Microbiological research has uncovered the basis of fermentation, infectious disease, vaccination and antibiotics. Now, a technological revolution leveraging DNA, the code of life, has allowed us to unravel cellular and evolutionary processes in exquisite detail. Today our need for new innovation is still great. The modern world is a challenging environment: over-population, climate change and highly mobile populations create a high risk of pandemic disease especially from viruses and many bacteria are now resistant to our life saving antibiotic drugs due to overuse. In hospitals, the spread of pathogens can be rapid and life threatening. Whole-genome sequencing has the power to identify the source of infections and determine whether clusters of cases belong to an outbreak. Portable, real-time nanopore sequencing enables sequencing to be performed near the patient, even in resource-limited settings. Integrating with existing datasets allows digital surveillance able to detect outbreaks earlier while they can still be contained. Early demonstrations of the power of whole-genome sequencing for outbreak surveillance have made it an area of intense interest and further development in laboratory methods and infrastructure will make it an important tool that can be deployed in response to future outbreaks.

Biosynthetic steps in the mupirocin (pseudomonic acids) biosynthetic pathway of Pseudomonas fluorescens NCIMB 10586 have already been deduced. Putative functions of most of the genes of \(mup\) cluster have been assigned although exact sequence of steps in the pathway and their timings are not yet known. Thiomarinols are another group of anti-bacterials produced by Pseudoalteromonas sp. SANK 73390. Very little is known about the biosynthesis of thiomarinols that share striking structural similarity with pseudomonic acids in their polyketide and fatty acid moieties. This similarity is reflected at genetic level as significant similarity in amino acid identity between the products of at least 27 ORFs in these biosynthetic clusters. This project aimed to learn more of biosynthetic steps in the biosynthesis of mupirocin and thiomarinol antibiotics by testing for functional cross-complementation between pair of genes or a group of genes whose products show significant homology. Surprisingly, only two genes \(tmlJ\) and \(tmlS\) out of nine studied showed complementation in the \(mup\) system. Findings suggested protein-protein interactions limited interchangeability of equivalent functions between two biosynthetic systems. It was shown by expressing related genes as groups for complementation in the \(mup\) system that it was possible to confirm specificities of such interactions.

DNA associates with proteins to form chromatin which is essential for the compaction of the DNA into the cell nucleus and is highly dynamic in order to allow the different biological processes of the DNA to occur. Chromatin compaction is achieved at different hierarchical levels: the 10nm fibre (DNA associates to nucleosomes formed by different histones), the Higher Order Chromatin fibre and the 300 nm chromosome structures. This study has shown that both H1 and H4 histones play a crucial role in preserving meiotic as well as mitotic chromosome structure and functional genome integrity in Arabidopsis. The role of the different linker histone H1 isoforms as well as the core histone H4 in Arabidopsis thaliana was investigated using T-DNA and RNAi mutant lines which showed different meiotic defects. Chromosomal breaks as well as non-homologous connections in the h4RNAi were linked to 45S/5S rDNA disorganisation, suggesting that H4 preserves chromosome integrity at these rDNA regions. Ath1.1 mutant presented univalents and reduced chiasma frequency at metaphase I, linked to a severe defect in ASY1 localisation on the meiotic chromosome axes. Thus, indicating that histone H1.1 is vital for proper chromatin axis organization that permit normal loading of recombination machinery proteins in Arabidopsis.

CD4\(^+\) and CD8\(^+\) T cell populations can be divided into subpopulations based on expression of surface markers CCR7 and CD45RA. The resulting populations are referred to as naive, central memory, effector memory and effector memory RA\(^+\) (EMRA). The aim of this study was to identify potential lineage relationships between these subpopulations for both CD4\(^+\) and CD8\(^+\) T cells through microarray analysis. The genes found to distinguish between these subpopulations include many molecules with known functions in T cell differentiation, including CCR7, CD45RA, granzymes, L-selectin and TNF receptors. Several genes from the tetraspanin family of proteins were found to be differentially expressed at mRNA and protein level; suggesting a possible role for these genes in CD4\(^+\) and CD8\(^+\) T cell activation, migration and lysosomal function. Other genes identified, such as LRRN3 and CXCR5 which were expressed highest on naive and CM T cells respectively, provide interesting gene targets to follow up on their function in these T cell populations. Microarray data was validated through Real Time PCR and suggests that both CD4\(^+\) and CD8\(^+\) T cells differentiate along a linear pathway of naive to central memory to effector memory. The transcriptional programmes responsible for these differentiation steps were distinct between CD4\(^+\) and CD8\(^+\) T cells, although additional elements were common to both subsets.

