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.

Evidence suggests haematopoietic stem cells (HSCs) can migrate to injured liver and influence tissue repair. However, the molecular adhesive mechanisms governing HSC recruitment to injured hepatic microcirculation are poorly understood. These mechanisms were investigated in vivo following murine hepatic ischemia-reperfusion (IR) injury. HSC adhesion was significantly enhanced in injured livers and could be reduced by blocking CD49d on HSCs or VCAM-1 in vivo. Blockade of HSC CD18, CD31 or CD44 did not alter adhesion. HSC adhesion in sham treated CD31-/- animals was raised compared to wild-type animals and IR injury did not further raise this adhesion. Studies in vitro demonstrated that HSC treatment with inflammatory cytokines or conditioned media/plasma did not upregulate adhesion molecule expression but CXCL12 and CXCL1 did significantly enhance HSC adhesion to endothelium. However, blockade of CXCR4 (CXCL12 receptor) failed to reduce HSC adhesion in vivo following IR injury. Furthermore, we demonstrated exogenous HSCs were identified primarily in the pulmonary circulation and intraportal injection raised recruitment within the liver irrespective of the presence of injury. This study provides novel evidence for the importance of the VLA-4/VCAM-1 pathway in HSC recruitment to IR injured liver, a pathway that may be manipulated in order to enhance hepatic engraftment of these cells clinically.

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a deficiency of the α-galactosidase-A (α-gal-A) enzyme. The lack of enzymatic activity results in the accumulation of glycosphingolipids (GSLs) in the lysosomes of various tissues and organs. Globotriaosylceramide (Gb3) and Globotriaosylsphingosine (Lyso-Gb3) and their isoforms/analogues have been identified and quantified as potential biomarkers. This study aimed to develop an HPLC-MS based method for the quantitation of plasma and urinary Lyso-Gb3 and its analogues in Fabry patients to evaluate its utility in diagnosis and monitoring FD. The results showed that plasma Lyso-Gb3 as a reliable diagnostic biomarker for FD, as plasma Lyso-Gb3 levels could easily discern classical Fabry patients from controls. Moreover, plasma Lyso-Gb3 could also distinguish male cardiac variant Fabry patients from control males. Nevertheless, cardiac variant Fabry females showed an overlap of Lyso-Gb3 levels with controls, hence a positive value in this group would be considered diagnostic but a negative value could not exclude FD. In a small cohort of our patients on ERT there was a trend towards falling Lyso-Gb3 levels with time suggesting that Lyso-Gb3 has a potential value in monitoring these patients. Urinary Lyso-Gb3 levels were substantially different between classical and cardiac variant Fabry patients, and the lack of detectable urinary Lyso-Gb3 and analogues in controls allowed us to differentiate between these patients and healthy controls. The total levels of urinary Lyso-Gb3 and its analogues proved particularly useful in differentiating between classical and atypical Fabry patients of both genders. In the course of the study, a novel rapid MALDI-TOF-MS based Method for measuring urinary Gb3 in Fabry patients has been established. Collectively, the final findings demonstrate that urinary Lyso-Gb3 is superior to urinary Gb3 as a diagnostic biomarker for FD, where the later has been shown to be found in healthy subjects. Our study subsequently led to development of regional laboratory service for testing Lyso-Gb3 in Queen Elizabeth Hospital, Birmingham, UK. This service is now open to Fabry patients across England. In conclusion both plasma and urinary Lyso-Gb3 levels are useful diagnostic and monitoring biomarker in classical and cardiac variant males patients, but have questionable utility in cardiac variant females due to overlap with healthy controls. Although we studied the role of Lyso-Gb3 in diagnosing FD further studies are needed to establish its role in disease severity assessment and a larger study required to test our initial finding related to monitoring disease in patients on treatment.

