Pathology - Theses
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ItemApplication of bayesian networks to a longitudinal asthma study( 2016)Asthma is a highly prevalent and often serious condition causing significant illness and sometimes death. It typically consumes between 1-3% of the medical budget in most countries and imposes a disease burden on society comparable to schizophrenia or cirrhosis of the liver. Its causes are as yet unknown but a significant number of risk factors, covering such diverse factors as viral infections during infancy, blood antibody titres, mode of birth and number of siblings have been identified. In recent years there has been increasing recognition of the role played by the microbiome in human health, with a growing understanding that our relationship with the microbes that colonise the different parts of the human body is symbiotic. Disruptions in the microbiome have been implicated in diseases such as obesity, autism and auto-immune diseases, as well as asthma. At the same time there has been an increasing awareness in asthma research that its multi-faceted and multi-factorial nature requires more sophistication than statistical association and regression. In this spirit we employ Bayesian networks, whose properties render them suitable for representing time-direct or even causal relationships, to gain insight into the nature of asthma. We begin with an example of the simplest Bayesian networks, a linear classifier, with which we predict outcomes in the fifth year-of-life according to the statistical distribution of variables from the first two years-of-life. (The qualification linear refers to the neglect of correlation and interaction among the predictive variables.)While classifiers have long been used for prognosis and diagnosis, we use them to identify useful asthma subtypes, called endotypes. Different endotypes often require different treatments and management programs, and driven by different biological factors. These different factors provide different predictors, and a predictor which separates one endotype from the healthy may not do so for a different endotype. We use this to mathematically construct an indicator of when a given predictor is exclusively predictive of a given endotype. Our so-called “exclusivity index” is quantitatively precise, unlike a significance threshold. The Cohort Asthma Study, whose longitudinal data we analyse, includes the relative abundances of genera present in the nasopharyngeal microbiome. In an apparent diversion, we use qq-plots to indicate relationships between the infant microbiome and fifth-year wheeze- and atopy- status. Interestingly, the relative abundance of Streptococcus under certain circumstances was found to be highly predictive of one of the endotypes we identified in the preceding chapter. Finally, we address the problem of mapping out the complicated interactions among multiple variables. Our model is an adaption of a package originally designed for inferring gene-interaction networks, called ARTIVA. This was a non-trivial matter requiring us to augment the discrete data values in order to make them compatible with the underlying mathematics of ARTIVA’s algorithm. With questions from the asthma literature and the posterior probabilities output by ARTIVA, we were guided to networks of the interactions between atopy, wheeze and infection, and could see the difference in the development of immunity-related variables between those who went on to exhibit wheeze in the fifth year-of-life and those who did not. Our model yielded networks indicating that sensitivity to viral infection is an effect and not a cause of atop and wheeze.
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ItemNorepinephrine transporter gene silencing by MeCP2 in postural tachycardia syndrome( 2016)The Postural Tachycardia Syndrome (POTS) is characterized by the clinical symptoms of orthostatic intolerance, light-headedness, tachycardia and syncope or near syncope with assumption of upright posture. While the aetiology remains largely unknown, faulty neuronal reuptake of the sympathetic nervous system signaling neurotransmitter has been implicated. The action of norepinephrine (NE) is terminated, in part, by its uptake into presynaptic noradrenergic neurons by the plasma-membrane NE transporter (NET) which is encoded by the SLC6A2 gene. The effectiveness of NE reuptake relies on the capacity of NET to tightly recapture NE released by sympathetic nerves, this being approximately 90% for the heart. Since inter-individual variation in NET function is likely to be common and, in light of the clinical importance of the transporter’s central role in NE regulation, rather than querying genetic variation, we investigated gene-environment interactions to determine which biological processes may be prime targets for SLC6A2 regulation. Experimental evidence from our group has previously shown chromatin-modifying events associated with SLC6A2 repression may augment epigenetic regulation in POTS. SLC6A2 repression is associated with substitution of active histone modifications with repressive modifications and binding of Methyl CpG binding protein-2 (MeCP2) co-repressor complex. MeCP2 is commonly assumed to function mainly as a silencing factor at methylated DNA sequences. Interestingly, DNA methylation is unremarkable between the case and control cohort. In context to these findings and given that non-coding RNAs (ncRNAs) interactions can induce structural changes to chromatin to regulate transcription, we hypothesized that MeCP2 binding is dependent on its interaction with ncRNAs. The purpose of this study was to investigate SLC6A2 silencing with the specific aim to understand epigenomic regulation as a mean to reactivate expression. We developed a novel RICh-seq (RNA of Isolated Chromatin combined with sequencing) method to identify SLC6A2 promoter bound RNAs. Analysis of RICh-seq data identified let-7i associated with SLC6A2 promoter with elevated expression in POTS patients. We show that let-7i miRNA interacts with MeCP2 co-repressor to silence SLC6A2 activity in POTS subjects. Furthermore, we demonstrate that in POTS subject’s histone modifying drugs such as histone deacetylase inhibitors and inhibitor for EZH2 (Enhancer of zeste homologue 2) restores epigenetic modifications associated with SLC6A2 gene expression. Our results represent the first pre-clinical description for the gain of NET function in POTS and a previously unknown target of pharmacological therapy.
