1,721,068 research outputs found
Investigation into the epigenetic mechanisms involved in microglial activation in the animal model of multiple sclerosis
Get PDFIn patients with multiple sclerosis (MS), microglia become activated due to the autoimmune inflammatory response which is directed against the central nervous system (CNS). Following the first disease relapse, microglia remain activated and do not return to a resting state during remissions. Chronically-activated microglia release inflammatory mediators that cause CNS tissue damage, and as such, MS progression has been associated with widespread, chronic microglial activation that correlates with neurodegeneration. To date, only one histone demethylase, Jmjd3, has been described to have a role in inflammation. In agreement with this, up-regulation of Jmjd3 expression was observed following microglial treatment with several pro-inflammatory stimuli, including a range of toll-like receptors ligands and cytokines, suggesting a universal role of Jmjd3 during microglial activation. Subsequent ChIP-qPCR assays revealed that Jmjd3 was recruited to the promoters of Il6, Ccl3, Ccl5 and Nos2 following activation, which, in turn, presented a decrease in their H3K27me3 levels. Using an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, Jmjd3 expression was shown to be increased in activated microglia from mice in the acute and late phases of disease. Immunization with complete Freud’s adjuvant (CFA) alone, also caused microglial activation with Jmjd3 induction, indicating a CFA-mediated TLR2 and TLR4 stimulation of microglia. Further investigation, in which primary microglia were isolated from mice deficient in Jmjd3 (Jmjd3-/-), however demonstrated that the absence of Jmjd3 alone had no resultant effect on the expression of a subset of immune response and inflammation related genes, including the Jmjd3 target genes Il6, Ccl3, Ccl5 and Nos2, before or after activation. This suggested that Jmjd3 acts in concert with a repertoire of other demethylases to facilitate microglia activation, and as such was rendered redundant in this setting. Deciphering the epigenetic profile of microglia in MS and determining whether it is involved in the maintenance of chronic microglial activation in the progressive phase of the disease remains an important line of investigation, and through a clearer understanding of its role in MS pathophysiology, could lead to the development of novel therapeutic interventions in the future
Functional characterisation of multiple sclerosis-associated single nucleotide polymorphisms in the Eomesodermin region
Get PDFMultiple sclerosis (MS) is a common inflammatory neurological disease affecting young adults, causing significant disability and increased mortality. Genome wide association studies (GWAS) have uncovered over 100 single nucleotide polymorphisms (SNPs) associated with MS susceptibility, most of which lie outside transcribed DNA and therefore do not have obvious consequences. SNPs in two such non-coding DNA segments upstream of the EOMES gene, encoding Eomesodermin (Eomes) have been consistently associated with MS, as well as with the two other autoimmune diseases rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Eomes has been shown to be important for embryonic and neural development, as well as in the immune system, the latter of which was the focus of this investigation. The association of genetic polymorphisms in the EOMES region with MS was interrogated using computational methods. Imputation analysis enabled detection of new variants, which aligned with regions of epigenetic significance and possible regions of DNA-interaction, implying an effect on regulation of gene expression. Subsequently, Eomes expression was characterised in peripheral blood cell populations, with the highest expression detected in natural killer cells and effector/memory T cell subsets, including MAIT cells, an innate-like lymphocyte population. To investigate potential genotype dependent changes in Eomes expression and the effect on downstream pathways, studies were performed using samples from healthy donors prospectively genotyped for the two MS associated loci. Despite observing significant differences in expression between genotype groups in a pilot study, findings in a larger cohort did not reach the threshold for significance. Finally, the presence of MAIT cells was investigated in the context of MS and these cells were detected in MS lesions. Collectively, in this D.Phil project I have illustrated potential ways in which the functional relevance of disease-associated SNPs in non-coding regions can be studied. This has been thoroughly investigated through computational studies, analysis of gene expression in peripheral blood cells, and in genotype-dependent studies of both expression and downstream function
Functional characterisation of a multiple sclerosis-associated genetic variant
Get PDFIn recent years, Genome-wide association studies (GWAS) have identified several susceptibility- modifying genetic variants to complex diseases such as multiple sclerosis (MS). An association for one such single nucleotide polymorphism (SNP) in the gene encoding the principle receptor for the cytokine TNF (TNFR1), TNFRSF1A, has been replicated several times since its initial identification by De Jager et al., 2009. This study demonstrates that the MS risk-associated G allele of this SNP, rs1800693, results in an alternative splicing event in which exon 6 is excluded from the mature TNFRSF1A transcript both in an in vitro splicing assay and ex vivo in several human immune cell types, and investigates the potential for creating an antisense-based mouse model for the effect of this SNP. Importantly, we show that the transcript lacking exon 6 is translated into protein, designated Δ6TNFR1, which can be detected in primary samples. Δ6TNFRl is