Formalizing languages for service oriented computing

Service Oriented Computing is a new programming paradigm for addressing distributed system design issues. Services are autonomous computational entities which can be dynamically discovered and composed in order to form more complex systems able to achieve different kinds of task. E-government, e-business and e-science are some examples of the IT areas where Service Oriented Computing will be exploited in the next years. At present, the most credited Service Oriented Computing technology is that of Web Services, whose specifications are enriched day by day by industrial consortia without following a precise and rigorous approach. This PhD thesis aims, on the one hand, at modelling Service Oriented Computing in a formal way in order to precisely define the main concepts it is based upon and, on the other hand, at defining a new approach, called bipolar approach, for addressing system design issues by synergically exploiting choreography and orchestration languages related by means of a mathematical relation called conformance. Choreography allows us to describe systems of services from a global view point whereas orchestration supplies a means for addressing such an issue from a local perspective. In this work we present SOCK, a process algebra based language inspired by the Web Service orchestration language WS-BPEL which catches the essentials of Service Oriented Computing. From the definition of SOCK we will able to define a general model for dealing with Service Oriented Computing where services and systems of services are related to the design of finite state automata and process algebra concurrent systems, respectively. Furthermore, we introduce a formal language for dealing with choreography. Such a language is equipped with a formal semantics and it forms, together with a subset of the SOCK calculus, the bipolar framework. Finally, we present JOLIE which is a Java implentation of a subset of the SOCK calculus and it is part of the bipolar framework we intend to promote.

Sostanzialismo e relazionalismo dello spaziotempo alla luce della relatività generale

Until recently the debate on the ontology of spacetime had only a philosophical significance, since, from a physical point of view, General Relativity has been made "immune" to the consequences of the "Hole Argument" simply by reducing the subject to the assertion that solutions of Einstein equations which are mathematically different and related by an active diffeomorfism are physically equivalent. From a technical point of view, the natural reading of the consequences of the "Hole Argument” has always been to go further and say that the mathematical representation of spacetime in General Relativity inevitably contains a “superfluous structure” brought to light by the gauge freedom of the theory. This position of apparent split between the philosophical outcome and the physical one has been corrected thanks to a meticulous and complicated formal analysis of the theory in a fundamental and recent (2006) work by Luca Lusanna and Massimo Pauri entitled “Explaining Leibniz equivalence as difference of non-inertial appearances: dis-solution of the Hole Argument and physical individuation of point-events”. The main result of this article is that of having shown how, from a physical point of view, point-events of Einstein empty spacetime, in a particular class of models considered by them, are literally identifiable with the autonomous degrees of freedom of the gravitational field (the Dirac observables, DO). In the light of philosophical considerations based on realism assumptions of the theories and entities, the two authors then conclude by saying that spacetime point-events have a degree of "weak objectivity", since they, depending on a NIF (non-inertial frame), unlike the points of the homogeneous newtonian space, are plunged in a rich and complex non-local holistic structure provided by the “ontic part” of the metric field. Therefore according to the complex structure of spacetime that General Relativity highlights and within the declared limits of a methodology based on a Galilean scientific representation, we can certainly assert that spacetime has got "elements of reality", but the inevitably relational elements that are in the physical detection of point-events in the vacuum of matter (highlighted by the “ontic part” of the metric field, the DO) are closely dependent on the choice of the global spatiotemporal laboratory where the dynamics is expressed (NIF). According to the two authors, a peculiar kind of structuralism takes shape: the point structuralism, with common features both of the absolutist and substantival tradition and of the relationalist one. The intention of this thesis is that of proposing a method of approaching the problem that is, at least at the beginning, independent from the previous ones, that is to propose an approach based on the possibility of describing the gravitational field at three distinct levels. In other words, keeping the results achieved by the work of Lusanna and Pauri in mind and following their underlying philosophical assumptions, we intend to partially converge to their structuralist approach, but starting from what we believe is the "foundational peculiarity" of General Relativity, which is that characteristic inherent in the elements that constitute its formal structure: its essentially geometric nature as a theory considered regardless of the empirical necessity of the measure theory. Observing the theory of General Relativity from this perspective, we can find a "triple modality" for describing the gravitational field that is essentially based on a geometric interpretation of the spacetime structure. The gravitational field is now "visible" no longer in terms of its autonomous degrees of freedom (the DO), which, in fact, do not have a tensorial and, therefore, nor geometric nature, but it is analyzable through three levels: a first one, called the potential level (which the theory identifies with the components of the metric tensor), a second one, known as the connections level (which in the theory determine the forces acting on the mass and, as such, offer a level of description related to the one that the newtonian gravitation provides in terms of components of the gravitational field) and, finally, a third level, that of the Riemann tensor, which is peculiar to General Relativity only. Focusing from the beginning on what is called the "third level" seems to present immediately a first advantage: to lead directly to a description of spacetime properties in terms of gauge-invariant quantites, which allows to "short circuit" the long path that, in the treatises analyzed, leads to identify the "ontic part” of the metric field. It is then shown how to this last level it is possible to establish a “primitive level of objectivity” of spacetime in terms of the effects that matter exercises in extended domains of spacetime geometrical structure; these effects are described by invariants of the Riemann tensor, in particular of its irreducible part: the Weyl tensor. The convergence towards the affirmation by Lusanna and Pauri that the existence of a holistic, non-local and relational structure from which the properties quantitatively identified of point-events depend (in addition to their own intrinsic detection), even if it is obtained from different considerations, is realized, in our opinion, in the assignment of a crucial role to the degree of curvature of spacetime that is defined by the Weyl tensor even in the case of empty spacetimes (as in the analysis conducted by Lusanna and Pauri). In the end, matter, regarded as the physical counterpart of spacetime curvature, whose expression is the Weyl tensor, changes the value of this tensor even in spacetimes without matter. In this way, going back to the approach of Lusanna and Pauri, it affects the DOs evolution and, consequently, the physical identification of point-events (as our authors claim). In conclusion, we think that it is possible to see the holistic, relational, and non-local structure of spacetime also through the "behavior" of the Weyl tensor in terms of the Riemann tensor. This "behavior" that leads to geometrical effects of curvature is characterized from the beginning by the fact that it concerns extensive domains of the manifold (although it should be pointed out that the values of the Weyl tensor change from point to point) by virtue of the fact that the action of matter elsewhere indefinitely acts. Finally, we think that the characteristic relationality of spacetime structure should be identified in this "primitive level of organization" of spacetime.

Il ruolo delle idee e del loro cambiamento nei processi di policy making universitario nel mondo anglosassone dagli anni ottanta ad oggi. I casi di Inghilterra e Nuova Zelanda in prospettiva comparata