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ItemCuproenzyme dysfunction in the pathogenesis of amyotrophic lateral sclerosis and multiple sclerosis( 2016)Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the selective death of motor neurons within the spinal cord and brain. Although the aetiology of the disease is not well understood, inherited genetic mutations account for a small proportion of cases, with Cu,Zn-superoxide dismutase (SOD1) mutations being the most extensively studied. Effective treatment options for ALS do not exist, however, pre-clinical outcomes indicate that therapeutically modulating copper bioavailability in the central nervous system (CNS) may be a feasible treatment strategy for ALS. Therefore, the initial objective of this study was to investigate the significance of copper dyshomeostasis in the progression of a mutant SOD1 mouse model of ALS. We hypothesised that age-related changes to cuproenzymes progress with disease symptoms in SOD1G37R mice compared to age-matched non-transgenic littermates and mice overexpressing wild-type human SOD1. To test this hypothesis, locomotor performance was assessed to track disease progression, then CNS and peripheral tissues were collected at distinct stages of disease for biochemical analyses. Data presented in Chapter 3 provide evidence for copper malfunction in the CNS of ALS mice and indicate that copper malfunction is an early feature of the disease which worsens as symptoms progress. Specifically, a disconnect exists between the abundance and copper-dependent activity of cuproenzymes SOD1 and ceruloplasmin. Next, the therapeutic significance of these changes to SOD1 and ceruloplasmin were assessed. In Chapter 4, data show that overexpressing CTR1 or treating ALS model mice with the copper compound CuII(atsm) extends survival and improves copper bioavailability to SOD1 and ceruloplasmin in the CNS. To ascertain the relevance of outcomes in a broader disease context, we next assessed human cases of sporadic ALS. Data presented in Chapter 5 show that SOD1 and ceruloplasmin dysfunction detected in mice is also evident in sporadic ALS. Significantly, changes to ceruloplasmin are associated with changes to iron homeostasis, where diminished copper- dependent ceruloplasmin activity may contribute to iron overload in the ALS-affected motor cortex and decreased transferrin bound iron in the cerebrospinal fluid. As such, we propose that changes to copper-dependent ceruloplasmin activity in ALS may be the mechanistic basis for two ALS biomarkers and represent the first biochemical evidence for the feasibility of treating ALS, including sporadic ALS, by therapeutically improving copper bioavailability to CNS cuproenzymes. Multiple sclerosis (MS) is a disease characterised by CNS demyelination, with evidence suggesting a link between demyelination and limited copper bioavailability. This is supported by data presented in Chapter 7 from both ALS model mice and MS-affected CNS tissue. We also show that changes to copper, SOD1, ceruloplasmin and myelin-associated proteins are common to ALS and MS, and that modulating copper bioavailability may provide a therapeutic intervention. Overall, data presented in this thesis indicate that: copper malfunction is a feature of ALS and MS; copper malfunction evident in sporadic cases of ALS are recapitulated in mutant SOD1 mouse models of familial ALS; and perturbations to the copper-dependent ceruloplasmin activity may be important to iron accumulation in the ALS-affected motor cortex. The therapeutic implications of these observations are discussed.
