Medicine & Health

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Now showing 1 - 10 of 25
  • (2012) Funnell, Alister; Norton, Laura; Mak, Ka Sin; Burdach, John; Artuz, Crisbel; Twine, Natalie; Wilkins, Marc; Hung, TT; Perdomo, Jose; Power, Carl; Koh, P; Bell Anderson, Kim; Orkin, S; Fraser, Stuart; Perkins, Andrew; Pearson, Richard; Crossley, Merlin
    Journal Article
    The CACCC-box binding protein erythroid Krüppel-like factor (EKLF/KLF1) is a master regulator that directs the expression of many important erythroid genes. We have previously shown that EKLF drives transcription of the gene for a second KLF, basic Krüppel-like factor, or KLF3. We have now tested the in vivo role of KLF3 in erythroid cells by examining Klf3 knockout mice. KLF3-deficient adults exhibit a mild compensated anemia, including enlarged spleens, increased red pulp, and a higher percentage of erythroid progenitors, together with elevated reticulocytes and abnormal erythrocytes in the peripheral blood. Impaired erythroid maturation is also observed in the fetal liver. We have found that KLF3 levels rise as erythroid cells mature to become TER119(+). Consistent with this, microarray analysis of both TER119(-) and TER119(+) erythroid populations revealed that KLF3 is most critical at the later stages of erythroid maturation and is indeed primarily a transcriptional repressor. Notably, many of the genes repressed by KLF3 are also known to be activated by EKLF. However, the majority of these are not currently recognized as erythroid-cell-specific genes. These results reveal the molecular and physiological function of KLF3, defining it as a feedback repressor that counters the activity of EKLF at selected target genes to achieve normal erythropoiesis.

  • (2013) Kavanagh, Tomas; Mills, James D; Kim, Woojin S; Halliday, Glenda; Janitz, Michael
    Journal Article
    Pathway analysis is a powerful method for discerning differentially regulated genes and elucidating their biological importance. It allows for the identification of perturbed or aberrantly expressed genes within a biological context from extensive data sets and offers a simplistic approach for interrogating such datasets. With the growing use of microarrays and RNA-Seq data for genome wide studies is growing at an alarming rate and the use of deep sequencing is revealing elements of the genome previously uncharacterised. Through the employment of pathway analysis, mechanisms in complex diseases may be explored, and novel causatives found primarily through differentially regulated genes. Further, with the implementation of next generation sequencing (NGS) a deeper resolution may be attained, particularly in identification of isoform diversity and SNP’s. Here we look at a broad overview of pathway analysis in the human brain transcriptome and its relevance in teasing out underlying causes of complex diseases. We will outline processes in data gathering and analysis of particular diseases in which these approaches have been successful.

  • (2012) Wong, Jenny; Garner, Brett; Halliday, Glenda; Sharpe, Laura J; Brown, Andrew J
    Journal Article
    Selective Alzheimer’s Disease Indicator-1 (Seladin-1) was originally identified by its down-regulation in the brains of Alzheimer’s Disease (AD) patients. Here, we re-examine existing data and present new gene expression data that refutes its role as a selective AD indicator. Furthermore, we caution against the use of the name “Seladin-1” and instead recommend adoption of the approved nomenclature, 3â-hydroxysterol Ä24-reductase (or DHCR24), which describes its catalytic function in cholesterol synthesis. Further work is required to determine what link, if any, exists between DHCR24 and AD.

  • (2020) Masand, Natasha
    Thesis
    DNA 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.

  • (2021) Begik, Oguzhan
    Thesis
    RNA 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.

  • (2021) Ross, Samuel
    Thesis
    Upon 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.

  • (2022) Young, Clara
    Thesis
    A 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.

  • (2022) Farrell, Megan
    Thesis
    T 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.

  • (2023) Green, Nicole
    Thesis
    The 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.

  • (2022) Mc Guinness, Conall
    Thesis
    Perfringolysin 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.