Graduation Year

2020

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

Laura Blair, Ph.D.

Committee Member

Ghanim Ullah, Ph.D.

Committee Member

Dmitri Voronine, Ph.D.

Keywords

Fluorescence, Lysozyme, Aggregate Species, Cytotoxicity

Abstract

Alzheimer’s disease, type II diabetes, and other amyloid diseases are known to be associated with the formation of amyloid aggregates. It has been thoroughly researched whether amyloid fibrils or oligomers are the main culprit for these diseases, and recent evidence has connected oligomers as the most disease relevant aggregate species. However, many difficulties have arose in confirming this hypothesis. Techniques for oligomer detection are often limited in their sensitivity, and in many cases are unable to distinguish oligomers from rigid fibrils. Additionally, the role oligomer splay in fibril assembly is still unclear, and has led to the belief that different types of oligomers may exist with potentially differing biological relevance.

The first part of my research aimed to develop an assay for effectively screening fluorescent dyes for selectivity toward a specific amyloid aggregate species. Lysozyme was shown to undergo amyloid formation along two pathways, with a clear difference in their kinetics. Fibril growth had a clear lag period followed by sigmodial growth, while early oligomer growth instead transitioned the kinetics to biphasic. This transition was utilized to screen various dyes for oligomer sensitivity, and found a promising candidate for an oligomer selective dye in crystal violet. Additionally, concurrent kinetics of both Thioflavin-T and crystal violet allowed me to separate the biphasic kinetics into its oligomer and fibril components.

The second part of my researched shows the formation of two distinct types of oligomers formed by Lysozyme. These two types of oligomers formed at different time points during amyloid fibril growth and showed a clear difference in their level of cytotoxicity. This difference highlights a main issue in determining the disease relevance of oligomers, as specific environmental conditions can lead to the formation of oligomers with differing levels of biological activity. Both oligomer types were characterized through a variety of techniques and were shown to share many similarities but were found to differ in secondary structure, and in their response to the oligomer selective antibody A11.

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