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Title: Optical traps for single cell analysis
Author: Phillips, John Robert
ISNI:       0000 0004 2732 4419
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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In order to better understand the development and spread of cancer, the methods by which cells communicate and disseminate information must be closely examined. As such, membrane proteins are an important target in modern cancer research due to their role in cellular signalling cascades. Disruption of these cascades can lead to abnormal cell growth and the onset of cancer. Therefore, characterising the recruitment and action of such membrane proteins is a key facet of the Single Cell Proteomics (SCP) initiative. This thesis lays out work on the use of optical trapping methods to study membrane proteins on single cancer cells. Optical traps provide a sterile, versatile toolset to manipulate single cells, and have been used here to manipulate cells both directly and indirectly. Direct manipulation of cells is used force them into contact to characterise the behaviour of the second messenger protein kinase Cε (PKCε). Particular attention is given both to the stage of the growth cycle of cells during recruitment, and to the effects of free calcium in both facilitating the accumulation of PKCε, and cell-to-cell adhesion strength. The indirect manipulation of cells takes a different approach to the study of membrane proteins, through the use of “Smart Droplet Microtools” as sampling agents. Comprised of micron-sized lipid-coated droplets with either a solid or oil core, SDMs exchange material with the cellular membrane when brought into contact with it. These SDMs can simultaneously remove membrane proteins from multiple sites on the same cell, providing spatial selectivity of the target protein. The SDMs can then transport the proteins downstream to interface with other techniques for further analysis. This project details not only improvements in stability and efficiency of these SDMs, but also provides the first examples of the transfer of their cargo onto solid-supported lipid bilayers and antibody microarrays for readout.
Supervisor: Neil, Mark ; Klug, David ; Willison, Keith Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral