Molecular variability of Meningococcal antigens in carriage and disease strains and in other Neisseria species

This PhD thesis is focused on the study of the molecular variability of some specific proteins, part of the outer membrane of the pathogen Neisseria meningitidis, and described as protective antigens and important virulence factors. These antigens have been employed as components of the vaccine developed by Novartis Vaccines against N. meningitidis of serogroup B, and their variability in the meningococcal population is a key aspect when the effect of the vaccine is evaluated. The PhD project has led to complete three major studies described in three different manuscritps, of which two have been published and the third is in preparation. The thesis is structured in three main chapters, each of them dedicated to the three studies. The first, described in Chapter 1, is specifically dedicated to the analysis of the molecular conservation of meningococcal antigens in the genomes of all species classified in the genus Neisseria (Conservation of Meningococcal Antigens in the Genus Neisseria. A. Muzzi et al.. 2013. mBio 4 (3)). The second study, described in Chapter 2, focuses on the analysis of the presence and conservation of the antigens in a panel of bacterial isolates obtained from cases of the disease and from healthy individuals, and collected in the same year and in the same geographical area (Conservation of fHbp, NadA, and NHBA in carrier and pathogenic isolates of Neisseria meningitidis collected in the Czech Republic in 1993. A. Muzzi et al.. Manuscript in preparation). Finally, Chapter 3 describes the molecular features of the antigens in a panel of bacterial isolates collected over a period of 50 years, and representatives of the epidemiological history of meningococcal disease in the Netherlands (An Analysis of the Sequence Variability of Meningococcal fHbp, NadA and NHBA over a 50-Year Period in the Netherlands. S. Bambini et al.. 2013. PloS one e65043).

Therapeutic microbiology: characterization of Bifidobacterium strains for the treatment of enteric disorders in newborns.

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastro-intestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to non tumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify 3 B. breve strains and a B. longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. The formulation of a synbiotic product with an appropriate prebiotic fiber capable of supporting the growth of the selected Bifidobacterium strains was also considered in this study. In this respect the ability of the 4 selected Bifidobacterium strains to use as the sole carbon source and energy source different polisaccharide fibers was investigated The last phase of the work has been dedicated to the evaluation of the gut microbial diversity in newborns whose mothers has been subjected to antibiotic therapy a few hours before the delivery because of a Streptococcus type B infection. These newborns can represent a possible target for the probiotic strains selected in this work.

Phenotype and genotype characterization of Monilinia spp. isolates and preformed antifungal compounds in peach peel fruit at different developmental stages

The brown rot fungi belong to a group of fungal pathogens that causes considerable damage to cultivated fruits trees, particularly stone fruits and apples in the temperate regions of the World and during the postharvest with an important economic impact. In particular in Italy, it is important to monitor the Monilinia population to control economic losses associated to the peach and nectarine market. This motivates the research steps presented in this dissertation on Monilinia Italian isolates. The Monilinia species collected from stone fruits have been identified using molecular analysis based on specific primers. The relevant role of M. fructicola was confirmed and, for the first time, it was found also on apple fruits. To avoid the development of resistant strains and implement valid treatment strategies, the understanding of the fruit natural resistance during different developmental stages and the assessment of the Monilinia sensitivity/resistance to fungicides are required. The relationship between the inhibition spots and the phenolic compounds in peach fruit peel was highlighted in this research. Three methods were used to assess isolate resistance/sensitivity, the amended medium, the Spiral Gradient Endpoint Method (SGD) and the Alamar Blue method. The PCR was used to find possible mutation points in the b-tubulin gene that is responsible for fungicide resistance. Interestingly, no mutation points were observed in resistant M. laxa isolates, suggesting that the resistance could be stimulated by environmental factors. This lead to the study of the effect of the temperature on the resistance and the preliminary results of in vitro tests showed that maximum inhibition was observed at 30°C.

Analysis of Two Component Systems in Group B Streptococcus Shows that RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness

Group B Streptococcus (GBS), in its transition from commensal to pathogen, will encounter diverse host environments and thus require coordinately controlling its transcriptional responses to these changes. This work was aimed at better understanding the role of two component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knock-out strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1-3% of the genome. Interestingly, two sugar phosphotransferase systems appeared differently regulated in the knock-out mutant of TCS-16, suggesting an involvement in monitoring carbon source availability. High throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16 with concomitant dramatic up-regulation of the adjacent operon encoding a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization and impaired growth/survival in the presence of vaginal mucoid components. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and also provide experimental evidence for TCS-17/RgfAC involvement in virulence.

