Enterotoxigenic Escherichia coli (ETEC) encompass a group of diverse bacterial pathogens that collectively cause hundreds of millions of diarrheal cases annually, mostly in developing countries. As part of its infection strategy, ETEC invades and colonizes small intestinal epithelial cells where it secretes heat-labile and/or heat-stable enterotoxins, inducing diarrhoea. The ability of ETEC to invade human epithelial cells is a hallmark of its pathogenicity and is controlled by a set of plasmid and chromosome encoded virulence factors. They include EtpA, a 170 kDa plasmid encoded autotransporter. During infection, EtpA functions as an adhesin linking flagellin at the tip of ETEC flagella to the host cell surface and allowing ETEC to deposit its toxins. Antibodies targeting either EtpA or the conserved regions of flagellin impair delivery of the heat-labile toxin in vitro, and prevent intestinal colonization of mice following gastrointestinal challenge with ETEC. EtpA is thus critical to the pathogenicity of ETEC. In this study, a truncated version of EtpA (35 kDa) termed N-terminal EtpA69-445 or N-EtpA69-445 was cloned and produced as an N-terminal GST-tagged cytoplasmic fusion protein in E. coli BL21 cells. The protein was purified by affinity chromatography on glutathione agarose beads. However, the yield of the pure protein was poor due to its limited solubility. As an alternative, a 57 kDa truncated version of EtpA (N-EtpA69-607) was produced as a secreted C-terminal His6-tagged fusion protein in E. coli TOP10 cells. The protein was purified to homogeneity by metal affinity chromatography (MAC) using Ni-NTA and ion exchange chromatography (IEC) on a Mono S 10/100 GL column. Biophysical characterization of N-EtpA69-607 using circular dichroism (CD) spectroscopy revealed the typical spectrum of a β-helical protein. The in silico modelled structure of the protein confirmed N-EtpA69-607 to be a β-helical protein. CD spectra recorded at increasing temperatures indicated N-EtpA69-607 to be thermally highly stable retaining its fold up to 95°C. Dynamic light scattering (DLS) experiments showed that N-EtpA69-607 is polydisperse in solution forming higher oligomers. Lead crystallization conditions of N- EtpA69-607 were determined but the crystals were too small for X-ray data collection. This study thus represents a significant step towards the characterization of the three dimensional structure of EtpA and understanding its structure-function relationship. / Dissertation (MSc)--University of Pretoria, 2016. / Biochemistry / MSc / Unrestricted

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<p> The presence of 0.10 moles/l. of glycine, glutamic acid, aspartic acid or leucine in a saturated bicarbonate solution will promote the formation of aragonitic calcium carbonate at 25.2°C. Magnesium ion, with or without an amino acid, also promotes the formation of aragonite. With increased amino acid concentration glutamic acid and alanine tend to promote the formation of more calcitic calcium carbonate, glycine and valine tend to promote the formation of more aragonitic calcium carbonate. Lysine and alanine show an increased tendency to form calcite at higher ionic strengths (.20) whereas glutamic acid shows the reverse. Some correlation with solubility and equilibrium constants for magnesium-amino acid complexes is indicated.</p> / Thesis / Candidate in Philosophy

Finding materials with high stability, high yield, and excellent catalytic performance has been an outstanding research challenge. Thiol-stabilized atomically precise metal nanoclusters (NCs) have attracted a lot of interest in recent years due to their unique properties and industrial applications, including catalysis, toxic metal-ion sensing, and magnetism. Unlike classical nanoparticles (NPs) which have larger sizes ranging between 10-100 nm, the size of nanoclusters typically ranges from 1 to 3 nm. Thus, nanoclusters represent good candidates to gain fundamental insights into optical and catalytic properties from the atomic to the molecular level. The majority of nanoclusters are based on Ag, Au, and more recently Cu. The major goal of this dissertation is the synthesis and structure determination of silver and copper nanoclusters. The first part of this dissertation addresses a shape-controlled synthesis strategy based on an isomeric dithiol ligand to yield the smallest crystallized silver nanocluster to date, formulated as Ag9. The second part discusses the preparation of different sizes of copper nanoclusters using a one-pot synthesis method and attempted to understand the structure-dependent catalytic pathways at the atomic level. Moreover, three novel Cu nanoclusters were developed: Cu29, Cu45, and Cu67. In terms of size, Cu67 represents a milestone towards high-nuclearity nanoclusters with interesting optical and structural properties. In terms of catalytic properties, Cu45 was found to be promising catalyst in hydroboration reactions of alkynes/alkenes and B2pin2, which affords hydroborated products with good to excellent yield. Mechanistic studies showed single-electron oxidation of an in-situ formed ate complex ([B2pin2]OH-) by the Cu45 catalyst, enabling hydroboration of unsaturated double bonds via a boryl-centred radical. These findings motivate us to extend the library of ligand-protected nanoclusters and focus deeply on exploiting metal nanoclusters based on Cu for catalysis.

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