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ItemMitochondria and energy metabolism in cell culture models of motor neuron disease( 2016)Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the selective loss of motor neurons. Although a relatively small proportion of all ALS cases are caused by familial mutations in proteins such as superoxide dismutase 1 (SOD1) and transactive response DNA binding protein 43 (TDP43), shared clinical and pathological features across sporadic and familial cases of ALS suggest that both may share a common underlying mechanism. The reasons why motor neurons are primarily affected in ALS, particularly in those cases caused by a ubiquitously expressed mutation, remain unknown. Given the disproportionately high energy demand of motor neurons when compared to other cell types, and the wealth of evidence demonstrating the role of impaired energy metabolism in both sporadic and familial ALS, it is possible that the selectivity of motor neuron death in ALS involves impaired energy metabolism. To investigate this possibility, a better understanding of the mechanisms leading to impaired energy metabolism in ALS is needed, and this is dependent upon the availability of valid models. Although neurons in the body are largely dependent on mitochondrial oxidative phosphorylation (OXPHOS) to meet the bulk of their energy demands, most cultured cells generate the bulk of their energy via glycolysis. Substituting medium glucose with galactose is one way of increasing OXPHOS in cultured cells, however galactose is not normally present in the central nervous system (CNS). Commonly used cell culture conditions, therefore, limit the ability to extrapolate results obtained under such conditions to in vivo neurons. As such, the hypothesis of this thesis was that increasing reliance on mitochondrial OXPHOS in cultured cells in a way that more closely replicates the in vivo energy metabolism of neurons will expose energy metabolism deficits due to ALS-causing mutations. To establish a cell culture model in which cells have an increased reliance on mitochondrial OXPHOS in a way that more closely replicates the in vivo energy metabolism of neurons (Aim 1), primary cortical neurons were initially utilised for this study. However, due to the slow consumption of extracellular glucose cultured neurons could not be easily driven towards dependence on extracellular lactate as the primary fuel of mitochondrial OXPHOS. In subsequent experiments an alternative cell type, primary mouse embryonic fibroblasts (pMEFs), were grown in medium containing 3.5 mM glucose and two phases of growth were identified: the initial glucose-consuming (i.e. glycolytic) phase followed by the lactate-consuming (i.e. OXPHOS) phase. The lactate-consuming phase was characterised by increased Mito-Tracker Deep Red staining intensity, increased expression of nuclear-encoded mitochondrial proteins, and increased sensitivity to the OXPHOS inhibitor rotenone. Thus, by inducing cell autonomous depletion of glucose from the culture medium cells were forced cells to utilise extracellular lactate via mitochondrial OXPHOS to supply their energy needs. To determine the effects of ALS-causing mutations on the mitochondria and energy metabolism of cells that have an increased reliance on mitochondrial OXPHOS (Aim 2), pMEFs derived from mice expressing mutant SOD1-G37R or mutant TDP43-A315T were grown under conditions established in Aim 1. No difference between control and mutant pMEFs (SOD1-G37R or TDP43-A315T) was observed with respect to any of the mitochondrial and energy metabolism parameters investigated. By contrast, when human fibroblasts derived from an ALS patient expressing mutant TDP43-M337V were grown under the same conditions, expression of TDP43-M337V suppressed upregulation of several nuclear-encoded mitochondrial proteins. However, this was also observed in human fibroblasts that were grown under conditions whereby they continued to be glucose-consuming (i.e. glycolytic) throughout the culture period. Common to both culture conditions was the fact that irrespective of metabolic state, cell proliferation markedly decreased with time in culture. This indicates that, in contrast to the need to grow cells under conditions whereby an increased reliance on OXPHOS is achieved via manipulating the availability of glucose, the proliferative state of the cells appeared to be a greater determinant of mutant TDP43-mediated effects than the relative availability of glucose or lactate. Therefore, increased reliance of cultured cells on mitochondrial OXPHOS in a way that more closely replicates the in vivo energy metabolism of neurons does not appear to be necessary to expose energy metabolism deficits due to ALS-causing mutations. Rather the findings presented in this thesis indicate that it is necessary to analyse less proliferative cells, which more closely replicate the non-proliferative state of terminally differentiated post-mitotic neurons, in order to expose mitochondrial changes due to ALS-causing mutations. This is the first study to show a connection between a TDP43 mutation and nuclear-encoded mitochondrial protein alterations. Given the high energetic demand of motor neurons, this finding may explain why terminally differentiated post-mitotic neurons are more sensitive to ubiquitously expressed mutant TDP43.
