Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are associated with increased risk of atherothrombotic disease. Lp(a) is a unique lipoprotein consisting of a low density lipoprotein-like moiety covalently linked to apolipoprotein(a) (apo(a)), a homologue of the fibrinolytic proenzyme plasminogen. Apo(a) is extremely heterogeneous in size with small isoforms being independently associated with increased cardiovascular risk. Several in vitro and in vivo studies have shown that Lp(a)/apo(a) can inhibit tissue-type plasminogen activator (tPA)-mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apo(a) remains controversial. Essential to fibrin clot lysis are a number of plasmin-dependent positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin-mediated conversion of Glu1-plasminogen to Lys78-plasminogen. Additionally, abnormal fibrin clot structures have been associated with both an increased risk of cardiovascular disease and elevated Lp(a) levels. Similarly, oxidized phospholipids have been implicated in the development of cardiovascular disease, and are not only preferentially carried by Lp(a) in the plasma but have also been shown to covalently-modify both apo(a) and plasminogen. In this thesis, we built upon the understanding of the role of apo(a) in plasminogen activation on the fibrin/degraded fibrin surface by determining that: (i) apo(a) inhibits plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion and identifying the critical domains in apo(a) responsible for this effect, (ii) apo(a) isoform size does not affect either the inhibition of tPA-mediated plasminogen activation or the inhibition of plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion, (iii) apo(a) modifies fibrin clot structure to form more dense clots with thinner fibers and reduced permeability, modifications that enhance the ability of apo(a) to inhibit tPA-mediated plasminogen activation and (iv) the phosphorus content of apo(a) affects its ability to inhibit tPA-mediated plasminogen activation and the phosphorus content of plasminogen affects its ability to be activated by tPA. By understanding these individual reactions, each of which has the potential to affect the broader fibrin clot lysis process, we have expanded our understanding of the overall effect of Lp(a)/apo(a) in the inhibition of plasminogen activation on the fibrin/degraded fibrin surface and thus broadened our understanding of how Lp(a)/apo(a) may mediate the inhibition of thrombolysis in vivo. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-12-14 08:26:54.99

We detail for the first time the uniquely altered fibrin polymerization of homophenotypic Aα R16H dysfibrinogen. By polymerase chain reaction amplification and DNA sequencing, our new proposita's genotype consisted of a G>A transition encoding for Aα R16H, and an 11 kb Aα gene deletion. High-performance liquid chromatography disclosed fibrinopeptide A release approximately six times slower than its fibrinopeptide B. Turbidimetric analyses revealed unimpaired fibrin repolymerization, and abnormal thrombin-induced polymerization (1-7 μmol/l fibrinogen, > 96% coagulable), consisting of a prolonged lag time, slow rate, and abnormal clot turbidity maxima, all varying with thrombin concentration. For example, at 0.2-3 U/ml, the resulting turbidity maxima ranged from lower to higher than normal control values. By scanning electron microscopy, clots formed by 0.3 and 3 thrombin U/ml displayed mean fibril diameters 42 and 254% of the respective control values (n = 400). Virtually no such differences from control values were demonstrable, however, when clots formed in the presence of high ionic strength (μ = 0.30) or of monoclonal antiβ(15-42)IgG. The latter also prolonged the thrombin clotting time approximately three-fold. Additionally, thrombin-induced clots displayed decreased elastic moduli, with G′ values of clots induced by 0.3, 0.7 and 3 thrombin U/ml corresponding to 11, 34, and 45% of control values. The results are consistent with increased des-BB fibrin monomer generation preceding and during polymerization. This limited the inherent gelation delay, decreased the clot stiffness, and enabled a progressively coarser, rather than finer, network induced by increasing thrombin concentrations. We hypothesize that during normal polymerization these constitutive des-BB fibrin monomer properties attenuate their des-AA fibrin counterparts.

