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dc.contributor.authorFries, Maximilian Werner
dc.date.accessioned2018-03-09T10:02:27Z
dc.date.available2018-03-09T10:02:27Z
dc.date.issued2018-04-28
dc.date.submitted2017-08-23
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/273868
dc.description.abstractGenetically identical cells show a heterogeneous response to a multitude of signals such as growth factors and DNA damage. While this heterogeneity has been shown to be a major determinant of treatment success in several diseases including cancer, little is known about how differences in biochemical signalling networks underlie such heterogeneity. State-of-the-art methodologies to study biochemical networks are often invasive and enable to quantify biochemical events only on cell populations or at a single point in time for a single cell, and therefore, cannot adequately quantify the fast, asynchronous and heterogeneous responses. In order to address these limitations, we have developed a unique sensing platform based on fluorescence lifetime imaging microscopy (FLIM) capable to multiplex at least three biosensors by utilizing Förster Resonance Energy Transfer (FRET) efficiently. After an overall introduction in Chapter 1, I describe the rational design and characterization of novel FRET pairs aiming to utilize the visible spectrum efficiently in combination with FLIM in Chapter 2. We combined blue, green and red donor fluorescent proteins that are excited at the same wavelength (840 nm for two-photon excitation) with genetically encoded quenchers, i.e. non-fluorescent chromoproteins as acceptors. This sensing platform enables the simultaneous detection of three biochemical reactions within single living cells providing new opportunities to characterize and understand non-genetic heterogeneity. In Chapter 3, I will demonstrate the first application of this novel platform by studying the activity of three key enzymes in DNA damage-induced cell death, caspase-2, -3, and -9. We confirm the heterogeneous nature of Cisplatin-induced cell death in genetically identical cells but reveal the existence of at least three subpopulations of cells characterized by distinct caspase dynamics. By combining biochemical and morphological information we infer the existence of different biochemical network topologies that are associated with alternative death phenotypes each cell adopts, such as apoptosis and programmed necrosis. Finally, deconvolution of cellular populations and direct measurement of a three-node caspase network - formerly impossible - permitted us to design perturbations of cell fate choices utilizing clinically relevant inhibitors. These perturbations resulted in changes in cell fate in response to Cisplatin, a clinically desirable outcome that suggests new avenues for combinatorial drugging and a new strategy to reveal cancer vulnerabilities that may be otherwise confounded by typical genetic and non-genetic heterogeneity.
dc.description.sponsorshipGates Cambridge Trust
dc.language.isoen
dc.rightsNo Creative Commons licence (All rights reserved)
dc.rightsAll Rights Reserveden
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/en
dc.subjectApoptosis
dc.subjectCaspase
dc.subjectFLIM
dc.subjectMICROSCOPY
dc.subjectFRET
dc.subjectMultiplexing
dc.subjectDNA Damage
dc.subjectFluorescent proteins
dc.titleMultiplexed biochemical imaging reveals the extent and complexity of non-genetic heterogeneity in DNA damage-induced caspase dynamics.
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.publisher.departmentMRC Cancer Unit
dc.date.updated2018-03-08T22:23:26Z
dc.identifier.doi10.17863/CAM.20943
dc.publisher.collegeKing's College
dc.type.qualificationtitlePhD in Medical Sciences
cam.supervisorVenkitaraman, Ashok R.
rioxxterms.freetoread.startdate2019-03-09


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