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Regenerative medicine has the potential to establish or restore normal function in defective tissues and organs. The realisation of such therapies is restricted due to costs, lack of scalability and inefficient manufacturing process controls. A major contributor to cost is the use of expensive growth factors supplemented into media at high concentrations. In vivo, growth factors exist in soluble, immobilised and transmembrane forms, expressed in a spatiotemporal fashion within the stem cell niche. In comparison to soluble equivalents, immobilised growth factors exhibit increased potency, distinct functional activities, improved cell phenotypic control and act in synergy with other soluble and immobilised ligands. To date, most research into immobilised growth factors has been restricted to planar cell culture surfaces such as tissue culture plastics which have limited scalability. To address the scalability limitations, a novel growth factor immobilisation technology was developed using magnetic microparticles which can be scaled with respect to surface area to volume ratio in standard stirred tank bioreactors. Three clinically relevant growth factors, SCF, TPO and GM-CSF were immobilised and were shown to remain functionally active where surface concentration could be manipulated in a number of ways. Through a series of experiments, it was demonstrated that immobilised growth factors exhibited ~10-fold increase in potency compared with soluble equivalents and remain stable for up to 192 hours following recycling during multiple media passages. Immobilised growth factors were able to expand more cells over a longer period of time after transient exposure and finally, the immobilisation technique was successfully applied to the expansion of umbilical cord derived haematopoietic stem cells using immobilised SCF. The immobilisation method described here has the potential to significantly reduce media costs in large scale cell manufacturing processes.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: During the last two and a half decades a large body of research has accumulated indicating the presence of various natural and synthetic chemical compounds within the environment capable of inducing hormone-like responses in humans and animals. Such compounds, termed endocrine disruptors, have been implicated in a variety of developmental, reproductive and physiological abnormalities which have been shown to converge on the endocrine system. Given that endocrine disrupters are comprised of a diverse group of molecules with dissimilar chemical structures, general screening techniques are not feasible for effective environmental monitoring. A primary method of action by which these exogenous molecules affect the homeostatic regulation of the endocrine system is believed to be via the modulation of gene transcription. It is now well established that many endocrine disrupting compounds act upon a principal group of transcription factors, the nuclear receptors, by chance interaction with the ligand binding domains of these proteins. With a view to ultimately design a portable kit for the detection of endocrine disrupting compounds in water based on the bio-specific immobilisation of nuclear receptor ligand binding domains to a stationary membrane matrix, this study specifically describes: 1. The effects on recombinant protein expression by the addition of small molecules to the cultivation media of bacteria. 2. The optimisation of conditions for the lysis of bacterial cells to increase the solubility of heterologously expressed proteins. 3. The purification of recombinant proteins from bacterial cell lysates by means of a two-step chromatographic methodology. 4. The cloning of the genes for the human androgen and estrogen receptors’ ligand binding domains into baculovirus transfer plasmids. 5. Transfer of genetic material from the created baculovirus transfer plasmids to a linearised baculovirus genome for the generation of recombinant viruses. 6. The cultivation, and baculoviral infection, of Spodoptera frugiperda and Trichoplusia ni cell lines. 7. Expression and purification of N-terminal hexahistidine-tagged human nuclear receptor LBDs from insect cell lysates by means of immobilised metal affinity chromatography. / AFRIKAANSE OPSOMMING: Die teenwoordigheid van natuurlike en sintetiese chemiese middels wat oor die vermoë beskik om die aksies van hormone in die mens en dier na te boots het toenemend aftrek gekry in navorsing gedurende die laaste twee en ’n halwe dekades. 'n Verskeidenheid van ontwikkelings-, reproduktiewe- en fisiologiese abnormaliteite ontstaan as gevolg van die aksies van hierdie molekule, genaamd endokriene-ontwrigters, op die natuurlike funksionering van die endokriene-sisteem. Gegewe dat die groep chemiese middels waaruit endokriene-ontwrigters bestaan van diverse oorsprong afkomstig is lei dit daartoe dat algemene analitiese tegnieke nie in alle gevalle geskik is vir effektiewe omgewingsmonitering is nie. Die modulasie van geentranskripsie is een van die metodes wat voorgestel word as ’n metode waarop hierdie eksogene molekule die homeostatiese regulering deur die endokriene-sisteem omverwerp. ’n Algemene metode waarop vele endokrien-ontwrigtende stowwe geentranskripsie beïnvloed, is deur interaksie met die hormoon-bindende gedeeltes van ’n belangrike groep transkripsiefaktore, die nukluêre reseptore. Hierdie studie, met die uiteindelike ontwikkeling van ’n draagbare toetsstelsel vir die opsporing van endokrien-ontwrigtende-stowwe in water, gebasseer op die bio-spesifieke immobilisering van nukluêre reseptor ligand bindingsdomeins op ’n stasionêre membraanmatriks, het ten doel om die volgende te beskryf: 1. Die effek wat die byvoeging van klein molekule tot die groeimedium van bakteriëe het op die uitdrukking van rekombinante proteïene. 2. Die optimisering van bakteriese sel-lisering in terme van verhoging in die oplosbaarheid van heteroloë proteïene. 3. Die suiwering van rekombinante proteïen vanuit bakteriese sellisate deur middel van ’n twee-stap chromatografiese sisteem. 4. Die klonering van die gene vir die menslike androgeen en estrogeen reseptore se ligand bindingsdomeine in bakulovirus oordragplasmiede. 5. Die oordrag van genetiese materiaal vanaf hierdie bakulovirus oordragplasmiede na ’n gelineariseerde bakulovirus genoom deur middel van homoloë rekombinasie vir die produksie van rekombinante virusse. 6. Die groei en infeksie van Spodoptera frugiperda en Trichoplusia ni sellyne wat lei tot die uitdrukking van menssoortgelyke nukluêre reseptor ligandbindingsdomains. 7. Suiwering van N-terminaal heksahistidien-etiket-gekoppelde menslike nukluêre reseptor ligandbindingsdomeins vanuit inseksellisate deur middel van geïmmobiliseerde metaal affiniteitschromatografie.

