No description available.

No description available.

No description available.

No description available.

Microsporidia are an enormously successful group of obligate intracellular fungal parasites that infect most eukaryotes including humans. In my thesis I have analysed and compared microsporidian genomes to identify which genes have been conserved, and which lost, during the transition of the group to parasitism, sequenced the transcriptome of a mixed infection of Trachipleistophora hominis in a rabbit kidney cell line, and provided a description of the T. hominis intracellular lifecycle. My results demonstrate that microsporidian genome evolution is extremely dynamic; with huge loss of genes in the microsporidian common ancestor balanced by group and lineage-specific gene family expansion and innovation. Genes that are conserved among microsporidians are generally expressed at higher than average levels in the transcriptome of T. hominis. Lineage-specific genes show greater variation in expression, but some are very highly expressed suggesting that they play important roles in T. hominis biology. The transcriptomics data for T. hominis confirmed that it contains one of the largest microsporidian genomes in terms of gene content and also identified previously unannotated genes, some of which may have important roles in the parasite. Detailed analysis of my gene expression data demonstrated that differential expression of duplicate genes is a general feature of T. hominis gene families, including nucleotide transport proteins that are already known to have important roles in the microsporidian lifestyle. A method for partially synchronising the T. hominis infection was established and used to investigate the development of the infectious cycle using light microscopy and antibodies to T. hominis proteins. My observations suggest that the infection proceeds in a reproducible and predictable pattern under the experimental conditions used; providing a tool for future detailed study of how T. hominis infects and exploits eukaryotic host cells.

No description available.

Microsporidia are eukaryotes with compacted genomes, due to an obligate intracellular lifestyle. Microsporidia may provide valuable models for understanding the minimum requirements for sexual reproduction; however, their sexual status is debated, with most evidence limited to unreliable morphological inferences. Molecular biological investigations into sexual reproduction are sparse: observations of recombination are attributable to mitotic processes and searches of meiosis-related genes are limited in scope. Current proposals of extant sexuality are based on genes similar to fungal high-mobility-group or homeodomain mating-type determinants. Here, molecular genome data were used to infer the sexual status of microsporidia. PCR, cloning and HAPPY mapping were used to investigate variability at the proposed mating-type loci of Antonospora locustae and Paranosema grylli, followed by orthology comparisons with fungal mating-types. No intraspecies or interspecies variability in either locus was found and no orthological relationship to fungal mating-type proteins, refuting suggestions that they determine mating-types. Thus, inferred relationships to the Mucoromycotina are invalid. Alternative microsporidian mating-type determinants may exist. A novel microsporidian infecting commercial cultures of Gryllus bimaculatus was discovered and characterised. Characters of Microsporidium sp. supported the erection of a genus, sister to Trachipleistophora and Vavraia. Within-species variability in RPB1 sequences coincided with an indel used to define Trachipleistophora and Vavraia, thus their generic diagnosis requires review. Host ranges of Vavraia and Microsporidium sp. suggested that insects are natural reservoirs for human infections by Trachipleistophora. Finally, a robust methodology was implemented to identify meiosis-related gene orthologs in eleven microsporidian genome surveys. Observed distributions of meiosis-specific gene orthologs indicate that sex is an ancestral feature of microsporidia, recently lost in Encephalitozoon intestinalis and E. cuniculi. This supports current evolutionary theory that asexual species arise frequently, but are evolutionarily short-lived. A. locustae and Vavraia culicis appear capable of sexual reproduction and are proposed as model species for future investigations into microsporidian sexual processes.

