Microfluidics and chemical kinetics to analyse protein interactions, aggregation, and physicochemical properties
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Authors
Advisors
Date
2019-05-10Awarding Institution
University of Cambridge
Author Affiliation
Chemistry
Qualification
Doctor of Philosophy (PhD)
Language
English
Type
Thesis
Metadata
Show full item recordCitation
Lapinska, U. (2019). Microfluidics and chemical kinetics to analyse protein interactions, aggregation, and physicochemical properties (Doctoral thesis). https://doi.org/10.17863/CAM.32299
Abstract
Proteins play a major role in living systems and present a wide spectrum of functionalities.
Many different types of proteins are involved into biological processes, such as
the catalysis of biochemical reactions, cellular membrane transport, immune system
response and DNA replication. However, some proteins and peptides might become
harmful to living organisms; for example, their abnormal aggregation causes neurodegenerative
disorders including Alzheimer disease (AD). One of the causes of AD is
the presence of amyloid beta peptides Aβ(1-42), Aβ(1-40), which self-assemble into
insoluble fibrils and plaques, which surround neuronal cells impeding synapsis. The
number of AD patients is increasing, but a cure has not been founded yet. Therefore, it
is crucial to investigate the mechanisms underlying amyloid aggregation and screening
for compounds able to prevent this irreversible process.
Microfluidics permits characterising the physicochemical properties of proteins, investigate
their aggregation and study their interactions with other molecules. Chemical
kinetics allows studying the microscopic events occurring during protein self-assembly.
The combination of these two techniques provides a powerful tool for the identification
of compounds inhibiting the aggregation process. In this thesis by using microfluidics,
chemical kinetics and other biophysical assays, I have investigated the proteins
isoelectric point (pI) and the inhibition of aberrant Aβ(1-42) self-assembly process.
Firstly, I describe the development of a microfluidic platform allowing for the
measurement of the protein pI, in a gradient-free manner. This approach overcomes a
fundamental limitation of convectional techniques that is the achievement of a stable
and well-controlled pH gradient.
Secondly, I investigate the inhibiting effect of llama nanobodies on Aβ(1-42)
aggregation. The findings from this study show that nanobodies target monomeric
species with high affinity whereas interactions with fibril surfaces are weak.
Finally, I discuss the use of other compounds inhibiting specific nucleation stages.
These include the chaperones clusterin and brichos, as well as soot and pure carbon
nanoparticles. Importantly, the addition of both chaperones to Aβ(1-42) solutions has an additive inhibitory effect on aggregation. My findings will improve the characterization of the physicochemical properties of proteins as well as providing promising candidates for the inhibition of specific stages of amyloid beta aggregation opening the way to possible cures for AD disease.
Keywords
amyloid, misfolding, protein, aggregation, chemical kinetics, microfluidics, Alzheimer's disease, solubility, chaperons, amyloid beta, isoelectric point, BSA, electrophoresis, nanoparticles
Sponsorship
Departmental Funding (Department of Chemistry, University of Cambridge)
Embargo Lift Date
2023-11-12
Identifiers
This record's DOI: https://doi.org/10.17863/CAM.32299
Rights
All rights reserved, All Rights Reserved
Licence URL: https://www.rioxx.net/licenses/all-rights-reserved/
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