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(2018)ThesisCardiovascular disease affects about 1.4 million Australians and was responsible for more than 45000 deaths in 2011. Compelling evidence from human population studies has shown a positive correlation between elevated plasma high density lipoprotein cholesterol (HDL-C) levels and a reduced risk of major cardiovascular events. HDLs have several potential cardioprotective functions, the most extensively studied of which is their ability to remove excess cholesterol from macrophages. They also have anti-inflammatory properties, enhance endothelial repair and improve endothelial function. HDLs also have anti-oxidant, anti-apoptotic, anti-thrombotic and anti-diabetic functions. Therapies that increase HDL-C levels reduce atherosclerotic lesion development in animals. These observations have led to the hypothesis that increasing HDL-C levels will reduce cardiovascular events in humans. Although CETP inhibitors have been shown to markedly increase HDL-C levels and reduce major cardiovascular events in a recent clinical outcome trial, there remains doubt about the effects of CETP inhibition on the cardioprotective properties of HDLs. The work in this thesis examines the effects of inhibiting CETP on the cardioprotective functions of HDLs. The studies investigate the effects of a novel CETP inhibitor, AMG-899, on HDL composition, size and several of their cardioprotective properties. Subjects were treated with placebo or AMG-899 (2.5 mg/day or 10 mg/day) over 12 weeks. HDLs were isolated from subjects before and after treatment. AMG-899 treatment increased plasma HDL-C levels and decreased LDL-C levels (Chapter 3). Treatment with AMG-899 also changed the composition of HDLs, increased HDL size (Chapter 3) and increased HDL-mediated ABCA1- and ABCG1-specific cholesterol efflux (Chapter 4). Treatment with 10 mg/day AMG-899 also improved the anti-inflammatory properties of HDLs (Chapter 5), while HDLs isolated from subjects after treatment with 2.5 mg/day and 10 mg/day AMG-899 were equally effective at increasing endothelial proliferation and migration relative to HDLs isolated prior to treatment (Chapter 6). These studies establish that inhibiting CETP activity with AMG-899 does not impair, and in some cases, improves the potentially cardioprotective functions of HDLs. In conclusion, as AMG-899 treatment increases HDL-C levels more effectively than other CETP inhibitors, and does not impair the major cardioprotective functions of HDLs, it is a potential candidate for future clinical development.
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(2018)ThesisExcitotoxicity refers to the damaging and toxic effects inflicted upon neuronal tissue resulting from excessive or prolonged activation of excitatory receptors. It has been implicated in many neurological conditions ranging from epilepsy to stroke and chronic neurodegenerative conditions like Alzheimer’s disease. In the first part of this thesis, we utilised a tau deficient mouse to demonstrate that the lack of tau protects against excitotoxic brain injury after experimental stroke. Furthermore, we showed that this protection is due to site-specific inhibition of Ras/ERK-mediated toxicity by the post-synaptic accumulation of a Ras-inhibiting protein known as SynGAP1. We were able to reverse this protection via viral-mediated knockdown of SynGAP1 in tau deficient mice. Conversely, in vitro over-expression of SynGAP1 prevented excitotoxic ERK signalling in wild-type neurons. This makes tau relevant in acute excitotoxic conditions like stroke making tau relevant beyond neurodegenerative diseases such as Alzheimer’s disease. Next, we utilised a new murine genetic reference population known as the Collaborative Cross to screen for genes implicated in excitotoxicity via a chemical induced epileptic seizure model. After successful identification of two potential candidates, Lamp5 & 4933407L21Rik, we utilised CRISPR-mediated genetic editing to delete our candidate genes and confirmed the observed phenotype in both strains. To our knowledge, this is the first time susceptibility genes for a complex trait have been identified and confirmed in the Collaborative Cross mice using both forward and reverse genetic approaches. In summary, we demonstrated the role tau plays in mediating excitotoxicity and stroke; as well as identified and confirmed two new seizure susceptibility genes that have not been previously described.
