Melbourne Medical School Collected Works - Theses
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ItemThe role of the renin-angiotensin system in portal hypertension and hepatic fibrosis(University of Melbourne, 2008)
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ItemCoagulation and inflammation in experimental colitis(University of Melbourne, 2008)
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ItemFactors controlling the differentiation of macrophage lineage cells into osteoclasts(University of Melbourne, 2007)
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ItemSuperantigens and the innate immune response in human sepsis(University of Melbourne, 2006)
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ItemSuperantigens and the innate immune response in human sepsis(University of Melbourne, 2006)
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ItemThe role of Dlk1 in haematopoiesis, leukaemia and angiogenesis( 2017)Delta-Like Homologue 1 (DLK1), also known as Preadipocyte Factor 1 (PREF-1), is a non-canonical EGF-like NOTCH ligand. It is maternally imprinted at the Dlk1-Dio3 imprinted locus, and has been shown to regulate embryonic growth, lipid metabolism and skeletal development. However, the precise role of Dlk1 in haematopoiesis, leukaemia and angiogenesis, processes in which it has been previously implicated, is unknown. We generated Dlk1 knockout and conditional knockout mice and used a constitutive overexpression system via retroviral transduction to specifically study Dlk1 in these contexts. Dlk1 knockout mice showed distinctive phenotype of increased perinatal mortality and growth retardation. In foetal livers, significant expression of Dlk1 was detected in the haematopoietic cells, with higher level of expression in the haematopoietic stem cells compared to lineage positive mature cells, with overall expression decreasing with embryonic age. Dlk1 knockout mice were not significantly different from wild type mice in the mature haematopoietic lineages, but serial competitive transplant assays demonstrated Dlk1 knockout bone marrow cells were inferior to controls in reconstituting lethally irradiated recipient mice in short term haematopoietic reconstitution assays, suggesting that Dlk1 knockout led to a defect in adult short term haematopoietic stem cells. Despite frequent overexpression of DLK1 found in many human acute myeloid leukaemias, constitutive overexpression of Dlk1 did not lead to increase in acute leukaemia or death in reconstituted mice. However, Dlk1-overexpressing haematopoietic cells demonstrated competitive repopulation advantage compared to MIG-transduced controls. Using a retinal model of angiogenesis, Dlk1 was found to be expressed by the pericytes rather than endothelium of newly developing blood vessels in postnatal murine pups, in contrast to the published data. Conditional knockout of Dlk1 in endothelial cells using the endothelial specific Tie2 Cre transgene did not lead to significant abnormality in postnatal retina, confirming that Dlk1 did not have a functional role in the retinal endothelium. These new findings add to our current knowledge of stem cell biology and leukaemia, and the role of Dlk1 in angiogenesis.
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ItemTargeting cyclin-dependent kinase 9 and myeloid cell leukaemia 1 in MYC-driven B-cell lymphoma( 2016)Aggressive B-cell lymphomas include diffuse large B-cell lymphoma, Burkitt lymphoma and intermediate forms. Despite high response rates to conventional immuno-chemotherapeutic approaches, an unmet need for novel therapeutic strategies is required in the setting of relapsed and refractory disease, typified by resistance to chemotherapy and radiotherapy. The proto-oncogene MYC is frequently dysregulated in the aggressive B-cell lymphomas, however, it has proven an elusive direct therapeutic target. A significant body of evidence is accumulating to suggest that MYC-dysregulated disease maintains a ‘transcriptionally-addicted’ state, whereby perturbation of RNA polymerase II activity may indirectly antagonise MYC activity. Furthermore, very recent studies implicate anti-apoptotic myeloid cell leukaemia 1 (MCL-1) as a critical survival determinant of MYC-driven lymphoma. This thesis utilises pharmacologic and genetic techniques in MYC-driven models of aggressive B-cell lymphoma to demonstrate that cyclin-dependent kinase 9 (CDK9) and MCL-1 are oncogenic dependencies of this subset of disease. The cyclin-dependent kinase inhibitor, dinaciclib, and more selective CDK9 inhibitors are used to demonstrate efficient apoptosis induction conferred at least in part by downregulation of MCL1 transcription. Furthermore, a genetic screen identifies other transcriptional cyclin-dependent kinases that are required for viability of MYC-driven lymphoid disease. Finally, having established MCL-1 as a critical oncogenic dependency of MYC-driven lymphoma, this thesis demonstrates the significant activity that is conferred by direct pharmacologic antagonism of MCL-1 using a small molecule BH3-mimetic inhibitor of MCL-1. These findings confirm a druggable pathway of oncogenic cMYC dependency involving CDK9 regulated RNA polymerase II-mediated transcription of MCL-1, and proposes pharmacologic inhibition of CDK9 and MCL-1 as novel anti-lymphoma strategies.
