Year: 2015
Type: dissertation
Abstract: Amyloid fibrils are self-assembled protein homopolymers that play\ncentral roles in the pathology of several human diseases, most notably in progressive\nneurodegenerative diseases such as Alzheimer’s disease (AD). In AD, fibrils are\nformed by the amyloid-β (Aβ) peptide, an enzymatic cleavage product that is naturally\nproduced in human brain tissue. The fibrillar Aβ accumulates in deposits known as\nsenile plaques in the diseased AD brain, and the processes by which this occurs are\npostulated to be primary causes of the neuronal loss that is associated with AD\nprogression. As such, it is of great importance to understand how Aβ fibrils are\nformed, and why they exert neurotoxic effects in AD patients.\nIn pursuit of this goal, there is a need for more effective and precise methods of\nanalysing amyloid material. In this work, the aim was to improve the read outs of\ntechniques that are currently used, and to develop novel methods for amyloid\ndetection. The main focus has been on small molecules that work as light-switch\nmolecules in presence of amyloids, i.e. they exhibit greatly enhanced fluorescence\nintensity upon binding to amyloid fibrils. I have characterised the classic amyloid stain\nthioflavin-T (ThT), and showed how it can differentiate between different amyloid\nsamples based on their morphological attributes, particularly upon detection of its\nfluorescence lifetime. I have also characterised the binding and fluorescent properties\nof YOYO-1; a traditional DNA stain that proved to be an attractive alternative to ThT\nand that therefore may function as a novel amyloid stain, extending the amyloid\nrecognition toolbox.\nThe Thesis also outlines the development of an existing protocol for expression and\npurification of monomeric seed-free amyloid-β(1-42) that has been setup in our\nlaboratory in order to facilitate reproducible investigation into the formation kinetics\nof Aβ(1-42) fibrils.\nThis work is a stepping stone towards studies of amyloid-β formation kinetics and\nneurotoxicity. I intend to utilise the fluorescence techniques described herein, together\nwith the developed protocol for Aβ production, to explore different stages of Aβ\naggregation; how they exert their neurotoxic effects, and if these properties are\nmodulated by lipid membranes and molecular crowding effects.
Author: David J. Lindberg
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
Funders: Lunds Universitet, Karolinska Institutet
OpenAlex ID: W2147766300