Browsing Inaugural Addresses (Biochemistry) by Title
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- ItemComparative characterization and mutational analysis of type III pantothenate kinases(Stellenbosch : University of Stellenbosch, 2006-03) Brand, Leisl Anne; Strauss, Erick; Swart, Pieter; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.This thesis reports the cloning, overexpression and characterization of the coaX gene product from Bacillus subtilis and its homologue from Helicobacter pylori. It demonstrates that these proteins have pantothenate kinase activity. Compared to the two pantothenate kinase analogues classified to date, these two enzymes exhibit distinctly different characteristics, suggesting that they are the first characterized examples of a third pantothenate kinase analogue. In addition, mutational studies are presented that probe the importance of conserved aspartate residues within the active sites of these newly characterized analogues. The results show that these residues are important for the activity of the protein.
- ItemDevelopment of a synthetic affinity membrane for the purification of recombinant maltose binding proteins(Stellenbosch : Stellenbosch University, 2008-12) Asongwe, Lionel Ateh Tantoh; Swart, P.; Jacobs, E. P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.The aim of this project was to fabricate a new affinity membrane-based system that is biospecific and biocompatible, and which could be used as an adsorption matrix for the immobilization of the recombinant protein maltose binding protein human estrogen receptor alpha ligand binding domain (MBP-hER LBD). The viability of the affinity membrane system (AMS) for the detection of estrogenic compounds (ECs) in drinking water, using affinity principles was determined. This affinity separation was based on the interaction between the analyte 17 -estradiol (E2) and the recombinant protein MBP-hER LBD. The MBP-hER LBD was immobilized on a solid matrix support. The alpha human estrogen hormone receptor (hER ) was used to test for the binding affinity of the fusion protein to a ligand, radiolabelled E2. Each component of this bioaffinity system, from the membrane matrix to the expression/purification of the bioligand, and raising of antibodies against the purified bioligand, was studied with the aim of producing a well-characterized system with the following advantages: robust in nature, cost effective and high loading capacity. Specifically, this study describes: 1. Expression of the bioligand maltose-binding protein (MBP) to be used as an affinity ligand for immobilization onto a solid membrane matrix. 2. Expression of MBP as a fusion protein to the human estrogen receptor alpha ligand binding domain (hER LBD). 3. The affinity purification of biospecific bioligands (MBP and MBP-hER LBD) using a one-step affinity purification system with amylose forming the solid phase of the affinity chromatographic column. 4. Generation of anti MBP-hER LBD antibodies to be used for the characterization of the bioligands by means of western blotting. 5. The fabrication and characterization of a flat-sheet membrane as a model affinity-matrix. 6. Developing an affinity immobilization protocol for the immobilization of the bioligand onto the affinity membrane (AM) matrix. 7. Quantitative analysis of the immobilized bioligand present on the surface of the membrane matrix using tritiated E2. The recombinant protein (MBP-hER LBD) was successfully expressed and purified to form a bio-specific ligand for its immobilization onto a cellulose acetate (CA)/amylose functionalized affinity membrane. Polyclonal antibodies were successfully raised against the purified recombinant protein. The anti-MBP-hER LBD antibodies were subsequently used as a potential ‘marker’ to confirm the immobilization of the recombinant protein onto the CA/amylose functionalized membrane. Attempts to utilize the protein-coated membrane for the selective recovery of E2 were, however, unsuccessful.
