TitleModels for investigating functional roles of osteopontin
NameCifelli, Dana M. (author), Axelrod, David (chair), Ron, Yavoc (internal member), Chen, Chiann-Chyi (internal member), Denhardt, David (internal member), Rutgers University, Graduate School - New Brunswick,
SubjectMicrobiology and Molecular Genetics,
DescriptionOsteopontin 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.
NoteIncludes bibliographical references (p. 55-60)
Noteby Dana M. Cifelli
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.