eprintid: 10192676 rev_number: 16 eprint_status: archive userid: 699 dir: disk0/10/19/26/76 datestamp: 2024-10-04 12:01:19 lastmod: 2025-06-01 06:10:37 status_changed: 2024-10-04 12:01:19 type: thesis metadata_visibility: show sword_depositor: 699 creators_name: Wilson, Jo-Anne Alice Maud title: Dissecting the role of mTOR during fibrogenesis ispublished: unpub divisions: UCL divisions: B02 divisions: C10 divisions: D17 divisions: K71 note: Copyright © The Author 2024. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). abstract: Fibrosis is the concluding pathological outcome and major cause of morbidity and mortality in various chronic inflammatory, metabolic and immune-mediated diseases, as well as cancers. The most rapidly progressive and fatal of all the fibroproliferative diseases is idiopathic pulmonary fibrosis (IPF), with a median survival of only 3.5 years post-diagnosis with current anti-fibrotic approaches. The histological hallmark of IPF is the fibrotic focus, consisting of dense aggregations of activated hyper synthetic myofibroblasts which deposit a collagen-rich extracellular matrix (ECM) in response to the profibrotic cytokine transforming growth factor-beta (TGF- ß1). Recent studies from our centre provide strong support for the mechanistic target of rapamycin (mTOR) signalling hub in regulating TGF-ß 1-induced production of collagen I, a major and critical component of the ECM. mTOR is a nodal serine/threonine kinase that is present in two distinct complexes, that have distinct inputs and downstream effectors. mTOR assembly with its accessory protein, raptor (encoded by the RPTOR gene), forms mTOR complex 1 (mTORC1), while raptor is replaced by rictor (encoded by the RICTOR gene) to form mTOR complex 2 (mTORC2). The work presented in this thesis aims to extend current understanding of the role of mTOR during TGF- ß1-induced fibrogenesis at both the transcriptional and translational levels. Using RNA-Seq analysis of RPTOR and RICTOR CRISPR/Cas9 gene edited primary human lung fibroblasts (pHLFs) to disrupt mTORC1 and mTORC2 signalling respectively, in combination with pharmacological mTOR inhibitors (rapamycin and AZD8055), a critical role is revealed for rapamycin-insensitive mTORC1 signalling in regulating multiple ECM genes upregulated in response to TGF- ß1. These genes encode several collagens (including collagen I), glycoproteins and proteoglycans. In contrast, mTORC2 was found to be dispensable in the context of TGF- ß1-induced fibrogenesis. TGF- ß1-induced collagen I production was confirmed to be regulated via mTORC1 and its downstream effector protein 4E-BP1, a major repressor of cap-dependent translation initiation. Using RIP-Seq analysis to capture the translation initiation complex and associated mRNAs of pHLFs expressing a dominant-negative 4E-BP1 phosphomutant, novel 4E-BP1-dependent transcription factors were identified and demonstrated to regulate TGF- ß1-induced collagen I expression during the fibrotic response. Taken together, this work demonstrates that the mTORC1/4E-BP1 axis plays a critical role in regulating both transcriptional and translational control mechanisms to promote the potent fibrogenic effects of TGF- ß1. These findings hold potential implications for the development of novel anti-fibrotic strategies. date: 2024-05-28 date_type: published oa_status: green full_text_type: other thesis_class: doctoral_embargoed thesis_award: Ph.D language: eng primo: open primo_central: open_green verified: verified_manual elements_id: 2277919 lyricists_name: Wilson, Jo-Anne lyricists_id: JWILS35 actors_name: Wilson, Jo-Anne actors_id: JWILS35 actors_role: owner full_text_status: public pages: 277 institution: UCL (University College London) department: Respiratory Medicine thesis_type: Doctoral citation: Wilson, Jo-Anne Alice Maud; (2024) Dissecting the role of mTOR during fibrogenesis. Doctoral thesis (Ph.D), UCL (University College London). Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/10192676/13/Wilson_10192676_Thesis_sig_removed.pdf