Histone modifications have been closely associated with changing levels of gene expression, but their role in determining, or possibly predicting, patterns of expression is uncertain. Here, the link between histone modifications and Hoxb gene expression in mouse embryonic stem (ES) cells was explored. Levels of the “active” modifications H3K9ac and H3K4me3 at Hoxb promoters varied widely from gene to gene, but were closely correlated in ES cells. Contrastingly, the repressive modification H3K27me3 was found at equivalent levels across the cluster. Treatment with the histone deacetylase inhibitor valproate induced a coordinate increase in the levels of H3K9ac and H3K4me3 at all Hoxb promoters, but not other genes, whilst H3K27me3 was unaffected. Such increases were not maintained upon removal of the inhibitor. All Hoxb genes were silent in undifferentiated ES cells, but expression was activated at defined times of differentiation in the expected 3’ to 5’ sequence. The valproate induced increase in active modifications did not induce Hoxb expression from the cluster in undifferentiated cells, nor was there any major shift in the timing of Hoxb expression in cells transiently exposed to valproate (ie. hyperacetylated) during the start of differentiation. Thus, active histone modifications at the Hox genes are uncoupled from transcription.

Development of the human heart is a complex process controlled by multiple genes (in interacting pathways), many of which are still to be determined. Abnormal heart development results in a spectrum of congenital heart disease (CHD), occurring in isolation or part of a syndrome, and with or without a family history, implying a genetic basis in some individuals. In this project I investigated the molecular genetic basis of CHD, in 23 families with nonsyndromic CHD. Using autozygosity mapping, I initially investigated the molecular basis of CHD in a single large consanguineous family (CHD1), and identified a region of interest containing a candidate gene (GDF1). I proceeded to sequence GDF1 (and genes in the same developmental pathway - NODAL, CFC1, TDGF1, and FOXH1) in 9 kindreds, but did not identify any pathogenic mutations. I then utilised whole exome sequencing (WES) to identify candidate mutations in potential CHD genes (GMFG, WNT11 and DVL2), and investigated these further by conventional sequencing. A novel GMFG nonsense variant was validated in family CHD1 and was absent from ethnically matched controls. Bioinformatics analysis of WES data from 19 affected individuals from 9 kindreds did not identify a frequently mutated candidate gene (or further GMFG candidate mutations), though candidate variants in individual kindreds were identified. Further functional analysis using animal models is required to determine the pathophysiological effect of the GMFG truncating mutation in cardiac development.

An affordable vaccine against all serogroups of Neisseria meningitidis causing reoccurring epidemics in sub-Saharan Africa is needed. Native outer membrane vesicles (NOMV) with over-expressed factor H binding protein (fHbp) represent a promising vaccine approach. We investigated the contribution of fHbp variant 1 amino acidic sequence to the cross-reactivity of the antibody response generated by NOMV OE fHbp. Coupling structure-sequence analysis of fHbp with epidemiological data of meningococcal prevalence in Africa, we selected four fHbp v.1 IDs (1, 5, 9, 74), to generate isogenic mutants of a serogroup W African isolate, over-expressing each of the them. NOMV from the mutants were purified, characterized, and the antibody response generated in mice was investigated, and compared to the corresponding recombinant fHbp. This is the first study indicating that the amino acid sequence of fHbp influences the specificity of the antibody response generated, not only as recombinant protein, but also when over-expressed on NOMV. In mice NOMV OE fHbp induced a fast, long-lasting antibody response, with high IgM and IgG antibody levels 7 days after immunization, and germinal centers induced. LipidA modifications do not impact primary antibody response, while precipitation of the antigen on alum attenuated the early antibody response, but enhance its longevity.

