This thesis presents a qualitative study of genetic counselling. Using a combination of semi-structured interviews and conversation analysis, it focuses on the role, function and structure of genetic counselling and on its status as medical or counselling work. Semi-structured interviews are used to ascertain genetic counsellors' accounts or perceptions of the nature of their role, their views on client expectations, and genetic counselling clients' perceptions and expectations of the same. Conversation analytic study of recorded genetic counselling consultations is used to identify whether or not they possess an overall shape and whether they appear conversationally as a counselling or a medical interaction. Rose's (1998, 1999) sociological work on the growth of the therapeutic community and the techne of 'psy' provides a framework for a discussion on the strength of the genetic counselling profession's association with a Rogerian counselling philosophy and on the potential difficulties this may bring. The questions are raised; does genetic counselling have many similarities to "personal, emotional or psychological" 'counselling' at all? And is this alliance with the counselling community either fair or possible for the professionals involved? The results were as follows. First, that the genetic counselling consultations in this corpus do not present with one unique overall shape that can encompass all interactions. Second, that the accounts of the genetic counsellors and clients in this sample, and the conversation analytic study of the recorded consultations, suggest that genetic counselling is primarily a medical-based activity and that this is what clients want. Third, that genetic counselling has a number of dissimilarities to psychotherapeutic counselling that suggest it is not so much 'counselling' as using counselling skills, and finally, that the tensions incurred in fulfilling medical-type tasks within what is ostensibly a 'counselling' role are neither fair nor practical for the professionals involved.

The hormone gastrin regulates gastric acid secretion and through its effects on cell proliferation, apoptosis and angiogenesis also regulates gastric epithelial and enterochromaffin-like (ECL) cell growth. The influence of various factors (host, bacterial and environmental) upon fasting serum gastrin concentrations and to what extent these factors interact to influence the progression of gastric preneoplastic pathology is not fully understood. Long standing hypergastrinaemia secondary to hypochlorhydria resulting from autoimmune gastritis can result in the development of ECL-cell hyperplasia. In some patients this progresses to type-1 gastric neuroendocrine tumour formation. The factors that influence this progression have not been fully characterised. The management of type-1 gastric neuroendocrine tumours is dependent on their size. However, there is still controversy regarding the optimal management of larger (> 1cm) tumours. Antrectomy is one option and the results of an octreotide suppression test (to determine gastrin dependency of type-1 gastric neuroendocrine tumours in order to predict response to antrectomy) have been reported in a single patient. This aims of this thesis were to assess: 1. The interaction between various factors (host, bacterial and environmental) that may influence fasting serum gastrin concentrations and the development of gastric preneoplastic pathology. 2. The roles of certain factors in the progression of ECL-cell hyperplasia to type-1 gastric neuroendocrine tumours. 3. The role of an octreotide suppression test in identifying patients with type-1 gastric neuroendocrine tumours who may benefit from antrectomy. In a large cohort of >1000 prospectively recruited patients, we demonstrated that in addition to H. pylori infection, the presence of a host factor (advancing age), a bacterial virulence factor (cagA) and elevated fasting serum gastrin concentrations (>100pM) were significantly associated with the presence of gastric preneoplastic pathology. Concurrent proton pump inhibitor therapy was however not associated with the presence of gastric preneoplastic pathology. The interactions between H. pylori infection, proton pump inhibitor use and the presence of gastric preneoplastic pathology in determining fasting serum gastrin concentrations were found to be complex. In addition, other host and environmental factors also influenced fasting serum gastrin concentrations. Although results from our study did not demonstrate any statistically significant associations, there did appear to be correlations between the presence of factors such as hypothyroidism, positive anti-gastric parietal and intrinsic factor antibodies and extent of gastric atrophy with the presence of more advanced degrees of gastric ECL-cell hyperplasia. Although a positive octreotide suppression test was associated with tumour regression following antrectomy in four patients with type-1 gastric neuroendocrine tumours, a fifth patient who had a positive test did not show tumour regression and needed additional surgery. In conclusion, gastrin appears to act as an important co-factor in the pathogenesis of epithelial and neuroendocrine neoplasia in the stomach, but interactions with other factors are complex.