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

Made available in DSpace on 2014-06-11T19:27:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-29Bitstream added on 2014-06-13T20:17:05Z : No. of bitstreams: 1 oliveira_gjpl_me_arafo.pdf: 645436 bytes, checksum: 36e8a12c99620e4820f3710a3bb8b0d3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Foram utilizados 60 dentes humanos nessa pesquisa. No primeiro experimento, 15 dentes extraídos por doença periodontal foram selecionados para avaliar a influência da irradiação com laser de Er,Cr: YSGG sobre a morfologia e adesão de células sanguíneas sobre as superfícies radiculares. As 60 amostras provenientes desses dentes foram divididos em 3 grupos de acordo com o tipo de tratamento aplicado: Grupo 1- RAR; Grupo 2- Irradiação com laser de Er,Cr: YSGG; Grupo 3- RAR e irradiação com o laser de Er,Cr: YSGG. 10 amostras de cada grupo foram avaliados quanto a adesão de elementos sanguíneos, e as outras 10 amostras foram avaliados quanto a morfologia da superfície radicular por MEV. Os testes de Kruskall-Wallis e Mann-Whitney foram utilizados para avaliar os resultados. Em relação à adesão de elementos sanguíneos, este estudo não demonstrou diferenças estatísticas entre os grupos (p=0.359), a análise morfológica demonstrou que as superfícies radiculares irradiadas com o laser de Er-Cr:YSGG foram mais rugosas que as do grupo controle (G2-G1: p=0.0003 e G3-G1: p=0.0003). No segundo experimento, 20 dentes foram utilizados para avaliar a influência do ângulo de irradiação do laser de Er,Cr:YSGG sobre a rugosidade e o desgaste das superfícies radiculares. Cada face proximal foi dividida em 3 áreas, sendo que a área superior foi tratada com raspagem e alisamento radicular, a área média não foi submetida a nenhum tipo de tratamento e a área inferior foi irradiada com o laser de Er,Cr:YSGG. Os dentes foram divididos em 4 grupos ,com 5 dentes cada, a depender da angulação da aplicação da irradiação do laser de Er,Cr:YSGG na área ( 30º, 45º, 60º, 90º). A rugosidade das áreas foram avaliadas através de um rugosímetro e posteriormente os dentes foram submetidos a processamento histológico... / 60 human teeth were used in that research. In the first experiment, 15 extracted teeth for periodontal disease were selected to evaluated the effect of Er,Cr:YSGG irradiation on root surfaces for adhesion of blood components and morphology. 60 root surface specimens were obtained by selecting four from each tooth. Samples were divided into three groups of 20 each, according to treatments. Group 1 (G1) was treated by scaling and root planing (SRP), Group 2 (G2) was irradiated by Er,Cr:YSGG laser and Group 3 (G3) was treated by SRP and Er,Cr:YSGG laser irradiation. Blood was placed on each of 10 specimens from each of the three groups, to evaluate adhesion of blood components to the root surfaces. A morphological analysis was made of the root surfaces of the other 10 specimens from each group by scanning electron microscope (SEM). Statistical processing was done with the Kruskal-Wallis and Mann-Whitney tests. No statistical differences for adhesion of blood components to root surfaces were found between the groups (p = 0.359). However, morphological analysis disclosed that all root surfaces irradiated by Er,Cr:YSGG laser (100%) were rougher than surfaces that were not irradiated (G1-G2: p = 0.0003 and G1-G3: p = 0.0003). In the second experiment, 20 teeth were used to evaluated the effect of the working tip angulations on root roughness and substance removal using Er,Cr:YSGG radiation. The distal and mesial surfaces of each tooth was divided in 3 areas. The upper area was treated with scaling and root planing. The medium area was not submitted to any treatment and the lower area was irradiated with Er,Cr:YSGG laser. The teeh were divided in 4 groups, with 5 teeth each depending the working tip angulations using Er,Cr:YSGG at the lower area (30º, 45º, 60º, 90º).The roughness surfaces were evaluated by a profilometer, and... (Complete abstract, click electronic access below)

