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Development of the high throughput mammalian PIG-A gene mutation assay in vitro. / Benjamin James Rees

Swansea University Author: Benjamin James Rees

Abstract

The field of genetic toxicology has recently undergone reform which has limited or banned the use of animal models within a number of different industries (cosmetics). Consequently, greater emphasis has been placed on developing novel, highly sensitive, in vitro test systems which can generate robus...

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Published: 2015
Institution: Swansea University
Degree level: Doctoral
Degree name: Ph.D
URI: https://cronfa.swan.ac.uk/Record/cronfa43011
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first_indexed 2018-08-02T18:56:04Z
last_indexed 2019-10-21T16:48:52Z
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spelling 2018-08-29T15:13:13.2425214 v2 43011 2018-08-02 Development of the high throughput mammalian PIG-A gene mutation assay in vitro. 3a9cdc8c95423587e76dc341991844c7 NULL Benjamin James Rees Benjamin James Rees true true 2018-08-02 The field of genetic toxicology has recently undergone reform which has limited or banned the use of animal models within a number of different industries (cosmetics). Consequently, greater emphasis has been placed on developing novel, highly sensitive, in vitro test systems which can generate robust data to aid regulatory hazard and risk assessment.The main aims of this project were i) to develop a highly sensitive and specific, high throughput mammalian in vitro PIG-A gene mutation assay to enable quantitative dose response modelling and further investigate the potential use of in vitro data within human health assessment, ii) Investigate the genotype to phenotype relationship, a potentially delaying step within future OECD guideline drafting for the current in-vivo Pig-a mutation assay and iii) help develop and optimise a preliminary comprehensive human PIG-A bio­monitoring platform.During in-vitro and ex-vivo PIG-A assay development, flow cytometry was the fundamental technique utilised. Multiple additional laser excitation platforms were evaluated for use, including Amnis ImageStream ™ and laser scanning confocal. Proteomic as well as genomic techniques were used during the supplementary investigations surrounding assay development, with microbiological groundings throughout.The finalised in-vitro assay protocol was established within human, metabolically active, MCL-5 cells. Using the refined assay design, proof of principle experimentations were able to show the potential for future quantitative work and the general promise with this novel approach. The genotype to phenotype relationship validation is currently still on-going following the preliminary work described herein and recent publications. The ex-vivo human PIG-A assay platforms were shown to require further optimisation in terms of sensitivity, excluding red blood cells, but showed good aptitude for future use.Currently it looks promising that further refinement could lead to a comprehensive high content, high-throughput assay system with the potential to be used within future hazard and risk assessment E-Thesis Genetic toxicology 31 12 2015 2015-12-31 COLLEGE NANME Swansea University Medical School COLLEGE CODE Swansea University Doctoral Ph.D 2018-08-29T15:13:13.2425214 2018-08-02T16:24:31.0538053 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine Benjamin James Rees NULL 1 0043011-02082018162539.pdf 10821401.pdf 2018-08-02T16:25:39.6800000 Output 41033545 application/pdf E-Thesis true 2018-08-02T16:25:39.6800000 false
title Development of the high throughput mammalian PIG-A gene mutation assay in vitro.
spellingShingle Development of the high throughput mammalian PIG-A gene mutation assay in vitro.
Benjamin James Rees
title_short Development of the high throughput mammalian PIG-A gene mutation assay in vitro.
title_full Development of the high throughput mammalian PIG-A gene mutation assay in vitro.
title_fullStr Development of the high throughput mammalian PIG-A gene mutation assay in vitro.
title_full_unstemmed Development of the high throughput mammalian PIG-A gene mutation assay in vitro.
title_sort Development of the high throughput mammalian PIG-A gene mutation assay in vitro.
author_id_str_mv 3a9cdc8c95423587e76dc341991844c7
author_id_fullname_str_mv 3a9cdc8c95423587e76dc341991844c7_***_Benjamin James Rees
author Benjamin James Rees
author2 Benjamin James Rees
format E-Thesis
publishDate 2015
institution Swansea University
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
document_store_str 1
active_str 0
description The field of genetic toxicology has recently undergone reform which has limited or banned the use of animal models within a number of different industries (cosmetics). Consequently, greater emphasis has been placed on developing novel, highly sensitive, in vitro test systems which can generate robust data to aid regulatory hazard and risk assessment.The main aims of this project were i) to develop a highly sensitive and specific, high throughput mammalian in vitro PIG-A gene mutation assay to enable quantitative dose response modelling and further investigate the potential use of in vitro data within human health assessment, ii) Investigate the genotype to phenotype relationship, a potentially delaying step within future OECD guideline drafting for the current in-vivo Pig-a mutation assay and iii) help develop and optimise a preliminary comprehensive human PIG-A bio­monitoring platform.During in-vitro and ex-vivo PIG-A assay development, flow cytometry was the fundamental technique utilised. Multiple additional laser excitation platforms were evaluated for use, including Amnis ImageStream ™ and laser scanning confocal. Proteomic as well as genomic techniques were used during the supplementary investigations surrounding assay development, with microbiological groundings throughout.The finalised in-vitro assay protocol was established within human, metabolically active, MCL-5 cells. Using the refined assay design, proof of principle experimentations were able to show the potential for future quantitative work and the general promise with this novel approach. The genotype to phenotype relationship validation is currently still on-going following the preliminary work described herein and recent publications. The ex-vivo human PIG-A assay platforms were shown to require further optimisation in terms of sensitivity, excluding red blood cells, but showed good aptitude for future use.Currently it looks promising that further refinement could lead to a comprehensive high content, high-throughput assay system with the potential to be used within future hazard and risk assessment
published_date 2015-12-31T03:54:05Z
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score 11.037603

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