PhD Defense: Adam Prasanphanich

Date:        12/16/14
Time:        2:00 PM
Location:        Pettit Microelectronics Research Building, Room 102
Advisor:        Melissa Kemp, PhD
Committee:    Thomas Barker, PhD
           Hanjoong Jo, PhD
           Russell Medford, MD, PhD
           Eberhard Voit, PhD

 Dynamic Redox Signaling During TGF-Beta-Induced Epithelial-Mesenchymal Transition

 
   The morphogen transforming growth factor β (TGFβ) can initiate diverse cellular responses associated with development, cancer, and fibrosis.  TGFβ signaling results in down-regulation of numerous antioxidant species but itself has been shown to exhibit redox sensitivity. In the context of TGFβ-mediated epithelial-mesenchymal transition (EMT), there exists a possibility of a positive feedback loop operating over multiple temporal and biological scales to stabilize a mesenchymal phenotype. Additionally, drug-resistant side populations (SP) arise in populations that exhibit heterogeneity of activity of a glutathione transporter, ABCG2, which is regulated within the same cellular program as antioxidants. Therefore, it is possible that SPs reflect heterogeneity in redox regulation within a population; however, how single-cell ABCG2 activity heterogeneity manifests population level characteristics is not known. The overall objective of this research was to investigate the relationship of redox-regulated processes to the complex phenotypes that arise in the context of TGFβ-mediated EMT using multivariate modeling approaches.
   The dynamics of redox regulation were investigated in the context of EMT, based upon the hypothesis that decreased nucleophilic tone acquired during EMT strengthens TGFβ signaling, enhancing acquisition and stabilization of the mesenchymal phenotype. Customized in-cell western assays were developed to evaluate multivariate phenotype states as they developed during EMT. TGFβ treatment decreased H2O2 degradation rates and increased glutathione (GSH) redox potential, indicating decreased nucleophilic tone. Epithelial/mesenchymal differentiation markers and redox time course data were paired using principal component analysis (PCA) to construct a multivariate representation of phenotype over the time course of EMT. Decreased nucleophilic tone during EMT coincided with acquisition of a mesenchymal phenotype over time scales too large to enable enhancement of EMT.
   Next, the role of heterogeneity in the activity levels of a GSH transporter, ABCG2, was investigated at the single cell level for the emergence of drug-resistant SPs at the population level. The objective was to develop a multiscale ensemble model consisting of a heterogeneous population of individual cells to interrogate multiple kinetic schemas and determine the means by which TGFβ signaling modulates heterogeneity to affect SP size. TGFβ was found to decrease the size of SPs as well as the magnitude of response. A highly active subpopulation juxtaposed by an inactive main population was identified, suggesting the SP cells may exhibit a distinct redox profile from main cells, the frequency of which was decreased with TGFβ.
   In summary, this work represents systems approaches to investigate the dynamics of redox regulation during TGFβ-mediated EMT from the perspective of a multivariate phenotype, simultaneously accounting for changes in epithelial/mesenchymal differentiation and to the intracellular redox environment. Additionally, an outcome of this project is a multiscale ensemble modeling methodology that is generalizable for future mechanistic studies of drug intracellular interactions that influence multivariate population characteristics as observed in flow cytometry.