Macquarie University
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Generation of tools for real-time observation of neurodegenerative disease related cellular pathologies

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posted on 2022-10-25, 02:15 authored by Nicholas Kakaroubas

Currently, the pathological biochemistry of Amyotrophic Lateral Sclerosis (ALS) is poorly understood resulting in limited treatment options. Emerging research has implicated aberrant stress granules in ALS pathology. Stress granules are accumulations of non-membrane bound RNA-protein assemblies which aggregate in the cytosol of cells as a natural response to external stimuli. These stress granule assemblies are a dynamic biological response to cellular stress, limiting mRNA translation initiation to help the cell survive short-term stresses such as thermal, metabolic, and oxidative stress. Chronic stress granules form when the cell does not disperse the RNA-protein assemblies leading to cell death. The objective of this thesis is to create models to study stress granule formation and disassembly in ALS in real time, in vivo. Zebrafish are the most suitable model organism for this study. Their high reproduction rate, a well characterised gene altering ‘toolkit’, and transparency in their embryonic and larval stages allow for a high number of samples to be genetically altered and screened over short time frames. Zebrafish share up to 70% of exons and major organs of interest with humans which should enable sound comparison at the cellular level of stress granule dynamics. To study stress granule formation, this project aims to link known stress granule associated proteins with fluorescent markers to visualise stress granule dynamics, in real time, in vivo, through confocal microscopy. In this thesis we have generated mRNA stress granule reporters. These mRNA stress granule reporters were injected into zebrafish embryos, allowing the visualisation of the formation of protein aggregates determined to be stress granules. These puncta were quantified and analysed with morphological assays to determine the effect on the zebrafish model. We have characterised and established the mVenus-G3bp1 mRNA transcript as a stress granule reporter and established direction for the future of this research.


Table of Contents

1. Introduction -- 2. Aims of thesis -- 3. Methods and materials -- 4. Results -- 5. Discussion -- 6. References -- 7. Appendix -- 8. Ethics approval


A thesis submitted for the partial fulfilment of the requirements for the degree of Master of Research Additional Supervisor 3: Angela Laird

Awarding Institution

Macquarie University

Degree Type

Thesis MRes


Thesis (MRes), Macquarie University, Faculty Medicine, Health, and Human Sciences, 2021

Department, Centre or School

Department of Biomedical Sciences, Macquarie University Centre for Motor Neuron Disease Research

Year of Award


Principal Supervisor

Emily Don

Additional Supervisor 1

Alison Hogan

Additional Supervisor 2

Marco Morsch


Copyright: Nicholas Kakaroubas Copyright disclaimer:




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