Catalysis plays a key role in chemical conversions by making them faster and more selective. Despite its widespread use and decades of academic and industrial research, limited catalyst selectivity and stability still call for major improvements in catalyst performance to meet the demands of a sustainable society. Phosphine ligands are ubiquitous in transition metal chemistry and lead to extremely reactive and versatile homogeneous catalysts. Fast development of tailor-made catalysts and catalyst recovery are key issues in (asymmetric) homogeneous catalysis. Therefore libraries of ligands have to be synthesised and screened in an efficient way, which could be facilitated by Solid Phase Synthesis (SPS). Currently, most polymer bound ligands are anchored to the support after the synthesis in solution. However, the main advantages of synthesising the ligands directly on the polymeric support are not only easy catalyst recycling and product separation, but also the ease of purification during the synthesis steps, namely by simple washing and filtration. The use of SPS is very efficient for high throughput synthesis and screening of ligand libraries, however applications of SPS towards libraries of phosphorus ligands are rare, because the synthetic methodologies are still lacking. Here we present the development of methodologies towards novel immobilised bis(phosphine) ligands synthesised on polystyrene and JandaJel™ resin. By performing the synthesis steps on a solid support, the advantages of SPS are fully utilised. Successful routes have been developed towards immobilised secondary phosphine-boranes, which were versatile synthons to prepare a variety of new polymer-supported (C-chiral) bis(phosphine) ligands. These ligands were then tested for their catalytic activity in rhodium catalysed hydrogenation reactions.

Molecularly Imprinted Polymers (MIPs) are potential generic alternatives to antibodies in diagnostics and separations. To compete with biomolecules in these technological niches, MIPs need to share the characteristics of antibodies (solubility, size, specificity and affinity) whilst maintaining the advantages of MIPs (low cost, short development time and high stability). For this reason the interest in preparing MIPs as nanoparticles (MIP NPs) has increased exponentially in the last decade. Cont/d.

>Magister Scientiae - MSc / In recent times, the occurrence and presence of complex recalcitrant toxic contaminants in water and wastewater is increasing and consequently contributes to the non-availability of clean and safe drinking water. Water treatment is complex, time demanding and energy intensive due to the physico-chemical structural complexity and diversity of the pollutants. Non-availability of good drinking water has negatively affected human health and the ecosystem. Over the years, numerous conventional treatment techniques were used to degrade and remove these pollutants, but investigations indicated that some of the pollutants are not susceptible to conventional treatment. Advanced oxidation technology, among which heterogeneous photocatalysis (involving the use of a semiconductor) has emerged as one of the more promising techniques to remediate contaminated water. Titanium dioxide (TiO2) semiconductor photocatalysis is considered to be a good option due to its cost effectiveness, chemical and thermal stability, and inertness in the area of wastewater reclamation and re-use. However the post separation of the titania particles poses a threat to the wastewater remediation. Hence there is a need to develop a supported high surface area photocatalyst that will resolve the post separation challenge. This present study aimed to prepare high surface area TiO2 anatase nanocrystals supported on a stainless steel mesh. These new composite materials were used to remove methylene blue (MB) from aqueous solutions. The supporting procedure involved the thermal decomposition of a sol gel solution coated upon stainless steel mesh. The nanocrystalline anatase phase was formed by thermal decomposition on a stainless steel mesh coated with 8 % PAN/DMF/TiO2 sol gel formation calcined at varying temperatures of 300 °C, 400 °C, 500 °C and 600 °C. The heating rate of 50 °C/min and independent holding time of 1 h, 2 h, 3 h and 4 h were applied to find the optimum supporting conditions. The synthesised TiO2 nanocomposites materials were characterised using the following analytical techniques: XRD, HRSEM, EDS, HRTEM, SAED, FTIR and UV-Vis absorption spectroscopy materials were characterised, and the results indicate that synthesised TiO2 nanocrystals were in the anatase form, polycrystalline in nature, and contained additional carbon-carbon bonds from the polymer used during preparation with TiO2 particle sizes range from 13.6 nm to 2285 nm.

