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Impatto di rifaximina sul microbiota intestinale: selezione di bifidobatteri antibiotico resistenti

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

Processi di biorefining per l'estrazione di secondary chemical building blocks da sottoprotti dell'agro-industria

Phenol and cresols represent a good example of primary chemical building blocks of which 2.8 million tons are currently produced in Europe each year. Currently, these primary phenolic building blocks are produced by refining processes from fossil hydrocarbons: 5% of the world-wide production comes from coal (which contains 0.2% of phenols) through the distillation of the tar residue after the production of coke, while 95% of current world production of phenol is produced by the distillation and cracking of crude oil. In nature phenolic compounds are present in terrestrial higher plants and ferns in several different chemical structures while they are essentially absent in lower organisms and in animals. Biomass (which contain 3-8% of phenols) represents a substantial source of secondary chemical building blocks presently underexploited. These phenolic derivatives are currently used in tens thousand of tons to produce high cost products such as food additives and flavours (i.e. vanillin), fine chemicals (i.e. non-steroidal anti-inflammatory drugs such as ibuprofen or flurbiprofen) and polymers (i.e. poly p-vinylphenol, a photosensitive polymer for electronic and optoelectronic applications). European agrifood waste represents a low cost abundant raw material (250 millions tons per year) which does not subtract land use and processing resources from necessary sustainable food production. The class of phenolic compounds is essentially constituted by simple phenols, phenolic acids, hydroxycinnamic acid derivatives, flavonoids and lignans. As in the case of coke production, the removal of the phenolic contents from biomass upgrades also the residual biomass. Focusing on the phenolic component of agrifood wastes, huge processing and marketing opportunities open since phenols are used as chemical intermediates for a large number of applications, ranging from pharmaceuticals, agricultural chemicals, food ingredients etc. Following this approach we developed a biorefining process to recover the phenolic fraction of wheat bran based on enzymatic commercial biocatalysts in completely water based process, and polymeric resins with the aim of substituting secondary chemical building blocks with the same compounds naturally present in biomass. We characterized several industrial enzymatic product for their ability to hydrolize the different molecular features that are present in wheat bran cell walls structures, focusing on the hydrolysis of polysaccharidic chains and phenolics cross links. This industrial biocatalysts were tested on wheat bran and the optimized process allowed to liquefy up to the 60 % of the treated matter. The enzymatic treatment was also able to solubilise up to the 30 % of the alkali extractable ferulic acid. An extraction process of the phenolic fraction of the hydrolyzed wheat bran based on an adsorbtion/desorption process on styrene-polyvinyl benzene weak cation-exchange resin Amberlite IRA 95 was developed. The efficiency of the resin was tested on different model system containing ferulic acid and the adsorption and desorption working parameters optimized for the crude enzymatic hydrolyzed wheat bran. The extraction process developed had an overall yield of the 82% and allowed to obtain concentrated extracts containing up to 3000 ppm of ferulic acid. The crude enzymatic hydrolyzed wheat bran and the concentrated extract were finally used as substrate in a bioconversion process of ferulic acid into vanillin through resting cells fermentation. The bioconversion process had a yields in vanillin of 60-70% within 5-6 hours of fermentation. Our findings are the first step on the way to demonstrating the economical feasibility for the recovery of biophenols from agrifood wastes through a whole crop approach in a sustainable biorefining process.

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.

Mechanism of colonization in the Mediterranean-sea. Asperarca nodulosa: morphological and genetic analysis

Il mar Mediterraneo è un bacino acquifero peculiare per la recente colonizzazione di specie aliene, per l’evento geologico legato alla Crisi di salinità del Messiniano e per l’ampio range di salinità. L’individuazione dei meccanismi di colonizzazione si è incentrata sullo studio morfologico, istologico e molecolare delle specie Asperarca nodulosa ed Anadara demiri (Arcidae-Bivalvia-Mollusca). La ricerca si è basata sulla caratterizzazione morfologica, con utilizzo del microscopio elettronico a scansione, al fine di individuare il tipo di sviluppo larvale. Successivamente i dati rilevati al S.E.M. sono stati supportati dall’indagine istologica che ha evidenziato la presenza di gonadi a sessi distinti e la non incubazione larvale. L’ulteriore analisi filogenetica ha permesso di evidenziare la netta suddivisione tra le tre popolazioni studiate, indagine effettuata tramite marcatori arbitrari (RAPDs) e nucleari specifici (ITS). I risultati ottenuti trovano supporto da quanto noto su base morfologica. I dati, nel complesso, mostrano una perdita delle capacità di diffusione della specie tramite sviluppo larvale plantotrofico a favore di quello lecitotrofico o diretto; tale tesi è ulteriormente supportata dai dati molecolari che mostrano una netta separazione delle popolazioni prese in esame ed un conseguente isolamento tra individui appartenenti a zone di profondità del Mediterraneo (sub-bacini abissali). La ricerca ha, inoltre,esaminato i meccanismi di introduzione attuali nel bacino acquifero che è soggetto ad una nuova invasione da parte specie aliene dovuta all’apertura del canale di Suez. L’analisi si è focalizzata sullo studio per l’ individuazione dell’origine della specie aliena A. demiri , di presunta derivazione Indo-Pacifica, ma rivelatasi, nei dati preliminari, di origine Atlantica.

