Advanced glycated albumin impairs HDL anti-inflammatory activity and primes macrophages for inflammatory response that reduces reverse cholesterol transport

Objective: We investigated the effect of advanced glycated albumin (AGE-albumin) on macrophage sensitivity to inflammation elicited by S100B calgranulin and lipopolysaccharide (LPS) and the mechanism by which HDL modulates this response. We also measured the influence of the culture medium, isolated from macrophages treated with AGE-albumin, on reverse cholesterol transport (RCT). Methods and results: Macrophages were incubated with control (C) or AGE-albumin in the presence or absence of HDL, followed by incubations with S100B or LPS. Also, culture medium obtained from cells treated with C- or AGE-albumin, following S100B or LPS stimulation was utilized to treat naive macrophages in order to evaluate cholesterol efflux and the expression of HDL receptors. In comparison with C-albumin, AGE-albumin, promoted a greater secretion of cytokines after stimulation with S100B or LPS. A greater amount of cytokines was also produced by macrophages treated with AGE-albumin even in the presence of HDL Cytokine-enriched medium, drawn from incubations with AGE-albumin and S100B or LPS impaired the cholesterol efflux mediated by apoA-I (23% and 37%, respectively), HDL2 (43% and 47%, respectively) and HDL3 (20% and 8.5%, respectively) and reduced ABCA-1 protein level (16% and 26%, respectively). Conclusions: AGE-albumin primes macrophages for an inflammatory response impairing the RCT. Moreover, AGE-albumin abrogates the anti-inflammatory role of HDL, which may aggravate the development of atherosclerosis in DM. (C) 2012 Elsevier BM. All rights reserved.

Longitudinal bar buckling behavior

Reinforced concrete columns might fail because of buckling of the longitudinal reinforcing bar when exposed to earthquake motions. Depending on the hoop stiffness and the length-over-diameter ratio, the instability can be local (in between two subsequent hoops) or global (the buckling length comprises several hoop spacings). To get insight into the topic, an extensive literary research of 19 existing models has been carried out including different approaches and assumptions which yield different results. Finite element fiberanalysis was carried out to study the local buckling behavior with varying length-over-diameter and initial imperfection-over-diameter ratios. The comparison of the analytical results with some experimental results shows good agreement before the post buckling behavior undergoes large deformation. Furthermore, different global buckling analysis cases were run considering the influence of different parameters; for certain hoop stiffnesses and length-over-diameter ratios local buckling was encountered. A parametric study yields an adimensional critical stress in function of a stiffness ratio characterized by the reinforcement configuration. Colonne in cemento armato possono collassare per via dell’instabilità dell’armatura longitudinale se sottoposte all’azione di un sisma. In funzione della rigidezza dei ferri trasversali e del rapporto lunghezza d’inflessione-diametro, l’instabilità può essere locale (fra due staffe adiacenti) o globale (la lunghezza d’instabilità comprende alcune staffe). Per introdurre alla materia, è proposta un’esauriente ricerca bibliografica di 19 modelli esistenti che include approcci e ipotesi differenti che portano a risultati distinti. Tramite un’analisi a fibre e elementi finiti si è studiata l’instabilità locale con vari rapporti lunghezza d’inflessione-diametro e imperfezione iniziale-diametro. Il confronto dei risultati analitici con quelli sperimentali mostra una buona coincidenza fino al raggiungimento di grandi spostamenti. Inoltre, il caso d’instabilità globale è stato simulato valutando l’influenza di vari parametri; per certe configurazioni di rigidezza delle staffe e lunghezza d’inflessione-diametro si hanno ottenuto casi di instabilità locale. Uno studio parametrico ha permesso di ottenere un carico critico adimensionale in funzione del rapporto di rigidezza dato dalle caratteristiche dell’armatura.

Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

Reverse Charge nell'Iva comunitaria

Oggetto della tesi è il reverse charge, ovvero inversione contabile, che rappresenta un particolare metodo impositivo dell’Iva, derogatorio rispetto a quello tipico ed ordinariamente applicabile. L’imposta sul valore aggiunto è un’imposta plurifase, non cumulativa, basata sulle figure della rivalsa e della detrazione, attraverso la cui combinazione viene garantita la neutralità tipica di tale tributo, affinché l’onere conclusivamente sopportato dal consumatore finale non venga condizionato dal numero degli stadi di lavorazione di cui consta un eventuale ciclo produttivo. Il reverse charge deroga al citato meccanismo impositivo in quanto, nelle operazioni in cui tale sistema trova applicazione, il fornitore o il prestatore non addebitano alcuna imposta in via di rivalsa al cessionario o al committente, ed il debito Iva generato dall’operazione da loro posta in essere viene assolto direttamente, e contrariamente a quanto dovrebbe accadere, dal cessionario o dal committente. La tesi offre sostanzialmente una riflessione ampia e generalizzata di tale particolare metodo impositivo, al fine di individuarne i tratti caratteristici, e verificare come detto metodo impositivo, comunque derogatorio, si ponga nell’ambito del sistema ordinario dell’Iva, e quale incidenza abbia rispetto alla finalità ultima e peculiare del tributo, ossia la tassazione del consumo finale, mediante un sistema impositivo che risulti neutrale per gli operatori commerciali, e quindi per i soggetti passivi d’imposta.

Reverse Engineering tools: development and experimentation of innovative methods for physical and geometrical data integration and post-processing

In recent years, the use of Reverse Engineering systems has got a considerable interest for a wide number of applications. Therefore, many research activities are focused on accuracy and precision of the acquired data and post processing phase improvements. In this context, this PhD Thesis deals with the definition of two novel methods for data post processing and data fusion between physical and geometrical information. In particular a technique has been defined for error definition in 3D points’ coordinates acquired by an optical triangulation laser scanner, with the aim to identify adequate correction arrays to apply under different acquisition parameters and operative conditions. Systematic error in data acquired is thus compensated, in order to increase accuracy value. Moreover, the definition of a 3D thermogram is examined. Object geometrical information and its thermal properties, coming from a thermographic inspection, are combined in order to have a temperature value for each recognizable point. Data acquired by an optical triangulation laser scanner are also used to normalize temperature values and make thermal data independent from thermal-camera point of view.

Reverse engineering di un'applicazione ad agenti nel contesto Smart Home

Relazione completa delle scelte progettuali e implementative di un applicativo ad agenti sviluppato nel contesto Smart Home. Essa contiene un veloce riassunto dello scenario e dello stato attuale dell'applicazione, unitamente a un'introduzione sul middeware su cui si appoggia l'applicativo (TuCSoN). Segue quindi un'analisi delle scelte di modeling delle entita da gestire, le metodologie di supporto alla persistenza e un'ampia descrizione su come gli agenti comunichino tra loro e attraverso quali mezzi (centri di tuple). Quindi viene analizzata l'implementazione partendo dalle scelte implementative sino ad esaminare cosa avviene nel programma a seguito dell'interazione con l'utente. Infine le conclusioni a cui si e giunti e due appendici sulla terminologia e le classi presenti nel prototipo attuale.

Viral suppression rates in salvage treatment with raltegravir improved with the administration of genotypic partially active or inactive nucleoside/tide reverse transcriptase inhibitors

Nucleoside reverse transcriptase inhibitors (NRTIs) are often administered in salvage therapy even if genotypic resistance tests (GRTs) indicate high-level resistance, but little is known about the benefit of these additional NRTIs.

Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine

The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for Tm and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct Tm's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher Tm closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function.