Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.816659
Title: Mapping of signalling pathways using a FACS-based forward genetic approach
Author: De Zan, Erica
ISNI:       0000 0004 9355 6226
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2020
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Abstract:
Since its inception, the central goal of functional genomics has been to gain a mechanistic understanding of how genes operate and interact with each other to regulate cellular and organismal phenotypes. To this end, screening approaches have been designed to systematically perturb the activity of every genetic element in the genome and identify mutants affecting defined phenotypic traits. Only recently, screenable phenotypes in mammalian cell systems moved past “fitness” and onto detection of molecular markers’ gradients at single cell resolution. This thesis describes the interrogation of regulatory gene networks behind key intracellular signalling pathways using a Fluorescence Activated Cell Sorting (FACS)-based approach coupled to haploid genetic screening. After establishing the technology’s robustness and reproducibility in the first result Chapter, a set of iterative screens is performed in the presence of pharmacological and genetic perturbations to dissect mammalian target of rapamycin complex 1 (mTORC1) activation and tumour necrosis factor α (TNFα) pro-inflammatory signalling. Data presented in the second result Chapter demonstrate that, contrary to previous assumptions, Ragulator subunit LAMTOR4 is not strictly required for in mTORC1 activation under steady-state conditions. Furthermore, LAMTOR4 is part of a “synthetic-sick” interaction with tumour suppressor gene FLCN, resulting in inhibition of cell proliferation which could be therapeutically relevant. The third result Chapter describes a novel regulatory mechanism of TNFα-dependent pro-inflammatory signalling through transcriptional modulation of its receptor TNFR1. The effect is mediated by the largely uncharacterised proteins QRICH1, SEPHS1 and ZNF652, the first two of which functionally, and possibly also physically, interact. Together, this thesis details how genetic screening for modulation of molecular makers can advance understanding of functional cellular signalling network.
Supervisor: Gyrd-Hansen, Mads ; Nijman, Sebastian ; Brennan, Paul Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.816659  DOI: Not available
Keywords: Forward genetic screening ; Functional genomics ; Molecular Biology
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