The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor WD-40 domains, also known as beta-transducin repeats, are highly conserved repeating amino acid units, which are found in a wide variety of eukaryotic proteins that have a range of different functions. In the late 1990s, the first WD-40 containing proteins were identified in prokaryotes, however the knowledge about their function is scarce. Streptomyces coelicolor is a gram-positive bacterium with complicated morphological and physiological differentiation in the course of its life cycle. The genome of Streptomyces coelicolor encodes 6 potential genes encoding proteins with WD-repeat motifs. To determine the function of two of these WD-40 genes (wdpB and wdpC), the deletion replacement mutants in both genes were prepared. Both mutants exhibited medium-dependent phenotypes, which are markedly evident on modified R3 plates. Phenotypic studies revealed that deletion of wdpB gene resulted in substantial reduction of aerial hyphae formation and reduced production of undecylprodigiosin. In addition, the hyphae of ΔwdpB mutant were unusually branched and showed the signs of precocious lysis. Delayed spore-containing hyphae were irregularly septated....

Sialinsäuren sind Teil der äußersten Komponente der Glykokalyx aller Wirbeltiere. Als solche sind sie grundlegende Marker in physiologischen und pathologischen Prozessen. Der „Hauptregulator“ der Sialinsäure-Biosynthese ist die bifunktionelle UDP-N-Acetylglucosamin-2-Epimerase/N-Acetylmannosamin-Kinase (GNE/MNK). Das Hauptziel dieser Dissertation war die Identifizierung neuer Inhibitoren der GNE-Subdomäne, die zur Hemmung der Sialinsäurebiosynthese in Zellen und zur Verringerung der Sialylierung der Zelloberfläche eingesetzt werden könnten. Zu diesem Zweck wurde GNE rekombinant exprimiert und eine Screening-Kampagne mit fast 70 000 Verbindungen gegen seine Aktivität durchgeführt. Die primären Treffer aus der Screening-Kampagne wurden anschließend in vitro und in cellulo charakterisiert. Die Inhibitoren haben nicht auf Zellen funktioniert, dafür haben sie in einem neu entwickelten NMR basierten Assay GNE Aktivität im Rattenleberzytosolextrakt inhibiert. / Sialic acids are part of the outermost component of the glycocalyx of all vertebrates. As such, they are fundamental markers in physiological and pathological processes. The "main regulator" of sialic acid biosynthesis is the bifunctional UDP-N-Acetylglucosamine-2-Epimerase/N-Acetylmannosamin Kinase (GNE/MNK). The main objective of this dissertation was the identification of new inhibitors of the GNE subdomain, which could be used to inhibit sialic acid biosynthesis in cells and reduce the sialylation of the cell surface. For this purpose, GNE was recombinantly expressed and a screening campaign against its activity was conducted with nearly 70,000 compounds. The primary hits from the screening campaign were subsequently characterized in vitro and in cellulo. The inhibitors did not work on cells, but they inhibited GNE activity in a rat liver cytosol extract in a newly developed NMR-based assay.

Obesity represents a serious threat to health which can be reduced by volitional control of eating and physical activity behaviour. Social cognition theories propose that such behaviour is influenced by cognitions regarding its desirability. The role of obesity outcome expectancies in predicting weight control behaviour has not been established and there are no psychometrically sound measures of these constructs. This thesis aimed to investigate the relationship between knowledge and beliefs regarding obesity's consequences and weight control Intentions in obese patients. The Obesity Risk Knowledge Scale (ORKS-10) was developed using item analysis and rigorously evaluated in a large population (n=965). The ORKS-10 scale proved to be a short, reliable and valid measure of knowledge regarding the health risks associated with obesity. In addition, thematic analysis of data from focus groups and structured interviews was used to identify 41 salient items for a scale to measure obesity outcome expectancy beliefs. Factor and item analysis were then used to develop the Obesity Outcome Expectancy Beliefs Scale (ObEx-15). The ObEx-15 comprises three reliable and unidimensional subscales; the Health Benefits of Weight Control (HBen), Social and Aesthetic Benefits of Weight Control (SABen) and Costs of and Barriers to Weight Control (Cost). Obese adults were recruited from weight management clinics (n=110, response rate=54.19%). Multiple regression analysis indicated that weight control intentions were most strongly associated with endorsement of the social and aesthetic consequences of obesity (B=0.117, t104=2.314, p<0.05) and rejection of the costs and barriers of weight control (B=0.088, t104=2.273, p<0.05). Participants had low levels of knowledge about obesity's health risks and neither ORKS-10 scores nor HBen scores were associated with intentions. Health promotion might, therefore, benefit from focusing upon obesity's non-health impacts and the costs and barriers of weight control. Future obesity outcome expectancies research will also profit from the availability of psychometrically sound measures.