Background: Development of ultra-short chemotherapy for tuberculosis (TB) is thwarted by drug-tolerant bacillary persistence and a lack of surrogate endpoints to predict outcome from early clinical studies. Characterising bacillary elimination amongst TB patients may provide important new information. Bacilli harbouring intra-cytoplasmic lipid bodies (LBs) may represent a drug-tolerant phenotype, responsible for delayed bacterial clearance. Methods: Malawian adults with pulmonary TB were treated with standard 6 month therapy. Two quantitative sputum culture methods were used to model bacillary elimination during the first 2 months; serial sputum colony counting (SSCC) and time to positivity (TTP) in BACTEC MGIT broth. Fluorescence microscopy was used to assess the proportion of LB positive bacilli on sputum smears. Plasma concentrations of anti-TB drugs were assayed at day 14 or 21. Patients were followed until one year post-treatment. Outcomes were defined as favourable (stable cure) or unfavourable (failure/relapse). The effect of microbiological and pharmacological factors on outcome was assessed. Results: 169 patients (59% with HIV co-infection) were recruited. Of 133 final outcomes, 15 (11%) were unfavourable. Partial likelihood non-linear mixed effects (NLME) modelling of SSCC data from 86 (64%) patients showed biphasic bacillary elimination; slow late-phase eradication of persisters was associated with unfavourable outcome (p=0.001). Linear mixed effects (LME) modelling of TTP data from 124 (93%) patients showed that a slower MGIT Bacillary Elimination Rate (MBER) was associated with unfavourable outcome (p=0.007). 28% (range 0-79%) of acid-fast bacilli in baseline sputum samples were LB positive. During the first month there was a trend towards higher LB counts in patients who went on to have unfavourable vs. favourable outcomes (p=0.085). Low plasma rifampicin and isoniazid concentrations were reported in 87% and 50% patients respectively. A lower isoniazid AUC(0-6hr) exposure was associated with unfavourable outcome (p=0.035). Conclusions: The two main findings were: 1. Modelling of bacillary elimination during the first 2 months of TB therapy predicted long-term outcome. The MBER, in particular, could be calculated for 93% of patients and should be considered as a surrogate marker for early clinical trials. 2. The observation of a higher proportion of LB positive bacilli in later sputum samples from patients with unfavourable outcomes suggests that these may be drug-tolerant persister cells, with negative implications for treatment efficacy.

