Importanza prognostica delle citochine e loro ruolo nell'immunomodulazione: risultati di studi sperimentali in vivo e in vitro

Citokines are proteins produced by several cell types and secreted in response to various stimuli. These molecules are able to modify the behaviour of other cells inducing activities like growth, differentiation and apoptosis. In the last years, veterinary scientists have investigated the role played by these factors; in fact, cytokines can act as intercellular communicative signals in immune response, cell damage repair and hematopoiesis. Up to date, various cytokines have been identified and in depth comprehension of their effects in physiology, pathology and therapy is an interesting field of research. This thesis aims to understand the role played by these mediators during natural or experimentally induced pathologies. In particular, it has been evaluated the genic and protein expressions of a large number of cytokines during several diseases and starting from different matrix. Considering the heterogeneity of materials used in experimentations, multiple methods and protocols of nucleic acids and proteins extractions have been standardized. Results on cytokines expression obtained from various in vitro and in vivo experimental studies have shown how important these mediators are in regulation and modulation of the host immune response also in veterinary medicine. In particular, the analysis of inflammatory and septic markers, like cytokines, has allowed a better understanding in the pathogenesis during horse Recurrent Airway Obstruction, foal sepsis, Bovine Viral Diarrhea Virus infection and dog Parvovirus infection and the effects of these agents on the host immune system. As experimentations with mice have shown, some pathologies of the respiratory and nervous system can be reduced or even erased by blocking cytokines inflammatory production. The in vitro cytokines expression evaluation in cells which are in vivo involved in the response to exogenous (like pathogens) or endogenous (as it happens during autoimmune diseases) inflammatory stimuli could represent a model for studying citokines effects during the host immune response. This has been analyzed using lymphocytes cultured with several St. aureus strains isolated from bovine mastitic milk and different colostrum products. In the first experiment different cytokines were expressed depending on enterotoxins produced, justifying a different behaviour of the microrganism in the mammal gland. In the second one, bone marrow cells derived incubated with murine lymphocytes with colostrum products have shown various cluster of differentiation expression , different proliferation and a modified cytokines profile. A better understanding of cytokine expression mechanisms will increase the know-how on immune response activated by several pathogen agents. In particular, blocking the cytokine production, the inhibition or catalyzation of the receptor binding mechanism and the modulation of signal transduction mechanism will represent a novel therapeutic strategy in veterinary medicine.

La protezione dei diritti sociali nell'ordinamento dell'Unione Europea

This study deals with the protection of social rights in Europe and aims to outline the position currently held by these rights in the EU law. The first two chapters provide an overview of the regulatory framework in which the social rights lie, through the reorganisation of international sources. In particular the international instruments of protection of social rights are taken into account, both at the universal level, due to the activity of the United Nations Organisation and of its specialized agency, the International Labour Organization, and at a regional level, related to the activity of the Council of Europe. Finally an analysis of sources concludes with the reconstruction of the stages of the recognition of social rights in the EU. The second chapter describes the path followed by social rights in the EU: it examines the founding Treaties and subsequent amendments, the Charter of Fundamental Social Rights of Workers of 1989 and, in particularly, the Charter of Fundamental Rights of the European Union, the legal status of which was recently treated as the primary law by the Treaty of Lisbon signed in December 2007. The third chapter is, then, focused on the analysis of the substantive aspects of the recognition of the rights made by the EU: it provides a framework of the content and scope of the rights accepted in the Community law by the Charter of Fundamental Rights, which is an important contribution to the location of the social rights among the fundamental and indivisible rights of the person. In the last section of the work, attention is focused on the two profiles of effectiveness and justiciability of social rights, in order to understand the practical implications of the gradual creation of a system of protection of these rights at Community level. Under the first profile, the discussion is focused on the effectiveness in the general context of the mechanisms of implementation of the “second generation” rights, with particular attention to the new instruments and actors of social Europe and the effect of the procedures of soft law. Second part of chapter four, finally, deals with the judicial protection of rights in question. The limits of the jurisprudence of the European Union Court of Justice are more obvious exactly in the field of social rights, due to the gap between social rights and other fundamental rights. While, in fact, the Community Court ensures the maximum level of protection to human rights and fundamental freedoms, social rights are often degraded into mere aspirations of EU institutions and its Member States. That is, the sources in the social field (European Social Charter and Community Charter) represent only the base for interpretation and application of social provisions of secondary legislation, unlike the ECHR, which is considered by the Court part of Community law. Moreover, the Court of Justice is in the middle of the difficult comparison between social values and market rules, of which it considers the need to make a balance: despite hesitancy to recognise the juridical character of social rights, the need of protection of social interests has justified, indeed, certain restrictions to the free movement of goods, freedom to provide services or to Community competition law. The road towards the recognition and the full protection of social rights in the European Union law appears, however, still long and hard, as shown by the recent judgments Laval and Viking, in which the Community court, while enhancing the Nice Charter, has not given priority to fundamental social rights, giving them the role of limits (proportionate and justified) of economic freedoms.