Background: The role of P2X7R in the immune response has been investigated and this receptor clearly has important roles in inflammation. However, the mechanisms which integrate P2X7R activation with biochemical changes in T lymphocytes such as: proliferation, migration and regulation of adhesion molecule expression are less well understood. Many of these processes are controlled by the PI3K pathway, which is an important signalling cascade involved in development and immunity that it is frequently altered in disease. This study sought to investigate if PI3K was responsible for integrating P2X7R dependent signalling in primary human T lymphocytes, with an emphasis on regulation of the adhesion molecule CD62L. Results: Whole cell patch clamp electrophysiology is an important technique for characterising ion channel expression. This technique was optimised for the first time in primary human naïve CD4+ T lymphocytes and used to show P2X7R expression in this study. The pharmacology of ATP in the process of CD62L down-regulation in these cells was explored using new P2X7R antagonists with improved selectivity over previous compounds. Remarkably, PI3K/mTOR, MAPK and PKC signalling was shown to be dispensable for this down-regulation of cell surface CD62L expression. However, while investigating novel mechanisms for ATP induced CD62L down-regulation, it was revealed that pharmacological modulation of mitochondrial complex I or III, but not inhibition of NADPH oxidase, enhanced P2X7R dependent CD62L down-regulation by increasing ATP potency. The mechanism for this was further explored and this effect may arise from enhanced superoxide generation in the mitochondria of rotenone and antimycin A treated cells. Crucially, although ATP alone did not cause apoptosis of cell, perturbation of the mitochondria of cell with these compounds followed by ATP treatment, revealed P2X7R exposure of phosphatidyl serine. Discussion: This major new finding may have implications for the clearance of naïve CD4+ T lymphocytes which have undergone mitochondrial damage. A novel protective mechanism for the potential removal of cells with damaged mitochondria is presented, whereby, P2X7R dependent PS exposure occurs only when cells have enhanced mitochondrial ROS generation. Given the potential role of P2X7R in a number of diseases with a mitochondrial element, the findings of this thesis are of great importance for the targeting of P2X7R in inflammation.

No description available.

Diets rich in cruciferous vegetables· are associated with a reduced risk of cancer. This reduction in risk may be attributed to isothiocyantes (lTCs), which are the degradation products of glucosinolates. Sulforaphane (4-methylsulfinylbutyl isothiocyanate, SF) is the predominant ITC formed from heading broccoli ('calabrese') and iberin (3-methylsulphinyl propyl isothiocyanate) is the predominant ITC formed from purple sprouting broccoli. While, there are many studies regarding the biological activity of SF, there are few studies associated with iberin. Moreover, the majority of studies have been undertaken with transformed cells, usually epithelial in origin, that have cancerous phenotypes. In this study, primary epithelial and fibroblast cells derived from benign prostatic hyperplasia tissue (BPH) were used as a more appropriate model of normal prostate tissue. There are thought to be. complex interactions between epithelial and fibroblast .cells during the initiation and progression of prostate cancer. Affymetrix array was used to report global gene expression changes in both primary fibroblast and epithelial cells on exposure to physiologically appropriate concentrations ofSF and iberin. The majority of genes altered between iberin and SF treated cells were different; however, according to GenMAPP analysis, these genes were involved in some ofthe same pathways. Genes altered by both iberin and SF fitted into four main gene ontology categories: xenobiotic metabolism, cell cycle arrest, apoptosis and oxidative stress. A number ofgenes from these categories were selected for real time RT-PCR analysis, which showed a novel increase in gene expression on iberin and SF exposure. These genes were eyelin-dependent kinase inhibitor lA (p21wafllCipl), kruppel-like factor 4 (KLF4), pleiomorphic adenoma gene-like 1 (PLAGLl), tumour necrosis factor receptor super family, member lOb (TNFRSFlOb) and thioredoxin reductase 1 (TrxRl). This study is also the first evidence that iberin and SF reduce the mRNA expression of interferon induced transmembrane protein (lFITMl), chondroitin sulfate proteoglycan 2 (CSPG2) and vimentin (VIM) in prostate epithelial cells. Histone acetylation was investigated as a mechanism of global gene control by SF and iberin, however there was inconsistent evidence that either isothiocyante controls gene expression through histone deacetylase inhibition.