Epidemiological and functional characterization of Streptococcus pneumoniae pili

Streptococcus pneumoniae is an important life threatening human pathogen causing agent of invasive diseases such as otitis media, pneumonia, sepsis and meningitis, but is also a common inhabitant of the respiratory tract of children and healthy adults. Likewise most streptococci, S. pneumoniae decorates its surface with adhesive pili, composed of covalently linked subunits and involved in the attachment to epithelial cells and virulence. The pneumococcal pili are encoded by two genomic regions, pilus islet 1 (PI-1), and pilus islet-2 (PI-2), which are present in about 30% and 16% of the pneumococcal strains, respectively. PI-1 exists in three clonally related variants, whereas PI-2 is highly conserved. The presence of the islets does not correlate with the serotype of the strains, but with the genotype (as determined by Multi Locus Sequence Typing). The prevalence of PI-1 and PI-2 positive strains is similar in isolates from invasive disease and carriage. To better dissect a possible association between PIs presence and disease we evaluated the distribution of the two PIs in a panel of 113 acute otitis media (AOM) clinical isolates from Israel. PI-1 was present in 30.1% (N=34) of the isolates tested, and PI-2 in 7% (N=8). We found that 50% of the PI-1 positive isolates belonged to the international clones Spain9V-3 (ST156) and Taiwan19F-14 (ST236), and that PI-2 was not present in the absence of Pl-1. In conclusion, there was no correlation between PIs presence and AOM, and, in general, the observed differences in PIs prevalence are strictly dependent upon regional differences in the distribution of the clones. Finally, in the AOM collection the prevalence of PI-1 was higher among antibiotic resistant isolates, confirming previous indications obtained by the in silico analysis of the MLST database collection. Since the pilus-1 subunits were shown to confer protection in mouse models of infection both in active and passive immunization studies, and were regarded as potential candidates for a new generation of protein-based vaccines, the functional characterization was mainly focused on S. pneumoniae pilus -1 components. The pneumococcal pilus-1 is composed of three subunits, RrgA, RrgB and RrgC, each stabilized by intra-molecular isopeptide bonds and covalently polymerized by means of inter-molecular isopeptide bonds to form an extended fibre. The pilus shaft is a multimeric structure mainly composed by the RrgB backbone subunit. The minor ancillary proteins are located at the tip and at the base of the pilus, where they have been proposed to act as the major adhesin (RrgA) and as the pilus anchor (RrgC), respectively. RrgA is protective in in vivo mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

Influenza del sistema immunogenetico KIR nell'alloreattività Natural Killer nel trapianto di rene

Kidney transplantation is the best treatment option for the restoration of excretory and endocrine kidney function in patients with end-stage renal disease. The success of the transplant is linked to the genetic compatibility between donor and recipient, and upon progress in surgery and immunosuppressive therapy. Numerous studies have established the importance of innate immunity in transplantation tolerance, in particular natural killer (NK) cells represent a population of cells involved in defense against infectious agents and tumor cells. NK cells express on their surface the Killer-cell Immunoglobulin-like Receptors (KIR) which, by recognizing and binding to MHC class I antigens, prevent the killing of autologous cells. In solid organ transplantation context, and in particular the kidney, recent studies show some correlation between the incompatibility KIR / HLA and outcome of transplantation so as to represent an interesting perspective, especially as regards setting of immunosuppressive therapy. The purpose of this study was therefore to assess whether the incompatibility between recipient KIR receptors and HLA class I ligands of the donor could be a useful predictor in order to improve the survival of the transplanted kidney and also to select patients who might benefit of a reduced regimen. One hundred and thirteen renal transplant patients from 1999 to 2005 were enrolled. Genomic DNA was extracted for each of them and their donors and genotyping of HLA A, B, C and 14 KIR genes was carried out. Data analysis was conducted on two case-control studies: one aimed at assessing the outcome of acute rejection and the other to assess the long term transplant outcome. The results showed that two genes, KIR2DS1 and KIR3DS1, are associated with the development of acute rejection (p = 0.02 and p = 0.05, respectively). The presence of the KIR2DS3 gene is associated with a better performance of serum creatinine and glomerular filtration rate (MDRD) over time (4 and 5 years after transplantation, p <0.05), while in the presence of ligand, the serum creatinine and MDRD trend seems to get worse in the long term. The analysis performed on the population, according to whether there was deterioration of renal function or not in the long term, showed that the absence of the KIR2DL1 gene is strongly associated with an increase of 20% of the creatinine value at 5 years, with a relative risk to having a greater creatinine level than the median 5-year equal to 2.7 95% (95% CI: 1.7788 - 2.6631). Finally, the presence of a kidney resulting negative for HLA-A3 / A11, compared to a positive result, in patients with KIR3DL2, showed a relative risk of having a serum creatinine above the median at 5 years after transplantation of 0.6609 (95% CI: 0.4529 -0.9643), suggesting a protective effect given to the absence of this ligand.

