Date of Award

1-1-2013

Document Type

Open Access Thesis

Department

Biomedical Engineering

First Advisor

Jay D Potts

Abstract

Connexin43 (Cx43) is a component of gap junctions and is involved in intercel- lular signaling following injury to tissues. The carboxyl terminus of Cx43 binds to the PDZ2 domain of ZO-1 in order to form gap junction plaques and connect to the cytoskeleton. A biomimetic peptide known as αCT-1, replicating the last 9 amino acids found in the carboxyl terminus of CX43, has been shown to improve wound healing by preferentially binding to the PDZ2 domain of ZO-1. A possible mecha- nism for its action is through the Epithelial-Mesenchymal Transformation (EMT). Scratch assays were performed on rat bone marrow stromal cells treated with the peptide and were then analyzed using qPCR, western blotting, confocal microscopy, and live cell imaging. The gene expression analysis showed up-regulation of F11r and Krt19 and down-regulation of Mmp3. Protein expression analysis indicated an increase in Krt19 and the complete absence of Snai2 in the αCT-1 treated samples. Confocal microscopy suggested increased actin remodeling, increased size of Cx43 gap junctions, and reduction and localization of Mmp3 to the nucleus. Live cell imaging showed an increase in migration for the first seven hours followed by a breakdown in cell to cell contact. The genes and proteins chosen in this study did not indicate the changes of EMT; however, migration and morphology of the cells were similar to that of cells undergoing EMT. This study provides a beginning look into the mechanism of αCT-1 and will aid in its future development as a treatment for enhancing healing capabilities.

Rights

© 2013, Adam Clay Vandergriff

Additional Files
C1819Control.avi (40117 kB)
Live cell Control #1
C1920Control.avi (42186 kB)
Live cell Control #2
C2021ACT.avi (41953 kB)
Live cell ACT #1
C2223ACT.avi (39880 kB)
Live cell ACT #2
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