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(2013)Journal ArticleA pathological hallmark of Alzheimer’s disease (AD) is the presence of amyloid-beta peptide (Aβ) plaques in the brain. Aβ is derived from a sequential proteolysis of the transmenbrane amyloid precursor protein (APP), a process which is dependent on the distribution of lipids present in the plasma membrane. Sphingomyelin is a major membrane lipid, however its role in APP processing is unclear. Here, we assessed the expression of sphingomyelin synthase (SGMS1; the gene responsible for sphingomyelin synthesis) in human brain and found that it was significantly elevated in the hippocampus of AD brains, but not in the cerebellum. Secondly, we assessed the impact of altering SGMS activity on Aβ generation. Inhibition of SGMS activity significantly reduced the level of Aβ in a dose and time dependent manner. The decrease in Aβ level occurred without changes in APP expression or cell viability. These results when put together indicate that SGMS activity impacts on APP processing to produce Aβ and it could be a contributing factor in Aβ pathology associated with AD.
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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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(2016)ThesisHLA-B27 is a Class I Major Histocompatability Molecule that is highly prevalent in patients with Acute Anterior Uveitis (AAU) and Seronegative Arthritis (SNA). Despite over 40 years of research, the pathogenic mechanism of HLA-B27-related disease remains elusive. The bacterium Chlamydia trachomatis has been implicated in disease pathogenesis, whereby molecular mimicry results in an antigen derived from the microorganism to cross-react with a molecule in the host, triggering an inflammatory response. Lumican is an extracellular molecule that is found in both the eye and joint and has important structural, functional and immunological functions. This study showed that lumican was present in the aqueous humor of eyes in patients with AAU. Furthermore, immunohistochemical studies did show it is present in the iris and associated blood vessels, the site of inflammation of AAU. Additionally it was found that lumican has sequence homology with the cell wall component of C. trachomatis. Given these properties of lumican, this study aimed to examine cellular responses to peptide antigens derived from C. trachomatis and lumican in the peripheral blood of patients with AAU and SNA and to compare the prevalence of positive serology to Chlamydia in patients who are HLA-B27-positive to those who are HLA-B27-negative. It was shown that HLA-B27-positive patients with both AAU and SNA were more likely to display positive IgG serology to Chlamydia compared to healthy controls, suggesting that past exposure to Chlamydia is important in disease pathogenesis. ELISPOT assays were employed to measure cellular responses to antigens. They showed that HLA-B27 patients had a higher percentage of responders to groups of peptides derived from C. trachomatis, lumican and other extracellular matrix components than both HLA-B27 negative patients and healthy controls. Using flow cytometric methods CD4+ and CD8+ T cell responses were measured which indicated that patients with HLA-B27-related disease responded more to some antigens, and these reached statistical significance when CD8+ T cell responses to some antigens were measured. In summary, this study does suggest that cross-reactivity between a bacterial trigger and a self-protein may be important in disease pathogenesis, lending weight to the hypothesis that there exists an arthritogenic or uveitogenic peptide.
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(2015)Thesisα1 adrenoceptors are three of the nine receptors that bind and respond to the hormones adrenaline and noradrenaline. α1 adrenoceptors mediate a number of physiological responses such as smooth muscle contraction as well as modulating cognition. The high homology between the nine adrenoceptors, as well as other biogenic amine receptors, results in a difficulty in finding highly selective drugs for these receptors. A potential target site for highly selective ligands is the extracellular domain, which has low sequence similarity across the receptors This thesis uses in silico homology modelling and molecular docking to identify and characterise potentially exploitable residues in the extracellular domain of the α1 adrenoceptors. Firstly, D191 in the second extracellular loop of the α1B adrenoceptor was identified in a homology model and shown, by mutagenesis, to have an impact on agonist binding to the receptor, with no effect on subsequent receptor activation. It appears that D191 is contributing direct contact with agonists, even though its position on the extracellular surface of the receptor suggests that it does not form a part of the canonical orthosteric binding site. Following this, a series of bisacridines, 9-aminoacridine moieties conjugated with increasing length methylene linkers, was used as a molecular ruler to investigate the optimum size of a drug to engage in critical contacts with the receptor. The 4-carbon linker was found to be ideal for selective binding, but the 9-aminoacridine based ligands displayed cooperative binding that was subsequently attributed to a bitopic mode of binding that engaged an allosteric site on the receptors. Docking was used, taking advantage of the bitopic mode of binding, to identify residues at the extracellular end of TMII that, when mutated, affected [3H]prazosin dissociation rates, and the magnitude of the allosteric effect of the 9-aminoacridines. All of the residues identified were on the extracellular surface of the receptor and are thought to contribute to the binding/debinding pathway for ligands of the α1 adrenoceptors and other closely related biogenic amine receptors. This structure data can be useful for identification and further development of highly selective, allosteric modulators of the α1 adrenoceptors.
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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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(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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(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.