La cessazione dell'attività economica nell'IVA: profili nazionali e comunitari

La ricerca oggetto della tesi dottorale verte sulla ricostruzione della disciplina della cessazione e delle fasi liquidatore dell’attività nell’ambito dell’imposta sul valore aggiunto, nel tentativo di individuare criteri certi ed omogenei per la sua individuazione, sia in prospettiva comunitaria che in prospettiva nazionale. In primo luogo, l’indagine si concentra sulla ricostruzione della nozione di attività economica e sui profili dinamici dell’attività in ambito comunitario, nell’intento di chiarire, sulla base della giurisprudenza, i criteri utilizzati per individuare la cessazione dell’attività rilevante ai fini dell’imposta, sfruttando in tal senso l’esperienza degli atti preparatori e l’applicabilità dell’ipotesi di applicazione dell’imposta per autoconsumo Vengono così valorizzati, accanto al profilo oggettivo costituito dalla presenza di una legame tra le operazioni della fase liquidatoria, il ruolo del soggetto passivo, quale portatore di una volontà che è in grado di influire sull’esistenza dell’attività stessa, e, conseguentemente, delle dichiarazioni formali imposte dalla Direttiva, quale manifestazione diretta di tale volontà ed elemento in grado di costituire un indice certo di cessazione. In seguito si esamina il regime impositivo nazionale ed in particolare le linee interpretative maggioritarie accolte da dottrina e giurisprudenza. Ricostruiti nozione e ruolo di attività economica, con riferimento all’esercizio d’impresa, vengono analizzati i diversi casi di cessazione contemplati dall’ordinamento –liquidazione volontaria, trasferimento dell’azienda e fallimento- analizzati sia nella prospettiva del diritto tributario che in quella del diritto commerciale. L’esame così sviluppato è volto a contestare l’interpretazione maggioritaria che vede nella sola presenza di beni residui alla liquidazione, o comunque la permanenza di un insieme di beni potenzialmente in grado di far riprendere l’esercizio, elemento sufficiente a mantenere in vita l’impresa e con essa la soggettività passiva all’imposta. Infine, vengono confrontati i risultati raggiunti nell’esame della disciplina comunitaria nazionale, ponendone in evidenza le possibilità incompatibilità ed i punti di contatto, con l’intenzione di dimostrare l’applicabilità anche a livello nazionale dei principi emersi in ambito comunitario.

Produzione di virus sintetici per lo studio dei meccanismi di interazione coinvolti nell'induzione di resistenza

Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV) are members of Benyvirus genus. BSBMV has been reported only in the United States while BNYVV has a worldwide distribution. Both viruses are vectored by Polymyxa betae, possess similar host ranges, particles number and morphology. Both viruses are not serologically related but have similar genomic organizations. Field isolates consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and replication while RNAs 3 and 4 play important roles on plant and vector interactions, respectively. Nucleotide and amino acid analyses revealed BSBMV and BNYVV are different enough to be classified in two different species. Additionally in BNYVV/BSBMV mixed infections, a competition was previous described in sugar beet, where BNYVV infection reduces BSBMV accumulation in both susceptible and resistant cultivars. Considering all this observations we hypothesized that BNYVV and BSBMV crossed study, exploiting their similarities and divergences, can improve investigation of molecular interactions between sugar beets and Benyviruses. The main achievement of our research is the production of a cDNA biologically active clones collection of BNYVV and BSBMV RNAs, from which synthetic copies of both Benyviruses can be transcribed. Moreover, through recombination experiments we demonstrated, for the first time, the BNYVV RNA 1 and 2 capability to trans-replicate and encapsidate BSBMV RNA 3 and 4, either the BSBMV RNA 1 and 2 capability to replicate BNYVV RNA2 in planta. We also demonstrated that BSBMV RNA3 support long-distance movement of BNYVV RNA 1 and 2 in B. macrocarpa and that 85 foreign sequence as p29HA, GFP and RFP, are successfully expressed, in C. quinoa, by BSBMV RNA3 based replicon (RepIII) also produced by our research. These results confirm the close correlation among the two viruses. Interestingly, the symptoms induced by BSBMV RNA-3 on C. quinoa leaves are more similar to necrotic local lesions caused by BNYVV RNA-5 p26 than to strongly chlorotic local lesions or yellow spot induced by BNYVV RNA- 3 encoded p25. As previous reported BSBMV p29 share 23% of amino acid sequence identity with BNYVV p25 but identity increase to 43% when compared with sequence of BNYVV RNA-5 p26. Based on our results the essential sequence (Core region) for the longdistance movement of BSBMV and BNYVV in B. macrocarpa, is not only carried by RNA3s species but other regions, perhaps located on the RNA 1 and 2, could play a fundamental role in this matter. Finally a chimeric RNA, composed by the 5’ region of RNA4 and 3’ region of RNA3 of BSBMV, has been produced after 21 serial mechanically inoculation of wild type BSBMV on C. quinoa plants. Chimera seems unable to express any protein, but it is replicated and transcript in planta. It could represent an important tool to study the interactions between Benyvirus and plant host. In conclusion different tools, comprising a method to study synthetic viruses under natural conditions of inoculum through P. Betae, have been produced and new knowledge are been acquired that will allow to perform future investigation of the molecular interactions between sugar beets and Benyviruses.

