Introduction: Cardiovascular disease (CVD) is the leading cause of global mortality and its prevalence is increasing among black South Africans in spite of their favourable lipid profile. Apolipoprotein E (ApoE) is a well-described risk factor for CVD and certain polymorphisms within this gene alter the lipid profile. The author hypothesised that there are population-specific effects within the ApoE gene that are responsible for the favourable lipid profile observed in black South Africans whose effects are being altered by environmental factors. Objectives: The main aim of this study was to investigate the associations between specific ApoE single nucleotide polymorphisms (SNPs) and the lipid profile of a black South African population, taking into account certain environmental and phenotypic factors in order to explore the interaction effects between these variables. Methods: Genotyping within this cross-sectional study (n=1 588), nested within the Prospective Urban and Rural Epidemiology (PURE) study, was achieved using Illumina‘s® GoldenGate Genotyping Assay with VeraCode® technology on the BeadXpress® platform (proprietary multiplex fluorescent hybridisation assays on a bead array substrate) or the Bio-Rad CFX Manager© (version 2.0). The Konelab20i™ auto analyser was used for quantitative determination of serum total cholesterol; high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) concentrations. Low-density lipoprotein cholesterol concentrations were estimated by the Friedewald equation. Results: All SNPs adhered to the assumptions of the Hardy-Weinberg equilibrium, yet the frequency of the SNPs often differed from that reported in other ethnic groups. The well-reported rs429358 and rs7412 SNPs (as the constituent SNPs of the haplotype-genotypes) presented with the strongest associations with various components of the blood lipid profile in the black South African cohort under investigation. Two gene-environment (rs405509 and rs7412) interaction effects on TG remained significant after conducting post hoc tests. Two genotype-phenotype interaction effects between the rs7412 SNP and body mass index and gamma-glutamyl transferase on the HDL-C concentrations remained significant after conducting post hoc tests. Conclusions: The variety of associations between these particular SNPs and the blood lipid profile determined in the present cohort strongly indicates that it is integral to any public health investigation into CVD development that these SNPs be investigated. This study further produced greater insight into the biological mechanisms underlying serum lipid and cholesterol concentrations in a black South African population. Therefore, from these results it is evident that the lipid profile of black South Africans is most definitely influenced by not only genetic variations in the ApoE gene and certain environmental factors, but by the interaction between these factors as well. The present study is the largest study to date to investigate the effect of polymorphisms in the ApoE gene on the lipid profile of black South Africans. / MSc (Nutrition), North-West University, Potchefstroom Campus, 2014

Skeletal muscles in broilers are generally characterised by pathological muscle damage, indicated by greater plasma creatine kinase (CK) activity, higher incidence of haemorrhages, lighter and less coloured breast muscles, compared with layers and traditional breeds of chicken. Muscle damage is further exacerbated by exposure to stressful conditions such as high ambient temperatures which results in a further decrease in the quality of broiler meat and leads to the production of pale, soft and exudative (PSE) meat. This growing incidence of poor quality poultry meat is causing substantial losses to the meat industry. However, in contrast to pork the genetics of poor muscle and meat quality in chicken is unknown. The present project was conducted to identify the underlying genetics of this low quality meat by using heat-stress as a tool to amplify muscle damage and expression of the relevant genes. Whole-genome expression studies in broiler and layer breast muscles were conducted before and after heat-stress and some phenotypic data were also recorded. From the gene expression studies, 2213 differentially expressed genes (P<0.05) were found. About 700 of these genes had no gene ontology (GO) terms associated with them for biological process or function. The significant gene set was analysed in BioLayout Express and interesting clusters of the genes, based on their positive correlation with each other, were selected for further investigation. Genes were grouped together in 6 different categories or clusters, on the basis of their expression pattern. The genes in the selected clusters were analysed in Ingenuity Pathway Analysis (IPA) software, for each category separately, and relevant biological pathways and networks for those genes were studied. Similarly, the genes filtered out by BioLayout Express at a Pearson threshold of 0.80 were also analysed in IPA separately and interesting pathways and networks were selected. From the pathways and networks analyses of these genes, it was discovered that genes involved in inflammatory, cell death, oxidative stress and tissue damage related functions were