of T cells to IL-2. However, the expression of SOCS1 in Ifngr1 mutant thymocytes is similar to that in NOD thymocytes and IFNgamma receptor deficiency did not affect thymocyte proliferation and development. IFNgamma receptor deficiency impaired immune-mediated suppression of T cell lymphoma development. In conclusion, IFNgamma receptor deficiency does not cause increased T cell lymphomagenesis but impairs immune-mediated suppression of T cell lymphoma development. My PhD work shows that cytokines working through the JAK-STAT pathway lead to increased proliferation of pathological islet specific T cells and autoimmune diabetes. JAK inhibitor administration can reduce T cell proliferation to prevent and reverse autoimmune diabetes in NOD mice. However, increased T cell lymphomas in Ifngr1 mutant mice is not due to increased JAK-STAT signalling but due to impaired immune-mediated suppression of T cell lymphoma.
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    A genome stability pathway with dual roles in meiosis and germ cell development
    Tsui, Vanessa ( 2022)
    Chromosome segregation errors because of meiotic recombination of are a leading cause of pregnancy loss. In the case of live births can cause serious congenital aneuploidy such as trisomy 21. Meiotic recombination pairs homologous chromosomes to promote large scale exchanges of genetic information, resulting in recombinant haploid gametes (sperm or eggs). Meiotic recombination begins with many programmed DNA double strand breaks (DSBs). The vast majority of these DSBs is repaired directly and only a small subset leads to genetic exchange between the two parental chromosomes, also known as crossing over. This suggests there are factors that actively promote DSB repair as non-crossovers. In my PhD project, I studied the role of a DNA repair protein, FANCM, and how crossover rates are altered in a Fancm knockout mouse model. My results indicate that FANCM deletion leads to two distinct but related outcomes: 1) increased genome instability in the all the cells of the body and 2) increased crossover rates between homologous chromosomes in meiotic cells. As a result of overall genome instability, there is increased spontaneous DNA damage leading to perturbed germ cell production, reducing spermatogenesis in Fancm-knockout males and reduced fertility in Fancm-knockout females. The absence of FANCM alters the DSB repair pathway choice in meiotic cells, resulting in more crossovers compared to wild-type conditions. Therefore, FANCM plays an important role in regulating crossover designation and balancing the exchange of genetic information during each reproductive generation. My work and further investigations into reproductive biology will provide more insight into how DNA repair defects may cause human congenital aneuploidies and could shed light on earlier diagnostic opportunities.
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    Behavioural treatment for functional bowel symptoms in inflammatory bowel disease
    Khera, Angela Jane ( 2022)
    Inflammatory bowel diseases (IBD) are chronic relapsing and remitting inflammatory diseases of the gastrointestinal tract, with the most common sub-types being Crohn’s disease and ulcerative colitis. Despite achieving disease remission patients with inflammatory bowel disease often experience persisting bowel symptoms, considered “functional in nature”, including increased bowel frequency, urgency, faecal incontinence, constipation (low bowel frequency or difficult rectal emptying), abdominal pain, bloating, and anorectal pain. Some patients with ulcerative colitis require major surgery, involving colectomy and construction of a neo-rectum or ileoanal pouch. Many continue to have similar long-term symptoms. Poor health-related quality of life, anxiety, depression, and health-seeking behaviour are associated with these functional bowel disorders in patients with IBD. Behavioural treatment for bowel dysfunction incorporates education, muscular and sensory rehabilitation, defaecation training, practical management strategies, and psychological support. It is of proven efficacy in the non-IBD population but the evidence in patients with IBD is extremely limited. The primary aim of this thesis is to determine the efficacy of behavioural treatment, including pelvic floor muscle training, for persistent bowel symptoms in patients with quiescent IBD or an ileoanal pouch. These studies have demonstrated that behavioural treatment is an effective management strategy for patients with quiescent IBD, in both public and private healthcare settings. It reduces symptom severity and improves health-related quality of life. The long-term findings of the prospective LIBERATE study found that most patients had maintained the improvement in symptoms at 12 months after completing treatment. The cross-sectional study of patients with an ileoanal pouch confirms that many do have long-term pouch dysfunction, and most have learned to live with it. The preliminary findings of the randomised controlled RAP trial, for patients undergoing pouch surgery, demonstrate statistically significant superiority of peri-operative physiotherapist-led behavioural intervention over standard or usual care, with regard to symptoms, psychological well-being, and quality of life. This thesis includes the first studies examining pelvic floor muscle function in patients with IBD using real-time ultrasound. They are also the first studies to use transperineal ultrasound as an outcome measure following pelvic floor behavioural treatment for faecal incontinence or defaecatory dysfunction. Transperineal ultrasound identified pelvic floor muscle dysfunction and accurate displacement, although these were not associated with the primary outcomes of treatment. Real-time ultrasound examination was well tolerated and an excellent non-invasive biofeedback tool for patients with IBD and pelvic floor muscle dysfunction. The findings from this thesis provide a clear rationale for behavioural treatment to be offered to patients, particularly when other therapies have failed. This work also suggests that identifying functional bowel symptoms earlier and treating them effectively may minimise the impact on psychological well-being and quality of life, particularly for those undergoing major pouch surgery. Improving the recognition of functional bowel symptoms and providing evidence for behavioural treatment as a safe and effective management option has the potential to alter the current model of care and reduce the personal and economic burden of IBD.
