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.

The work presented in this thesis involves the characterization of a small plasmid, pHD2, of approximately 2 kb in size from Bacillus thuringiansis subspecies kurstaki HDl-Dipel. The plasmid was cloned and sequenced and compared to other plasmids from Gram-positive bacteria for which sequence information was available and for which replication functions had been assigned. Homology between the predicted amino acid sequence of an open reading frame within pHD2 and the rep gene products of the pT181 group of staphylococcal plasmids suggested a common method of plasmid replication. The further identification of possible plus and minus origins indicated that pHD2 was a member of a family of plasmids replicating via a single-stranded DNA intermediate. Plasmid replication control in the staphylococcal plasmids pT181, pC221 and pS194 involves a negative control circuit using countertranscripts. Replication control in the case of pHD2 may utilize an alternative system involving the gene product of a second small open reading frame with homology to RepA of pLSl in which replication control is achieved by the binding of the repA gene product to the promoter region of the replication protein repB. pHD2 is the first plasmid from a Bacillus thuringiensis isolate to which replication functions have been assigned. The work presented here suggests pHD2 to be a member of the ssDNA family of plasmids and extends the range of such plasmids which have been characterized with the suggestion that this group contains, in addition to the highly related staphylococcal plasmids, a number of more distantly related members. Additionally, chimeric plasmids containing pHD2 and pBR322 have been demonstrated to show structural instability, although not segregational instability, in an alternative Bacillus thuringiensis host. Consequently, the use of such constructs in the cloning of heterologous genes in Bacillus thuringiensis in such a system may prove impractical at this stage with further work being required in order to overcome these problems and extend the exploitation of this industrially important family of entomopathogenic bacteria.

The control of cellular morphogenesis and differentiation at the molecular level in Rhodomicrobium vannlelii is investigated by biochemical and genetic analysis. Chemical characterization indicates that the R.vannlelll genome is 2.19 x 109 daltons, and is made up of 95 per cent unique DNA sequence. Five per cent of the DNA consists of short inverted repeat sequences, 400 bp in length. No extrachromoscmal DNA is detectable. The DNA from each of the cellular expressions in R.vannielil does not show any major differences in sequence composition. Kinetic analysis of nucleic acid synthesis during the obligate differentiation of the swarm cell shows a 'lag' or maturation period prior to the onset of DNA replication, whereas no lag occurs in RNA synthesis. The initiation of DNA replication during swarm cell differentiation occurs towards the completion of stalk synthesis. Studies with the protein synthesis inhibitor chloramphenicol during swarm cell differentiation demonstrate a requirement for protein synthesis in the initiation, but not the elongation step of DNA replication. Nalidixic acid inhibits DNA synthesis in the swarm cell, and although a new daughter cell is produced, cell division does not occur. This implies that chromosome replication and cell division cure directly linked in a 'dependent pathway' of events. Daughter cell synthesis is under the control of the mother cell genome, the daughter cell genome becoming metabolically active just prior to cell division. Further 'cells' produced in nalidixic acid-treated cultures show gross cellular distortion and no stalk formation. The development of a genetic system for R.vannielll is discussed. The promiscuous plasmid R.68.45 is transferred by conjugation from E.coll to R.vannlelll, where it expresses only one of three plasmid-bome antibiotic resistances, but may be maintained intact in the bacterium. This plasmid may now be used in mapping the R.vannielll chromosome. Analysis of the R.vannlelli genome by restriction enzyme cleavage is described. Restriction fragments containing the coding sequences for 16s rRNA and 23s rRNA are Identified by 'Southern' hybridization, and their sizes estimated. Attempts to locate the origin of replication on a single restriction fragment are discussed. Preliminary data on the physiology of nitrogen fixation in R.vannlelli shows that the enzyme system is inducible. The potential of this organism as a model system for further study on the molecular biology of microbial morphogenesis and differentiation is discussed.

