Abstract
dc:description.abstractThe behaviour and function of biomolecules represent a fundamental aspect in modulating the activity of micro and macro-scale complexes evolved in cells and tissues. The network of interactions of such biomolecules allow for the formation and regulation of the basic machinery of life, yet is commonly studied under non-physiological conditions. In order to characterise the behaviour and function of such biomolecules, they have to be analysed under relevant conditions, ideally in biofluids, cells or artificial systems which mainly imitate these properties. Recent microfluidic applications present an orthogonal approach for determining the interactions between a wide range of biomolecules, thus allow the study of molecular binding in the condensed phase with no need for extensive dilution, sample immobilisation or changes to the molecular environment from the liquid to the gas phase. As part of my PhD, I capitalised on microfluidic diffusion approaches, developed in the Knowles lab to systematically study the binding and thermodynamics of small heat shock proteins, such as clusterin, αB-crystallin and the Brichos chaperone domain, to aggregated forms of amyloid-beta and α-synuclein, protein aggregates that are associated with a wide range of neurodegenerative diseases. The three chaperones are crucial components of the cellular proteostasis network and characteristically overexpressed during cell stress. Each chaperone type shows distinct binding behaviour to protein aggregates, which can be related to its inhibitory function. While αB-crystallin binding to α-synuclein is entropically driven by conformational rearrangement, the binding of Brichos to amyloid-beta fibrils is shown to be enthalpically driven as it inhibits specifically secondary nucleation processes. In contrast to the specific secondary nucleation inhibition by Brichos, clusterin inhibits specifically fibril elongation of amyloid-beta. I could show that these two specific aggregation processes are affected by the two chaperones, Brichos and clusterin, in a non-cooperative manner. These molecular details are particularly relevant in the context of the rational design of drug molecules that could, potentially in combination, target multiple specific aggregation steps in a selective manner. Therefore, I further screened the binding of a wide range of monoclonal antibodies to either amyloid-beta monomers or fibrils, which are currently at different stages of clinical phase trials for Alzheimer's therapy. I thus show that the obtained stoichiometry and affinity information of the drug correlates with the distinct inhibition mechanisms and consequently provides mechanistic and structural information. In contrast to studying disease related model systems in vitro under homogeneous conditions, measurements in complex body fluids are key in medical applied science, e.g. cancer treatment or immunological characterisation. In my research, I have undertaken the challenge of extending the platform developed above to characterise the binding of a wide range of molecules under complex solution conditions. Preliminary data obtained during my PhD underlines the extraordinary capability of the diffusion-based microfluidics to being applicable for investigating the binding parameters of molecules involved in alloimmunisation in human serum. Along with my main focus on measuring protein interactions with diffusion-based microfluidics, I further developed a technique using selective separation properties, such as particle charge, hydrophobicity, size or immunoaffinity and coupled it with a series of microfluidic devices for an instantaneous and full biophysical characterisation of heterogeneous solutions. This new technique can be used to explore the formation of protein oligomers or protein complexation, characterisation and identification of complex mixtures in the context of amyloid formation and protein homeostasis.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2019
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Scheidt, Tom
- Advisor dc:contributor.advisor
-
- Knowles, Tuomas
Subjects
dc:subject × 5Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.55494
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/308407