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Title: Development of a cell free system for analysis of the mechanisms of exocytosis using total internal reflectance microscopy
Author: Don-Wauchope, A. C.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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Regulated exocytosis occurs in a number of cell types and has been studied in many of these. There appears to be a commonality of function in the cells that exhibit regulated exocytosis. Many proteins have been implicated in the process of regulated exocytosis and a number of trigger signals have been identified. There are various means of investigating regulated exocytosis and these include capacitance measurements, amperometry and single-cell imaging. Reconstitution from defined components of the steps leading to exocytosis has not yet been achieved, whereas the investigation of the actual exocytotic event is well described. Access to the cytosolic components of the cell is a requirement of any assay that attempts to investigate the process leading to the final ‘fusion event’ of regulated exocytosis. The aim of this research was to develop an in-vitro assay that would allow investigation of the steps leading to regulated exocytosis on a plasma membrane. The method was based on the use of ‘unroofed’ bovine adrenal chromaffin cells, viewed by total internal relfection fluorescence microscopy. Development of the assay posed a number of challenges, not all of which were overcome during the study. The basic platform for the assay, a cell membrane patch with adhering secretory vesicles, was produced with some degrees of success. However, the imaging method had a number of limitations and this frustrated the method’s development. The results, although encouraging, were judged not to be robust enough to permit development of a reconstituted system and a more reliable imaging procedure is recommended for further study.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available