La cultura architettonica del periodo normanno e l'influenza bizantina in Sicilia

The arguments of the thesis is the relationship between the Norman domination and the Greek-speaking people living in Sicily and Southern Italy. Particularly the ascendancy of the greek culture on the norman architecture and his role in the construction of the Norman Kingdom.

Analysis of the memory B cell response against glycoconjugate vaccines

The development of vaccines directed against polysaccharide capsules of S. pneumoniae, H. influenzae and N. meningitidis have been of great importance in preventing potentially fatal infections. Bacterial capsular polysaccharides are T-cell-independent antigens that induce specific antibody response characterized by IgM immunoglobulins, with a very low IgG class switched response and lack of capability of inducing a booster response. The inability of pure polysaccharides to induce sustained immune responses has required the development of vaccines containing polysaccharides conjugated to a carrier protein, with the aim to generate T cell help. It is clear that the immunogenicity of glycoconjugate vaccines can vary depending on different factors, e.g. chemical nature of the linked polysaccharide, carrier protein, age of the target population, adjuvant used. The present study analyzes the memory B cell (MBC) response to the polysaccharide and to the carrier protein following vaccination with a glycoconjugate vaccine for the prevention of Group B streptococcus (GBS) infection. Not much is known about the role of adjuvants in the development of immunological memory raised against GBS polysaccharides, as well as about the influence of having a pre-existing immunity against the carrier protein on the B cell response raised against the polysaccharide component of the vaccine. We demonstrate in the mouse model that adjuvants can increase the antibody and memory B cell response to the carrier protein and to the conjugated polysaccharide. We also demonstrate that a pre-existing immunity to the carrier protein favors the development of the antibody and memory B cell response to subsequent vaccinations with a glycoconjugate, even in absence of adjuvants. These data provide a useful insight for a better understanding of the mechanism of action of this class of vaccines and for designing the best vaccine that could result in a productive and long lasting memory response.

Funzioni della subunità θ e del dominio PHP della subunità α nel core catalitico della DNA polimerasi III di Escherichia coli

Il core catalitico della DNA polimerasi III, composto dalle tre subunità α, ε e θ, è il complesso minimo responsabile della replicazione del DNA cromosomiale in Escherichia coli. Nell'oloenzima, α ed ε possiedono rispettivamente un'attività 5'-3' polimerasica ed un'attività 3'-5' esonucleasica, mentre θ non ha funzioni enzimatiche. Il presente studio si è concentrato sulle regioni del core che interagiscono direttamente con ε, ovvero θ (interagente all'estremità N-terminale di ε) e il dominio PHP di α (interagente all'estremità C-terminale di ε), delle quali non è stato sinora identificato il ruolo. Al fine di assegnare loro una funzione sono state seguite tre linee di ricerca parallele. Innanzitutto il ruolo di θ è stato studiato utilizzando approcci ex-vivo ed in vivo. I risultati presentati in questo studio mostrano che θ incrementa significativamente la stabilità della subunità ε, intrinsecamente labile. Durante gli esperimenti condotti è stata anche identificata una nuova forma dimerica di ε. Per quanto la funzione del dimero non sia definita, si è dimostrato che esso è attivamente dissociato da θ, che potrebbe quindi fungere da suo regolatore. Inoltre, è stato ritrovato e caratterizzato il primo fenotipo di θ associato alla crescita. Per quanto concerne il dominio PHP, si è dimostrato che esso possiede un'attività pirofosfatasica utilizzando un nuovo saggio, progettato per seguire le cinetiche di reazione catalizzate da enzimi rilascianti fosfato o pirofosfato. L'idrolisi del pirofosfato catalizzata dal PHP è stata dimostrata in grado di sostenere l'attività polimerasica di α in vitro, il che suggerisce il suo possibile ruolo in vivo durante la replicazione del DNA. Infine, è stata messa a punto una nuova procedura per la coespressione e purificazione del complesso α-ε-θ