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ItemLynch syndrome in the Asian populations( 2016)Lynch syndrome is an autosomal dominant genetic disorder. Mutation carriers are at significant risk of developing colorectal and a variety of extra-colonic cancers, often at a younger age compared to sporadic cancers. The current guidelines on detection and management of Lynch syndrome are based upon studies in Caucasian populations with Lynch syndrome. The applicability of these guidelines to Asian populations is not known, as the prevalence and risks associated with mutations in mismatch repair genes may be different compared to Caucasian populations. The aims of this thesis were: 1. To assess the applicability of the current guidelines for the screening of Lynch syndrome and cancer surveillance strategies in the Asian populations (Chapter 2). 2. To study the phenotype of Lynch syndrome in Asian populations (Chapter 3). 3. To describe the mutation profile of novel pathogenic Asian variants and the associated phenotype of Lynch syndrome (Chapter 4). 4. To examine the applicability of next generation sequencing technology in microsatellite instability testing (Chapter 5). Chapter 2 describes the performance of seven predictive models (PREMM1,2,6, MMRpro, MMRpredict, Wijnen, Myriad, Amsterdam Plus, and Amsterdam Alternative), which were derived from European/Caucasian populations, on the screening of Lynch syndrome in the Asian populations with colorectal cancer. There was no evidence that these models will perform poorly in Asian families with performance similar to that reported for Caucasian families. However, many mutation carriers were not detected when these models were tested on Asian families simulated under the one-child policy (China). In addition, similar inferior performance was observed when these models were applied to families with a low penetrant gene associated with Lynch syndrome (e.g.: PMS2). These findings provide strong evidence that these models are applicable to Asian families where the size is not restricted. Their superiority over existing clinical guidelines indicates that case detection of Lynch syndrome can be maximised while minimizing false positive results when these models are applied instead of simply the Bethesda Guidelines or Amsterdam Criteria. However, alternative methods for assessing who should be tested for mutations need to be developed apart from family history in settings where small families are common. All models in this study may be deficient for the families with a PMS2 mutation, and alternative methods should be explored. Chapter 3 examines the phenotype of Lynch syndrome in Asian populations. To date, many conflicting results are observed about phenotype of Lynch syndrome in the Asian populations amongst the published studies. While the majority of Asian studies indicated that Lynch syndrome phenotype in Asian and Caucasian populations share many similarities, other studies have disagreed. In Chapter 3, no differences in the risk of cancers related to Lynch syndrome were observed when the phenotype of Asian and Caucasian mutation carriers were compared. These findings suggest that Lynch syndrome in the Asian and Caucasian populations are indeed quite similar, and raise the possibility of a similar carcinogenesis pathway that is not affected by ethnicity. Consequently, Asian mutation carriers with Lynch syndrome should be managed according to the current cancer surveillance programs, which are based on data of Lynch syndrome in Caucasian populations, until an ethnicity-specific program becomes available. The findings of this chapter also explain why the predictive models, which were developed using European/Caucasian populations, performed well in Asian populations as demonstrated in Chapter 2. The discrepancy of Lynch syndrome phenotypes reported amongst the Asian studies can be explained by their recruitment strategies. Many of these studies derived their phenotypic data from families that only fulfilled clinical guidelines without germline testing, and many sporadic cancers may have been included in their analyses. In contrast, all the families with Lynch syndrome in the present study have been confirmed to carry a pathogenic mismatch repair (MMR) gene mutation. Chapter 4 describes the phenotype of Lynch syndrome in Asian families with a novel pathogenic mutation. These mutations are yet to be described in the literature pertaining to Lynch syndrome in Caucasian populations. The reported phenotypic manifestation of Lynch syndrome in the Asian populations varies widely amongst the available studies, with some studies concluding that the cancer risk of Asian and Caucasian populations with Lynch syndrome is similar while other studies disagree. It remains uncertain if these novel variants confer a different cancer risk compared with Caucasian populations with Lynch syndrome. In Chapter 4, Asian families with a novel pathogenic MMR gene mutation were demonstrated to share many phenotypic manifestations as described in the Lynch syndrome literature based on Caucasian populations. This strengthens the hypothesis that Lynch syndrome mutation carriers of different ethnicities progress through similar carcinogenesis pathway despite the presence of novel mutations. These findings also strengthen the conclusions derived from the study of Lynch syndrome phenotype in the Asian populations in Chapter 3, and explain why the MMR predictive models, that were developed and validated in the Caucasian populations, performed well in Asian populations with colorectal