<p> Most of the colon cancer patient tumors progress to metastases, despite undergoing surgical resection or adjuvant chemotherapy. Predicting which patients will progress to metastases has been extremely challenging. There is an urgent need to identify early novel prognostic biomarkers that can early on predict the patient outcome. Vitamin C has been shown to have a pro-oxidant effect on cancer that enhances tumor growth and survival. Vitamin C is transported into mammalian cells via two isoforms of sodium-dependent vitamin C transporters (SVCTs), SVCT1 and SVCT2. The expression and clinical implications of SVCTs in tumor tissues could help us investigate its prognostic value in predicting patient outcome. In this report, we performed immunohistochemistry to determine SVCT1 and SVCT2 expression on primary tumors of 178 colon cancer patients. Colon cancer cells selectively expressed SVCT2 but not SVCT1. Moreover, poorly differentiated and metastatic tumors correlated with higher SVCT2 expression. Furthermore, increased SVCT2 expression was associated with shorter progression-free survival in patients with no or little lymph node invasion. We confirmed that SVCT2 could be an early stage prognostic biomarker that can predict colon cancer disease progression and survival.</p><p>

Helicobacter felis (H. felis) infection has been used for numerous years as a standard model of gastric carcinogenesis and recapitulates the pathogenesis seen with human Helicobacter pylori infection. H. felis induces a a wide array of chronic low grade inflammatory responses, some of which directly contribute to carcinogenesis and some of which do not. To tease out the role of various processes, we have compared and contrasted models that recapitulate H. felis infection or synergize with it, namely, the K19kras transgenic mouse, and diet-induced obesity, respectively. While much attention has been rightly paid to the behavior of transformed cells, some focus has recently shifted to the components of the soil in which tumors develop and grow, commonly referred to as the microenvironment. Here, we investigate the gastric tumor microenvironment and shed light on the role of myofibroblasts, T lymphocytes and myeloid cells. K19kras mice express a mutant K-ras transgene in K19+ cells of the stomach; while K19 (and therefore the transgene) is not expressed in candidate stem/progenitor cells of the gastric corpus (marked by Dcamkl1 expression), a severe expansion and repositioning of Dcamkl1+ cells is observed in this model, and correlates with the accumulation of αSMA+ myofibroblasts and recruitment of bone marrow-derived inflammatory cells surrounding glands. These transgenic mice also progress to high grade dysplasia at a similar rate as H. felis-infected mice, and this correlates with expression of select cytokines and chemokines expressed in both models, namely IL-6, IL-1β, and CXCL1. While H. felis infection had no effect on the phenotype of K19kras mice, epidemiological studies have shown that in humans, obesity increases one's risk of a wide variety of cancers, including gastric cancer, and indeed, we noted a synergy when recapitulating this in the mouse model. Acceleration in dysplasia progression was correlated with elevated levels of serum IL-6 and Leptin, which translated to excessive Stat3 activation in both epithelial and stromal compartments of the gastric epithelium, and excessive production of IL-17A in obese, H. felis-infected mice. As some reports indicate the TH17 response (characterized by IL-17A production) can, in certain contexts, promote tumorigenesis, it may be fundamental to H. felis-induced gastric carcinogenesis, and be a mechanism by which obesity accelerates cancer development. Another important component of the tumor microenvironment, as described previously in the H. felis and other cancer models are myeloid cells. Namely, a group of phenotypically immature myeloid cells have been termed IMCs (immature myeloid cells), and can both suppress anti-tumor immunity and express mediators that directly promote carcinogenesis. IMCs were recruited from the bone marrow in H. felis-infected mice, although unexpectedly, these cells were intercepted by expanded adipose tissue (in obese mice), where they differentiated into mature macrophages. This led to an overall increase in the number of myeloid cells in the adipose tissue of obese H. felis-infected mice when compared to uninfected obese mice, and most importantly, an increase in adipose CD11b+F4/80+CD11c+ myeloid cells, which are known to be a primary source of insulin resistance-inducing adipokines. Fittingly, obese H. felis-infected mice had significantly higher levels of IL-6, resistin, and PAI-1, and were more glucose intolerant than uninfected obese mice. Myeloid proliferation was similar between the two groups (as measured by KI67 and BrdU), as well as myeloid chemotaxis from the blood into adipose tissue (as determined by CCL chemokine production). Therefore, excessive myeloid recruitment to the adipose tissue could be due to elevated myeloid cells in the blood, which in turn is due to mobilization from the bone marrow. Indeed, we noted slightly elevated levels of blood IMCs in uninfected obese mice and H. felis-infected lean mice; in an additive fashion, this led to significantly elevated levels in obese H. felis-infected mice. Blood myeloid concentration was correlated with serum levels of the recently identified IMC mobilizer CXCL1, which was produced by H. felis-infected gastric tissue, and obese adipose tissue. Collectively, these results further stress the role of desmoplasia in gastric carcinogenesis. The recruitment of bone marrow derived cells may lead to the engraftment of myofibroblasts in the stem cell niche and permanently alter signals regulating stem cell homeostasis. This, in turn, may increase the number of long-lived undifferentiated progenitors which can accumulate multiple mutations and initiate tumors. Furthermore, interaction and cytokine production by components of the inflammatory microenvironment, namely IL-6, IL-1β, and IL-17 (Stat3 and NFκB activators) initiate and maintain cell transformation and promote their progression. Finally, the recruitment of cells that participate in the tumor microenvironment may have unintended side effects if they are intercepted by other tissues, as exemplified here in the exacerbation of insulin resistance.

