Summary of Research
Tissue oxidative stress is an important underlying cause in many malignant transformations and degenerative diseases. ROS  derived from cell metabolism, inflammation and exogenous sources can disrupt cellular oxidation-reduction (redox) balance which governs metabolic fidelity and cell fate. Our laboratory examines the role of GSH redox in regulating cell death/survival and the mechanisms in cytoprotection against oxidative challenge. Ongoing research employs cell culture and animal models and various biochemical, analytical and molecular techniques to address two areas:
Project 1
Mitochondria are sentinel in cell survival and are vulnerable to oxidative damage. This research investigates the relationship between oxidative stress and mitochondrial protein and DNA damage, the role of GSH redox in repair mechanisms, and how loss of mitochondrial function orchestrates apoptotic death.
Project 2
Chronic oxidative stress results in tissue death, transformation or adaptation. This research investigates the role of GSH redox in controlling cell signaling and gene transcription and mechanisms in enzyme expression and function in maintaining GSH balance and cell survival.
Techniques/Procedures
Experimental models: Cellular Models - A variety of human colonic cells (CaCo-2, HT-29, NCM-460 ), human umbilical vein endothelial cells (HUVEC), HeLa cells, subcellular fractions of nuclei and mitochondria
Animal models - Rat models: lymph fistula/bile fistula, chronic peroxide consumption, chronic hypoxia; Mouse models: in situ liver perfusion (periportal/perivenous perfusion), in vivo partial ischemia/reperfusion liver perfusion
Techniques
a. HPLC with uv-vis and radiomatic detection; electrochemical detection
b. Agarose gel electrophoresis; polyacrylamide gel electrophoresis, Westerns, Northerns, RNAse protection assay,
c. Dual wavelength spectrometry, fluorescence spectrometry
d. Induction of graded hypoxia and anoxia and anoxia/reoxygenation
e. Biochemical assays for: oxidants, GSH/GSSG, metabolic and detoxication enzymes, cell metabolites and bioenergetics, cell proliferation and apoptosis, oxidative DNA damage, leukocyte-endothelial cell adherence
f. Cell elutriation
g. Molecular biology assays for: transcription factor activation; protein and mRNA expression of adhesion molecules, apoptotic and proliferative markers; overexpression of MnSOD, CuZn SOD in cells, generation of transient and stable transfectants. |
