Philosophiae Doctor - PhD / Actinomycetes are ubiquitous in many environments such as soil, activated sludge and water.Besides the genus Streptomyces, which has been extensively exploited, members of other genera including Micromonospora have been shown to be a promising source of novel secondary metabolites and enzymes.The biocatalytic conversion of 5-monosubstituted hydantoin derivatives to optically pure amino acids involves two reaction steps. The first step, catalysed by a hydantoinase, yields an N-carbamylamino acid intermediate, which is subsequently broken down by an Ncarbamoylase to the amino acid. This process has been successfully applied in industry for the production of optically pure amino acids which are used in the synthesis of pharmaceuticals,insecticides, hormones, and food additives. The need for novel hydantoinases to hydrolyse a wider variety of substrates is increasing. This thesis describes the search for a novel hydantoinase from environmental isolates obtained from two Zambian hot-springs. The aim of this study was to isolate, characterise and screen novel actinobacteria for industrially relevant enzymes including hydantoinases. Fifty one actinobacteria were isolated. Isolates were characterized by a polyphasic approach using standard methods, combining phylogenetic analysis of the 16S rRNA gene, chemotaxonomic and phenotypic characterization. Results revealed that these sites were dominated by actinobacteria belonging to the family Micromonosporaceae, and a potentially novel Verrucosispora species was identified. Screening the isolate identified a Streptomyces species which has hydantoinase, carbamoylase, amidase and nitrilase activities.The Streptomyces sp. hydantionase was cloned and functionally expressed in E.coli. The recombinant enzyme showed 49 % similarity to a crystallised hydantoinase from a Bacillus species. Homology modelling revealed that the enzyme had the TIM barrel topology which is characteristic of hydantoinases. Amino acid residues predicted to be involved in the catalytic activity as well as substrate orientation were identified. The partially purified hydantoinase was characterised and showed optimally activity at 45 °C and pH 8. This study revealed that hot springs may represent a previously unexplored source of novel actinobacterial diversity. However, it also revealed that novel secondary metabolites are not only limited to novel organisms but that some of the answers for the challenges we face today maybe found in organisms we have already encountered and characterised.

M. Ing.

No description available.

The full text of this thesis/dissertation is not available online. Pending permission from the author/supervisor access may be provided on <a href="mailto:upetd@up.ac.za">request.</a> Read the abstract in the section 00front of this document. / Dissertation (M Eng (Metallurgical Engineering))--University of Pretoria, 2006. / Materials Science and Metallurgical Engineering / unrestricted

Plants possess integrated signalling networks that mediate the responses to various environmental conditions. Mitogen-activated protein kinases (MAPKs) constitute a highly conserved family of enzymes in eukaryotes, and in plants MAPK-based signal transduction modules regulate a large number of physiological processes, including responses to environmental stresses and phytohormones. Regulated dephosphorylation of active MAPKs is a key component of the control of MAPK signalling cascades, and in mammals, members of the MAPK phosphatase (MKP) sub-class of dual-specificity tyrosine phosphatases have been recognized as key players for inactivating MAPKs. Five MKP homologues are found in Arabidopsis thaliana, but only limited information is available concerning their properties and biological roles. Based on initial data derived from my reverse genetics and protein interaction studies of these five potential MKPs, as well as gene function information in the literature, I chose to focus on two putative Arabidopsis MKPs, AtMKP2 and Indole-3-Butyric Acid-response 5 (IBR5). By using a combination of genetic and biochemical studies, I established that the previously uncharacterized MKP designated AtMKP2, participates in the regulation of cellular homeostasis in ozone-challenged tissue, and can influence the activation state of two MAPKs, MPK3 and MPK6. AtMKP2-suppressed plants displayed significantly prolonged MPK3 and MPK6 activation during ozone treatment, and recombinant AtMKP2 was able to dephosphorylate both phospho-MPK3 and phospho-MPK6 in vitro, providing direct evidence that AtMKP2 may target these oxidant-activated MAPKs. A mutation in IBR5, one of the five potential AtMKPs, was previously reported to confer reduced sensitivity to auxin and ABA in Arabidopsis. My protein interaction studies demonstrated that IBR5 and MPK12 are physically coupled and that the C-terminus of MPK12 is essential for its interaction with IBR5. In vitro dephosphorylation assays indicated that recombinant phosphoMPK12 is efficiently dephosphorylated by IBR5. In transgenic plants with reduced expression of the MPK12 gene, root growth is hypersensitive to exogenous auxins, consistent with the lower auxin sensitivity reported for ibr5 mutants. Taken together, my data demonstrate for the first time that both AtMKP2 and IBR5 are bona fide Arabidopsis MAPK phosphatases and that they serve as important regulators of oxidative stress and auxin signalling, respectively, in Arabidopsis. / Science, Faculty of / Botany, Department of / Graduate

