Phase I of the program will focus on acquiring fundamental knowledge and developing basic laboratory skills required for advanced work in cell and molecular biology. This component will be guided and directly supervised by Dr. Mooseker in his laboratory on the main campus of Yale University. Selected graduate level coursework and seminars, completed in both the Graduate School of Arts and Sciences and School of Medicine, will compliment the initial basic research experience. Phase I research will focus on the expression, assembly, and targeting cytoskeletal proteins to the brush border of the enterocyte during embryogenesis and in chick epithelial cells in vitro. In addition initial studies of the role of a candidate protein in ileal bile acid transport will be undertaken. The effect of antibodies against this candidate protein on ileal bile acid transport will be studied. Alternatively efforts will be initiated in cloning the gene for this protein and expressing it in oocytes or a well differentiated Caco cell line. The issues addressed in these studies and the newly acquired technical expertise will be highly relevant to Dr. Shneider's longterm goal of characterizing another ontogenically regulated brush border protein, i.e., the ileal bile acid transporter. Phase II will be accomplished under the guidance of Dr. Suchy and with the continuing involvement of Dr. Mooseker. The objective of the applicant will be to now employ cell and molecular biological approaches to define the mechanisms underlying the ontogenic expression and regulation of the ileal, brush border membrane transport of bile acids. To this end specific antibodies against the transporter will be used to demonstrate species and tissue specificity of the transport protein, to define the expression of the protein along the crypt/villus axis, and to localize the carrier to the apical domain of the enterocyte by immunofluorescence microscopy. Moreover it will also be determined at what time during postnatal development the transport protein can be first detected and is sorted to the brush border membrane (versus accumulation intracellularly or on the basolateral domain). A cDNA probe encoding the transporter will be cloned and used to study gene expression during ileal development and enterocyte differentiation.
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