Date of Award

12-2019

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Ocean Engineering and Marine Sciences

First Advisor

Shaohua Xu

Second Advisor

Eric Guisbert

Third Advisor

Andrew Palmer

Fourth Advisor

Ted Conway

Abstract

The presence of plaques and tangles in the human brain is a major feature observed in several neuropathological diseases including Parkinson’s and Alzheimer’s. Due to the high morbidity and mortality rates of such diseases, research on the characteristics and functions of these highly ordered structures aims to expand the understanding of how the presence of these plaques lead to cognitive decline. Many studies have led to the development of models that suggest that the presence of amyloid plaques and tangles within the brain induce a cytotoxic cycle that results in the suffocation and death of neurons within the brain. A model to study plaques in vivo by using the protein lysozyme which can undergo gelation to form amyloid fibers has been established in previous studies in our laboratory. Looking at proteins normally found within systemic circulation and their effect on plaque formation and gelation might also signify a correlation between breakage of the blood-brain barrier seen during physiological stress such as during transient ischemic attacks, or mini-stokes. Other molecules of interest in this study include neuromelanin, a protein found in the central nervous system. Isolation of this molecule was achieved by dissection of bovine brains. Further studies should also be conducted on the effect that neuromelanin and other molecules have on amyloid fiber formation by using the lysozyme model described here. The aim of this study was to use and optimize lysozyme as a protein model for amyloid fiber formation and characterization. The lysozyme model enables a universal system that can be accessible to different laboratories. Using previously studied conditions for lysozyme, amyloid fibers were able to be synthesized in vitro and were therefore able to be studied in the presence of a copious number of variables. Specifically, this study looks at the presence of human plasma and its effect on amyloid fiber synthesis in vitro through the use of lysozyme protein gelation. With this, we aim to expand on the understanding of possible risk factors associated with Alzheimer’s disease as well as possible therapeutic opportunities. Amyloid fibers were synthesized by incubating lysozyme in glycine buffer. Samples were stored at a shaking speed for at 55°C until gelation occurs. The presence formation of a gel in our lysozyme model indicates the formation of amyloid fibers. These amyloid fibers mimic those seen in Alzheimer’s disease and allowed for study of the structure of the fibers via a plethora of biochemical assays. One goal was to achieve isolation of the amyloid fibers through common laboratory techniques – such as ultracentrifugation – to optimize the lysozyme model. In the presence of increasing concentrations of human plasma, amyloid fiber formation was observed to be significantly slowed. While whole plasma slows amyloid formation, the exact effect of each individual protein on amyloid synthesis is unknown. The use of whole human plasma should therefore be followed up with further study of specific plasma proteins (i.e. albumin, hemin) and how they individually effect amyloid fiber synthesis.

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