| 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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