Research Proposal

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===Background===
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'''Background'''
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The cornea is the transparent, outermost part of the eye which protects the fragile intraocular contents from mechanical and pathogenic perturbations. It consists of three distinct layers:  a tear-bathed, stratified layer of epithelial cells; a thick stroma of collagen fibrils embedded with quiescent fibroblasts; and an inner epithelial layer, called the endothelium. Poor cell growth in any one of these layers can result in a loss of corneal transparency and thus blindness. The endothelium in particular lacks a robust proliferative response to cell loss, and this can hinder recovery after eye surgery and in diseases such as keratoconus, which affects between 1 in 500 and 1 in 2000 people in the U.S. <issue of endothelial to mesenchymal (fibroblast?) transfer>.
'''Project Idea'''
'''Project Idea'''
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A library of Hsp90 mutants will be constructed with ePCR.  SDM mutants may be made if there are any particularly compelling regions that we can specifically identify to mutate.  The rationale is that more promiscuous Hsp90, or more actively expressed Hsp90 (or non-degraded), will be able to buffer a larger amount of phenotypic variation than standard strains.  This underlying abnormally large phenotypic diversity will be exposed in subsequent screens of growth under industrially relevant concentrations of sugar and ethanol.  The rate of succesful mutants will be compared to standard random mutagenesis techniques.  Particularly succesful mutants will be sequenced and undergo DNA microarray analysis, to determine the gene clusters up or downregulated in the mutants.
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Our goal is to determine whether we can enhance the proliferation of human corneal endothelial cells (HCECs) while maintaining their phenotype. We will use ROCK inhibitor to increase the proliferation of HCECs and study whether the presence of TGF-Beta will allow the cells to maintain their phenotype in culture.  
'''References'''
'''References'''
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''Okumura, Naoki, EunDuck Kay, et al. "Inhibition of TGF-β Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine." PLoS One. 8.2 (2013): n. page. Web. 5 May. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581499/>.''
''Okumura, Naoki, EunDuck Kay, et al. "Inhibition of TGF-β Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine." PLoS One. 8.2 (2013): n. page. Web. 5 May. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581499/>.''
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This paper ...
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-->This paper demonstrated that the use of SB431542, a selective inhibitor of the TGF-β receptor, in culture media counteracted the development of fibroblastic phenotypes in HCECs.  
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''Pipparelli, Aurélien, Yvan Arsenijevic, et al. "ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells." PLoS One. 8.4 (2013): n. page. Web. 5 May. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633902/>. ''
''Pipparelli, Aurélien, Yvan Arsenijevic, et al. "ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells." PLoS One. 8.4 (2013): n. page. Web. 5 May. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633902/>. ''
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This paper...
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--> This paper...

Revision as of 23:41, 5 May 2013

Background

The cornea is the transparent, outermost part of the eye which protects the fragile intraocular contents from mechanical and pathogenic perturbations. It consists of three distinct layers: a tear-bathed, stratified layer of epithelial cells; a thick stroma of collagen fibrils embedded with quiescent fibroblasts; and an inner epithelial layer, called the endothelium. Poor cell growth in any one of these layers can result in a loss of corneal transparency and thus blindness. The endothelium in particular lacks a robust proliferative response to cell loss, and this can hinder recovery after eye surgery and in diseases such as keratoconus, which affects between 1 in 500 and 1 in 2000 people in the U.S. <issue of endothelial to mesenchymal (fibroblast?) transfer>.

Project Idea

Our goal is to determine whether we can enhance the proliferation of human corneal endothelial cells (HCECs) while maintaining their phenotype. We will use ROCK inhibitor to increase the proliferation of HCECs and study whether the presence of TGF-Beta will allow the cells to maintain their phenotype in culture.

References

Okumura, Naoki, EunDuck Kay, et al. "Inhibition of TGF-β Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine." PLoS One. 8.2 (2013): n. page. Web. 5 May. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581499/>.

-->This paper demonstrated that the use of SB431542, a selective inhibitor of the TGF-β receptor, in culture media counteracted the development of fibroblastic phenotypes in HCECs.


Pipparelli, Aurélien, Yvan Arsenijevic, et al. "ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells." PLoS One. 8.4 (2013): n. page. Web. 5 May. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633902/>.

--> This paper...

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