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D.Phil. (Chemistry) / Please refer to full text to view abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disorder. Its cause(s) are predominantly unknown; however, a subset of cases has a genetic origin. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of PD. Cases are clinically indistinguishable from idiopathic PD and display incomplete penetrance. Thereby, it is predicted that genetic vulnerability combined with environmental factors cause pathogenesis. However, the identity of these factors is unknown. Unfortunately, LRRK2`s native and pathogenic biological function(s) remain to be defined; owing to obstacles including a complex protein structure and the lack of pathological phenotypes in LRRK2 research models. To address the knowledge gap in LRRK2 biology, we set out to investigate the role of LRRK2 in the central nervous system (CNS). We generated and characterized a disease-mimicking D. melanogaster model of LRRK2-linked PD. This system was utilized to perform an in vivo, unbiased, high-throughput genetic screen to identify candidate interactors of LRRK2. Successful identification of a discrete number of genetic interactors was accomplished and, coupled with published evidence, highlighted the pursuit of subsequent mitochondrial-related investigations of LRRK2. These studies were performed using the M. musculus model system. Since LRRK2 murine models lack disease-relevant phenotypes, and LRRK2’s incomplete penetrance is predicted to be the result of gene-environment interaction, we employed the mitochondrial-targeting exogenous neurotoxin – MPTP/MPP+, to investigate neuronal mitochondrial phenotypes and subsequent survival in the context of LRRK2. Using the pathogenic R1441 GTPase-linked mutation, we did not observe altered neuronal mitochondrial length phenotypes or enhanced CNS sensitization to MPTP/MPP+-induced death; highlighting that MPTP-mediated, mitochondrial-centered mechanisms of action should be approached cautiously in the context of R1441-LRRK2. Collectively, the work presented herein has unveiled novel targets for the exploration of LRRK2 biological function and encourages the investigation of alternative pathogenic trigger mechanisms in the context of LRRK2-linked PD.