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ItemLinguistic change in an online support group( 2016)Online support groups (OSGs) are popular sources of both health information and social support. Though early research into OSGs highlighted a concern that non-expert members may give harmful advice, more recent studies have typically shown that engagement with OSGs can increase consumer satisfaction with the treatment process, enhance wellbeing, and ultimately improve health outcomes. OSGs are well-researched within applied linguistics. Qualitative studies have focussed on member roles within OSGs, as well as the ways in which group members discursively construct their identities, especially with respect to their illness. These approaches have generated rich insights into consumer healthcare discourse that can inform strategies for fostering consumer-centred care. Qualitative approaches, however, are resource-intensive, di cult to reproduce, and limited in terms of generalisability and representativeness. Quantitative and computational approaches are able to overcome these shortcomings, adding transparency, reproducibility, scalability, and reducing the potential for researcher bias. Current computational approaches to consumer healthcare discourse, however, tend to rely on simplified conceptualisations of language, prioritising lexis over grammar, and thus ignoring the central role played by grammar in the meaning-making process. To address current methodological shortcomings in OSG discourse research, this thesis presents an interdisciplinary, corpus-based investigation of lexicogrammatical and discourse-semantic choices made by members over the course of membership in an online bipolar disorder support community. 8.2 million words in over 66,000 posts from approximately 3,500 members were transformed into a metadata-rich, grammatically annotated corpus and investigated from a systemic-functional linguistic (SFL) perspective using purpose-built corpus/computational linguistic tools. An analysis of MOOD and MODALITY choices made over ten stages of membership highlights differences in the ways members negotiate role-relationships, with changes in Mood Type, Modality and Speech Function reflecting a longitudinal increase in the provision of advice and social support. An analysis of the TRANSITIVITY system shows longitudinal changes in the kinds of participants and processes construed by Forum members, as well as changes in how these participants and processes behave lexicogrammatically. The diagnosis Event, for example, is represented by newcomers as a process and modi ed temporally; at later stages of membership, it is more often reconstrued as a participant in discourse, framed in terms of veracity. Longitudinal shifts were also observed in the preferred ways of ascribing/attributing bipolar disorder to Forum members: new members use bing forms (I’m bipolar), while veteran members prefer having constructions (I have bipolar). The thesis has implications for corpus linguistics, systemic-functional linguistic theory, and healthcare communication research. For corpus linguistics and corpus-assisted discourse studies, the main contribution is corpkit, an open-source software tool designed to build and analyse parsed and metadata-rich corpora. it is suggested that the developed tools and methods can circumvent theoretically problematic current practices, and increase the accuracy and automatability of the analytical process. For healthcare communication research, the case study demonstrates the importance of expanding the conceptualisation and analysis of the consumer healthcare journey to include intra–consumer communication that occurs outside of hospitals and clinics. The thesis also advances an argument that the emerging eld of clinical natural language processing stands to benefit from increased engagement with functional linguistic theory and insights generated within the qualitative paradigm. I argue that combining the insights from functional linguistics and discourse analysis with automated computational workflows is a step toward an important future goal of improvement of consumer health outcomes through analysis of large, digital collections of spoken and written healthcare discourse.
