First Name:
Alicia
Last Name:
Woock
Major Department:
Chemistry
Thesis Director:
Jennifer Cecile
Date of Thesis:
May 2012
Organic anion transporters (OATs), members ofthe major facilitator superfamily, promiscuously excrete negatively charged xenobiotic and antiviral drugs in the kidney and liver of mammals. Investigation in this project focuses on a single mammalian OA T homologue present in Caenorhabditis elegans (C. elegans). Use of anionic fluorescein has previously been successful in assays to verify interaction ofmammalian OAT substrates and inhibitors with the C. elegans OAT (CeOAT). In this research, the accumulation offluorescein is examined by fluorescence microscopy. This approach provides localization of fluorescein not available in previous experiments. In addition, microscopy techniques may validate where the OATs function in the C. elegans body, how fast substrates are actively transported, how well stimulation of the CeOAT occurs, and any additional gradients that may affect OAT function. Use ofthree strains ofwild-type and knockout (mutant) C. elegans will be essential in order to test inhibitors that vary in affecting not only the OATs but also the efflux transporters present on the luminal side of the same cells. Fluorescent and confocal microscopy methods have been tested and preliminary data guided the development of the protocol we used. Experimental conditions and complications with imaging the C. elegans are discussed for determining protocol. Results and images from several parameters used are presented, including the use of inhibitors and stimulants on uptake. We show inhibition by probenecid, possible stimulation by the dicarboxylate fumarate, and in ongoing research how the physiological sodium gradient affects OAT transport. This work will be used to determine the function ofthe CeOAT and how it relates to mammalian OATs, increasing the body of knowledge for this particular whole animal model system.