Valutazione delle condizioni di rischio idraulico per il Rio Ponticelli

Applicazione di metodi indiretti per la stima delle portate di progetto, aventi tempi di ritorno pari a: 30, 100 e 200 anni. Simulazione in moto permanente mediante programma di calcolo noto come hec-Ras. individuazione su carta delle fasce inondabili in seguito ad un evento di piena con tempo di ritorno assegnato.

Ricostruzione degli sforzi in elementi finiti di piastra

Nell’ambito dell’analisi computazionale delle strutture il metodo degli elementi finiti è probabilmente uno dei metodi numerici più efficaci ed impiegati. La semplicità dell’idea di base del metodo e la relativa facilità con cui può essere implementato in codici di calcolo hanno reso possibile l’applicazione di questa tecnica computazionale in diversi settori, non solo dell’ingegneria strutturale, ma in generale della matematica applicata. Ma, nonostante il livello raggiunto dalle tecnologie ad elementi finiti sia già abbastanza elevato, per alcune applicazioni tipiche dell’ingegneria strutturale (problemi bidimensionali, analisi di lastre inflesse) le prestazioni fornite dagli elementi usualmente utilizzati, ovvero gli elementi di tipo compatibile, sono in effetti poco soddisfacenti. Vengono in aiuto perciò gli elementi finiti basati su formulazioni miste che da un lato presentano una più complessa formulazione, ma dall’altro consentono di prevenire alcuni problemi ricorrenti quali per esempio il fenomeno dello shear locking. Indipendentemente dai tipi di elementi finiti utilizzati, le quantità di interesse nell’ambito dell’ingegneria non sono gli spostamenti ma gli sforzi o più in generale le quantità derivate dagli spostamenti. Mentre i primi sono molto accurati, i secondi risultano discontinui e di qualità scadente. A valle di un calcolo FEM, negli ultimi anni, hanno preso piede procedure di post-processing in grado, partendo dalla soluzione agli elementi finiti, di ricostruire lo sforzo all’interno di patch di elementi rendendo quest’ultimo più accurato. Tali procedure prendono il nome di Procedure di Ricostruzione (Recovery Based Approaches). Le procedure di ricostruzione qui utilizzate risultano essere la REP (Recovery by Equilibrium in Patches) e la RCP (Recovery by Compatibility in Patches). L’obbiettivo che ci si prefigge in questo lavoro è quello di applicare le procedure di ricostruzione ad un esempio di piastra, discretizzato con vari tipi di elementi finiti, mettendone in luce i vantaggi in termini di migliore accurattezza e di maggiore convergenza.

Il diritto di asilo tra ordinamento costituzionale e sistema europeo di protezione multilivello

