Osteoarthrose ist eine häufige Erkrankung des Menschen, die mit einer deutlichen Morbidität und körperlichen Einschränkungen assoziiert ist. Weil Knorpelgewebe avaskulär ist und die Chondrozyten sich in einem postmitotischen Zustand befinden, besitzt Knorpel nur sehr geringes Selbstheilungspotenzial. Es gibt momentan keine effektive Therapie der Arthrose. Obwohl regenerative Ansätze mit dem Tissue Engineering von Knorpelgewebe vielversprechende Therapiealternativen darstellen, stellt die mangelnde laterale Integration von Knorpelgewebe ein chronisches Problem dar, das die Implantation von Knorpelkonstrukten vor Schwierigkeiten stellt. Die optimale Integrationsmethode sollte das Gewebe stark verbinden, klinisch schnell und einfach angewendet werden können, ein hohes Maß an Biokompatibilität besitzen und außerdem die Gewebereparatur fördern. In dieser Arbeit wurden natives Fibrinogen und unmodifizierte Gelatine in gelöster Form mittels einer neuartigen und schnellen Photooxidationsmethode unter Verwendung von Rutheniumkomplexen und Licht aus dem sichtbaren Spektrum zu Klebern vernetzt. Dabei ließ sich feststellen, dass insbesondere der Kleber aus Ruthenium und Gelatine Potenzial besitzt, Einsatz als Bioadhäsivum im Bereich Tissue Engineering von Knorpelgewebe zu finden. Ausschlaggebend dafür ist die Herstellung einer suffizienten Sofortadhäsion zwischen gegenüberliegenden Knorpelflächen einerseits, sowie die Förderung der Langzeitintegration andererseits, die für eine Stimulierung der Gewebereparatur im echten Knorpeldefekt vielversprechend ist. Weitere Forschung ist jedoch nötig, um die Abgrenzung der mechanischen Integration gegenüber der Kontrollgruppe besser zu unterstreichen und das Material weiteren Untersuchungen wie einer Analyse des Quellverhaltens zu unterziehen. Schließlich sollte das regenerative Potenzial des Gewebeklebers in in vivo Tiermodellen weiter systematisch untersucht werden. Zudem lieferte diese Arbeit vielversprechende Ergebnisse für den potenziellen Einsatz von RuGel im Bereich hydrogelbasiertes Tissue Engineering. / Osteoarthritis is a common human disease associated with significant morbidity and physical limitations. Because cartilage tissue is avascular and chondrocytes are in a post-mitotic state, cartilage has very little self-healing potential. There is currently no effective therapy for osteoarthritis. Although regenerative approaches using tissue engineering of cartilage tissue are promising therapeutic alternatives, the lack of lateral integration of cartilage tissue is a chronic problem that poses difficulties for the implantation of cartilage constructs. The optimal integration method should strongly connect the tissue, be clinically fast and easy to apply, have a high degree of biocompatibility, and also promote tissue repair. In this work, native fibrinogen and unmodified gelatin in dissolved form were crosslinked into adhesives by a novel and rapid photooxidation method using ruthenium complexes and visible spectrum light. It was found that the adhesive made of ruthenium and gelatin in particular has the potential to be used as a bioadhesive in the field of tissue engineering of cartilage tissue. Crucial for this is the establishment of a sufficient immediate adhesion between opposing cartilage surfaces on the one hand, and the promotion of long-term integration on the other hand, which is promising for stimulating tissue repair in real cartilage defects. However, further research is needed to better emphasize the delineation of mechanical integration versus the control group and to subject the material to further investigations such as an analysis of swelling behavior. Finally, the regenerative potential of the tissue adhesive should be further systematically investigated in in vivo animal models. In addition, this work provided promising results for the potential use of RuGel in hydrogel-based tissue engineering.