<p>Genetic profiling is commonly performed on the autosomes using multiple DNA markers. Although routine forensic DNA analysis is robust and based on reliable technologies, samples with degraded or limited amounts of DNA often fail. In these cases, the analysis of mitochondrial DNA (mtDNA) can be very valuable due to the high copy number per cell. This thesis describes evaluation and modifications of existing technologies that are useful in forensic DNA typing, mainly focusing on mtDNA.</p><p>DNA quantities isolated from common evidence materials such as hairs, fingerprints and accessories were estimated using a real-time quantification assay. Knowledge of quantitative differences between materials can guide forensic scientists to perform the best analysis (Paper I).</p><p>The current mtDNA analysis is based on hypervariable region (HVI/HVII) sequencing, which is the most rigorous and time-consuming forensic DNA analysis. Therefore, we evaluated the possibility to exclude individuals by screening for non-matching samples using the rapid and easy mtDNA Linear Array Assay (Paper II). </p><p>The major disadvantage using mtDNA is the lower discrimination power compared to multiple nuclear DNA markers. In contrast to the nuclear genome, due to the uniparental (maternal) mode of inheritance, no individual has unique mtDNA. We investigated the possibility of increasing the discrimination power by using pyrosequencing technology to analyse parts of the coding region in addition to HVI/HVII (Paper III). Furthermore, the addition of coding mtDNA information was evaluated by comparing several recently published mtDNA coding region assays (Paper IV). </p><p>Mixtures of DNA are common in forensic genetics due to contribution of DNA from several individuals, contamination or heteroplasmy. To resolve mixtures we have developed a pyrosequencing-based assay for the accurate quantification of the mtDNA mixture components (Paper V).</p><p>In conclusion, this thesis describes several assays that are valuable in forensic genetics for DNA quantification, improved mtDNA analysis, and mtDNA mixture interpretation.</p>

Genetic profiling is commonly performed on the autosomes using multiple DNA markers. Although routine forensic DNA analysis is robust and based on reliable technologies, samples with degraded or limited amounts of DNA often fail. In these cases, the analysis of mitochondrial DNA (mtDNA) can be very valuable due to the high copy number per cell. This thesis describes evaluation and modifications of existing technologies that are useful in forensic DNA typing, mainly focusing on mtDNA. DNA quantities isolated from common evidence materials such as hairs, fingerprints and accessories were estimated using a real-time quantification assay. Knowledge of quantitative differences between materials can guide forensic scientists to perform the best analysis (Paper I). The current mtDNA analysis is based on hypervariable region (HVI/HVII) sequencing, which is the most rigorous and time-consuming forensic DNA analysis. Therefore, we evaluated the possibility to exclude individuals by screening for non-matching samples using the rapid and easy mtDNA Linear Array Assay (Paper II). The major disadvantage using mtDNA is the lower discrimination power compared to multiple nuclear DNA markers. In contrast to the nuclear genome, due to the uniparental (maternal) mode of inheritance, no individual has unique mtDNA. We investigated the possibility of increasing the discrimination power by using pyrosequencing technology to analyse parts of the coding region in addition to HVI/HVII (Paper III). Furthermore, the addition of coding mtDNA information was evaluated by comparing several recently published mtDNA coding region assays (Paper IV). Mixtures of DNA are common in forensic genetics due to contribution of DNA from several individuals, contamination or heteroplasmy. To resolve mixtures we have developed a pyrosequencing-based assay for the accurate quantification of the mtDNA mixture components (Paper V). In conclusion, this thesis describes several assays that are valuable in forensic genetics for DNA quantification, improved mtDNA analysis, and mtDNA mixture interpretation.

The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli. A positively charged purification tag, Zbasic, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Zbasic fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified Escherichia coli homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Zbasic moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Zbasic, was effected by adsorption to a second cation-exchanger. Using a similar strategy, a purification tag with a negatively charged surface, denoted Zacid, was constructed and thoroughly characterised. Contrary to Zbasic, the negatively charged Zacid was highly unstructured in a low conductivity environment. Despite this, all Zacid fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed in vivo by the use of a flow cytometer. The positively charged domain, Zbasic, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Zbasic as a reversible linker to the cation-exchanger resin. / QC 20101020