To determine the expression pattern of XPAT (Xenopus primordial germ cell associated transcript) protein in Xenopus oocytes, XPAT-GFP fusion proteins were generated. When XPAT was amino-terminally tagged with GFP it became localised to the nuclei of stage VI Xenopus oocytes. However, when carboxy-terminally tagged with GFP, XPAT also translocated to the vegetal pole of stage VI oocytes. XPAT-GFP formed particles (1 to 2.5mm in diameter) which aggregated into large (10 to 50mm) granular structures at the vegetal pole. These particles looked exactly like those seen after in situ hybridisation to germ plasm RNAs. The granules of XPAT-GFP were larger than endogenous germ plasm granules seen in stage VI oocytes; they were more consistent with those observed in 2-cell embryos during germ plasm aggregation. Studies involving the use of the anticytoskeletal drugs colcemid, nocodazole and cytochalasin D and the microtubule stabilising agent taxol indicated that microtubular transport was important in the location of XPAT-GFP. Several attempts were made to raise antibodies to XPAT peptides, but at present the endogenous expression pattern of XPAT protein is unresolved. To investigate possible domain structure of XPAT, one carboxy-terminal and three amino-terminal deletion variants of XPAT-GFP were constructed. An N-terminal deletion protein lacking the first 61 amino acids of XPAT was able to form small particles, but none of the deletion proteins exhibited vegetal localisation or formed large aggregates in Xenopus oocytes. The N-terminal deletion proteins all became predominantly localised to the nucleus; protein motif analysis revealed that XPAT contains a putative bipartite NLS in its carboxy-terminal region. The C-terminal deletion protein, which lacked the putative NLS, was evenly distributed throughout the nucleus and cytoplasm of Xenopus oocytes. XPAT was shown to be able to bind to homopolymeric RNAs in vitro. When Xpat mRNA was depleted from stage VI Xenopus oocytes (by injection of an antisense oligo) levels of DEADSouth and XVLG1 mRNAs decreased substantially.

Myxococcus xanthus is a soil-dwelling bacterium which produces carotenoids upon irradiation with blue light. Genetic analysis has allowed elucidation of transduction of the light signal to the carotenogenic machinery within the cell. The primary element within the carotenogenic regulon is the genetic switch manifested by CarR and CarQ. CarR is an integral membrane protein which binds to the sigma factor CarQ and holds it in an inactive state at the cell membrane. Illumination of the cell with blue light excites the photosensitiser protoporphyrin IX (PPIX) within the bacterial membrane, which then excites molecular oxygen to the excited singlet state. Both singlet oxygen and excited triplet state PPIX can cause large amounts of cellular damage. Carotenoids prevent this damage by absorbing the excess energy from these excited species and dissipating it harmlessly as heat. The presence of singlet oxygen within the bacterial membrane causes the inactivation/degradation of CarR. Removal of CarR releases CarQ from the membrane enabling it to mediate transcription from various promoters. CarQ causes transcription of the crtI gene and of the carQRS operon which produces further CarQ and CarS. CarS causes de-repression of the crtEBDC cluster. The carotenogenic enzymes encoded by crtI and the crtEBDC cluster catalyse the production of carotenoids which quench the initial signalling molecules, singlet oxygen and triplet PPIX. This causes down-regulation of the regulon as a whole as CarR is no longer degraded and once again carries nascent CarQ to the membrane in an inactive state. The negative feedback loop described above is an important consideration when assessing mutants which produce carotenoids either constitutively (Car c phenotype), or under no conditions (Car- phenotype). This work investigates the consequences of Carc and Car- mutations on the activity of promoters within the Car regulon in order to clarify the roles of various genetic loci. It is demonstrated that CarA has no regulatory role in expression of crtI or carQRS and that the expression of crtI has no regulatory consequences. Sequencing downstream of crtI revealed a novel gene gufB (gene of unknown function B) which has homologues of no known function. The critical event in the activation of the carotenogenic system is expression of the carQRS operon allowed by the release of CarQ from its complex with CarR at the membrane. Attempts were made to extract information about the interaction of CarQ with its cognate promoter at carQRS through a variety of in vivo and in vitro molecular and genetic techniques. Site-directed mutations within pcarQRS were assessed in vivo through the use of lacZ transcriptional fusions, enabling identification of important regions within the carQRS promoter. In vitro experiments provided information about the possibility of using molecular methods to assess interactions between CarQ and the pcarQRS promoter.

