| The two goals of this project were to reproduce a c-Src kinase assay that had been claimed to measure Ah receptor-dependent increase in kinase activity, and to produce a capillary electrophoresis version of the gel mobility shift assay for ALBS binding to DNA. Previous reports have suggested that an increase in tyrosine kinase activity, namely c-Src activity, accompanies Ah receptor activation. The potential of c-Src signalling to mediate toxic endpoints of TCDD exposure is largely unexplored. The goal of this project was to reproduce the c-Src assay of Enand & Matsumura (1995) in order to evaluate the effects of mixtures of ligands on this activity. However the results were not reproducible, both in vitro and in vivo, and the goal was changed to identify reasons for this. Various parameters were altered in order to correct the assay, such as the homogenization procedure, the buffers, tissues, incubation times, sample handling and protein concentration. Changing these parameters did not allow for increases in c-Src kinase activity to be measured by the assay. The capillary electrophoresis method development involved producing a capillary electrophoresis mobility shift assay capable of measuring the affinity of the Ah receptor ligand binding subunit (ALBS) to DNA binding upon ligand occupancy. CE was unable to quantify the ALBS/DNA analyte either because the peak was not separated or because the quantity of ALBS/DNA complex was below the detection limit. Further experiments are needed for this determination.;Secondary goals of the project were to summarize what is currently known about dioxin toxicology and to propose hypotheses that might help relate dioxin-like compounds to diabetes. The introduction of the thesis summarizes the web of effects initiated by Ah receptor activation. Chapter 5 explores the similarities between the Ah receptor and peroxisome proliferator activated receptors and relates these similarities to diabetes. |
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