Polyadenylation is an essential post-transcriptional modification resulting in a mature mRNA in eukaryotes. Three cis-elements the Far Upstream Element (FUE), Near Upstream Element (NUE), and Cleavage Site (CS) - guide the process of cleavage and polyadenylation with the help of multi-subunit protein complexes cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF) along with cleavage factors and poly(A) polymerase. Protein-protein interactions play an important role in the cleavage and polyadenylation process. WD repeat proteins play an important role in protein-protein interactions and have diverse functions in plant system. In the present study WD repeat proteins AtCstF50 and AtFY were studied for their role in polyadenylation process. Mammalian CstF50 is a WD repeat protein that is one of the subunit of CstF that aids in the cleavage step by associating with CPSF and cleavage factors. AtCstF50 was functionally characterized using T-DNA knock-out lines and by identifying the proteins that interacts with it in the process. Results shows that AtCstF50 is essential and was identified as part of CPSF complex, which is different from its mammalian counter part. CPSF was known to interact with Fip (factor interacting with PAP), Poly(A) polymerase and Poly(A) binding protein and AtCstF50 also interacts with these complexes. AtFY is a 3’ end processing factor which contains WD repeats is one of the subunits of the CPSF complex in Arabidopsis polyadenylation machinery. The AtFY interacts with FCA and promotes the alternative polyadenylation and also plays a role in polyadenylation site choice of FCA mRNA. We characterized the FY expression and localization of FY in the cell by fusing with RFP reporter. Results show that FY accumulates in the nucleus while FY with deleted calmodulin binding domain localizes both to the nucleus and outside the nucleus. The individual N-terminal and C-terminal domains also localized in the nucleus suggesting that they are multiple nuclear localization signals in FY and calmodulin might play a direct or indirect role in FY localization. Using a tethering assay we proved that AtFY is able to recruit the 3’ end processing complex in the proximal polyadenylation site choice of the reporter mRNA.

Classical coeliac disease (CD) is a well-defined syndrome of small bowel villous atrophy associated with abdominal pain, malabsorption, and weight loss as a result of gluten-sensitivity, reversed rapidly by gluten exclusion diet. Disease associations include dermatitis herpetiformis (DH), Addison’s disease, type 1 diabetes mellitus, autoimmune thyroid disease and a variety of neurological disorders. This thesis aims to investigate the hypothesis of the existence of a gluten sensitive neurological disease CD with coexistent neurological dysfunction is only rarely reported in a neurological setting. 23 cases were reported from the British Neurological Surveillance Unit (BNSU) over 24 months and 13 locally over 31 months. 18 sets of notes (50%) were reviewed. These patients comprise a heterogeneous group of neurological disorders including epilepsy, myelopathy, axonal neuropathy and migraine. Neurological disorders in patients with confirmed gluten sensitivity may occur simply by chance. In a cohort of 801 CD patients, 54 neurological disorders were identified in 177 patients including stroke (2.9%), migraine (2.7%), epilepsy (2.6%) and carpal tunnel syndrome (2.0%). More detailed investigation of 35 patients with DH and 53 patients with CD, confirmed a low prevalence of idiopathic neurological abnormalities (DH 11%; CD 25%). Analysis of sera from these patients did not identify the presence of anti-neuronal antibodies. A novel anti-spinal antibody was identified in over 50% of the subjects with DH but this requires further characterisation. It has been postulated that patients with idiopathic neurological disease and anti-gliadin antibody (AGA) seropositivity are gluten sensitive. However, AGA lacks disease specificity being found in 10% of healthy blood donors. Screening of 49 unselected multiple sclerosis cases found IgG AGA in 12% of patients and 13% of blood donors confirming that AGA (especially IgG isotype) can be a non-specific finding. AGA, other food antibodies and tissue transglutaminase antibody (TTG) were measured in patients with idiopathic ataxia (20), hereditary ataxia (7) and idiopathic peripheral neuropathy (32). None of the cases was positive for IgA TTG making occult CD unlikely. Cerebellar ataxia with positive AGA (so-called ‘gluten ataxia’) was rare (4 cases in 2 years from a population of 2 million). All food antibodies tested (AGA, hen’s egg albumen, and cow’s milk lactoglobulin), particularly IgG, were a common finding in both ataxia and peripheral neuropathy groups. This study found no evidence for gluten neurotoxicity. Serological tests, particularly AGA, need to be interpreted with caution. Further study is required regarding the nature of the association between neurological illness and gluten sensitivity.

