Includes bibliographical references (leaves 92-109). / The precise mechanisms of action of the quinoline anti-malarial drugs are uncertain, although they have been found to influence endocytosis, vesicular processing and secretion in malarial parasites and mammalian cells. In this study, the effects of chloroquine, amadiaquine, halofantrine, mefloquine and quinine on the endolysosomal systems in Plasmodium falciparum 3D7, Dictyostelium discoideum and A549 pulmonary cancer cells were examined.

A novel high performance liquid chromatographic assay has been developed for the simultaneous determination of isoniazid and N-acetylisoniazid in plasma. Solid phase extraction involving C18 columns is used to extract the drug and the metabolite from 0.5 ml plasma. The analyte peaks are resolved using a CB Spherisorb analytical column and ultraviolet detection at 270 nm. The assay is specific to the compounds, with consistent recovery of greater than 75% for isoniazid and over 90% for N-acetylisoniazid. The limits of detection in plasma are 300 ng/ml and 150 ng/ml for isoniazid and N-acetylisoniazid respectively. Linearity was conserved down to these concentrations. This assay was used to generate pharmacokinetic data on 114 tuberculosis patients recruited for this study at Brewelskloof hospital, Worcester, South Africa. Using these data, various markers were investigated for the determination of acetylator phenotype, namely isoniazid half-life, isoniazid plasma level at three hours, and the ratio of metabolite to drug at three hours. The ratio of metabolite to drug at three hours proved to be the most reliable method for phenotype classification, this being confirmed during the genotypic portion of the study. Trimodality was evident, although the nondiscrete separation of intermediate and rapid acetylators made this tentative. The mean values of area under the concentration-time curve for each acetylator type were found to be significantly different, with rapid acetylators being potentially compromised in terms of exposure to isoniazid (slow 32.39 mg. l⁻¹.hr, intermediate 21.25 mg. l⁻¹.hr and rapid 16.04 mg. l⁻¹.hr). Other pharmacokinetic parameters were bimodally distributed, homozygous and heterozygous rapid acetylators forming a single acetylator group. Codominance of the rapid and slow alleles was confirmed, the estimation of a mean intermediate elimination rate constant being within 7% of the observed mean. The correlation of genotype to phenotype was found to be 88.2% and the allelic distribution was determined to be acceptable using the Hardy Weinberg equation. The incidence of raised liver enzyme levels was low in the study population with no relation to acetylator phenotype. Age and weight gain after two months of daily therapy did not correlate with phenotype. The slow acetylator population comprised of a greater proportion of men, while women exhibited twice the number of rapid acetylators. No patient factors could be implicated in the apparent discordance of phenotype with genotype, and this suggests that there may be new allelic variants in this population. This report provides validation and proves the usefulness of a novel HPLC plasma-based assay for determining isoniazid and N-acetylisoniazid levels in patients with tuberculosis.

Includes bibliographical references.

The Hippo tumor suppressor pathway is a highly conserved signaling cascade initially identified in Drosophila which acts to regulate organ size and cellular proliferation. The Hippo pathway integrates extracellular and intracellular cues such as cytoskeletal tension, growth factor signaling, and nutrient availability to ultimately activate the LATS kinases. Activated LATS kinases then inhibit the downstream oncoproteins YAP and TAZ via a phosphorylation-dependent mechanism, in which 14-3-3 dependent cytoplasmic sequestration promotes YAP/TAZ degradation via the ubiquitin-proteasome pathway. Upstream regulators of LATS activation remain poorly characterized. MST1/2, which are mammalian orthologs of the Drosophila Hpo kinase, appear to be largely dispensable for Hippo pathway activation, suggesting evolutionary redundancy arising as a result of divergence and diversification of MSTs in human cells. We identified MST4, a close cousin of MST1/2, as a potential novel regulator of Hippo signaling in non-transformed, non-polarized human cells. Loss of MST4 resulted in decreased YAP phosphorylation in response to actin disruption, and also increased total abundance of TAZ, but interestingly did not affect levels of phosphorylated LATS. Overexpression of wild-type MST4 activated Hippo signaling and promoted TAZ degradation, which correlated to the effects MST4 had on levels of HIF1α. MST4 may be playing a previously unappreciated role in regulation of Hippo tumor suppressor signaling via a LATS1/2-independent pathway.

An enzyme immunoassay specific for several B-lactam antibiotics rather than individual antibiotics was investigated. The goal to develop an enzyme immunoassay for analysis of a whole class of compounds at one time is different than the goal of most enzyme immunoassays which desire specificity for drugs or hormone levels. Detection of the presence of all B-lactam antibiotics is wanted and identification of specific antibiotics is not needed. 6-Aminopenicillanic acid, the common structural moiety of B-lactam antibiotics was used tin this investigation. Methods of preparation of 6-aminopencillanic ac id conjugates 2nd antibodies needed for enzyme immunoassays have been developed. 6-Aminopenicillanic acid was conjugated to ovalbumin and bovine gamma globulin for production of antibodies with specificity to 6-aminopenicillanic acid. 6-Aminopenicillanic acid was also linked to the enzyme· horseradish peroxidase for future use in enzyme immunoassays. Antibodies produced against 6-aminopenicillanic acid are antigenic towards the thiazolidine ring of penicillins as shown by their affinity to ampicillin and penicillin. Anti-6-aminopenicillanic acid antibodies should therefore be antigenic towards other semisynthetic penicillins because 6-aminopenicillanic acid is usually used in their synthesis.

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