Epstein Barr Virus driven post transplant lymphoproliferative disease (PTLD) is a serious complication following either stem cell or solid organ transplantation (SOT), occurring as a result of suppressed cellular immunity. Adoptive transfer of EBV specific cytotoxic T cells (EBV-CTLs) is effective as both prophylaxis and treatment for PTLD following stem cell transplantation, but is less effective when applied to PTLD after SOT. It is likely that the continued pharmacologic immunosuppression designed to prevent graft rejection compromises the function of infused EBV-CTLs. To address this issue, we have generated calcineurin (CN) mutants that do not bind the immunosuppressants Tacrolimus (FK506) and Cyclosporin A (CsA), therefore conferring resistance to these CN inhibitors. A panel of 54 such mutants was designed, generated and screened in a Luciferase expression assay, with identification of those capable of resisting suppression with FK506, CsA or both. Subsequently, in assays utilising both the Jurkat T cell line and primary human EBV-CTL lines, mutant CNa12 conferred resistance to CsA, mutant CNa22 conferred resistance to FK506 and mutant CNb30 rendered cells resistant to both calcineurin inhibitors. EBV-CTL lines transduced with a retroviral vector encoding these mutants retained the ability to both proliferate and secrete normal levels of interferon-\gamma in the presence of therapeutic and supratherapeutic levels of FK506 (CNa12), CsA (CNa22), or both (CNb30). The cytotoxicity, phenotype and antigen dependence of EBV-CTL lines were unaffected by expression of mutant CN. A xenogenic murine model of PTLD was established in the RAG2^{-/-}/\gamma c^{-/-}/C5^{-/-} triple knockout SCID mouse strain and the ability of EBV-CTL lines to induce tumour regression was examined. Administration of EBV-CTLs caused regression of subcutaneous LCL tumours in vivo in some donors. This model will be utilised in further investigations of transduced EBV-CTLs in vivo. The generation of EBV-CTLs that are resistant to CN inhibitors should allow effective immunotherapy in the SOT setting without risking rejection of the graft by reducing immunosuppression. Additionally this represents a generic approach to improving immunotherapy in the face of immunosuppression in other settings.

Mycobacterium tuberculosis, the causative agent of tuberculosis exists in a number of different environmental states. It must therefore have gene regulatory systems which are specific for virulence. One major signalling method is through reversible phosphorylation of proteins, mediated by protein kinases and phosphatases. This study focuses on the function of one serine-threonine protein kinase, PknF, and its substrate, the ABC transporter Rv1747 which is necessary for growth in a virulent infection. This kinase is known to interact with both of the fork-head associated (FHA) domains of Rv1747 in a phosphorylation dependent manner. The aims of this study were to analyse the function of Rv1747 particularly in relation to its requirement for a virulent infection and to investigate how PknF is controlling Rv1747 function. pknF was shown to be co-transcribed with Rv1747 and the stimulus sensed by the kinase was investigated. Phenotypic analysis linked the function of Rv1747 to properties of the cell wall. Transcriptional microarray analysis of pknF and Rv1747 mutants showed altered expression levels of genes involved in cell wall functions. Moreover, thin layer chromatography revealed changes in lipid profiles between wild type and mutant, but these differences could not be confirmed to be due to the mutation since they were not restored by complementation. Cell wall structure, however, appeared normal by transmission electron microscopy. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues; mutation analysis indicated that these are likely to be the only residues phosphorylated. The involvement of the FHA domains in this regulation was demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. Furthermore, FHA-1 domain mutation resulted in attenuation in macrophages highlighting its critical role in Rv1747 function. Infection experiments in macrophages and in mice have been performed using the threonine mutants to determine the in vivo consequences of phosphorylation and hence construct a model of how this regulation takes place. This study has revealed that PknF positively regulates the function of Rv1747 which is required for growth in both the lungs and spleens of mice.

In this thesis, I give a metascientific account of causality in medicine. I begin with two historical cases of causal discovery. These are the discovery of the causation of Burkitt's lymphoma by the Epstein-Barr virus, and of the various viral causes suggested for cervical cancer. These historical cases then support a philosophical discussion of causality in medicine. This begins with an introduction to the Russo-Williamson thesis (RWT), and discussion of a range of counter-arguments against it. Despite these, I argue that the RWT is historically workable, given a small number of modifications. I then expand Russo and Williamson's account. I first develop their suggestion that causal relationships in medicine require some kind of evidence of mechanism. I begin with a number of accounts of mechanisms and produce a range of consensus features of them. I then develop this consensus position by reference to the two historical case studies with an eye to their operational competence. In particular, I suggest that it is mechanistic models and their representations which we are concerned with in medicine, rather than the mechanism as it exists in the world. I then employ these mechanistic models to give an account of the sorts of evidence used in formulating and evaluating causal claims. Again, I use the two human viral oncogenesis cases to give this account. I characterise and distinguish evidence of mechanism from evidence of difference-making, and relate this to mechanistic models. I then suggest the relationship between types of evidence presents us with a means of tackling the reference-class problem. This sets the scene for the final chapter. Here, I suggest the manner in which these two different classes of evidence become integrated is also reflected in the way that developing research programmes change as their associated causal claims develop.