L'infiammazione intestinale nell'animale sperimentale come modello per lo sviluppo di nuovi farmaci: ruolo della via tachichininergica nelle malattie infiammatorie intestinali

Background: Several lines of evidence showed that inflammation is associated with changes in the expression of tachykinins both in human and animal models. Tachykinins, including substance P (SP), are small peptides expressed in the extrinsic primary afferent nerve fibres and enteric neurons of the gut: they exert their action through three distinct receptors, termed NK1, NK2 and NK3. SP modulates intestinal motility and enteric secretion, acting preferentially through the NK1 receptor. SP neural network and NK1 receptor expression are increased in patients with inflammatory bowel disease, and similar changes were observed in experimental models of inflammation. The 2,4 Dinitrobenzene Sulphonic Acid (DNBS) model of colitis is useful to study innate immunity, non-specific inflammation and wound healing; it has been suggested that the transmural inflammation seen in this model resembles that found in Crohn’s disease and can therefore be used to study what cells and mediators are involved in this type of inflammation. Aim: To test the possible protective effect of the NK1 receptor antagonist SSR140333 on: 1) acute model of intestinal inflammation; 2) reactivation of DNBS-induced colitis in rats. Methods: Acute colitis was induced in male SD rats by intrarectal administration of DNBS (15 mg/rat in 50% ethanol). Reactivation of colitis was induced by intrarectal injections of DNBS on day 28 (7.5 mg/rat in 35% ethanol). Animals were sacrificed on day 6 (acute colitis) and 29 (reactivation of colitis). SSR140333 (10 mg/kg) was administered orally starting from the day before the induction of colitis for 7 days (acute colitis) or seven days before the reactivation of colitis. Colonic damage was assessed by means of macroscopic and microscopic scores, myeloperoxidase activity (MPO) and TNF-α tissue levels. Enzyme immunoassay was used to measure colonic substance P levels. Statistical analysis was performed using analysis of variance (one-way or two-way, as appropriate) with the Bonferroni’s correction for multiple comparisons. Results: DNBS administration impaired body weight gain and markedly increased all inflammatory parameters (p<0.01). Treatment with SSR140333 10 mg/kg significantly counteracted the impairment in body weight gain, decreased macroscopic and histological scores and reduced colonic myeloperoxidase activity (p<0.01). Drug treatment counteracted TNF-α tissue levels and colonic SP concentrations (acute model). Similar results were obtained administering the NK1 receptor antagonist SSR140333 (3 and 10 mg/kg) for 5 days, starting the day after the induction of colitis. Intrarectal administration of DNBS four weeks after the first DNBS administration resulted in reactivation of colitis, with increases in macroscopic and histological damage scores and increase in MPO activity. Preventive treatment with SSR140333 10 mg/kg decreased macroscopic damage score, significantly reduced microscopic damage score but did not affect MPO activity. Conclusions: Treatment with SSR140333 significantly reduced intestinal damage in acute model of intestinal inflammation in rats. The NK1 receptor antagonist SSR140333 was also able to prevent relapse in experimental colitis. These results support the hypothesis of SP involvement in intestinal inflammation and indicate that NK receptor antagonists may have a therapeutic potential in inflammatory bowel disease.

Caratteristiche e vulnerabilità della rete di trasporto di Casalecchio di Reno nell'ambito dello sviluppo del Piano di Segnalamento per le emergenze previsto dal PGTU del Comune

Per migliorare la lettura del territorio, al fine di facilitare gli interventi di pronto intervento, in seguito a sopralluoghi sul territorio, sono stati eseguiti interventi di riqualificazione della segnaletica d'indicazione e di miglioramento dell'accessibilità in alcune zone di Casalecchio di Reno, in collaborazione con 118 e 115.