All the people I worked with gave me incredible support during these years, technical and emotional. From the first day, with my shirt decorated with bananas, to the lasts, when they had to bear my nervous and stressed mood. I thank Anna, Linda and Pierre, for the funny moments, the laughs and the many beers together. And for the precious scientific discussions, of course! Thank you also to Eethan, the living scientific encyclopedia, with (almost) always the right answer to my problems in the lab. Thank you, Enoch, for the discussions, especially those in the microscopy room. Emir, thank you for your help with the cloning, microscopy and the bacteriographs. I like your enthusiasm toward science, do not lose it! Thank you, Mehmet, for the support to the protein function prediction of my project, I appreciated the point of view from someone with a "dry biology" background and the comments on my thesis. Jeremy, Mirko, Giada, and Jara, thank you for the daily chats and jokes together. The daily routine was lighter with you around. Thank you, Dagmar, oh Dagmar, for your patience and your smiles. Lastly, thanks to the grumpy Daniel, my dear deskmate and benchmate, fellow of complains.Vorrei anche ringraziare i miei amici rimasti in Italia. Anche se ci vediamo poco, quando ci troviamo è sempre una festa. Sono fortunato a non averli persi dopo più di 5 anni passati all'estero: è anche grazie a loro che ho mantenuto la tenacia per arrivare fino alla fine. In particolare, Claudio, Stefano e Vito, grazie per la vostra vicinanza nonostante la lontananza. Grazie anche a Laura, la mia supervisor durante il mio tirocinio di tesi magistrale in Unimi, che mi ha insegnato come pensare e come muovermi nell'ambito di un progetto di ricerca: quello che ho imparato in quell'anno mi è stato molto utile durante il dottorato.Un grazie immenso alla mia famiglia e al nonno, senza i quali non sarei potuto arrivare dove sono ora, incoraggiandomi e sostenendomi sempre. E infine, Margherita, grazie per essere sempre stata al mio fianco in questi anni, anche quando non avevo occhi che per la tesi, risollevandomi dai momenti di sconforto e spazzando via i cattivi pensieri. ContributionsLeonard Ernst, a former bachelor student of the Di Ventura lab, cloned a 2-plasmid-based library composed of 5 VVD-AraC fusion proteins under IPTG induction and performed the first explorative experiments to demonstrate the light-responsiveness of the system. Emir Bora Akmeriç, technical assistant in our laboratory, helped me during the last year of my doctorate in the cloning process of some constructs and with the induction and imaging of the genes with poor or unknown function. Moreover, he took care of the Blade Runner bacteriograph, repeating it until he made it flawless.Mehmet Ali Öztürk, postdoc in our laboratory, contributed with structural biology analysis of the I1-I2 sequences rearrangements and collected, interpreted and catalogued the function and localization prediction outputs of the genes with poor or unknown function. He also provided valuable sug...

Vascular endothelial (VE)-cadherin in endothelial adherens junctions is an essential component of the vascular barrier, critical for tissue homeostasis and implicated in diseases such as cancer and retinopathies. Inhibitors of Src cytoplasmic tyrosine kinase have been applied to suppress VE-cadherin tyrosine phosphorylation and prevent excessive leakage, edema and high interstitial pressure. Here we show that the Src-related Yes tyrosine kinase, rather than Src, is localized at endothelial cell (EC) junctions where it becomes activated in a flow-dependent manner. EC-specific Yes1 deletion suppresses VE-cadherin phosphorylation and arrests VE-cadherin at EC junctions. This is accompanied by loss of EC collective migration and exaggerated agonist-induced macromolecular leakage. Overexpression of Yes1 causes ectopic VE-cadherin phosphorylation, while vascular leakage is unaffected. In contrast, in EC-specific Src deficiency, VE-cadherin internalization is maintained and leakage is suppressed. In conclusion, Yes-mediated phosphorylation regulates constitutive VE-cadherin turnover, thereby maintaining endothelial junction plasticity and vascular integrity.

In Escherichia coli, the operon responsible for the catabolism of L-arabinose is regulated by the dimeric DNA-binding protein AraC. In the absence of L-arabinose, AraC binds to the distal I1 and O2 half-sites, leading to repression of the downstream PBAD promoter. In the presence of the sugar, the dimer changes conformation and binds to the adjacent I1 and I2 half-sites, resulting in the activation of PBAD. Here we engineer blue light-inducible AraC dimers in Escherichia coli (BLADE) by swapping the dimerization domain of AraC with blue light-inducible dimerization domains. Using BLADE to overexpress proteins important for cell shape and division site selection, we reversibly control cell morphology with light. We demonstrate the exquisite light responsiveness of BLADE by employing it to create bacteriographs with an unprecedented quality. We then employ it to perform a medium-throughput characterization of 39 E. coli genes with poorly defined or completely unknown function. Finally, we expand the initial library and create a whole family of BLADE transcription factors (TFs), which we characterize using a novel 96-well light induction setup. Since the PBAD promoter is commonly used by microbiologists, we envisage that the BLADE TFs will bring the many advantages of optogenetic gene expression to the field of microbiology.