In order to enhance the sensing capabilities of DNA, an array of tags and modified nucleic acids have been synthesised. This thesis studies both the sensing capabilities and the stability of electrochemical DNA reporters based on the redox active molecule ferrocene. The topics covered are as follows: 1. Two synthetic ferrocene nucleic acid (FcNA) DNA mimics and a redox-active tag based on two covalently connected ferrocene molecules were incorporated into the backbone of a DNA strand in a central position and the 5’ end of a DNA strand, respectively. These probes were then used to form mixed monolayers and their stability was assessed. DNA target detection was possible with square wave voltammetry due to the unusual reliance of this technique on the ‘critical frequency’ of the redox active molecule. 2. DNA probes containing thymine-modified FcNA reporter groups were investigated further due the ability of mercuric ions (Hg2+) to bind covalently to two opposite thymine groups. Detection of these ions was possible through both cyclic voltammetry and square wave voltammetry. 3. The FcNA-conjugated DNA probes are assessed for their potential as therapeutic antisense agents by exposing the single stranded and duplexed probes to a DNase and two types of exonuclease enzyme.

Gene expression is compartmentalised in eukaryotes due to the nuclear envelope separating the nuclear processes of transcription and pre-mRNA processing from cytoplasmic translation. While ribosomes are synthesised in the nucleus, it is understood that a number of mechanisms keep them inactive until they reach the cytoplasm, where they mature to become translation-competent. However, this consensus view is being challenged by a growing body of evidence in support of nuclear translation. A newly developed technique, known as ribopuromycylation (RPM), had reported the presence of puromycin-bound nascent peptides on immobilised ribosomes in the nuclei of human cells. I investigated whether this method could be used, combined with chromatin immunoprecipitation, to determine whether nuclear ribosomes can cotranscriptionally translate nascent transcripts in Schizosaccharomyces pombe. Surprisingly, I discovered that, in contrast to that reported in the original study, immobilising ribosomes with translation elongation inhibitors does not lead to retention of puromycylated peptides on ribosomes in either S. pombe, Drosophila melanogaster or HeLa cells. However, I show here preliminary data which suggest that despite puromycylated peptides being released from the ribosome, puromycin immunostaining might still be used to visualise the sub­ cellular localisation of ribosomes inS. pombe, along with other approaches which I also describe.

Nephrolithiasis is a common disorder of multifactorial aetiology. Although most patients have a family history, the underlying genetic causes are largely unknown. To identify novel genes for nephrolithiasis, large-scale radiological and biochemical screens of male mice derived from an N-ethyl-N-nitrosourea-mutagenesis (ENU) programme were performed. This identified models for genetic renal calcification (Rcalc1) and genetic hypercalciuria (Hcalc1). Rcalc1, an ENU-induced mutant with renal opacities on X-ray and histological renal papillary calcification, exhibited autosomal dominant transmission with reduced penetrance. Linkage analysis mapped the Rcalc1 locus to a ~1.1-Mbp region on mouse chromosome 17A3.3, a novel calcification locus containing 30 genes. Urine and plasma biochemistry did not identify a biochemical abnormality associated with Rcalc1, but cDNA microarrays identified transcriptional alterations in genes involved in apoptosis and lipid metabolism. Hcalc1, an ENU-mutant with 24-hour urinary calcium 10x normal, renal stones and diffuse renal calcification on histological analysis, was found to be due to a dominant Ser682Pro transient receptor potential subfamily V, member 5 (Trpv5) mutation. Trpv5 is involved in Vitamin D-mediated renal calcium reabsorption. In summary, this work has identified two novel ENU mouse models for autosomal dominant renal calcification which will further contribute to the study of the genetic basis of renal stones.

The role of Placenta growth factor (PlGF) in physiological angiogenesis is quiescent, preclinical and human studies have established PlGF angiogenic activity to be regulated in pathologies. PlGF elicits its cellular functions via (vascular endothelial growth factor receptor) VEGFR-1 alone, and has gained more attention for its safety profile, especially when compared to the risks involved in VEGF-derived therapies. Loss and gain studies of PlGF function highlight the contribution of PlGF in disease aggravation or alleviation; however, factors regulating PlGF expression remains elusive.