Analysis of the immunological and functional features of the Neisserial Heparin Binding Antigen (NHBA)

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by genetically diverse Neisseria meningitidis strains and is an antigen of the multicomponent protein-based 4CMenB vaccine, able to induce bactericidal antibodies in humans and to bind heparin-like molecules. The aim of this study is to characterize the immunological and functional properties of NHBA. To evaluate immunogenicity and the contribution of aminoacid sequence variability to vaccine coverage, we constructed recombinant isogenic strains that are susceptible to bactericidal killing only by anti-NHBA antibodies and engineered them to express equal levels of selected NHBA peptides. In these recombinant strains, we observed different titres associated with the different peptide variants. These recombinant strains were then further engineered to express NHBA chimeric proteins to investigate the regions important for immunogenicity. In natural strains, anti-NHBA antibodies were found to be cross-protective against strains expressing different peptides. To investigate the functional properties of this antigen, the recombinant purified NHBA protein was tested in in vitro binding studies and was found to be able to bind epithelial cells. The binding was abolished when cells were treated specifically with heparinase III, suggesting that the interaction with the cells is mediated by heparan sulfate proteoglycans (HSPG). Mutation of the Arg-rich tract of NHBA abrogated the binding, confirming the importance of this region in mediating the binding to heparin-like molecules. In a panel of N. meningitidis strains, the deletion of nhba resulted in a reduction of adhesion with respect to each isogenic wild type strain. Furthermore, the adhesion of the wild-type strain was prevented by using anti-NHBA polyclonal sera, demonstrating the specificity of the interaction. These results suggest that NHBA could be a novel meningococcal adhesin contributing to host-cell interaction. Moreover, we analysed NHBA NalP-mediated cleavage in different NHBA peptides and showed that not all NHBA peptides are cleaved.

Identification and genetic diversity in phytoplasmas associated with diseases of cassava and other agronomic relevant crops in south-east Asia and Latin America

Identification and genetic diversity of phytoplasmas infecting tropical plant species, selected among those most agronomically relevant in South-east Asia and Latin America were studied. Correlation between evolutionary divergence of relevant phytoplasma strains and their geographic distribution by comparison on homologous genes of phytoplasma strains detected in the same or related plant species in other geographical areas worldwide was achieved. Molecular diversity was studied on genes coding ribosomal proteins, groEL, tuf and amp besides phytoplasma 16S rRNA. Selected samples infected by phytoplasmas belonging to diverse ribosomal groups were also studied by in silico RFLP followed by phylogenetic analyses. Moreover a partial genome annotation of a ‘Ca. P. brasiliense’ strain was done towards future application for epidemiological studies. Phytoplasma presence in cassava showing frog skin (CFSD) and witches’ broom (CWB) diseases in Costa Rica - Paraguay and in Vietnam – Thailand, respectively, was evaluated. In both cases, the diseases were associated with phytoplasmas related to aster yellows, apple proliferation and “stolbur” groups, while only phytoplasma related to X-disease group in CFSD, and to hibiscus witches’ broom, elm yellows and clover proliferation groups in CWB. Variability was found among strains belonging to the same ribosomal group but having different geographic origin and associated with different disease. Additionally, a dodder transmission assay to elucidate the role of phytoplasmas in CWB disease was carried out, and resulted in typical phytoplasma symptoms in periwinkle plants associated with the presence of aster yellows-related strains. Lethal wilt disease, a severe disease of oil palm in Colombia that is spreading throughout South America was also studied. Phytoplasmas were detected in symptomatic oil palm and identified as ‘Ca. P. asteris’, ribosomal subgroup 16SrI-B, and were distinguished from other aster yellows phytoplasmas used as reference strains; in particular, from an aster yellows strain infecting corn in the same country.