truncated in several important domains of the full-length receptor and is demonstrated to be unable to traffick to and anchor at the cell membrane, instead being constitutively secreted. Moreover, in contrast with the full-length receptor, the MS-associated splice isoform is unable to oligomerise either homotypically or with full-length receptors, or to engage the NF-KB-mediated or apoptosis signalling pathways in vitro. Δ6TNFRl does, however, retain the ability to bind and neutralise TNF, albeit to a lesser extent than the full-length protein. This is striking in light of the results of clinical trials for the use of TNF antagonists in MS patients, in which neutralisation of TNF was associated with relapses and exacerbation of symptoms, and rare MS symptom-like side-effects in patients being treated with anti-TNF agents for the management of other inflammatory autoimmune diseases. In conclusion, this work provides a functional explanation for the association of an MS-risk SNP with the disease and provides support for a detrimental effect of broad-spectrum TNF inhibition in MS. Moreover, it reveals a novel use for GWAS; to inform clinical practice.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
The functional consequences of autoimmune variants in the tyrosine kinase 2 gene region
Get PDFThe tyrosine kinase 2 (TYK2) gene was first implicated in autoimmune disease in 2009 when a nonsynonymous single nucleotide polymorphism (nsSNP) in TYK2 was reported to be associated with multiple sclerosis (MS). The immunological function of TYK2, as a kinase involved in signal transduction downstream of numerous different cytokine receptors, further strengthened the candidacy of the gene as an MS-relevant risk factor. More recently, this nsSNP has been associated with several other autoimmune conditions. In addition, another three different SNPs in the region have been found to be associated with a number of autoimmune diseases, sometimes in opposing directions. Considering the complex genetic association pattern that is emerging for the TYK2 region across autoimmune conditions, it was hypothesised that this complexity reflects shared but also distinct pathogenic mechanisms, with the consequences of disease-associated SNPs being unlikely to all be restricted to genotype-dependent effects influencing TYK2. Therefore, the main aim of the work presented in this thesis has been to address this hypothesis by investigating the functional consequences of the disease-associated SNPs in the TYK2 gene region. Using an in vitro cell line system and primary human immune cells, obtained from a genotype-selectable donor cohort, protection at the MS-associated nsSNP was found to correlate with reduced TYK2-mediated signalling downstream of the type I interferon receptor. However, other cytokine signalling pathways were not affected, indicating a greater specificity to the function of TYK2 than has previously been appreciated. For the other SNPs in the region, substantial effects on TYK2 were not observed but immune cell subset-specific correlations with RNA-level expression of other genes in the region were identified. Thus, this is the first study to support the concept that a careful cross-comparative analysis of SNP association patterns in a single genomic region across multiple autoimmune diseases can have significant implications for enabling the delineation of pathways common or specific to different conditions, and this is of particular importance for drug repositioning strategies
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The functional analysis of rare genetic variants in a familial neurodegenerative spectrum disorder
Get PDFWhole genome sequencing can enable an unbiased approach to the diagnosis and to the understanding of genetic aetiology of neurodegenerative diseases, which are a clinically and genetically heterogeneous group of disorders. Two brothers, originally
diagnosed with primary progressive multiple sclerosis (PPMS), displayed an atypical disease progression over a 30-year period with a clinical picture of paresis, ataxia, mental impairment and dysarthria. Late stage fasciculations, absence of sensory symptoms and a follow-up magnetic resonance imaging (MRI) scan showing severe atrophy of the cerebrum, cerebellum and brainstem, but no white matter lesions, called the original diagnosis into question. Given the familial nature and the rarity of this very similar clinical profile, we hypothesised that a rare variant could be contributing to disease. Whole genome sequencing identified three rare variants segregating with disease: a homozygous non-frameshift deletion in the fatty acid 2-hydroxylase (FA2H) gene, an X-bound non-synonymous single nucleotide variant in the adaptor protein-1 complex subunit sigma-2 (AP1S2) gene, and a homozygous frameshift deletion in the mixed lineage kinase domain-like
protein (MLKL) gene. Lipidomic analysis using liquid chromatography-tandem mass spectrometry of FA2H variant-expressing cell lines demonstrated no detrimental impact of the deletion in FA2H on protein function. Bioinformatic and computational structural analysis suggested the variant in AP1S2 to be benign. In contrast, the frameshift deletion in MLKL prevents expression of MLKL protein as demonstrated in patient-derived primary cells. The absence of MLKL was shown ex vivo to cause
complete deficiency in necroptosis, a recently identified form of programmed cell death that is involved in a spectrum of neurodegenerative diseases. This thesis provides the first clinical case report implicating MLKL and necroptosis deficiency in a neurodegenerative spectrum disorder. The results suggest that current strategies to pursue MLKL inhibition for human therapy should be approached with caution.</p
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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