http://hdl.handle.net/10468/1244 Mon, 30 Oct 2017 17:17:21 GMT 2017-10-30T17:17:21Z http://hdl.handle.net/10468/3566 The mucosal microbiome of inflammatory bowel diseases Ryan, Feargal The human microbiome is the diverse community of micro-organisms that live with humans. The gut microbiome contains the densest collection of microbial cells in the body and has been described as functioning as an organ. Alterations in these microbial communities are associated with disease. This work provides a new methodology for the taxonomic assignment of 16S rRNA gene sequencing data, then utilises this and other cutting edge techniques to elucidate the mucosal associated microbiome in Inflammatory Bowel Disease and finally examines the viability of using mucosal samples for shotgun metagenomics or metatranscriptomics. Taxonomic classification is a corner stone for the characterisation of microbial communities, but currently many studies are limited to genus level classification. Thus, we developed SPINGO, a flexible and stand-alone software dedicated to species level assignment of 16S rRNA gene sequences. SPINGO outperforms other methods in terms of classification accuracy, and is as fast or faster than those that have higher error rates. Crohn’s Disease (CD) and Ulcerative Colitis (UC) are Inflammatory Bowel Diseases (IBD) which cause inflammation along the gastrointestinal tract of millions of people worldwide. By collecting paired colonic pinch biopsies from inflamed and noninflamed tissue from IBD patients and paired healthy biopsies from healthy individuals we reveal a gradient between IBD and healthy controls that is associated with inflammation. Differential abundance analysis reveals a total of 89 taxa in this cohort to be different between CD and UC when compared to healthy controls. Clustering of these samples reveals discrete groups which may provide a frame-work for sub-typing IBD based on the microbiota. Microbiome scientists frequently rely on faecal samples as a proxy for the colonic environment despite numerous studies finding that it is not completely representative. Here we assess the potential of using colonic pinch biopsies for profiling the metagenome and metatranscriptome of the mucosal associated microbiota. We find that current methodologies are not capable of adequately separating microbial and human DNA or RNA resulting in as few as 1 – 2% of microbial reads after sequencing. Fri, 01 Jan 2016 00:00:00 GMT http://hdl.handle.net/10468/3566 2016-01-01T00:00:00Z http://hdl.handle.net/10468/2132 An examination of the impact of dietary lipids on behaviour and neurochemistry Pusceddu, Matteo The molecular and cellular basis of stress pathology remains an important research question in biological science. A better understanding of this may enable the development of novel approaches for the treatment of stress-related disorders. There is a considerable body of scientific evidence suggesting that dietary lipids, phospholipids and omega-3 polyunsaturated fatty acids (n-3 PUFAs), have therapeutic potential for certain psychiatric disorders. Thus, we proposed n-3 PUFAs as a novel strategy for the prevention or amelioration of stress-related disorders. We hypothesised that these compounds would improve behavioural and neurobiological responses and alter gut microbial composition. Furthermore, we proposed a new mechanism of action exerted by n-3 PUFAs using an in vitro model of stress. Lastly, we explored the protective effects of both phospholipids and n-3 PUFAs against neuroinflammation, which has been shown to contribute to the development of stress-related disorders. We provide further evidence that glucocorticoids, inflammation and early-life stress induce vulnerability to psychopathologies. Specifically, we have demonstrated that corticosterone (CORT) alters cortical neuron and astrocyte percentage composition, reduces brain-derived-neuronal factor (BDNF) expression, and induces glucocorticoid receptor (GR) down-regulation in mixed cortical cultures. Interestingly, we found that lipopolysaccharide (LPS) treatment resulted in an over-expression of pro-inflammatory cytokines in cortical astrocyte cultures. Moreover, we demonstrate that early-life stress induces changes to the monoaminergic and immune systems as well as altered neuroendocrine response to stressors later in life. In addition, we found that early-life stress alters the gut microbiota in adulthood. These data demonstrate that n-3 PUFAs can attenuate CORT-induced cellular changes, but not those caused by LPS, within the cerebral cortex. Similarly, phospholipids were unable to reverse LPS-induced inflammation in cultured astrocytes. In addition, this thesis proposes that n-3 PUFAs may prevent the development or lessen the symptoms of mental illnesses, ameliorating anxiety- and depressive-like symptoms as well as cognitive effects, particularly when administered during neurodevelopment. Such effects may be mediated by GR activation as well as by modification of the gut microbiota composition. Taken together, our findings suggest that n-3 PUFAs have therapeutic potential for stress-related disorders and we provide evidence for