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ItemImproving epigenetic therapies for haematological malignancies( 2016)The work presented here is the product of several years of clinical trial and laboratory endeavour focused on the theme of understanding and improving currently available epigenetic therapies for blood cancers, and understanding fundamental aspects of clinical trial design. Several classes of epigenetically active drugs with proven clinical usefulness have received regulatory approval for the therapy of haematological malignancies in the last 5 years. At the same time, advances in high throughput sequencing technologies have provided landmark discoveries that reinforce the hypothesis that epigenetic mechanisms play a central role in the neoplastic process, as well as giving us new opportunities to develop targeted therapies. Some of the most exciting new targeted biological therapies arising from these discoveries such as inhibitors of histone methyl-transferases, the BET bromodomain inhibitors and the isocitrate dehydrogenase inhibitors are only now in the earliest phase of clinical development with initial signals that many of these agents will be both tolerable and active therapies for blood and solid tumours. In the context of the field at the start of 2010, only two classes of agent were available in clinical trials and for early investigator-initiated development. Even today, only these classes of epigenetic agents have marketing approval. As a consequence, histone deacetylase inhibitors and DNA methyl-transferase inhibitors are the focus of this thesis. The thesis presents investigator-initiated trials of agents in these two drug classes as well as the theory associated with the design of the trials. The literature review in Chapter 1 establishes the foundation for the clinical trial work. Here, the mechanism of action of the histone deacetylase inhibitors and the DNA-demethylating agents is reviewed. Activity of these agents in industry-sponsored clinical trials is presented as well as key toxicity data that informs the hypotheses and design of the clinical research presented in the thesis. The theories underlying the combination therapies that form the backbone of the thesis are discussed. Eltrombopag, a thrombopoietin agonist, is given particular prominence for its potential as a supportive care agent. The clinical trials that represent the majority of the thesis were designed to explore the critical questions of improving the tolerability of the DNA-demethylating agent azacitidine, and developing novel combination therapies for HDAC inhibitors. As experimental design is a critical question, a discussion on clinical trial design is presented in Chapter 2, which closes with a relevant case study. Several clinical trials were developed during the conduct of this higher degree from concept through to funding and clinical recruitment. Three, presented in Chapters 3, 4, and 7 have completed accrual and a further trial (Chapter 5) has been interrupted due to administrative challenges and is currently being reworked. The protocol for each study was in itself an extensive body of novel work contributing to the thesis. These are included in the appendices but summarised within the body of the thesis. Chapter 3 presents finalised and novel data analysed in depth of a trial of the combination of eltrombopag, a thrombopoietin-receptor agonist and azacitidine. The purpose of the study was to develop a supportive care therapy for patients with myelodysplastic syndromes, a disease that causes low platelets (thrombocytopenia), and has a risk of transforming to acute myeloid leukaemia, an almost invariably fatal event. Thrombopoietin mimetic agents have a somewhat complicated relationship with myeloid malignancies in the minds of researchers and while the receptor (mpl) and the role of the mpl receptor in haematopoietic stem cell survival and fate has been the subject of the research for at least 20 years, many questions about their theoretical safety remain open. There is considerable debate over the theoretical safety of mpl in agonists in patients with myeloproliferative diseases. Hence, attention is given to the rationale for the choice of eltrombopag for myelodysplastic syndrome in Chapter 1; also, the established body of preclinical data is presented to provide a context. This particular trial, “AzaE”, has been presented in international fora and the data has provided a foundation for a 170-centre, 43-country international randomised study that, if positive, may change clinical practice for patients with myelodysplastic syndrome and thrombocytopenia. Hence Chapter 3 is a central part of the novel data in this thesis. Based on the hypothesis that HDAC inhibition would up-regulate CD20 on the surface of malignant B-cells, a collaborating colleague, Mark Bishton, developed a phase 1 clinical trial of the combination of the radio labelled rituximab and the HDACi panobinostat. Dr Bishton and Prof Seymour wrote the trial protocol. As Principal