The lower mean levels of total cholesterol, LDL cholesterol, fibrinogen; the higher likelihood of being from more affluent social class; and the small but significant rise in HDL cholesterol and reduction in blood pressure amongst coeliacs presenting with gastrointestinal symptoms observed following treatment with a gluten-free diet suggests coeliacs have favourable vascular risk profile features in comparison to the general population. However, the higher likelihood of having abdominal truncal obesity amongst incident coeliacs that only worsens following treatment with a gluten-free diet together with the higher proportion of measured systolic hypertension amongst male coeliacs suggests that there are also potentially adverse vascular risk profile features associated with celiac disease. Though incident coeliacs with silent disease reported no change in their quality of life prior to diagnosis of coeliac disease, silent coeliacs were as likely to have villous atrophy and physiological derangement to those coeliacs presenting with symptoms or with classic features of coeliac disease. The quality of life reported by coeliacs presenting with silent disease, classic disease and with gastrointestinal symptoms was worse than that observed in the general population. A year's treatment with a gluten-free diet resulted in coeliacs having similar or in some components better quality of life than that observed in the general population. The rate of change of quality of life was similar amongst those coeliacs with silent, classic or symptomatic disease. The breast cancer risk profile suggests both protective and adverse associations of coeliac disease. The higher proportion of women being parous, having their first full-term pregnancy before 30 years and breastfeeding in addition to the younger mean age at menopause suggests women with coeliac disease have favourable breast cancer risk profile features in comparison to the general population. However, the higher likelihood of being Caucasian and of affluent social class together with higher proportion having early menarche and irregular menstrual cycles suggests there are also potentially adverse breast cancer risk profile features associated with celiac disease. Conclusions Persons with mild enteropathy disease have few physiological derangements at diagnosis of coeliac disease and show no important biochemical change following treatment with a gluten-free diet in comparison to those with severe enteropathy coeliac disease. The prevalence of hypertransaminasaemia is lower than previously reported which may be reflective of differences in study design or contemporary coeliac disease involves a milder spectrum of disease. The observed vascular and breast cancer risk profile suggests both protective and adverse associations of celiac disease and on treatment with a gluten-free diet results in an attenuation or indeed reversal of the vascular risk profile in some co-variates. Silent coeliac disease is associated with a reduction with quality in life which improves like in symptomatic and classic disease with treatment with a gluten-free diet. Incident coeliac disease is associated with more affluent social class.

The evidence for genetic contributions to the development of asthma and atopy has been well established. Refining the major genetic factors underlying these contributions will lead to a greater understanding of the pathophysiology of these conditions and potentially identify novel therapeutic targets. This thesis describes a series of studies designed primarily using genome-wide association (GWA) approaches to examine common single nucleotide polymorphisms (SNPs) contributing to these phenotypes in the Caucasian population. Susceptibility to atopy was assessed using both non-parametric association tests of SNPs across the genome and focused analysis of two regions on chromosomes 3p22.1-q22.1 and 17p12-q24.3 previously identified through a meta-analysis of genome-wide linkage studies (GSMA). The discovery cohort consisted of 1,083 cases and 2,770 controls, replication analyses were undertaken in four independent population cohorts. A GWA study of severe asthma was carried out in 933 cases and 3,346 controls with replication in a further 231 cases and 1,345 controls. The contribution of SNPs within all previously reported asthma susceptibility loci identified using a comprehensive literature search was also evaluated. Overall, there is evidence for a large number of loci influencing both atopy and severe asthma, each harbouring modest effects. A number of potentially novel loci meeting nominal significance in both GWA studies have been identified requiring further work. Strong evidence was found to support the IL1RL1-IL33 signalling pathway in asthma pathogenesis. Molecular characterisation of the 5’ untranslated regions of IL1RL1 and IL33 suggest a complex regulatory role of identified common variants involving multiple promoters for IL1RL1. A number of asthma specific variants within the chromosome 2q12 and 9p24.1 regions were detected using next generation re-sequencing in homogenised pools of cases and controls warranting further analyses. This work has identified a potentially important pathway in which to focus the development of effective asthma therapies. Future directions will include functional analysis of replicated variants and tests of interactions between the multiple genetic and environmental factors likely to be involved in disease.