Characterization of West Nile virus strains isolated in Italy

West Nile virus (WNV) is a neurotropic flavivirus that is maintained in an enzootic cycle between mosquitoes and birds, but can also infect and cause disease in humans and other vertebrate species. Most of WNV infections in humans are asymptomatic, but approximately 20% of infected people develop clinical symptoms, although severe neurological diseases are observed in less than 1% of them. WNV is the most widely distributed arbovirus in the world and has been recently associated with outbreaks of meningo-encephalitis in Europe, including Italy, caused by different viral strains belonging to distinct lineages 1 and 2. The hypothesis is that genetic divergence among viral strains currently circulating in Italy might reflect on their pathogenic potential and that the rapid spread of WNV with increased pathogenicity within naïve population suggest that epidemic forms of the virus may encode mechanisms to evade host immunity. Infection with WNV triggers a delayed host response that includes a delay in the production of interferon-α (IFN-α). IFNs are a family of immuno-modulatory cytokines that are produced in response to virus infection and serve as integral signal initiators of host intracellular defenses. The increased number of human cases and the lack of data about virulence of European WNV isolates highlight the importance to achieve a better knowledge on this emerging viral infection. In the present study, we investigate the phenotypic and IFN-α-regulatory properties of different WNV lineage 1 and 2 strains that are circulating in Europe/Italy in two cell lines: Vero and 1321N1. We demonstrate that: Vero and 1321N1 cells are capable of supporting WNV replication where different WNV strains show similar growth kinetics; WNV lineage 2 strain replicated in Vero and 1321N1 cells as efficiently as WNV lineage 1 strains; and both lineages 1 and 2 were highly susceptible to the antiviral actions of IFN-α.

Development of new molecular methods for the diagnosis and the study of viral diseases of fish

The increase in aquaculture operations worldwide has provided new opportunities for the transmission of aquatic viruses. The occurrence of viral diseases remains a significant limiting factor in aquaculture production and for the sustainability. The ability to identify quickly the presence/absence of a pathogenic organism in fish would have significant advantages for the aquaculture systems. Several molecular methods have found successful application in fish pathology both for confirmatory diagnosis of overt diseases and for detection of asymptomatic infections. However, a lot of different variants occur among fish host species and virus strains and consequently specific methods need to be developed and optimized for each pathogen and often also for each host species. The first chapter of this PhD thesis presents a complete description of the major viruses that infect fish and provides a relevant information regarding the most common methods and emerging technologies for the molecular diagnosis of viral diseases of fish. The development and application of a real time PCR assay for the detection and quantification of lymphocystivirus was described in the second chapter. It showed to be highly sensitive, specific, reproducible and versatile for the detection and quantitation of lymphocystivirus. The use of this technique can find multiple application such as asymptomatic carrier detection or pathogenesis studies of different LCDV strains. The third chapter, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD) in a single assay. This method was able to efficiently detect the viral RNA in tissue samples, showing the presence of single infections and co-infections in rainbow trout samples. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.

Metabolismo di oligosaccaridi prebiotici in Bifidobacterium per il potenziale sviluppo di nuovi prodotti alimentari funzionali

