No description available.

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

Revascularização é uma técnica utilizada em dentes jovens, que apresentem rizogênese incompleta e danos irreversíveis ao tecido pulpar, necessitando de tratamento endodôntico, para formar novo tecido em lugar da polpa perdida. Clinicamente os resultados mostram a continuidade da rizogênese e a devolução da vitalidade dental. Porém, pouco se sabe sobre o novo tecido formado e não está estabelecido se este é capaz de desempenhar todas as funções da polpa dentária. Para melhorar as características do tecido formado pela técnica da revascularização, podemos utilizar ferramentas de engenharia tecidual, como célulastronco, fatores de crescimento e arcabouços de sustentação celular (scaffolds). As célulastronco (CTs) já estão presentes quando o sangue invade o canal radicular, e utilizar essa reserva de CTs que o hospedeiro possui, procedimento conhecido como homing, é uma vantagem em comparação com outras técnicas que injetam CTs obtidas por cultivo em laboratório. Entretanto os aspectos físicos do coágulo sanguíneo formado no interior do canal radicular podem ser melhorados com a adição de hidrogel de quitosana, que interage quimicamente com o sangue e forma um scaffold híbrido mais estável. Então, o objetivo deste estudo foi testar a hipótese de que o scaffold híbrido, composto por hidrogel de quitosana e sangue, ofereceria maior estabilidade física estrutural, bem como condições favoráveis à adesão e proliferação de células-tronco da papila apical humana (SCAPs; do inglês, Stem Cell from Apical Papila). Para isso, investigamos in vitro se a incorporação do sangue ao hidrogel de quitosana gera um scaffold mais estável, se este é favorável à adesão e proliferação de células-tronco da papila apical e se a fotobiomodulação potencializa essas características celulares. Para isso, SCAPs foram isoladas e caracterizadas por citometria de fluxo, tempo de dobra populacional, e contagem de unidades formadoras de colônias fibroblásticas (CFU-F; do inglês, Colony Forming Units - Fibroblastic). Ensaios de incorporação sanguínea, dissolução e embebição foram realizados para determinar o comportamento dos scaffolds híbridos. A adesão celular foi observada pela coloração PHK26® (do inglês, Red Fluorescent Cell Linker) e por microscopias eletrônicas de varredura (MEV); e a proliferação foi investigada pelo ensaio de alamarBlue®. Adicionalmente, a sobrevivência das SCAPs após a degradação do scaffold híbrido foi avaliada pela coloração Live/Dead®. A população celular estudada apresentou características de células tronco. O scaffold híbrido, constituído de densa rede alveolar com poros interconectados, embebeu e dissolveu rapidamente. De acordo com o PKH26® e alamarBlue® SCAPs aderiram e proliferaram no scaffold híbrido. SCAPs fotobiomoduladas exibiram maior taxa de proliferação e o ensaio Live/Dead® mostrou células vivas após 12 dias de cultivo. Concluiu-se que o scaffold híbrido apresenta biocompatibilidade e condições favoráveis de sobrevivência para SCAPs, que são potencializadas pela fotobiomodulação. / Revascularization is a technique used to form a new tissue, replacing the lost pulp, in young permanent teeth presenting incomplete rhizogenesis and irreversible damage, where endodontic treatment is needed. Clinically, the results show the continuity of the root formation and the return of dental vitality. However, little is known about the newly formed tissue and it has not been established if it is able to perform all functions of the dental pulp. To improve the characteristics of the newly formed tissue by the technique of revascularization, tissue engineering tools can be used, represented by stem cells, growth factors and scaffolds for cell supportting. Stem cells (SCs) are already present when the blood invades the root canal, and to use this SCs reserve that the host possesses, procedure known as homing, is an advantage compared with other techniques that inject SCs obtained by cultivation in the laboratory. However the physical aspects of blood clot in the root canal can be improved with the addition of chitosan hydrogel that chemically interacts with the blood and forms a more stable hybrid scaffold. So the aim of this study was to test the hypothesis that the hybrid scaffold, composed of hydrogel chitosan and blood clot, provides greater structural physical stability as well as favorable conditions for adhesion and proliferation of Stem Cells from Apical Papilla (SCAPs). For this, we investigated in vitro if the incorporation of blood to chitosan hydrogel, generates a more stable scaffold and if it supports the stem cell adhesion and proliferation, in addition, if photobiomodulation potentiates these cell characteristics. For this, SCAPs were isolated and characterized by flow cytometry, population doubling time, and counting colony forming units - fibroblastic (CFUF). Blood incorporation assays, dissolution and swelling were conducted to determine the behavior of hybrid scaffolds. Cell adhesion was observed by PHK26® (Red Fluorescent Cell Linker) and scanning electron microscopy (SEM); and proliferation was investigated by alamarBlue® assay. In addition, the survival of SCAPs after degradation of the scaffold was assessed by Live/Dead® staining. The cell population showed stem cell characteristics. The hybrid scaffold, constituted of dense cellular network with interconnected pores, soaked and dissolved quickly. According to PKH26® and alamarBlue® assays, the SCAPs adhered and proliferated in the hybrid scaffold. Photobiomodulation leads to SCAPs higher proliferation rate and the Live/Dead® test showed live cells after 12 days of cultivation. It was concluded that the hybrid scaffold is biocompatible and favors survival of SCAPs, which was enhanced by photobiomodulation.

