Investigation of genomic diversity can provide insight into the evolutionary history of bacterial species. However, complete genome sequencing is not yet practical for large strain collections at the beginning of this project. In this project PCR-based methods to investigate the genomic diversity of non-sequenced strains were successfully developed. In \(Escherichia\) \(coli\), the distribution of two Type III secretion system, ETT2 (\(E.\) \(coli\) Type Three Secretion 2) and Flag-2 (\(E.\) \(coli\) Flagellar system 2), were surveyed among a collection of 79 strains. Remnants of both clusters were found to be present in most strains, suggesting that both have a long evolutionary history within \(E.\) \(coli\). The PCR-based methods were also developed for application as part of genome sequencing projects. They were used to explore the co-linear and variable regions between \(Campylobacter\) \(jejuni\) M1 and the genome sequenced strain \(C.\) \(jejuni\) strain 11168. The \(C.\) \(jejuni\) M1 genome was assembled into thirty-four genomic contigs relative to strain 11168, and the size and position of insertions/deletions were characterised. Similar methods were used to facilitate the finishing of the genome of \(Francisella\) \(tularensis\) strain FSC198, using sequence information from strain Schu S4. The completed genome sequence of FSC198 showed it to be almost identical to that of Schu S4. The two genomes differ at only 11 loci, eight SNPs (single nucleotide polymorphisms) and 3 VNTRs (variable number tandem repeats). This surprising finding suggested that the European isolate FSC198 may be derived from the US laboratory strain Schu S4. Two virulence factors, IglA and IglB, from a pathogenicity island of strain FSC198 were further investigated and found to interact at the protein level. These proteins are possibly involved in Type VI secretion, and may represent potential vaccine candidates.

Enterotoxigenic Escherichia coli is a gram negative bacterium that is responsible for acute watery diarrhoea in humans. Virulence factors of ETEC have been characterized but underlying gene regulatory mechanisms are not yet understood. Moreover, antibiotic resistance in bacteria is a serious problem worldwide. Multiple antibiotic resistance in bacteria can be driven by transcriptional regulators in the AraC/XylS family. The E. coli mar regulon is considered a paradigm for such systems. The mar locus consists of 3 genes; marR, marA, and marE. A transcriptional activator encoded by marA enhances drug resistance by binding to "marbox" sequences at target promoters. The best characterised MarA targets encode the AcrAB-TolC drug efflux pump. In this work, we have defined a molecular mechanism controlling expression of ETEC heat stable enterotoxin. We show that the CRP directly activates expression of heat-stable toxin and H-NS can exclude CRP from the activation-binding site. We also show that heat-stable toxin expression can be controlled by osmo-metabolic flux; CRP and H-NS allow the toxin gene promoter to respond to both glucose and salt. These conditions are encountered on host cell attachment and during oral rehydration therapy. We also identify 33 MarA targets to reveal novel mechanisms of antibiotic resistance.

All chronic liver diseases can induce fibrosis and lead to liver cirrhosis. Within liver disease, hepatic stellate cells (HSC) are accepted as the major effectors of fibrogenesis and changes in the extracellular matrix (ECM). Cytoglobin (CYGB), a hexacoordinated globin, is upregulated in liver disease, and expression has been reported to be specific to HSCs in the liver, though this is disputed. Data presented in Chapter 3 of this thesis confirm upregulation of Cygb in murine models of liver disease and diseased human liver tissue. Chapter 4 shows how ECM can effect HSC morphology, behaviour and phenotype with collagen I, an important component of the hepatic scar conferring an activated HSC phenotype, and laminin, a basal protein in a normal liver, inducing a more quiescent phenotype in HSC cell lines HSC-T6 and LX-2. Chapter 5 demonstrates the novel observation of collagen I-induced downregulation, and laminin-induced upregulation, of Cygb in HSC-T6s. Chapter 6 explores the role of cell signalling through membrane receptors and regulation of Cygb expression, identifying phosphorylated focal adhesion kinase as a mechanism of signal transduction through integrin activation. These findings suggest that Cygb expression is modulated by ‘outside-in signalling’ and this is important in the activation status of HSCs.