THE TITLE OF MY THESIS IS THE ROLE OF THE IDEAS AND THEIR CHANGE IN HIGHER EDUCATION POLICY-MAKING PROCESSES FROM THE EIGHTIES TO PRESENT-DAY: THE CASES OF ENGLAND AND NEW ZEALAND IN COMPARATIVE PERSPECTIVE UNDER A THEORETICAL POINT OF VIEW, THE AIM OF MY WORK IS TO CARRY OUT A RESEARCH MODELLED ON THE CONSTRUCTIVIST THEORY. IT FOCUSES ON THE ANALYSIS OF THE IMPACT OF IDEAS ON THE PROCESSES OF POLICY MAKING BY MEANS OF EPISTEMIC COMMUNITIES, THINK TANKS AND VARIOUS SOCIOECONOMIC CONTEXTS THAT MAY HAVE PLAYED A KEY ROLE IN THE CONSTRUCTION OF THE DIFFERENT PATHS. FROM MY POINT OF VIEW IDEAS CONSTITUTE A PRIORITY RESEARCH FIELD WHICH IS WORTH ANALYSING SINCE THEIR ROLE IN POLICY MAKING PROCESSES HAS BEEN TRADITIONALLY RATHER UNEXPLORED. IN THIS CONTEXT AND WITH THE AIM OF DEVELOPING A RESEARCH STRAND BASED ON THE ROLE OF IDEAS, I INTEND TO CARRY ON MY STUDY UNDER THE PERSPECTIVE OF CHANGE. DEPENDING ON THE DATA AND INFORMATION THAT I COLLECTED I EVALUATED THE WEIGHT OF EACH OF THESE VARIABLES AND MAYBE OTHERS SUCH AS THE INSTITUTIONS AND THE INDIVIDUAL INTERESTS, WHICH MAY HAVE INFLUENCED THE FORMATION OF THE POLICY MAKING PROCESSES. UNDER THIS LIGHT, I PLANNED TO ADOPT THE QUALITATIVE METHODOLOGY OF RESEARCH WHICH I BELIEVE TO BE VERY EFFECTIVE AGAINST THE MORE DIFFICULT AND POSSIBLY REDUCTIVE APPLICATION OF QUANTITIVE DATA SETS. I RECKON THEREFORE THAT THE MOST APPROPRIATE TOOLS FOR INFORMATION PROCESSING INCLUDE CONTENT ANALYSIS, AND IN-DEPTH INTERVIEWS TO PERSONALITIES OF THE POLITICAL PANORAMA (ÉLITE OR NOT) WHO HAVE PARTICIPATED IN THE PROCESS OF HIGHER EDUCATION REFORM FROM THE EIGHTIES TO PRESENT-DAY. THE TWO CASES TAKEN INTO CONSIDERATION SURELY SET AN EXAMPLE OF RADICAL REFORM PROCESSES WHICH HAVE OCCURRED IN QUITE DIFFERENT CONTEXTS DETERMINED BY THE SOCIOECONOMIC CHARACTERISTICS AND THE TRAITS OF THE ÉLITE. IN NEW ZEALAND THE DESCRIBED PROCESS HAS TAKEN PLACE WITH A STEADY PACE AND A GOOD GRADE OF CONSEQUANTIALITY, IN LINE WTH THE REFORMS IN OTHER STATE DIVISIONS DRIVEN BY THE IDEAS OF THE NEW PUBLIC MANAGEMENT. CONTRARILY IN ENGLAND THE REFORMATIVE ACTION OF MARGARET THATCHER HAS ACQUIRED A VERY RADICAL CONNOTATION AS IT HAS BROUGHT INTO THE AMBIT OF HIGHER EDUCATION POLICY CONCEPTS LIKE EFFICIENCY, EXCELLENCE, RATIONALIZATION THAT WOULD CONTRAST WITH THE GENERALISTIC AND MASS-ORIENTED IDEAS THAT WERE FASHIONABLE DURING THE SEVENTIES. THE MISSION I INTEND TO ACCOMPLISH THORUGHOUT MY RESEARCH IS TO INVESTIGATE AND ANALYSE INTO MORE DEPTH THE DIFFERENCES THAT SEEM TO EMERGE FROM TWO CONTEXTS WHICH MOST OF THE LITERATURE REGARDS AS A SINGLE MODEL: THE ANGLO-SAXON MODEL. UNDER THIS LIGHT, THE DENSE ANALYSIS OF POLICY PROCESSES ALLOWED TO BRING OUT BOTH THE CONTROVERSIAL AND CONTRASTING ASPECTS OF THE TWO REALITIES COMPARED, AND THE ROLE AND WEIGHT OF VARIABLES SUCH AS IDEAS (MAIN VARIABLE), INSTITUTIONAL SETTINGS AND INDIVIDUAL INTERESTS ACTING IN EACH CONTEXT. THE CASES I MEAN TO ATTEND PRESENT PECULIAR ASPECTS WORTH DEVELOPING AN IN-DEPTH ANALYSIS, AN OUTLINE OF WHICH WILL BE PROVIDED IN THIS ABSTRACT. ENGLAND THE CONSERVATIVE GOVERNMENT, SINCE 1981, INTRODUCED RADICAL CHANGES IN THE SECTOR OF HIGHER EDUCATION: FIRST CUTTING DOWN ON STATE FUNDINGS AND THEN WITH THE CREATION OF AN INSTITUTION FOR THE PLANNING AND LEADERSHIP OF THE POLYTECHNICS (NON-UNIVERSITY SECTOR). AFTERWARDS THE SCHOOL REFORM BY MARGARET THATCHER IN 1988 RAISED TO A GREAT STIR ALL OVER EUROPE DUE TO BOTH ITS CONSIDERABLE INNOVATIVE IMPRINT AND THE STRONG ATTACK AGAINST THE PEDAGOGY OF THE ‘ACTIVE’ SCHOOLING AND PROGRESSIVE EDUCATION, UNTIL THEN RECOGNIZED AS A MERIT OF THE BRITISH PUBLIC SCHOOL. IN THE AMBIT OF UNIVERSITY EDUCATION THIS REFORM, TOGETHER WITH SIMILAR MEASURES BROUGHT IN DURING 1992, PUT INTO PRACTICE THE CONSERVATIVE PRINCIPLES THROUGH A SERIES OF ACTIONS THAT INCLUDED: THE SUPPRESSION OF THE IRREMOVABILITY PRINCIPLE FOR UNIVERSITY TEACHERS; THE INTRODUCTION OF STUDENT LOANS FOR LOW-INCOME STUDENTS AND THE CANCELLATION OF THE CLEAR DISTINCTION BETWEEN UNIVERSITIES AND POLYTECHNICS. THE POLICIES OF THE LABOUR MAJORITY OF MR BLAIR DID NOT QUITE DIVERGE FROM THE CONSERVATIVES’ POSITION. IN 2003 BLAIR’S CABINET RISKED TO BECOME A MINORITY RIGHT ON THE OCCASION OF AN IMPORTANT UNIVERSITY REFORM PROPOSAL. THIS PROPOSAL WOULD FORESEE THE AUTONOMY FOR THE UNIVERSITIES TO RAISE UP TO 3.000 POUNDS THE ENROLMENT FEES FOR STUDENTS (WHILE FORMERLY THE CEILING WAS 1.125 POUNDS). BLAIR HAD TO FACE INTERNAL OPPOSITION WITHIN HIS OWN PARTY IN RELATION TO A MEASURE THAT, ACCORDING TO THE 150 MPS PROMOTERS OF AN ADVERSE MOTION, HAD NOT BEEN INCLUDED IN THE ELECTORAL PROGRAMME AND WOULD RISK CREATING INCOME-BASED DISCRIMINATION AMONG STUDENTS. AS A MATTER OF FACT THE BILL FOCUSED ON THE INTRODUCTION OF VERY LOW-INTEREST STUDENT LOANS TO BE SETTLED ONLY WHEN THE STUDENT WOULD HAVE FOUND A REMUNERATED OCCUPATION (A SYSTEM ALREADY PROVIDED FOR BY THE AUSTRALIAN LEGISLATION). NEW ZEALAND CONTRARILY TO MANY OTHER COUNTRIES, NEW ZEALAND HAS ADOPTED A VERY WIDE VISION OF THE TERTIARY EDUCATION. IT INCLUDES IN FACT THE FULL EDUCATIONAL PROGRAMME THAT IS INTERNATIONALLY RECOGNIZED AS THE POST-SECONDARY EDUCATION. SHOULD WE SPOTLIGHT A PECULIARITY OF THE NEW ZEALAND TERTIARY EDUCATION POLICY THEN IT WOULD BE ‘CHANGE’. LOOKING AT THE REFORM HISTORY RELATED TO THE TERTIARY EDUCATION SYSTEM, WE CAN CLEARLY IDENTIFY FOUR ‘SUB-PERIODS’ FROM THE EIGHTIES TO PRESENT-DAY: 1. BEFORE THE 80S’: AN ELITARIAN SYSTEM CHARACTERIZED BY LOW PARTICIPATION RATES. 2. BETWEEN MID AND LATE 80S’: A TREND TOWARDS THE ENLARGEMENT OF PARTICIPATION ASSOCIATED TO A GREATER COMPETITION. 3. 1990-1999: A FUTHER STEP TOWARDS A COMPETITIVE MODEL BASED ON THE MARKET-ORIENTED SYSTEM. 4. FROM 2000 TO TODAY: A CONTINUOUS EVOLUTION TOWARDS A MORE COMPETITIVE MODEL BASED ON THE MARKET-ORIENTED SYSTEM TOGETHER WITH A GROWING ATTENTION TO STATE CONTROL FOR SOCIAL AND ECONOMIC DEVELOPMENT OF THE NATION. AT PRESENT THE GOVERNMENT OF NEW ZEALAND OPERATES TO STRENGHTHEN THIS PROCESS, PRIMARILY IN RELATION TO THE ROLE OF TERTIARY EDUCATION AS A STEADY FACTOR OF NATIONAL WALFARE, WHERE PROFESSIONAL DEVELOPMENT CONTRIBUTES ACTIVELY TO THE GROWTH OF THE NATIONAL ECONOMIC SYSTEM5. THE CASES OF ENGLAND AND NEW ZEALAND ARE THE FOCUS OF AN IN-DEPTH INVESTIGATION THAT STARTS FROM AN ANALYSIS OF THE POLICIES OF EACH NATION AND DEVELOP INTO A COMPARATIVE STUDY. AT THIS POINT I ATTEMPT TO DRAW SOME PRELIMINARY IMPRESSIONS ON THE FACTS ESSENTIALLY DECRIBED ABOVE. THE UNIVERSITY POLICIES IN ENGLAND AND NEW ZEALAND HAVE BOTH UNDERGONE A SIGNIFICANT REFORMATORY PROCESS SINCE THE EARLY EIGHTIES; IN BOTH CONTEXTS THE IMPORTANCE OF IDEAS THAT CONSTITUTED THE BASE OF POLITICS UNTIL 1980 WAS QUITE RELEVANT. GENERALLY SPEAKING, IN BOTH CASES THE PRE-REFORM POLICIES WERE INSPIRED BY EGALITARIANISM AND EXPANSION OF THE STUDENT POPULATION WHILE THOSE BROUGHT IN BY THE REFORM WOULD PURSUE EFFICIENCY, QUALITY AND COMPETITIVENESS. UNDOUBTEDLY, IN LINE WITH THIS GENERAL TENDENCY THAT REFLECTS THE HYPOTHESIS PROPOSED, THE TWO UNIVERSITY SYSTEMS PRESENT SEVERAL DIFFERENCES. THE UNIVERSITY SYSTEM IN NEW ZEALAND PROCEEDED STEADILY TOWARDS THE IMPLEMENTATION OF A MANAGERIAL CONCEPTION OF TERTIARY EDUCATION, ESPECIALLY FROM 1996 ONWARDS, IN ACCORDANCE WITH THE REFORMATORY PROCESS OF THE WHOLE PUBLIC SECTOR. IN THE UNITED KINGDOM, AS IN THE REST OF EUROPE, THE NEW APPROACH TO UNIVERSITY POLICY-MAKING HAD TO CONFRONT A DEEP-ROOTED TRADITION OF PROGRESSIVE EDUCATION AND THE IDEA OF EDUCATION EXPANSION THAT IN FACT DOMINATED UNTIL THE EIGHTIES. FROM THIS VIEW POINT THE GOVERNING ACTION OF MARGARET THATCHER GAVE RISE TO A RADICAL CHANGE THAT REVOLUTIONIZED THE OBJECTIVES AND KEY VALUES OF THE WHOLE EDUCATIONAL SYSTEM, IN PARTICULAR IN THE HIGHER EDUCATION SECTOR. IDEAS AS EFFICIENCY, EXCELLENCE AND CONTROL OF THE PERFORMANCE BECAME DECISIVE. THE LABOUR CABINETS OF BLAIR DEVELOPED IN THE WAKE OF CONSERVATIVE REFORMS. THIS APPEARS TO BE A FOCAL POINT OF THIS STUDY THAT OBSERVES HOW ALSO IN NEW ZEALAND THE REFORMING PROCESS OCCURRED TRANSVERSELY DURING PROGRESSIVE AND CONSERVATIVE ADMINISTRATIONS. THE PRELIMINARY IMPRESSION IS THEREFORE THAT IDEAS DEEPLY MARK THE REFORMATIVE PROCESSES: THE AIM OF MY RESEARCH IS TO VERIFY TO WHICH EXTENT THIS STATEMENT IS TRUE. IN ORDER TO BUILD A COMPREHENSIVE ANALYLIS, FURTHER SIGNIFICANT FACTORS WILL HAVE TO BE INVESTIGATED: THE WAY IDEAS ARE PERCEIVED AND IMPLEMENTED BY THE DIFFERENT POLITICAL ELITES; HOW THE VARIOUS SOCIOECONOMIC CONTEXTS INFLUENCE THE REFORMATIVE PROCESS; HOW THE INSTITUTIONAL STRUCTURES CONDITION THE POLICY-MAKING PROCESSES; WHETHER INDIVIDUAL INTERESTS PLAY A ROLE AND, IF YES, TO WHICH EXTENT.