up-regulated in control broilers compared with control and similar to heat-stressed layers. After exposure to heat-stress the expression levels of these genes were further increased in broilers. These results led us to develop the hypothesis that breast muscles in broilers are under stress-related damage even under the normal rearing conditions. This hypothesis was tested by rearing the broilers birds at normal/conventional and comparatively low ambient temperature and its effects on breast muscle quality and meat quality were studied. Significant improvement of breast muscle redness was observed. Additionally substantial numerical improvements for other meat and muscle quality traits like breast muscle lightness and histopathology were observed. From the key positions of interesting significant pathways and networks, candidate genes were selected for further investigation. In total, 25 candidate genes were selected for SNP genotyping: 19 genes were selected from the interesting pathways and networks and 6 genes were selected on the basis of their GO terms. For each gene 4-5 SNPs were selected, where possible, that were present in exons and promoter regions of the candidate genes. The selected SNPs were genotyped for muscle and meat quality traits in 34 breeds of chicken and significant causative SNPs for each trait including plasma CK activity, pHi and pHu for breast muscles, colour (L*, a*, and b*) traits for breast and thigh muscles were found. These SNPs were responsible for explaining a moderate to high (15-55%) percentage of phenotypic variance for these traits. To our knowledge this is the first study in which gene-expression in chicken breast muscle was conducted in response to heat-stress and additionally, for the first time, a set of novel SNPs for all of these traits were identified. Some of the significant causative SNPs were lying in the protein coding sequences and some were present in the promoter regions of the candidate genes.

This thesis addresses the dissection and characterisation of quantitative trait loci (QTL) affecting production traits in sheep. Firstly, the association between specific genetic polymorphisms and complex variation in weight, muscle and fat depositions was investigated. Research concentrated on assessing the presence, correspondence and significance of two single nucleotide polymorphisms (SNPs) in the GDF8 region of ovine chromosome 2, reportedly affecting muscle production. Commercial populations of British Texel, Suffolk and Charollais sheep were studied. The SNPs were absent in Suffolk and almost fixed in Texel breeds. In the Charollais population, the SNPs segregated at intermediate frequencies and a significant association was found between these polymorphisms and muscle depth. The previously proposed causative allele at one of the loci resulted in increased muscle depth and, at allele frequency of 0.5, this locus would explain one third of the additive genetic variance for the trait. Partial recessive allelic expression is proposed by genotypic value predictions and is consistent with the previously postulated molecular mechanism by which it gives rise to muscle changes. Secondly, the thesis focused on detection of QTL associated with growth. Live weight is a composite of growth rates over time, with inter-age genetic correlations for live weight decreasing as time between weight measurements increases. To explore whether observed genetic correlation patterns translate into distinct loci acting on weight at different growth stages, a novel method was developed and the applicability of a second proposed method was explored. Both methods allowed simultaneous analysis of multiple live weights per animal, while accounting differently for the correlation among measurements ordered in time. In the first approach, a growth curve technique was developed and employed to map growth QTL for curve parameters and predicted growth descriptors. A study of actual live weights identified significant QTL at different ages on distinct chromosomes, with QTL significance and variance changing over time. Further application of this technique on a simulated dataset validated its effectiveness in detecting age-dependent QTL. An extension of the procedure resulted in a novel technique for genomic evaluation of longitudinal traits. In the second method examined, random regression (RR) models were applied for dissection of growth QTL. Systematic model selection and inclusion of relevant random effects resulted in apparently significant QTL, but the method was computationally demanding, model choice proved challenging and the results were questioned. To further explore the method, RR models were applied to various simulated growth phenotypes composed of time-dependent QTL trajectories, polygenic and environmental effects. Statistically optimal RR models succeeded in identifying significant QTL and predicting the simulated time-dependence for most scenarios. However, the issue of model choice was again prominent, as suboptimal models resulted in unreliable QTL variance trajectories and pronounced confounding between different time-dependent effects. Thus, the growth curve approach appeared to be the more flexible and robust process for analysing longitudinal data to map agedependent QTL.

