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INTRODUCTION AND AIM Black South Africans are experiencing an increase in the prevalence of cardiovascular disease (CVD). Fibrinogen functionality, including total and gamma prime (y’) fibrinogen concentration, as well as fibrin network structure, play an important role in CVD development and events. Several genetic and environmental factors influence fibrinogen functionality, and in turn, known CVD risk factors associated with total and y’ fibrinogen concentration have also been associated with altered fibrin clot structure. However, the main body of evidence regarding the role of fibrinogen functionality in CVD is based on studies conducted in white ethnicities and/or in vitro. The main aim of this study was, therefore, to determine the relationship between fibrinogen functionality and CVD in black South Africans in a plasma setting. Since there is greater genetic diversity in Africans than in non-black ethnicities, it was also our objective to investigate the influence of genetic polymorphisms in determining fibrinogen synthesis and plasma clot properties, and to determine possible gene-environment interactions altering clot properties. PARTICIPANTS AND METHODS The South African arm of the Prospective Urban and Rural Epidemiology (PURE) study included 2010 apparently healthy black men and women between the ages of 35 and 65 years, residing in rural or urban settlements. Blood samples were collected from the participants during a 12-week period in 2005. The following variables were analysed: total and y’ fibrinogen concentration, CVD risk factors and genetic polymorphisms in the fibrinogen and Factor XIII genes as well as turbidimetric analysis of clot formation and lysis (expressed as clot lysis time (CLT)). RESULTS Increased plasma levels of both total (largest contribution of 33%) and y’ fibrinogen were associated with increased fibre diameter while y’/total fibrinogen ratio had the opposite effect. The rate of lateral aggregation of fibrin fibres (slope) increased with an increase in total fibrinogen concentration, but not fibrinogen y’. Increasing fibrinogen y’ concentration was associated with longer CLTs and was the largest contributor to its variance (12%). Increased total and y’ fibrinogen were significantly associated with increased waist circumference, body mass index, C-reactive protein (CRP), glycosylated haemoglobin, metabolic syndrome (MetS) and low high-density lipoprotein (HDL) cholesterol levels. Furthermore, the association of fibrinogen y’ with these CVD risk factors was independent of total fibrinogen levels. C-reactive protein was the largest contributor to variance in fibrinogen y’ levels and y’/total fibrinogen ratio (apart from total fibrinogen). We observed significant associations between single nucleotide polymorphisms (SNPs) at rs1049636 and rs2070011 loci and increased total and y’ fibrinogen levels, respectively. Only SNP rs1800787 was associated with clot properties (increased maximum absorbance). Significant gene-environment interactions were observed between SNPs rs2227385, rs1800787, rs1800788, rs4220 and rs5985 and total and/or y’ fibrinogen levels in determining clot properties. The CVD risk factors age, MetS, CRP, HDL-cholesterol and homocysteine associated significantly with clot properties, independent of total and/or y’ fibrinogen plasma concentration. CONCLUSION The results of this thesis provide several novel insights relevant to this research field. Plasma y’ fibrinogen concentration and y’ ratio were found to be associated with altered clot properties in a plasma setting, and are also influenced by CVD risk factors other than fibrinogen. The associations between SNPs, total and y’ fibrinogen and clot properties differ somewhat from evidence reported in white populations. Significant gene-environment interactions between SNPs and total and y’ fibrinogen in determining clot properties existed and had opposing effects, i.e. both prothrombotic and antithrombotic, suggesting that the influence of genetic factors on fibrinogen should focus not only on concentration, but also on functionality. Cardiovascular disease risk factors also influence clot properties in vivo, through mechanisms independent of total and/or y’ fibrinogen concentration. / PhD (Nutrition), North-West University, Potchefstroom Campusm, 2015