Latest epidemiological data suggests that 1.5 billion adults worldwide are obese or overweight. Excess weight and adipocyte hypertrophy have long been associated with contributing to low-grade systemic inflammation through elevated adipokine secretion. These increased endocrine signals further augment the metabolic dysfunction related to the presence of obesity. A chronic exposure to obesity mediated inflammation is also suggested to be responsible for progression of renal pathology and eventual end-stage organ failure. In human clinical statistics, these factors indicate a gender disparity, as males demonstrate much faster progression rates of obesity-linked renal disease than females. Therefore, the aim of this thesis was to investigate the role of gender in obesity mediated inflammation in the development of renal disease using a large animal model i.e. sheep. Post-natal female and male sheep were exposed to a lean or obesogenic environment by restricting physical activity from ≈3 months to ≈17 months of age. Analysis of body composition and adipose tissue physiology, morphology and deposition identified the development of moderate obesity following chronic exposure to a low physical environment, although no differences were observed with gender. With obesity, both genders demonstrated metabolic irregularities; males showed hyperinsulinaemia and females displayed hypercortisolism. Gene expression analysis identified an up-regulation of inflammatory related genes in perirenal adipose tissue (PAT) and kidney in obese males, a finding not seen in females, although obese females exhibited an up-regulation in glucocorticoid receptor abundance in PAT. Furthermore, the males demonstrated adaptations in renal structure and function with obesity, modifications not observed in females. The main conclusion of my thesis is that after the development of obesity, males appear much more sensitive to the metabolic, inflammatory and renal adaptations associated with an obese condition. Females displayed a down-regulation of inflammatory genes with obesity which I propose acts as a protective mechanism against the progression of renal disease, perhaps mediated by an immunosuppressive glucocorticoid action in adipose tissue. It is also possible that sex hormones play a role in obesity inflammatory renal disease development, postulated to occur through HPA activation and epigenetic alterations.

Obesity continues to be a major cause of morbidity and mortality and worldwide prevalence rates continue to rise. The cornerstone for treating obesity remains diet and lifestyle, with the ultimate goal being normalising those parameters that are associated with ill health, for example hyperinsulinaemia and insulin resistance. Because obesity predominantly develops due to a mismatch between energy intake and utilisation, this thesis looked at the effects of dietary interventions upon Resting Energy Expenditure (REE) and substrate oxidation. In addition, the impact of popular dietary interventions upon body composition and insulin resistance was examined. When phenotypic characteristics were investigated before and after weight loss by using hypocaloric diets, which differed in fat and carbohydrate content, Fat-Free Mass (FFM) and Fat Mass (FM), were strong predictors of REE before and after the intervention and weight loss rather than the specific dietary intervention, significantly predicted post intervention REE. Fasting fat oxidation was found to be lower in obese subjects and they had a lower postprandial response to a high fat challenge. This implied that a diet high in fat is more likely to promote a positive energy balance an ultimate weight gain. The final study compared 4 popular dietary interventions. Each was equally effective at achieving clinically significant weight loss and improvements in insulin sensitivity. Although none was significantly more superior, there was a trend supporting three of the diets (Atkins’, Weight Watchers and Rosemary Conley) above the other (Slim-Fast) and it was the pattern of weight loss, i.e. mainly loss of FM, which proved beneficial with regards to improving insulin sensitivity. In summary, this thesis confirms that REE is mainly predicted by FFM and FM and that there is diminished fat oxidation on obese subjects. What this thesis also adds to previous research that it if a specific diet can improve the pattern of weight loss, this can be clinically beneficial.