Persistent accumulation of free radicals in cells leads to oxidative stress, which plays a causative role in the induction and progression of various chronic diseases. Therapeutic focus has therefore shifted towards the use of antioxidants, with recent interest in those of plant origin. This study investigated radical scavenging and cytoprotective activities of phytochemicals (quercetin, curcumin, sulforaphane, rosmarinic acid, caffeic acid, danshensu (3,4-dihydroxyphenyllactic acid), ferulic acid and m-coumaric acid) against DPPH free radical in a non-cellular assay, and oxidative damage in hepatic (HepG2) and pancreatic (1.1B4) cells, elicited by an organic hydroperoxide (tert-butylhydroperoxide - tBHP) and a more physiologically relevant stressor (palmitate). Direct and indirect cytoprotective activities were assessed by neutral red viability assay after 5 h co-exposure and 20 h pre-exposure conditions, respectively. Radical scavenging activities of three well-known phytochemicals - quercetin, curcumin and sulforaphane - were initially validated against DPPH (non-cellular assay), where quercetin was shown to be more potent than curcumin; sulforaphane was without effect. With quercetin as positive control, radical scavenging activities of rosmarinic acid and three of its principal metabolites (caffeic acid, danshensu and ferulic acid) were comparable, while m-coumaric acid lacked antiradical activity against DPPH radical. Subsequently in HepG2 hepatoma cells, quercetin and curcumin were confirmed to possess direct and indirect cytoprotective acitivities against 0.5 mM tBHP while, sulforaphane only had indirect cytoprotective acitivities. Additionally, co-treatment of HepG2 cells with low concentrations of quercetin and curcumin (used together) exhibited direct cytoprotective activities against tBHP. However, direct cytoprotective potencies of rosmarinic acid and caffeic acid were less than quercetin. Similar pattern was observed for indirect cytoprotective activities; with danshensu, ferulic acid and m-coumaric acid lacking hepatoprotective activity in co-exposure and pre-exposure conditions. These results highlight the discrepancy between non-cellular and cellular antioxidant activities, which could be accounted to the poor lipophilicity profiles of rosmarinic acid and its principal metabolites. Cytotoxicity assay in 1.1B4 human pancreatic β-cells revealed that these cells were more vulnerable to tBHP-induced oxidative damage than HepG2 cells. An investigation of selected phytochemicals in 1.1B4 cells produced novel findings, with quercetin exhibiting direct and indirect cytoprotective activities against tBHP (0.125 mM and 0.5 mM). Curcumin and caffeic acid were also cytoprotective against 0.125mM tBHP but only exhibited direct cytoprotection against 0.5mM tBHP. Sulforaphane lacked both direct and indirect cytoprotective activities in 1.1B4 cells, exhibiting marked cytotoxic effects in both conditions. Further analysis in both HepG2 and 1.1B4 cells proved that indirect cytoprotective activities of selected phytochemicals were not dependent on pro-proliferative activities of quercetin, curcumin, caffeic acid and sulforaphane. Moreover, it was observed that high concentrations of curcumin and sulforaphane caused necrosis in both cell types, rather than apoptosis; caffeic acid also produced necrotic effect in 1.1B4 cells. Whilst prolonged exposure of HepG2 and 1.1B4 cells to high glucose concentrations failed to elicit any evidence of glucotoxicity, sodium palmitate caused concentration-dependent cytotoxicity after short-term (5 h) and long-term (20 h) exposure to both cell types. Overall, selected phytochemicals caused additive cytotoxicity in the presence of palmitate, although quercetin demonstrated direct cytoprotection alone in HepG2 cells. Using Western blot, curcumin, caffeic acid and sulforaphane did not upregulate NQO1, but 20 h exposure to 0.1 mM quercetin resulted in upregulation in HepG2 cells, amidst high basal levels of NQO1 in this cell type. However, both basal and inductive expression of NQO1 has not been observed in 1.1B4 cells. Thus, although rosmarinic acid, danshensu, caffeic acid and ferulic acid may possess good intrinsic antioxidant properties, their physicochemical properties may limit pharmacological activities at the cellular level. Moreover, the additive cytotoxicity resulting from treatment with selected phytochemicals and sodium palmitate highlights a discrepancy between mechanisms of cytotoxicities by tBHP and palmitate.

Type-III secretion systems (T3SSs) are responsible for the biosynthesis of flagella, and the interaction of many animal and plant pathogens with eukaryotic cells. T3SSs consist of multiple proteins which assemble to form an apparatus capable of exporting proteins through both membranes of Gram-negative bacteria in one step. Proteins conserved amongst T3SSS can be used for analysis of these systems using computational homology searching. By using tools including BLAST and HMMER in conjunction phylogenetic analysis this thesis examines the range of T3SSs, both in terms of the proteins they contain, and also the bacteria which contain them. In silico analysis of several of the conserved components of T3SSs shows similarities between them and other secretion systems, as well as components of ATPases. Use of conserved components allows for identification of T3SS loci in diverse bacteria, in order to assess in the different proteins used by different T3SSs, and to see where, in evolutionary space, these differences arose. Analysis of homology data also allows for comprehensive re-annotation of T3SS loci within Desulfovibrio, Lawsonia and Hahella, and subsequent comparison of these T3SSs with related Yersinial T3SSs, and also (in conjunction with in vitro assays) for identification of many novel effectors in E. coli.