INTRODUCTION AND AIM Black South Africans are experiencing an increase in the prevalence of cardiovascular disease (CVD). Fibrinogen functionality, including total and gamma prime (y’) fibrinogen concentration, as well as fibrin network structure, play an important role in CVD development and events. Several genetic and environmental factors influence fibrinogen functionality, and in turn, known CVD risk factors associated with total and y’ fibrinogen concentration have also been associated with altered fibrin clot structure. However, the main body of evidence regarding the role of fibrinogen functionality in CVD is based on studies conducted in white ethnicities and/or in vitro. The main aim of this study was, therefore, to determine the relationship between fibrinogen functionality and CVD in black South Africans in a plasma setting. Since there is greater genetic diversity in Africans than in non-black ethnicities, it was also our objective to investigate the influence of genetic polymorphisms in determining fibrinogen synthesis and plasma clot properties, and to determine possible gene-environment interactions altering clot properties. PARTICIPANTS AND METHODS The South African arm of the Prospective Urban and Rural Epidemiology (PURE) study included 2010 apparently healthy black men and women between the ages of 35 and 65 years, residing in rural or urban settlements. Blood samples were collected from the participants during a 12-week period in 2005. The following variables were analysed: total and y’ fibrinogen concentration, CVD risk factors and genetic polymorphisms in the fibrinogen and Factor XIII genes as well as turbidimetric analysis of clot formation and lysis (expressed as clot lysis time (CLT)). RESULTS Increased plasma levels of both total (largest contribution of 33%) and y’ fibrinogen were associated with increased fibre diameter while y’/total fibrinogen ratio had the opposite effect. The rate of lateral aggregation of fibrin fibres (slope) increased with an increase in total fibrinogen concentration, but not fibrinogen y’. Increasing fibrinogen y’ concentration was associated with longer CLTs and was the largest contributor to its variance (12%). Increased total and y’ fibrinogen were significantly associated with increased waist circumference, body mass index, C-reactive protein (CRP), glycosylated haemoglobin, metabolic syndrome (MetS) and low high-density lipoprotein (HDL) cholesterol levels. Furthermore, the association of fibrinogen y’ with these CVD risk factors was independent of total fibrinogen levels. C-reactive protein was the largest contributor to variance in fibrinogen y’ levels and y’/total fibrinogen ratio (apart from total fibrinogen). We observed significant associations between single nucleotide polymorphisms (SNPs) at rs1049636 and rs2070011 loci and increased total and y’ fibrinogen levels, respectively. Only SNP rs1800787 was associated with clot properties (increased maximum absorbance). Significant gene-environment interactions were observed between SNPs rs2227385, rs1800787, rs1800788, rs4220 and rs5985 and total and/or y’ fibrinogen levels in determining clot properties. The CVD risk factors age, MetS, CRP, HDL-cholesterol and homocysteine associated significantly with clot properties, independent of total and/or y’ fibrinogen plasma concentration. CONCLUSION The results of this thesis provide several novel insights relevant to this research field. Plasma y’ fibrinogen concentration and y’ ratio were found to be associated with altered clot properties in a plasma setting, and are also influenced by CVD risk factors other than fibrinogen. The associations between SNPs, total and y’ fibrinogen and clot properties differ somewhat from evidence reported in white populations. Significant gene-environment interactions between SNPs and total and y’ fibrinogen in determining clot properties existed and had opposing effects, i.e. both prothrombotic and antithrombotic, suggesting that the influence of genetic factors on fibrinogen should focus not only on concentration, but also on functionality. Cardiovascular disease risk factors also influence clot properties in vivo, through mechanisms independent of total and/or y’ fibrinogen concentration. / PhD (Nutrition), North-West University, Potchefstroom Campusm, 2015

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

Discriminant analysis is a procedure for identifying the relationships between qualitative criterion variables and quantitative predictor variables. Data bases of genetic polymorphisms are currently available that group such polymorphisms by ethnic origin or nationality. Such information could be useful to entities that base financial determinations upon predictions of disease or to medical researchers who wish to target prevention and treatment to population groups. While the use of genetic information to make such determinations is unlawful in states and confidentiality and privacy concerns abound, methods for human “redlining” may occur. Thus, it is necessary to investigate the efficacy of the relationship of certain genetic information to ethnicity to determine if a statistical analysis can provide information concerning such relationship. The use of the statistical technique of discriminant analysis provides a tool for examining such relationship.