Il conferimento di azienda

Affrontare il tema del conferimento di azienda, così come esaminare ogni aspetto inerente, più in generale, l'azienda, può apparire estremamente arduo e complicato, stante il rischio di ripetere, in modo quasi scontato, "cose già dette" nel corso degli anni, prendendo le mosse da contributi noti ed arcinoti, soprattutto con riferimento alla disciplina dell'imprenditore commerciale, ossia alla prima tematica che viene presa in considerazione da parte del neofita del diritto commerciale. Tuttavia, a ben vedere, l'esaustività e completezza di tali studi non impedisce di affrontare, nell'attualità, alcuni aspetti che, anche grazie ai continui interventi del legislatore, presentano elementi degni di approfondimento. In tale ottica, quindi, è stato impostato il presente lavoro di ricerca. Così, ad un primo capitolo prevalentemente compilativo e volto ad individuare, nell'ordinamento giuridico, tutte le disposizioni che possono assumere rilevanza nell'inquadramento della disciplina del conferimento di azienda, nonchè le nozioni base di tale operazione, ne seguono altri due che si caratterizzano, rispettivamente: a) l'uno (il secondo capitolo), quale concreta individuazione delle modalità di esecuzione dell'operazione di conferimento di azienda, dalla decisione di procedere in tal senso, alla concreta esecuzione dell'apporto; b) l'altro (il terzo capitolo), quale esame di alcuni aspetti problematici che emergono a seguito dell'esecuzione dell'apporto, sia con riferimento agli effetti prodotti nei rapporti ricompresi nell'oggetto dell'apporto, sia con riferimento al ruolo dei diversi soggetti che, direttamente e/o indirettamente, ne subiscono i riflessi. In entrambi i capitoli (due e tre), peraltro, si affrontano non solo aspetti connessi alla disciplina dell'impresa ancora in bonis, ma anche, data l'attualità degli interventi operati dal legislatore nell'ambito della risoluzione della crisi dell'impresa, le questioni inerenti l'utilizzo di tale operazione quale forma di liquidazione dell'attivo. Si chiude l'elaborato con alcune riflessioni inerenti le forme di reazione agli abusi che, sovente, caratterizzano l'apporto dell'azienda.

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.

