The subcellular fate of ouabain digoxin and digitoxin after binding to HeLa cell surface was studied using a conventional ultracentrifugation technique, Ouabain, digoxin and digitoxin codistributed with the plasma membrane marker 5'-nucleotidase immediately after bindings and cells chilled at 2°C for 24 hours retained ouabain in the plasma membrane fractions with time these glycosides are internalized to a subcellular compartment apparently the lysosomes. Internalized ouabain showed a distribution pattern throughout the linear sucrose gradient which was similar to that of the lysosomal marker B-hexosaminidase and both activity were dependent on lysosomal integrity by showing similar response to shearing forceo Internalized digoxin although showed a peak of activity in the same fraction as the lysosomal marker, but its sedimentability was insensitive to shear. Both ouabain and digoxin also responded differently to chloroquine. Chloroquine slightly reduced the accumulation of ouabain by HeLa cells, but did not much alter its excretion rate. On the other hand the amount of digoxin retained by the cell depends on the lysosomal activity. Chloroquine which reduces the lysosomal enzyme activity and inhibits lysosomal degradation result in larger accumulation of digoxin by the cell. Antitubular agents (vinblastine and nocodazol), were found to have no detectable effect on the excretion rate of ouabains while the excretion rate of digoxin was reduced by vinblastine. The results are consistent with a process of internalization and turnover of sodium pumps by HeLa cells through receptor mediated endocytosis. The lipophilic property of digoxin seems to account for the differences between ouabain and digoxin handling by HeLa cells.

The major issue that hinder the widespread use of biodiesel is its poor cold weather stability and operability. This is attributed minor components identified as monoglycerides (MG), diglycerides (DG) and sterol glycosides (SG). There is currently no standard method to determine SG levels in biodiesel. A method to isolate and measure SG concentration in biodiesel and FAME was first developed. This was accomplished by decompatibilizing SG from the biodiesel matrix using n-dodecane and purifying the solids using a Folch liquid-liquid extraction. The extracted SG was analyzed by GC-FID; the tricaprin internal standard was detected at 21.5 min and SG from 26-26 min. Recovery using this method was 100% ± 2.5% when 3 commercial canola biodiesel samples were spiked with 38 ppm SG and extracted. This method was used to measure SG concentration of filtered FAME produced using 0.3wt%, 0.5wt% and 0.7wt% at a MeOH:Oil (mol/mol) ratio of 4:1, 5:1, 6:1, 7:1 and 9:1. The biodiesel produced was characterized according to ASTM D6584; MG, DG and TG decreased with increasing catalyst concentration and MeOH:Oil ratios. The SG solubility in reactive FAME was found to be lowest at high glycerol catalyst concentration. High levels of TG were found to solubilize SG in the reactive FAME. Finally, the solubility of SG in reactive FAME increased when high ratios of methanol were used.

D.Sc. (Chemistry) / Two different projects with the central theme of oxidation are discussed, namely the ozonation of saturated hydrocarbons and the total synthesis of an unusual steroid glycoside. The oxidation of saturated hydrocarbons by ozone with the so-called dry ozonation technique was investigated. This study led to the development of improved procedures in which the rate, yield and selectivity of the reaction between ozone and various saturated substrates could be controlled. Subsequently the dry ozonation of a broadspectrum of long straight chain saturated hydrocarbons, including waxes, was investigated. Reaction with these highly unreactive compounds gave almost exclusively ketones as products in high yields. The average amount of keto functionalities in the products is dependent on the reaction conditions as well as the chain length of the substrate. Therefore, a reliable method has been developed for the selective oxidation of straight chain saturated hydrocarbons, a process that is of great chemical, industrial as well as commercial importance. The dry ozonation of various straight chain monoketo compounds was also studied. Poliketo compounds are mainly formed in these reactions. This investigation showed that keto functionalities in the substrates exert a deactivating effect on neighbouring methylene groups, so that oxidations do not take place at these positions. Therefore, it was possible to predict the product distribution formed during the dry ozonation of shorter chain length monoketo compounds (Cg) . An effective synthesis of an unusual steroid glycoside similar to orbicusides A-C, compounds that contain extraordinary carbohydrate and A-ring moieties, was developed. Oxidation reactions played a prominent role in this reaction sequence. A substantial effort was also directed towards obtaining the correct stereochemistry at the various stereocentres of the carbohydrate moiety. This facilitated ring closure to a final product that contained carbohydrate and A-ring moieties similar to orbicusides A-C. As a result of this work, a method was developed for the introduction of a vicinal cis-diol on the sterically hindered face of an alkene. This method is especially suited for substrates where acid labile functional groups are present.

Van Heerden, F.R., Prof. / Please refer to full text to view abstract

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One important goal of this thesis is the hydrogenation of the glycosyl cyanide, which has never been mentioned by any of the groups who have done work in this area, except for B. Coxon and G. Fletcher, who in 1964, reduced a tetra-O-acetyl-β-D-galactopyranosyl cyanide with lithium aluminum hydride. We were hoping to obtain, by reduction with hydrogen on Pd/C, an aminomethyl C-glycoside. It is believed that these aminomethyl C-glycosides are of potential biological importance especially in the area of AIDS and HIV therapy.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy