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(2020)ThesisDNA cytosine methylation is an important epigenetic modification that plays a key role in gene expression. DNA methylation has been shown to be involved in numerous processes, including X-chromosome inactivation in mammals, retrotransposon silencing, genomic imprinting, carcinogenesis and the regulation of tissue specific gene expression during development. Gene expression is tightly regulated via DNA methylation (5mC) and the aberrant expression of meiotic genes in mitotic cells via CpG promoter hypomethylation has been proposed to cause cancer. Cancer/Testis Antigens (CTAs) are a group of genes that encode tumour specific antigens and are expressed in the testis, certain cancers but not in normal post-natal somatic tissues. CpG island methylation and histone modifications appear to play a role in the epigenetic regulation of CTA expression, however, very little is known about their functions in vivo. A widely studied but poorly understood question to date is the mechanisms behind aberrant CTA reactivation in cancer. Given that 5mC mediated gene repression has been found to exist in vertebrate genomes and CTAs have also been identified to be a subset of highly evolutionarily conserved genes, it is critical to understand the role of 5mC mediated CTA silencing in vertebrates. By gaining a deeper understanding into the mechanisms behind this highly conserved pattern of gene repression on a specific subset of genes, we would be able to identify methods to prevent aberrant gene expression. In this study, I analysed publicly available whole genome bisulfite sequencing (WGBS), RNA-seq and chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq) data of developing embryonic and adult somatic tissue of 3 vertebrate species to elucidate the evolutionary epigenetic regulation of CTAs in vertebrate genomes. Integrative WGBS, RNA-seq and ChIP-seq analysis revealed that CTAs are evolutionarily conserved in zebrafish, mice and humans and mechanisms of their epigenetic regulation are also conserved. I observed that histone modifications could potentially serve as an indicator of the methylation status of CTA gene promoters and that the expression of CTAs was inversely related to gene promoter 5mC levels. I demonstrate that CTAs when over-expressed cause embryonic lethality in zebrafish and the same genes are aberrantly hypomethylated at their CpG islands in a subset of human cancers. Overall, my work shows that CTAs are epigenetically regulated in an evolutionarily conserved manner and possibly via a conserved transcription factor, ETS1, that is expressed both in embryonic and cancerous tissue.
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(2021)ThesisRNA modifications, collectively referred to as the ‘epitranscriptome’, are not mere decorations of RNA molecules, but can be dynamically regulated upon environmental queues and changes in cellular conditions. This dynamic behaviour is achieved through the RNA modification machinery, which comprises “writer”, “reader” and “eraser” proteins that modify, recognize and remove the modification, respectively. Chapter1 presents a comprehensive analysis of the RNA modification machinery (readers, writers and erasers) across species, tissues and cancer types, revealing gene duplications during eukaryotic evolution, changes in substrate specificity and tissue- and cancer-specific expression patterns. Chapters 2 and 3 presents the exploration and development of novel methods to map and analyze RNA modifications transcriptome-wide. Nanopore direct-RNA sequencing technology was used to provide RNA modification maps in full-length native RNA molecules. Firstly, it is shown that RNA modifications can be detected in the form of base-calling ‘errors’, thus allowing us to train Support Vector Machine models that can distinguish m6A-modified from unmodified sites, both in vitro and in vivo. Secondly, it is demonstrated that distinct RNA modification types have unique base-calling ‘error’ signatures, allowing us to exploit these signatures to distinguish different RNA modification types. It is found that pseudouridine has one of the most distinct signatures, appearing in the form of C-to-U mismatches. Finally, this information was used to predict novel pseudouridine sites on ncRNAs and mRNAs transcriptome-wide, as well as to obtain quantitative measurements of the stoichiometry of modified sites. Chapter 4 presents the development of a novel nanopore-based method, which is termed ‘Nano3P-seq’, to simultaneously quantify RNA abundance and tail length dynamics in individual molecules in both the coding and non-coding transcriptome, from cDNA reads. It is demonstrated that Nano3P-seq offers a simple approach to study the coding and non-coding transcriptome at single molecule resolution regardless of the tail ends. Together, this work provides a comprehensive framework for the study of RNA modifications and polyA tail dynamics using third generation sequencing technologies, opening novel avenues for future works that aim to characterize their dynamics and biological roles both in health and in disease.
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(2021)ThesisUpon fertilisation of vertebrate embryos, the epigenomes of the responsible gametes need to be reconfigured into a state that is compatible with totipotency and zygotic transcriptional programs. Furthermore, the epigenomes of differentiating cells then need to be remodelled again in order to form the complex structures of the body, such as the vastly intricate nervous system. This includes, but is not limited to, the remodelling of DNA methylation, the most abundant DNA modification in vertebrates with critical roles in embryogenesis and neurodevelopment. In mammals, methylation of cytosines in cytosine-guanine dinucleotides (mCG) is almost completely erased after fertilization before it is re-established during gastrulation. Similarly, methylation of cytosines outside the CG context (mCH; H = A,T,C) is diluted in the early mammalian embryo before it is re-established mainly in the nervous system. However, in non-mammalian vertebrates, it appears that no global erasure of mCG takes place, raising questions about their propensity for transgenerational epigenetic inheritance. Additionally, the conservation of mCH in non-mammalian vertebrates is largely unexplored. In this thesis, I look to expand our knowledge on the developmental dynamics, evolutionary conservation and the molecular components of DNA methylome remodelling in vertebrates by studying methylome dynamics in two distantly related teleost species (ray-finned, protruding jawed fish). I functionally explore how DNA methylation is regulated during the development of zebrafish (Danio rerio), medaka (Oryzias latipes), and zebrafish-medaka hybrids, in both the CG and CH context. I employ CRISPR/cas9 technology, whole-genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and RNA sequencing (RNA-seq), to interrogate a wide range of developmental time points and adult tissues. Overall, I have: i) developed a system to functionally test for regulators of developmental DNA methylation; ii) revealed a novel form of developmentally remodelled mCH in zebrafish and medaka which is deposited by the teleost specific DNMT3BA enzyme, iii) demonstrated evolutionary conservation of mammalian-like mCH features in the developing zebrafish nervous system, and iv) shown that DNA methylome dynamics in medaka and zebrafish embryos are highly comparable and compatible during the first 24 hours of zebrafish-medaka hybrid development. Altogether, this work greatly expands our understanding of the form and function of a critical DNA modification during development.
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(2022)ThesisA breakdown in B cell self-tolerance can lead to antibody-mediated autoimmune disease. This thesis aims to explore how B cell tolerance can be broken in two distinct, but complementary projects within the context of the Goodnow somatic mutation hypothesis. In both scenarios, B cells that escape self-tolerance and generate autoantibodies are referred to as “rogue” B cells. First, this thesis aimed to elucidate the precise steps undertaken by expanded rogue B cell clones in patients with chronic Hepatitis C virus (HCV)-associated cryoglobulinemic vasculitis, an autoimmune disease characterised by the production of a rheumatoid factor cryoglobulin autoantibody. The rogue B cell clones in the HCV cryoglobulinemic vasculitis patients were confirmed to be the source of the autoantibody. The rogue B cell clone precursor antibodies failed to bind the HCV envelope glycoprotein E2, yet bound multimerised self-antigen IgG relative to membrane IgM density. These findings disfavour a molecular mimicry hypothesis, and instead indicate IgG immune complexes may be sufficient to drive recruitment of the rogue B cell clone precursors. Finally, the rogue B cells clones were found to carry somatic lymphoma-associated, non-immunoglobulin gene mutations and chromosomal aberrations, predicted to cause hyperactivation of the NF-kB signalling pathway and escape of B cell tolerance. This finding provides additional evidence in support of the Goodnow somatic mutation hypothesis. Second, this thesis examined rogue germinal centre (GC) B cells that arise in the absence of the receptor FAS. Rogue GC B cells loose specificity for the foreign antigen and incidentally generate autoantibodies. However, the accumulation of rogue GC B cells cannot be explained by our current understanding of affinity-based selection in the GC. This work revealed rogue GC B cells, unlike “conventional” GC B cells undergoing affinity maturation to the foreign antigen, can be identified by low expression of CD21 and high expression of B220 (CD21loB220hi). Moreover, rogue GC B cells were found to be rapidly entering cell cycle, enriched for a dark zone phenotype and T-cell dependent, reminiscent of positively selected GC B cells. Thus, rogue GC B cells typically removed by FAS, likely persist in the competitive GC microenvironment despite their loss of BCR specificity to foreign antigen, because they retain the capacity to undergo T-cell dependent positive GC selection.