La costruzione razionale della casa. Scritti e progetti di Giuseppe Pagano

The rational construction of the house. The writings and projects of Giuseppe Pagano Description, themes and research objectives The research aims at analysing the architecture of Giuseppe Pagano, which focuses on the theme of dwelling, through the reading of 3 of his house projects. On the one hand, these projects represent “minor” works not thoroughly known by Pagano’s contemporary critics; on the other they emphasise a particular methodological approach, which serves the author to explore a theme closely linked to his theoretical thought. The house project is a key to Pagano’s research, given its ties to the socio-cultural and political conditions in which the architect was working, so that it becomes a mirror of one of his specific and theoretical path, always in a state of becoming. Pagano understands architecture as a “servant of the human being”, subject to a “utilitarian slavery” since it is a clear, essential and “modest” answer to specific human needs, free from aprioristic aesthetic and formal choices. It is a rational architecture in sensu stricto; it constitutes a perfect synthesis between cause and effect and between function and form. The house needs to accommodate these principles because it is closely intertwined with human needs and intimately linked to a specific place, climatic conditions and technical and economical possibilities. Besides, differently from his public and common masterpieces such as the Palazzo Gualino, the Istituto di Fisica and the Università Commerciale Bocconi, the house projects are representative of a precise project will, which is expressed in a more authentic way, partially freed from political influences and dogmatic preoccupations and, therefore, far from the attempt to research a specific expressive language. I believe that the house project better represents that “ingenuity”, freshness and “sincerity” that Pagano identifies with the minor architecture, thereby revealing a more authentic expression of his understanding of a project. Therefore, the thesis, by tracing the theoretical research of Pagano through the analysis of some of his designed and built works, attempts to identify a specific methodological approach to Pagano’s project, which, developed through time, achieves a certain clarity in the 1930s. In fact, this methodological approach becomes more evident in his last projects, mainly regarding the house and the urban space. These reflect the attempt to respond to the new social needs and, at the same time, they also are an expression of a freer idea of built architecture, closely linked with the place and with the human being who dwells it. The three chosen projects (Villa Colli, La Casa a struttura d’acciaio and Villa Caraccio) make Pagano facing different places, different customers and different economic and technical conditions, which, given the author’s biography, correspond to important historical and political conditions. This is the reason why the projects become apparently distant works, both linguistically and conceptually, to the point that one can define them as ”eclectic”. However, I argue that this eclecticism is actually an added value to the architectural work of Pagano, steaming from the use of a method which, having as a basis the postulate of a rational architecture as essence and logic of building, finds specific variations depending on the multiple variables to be addressed by the project. This is the methodological heritage that Pagano learns from the tradition, especially that of the rural residential architecture, defined by Pagano as a “dictionary of the building logic of man”, as an “a-stylistic background”. For Pagano this traditional architecture is a clear expression of the relationships between a theme and its development, an architectural “fact” that is resolved with purely technical and utilitarian aims and with a spontaneous development far from any aprioristic theoretical principle. Architecture, therefore, cannot be an invention for Pagano and the personal contribution of each architect has to consider his/her close relationship with the specific historical context, place and new building methods. These are basic principles in the methodological approach that drives a great deal of his research and that also permits his thought to be modern. I argue that both ongoing and new collaborations with younger protagonists of the culture and architecture of the period are significant for the development of his methodology. These encounters represent the will to spread his own understanding of the “new architecture” as well as a way of self-renewal by confronting the self with new themes and realities and by learning from his collaborators. Thesis’ outline The thesis is divided in two principal parts, each articulated in four chapters attempting to offer a new reading of the theory and work of Pagano by emphasising the central themes of the research. The first chapter is an introduction to the thesis and to the theme of the rational house, as understood and developed in its typological and technical aspects by Pagano and by other protagonists of the Italian rationalism of the 1930s. Here the attention is on two different aspects defining, according to Pagano, the house project: on the one hand, the typological renewal, aimed at defining a “standard form” as a clear and essential answer to certain needs and variables of the project leading to different formal expressions. On the other, it focuses on the building, understood as a technique to “produce” architecture, where new technologies and new materials are not merely tools but also essential elements of the architectural work. In this way the villa becomes different from the theme of the common house or from that of the minimalist house, by using rules in the choice of material and in the techniques that are every time different depending on the theme under exploration and on the contingency of place. It is also visible the rigorous rationalism that distinguishes the author's appropriation of certain themes of rural architecture. The pages of “Casabella” and the events of the contemporary Triennali form the preliminary material for the writing of this chapter given that they are primary sources to individuate projects and writings produced by Pagano and contemporary architects on this theme. These writings and projects, when compared, reconstruct the evolution of the idea of the rational house and, specifically, of the personal research of Pagano. The second part regards the reading of three of Pagano’s projects of houses as a built verification of his theories. This section constitutes the central part of the thesis since it is aimed at detecting a specific methodological approach showing a theoretical and ideological evolution expressed in the vast edited literature. The three projects that have been chosen explore the theme of the house, looking at various research themes that the author proposes and that find continuity in the affirmation of a specific rationalism, focussed on concepts such as essentiality, utility, functionality and building honesty. These concepts guide the thought and the activities of Pagano, also reflecting a social and cultural period. The projects span from the theme of the villa moderna, Villa Colli, which, inspired by the architecture of North Europe, anticipates a specific rationalism of Pagano based on rigour, simplicity and essentiality, to the theme of the common house, Casa a struttura d’acciaio, la casa del domani, which ponders on the definition of new living spaces and, moreover, on new concepts of standardisation, economical efficiency and new materials responding to the changing needs of the modern society. Finally, the third project returns to the theme of the, Villa Caraccio, revisiting it with new perspectives. These perspectives find in the solution of the open plant, in the openness to nature and landscape and in the revisiting of materials and local building systems that idea of the freed house, which express clearly a new theoretical thought. Methodology It needs to be noted that due to the lack of an official Archive of Pagano’s work, the analysis of his work has been difficult and this explains the necessity to read the articles and the drawings published in the pages of «Casabella» and «Domus». As for the projects of Villa Colli and Casa a struttura d’acciaio, parts of the original drawings have been consulted. These drawings are not published and are kept in private archives of the collaborators of Pagano. The consultation of these documents has permitted the analysis of the cited works, which have been subject to a more complete reading following the different proposed solutions, which have permitted to understand the project path. The projects are analysed thought the method of comparison and critical reading which, specifically, means graphical elaborations and analytical schemes, mostly reconstructed on the basis of original projects but, where possible, also on a photographic investigation. The focus is on the project theme which, beginning with a specific living (dwelling) typology, finds variations because of the historico-political context in which Pagano is embedded and which partially shapes his research and theoretical thought, then translated in the built work. The analysis of the work follows, beginning, where possible, from a reconstruction of the evolution of the project as elaborated on the basis of the original documents and ending on an analysis of the constructive principles and composition. This second phase employs a methodology proposed by Pagano in his article Piante di ville, which, as expected, focuses on the plant as essential tool to identify the “true practical and poetic qualities of the construction”(Pagano, «Costruzioni-Casabella», 1940, p. 2). The reading of the project is integrated with the constructive analyses related to the technical aspects of the house which, in the case of Casa a struttura d’acciaio, play an important role in the project, while in Villa Colli and in Villa Caraccio are principally linked to the choice of materials for the construction of the different architectural elements. These are nonetheless key factors in the composition of the work. Future work could extend this reading to other house projects to deepen the research that could be completed with the consultation of Archival materials, which are missing at present. Finally, in the appendix I present a critical selection of the Pagano’s writings, which recall the themes discussed and embodied by the three projects. The texts have been selected among the articles published in Casabella and in other journals, completing the reading of the project work which cannot be detached from his theoretical thought. Moving from theory to project, we follow a path that brings us to define and deepen the central theme of the thesis: rational building as the principal feature of the architectural research of Pagano, which is paraphrased in multiple ways in his designed and built works.