An investigation of amino acid metabolism in Streptomyces coelicolor, including the anabolism of tryptophan, histidine, the branched-chain amino acids and proline, as well as the catabolism of the latter, is reported. The experiments reported herein were conceptually conceived within a functional genomics framework. For this purpose the complete genome sequence of S. coelicolor was systematically exploited. Moreover, the current knowledge on the physiology of Streptomyces was taken onboard, as well as the prevailing and emerging notions on the evolution of proteins and metabolic pathways. Some of the results obtained using S. coelicolor as a model organism were expanded to other actinomycetes, such as Mycobacterium tuberculosis. This was aided by a comparative genomics analysis of the actinomycetes whose genomes have been sequenced. The theoretical principles that give support to this thesis are introduced in Chapter 1. This study was greatly facilitated by the development of a novel PCRtargeting mutagenesis method of which details can be found in Chapter VII. The discovery of a common isomerase for tryptophan and histidine biosynthesis is reported in Chapter II. This discovery arose from efforts aimed at reconstructing the tryptophan biosynthetic pathway of S. coelicolor, since the genome sequence project of this organism failed to identifiy a trpF gene coding for the enzyme phosphoribosyl anthranilate isomerase. The solution of this functional genomics discrepancy led to the discovery of a putative (~a)8-barrel enzyme, termed PriA, whose preliminary functional and structural characterisation is reported in Chapter III. The evolutionary implications of the discovery of PriA are discussed within Chapters III and N. A comparative genomics analysis of actinomycetes centred on the priA gene is presented in the latter Chapter, supporting the notion that this novel protein is spread across the high (0 + C) content Gram-positive organisms. Indeed, it was predicted that a priA orthologue accounts for the lack of a trpF gene from the genome of M tuberculosis, a hypothesis that proved to be correct. Finally, evidence to support the notion that the histidine and tryptophan biosynthetic pathways co-evolved is presented. In contrast to the isomerisation catalysed by PriA, in which an enzyme is shared by two amino acid biosynthetic pathways, several paralogous enzymes with the potential to account for the first step of tryptophan biosynthesis from chorismate were found on the genome of S. coelicolor. These chorismate-utilising enzymes are investigated in Chapter V. Mutational analysis of some of this paralogues is reported and it is anticipated that the analysis and results reported therein will serve to direct future experiments aimed at identifying the trpE paralogue encoding the enzyme anthranilate synthase. Chapter VI reports on the identification of the proC gene involved in the last step of proline biosynthesis in S. coelicolor. The pyrroline-5-carboxylate reductase activity of the enzyme encoded by the putative proC gene was extensively characterised, with particular emphasis on the interaction between primary and secondary metabolism. Furthermore, mutational analysis of proC suggested that paralogues of this gene are present on the genome of this organism, since its deletion did not lead to an auxotrophic phenotype. Investigation of this observation showed that two paralogous enzymes encoded by i1vC-like genes, involved in biosynthesis of the branched-chain amino acids, are capable of compensating for the lack of proC. This is the first example of a physiological link between the biosynthesis of proline and the branched-chain amino acids. To sum up, the results reported in this thesis represent an advancement towards understanding the physiology of S. coelicolor as a model actinomycete, within a functional and structural genomics framework. They also offer evidence on the evolutionary principles that lead to the appearance of novel proteins and metabolic pathways in bacteria.

Molecular detection methods were developed to study the distribution of type II polyketide synthase (PKS) genes in Cuban soils. A PCR based detection method targeting the α and β ketosynthase genes was applied to a number of different total community DNA samples. These genes were detected in 43% of samples tested from a number of different locations. A botanical garden site located in Havana, Cuba, was found to show the greatest distribution of type II PKS genes across the sites tested. It was not possible to amplify type II PKS genes from a pristine island site off the coast of Cuba. Further investigation revealed that actinornycetes containing type II PKS were present in the soil community at a level above the detection limit of the PCR protocol. Further total community DNA cleanup steps failed to allow the detection of type II PKS genes within the DNA samples suggesting PCR inhibition was responsible for negative results. The molecular detection of type II PKS genes in total community DNA was compared to the detection of type II PKS genes in actinomycete isolates. A lack of correlation between these two approaches was observed with the molecular detection limit unable to amplify type II PKS genes in <50% of crop soils tested. Actinomycetes containing type II PKS genes could be isolated from all crop soils tested. No difference was seen in the detection of type II PKS genes between rhizosphere and bulk soil samples. Actinomycetes were isolated using a selective isolation procedure at a level of approximately 10(7) cfu g-1 soil compared to 10(8) cfu g-1 for total bacterial counts. Actinomycetes were isolated from Cuban crop soils and screened for the presence of type II PKS genes. Out of 100 isolates 26 were found to contain the genes of interest. Phylogenetic analysis of these isolates based on 16S rDNA and recA sequence data showed them to be closely grouped within the streptomycetes. Sequence data based on KSα genes from Cuban isolates showed them to be representative of both spore pigment and antibiotic polyketide genes. A representative clone library was constructed containing type II PKS genes amplified from total community DNA. Rhizosphere and bulk soil samples were compared from the same site. Sequences obtained from rhizosphere total community DNA appeared to be widely distributed when compared to published sequences and included examples of both spore pigment and antibiotic polyketide genes. A molecular method was developed to amplify near full length α and β KS genes from type II PKS gene clusters. Expression vectors were constructed to allow these genes to be expressed along with an ACP to give a functional minimal PKS for polyketide chain production. This method was used on total community DNA in an attempt to extract diverse genes from as yet uncultured organisms.