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(2018)ThesisThe mammary gland is a unique model to study regulation of stem cell homeostasis and differentiation due to its postnatal development. Embedded within the bilayered mammary epithelium are populations of mammary stem cells (MaSCs), which give rise to inner luminal and outer basal/myoepithelial cells. Developmental transcription factors (TFs) control stem cell self-renewal and differentiation. TFs driving luminal differentiation are well studied, however the regulators of MaSC maintenance and myoepithelial differentiation of basal cells are poorly understood. The helix-loop-helix (HLH) TF Inhibitor of Differentiation 4 (ID4) is exclusive to basal cells and is essential for appropriate mammary gland development. ID proteins lack DNA binding domains, and function by binding to and inhibiting basic HLH (bHLH) TFs. The overarching aims of this thesis were to i) isolate and characterise mammary epithelial cells expressing ID4, and ii) integrate multi-omic approaches to define the cis-regulatory mechanism through which ID4 controls cell fate. I found that selecting for cells with high ID4 expression enriched for basal cells with stem cell characteristics. Moreover, basal cells with low ID4 expression exhibited a differentiated myoepithelial phenotype. ID4 was required to inhibit expression of genes involved in muscle contraction, and to activate genes involved in cell growth, metabolism and cell cycle, suggesting that ID4 blocks myoepithelial specialisation and maintains proliferation of MaSCs. ID4 was found to interact with ubiquitously expressed bHLH TFs, E2A, HEB, and ITF-2 (E-proteins) using an unbiased proteomics approach. E-proteins co-regulated a large subset of ID4 target genes. This was unexpected, due to the typical role of ID proteins in antagonising E-protein activity. Motif enrichment revealed E-protein binding motifs in the promoters of genes co-regulated by both ID4 and E-proteins. This raises the possibility of a novel mechanism for ID4 in acting as a chromatin-bound co-factor for E-proteins, rather than simply sequestering E-proteins in the nucleoplasm. Chromatin Immunoprecipitation followed by DNA-Sequencing (ChIP-Seq) experiments will directly test this hypothesis. I discovered that ID4 dictates the MaSC fate by inhibiting myoepithelial differentiation, potentially by cooperating with E-proteins at the level of chromatin. Lessons learnt from the mammary gland may have broader implications in the regulation of adult stem cells by HLH TFs.
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(2019)ThesisCancer immune evasion is recognised as a central hallmark of tumour development. One mechanism that cancer cells use to protect themselves from anti-tumour immune responses is the over-expression of Programmed Death Ligand 1 (PD-L1). The immune checkpoint protein Programmed Death receptor 1 (PD-1) expressed by lymphocytes negatively regulates T-cells effector functions against target cells, including tumour cells. Several therapeutic monoclonal antibodies targeting PD-L1/PD-1 have been approved by the FDA for adult melanoma and lung cancer. However, their efficacy is limited by acquired resistance and immune-related adverse events in many patients. Additionally, the regulation of PD-L1 expression on tumour cells is still poorly understood. Copper transporter 1 (CTR-1) and copper levels are elevated in tumours and the use of copper targeting agents is currently under intense investigations. It has been also reported that copper plays a major role in the immune system, but its activity is unclear. In this study, we demonstrated for the first time that copper plays a key role in the expression of PD-L1 in cancer cells. Tissue microarrays from neuroblastoma and glioblastoma patients showed a significant correlation between CTR-1 and PD-L1 expression (p<0.0001 and p=0.006 respectively). Deep analysis of the TCGA database showed that CTR-1 and PD-L1 association also occurs across many cancer types characterised by high copper levels, but not in the corresponding normal tissues. In vitro experiments showed that copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells. Consistently, Dextran-Catechin (DC) and TEPA, copper-lowering drugs, were able to down-regulate PD-L1 expression in cancer cells, both post-transcriptionally by inhibiting EGFR-phosphorylation and promoting ubiquitin-mediated PD-L1 degradation and transcriptionally by reducing STAT signalling pathway. In vivo studies showed that copper-lowering drugs slowed tumour growth and improved mice survival, by down-regulating PD-L1 expression, which in turn caused a significantly increase in number of tumour-infiltrating CD8+ T cells and natural killer cells. This study reveals an important role for copper in regulating PD-L1 expression and it raises the potential of repurposing copper-lowering drugs to enhance immune checkpoint blockade. This novel therapeutic approach could target a wide spectrum of copper-dependent cancers.