NMR, Metabonomics and Molecular Profiles: Applications to the Quality Assessment of Foodstuffs

Nuclear Magnetic Resonance (NMR) is a branch of spectroscopy that is based on the fact that many atomic nuclei may be oriented by a strong magnetic field and will absorb radiofrequency radiation at characteristic frequencies. The parameters that can be measured on the resulting spectral lines (line positions, intensities, line widths, multiplicities and transients in time-dependent experi-ments) can be interpreted in terms of molecular structure, conformation, molecular motion and other rate processes. In this way, high resolution (HR) NMR allows performing qualitative and quantitative analysis of samples in solution, in order to determine the structure of molecules in solution and not only. In the past, high-field NMR spectroscopy has mainly concerned with the elucidation of chemical structure in solution, but today is emerging as a powerful exploratory tool for probing biochemical and physical processes. It represents a versatile tool for the analysis of foods. In literature many NMR studies have been reported on different type of food such as wine, olive oil, coffee, fruit juices, milk, meat, egg, starch granules, flour, etc using different NMR techniques. Traditionally, univariate analytical methods have been used to ex-plore spectroscopic data. This method is useful to measure or to se-lect a single descriptive variable from the whole spectrum and , at the end, only this variable is analyzed. This univariate methods ap-proach, applied to HR-NMR data, lead to different problems due especially to the complexity of an NMR spectrum. In fact, the lat-ter is composed of different signals belonging to different mole-cules, but it is also true that the same molecules can be represented by different signals, generally strongly correlated. The univariate methods, in this case, takes in account only one or a few variables, causing a loss of information. Thus, when dealing with complex samples like foodstuff, univariate analysis of spectra data results not enough powerful. Spectra need to be considered in their wholeness and, for analysing them, it must be taken in consideration the whole data matrix: chemometric methods are designed to treat such multivariate data. Multivariate data analysis is used for a number of distinct, differ-ent purposes and the aims can be divided into three main groups: • data description (explorative data structure modelling of any ge-neric n-dimensional data matrix, PCA for example); • regression and prediction (PLS); • classification and prediction of class belongings for new samples (LDA and PLS-DA and ECVA). The aim of this PhD thesis was to verify the possibility of identify-ing and classifying plants or foodstuffs, in different classes, based on the concerted variation in metabolite levels, detected by NMR spectra and using the multivariate data analysis as a tool to inter-pret NMR information. It is important to underline that the results obtained are useful to point out the metabolic consequences of a specific modification on foodstuffs, avoiding the use of a targeted analysis for the different metabolites. The data analysis is performed by applying chemomet-ric multivariate techniques to the NMR dataset of spectra acquired. The research work presented in this thesis is the result of a three years PhD study. This thesis reports the main results obtained from these two main activities: A1) Evaluation of a data pre-processing system in order to mini-mize unwanted sources of variations, due to different instrumental set up, manual spectra processing and to sample preparations arte-facts; A2) Application of multivariate chemiometric models in data analy-sis.

Ricerca, mediante tecniche di proteomica, di biomarcatori proteici utili nella valutazione della qualità degli alimenti di origine animale

In the last decades, the increase of industrial activities and of the request for the world food requirement, the intensification of natural resources exploitation, directly connected to pollution, have aroused an increasing interest of the public opinion towards initiatives linked to the regulation of food production, as well to the institution of a modern legislation for the consumer guardianship. This work was planned taking into account some important thematics related to marine environment, collecting and showing the data obtained from the studies made on different marine species of commercial interest (Chamelea gallina, Mytilus edulis, Ostrea edulis, Crassostrea gigas, Salmo salar, Gadus morhua). These studies have evaluated the effects of important physic and chemical parameters variations (temperature, xenobiotics like drugs, hydrocarbons and pesticides) on cells involved in the immune defence (haemocytes) and on some important enzymatic systems involved in xenobiotic biotransformation processes (cytochrome P450 complex) and in the related antioxidant defence processes (Superoxide dismutase, Catalase, Heat Shock Protein), from a biochemical and bimolecular point of view. Oxygen is essential in the biological answer of a living organism. Its consume in the normal cellular breathing physiological processes and foreign substances biotransformation, leads to reactive oxygen species (ROS) formation, potentially toxic and responsible of biological macromolecules damages with consequent pathologies worsening. Such processes can bring to a qualitative alteration of the derived products, but also to a general state of suffering that in the most serious cases can provoke the death of the organism, with important repercussions in economic field, in the output of the breedings, of fishing and of aquaculture. In this study it seemed interesting to apply also alternative methodologies currently in use in the medical field (cytofluorimetry) and in proteomic studies (bidimensional electrophoresis, mass spectrometry) with the aim of identify new biomarkers to place beside the traditional methods for the control of the animal origin food quality. From the results it’s possible to point out some relevant aspects from each experiment: 1. The cytofluorimetric techniques applied to O. edulis and C. gigas could bring to important developments in the search of alternative methods that quickly allows to identify with precision the origin of a specific sample, contributing to oppose possible alimentary frauds, in this case for example related to presence of a different species, also under a qualitative profile, but morpholgically similar. A concrete perspective for the application in the inspective field of this method has to be confirmed by further laboratory tests that take also in account in vivo experiments to evaluate the effect in the whole organism of the factors evaluated only on haemocytes in vitro. These elements suggest therefore the possibility to suit the cytofluorimetric methods for the study of animal organisms of food interest, still before these enter the phase of industrial working processes, giving useful information about the possible presence of contaminants sources that can induce an increase of the immune defence and an alteration of normal cellular parameter values. 2. C. gallina immune system has shown an interesting answer to benzo[a]pyrene (B[a]P) exposure, dose and time dependent, with a significant decrease of the expression and of the activity of one of the most important enzymes involved in the antioxidant defence in haemocytes and haemolymph. The data obtained are confirmed by several measurements of physiological parameters, that together with the decrease of the activity of 7-etossi-resourifine-O-deetilase (EROD linked to xenobiotic biotransformation processes) during exposure, underline the major effects of B[a]P action. The identification of basal levels of EROD supports the possible presence of CYP1A subfamily in the invertebrates, still today controversial, never identified previously in C. gallina and never isolated in the immune cells, as confirmed instead in this study with the identification of CYP1A-immunopositive protein (CYP1A-IPP). This protein could reveal a good biomarker at the base of a simple and quick method that could give clear information about specific pollutants presence, even at low concentrations in the environment where usually these organisms are fished before being commercialized. 3. In this experiment it has been evaluated the effect of the antibiotic chloramphenicol (CA) in an important species of commercial interest, Chamelea gallina. Chloramphenicol is a drug still used in some developing countries, also in veterinary field. Controls to evaluate its presence in the alimentary products of animal origin, can reveal ineffective whereas the concentration results to be below the limit of sensitivity of the instruments usually used in this type of analysis. Negative effects of CA towards the CYP1A- IPP proteins, underlined in this work, seem to be due to the attack of free radicals resultant from the action of the antibiotic. This brings to a meaningful alteration of the biotransformation mechanisms through the free radicals. It seems particularly interesting to pay attention to the narrow relationships in C. gallina, between SOD/CAT and CYP450 system, actively involved in detoxification mechanism, especially if compared with the few similar works today present about mollusc, a group that is composed by numerous species that enter in the food field and on which constant controls are necessary to evaluate in a rapid and effective way the presence of possible contaminations. 4. The investigations on fishes (Gadus morhua, and Salmo salar) and on a bivalve mollusc (Mytilus edulis) have allowed to evaluate different aspects related to the possibility to identify a biomarker for the evaluation of the health of organisms of food interest and consequently for the quality of the final product through 2DE methodologies. In the seafood field these techniques are currently used with a discreet success only for vertebrates (fishes), while in the study of the invertebrates (molluscs) there are a lot of difficulties. The results obtained in this work have underline several problems in the correct identification of the isolated proteins in animal organisms of which doesn’t currently exist a complete genomic sequence. This brings to attribute some identities on the base of the comparison with similar proteins in other animal groups, incurring in the possibility to obtain inaccurate data and above all discordant with those obtained on the same animals by other authors. Nevertheless the data obtained in this work after MALDI-ToF analysis, result however objective and the spectra collected could be again analyzed in the future after the update of genomic database related to the species studied. 4-A. The investigation about the presence of HSP70 isoforms directly induced by different phenomena of stress like B[a]P presence, has used bidimensional electrophoresis methods in C. gallina, that have allowed to isolate numerous protein on 2DE gels, allowing the collection of several spots currently in phase of analysis with MALDI-ToF-MS. The present preliminary work has allowed therefore to acquire and to improve important methodologies in the study of cellular parameters and in the proteomic field, that is not only revealed of great potentiality in the application in medical and veterinary field, but also in the field of the inspection of the foods with connections to the toxicology and the environmental pollution. Such study contributes therefore to the search of rapid and new methodologies, that can increase the inspective strategies, integrating themselves with those existing, but improving at the same time the general background of information related to the state of health of the considered animal organism, with the possibility, still hypothetical, to replace in particular cases the employment of the traditional techniques in the alimentary field.

