Introduction: The prevalence of multiple sclerosis (MS) shows a latitude gradient which may be related to exposure to ultraviolet radiation (UVR). The effects of UVR may be mediated through 1,25-dihydroxycholecalciferol which exerts its effects via the vitamin D receptor. We hypothesised that polymorphisms in the vitamin D receptor gene (VDR) are associated with risk and severity of multiple sclerosis. We also hypothesised that MS cases may be clustered in space as a reflection of shared aetiological exposures. Methods: We examined g. 1229A>G, g. 3436C>G, g. 3944G>A, g. 20965C>T, g. 30056C>T, g. 30875T>C, g. 48200C>T, and g. 65013C>T single nucleotide polymorphisms in patients with multiple sclerosis (Poser criteria) and controls using pyrosequencing technology. Disability was assessed with the expanded disability status scale (EDSS) and was dichotomised into mild to moderate (EDSS <6) and severe (EDSS>6) disability. Genotypes and haplotypes at the 8 sites were analysed for association with disability (in patients with disease duration >10 years) and risk (in the case and control groups) using logistic regression with corrections for age, gender and disease duration. Results: U30875 genotype was significantly associated with less severe disability (EDSS<6) (odds ratio=0.37,95% CI=0.20-0.70, p=0.048). Genotype combinations including TT3087w5 ere also associated with less severe disability. Non-significant associations with MS risk (after correction formultiple comparisons) were found for AA 3944a nd CT3 0875 . Haplotype G3436_ G3 944w as associated with reduced risk of MS whereas A3944C 2096h5a plotype was risk conferring. Conclusions: We demonstrate associations between VDR polymorphisms and MS severity and risk. These preliminary results will need confirmation in independent cohorts.

Fanconi Anaemia (FA) is an inherited genomic instability disorder caused by mutations in one of 13 FANe genes, the products of which interact to form the FABRCA pathway, which is involved in the cellular response to DNA damage. FA patients have a high risk of acute myeloid leukaemia (AML), implying that the FABRCA pathway has an important role in suppressing leukaemic transformation. A detailed understanding of AML arising in FA is lacking, but chromosome aberrations leading to gains of material from the long arm of chromosome 3 (3q) are a characteristic and recurrent finding in FA bone marrow and appear to indicate imminent malignant transformation. Analysis of two FA-derived AML cell lines, SB1685CB and SB1690CB, pointed to 3q26.2-q26.31 as the minimal amplified region in FA-derived AML. This region encompasses the EVIl proto-oncogene. EVIl encodes a DNA binding transcription factor that is overexpressed in approximately 10% of sporadic AML, where it confers an extremely poor prognosis. To investigate the impact of FA-associated 3q gains on EVIl expression I analysed EVIl transcription in all FA-derived AML cell lines. In each one EVIl is expressed. In SB1685CB and SB1690CB expression of EVIl, at both the transcript and the protein level, exceeds that in all other cell lines investigated in this study, including MUTZ-3 that harbour an inv(3)(q21;q26), which clinically is associated with the highest levels of EVIl overexpression. Analysis of preleukaemic FA bone marrow demonstrated elevated EVll expression in samples with 3q gains compared to FA marrow without 3q gains and normal controls. This suggests that FA-associated 3q gains commonly lead to deregulated EVIl expression as a key event in leukaemic transformation. The high protein levels in SBI690CB cells allowed further investigation of the EVIl protein, which focused on EVIl protein phosphorylation, as this post-translational modification has a central role in regulating the function of many proteins. Mass spectrometric analysis of endogenous EVIl immunoprecipitated from SB1690CB cells detected a novel site of phosphorylation on serine-196 (S 196), as well as phosphorylation on S860, which has also been identified by other groups during the course of this project. EVIl phosphorylation on S858 was also detected, but only after treatment with ionizing radiation (IR), and then only in conjunction with phosphorylation on S860. Employing purpose made phosphospecific antibodies that specifically recognise EVIl phosphorylated on S860 alone and EVIl phosphorylated on both S858 and S860, I show that IR leads to a reduction of the cellular pool of EVIl phosphorylated on S860 alone, whilst leading to a concomitant increase in EVIl phosphorylated on both S858 and S860, implying that S860 phosphorylation primes EVIl for phosphorylation on S858 in response to DNA damage. The S196 phosphorylation site lies within the first DNA binding zinc finger domain of EVI I. A mutant EVIl protein mimicking S196 phosphorylation showed significantly reduced DNA binding in vitro. Moreover, the ability of EVIl to promote colony growth of Rat I fibroblasts in soft agar, a measure of the transforming capacity of EVIl, was abrogated by the substitution of S196 with a phosphomimetic mutation. Activation of EVIl is an important event in FA-associated AML and EVIl protein function is critically regulated by phosphorylation.