Cytomegalovirus (CMV) infection is the most frequent and significant opportunistic infection in kidney transplant recipients. It is associated with direct (CMV disease) and indirect (rejection, poor graft survival) effects with resultant increases in morbidity and mortality. The mechanisms responsible for the indirect effects of CMV infection remain unclear. In this thesis, the indirect effects of cytomegalovirus infection in kidney transplantation are studied. Firstly, the mechanism of CMV infection is investigated. Secondly, the mechanism of CMV associated kidney transplant damage is explored. Thirdly, an assessment for the role of CMV in causing immunosenescence within the kidney transplantation cohort is undertaken. This thesis provides previously undescribed and direct evidence of immune hypo- responsiveness to latent CMV. I have shown CD4⁺CD27⁻CD28^null cells are pathognomonic of prior CMV exposure and have a role in glomerular endothelial cell damage, an effect which may be mediated by NKG2D. Higher CD4⁺CD27⁻CD28^null cell counts at 12 months post-transplantation predict a steeper decline in kidney allograft function thereafter. I provide novel insight into the ‘indirect’ effect of CMV in the pathogenesis of CD8⁺CD28^null cells. My study is the first to demonstrate a temporal association between elevated CD8⁺CD28^null cell frequencies and subsequent development of clinically relevant episodes of infection. The findings from this thesis set the scene for future interventional research and therapeutic strategies.

The aim of this thesis was to determine the diagnostic accuracy of outpatient endometrial evaluation using endometrial biopsy (EB), ultrasound scan (USS) and hysteroscopy (OPH) by conducting systematic quantitative reviews of the published literature. The optimum diagnostic strategy in terms of cost-effectiveness (cost per life year gained), was then established for the investigation of women with post-menopausal bleeding (PMB) for endometrial cancer, using the review data in a decision analysis designed to reflect current service provision. Meta-analyses showed that a positive test result following EB or OPH was more useful for predicting endometrial disease than USS, whereas a negative test result following USS was more useful for excluding endometrial disease than EB or OPH. The economic model included 12 diagnostic strategies and indicated that a strategy based on initial diagnosis with USS, using a 5mm double layer endometrial thickness cut-off, was the most cost-effective. Sensitivity analyses showed that initial investigation with EB or USS using a 4mm cut-off were also potentially cost-effective (incremental cost-effectiveness ratios under £30,000 per life year gained) at their most favorable estimates of diagnostic performance, in women under 65 years and at disease prevalence of 10% or more. The choice between initial testing with EB or USS will therefore depend upon patient age and preference, disease prevalence and the availability of high quality USS. In most circumstances women presenting for the first time with PMB should undergo initial evaluation with pelvic ultrasound using a threshold of 4mm or 5mm to define abnormal results.