Biomass Gasification - Process analysis and dimensioning aspects for downdraft units and gas cleaning lines

In such territories where food production is mostly scattered in several small / medium size or even domestic farms, a lot of heterogeneous residues are produced yearly, since farmers usually carry out different activities in their properties. The amount and composition of farm residues, therefore, widely change during year, according to the single production process periodically achieved. Coupling high efficiency micro-cogeneration energy units with easy handling biomass conversion equipments, suitable to treat different materials, would provide many important advantages to the farmers and to the community as well, so that the increase in feedstock flexibility of gasification units is nowadays seen as a further paramount step towards their wide spreading in rural areas and as a real necessity for their utilization at small scale. Two main research topics were thought to be of main concern at this purpose, and they were therefore discussed in this work: the investigation of fuels properties impact on gasification process development and the technical feasibility of small scale gasification units integration with cogeneration systems. According to these two main aspects, the present work was thus divided in two main parts. The first one is focused on the biomass gasification process, that was investigated in its theoretical aspects and then analytically modelled in order to simulate thermo-chemical conversion of different biomass fuels, such as wood (park waste wood and softwood), wheat straw, sewage sludge and refuse derived fuels. The main idea is to correlate the results of reactor design procedures with the physical properties of biomasses and the corresponding working conditions of gasifiers (temperature profile, above all), in order to point out the main differences which prevent the use of the same conversion unit for different materials. At this scope, a gasification kinetic free model was initially developed in Excel sheets, considering different values of air to biomass ratio and the downdraft gasification technology as particular examined application. The differences in syngas production and working conditions (process temperatures, above all) among the considered fuels were tried to be connected to some biomass properties, such elementary composition, ash and water contents. The novelty of this analytical approach was the use of kinetic constants ratio in order to determine oxygen distribution among the different oxidation reactions (regarding volatile matter only) while equilibrium of water gas shift reaction was considered in gasification zone, by which the energy and mass balances involved in the process algorithm were linked together, as well. Moreover, the main advantage of this analytical tool is the easiness by which the input data corresponding to the particular biomass materials can be inserted into the model, so that a rapid evaluation on their own thermo-chemical conversion properties is possible to be obtained, mainly based on their chemical composition A good conformity of the model results with the other literature and experimental data was detected for almost all the considered materials (except for refuse derived fuels, because of their unfitting chemical composition with the model assumptions). Successively, a dimensioning procedure for open core downdraft gasifiers was set up, by the analysis on the fundamental thermo-physical and thermo-chemical mechanisms which are supposed to regulate the main solid conversion steps involved in the gasification process. Gasification units were schematically subdivided in four reaction zones, respectively corresponding to biomass heating, solids drying, pyrolysis and char gasification processes, and the time required for the full development of each of these steps was correlated to the kinetics rates (for pyrolysis and char gasification processes only) and to the heat and mass transfer phenomena from gas to solid phase. On the basis of this analysis and according to the kinetic free model results and biomass physical properties (particles size, above all) it was achieved that for all the considered materials char gasification step is kinetically limited and therefore temperature is the main working parameter controlling this step. Solids drying is mainly regulated by heat transfer from bulk gas to the inner layers of particles and the corresponding time especially depends on particle size. Biomass heating is almost totally achieved by the radiative heat transfer from the hot walls of reactor to the bed of material. For pyrolysis, instead, working temperature, particles size and the same nature of biomass (through its own pyrolysis heat) have all comparable weights on the process development, so that the corresponding time can be differently depending on one of these factors according to the particular fuel is gasified and the particular conditions are established inside the gasifier. The same analysis also led to the estimation of reaction zone volumes for each biomass fuel, so as a comparison among the dimensions of the differently fed gasification units was finally accomplished. Each biomass material showed a different volumes distribution, so that any dimensioned gasification unit does not seem to be suitable for more than one biomass species. Nevertheless, since reactors diameters were found out quite similar for all the examined materials, it could be envisaged to design a single units for all of them by adopting the largest diameter and by combining together the maximum heights of each reaction zone, as they were calculated for the different biomasses. A total height of gasifier as around 2400mm would be obtained in this case. Besides, by arranging air injecting nozzles at different levels along the reactor, gasification zone could be properly set up according to the particular material is in turn gasified. Finally, since gasification and pyrolysis times were found to considerably change according to even short temperature variations, it could be also envisaged to regulate air feeding rate for each gasified material (which process temperatures depend on), so as the available reactor volumes would be suitable for the complete development of solid conversion in each case, without even changing fluid dynamics behaviour of the unit as well as air/biomass ratio in noticeable measure. The second part of this work dealt with the gas cleaning systems to be adopted downstream the gasifiers in order to run high efficiency CHP units (i.e. internal engines and micro-turbines). Especially in the case multi–fuel gasifiers are assumed to be used, weightier gas cleaning lines need to be envisaged in order to reach the standard gas quality degree required to fuel cogeneration units. Indeed, as the more heterogeneous feed to the gasification unit, several contaminant species can simultaneously be present in the exit gas stream and, as a consequence, suitable gas cleaning systems have to be designed. In this work, an overall study on gas cleaning lines assessment is carried out. Differently from the other research efforts carried out in the same field, the main scope is to define general arrangements for gas cleaning lines suitable to remove several contaminants from the gas stream, independently on the feedstock material and the energy plant size The gas contaminant species taken into account in this analysis were: particulate, tars, sulphur (in H2S form), alkali metals, nitrogen (in NH3 form) and acid gases (in HCl form). For each of these species, alternative cleaning devices were designed according to three different plant sizes, respectively corresponding with 8Nm3/h, 125Nm3/h and 350Nm3/h gas flows. Their performances were examined on the basis of their optimal working conditions (efficiency, temperature and pressure drops, above all) and their own consumption of energy and materials. Successively, the designed units were combined together in different overall gas cleaning line arrangements, paths, by following some technical constraints which were mainly determined from the same performance analysis on the cleaning units and from the presumable synergic effects by contaminants on the right working of some of them (filters clogging, catalysts deactivation, etc.). One of the main issues to be stated in paths design accomplishment was the tars removal from the gas stream, preventing filters plugging and/or line pipes clogging At this scope, a catalytic tars cracking unit was envisaged as the only solution to be adopted, and, therefore, a catalytic material which is able to work at relatively low temperatures was chosen. Nevertheless, a rapid drop in tars cracking efficiency was also estimated for this same material, so that an high frequency of catalysts regeneration and a consequent relevant air consumption for this operation were calculated in all of the cases. Other difficulties had to be overcome in the abatement of alkali metals, which condense at temperatures lower than tars, but they also need to be removed in the first sections of gas cleaning line in order to avoid corrosion of materials. In this case a dry scrubber technology was envisaged, by using the same fine particles filter units and by choosing for them corrosion resistant materials, like ceramic ones. Besides these two solutions which seem to be unavoidable in gas cleaning line design, high temperature gas cleaning lines were not possible to be achieved for the two larger plant sizes, as well. Indeed, as the use of temperature control devices was precluded in the adopted design procedure, ammonia partial oxidation units (as the only considered methods for the abatement of ammonia at high temperature) were not suitable for the large scale units, because of the high increase of reactors temperature by the exothermic reactions involved in the process. In spite of these limitations, yet, overall arrangements for each considered plant size were finally designed, so that the possibility to clean the gas up to the required standard degree was technically demonstrated, even in the case several contaminants are simultaneously present in the gas stream. Moreover, all the possible paths defined for the different plant sizes were compared each others on the basis of some defined operational parameters, among which total pressure drops, total energy losses, number of units and secondary materials consumption. On the basis of this analysis, dry gas cleaning methods proved preferable to the ones including water scrubber technology in al of the cases, especially because of the high water consumption provided by water scrubber units in ammonia adsorption process. This result is yet connected to the possibility to use activated carbon units for ammonia removal and Nahcolite adsorber for chloride acid. The very high efficiency of this latter material is also remarkable. Finally, as an estimation of the overall energy loss pertaining the gas cleaning process, the total enthalpy losses estimated for the three plant sizes were compared with the respective gas streams energy contents, these latter obtained on the basis of low heating value of gas only. This overall study on gas cleaning systems is thus proposed as an analytical tool by which different gas cleaning line configurations can be evaluated, according to the particular practical application they are adopted for and the size of cogeneration unit they are connected to.