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(2022)ThesisT cells are critical in the body's defence against viruses and cancerous cells. They specifically recognise viral or tumour antigens presented on antigen presenting cells using their T cell receptor (TCR). Antigen binding triggers the TCR, transmitting signal intracellularly and resulting in the recruitment of a plethora of signalling proteins to the membrane. The signal is transmitted by post-translational modifications, such as the phosphorylation of tyrosine residues in intracellular tails of receptors, resulting in the recruitment of signalling proteins via their interaction domains. The spatial organisation of signalling proteins at the membrane determines the effector response of the T cell and is therefore critical for understanding the complex array of T cell responses. In this thesis, I develop a novel microscopy technique that reports on the nanoscale locations of signalling proteins and their binding kinetics to receptors at the T cell membrane. This technique utilizes the SH2 interaction domains of various signalling proteins which selectively and transiently bind to phosphorylated tyrosines on receptors. This transient binding results in the stochastic blinking necessary for super-resolution microscopy. Using this technique, termed protein point accumulation in nanoscale topography (pPAINT), I investigate the binding of multiple signalling proteins, achieving multiplexed imaging both simultaneously and sequentially with a combined microfluidic and microscopy approach. In the second half of this thesis, I apply pPAINT to study how chimeric antigen receptors (CARs) signal in T cells. In CAR-T therapy, patient cytotoxic T cells are isolated and transduced with a CAR construct that recognises tumour antigens, and are then reintroduced into the patient where they find and eliminate cells expressing the CAR target antigen. CAR constructs are made up of an antigen recognition domain fused to various intracellular signalling motifs from the TCR complex and co-stimulatory receptors, such as CD28, which are crucial for T cell activation. The first-generation of CARs contained an intracellular tail of the TCR, the CD3ζ chain, but it has been the second-generation CARs, with the addition of co-stimulatory receptor signalling domains, that have proven clinically effective. However, CAR therapy is not successful in all patients; limitations reducing their efficacy include inefficient recognition of low antigen densities, finite persistence in the body and off-target side effects in patients. It follows that a detailed knowledge and understanding of CAR activation and signalling is needed to optimise CAR design. In this thesis I use pPAINT to gain a unique perspective on how different generations of CARs signal upon activation, identifying key similarities and differences to signalling from the standard TCR. Signalling was investigated in CAR-T cells generated in a similar way to clinically used CAR-T therapies. In doing so, unique signalling mechanisms utilized by CARs were identified that will be valuable for the development of more effective chimeric antigen receptors. The results demonstrate that although CARs utilise the signalling domains of the TCR and co-stimulatory receptors, the pattern of adaptor protein recruitment is different from that of T cells stimulated through the TCR and co- stimulatory receptors. Specifically, I found that whilst hubs of signalling proteins spatially diverged from clusters of activated TCR, they were instead closely colocalised with activated CARs. The incorporation of the CD28 coreceptor in CAR design improves signalling protein recruitment patterns, however, the patterns of protein binding were still vastly different to co-stimulated T cells. Collectively, the results indicate that CARs utilize a signalling pathway unique to that of costimulated T cells, in a mechanism that may have ramifications in the functional responses exhibited by cells used in CAR-T therapy.