Mathematical models elaborated for removal of microconstituents (MCs) in activated sludge (AS) system have not incorporated developments in International Water Association (IWA) models. In this thesis, sorption and biodegradation kinetic models that can be applied to describe transformation of MCs in an AS process were evaluated (volatilization and photodegradation were considered negligible). Bisphenol-A (BPA), 17α-ethinylestradiol (EE2), and triclosan (TCS) have been selected as target compounds in this thesis. Sorption batch tests were conducted to retrieve kinetic and equilibrium data. Nine lab-scale continuous flow porous-pot bioreactors operating at various solids retention times (SRTs) and hydraulic retention times (HRTs) were studied for biodegradation of MCs. The effects of SRT, HRT and the biomass concentration on sorption and removal of MCs were also investigated and the results of each phase were incorporated into MCs fate models. Freundlich and linear sorption isotherms and pseudo-second-order kinetic models with different kinetic rates were found to best fit the sorption and desorption results. The result of biodegradation study in the presence of 20 µg/L of MCs demonstrated 90-98, 63-91 and 97-98% mean removal rates of BPA, EE2 and TCS, respectively, in systems operating at SRT of 5–15 d and HRT of 4-10 h. Calculation of mass fluxes of selected MCs in the dissolved and particulate phases showed that biotransformation was the principal removal mechanism of targeted MCs. The fate models for the degradation mechanism of selected MCs were evaluated by applying various mathematical models. The pseudo-second-order model was found to best fit the results when active MCs degraders (XC) were used in the model. It was found that biodegradation studies should incorporate XC and not mixed liquor suspended solids concentration in their kinetic formulations. Therefore, the result of this study could be seen in the context, where the active MCs degraders are proposed to reduce the variability of biodegradation kinetic rates in AS systems operating at different operational conditions. Finally nitrification inhibitors, allylthiourea (ATU) were added to reactors and it was found that although nitrification process affects the fate of MCs in AS system, heterotrophs were most likely responsible for the biotransformation of the targeted MCs.

Endometrial cancer is a common gynaecological cancer. Improving outcomes for women with advanced disease remains a challenge and there is also a need to develop preventative strategies in those women at highest risk of developing disease. Peroxisome proliferator-activated receptors (PPARs) comprise of a group of transcription factors belonging to the nuclear hormone receptor subfamily. PPAR sub-types are involved in metabolic homeostasis and have been implicated in malignancy, particularly breast and colo-rectal malignancies both of which are associated with obesity. Endometrial cancer is also closely associated with both obesity and insulin resistance. The work described in this thesis examined the expression of PPARs in endometrioid endometrial cancer and investigated their effects on key pathways implicated in this disease. Immunoblotting revealed over expression of PPARα and loss of PPARγ in human endometrioid endometrial cancer tissues. Pull-down assays also demonstrated differential selectivity of different PPARs for heterodimerisation with different isoforms of the RXR family of transcription factors. PPARα was localized to tumour cells and vascular endothelium and ELISA demonstrated an increase in VEGF-A in PPARα silenced cells suggesting that PPARα may promote tumour angiogenesis. PPARγ was largely seen in epithelial cells and also macrophages within benign endometrium. Reduction of PPARγ expression in cultured endometrial cells led to increased proliferation and decreased apoptosis. Loss of PPARγ was correlated with a loss of the tumour suppressor PTEN in endometrial tissues. Furthermore, PPARγ silencing led to diminished expression of PTEN and a concomitant increase in phosphorylated AKT suggesting that PPARγ is protective against deregulated growth within the endometrium. Synthetic PPAR-specific ligands reduced proliferation and increased apoptosis in endometrial cell lines. These effects were present in PPAR-silenced cells too although reduced in magnitude, indicating that the actions of specific PPAR ligands are mediated via both receptor dependent and receptor independent pathways.In conclusion, this work has demonstrated the differential expression of PPARs and RXRs in endometrial cancers and identified possible mechanisms, both direct and indirect, by which these may modulate endometrial cancer growth. Different PPAR family members may provide targets for therapeutic intervention in endometrial cancer care and require further study in this regard.

