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Microfluidics and chemical kinetics to analyse protein interactions, aggregation, and physicochemical properties 10.17863/CAM.32299 Lapinska, Urszula 0000000335939248 Knowles, Tuomas 0000000278790140 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. University of Cambridge 2019-05-10 Thesis Doctoral Doctor of Philosophy (PhD) en Departmental Funding (Department of Chemistry, University of Cambridge) https://www.repository.cam.ac.uk/handle/1810/284929 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/173ea4f1-a08a-4ee5-879f-5cdd43b437a7/download 2dc4a7aaab9080826beabb7ca4eb1780 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/43122d5e-de17-4743-b614-332b00a3f358/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ amyloid misfolding protein aggregation chemical kinetics microfluidics Alzheimer's disease solubility chaperons amyloid beta isoelectric point BSA electrophoresis nanoparticles