cancer as described in Chapter 2. Chapter 5 describes the testing of microsatellite instability (MSI) using next generation sequencing (NGS) technology. The current family history-based screening strategies are inadequate in the detection of Lynch syndrome in small Asian families and families harbouring a gene with reduced penetrance (e.g.: PMS2) as described in Chapter 2. An alternative screening strategy would be to implement universal MSI testing, regardless of age of diagnosis or family history. The current MSI testing platform is based on multiplex polymerase chain reaction (PCR) technology, which may not meet the demand if MSI testing of cancers related to Lynch syndrome becomes universal. In chapter 5, NGS technology has been demonstrated to be a feasible platform for MSI testing. The main advantage of NGS over the traditional multiplex PCR-based method is that it allows simultaneous testing of large batches of cancer samples, thus improving the efficiency of testing. In addition, compared to other NGS-based MSI testing approaches, the method described in the present study obviates the need for genome-wide alignment in data analysis. Therefore, complex data processing pipelines are not required and data analysis can be performed using standard computing resources. In conclusion, this thesis describes the characteristics of various cancers related to Lynch syndrome and the screening of Lynch syndrome using the prediction models in the Asian populations. The data suggests that Asian and Caucasian populations with Lynch syndrome share many clinical characteristics, and the current Caucasian-based clinical guidelines and screening models are applicable to the Asian populations. NGS technology is applicable to MSI testing, and this would be a promising platform if universal colorectal cancer screening for Lynch syndrome becomes the standard of practice.
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ItemTemporal and spatial regulation of Yorkie levels and activity throughout Drosophila melanogaster larval development( 2016)The regulation of tissue growth is a fundamental process in normal development, ensuring correct scaling of organs and limbs and ensuring growth does not exceed that which can be supported by nutritional availability. The Hippo signalling pathway is a recently discovered, yet ancient and highly conserved regulator of growth that was initially identified in the vinegar fly Drosophila melanogaster. A key role of Hippo signalling is to regulate growth through the activity of the transcriptional coactivator Yorkie. The main mechanism by which this is achieved is by regulating Yorkie's nuclear and cytoplasmic localisation. Importantly, deregulation of the Hippo pathway has been found to drive the development of a wide range of cancers. Therefore understanding the mechanisms by which this pathway regulates growth both in normal development and in disease set-tings is of paramount importance. The high degree of conservation between D. melanogaster and mammals make D. melanogaster a key model system for unravelling the intricacies of this pathway. I sought to increase our understanding of the mechanisms by which the Hippo pathway controls development and chose to focus on the role of Yorkie, the key transcriptional activator of Hippo signalling. I asked a number of questions to address this aim: 1) Does Yorkie localisation correlate with its function throughout development in D. melanogaster? 2) What are the dynamics of the subcellular localisation of Yorkie during D. melanogaster epithelial tissue development? 3) Which components of the ubiquitination machinery are responsible for regulating Yorkie-mediated tissue growth? In addressing these questions I came to the following conclusions: 1) Yorkie sub-cellular localisation changes throughout development of larval epithelial tissues, and the nuclear localisation of Yorkie does not necessarily correlate with increased Yorkie transcriptional activity, as previously assumed. The transcriptional output of Yorkie is regulated in certain contexts by Notch signalling and histone modifications, and Yorkie also has a role in specification of a key developmental compartment boundary. 2) Using live imaging I showed that Yorkie localises strongly to chromatin during the mitotic phase of the cell cycle, and at this time displays an increased interaction with its key transcription factor partner Scalloped. I also demonstrated that at other times in the cell cycle, when Yorkie appears to be sequestered in the cytoplasm, it is in fact being continuously shuttled in and out of the nucleus and its predominant cytoplasmic localisation is likely the net result of a higher rate of nuclear export than import. I propose a dynamic model that accounts for different contributions of upstream regulators in regulating Yorkie localisation. 3) An RNA interference screen showed that depletion of a number of components of the ubiquitination machinery altered Yorkie-induced growth, and further analyses demonstrated that a number of these components regulate the localisation, protein levels and transcriptional activity of Yorkie. This comprehensive survey of the ubiquitin machinery in D. melanogaster is a rich resource for the study of the role of ubiquitination in growth control and Hippo pathway regulation. These results provide new insights in to the mechanisms by which the key effector of the Hippo pathway is regulated in a normal developmental setting and reveal added levels of complexity to the role and regulation of the nuclear localisation of Yorkie.