A lab-scale study of anoxic-aerobic digestion of waste activated sludge was performed, using 6 litre digesters, and operated in a semi - continuous (fed-once-a-day) manner with solids retention times (SRTs) of 20, 15 and 10 days and mixed-liquor temperatures of 20 °C and 10 °C. Raw sludge was obtained from a pilot-scale biological phosphorus removal facility operating at U.B.C. Fresh sludge was obtained daily and digested by three different digestion modes: anoxic-aerobic, aerobic with lime addition and aerobic. Two aerobic control digesters were run in parallel with the anoxic-aerobic digesters. One of the aerobic digesters received a daily dose of lime slurry. All three digesters were operated under identical conditions (except for the cycling of air supply to the anoxic-aerobic digesters) so that direct comparison could be made between the three digestion modes. Comparisons were made on the basis of five main parameters related to: (1) digestion kinetics, (2) digested sludge characteristics, (3) supernatant quality, (4) ORP monitoring, and (5) an overall rating system. Percent volatile suspended solids (VSS) reduction was used as one performance variable. Despite using only 42 percent of the air required by the two controls, anoxic-aerobic digestion showed comparable percent VSS reductions. All three digestion modes showed increased solids reduction with increasing SRT and temperature. There was a linear relationship between percent TVSS and the product of SRT and temperature. All three digestion modes had a propensity to retain their percent nitrogen and phosphorus within their solids. However, with respect to retaining phosphorus, the aerobic controls were the least effective. Anoxic-aerobic digestion maintained neutral mixed-liquor pH (MLpH) throughout. Lime controls were maintained at MLpH close to neutral. Aerobic digestion, in general, resulted in MLpH levels below 5.0, however, there were periods when the MLpH of the aerobic digesters varied widely between 4.2 and 6.8. Supernatant quality was superior for the anoxic-aerobic digesters. Due to the incorporation of non-aerated periods, there was almost 100 percent denitrification of nitrates produced during the aerated time. This nitrification-denitrification resulted in very low soluble nitrogen levels in the effluent, as well as considerable removal of nitrogen gas. Neither of the controls showed this ability. The lime and aerobic controls produced high levels of effluent nitrates, as well as occasional measurements of ammonia and nitrite. Phosphorus levels were lowest for the lime control and anoxic-aerobic digesters. Presumably, due to reduced pH levels, the soluble phosphorus levels from the aerobic digesters were 2 to 3 times those in the lime or anoxic-aerobic digesters. Alkalinity was conserved in the anoxic-aerobic digesters as well as the lime control. However, the purely aerobic digesters consumed alkalinity until very little buffering capacity remained. Oxidation-reduction potential (ORP) was used as a means of monitoring the anoxic-aerobic digesters on a real time basis. ORP was particularly useful during the non-aerated periods, due to the fact that, at those times, dissolved oxygen was undetectable. Characteristic real time ORP profiles were revealed. Slope changes correlated well with events of theoretical and engineering interest; the' disappearance of ammonia and nitrates, as well as the (dis)appearance of detectable dissolved oxygen, could be predicted by these slope changes. As a result of the findings, ORP may prove to be an ideal parameter for the control of the anoxic-aerobic digestion process. Finally, an overall rating system was developed. The results of this study suggest that, for the digestion of waste activated sludge, anoxic-aerobic digestion out-performed both lime-control and conventional digestion modes. / Applied Science, Faculty of / Civil Engineering, Department of / Graduate

Diploma thesis Effect of emergent micropolutants on the denitrification process through activated sludge is focused on three specific substances. The substances under investigation are atrazine, terbutylazine and sulfametaxazole. Atrazine and terbutylazine are pesticides, particularly herbicides. Sulfametaxazole was chosen as a common antibiotic. The thesis is summarized into 10 chapters and conclusion. The work consists essentially of theoretical and practical part. Part of the theoretical part of the thesis is legislation that deals with the matters of the problem with micropolutants. The next chapter is an introduction to the issue, where we want to familiarize the reader with the current situation. There are also chapters on emergent micropolutants, denitrification and investigated substances. If we look at the practical part, there are chapters on sampling, evaluation of BATCH tests and evaluation of inhibition tests. There are also chapters on BATCH tests and respiratory inhibition test, which describe procedures of how we performed this test. Laboratory test reports are included in the Annex.

Upset events due to the inflow of toxic chemicals are a critical issue for wastewater treatment facilities. Understanding the source-effect relationship of toxic chemicals can facilitate the prevention or improved reaction to upset events. Part one of this study was conducted to investigate the source of upset events at a regional industrial wastewater treatment plant (WWTP). Part two of this study determined the process performance effects of two chemical shocks, cyanide (zinc-cyanide complex) and pH, on nitrifying and non-nitrifying activated sludge. A modified respirometric assay protocol was developed to allow the industrial WWTP to screen industrial wastewaters for inhibitory properties. All five industrial wastewaters tested revealed inhibitory properties. Large day-to-day variations were found, illustrating the need for a large database of results for comparison over time. Additionally, a small volume contributor, that was thought by the utility to be an unlikely source of problems, contributed significantly to the wastewater oxygen demand and demonstrated inhibitory properties. The modified respirometric procedure enabled the WWTP to identify possible industrial sources that could cause an upset event. Lab-scale sequencing-batch reactors were used to determine the effects of cyanide and pH shock on activated sludge. Three reactors were shocked with increasing weak-acid complexed zinc cyanide or pHs of 5, 9, and 11. The resulting effects were compared to an un-shocked control reactor. It was found that respiration and nitrification were affected by the zinc cyanide complex, while COD removal, effluent TSS and dewaterability were not. Recovery was seen in less than 2 X solids residence time (SRT) for the nitrifying biomass and within 3 X SRT for the non-nitrifying biomass. The results of the pH experiment showed that the pH 11 shock affected the settleability, nitrification, COD removal, and effluent TSS levels of the reactors, while pH 5 and pH 9 shocks had no effect. Recovery was seen within 3 X SRT for both the nitrifying and non-nitrifying systems. / Master of Science