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(2019)ThesisT cell activation is an essential part of the immune response and requires the activation of the T cell receptor (TCR) and co-receptors, including CD28. After ligand binding, these receptors are internalised into intracellular compartments, sorted for degradation or recycling back to the plasma membrane (PM). Endocytic trafficking plays a central role in the modulation of the immune response, as it regulates intensity and duration of receptor signalling. Much remains to be understood about how these processes are regulated for the TCR and CD28, which are crucial for an effective and sustained T cell activation. In this work, I developed and used a wide range of fluorescence microscopy approaches to investigate the endocytic pathways of TCR and CD28, which are detailed in chapter three. In chapter four, I contributed to decipher the regulation of TCR endocytic trafficking. My data showed, that TCR internalisation is clathrin independent and relies on dynamin for scission of the vesicle from the PM. My results further revealed that after endocytosis, TCR accumulates into an endosomal network which relies on the membrane-organising proteins flotillins. Finally, I contributed to show that flotillins promote sorting of TCR into a fast recycling pathway composed of Rab5 and Rab11-positive endosomes. In chapter five I aimed to understand the role of sorting nexin 9 (SNX9) in CD28 endocytosis. However, the results obtained point towards a different role for SNX9 in the regulation of the spatial organisation of CD28. My data showed that T cell activation leads to the de novo formation of an endosomal network demarked by SNX9. This network is proximal to the immunological synapse and does not extend inside the cell. Furthermore, SNX9 is also present at the PM where it strictly localises within CD28 clusters. I further showed that SNX9 is not required for CD28 clustering, recruitment to the PM or internalisation. Instead, SNX9 is necessary for the stability of CD28 clusters, phosphorylation and cellular signalling events downstream of CD28 activation such as T cell activation and IL-2 secretion. Altogether, my data shows that various endosomal networks regulate the spatial distribution of key T cell receptors.
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(2019)ThesisThe development of drug resistance remains a major limitation to the successful treatment of high-risk neuroblastoma, with the survival rate still below 50%. Multidrug resistance is the simultaneous resistance to multiple unrelated drugs and can be conferred by the expression of proteins from the ABC transporter superfamily. Multidrug resistance-associated protein 1 (MRP1) is frequently overexpressed in tumours and effluxes chemotherapeutic agents from tumour cells, protecting them from chemotherapy. In neuroblastoma, elevated MRP1 expression is highly prognostic of poor clinical outcome and contributes to chemoresistance in the Th-MYCN transgenic mouse model of high-risk neuroblastoma. Based on the previously identified MRP1 modulator Reversan, a series of highly promising new MRP1 modulators were developed with a view to identifying candidate molecules suitable for clinical development as chemosensitising agents. These second-generation modulators were highly selective for MRP1 over P-glycoprotein, ABCG2, MRP2 and MRP3, and sensitised the neuroblastoma cell lines Kelly and IMR32 at least two-fold to the substrate drugs etoposide, vincristine and arsenic trioxide in vitro (P<0.001). The second-generation modulators were active in the Th-MYCN transgenic mouse neuroblastoma model, doubling survival of etoposide-treated animals without impacting on etoposide pharmacokinetics, but conferred only minimal survival advantage in Kelly human cell line xenografts. Additional studies with independent MRP1 inhibitors suggest that inhibition of MRP1 alone is not sufficient to sensitise human neuroblastoma xenografts to standard-of-care agents. In addition to blocking efflux of chemotherapy, the second-generation modulators stimulated MRP1-dependent efflux of reduced glutathione (GSH), leading to depletion of intracellular GSH. MRP1 modulators synergised with the GSH synthesis inhibitor L-buthionine sulfoximine (BSO) in MRP1-expressing cells, leading to near-complete intracellular GSH depletion, abolition of colony formation and sensitisation to chemotherapeutics. This dual activity may be beneficial for the treatment of neuroblastoma and other MRP1-expressing tumours. In conclusion, MRP1 inhibition alone may be insufficient to sensitise high-risk neuroblastoma to chemotherapy, however highly selective second-generation MRP1 modulators may increase the therapeutic window for standard-of-care drugs through combined inhibition of drug efflux and depletion of GSH.