Aims: The p53 regulatory network is crucial in directing the suppression of cancer formation and mediating the response to commonly used cancer therapies. Functional genetic variants in the genes comprising this network could help identify individuals at greater risk for cancer and patients with poorer responses to therapies, but few such variants have been identified as yet. Methods: We first develop and apply three different screens that utilize known characteristics of functional single nucleotide polymorphisms (SNPs) in the p53 network to search for variants that associate with allelic differences in (i) recent natural selection, (ii) chemosensitivity profiles, and (iii) the gender- and age- dependent incidence of soft-tissue sarcoma. Secondly, we study and explore the functional mechanisms associated with the identified variants. Results: We identify SNPs in the PPP2R5E, CD44, YWHAQ and ESR1 genes that associate with allelic differences in the age of tumour diagnosis (up to 32.5 years, p=0.031), cancer risk (up to 8.1 odds ratio, p=0.004) and overall survival (up to 2.85 relative risk, p=0.011) in sarcomas, ovarian and pancreatic cancers, and exhibit allelic differences in the cellular responses to cytotoxic chemotherapeutic agents (up to 5.4-fold, p=5.6x10^-47). Lastly, we identify candidate causal SNPs in those genes and describe the regulatory mechanisms by which they might affect human cancer. Conclusions: Together, our work suggests that the inherited genetics of the p53 pathway have a great potential to further define populations in their abilities to react to stress, suppress tumor formation and respond to therapies.

Abstract FUNCTIONAL AND BIOCHEMICAL CONSEQUENCES OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE HUMAN VESICULAR MONOAMINE TRANSPORTER 1 GENE (SLC18A1) By Sally Gamal Shukry, B.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology at Virginia Commonwealth University. Virginia Commonwealth University, 2012 Major Advisor: Jennifer K. Stewart Associate Professor and Graduate Director, Department of Biology Single nucleotide polymorphisms (SNP) in the human VMAT1 gene (SLC18A1) have been associated with schizophrenia in three different populations: Han Chinese, Western European and Japanese. Effects of these mutations on transport function of the hVMAT1 protein have not been reported. The goal of this study was to investigate functional and biochemical differences in human VMAT1 proteins with a threonine or proline at amino acid position 4 (Thr4Pro) and a serine or threonine at position 98 (Ser98Thr). COS1 cells were transfected with variant SNPs coding for 4Thr/98Ser, 4Pro/98Ser, or 4Thr98Thr. Western blotting demonstrated robust over expression of the genes and no differences in electrophoretic mobility of the proteins. Maximal transport of serotonin by the VMAT1 protein with 4Pro/98Ser was less than that of the 4Thr/98Ser or the 4Thr/98Thr. Response of the 4Pro/Ser98 to the VMAT inhibitor reserpine was lower than that of the 4Thr/98Thr. These findings suggest mechanisms for human VMAT1 links to schizophrenia.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Jana Jeřábková Supervisor: Doc. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Interaction of selected anthocyanidins with farnesoid X receptor Human farnesoid X receptor (FXR) is a member of nuclear receptor superfamily that act as ligand-activated transcription factors. FXR binds to specific regulatory DNA regions and induces expression of many target genes. These regulated genes are involved in bile acid metabolism and transport, maintaining blood lipids, liporoteins and glucose homeostasis and also contribute to maintain intestinal bacterial balance, hepatoprotection and liver regeneration. The interest of recent studies is to test the range of FXR ligands for treatment and prevention of many diseases such as cholestais, cholesterol gallstone disease, steato-hepatitis, dyslipidemia, atherosclerosis, type 2 diabetes mellitus, metabolic syndrome, liver cancer and other forms of cancer such as breast cancer. In this experimental diploma thesis we are focused on testing of potencial ligands of human farnesoid X receptor from the group of natural plant pigments anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin) using the human hepatoma cell line...

