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Date of Award

2011

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Lisa A. Wrischnik

First Committee Member

William K. Chan

Second Committee Member

Craig A. Vierra

Third Committee Member

Gregg D. Jongeward

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated bHLH-PAS protein that binds its partner, the aryl hydrocarbon receptor nuclear translocator (Arnt), in the nucleus to initiate the expression of proteins involved with detoxification. Published work suggests cross-talk between both proteins and cellular pathways involving the transcription factors, HIF -1 , ER, and NFKB, whose activity is typically upregulated in cancer. This thesis focuses on using a truncated form of AhR, AhR CΔ553, which is thought to act as a dominant-negative to sequester Arnt from its other binding partners. To test this hypothesis, we transfected HeLa cells with AhR CΔ553 fused to pEGFP or a vector under a tetracycline-inducible promoter. Stable cell lines expressing pEGFP-AhR CΔ553 have been generated and confirmed to have nuclear localization. We were also interested in confirming endogenous localization patterns of AhR and Arnt to study the role of p23 in the nuclear translocation of AhR. While we were successful in showing AhR translocating to the nucleus in treated MCF-7 cells, we couldn't clearly see nuclear AhR in Hepalclc7 cells, the cell line with knockdown levels of p23. To compare DNA damage generated in Jm·kat and Hepalclc7 cells, we looked for reactive oxygen species (ROS) production and quantified DNA damage after exposure to benzo[a]pyrene (B[a]P) and some of its derivatives. Hepalclc7 cells were prone to a wide variety of DNA damage, but Jurkat cells did not appear to undergo damage specifically through ROS production. Finally, we wanted to confirm apoptosis in HeLa cells after being cocultured with Trichomonas vaginalis. The G3 lab strain was more aggressive than Tl , but Parp, and apoptotic marker, was not observed in HeLa cells, suggesting that experimental conditions need to be further optimized.

Pages

73

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