Research proposal

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(Research Problem and Goals)
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<center>'''Isaac Businger and Ryan Alexander'''</center>
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<center>'''Ebaa Al-Obeidi and Dominique Forestier'''</center>
==Project Overview==
==Project Overview==
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The goal of this project is to propose mechanisms that can optimize an existing biofuel-producing E. coli strain. Specifically we're looking at the biosynthesis of butanol.
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The aim of this project is to identify media additives that promote human corneal endothelial cell (HCEC) proliferation and preservation of phenotype during ''in vitro''culturing.  
==Background Information==
==Background Information==
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The cornea is the transparent, outermost part of the eye. It consists of three distinct layers: an outer layer of epithelial cells; a thick stroma of collagen fibrils embedded with quiescent fibroblasts; and an inner epithelial layer, called the endothelium. The transparency of the cornea depends mainly on the endothelium, which can be damaged by surgery or disease, leading to a permanent loss of visual acuity. This deterioration is permanent because the endothelial cells do not inherently proliferate, and once they are damaged they are not replaced. Thus, research in the field has sought to identify growth factors that can stimulate proliferation. Progress has been hindered, however, by the transformation of endothelial cells to fibroblastic cells in culture, which abolishes the endothelial phenotype.
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==Research Problem and Goals==
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==Research idea and Goals==
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Goal is to create an efficient way to biosynthesize butanol from cellulose. Existing systems have been able to biosynthesize ethanol from cellulose, but not butanol which is more energy-rich.
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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 SB431542, an inhibitor of the TGF-β pathway, and upregulation of nerve growth factor (NGF) to examine whether these proteins will enable HCECs to proliferate and avoid endothelial-mesenchymal transformation.  
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==Project Methods==
==Project Methods==
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''(We will use'' 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/> ''to design our methods because these researchers were asking a similar question about their inhibitor of interest -- specifically, whether it would allow for HCEC proliferation.)''
==Predicted Outcomes==
==Predicted Outcomes==
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==References==
==References==
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http://www.harvestcleanenergy.org/enews/enews_0505/enews_0505_Cellulosic_Ethanol.htm
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Hong, J., Qian, T., NGF promotes cell cycle progression by regulating D-type cyclins via PI3K/Akt and MAPK/Erk activation in human corneal epithelial cells, Molecular Vision, Vol 18, Mar 30, 2012, Pages 758-764, <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324366/>.
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Researchers can now transform straw, and other plant wastes, into cellulosic ethanol.  While chemically identical to ethanol produced from corn or soybeans, cellulose ethanol exhibits a net energy content three times higher than corn ethanol and emits a low net level of greenhouse gases. Recent technological developments are not only improving yields but also driving down production cost, leading to a day when cellulosic ethanol could replace expensive, imported "black gold" with a sustainable, domestically produced biofuel.
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--> Nerve growth factor is a protein that promotes cell survival through the MAPKcascade. Without NGF the expression of pro-apoptotic proteins are increased, leading to cell death. NGF positively regulates HCEC cell cycle progression which can enhance cell proliferation. We would like to up-regulate NGF expression in order to allow the HCECs to proliferate during wound healing in vitro.
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http://www.ncbi.nlm.nih.gov/pubmed/19325807?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=9
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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/>.
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Consolidated bioprocessing (CBP) is a system in which cellulase production, substrate hydrolysis, and fermentation are accomplished in a single process step by cellulolytic microorganisms. CBP offers the potential for lower biofuel production costs due to simpler feedstock processing, lower energy inputs, and higher conversion efficiencies than separate hydrolysis and fermentation processes, and is an economically attractive near-term goal for "third generation" biofuel production
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http://www.springerlink.com/content/p48g1n2nq88rrx1r/fulltext.pdf
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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. This suggests that SB431542 would be a useful additive to our HCEC growth media because it may allow the cells to maintain their phenotype as they proliferate.
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Modified E. coli was used to metabolize cellulose and use cellulose as its sole carbon source.
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Revision as of 09:38, 7 May 2013

Contents

Ebaa Al-Obeidi and Dominique Forestier

Project Overview

The aim of this project is to identify media additives that promote human corneal endothelial cell (HCEC) proliferation and preservation of phenotype during in vitroculturing.

Background Information

The cornea is the transparent, outermost part of the eye. It consists of three distinct layers: an outer layer of epithelial cells; a thick stroma of collagen fibrils embedded with quiescent fibroblasts; and an inner epithelial layer, called the endothelium. The transparency of the cornea depends mainly on the endothelium, which can be damaged by surgery or disease, leading to a permanent loss of visual acuity. This deterioration is permanent because the endothelial cells do not inherently proliferate, and once they are damaged they are not replaced. Thus, research in the field has sought to identify growth factors that can stimulate proliferation. Progress has been hindered, however, by the transformation of endothelial cells to fibroblastic cells in culture, which abolishes the endothelial phenotype.

Research idea and Goals

Our goal is to determine whether we can enhance the proliferation of human corneal endothelial cells (HCECs) while maintaining their phenotype. We will use SB431542, an inhibitor of the TGF-β pathway, and upregulation of nerve growth factor (NGF) to examine whether these proteins will enable HCECs to proliferate and avoid endothelial-mesenchymal transformation.

Project Methods

(We will use 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/> to design our methods because these researchers were asking a similar question about their inhibitor of interest -- specifically, whether it would allow for HCEC proliferation.)

Predicted Outcomes

Resources Required

References

Hong, J., Qian, T., NGF promotes cell cycle progression by regulating D-type cyclins via PI3K/Akt and MAPK/Erk activation in human corneal epithelial cells, Molecular Vision, Vol 18, Mar 30, 2012, Pages 758-764, <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324366/>.

--> Nerve growth factor is a protein that promotes cell survival through the MAPKcascade. Without NGF the expression of pro-apoptotic proteins are increased, leading to cell death. NGF positively regulates HCEC cell cycle progression which can enhance cell proliferation. We would like to up-regulate NGF expression in order to allow the HCECs to proliferate during wound healing in vitro.


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. This suggests that SB431542 would be a useful additive to our HCEC growth media because it may allow the cells to maintain their phenotype as they proliferate.

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