The fumonisins are a class of polyketide mycotoxins produced by Fusarium verticilliodes (formerly Fusarium monoliforme) which commonly affects maize. Ingestion of these toxins has been associated with leukoencephalomalacia in equine species, pulmonary oedema in swine, hepatocarcinogenesis in rats and have been linked to oesophageal cancer in humans. The structurally related AAL toxins are host specific mycotoxins produced by Alternaria alternata f. sp. lycopersici, producing stem canker disease in susceptible tomato cultivars. Examination of the C-11-C-20 fragment of the fumonisin B1 backbone [(2S,3S,5R,10R,12S,14S, 15R,16R)-2-amino-3,5,10,14,15-hydroxy-12,16-dimethyleicosane] and the C-10-C-17 fragment of the AAL toxin TA backbone[(2S,4S,5R,11S,13S,14R,15R)-1-amino-2,4,5,13,14-hydroxy-11,15- dimethylheptadecane], reveals four common stereogenic centres, with the only difference between the two fragments being the length of the alkyl chain. It is thought that the position and configuration of these four stereogenic centres is conserved among all members of the fumonisin and AAL classes of toxins. Retrosynthetic analysis of the backbones reveals a common intermediate aldehyde, which can be synthesised from methyl (S)-3-hydroxy-2-methylpropionate. A simple synthetic route to access the C-11-C-20 fragment for the fumonisins and the C-10-C-17 fragment of the AAL toxins was devised utilising Sharpless asymmetric epoxidation and an Evans aldol reaction as key transformations. In practice, it was found that although the Sharpless asymmetric epoxidation produced the desired epoxide in low enantiomeric excess, the two diastereomers produced could be separated by two consecutive flash chromatography silica gel columns. In pursuit of a more efficient method for introduction of the stereogenic centre in the target, other synthetic routes and key transformations were considered. Jacobsen’s kinetic resolution of terminal racemic epoxides was explored, requiring a terminal alkene from which the racemic epoxide was synthesised. An attempt to synthesise the terminal alkene from the appropriate tosylate and vinyl-MgBr, mediated by copper (I) iodide, failed. The synthetic route was redesigned, and the terminal alkene was synthesised by two one-carbon additions: the first a nucleophilic substitution with cyanide, and the second a Wittig olefination. The resolution of the terminal epoxide was also unsuccessful with no significant kinetic resolution occurring. Sharpless asymmetric dihydroxylation was also investigated; however, this reaction too failed to produce products of high diastereomeric excess. As a consequence, it was decided to pursue the asymmetric epoxidation route as the diastereomeric products could at least be separated. The second key transformation, the Evans aldol reaction, also provided an interesting result. When the aldol reaction was attempted with benzaldehyde and enolates derived from (4R,5S)-3-butanoyl-4-methyl-5-phenyl-oxazolidin-2-one and (4R,5S)-3-hexanoyl-4-methyl-5-phenyl-oxazolidin-2-one, the butanoyl derivative was found to give the expected Evans syn product, while the hexanoyl derivative was found to give the non-Evans syn product, with proof provided by single crystal X-ray diffraction analysis. It is proposed that the aldol reaction with the hexanoyl derivative does not proceed through the expected Zimmerman-Traxler-type transition state, but rather through an open chain transition state similar to that seen for asymmetric alkylation reactions. Synthesis of the pentanoyl derivative, and subjecting it to the same aldol reaction gave the expected syn Evans product, as deduced from spectroscopic properties. When the aldol reaction was attempted with the appropriate aldehyde intermediate, it was found that the dibutylboron triflate in the reaction medium caused the cleavage of the O-TBS ether protection, resulting in the formation of (3S,5R)-3-(4-methoxybenzyloxy)-5-methyl-tetrahydropyran-2-ol, before the aldehyde could undergo the aldol reaction. In order to avoid this problem, it is suggested that an alternative protecting group strategy using a more robust protecting group, such as a benzyl group which is stable to Lewis acids, could be substituted for the O-TBS group. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Chemistry / unrestricted

In this thesis I describe a toxic protein, diamphotoxin, that is present in the pupae of the beetle Diamphidia nigro-ornata. This insect is used as an arrow poison by the !Kung Bushmen inhabiting the savannah of eastern Namibia and western Botswana. Preliminary investigations showed that the pupae contained a 3,7 S cationic protein which caused haemolysis and, after intramuscular injection, local paralysis followed by death. By intravenous lethality assay, one 200 mg pupa contained 20 000 mouse lethal doses. Assays for the toxin were developed based upon haemolysis in vitro and lethality in vivo. These assays were used to monitor purification of the toxin. Diamphotoxin was purified by acid extraction in 0,1M glycine-HCl pH 3,0 followed by ammonium sulphate fractionation, chromatography on hydroxyl apatite, phosphocellulose and, finally, on DEAE cellulose. A consistent increase in activity after the hydroxyl apatite chromatography pointed to the removal of an inhibitor during this step. A subsequent severe loss of activity after chromatography on phosphocellulose could neither be explained nor overcome. The phosphocellulose chromatography step yielded three peaks of toxic activity. Immunological studies revealed cross-reactivity but not identity between these three toxin species. The toxin in the first peak to elute from the phosphocellulose column was purified to electrophoretic homogeneity by chromatography on DEAE cellulose. Attempts to purify the toxin in the other two phosphocellulose peaks were not successful. The isolated molecule was confirmed to be the toxin by haemolysis in a blood-agarose underlay after SDS-gel electrophoresis. The molecular weight estimate for the toxin by SDS-gel electrophoresis was 60 700 daltons and by analytical ultracentrifugation 62 100 daltons. The molecule appeared to exist as a single polypeptide chain. The amino acid composition showed a high proportion of hydrophobic amino acids. Isoelectric focussing showed an isoelectric point of pH 9,45. Toxin mediated haemolysis was studied in detail. The haemolytic event could be broken down into two stages. In the first stage toxin bound irreversibly to the cell but, provided no divalent cations were present, no damage to the cell could be detected. The second stage required the presence of free calcium (or certain other divalent cations), with an optimum concentration at 1 mM. The interaction of calcium with the cell-bound toxin resulted in the cell membrane becoming highly permeable to Na⁺ and K⁺ ions. Experiments designed to detect phospholipase or protease activity in toxin solutions gave negative results. Erythrocytes incubated with ¹²⁵I-labelled pure toxin in calcium-free medium retained a quantity of bound toxin which could not be removed by repeated washing. Incubation of erythrocytes with calcium-free toxin resulted in depletion of the activity of the toxin solution. The kinetics of the haemolytic action of the toxin were shown to be stoichiometric rather than catalytic. It was estimated that haemolysis by the toxin required a minimum of approximately 100 molecules per cell. Studies using circular dichroism measurements and the fluorescent probe 8-anilino-1-naphthalene sulphonic acid (ANS) indicated that a conformational change occurred in the toxin upon exposure to calcium. The ANS studies indicated that upon the addition of calcium the toxin molecule became more hydrophobic. It was concluded that the toxin functions as a calcium regulated Na⁺ and K⁺ ionophore in that it binds to the cell membrane and, in the presence of calcium or certain other divalent cations, assumes a conformation which mediates the free passage of Na⁺ and K⁺ ions. The resultant disruption of normal transmembranous ionic concentration gradients leads to cell lysis by loss of osmoregulation and, in the case of excitable membranes, disruption of electrophysiological activity.