No description available.

INTRODUCTION AND AIM The prevalence of cardiovascular disease (CVD) has increased significantly in the black South African population in recent years. Early in the development of CVD, atherosclerotic plaques form in the vessel wall. When this plaque becomes unstable and ruptures, the coagulation cascade is activated and a blood clot forms. The function of this clot is to stop bleeding. However, it cannot remain in the vasculature indefinitely and has to be lysed again. The ability of the body to lyse clots can be measured with global fibrinolytic potential (GFP) assays and expressed as lysis time. Increased clot lysis time (CLT) has been shown to be significantly associated with various CVD risk factors and CVD events in Caucasian populations while very little information is available for other ethnicities. In this study we investigated plasma GFP and its relation to CVD risk factors in a large black African population. We also determined the effect of three polymorphisms in the promoter area of the plasminogen activator inhibitor-1 (PAI-1) gene on PAI-1act (activity) levels (a main determinant of CLT) and CLT, together with gene-environment interactions and the effect of urbanisation on these interactions. PARTICIPANTS AND METHODS Apparently healthy men and women between the ages of 35 and 65 years were recruited to take part in the South African arm of the Prospective Urban and Rural Epidemiology (PURE) study. Approximately 1000 rural and 1000 urban black African individuals participated. Data and samples were collected during a 12-week collection period in 2005 for cross-sectional analysis. RESULTS Increased PAI-1act levels, body mass index (BMI), glycosylated haemoglobin (HbA1c), triglycerides, fibrinogen concentration, C-reactive protein, female sex, positive HIV-status and the metabolic syndrome were all associated with prolonged CLTs, while increased habitual alcohol consumption was associated with shorter iv CLTs. Urban-rural differences for CLT existed in women only. This is likely due to the larger extent of rural-urban differences in other CVD risk factors observed in women compared to what was observed in men. Of the CVD risk factors measured, PAI-1 explained the largest proportion of the variance in CLT (27%). Owing to the important role PAI-1act plays in CLT, we investigated three polymorphisms in the PAI-1 gene promoter area (the 4G/5G polymorphism, the novel SNP C428T and SNP G429A (previously identified)), and the influence of these polymorphisms on PAI-1act levels and CLT. The frequency of the 5G allele was high (0.85) in comparison with previously reported literature. PAI-1act increased significantly across genotypes in the urban (5G/5G: 3.84 U/ml; 4G/5G: 4.85 U/ml; 4G/4G: 5.96 U/ml p=0.009) but not the rural subgroup, while CLT did not differ. We found significant interactions between the 4G/5G polymorphism and BMI, waist circumference and triglycerides in determining PAI-1act, and between the 4G/5G polymorphism and fibrinogen and fibrinogen gamma prime in determining CLT. Direct relationships with PAI-1act or CLT were not found for the C428T and G429A polymorphisms; they did, however, influence associations of other environmental factors with PAI-1act and CLT. Several of these interactions differed significantly between rural and urban subgroups, particularly in individuals harbouring the mutant alleles. CONCLUSION CLT associated with many of the same CVD risk factors described in the literature for Caucasian populations, but also with other risk factors. Rural-urban differences in CLT are dependent on the association of CLT with other CVD risk factors in the rural-urban setting. Genetic polymorphisms of the PAI-1 gene did not directly influence CLT, despite influencing PAI-1act. The main contributor to PAI-1act variance, however, was (central) obesity. The effect of the 4G/5G polymorphism on PAI-1act, as well as gene–environment interactions for the C428T and G429A genotypes in determining PAI-1act and CLT, were significantly influenced by urbanisation. / Thesis (PhD (Nutrition))--North-West University, Potchefstroom Campus, 2013.

