| Summary: | The Nrf2 system plays an important role in the regulation of the redox state of the cell. Protection from oxidative and genotoxic damage is achieved by Nrf2-mediated expression of antioxidant and phase-2 detoxification enzymes. The Nrf2 network topology possesses several feedback mechanisms that could play a role in the dynamics of Nrf2 protein. To address this issue, a range of molecular reagents were generated, including plasmid and Bacterial Artificial Chromosome (BAC) expression systems. These allowed the localization and dynamics of Nrf2 to be analysed at near endogenous expression levels. Specifically, we generated and characterised an Nrf2-venus BAC stable SK-N-AS cell line using state-of-the-art molecular biology and microscopy techniques including Fluorescence In Situ Hybridization (FISH) and Fluorescence Correlation Spectroscopy (FCS). Single cell imaging showed oscillations in Nrf2 levels that were heterogeneous amongst the cell population. These novel tools were applied to investigate the reciprocal effects of crosstalk between the Nrf2 and NF-κB signalling networks. These interactions have direct implications for a range of physiological and pathological processes including cancer, ageing and inflammation. While systems biology approaches have made important contributions to our understanding of the NF-κB dynamics, we still have a relatively poor understanding of how this system integrates with other regulatory networks in different cell types. Single live-cell imaging of the dynamics of these pathways suggested reciprocal dynamics, where stimulation of Nrf2 delays rather than inhibits NF-κB dynamics and Nrf2 oscillates out-of-phase to robust p65 oscillations. The tools developed during this study will be an important resource for further investigations of the Nrf2 system and its crosstalk with other signalling pathways. The BAC could also be used to generate novel reporter transgenic mice. Such data could be used to inform predictive mathematical models, which have direct implications for rational drug evaluation in either normal or diseased cells and tissues.
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