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Master of Science / Department of Biochemistry / Michal Zolkiewski / Early-onset dystonia (EOTD, also known as DYT1 or Oppenheim’s dystonia is the most severe and common form of hereditary dystonia, a neurological disorder characterized by abnormalities in the control of movement. It is linked to the deletion of a single GAG codon in the gene DYT1 that leads to the loss of a single glutamic acid residue in the C-terminal region of the protein torsinA (ΔE-torsinA). It is not known how the GAG deletion alters the torsinA structure and function. In this thesis, the expression and purification of recombinant torsinA variants from E. coli is reported. Wild type torsinA is not soluble after its expression in E. coli, possibly due to misfolding caused by cysteine. We produced Cys-less torsinA, and established a purification procedure to produce this mutant torsinA. Furthermore, because of the critical role likely to be played by the C-terminal domain of torsinA that contains the glutamate deletion, we produced fragments encoding the C-terminal domain of torsinA, and attempted to purify it. However, we failed to obtain appreciable amount of active proteins by both of the strategies. A novel SUMO fusion technology was also used in this study. We demonstrated that SUMO, when fused with torsinA variants, was able to enhance its expression and solubility in E. coli. A satisfactory yield of the fusion protein was successfully purified. Once we get appreciable quantities of folded torsinA variants, it is our future goal to study their function by using biochemical and high-resolution structural approaches.

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A research proposal submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science in Engineering. Johannesburg, 2016 / In the past three decades there has been an increased interest in constructed wetlands (CW) and their effectiveness in treating water. The hydraulic efficiency of a CW can be determined by using chemical reactor theory to develop residence time distribution (RTD) parameters such as effective volume (Veff), normalized variance (σ2) and mean residence time (τm). Five experiments were conducted to study the effects on these RTD parameters in a CW by using a glass pilot-scale laboratory rig and varying the inlet-outlet positions. The rig made use of a glass tank 250x250x500mm filled with clear superabsorbent polymer balls as a packing. The clear tank and balls made it possible for the flow to be observed when a FWT red impulse tracer dye was inserted into the system. The flow was photographed at specific time intervals for visual analysis and comparison. . The visual results showed the formation of a hull-shaped velocity profile in all the experiments. The RTD was obtained by collecting tracer samples at specific outlet positions during the course of each experiment. The five inlet-outlet configurations RTD parameters results showed; a straight flow path from a single inlet to outlet yielded the lease desirable hydraulic performance with dead volumes contributing to up to 67% of the CW. An increase in the number of outlets and changing the direction of flow diagonally showed up to a 96% improvement to the effective volume of the system could be achieved when compared with single inline inlet-outlet flow. The best result was achieved by combining the visual and RTD data to make changes to the rigs geometry in order to eliminate dead zones and yielded up to a 148% improvement in the effective volume of the system when compared with single inline inlet-outlet flow. A well designed CW with respect to inlet-outlet position can result in reduced land requirements and construction costs by minimizing the dead volume and improving hydraulic efficiency. / MT2016

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment for the degree of Master of Science in Chemistry. May 2013. / In relation to conventional water treatment methods, membrane separation has acquired a great audience due to its wide applicability, reliability, low cost, low energy demands, and ease of use. However, membrane fouling has been identified as the main downsizing factor in the application of this technology. To address this issue, several studies have suggested the use of inorganic additives for enhancement of the membrane antifouling properties. In this study, silver (Ag) decorated nitrogen doped carbon nanotubes (N-CNTs) dispersed into polyethersulphone (PES) membranes for potential use in water treatment. Firstly, N-CNTs were synthesised using the chemical vapour deposition (CVD) method. The black soot was functionalised and characterised using transmission electron microscopy (TEM), scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and Brunner-Emmett-Teller (BET). The N-CNTs were found to have an average diameter of 15 nm. The functionalised N-CNTs were then decorated with Ag nanoparticles (AgNPs) using the Polyol method. The resultant product was also characterised using TEM, the AgNPs were found to have an average diameter of 6 nm. The N-CNTs and Ag/N-CNTs were then uniformly dispersed into (PES) membranes to form N-CNT/PES and Ag/N-CNT/PES blend membranes, respectively. The membranes were then characterised using several series of techniques including scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle analyser and a cross-flow filtration system. The blend membranes were investigated for any improved properties and tested for their efficiency in removing model pollutants (polyethylene glycol, humic acids, and bacteria) from water. The AFM results revealed a reduction in surface roughness from 23.9 nm for the pristine PES to 12.7 nm in the N-CNT/PES blend membranes. The mechanical stability increased from 3.7 MPa for the pristine PES to 4.4 MPa with a small addition of N-CNTs. Furthermore, the performance studies showed a 46% increase in pure water flux and a 13% increase in rejections for N-CNT blend PES membranes as compared to the pristine PES membrane. Antibacterial studies were also performed where Ag modified N-CNTs were found to inactivate Enterohaemorrhagic E. coli by almost twice the initial concentration in the bacterial suspensions. Finally, Ag/N-CNTs were immersed into PES membranes. The Ag/N-CNTs/PES membranes were then tested for their activity towards the bacteria.