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis and is the target of several antitubercular drugs. Arabinofuranosyltransferase enzymes, such as EmbA, EmbB, and AftA, play pivotal roles in the biosynthesis of arabinogalactan. The anti-tuberculosis agent ethambutol (EMB) targets arabinogalactan biosynthesis through inhibition of Mt-EmbA and Mt-EmbB and also targets the biosynthesis of the important immunomodulatory molecule lipoarabinomannan (LAM), through inhibition of Mt-EmbC. A bioinformatics approach identified putative integral membrane proteins in Mycobacterium smegmatis, M. tuberculosis and the closely related species Corynebacterium glutamicum, with features common to the GT-C superfamily of glycosyltransferases. A novel arabinofuranosyltransferase, AftC, was deleted from both M. smegmatis and C. glutamicum and shown to be an internal branching α(1→3) arabinofuranosyltransferase involved in arabinogalactan biosynthesis. Further studies revealed a truncated LAM whereby the arabinan domain was severely reduced and consisted of a simple linear arabinan of approximately 12-15 α(1→5) linked Araf residues. This mutant LAM was also shown to be a potent stimulator of TNF-α production using a human macrophage cell line, thus illustrating that masking of the mannan core by arabinan in wild type LAM alters its ability in the production of this cytokine. We also describe a further arabinofuranosyltransferase, AftB. Deletion of its orthologue in C. glutamicum resulted in a viable mutant and biochemical analysis revealed the complete absence of terminal β(1→2)-linked arabinofuranosyl residues. Further analysis confirmed AftB as a terminal β(1→2) arabinofuranosyltransferase, which was also insensitive to EMB. The bioinformatic search for cell wall glycosyltransferases led to the identification of a rhamnosyltransferase in C. glutamicum, RptA. Deletion resulted in a reduction of terminal-rhamnopyranosyl linked residues and as a result, a corresponding loss of branched 2,5-linked arabinofuranosyl residues. Furthermore, analysis of base-stable extractable lipids from C. glutamium revealed the presence of decaprenyl-monophosphorylrhamnose, a putative substrate for the cognate cell wall transferase. Altogether, these studies have shed further light on the complexities of Corynebacterianeae cell wall biosynthesis, and represent potential new drug targets.

Eukaryotic cells are highly compartmentalized, so that some steps of gene expression occur in the nucleus and others in the cytoplasm, i.e., transcription and pre-mRNA processing are spatially separated from translation which occurs in the cytoplasm. Ribosome subunits and several translation factors are present in the nucleus but it is understood that they can interact to form the functional ribosome only during translation in the cytoplasm. Recent studies, however, have suggested that translation may occur also in the nucleus. That functional ribosomes may exist also in the nucleus has been suggested by studies in the field of nonsense mediated mRNA decay (NMD) and, more directly, by reports that ribosomal proteins are found associated with nascent transcripts on Drosophila polytene chromosome and that amino acids are incorporated at transcription sites. My project aimed to investigate further the question of whether there are functional ribosomes in the nucleus; and also to confirm the presence of ribosomal proteins at active transcription sites. To address these issues, I have developed a system to visualize ribosomal subunits interaction at the molecular level. The technique consists in tagging pair of ribosomal proteins, located at interaction surface of the 40S and 60S subunits, with split fragments of yellow fluorescent protein so that bimolecular fluorescence complementation (BiFC) occurs only when the subunits join to form a 80S ribosome. With this technique I was able to visualize translation sites in Drosophila S2 cells and in transgenic flies. Translation sites are most apparent in the cytoplasm, however in cells in which export of ribosomal subunits was blocked by drug treatment a clear signal is visible also in the nucleoplasm. Notably, I also I 2 detected a strong signal in the nucleolus. These observations suggest that either there is translation in the nucleolus or that, contrary to what is currently understood, ribosome subunits can join together during ribosome biogenesis. In summary, with the technique I have developed I was able to find further evidence that functional ribosomes are present in the nucleus. This technique and these first results shall aid future investigations into the fundamental issue of whether ribosomes have a function in the nucleus and also to monitor translation changes in living cells.

Long terminal repeat (LTR) elements are wide-spread in the human genome and have the potential to act as alternative promoters and enhancers. Their expression is therefore under tight epigenetic control. We previously reported that a member of a specific class ofLTR elements (THE I B) in Hodgkin's Lymphoma (HL) acted as a promoter for the growth factor receptor gene CSF 1 R and that this gene is required for HL cell survival. However, to which extent and how such elements participate in shaping the unique gene expression program of HL is unknown. To address this question we mapped the genome-wide activation ofLTRs in HL using a novel targeted next generation sequencing approach (RACE-Seq). Integration of such data with global gene expression as well as chromatin profiling data from HL and non-HL cell lines discovered a unique pattern of LTR activation impacting on gene expression, including a number of genes associated with the HL phenotype. We also show that global LTR activation is induced by activation of inflammatory signaling pathways. Together these results demonstrate that LTR activation presents an additional layer of gene expression deregulation in HL and highlight the potential for the impact of genome-wide L TR activation in other inflammatory diseases.

