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Development of high-throughput screening methods and their application to a C. elegans model of alpha-1 antitrypsin deficiency

Brown, Anwen Elinor; (2019) Development of high-throughput screening methods and their application to a C. elegans model of alpha-1 antitrypsin deficiency. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder which involves the toxic aggregation of misfolded mutant alpha-1 antitrypsin (AAT) protein, within the endoplasmic reticulum of hepatocytes, where it is synthesised. AAT is a protease inhibitor, which regulates lung tissue turnover. Loss of AAT protection in the lung leads to pulmonary disease (COPD and emphysema), and accumulation of mutant AAT in the liver can lead to inflammation, cirrhosis and hepatocellular carcinoma. Current treatments are limited to liver transplantation and symptomatic relief, meaning there is an urgent need for novel therapies. This thesis study aimed to recapitulate the liver component of AATD in the genetic model organism C. elegans. The transgenic worm expressed the mutant form of the protein (ZAAT) in the muscle, which gave rise to a growth/motility deficient phenotype. The mutant phenotype was characterised using the in-house automated high-throughput imaging system INVertebrate Automated Phenotyping Platform (INVAPP). The capabilities of this system as a high-throughput screening tool are illustrated using nematode growth and motility and by screening of an open source (Pathogen Box) small molecule library on C. elegans. Hits included auranofin, currently indicated for rheumatoid arthritis, isradipine and tolfenpyrad. The powerful combination of the worm model of AATD displaying a growth/motility deficient phenotype and the ability to quantify this phenotype using the INVAPP imaging system allowed the development of high-throughput screening protocols to search for novel modifiers of AATD. Three such screens were developed and carried out in this study. A drug library screen was undertaken to search for small molecules which rescue the transgenic worm movement deficiency phenotype. A high-throughput genome-wide RNAi screen was performed for 2 of the 6 chromosomes of C. elegans. A chemically-induced forward genetic screen was also undertaken to search for novel genetic modifiers of the mutant phenotype. Preliminary hits from the screens were investigated and validated for their relevance to AATD.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Development of high-throughput screening methods and their application to a C. elegans model of alpha-1 antitrypsin deficiency
Event: UCL
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10067367
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