Characterization of the Staphylococcus aureus bone sialoprotein-binding protein SdrE and the serine protease EpiP

In an attempt to develop a Staphylococcus aureus vaccine, we have applied reverse vaccinology approach, mainly based on in silico screening and proteomics. By using this approach SdrE, a protein belonging to serine-aspartate repeat protein family was identified as potential vaccine antigen against S. aureus. We have investigated the biochemical properties as well as the vaccine potential of SdrE and its highly conserved CnaBE3 domain. We found the protein SdrE to be resistant to trypsin. Further analysis of the resistant fragment revealed that it comprises a CnaBE3 domain, which also showed partial trypsin resistant behavior. Furthermore, intact mass spectrometry of rCnaBE3 suggested the possible presence of isopeptide bond or some other post-translational modification in the protein.However, this observation needs further investigation. Differential Scanning Fluorimetry study reveals that calcium play role in protein folding and provides stability to SdrE. At the end we have demonstrated that SdrE is immunogenic against clinical strain of S. aureus in murine abscess model. In the second part, I characterized a protein, annotated as epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. The crystal structure of the rEpiP was solved at 2.05 Å resolution by x-ray crystallography . The structure showed that rEpiP was cleaved somewhere between residues 95 and 100 and cleavage occurs through an autocatalytic intra-molecular mechanism. In addition, the protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine if the protein acts as a serine protease, we mutated the catalytic serine 393 residue to alanine, generating rEpiP-S393A and solved its crystal structure at a resolution of 1.95 Å. rEpiP-S393A was impaired in its protease activity, as expected. Protective efficacy of rEpiP and the non-cleaving mutant protein was comparable, implying that the two forms are interchangeable for vaccination purposes.

Cleavage Politics in Changing Times. Political Realignment Processes in Western European Countries

The dissertation fits the political realignment literature and aims to pro-vide further insights into cleavage politics by investigating voting behaviour in the Western European countries’ national elections. In particular, the dis-sertation focuses on the class and value voting patterns and on the change of these patterns in different countries and over the course of time. Peculiar pro-cesses affected all Western European party systems: whilst the «traditional» cleavage theory accounts for National and Industrial revolutions, those pro-cesses assumed to constitute the «societal modernization» determined chang-es in electoral competitions that questioned the relevance of individuals’ so-cial positions to study electoral preferences. Since the associations between social positions and voting behaviour underpin the so-called political cleav-age, the dealignment perspective assumes them to have been eroding since the second half of the XX century. On the other hand, the realignment perspective argues that the cleavage theory still accounts for individuals’ vote choices: of the four «traditional» cleavages, this perspective hypothesizes new class vot-ing patterns and alignments between electoral preferences and a new line of conflict, that is based on values. The dissertation provides a theoretical ac-count of the realignment of the class cleavage and a new conceptualization of value voting. Then, class and value voting patterns are explored. The analyses employ European Social Survey data and detect general and country-specific patterns. The dissertation adopts a mediation perspective and aims to observe how class voting patterns change when controlling for value orientations. The results are provided with a sensitivity analysis, indeed two versions of the measures computed for value orientations are compared. The findings show that social class continues to affect voting behaviour and that value orienta-tions both mediate this effect and affect electoral preferences.