Investigator of the study, I set up the study, moving it from protocol to execution. I oversaw the conduct of the study as the principal investigator and reviewed and approved the clinical data. The combination was toxic and the lessons learned are cogent and argue against further developing this sort of combination therapy. This study is presented in Chapter 4. In order to address the moderate activity of the HDACi inhibitor romidepsin and to broaden its clinical indications, a phase I/II study was designed in collaboration with academic partners from Yale University. The strategy behind the design of that study, the protocol itself, and initial data from six patients that have been recruited is presented in Chapter 5. That study has been halted due to regulatory challenges in the USA and Yale University that interrupted the research without warning. The trial strategy is discussed and preliminary results are presented. HDACi are highly active in cancers of the immune system, where the symptomatic and physical manifestations are to a considerable extent driven by cytokine changes which serve a both disease epiphenomena and drivers of the malignancy itself. In Chapter 6, I investigate the effect of the HDACi on human monocyte derived dendritic cells, identifying a mechanism by which these agents are likely shift the T-cell populations, altering patient symptoms and potentially inducing clinical responses. The findings support the contention that HDACi are immunosuppressive and do not make suitable partner drugs for treatments that benefit from an intact antigen presentation apparatus. Histone deacetylase inhibitors and DNA demethylating agents are often conceptually explained as driving a single cellular mechanism. However, the effects within the cell are pleiotropic and there are very few studies that provide data on predictors for response, especially so for the HDAC inhibitors. Therefore, a part of this work was to design a clinical trial that enables future scientific studies on predictors of response to HDAC inhibitors. Several academic questions were faced in developing the approach to this study. Chapter 7 presents the approach to the design of the clinical study, and presents the clinical features of the patients who have been recruited and key response data. An overview of planned experiments is presented. Having concerned itself with the design of investigator-initiated clinical trials of novel combinations and presented successes and failures of trial design and execution as well as original laboratory work that may inform combinations, the thesis concludes with a discussion on future directions for the projects arising from this work and epigenetic agents in general.
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ItemAcupuncture for menopausal hot flushes: an individually randomised sham-controlled trial( 2016)Menopausal hot flushes affect up to 90% of women and pose a significant health and financial burden. Many women are reluctant to use hormone replacement therapy, an effective treatment, because of fear of serious adverse events. Acupuncture is a safe treatment with conflicting evidence for efficacy as a hot flush treatment. The aim of this thesis was to assess the efficacy of acupuncture for menopausal hot flushes. The principal study was a fully powered randomised sham-controlled trial which enrolled women aged over 40 in the late menopausal transition or postmenopause with a moderate hot flush burden, and who met criteria for the Chinese medicine diagnosis of Kidney Yin deficiency. Women were randomised to receive eight weeks of either Chinese medicine needle acupuncture or non-insertive sham acupuncture. The primary outcome was hot flush score at the end of treatment. Secondary outcomes included quality-of-life, anxiety, depression, and adverse events. Women were followed up at 4 and 8 weeks (end of treatment) and at 3 and 6 months post end of treatment. Analysis was by intention-to-treat using mixed effects modelling. 327 women were randomised to real (n=163) or sham acupuncture (n=164). There was no evidence for a between-group difference in hot flush scores at end-of-treatment (mean difference 0·33; 95% CI -1·87 to 2·52; p=0·77) or for secondary outcomes, and no serious adverse events. Both groups improved by an average of 40% from baseline for hot flush score. There was a small, clinically irrelevant difference for hot flush severity at end of treatment. Limitations of the study are that participants were predominantly Caucasian and none had breast cancer or surgical menopause, conditions which predispose to more severe hot flushes. In conclusion, an eight week course of Chinese medicine acupuncture was not superior to non-insertive sham needling for menopausal hot flushes. Women may have improved because of natural resolution of symptoms, frequent therapist interaction, or the placebo effect. A specific effect from acupuncture needling was not demonstrated. Future research may focus on the role of acupuncture in breast cancer survivors.