First Name:
Amber
Last Name:
Harold
Major Department:
Chemistry
Thesis Director:
Jennifer Cecile
Date of Thesis:
May 2013
Organic anion transporters (OATs) are known to aid in the elimination of negatively charged xenobiotics from the body through excretory organs such as the liver, kidney, and intestine. The non-mammalian Caenorhabditis elegans (C. elegans) nematode serves as a model to study mammalian OATs due to homologous amino acid sequences and similar substrate interactions in the conserved active site. Competition assays illustrate the inhibition of the negatively charged fluorescein uptake into the C. elegans intestine in the presence of increasing concentrations of nonfluorescent anions. While this model is useful in screening anionic pharmaceuticals for interactions with these transporters, a high-throughput screening method is more desirable to maximize benefits and to commercialize this model. We have employed two methods to gain efficiency in these competitive inhibition assays. First, we have utilized liquid culture to synchronize C. elegans ages in these assays to provide similar nematode counts and provide higher levels of exposure to substrates in replicate experiments. Secondly, we have begun to employ 96-well microplates for culturing and assaying the nematodes. Both methods allow for various physiological states, such as regulation, to be observed. In addition to these two experimental methods, computational analysis has been exploited in order to better understand the activity of the OAT interactions with substrates and inhibitors. These molecular models have been useful in identifying key characteristics of the most efficient substrate molecules and should aid in identifying and quantifying downstream applications in tandem with the experimental methods.