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Models for investigating functional roles of osteopontin
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Text
TitleInfo
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Title
Models for investigating functional roles of osteopontin
SubTitle
characterization of anti-OPN monoclonal antibodies
Identifier
ETD_1761
Identifier
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http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000051189
Language
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eng
Genre
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theses
Subject
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Topic
Microbiology and Molecular Genetics
Subject
(ID = SBJ-1);
(authority = ETD-LCSH)
Topic
Osteopontin
Subject
(ID = SBJ-1);
(authority = ETD-LCSH)
Topic
Monoclonal antibodies
Abstract
Osteopontin is an integrin-binding phosphorylated glycoprotein. It is found in many tissues and can be found in all body fluids. It is associated with several cellular processes such as cell survival, cancer progression and stress response. These particular cellular processes were of interest in my research.
This thesis is divided into two parts. Part I being about the purification of OPN and characterization of anti-OPN monoclonal antibodies. In order to accomplish part I of this thesis, OPN was purified by ion-exchange and desalting chromatography, and analyzed by SDS-PAGE followed by Coomassie Blue staining. The methods for purifying anti-OPN mAbs from hybridomas by ascites consisted of ammonium sulfate precipitation, ion-exchange chromatography, and protein A/G purification. Anti-OPN mAbs were characterized by SDS-PAGE analysis, Western blotting and ELISAs. The results from these analyses were then compiled into our database. The OPN and mAbs that were made were used in several experiments by my fellow lab mates and me.
The second part of my thesis was to investigate potential models for characterizing the functional roles of OPN. This was accomplished by studying cell survival with human umbilical vein endothelial cells (HUVECs) and cancer cell phenotypes with the pancreatic cancer cell line HS766T. Flow cytometry was used to analyze cell survival in HUVECs. Transfection with specific plasmids designed to increase or decrease OPN expression was used to modulate the cancer cell phenotype of HS766T cells.
In conclusion, I was able to successfully generate several pure stable preparations of OPN and anti-OPN mAbs. Also, I was able to reproduce cell survival in HUVECS by OPN a few times but because it was not consistently reproducible, I was unable to tests the effects of mAbs on the survival of HUVECs by OPN. Also, since our research was already successfully carried out and published by another group, I decided not to purse this project further. Lastly, I was able to create several clones of HS766T that exhibited increased or decreased OPN expression as determined by Real-Time PCR analysis.
This thesis is divided into two parts. Part I being about the purification of OPN and characterization of anti-OPN monoclonal antibodies. In order to accomplish part I of this thesis, OPN was purified by ion-exchange and desalting chromatography, and analyzed by SDS-PAGE followed by Coomassie Blue staining. The methods for purifying anti-OPN mAbs from hybridomas by ascites consisted of ammonium sulfate precipitation, ion-exchange chromatography, and protein A/G purification. Anti-OPN mAbs were characterized by SDS-PAGE analysis, Western blotting and ELISAs. The results from these analyses were then compiled into our database. The OPN and mAbs that were made were used in several experiments by my fellow lab mates and me.
The second part of my thesis was to investigate potential models for characterizing the functional roles of OPN. This was accomplished by studying cell survival with human umbilical vein endothelial cells (HUVECs) and cancer cell phenotypes with the pancreatic cancer cell line HS766T. Flow cytometry was used to analyze cell survival in HUVECs. Transfection with specific plasmids designed to increase or decrease OPN expression was used to modulate the cancer cell phenotype of HS766T cells.
In conclusion, I was able to successfully generate several pure stable preparations of OPN and anti-OPN mAbs. Also, I was able to reproduce cell survival in HUVECS by OPN a few times but because it was not consistently reproducible, I was unable to tests the effects of mAbs on the survival of HUVECs by OPN. Also, since our research was already successfully carried out and published by another group, I decided not to purse this project further. Lastly, I was able to create several clones of HS766T that exhibited increased or decreased OPN expression as determined by Real-Time PCR analysis.
PhysicalDescription
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electronic resource
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viii, 60 p. : ill.
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Note
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M.S.
Note
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Includes bibliographical references (p. 55-60)
Note
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by Dana M. Cifelli
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Cifelli
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Dana M.
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1984
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Dana M. Cifelli
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Axelrod
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David
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chair
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Advisory Committee
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David Axelrod
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Ron
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Yavoc
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internal member
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Yavoc Ron
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Chen
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Chiann-Chyi
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Chiann-Chyi Chen
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Denhardt
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David
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David Denhardt
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Rutgers University
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Graduate School - New Brunswick
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2009
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2009-05
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xx
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Rutgers University Electronic Theses and Dissertations
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ETD
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Title
Graduate School - New Brunswick Electronic Theses and Dissertations
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rucore19991600001
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NjNbRU
Identifier
(type = doi)
doi:10.7282/T30C4W05
Genre
(authority = ExL-Esploro)
ETD graduate
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The author owns the copyright to this work.
Copyright
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Copyright protected
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Open
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Non-exclusive ETD license
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Author Agreement License
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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