Optical absorption and nonradiative decay mechanism of E′ center in silica

We report ab initio configuration interaction calculations on the optical transitions of the E′ center, a hole trapped at an oxygen vacancy, ( - O)3Si• +(O - )3, in silica. We found two competing excitation mechanisms: (1) promotion of one electron from an O(2p) valence band orbital to the singly occupied Si dangling bond; (2) charge transfer (CT) transition from ( - O)3Si• to +Si(O - )3. The two excitations occur at similar energies, ≈5.8-6 eV (5.85 eV in the experiment), but only the CT has a strong intensity. The excitation is followed by a complex nonradiative decay process which may explain the absence of luminescence for this cente

Diabetes Mellitus and Cardiovascular Prevention: The Role and the Limitations of Currently Available Antiplatelet Drugs

Diabetes mellitus (DM) is associated with macrovascular and microvascular complications. Platelets have a “key role” in atherogenesis and its thrombotic complications in subjects with DM. Moreover, the concomitant presence of multiple “classical” cardiovascular risk factors in diabetic subjects contributes to enhanced atherothrombotic risk. Antiplatelet agents are effective in primary and secondary prevention of arterial thrombosis (cardiovascular events, ischaemic stroke, and peripheral arterial occlusive disease). The role of chronic administration of antiplatelet drugs in primary prevention of arterial vascular events is known to be less clear than in secondary prevention, and, also in diabetic patients, the decision to give primary prophylaxis should be taken on an individual-patient basis, after a careful evaluation of the balance between the expected benefits and the risk of major bleedings. Although, currently, treatment has proven useful in reducing vascular events, diabetic patients continue to have a higher risk of adverse cardiovascular events compared with those in nondiabetic patients. This paper reviews the role of currently available antiplatelet drugs in primary and secondary prevention of vascular events in diabetic patients and the limitations of these drugs, and it discusses the role of novel and more potent antiplatelets and of new agents currently under clinical development

Treatment of hemophilia: a review of current advances and ongoing issues

Replacement of the congenitally deficient factor VIII or IX through plasma-derived or recombinant concentrates is the mainstay of treatment for hemophilia. Concentrate infusions when hemorrhages occur typically in joint and muscles (on-demand treatment) is able to resolve bleeding, but does not prevent the progressive joint deterioration leading to crippling hemophilic arthropathy. Therefore, primary prophylaxis, ie, regular infusion of concentrates started after the first joint bleed and/or before the age of two years, is now recognized as first-line treatment in children with severe hemophilia. Secondary prophylaxis, whenever started, aims to avoid (or delay) the progression of arthropathy and improve patient quality of life. Interestingly, recent data suggest a role for early prophylaxis also in preventing development of inhibitors, the most serious complication of treatment in hemophilia, in which multiple genetic and environmental factors may be involved. Treatment of bleeds in patients with inhibitors requires bypassing agents (activated prothrombin complex concentrates, recombinant factor VIIa). However, eradication of inhibitors by induction of immune tolerance should be the first choice for patients with recent onset inhibitors. The wide availability of safe factor concentrates and programs for comprehensive care has now resulted in highly satisfactory treatment of hemophilia patients in developed countries. Unfortunately, this is not true for more than two-thirds of persons with hemophilia, who live in developing countries

Pseudomonas aeruginosa Exploits Lipid A and Muropeptides Modification as a Strategy to Lower Innate Immunity during Cystic Fibrosis Lung Infection

Pseudomonas aeruginosa can establish life-long airways chronic infection in patients with cystic fibrosis (CF) with pathogenic variants distinguished from initially acquired strain. Here, we analysed chemical and biological activity of P. aeruginosa Pathogen-Associated Molecular Patterns (PAMPs) in clonal strains, including mucoid and non-mucoid phenotypes, isolated during a period of up to 7.5 years from a CF patient. Chemical structure by MS spectrometry defined lipopolysaccharide (LPS) lipid A and peptidoglycan (PGN) muropeptides with specific structural modifications temporally associated with CF lung infection. Gene sequence analysis revealed novel mutation in pagL, which supported lipid A changes. Both LPS and PGN had different potencies when activating host innate immunity via binding TLR4 and Nod1. Significantly higher NF-kB activation, IL-8 expression and production were detected in HEK293hTLR4/MD2-CD14 and HEK293hNod1 after stimulation with LPS and PGN respectively, purified from early P. aeruginosa strain as compared to late strains. Similar results were obtained in macrophages-like cells THP-1, epithelial cells of CF origin IB3-1 and their isogenic cells C38, corrected by insertion of cystic fibrosis transmembrane conductance regulator (CFTR). In murine model, altered LPS structure of P. aeruginosa late strains induces lower leukocyte recruitment in bronchoalveolar lavage and MIP-2, KC and IL-1β cytokine levels in lung homogenates when compared with early strain. Histopathological analysis of lung tissue sections confirmed differences between LPS from early and late P. aeruginosa. Finally, in this study for the first time we unveil how P. aeruginosa has evolved the capacity to evade immune system detection, thus promoting survival and establishing favourable conditions for chronic persistence. Our findings provide relevant information with respect to chronic infections in CF