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ItemIdentifying the key Aβ oligomer species by correlating cell neurotoxicity and binding properties( 2016)Alzheimer’s disease (AD) represents 60-80% of dementia cases and predominantly affects elderly people above 65 years of age. While it is the second most leading cause of death in Australia, there is no cure or effective treatment available. Amyloid beta (Aβ) peptide which aggregates into fibrils and is deposited as extracellular amyloid plaques in the brain, were originally hypothesized as the pathogenic agent causing AD. Today, there is strong agreement that the lower molecular weight Aβ oligomers correlates best with AD clinical symptoms and are most likely the toxic species causing AD. Different oligomeric Aβ species have been claimed as the toxic Aβ species but the true identity of the toxic species is yet to be resolved. The major challenge working with the Aβ peptide oligomers has been its isolation, preparation, or purification of either synthetic material or brain tissue because of its ability to continually self-aggregate into larger species. In addition, previous reports from our laboratory demonstrated that Aβ binding to neurons was critical for Aβ to induce toxicity. Taken together, my hypothesis for this PhD thesis is that there is a specific oligomeric Aβ species that binds to neurons in order to induce neuronal cell death associated with AD. Therefore, the major aim of my thesis was to identify the specific neurotoxic oligomeric Aβ species that will bind and kill neurons. To address this aim, mouse cortical neuronal cultures were treated with soluble monomerised synthetic Aβ40 and Aβ42 peptides. We identified increasing Aβ trimer and tetramer bound to treated neuronal cultures which correlated with increased cell death. Photo induced cross linking of unmodified peptides (PICUP) technique was utilized to prepare individual Aβ 2mer, 3mer and 4mer. The neurotoxicity of these purified Aβ40 3mer and 4mer was up to 50-fold more potent compared to soluble uncross-linked Aβ peptides. Biophysical and biochemical analyses demonstrated that PICUP crosslinking significantly halted further aggregation of these species, they adopted an identical compact physical arrangement and cross-linking occurred via a di-tyrosine bond. Furthermore, purified PICUP cross-linked Aβ40 2mer, 3mer and 4mer but not 1mer, were able to induce significant inhibition of long term potentiation in mouse hippocampal brain slices indicating that these oligomers can disrupt synaptic function. Aβ peptide immunofluorescence staining of treated neurons showed predominant punctate binding along the axonal and neurite outgrowth and the observation of retrograde trafficking of purified Aβ40 3mers and soluble uncross-linked together support the notion that these oligomers directly target the synaptic membrane. Brilliant blue G dye was used in identifying PICUP cross-linked Aβ peptide bands in SDS-PAGE gels and when bound to Aβ peptide, this resulted in diminished Aβ cell binding and neurotoxicity. However, blocking potential Aβ binding receptor sites with chemical antagonists or deleting PrPc expression did not reduce Aβ cellular binding and neurotoxicity. In conclusion, the results presented in this thesis have demonstrated that Aβ 3mer and 4mer are the key toxic Aβ oligomers causing the AD pathogenesis. Based on these findings, future therapeutic strategies that target these oligomeric species and modulate their toxic or cell binding behaviours will greatly assist in the treatment AD.
