APC Microbiome Ireland - Masters by Research Theses

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    Natural transformation in lactic acid bacteria
    (University College Cork, 2020-10-16) O'Connell, Laura Michelle; van Sinderen, Douwe; Mahony, Jennifer; Science Foundation Ireland
    Fermentation has been commercially exploited to produce food products for millennia. The most intensely exploited microorganisms used in modern industry to commercially produce fermented products are yeasts and lactic acid bacteria (LAB). LAB are most widely employed in dairy processing, where they contribute to the biochemical changes that convert milk to cheese and yoghurt. To ensure consistency, large-scale, industrial fermentations use starter cultures of LAB, meaning a known strain or a sometimes-undefined combination of multiple strains with well-defined characteristics. Hypothetically, through natural genetic modification, which exploits DNA transfer systems occurring in nature, desirable traits could be introduced to strains to improve the efficacy of the starter culture. The focus of this thesis is natural competence. Competence refers to the specialised physiological state in which bacteria are able to undergo transformation through the internalisation of exogenous DNA. Recently an early com system was described for the industrially relevant LAB, Streptococcus thermophilus and a late com system homologous to that of S. pneumoniae was described in another LAB widely employed in cheese production, Lactococcus lactis. This thesis aimed to explore the functionality of the proven and predicted natural com systems encoded these LAB. In the case of S. thermophilus, this was achieved by defining the nutritional requirements of competence development, which involved defining a minimal cultivation medium. This medium was used in competence assays involving a model strain, S. thermophilus LMD-9, to confirm the functionality of the competence system in the minimal medium. The optimised medium and assay were then applied to a panel of industrial strains to determine the transferability of the transformation protocol. While it was observed that the minimal medium could support the growth of the industrially relevant strains, no transformation was detected in any industrial strains. A different approach was taken to study competence in L. lactis, as the system is far less defined compared to that of S. thermophilus. For this reason, an investigation of the gene activity of the lactococcal competence components was conducted, as opposed to competence assays. Specifically, the activity of the com-promoters was assessed via blue/white selection, using a promoter-probe vector constructed in this study. Several conditions were trialled to identify those optimal for competence development. This included the nisin-induction of the master competence regulator, ComX, which was the only condition that proved capable of activating the com-promoters. Chemical mutagenesis was employed in an attempt to generate random mutants that did not require comX-induction; however, this method did not prove effective, and no such mutants were isolated and the mechanism of lactococcal competence remains elusive.
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    Deciphering the role of microbially-derived metabolites on the microbiota-gut-brain axis
    (University College Cork, 2021-01-25) Spichak, Simon; Cryan, John; Dinan, Timothy G.; Science Foundation Ireland; Irish Research Council
    The trillions of microbial organisms residing in the gut, microbiota, are now recognized as major modulators of physiology and health, quickly becoming one of the most exciting emerging areas in neuroscience. Preclinical and clinical research alike suggests that the metabolites produced by these gut microbes modulate brain, behavior and disease. Short-chain fatty acids, tryptophan metabolites and bile acids are promising targets for new microbiome-based therapies. But, little is known about their mechanisms. To this end, the second chapter of the thesis collates 278 studies relating to the human microbiota-gut-brain axis, identifying trends and technical/bioinformatics limitations. These studies across different disorders of the brain as well as healthy human behavioral functions. Then a 35 of these studies was reanalyzed with an up-to-date bioinformatics pipeline. New tools, mainly the gut-brain modules provide a predictive framework for identifying whether these gut microbial metabolic pathways are dysregulated in brain diseases and disorders. We uncovered evidence of disease-related alterations in microbial metabolic pathways in Alzheimer’s Disease, schizophrenia, anxiety and depression. Previous human studies suggest that astrocyte immunity and metabolism is affected by short-chain fatty acids. Thus we grew primary male and female mouse astrocyte cultures, treating them with acetate, butyrate and propionate. Butyrate treatment (0 – 25μM) increased gene expression of Bdnf and Pgc1-α expression, implicating histone-deacetylase inhibitor pathways only in female cells. Acetate (0 – 1500 μM) positively correlated with Ahr and Gfap expression in males, suggesting an immune modulatory role. These findings show a novel sex-dependent impact of acetate and butyrate, but not propionate on astrocyte gene expression. These studies increase understanding of microbial metabolites and how they might impact the brain. It also provides guidance to improve and direct future investigations aimed at identifying the mechanisms of other metabolites.
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    The effects of stressors during early life on hippocampal neurogenesis and microglial activation in the male and female brain
    (University College Cork, 2021-04-05) McGovern, Andrew Joseph; Nolan, Yvonne M.; O'Leary, Olivia
    Stress during critical periods of brain development and maturation such as adolescence is associated with an increased risk of developing stress-related psychiatric disorders which are more common in women than men. Early life stress such as maternal separation (MS), juvenile stress (JS) and inflammatory insults like lipopolysaccharide (LPS), have been found to induce anxiety and depressive-like behaviours and decrease adult hippocampal neurogenesis in rodents. However, the effects of early life stress on adult hippocampal neurogenesis and associated function have been mostly assessed in male rodents. The impact of early life stress on microglia, which are involved in the regulation of adult hippocampal neurogenesis and dendritic remodelling, has also been predominantly examined in male rodents. Thus, in this study we assessed adult hippocampal neurogenesis and hippocampal microglia following LPS administration in MS juvenile female Sprague-Dawley rats and following JS in male and female Sprague-Dawley rats in adulthood. MS increased the number of newly born hippocampal neurons in the ventral hippocampus, reduced the dendritic complexity of newly born neurons in the whole hippocampus and increased the soma size of microglia, indicating activation. LPS reduced newly born hippocampal dendritic complexity and increased the number of microglia in the dorsal hippocampus. Conversely, LPS administration in MS rats reduced the number of microglia in the dorsal hippocampus and MS attenuated microglial activation in response to LPS. LPS administration in MS increased dendritic complexity in the granule cell layer (GCL) and further reduced dendritic complexity in the ventral but not dorsal hippocampus of juvenile female rats. JS did not affect hippocampal neurogenesis in adult male or female rats but reduced the cell soma size of microglia in the GCL in the dorsal hippocampus of females. We observed significant sex differences in adult rats; females had fewer newly born neurons with less dendritic complexity in the dorsal hippocampus than males. There were also fewer microglia in the molecular layer (ML) of the hippocampus in adult female than male rats. Together the data here shows that the effect of early life stressors differentially affects hippocampal neurogenesis and hippocampal microglia dependent on age, sex and subregion of the hippocampus analysed.