The mammary gland of lactating transgenic animals has several advantages for production of heterologous proteins including a high cell density that results in high concentrations of secreted protein and the ability to perform several types of post-translational modifications. Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the Aα, Bβ and γ fibrinogen chains to evaluate the requirements of the transgenic murine mammary gland for high level secretion of fully assembled human fibrinogen. After introducing the constructs into the murine zygotes by microinjection, secretion of fully assembled fibrinogen into milk was measured at concentrations between 10 ug/ml to 200 ug/ml. In one line of mice the total secretion of fibrinogen and unassembled subunits approached 700 ug/ml in milk. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the Bβ and γ chains were rate limiting. Also, the subunit complexes γ₂, Aαγ₂ and the individual subunits Aα, Bβ and γ were found as secretion products. This is the first time that secretion of individual Bβ-subunits by any cell type has been reported and suggests the organization of the secretion pathway in mammary epithelia is different from that in liver. Glycosylated forms of individual Bβ-chain contained a complex saccharide with low mannose. Glycosylation of the γ-chain was also observed. These results suggest the 4.1 mWAP promoter can drive expression of fibrinogen cDNAs to high levels and that the amount of fully assembled fibrinogen secreted is equal to the level of the lowest expressing chain. / Master of Science

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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

Duchenne muscular dystrophy (DMD) is a fatal degenerative disorder of locomotor and respiratory muscles, in which myofibers are progressively replaced by non-muscular fibrotic tissue. Here, we show that fibrin/ogen accumulates in dystrophic muscles of DMD patients and of the mdx mouse model of DMD. Genetic loss or pharmacological depletion of fibrin/ogen in mdx mice attenuated muscular dystrophy progression and improved locomotor capacity. More importantly, fibrin/ogen depletion reduced fibrosis in mdx mouse diaphragm. Our data indicate that fibrin/ogen, through induction of IL-1 Ò, drives the synthesis of TGF Ò by mdx macrophages, which in turn, induces collagen production in mdx fibroblasts. Fibrin/ogen-produced TGF Ò further amplifies collagen accumulation through recruitment and activation of pro-fibrotic alternatively activated macrophages. Fibrin/ogen also stimulated collagen synthesis directly in mdx fibroblasts, via Ñv Ò3 integrin engagement. In addition, when analyzing a group of 39 DMD patients, fibrin/ogen accumulation in locomotor muscles was found associated with fibrosis and disease severity. These data unveil a novel role of fibrin/ogen in muscular dystrophy and, importantly, in the replacement of muscle by fibrotic tissue.

Prothrombin, a protein involved in blood coagulation, is a plasma glycoprotein composed of the Gla domain, two adjacent kringle domains, and a serine protease domain. Prothrombin is a thrombin precursor playing the important role in the coagulation physiological as well as pathological condition. Thrombin is the key to convert the fibrinogen into fibrin by switching activation of XIII factor, pushed plasminogen into plasmin, the develope of the fibroblast and helps the stabilization of thrombolysis. In this study, the prothrombin gene was 936 bp in lengths and encoded 312 amino acids from bovine lung was optimized codon, was cloned in pET21a+ vector and expression in E. coli, in order to replace traditional bandages having slow affect, reduce the cost of products, cater the comunity health. The results showed that initially the successful cloning and expression of recombinant prothrombin in E. coli JM109(DE3). / Prothrombin, 1 glycoprotein huyết tương liên quan tới quá trình đông máu gồm 2 vùng Gla, 2 vùng Kringle và 1 vùng serine protease. Prothrombin là tiền chất của thrombin có vai trò quan trọng trong sinh lý đông máu cũng như tình trạng bệnh lý. Thrombin được xem như chìa khóa để chuyển hóa fibrinogen thành fibrin bằng cách hoạt hóa các yếu tố đông máu như XIII, thúc đẩy chuyển plasminogen thành plasmin và kích thích tăng sinh các tế bào tơ (fibroblast), giúp ổn định quá trình làm tan huyết khối. Trong nghiên cứu, các gen prothrombin được tách dòng từ phổi bỏ có kích thước 936 bp, mã hóa cho 312 axit amin được tối ưu hóa codon, nhân dòng vào vector pET21a+ và biểu hiện trong E. coli. Mục đích của nghiên cứu nhằm tạo ra băng gạc cầm máu nhanh, giá thành rẻ, phục vụ sức khỏe cộng đồng và thay thể băng gạc truyền thống. Kết quả nghiên cứu bước đầu cho thấy đã nhân dòng và biểu hiện thành công prothrombin tái tổ hợp ở chủng E. coli JM109(DE3).