Movement and positional control of mitochondria and other organelles are coordinated with cell cycle progression in the budding yeast, Saccharomyces cerevisiae. Recent studies have revealed a checkpoint that inhibits cytokinesis when there are severe defects in mitochondrial inheritance. An established checkpoint signaling pathway, the mitotic exit network (MEN), participates in this process. Here, we describe mitochondrial motility during inheritance in budding yeast, emerging evidence for mitochondrial quality control during inheritance, and organelle inheritance checkpoints for mitochondria and other organelles.

Stroke has been a major source of morbidity and mortality for centuries. Eight-five percent of all strokes are ischemic in nature, meaning they are caused by the occlusion of a major cerebral artery. Despite extensive research to develop effective treatments for ischemic stroke, therapeutic options remain limited. Apoptosis (also termed "programmed cell death") is a process by which a stressed or damaged cell commits "suicide". In stroke, runaway apoptosis contributes to stroke neurodegeneration and neurological decline for days to weeks after disease onset. Cysteine-ASPartic proteASEs (caspases) are key mediators of apoptosis that are activated in distinct molecular pathways, but their impact in stroke is poorly defined. Direct evidence for caspase activation in stroke and the functional relevance of this activity has not been previously characterized. For this dissertation, we developed an unbiased technique for in vivo trapping of active caspases in rodent models of ischemic stroke. We isolated active caspase-9 as a principal contributor to ischemic neurodegeneration in rodents (Rattus norvegicus and Mus musculus). Caspase-9 is the initiator caspase for the intrinsic cell death pathway. Intranasal delivery of a novel, cell membrane-penetrating inhibitor for caspase-9 confirmed the pathogenic relevance of caspase-9 activity in stroke. Caspase-9 inhibition provided neurofunctional protection and established caspase-6 as its downstream target. Caspase-6 is an effector caspase and a member of the intrinsic death pathway that has never been implicated in stroke until now. Coincidentally, we discovered that caspase-6 is specifically activated within the axonal compartment. The temporal and spatial pattern of activation demonstrates that neuronal caspase-9 activity induces caspase-6 activation, which mediates axonal loss in the early stages of stroke (+/- 24 hours). We developed a novel inhibitor for caspase-6, based on a catalytically inactive clone, which demonstrated neuroprotective and axoprotective efficacy against ischemia. Collectively, these results assert that selective inhibition of caspase-9 and caspase-6 is an effective translational strategy for stroke. The impact of caspase activity is not restricted to neuronal death, as caspases can exacerbate inflammation and alter glial function. Thus, caspases are logical therapeutic targets for stroke. However, they have never been clinically evaluated due to a paucity of ideal drug candidates. This dissertation outlines fresh insights into the mechanisms of stroke neurodegeneration and offers novel caspase-based therapeutic strategies for clinical evaluation.