Mycobacterium tuberculosis is the causative agent of Tuberculosis (TB). Two billion people are currently infected with M. tuberculosis bacilli, one in ten of whom will develop active TB in their lifetime. M. tuberculosis is able to survive within macrophages but the exact mechanisms used for intracellular survival are poorly understood. DNA-dependent RNA polymerase is the enzyme responsible for transcription in all living organisms. In bacteria this enzyme recognises different promoters by binding to sigma factors that recognise those promoters. This study focuses on the role and regulation of the M. tuberculosis extracytoplasmic function (ECF) sigma factor, SigG. ECF sigma factors are responsible for upregulating genes necessary for bacterial stress responses. SigG has previously been shown to be upregulated in response to DNA damage and during macrophage infection. It has been demonstrated that sigG is expressed from at least 2 promoters and that only promoter P1 is DNA-damage inducible. sigG is co-transcribed with the two downstream genes Rv0181c and Rv0180c, which were hypothesised to be anti- and anti-anti-sigma factors to SigG. Protein-protein interaction studies showed that SigG and Rv0181c do not interact. Potential anti-sigma factors to SigG were identified, the most promising of which was the thioredoxin family protein Rv1084. Two potential SigG-dependent genes had previously been identified, Rv0887c and Rv0911. It has been demonstrated that SigG is able to bind to the promoter regions of these genes but this interaction was not specific. An M. tuberculosis sigG-Rv0180c deletion strain was constructed and complemented with the whole operon as well as with sigG alone. The phenotype of the mutant strain was examined in vitro as well as in vivo to test the hypothesis that SigG has a role during infection. Use of a phenotype microarray revealed an enhanced susceptibility of the mutant strain to oleic acid and its ester, Tween 80, leading to investigation of the sensitivity of the strains to a range of fatty acids.

Genome-wide association studies (GWAS) have identified germline single nucleotide polymorphisms (SNPs) that are associated with colorectal cancer (CRC) susceptibility. This thesis applies the same approach to the identification of germline determinants of prognosis in CRC, attempts to verify potential susceptibility loci, and examines the relationship between SNPs and some forms of non-SNP based germline variation. The GWAS for prognosis used 931 patients enrolled in the VICTOR trial in the discovery phase, screening 309,200 autosomal SNPs for an association with disease-free survival (DFS). Following the application of selection filters based on statistical significance levels and performance of the genotyping, 40 SNPs were identified to be examined in further cohorts. The verification phase consisted of 1338 patients in the PETACC 3 trial and three population based cohorts: 899 patients from Scotland, 599 patients from Denmark, and 962 patients from Finland. The SNPs that came closest to genome-wide significance in stage 2 and 3 CRC was rs7556894, 15kb from Actin-related protein 2 (ARP2) on chromosome 2, part of the ARP2/3 complex essential for cell shape and motility, with p=8.96e-07. The impact on prognosis of rs7556894 was estimated as HR=1.52 (95% CI 1.17-1.96). Because of the failure to reach genome-wide significance (p<1e-07), two further approaches to the discovery phase are presented: the meta-analysis of two discovery cohorts to increase event rate and subject numbers and a GWAS for predictive markers for the benefit of adjuvant 5-FU chemotherapy. Formal verification of either approach was not undertaken as part of this thesis. Further loci were subjected to specific analyses of association with prognosis or CRC susceptibility: rs6983267 and the previously identified CRC susceptibility loci to a survival analysis, and not found to be associated; rs6687758, previously identified as a potential CRC risk locus to a susceptibility verification, confirming a significant association with HR=1.15, 95% CI 1.10-1.21, p=5.04e-08; and a variety of hypothesis driven potential risk loci to a screen for an association with CRC susceptibility, none was found but the LD relationship between tagSNPs and insertion/deletion polymorphisms appears to be the same as for ‘normal’ SNPs. Overall, the data presented in this thesis quantify further the contribution of germline variation to CRC susceptibility, exclude a major effect of such variation on prognosis, and verify rs6687758 as a further low-penetrance CRC susceptibility locus.

No description available.

Colorectal cancer (GRG) is the 3rd common cancer, and second leading cause of cancer associated mortality. Oncogenic mutations in the BRAF gene results in an altered protein structure, leading to constitutively activated MAPK signalling. Beneficial treatment strategies for this poor prognostic subgroup of GRG patients have yet to be identified. Hence this objective of this study was to identify novel treatment strategies in BRAF mutant GRG models. Our data has shown that MEK inhibition results in acute expression of pSTAT3, regulated through the c-METI JAK signalling axis. Taking a combined approach of JAKlMEK, or c-MET/MEK inhibition we have shown that these combinations results in significant increased apoptosis in BRAFMT GRG, and have potential as novel combinations. Furthermore, we are the first to show that MEK inhibition results in increased expression of the caspase 8 inhibitor c-FLlP, as a mechanism of resistance to apoptosis induction. Using a gene silencing and small-molecule inhibitor approach, we have identified that combined c-FLlP/MEK inhibition is a novel treatment strategy that may provide benefit for this subgroup of GRG patients.

No description available.

No description available.

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