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ItemPerforin biochemistry: function and dysfunction( 2016)Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), collectively referred to as cytotoxic lymphocytes (CLs), are responsible for clearing virus infected and cancerous cells. The predominant manner by which CLs do this is through the delivery of the pore forming protein, perforin, and pro-apoptotic granzymes that synergise to induce apoptosis in a conjugated target cell. Humans who inherit bi-allelic inactivating perforin mutations develop the immunoregulatory disease familial haemophagocytic lymphohistiocytosis (FHL) and/or haematological malignancies, demonstrating the critical importance of expressing functional perforin for the maintenance of immune homeostasis and tumour immune surveillance. While most disease associated perforin mutations are rare, 8-9% of the Caucasian population are carriers of polymorphism A91V (rs35947132, 272C>T). It has been suggested that >50% of individuals homozygous for the A91V allele develop FHL and/or cancer and that individuals heterozygous for A91V have an increased susceptibility to ALL. Despite its frequency and disease association, it remained unknown whether heterozygous inheritance of the A91V allele impairs human CL cytotoxicity and, more broadly, whether perforin is rate limiting in CL cytotoxicity. Here, it has been demonstrated that NK cells from healthy humans heterozygous for the A91V allele show an almost 50% reduction in cytotoxicity compared to individuals homozygous for WT perforin. This reduction in function was due to A91V perforin protein being misfolded within human primary NK cells. Moreover, it was also observed that heterozygous perforin knockout mouse CTLs showed an ~50% reduction in cytotoxicity. Taken together, these data demonstrate that perforin is indeed rate limiting for CLs cytotoxicity and therefore, individuals heterozygous for defective perforin alleles have impaired CL function. Although FHL predominantly presents shortly after birth, a subset of patients present at an age greater than three years, owing to the expression of misfolded perforin variants. Previous studies have shown that when transiently expressed in CTLs, perforin variants associated with late onset disease failed to traffic within CTLs and the cells remained non-functional. Therefore, it was unknown how patient CTLs expressing these variants could avoid FHL in infancy, and maintain a level of immune homeostasis for many years, or even decades. Here, it is shown that perforin variants associated with late onset disease can fold correctly and traffic within CTLs, and thus provide a significant level of cytotoxic function. However, this function was found to be lost if CTLs were cultured at an increased temperature (39 ̊C). Taken together, these data suggest that the CTLs of late onset FHL patients may have sufficient cytotoxicity to delay FHL onset in infancy. However, prolonged fever and, potentially, a more rapid exhaustion of the limited pool of correctly folded perforin mutants may result in the loss of CTL function, leading to FHL and cancer later in life. Prior to its secretion from the CL, the evolutionarily conserved C-terminal residues of perforin are proteolytically cleaved. The functional significance of C- terminal processing has remained controversial. Here it is shown that perforin enriched from human NK cell with an intact and glycosylated C-terminus was not cytotoxic. However, removal of the C-terminal glycan from the protein was found to completely restore function. As full-length deglycosylated perforin has wild type activity, these data suggest that C-terminal cleavage of perforin is permissive for cytotoxic function due to removal of an inhibitory N-linked glycan moiety at the C-terminus of the protein. These findings position the protease(s) responsible for perforin cleavage as critical to CL function. In summary, the studies described in this thesis have added to the understanding to how perforin mutations affect CL cytotoxicity and described a critical final step in perforin maturation. Together, these advancements in perforin biology may contribute to the treatment of disease arising from perforin deficiency and also define new factors critical for CL function and human health.
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ItemBiomarkers in ductal carcinoma in situ( 2016)Ductal carcinoma in situ (DCIS), a non-invasive form of breast cancer and a non-obligate precursor of invasive carcinoma of the breast, displays heterogeneous behaviour. Most DCIS are adequately managed by local surgical excision alone, but in 20-30% of cases, disease recurrence occurs after local surgical excision either as DCIS or invasive carcinoma. Accurate identification of these two clinical outcome groups at the time of diagnosis is desirable to allow appropriate treatment allocation. In this thesis, genomic and epigenetic alterations in DCIS epithelium, including copy number aberrations, somatic mutations, and DNA methylation were investigated as markers of DCIS biology and outcome. In addition, the expression and significance of LRH-1, a nuclear receptor which acts as a transcription factor, was investigated in both invasive carcinoma and DCIS. Copy number analysis of DCIS of known clinical outcome identified amplification of 20q13 to be associated with disease recurrence, but this was unable to be validated on an independent cohort. Targeted next generation sequencing of a panel of breast cancer-relevant genes revealed that the mutational profile of DCIS was similar to that reported for invasive carcinomas, with the most frequently mutated genes being GATA3, PIK3CA, and TP53. A high prevalence of GATA3 mutations in DCIS was observed and TP53-mutant DCIS was associated with high stromal tumour infiltrating lymphocytes. Mutations of RUNX1 were a novel finding, not previously reported in DCIS. Promoter methylation of a candidate gene panel, consisting predominantly of known tumour suppressor genes, was associated with adverse tumour features in DCIS. Methylation load varied among DCIS cases, suggesting that methylation differs in importance in the tumorigenesis of DCIS, and that assessment of methylation may be useful as a biological classifier of DCIS. Finally, LRH-1 mRNA expression patterns in breast cancers was similar to that reported for breast cancer cell lines and distinct LRH-1 immunohistochemical staining patterns were associated with tumour phenotype in both invasive breast carcinoma and DCIS. The results of this thesis demonstrate that copy number alterations, somatic mutations, DNA methylation, and LRH-1 expression are indicative of DCIS phenotype and hence biology. These markers showed promise as prognostic biomarkers, although validation of their utility was hampered by the small number of pure DCIS cases with both adequate genomic material and long-term clinical outcome data. Nonetheless, the findings of this thesis indicate that assessment of these biomarkers can be performed in routine diagnostic tissue material and provide several avenues for future research.
