Raf Kinase Inhibitor Protein (RKIP) was originally described as an inhibitor of the Ras-Raf-MEK-ERK pathway, exerting its action by the physical inhibition of the interaction of Raf with MEK. It has subsequently been shown to play important roles in a number of other signalling pathways, including the NFκB pathway and in the stability of the mitotic spindle. Not surprisingly given that it impacts on many important signalling pathways RKIP levels have been shown to be important in the progression of a number of different cancers. RKIP expression is lost or decreased in a number of common human cancers and decreased still further in tumour metastases. One of the tumours in which RKIP is downregulated is colorectal cancer (CRC). Importantly it has been shown that not only is RKIP depleted in tumour tissue when compared with normal tissue but that the level of RKIP within a tumour is inversely correlated with the likelihood of metastatic relapse and with patient prognosis. Although we already have a number of very good prognostic indicators in CRC, one group of patients for whom new prognostic indicators would be useful are patients with Dukes B CRC. These are patients with locally advanced but non-metastatic disease and at present there is no firm consensus on their correct post-operative management. Therefore we set out to examine whether RKIP is a useful prognosticator in this particular group using a tissue microarray (TMA) with samples from over 200 patients with Dukes B CRC. The analysis revealed a strong inverse correlation between RKIP levels and disease specific survival. Moreover, in a multivariate analysis RKIP emerged as an independent prognostic indicator along with lympho-vascular invasion and peritoneal invasion, two well-known and powerful prognosticators. This allowed for the generation of a simple prognostic index, using information from the different independent indicators, allowing for improved patient risk stratification. This led us to examine whether RKIP could also function as a predictive marker in CRC. To do this we again used a TMA, this time consisting of a much larger cohort of patients across the whole range of tumour stages. The results confirmed the prognostic utility of RKIP and indicated that patients whose tumours have low levels of RKIP may derive a greater benefit from chemotherapy than those patients whose tumours have high levels, although this result did not reach statistical significance. In the second part of the thesis I have examined the effect of RKIP in previously characterised mouse models of CRC. To do this I have used a germline RKIP knockout mouse and in the first instance crossed it to the APC580S mouse. In this mouse APC is lost conditionally within the intestine and liver. RKIP knockout did not have any effect on the rate of tumourigenesis or on the invasiveness of tumours in this model. However, in the setting of acute homozygous deletion of APC, RKIP knockout resulted in a decrease in apoptoses in the small intestine and an increase in aberrant mitotic activity in the liver. To follow this up I have examined the effect of RKIP knockout in a mouse model of superficially invasive CRC, specifically to see if RKIP knockout can promote invasive and metastatic behaviour. In this model the APC580S mouse is crossed to mice which conditionally express oncogenic KRas. Although RKIP knockout did not result in an increase in invasive tumours in this model there was a shift in tumour location from the small intestine to the colon. This shift appeared to be due, at least in part to an increase in chromosomal instability in the tumours. The final aim of the thesis was to develop a mouse model of CRC which more closely recapitulates the late stages of the human disease, specifically invasion and metastasis. To do this we have crossed the APC580S mouse with either a conditional p53 knockout or with a mouse that conditionally expresses a point mutation of p53 (p53R172H). In human tumours the majority of abnormalities of p53 are point mutations that result in the production of mutant protein that accumulates in tumour cells. There is evidence that this mutant protein may have oncogenic properties beyond the simple loss of normal p53 protein function. Therefore we have also used this model to study the differing effects of p53 loss and point mutation in CRC. We found that mice homozygous for p53 deletion (p53fl/fl) and those expressing a single copy of the mutant allele with loss of the second copy (p53R172H/fl) developed invasive tumours with nearly 100% penetrance and indeed metastasis was observed. Remarkably, although mice that were heterozygous for p53 deletion (p53fl/+) only rarely developed invasive tumours almost 100% of mice expressing a single copy of the mutant allele (p53R172H/+) developed invasive tumours. We went on to show that the increase in invasion seen in this model is related to an increase in Wnt signalling, which is associated with increased expression of pro-invasive Wnt targets such as fascin. We also showed a novel pro-invasive role for ARF in this process. This is also an excellent model of Dukes B CRC and therefore the ideal model to test the effect of RKIP deletion on invasion and metastasis. These studies led us to examine the differences in effect between knockout and mutant p53 in another tumour model. In this we used a novel model of the aggressive tumour pleomorphic rhabdomyosarcoma to demonstrate that mutant p53 can both promote both tumourigenesis and metastasis more potently than p53 knockout. These studies have demonstrated the value of RKIP in the clinically important Dukes B CRC population and shown its possible utility as a predictive marker in this group. Although we have not seen an effect of RKIP knockout in traditional mouse models of CRC we have developed a novel model which closely recapitulates Dukes B CRC and may be useful in elucidating the effect of RKIP knockout. We have also used this model to gain novel insights into the invasive process, in particular into the role played by mutant p53.