Vitamin D is central in calcium turnover, and adequate levels are important for skeletal health. It is not clear how large contributions from food and sunlight are in Swedish primary care patients, considering the low radiation of UVB in Sweden and fortification of some foods, and whether differences exist between patients of immigrant and Swedish origin. Increasing incidence of osteoporosis-related fractures is a major global health problem. Genetic variations in metabolising enzymes and in the Vitamin D receptor (VDR) have also been shown to be of importance to the overall effect of vitamin D. Polymorphic variation in the gene CYP2R1 encoding the 25-hydroxylase has previously been reported to correlate with circulating levels of 25(OH)D3. Results of association studies between genetic variants of the VDR and muscle strength, as well as falls have been contradictory. The purposes of this thesis were to examine possible differences in plasma-25(OH)D3 levels and intake of vitamin D between Swedish and immigrant female primary care patients, to estimate what foods contribute the most, and to identify contributors to vitamin D status (Paper I-II). Furthermore, the relationship between polymorphisms in the CYP2R1 gene and levels of 25(OH)D3 as well as other biochemical parameters (parathyroid hormone, calcium, phosphate and fibroblast growth factor 23) of skeletal homeostasis, bone mineral density and incidence of fractures was investigated (Paper III). Also, the association between genetic variations in the gene for the vitamin D receptor and measures of muscle strength, physical performance and falls (Paper IV), was investigated by using data from a Swedish multicenter study of elderly men (MrOS). Most important results: Vitamin D deficiency was common, with significant difference between Swedish born and immigrant patients (Paper I). Food intake of vitamin D is associated with circulating vitamin D, but the factors most strongly affecting vitamin D levels were reported sun holiday and origin (Paper II). CYP2R1 polymorphisms are associated with circulating levels of 25(OH)D3 and bone mineral density (Paper III). VDR genetic variants do not appear to have a direct effect on muscle strength or physical performance and incidence of falls in elderly Swedish men (Paper IV).

A number of polymorphisms in the human VDR gene (located on chromosome 12q12-14) have previously been associated with TB susceptibility, of which most commonly, FokI, BsmI, ApaI and TaqI. The active metabolite of vitamin D, 1,25 dihydroxyvitamin D3, is an important immunoregulatory hormone. Its effects are exerted via the VDR, which is present on human monocytes and activated T and B-lymphocytes. Considering the role of the VDR and it specific immunological functions, including activation of monocytes, stimulation of cell-mediated immunity, suppression of lymphocyte proliferation, immunoglobulin production and cytokine synthesis, variation in the VDR genes may contribute to disease susceptibility. Hypothesis and objectives: In this study it was hypothesized that polymorphisms in the vitamin D receptor (VDR) gene influence TB susceptibility in the Venda population of South Africa. The role of VDR polymorphisms in TB susceptibility was investigated by setting the following objectives: 1. Typing of VDR gene polymorphisms (FokI, BsmI, ApaI and TaqI) in controls and TB cases as well as family samples from a Venda population of the Northern Province of South Africa. Comparing VDR allele and genotype frequencies between the Venda population and other populations as reported in the literature. 2. Analysis of possible associations in the case-control and family-based study 3. Studying Linkage disequilibrium (LD) in the case-control and the family samples. 4. Screening VDR haplotypes in the case-control and family-based study for possible association with TB. Patient and laboratory methods: VDR polymorphisms were studied in controls (n=110), TB cases (n=113) and 25 families in the Venda population inhabiting the Northern Province of South Africa. Case-control studies are vulnerable to false positive results caused by inadequate matching of the two groups. For this reason, a withinfamily association test was also implemented (the transmission disequilibrium test, TDT), as this is robust to this possible source of error. ARMS-PCR was used to type the VDR allele polymorphisms. The Statistical Package for Social Sciences (SPSSv10) was used to analyze possible association in case-controls, while TRANSMIT was used to analyze transmission of VDR alleles to affected offspring. LD between the single nucleotide polymorphisms (SNPs) of the VDR gene was investigated using the software “Graphical Overview of Linkage Disequilibrium” (GOLD) in both case-control and family studies. Possible association of TB with haplotypes comprising the VDR SNPs, ApaI and TaqI, were determined using SPSSv10 and TRANSMIT respectively for case-controls and the family based study. Results: 1. The comparative analysis indicated that the allele frequencies of the VDR SNPs related to the African American population. Genotype frequencies for FokI, BsmI and TaqI were similar to the West African population, whereas ApaI did not relate to any of the populations investigated. 