The growth and the metabolism of Bifidobacterium adolescentis MB 239 fermenting GOS, lactose, galactose, and glucose were investigated. An unstructerd unsegregated model for growth of B. adolescentis MB 239 in batch cultures was developed and kinetic parameters were calculated with a Matlab algorithm. Galactose was the best carbon source; lactose and GOS led to lower growth rate and cellular yield, but glucose was the poorest carbon source. Lactate, acetate and ethanol yields allowed calculation of the carbon fluxes toward fermentation products. Similar distribution between 3- and 2-carbon products was observed on all the carbohydrates (45 and 55%, respectively), but ethanol production was higher on glucose than on GOS, lactose and galactose, in decreasing order. Based on the stoichiometry of the fructose 6-phosphate shunt and on the carbon distribution among the products, ATP yield was calculated on the different carbohydrates. ATP yield was the highest on galactose, while it was 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondance among ethanol production, low ATP yields, and low biomass production was established demonstrating that carbohydrate preferences may result from different sorting of carbon fluxes through the fermentative pathway. During GOS fermentation, stringent selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were first to be consumed, and a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β-(1-4) galactosides can be hydrolysed before they are taken up. The physiology of Bifidobacterium adolescentis MB 239 toward xylooligosaccharides (XOS) was also studied and our attention was focused on an extracellular glycosyl-hydrolase (β-Xylosidase) expressed by a culture of B. adolescentis grown on XOS as sole carbon source. The extracellular enzyme was purified from the the supernatant, which was dialyzed and concentrated by ultrafiltration. A two steps purification protocol was developed: the sample was loaded on a Mono-Q anion exchange chromatography and then, the active fractions were pooled and β-Xylosidase was purified by gel filtration chromatography on a Superdex-75. The enzyme was characterized in many aspects. β- Xylosidase was an homo-tetramer of 160 kDa as native molecular mass; it was a termostable enzyme with an optimum of temperature at 53 °C and an optimum of pH of 6.0. The kinetics parameter were calculated: km = 4.36 mM, Vmax = 0.93 mM/min. The substrate specificity with different di-, oligo- and polysaccharides was tested. The reactions were carried out overnight at pH 7 and at the optimum of temperature and the carbohydrates hydrolysis were analyzed by thin layer chromatography (TLC). Only glycosyl-hydrolase activities on XOS and on xylan were detected, whereas sucrose, lactose, cellobiose, maltose and raffinose were not hydrolyzed. It’s clearly shown that β-Xylosidase activity was higher than the Xylanase one. These studies on the carbohydrate preference of a strain of Bifidobacterium underlined the importance of the affinity between probiotics and prebiotics. On the basis of this concept, together with Barilla G&R f.lli SpA, we studied the possibility to develop a functional food containing a synbiotic. Three probiotic strains Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 were studied to assess their suitability for utilization in synbiotic products on the basis of antioxidative activity, glutathione production, acid and bile tolerance, carbohydrates fermentation and viability in food matrices. Bile and human gastric juice resistance was tested in vitro to estimate the transit tolerance in the upper gastrointestinal tract. B. lactis and L. plantarum were more acid tolerant than S. thermophilus. All the strains resisted to bile. The growth kinetics on 13 prebiotic carbohydrates were determined. Galactooligosaccharides and fructo-oligosaccharides were successfully utilized by all the strains and could be considered the most appropriate prebiotics to be used in effective synbiotic formulations. The vitality of the three strains inoculated in different food matrices and maintained at room temperature was studied. The best survival of Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 was found in food chocolate matrices. Then an in vivo clinical trial was carried out for 20 healthy volunteers. The increase in faecal bifidobacteria and lactobacilli populations and the efficacy of the pre-prototype was promising for the future develop of potential commercial products.

Composti organostannici nell'ambiente marino e membrane biologiche: risposte molecolari e biochimiche nei molluschi bivalvi

