Pathology - Theses
Permanent URI for this collection
Search Results
1 - 10 of 98
-
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.
-
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.
-
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.
-
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.
-
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.
-
ItemThe genomic landscape of phaeochromocytoma( 2015)Phaeochromocytomas (PCC) and paragangliomas (PGL) (collectively PPGL) are rare neural crest-derived tumours originating from adrenal chromaffin cells or extra-adrenal sympathetic and parasympathetic tissues. More than a third of PPGL cases are associated with heritable syndromes involving 18 or more known genes. These genes have been broadly partitioned into two groups based on pseudo-hypoxic and receptor tyrosine kinase (RTK) signalling pathways. Many of these genes can also become somatically mutated, although up to one third of sporadic cases have no known genetic driver. Furthermore, little is known of the genes that co-operate with known driver genes to initiate and drive tumourigenesis. To explore the genomic landscape of PPGL, exome sequencing, high-density SNP-array analysis, and RNA sequencing was applied to 36 PCCs and four PGL tumours. All tumours displayed a low mutation frequency in combination with frequent large segmental copy-number alterations and aneuploidy, with evidence for chromothripsis seen in a single case. Thirty-one of forty (77.5%) cases could be explained by germline or somatic mutations or structural alterations affecting known PPGL genes. Deleterious somatic mutations were also identified in known tumour-suppressor genes associated with genome maintenance and epigenetic modulation (e.g. TP53, STAG2, KMT2D). A multitude of other genes were also found mutated that are likely important for normal neuroendocrine cell function (e.g. ASCL1, NCAM1, GOLGA1). In addition, the existing paradigm for gene-expression subtyping of PPGL was further refined by applying consensus clustering to a compendium of previously published microarray data, enabling the identification of six robust gene-expression subtypes and subsequent cross-platform classification of RNA-seq data. The majority of cases in the cohort with no identifiable driver mutation were classified into a gene-expression subtype bearing similarity to MAX mutant PPGL, suggesting there are yet unknown PPGL cancer genes that can phenocopy MAX mutations. The cross-platform classification model was then further refined to develop a 46-gene Nanostring-based diagnostic tool capable of classifying PPGL tumours into gene-expression subtypes. The strong genotype-to-subtype relationship in PPGL makes subtyping a powerful tool that can be used clinically to guide and interpret genetic testing, determine surveillance programs and aid in better elucidation of PPGL biology. In applying the diagnostic assay to a test set of 38 cases, correct classification into one of the six subtypes was achieved for 34 (90%) samples based on the known genotype to gene-expression subtype association. The observation that at least one of the six subtypes is likely defined by the presence of non-neoplastic cells led to further refinement into five, four, and three-class architectures, further improving classification accuracy. Increasingly tumour heterogeneity is being recognised as one of the most significant challenges facing modern oncology. Genomically diverse tumour regions create additional complexity in predicting treatment response and metastatic potential through biopsy. Multi-region sampling of multiple synchronous primaries from patients with a predisposing germline mutation was used to explore tumour evolution and heterogeneity in PPGL and concomitant medullary thyroid carcinoma. Evolutionary reconstruction of a single primary PPGL demonstrated periods of both branched and linear evolution resulting in a high degree of intratumoural heterogeneity. Comparison of multiple synchronous primaries provided strong evidence of convergent evolution through recurrent chromosomal aberrations, indicating these may be obligate events in tumourigenesis, and as such, may indicate potential novel therapeutic targets.
-
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.
-
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.
-
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.
-
ItemInvestigating the role of hypoxia in tumour progression in breast cancer( 2015)Metastasis is a major cause of morbidity and mortality in breast cancer patients. The molecular processes and mediators that underpin this process have yet to be completely delineated. Hypoxia, the state of reduced oxygen conditions, occurs frequently in solid tumours and is a factor of poor prognosis for patient outcome. The upregulation of HIF-1α, the main mediator of the hypoxic response pathway, has been implicated in several different facets of tumour progression, including tumour growth, angiogenesis, therapy resistance and metastasis. Hypoxia has been shown to induce Epithelial-to-Mesenchymal Transition (EMT), a highly conserved developmental program that facilitates tumour cell dissemination. It is thought that EMT is co-opted by epithelial tumour cells in order to acquire a degree of plasticity, allowing them to undergo a number of genetic, biochemical and morphological changes to adopt a mesenchymal phenotype. This results in the loss of polarity, and the gain of migratory and invasive capabilities. EMT is regulated by a core cassette of transcription factors, including Snail, Slug, Twist, Zeb1 and Zeb2. Zeb1 is the most proximal transcription factor, however, how hypoxia modulates Zeb1 expression is not known. This study demonstrates that Siah, a family of E3 ubiquitin ligases and a master regulator of HIF-1α protein expression, binds to and targets Zeb1 for proteasomal degradation. Loss of Siah2 is sufficient to cause spontaneous EMT in tumour cells derived from the PyMT murine model of breast cancer. On the other hand, EMT induction led to the decrease in Siah protein expression. This work is the first to describe a post-translational mechanism of regulation of Zeb1 and further defines the relationship between hypoxia and EMT. There are, in fact, two forms of hypoxia in a growing tumour, chronic hypoxia and intermittent hypoxia. Chronic hypoxia describes the long-term limitations on oxygen diffusion caused by abnormal tumour vasculature. While intermittent hypoxia refers to the fluctuations of oxygen tension in a tumour, caused by the aberrant and temporary closing and reopening of tumour-supplying blood vessels. The consequences of these two different types of hypoxia in breast cancer have not yet been well characterised. Using the orthotopic, syngeneic PyMT murine model of breast cancer, it was found that intermittent hypoxia-treated cells gave rise to a greater number of larger lung metastases in vivo. This was facilitated by an enhanced ability for anchorage-independent growth, increased clonogenicity, the induction of a pro-tumourigenic gene expression and secretory profile, and the increase in tumour-initiating capacity through the gain of cancer stem cell properties. RNA sequencing of hypoxia-treated cells found distinct gene expression patterns between treatment groups. While, pathway analysis revealed a marked enrichment of immune-related pathways and a downregulation of DNA replication and cell cycle pathways, by both chronic and intermittent hypoxia. Interestingly, chronic hypoxia also upregulated extracellular matrix degradation pathways, in spite of the lack of an overt EMT in cells. These results unveil novel mechanisms and pathways involved in hypoxia-mediated metastasis while highlighting the extensive effects of hypoxia signalling in cancer. Taken together, this work demonstrates the complexity of hypoxia signalling in tumour progression. Not only does it endow tumour cells with an aggressive, tumour-initiating phenotype, but it also contributes to the priming of the tumour microenvironment to be pro-inflammatory and immunosuppressive and ultimately, tumour-promoting.