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Are tumours killing time? : understanding the role of circadian genes in cancer biology
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Stephenson, Ewan (2022) Are tumours killing time? : understanding the role of circadian genes in cancer biology. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3878448~S15
Abstract
Circadian rhythms link internal biological processes to the external, geophysical day and act as a temporal anchor by which organisms can structure their endogenous processes. These oscillations regulate a huge proportion of the human transcriptome and proteome, and by extension have influence over vast swathes of physiology. Epidemiologic studies have linked circadian rhythms with cancer, one of the most dangerous health problems our species currently faces. The role of molecular clock dysfunction in these malignant and maligned cells remains poorly understood, with current literature suggesting a complex gene-specific and cell-specific role for each of the clock components.
We wished to systematically probe this link between circadian genes and cancer, along the way developing tools to facilitate future research in this area. We developed hTOK, a circadian CRISPR screen which targets circadian and circadian-related genes in human cells. We demonstrated the efficacy of the screen in both breast cancer cells (MDA-MB-231) and lung cancer cells (A549). Due to the simplicity of implementation, it is likely that the screen would be straightforward to transfer to any cell type which is readily transduced.
We have used this screen in an assay of in vitro survivability over the space of a month, and in vivo over the space of ~2 months. However, our in vivo data has some issues of bias and likely requires repeating with a modified experimental protocol. Nevertheless, the in vitro screen demonstrated a number of interesting genes which are likely important to the survivability of MDA-MB-231 cells in vitro, including PARP-1, CSNK1A and DHX9.
We also utilised the hTOK screen in an assay of NFκB mediated apoptosis in MDA-MB-231 cells, investigating which sgRNAs conferred the ability to survive this treatment. We used two different experimental designs for this assay, the first showing potentially skewed data and the second fixing some of the issues with the first. The second screen uncovered five probable hits including CSNK1E, AhR and DYRK1A.
In addition to the CRISPR screen, we developed the EREBOS machine, a device capable of measuring light produced by luciferase constructs in a mouse and the activity of that mouse simultaneously. As a proof-of-concept we used EREBOS to measure the light from a PER2::LUC transgenic mouse and concurrently measured its activity, demonstrating that they were in phase as would be expected. The machine did demonstrate the need for greater light-tightness mechanisms, though it can be constructed for <10% the cost of the current market leading machines while performing the same experimental function.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QH Natural history > QH426 Genetics Q Science > QP Physiology R Medicine > RC Internal medicine |
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Library of Congress Subject Headings (LCSH): | Circadian rhythms, Genetic transcription -- Regulation, Cancer -- Research | ||||
Official Date: | August 2022 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Warwick Medical School | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Dallmann, Robert ; Virshup, David ; Tergaonkar, Vinay | ||||
Extent: | 266 pages : illustrations, charts | ||||
Language: | eng |
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