Brassica crops are grown worldwide for food, oil, medicinal and crop rotation properties. They suffer from insect pests which cause large yield and economic losses. Application of insecticides is the preferred way of dealing with insect problems, but it is not only hazardous to the environment, it also affects humans as the chemicals easily get incorporated into the food chain. As a result, new more resistant varieties are urgently needed to meet the demand of growing populations. A set of 200 accessions were classified as resistant (non-preferred) or susceptible (preferred) in response to cabbage aphid feeding in the field. Fifteen accessions were further assessed to characterize and identify the level and location of resistance factors by investigating feeding behaviour of cabbage aphid using the Electrical Penetration Graph (EPG) technique. The feeding behaviour assessment revealed the presence of interspecific & intraspecific variation and presence of resistance factors at multiple levels. The transcriptional response of these accessions under presence and absence of aphid feeding for 24h showed that gene expression is highly regulated in response to aphid feeding. Gene ontology (GO) enrichment study helped identify strong candidate genes for aphid resistance. In addition to this, the gene expression differences between CWR and landraces indicated adaptations of landraces during the process of domestication. Lastly, Gene expression data was used to develop models to predict insect resistance status. In conclusion, the combination of EPG and transcriptomics provides an opportunity to assess brassica germplasm for further research into defence mechanisms of cabbage aphids.

The process of angiogenesis in which new blood vessels form from pre-existing vessels, can be intensively studied through the use of in vitro and in vivo models. The in vitro co-culture tube formation assay is used to assess the ability of endothelial cells to develop into three dimensional tubular structures which mimics the growth of capillaries. Different fluorescent labelling techniques were developed and utilised alongside confocal microscopy to visualise endothelial tubulogenesis and investigate the mechanisms of lumenogenesis. Imaging the actin cytoskeletal organisation by expressing the lifeact peptide conjugated to fluorescent proteins revealed that Factin fibres outline lumens within endothelial tubules and enabled clear visualisation of filopodia formation. Further studies presented in this thesis aimed to develop, test and evaluate computational tools for analysing endothelial sprouting from fluorescently labelled spheroids generated using the in vitro hanging drop spheroid assay and quantify blood vessel formation in the in vivo zebrafish model. The results confirmed that both analysis tools were able to rapidly quantify a wide range of angiogenic images and generated comparable results to frequently used manual methods. The developed computational analysis tools are user friendly and can be used to assess the effects of inhibitor compounds and silencing vascular related genes.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, has a unique cell envelope which accounts for its unusual low permeability and contributes to resistance against common antibiotics. The mycobacterial cell wall consists of a cross-linked network of peptidoglycan (PG) in which some of the muramic acid residues are adorned with a complex polysaccharide, arabinogalactan (AG), via a unique α-L-rhamnopyranose–(1→3)-α-D-GlcNAc-(1→P) linker unit. Whilst the cytoplasmic steps of mycobacterial cell wall biosynthesis have been largely delineated, the molecular processes that govern the flux of PG intermediates and the mechanism by which PG and AG pathways converge has remained elusive. We identified key conserved serine/threonine residues of MurC, as potential candidates for phospho-regulation by the cognate protein kinase, PknA. Pseudo-phosphorylated MurC mutants exhibited differential enzyme activity, suggesting that M. tuberculosis is capable of tight control of PG biosynthesis through phosphorylation of MurC. In addition, we have identified Lcp1, a mycobacterial orthologue of the LytR-CpsA-Psr (LCP) family of proteins found in Gram-positive bacteria, responsible for ligating cell wall teichoic acids to PG. We demonstrate that lcp1 is an essential gene required for cell viability and show that recombinant Lcp1 is a phosphotransferase capable of ligating AG to PG in a cell free radiolabelled assay.

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.

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.

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.