This study set out to sequence the genome of Corynebacterium pseudotuberculosis (Cp) 3/99-5, an ovine strain isolated from a naturally-occurring case of caseous lymphadenitis (CLA) in Scotland. The isolate was sequenced and assembled by 454 Life Sciences, and then gap closure performed by ‘PCR bridging’. The resulting sequence consisted of three contigs with a length of 2,319,079 bp and a G+C content of 52.18%. The genome was then annotated and predicted to contain 2,153 coding sequences. Analysis of the coding sequences revealed the presence of several putative virulence factors, including four sortases with multiple sortase target proteins containing LPXTG motifs. A further two Cp strains, an Australian ovine and a North American equine isolate, as well as C. ulcerans NCTC 12077 were sequenced for comparison. Comparative genomics, both intra- and inter-species showed all the genomes to be highly homologous. However, the C. ulcerans genome is larger than the Cp genomes and is more distinct; it was found to be more similar to the equine Cp 1/06-A isolate which is the most diverged of the Cp isolates. Phylogenetic analyses of the Corynebacterium genus were performed using house-keeping loci but also secreted protein loci from Cp 3/99-5. Bayesian analysis of house-keeping loci distinguished the bacteria to a species level. Inclusion of secreted protein loci did not distinguish the isolates any further. The main objective of this work was to utilise the Cp genome sequence to identify potential diagnostic targets which could be used to augment the available ELITEST CLA or replace it. The ELITEST CLA is the only diagnostic test for CLA that exists on the commercial market in the UK. However, due to low specificity and sensitivity, it is only operated on a flock/group basis. Analyses of the Cp 3/99-5 genome identified several potential diagnostic candidates and seven protein targets were investigated further. Attempts were made to express these candidates as recombinant proteins, however, only two recombinants were successfully expressed and purified, Cp3995_0570 and CP40. The seroreactivity of these were then assessed by IgG ELISA using a panel of ten positive and ten negative CLA ovine sera. The sera were previously defined as positive or negative by PLD and whole cell ELISAs; both of which showed a significant difference between sera types. However, neither Cp3995_0570 nor CP40 distinguished between sera originating from Cp-infected and Cp-naïve animals.

Background: Host-associated microbial communities play a significant role in a species’ environmental interactions, often performing functions unachievable by the eukaryotic host, and is essential in developing a comprehensive understanding of the species and its impact on the local and global ecosystem. Earthworms (Lumbricina) habituate almost every type of soil environment globally, including sites of severe environmental stress and is an essential ecosystem engineer, central to healthy natural and agricultural soils. To date, only a singular symbiotic species (Verminephrobacter sp.) has been identified, but the earthworm impact on transient microbial communities and the surrounding soil microbiome is profound. Methods: Previous culture and molecular based studies found earthworm-associated microbiota unlikely however, this has not been explored using High Throughput Sequencing. Utilisation of Illumina, 454 and Ion Torrent sequencing has enabled production of the highest resolution microbial analysis of host-associated bacteria of any single eukaryotic species to date, including spatial bacterial localisation of the entire Lumbricus rubellus organism and impact analysis of a wide range of anthropogenic contaminants and environmental stressors on the basal microbiomic community. Results: A core bacterial community has been described which is distinct from the surrounding soil. A number of novel species have been associated with the earthworm crop, body wall and hindgut, contravening claims that the earthworm has limited or no impact on ingested soil bacteria. This demonstrate that the host properties impart significant effects on the transient population, demanding further analysis to determine potential symbiotic functionality. However, while a biologically important community has been described, the significant impact of anthropogenic contamination on the host microbiome must be considered given the observed eradication of the Verminephrobacter symbiont during the host’s exposure to arsenic and the potential subsequent implications on host health.

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.

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.