Troponin C is a regulatory protein of the myofilament which binds to calcium to trigger the process of contraction. This protein exists in two isoforms, skeletal and cardiac, which are spatially and temporally regulated. Work in this project builds the primary stage of a long-term project, for using the gene transfer method to overexpress the skeletal isoform of Troponin C in cardiomyocytes. The long-term aim is to achieve complete or partial substitution of the native cardiac isoform and study the effects on contractile force produced, in normal and ischemic cardiomyocytes, both in vitro and in vivo. This project has involved designing, constructing and analyzing expression of adenoviral gene transfer vectors overexpressing the sTnC isoform. Several adenoviral vectors were generated with the wild type sTnC gene under the control of muscle-specific promoters. To facilitate analysis of protein expression and its subcellular localization, the sTnC protein was tagged with epitope tags and adenovirus generated, with this gene under the control of constitutive (CMV) and cardiac-specific (HCA) promoters. Epitope-tagged adenoviruses were expressed in vitro using mouse fibroblast (NIH3T3) cells and analyzed by western blot analysis, showing successful constitutive expression. Recombinant adenoviruses containing epitope-tagged-sTnC under the control of the human cardiac actin promoter showed cardiac-specific expression in cultured cardiomyocytes, in situ, using immunocytochemistry. The constitutively-expressing sTnC adenoviral vector showed successful expression in cardiomyocytes in culture, using northern blot analysis. A range of adenoviral vectors have been successfully generated, and constitutive and tissue-specific expression has been established for some of these vectors. Successes attained in this project have established the initial requirements to achieve the long-term goal to alter calcium sensitivity of myofilaments, by overexpression of sTnC isoform in cardiomyocytes, both in vitro and in vivo.

Although the starvation or non-growth state is probably the most common physiological state of bacteria, it has been studied in relatively few organisms. In spite of its importance in pathogenesis, bioremediation and several industrial processes, limited research has been performed on Rhodococcus under starvation/stationary phase conditions. The objectives of this study were to analyse the physiological adaptation of Rhodococcus erythropolis SQ1 to starvation/stationary phase, and to generate and screen a bank of mutants to identify genetic elements involved in this adaptation. It was found that R. erythropolis SQ1 can survive for at least 43 days in LB and distilled water, and 65 days in chemically defined medium (CDM) containing high (1 % w/v) or low (0.1 % w/v) glucose concentrations. Early stationary phase R. erythropolis SQ1 cells grown in 0.1 % glucose also exhibited enhanced resistance to heat and oxidative stress compared with exponential phase cells. A mutant bank of 898 R. erythropolis SQ1 mutants was generated and screened; four mutants were of particular interest. The culturability of mutants 4G6 and 10D3 dropped to <0.1 % of the maximum CFU/ml at 27 days incubation, and to <3 % of the maximum CFU/ml for mutants 1B2 and 1H1, when grown in 1 % glucose medium. No drop in culturability was observed when mutants were grown in 0.1 % glucose. Mutant 4G6 had a transposon insertion in uvrB (UvrB, part of the DNA excision repair mechanism), while the insertion for mutant 10D3 was immediately downstream of a putative guaB gene, which, based on bioinformatic analyses, is followed by another putative IMP dehydrogenase (guaB-like) and/or a cholesterol oxidase gene. In mutant 1H1 the transposon inserted 272 nucleotides downstream of a gene encoding a putative phosphoglycerate mutase and upstream of putative thioredoxin and cytochrome c biogenesis genes. In conclusion, R. erythropolis SQ1 was shown to present a classic starvation/stationary phase survival response, with the associated increase in resistance to various external stresses. A mutant bank has been generated which can be used in the future to analyse other phenotypes of interest. Several genes linked to starvation/stationary phase survival were identified. These findings show that a wide variety of genes are involved in starvation/ stationary phase survival. Indeed, over 100 such genes have been identified in Escherichia coli and Mycobacterium tuberculosis.