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

Fatores microbianos, mecânicos e intrínsecos ao hospedeiro são os responsáveis pelo insucesso endodôntico e necessidade de reintervenção. Polimorfismos genéticos são diferenças na sequência do DNA humano que influenciam na susceptibilidade do organismo frente a doenças e nas suas respostas ao meio ambiente. O objetivo do presente estudo foi investigar o envolvimento de aspectos moleculares e clínicos na resposta do hospedeiro frente ao tratamento endodôntico, por meio da análise da expressão e frequência de polimorfismos dos genes reguladores do processo de remodelação óssea em pacientes submetidos ao tratamento endodôntico. Pacientes que apresentaram necrose pulpar e lesão periapical instalada no momento do tratamento endodôntico foram chamados para consulta de acompanhamento. Foram incluídos no presente estudo 150 pacientes, que apresentaram tratamento concluído há no mínimo um ano antes da consulta de acompanhamento. Desse total, 64 pacientes apresentaram sinais e sintomas clínicos/radiográficos indicativos de lesões periapicais persistentes, e 86 indivíduos apresentaram reparação da lesão. No momento do acompanhamento, foram coletas amostras de saliva dos pacientes como fonte de DNA genômico, o qual foi extraído a partir do pellet de células sedimentado e genotipado para RANK (rs3826620), RANKL (rs9594738) e OPG (rs2073618) por PCR em tempo real. A frequência dos genótipos e alelos foi avaliada por meio da razão de chance (odds ratio), teste do qui-quadrado ou teste exato de Fisher, utilizando os softwares Epi Info 3.5.2 e Graphpad Prism. O tempo de acompanhamento após o tratamento foi utilizado como covariável na análise de regressão logística múltipla para cada um dos polimorfismos. O nível de significância estabelecido foi de 5%. Foi observada associação na distribuição dos alelos do polimorfismo em RANK entre os grupos (p=0,04). No polimorfismo em RANKL, a distribuição dos genótipos apresentou diferença estatisticamente significante entre os grupos (p=0,05). O tempo de acompanhamento esteve associado aos casos de lesões periapicais persistentes para cada um dos polimorfismos analisados (p<0,05), sendo que na análise ajustada pelo tempo como covariável, os polimorfismos rs3826620 em RANK (p=0.02) e rs9594738 em RANKL (p=0.03) continuaram associados a lesões periapicais persistentes. O polimorfismo rs2073618 em OPG não foi associado a distribuição dos grupos em nenhuma das análises (p>0,05). Conclui-se que polimorfismos em RANK e RANKL estão associados ao risco do desenvolvimento de lesões periapicais persistentes / Microbial, mechanical and related to host response factors are responsible for endodontic failure and requirement for reintervention. Genetic polymorphisms are differences in the sequence of human DNA that influence in the susceptibility to diseases and host response to the environment. The aim of this study was to evaluate the association between molecular and clinical aspects in the host response to root canal therapy (RCT), by analyzing the expression and frequency of polymorphisms of genes regulating the bone remodeling process in patients submitted to endodontic treatment. Patients that presented pulp necrosis and apical periodontitis at the time of RCT, with at least 1 year of follow-up after RCT were recalled. Sixty-four subjects with signs/symptoms of PAP and 86 subjects with root canaltreated teeth exhibiting healthy perirradicular tissues (healed) were included. At the time of follow-up visit, saliva samples from patients were collected as a source of genomic DNA, which was extracted from the pellet of sedimented cells and used for RANK (rs3826620), RANKL (rs9594738) and OPG (rs2073618) genotyping by real-time PCR. Genotype and allele frequencies were compared by chi-square test or Fishers exact tests and odds ratio was implemented, using Epi Info 3.5.2 and Graphpad Prism. A logistic regression analysis was also performed using time of follow-up as co-variate. All tests were performed with an established alpha of 0.05. An association between allele distribution and the polymorphism in RANK was observed. Subjects that carry the allele T had a lower risk to have PAP (p=0.04). In RANKL polymorphism, the genotype distribution was statistically significant different between PAP and healed groups (p=0.05). Time of follow-up was associated with PAP (p<0.05). In the logistic regression analysis using time as a co-variant, RANK rs3826620 (p=0.02) and RANKL rs9594738 (p=0.03) were associated with PAP. The polymorphism rs2073618 in OPG was not associated with PAP (p>0.05). We conclude that polymorphisms in RANK and RANKL are associated with the risk of developing persistent periapical lesions