Use of solvents and environmental friendly materials for applications in Green Chemistry

The present Thesis studies three alternative solvent groups as sustainable replacement of traditional organic solvents. Some aspects of fluorinated solvents, supercritical fluids and ionic liquids, have been analysed with a critical approach and their effective “greenness” has been evaluated from the points of view of the synthesis, the properties and the applications. In particular, the attention has been put on the environmental and human health issues, evaluating the eco-toxicity, the toxicity and the persistence, to underline that applicability and sustainability are subjects with equal importance. The “green” features of fluorous solvents and supercritical fluids are almost well-established; in particular supercritical carbon dioxide (scCO2) is probably the “greenest” solvent among the alternative solvent systems developed in the last years, enabling to combine numerous advantages both from the point of view of industrial/technological applications and eco-compatibility. In the Thesis the analysis of these two classes of alternative solvents has been mainly focused on their applicability, rather than the evaluation of their environmental impact. Specifically they have been evaluated as alternative media for non-aqueous biocatalysis. For this purpose, the hydrophobic ion pairing (HIP), which allows solubilising enzymes in apolar solvents by an ion pairing between the protein and a surfactant, has been investigated as effective enzymatic derivatisation technique to improve the catalytic activity under homogeneous conditions in non conventional media. The results showed that the complex enzyme-surfactant was much more active both in fluorous solvents and in supercritical carbon dioxide than the native form of the enzyme. Ionic liquids, especially imidazolium salts, have been proposed some years ago as “fully green” alternative solvents; however this epithet does not take into account several “brown” aspects such as their synthesis from petro-chemical starting materials, their considerable eco-toxicity, toxicity and resistance to biodegradation, and the difficulty of clearly outline applications in which ionic liquids are really more advantageous than traditional solvents. For all of these reasons in this Thesis a critical analysis of ionic liquids has been focused on three main topics: i) alternative synthesis by introducing structural moieties which could reduce the toxicity of the most known liquid salts, and by using starting materials from renewable resources; ii) on the evaluation of their environmental impact through eco-toxicological tests (Daphnia magna and Vibrio fischeri acute toxicity tests, and algal growth inhibition), toxicity tests (MTT test, AChE inhibition and LDH release tests) and fate and rate of aerobic biodegradation in soil and water; iii) and on the demonstration of their effectiveness as reaction media in organo-catalysis and as extractive solvents in the recovery of vegetable oil from terrestrial and aquatic biomass. The results about eco-toxicity tests with Daphnia magna, Vibrio fischeri and algae, and toxicity assay using cultured cell lines, clearly indicate that the difference in toxicity between alkyl and oxygenated cations relies in differences of polarity, according to the general trend of decreasing toxicity by decreasing the lipophilicity. Independently by the biological approach in fact, all the results are in agreement, showing a lower toxicity for compounds with oxygenated lateral chains than for those having purely alkyl lateral chains. These findings indicate that an appropriate choice of cation and anion structures is important not only to design the IL with improved and suitable chemico-physical properties but also to obtain safer and eco-friendly ILs. Moreover there is a clear indication that the composition of the abiotic environment has to be taken into account when the toxicity of ILs in various biological test systems is analysed, because, for example, the data reported in the Thesis indicate a significant influence of salinity variations on algal toxicity. Aerobic biodegradation of four imidazolium ionic liquids, two alkylated and two oxygenated, in soil was evaluated for the first time. Alkyl ionic liquids were shown to be biodegradable over the 6 months test period, and in contrast no significant mineralisation was observed with oxygenated derivatives. A different result was observed in the aerobic biodegradation of alkylated and oxygenated pyridinium ionic liquids in water because all the ionic liquids were almost completely degraded after 10 days, independently by the number of oxygen in the lateral chain of the cation. The synthesis of new ionic liquids by using renewable feedstock as starting materials, has been developed through the synthesis of furan-based ion pairs from furfural. The new ammonium salts were synthesised in very good yields, good purity of the products and wide versatility, combining low melting points with high decomposition temperatures and reduced viscosities. Regarding the possible applications as surfactants and biocides, furan-based salts could be a valuable alternative to benzyltributylammonium salts and benzalkonium chloride that are produced from non-renewable resources. A new procedure for the allylation of ketones and aldehydes with tetraallyltin in ionic liquids was developed. The reaction afforded high yields both in sulfonate-containing ILs and in ILs without sulfonate upon addition of a small amount of sulfonic acid. The checked reaction resulted in peculiar chemoselectivity favouring aliphatic substrates towards aromatic ketones and good stereoselectivity in the allylation of levoglucosenone. Finally ILs-based systems could be easily and successfully recycled, making the described procedure environmentally benign. The potential role of switchable polarity solvents as a green technology for the extraction of vegetable oil from terrestrial and aquatic biomass has been investigated. The extraction efficiency of terrestrial biomass rich in triacylglycerols, as soy bean flakes and sunflower seeds, was comparable to those of traditional organic solvents, being the yield of vegetable oils recovery very similar. Switchable polarity solvents as been also exploited for the first time in the extraction of hydrocarbons from the microalga Botryococcus braunii, demonstrating the efficiency of the process for the extraction of both dried microalgal biomass and directly of the aqueous growth medium. The switchable polarity solvents exhibited better extraction efficiency than conventional solvents, both with dried and liquid samples. This is an important issue considering that the harvest and the dewatering of algal biomass have a large impact on overall costs and energy balance.

Development of muliplexed analytical methods based on bioluminescent whole-cell biosensors

The subject of this Ph.D. research thesis is the development and application of multiplexed analytical methods based on bioluminescent whole-cell biosensors. One of the main goals of analytical chemistry is multianalyte testing in which two or more analytes are measured simultaneously in a single assay. The advantages of multianalyte testing are work simplification, high throughput, and reduction in the overall cost per test. The availability of multiplexed portable analytical systems is of particular interest for on-field analysis of clinical, environmental or food samples as well as for the drug discovery process. To allow highly sensitive and selective analysis, these devices should combine biospecific molecular recognition with ultrasensitive detection systems. To address the current need for rapid, highly sensitive and inexpensive devices for obtaining more data from each sample,genetically engineered whole-cell biosensors as biospecific recognition element were combined with ultrasensitive bioluminescence detection techniques. Genetically engineered cell-based sensing systems were obtained by introducing into bacterial, yeast or mammalian cells a vector expressing a reporter protein whose expression is controlled by regulatory proteins and promoter sequences. The regulatory protein is able to recognize the presence of the analyte (e.g., compounds with hormone-like activity, heavy metals…) and to consequently activate the expression of the reporter protein that can be readily measured and directly related to the analyte bioavailable concentration in the sample. Bioluminescence represents the ideal detection principle for miniaturized analytical devices and multiplexed assays thanks to high detectability in small sample volumes allowing an accurate signal localization and quantification. In the first chapter of this dissertation is discussed the obtainment of improved bioluminescent proteins emitting at different wavelenghts, in term of increased thermostability, enhanced emission decay kinetic and spectral resolution. The second chapter is mainly focused on the use of these proteins in the development of whole-cell based assay with improved analytical performance. In particular since the main drawback of whole-cell biosensors is the high variability of their analyte specific response mainly caused by variations in cell viability due to aspecific effects of the sample’s matrix, an additional bioluminescent reporter has been introduced to correct the analytical response thus increasing the robustness of the bioassays. The feasibility of using a combination of two or more bioluminescent proteins for obtaining biosensors with internal signal correction or for the simultaneous detection of multiple analytes has been demonstrated by developing a dual reporter yeast based biosensor for androgenic activity measurement and a triple reporter mammalian cell-based biosensor for the simultaneous monitoring of two CYP450 enzymes activation, involved in cholesterol degradation, with the use of two spectrally resolved intracellular luciferases and a secreted luciferase as a control for cells viability. In the third chapter is presented the development of a portable multianalyte detection system. In order to develop a portable system that can be used also outside the laboratory environment even by non skilled personnel, cells have been immobilized into a new biocompatible and transparent polymeric matrix within a modified clear bottom black 384 -well microtiter plate to obtain a bioluminescent cell array. The cell array was placed in contact with a portable charge-coupled device (CCD) light sensor able to localize and quantify the luminescent signal produced by different bioluminescent whole-cell biosensors. This multiplexed biosensing platform containing whole-cell biosensors was successfully used to measure the overall toxicity of a given sample as well as to obtain dose response curves for heavy metals and to detect hormonal activity in clinical samples (PCT/IB2010/050625: “Portable device based on immobilized cells for the detection of analytes.” Michelini E, Roda A, Dolci LS, Mezzanotte L, Cevenini L , 2010). At the end of the dissertation some future development steps are also discussed in order to develop a point of care (POCT) device that combine portability, minimum sample pre-treatment and highly sensitive multiplexed assays in a short assay time. In this POCT perspective, field-flow fractionation (FFF) techniques, in particular gravitational variant (GrFFF) that exploit the earth gravitational field to structure the separation, have been investigated for cells fractionation, characterization and isolation. Thanks to the simplicity of its equipment, amenable to miniaturization, the GrFFF techniques appears to be particularly suited for its implementation in POCT devices and may be used as pre-analytical integrated module to be applied directly to drive target analytes of raw samples to the modules where biospecifc recognition reactions based on ultrasensitive bioluminescence detection occurs, providing an increase in overall analytical output.