Colorectal cancer (CRC) is the fourth most common cancer in the UK. Despite intensive research that identified the key driver mutations, the precise consequences of each mutation and how they modify therapeutic response is unclear. More recently, it has been shown that the serrated subgroup of CRC, which is driven by oncogenic KRAS or BRAF mutations, is associated with the poorest survival. Germline mutations in either Phosphatase and tensin homologue (PTEN) and Liver kinase B1 (LKB1) cause intestinal hamartomas that can progress to CRC. However, there is an ongoing debate whether tumourigenesis arises from the epithelial or the mesenchymal compartment of the gut and the contribution of these mutations to sporadic CRC. The aim of this thesis was to: • Address the role of Pten and Lkb1 in the murine intestinal epithelium • Determine if these mutations cooperate with other driver mutations such as Apc and KRas. • Understand the mechanistic basis that drives changes in homeostasis and tumourigenesis. • Use recently developed small molecule inhibitors that target these aforementioned candidate pathways. Neither Pten nor Lkb1 deficiency was sufficient to drive neoplasia in the murine intestinal epithelium. Loss of Pten in the murine epithelium does not alter intestinal homeostasis and appears to be redundant. Lkb1 deficiency causes an expansion of the goblet cells linage and activation of the Hippo pathway but only when either KRas or Apc mutations are present does this result in accelerated tumourigenesis. Pten and KRas cause MAPK and PI3K pathway hyperactivation that results in hyperproliferation and serration of the murine gut. These precursor lesions are sensitive to MEK, PI3K/mTOR and surface Wnt inhibition. KRas driven tumours from either Lkb1 or Pten deficient mice acquire a Wnt pathway hyperactivation that drives invasion and metastasis in mice and leads to resistance of PI3K/mTOR and surface WNT inhibition. Taken together my data has shown that KRAS mutation can initiate tumours via a serrated route which on the further deregulation of Wnt signalling convert to tumours resembling classical CRC. Importantly these tumours are now Wnt ligand independent and appear treatment resistant, analogous to the human tumours that are adenocarcinoma that bear a serrated signature. Importantly loss of either LKB1 or PTEN accelerated tumourigenesis down either the serrated or the classical route and suggests key roles for these proteins in sporadic colorectal carcinogenesis. Given the drug resistance of our models, they could be utilized as excellent therapeutic testing models that may more closely recapitulate the human disease.

