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Potential Biomarkers for Preterm Delivery in Amniotic Fluid

Ashtaputre, Ravina M.
http://rave.ohiolink.edu/etdc/view?acc_num=wright1464186519

Abstract Details

2016, Master of Science (MS), Wright State University, Pharmacology and Toxicology.
Preterm birth still remains a major unresolved issue accounting for over 75% of the perinatal mortality. Various maternal factors like demographics, behavioral and clinical characteristics including maternal race/ethnicity, age, psychosocial stress, intrauterine infection, previous history of preterm birth have been associated with preterm birth. The pathogenesis of preterm birth is multifactorial and hence the exact cause of preterm labor is unclear. In the project presented here, amniotic fluid was used to search for potential new biomarkers as predictors of risk for preterm labor. In the first aim of this project, we focused on proteins isolated with microvesicles and exosomes in amniotic fluid. Microvesicles/exosomes were extracted from amniotic fluid by ultracentrifugation at 100,000 x g for 2 hours and analyzed for size on a NanoSight system. Concentration of proteins in the extracellular vesicles was set to ~50 µg, labeled with Cy3 or Cy5 fluorescent reagent (preterm and term, respectively), and loaded onto isoelectric focusing (IEF) gels at a pH range of 3-10, followed by separation in an IEF cell. The proteins separated by isoelectric point were then separated on a 4-20% SDS-PAGE gel by size. Images of each gel were captured under appropriate excitation conditions for the fluor, and the images saved for further analysis. Our results show altered proteomic numbers in amniotic fluid of term samples compared to preterm, i.e., 50-103 matched proteins in the gels versus 1-54 unmatched proteins. The images were analyzed to visualize as many protein spots as possible, matched to each other for comparison of preterm and term proteins and 350 gel spots excised from the all the gels using a manual gel cutter. The proteins in the gel spots were digested with trypsin, followed by extraction of the peptides for future analysis by mass spectrometry fragmentation to identify the proteins in control versus preterm samples. In a second aim of this study, we have tested for differences in a common enzyme activity on a specific substrate in the amniotic fluid. Primary analysis by MALDI-TOF MS showed an increase in Ang (1-7) forming enzyme in the preterm samples compared to the term samples. To further elucidate the activity we used a fluorogenic assay system that had better specificity and could be analyzed in bulk. The results from this assay appeared to show a faster reaction time (minutes instead of hours), but still showed that the preterm amniotic fluid had a significantly higher concentration of the enzyme activity (p<0.05). The results from our study are preliminary at this point, until we can obtain identification of proteins by mass spectrometry. The enzyme assay needs to be repeated with more patients in comparison with fetal fibronectin, and normalized to creatinine to determine if it could be a viable predictor of risk for preterm labor.
David Cool, Ph.D. (Advisor)
Khalid Elased, Pharm. D., Ph.D. (Committee Member)
Ji Bihl, Ph.D. (Committee Member)
66 p.
Pharmacology
pharmacology

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Citations

  • Ashtaputre, R. M. (2016). Potential Biomarkers for Preterm Delivery in Amniotic Fluid [Master's thesis, Wright State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=wright1464186519

    APA Style (7th edition)

  • Ashtaputre, Ravina. Potential Biomarkers for Preterm Delivery in Amniotic Fluid. 2016. Wright State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=wright1464186519.

    MLA Style (8th edition)

  • Ashtaputre, Ravina. "Potential Biomarkers for Preterm Delivery in Amniotic Fluid." Master's thesis, Wright State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1464186519

    Chicago Manual of Style (17th edition)

wright1464186519
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© 2016, all rights reserved.
This open access ETD is published by Wright State University and OhioLINK.