All ETDs from UAB

Advisory Committee Chair

Om P Srivastava

Advisory Committee Members

Roswell R Pfister

Kent T Keyser

Rodrick Fullard

Dennis J Pillion

David R Whikehart

Document Type

Dissertation

Date of Award

2012

Degree Name by School

Doctor of Philosophy (PhD) School of Optometry

Abstract

DIFFERENTIAL EXPRESSION OF PROTEINS IN KERATOCONUS: POTENTIAL ROLE OF HUMAN ANTIGEN R (HuR) IN REGULATION OF ß-ACTIN ROY JOSEPH VISION SCIENCES ABSTRACT Keratoconus (KC) is a condition of unknown cause in which the cornea assumes a conical shape as a result of non-inflammatory thinning of the corneal stroma. The disease progresses at a variable speed with corneal thinning inducing irregular astigmatism, myopia, and corneal protrusion. Contact lenses, and ultimately keratoplasty, are often required to restore vision. Despite intensive investigations into the pathogenesis of KC, the exact cause of the disease is presently poorly understood. Keratoconus apparently arises due to number of factors, which include changes at cellular, biochemical, physiological and genetic levels. With the progression of KC, both epithelial cells and stromal keratocytes are affected. The purpose of this research was to identify a potential molecular mechanism of the keratoconus disease process. For this purpose, we used two different proteomic methods, shotgun proteomics and 2D-DIGE methods to identify relative changes in protein levels in both epithelium and stroma of KC corneas compared to normal corneas. Major changes were seen in the structural proteins of both epithelium and stroma of KC corneas compared to normal corneas, suggesting structural remodeling of both the tissues during the development and progression of keratoconus. The proteins that are involved in proliferation, growth and migration were down-regulated in KC epithelium. Based on the protein level changes and systems biology approach, two unique models were generated; one for epithelium and the other for stroma of keratoconus. 1) The epithelium showed a disruption of iron homeostasis in the KC corneas could lead to increased oxidative damage. 2) Changes in the cytoskeletal proteins of keratocytes could lead to cellular apoptosis. The above results led us to focus on one of the cytoskeletal protein, ß-actin. Our molecular analysis showed that ß-actin is down-regulated in the corneal stroma of patients with keratoconus, due to reduced levels of a stabilizing factor Human Antigen R (HuR) for ß-actin mRNA. In order to determine the functional significance of the down-regulation of ß-actin and HuR we used siRNA-mediated gene silencing in stromal keratocytes of normal corneas. Knockdown of HuR gene led to reduced expression of ß-actin mRNA. This in turn significantly reduced keratocytes migration and proliferation.

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Optometry Commons

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