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Investigating chromatin domain structures in living cells using histone pulse-labelling approaches
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Fernandes Duarte, Filipe Alexandre (2021) Investigating chromatin domain structures in living cells using histone pulse-labelling approaches. PhD thesis, University of Warwick.
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WRAP_Theses_FernandesDuarte_2021.pdf - Submitted Version - Requires a PDF viewer. Download (82Mb) | Preview |
Official URL: http://webcat.warwick.ac.uk/record=b3765750
Abstract
3D genome organisation has been predominantly studied through next generation sequencing methodologies with conclusions often being made from population averages rather than single cells, or by fixed cell imagining techniques such as FISH. Studying its dynamics has been mostly limited to tracking one or two foci, integrated repetitive arrays, or has involved indiscriminate labelling of bulk chromatin. As the domain structure of the genome is mirrored by its replication timing, I was able to use a recently developed technique (RAPID-release) where a short pulse of EGFP-tagged histones, during S-phase, is incorporated into distinct chromatin domains. As EGFP labeled H3.1 has a slow turnover in human chromatin, it was used as a location marker, allowing for the tracking of its position over time. I capitalised on this unique labelling ability to investigate the behaviour of chromatin domains in living cells, and across different time-scales using lattice light sheet microscopy. I was able to characterise the dynamic characteristics of late-replicating domains, observing the presence of two populations with different dynamic features: high mobility domains and low mobility domains. I also investigated the effect of nuclear perturbation on the kinetics of the labelled chromatin by inducing DNA damage, where I observed a generalised increase in dynamic behaviour. Finally, I adapted the RAPID-release technique to improve its kinetics and attempted to develop a parallel system that used orthologous components in order to expand on the capabilities of this system.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QH Natural history Q Science > QH Natural history > QH426 Genetics |
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Library of Congress Subject Headings (LCSH): | Genomics, Chromatin, Convex domains, Domain structure, DNA damage | ||||
Official Date: | July 2021 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Warwick Medical School | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Bowman, Andrew ; Burroughs, Nigel John | ||||
Format of File: | |||||
Extent: | 223 leaves : illustrations | ||||
Language: | eng |
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