Orientador: Rosemary Adriana Chiérici Marcantonio / Banca: Márcio Zafalon Casati / Banca: Estela Sasso Cerri / Resumo: Foram utilizados 60 dentes humanos nessa pesquisa. No primeiro experimento, 15 dentes extraídos por doença periodontal foram selecionados para avaliar a influência da irradiação com laser de Er,Cr: YSGG sobre a morfologia e adesão de células sanguíneas sobre as superfícies radiculares. As 60 amostras provenientes desses dentes foram divididos em 3 grupos de acordo com o tipo de tratamento aplicado: Grupo 1- RAR; Grupo 2- Irradiação com laser de Er,Cr: YSGG; Grupo 3- RAR e irradiação com o laser de Er,Cr: YSGG. 10 amostras de cada grupo foram avaliados quanto a adesão de elementos sanguíneos, e as outras 10 amostras foram avaliados quanto a morfologia da superfície radicular por MEV. Os testes de Kruskall-Wallis e Mann-Whitney foram utilizados para avaliar os resultados. Em relação à adesão de elementos sanguíneos, este estudo não demonstrou diferenças estatísticas entre os grupos (p=0.359), a análise morfológica demonstrou que as superfícies radiculares irradiadas com o laser de Er-Cr:YSGG foram mais rugosas que as do grupo controle (G2-G1: p=0.0003 e G3-G1: p=0.0003). No segundo experimento, 20 dentes foram utilizados para avaliar a influência do ângulo de irradiação do laser de Er,Cr:YSGG sobre a rugosidade e o desgaste das superfícies radiculares. Cada face proximal foi dividida em 3 áreas, sendo que a área superior foi tratada com raspagem e alisamento radicular, a área média não foi submetida a nenhum tipo de tratamento e a área inferior foi irradiada com o laser de Er,Cr:YSGG. Os dentes foram divididos em 4 grupos ,com 5 dentes cada, a depender da angulação da aplicação da irradiação do laser de Er,Cr:YSGG na área ( 30º, 45º, 60º, 90º). A rugosidade das áreas foram avaliadas através de um rugosímetro e posteriormente os dentes foram submetidos a processamento histológico... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: 60 human teeth were used in that research. In the first experiment, 15 extracted teeth for periodontal disease were selected to evaluated the effect of Er,Cr:YSGG irradiation on root surfaces for adhesion of blood components and morphology. 60 root surface specimens were obtained by selecting four from each tooth. Samples were divided into three groups of 20 each, according to treatments. Group 1 (G1) was treated by scaling and root planing (SRP), Group 2 (G2) was irradiated by Er,Cr:YSGG laser and Group 3 (G3) was treated by SRP and Er,Cr:YSGG laser irradiation. Blood was placed on each of 10 specimens from each of the three groups, to evaluate adhesion of blood components to the root surfaces. A morphological analysis was made of the root surfaces of the other 10 specimens from each group by scanning electron microscope (SEM). Statistical processing was done with the Kruskal-Wallis and Mann-Whitney tests. No statistical differences for adhesion of blood components to root surfaces were found between the groups (p = 0.359). However, morphological analysis disclosed that all root surfaces irradiated by Er,Cr:YSGG laser (100%) were rougher than surfaces that were not irradiated (G1-G2: p = 0.0003 and G1-G3: p = 0.0003). In the second experiment, 20 teeth were used to evaluated the effect of the working tip angulations on root roughness and substance removal using Er,Cr:YSGG radiation. The distal and mesial surfaces of each tooth was divided in 3 areas. The upper area was treated with scaling and root planing. The medium area was not submitted to any treatment and the lower area was irradiated with Er,Cr:YSGG laser. The teeh were divided in 4 groups, with 5 teeth each depending the working tip angulations using Er,Cr:YSGG at the lower area (30º, 45º, 60º, 90º).The roughness surfaces were evaluated by a profilometer, and... (Complete abstract, click electronic access below) / Mestre

Le rôle physiologique du caillot est d’éviter un épanchement excessif de sang en présence d’une brèche vasculaire. Une fois cette fonction remplie, il doit pouvoir être facilement détruit, afin qu’il ne passe pas dans le système veineux et ne gêne la circulation sanguine. La formation d’un caillot de fibrine et sa lyse, processus clés de l’hémostase, impliquent à la fois la polymérisation des monomères de fibrinogène en un réseau de fibres de fibrine, et la résorption du réseau de fibres de fibrine constitué. Bien que ce réseau contrôle l’ensemble des propriétés physiques et mécaniques du caillot, sa structure aux échelles inférieures au micron est très mal caractérisée. Le principal verrou à la caractérisation physique du caillot en environnement clinique est l’absence de méthode de mesure quantitative, fiable, sensible et reproductible. Il est donc nécessaire de produire une méthode de mesure adéquate, couplée à un système de mesure sensible. Nous avons démontré dans ce travail, grâce à notre méthode utilisant plusieurs longueurs d’onde, que l’analyse du spectre de lumière visible transmis à travers un caillot permet de déterminer simultanément, quantitativement et en conditions quasi-physiologiques, plusieurs paramètres essentiels de structure du caillot de fibrine, à savoir le nombre de protofibrilles par fibre de fibrine, le rayon et la densité de ces fibres, ainsi que les temps de formation et de lyse du caillot. Cette technique a été validée via les résultats avec des CV inférieurs dans l’ensemble