Research Proposal: Difference between revisions

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===Project Idea===
'''Background'''


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.
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>.


'''References'''
'''Project Idea'''
 
 
'''Alper, H., and Stephanopoulos, G. Global transcription machinery engineering: A new approach for improving cellular phenotype. Metab. Eng. 2007; 9:258–267.'''
 
This paper shows how global modifications to the transcriptome can be made by engineering the cellular transcription machinery. In particular, s70 is modified using this technique of global transcription machinery engineering (gTME) which allows multiple gene modifications to be made simultaneously. This would be very useful when engineering phenotypes which are affected by many genes. This paper will also serve as a reference for a detailed protocol for phenotype selection (ethanol tolerance, for example).


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  and upregulation of NGF to increase the proliferation of HCECs and study whether the presence of these proteins allow the cells to maintain their phenotype in culture.


'''References'''


'''Queitsch C., Sangster T., Lindquist S. Hsp90 as a capacitor of phenotypic variation. Nature 2002; 417:618-624.'''
''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 talks about Hsp90's role in buffering genetic variation and how reducing Hsp90 function resulted in the observance of a variety of morphological phenotypes. This paper helps us identify how making modifications to Hsp90 can produce different phenotypes, given its role in chaperoning regulatory proteins, buffering genetic variation and influencing responses to environmental changes and stress.  
--> ROCK inhibitor Y-27632 promotes adhesion and cell proliferation in monkey CECs in vitro. The addition of ROCK inhibitor to HCECs was hoped to increase anti-apoptotic activity, and thereby increase endothelial cell density in the cornea. Unfortunately, it was not found to enhance cell proliferation in HCECs ex vivo. ROCK inhibitor does, however, promote cell adhesion and wound healing in HCECs.




'''Whitesell L, Lindquist S. HSP90 and the chaperoning of cancer. Nature Reviews Cancer 2005; 5:761-772'''  
''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/>.''


This paper provided us with more background on HSP90's chaperoning functions and its role in cancer. It talks about the function and structure of HSP90 in detail and will help us identify particular regions to mutate if we chose to do so.
--> 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.




'''Sangster T.A., Salathia N., Undurraga S., Milo R., Schellenberg K., Lindquist S., Queitsch C. HSP90 affects the expression of genetic variation and developmental stability in quantitative traits. Proc. Natl. Acad. Sci. USA 2008 105:2963-2968.'''
''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 is another source discussing the role of HSP90 in concealing genetic variation. The hypothesis discussed is that changing HSP90 function may lead to the evolution of new phenotypes in response to environmental stress. They checked to see changes in hypocotyl elongation in the dark in Arabidopsis thaliana. We want to take this idea further to check to see changes in ethanol and sugar tolerance after HSP90 modification.  The general idea is that the revelation of poymorphisms previously buffered by HSP90 may lead to some interesting evolutionary behavior in suboptimal environments. Another thing to note from this paper is that they found that changes in the traits they were looking at outweighed decrease in developmental stability  (affected  by the natural variation buffered by HSP90 as well).
-->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.

Latest revision as of 05:18, 7 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 and upregulation of NGF to increase the proliferation of HCECs and study whether the presence of these proteins allow the cells to maintain their phenotype in culture.

References

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/>.

--> ROCK inhibitor Y-27632 promotes adhesion and cell proliferation in monkey CECs in vitro. The addition of ROCK inhibitor to HCECs was hoped to increase anti-apoptotic activity, and thereby increase endothelial cell density in the cornea. Unfortunately, it was not found to enhance cell proliferation in HCECs ex vivo. ROCK inhibitor does, however, promote cell adhesion and wound healing in HCECs.


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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