Oxidative stress is a biological state that occurs due to imbalance between reactive oxygen species (ROS) and the antioxidant system leading to subcellular disturbance. The purpose of this thesis was to determine changes in protein abundance/distribution between nuclear and cytoplasmic cell compartments in the normal human IMR90 fibroblasts subjected to mild oxidative stress. The experimental design was to exert tert- butyl hydrogen peroxide upon the IMR90 cell lines to induce mild oxidative stress followed by fractionation of the cells into nucleus and cytoplasm. Cellular response was measured by the use of subcellular spatial razor approach based on quantitative shotgun LC-MS/MS-based proteomics with SILAC isotope labeling. It has been found that, in response to the treatment, proteins were redistributed between nucleus and cytoplasm including numerous proteins that were not previously known to associate with oxidative stress. We found 121 most significant proteins with known function at unexpected subcellular location. These proteins were known to contribute to different cellular processes such as, transcription, iron/haem metabolism, TCA cycle, glycolysis, autophagy, signal transduction and ATP synthesis, and are consistent with functional networks that are spatially distributed across the cell. Specific metabolic pathways of NRF2 and proline regulatory axis were found to play an important part on the cellular response to mild oxidative stress. Iron metabolism with iron/haem as a cofactor and mitochondrial proteins were prominent into the response as well. By initial comparison of the oxidative stress fibroblasts with Cdc7 depleted fibroblasts after induction of origin activation checkpoint, it was found that both responses affect proteins related to glycolysis, TCA cycle and proline regulatory axis. Evidence suggested nuclear import/export of proteins induced by the treatment. Subcellular spatial razor results for response of fibroblasts to oxidative stress clearly suggested that, to obtain comprehensive pictures of cellular function, measurements of global changes in total protein abundance need to be combined with measurement of the dynamic subcellular spatial redistribution of proteins.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:654714 |
| Date | January 2015 |
| Creators | Baqader, N. O. A. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1469041/ |
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