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.

Understanding the origin of biodiversity is a central question in evolutionary biology. Ecological specialisation, including the repeated rapid and parallel evolution of ecological specialists (‘ecotypes’), is a major source of biodiversity. The parallel evolution of ecotypes in salmonid fishes, such as Arctic charr, brown trout and European whitefish, has resulted in extensive diversity in northern postglacial freshwater ecosystems. Despite their ecological diversity and importance for northern ecosystems, the knowledge on the genetic basis of ecological specialisation, the evolutionary history of adaptive divergence, and the factors shaping the underlying genetic architecture are still not well understood in salmonids. Over the last decade many studies have investigated the genetic basis of ecologically relevant phenotypic traits in a wide range of salmonid species using genetic mapping approaches. However, knowledge on the conservation of the genetic basis for particular traits, or suits of traits, across species is limited, mainly due to a lack of genomic resources. Similarly, little is known about the genomic architecture of phenotypic diversity within species, such as the organisation of species-specific quantitative trait loci across the genome and the frequency of potential pleiotropy or genetic linkage. To understand how conserved the genomic basis for particular traits is across species and how quantitative trait loci (QTL) are organised within the genome, we analysed the genetic basis for a wide range of phenotypic traits (N=18) in six salmonid species using a dataset comprising of 943 QTL markers. We developed a novel analytical approach to analyse the colocalisation and synteny of QTL within and across species using a hetero-specific reference genome, in this case the Atlantic salmon (Salmo salar) genome. We found that QTL were not randomly distributed across the genome and that gene-density determined the distribution of QTL across chromosomes. By comparing QTL across species, we further identified genomic regions that were enriched for QTL for morphological and physiological traits (synteny blocks) in a range of species. Within three of the species, we also detected the significant colocalisation of QTL for different traits. Overall, the detection of synteny blocks and colocalised traits suggests a small but detectable role of pleiotropy and genetic linkage in trait evolution in salmonids and a conserved genetic basis for some traits across species. However, the observed patterns of conserved genetic basis and colocalisation were relatively weak, as QTL were mostly not conserved across species or colocalised within species. In general, the repeated evolution of similar ecotypes across populations and species implies a certain predictability of evolution. However, it is not well understood how phenotypic evolution overcomes the contingencies of heterogeneous genomic backgrounds of natural populations. To investigate the repeatability and predictability of parallel evolution, we used eco-morphological, genome-wide SNP and transcriptome data within and across lakes and evolutionary lineages of Arctic charr (Salvelinus alpinus). We found significant parallelism across replicated ecological specialists in foraging-associated traits. This phenotypic parallelism evolved despite population-specific variation in demographic histories, varying genomic response to selection and the non-parallel genetic basis of ecotype divergence. However, the regulatory molecular basis of ecological specialisation, inferred from gene expression and biological pathways, was highly parallel across ecotypes, bridging non-parallel genomic patterns and parallel eco-morphology. These findings suggest that parallel phenotypic evolution is possible despite non-parallel evolutionary routes when the functional molecular basis of ecological specialisation compensates for non-parallel genomic basis and histories. Evolutionary and genomic contingencies, such as demographic histories and genomic features can strongly influence the genomic architecture of adaptive divergence and reproductive isolation. To investigate how genomic features and demographic history influence the genetic architecture of adaptation and reproductive isolation, we reconstructed the demographic history and analysed the genetic architecture of divergence in brown trout (Salmo trutta) from the Maree Catchment in Scotland. Brown trout display reproductively isolated and divergent life histories and ecological specialisation, including a large piscivorous life-history form (ferox trout) and a smaller benthivorous life-history form. We found that ferox trout and benthivorous brown trout most likely diverged under a secondary contact of at least two distinct postglacial lineages and identified 33 genomic islands across the genome differentiating life-history forms. We demonstrated that some of these genomic islands formed under selection, and contained genes and biological pathways related to growth, development and immune response. Overall, we found strong genomic signals of divergence that were partially driven by selection on divergent phenotypes, and not only caused by genetic drift or through underlying genomic features, such as reduced recombination. The identification of the underlying evolutionary history and genetic architecture highlights the strength of genomic studies using species pairs for understanding the driving factors of adaptive divergence and reproductive isolation. Despite extensive knowledge on the genomic mechanisms underlying adaptive divergence over longer time scales and under the influence of phases of geographic isolation, less is known about the mechanisms underlying rapid ecological and phenotypic divergence. Rapid evolution plays an important role in the adaptation of species to human-induced environmental changes. However, it has been shown that in some cases human-driven environmental changes can lead to rapid loss of species and functional diversity, e.g. through species collapse and hybridization. Even though theoretical models predict that species can rapidly re-diverge under the right conditions following a species collapse and hybridization, the underlying mechanisms of rapid re-divergence remain to be elucidated. Empirical evidence for re-divergence following a species collapse is also lacking. We found evidence for the rapid evolution of ecologically-relevant phenotypic diversity in a European whitefish subspecies from Lake Constance, the gangfisch (Coregonus lavaretus macrophthalmus) after the recovery of pristine ecosystem conditions, following human-driven eutrophication, and speciation reversal. We found that a key functional trait, gill raker number, rapidly diversified within less than 10 generations following ecosystem recovery, allowing the use of vacant trophic niches. Variation in gill raker number is controlled by a sparse genetic architecture, as predicted by theory, and we further found evidence suggesting that introgression potentially provided the underlying adaptive variants. Several biological pathways that are known to be involved to ecological specialisation in fishes, such as metabolism, immune response and neural development, were identified based on coexpressed gene modules and genes under selection associated with gill raker number. Overall, our results demonstrate that functional diversity can rapidly re-emerge, given the right combination of genetic architecture, genetic diversity, and selection. In summary, this thesis demonstrates the evolutionary and genomic routes underlying phenotypic evolution and ecological specialisation in salmonid fishes. Comparing across different study systems, we find that secondary contact and historical gene flow played an important role in the evolution of salmonid species. Despite strong variation in the genomic basis of phenotypic traits across species and the genomic patterns of divergence across populations within species, we find some molecular parallelism across populations and species. Parallel ecotypes most likely evolved through parallel regulatory evolution and involvement of similar functional biological pathways. Furthermore, we find biological pathways that are repeatedly involved in adaptive divergence in different species. / Overall, our results indicate that despite the flexibility of rapid and parallel phenotypic evolution on the genomic level, it is relatively conserved on the level of regulatory mechanisms and functional biological pathways.

