Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)

Department / School

Pharmaceutical Sciences

First Advisor

Suman Mukherjee


Studies have shown that t-butylhydroperoxide (t-BuOOH), a free radical generating toxin, induces significant cell death in human cortical neurons (HCN2 cells). Also, it was observed that the poly (ADP-ribose) polymerase (PARP) inhibitor nicotinamide protects HCN2 cells by preventing up regulation of the pro-apoptotic proteins p53 and p21/WAF-1 that is induced by t-BuOOH. Therefore the identification of the genes involved was important for understanding the mechanisms behind free radical induced neuronal injury and for the neuroprotection provided by nicotinamide. Thus HCN2 cells were treated with 100 µM t-BuOOH or with 1 mM nicotinamide plus 100 µM t-BuOOH for 6 hrs. Total RNA was isolated, reverse transcribed and subjected to cDNA microarray analysis with ResGen™ Genefilter® (GF211). Data from three replications were analyzed with Pathways™ 4 software. Cluster analyses were done with mean values of gene expression; clusters were formed depending on the putative function of the gene. Among various clusters we were particularly interested in genes involved in regulation of cell death. Therefore to further understand the process of cell death and cell protection in neurons, second-generation pathways specific arrays were performed to study genes involved in apoptosis. TUNEL staining studies were performed at different time intervals including 30 min 1, 3, 6 and 24hr. It was observed that the process of apoptosis starts as early as 1 hr. Therefore microarray studies were performed at 1 hr to observe the effects of t-BuOOH as well as nicotinamide at 1 hr on upstream genes. Data was analyzed using different softwares including ScanAlyze®, GEArray® and Significance Analysis of Microarrays (SAM). It appears that the PARP inhibitor, nicotinamide, protects HCN2 cells from apoptosis by up regulating the important class of genes 'Inhibitors of Apoptosis' (IAPs) that inhibit apoptosis by inhibiting caspases.

Library of Congress Subject Headings

Gene expression.
Free radicals (Chemistry)
NAD-ADP-ribosyltransferase -- Inhibitors.


South Dakota State University



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