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ItemInvestigating the mechanistic link between neuroinflammation and biometal homeostasis in neurodegenerative diseases( 2016)Neuroinflammation and biometal dyshomeostasis are two pathogenic features underlying a number of neurodegenerative diseases, however the mechanistic link between these two pathways has yet to be delineated. This study examined the hypothesis that impaired biometal homeostasis is associated with neuroinflammatory changes. To test this hypothesis I aimed to investigate the effects of key biometals on inflammatory processes in cultured microglia, and in turn, investigate how inflammatory activation of microglia affects homeostasis of biometals. These relationships were further examined in vivo to determine the effects of the type 1 interferon (IFN) pathway on biometal homeostasis in the CNS. In my in vitro study, primary murine microglial cultures were treated for 24h with maximal sub-toxic doses of biometals, delivered as ferric ammonium chloride (FAC), ZnCl2 and CuCl2 and the biometal chelators, diamsar or N,N,N_,N_-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) with and without concurrent interferon-_ (IFN_) and tumour necrosis factor-_ (TNF_) stimulation. Non-stimulated and IFN_/TNF_ stimulated microglia served as negative and positive controls for inflammatory activated microglia, respectively. I measured the levels of a number of key inflammatory cytokines to assess microglial inflammatory response to biometal and biometal chelator treatments. I found that FAC and CuCl2 treatment, significantly induced Fe and Cu uptake respectively, in both non-stimulated and stimulated microglia and that all biometal treatments, significantly reduced the expression of MCP-1 in stimulated and non-stimulated microglia, indicative of an anti-inflammatory role. In contrast, FAC treatment also induced TNF_ mRNA expression in these cultures, suggesting Fe may play a dual role in neuroinflammation. In addition, to investigate how inflammatory activation of microglia affects biometal homeostasis, the gene expression of the metal-binding protein, metallothionein-1 (MT-1) and the biometal transporter, ZRT/IRT-like transporter protein (Zip7) were also measured. I also found that IFN_/TNF_ stimulation inhibited Fe-induced MT-1 and Zip7 expression in microglia. These findings demonstrate that sub-toxic levels of key biometals have multiple modulatory actions on cultured microglia, with both inhibitory and stimulatory effects on cytokines. These changes may be associated with induction or inhibition of major metal response proteins, such as MT-1 and transporters. To examine the effects of the type 1 IFN pathway on biometal homeostasis in the CNS, I performed a spatio-temporal analysis of Fe, Zn, Cu and Mn levels in the CNS of interferon _ receptor-1 (Ifnar1) knock-out (-/-) mice and wild type (WT) mice at 6 and 10 months of age using ICP-MS analysis. A subset of 6-month-old Ifnar1-/- mice was also stimulated with lipopolysaccharide (LPS) treatment for 6h to determine the effects of Ifnar1-/- on biometals homeostasis under inflammatory conditions. I found reduced Cu and Mn levels in the cerebellum of aged (10-month-old) Ifnar1-/- mice, however, expression of key Cu and Mn transporter and regulatory proteins remained unchanged. I also found no significant alterations to biometals between WT and Ifnar1-/- mice at 6-month of age, however, when mice were challenged with LPS, I found a significant decrease in Fe levels in the cerebellum and cerebrum of WT mice and a significant decrease in Zn levels in the cerebrum of Ifnar1-/- mice compared to naïve mice of their respective genotypes. A significant increase and an upward trend in transferrin receptor1 (TfR1) levels in the cerebrum of LPS-challenged and naïve Ifnar1-/- mice, respectively was also observed. These data demonstrate that the type 1 IFN pathway is involved in the regulation of CNS biometal homoeostasis. The studies provide further evidence to support a major role for biometals in neuroinflammatory pathways, with important implications for neurodegenerative disease in which brain biometal homeostasis is altered.