published_or_final_version / Surgery / Master / Master of Philosophy

Thymidine oligomers were chemically synthesised and linked to available amino functions of transferrin in alternative orientations: (a) A CMP residue attached to the 3' end of (pT)₁₀ with terminal deoxynucleotidyl transferase was oxidised with NaI0 and linked to transferrin via a Schiff base formation. (b) The 5' terminal phosphate group of (pT)₅ was activated with imidazole and reacted with transferrin to form a phosphoramide bond. The (pT)₅ thus attached to the protein was elongated to (pT)₃₀₀ using terminal deoxnucleotidyl transferase and TTP. The latter conjugate was capable of hybridising poly(A) tailed linear PBR322 DNA. The binding of this hybridisation complex to the transferrin receptor on various cell types was investigated. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

>Magister Scientiae - MSc / Tuberculosis (TB) is a global health problem and rated as the second leading cause of death after HIV/AIDS. Transmission of TB from one person to the next is very rapid in crowded communities. Therefore, it is crucial to identify people who are infected as quickly as possible not only to provide treatment but also to prevent the spread of the disease. Current TB diagnostic tests such as the culture and sputum smear tests are time-consuming, while rapid tests make use of antibodies that are costly and have low sensitivity and stability. Great improvement has been observed when aptamers are used in place of antibodies in rapid diagnostic tests such as lateral flow devices (LFDs). Therefore, the current study aims to synthesize and identify aptamers against serum biomarkers for development of rapid TB diagnostic tests such as a lateral flow assay. Several TB serum biomarkers have been identified and can be used for the diagnosis of TB. TB biomarkers expressed in serum samples were identified through in silico approach. The biomarkers were expressed in bacterial systems using recombinant DNA technology. The recombinant proteins were purified by affinity chromatography and further used as targets for the selection of aptamers using Systemic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers for the selected biomarkers were synthesized based on magnetic-bead based SELEX and characterized by electrophoretic mobility shift assay (EMSA), Surface Plasmon resonance (SPR) and MicroScale Thermophoresis (MST). Six putative TB serum biomarker proteins were selected from literature, namely, Insulin-like Growth Factor Binding Protein 6 (IGFBP6), Interferon-stimulated Gene 15 (ISG15), Calcium Binding Protein (S100A9), Retinol Binding Protein 4 (RBP4), Granzyme A (GrA), and Transgelin-2 (TAGLN2). The biomarkers were recombinantly expressed and purified after which they were used as targets in SELEX for aptamers synthesis. Aptamers were analysed by in silico method and the ones with highly conserved motifs were selected. The selected aptamers were synthesized and later characterized. The aptamers that show high affinity and specificity for the biomarkers will be used for the fabrication of a rapid lateral flow device for TB screening. Such a test would allow for a short diagnostic turnaround time, and hence expedite treatment.

Novel pairs of Bet/Exo recombineering proteins were identified in the beta-proteobacterium Laribacter hongkongensis (LHK) and in the SXT genetic element isolated from Vibrio cholerae. In this research, these new recombineering proteins were functionally characterized using a variety of in vivo and in vitro techniques. The SXT-Exo and LHK-Exo proteins were both found to be alkaline exonucleases, with activities similar to those of Lambda-Exo. Both the SXT-Bet/Exo and LHK-Bet/Exo protein pairs had dsDNA recombination activity within E. coli. / Recombineering is a powerful tool used to manipulate or engineer DNA in vivo, which enables chromosomes and plasmids to be modified precisely and efficiently. It is of critical importance for research into genome and proteome function, and greatly facilitates metabolic engineering applications. The Lambda-Red (Bet and Exo) and RecET proteins constitute the most efficient bacterial recombineering systems characterized to date. However, they work only in E. coli or closely-related bacteria (e.g. Salmonella spp.), which limits their widespread application. / The Lambda-Red and RecET recombineering systems can use PCR products (double stranded DNA, dsDNA) or single stranded DNA (ssDNA, oligonucleotides) to create precise point mutations (substitutions), gene deletions and insertions in chromosomal or episomal DNA. The Exo/RecE exonuclease proteins digest dsDNA and produce long 3'-ssDNA tails. The Bet/RecT ssDNA annealing proteins bind to these 3'-ssDNA tails and promote their homologous recombination with complementary ssDNA regions on the chromosome or episome. / The results described in this thesis will be very useful in assisting the future development of novel recombineering systems that can be used for genetic engineering applications across a wide range of bacterial organisms. Such tools will greatly promote functional genomic and proteomic studies within these organisms, and may also be used for microbial engineering and biotechnological applications. / The ssDNA recombination activities of five different Bet/RecT recombinases were directly compared using an E. coli reporter system. The comparison revealed that Bet protein from LHK had a higher efficiency than Lambda-Bet or RecT. Based on their predicted secondary structure, a set of rationally-designed lambda-Bet protein truncations were prepared and their biological activity was examined, to investigate structure-function relationships within this recombinase. / Chen, Wenyang. / Adviser: Ho W.S. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 113-128). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