- ItemGeneric kinetic equations for modelling multisubstrate reactions in computational systems biology(Stellenbosch : University of Stellenbosch, 2006-03) Hanekom, Arno J.; Rohwer, J. M.; Hofmeyr, J-H. S.; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.Systems biology is a rapidly developing field, studying biological systems by methodically perturbing them either chemically, genetically or biologically. The system response is observed and incorporated into mathematical models. These computational models describe the system structure, predicting its behaviour in response to individual perturbations. Metabolic networks are examples of such systems and are modelled in silico as kinetic models. These kinetic models consist of the constituent enzyme reactions that make up the different pathways of a metabolic network. Each enzyme reaction is represented as a mathematical equation. The main focus of a kinetic model is to portray as realistically as possible a view in silico of physiological behaviour. The equations used to describe model reactions therefore need to make accurate predictions of enzyme behaviour. Numerous enzymes in metabolic networks are cooperative enzymes and many equations have been put forward to describe these reactions. Examples of equations used to model cooperative enzymes are the Adair equation, the uni-reactant Monod, Wyman and Changeux model, Hill equation, and the recently derived reversible Hill equation. Hill equations fit the majority of experimental data very well and have many advantages over their uni-substrate counterparts. In contrast to the abovementioned equations, the majority of enzyme reactions in metabolism are of a multisubstrate nature. Moreover, these multisubstrate reactions should be modelled as reversible reactions, as the contribution of the reverse reaction rate on the net conversion rate can not be ignored . To date, only the bi-substrate reversible MWC equation has been formulated to describe cooperativity for a reversible reaction of more than one substrate. It is, however, difficult to use as a result of numerous parameters, not all of which have clear operational meaning. Moreover, MWC equations do not predict realistic allosteric modifier behaviour [2, 3]. Hofmeyr & Cornish-Bowden  showed how the uni-reactant reversible Hill equation succeeds in predicting realistic allosteric inhibitor behaviour, compared to the uni-reactant MWC equation, which does not. The aim of this study was to therefore derive a reversible Hill equation that can describe multisubstrate cooperative reactions and predicts realistic allosteric modifier behaviour. In this work, we present a generalised multisubstrate reversible Hill (GRH) equation. The bi-substrate and three substrate cases of this equation were also extended to incorporate any number of independently binding allosteric modifiers. The derived GRH equation is evaluated against the above mentioned cooperative models and shows good correlation. Moreover, the predicted behaviour of the bi-substrate reversible Hill equation with one allosteric inhibitor is compared to the MWC equation with one allosteric inhibitor in silico. This showed how the bi-substrate reversible Hill equation is able to account for substrate-modifier saturation, unlike the MWC equation, which does not. Additionally, the bi-substrate reversible Hill equation behaviour was evaluated against in vitro data from a cooperative bi-substrate enzyme which was allosterically inhibited. The experimental data confirm the validity of the behaviour predicted by the bi-substrate reversible Hill equation. Furthermore, we also present here reversible Hill equations for two substrates to one product and one substrate to two products reactions. Reactions of this nature are often found in metabolism and the need to accurately describe their behaviour is as important as reactions with equal substrates and products. The proposed reversible Hill equations are all independent of underlying enzyme mechanism, they contain parameters that have clear operational meaning and all of the newly derived equations can be transformed to non-cooperative equations by setting the Hill coefficient equal to one. These equations are of great use in computational models, enabling the modeller to accurately describe the behaviour of a vast number of cooperative and non-cooperative enzyme reactions with only a few equations.
- ItemI go to seek a great perhaps: the quest to obtain an approximate understanding of steriod hormone receptor signalling(2015-04) Louw, AnnAnn Louw (née Ramsay) was born on 30 March 1957 in Vereeniging, where she grew up until the age of 13 when her parents relocated to Mexico City. She started her schooling at Drie Riviere Laerskool and Drie Riviere Hoërskool in Vereeniging, continued it at the Greengates School in Mexico City and completed Grade 13 at Forest Hill Collegiate Institute in Toronto, Canada. She returned to South Africa for her tertiary education, completing her BSc with majors in biochemistry and physiology in 1977 and her BSc Honours in 1978 at Stellenbosch University, the alma mater of both her mother, Una Ramsay (BA in 1947), and her grandfather, Eben Dönges (BA in 1918, MA in 1919). She then continued studying towards her MSc in Biochemistry while working for the Research Department of the Western Province Blood Transfusion Service (1979–1985), completing the degree cum laude in 1984. She married her husband Albé in 1980. In 1986 her son Niel was born, and she needed some time to acclimatise to parenthood. Thus she accepted part-time employment: first as part-time technical officer in the Department of Medical Biochemistry at the University of Cape Town (1987–1988) and in 1988 as part-time technical officer in the Department of Biochemistry at Stellenbosch University, where she has remained ever since. In 1990, the year her second son, Ramsay, was born, she was appointed as lecturer in the Department of Biochemistry and continued her studies under the supervision of Prof Kirsten van der Merwe and Prof Pieter Swart, receiving her PhD in 1998, the year her daughter Una was born. She was promoted to senior lecturer in 1999, to associate professor in 2009 and to full professor in 2014. Prof Louw’s research focuses on signal transduction via steroid hormone receptors, specifically the glucocorticoid receptor (GR) and the oestrogen receptor (ER), which mediate the intracellular actions of the stress hormone cortisol and the sex hormone oestrogen, respectively. Recent work on the phytooestrogenic activity of Cyclopia or honeybush tea has highlighted ER subtype-specific signalling, indicating a potential use in breast cancer prevention or treatment, while recent work on the GR is focussing on the implications of loss or gain of dimerisation.