Interleukin 6 (IL6) is associated with arterial disease development, progression and surgical outcome. Raised levels of IL6 may play a causal role in disease development or may be the effect of pathology. An IL6 single nucleotide polymorphism (SNP) G- 174C has been identified and reported to associate with IL6 expression. However, conflicting results have emerged and both the relationship between IL6 and vascular disease and the precise effect of SNP G-174C in vivo in humans remains obscure. The aim of this study was to establish the effect of SNP G-174C in humans, in vivo in different tissues. Varicose vein surgery patients donated adipose tissue, skeletal muscle, vein and blood samples. Patients were genotyped for SNP G-174C. A new pre-mRNA assay was developed, using gel electrophoresis, restriction digest and fluorescence quantification, to measure the ratio of heterozygous allelic pre-mRNA transcription. IL6 mRNA expression in different tissues was also measured using relative real time PCR (RT-PCR) to assess effect of tissue type on expression profiles. mRNA expression within tissues was compared between G-174C genotypes, to further quantifying the association of SNP G-174C with transcript levels. The pre-mRNA assay showed higher expression for the C-allele, though not statistically significant. The pre-mRNA assay needed to detect low levels of intron retaining allelic pre-mRNA isoforms. Replicates and controls for residual genomic DNA were used to monitor assay precision. Adipose tissue gave the greatest precision in the pre-mRNA assay. In the RT PCR assay adipose tissue expressed significantly more IL6 mRNA than all other tissues examined. In vein and leukocytes subjects with the CC genotype expressed significantly higher levels of IL6 mRNA than subjects with GC or GG genotypes. There was a trend towards higher expression for the CC genotype in all tissue types. A significant though weak correlation between IL6 mRNA expression and age was demonstrated for vein and leukocytes. Adipose tissue may be an important source of IL6 compared to other tissues. This may be relevant for obesity associated diseases. Subjects with G-174C genotype CC showed a trend towards higher IL6 RNA expression. Further studies are necessary to clarify the effect of genotype on IL6 expression.

The Ubiquitin Conjugating System (UCS) describes a system in which the 96-amino acid residue Ubiquitin can be selectively covalently linked to intracellular proteins. This endows cells with an indispensable level of regulation to determine protein fate in a wide range of basic cellular events. The abundant, neuron specific Ubiquitin Carboxyl-Terminal Hydrolase-L1 (UCH-L1) is intimately involved with the UCS – both in a hydrolase and ligase capacity. Mutations in UCH-L1 have clearly been associated with various neurodegenerative disorders, including Alzheimer’s, Huntington’s and particularly Parkinson’s disease. The main and unique objective of this study was to identify any common Caucasian sequence variants in UCH-L1’s promoter, and to investigate whether they are associated with neurodegenerative symptoms, and any change in UCH-L1 transcriptional activity. Seven novel UCH-L1 Single Nucleotide Polymorphisms (SNPs), as well as the C54A documented coding region polymorphism (Ser18Tyr), were identified using both denaturing High Performance Liquid Chromatography (dHPLC) and DNA sequencing analysis. In relation to the translational start site, the novel SNPs elucidated were: A-307G, A-306G, G-234A, A-24G, C-16T, G12A and G21A. Restriction Fragment Length Polymorphism (RFLP) genotyping analysis was then employed within Caucasian DNA sample sets of 31 and 480 individuals, to firstly elucidate the common UCH-L1 promoter haplotypes that exist within the population, and secondly, in an attempt to uncover any association between the polymorphic alleles and general neurodegenerative symptoms - no association was uncovered. Using pGEM-T Easy as an initial ‘holding vector’, the three common UCH-L1 promoter haplotypes elucidated – AAGAC, GAGGT and AGAAC - were incorporated into a modified pGL3 vector to ascertain transcriptional activity rates. This was done by Luciferase expression analysis, and the results identified the GAGGT promoter haplotype as having a significantly increased transcriptional activity in all human cell lines tested. It is my contention, that the pronounced increase in transcriptional activity elucidated for the GAGGT UCH-L1 promoter haplotypes, potentially indicates a primary genetic risk factor for sporadic Parkinson’s disease in the Caucasian population – a novel pathogenic model of which is proposed in this thesis. The fact that RFLP genotyping analysis uncovered no association of the promoter polymorphic alleles with more general neurodegenerative symptoms, indicates the need for further studies to be focused more specifically towards Parkinson’s disease.