The activity of the major pathogenicity determinant of enterohaemorrhagic Escherichia coli is primarily coordinated by expression of the LEE1 operon which is part of the locus of enterocyte effacement (LEE). The LEE1 operon regulatory region has been dissected. LEE-encoded transcription factor, GrlA, activates the LEE1 P1 promoter by binding to a target located within the 18 base pair spacer between the promoter 10 and 35 elements. Shortening this spacer to 17 base pairs increases P1 promoter activity and short-circuits GrlA dependent activation, suggesting that GrlA functions like MerR family transcription activators. It was also found that the LEE1 P1 promoter is overlapped by a cryptic promoter, designated P1A. A single base substitution in the P1 consensus -35 element unmasks P1A promoter activity. In contrast, P1A activity is much less when P1 is inactivated by a mutation in its -10 hexamer element. Hence, even when P1 is inactive, the consensus -35 element sequesters RNA polymerase and prevents its access to the P1A promoter. The LEE1 leader sequence also contains a mini-gene that encodes a dipeptide. Genetic studies showed that expression of this mini-gene is important for optimal expression of downstream genes.

Streptococcus pneumoniae is the primary cause of community acquired pneumonia and represents a substantial disease burden worldwide. The PatAB ABC transporter is a multidrug efflux pump in this organism, encoded by the patA and patB genes, and over-expression of PatAB confers resistance to fluoroquinolone antibiotics. This thesis investigates the clinical relevance and the regulation of PatAB expression. A strong association was found between cross-resistance to fluoroquinolones and dyes and over-expression of patAB in pneumococcal clinical isolates, supporting previous assertions that PatAB is the main fluoroquinolone efflux pump in S. pneumoniae. Two mechanisms that caused increased expression of patA and patB were identified by whole genome sequencing of multidrug resistant mutants of S. pneumoniae R6. Firstly, a novel duplication of a nine kilobase genomic region, including patA and patB, was identified in one mutant. Further investigation suggested that duplication caused high expression of the second copy of patAB by bringing these genes under the control of a tRNA gene promoter. Secondly, mutations were identified in the terminator stem-loop of a predicted transcriptional attenuator upstream of patA in three other mutants. Transcriptional fusion of mutated attenuator sequences with a GFP reporter gene suggested that these mutations increased transcription from the patA promoter.

Within the female reproductive tract mammalian sperm gradually develop hyperactivated motility, triggered by increased flagellar Ca2+ resulting in deep flagellar bends. Two sources of Ca2+ may contribute to the regulation of hyperactivation. Ca2+ influx at the plasma membrane is crucial and the sperm specific CatSpers channels are of great importance. In addition, studies on sperm have provided strong evidence that Ca2+ stored in the region of the sperm neck contributes to regulation of flagellar activity. 4-aminopyridine caused robust and persistent hyperactivation of motility in human sperm. Ca2+ imaging showed that treatment with 4-aminopyridine induced a parallel elevation of [Ca2+]i, which initiated at the sperm neck/midpiece and was associated with asymmetric flagellar bending in this region. This report demonstrates that 4-aminopyridine induced hyperactivation in human sperm is not solely pH/CatSper channel dependent and that mobilisation of stored Ca2+, possibly causing activation of store-operated Ca2+ influx, is essential for hyperactivation in a population of human sperm. 4-aminopyridine induced IP3R and RyR store release in sacroplasmic reticulum and brain microsomal vesicles. Although the physiological factor(s) that activate hyperactivation in vivo remains elusive we can conclude that release of the neck/midpiece intracellular Ca2+ store was sufficient to initiate hyperactivation in a population of human sperm.

Methionine mutants of Neurospora crassa were isolated by a filtration concentration technique following ultra-violet irradiation. These and other mutants were classified by the physiological tests of precursor utilisation and heterocaryon complementation. Precursor utilisation data are in agreement with the present outline of methionine synthesis from cysteine but provide little information on the pathway of sulphate reduction and incorporation of sulphur within a carbon skeleton to yield cysteine. Seven cysteine and eight methionine loci are represented on the basis of heterocaryon complementation tests. Four of the cysteine loci and either one or two of the methionine loci, associated with the methylation of homocysteine, have not been reported previously. Two of the eight methionine loci had already been located but five of the remainder were associated with definite chromosome regions. No evidence for the tight clustering of either these or the cysteine loci was obtained. Inter-allelic complementation has been observed at two loci and the fine structure of one of these loci (me-2) has been studied in some detail by analysis of prototroph formation in inter-allelic crosses. The use of "outside markers" has facilitated the demonstration of intra-genic recombination and "conversion". Furthermore, these studies provide evidence for a linear arrangement of mutable sites within the locus with additive prototroph frequencies to yield a site map which can be correlated with a complementation map. The results also show that some alleles are more prone to "conversion" than others.