Reactive oxygen species (ROS) have been recently qualified as intracellular second messengers essential to many growth factors signalling. However, excess of ROS production by the newly discovered integral membrane NADPH oxidases (NOXs) results in or accompanies a large spectrum of life-threatening human diseases including cancer, diabetes, vascular diseases, neurodegeneration and aging. Although NOXs are the main ROS producing enzyme in vascular cells where they are involved angiogenesis and in the atherosclerosis pathology (through endothelial senescence), a lot is unknown about the pathways regulating NOX activity in endothelial cells.Therefore, I used the yeast two-hybrid system to identify TEM8 as a novel protein interacting with NOXO1, an essential adaptor and regulatory protein of NOXs. TEM8 is expressed in the vessels of tumours and early developing mouse embryos. I propose a multidisciplinary approach using specific ROS probes, shRNA, live imaging and physiological readouts to functionally character ize the TEM8/NOXO1 interaction in what could be a new angiogenic pathway. Furthermore, I will use the new and innovative yeast membrane split-ubiquitin system to identify proteins interacting with (and regulating) NOX1 and NOX4, the membrane proteins within the NOX complex. In contrast to the traditional yeast-two hybrid system, the split-ubiquitin system allows for the identification of protein interacting with transmembrane proteins. Moreover, the split-ubiquitin system will be modified to allow co-expression in yeast of NOX1 (or NOX4) with p22phox the essential component of the complex.Then, I will identify proteins interacting with the NOX/p22phox complex. This is especially relevant since known NOXs effectors, NOXA1 and NOXO1, interact preferentially with the complex rather than with a single subunit. Importantly, the proteins identified will be potential targets for the development of new valuable medicinal drugs to help treat ROS-related diseases.