The myeloproliferative disorders (MPDs) are a group of haematopoietic stem cell diseases, characterised by proliferation of one or more cells of the myeloid lineage. Several lines of evidence have highlighted the importance of aberrant tyrosine kinase signalling in the pathogenesis of these disorders. Cloning of rare chromosomal translocations and point mutation analysis in the MPDs has identified diverse deregulated tyrosine kinase genes, notably PDGFRA, PDGFRB, FGFR1 and JAK2. However the majority of atypical MPDs still remain to be characterised and identification of patients harbouring fusions, particularly those involving the PDGF receptors is of increasing importance, as they are likely to be responsive to targeted therapy with imatinib. I am investigating MPD patients for abnormalities affecting tyrosine kinase signalling, and have used three approaches, translocation cloning, expression analysis and SNP array analysis to detect regions of loss of heterozygosity (LOH). Thus far, by translocation cloning I have identified a previously undescribed partner gene fused to PDGFRB and two new PDGFRA fusion genes. I have also designed two reverse transcriptase PCR (RT-PCR) assays and a cDNA MLPA assay to detect over-expression of specific tyrosine kinases screening approximately 200 patients. Each assay identified all patients previously diagnosed with known fusions. Additionally, two patients identified with overexpression of PDGFRB have been found to have cryptic ETV6-PDGFRB fusions and overexpression of PDGFRA in one patient lead to the discovery of a previously undescribed fusion involving a novel partner gene (KIF5B). Recent evidence has indicated that acquired isodisomy is a novel mechanism by which mutations in cancer may be reduced to homozygosity. Typically, acquired isodisomy is associated with oncogenic changes rather than tumour suppressor genes, eg. the activating JAK2 V617F mutation and 9p aUPD. I have undertaken a screen using Affymetrix 50K SNP arrays for regions of acquired isodisomy as a means to identify genomic regions that may harbour novel oncogenes in different subgroups of MPD patients. Large tracts of homozygosity (defined as >20Mb running to a telomere), strongly suggesting acquired isodisomy, were seen in 40% aMPD patients. The homozygous tracts encompassed diverse genomic regions in aMPD, but two common regions (3 cases for each region) were identified at 7q and 11q. Mutations in the CBL ubiquitin ligase gene were discovered in all three aCML patients with 11q aUPD as well as in an additional 23 MPD patients following further screening.

Hodgkin lymphoma (HL) is a malignant lymphoma that is diagnosed mostly in young adults, and is the second most common malignancy to affect this age group. This disease is subdivided into two entities with different aetiologies: classical HL (cHL) (~95% of cases) and nodular lymphocyte-predominant HL. In Europe, ~82% of young adults with cHL are non-Epstein-Barr virus associated and epidemiological studies have suggested that a common infectious agent may play a key role in the aetiology of these cases. The molecular biology of HL is not well understood, primarily due to the low number of Hodgkin and Reed-Sternberg (HRS) cells present within these tumours. However, recently developed techniques for the selection and micromanipulation of single HRS cells from tumours, and the development of molecular cytogenetic techniques (i.e. array-comparative genomic hybridisation (CGH)) are overcoming these difficulties. To investigate a potential candidate virus, DNA samples from cHL biopsies were screened for the measles virus (MV) and polyomaviruses (PyV), using immunohistochemistry and highly sensitive PCR assays. Chromosomal imbalances in six well-established cHL-derived cell lines and a cHL case were analysed by array-CGH. To obtain sufficient DNA for array-CGH from the cHL case, single HRS cells were isolated using laser microdissection. DNA was extracted then amplified by degenerate oligonucleotide primer polymerase chain reaction. MV and PyV genomes were not detected within cHL biopsies. Recurrent chromosomal imbalances were confirmed within the cHL-derived cell lines and cHL case, in addition to several novel imbalances. This is the first time that a cHL case has been analysed by array-CGH.

Enterohaemorrhagic E. coli (EHEC) are important gastrointestinal pathogens of humans. E. coli serotype O157:H7 is the EHEC most commonly associated with human illness. E. coli O157:H7 is carried asymptomatically by cattle which form an important reservoir for the bacterium. E. coli O157:H7 has been found to colonise at the terminal rectum of cattle in preference to other sites in the bovine gastrointestinal tract. The first objective of this work was to characterise the roles of bacterial secreted components responsible for key functions in the modulation of host defences against EHEC. Data presented here reaffirms the role of flagellin in the elicitation of a proinflammatory response in a cultured human epithelial cell line; however, the response of a bovine epithelial cell line to bacterial secreted products was not affected by the presence or absence of flagellin. A role in the modulation of the host response for the StcE protease was also investigated: although its role in interaction with the bovine host was not established, bovine secretory antibodies to StcE were detected in rectal mucosal scrapings from an E. coli O157:H7-challenged calf, suggesting that StcE is expressed and recognised in vivo. The second key objective was to isolate E. coli from the bovine intestinal tract in order to define the colonisation patterns of E. coli within the bovine intestinal tract and relate this to bacterial genotype and to provide bovine E. coli isolates to test for inhibitory activity against E. coli O157:H7 which may yield bacteria with potential as probiotic agents with a view to reducing the prevalence of EHEC in cattle. Genotypic analysis of bovine resident E. coli confirmed that these strains carry a variety of virulence factor-encoding genes; however, certain dominant genotypes were identified and the genomic structure of representative isolates was predicted by genomic microarray. EHEC-related genotypes were found to be positively associated with colonisation at the rectum, whereas non-EHEC genotypes were found to colonise multiple intestinal sites without showing any apparent site-specificity. The third and final objective of this analysis was to carry out genotypic analysis of Scottish EHEC strains in order to predict whether increased incidence of EHEC infection in Scotland may be related to the presence of EHEC strains carrying altered complement of virulence factor-encoding genes. The analysis of EHEC isolated in Scotland revealed that these strains exhibit a genomic profile which is largely typical of EHEC isolated elsewhere, although there were certain differences in the carriage of a certain genomic elements. The results presented here support the proposal that bacteriophages are the key mediators of genetic variability among E. coli isolates.