2. No significant differences existed concerning the allele frequencies or genotype distribution between controls and TB cases with regard the VDR SNPs; FokI, BsmI and TaqI. The ApaI allele ‘A’ differed between cases and controls (p=0.041) but, no significant differences were obtained for genotype distribution between cases and controls for this SNP. All SNPs were in Hardy-Weinburg equilibrium except for ApaI. No differences between controls and cases were observed concerning allele presence or absence. In the family-based study, no significant difference was observed between observed and expected numbers of transmission for neither alleles nor genotypes, although, a borderline association was observed between TaqI ‘T’ allele and TB susceptibility (p=0.084). 3. LD in the case-control study was found between the VDR SNPs: BsmI and ApaI (p=0.051, D’=0.478), BsmI and TaqI (p=0.007, D’=0.222) and ApaI and TaqI (p=0.003, D’=0.479). In the family-based study, LD was only observed between ApaI and TaqI (p=0.049, D’=0.001) 4. Haplotype analysis in the case-control study indicated that no significant difference between controls and TB cases was observed. The family-based study indicated that transmission of the VDR haplotype ‘AT’ and ‘at’ differed from the expected transmission, but was of borderline significance. ‘AT’ was transmitted more than expected (P=0.090) and ‘at’ was transmitted less than expected (P=0.075), indicating that ‘AT’ may play a role in TB susceptibility and ‘at’ may play a protective role. Conclusion: The current study supports a possible role for VDR polymorphisms, ApaI (‘A’, p=0.041), or nearby yet unidentified markers in TB susceptibility in African populations (FokI-ApaI-BsmI-TaqI) (p=0.049). A larger sample size may clarify associations found to be of borderline significance. Results obtained may contribute to an improved understanding of genetic susceptibility factors in Africans. / Prof. L. Bornman

La coévolution ainsi que les différentes interactions entre hôte et pathogène contribuent à former la diversité génétique de ces deux organismes. Dans le cadre de cette thèse, nous nous sommes intéressés à l’étude de la variabilité génétique de 1760 gènes immunitaires choisis suivant des critères définis, pour essayer d’expliquer pourquoi il existe une variation individuelle face aux infections. L’objectif principal de ce projet était alors de caractériser et d'analyser de nouveaux gènes et polymorphismes immunitaires pouvant expliquer le contrôle ou la susceptibilité à certaines infections. Deux études pilotes nous ont permis de développer le pipeline de détection de polymorphismes. Pour la première, le polymorphisme des 3 gènes CD28, CTLA4, et ICOS a été caractérisé. Dans la deuxième, nous avons caractérisé le polymorphisme de 10 gènes impliqués dans la réponse immunitaire contre M. tuberculosis. Ces gènes ne sont pas très polymorphes et trois d’entre eux sont très conservés. Ces deux études nous ont aidés à préparer l’analyse à grande échelle avec les mises au point et l’amélioration du pipeline. Nous avons sélectionné 1760 gènes en se basant sur des critères définis. La variabilité génétique a été étudiée dans les populations humaines par une analyse minutieuse in silico de données de séquençage d’exomes générées par différents projets et consortiums pour plus de 700 individus représentant 20 populations à travers le monde. 30 gènes les plus polymorphes ont été ainsi identifiés. Ces gènes pourront être entièrement caractérisés et les données produites pourraient être comparées avec des données de résistance/sensibilité de certaines maladies infectieuses. / Host-pathogen co-evolution and interactions contribute in shaping the genetic diversity of both organisms. The objective of this thesis is to define the genetic basis of variability in disease resistance/susceptibility through the development of large-scale in silico screens to identify novel gene candidates implicated in host-pathogen interactions (such as tuberculosis).A pilot study was conducted on CD28, CTLA4, and ICOS to investigate their polymorphism. As a first step in our study based on data available in the literature, we selected a set of ten genes relevant for the immune response against M. tuberculosis. Seven of these genes were moderately polymorphic, while three of them were highly conserved. This analysis was used to prepare and setup the large scale analysis using the same developed pipeline for polymorphism detection and allele reconstruction. For our in silico, we used sequence data from several projects and consortiums to isolate most polymorphic human genes amongst a list of over 1760 candidates selected based on already established relevance for infections and on evolutionary considerations. A first screen of 64 individuals from eight different populations from several regions of the world was performed and most variable genes were selected for further extensive analyses on a larger panel (715 individuals). 30 most polymorphic genes were thus identified. The extent of polymorphism and the allelic worldwide variants of each of these 30 genes are ready to be fully characterized. The data generated could be compared against infectious disease resistance/susceptibility data to identify potentially relevant gene variation.

No description available.