- ItemMore than a vitamin : walks along an essential pathway(Stellenbosch : Stellenbosch University, 2014-03) Strauss, ErickErick Strauss, born on 4 November 1975 and raised in Pretoria, attended the University of Pretoria from 1994 to 1997 where he obtained his BSc (majoring in chemistry and biochemistry) and BSc(Hons) (in chemistry) degrees, both cum laude. He subsequently moved to the USA in 1998 to pursue his graduate studies at Cornell University in Ithaca, NY, where he worked on the biosynthesis of coenzyme A with Prof Tadhg Begley. He obtained a PhD in Chemistry and Chemical Biology from Cornell in 2003. He returned to South Africa in the same year to accept an offer from Stellenbosch University to establish his own research group in the Department of Chemistry and Polymer Science. In 2008 he moved to the Department of Biochemistry (also at Stellenbosch University) as associate professor, and was promoted to full professor in 2013. Since the start of his independent career he has trained 11 MSc students (one as co-supervisor) and nine PhD students (one as co-promotor); he currently leads a group of four PhD students and four postdoctoral fellows. Erick is regarded as a leading authority on the biosynthesis and enzymology of the essential metabolic cofactor coenzyme A, as well as of the design, discovery and development of antimicrobial agents that target this pathway. His publications, which have attracted a total of more than 680 citations (h-index of 12), include articles in prestigious journals such as the Proceedings of the National Academy of the USA (PNAS), the Journal of the American Chemical Society (JACS) and two articles in Nature Chemical Biology. One of these is a commentary piece chosen as one of ten winning entries (one of only two from outside the United States) submitted by young scientists active in the field in which they expounded their vision of the future ‘Grand Challenges’ in the field of Chemical Biology. Erick currently holds a B3-rating from the NRF. Erick is a recipient of the DuPont Prize for Excellence in Teaching from Cornell University (1999); the Rector’s Award for Excellence in Teaching from Stellenbosch University (2007); the President’s Award from the South African National Research Foundation (2008); the Beckman-Coulter Silver Medal from the South African Society for Biochemistry and Molecular Biology (2010); and the Raikes Medal from the South African Chemical Institute (2013). In 2012 he was elected as a founding member of the South African Young Academy of Science. Since 2009 he is also husband to Suzanne and since 2012 father to Matteo – perhaps his biggest (and ongoing!) achievements to date.