Tailoring the Structure of Cell Penetrating DNA and RNA Binding Nucleopeptides

Synthetic nucleic acid interactors represent an exciting research field due to their biotechnological and potential therapeutic applications. The translation of these molecules into drugs is a long and difficult process that justifies the continuous research of new chemotypes endowed with favorable binding, pharmacokinetic and pharmacodynamic properties. In this scenario, we describe the synthesis of two sets of homo-thymine nucleopeptides, in which nucleobases are inserted in a peptide structure, to investigate the role of the underivatized amino acid residue and the distance of the nucleobase from the peptide backbone on the nucleic acid recognition process. It is worth noting that the CD spectroscopy investigation showed that two of the reported nucleopeptides, consisting of alternation of thymine functionalized L-Orn and L-Dab and L-Arg as underivatized amino acids, were able to efficiently bind DNA and RNA targets and cross both cell and nuclear membranes

"Structure and bioactivity of bacterial glycolipids as targets for biomedical applications" - "Struttura e attività di glicoconiugati di origine batterica quali principi attivi per applicazioni in campo biomedico"

This thesis is focused on the characterization of Gram negative endotoxin as they play a key role in bacterial pathogenesis. Gram negative endotoxins or lipopolysaccharides (LPS) are glycoconjugates molecules exposed toward the external environment as they are the major components of Gram negative external leaflet. They have a structural role since they contribute to the cellular rigidity increasing the strength of cell wall and mediate the contacts with the external environment. Moreover, LPS can modulate the host immune response as they are recognised by immune mechanisms of defence. Structural elucidation of these molecules is an essential pre requisite in the comprehension of chemical structures that are responsible for bacterial pathogenesis. Lipopolysaccharides are build up according to a common structural architecture. They are composed of a hydrophilic hetero-polysaccharide (formed by core oligosaccharide and O-specific polysaccharide or O-chain) covalently linked to a lipophilic domain termed lipid A, which is embedded in the outer leaflet and anchors these macromolecules to the membrane through electrostatic and hydrophobic interactions. These three major domains are genetically, biologically and chemically distinct. The endotoxins analysed in this work are extracted from opportunistic Cystic Fibrosis pathogens. These bacteria are responsible for feared infections that reduce life expectancy in CF patients. The first part of the project was centred on the study of LPS involvement in bacterial adaptation after lung transplantation to a healthy not CF tissue. LPS were extracted from the most representative and problematic group of CF pathogens, the Burkholderia cepacia complex (Bcc). Burkholderia multivorans (genomovar II) and Burkholderia vietnamiensis (genomovar V) were respectively isolated from CF patients that underwent lung transplantation. For each clinical isolated, two paired clonal strains were recovered: one pre and the other post transplantation. Thus, in order to investigate the possible structural changes that possible occurred during the adaptation, for the first time LPSs from each strain, isolated in two different moments of the infection, were extracted and characterized. B. multivorans LPS isolated pre and post transplantation lacking the polysaccharidic moiety of the O-chain, revealed to be a lipooligosaccharide (LOS). The differences between the LOSs isolated from the two clonal strains were mainly found in the lipid A moiety, even though in the post transplantation strain there was an additional residue of Ara4N on the core portion. These chemical differences were responsible for the different biological activities found for LOSs. Generally, it was found a loss of inflammatory activity after lung transplantation when the two purified LOSs were tested as elicitors of TNF-alpha induction in human myelomonocytic U937 cells and of NF-KB induction in transfected TLR 4/MD2/CD 14 HEK cell lines. Even B. vietnamiensis, producing in both pre- and post transplantation strains LOSs, showed substantial differences in the lipid A moieties that justified an increased inflammatory activity for post transplantation strain when it was tested for TNF-alpha and NF-KB inductions. The features found in both studies revealed that lipid A moiety changes during bacterial adaptation. These data are not surprising since this LPS portion accounts for endotoxin virulence as it is recognized by the innate immune system of the host. Further, from a post transplanted allograft, B. cenocepacia (genomovar III) the most virulent of Bcc members, was also collected, and its endotoxin characterized. This structural investigation constituted a complementary work to the previous study on B. cenocepacia ET-12 endotoxin already performed in this laboratory. Thus, it gives a clear aspect of the endotoxin phenotype produced by the most pathogenic strain of Bcc, when it colonized a survived CF patient. An additional structural investigation was also performed on the second most pathogenic Bcc member, namely B. multivorans. The endotoxin was recovered from the most virulent strain of genomovar II, the C1576, that was responsible of a fatal outbreak in Glasgow, when only few children survived to adulthood. This strain produces a smooth type LPS, and its lipid A and O-chain moieties were characterized. In this case, differently from the other Burkholderia lipid A analyzed, bacteria produced a less phosphorylated blend of lipid A species. Even though lipid A is the highest conserved portion of LPS, little changes, as the presence or absence of charged residues or the number and the distribution of fatty acids, may occur in response to bacterial exigencies of adaptation. The O-chain portion was instead constituted by two polysaccharides characterized by two different repeating units. Moreover, a conformational study was executed on these polysaccharides in order to investigate about their supra molecular arrangements. A conformational study and a MD simulation was also performed on the oligosaccharidic region of the LOS molecule extracted from the clinical isolated strain of B. cenocepacia ET-12, previously characterized. This analysis was performed with AMBER package in a solvent explicit model, thus mimicking real conditions. The investigation on three dimensional structure adopted by the most exposed moiety of B. cenocepacia endotoxin better clarified its spatial orientation. Moreover, data obtained give a real idea of the residues that are exposed toward the external environment, thus playing a key role in molecular recognition. The results carried out also gave information about the relative orientation of sugar rings and the conformational spaces energetically permitted for glycosidic bond rotations. These data can also contribute to justify and explain LPS role in increasing membrane rigidity and cell-wall strength. The last part of this project was focused on the investigation of whether and, possibly, how P. aeruginosa genetic adaptation resulted in a bacterial strategy to evade the host immune sensing. With this aim, the impact of lipopolysaccharide, purified by P. aeruginosa strains isolated from acute and chronic infection of a CF patient, was analysed. Typically, P. aeruginosa infections are followed by a chronic persistence of the disease that cause permanent respiratory symptoms and decline in lung functions. The long term colonization of CF airways selects pathoadaptive variants with several features which differentiate late P. aeruginosa isolates from the initially acquired strain. Even though P. aeruginosa strains that initiates infections are characterized by a large arsenal of virulence factors, CF chronic infection are characterized by less virulent but more persistent phenotypes. LPS modification appears to be one of the main factors in the adaptation of this pathogen during chronic infections but no conclusive information were present so far on the putative lipid A changes in the acute to chronic evolution of the infection. The results obtained emphasize the reduced immunopotential of LPS extracted from late colonizer P. aeruginosa strains, demonstrating the lost of large arsenal of virulence factors during chronic infection

