Date of Award

Fall 2024

Document Type

Open Access Thesis

Department

Biomedical Engineering

First Advisor

Melissa Moss

Abstract

Amyloid-β protein (Aβ) is widely studied due to its key role in Alzheimer’s disease (AD) pathology. One characteristic of patients suffering from AD is the deposition of amyloid plaques comprised of Aβ aggregates, which are associated with synapse damage. Thus, studying Aβ aggregation and aggregation inhibition may provide pathological and therapeutic insights. Aggregation of Aβ is a nucleation dependent process that may be influenced by the presence of pre-formed Aβ aggregate seeds that can facilitate formation of protein aggregates. These seeds may appear during preparation or storage of monomer, posing impact on experiments that study the aggregation process and the ability of prospective therapeutics to disrupt that process. To understand the limitations of solution storage for monomeric Aβ, this systematic study examines the impact of Aβ monomer storage time and temperature on aggregation for SEC-purified monomer using aggregation assays analyzed via fluorophore thioflavin T (ThT) to observe changes in the lag time to aggregate formation and the extent of aggregation. Generally, results demonstrate a shift towards increased lag times and reduced extent of aggregation as storage time increases. Results indicate that purified Aβ monomer is most stable when stored at -80°C and least stable at -20°C, with significant differences in lag time observed following storage at -20°C for 14 and 28 days. Additionally, when transmission electron microscopy (TEM) was used to examine fibril morphology, aggregates formed from monomer stored at -20°C exhibited an additional unique short, straight morphology. These results illustrate that storage temperature and duration can impact aggregation experiments and establish guidelines for storage of Aβ monomer in solution.

Rights

© 2025, Amy Veihdeffer

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