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(2023)ThesisThe onset of psychiatric disorders, such as autism spectrum disorder, schizophrenia, anxiety, and major depressive disorder, can be associated with both genetic and environmental factors. This thesis examines transcriptional changes caused by either genetic or environmental factors linked to psychiatric disorders. We investigated regulatory regions which harbour mutations associated with psychiatric disorders, and the effects of early life stress on the developing brain and its consequent adverse mental health outcomes. Here, we assessed the function of active enhancers in the brain to understand their regulatory roles, and their link to mutations associated with psychiatric disorders. This was achieved using a CRISPR interference (CRISPRi) screen to repress almost one thousand enhancers in primary human astrocyte cells. The effect of enhancer repression on transcription was measured using single cell mRNA sequencing (scRNA-seq), a which measures gene expression from individual cells. This thesis describes the first CRISPRi screen to be performed in brain cells, and the first to be performed in primary cells. Active enhancers targeted in this screen were characterised based on their chromatin state and through analysing enhancer marks from relevant published data. Regions of open chromatin are a marker for enhancers and were determined in primary human astrocytes using ATAC-seq, a method which transposes DNA in an open chromatin state, before using sequencing to determine regions of open chromatin. Candidate enhancers were filtered based on the ability to detect potential target genes of the enhancer by scRNA-seq, to which gene expression was required to be above an expression threshold. The expression of potential target genes was determined by mRNA-seq, and candidate enhancers were required to be in the same topologically associated domain as a detectable target gene expressed above the threshold. The CRISPRi screen was optimised with a pilot screen to determine the degree of silencing due to CRISPRi and the detectability of the transcriptional effect of silencing enhancers by CRISPRi using scRNA-seq. A large-scale CRISPRi screen was then performed to investigate and validate candidate enhancers in primary human astrocytes. This identified almost one hundred enhancers active in the human brain that have disease-associated variants, and 140 enhancer target genes associated with disease. We also investigated the transcriptional effect of early life stress using murine models. A limited nesting (LN) model was used, as this emulates human maternal neglect and has been previously shown to increase measures of anxiety like-behaviours in rats. We performed RNA sequencing on the prefrontal cortex (PFC) of rats that underwent an LN paradigm as a model for early life stress. Our results indicated that this primarily led to transcriptional repression in the PFC. We further identified a molecular signature of LN in rat PFC that, when compared with other studies, is observed across ELS protocols and replicable in other murine models.
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(2022)ThesisPerfringolysin O (PFO) is a cholesterol dependent cytolysin (CDC) secreted by Clostridium perfringens, which forms pores in cholesterol containing membranes. CDCs are part of the larger Membrane attack complex-Perforin/CDC (MACPF/CDC) superfamily, containing pore formers responsible for controlling infectious disease and cancer in humans. CDCs are secreted as soluble hydrophilic monomers which oligomerise on lipid bilayers, ultimately forming bilayer spanning closed ring or open arc-shaped β-barrel pores. Perfringolysin O (PFO) was the first CDC to have its crystallographic structure resolved in its soluble monomeric form and has since become the prototypical CDC for investigating pore-forming mechanism. Previous studies on PFO have revealed a general outline of the steps involved in CDC pore formation; recognition of cholesterol and membrane binding, oligomerisation and ultimately membrane insertion to form large amphipathic pores. These steps have been elucidated using bulk assays and static imaging techniques such as electron microscopy or atomic force microscopy, however key mechanistic details remain uncharacterised due to the lack of time resolved data at a single pore level. Here we present a novel assay using microfluidics and total internal reflection microscopy to track PFO pore formation dynamics. Fluorescently labelled PFO and dye encapsulating liposomes were employed in conjunction to measure the kinetics of PFO binding from solution, nucleation, and oligomerisation on the surface of cholesterol containing liposomes. By visualising fluorescent dye release from our liposomes, we were able to determine the number of molecules necessary for an oligomer to insert and form a bilayer spanning pore. This information was collected from a large number of liposomes and used to build a mathematical model to quantify parameters controlling PFO accumulation and insertion kinetics on liposomes. We have also demonstrated how a different membrane composition affects these parameters by performing the assay on virus-like particles derived from HIV. This also displayed the adaptability of the assay which can be applied to different membranes and other pore forming proteins.