Organotin compounds are worldwide diffused environmental contaminants, mainly as consequence of their extensive past use as biocides in antifouling paints. In spite of law restrictions, due to unwanted effects, organotin still persist in waters, being poorly degraded, easily resuspended from sediments and bioaccumulated in exposed organisms. The widespread toxicity and the possible threat to humans, likely to be organotin-exposed through contaminated seafood, make organotin interactions with biomolecules an intriguing biochemical topic, apart from a matter of ecotoxicological concern. Among organotins, tributyltin (TBT) is long known as the most dangerous and abundant chemical species in the Mediterranean Sea. Due to its amphiphilic nature, provided by three lipophilic arms and an electrophilic tin core, TBT can be easily incorporated in biomembranes and affect their functionality. Accordingly, it is known as a membrane-active toxicant and a mitochondrial poison. Up to now the molecular action modes of TBT are still partially unclear and poorly explored in bivalve mollusks, even if the latter play a not neglectable role in the marine trophic chain and efficiently accumulate organotins. The bivalve mollusk Mytilus galloprovincialis, selected for all experiments, is widely cultivated in the Mediterranean and currently used in ecotoxicological studies. Most work of this thesis was devoted to TBT effects on mussel mitochondria, but other possible targets of TBT were also considered. A great deal of literature points out TBT as endocrine disrupter and the masculinization of female marine gastropods, the so-called imposex, currently signals environmental organotin contamination. The hormonal status of TBT-exposed mussels and the possible interaction between hormones and contaminants in modulating microsomal hydroxilases, involved in steroid hormone and organotin detoxification, were the research topics in the period spent in Barcelona (Marco Polo fellowship). The variegated experimental approach, which consisted of two exposure experiments and in vitro tests, and the choice of selected tissues of M. galloprovincialis, the midgut gland for mitochondrial and microsomal preparations for subsequent laboratory assays and the gonads for the endocrine evaluations, aimed at drawing a clarifying pattern on the molecular mechanisms involved in organotin toxicity. TBT was promptly incorporated in midgut gland mitochondria of adult mussels exposed to 0.5 and 1.0 μg/L TBT, and partially degraded to DBT. TBT incorporation was accompanied by a decrease in the mitochondrial oligomycin-sensitive Mg-ATPase activity, while the coexistent oligomycin-insensitive fraction was unaffected. Mitochondrial fatty acids showed a clear rise in n-3 polyunsaturated fatty acids after 120 hr of TBT exposure, mainly referable to an increase in 22:6 level. TBT was also shown to inhibit the ATP hydrolytic activity of the mitochondrial F1FO complex in vitro and to promote an apparent loss of oligomycin sensitivity at higher than 1.0 μM concentration. The complex dose-dependent profile of the inhibition curve lead to the hypothesis of multiple TBT binding sites. At lower than 1.0 μM TBT concentrations the non competitive enzyme inhibition by TBT was ascribed to the non covalent binding of TBT to FO subunit. On the other hand the observed drop in oligomycin sensitivity at higher than 1.0 μM TBT could be related to the onset of covalent bonds involving thiolic groups on the enzyme structure, apparently reached only at high TBT levels. The mitochondrial respiratory complexes were in vitro affected by TBT, apart from the cytocrome c oxidase which was apparently refractory to the contaminant. The most striking inhibitory effect was shown on complex I, and ascribed to possible covalent bonds of TBT with –SH groups on the enzyme complexes. This mechanism, shouldered by the progressive decrease of free cystein residues in the presence of increasing TBT concentrations, suggests that the onset of covalent tin-sulphur bonds in distinct protein structures may constitute the molecular basis of widespread TBT effects on mitochondrial complexes. Energy production disturbances, in turn affecting energy consuming mechanisms, could be involved in other cellular changes. Mussels exposed to a wide range of TBT concentrations (20 - 200 and 2000 ng/L respectively) did not show any change in testosterone and estrogen levels in mature gonads. Most hormones were in the non-biologically active esterified form both in control and in TBT-treated mussels. Probably the endocrine status of sexually mature mussels could be refractory even to high TBT doses. In mussel digestive gland the high biological variability of microsomal 7-benzyloxy-4-trifluoromethylcoumarin-O-Debenzyloxylase (BFCOD) activity, taken as a measure of CYP3A-like efficiency, probably concealed any enzyme response to TBT exposure. On the other hand the TBT-driven enhancement of BFCOD activity in vitro was once again ascribed to covalent binding to thiol groups which, in this case, would stimulate the enzyme activity. In mussels from Barcelona harbour, a highly contaminated site, the enzyme showed a decreased affinity for the 7-benzyloxy-4-trifluoromethylcoumarin (BCF) substrate with respect to mussel sampled from Ebro Delta, a non-polluted marine site. Contaminant exposure may thus alter the kinetic features of enzymes involved in detoxification mechanisms. Contaminants and steroid hormones were clearly shown to mutually interact in the modulation of detoxification mechanisms. The xenoestrogen 17α-ethylenyl estradiol (EE2) displayed a non-competitive mixed inhibition of CYP3A-like activity by a preferential bond to the free enzyme both in Barcelona harbour and Ebro Delta mussels. The possible interaction with co-present contaminants in Barcelona harbour mussels apparently lessened the formation of the ternary complex enzyme-EE2-BCF. The whole of data confirms TBT as membrane toxicant in mussels as in other species and stresses TBT covalent binding to protein thiols as a widespread mechanism of membrane-bound-enzyme activity modulation by the contaminant.

