The proteome is in constant flux and therefore it is important to understand the contribution of protein dynamics to the function of an organism. The zebrafish (Danio rerio) is a recognised model organism that is widely used to investigate physiological processes. The focus of this thesis was to develop a method to calculate the rates of synthesis of heart proteins of zebrafish on a proteome-wide scale. The initial stage of the project involved the optimisation of a method to characterise the protein complement of individual zebrafish hearts. It was concluded that for the rapid screening of proteins 1-dimensional gel electrophoresis in conjunction with high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) was the most appropriate experimental approach. In order to determine the rates of protein synthesis, zebrafish were administered with a stable isotope-labelled amino acid ([2H7] L-leucine) via the diet and its incorporation into heart proteins was monitored over an 8 week time course. Using this method it was possible to calculate the synthesis rates of over 600 proteins. The experimental strategy was then applied to define the changes in protein synthesis rates in hearts from zebrafish that were subjected to chronic unpredictable stress (CUS). A variety of stressors on zebrafish comprising air emersion, net chasing or net confinement were employed to model CUS. These approaches were validated by a parallel behavioural analysis. The results revealed that glycolytic and gluconeogenic enzymes as well as proteins involved in hypoxia had significantly altered synthesis rates in response to induced stress. This thesis describes for the first time a proteomics approach to determine the rates of synthesis of individual proteins in the zebrafish and its application to investigate the effects of stress conditions on heart proteome dynamics.
|Date of Award||31 Jul 2016|
|Supervisor||Mary Doherty (Supervisor), Phillip D Whitfield (Supervisor), Iain S Young (Supervisor) & Phillip Cash (Supervisor)|