Publication Date

2019

Document Type

Dissertation/Thesis

First Advisor

Gaillard, Elizabeth

Degree Name

Ph.D. (Doctor of Philosophy)

Legacy Department

Department of Chemistry and Biochemistry

Abstract

Age-related biochemical and biophysical changes to the structural properties of ocular lens membrane proteins and retinal pigment epithelium (RPE) cells are thought to play an underlying role in the etiology and progression of age-related cataract and macular degeneration. This dissertation work studies biophysical properties of biomimetic membranes to aid in further understanding of the role of aquaporin-0 in cataract pathogenesis and the biochemical composition of age-related pigmented granules, melanolipofuscin, and their implications in macular degeneration.

Cataract is the leading cause of blindness globally and is characterized by the opacification of the ocular lens. As a key optical component of the eye, the lens must remain transparent and elastic throughout life which requires proper water homeostasis through lens specific integral membrane protein aquaporin-0 (AQP0). Structures of AQP0 lack protein-lipid bilayer structural information. Since AQP0 function depends on its lipid bilayer environment,

this work investigated structural properties of oriented biomimetic lipid membranes using x-ray reflectivity and compared the results to literature. The data were fit with physically meaningful parameters such as molecular area and bilayer thickness. Results from the fits show a general decrease in molecular area which is consistent with the condensation effect of cholesterol on lipid bilayers. Some parameters for the highest cholesterol mol% tended to deviate from what was expected based on general trends. This may possibly be due to phase separation of cholesterol into cholesterol-rich domains as observed in lens fiber cell membranes. These data provide important information to understanding AQP0/lipid bilayer structural properties upon insertion of AQP0 into the biomimetic membranes.

Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries including the United States. AMD presents in two distinct forms, exudative and non-exudative, with the exudative form being the less common but most severe form. Both forms are characterized by the accumulation of membrane-bound fluorescent pigment granules in the retinal pigment epithelium (RPE), a monolayer of cells that line the back of the retina and form the blood-retinal barrier. The pigment granules form from lysosomal uptake and storage of undigested material that is of photoreceptor origin and are termed lipofuscin. The lipofuscin granules appear to correlate with retinal degeneration and have received considerable attention in the literature. Over time, some of the lipofuscin granules fuse with the protective melanin containing vesicles (melanosomes) to form melanolipofuscin which is potentially very harmful to the tissue because it likely inhibits the ability of melanin to protect against oxidative and photooxidative stress. However, almost nothing is known about the chemical composition or reactivity of these granules. Therefore, we have undertaken a systematic analysis of the organic soluble portion of human retinal melanolipofuscin using liquid chromatography-mass

spectrometry. A2E is a by-product of the visual cycle, a known component of retinal lipofuscin, and implicated in oxidative stress in the RPE due its ability to produce reactive oxygen species. A2E was identified in organic soluble melanoplipofuscin based on characteristic absorption maximum around 440 nm and tandem mass spectrometry fragments. Two abundant ions with m/z 618 and 646 were investigated. Tandem mass spectrometry data suggest that m/z 618 and 646 could result from the reaction of an aldehyde derivative of oxidized A2E with lysine and arginine, respectively. The proposed structures for m/z 618 and 646 will be synthesized and analyzed with these methods to confirm this hypothesis.

Extent

176 pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

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