Masters Degrees (Haematology and Cell Biology)
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Browsing Masters Degrees (Haematology and Cell Biology) by Author "De Bruin, Karen"
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Item Open Access Construction of cDNA libraries, and the selection and expression of proteins and peptides involved in haemostasis(University of the Free State, 2004-06) De Bruin, Karen; Meiring, S. M.; Deckmyn, H.English: The need to find new manners in which to combat cardiovascular disease and associated thrombotic complications, remains a high priority in industrialised countries. Even in third-world countries the implications and associated risks of these diseases are being felt more and more. The advent of the biotechnology era and employment of recombinant DNA techniques has brought about exponential advances in understanding the complex mechanisms of haemostasis, and is employed to find new ways to combat pathological thrombotic complications. The challenge is to harness the many tools and techniques produced by the ongoing biotechnology explosion, and apply them to elucidate questions still unanswered and explore areas still unknown. In this study it was illustrated that modern molecular biology techniques can be applied in many areas of thrombosis and haemostasis research. The display of cDNA libraries on the surfaces of filamentous bacteriophages was used in the search for novel antithrombotic compounds from a haematophagous insect Hippobosca rufipes. Phages displaying the cDNA libraries were panned against human a-thrombin and selected according to their binding affinity and inhibition ability. To illustrate the use of a Escherichia coli expression system, a domain of a enzyme was cloned, expressed, and the recombinant peptide isolated and refolded. ADAMTS-13 was recently identified as an important role player in the realm of von Willebrand factor activity, including primary haemostasis and pathological disorders. The second carboxy-terminal CUB domain of ADAMTS-13 was amplified from full-length cDNA, cloned into a expression vector system, and expressed as insoluble inclusion bodies in the cytoplasm of E. coli, from where it was isolated and refolded. In this study, molecular techniques were used in different phases of research into the specific activity and interactions of a particular component of the haemostatic system. This illustrated the marriage of biotechnology with fundamental medical research in an era of interdisciplinary sciences.