Working equids, vital to many of the world’s most economically vulnerable people, face many challenges to their health, welfare and productivity. In The Gambia, West Africa, appropriate nutrition, husbandry and veterinary assistance are limited, while infectious disease is a constant threat, particularly the parasitic disease trypanosomosis. The prevalence of generalised trypanosomosis in working equids attending the Gambia Horse and Donkey Trust show in 2013 using PCR was 55.4%. Trypanosoma congolense was most prevalent (47.0%), followed by T. vivax (15.7%) and T. brucei s.l. (2.4%). Mixed infections were common (9.4%) and T. congolense/ T. vivax coinfection appeared to have the greatest clinical effect. Spread of T. brucei parasites to the central nervous system (CNS), confirmed using immunohistochemistry and PCR, causes severe CNS dysfunction. Horses showed spastic paraparesis that rapidly progressed to recumbency, while donkeys more often displayed somnolence and cranial nerve dysfunction with a slower deterioration. The disease was fatal in all cases. Histopathology revealed diffuse lymphocytic-plasmacytic meningoencephalo-myelitis with marked perivascular cuffing, particularly in the white matter. T cells were prominent in this first study of lymphocyte distribution in equine CNS trypanosomosis. Extensive reactive astrocytosis was also demonstrated. Currently, a reliable diagnosis of equine CNS requires post mortem samples. The loop-mediated isothermal amplification (LAMP) assay was assessed for the diagnosis of equine T. brucei infection for the first time in both blood and cerebrospinal fluid (CSF). An entomological survey showed that Glossina morsitans submorsitans was common in dry woodland areas while G. palpalis gambiensis was found in riverine habitats. The prevalence of T. brucei in the midguts of Glossina specimens was 1.7% and equine DNA was found in tsetse bloodmeals, providing evidence for ongoing interaction between host, parasite and vector. Atylotus agrestis, vector of T. vivax and T. congolense, was present in large numbers in village areas. Equine DNA was detected in one A. agrestis specimen, however, no evidence of T. brucei in association with these flies was found. Finally, microsatellite genotyping was used for the first time to investigate T. brucei populations in equine trypanosomosis in The Gambia. The results revealed a heterogenous population, providing further evidence for a tsetse-transmitted mode of transmission. No evidence of population clustering by disease type or host species was detected, suggesting that host factors determine pathogenesis. Initial evidence for the involvement of the tsetse vector supports evaluation of vector control methods although further analysis of T. brucei populations in insect vectors and their relationships with those infecting equids is recommended. The clinicopathological descriptions will be of use in further study of equine CNS trypanosomosis and the development of new therapeutics and LAMP has the potential to facilitate research, especially in the study of CNS infection which has, up to now, relied on post mortem confirmation.

Duchenne muscular dystrophy (DMD) is an X chromosome-linked disease caused by the absence of the sarcolemmal protein dystrophin. The skeletal muscles of DMD have disrupted dystrophin-glycoprotein complex (DGC) and impaired sarcolemma integrity. In this study, we show that clonally derived dystrophin-deficient myoblasts PD50A are differentiation impaired. Coculture with osteoblasts improves the differentiation efficiency of PD50A myoblasts. We also establish that supplementation of combinations of IGF-1/IGF-2, IGF-1/LIF and IGF- 2/LIF in cultured PD50A myoblasts ameliorates the differentiation impairment. We establish that there are elevated levels of Cav-3 and Cav-1 proteins in dystrophin-deficient myoblasts and mdx mouse embryos and that Cav-3 and Cav-1 form heterooligomers in adult skeletal muscles. We show that overexpression of Pax7 suppresses Cav-3 in dystrophin-deficient myoblasts. Using a genetic mouse model (mdx/cav3\(^{+/-}\)) embryo we further establish that immunohistochemistry staining of Cav-1 and Cav-3 coincides with the mouse heart development. The DGC of skeletal muscles plays a role in signal transduction and mechanical response. Here we show that AKT/mTOR and IGF-2/p57\(^{kip2}\) (but not ERK) signalling pathways are upregulated in dystrophin-deficient myoblasts and mouse embryos. Using atomic force microscope we show that Cav-1 helps maintain the stiffness of dystrophindeficient myotubes while Cav-3 help maintain that of dystrophin-deficient myoblasts. This study suggests that Cav-1 and Cav-3 have both compensatory and compromising roles in mdx.