Eterogeneità delle proprietà fisico – chimiche della proteina prionica patologica e variabilità fenotipica ceppo specifica nella malattia di Creutzfeldt – Jakob

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are neurodegenerative disorders that affect humans and mammals. Creutzfeldt-Jakob disease (CJD), the most common TSE in humans, can be sporadic (sCJD), genetic (gCJD), or acquired by infection. All TSEs are characterised by the accumulation of PrPSc, a misfolded form of the cellular protein PrPC. PrPSc is insoluble in detergents, partially resistant to proteolysis and shows a highly enriched β-sheet secondary structure. Six clinico-pathological phenotypes of sCJD have been characterized which correlate at the molecular level with two types (1 or 2) of PrPSc with distinctive physicochemical properties and the genotype at the polymorphic (methionine or valine) codon 129 of the prion protein gene. According to the protein-only hypothesis, which postulates that prions are composed exclusively of PrPSc, the strains of prions that are largely responsible for the wide spectrum of TSE phenotypes are enciphered in PrPSc conformation. In support to this view, studies mainly conducted in experimental scrapie, have shown that several prion strains can be identified based on distinguishing PrPSc biochemical properties. To further contribute to the understanding of the molecular basis of strains and to develop more sensitive strain typing assays in humans we have analyzed PrPSc biochemical properties in two experimental setting. In the first we compared the size of the core after protease digestion and the glycoform pattern of PrPSc before and after transmission of human prions to non human primates or bank voles, whereas in the second we analyzed the conformational stability of PrPSc associated with sCJD, vCJD or fCJD using guanidine hydrochloride (GdnHCl) as denaturant. Combining the results of the two studies, we were able to distinguish five human strains for at least one biochemical property. The present data extend our knowledge about the extent of strain variation and its relationship with PrPSc properties in human TSEs.

La città divisa. Conflittualità, confini, prove di comunità

Il presente lavoro approfondisce le tematiche della conflittualità e della separazione etnica, sociale, religiosa, generazionale e culturale. In particolare, riporta i risultati di ricerche condotte in alcuni degli attuali contesti urbani più carichi di tensioni conflittuali, cercando di individuare i motivi vicini e remoti del confligere, le attività messe in atto per contenere le tensioni e le relative necessità educative poste in primo piano. L’elaborato si compone di cinque parti. La prima parte consiste in una riflessione teorica sulle dinamiche che caratterizzano le interazioni sociali nello spazio cittadino. Nella seconda parte viene trattato il tema del settarismo in Scozia, che vede contrapposti i cattolici di origine irlandese (generalmente tifosi della squadra di calcio dei Celtic) e i protestanti di sangue scozzese (generalmente tifosi dei Rangers). La terza parte ricostruisce la storia e il presente della lunga convivenza tra tatari musulmani e russi cristiano-ortodossi nei territori dell’attuale Repubblica etnica del Tatarstan, situata nel cuore della Russia europea, ponendo particolare attenzione agli aspetti religiosi, linguistici, culturali ed educativi. La quarta parte parla del disagio nelle periferie europee, manifestatosi in modo eclatante con le rivolte giovanili in Francia (2005) e nel Regno Unito (2011). La parte conclusiva, infine riprenderà alcuni degli elementi emersi per proporre una riflessione di tipo pedagogico atta ad affrontare in tutta la sua complessità, e con approcci nuovi, il tema della città divisa, con le relative conflittualità, i confini e le prove di comunità.

Una grande narrazione del capitalismo: potere e scienze sociali nel pensiero politico di Daniel Bell

Questa tesi punta a ricostruire il pensiero politico di Bell tra il secondo dopoguerra e la metà degli anni Settanta. In tale arco cronologico, la riflessione politica di Bell si profila, per usare una formula di Jean-François Lyotard, come una «grande narrazione» del capitalismo. Nel complesso, cioè, l’opera di Bell appare come una storia sociologica del capitalismo, che nella fine delle ideologie registra l’apogeo del fordismo e, in seguito, ne mette in luce le trasformazioni in senso post-industriale, indagando le ricadute che tali mutamenti implicano sul piano dei rapporti di potere e della legittimazione del sistema. Nell’ottica di Bell, pertanto, il capitalismo non costituisce soltanto un sistema economico, ma la forma specifica attraverso cui si dispiega la società nel suo complesso, attivando una serie di rapporti di potere mediante i quali gli individui vengono coordinati e subordinati. Una siffatta concezione del capitalismo agisce immediatamente la questione del potere e solleva un interrogativo a esso connesso: «che cosa tiene insieme una società?». Una domanda che attraversa la traiettoria intellettuale di Bell e, sia pure declinata mediante una terminologia sociologica, riflette in realtà l’ambizione delle scienze sociali di farsi teoria politica. Esse si presentano quindi come teoria politica della modernità, nella misura in cui distinguono il potere sociale dal potere politico e, al tempo stesso, instaurano tra i due poli una tensione dialettica produttiva. Mettendo a fuoco la concettualizzazione del potere nell’opera di Bell si analizzeranno le mutazioni nel rapporto tra Stato e società negli Stati Uniti durante la Golden Age del capitalismo. In particolare, si metterà in luce nella grande narrazione di Bell l’ascesa e il declino di un ordine istituzionale che, alla metà degli anni Settanta, appare percorso da molteplici tensioni politiche e sociali che preannunciano l’avvento dell’età globale e il bisogno di una nuova “scala” di governo.