<p>This thesis examines the Swedish political response to the challenges posed by gene technology, seen through the prism of governmental commissions. It discerns and analyses continuities and changes in the Swedish political conception of gene technology, over the course of two decades, 1980–2000. This is done by thematically following ideas of “risks” and “ethics” as they are represented in the inner workings and reception of three governmental commissions. The Gene-Ethics Commission (1981–1984), the Gene Technology Commission (1990–1992) and the Biotechnology Commission (1997–2000) form the empirical focal points of this analysis. The first two provided preparatory policy proposals that preceded the implementation of the Swedish gene technology laws of 1991 and 1994. The last one aimed at presenting a comprehensive Swedish biotechnology policy for the new millennium.</p><p> The study takes into account the role of governmental commissions as arenas where science and politics intersect in Swedish political life, and illuminates how this type of “boundary organisation”, placed on the border of science and politics, impinges on the understanding of the gene technology issue. The commissions have looked into the limits, dangers, possibilities and future applications of gene technology. They have been appointed to deal with the problematic task of distinguishing between what is routine and untested practices, realistic prediction and “science fiction”, what are unique problems and what are problems substantially similar to older ones, what constitutes a responsible approach as opposed to misconduct and what it means to let things “get out of hand” in contrast to being “in control”. Throughout a period of twenty years, media reports have continued to frame the challenges posed by gene technology as a task of balancing risks and benefits, walking the fine line between “frankenfoods” and “miracle drugs”. </p><p>One salient problem for the commissions to solve was that science and industry seemed to promote a technology the public opposed and resisted, at least in parts. For both politics and science to gain, or regain, public trust it needed to demonstrate that risks – be it environmental, ethical or health related ones – were under control. Under the surface, it was much more complicated than “science helping politics” to make informed and rational decisions on how to formulate a regulatory policy. Could experts be trusted to participate in policy-making in a neutral way and was it not important, in accordance with democratic norms, to involve the public? </p>

Thesis (D.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 xi, 209 leaves / Protein engineering technologies, such as directed evolution and DNA recombination, are often used to modify enzymes on a genetic level for the creation of useful industrial catalysts. Pre-treatment of paper pulps with xylanases have been shown to decrease the amounts of toxic chlorine dioxide used to bleach pulp. This study was undertaken to improve the thermal and alkaline stabilities of the xylanase from the fungus Thermomyces lanuginosus using ep-PCR and DNA shuffling.

Thesis (Ph. D.)--University of Sydney, 2009. / Title from title screen (viewed 31 March 2009). Submitted in fulfilment of the of the requirements for the degree of Doctor of Philosophy to the Discipline of Medicine, Faculty of Medicine. Degree awarded 2009; thesis submitted 2008. Includes bibliographical references. Also available in print form.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 133-150) Also available in print.

Thesis (Ph.D.)--University of Wollongong, 2008. / Typescript. Includes bibliographical references.

Thesis (D.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 xi, 209 leaves / Protein engineering technologies, such as directed evolution and DNA recombination, are often used to modify enzymes on a genetic level for the creation of useful industrial catalysts. Pre-treatment of paper pulps with xylanases have been shown to decrease the amounts of toxic chlorine dioxide used to bleach pulp. This study was undertaken to improve the thermal and alkaline stabilities of the xylanase from the fungus Thermomyces lanuginosus using ep-PCR and DNA shuffling.