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(2018)ThesisEstrogen receptor positive (ER+) breast cancer constitutes 70% of all breast cancers and anti-ER therapies such as aromatase inhibitors and tamoxifen represent the main therapeutic strategies in the treatment of this disease. Unfortunately, up to 30% of all primary ER+ tumours will ultimately develop endocrine-resistance and progress on ER-targeted therapies resulting in disease-related morbidity. As a result, there is an urgent medical need for novel therapeutic strategies capable of managing endocrine-resistant breast cancer. Androgen receptor (AR) is expressed in up to 90% of ER+ breast cancers. AR functions as a tumour suppressor in primary ER+ breast cancer and high AR positivity is strongly associated with a favourable patient outcome in the ER+ setting. However, the role of AR in endocrine-resistant breast tumours is highly controversial with data supporting both oncogenic and tumour suppressive functions reported in the literature. Here I have used different modulatory approaches on in vitro and in vivo preclinical models to dissect the functions of AR and determine the best approach to target AR in endocrine-resistant breast cancer. I use an siRNA-mediated approach to knock down AR in cell line models and discover that the basal expression of AR contributes to endocrine-resistance and that loss of AR restores classical ER signalling and reverses endocrine-resistance. However, inhibiting the transcriptional activity of AR with enzalutamide does not recapitulate this effect, suggesting that it is the non-canonical activity of AR which contributes to endocrine-resistance. In contrast, I show that activation of AR by either 5-α dihydrotestosterone (DHT) or selective AR modulator enobosarm in vitro and in patient derived (PDX) models of endocrine-resistance results in significant growth suppression. Mechanistically, this growth-inhibitory effect of AR activation is associated with downregulation of ER signalling. Moreover, I identify AR-regulated genes from the global gene expression of an ER+AR+ endocrine-resistant PDX model treated with DHT and establish a highly prognostic AR gene signature based on primary ER+ patients in the METABRIC dataset. This suggests that activity of AR is tumour-suppressive independent of endocrine-sensitivity. In summary, I demonstrate that activation, not antagonism, is the optimal AR-targeted therapeutic strategy in the management of endocrine-resistant breast cancer.
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(2019)ThesisThe skeleton is a multifunctional organ-system, providing structural support to the body and maintaining mineral homeostasis through endocrine interactions with distant organs. Balance between these functions is critical to skeletal health and is regulated by a network of cells distributed throughout bone tissue - the osteocyte network. Little is known of the genes with which the osteocyte network performs this specialised function. As a consequence, understanding of its contribution to skeletal disease is very limited. I hypothesised that defining gene expression in the osteocyte network would reveal genes important to its function and provide insights into skeletal disease. To test this, I developed techniques to perform transcriptome sequencing on the osteocyte network and analysed how gene expression is influenced by skeletal-site, age and sex. I established experimental and analytical strategies to identify a signature of genes enriched for expression in the osteocyte network and reveal molecular processes enabling its specialised function. These genes were examined for their association with skeletal dysplasia and clinically relevant skeletal traits. This work revealed that gene expression in osteocytes is highly conserved between skeletal sites, with the exception of a limited number of developmental transcription factors differentially active between adult bone types. Dynamic changes in the osteocyte transcriptome during skeletal maturation were also identified, including the sexually dimorphic regulation of genes associated with perilacunar-remodelling. An osteocyte transcriptome signature was defined - 830 genes enriched for expression within the osteocyte network. Enriched expression in the osteocyte network was the first evidence of skeletal involvement for the majority of signature genes, including novel genes with skeletally-restricted activity alongside known osteocyte markers. This work identified a range of signalling pathways significantly enriched in the osteocyte network, including neuron-like network formation pathways upregulated early in osteocytic differentiation. This osteocyte signature is enriched for gene-orthologs known to cause human skeletal dysplasias and influence bone mineral density. These discoveries identify the genes and molecular processes that define the osteocyte network and demonstrate that specific expression in the osteocyte network may be a powerful filter to identify genes that cause skeletal disease.