the mechanisms by which they may exert these effects. These findings contribute to an exciting and growing body of research suggesting that nutritional interventions may have an important role to play in the treatment of stress-related psychiatric conditions. Thu, 01 Jan 2015 00:00:00 GMT http://hdl.handle.net/10468/2132 2015-01-01T00:00:00Z http://hdl.handle.net/10468/2061 Metabolism of host-derived carbohydrates by Bifidobacterium breve UCC2003 Egan, Muireann Bifidobacteria are Gram positive, anaerobic, typically Y-shaped bacteria which are naturally found in the digestive tract of certain mammals, birds and insects. Bifidobacterium breve strains are numerically prevalent among the gut microbiota of many healthy breast-fed infants. The prototypical B. breve strain UCC2003 has previously been shown to utilise numerous carbohydrates of plant origin. Various aspects of host-derived carbohydrate metabolism occurring in this bacterium will be described in this thesis. Chapter II describes B. breve UCC2003 utilisation of sialic acid, a nine-carbon monosaccharide, which is found in human milk oligosaccharides (HMOs) and the mucin glycoprotein. B. breve UCC2003 was also shown to cross-feed on sialic acid released from 3’ sialyllactose, a prominent HMO, by the extracellular sialidase activity of Bifidobacterium bifidum PRL2010. Chapter III reports on the transcriptional regulation of sialic acid metabolism in B. breve UCC2003 by a transcriptional repressor encoded by the nanR gene. NanR belongs to the GntR-family of transcriptional regulators and represents the first bifidobacterial member of this family to be characterised. Chapter IV investigates B. breve UCC2003 utilisation of mucin. B. breve UCC2003 was shown to be incapable of degrading mucin; however when grown in co-culture with B. bifidum PRL2010 it exhibits enhanced growth and survival properties. A number of methods were used to investigate and identify the mucin components supporting this enhanced growth/viability phenotype. Chapter V describes the characterisation of two sulfatase-encoding gene clusters from B. breve UCC2003. The transcriptional regulation of both sulfatase-encoding gene clusters was also investigated. The work presented in this thesis represents new information on the metabolism of host-derived carbohydrates in bifidobacteria, thus increasing our understanding of how these gut commensals are able to colonise and persist in the gastrointestinal tract. Thu, 01 Jan 2015 00:00:00 GMT http://hdl.handle.net/10468/2061 2015-01-01T00:00:00Z http://hdl.handle.net/10468/3811 A study into the properties and applications of bacterial microcompartments and polyphosphate metabolism McCarthy, Karen Christine Bacterial microcompartments were initially observed in the 1950s and since that time, research in the field has focused on the unique properties of their proteinaceous structures and their role in the bacterial cell. This thesis investigates the properties of the propanediol utilization (Pdu) and ethanolamine (Eut) utilization microcompartments and applications of their components in the cells of Lactobacillus reuteri, and Escherichia coli predominantly, with additional investigation into Yersinia spp. The experimental chapters of this thesis initially investigate the application of bacterial microcompartments as tools for compartmentalizing metabolic reactions, in particular regarding the uptake of phosphate from extracellular environment and the retention of polyphosphate intracellularly. This was observed experimentally through the engineering of recombinant constructs containing genes encoding microcompartment-directed polyphosphate kinase enzyme. Bacterial phenotypes conferred by these constructs with respect to enhanced net uptake of phosphate from the environment were defined, and potential applications as a clinical therapy addressed in an animal trial, which utilized the phosphate-uptake constructs which had been created. In the trial, recombinant bacteria expressing these constructs were successfully delivered in therapeutic quantities to the target (small intestine) of rats experiencing induced renal failure, with no adverse effects were observed. However inter-group animal variation regarding baseline blood mineral content was larger than the expected therapeutic effect and therefore no therapeutic effect could be demonstrated. Additionally, novel phenotypic differences conferred by induction of Pdu and Eut microcompartments and empty recombinant microcompartments on host cells were identified. These identified an observable phenotypic difference in buoyant density between strains expressing the Pdu and Eut microcompartments. Further investigation into the potential function and phenotypic differences of strains expressing microcompartments were supported by methodological developments including the use of imaging instruments such as the FIB-SEM, and microcompartment extraction from cells. These technical developments enabled additional observations regarding the characteristics of microcompartments and provided novel information. Thu, 01 Jan 2015 00:00:00 GMT http://hdl.handle.net/10468/3811 2015-01-01T00:00:00Z