Mechanistic insights into the aggregation of [beta]-amyloid

Many of the fundamental characteristics of amyloid formation are still unknown, largely due to the limitations present in currently available techniques. This is particularly problematic for disease-associated amyloid-forming species such as betaamyloid (A/3) , due to the tremendous impact that they cause on human well-being (Alzheimer's disease). In this dissertation three new methodologies were developed in order to provide mechanistic insights into A/3 amyloid formation. In Chapter 3 the optimisation of an A/3 purification methodology was achieved, greatly improving the reproducibility and purity of the samples obtained. This methodology was then applied to the production of a tandem A/3 construct, a dimeric form of A/3 that has been previously studied in a D. melanogaster model, which had not been recombinantly produced before. A preliminary biophysical characterisation was performed, providing some surprising insights into the toxicity of its aggregates. In Chapter 4 a novel methodology for using Tyr10 solvent exposure as a structural probe of A/3 formation was created using acrylamide quenching of tyrosine fluorescence. The data obtained exhibited similar solvent exposures between free tyrosine, monomers of both the A/31_ 40 and A/31_ 42 variants, and ADDLs of A/31_ 42 , results that agree with our current understanding of their structure. Fibrils of A/31_ 40 and A/31_ 42 exhibited solvent exposures similar to each other, but both.had significant reductions compared to the other species, in agreement with fibril structure models. � Lastly, in Chapter 5, the development of a technique for the creation, long-term storage and observation of aggregation events inside micro droplets was achieved. This technique exhibits remarkable temporal and spatial resolution for sample sizes that are ten orders of magnitude smaller than standard biophysical techniques. The methodology was applied to the study of glucagon, and the versatility of this technique was demonstrated by the collection of data including the total THT fluorescence evolution, the spatial growth of the aggregates and information on aggregate morphology, in conformity with spherulite formation.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2014

Author dc:creator

• Aran Terol, Pablo

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f4cab74e-10fa-4240-bf7b-766673289d2c/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.11752

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/265574