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(2023)ThesisCurrent healthcare infection surveillance rarely monitors the distribution of antimicrobial resistance (AMR) in bacteria beyond clinical settings in Australia and overseas. This results in a significant gap in our ability to fully understand and manage the spread of AMR in the general community. This thesis explores whether wastewater-based monitoring could reveal geospatial-temporal and demographic trends of antibiotic-resistant bacteria in the urban area of Greater Sydney, Australia. Untreated wastewater from 25 wastewater treatment plants sampled between 2017 and 2019 consistently contained extended-spectrum β-lactamases-producing Enterobacteriaceae (ESBL-E) isolates, suggesting its endemicity in the community. Carbapenem-resistant Enterobacteriaceae (CRE), vancomycin-resistant enterococci (VRE), and methicillin-resistant Staphylococcus aureus (MRSA) isolates were occasionally detected. Demographic and healthcare infection-related factors correlated with the ESBL-E load, and demographic variables influenced the VRE load. In contrast, the healthcare infection-related factor mainly drove the CRE load. These findings demonstrate the potential of wastewater-based surveillance to understand the factors driving AMR distribution in the community. The subsequent thesis work covers the genomic characterisation of selected ESBL-E and CRE wastewater isolates to reveal their nature, origin, and underlying resistance mechanisms. Phylogenetic analysis showed that Escherichia coli isolates were related to high-risk human-associated pandemic clones and non-human-associated clones. The Klebsiella pneumoniae and K. variicola isolates were related to globally disseminated and emerging human-associated clones, and some were detected for the first time in Australia. Genomic analysis also indicated novel resistance mechanisms against nitrofurantoin in E. coli, and against piperacillin/tazobactam and ticarcillin/clavulanic acid in Klebsiella isolates. The virulence gene content indicated that some E. coli and Klebsiella isolates were likely associated with infections, while the asymptomatic carriage was suggested for other isolates. These results demonstrate a clear potential for wastewater-based surveillance to monitor the emergence and dissemination of resistance in non-clinical isolates, and in particular, isolates from the community and non-human sources. The findings of this study can complement healthcare infection surveillance to inform management strategies to mitigate the emergence and dissemination of AMR and important human pathogens in the general community.
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(2023)ThesisCytotoxic T lymphocytes (CTLs) can target and induce apoptosis in cancer cells during the anti-tumour immune response. However, the cytotoxicity (or killing) function of CTLs can be perturbed directly by cancer cells or via the tumour microenvironment (TME). Among the various factors in TME that can influence T cell function, the effect that mechanical properties of the extracellular matrix (ECM) have on CTL responses is unclear. Research into CTL-mediated cytotoxicity is typically performed in either two-dimensional (2D) matrix-free culture or in complex in vivo animal models. In vitro, 2D studies are limited in recapitulating the CTL response in vivo, whereas it is very difficult to manipulate the TME and perform high-throughput experiments using in vivo models. Recently 3D culture models have been introduced to fill the gap between 2D and in vivo studies. In this study, we used an automated 3D bioprinter to incorporate OT-I T cells and cognate and non-cognate target cells in a polyethylene glycol (PEG)-based hydrogel and studied the killing efficiency in comparison with 2D culture and manually-prepared gels. Here, we showed that the 3D bioprinter embeds both CTLs and target cells in the hydrogel and enables control over the dimensions of the embedding matrix as well as the number and spatial organisation of cells. Moreover, the ability to digest the gel and release the cells allowed us to perform killing efficiency comparisons and downstream high-throughput CTL functional analyses using flow cytometry. This novel 3D cell culture system allowed us to investigate the effects of tunable ECM mechanical properties in a reproducible cytotoxicity model of matrix-embedded CTL and target cells. Our results demonstrate that in