- ItemSelective extraction of Cyclopia for enhanced in vitro phytoestrogenicity(Stellenbosch : University of Stellenbosch, 2008-03) Mfenyana, Ciko; Louw, Ann; Joubert, Lizette; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.Phytoestrogens are plant compounds whose ability to mimic the action of estrogens has resulted in their usage for the treatment of menopausal symptoms. Despite uncertainties about the safety and effectiveness of phytoestrogens in humans, the use of market phytoestrogenic nutraceuticals and botanicals is on the increase. Positive epidemiological study findings coupled to an entrenched belief in many societies about the superiority of what they view as “natural” remedies, as well as the reluctance of women to use the traditional hormone replacement therapy due to its association with detrimental health effects as reported by studies such as the World Health Initiative, the Million Women and the Kronos Early Estrogen Prevention studies, are thought to be instrumental in the growth of the phytoestrogen market. As the subject of the current thesis, we investigated the candidacy of extracts of the honeybush plant (genus Cyclopia), which is used for the manufacture of popular tea beverages, for use in the formulation of a high quality phytoestrogenic nutraceutical with a competitive market edge. We evaluated four harvestings of Cyclopia (M6-9) available in bulk and selected 2 harvestings (M6 and M7) for further extraction using solvents of differing polarity and also mimicking the preparation of a cup of tea. Our findings clearly demonstrate that of the resultant 22 extracts the SM6Met and SM6EAc extracts had the highest in vitro potency and efficacy, respectively. Another exciting finding from our study is the unequivocal demonstration of phytoestrogenic activity by extracts prepared in the same manner as the traditional cup of honeybush tea. Additionally, our study has highlighted the importance and the influence of experimental variables such as the specific harvesting evaluated and the characteristics of the extraction solvent (e.g. polarity and temperature) on the yield and the estrogenic activity of the extracts. In addition, the advantage of certain in vitro assays over others for discriminating between estrogenic substances based on their efficacies and potencies was demonstrated with the alkaline phosphatase assay being most suitable for discriminating efficacy and the E-screen most suitable for discriminating potency. Furthermore, our study has imparted a valuable lesson about the pharmacological behavior of estrogenic substances by presenting a conundrum in the form of the two desirable pharmacological parameters (potency and efficacy) occurring in different extracts, an outcome that complicates the central aim of our study, which is the preparation of an extract that embodies both parameters. Additionally, the low quantity of known putative phytoestrogens and the presence of unidentified polyphenols in M6, the source of our choice extracts (SM6Met and SM6EAc), makes the high estrogenic potency and efficacy of the choice extracts that much more intriguing. Nonetheless, benchmarking against four market phytoestrogen extracts indicate that the Cyclopia extracts have comparable estrogenicity suggesting potential as marketable phytoestrogenic preparations. The combination of the achievement of aims and the birth of new questions from that very achievement, which are the hallmark of scientific endeavors, have made this study a rewarding experience and we hope to share the feeling in its entirety with the reader.
- ItemUnravelling the interconnections of cellular regulation(Stellenbosch : Stellenbosch University, 2012-10) Rohwer, Johann M.Johann Rohwer was born in Greytown on 25 May 1968 and grew up in the German settlement Hermannsburg in the KwaZulu-Natal Midlands. He received all his schooling at the Deutsche Schule Hermannsburg. After matriculating in 1985, a post-matric year (the German ‘Abitur’) at the Deutsche H¨ohere Privatschule inWindhoek followed. From 1987 to 1989 he studied for a BSc degree at Stellenbosch University, majoring in biochemistry, chemistry and mathematics. This was followed by a BScHons and an MSc in biochemistry (all cum laude), the latter under the supervision of Prof Jannie Hofmeyr, studying the regulation of serine biosynthesis in Escherichia coli. For his doctoral studies, Johann went to the Netherlands and investigated the regulation of bacterial sugar transport under the supervision of Prof HansWesterho and Dr Pieter Postma at the University of Amsterdam, graduating in April 1997. Upon his return to South Africa in 1997, Johann took up a position as Senior Lecturer in the Department of Biochemistry at Stellenbosch University and has been with the department ever since. He was promoted to Associate Professor in 2002 and to Full Professor in 2011. Under his supervision and co-supervision fourteen MSc students and seven PhD students obtained their degrees, and he has co-authored 45 peer-reviewed articles in international journals. Prof Rohwer was fortunate to spend two sabbaticals in overseas laboratories. During 2001, he visited Prof Philip Kuchel at the University of Sydney and learnt about applying the technique of NMR spectroscopy to study living cells in a non-invasive way. During 2008, he spent a year in Germany with his family as a research fellow of the Alexander von Humboldt Foundation, collaborating with Prof Mark Stitt at the Max Planck Institute of Molecular Plant Physiology on the modelling of central metabolism in plants. Johann’s research interests are computational and experimental systems biology, focusing on the analysis of the central metabolism of microbes and plants. He has received numerous awards, among others the Stellenbosch University Chancellor’s Medal (1993), the President’sAward fromthe South African National Research Foundation (2001), the Silver Medal of the South African Society of Biochemistry and Molecular Biology (2003), and the Vice Chancellor’s Award for Excellent Research from Stellenbosch University (2010). He serves on the international STRENDA Commission and on the editorial boards of BMC Systems Biology and Frontiers in Plant Systems Biology. Johann is married to Christa and they have three children–Nicola (9), Saskia (7) and Martin (19 months).