INTRODUCTION AND AIM The prevalence of cardiovascular disease (CVD) has increased significantly in the black South African population in recent years. Early in the development of CVD, atherosclerotic plaques form in the vessel wall. When this plaque becomes unstable and ruptures, the coagulation cascade is activated and a blood clot forms. The function of this clot is to stop bleeding. However, it cannot remain in the vasculature indefinitely and has to be lysed again. The ability of the body to lyse clots can be measured with global fibrinolytic potential (GFP) assays and expressed as lysis time. Increased clot lysis time (CLT) has been shown to be significantly associated with various CVD risk factors and CVD events in Caucasian populations while very little information is available for other ethnicities. In this study we investigated plasma GFP and its relation to CVD risk factors in a large black African population. We also determined the effect of three polymorphisms in the promoter area of the plasminogen activator inhibitor-1 (PAI-1) gene on PAI-1act (activity) levels (a main determinant of CLT) and CLT, together with gene-environment interactions and the effect of urbanisation on these interactions. PARTICIPANTS AND METHODS Apparently healthy men and women between the ages of 35 and 65 years were recruited to take part in the South African arm of the Prospective Urban and Rural Epidemiology (PURE) study. Approximately 1000 rural and 1000 urban black African individuals participated. Data and samples were collected during a 12-week collection period in 2005 for cross-sectional analysis. RESULTS Increased PAI-1act levels, body mass index (BMI), glycosylated haemoglobin (HbA1c), triglycerides, fibrinogen concentration, C-reactive protein, female sex, positive HIV-status and the metabolic syndrome were all associated with prolonged CLTs, while increased habitual alcohol consumption was associated with shorter iv CLTs. Urban-rural differences for CLT existed in women only. This is likely due to the larger extent of rural-urban differences in other CVD risk factors observed in women compared to what was observed in men. Of the CVD risk factors measured, PAI-1 explained the largest proportion of the variance in CLT (27%). Owing to the important role PAI-1act plays in CLT, we investigated three polymorphisms in the PAI-1 gene promoter area (the 4G/5G polymorphism, the novel SNP C428T and SNP G429A (previously identified)), and the influence of these polymorphisms on PAI-1act levels and CLT. The frequency of the 5G allele was high (0.85) in comparison with previously reported literature. PAI-1act increased significantly across genotypes in the urban (5G/5G: 3.84 U/ml; 4G/5G: 4.85 U/ml; 4G/4G: 5.96 U/ml p=0.009) but not the rural subgroup, while CLT did not differ. We found significant interactions between the 4G/5G polymorphism and BMI, waist circumference and triglycerides in determining PAI-1act, and between the 4G/5G polymorphism and fibrinogen and fibrinogen gamma prime in determining CLT. Direct relationships with PAI-1act or CLT were not found for the C428T and G429A polymorphisms; they did, however, influence associations of other environmental factors with PAI-1act and CLT. Several of these interactions differed significantly between rural and urban subgroups, particularly in individuals harbouring the mutant alleles. CONCLUSION CLT associated with many of the same CVD risk factors described in the literature for Caucasian populations, but also with other risk factors. Rural-urban differences in CLT are dependent on the association of CLT with other CVD risk factors in the rural-urban setting. Genetic polymorphisms of the PAI-1 gene did not directly influence CLT, despite influencing PAI-1act. The main contributor to PAI-1act variance, however, was (central) obesity. The effect of the 4G/5G polymorphism on PAI-1act, as well as gene–environment interactions for the C428T and G429A genotypes in determining PAI-1act and CLT, were significantly influenced by urbanisation. / Thesis (PhD (Nutrition))--North-West University, Potchefstroom Campus, 2013.

Carboxypeptidase N (CPN) is a plasma carboxypeptidase that was discovered in the 1960s as a regulator of inflammation and vascular tone. Through the removal of carboxy-terminal basic residues, CPN alters the activity or binding specificity of inflammatory mediators and vasoactive peptides. CPN shares significant homology with carboxypeptidases known to mediate antifibrinolysis through the removal of basic residues from fibrin clots, which would otherwise stimulate fibrinolysis. Despite the similarity of these enzymes, CPN is generally regarded as lacking a role in fibrinolysis. This thesis demonstrates that CPN is indeed a capable antifibrinolytic enzyme, and that the antifibrinolytic activity of CPN was previously undisclosed due to the presence of a circulating CPN inhibitor, which is likely the free CPN2 subunit. This inhibitor is described for the first time here. Furthermore, potential mechanisms of inhibition and mechanisms of enhancing activity of CPN are proposed based upon the additional structural characterization of CPN presented here.