Acute tubulointerstitial nephritis (ATIN) is an important cause of renal morbidity. This study showed that it represents up to 8% of acute renal failure where biopsy material was available and accounted for 1% of all renal biopsy material. This study, which is believed to be the largest single retrospective study carried out to date, consists of 78 cases over nineteen years (1984-2002). Forty one cases were males and 37 were females. Acute tubulointerstitial nephritis (ATIN) was divided into three groups according to the cause: drug-induced ATIN, idiopathic ATIN and TINU syndrome. Drug-induced ATIN has come to dominate this area of medicine and in this study it represented 85% of all the cases of ATIN. Comparing the creatinine level at different time points among the the diagnosis groups shows that the creatinine level at presentation was high in patients with drug-induced ATIN compared to patients with TINU syndrome or idiopathic ATIN and the P value was 0.020. Comparison of clinical features investigations with the reversibility of renal function (cr level < 150μmol/l) shows that patients with fever, normal or high haemoglobin level, normal or low potassium level, and normal or low level of phosphate tended to have reversible renal function with P values of 0.021, 0.018, 0.002 and 0.03 respectively. Comparing the result of investigations between the different diagnosis groups showed that the lymphocyte count tended to be lower in drug induced ATIN and TINU syndrome than in idiopathic acute tubulointerstitial nephritis and this difference was statistically significant (P value = 0.026). By one year follow-up, 75% of patients had an improvement in renal function(creatinine level < 150 μmol/l). Comparing the outcome for renal function among the different diagnostic groups showed a significant statistical difference (P values 0.017-30.020). ATIN due to non steroidal anti-inflammatory drugs carried a bad prognosis in comparison to other groups. Histologically, sections of ATIN tissue biopsies showed a strong staining for CD3+ cells, CD4+ cells, CD8+ cells, CD68+ cells, eotaxin, CCR3, VCAM-1, IL-4 and eosinophil proteins. These results suggest that there is a Th 2 type of inflammatory and immune response in acute tubulointerstitial nephritis. Comparison between the infiltrating cells showed no significant difference (P values were between 0.549 and 1.00). Comparison between the infiltrating cells and the renal function outcome shows no relationship. On the other hand, there was a correlation between the CD68 positive cells and the creatinine level at presentation which indicated that there was a tendency for a greater CD68 positive cell infiltration to be associated with a higher creatinine level at presentation, and the P value was 0.003 (r =0.651). Comparison between the index of chronic damage and the reversibility of renal function shows a significant relationship at three months and one year time and the P values were 0.002 and 0.001 respectively (low index of chronic damage associated with low creatinine level). This study also showed no relationship between idiopathic acute tubulointerstitial nephritis and Epstein-Barr virus. This study confirms that ATIN remains an important cause of acute renal failure, that is predominantly drug-related, and that renal biopsy has diagnostic and prognostic significance. Immunological mechanisms are important in pathogenesis with macrophagedependent processes correlating with renal function at presentation. Prognosis is good providing diagnosis and therapeutic intervention occurs before irreversible chronic damage has developed.

Long terminal repeat (LTR) elements are wide-spread in the human genome and have the potential to act as alternative promoters and enhancers. Their expression is therefore under tight epigenetic control. We previously reported that a member of a specific class ofLTR elements (THE I B) in Hodgkin's Lymphoma (HL) acted as a promoter for the growth factor receptor gene CSF 1 R and that this gene is required for HL cell survival. However, to which extent and how such elements participate in shaping the unique gene expression program of HL is unknown. To address this question we mapped the genome-wide activation ofLTRs in HL using a novel targeted next generation sequencing approach (RACE-Seq). Integration of such data with global gene expression as well as chromatin profiling data from HL and non-HL cell lines discovered a unique pattern of LTR activation impacting on gene expression, including a number of genes associated with the HL phenotype. We also show that global LTR activation is induced by activation of inflammatory signaling pathways. Together these results demonstrate that LTR activation presents an additional layer of gene expression deregulation in HL and highlight the potential for the impact of genome-wide L TR activation in other inflammatory diseases.

GPVI activates platelets through an ITAM pathway by activation of Src and Syk kinases leading to activation of PLC\(_y\)2. CLEC-2 has been shown to activate platelets using an ITAM-like sequence in its cytoplasmic tail that is also dependent on Src and Syk kinases, but shows a partial rather than an absolute dependence on adapter SLP-76 for activation of PLC\(_y\)2. The aim of this thesis is to understand some of the key differences in these signalling pathways. GPVI is in complex with FcRwhich contains the ITAM sequence (Yxx(L/I)x\(_{6-12}\)Yxx(L/I)). These two tyrosines provide a docking site for the tandem-SH2 domains of Syk. In this thesis I show that CLEC-2 signalling through Syk is mediated by phosphorylation of the CLEC-2 YxxL sequence, receptor dimerisation and cross-linking by the Syk SH2 domains. I also show that the differential requirement for SLP-76 is not mediated by Gads. Both signalling pathways also show partial dependency for LAT. I also show that a novel protein, G6f, is not able to substitute for LAT in this signalling pathway and also exclude the LAT-family proteins PAG, LIME, LAX and NTAL as potential LAT replacements in platelet activation by GPVI. These results extend our understanding of platelet activation by CLEC-2.