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theses

Extensive experimental studies and several human epidemiological surveys have revealed that exposure to high doses of ionizing radiation (>100 mSv) causes significant adverse health outcomes. The mechanisms underlying these effects have been well characterized. In contrast, the biological effects and health risks of exposure to low doses of radiation (<100 mSv) continue to be unclear and are a current subject of conflicting considerations. Due to insufficient statistical power in the limited number of available epidemiological studies evaluating health risks of human exposures to effective doses less than 100 mSv, mechanistic studies in cultured cells and animal models have been considered to be vital for understanding biological effects, and reducing the uncertainty in predicting health risks. This project builds on the body of studies characterizing the biochemical and biological effects of low dose ionizing radiation, but the emphasis is on characterizing post translational modification of proteins, namely S-nitrosylation, an area that is under-studied, even though it could greatly impact radiation sensitivity. Here, changes in S-nitrosylation were studied following in vivo exposure to either a low (0.1 Gy) or high doses (4 Gy) of 137Cs g rays, which mimics doses received in diagnostic and therapeutic radiation, respectively. The goal was to investigate whether similar or distinct S-nitrosylation events are induced in brain tissue following low and high dose g ray irradiation to test the following hypothesis: “Depending on radiation dose, S-nitrosylation triggers groups of proteins into participating in specific pathways that are protective (e.g. DNA repair, antioxidation reactions) or detrimental (loss of healthy cells, oxidative stress)”. Young adult C57BL/6J male mice were exposed to either 0, 0.1 or 4 Gy of Cesium- 137 g rays delivered uniformly to the whole body. Thirteen days after irradiation, the animals were euthanized, and the brains were harvested. The proteins were immediately extracted and processed for mass spectrometry analyses of global changes in Snitrosylation. Using Bioinformatic tools, the mass spectrometry results were analyzed by the R/Bioconductor statistical package. Several clustering approaches were used in order to create groups of proteins showing similar levels of S-nitrosylation for dose independent responses to radiation exposure, and dissimilar levels of S-nitrosylation for dose dependent responses. Clustering methods used a range of methods from purely mathematical to more intuitive manual approaches with somewhat arbitrary cutoff to analyze the proteomic data from irradiated and control samples. Additional clustering techniques like k-means and hierarchical clustering with several different numbers of clusters were applied to eliminate the cutoff bias. In addition, Ingenuity Pathway Analysis (IPA) software package was used to elucidate biological significance of the different groups of proteins. Depending on the clustering approaches used, several significant pathways were identified. For example, relative to control, the neuronal nitric oxide synthase (nNOS) pathway showed inactivation under low dose irradiation and activation under high dose irradiation. This pathway is under control of the N-methyl-D-aspartate receptor (NMDAR) activity, which becomes hyper-activated under high dose irradiation resulting in neurotoxicity. In conclusion, our results suggest that mouse exposure to low doses of Cesium-137 g rays may result in modulation of signaling pathways that promote protective effects through S-nitrosylation of certain key proteins and de-S-nitrosylation of others. This is an area that needs further investigation to elucidate the exact mechanism by which Snitrosylation occurs, and to confirm the role of the modulated pathways suggested by IPA in regulating cellular/tissue responses that impact sensitivity to radiation.

ETD_8914

Ph.D.

Includes bibliographical references

by Fadia Nicolas

doi:10.7282/T3Q52SZ5

ETD doctoral

The author owns the copyright to this work.