No description available.

The Keap1-Nrf2 pathway is a major upstream regulator of xenobiotic detoxification. In Drosophila, directed activation of the protein complex of Keap1 and CncC (the homolog of human Nrf2) in principal and stellate cells of the Malpighian (renal) tubules confers resistance to lethal doses of the pesticide malathion, which is metabolized into organic anions. Dietary exposure to organic anions such as salicylate (10 mM) causes increases in fluid secretion rate and salicylate flux across Malpighian (renal) tubules. Here we used salicylate-selective microelectrodes and Ramsay assays to determine the role of Keap1/CncC in regulating these responses. Fluid secretion rate and salicylate flux across tubules isolated from adults with directed activation of Keap1/CncC in the principal cells are comparable to the values from salicylate-fed controls. Fluid secretion rate, concentration of salicylate in the secreted fluid and salicylate flux did not differ significantly between tubules isolated from adults with directed activation of Keap1/CncC in the principal cells reared on a diet containing salicylate and those reared on control media, indicating that the detoxification pathway was activated regardless of the presence of dietary salicylate. This is in contrast to the significant increase in fluid secretion rate and salicylate flux between tubules isolated from salicylate-fed adults and adults reared on a control diet with directed activation of Keap1/CncC in the stellate cells, supporting previous studies that demonstrated the inability of stellate cells to transport organic anions. Taken together, these results suggest a role for Keap1/CncC in upregulating fluid secretion in response to the presence of dietary organic anions. / Thesis / Master of Science (MSc) / The Keap1-Nrf2 pathway is a major upstream regulator of xenobiotic detoxification. In Drosophila, directed activation of the protein complex of Keap1 and CncC (the Nrf2 homolog) in principal and stellate cells of the Malpighian (renal) tubules confers resistance to lethal doses of the pesticide malathion, which is metabolized into organic anions. Dietary exposure to organic anions such as salicylate (10 mM) causes increases in fluid secretion rate and salicylate flux across Malpighian (renal) tubules that are comparable to tubules isolated from adults with activated Keap1/CncC reared on a salicylate-free diet. This suggests a role for Keap1/CncC in upregulating fluid secretion in response to the presence of dietary organic anions.

No description available.

No description available.