Background The development of serological tests for the diagnosis of coeliac disease, including tests for endomysial and tissue transglutaminase antibodies, has made population screening for coeliac disease a realistic possibility. Several serological screening studies from European countries have shown that as many as 1% of the general population may have undetected coeliac disease. The implications of this diagnosis are unclear since the only data on the morbidity and physiological characteristics associated with previously undetected disease come from small, selected, case series. Most adult screening studies in the general population have identified only small numbers (i.e. less than 20 cases) of previously undetected cases and have therefore been unable to examine these issues through lack of statistical power. Clinically diagnosed coeliac disease has traditionally been linked with a variety of adverse co-morbid conditions including osteoporosis, non-Hodgkin’s lymphoma and an increased mortality in general. These conditions are thought to be partly a consequence of the altered nutritional status associated with the malabsorption that occurs with villous atrophy of the small bowel in coeliac disease. Although some of the adverse effects of, for example, vitamin and calcium deficiencies in coeliac disease have previously been explored whether there may be potentially beneficial effects of mild malabsorption have not. There are two main aspects in this thesis. The first is to estimate the prevalence of undetected coeliac disease in England and explore the important physiologic correlates of this condition. The second is to examine the risk of fracture, vascular disease, malignancy and mortality in people with diagnosed coeliac disease compared to the general population. Objectives 1. To estimate the seroprevalence of undetected coeliac disease in England. 2. To explore the relationship between undetected coeliac disease and various socio-demographic characteristics and physiological measures. 3. To quantify the impact of diagnosed coeliac disease (compared to the general population) on the risk of: a. Fracture b. Vascular disease (hypertension, high cholesterol, atrial fibrillation, myocardial infarction and stroke) c. Malignancy and mortality Methods To examine objectives 1 and 2 I utilised the Cambridge General Practice Health Study. This study identified individuals aged 45-76 registered with 12 general practices and invited them to complete a health survey, have a bone density measurement and submit a blood sample between 1990 and 1995. Serum samples from 7550 participants were tested for antiendomysial antibody (EMA). Seroprevalence of undetected coeliac disease was defined by EMA positivity. Differences between EMA positive and negative participants of various physiological measures and reported characteristics were estimated using multivariate logistic and linear regression and adjusted for age, gender, social class and smoking behaviour. To examine objective 3 I performed a population based cohort study using the General Practice Research Database to quantify the risk of fracture, vascular disease, malignancy and mortality in people with coeliac disease compared to the general population. I identified 4732 people with coeliac disease and 23620 age and sex matched control subjects. I used Cox regression to estimate hazard ratios for fracture, myocardial infarction, stroke, malignancy and mortality, and conditional logistic regression to estimate the risk of diagnosed hypertension, hypercholesterolaemia and atrial fibrillation, in people with coeliac disease compared to the general population. Findings The studies show that undetected coeliac disease is likely to affect about 1% of the population of England aged 45-76, a figure similar to several other countries. Those affected more commonly reported “good or excellent health”, however they do have an increased risk of osteoporosis and mild anaemia. In contrast they have a favourable cardiovascular risk profile including lower serum cholesterol and blood pressure. In people with clinically diagnosed coeliac disease, compared to the general population, there were small increases in both the absolute and relative overall fracture incidence with a 2-fold increase in the risk of hip fracture. Adults with treated coeliac disease did have a favourable vascular disease risk factor profile but numbers having heart attacks or strokes were modest and rates of heart attack and stroke were not reduced. There were modest increases in the overall risks of malignancy and mortality in people with coeliac disease and most of this excess risk occurred in the first year of follow up after diagnosis, suggesting ascertainment bias. I found a marked reduction in the risk of breast and lung cancer in people with coeliac disease and the mechanism of this merits further attention as it may provide insight into the aetiology of these common malignancies. Conclusions I found that approximately 1% of general adult population of the UK has undetected coeliac disease. The findings suggest that although coeliac disease is associated with some adverse conditions; it may also have some beneficial health effects. Please note: This version does not include the copies of journal articles which were included in the original thesis, but just details of the articles.