Prothrombin, a protein involved in blood coagulation, is a plasma glycoprotein composed of the Gla domain, two adjacent kringle domains, and a serine protease domain. Prothrombin is a thrombin precursor playing the important role in the coagulation physiological as well as pathological condition. Thrombin is the key to convert the fibrinogen into fibrin by switching activation of XIII factor, pushed plasminogen into plasmin, the develope of the fibroblast and helps the stabilization of thrombolysis. In this study, the prothrombin gene was 936 bp in lengths and encoded 312 amino acids from bovine lung was optimized codon, was cloned in pET21a+ vector and expression in E. coli, in order to replace traditional bandages having slow affect, reduce the cost of products, cater the comunity health. The results showed that initially the successful cloning and expression of recombinant prothrombin in E. coli JM109(DE3). / Prothrombin, 1 glycoprotein huyết tương liên quan tới quá trình đông máu gồm 2 vùng Gla, 2 vùng Kringle và 1 vùng serine protease. Prothrombin là tiền chất của thrombin có vai trò quan trọng trong sinh lý đông máu cũng như tình trạng bệnh lý. Thrombin được xem như chìa khóa để chuyển hóa fibrinogen thành fibrin bằng cách hoạt hóa các yếu tố đông máu như XIII, thúc đẩy chuyển plasminogen thành plasmin và kích thích tăng sinh các tế bào tơ (fibroblast), giúp ổn định quá trình làm tan huyết khối. Trong nghiên cứu, các gen prothrombin được tách dòng từ phổi bỏ có kích thước 936 bp, mã hóa cho 312 axit amin được tối ưu hóa codon, nhân dòng vào vector pET21a+ và biểu hiện trong E. coli. Mục đích của nghiên cứu nhằm tạo ra băng gạc cầm máu nhanh, giá thành rẻ, phục vụ sức khỏe cộng đồng và thay thể băng gạc truyền thống. Kết quả nghiên cứu bước đầu cho thấy đã nhân dòng và biểu hiện thành công prothrombin tái tổ hợp ở chủng E. coli JM109(DE3).

The need for tissue and organ replacements cannot be satisfied by autograft and allografts alone. The purpose of this study was to investigate the feasibility of electrospinning a blend of polydioxanone and fibrinogen to produce an engineered tissue scaffold. Fiber diameter and pore size of blends were characterized, as well as mechanical strength. Cell proliferation assays for 1 and 7 day cultures were preformed, and a histological evaluation was performed to determine how favorable the various blends were to cell infiltration and proliferation. Some ratios of blends were identified that contained both acceptable mechanical properties and properties that facilitated cell infiltration. These findings pave the way for future refinement and use of these scaffolds for a variety of tissue engineered targets.

Resumo:Os dímeros-D constituem os menores fragmentos dos produtos de degradação da fibrina e são produzidos após a lise pela plasmina da ligação cruzada da fibrina. Desta forma, os objetivos deste estudo foram determinar as concentrações plasmáticas de dímero-D e fibrinogênio de bezerros recém-nascidos e em vacas pós-parto e comparar as concentrações plasmáticas deste marcador hemostático entre os dois grupos. Aumentos significativos nas concentrações plasmáticas de dímero-D estão ligadas a ocorrência processo inflamatórios (como peritonite, sepse), coagulação intravascular disseminada, trombose de veia cava como por exemplo. As concentrações plasmáticas de dímero-D e de fibrinogênio não foram diferentes entre vacas pós-parto e bezerros com até 24 horas de idade. Houve correlação positiva significativa entre as concentrações plasmáticas de fibrinogênio entre os dois grupos. Não houve correlação entre as concentrações plasmáticas de dímero-D entre os dois grupos. Sendo assim, foi possível estabelecer intervalos de referência para as concentrações plasmáticas de dímero-D em vacas pós-parto e em bezerros recém-nascidos. / Abstract:The D-dimers are the smallest fragments of fibrin degradation products and are produced by plasmin after lysis of fibrin cross-connection. Thus, the objectives of this study were to determine plasma concentrations of D-dimer and fibrinogen in newborn calves and postpartum cows and to compare plasma concentrations of this hemostatic marker between these two groups. Significant increases in plasma concentrations of D-dimers occur during inflammatory processes (such as peritonitis, sepsis), disseminated intravascular coagulation and vena cava thrombosis for example. Plasma concentrations of D-dimer and fibrinogen were not different between groups. There was a significant positive correlation between plasma fibrinogen concentrations between groups. There was no correlation between plasma D-dimer between groups. Therefore, it was possible to establish reference intervals for plasma concentrations of D-dimer in postpartum cows and their newborn calves. / Orientador:Juliana Regina Peiró / Banca:Lina Maria Wehrle Gomide / Banca:Raimundo Souza Lopes / Mestre