La tesi della candidata presenta - attraverso lo studio della normativa e della giurisprudenza rilevanti in Italia, Francia e Germania – un’analisi dell'ambito soggettivo di applicazione del diritto costituzionale d'asilo e del suo rapporto con il riconoscimento dello status di rifugiato ai sensi della Convenzione di Ginevra del 1951, nonchè della sua interazione con le altre forme di protezione della persona previste dal diritto comunitario e dal sistema CEDU di salvaguardia dei diritti fondamentali. Dal breve itinerario comparatistico percorso, emerge una forte tendenza alla neutralizzazione dell’asilo costituzionale ed alla sua sovrapposizione con la fattispecie del rifugio convenzionale quale carattere comune agli ordinamenti presi in esame, espressione di una consapevole scelta di politica del diritto altresì volta ad assimilare la materia alla disciplina generale dell’immigrazione al fine di ridimensionarne le potenzialità espansive (si pensi alla latitudine delle formule costituzionali di cui agli artt. 10, co. 3 Cost. it. e 16a, co. 1 Grundgesetz) e di ricondurre l'asilo entro i tradizionali confini della discrezionalità amministrativa quale sovrana concessione dello Stato ospitante. L'esame delle fonti comunitarie di recente introduzione illumina l’indagine: in particolare, la stessa Direttiva 2004/83CE sulla qualifica di rifugiato e sulla protezione sussidiaria consolida quanto stabilito dalle disposizioni convenzionali, ma ne estende la portata in modo significativo, recependo gli esiti della lunga evoluzione giurisprudenziale compiuta dalle corti nazionali e dal Giudice di Strasburgo nell’interpretazione del concetto di “persecuzione” (specialmente, in relazione all’individuazione delle azioni e degli agenti persecutori). Con riferimento al sistema giuridico italiano, la tesi si interroga sulle prospettive di attuazione del dettato dell’art. 10, terzo comma della Costituzione, ed inoltre propone la disamina di alcuni istituti chiave dell’attuale normativa in materia di asilo, attraverso cui si riscontrano importanti profili di incompatibilità con la natura di diritto fondamentale costituzionalmente tutelato, conferita al diritto di asilo dalla volontà dei Costituenti e radicata nella ratio della norma stessa (il trattenimento del richiedente asilo; la procedura di esame della domanda, l’onere probatorio e le cause ostative al suo accoglimento; l’effettività della tutela giurisdizionale). Le questioni più problematiche ancora irrisolte investono proprio tali aspetti del procedimento - previsto per ottenere quello che alcuni atti europei, tra cui l'art. 18 della Carta di Nizza, definiscono right to asylum - come rivela la disciplina contenuta nella Direttiva 2005/85CE, recante norme minime per le procedure applicate negli Stati membri ai fini del riconoscimento e della revoca dello status di rifugiato. Infine, il fenomeno della esternalizzazione dei controlli compromette lo stesso accesso alle procedure, nella misura in cui rende "mobile" il confine territoriale dell’area Schengen (attraverso l'introduzione del criterio dello "Stato terzo sicuro", degli strumenti dell'esame preliminare delle domande e della detenzione amministrativa nei Paesi di transito, nonché per mezzo del presidio delle frontiere esterne), relegando il trattamento dei richiedenti asilo ad uno spazio in cui non sempre è monitorabile l'effettivo rispetto del principio del non refoulement, degli obblighi internazionali relativi all’accoglienza dei profughi e delle clausole di determinazione dello Stato competente all'esame delle domande ai sensi del Regolamento n. 343/03, c.d. Dublino II (emblematico il caso del pattugliamento delle acque internazionali e dell'intercettazione delle navi prima del superamento dei confini territoriali). Questi delicati aspetti di criticità della disciplina procedimentale limitano il carattere innovativo delle recenti acquisizioni comunitarie sull’ambito di operatività delle nuove categorie definitorie introdotte (le qualifiche di rifugiato e di titolare di protezione sussidiaria e la complessa nozione di persecuzione, innanzitutto), richiedendo, pertanto, l’adozione di un approccio sistemico – piuttosto che analitico – per poter rappresentare in modo consapevole le dinamiche che concretamente si producono a livello applicativo ed affrontare la questione nodale dell'efficienza dell'attuale sistema multilivello di protezione del richiedente asilo.

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.

Variabilità delle caratteristiche meccaniche del Marmo delle Alpi Apuane

Lo svolgimento di questa tesi ha riguardato la caratterizzazione di una roccia carbonatica metamorfica, marmo proveniente da un bacino estrattivo delle Alpi Apuane, mediante prove di laboratorio: in particolare, si è esaminato il comportamento di tale materiale sia mediante prove distruttive, sia mediante prove non distruttive. Lo studio è stato condotto su diverse tipologie di campioni (cilindri di diametro differente ma con uguale rapporto tra diametro e altezza, campioni informi di materiale), tutti ottenuti da blocchi informi prelevati nel bacino estrattivo dell’Arnetola, sito nel comune di Vagli Sotto (LU). Sui campioni carotati sono state eseguite più prove standard (distruttive e non), mentre i campioni informi sono stati utilizzati per la determinazione, mediante prova distruttiva, dell’indice di resistenza R.I.H.N. (acronimo di Rock Impact Hardness Number). Tale prova è in grado di fornire utili informazioni sulla resistenza dei materiali rocciosi, risultando particolarmente utile nell’industria estrattiva grazie alla semplice apparecchiatura che dà la possibilità di eseguire un numero elevato di determinazioni nei centri stessi di produzione, con bassi costi unitari. Data la variabilità dei risultati ottenuti, sono state eseguite ulteriori analisi (studio tessiturale in sezione sottile, analisi calcimetrica e analisi diffrattometrica) che mettessero in luce la composizione mineralogica e la struttura microscopica del materiale testato, in modo da interpretare i risultati delle prove di laboratorio effettuate. Alla luce dei risultati ottenuti, sono state elaborate alcune correlazioni tra le varie prove, facendo anche uso di alcuni abachi.

Triggers per attività di test su fascio di particelle. Realizzazione di un progetto in VHDL e sintetizzazione su FPGA

Progettazione e realizzazione di un dispositivo elettronico con lo scopo di coordinare e sincronizzare la presa dati del beam test del LUCID (CERN, luglio 2009) e tener traccia di tali eventi. Il circuito è stato progettato in linguaggio VHDL, simulato con il programma ModelSim, sintetizzato con il programma Quartus e implementato su un FPGA Cyclone residente su scheda di tipo VME 6U della CAEN. Infine la scheda è stata testata in laboratorio (verificandone il corretto funzionamento) assieme all'intero sistema di presa dati, e confermata per il beam test del LUCID.