Leishmania spp. are protozoan parasites that infect humans and other vertebrates to cause a spectrum of disease, ranging from cutaneous ulceration to visceral dissemination dependent on the species. Leishmaniasis is prevalent across the developing world and is a major global health issue, yet difficulties in the efficacy and administration route of current anti-leishmanial treatments means the existing drug repertoire is inadequate. To address this, further research and development measures are necessary to identify Leishmania proteins representing useful targets for drug inhibition. Essential genes encode proteins that are necessary for parasite survival and therefore represent suitable drug targets, but the study of such genes is limited by the absence of a conditional deletion system. A family of proteins which has previously been shown to regulate crucial aspects of Leishmania biology are the protein kinases. Protein kinases have been validated in mammalian systems as drug targets in cancer therapy, therefore they represent a promising avenue for research into anti-leishmanial drugs. The cdc-related kinases CRK3 has been studied in particular depth in Leishmania, and current reverse genetic techniques have implicated expression of CRK3 as essential to promastigote survival. CRK3 regulates the cell cycle as demonstrated by treatment of cdc2 inhibitors, but a lack of a system to regulate expression prevents more specific phenotypic dissection of the role of CRK3. In addition the validation of CRK3 as a drug target has been limited by an absence of a conditional genetic system to ablate the gene in mammalian infective amastigotes. To regulate CRK3 expression in a conditional manner to assess its function in the cell cycle of promastigotes and validate it as essential for amastigotes, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) for use in L. mexicana. Cre recombinase mediates the excision of DNA sequences flanked by 34bp loxP sites (‘floxed’). diCre is encoded as two separate subunits each linked to rapamycin binding domains (FRB and FKBP12); therefore recombinase activity is induced by rapamycin treatment which causes dimerisation of the subunits. Our method involves replacing both CRK3 alleles with a ‘floxed’ CRK3 open reading frame and the diCre coding sequence through promastigote transfection and homologous recombination. Induction of diCre through rapamycin treatment of promastigotes results in highly efficient deletion of CRK3 and a distinct growth arrest phenotype corresponding to a block in G2/M. Induced loss of CRK3 can be complemented by expression of a CRK3 transgene but not by expression of an inactive site (T178E) CRK3 mutant, showing that protein kinase activity is crucial for CRK3 function. Significantly, inducible deletion of CRK3 in stationary phase promastigotes prevents the establishment of murine infection, thereby demonstrating an essential role in the amastigote cell cycle to further validate CRK3 as a drug target. Promisingly, inducible deletion is functional in lesion-derived amastigotes and will enable direct phenotypic assessment following essential gene loss in this life cycle stage. To establish a basis for future in vivo application of diCre in Leishmania, a murine infection model was developed with which to track bioluminescent parasite burden by in vivo imaging and assess innate immune cell recruitment to the site of infection by flow cytometry analysis. The combination of functional gene regulation in amastigotes and measures of parasite burden and immune response will yield a powerful tool for the further study of Leishmania genes encoding suitable drug targets. The application of the diCre technique to Leishmania would be greatly benefitted by targeting genes where there is evidence of a regulatory role of orthologous genes in model organisms. The utilisation of genome or protein family-wide RNAi screens in Trypanosoma brucei has identified a number of protein kinases which regulate the differentiation of the parasite between life cycle stages. The repressor of differentiation (RDK1) protein regulates bloodstream form to procyclic form differentiation in T. brucei, and the identification of a protein in L. mexicana with high sequence identity suggested a potentially analogous role in preventing Leishmania from undergoing amastigote to promastigote differentiation in vivo. To assess this, a cell line was generated deficient in RDK1 but no effect on differentiation was identified, as parasites were able to maintain murine infection and differentiate between life cycle stages. This study represents an important addition to the reverse genetic toolkit to study aspects of cell cycle regulation in vitro, and further assess essential genes as drug targets by deletion in amastigotes. The application of the diCre conditional deletion method will enhance the discovery and evaluation of suitable drug targets in Leishmania by phenotypic analysis.