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Title: Association and segregation : fluorescence studies of protein organisation in the membranes of living cells
Author: Dunne, P. D.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2010
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This thesis details efforts to develop and adapt fluorescence microscopy techniques for the study of membrane protein association and segregation in live cells, both within membranes and between different membranes. After an initial introduction to the cell membrane and fluorescence microscopy, the experimental section of this thesis is split into two parts. Part A concerns the development of techniques to explore the unresolved triggering mechanism through which T-cells, a cell of the adaptive immune system, first recognise threats. After a brief introduction to T-cell biology, association of key proteins are examined at the single molecule level through two new techniques- Two Colour Coincidence Detection (TCCD) and Dynamic Single Molecule Colocalisation (DySCo). Association of proteins between different membranes also provides important clues relating to the triggering mechanism. To this end, an artificial bilayer system is created where the re-distribution of bilayer-bound fluorophore-tagged receptors upon encounter of a ligand-bearing cell provides kinetic and thermodynamic data about binding. Part B details aspects of TCCD and DySCo that have been adapted to examine protein association in other biological systems. An experimental protocol and data analysis software are developed to allow examination of the colocalisation of stationary, surface-adsorbed molecules; specifically those related to neurodegenerative conditions such as amyloid diseases. Following this, results are shown demonstrating segregation of membrane components in spermatozoa- a compartmentalised polarised cell type. Early results suggesting the passive size-dependent segregation of T-cell proteins attached to artificial bilayers upon interaction with B-cells are presented.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available