Exploring the biofilm of Streptococcus agalactiae to identify virulence factors

Streptococcus agalactiae, also known as Group B Streptococcus (GBS) is the primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30-76% of the cases of neonatal meningitis. Biofilms are dense aggregates of surface-adherent microorganisms embedded in an exopolysaccharide matrix. Centers for Disease Control and Prevention estimate that 65% of human bacterial infections involve biofilms (Post et al., 2004). In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and/or virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low- and non- biofilm forming strains and reduce ambiguous data interpretation. This protocol was applied to screen the in vitro biofilm formation ability of more than 350 GBS clinical isolates from pregnant women and neonatal infections belonging to different serotype, in relation to media composition and pH. The results showed the enhancement of GBS biofilm formation in acidic condition and identified a subset of isolates belonging to serotypes III and V that forms strong biofilms in these conditions. Interestingly, the best biofilm formers belonged to the serotype III hypervirulent clone ST-17.It was also found that pH 5.0 induces down-regulation of the capsule but that this reduction is not enough by itself to ensure biofilm formation. Moreover, the ability of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm formation and contribute to the biofilm structural stability. Finally, a set of proteins potentially expressed during the GBS in vitro biofilm formation were identified by mass spectrometry.

Variabilita' ed evoluzione dei prioni: Mutabilita' in vitro

I prioni, privi di acidi nucleici, esistono come ceppi e possono mutare, in particolare quando attraversano una barriera di specie. Numerosi studi convergono sulla conclusione che le caratteristiche ceppo-specifiche siano inscritte nella conformazione della PrPSc, con la variabilità di ceppo associata a varianti conformazionali della PrPSc. In questo studio ci siamo avvalsi del PMCA, tecnica che riproduce in vitro molti aspetti della biologia dei prioni, per mettere a punto condizioni sperimentali di replicazione che permettessero di osservare fenomeni di mutazione e selezione, onde investigare i meccanismi molecolari e di popolazione alla base della mutabilità dei prioni. In condizioni di replicazione eterologa, che mima la trasmissione tra diverse specie, è stato inizialmente possibile identificare un mutante difettivo della scrapie, caratterizzato da una diversa conformazione della PrPSc e capace di replicare in vitro ma non più in vivo. Le condizioni in cui tale mutante è emerso hanno permesso di sviluppare ulteriori ipotesi di lavoro, basate sul concetto della quasi-specie. Impartendo diversi regimi di replicazione e seguendo l’evoluzione di due ceppi, è stato possibile evidenziare fenomeni di mutazione anche in condizioni di replicazione omologa, in assenza di forti pressioni selettive. In entrambi i ceppi sono emerse varianti conformazionali di PrPSc durante passaggi replicativi ad ampia popolazione, mentre le popolazioni sottoposte a ripetuti colli di bottiglia hanno mostrato un rapido declino del tasso di replicazione. Sono stati infine investigati l’efficacia e il potenziale mutageno di molecole anti-prioniche, ottenendo importanti risultati preliminari sull’efficacia di molecole che legano la PrPC. Questi risultati evidenziano come la mutabilità sia una caratteristica intrinseca dei prioni e supportano l’idea che i prioni siano molto variabili, similmente alle quasi-specie virali, e perciò adattabili e proni a fenomeni di mutazione e selezione. Tali conclusioni hanno impatto su problematiche sanitarie quali lo studio del potenziale zoonotico e i fenomeni di farmaco-resistenza dei prioni.