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    Enhancing the hepatitis B care cascade in Australia: addressing barriers in the Chinese community and general practice, and evaluating cost-effectiveness
    Xiao, Yinzong ( 2021)
    Chronic hepatitis B infection is a significant global public health concern, with untreated chronic hepatitis B leading to cirrhosis, liver failure and liver cancer. In 2015, an estimated 257 million people worldwide were living with chronic hepatitis B, of whom only around 10% were diagnosed, and fewer than 1% received treatment. An estimated 870,000 deaths are attributed to hepatitis B each year. In 2016, the World Health Organization launched the global health sector strategy on eliminating viral hepatitis, which provides guidance and frameworks for country efforts to eliminate viral hepatitis as a major public health threat by 2030. In Australia, despite a high hepatitis B vaccination rate by global standards, there are significant gaps in the coverage of service delivery among people living with chronic hepatitis B. One in three people with chronic hepatitis B are not diagnosed, and 78% are not receiving ongoing clinical care. One of the priority populations of hepatitis B infection are people born in endemic countries, with China being the most common country of birth among overseas-born people living with hepatitis B in Australia. To increase hepatitis B diagnosis, care and treatment (that is, to enhance the hepatitis B care cascade) in Australia, several critical areas for improvement have been identified, including adapting services to the affected population, building health workforce capacity to meet demands, and building the investment case to support models of care that will promote hepatitis B elimination. The overarching aim of the research presented in this thesis was to generate evidence targeting each of these three aspects, with particular emphasis on: 1) engaging the Chinese community in hepatitis B testing and clinical management; 2) engaging general practitioners (GPs) in the provision of hepatitis B-related care; and 3) formulating a robust hepatitis B investment case. To accomplish these objectives, five interlocking studies were conducted. The first two studies focused on an affected community (the Australian Chinese community), the second two on health service providers, and the final study on the cost effectiveness of hepatitis B care. More specifically: Study 1 was a randomised controlled pilot and feasibility study of the impacts of an educational program designed to improve hepatitis B testing uptake in the Australian Chinese community; Study 2 was a qualitative study to explore enablers of hepatitis B clinical management among the Australian Chinese community; Study 3 was a before-and-after evaluation of a self-guided learning package among GPs practising in Victoria; Study 4 was a nationwide survey of knowledge, attitudes, barriers and enablers to the provision of hepatitis B care among GPs in Australia; and Study 5 was a cost-effectiveness study analysing the impacts of enhancing hepatitis B care cascade to reach global and national targets in Australia. The findings of this research indicated that a culturally tailored education program can contribute to improving hepatitis B-related knowledge among the Australian Chinese community. Key messages identified to resonate most strongly among people living with chronic hepatitis B included availability of effective and cheap treatment, and that long-term engagement with clinical management has substantial benefits. A holistic response from community, healthcare providers and the public health sector is required to motivate testing and clinical management among the Australian Chinese community at risk of hepatitis B infection. Additionally, this research showed that concise, clear and practical resources can support GPs to identify who to test for hepatitis B. It also showed that GPs lack of awareness, knowledge, confidence and intention to prescribe treatment for hepatitis B, highlighting the need for interventions to increase their interest and skills in the provision of hepatitis B-related care. Economic evaluation findings suggest that an improvement in the hepatitis B care cascade is required for Australia to reach the global 2030 targets, and that it is cost-effective to spend up to AUD328 million to AUD538 million per year on demand generation activities to reach the national and global targets. Overall, this research provides novel evidence about feasible and effective interventions for improving the hepatitis B care cascade in Australia. It also provides insights into ways to enhance the global hepatitis B care cascade.