matrices with higher density, CTL killing efficacy was compromised. This demonstrates that matrix stiffness, independent of matrix porosity or other variable characteristics, has a large impact on CTL function. From another perspective, cancer cells can directly induce dysfunctional programming in CTLs. Repeated stimulation of the T cell receptor (TCR) on CTLs with the tumour-associated antigen leads to overexpression of inhibitory receptors such as programmed cell death (PD)-1 on the surface of T cells, leading to aberrant response and eventually tumour escape. TCR signalling machinery can be affected by the expression of inhibitory receptors, but it is not clear whether inhibitory receptors alone are responsible for the dysfunction of exhausted T cells or to what degree other mechanisms contribute. To address this, we used a mouse model of T cell dysfunction, finding that T cells could exhibit a dysfunctional phenotype with minimal upregulation of inhibitory receptors and without downregulation of TCR. Instead, we found a decrease in the proximal signalling kinases Lck and ZAP70, specifically in dysfunctional cells. To confirm these results, we developed a human primary in vitro CD8+ T cell dysfunction model, which allowed us to study the effect of repeated antigen stimulation on the inhibitory receptors expression and expression of Lck and ZAP70 in human T cells. In this model, we again found that dysfunctional T cells had lower expression of Lck and ZAP70, confirming the results from the mouse model. Future experiments could be performed in which Lck and/or ZAP70 expression is enhanced in dysfunctional T cells. If this restores the functional phenotype, it may confirm that a low level of Lck and ZAP70 protein expression is a cause of T cell dysfunction. This research sheds light on how the external (matrix stiffness) and internal factors (TCR stimulation) affect the CTL response.
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(2022)ThesisBipolar disorder (BD) is a complex mental illness for which both environmental and biological (genetic and epigenetic) factors play an aetiological role. This thesis aimed to find factors behind risk of psychopathology, using a multi-site cohort with parents or siblings with BD (high-risk; HR) and offspring of healthy control parents. My aim was to explore the trajectory towards development of BD and related psychopathology, guided by the hypothesis that DNA methylation (DNAm), a key epigenetic process, may provide a molecular link of relevant gene-environment interactions. Firstly, association of stressful life events and family environment with clinical status was investigated. Next, blood-derived DNAm of a subset with available genotype data (n=415) was used to investigate epigenetic signals. Epigenome-wide association studies were used to identify differentially methylated CpG sites based on familial risk and emergent clinical status, and polygenic risk. Finally, methylation-derived markers, indexing C-reactive protein (CRPm) and neutrophil-to-lymphocyte ratio (NLRm), were used to investigate the influence of inflammatory processes in the development of psychopathology. Stressful life events and an unbalanced family environment increase risk of emergent psychopathology, providing targets for early intervention. Nominally differentially methylated CpG sites that were related to family history, clinical transition and polygenic risk showed enrichment in neurological pathways and functions. Family environment influenced DNAm signatures of clinical transition, and methylation profile scores showed partial prediction of clinical transition in independent validation. The influence of polygenic risk scores for BD revealed wide-ranging nominal effects on DNAm signatures and an epigenome-wide significant signal at mitochondrial gene VARS2, within the major histocompatibility complex (MHC) region. The relationship between DNAm signatures and polygenic background was independently validated. While the role of inflammation on psychopathology was not strong, CRPm could potentially predict psychopathology in HR individuals, and a trend of higher NLRm and CRPm was evident in HR with suicidal ideation. My findings showed that epigenetic processes are impacted by both familial background and genetic predisposition, may reflect underlying environmental exposures, and influence emergent psychopathology, providing insights into biological dysregulation leading to BD.