Wound healing is governed by a complex cascade of related processes, involving cells, extracellular matrix and cytokines. In adults this always results in a scar whilst embryonic wound healing is scarless and extensive research worldwide is aimed at reducing scarring in adults. A mathematical framework for problems in dermal wound healing is developed that incorporates models of the individual processes involved. Cells are modelled as discrete individuals. Cytokines and other biologically active factors are modelled as continua. A novel tensorial approach is taken to modelling the extracellular matrix. The numeric and computational challenges associated with combining models for the individual processes are identified and investigated. These include the development of data structures and numeric methods for the continuous and discrete species. Effective visualisation methods for the large amounts of data generated by the model are also discussed. The possibilities offered by high performance computing in mathematical biology are highlighted in this work. The final part of this thesis gives an example of a combined model of the inflammatory and proliferative phases of dermal wound healing using the new computational framework. Both quantitative and qualitative methods are used to analyse the information-rich data sets generated by the model, offering insight into the dynamic systems that can be modelled using the new approach.

La formation du caillot de fibrine, processus clé de la coagulation sanguine, implique la polymérisation des monomères de fibrine en un réseau de fibres. Ce réseau contrôle les propriétés mécaniques du caillot et constitue le squelette sur lequel se base la cicatrisation. Si l’influence des conditions de réaction (pH, concentration, …) est bien connue, le rôle de la composition du fibrinogène sur la structure de la fibrine est inexploré. Cet aspect pourrait être important pour les pathologies cardiovasculaires qui présentent toutes une structure de fibrine anormale.Nous avons étudié la relation entre la composition de plusieurs fibrinogènes et les propriétés structurelles nano- et micro-métriques ainsi que la mécanique des caillots de fibrine. La composition en protéines co-purifiées de ces fibrinogènes a peu d’influence, alors que le profil de polydispersité contrôle la structure multi-échelle de la fibrine. Des mesures de diffusion des rayons x, de spectrophotométrie multi-longueur d’ondes et de microscopie confocale ont mis en évidence que les fibres provenant des fibrinogènes monodisperses sont quasi-cristallines, droites et rigides. Les fibres provenant de fibrinogènes polydisperses sont, elles, beaucoup moins organisées, courbées, avec un module de rigidité faible. Enfin, les propriétés mécaniques de la fibrine ont montré que la réponse des caillots aux déformations, aussi que les scenarios de rupture, sont directement liés à sa structure et donc significativement dépendants du profil de polydispersité des fibrinogènes. Ces résultats ouvrent de nouvelles perspectives dans plusieurs domaines, que ce soit pour l’utilisation optimale des fibrinogènes pour les dysfibrinogénémies et hémorragies, mais également pour la reconstruction tissulaire, ainsi que la compréhension du lien entre la structure anormale des caillots et les maladies cardiovasculaires. / Fibrin clot formation is one of the major processes leading to blood clotting. It involves the polymerization of fibrin monomers into a network of fibrin fibres. This network controls the mechanical properties of the clot and serves as a skeleton for wound healing. Environmental factors (pH, concentration, …) have been proved to influence polymerization, however the role of fibrinogen composition on the structure of fibrin remains unexplored. This aspect might be important for the case of cardiovascular pathologies, which present abnormal fibrin structures.We have determined the relation between different sources of fibrinogen with the nano- and micro-metric structural and mechanical properties of fibrin clots. The composition in co-purified proteins of the fibrinogens has no significant importance, however the polydispersity profile controls the multiscale properties of fibrin. Indeed, x-ray scattering, multi-wavelength spectrophotometry and confocal microscopy measurements have proved that fibres from monodisperse fibrinogens are quasi-crystalline, straight and rigid. Fibres from polydisperse fibrinogens are less organised, curbed and less rigid. Finally, the mechanical properties of fibrin showed that the response of clots to deformation, as well as the scenarios of rupture are closely related to the structure, and consequently related to the profiles of polydispersity. This opens outstanding perspectives in many fields such the optimisation of fibrinogen’s use on dysfibrinogenemias or haemorrhages, tissue regeneration or the understanding between the abnormal structure of clots and cardiovascular diseases.