Graduation Year
2024
Document Type
Dissertation
Degree
Ph.D.
Degree Name
Doctor of Philosophy (Ph.D.)
Degree Granting Department
Physics
Major Professor
Martin Muschol, Ph.D.
Committee Member
Ghanim Ullah, Ph.D.
Committee Member
Jianjun Pan, Ph.D.
Committee Member
Piyush Koria, Ph.D.
Keywords
Amyloidoses, Cytotoxicity, Amyloid fibril bundling, Fluorescence, Binding site
Abstract
Amyloid proteins are a large group of proteins that tend to aggregate and form insoluble fibrils with a characteristic cross-β sheet structure. Intriguingly, this process of “amyloid formation” has been simultaneously linked to devastating human diseases called amyloidoses, ranging from Alzheimer’s Disease, cardiac amyloidoses to type II diabetes, as well as functional biological responses such as hormone storage and sperm quality control. Amyloid fibrils have also attracted significant attention for their unique mechanical, chemical, and optical characteristics, establishing them as promising tough biomaterials. The urgent need to find treatments for Alzheimer’s disease, and the demand for safe and renewable biomaterials requires that we unravel why amyloids can be either pathogenic or beneficial. This is the overarching motivation of my research work. I have pursued this overall aim by investigating two specific aspects of amyloid formation.
My project investigates the origin and biological activity of so-called “oligomers” formed by the amyloid protein hen egg white lysozyme (HEWL). Oligomers are small aggregates emerging in the early stages of amyloid formation and are believed to be major contributors to the toxicity associated with amyloid formation. I showed that there are at least two different types of oligomers that can emerge during amyloid formation. Specifically, we found that early-stage oligomeric species are less toxic and form gel-like clusters similar to diffuse plaques found in Alzheimer’s patients. We also uncovered a second and highly toxic late-stage form of amyloid oligomers.
The main focus of my thesis research, though, has been on the late stages of amyloid fibril assembly involving the formation of large fibrillar suprastructures such as gels and plaques. This research is motivated by the question of why and how individual amyloid fibrils assemble into large suprastructures. It is those fibrillar suprastructures that are the prominent histological markers of amyloid diseases in tissues. They are also implicated as the main cause of many non-neurological amyloid diseases. Finally, they are of immediate interest to the development of self-assembled biomaterials. My first aim was to determine which suprastructures emerged from preformed and isolated fibrils, i.e. in the absence of any fibril growth process. Using isolated amyloid fibrils, I investigated the supramolecular fibril networks they formed as a function of charge screening (salt concentration) or fibril charge (solution pH). We observed that individual amyloid fibrils assemble into either 3-D disordered gel clusters or highly ordered 2-D fibril sheets. The latter showed optical birefringence, which is characteristic for amyloid plaques. We proposed a model to explain the transition from disordered gel to ordered plaque formation for fibrils.
In my second project, I followed up on preliminary suggestions that the amyloid dye, Thioflavin T (ThT), in addition to responding to fibril formation by monomers, also responded to the dynamics of fibril self-assembly. I observed that ThT fluorescence increased to an extent and at a rate comparable observed for fibril self-assembly in microscopy, suggesting that ThT also monitors supramolecular fibril assembly. To understand the mechanisms of the observed fluorescence enhancement during fibril self-assembly, we introduced ThT at different time points during fibril assembly and performed fluorescence quenching experiment by acrylamide. Both experiments suggest that ThT fluorescence enhancement during fibril self-assembly arises from the unquenching of ThT fluorescence at binding sites that become partially covered upon fibril network formation. These results improve our understanding of how large amyloid plaques emerge and inform the design of highly ordered biomaterials using fibril self-assembly.
Scholar Commons Citation
Bushra, Nabila, "Investigation of Self-assembly of Hen Egg White Lysozyme Fibrils" (2024). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/10749