Analisi sperimentale dell'influenza dei parametri di processo sulla resistenza di contatto tra polimero e stampo durante la formatura

Con gli strumenti informatici disponibili oggi per le industrie, in particolar modo coi software CAE, le possibile simulare in maniera più che soddisfacente i fenomeni fisici presenti in natura. Anche il raffreddamento di un manufatto in polimero può venire simulato, a patto che si conoscano tutti i dati dei materiali e delle condizioni al contorno. Per quanto riguarda i dati dei materiali, i produttori di polimeri sono molto spesso in grado di fornirli, mentre le condizioni al contorno devono essere padroneggiate dal detentore della tecnologia. Nella pratica, tale conoscenza è al più incompleta, quindi si fanno ipotesi per colmare le lacune. Una tra le ipotesi più forti fatte è quella di una perfetta conduzione all'interfaccia tra due corpi. Questo è un vincolo troppo forte, se paragonato alla precisione di tutti gli altri dati necessari alla simulazione, e quindi si è deciso di eseguire una campagna sperimentale per stimare la resistenza al passaggio flusso termico all'interfaccia polimero-stampo ovvero determinare la conduttanza termica di contatto. L'attività svolta in questa tesi di dottorato ha come scopo quello di fornire un contributo significativo allo sviluppo e al miglioramento dell'efficienza termica degli stampi di formatura dei polimeri termoplastici con tecnologia a compressione.

Comportamento dinamico fuori del piano di pareti murarie: influenza della deformabilità degli impalcati

The aim of this study was to investigate the influence of the diaphragm flexibility on the behavior of out-of-plane walls in masonry buildings. Simplified models have been developed to perform kinematic and dynamic analyses in order to compare the response of walls with different restraint conditions. Kinematic non linear analyses of assemblages of rigid blocks have been performed to obtain the acceleration-displacement curves for walls with different restraint conditions at the top. A simplified 2DOF model has been developed to analyse the dynamic response of the wall with an elastic spring at the top, following the Housner rigid behaviour hypothesis. The dissipation of energy is concentrated at every impact at the base of the wall and is modelled through the introduction of the coefficient of restitution. The sets of equations of the possible configurations of the wall, depending on the different positions of the centre of rotation at the base and at the intermediate hinge have been obtained. An algorithm for the numerical integration of the sets of the equations of motion in the time domain has been developed. Dynamic analyses of a set of walls with Gaussian impulses and recorded accelerograms inputs have been performed in order to compare the response of the simply supported wall with the one of the wall with elastic spring at the top. The influence of diaphragm stiffness Kd has been investigated determining the variation of maximum displacement demand with the value of Kd. A more regular trend has been obtained for the Gaussian input than for the recorded accelerograms.

Development of new therapeutic approaches in the treatments of Inflammatory Bowel Diseases: functional food and nutraceuticals vs synthetic peptides based vaccines