TNF receptor/ligand superfamily members signal through pathways giving rise to proteins that regulate lymphocyte proliferation, activation, differentiation and survival. Absence of TNF ligands OX40L and CD30L impairs survival of GC T cells and affinity maturation of antibody responses. Direct effects of these molecules on B cells in antibody responses are not characterised. I dissected roles of OX40 and CD30 for B cells using T-independent type II (TI-II) antigen NP-Ficoll. Humoral immunity is impaired in OX40 deficiency. Defects in class switched and non-class switched antibody production are due to reduced development of antigen-specific switched and non-switched plasma cells. CD30 has an opposing role, deficiency results in similar or higher switched and non-switched antibody titres and higher numbers of antigen-specific plasma cells that develop rapidly. This may explain why in OX40/CD30 double deficiency, there is a less pronounced defect than in OX40 single deficiency. B cell intrinsic roles are revealed for OX40 and CD30 that suggest OX40 on B cells is critical for TI-II plasmablast differentiation or survival and B cell CD30 inhibits onset of plasmablast differentiation.

The Major Histocompatibility Complex (MHC) is a multi-gene family that includes most vertebrate immune genes. Life history traits have been associated with MHC allelic variation, including offspring survival, reproductive success, kin recognition, inbreeding avoidance, body mass gain, mate choice and parasite resistance. The studies reported in this thesis used laboratory and field investigations to identify differences in MHC genetic variation between truly wild, wild type and domesticated conspecifics and the implications of this for fitness, across the entire life history of a vertebrate, the guppy (Poecilia reticulata). Specifically, the effects of host inbreeding and domestication on parasite susceptibility are assessed in relation to MHC allelic and supertype composition. Laboratory studies showed that inbreeding and domestication lead to increased susceptibility to Gyrodactylus turnbulli, which was also linked to the presence of particular functional groups of MHC. A multi-site field sampling supported this finding; revealing that natural parasite communities reflected host MHC functional groups, as well as the river of origin. Truly wild fish had greater MHC genetic diversity than wild type (wild population maintained in the laboratory for ~ 3 years), which, in turn, were more genetically diverse than ornamental (domesticated) conspecifics. The accidental and deliberate release, into the wild, of domesticated fish is common. The release of infected and uninfected ornamental guppies into a wild type laboratory population increased parasite prevalence and abundance, due to the integration of a more susceptible individual into the social group. Mate preference is often linked to MHC similarity, whereby individuals select mates that are dissimilar or optimally similar at the MHC. The effects of sexual selection, MHC similarity and parasitism on mate choice, were assessed, indicating that a combination of factors are important in a female’s preference. Female guppies spent more time interacting with males with redder colouration and less MHC alleles in common. An experimental F1 generation revealed that offspring with parents sharing more MHC alleles and supertypes were more susceptible to parasitic infection. This research suggests that MHC functionality is at least as important as allelic and supertype diversity, with regards to individual fitness and life history traits.

Clostridium difficile is the leading cause of health care associated diarrhoea and remains a burden for the NHS. Disease symptoms can range from mild diarrhoea through to fulminant pseudomembranous colitis, resulting in mortality for some patients. Recurrence is a major problem and estimates are that 20% of all patients with disease will either relapse (with the same strain) or have a re-infection (with a different strain). Arguably, the main virulence factors are toxins A (TcdA) and toxin B (TcdB) which cause disease symptoms. The genes encoding TcdA and TcdB are located within the pathogenicity locus (PaLoc) along with three accessory genes; tcdR, tcdE and tcdC. The regulatory network has been studied but we aimed to add to this knowledge by using two under investigated strains R20291 a so-called hypervirulent strain and VPI 10463 a strain known to produce higher levels of toxin. Two different methods of investigation were employed during this study to improve our understanding of both the regulation of TcdA / TcdB but also the genetic mechanisms behind clinical relapse. These methods were; using forward and reverse genetic analysis to assess phenotypic differences and using bioinformatics to identify genes and / or single nucleotide variants (SNP) that may play a role. Using a combination these methods we have identified potential regulators of toxin production in both strains. We have also identified unique genes and SNPs that might provide a fitness benefit to strains of C. difficile that were isolated from patients who had suffered relapse episodes.