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Title: Towards single cell protein analysis of cardiac progenitor cells
Author: Milman, Gemma
ISNI:       0000 0004 5917 6385
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2016
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Studying single cell protein expression is becoming increasingly important; however it is limited by the availability of highly sensitive technology. In adult cardiac progenitor cells (CPCs), expression of all the key cardiac transcription factors can be found, and both in vivo and in vitro studies have shown they can form cells of the cardiac lineage. However, effective mobilisation of these cells in situ for heart regeneration remains elusive. Close examination of CPCs for the important interactions and modifications may reveal the control mechanisms holding them in their arrested state, and therefore could lead to more effective cell therapies for heart failure. Total internal reflection microscopy is a super-resolution technique, allowing the visualisation of individual protein molecules through fluorescent labelling. It uses and immunoassay to pull down the protein of interest, detecting the protein with a secondary fluorescently labelled antibody. Whilst the assay sensitivity relies heavily on the antibody kinetics, it avoids the need to genetically modify proteins with fluorescence. Thus the assay can be adapted to look at any protein, in any cell, and can be multiplexed to examine many proteins simultaneously. In this thesis, an assay for the detection of the cardiac transcription factor, Gata-4, is developed. Gata-4 is pivotal for cardiac development; it is a part of an extensive gene regulatory network, interacting with many other key transcription factors to confer precise heart morphogenesis. It is expressed in cardiac progenitor cells, and the aim here was to quantify protein expression from single CPCs. Another facet examined here was the potential use of alternative protein capture agents. DNA oligonucleotides, encoding the consensus binding sequence for Gata-4 was investigated for its suitability in an assay for single cell protein expression. Whilst this yielded promising results it was constrained by the lack of a suitable negative control.
Supervisor: Klug, David ; Schneider, Michael Sponsor: British Heart Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral