Academic & Professional Updates
My PhD project focused on the cloning and characterization of mRNA alternative splice variants of the human prostaglandin F2 alpha (FP) receptor. Initially, our hypothesis was based on identification of an ovine FPB but includes any splice variant of the FP receptor. Interestingly, we identified a 6-TM mRNA splice variant designated hFPs. hFPs is the first putative 6-TM prostaglandin receptor splice variant identified in humans and was cloned from human heart and placenta. Methods include utilizing Rapid Amplification of cDNA Ends (RACE) PCR, subcloning, southern and northern blot analysis, and DNA sequencing to obtain FP receptor clones. DNA sequences were analyzed using MacVector computer software. Once FP isoforms were identified, sequences were confirmed using RT-PCR and receptor characterization was determined utilizing radioligand binding assays, functional studies as well as protein chemistry and immunohistochemical labeling of human tissue. Radioligand binding assays were done using cell lines that either endogenously expressed or were stably transfected with the receptor of interest. The binding studies were done using either whole cell binding or membrane enrichment protocols depending on receptor expression. Functional studies include fluorescence imaging plate reader (FLIPR) experiments measuring; Ca+2 mobilization, pH fluctuation, membrane potential variation utilizing dose response and high throughput screening analysis, reporter gene assays, microarray analysis, total inositol phosphate accumulation assay and cell morphology assay. Protein chemistry utilized by western blot to identify receptor expression within a stable cell line using a custom antibody designed to the unique amino acid sequence. The custom antibody was also used for immunohistochemical labeling of human tissue to confirmhuman expression and localization of tissue from which it was cloned.
I currently work as an assistant professor at Kansas University.