Inflammatory Bowel Diseases (IBD) are intestinal chronic relapsing diseases which ethiopathogenesis remains uncertain. Several group have attempted to study the role of factors involved such as genetic susceptibility, environmental factors such as smoke, diet, sex, immunological factors as well as the microbioma. None of the treatments available satisfy several criteria at the same time such as safety, long-term remission, histopatological healing, and specificity. We used two different approaches for the development of new therapeutic treatment for Inflammatory Bowel Disease. The first is focused on the understanding of the potential role of functional food and nutraceuticals nutrients in the treatment of IBD. To do so, we investigated the role of Curcuma longa in the treatment of chemical induced colitis in mice model. Since Curcma Longa has been investigated for its antinflammatory role related to the TNFα pathway as well investigators have reported few cases of patients with ulcerative colites treated with this herbs, we harbored the hypothesis of a role of Curcuma Longa in the treatment f IBD as well as we decided to assess its role in intestinal motility. The second part is based on an immunological approach to develop new drugs to induce suppression in Crohn’s disease or to induce mucosa immunity such as in colonrectal tumor. The main idea behind this approach is that we could manipulate relevant cell-cell interactions using synthetic peptides. We demonstrated the role of the unique interaction between molecules expressed on intestinal epithelial cells such as CD1d and CEACAM5 and on CD8+ T cells. In normal condition this interaction has a role for the expansion of the suppressor CD8+ T cells. Here, we characterized this interaction, we defined which are the epitope involved in the binding and we attempted to develop synthetic peptides from the N domain of CEACAM5 in order to manipulate it.

Influenza della conservazione delle olive sulla qualita dell'olio vergine prodotto

Il primo scopo di questo progetto sperimentale è stato quello di valutare la qualità chimica e sensoriale di oli vergini d’oliva ottenuti da olive conservate per tempi diversi prima della loro lavorazione. Spesso, infatti, quando le olive arrivano in frantoio, la loro trasformazione può essere ritardata a causa della ridotta capacità di lavorazione degli impianti rispetto alla quantità di prodotto da lavorare. Il tempo e le modalità di conservazione delle drupe risultano fattori molto importanti ai fini della qualità organolettica dell’olio prodotto. Per questa finalità sono state acquistate olive di due diverse varietà (Correggiolo e Canino) e divise in due aliquote. La prima aliquota di ogni partita di olive è stata franta al momento della consegna (tempo 0), la seconda invece è stata stoccata in condizioni non ideali di conservazione ed umidità per 7 giorni (varietà Correggiolo) e 15 giorni (varietà Canino), quindi franta. Per tutti i campioni sono stati valutati i parametri di qualità stabiliti dal Reg. CEE n. 2568/91 e successive modifiche: acidità libera e numero di perossidi con valutazioni titrimetriche, estinzioni specifiche nell'ultravioletto mediante spettrofotometria, determinazione gascromatografica degli alchil esteri degli acidi grassi ed analisi sensoriale secondo “Panel test”. Sono stati inoltre valutati parametri compositivi che attualmente non sono contemplati nei regolamenti ufficiali: il profilo qualitativo e quantitativo gascromatografico in digliceridi e quello in componenti volatili (SPME). I risultati ottenuti per i campioni ottenuti da olive “fresche” e da olive conservate per tempi diversi sono stati comparati con i limiti di legge che definiscono l'appartenenza alle categorie merceologiche degli oli di oliva vergini (extravergine, vergine o lampante) e, per i parametri non normati, sono state valutate le principali differenze in relazione alla qualità del prodotto. Il secondo scopo del progetto è stato quello di valutare la possibilità di una rapida discriminazione di oli d’oliva caratterizzati da differenti concentrazioni di alchil esteri degli acidi grassi mediante Riflettometria nel Dominio del Tempo (TDR, Time Domain Reflectometry). Il metodo chimico proposto dal Reg. UE n. 61/2011 risulta, infatti, lungo e dispendioso prevedendo una separazione preparativa seguita da analisi gascromatografica. La TDR si presenta, invece, come un metodo alternativo rapido, economico e non distruttivo per la valutazione e discriminazione degli oli d’oliva sulla base di questo parametro qualitativo.

L'impatto della geometria urbana per applicazioni del telerilevamento in ambito energetico: l'influenza dello sky view factor.

Comprensione delle formulazioni analitiche e dei metodi tradizionali per il calcolo puntuale dello Sky View Factor (SVF), un parametro adimensionale in grado di esprimere con un singolo valore l’influenza dei fattori morfologici sulla porzione di volta celeste visibile da un punto. Valutazione delle performance dei software di mappatura dello SVF in area urbana al variare di tipologia e qualità del dato di input e dei parametri utilizzati. Al fine di studiare degli aspetti algoritmici dei modelli esistenti, è stato sviluppato uno script in linguaggio MATLAB per apportare miglioramenti alle tecniche esaminate.