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This thesis explores the pathophysiological mechanisms underlying the myeloproliferative disease, Polycythaemia Vera (PV). PV is characterised by clonal red cell expansion with a tendency for leukaemic transformation. This study focuses on primary erythroid progenitor cells derived from 27 PV patients and 49 control subjects with non-clonal red cell expansion or normal red cell mass providing insight into survival, signal transduction & the molecular signature of these cells. The erythropoietin independent colonies (EECs) characteristic of PV were detected in 93% PV, the V617F Jak2 mutation was present in 93% PV and marked upregulation of PV1 mRNA which can be associated with PV was found in 89% PV. None of the control subjects showed these features. PV erythroid progenitors showed increased proliferation with relative resistance to cytokine deprivation when 14 PV samples were compared with 10 controls. Apoptosis was not found to be increased in PV. In over 40 experiments PV erythroid progenitors showed aberrant signalling with constitutive and stimulated increases in activation of the PI3K, MAPK and Jak/STAT pathways. Activation of each pathway was reduced with specific small molecule inhibitors. Use of PI3K & Jak2 inhibitors caused comparable reduction between PV & control samples in 40 erythroid colony assays and 20 survival experiments using erythroid progenitors. However, there was some evidence to suggest that the Jak2 inhibitors used were less effective in PV with homozygous expression of V617F Jak2. EECs from PV samples were, however, greatly reduced with inhibitors. RNA from 14 erythroid progenitor samples including 6 PV were hybridised onto Affymetrix GeneChip microarrays. There was segregration of signature between subject groups. The array data was validated by real-time quantitative PCR and this technique was further used to show that 2/4 genes identified as upregulated in PV were not Jak2 dependent but that the well recognised Jak/STAT target, Pim-1 was.

Haemoglobin and nitric oxide (NO) strongly induce the stress protein haem oxygenase-1 (HO-1), which degrades haem to carbon monoxide (CO), a signalling and vasoactive molecule, the anti-oxidant biliverdin (BV) and iron. Raised HO-1 levels have been detected in the endothelium and kidneys of animals, as well as leukocytes of patients, suffering from sickle cell disease (SCD). A genetic mutation of haemoglobin causes SCD, pathologic symptoms of which include vaso-occlusive crises and high concentrations of free haemoglobin liberated during red blood cell haemolysis. Although HO-1 and its products have been linked to SCD, their potential role in this condition has not been examined. The work of this thesis aimed to: 1) investigate the interaction of haemoglobin and NO in modulating endothelial HO-1 expression 2) examine the effect of human sickle cell blood on HO-1 induction and vascular function and 3) assess the role of HO-1 and its products on sickle cell blood adhesion and regulation of vessel contractility. Experiments were performed using a combination of in vitro (cell culture and biochemical assays) and ex-vivo (isolated aortic rings) models. The results of the study indicated that NO synergises with haemoglobin to amplify HO-1 expression and haem incorporation by endothelial cells, suggesting that similar mechanisms might contribute to changes in vascular function occurring in haemolytic disorders. It was also found that sickle cell blood induces haem oxygenase to a greater extent than normal blood, an effect which is magnified under hypoxia. The increased haem oxygenase elicited by sickle cell blood depends on the time elapsed since the last vaso-occlusive crisis experienced by the patient. Finally, CO and BV diminish sickle blood adhesion to the endothelium and sickle blood can alter CO-mediated vessel relaxation. These findings support a functional role for the HO-1 pathway in SCD and may help to identify therapeutic strategies to counteract the vascular damage caused by SCD.

Iron overload is a major cause of morbidity and subsequent mortality in patients with thalassaemia major, its effects in thalassaemia intermedia and sickle cell anaemia are however less well known. The presence of non transferrin bound iron is well described in adult thalassaemia patients but it is unclear as to when NTBI appears and what relationship it has to ineffective erythropoiesis and end organ damage. Data is presented on children with thalassaemia from a five-year prospective study showing that NTBI is present early in thalassaemia syndromes and this is probably due to ineffective erythropoiesis. In addition results from this study show that there is no relationship between markers of oxidative damage and NTBI in early childhood. Following this a comparison of adult patients with sickle cell anaemia and thalassaemia is undertaken looking at NTBI and cardiac iron burdens assessed by MRI. The thalassaemia patients at high liver iron burdens have a significant risk of cardiac iron loading and when patients with sickle cell anaemia and thalassaemia major are matched for liver iron it is seen that cardiac iron loading is not seen in sickle patients and this may be because NTBI is lower in this group. In the last chapter data is presented showing that serum pro-hepcidin is down regulated by NTBI, anaemia and erythropoietin in thalassaemia but not sickle syndromes. There is no clear relationship between pro-hepcidin and liver iron but hepcidin mRNA is down regulated by iron burden supporting the important role of this protein in iron regulation.

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