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Title: Developing Dictyostelium discoideum as a model for the investigation of structurally diverse tastants
Author: Cocorocchio, Marco
ISNI:       0000 0004 8500 0885
Awarding Body: Royal Holloway, University of London
Current Institution: Royal Holloway, University of London
Date of Award: 2018
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The activity of many tastant compounds is poorly understood at a cellular level due to limited understanding of the mechanisms of taste. The multiple cellular effects and complex pharmacology of tastants make challenging the characterisation of their biological activities through conventional approaches. In addition, taste research often employs animal models that are difficult to manipulate at a molecular level and have associated ethical concerns. The potential of employing Dictyostelium discoideum as an animal replacement model for the study of poorly characterised natural and synthetic tastants was investigated in this thesis. Initially, an assay was developed using D. discoideum to monitor the effects of tastants at a cellular level employing the pungent alkaloid capsaicin. In this assay, live cell imaging was employed to monitor cell behaviour for 15 minutes including pre (5 minutes) and post (10 minutes) addition of capsaicin and computer-aided cell tracking was used to monitor behavioural changes. This study suggests that capsaicin may modulate D. discoideum behaviour through a novel mechanism involving Guanine Nucleotide Exchange Factors proteins (Rac-GEF). The method was then applied to the analysis of eight known and five blinded bitter compounds with diverse chemical structures. Data obtained were compared to the rat in vivo Brief Access Taste Aversion (BATA) assay and human taste panels. This analysis showed a positive linear correlation between the three models, suggesting that D. discoideum may provide a new model for the investigation of bitter tastants. The same approach was also employed to investigate changes in D. discoideum cell behaviour after exposure to the natural polyphenol curcumin and a range of structurally related derivatives. These compounds were also tested on cell growth and development to obtain a more in-depth understanding of their chronic cellular effects. Indeed, results have demonstrated that curcumin and related compounds have distinct effects on acute cell behaviour, growth and development. A mutant screen was then carried out to identify potential molecular targets of curcumin and structurally related molecules. This screen identified the Protein Phosphatase 2A regulatory subunit and the Presenilin B protein which controlled resistance to curcumin and a synthetic analogue, respectively. These studies demonstrate that D. discoideum may be employed as an animal reduction model to investigate the cellular effects of poorly characterised complex tastants and propose novel targets and mechanisms of action.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Marco Cocorocchio ; Dictyostelium discoideum ; pp2a ; Presenilin ; cancer ; Alzheimer's disease ; Bitter ; curcumin ; turmeric ; animal free research ; Taste