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(2018)ThesisDevelopment of a protective vaccine against hepatitis C (HCV) infection is essential to combat the global epidemic. In primary HCV infection development of neutralising antibody responses are associated with clearance, and therefore induction of such responses should be a key element of vaccine design. Very little is known about the induction of the HCV-specific B cell responses, which underpin neutralising antibody production. Several approaches to identification of HCV-specific B cells were explored, before a tetramer-based assay involving fluorescently-labelled Envelope (E2) glycoproteins was established. A multi-parameter flow cytometry panel was developed to identify B cell sub-populations (CD19, CD20, CD10, CD21, CD27, CD38, IgD, IgG) and activation / inhibition markers (CD86, CD95 and FcRL4), as well as tetramer. This panel was applied to three HCV sample sets: a cross-sectional, case-control series of subjects with cleared (Clearers) or chronic infections (Chronics); longitudinally collected samples from 19 subjects followed from the early acute phase of infection through to clearance or chronicity and 7 subjects during reinfection. The HCV E2 tetramers provided a sensitive and specific detection system for E2- specific B cells, with comparable frequencies detected in the majority of Clearers and Chronics in the cross-sectional study. In primary infection, an early accumulation of antibody-secreting cells (ASCs) was found, associated with an increase in the IgD-IgG- memory B cell subset, raising the possibility of an IgM (or IgA)-predominant isotype of the circulating memory B cell precursors to ASCs. There was upregulation of CD86 in memory B cells expressing the IgD-IgG+ phenotype, whereas CD95 was increased in both IgG- and IgG+ subsets. Subjects who developed chronic infection had high expression of FcRL4 on several subsets, suggestive of functional impairment. E2-specific B cell responses were varied between subjects regardless of infection outcomes. In the reinfection samples, there was an apparent expansion of HCV-specific resting memory B cells (CD21+CD27+) and relative contraction of activated memory B cells (CD21-CD27+) in those who achieved repeated clearance. These data provide the first detailed characterisation of the pattern and kinetics of HCV E2-specific B cell responses in primary infection and reinfection, and will underpin further studies to resolve the optimal HCV-specific B cell response characteristics for vaccine design.
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(2019)ThesisThe importance of physical activity (PA) for optimal growth and development is well recognised, yet most Australian children do not achieve the recommended three hours per day. Fundamental motor skills (FMS) are the building blocks for complex movement patterns used in a range of sport and recreations including playground games and organised sport. The aim of this thesis is to determine effective methods of improving FMS and PA levels in preschool aged children. The literature review (chapter 2) examines how PA and FMS are defined and measured, and the factors associated with PA levels and FMS proficiency in preschool aged children. The systematic review and meta-analysis (chapter 3) examines the optimal intervention characteristics for increasing FMS proficiency and PA levels in children. Teacher-led interventions conducted three or more times a week are most effective at increasing FMS and showed trends towards increasing overall PA, especially moderate-vigorous PA levels in pre-school aged children. These optimal intervention characteristics were then applied in The PhysicaL ActivitY & FUNdamental motor skills Program (PLAYFun) presented in chapters 4 and 5. PLAYFun led to significant improvement in FMS proficiency between the PLAYFun and control groups at the completion of the intervention, however improvement was not maintained at 12-weeks post intervention, indicating the ongoing need for FMS practice. PLAYFun was time and resource intensive, limiting its widespread dissemination. Therefore, chapter 6 describes The Fundamental Motor Skills Movie Education for Parents Program (FUN MOVES), which was designed to overcome the resource intensive barrier of PLAYFun. FUN MOVES was developed to educate parents, through weekly videos, to teach their children FMS with an emphasis on skill development and acquisition. However, no significant differences were found between groups for measures of FMS or PA. The findings of the studies contained within this thesis found that face to face FMS program delivery is effective at increasing FMS proficiency in preschool children but is resource intense. Digital delivery of a FMS program to parents, whilst addressing the limitations of face to face delivery, may not be associated with significant improvement in FMS proficiency and warrants further study.