A proposito del Quijote: un excursus dalla novela de caballería al romanzo moderno

Questo elaborato affronta la questione, condivisa dalla critica, della modernità del Quijote di Miguel de Cervantes Saavedra. Per fare ciò è necessario, per prima cosa, risalire alla prosa narrativa in voga in Spagna tra Quattrocento e Cinquecento. È dai generi letterari di questo periodo che Cervantes attinge per creare il Quijote: il primo romanzo che riunisce l’idealismo e il realismo. È possibile vedere come Cervantes ha ripreso i cliché della narrativa idealista attraverso le novelle intercalate – variazioni delle novelas. Dopodiché il mio lavoro introduce la censura umoristica del genere cavalleresco analizzando il famoso episodio del rogo dei libri contenuti nella biblioteca di Don Quijote. Dopo questo processo inquisitoriale, l’invettiva del genere cavalleresco prosegue attraverso l’uso dell’espediente del manoscritto ritrovato e di un complesso sistema di narratori e autori. Si passa poi alla grande confusione tra realtà e finzione che avviene nella testa di Don Quijote. Vedremo che la sua pazzia in realtà deriva da un errore di interpretazione dei testi letterari che assume come storici. Ad aprire l’ultimo capitolo è invece la trattatistica da cui Cervantes attinse: la dottrina aristotelica, divulgata in Spagna da Alonso López detto “El Pinciano”. Ne seguirà una spiegazione del termine novela e del concetto di verosimiglianza, entrambi fondamentali per la nascita del romanzo moderno. Successivamente mi concentro sulla teoria del romanzo all’interno del Quijote per poter delineare il pensiero di Cervantes. L’ultima parte di questo lavoro si concentra sul metaromanzo. La Prima Parte del 1605 diventa, quindi, la base di riferimento per la Seconda Parte. Non manca l’analisi della risposta di Cervantes al Quijote Avellaneda del 1614. L’astuzia di Cervantes consiste nel mostrarci che la versione apocrifa non è degna della nostra attenzione e lo fa soprattutto attraverso il personaggio di Don Álvaro Tarfe. Questi esce dal Quijote apocrifo per entrare in quello di Cervantes incontrandosi, così, con il vero hidalgo