à 6% sous plusieurs conditions de tests et différents profils plasmatiques : normaux, hypo/hyper coagulants et hypo/hyper fibrinolytiques, attestant de la robustesse et de la fiabilité de la technique de mesure aussi bien pour le suivi de la coagulation que de la lyse. Cette méthode de spectrophotométrie a pu être implantée sur un automate modifié à des fins de diagnostic et à vocation hospitalière pour des plasmas de patients présentant des troubles de l’hémostase. Les informations cliniques et intérêts attendus de ce nouveau test, concernent à la fois la qualité du réseau de fibrine, sa lyse accélérée ou sa résistance à la fibrinolyse ainsi que la résultante de la balance coagulo-lytique. / The physiological role of the clot is to avoid excessive bleeding in the presence of a vascular breach. Once this function is filled, the clot must be able to be easily destroyed, so that it is not transported in the venous system and does not hamper blood circulation. The formation of a fibrin clot and its lysis are key processes of hemostasis, implying simultaneously the polymerization of the fibrinogen monomers in a fibrin fibers network, and the destruction of this constituted network.Although this network controls the physical and mechanical properties of the clot, its structure at scales smaller than the micron is poorly characterized. The main problem in the physical characterization of clot in clinical settings is the current absence of a quantitative, sensitive and reproducible measurement method.We demonstrated in this work, thanks to our method using several wavelengths, that the analysis of the visible spectra of light transmitted through a clot allows to determine simultaneously, quantitatively and in quasi-physiological conditions, several essential parameters of structure of the fibrin clot, namely the number of protofibrils per fibrin fibers, the radius and the density of fibers, and various times of clotting and lysis of the clot. This method was validated by the results with CV inferior to 6 % under all test conditions and various plasmatic profiles: normal, hypo / hyper coagulant and hypo / hyper fibrinolytic. This demonstrates the robustness and reliability of the measurement method when measuring both clotting and clot lysis.This spectrophotometric method was implemented on a modified automaton dedicated to diagnosis of patients presenting hemostatic disorders. The clinical information and the interests expected from this new test concern at the same time the quality of the fibrin network, its accelerated lysis or its resistance to fibrinolysis, and the resultant of the coagulo-lytic balance.

<p dir="ltr">Though mammalian spinal cord injury (SCI) has long been a topic of study, effective therapies that promote functional recovery are not yet available. The axolotl, <i>Ambystoma mexicanum</i>, is a valuable animal model in the investigation of spinal cord regeneration, as this urodele is able to achieve functional recovery even after complete spinal cord transection. Understanding the similarities and differences between the mammalian SCI response and that of the axolotl provides insight into the process of successful regeneration, and bolsters the fundamental knowledge used in the development of future mammalian SCI treatments. This thesis provides a detailed analysis of the ultrastructure of the axolotl meninges, as this has not yet been presented in existing literature, and reveals that the axolotl meninges consist of 3 distinct layers as does mammalian meninges; the dura mater, arachnoid mater, and pia mater. The role of reactive meningeal and ependymal cells is also investigated in regard to the deposition and remodeling of the fibrotic ECM, which is found to be similar in composition to hydrogel scaffolds being studied in mammalian SCI. It is shown that meningeal fibroblasts are the primary source of the extensive fibrillar collagen deposition that fills the entire spinal canal, peaking at approximately 3 weeks post transection and remaining until approximately 5 weeks post transection, and that there is no deposition of type IV collagen within the lesion site. Mesenchymal ependymal cells are shown to contribute to the ECM deposition through the production of glycosaminoglycans that are used in sidechains of both unsulfated and sulfated proteoglycans, while simultaneously remodeling the ECM through the production of MMPs and phagocytosis of cellular debris. Further, this study shows that mesenchymal ependymal cells and a population of foamy macrophages contribute to the degradation of the fibrin clot that forms in the acute phase of injury, and that this fibrin clot provides a necessary and permissive substrate for early mesenchymal outgrowth.</p>