Vector-borne disease emergence in recent decades has been associated with different environmental drivers including changes in habitat, hosts and climate. Lyme borreliosis is among the most important vector-borne diseases in the Northern hemisphere and is an emerging disease in Scotland. Transmitted by Ixodid tick vectors between large numbers of wild vertebrate host species, Lyme borreliosis is caused by bacteria from the Borrelia burgdorferi sensu lato species group. Ecological studies can inform how environmental factors such as host abundance and community composition, habitat and landscape heterogeneity contribute to spatial and temporal variation in risk from B. burgdorferi s.l. In this thesis a range of approaches were used to investigate the effects of vertebrate host communities and individual host species as drivers of B. burgdorferi s.l. dynamics and its tick vector Ixodes ricinus. Host species differ in reservoir competence for B. burgdorferi s.l. and as hosts for ticks. Deer are incompetent transmission hosts for B. burgdorferi s.l. but are significant hosts of all life-stages of I. ricinus. Rodents and birds are important transmission hosts of B. burgdorferi s.l. and common hosts of immature life-stages of I. ricinus. In this thesis, surveys of woodland sites revealed variable effects of deer density on B. burgdorferi prevalence, from no effect (Chapter 2) to a possible ‘dilution’ effect resulting in lower prevalence at higher deer densities (Chapter 3). An invasive species in Scotland, the grey squirrel (Sciurus carolinensis), was found to host diverse genotypes of B. burgdorferi s.l. and may act as a spill-over host for strains maintained by native host species (Chapter 4). Habitat fragmentation may alter the dynamics of B. burgdorferi s.l. via effects on the host community and host movements. In this thesis, there was lack of persistence of the rodent associated genospecies of B. burgdorferi s.l. within a naturally fragmented landscape (Chapter 3). Rodent host biology, particularly population cycles and dispersal ability are likely to affect pathogen persistence and recolonization in fragmented habitats. Heterogeneity in disease dynamics can occur spatially and temporally due to differences in the host community, habitat and climatic factors. Higher numbers of I. ricinus nymphs, and a higher probability of detecting a nymph infected with B. burgdorferi s.l., were found in areas with warmer climates estimated by growing degree days (Chapter 2). The ground vegetation type associated with the highest number of I. ricinus nymphs varied between studies in this thesis (Chapter 2 & 3) and does not appear to be a reliable predictor across large areas. B. burgdorferi s.l. prevalence and genospecies composition was highly variable for the same sites sampled in subsequent years (Chapter 2). This suggests that dynamic variables such as reservoir host densities and deer should be measured as well as more static habitat and climatic factors to understand the drivers of B. burgdorferi s.l. infection in ticks. Heterogeneity in parasite loads amongst hosts is a common finding which has implications for disease ecology and management. Using a 17-year data set for tick infestations in a wild bird community in Scotland, different effects of age and sex on tick burdens were found among four species of passerine bird (Chapter 5). There were also different rates of decline in tick burdens among bird species in response to a long term decrease in questing tick pressure over the study. Species specific patterns may be driven by differences in behaviour and immunity and highlight the importance of comparative approaches. Combining whole genome sequencing (WGS) and population genetics approaches offers a novel approach to identify ecological drivers of pathogen populations. An initial analysis of WGS from B. burgdorferi s.s. isolates sampled 16 years apart suggests that there is a signal of measurable evolution (Chapter 6). This suggests demographic analyses may be applied to understand ecological and evolutionary processes of these bacteria. This work shows how host communities, habitat and climatic factors can affect the local transmission dynamics of B. burgdorferi s.l. and the potential risk of infection to humans. Spatial and temporal heterogeneity in pathogen dynamics poses challenges for the prediction of risk. New tools such as WGS of the pathogen (Chapter 6) and blood meal analysis techniques will add power to future studies on the ecology and evolution of B. burgdorferi s.l.

Nucleosomes comprise the most basic repeating unit of chromatin and provide hubs for the regulation of DNA transcription, replication and repair. ATPase chromatin remodelling complexes establish nucleosome occupancy, positioning and structure in a dynamic fashion to allow fine-tuning of protein-DNA interactions. The ISWI and CHD families of remodelers possess the ability to sample DNA linker length between nucleosomes and space nucleosomes evenly. How these spacing remodelers combine their functions to maintain phasing of nucleosomal arrays, and re-organise these arrays during development remains poorly understood. Furthermore the relationship between nucleosomal array structure and transcriptional regulation is unclear. Dictyostelium discoideum provides a complex chromatin environment and remodeler repertoire, while retaining a compact genome and ease of genetic manipulation. Thus we have utilized this model to generate remodeler null mutants, and double mutants to observe phenotypic effects and interactions. We further compiled comprehensive nucleosome mapping and RNA sequencing data sets for all spacing remodelers in Dictyostelium. Bioinformatic analysis of these data provide novel insights into remodeler functions, and help to establish a paradigm to explain the relationship between remodeler-mediated chromatin organisation and transcriptional regulation.

Primordial germ cells (PGC) are the precursors of the gametes. The mechanisms of PGC induction, specification and development are very well characterized in rodents, however recent investigations have demonstrated that the mechanisms of germ cell development differ significantly between mice and humans. Since the knowledge of PGC development in non-rodents is very limited, and early human embryos cannot be accessed it is important to establish a new model for PGC development with relevance to humans. In this thesis, I use pig embryo as a model for investigating PGC development in non-rodent mammals. The expression profile of key transcription factors, epigenetic reprograming and the role of signalling pathways were investigated during specification and development of pig PGCs. The key findings are: A- Specification of porcine PGC occurs after the onset of gastrulation, requiring BMP4 signalling. B- WNT signalling is required for the generation of precursors competent for germline commitment; however it is downregulated after PGCs are specified. WNT downregulation could be modulated by SOX17, the earliest gene expressed in pig PGCs. C- Epigenetic reprogramming of DNA and histone marks starts in pre-migratory porcine PGC. Furthermore, chromatin dynamics in pig gonadal PGCs resemble that of humans but differs to that of mice. D- The expression profile of transcription factors of porcine PGC is similar to that of humans, but different to mouse PGC. In conclusion, this study has highlighted critical differences between mice and humans/pigs during germ cell specification. I provide evidence that the pig embryo is a useful model for the study of human development, and future studies will need to be directed to re-evaluate concepts of cell differentiation and early lineage commitment established in mice that may not apply to humans.