Title page for ETD etd-11072011-135711


Type of Document Dissertation
Author Picou, Ryan Andrew
URN etd-11072011-135711
Title Analysis of Amyloid Beta (AΒ) Peptides by Capillary Electrophoresis and Laser-Induced Fluorescence Anisotropy Detection
Degree Doctor of Philosophy (Ph.D.)
Department Chemistry
Advisory Committee
Advisor Name Title
Gilman, Samuel D. Committee Chair
Murray, Kermit K. Committee Member
Poliakoff, Erwin Committee Member
Warner, Isiah Committee Member
Lindau, Chuck Dean's Representative
Keywords
  • Capillary Electrophoresis
  • Fluorescence Anisotropy
  • Amyloid Beta Peptide
Date of Defense 2011-11-01
Availability unrestricted
Abstract
The goal of the research presented in this dissertation is to develop laser-induced fluorescence anisotropy (LIFA) detection for detecting and characterizing amyloid beta (Aβ) peptide aggregates separated by capillary electrophoresis (CE). Senile plaques composed primarily of aggregated Aβ (1-40) and Aβ (1-42) peptides have been found in the brains of Alzheimer’s disease patients. These peptides are thought to be responsible for the disease pathology. A CE method using UV absorbance detection was developed for the quantification of Aβ (1-40) monomer and testing of Aβ monomer preparations for undesired aggregates (Chapter 2). Analyses of both Aβ (1-40) and Aβ (1-42) monomer and aggregate samples were performed by CE-UV (Chapter 3). The CE-UV experiments demonstrated that the CE method can separate Aβ monomers from aggregated Aβ. A lab-constructed CE-LIFA instrument was built to separate and detect individual Aβ fibrils using Thioflavin T (ThT) in the separation buffer as a fluorescent probe (Chapter 4). Separating and detecting individual Aβ aggregates opens the door to a better understanding of amyloid aggregation, which is normally studied with bulk methods, i.e. all aggregates measured at once. Individual Aβ aggregates detected by the CE-LIFA instrument were shown to exhibit fluorescence anisotropy, but all of the aggregate peaks exhibited similar anisotropy values using ThT as a fluorescent probe. Plots of fluorescence anisotropy vs. time did not produce peaks as expected. A data treatment method to facilitate visualization of peaks in CE-LIFA data was developed (Chapter 5). Thioflavin T spectroscopy in the presence of polystyrene spheres was studied to address potential false positives observed in preliminary experiments from Chapter 4 using ThT as a fluorescence probe for Aβ aggregate detection (Chapter 6). It was found that polystyrene spheres can enhance ThT fluorescence similar to Aβ. A CE-LIFA study of Aβ peptides covalently labeled with a fluorophore was performed to overcome the apparent limitations of ThT. Aggregation of two different preparations of Aβ was studied as a function of time. Different forms of Aβ were separated and detected using CE-LIFA, and peaks resulting from different forms of Aβ exhibited different fluorescence anisotropy values.
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