Investigating the role of single point mutations in the human proteome: a computational study

In the post genomic era with the massive production of biological data the understanding of factors affecting protein stability is one of the most important and challenging tasks for highlighting the role of mutations in relation to human maladies. The problem is at the basis of what is referred to as molecular medicine with the underlying idea that pathologies can be detailed at a molecular level. To this purpose scientific efforts focus on characterising mutations that hamper protein functions and by these affect biological processes at the basis of cell physiology. New techniques have been developed with the aim of detailing single nucleotide polymorphisms (SNPs) at large in all the human chromosomes and by this information in specific databases are exponentially increasing. Eventually mutations that can be found at the DNA level, when occurring in transcribed regions may then lead to mutated proteins and this can be a serious medical problem, largely affecting the phenotype. Bioinformatics tools are urgently needed to cope with the flood of genomic data stored in database and in order to analyse the role of SNPs at the protein level. In principle several experimental and theoretical observations are suggesting that protein stability in the solvent-protein space is responsible of the correct protein functioning. Then mutations that are found disease related during DNA analysis are often assumed to perturb protein stability as well. However so far no extensive analysis at the proteome level has investigated whether this is the case. Also computationally methods have been developed to infer whether a mutation is disease related and independently whether it affects protein stability. Therefore whether the perturbation of protein stability is related to what it is routinely referred to as a disease is still a big question mark. In this work we have tried for the first time to explore the relation among mutations at the protein level and their relevance to diseases with a large-scale computational study of the data from different databases. To this aim in the first part of the thesis for each mutation type we have derived two probabilistic indices (for 141 out of 150 possible SNPs): the perturbing index (Pp), which indicates the probability that a given mutation effects protein stability considering all the “in vitro” thermodynamic data available and the disease index (Pd), which indicates the probability of a mutation to be disease related, given all the mutations that have been clinically associated so far. We find with a robust statistics that the two indexes correlate with the exception of all the mutations that are somatic cancer related. By this each mutation of the 150 can be coded by two values that allow a direct comparison with data base information. Furthermore we also implement computational methods that starting from the protein structure is suited to predict the effect of a mutation on protein stability and find that overpasses a set of other predictors performing the same task. The predictor is based on support vector machines and takes as input protein tertiary structures. We show that the predicted data well correlate with the data from the databases. All our efforts therefore add to the SNP annotation process and more importantly found the relationship among protein stability perturbation and the human variome leading to the diseasome.

Biomechanics of sprint running: a methodological contribution

Sports biomechanics describes human movement from a performance enhancement and an injury reduction perspective. In this respect, the purpose of sports scientists is to support coaches and physicians with reliable information about athletes’ technique. The lack of methods allowing for in-field athlete evaluation as well as for accurate joint force estimates represents, to date, the main limitation to this purpose. The investigations illustrated in the present thesis aimed at providing a contribution towards the development of the above mentioned methods. Two complementary approaches were adopted: a Low Resolution Approach – related to performance assessment – where the use of wearable inertial measurement units is exploited during different phases of sprint running, and a High Resolution Approach – related to joint kinetics estimate for injury prevention – where subject-specific, non-rigid constraints for knee joint kinematic modelling used in multi-body optimization techniques are defined. Results obtained using the Low Resolution Approach indicated that, due to their portability and inexpensiveness, inertial measurement systems are a valid alternative to laboratory-based instrumentation for in-field performance evaluation of sprint running. Using acceleration and angular velocity data, the following quantities were estimated: trunk inclination and angular velocity, instantaneous horizontal velocity and displacement of a point approximating the centre of mass, and stride and support phase durations. As concerns the High Resolution Approach, results indicated that the length of the anterior cruciate and lateral collateral ligaments decreased, while that of the deep bundle of the medial collateral ligament increased significantly during flexion. Variations of the posterior cruciate and the superficial bundle of the medial collateral ligament lengths were concealed by the experimental indeterminacy. A mathematical model was provided that allowed the estimate of subject-specific ligament lengths as a function of knee flexion and that can be integrated in a multi-body optimization procedure.

Accretion disk winds in active galactic nuclei: an X-ray view

This Thesis focuses on the X-ray study of the inner regions of Active Galactic Nuclei, in particular on the formation of high velocity winds by the accretion disk itself. Constraining AGN winds physical parameters is of paramount importance both for understanding the physics of the accretion/ejection flow onto supermassive black holes, and for quantifying the amount of feedback between the SMBH and its environment across the cosmic time. The sources selected for the present study are BAL, mini-BAL, and NAL QSOs, known to host high-velocity winds associated to the AGN nuclear regions. Observationally, a three-fold strategy has been adopted: - substantial samples of distant sources have been analyzed through spectral, photometric, and statistical techniques, to gain insights into their mean properties as a population; - a moderately sized sample of bright sources has been studied through detailed X-ray spectral analysis, to give a first flavor of the general spectral properties of these sources, also from a temporally resolved point of view; - the best nearby candidate has been thoroughly studied using the most sophisticated spectral analysis techniques applied to a large dataset with a high S/N ratio, to understand the details of the physics of its accretion/ejection flow. There are three main channels through which this Thesis has been developed: - [Archival Studies]: the XMM-Newton public archival data has been extensively used to analyze both a large sample of distant BAL QSOs, and several individual bright sources, either BAL, mini-BAL, or NAL QSOs. - [New Observational Campaign]: I proposed and was awarded with new X-ray pointings of the mini-BAL QSOs PG 1126-041 and PG 1351+640 during the XMM-Newton AO-7 and AO-8. These produced the biggest X-ray observational campaign ever made on a mini-BAL QSO (PG 1126-041), including the longest exposure so far. Thanks to the exceptional dataset, a whealth of informations have been obtained on both the intrinsic continuum and on the complex reprocessing media that happen to be in the inner regions of this AGN. Furthermore, the temporally resolved X-ray spectral analysis field has been finally opened for mini-BAL QSOs. - [Theoretical Studies]: some issues about the connection between theories and observations of AGN accretion disk winds have been investigated, through theoretical arguments and synthetic absorption line profiles studies.