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Title: The role of cofilin in glioblastoma cell behaviour
Author: Yap, Celestial Therese Suen Mei
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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In vitro studies were performed on the human glioblastoma cell line U373 MG, originally derived from a patient. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the actin­-binding proteins cofilin, ADF and profilin are expressed in these cells. Using immunochemistry, the distribution of cofilin was investigated in cells cultured under standard culture conditions and after serum stimulation. The hypothesis was tested by overexpressing cofilin in the glioblastoma cell line and analysing for changes in cell motility compared to untransfected cells. Overexpression was achieved by transient and stable transfections with a plasmid vector, pCofilin-IRES2-EGFP, which was constructed by subcloning the coding sequence of human cofilin into pIRES2-EGFP. Transfected cells were identified by the expression of EGFP (enhanced green fluorescent protein) in timelapse experiments using confocal microscopy. In order to quantify the relative levels of cofilin overexpression in stable transfectants, cells were immunostained for cofilin using fluorescent detection and analysed by flow cytometry. Western blots confirmed the specificity of the anti-cofilin primary antibody used. The timelapse analyses indicated that overexpression of cofilin increases the motility of glioblastoma cells. The project was extended in an attempt to investigate whether variable levels of cofilin overexpression might affect cell motility to different extents. An inducible gene expression system based on the Tet-Off system (Clontech laboratories Inc.) was developed in order to control the level of cofilin overexpression. This system would enable the correlation between cofilin expression and motility to be examined over a wide range of overexpression.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available