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    The roles of retinoic acid receptors and target gene HOXA1 in regulating haematopoiesis and myelodysplastic syndromes
    Grace, Clea Simes ( 2022)
    Haematopoietic stem cells (HSCs) are multipotent cells responsible for the maintenance of the haematopoietic system throughout life. Dysregulation of the balance between HSC self-renewal, death and differentiation can have serious consequences such as myelodysplastic syndromes (MDS) or leukaemia. All-trans retinoic acid (ATRA), the biologically active metabolite of retinoic acid (RA) has pleiotropic effects on haematopoietic cells, maintaining haematopoiesis via complex intrinsic and extrinsic mechanisms involving retinoic acid receptor-alpha (RARalpha) and RARgamma signalling. Conditional murine Rar knockout models were utilised to determine the intrinsic and extrinsic impacts of Rara and/or Rarg deletion on HSCs and their progeny in vivo via global, postnatal deletion, haematopoietic cell-specific deletion (whole bone marrow transplants (WBMTs)) and HSC and progenitor-specific deletion (Lineage-c-Kit+Sca-1+ (LKS+) WBMTs). These data revealed novel roles of Rara in haematopoiesis, with postnatal deletion resulting in intrinsically-mediated myeloid-biased haematopoiesis, reduced numbers of CD8+ T cells and altered HSC reconstitution capacity, in addition to extrinsically-mediated aberrant BM erythropoiesis and B lymphopoiesis. Loss of either Rara and/or Rarg resulted in myeloid-biased haematopoiesis and concurrent deletion (expression of Rarb only) enhanced HSC exhaustion. Contrary to germline Rarg-/- studies, postnatal Rarg deletion had a limited impact on haematopoiesis. The observed phenotypical differences between germline and global, postnatal Rar deletions may be due to a combination of developmental and microenvironmental interactions, interactions between Rara and Rarg and their differential target genes and compensatory effects. Whilst the relative contribution of RA signalling to its haematopoietic regulation is unknown, homeoboxa1 (HOXA1) is an RARgamma target gene and its full-length isoform (HOXA1-FL) is frequently upregulated in human MDS. A series of studies utilising conditional murine haematopoietic Hoxa1 knockin mice revealed that whilst recipient mice developed MDS during serial transplantation, consistent with previous studies, the levels of Hoxa1-FL overexpression were insufficient to result in progression to secondary acute myeloid leukaemia (sAML). Additionally, Hoxa1 overexpression resulted in profound, persistent and serially transplantable macrothrombocytopenia despite a depleted yet highly megakaryocyte-primed HSC and progenitor compartment. The macrothrombocytopenia resulted from ineffective thrombopoiesis by dysfunctional, low ploidy megakaryocytes, potentially due to inflammation-mediated lineage skewing and/or late-stage developmental defect(s). Dysregulated BM erythropoiesis and granulopoiesis were also observed. These phenotypes were consistently evident early, persisted in non-transplanted pre-malignant mice and became more profound during serial transplantation and after MDS progression. Phenotypic severity also depended on Hoxa1 gene-dosage and isoform expression. Furthermore, the megakaryocyte-priming correlated to increased DNA damage and/or deficient DNA damage responses (DDR), increased expression of inflammatory markers and altered programmed cell death, suggesting that an altered inflammatory microenvironment and associated haematopoietic lineage skewing may have contributed to the Hoxa1-FL-mediated haematopoietic defects, although this remains under investigation. Whilst the precise mechanisms underlying these phenotypes therefore remain to elucidated, it is evident that Rara, Rarg and Hoxa1-FL have pleiotropic impacts on haematopoiesis that are both cell-type and maturation-stage dependent. Elucidation of these effects in murine models are critical steps in the development of targeted therapies for MDS patients with increased Hoxa1-FL expression and for other haematopoietic malignancies to which dysregulated Hoxa1 and/or RA signalling may contribute.