Characterization of L-type Calcium Channel Binding-Site of a new class of Calcium modulators by a Multidisciplinary approach

Many potential diltiazem related L-VDCC blockers were developed using a multidisciplinary approach. This current study was to investigate and compare diltiazem with to the newly developed compounds by mouse Langendorff-perfused heart, Ca2+-transient and on recombinant L-VDCC. Twenty particular compounds were selected by the ligand-based virtual screening procedure (LBVS). From these compounds, five of them (5b, M2, M7, M8 and P1) showed a potent and selective inotropic activity on guinea-pig left atria driven 1 Hz. Further assays displayed an interesting negative inotropic effect of M2, M8, P1 and M7 on guinea pig isolated left papillary muscle driven at 1 Hz, a relevant vasorelaxant activity of 5b, M2, M7, M8 and P1 on K+-depolarized guinea-pig ileum longitudinal smooth muscle and a significant inhibition of contraction of 5b, M2, M8 and P1 on carbachol stimulated ileum longitudinal smooth muscle. Wild-type human heart and rabbit lung α1 subunits were expressed (combined with the regulatory α2δ and β3 subunits) in Xenopus Leavis oocytes using a two-electrode voltage clamp technique. Diltiazem is a benzothiazepine Ca2+ channel blocker used clinically for its antihypertensive and antiarrhythmic effects. Previous radioligand binding assays revealed a complex interaction with the benzothiazepine binding site for M2, M7 and M8. (Carosati E. et al. J. Med Chem. 2006, 49; 5206). In agreement with this findings, the relative order of increased rates of contraction and relaxation at lower concentrations s(≤10-6M) in unpaced hearts was M7>M2>M8>P1. Similar increases in Ca2+ transient were observed in cardiomyocytes. Diltiazem showed negative inotropic effects whereas 5b had no significant effect. Diltiazem blocks Ca2+current in a use-dependent manner and facilitates the channel by accelerating the inactivation and decelerating the recovery from inactivation. In contrast to diltiazem, the new analogs had no pronounced use-dependence. Application of 100 μM M8, M2 showed ~ 10% tonic block; in addition, M8, M2 and P1 shifted the steady state inactivation in hyperpolarized direction and the current inactivation time was significantly decreased compared with control (219.6 ± 11.5 ms, 226 ± 14.5 vs. 269 ± 12.9 vs. 199.28 ± 8.19 ms). Contrary to diltiazem, the recovery from the block by M8 and M2 was comparable to control. Only P1 showed a significantly decrease of the time for the recovery from inactivation. All of the compounds displayed the same sensitivity on the Ca2+ channel rabbit lung α1 except P1. Taken together, these findings suggest that M8, M2 and P1 might directly decrease the binding affinity or allow rapid dissociation from the benzothiazepine binding site.

Structural and functional characterization of Group B Streptococcus class C sortases

In Group B Streptococcus (GBS) three structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies and in vivo mutagenesis we performed a broad characterization of GBS sortase C. The high resolution X-ray structure of the enzymes revealed that the active site, located into the β-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the “lid”. We show that the catalytic triad and the predicted N- and C-terminal trans-membrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild type protein.

Negoziazioni intergenerazionali dell'autonomia abitativa in contesti di working class

Nell’ambito della riflessione sui processi di riproduzione e trasmissione intergenerazionale delle disuguaglianze sociali, la presente tesi dottorale propone un’analisi in prospettiva intersezionale dei percorsi verso l’indipendenza abitativa dei giovani bolognesi di origine popolare, e il ruolo svolto dalle loro famiglie in questi percorsi. Nell’approfondimento teorico si offre una ricostruzione del dibattito sociologico nazionale e internazionale sul tema delle disuguaglianze sociali e abitative delle giovani generazioni, nonché dello studio della classe sociale e i principali approcci presenti in letteratura. La ricerca sul campo si concentra, in particolare, sui modi in cui le famiglie appartenenti ad una determinata classe sociale, intesa nel senso conferito al termine da Bourdieu, negoziano e sostengono la transizione all’indipendenza abitativa dei loro figli. L’approfondimento empirico consiste in una ricerca qualitativa longitudinale retrospettiva, condotta nel bolognese nel 2013-2014 su un campione di famiglie working class. Ai giovani-adulti coinvolti e ai loro genitori è stato chiesto di ricostruire le loro biografie attraverso lo strumento delle life histories. La ricerca evidenzia il delinearsi di “micro sistemi economici” familiari specifici e creativi. Le molteplici forme di sostegno genitoriale nei percorsi di autonomizzazione dei figli, individuate nel corso dell’analisi del materiale raccolto, si innestano quindi all’interno di un più ampio sistema di supporto intergenerazionale, che continua anche dopo l’uscita dalla famiglia di origine, a conferma dello stretto legame che caratterizza le famiglie italiane. Nello studio una particolare attenzione viene rivolta alle logiche di legittimazione del sostegno genitoriale, adottate dalle famiglie per orientare i propri interventi di aiuto. Infine, mettendo a confronto la concezione di indipendenza dei giovani adulti con quella dei loro genitori, l’indagine rileva l’esistenza di una apparente contraddizione tra le rappresentazioni dell’indipendenza e dell’autonomia abitativa e i comportamenti attuati nella vita quotidiana, contraddizione che trova una risoluzione nella negoziazione, tra le due diverse generazioni, del concetto stesso di indipendenza.