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La production de matériaux issus de matières premières renouvelables se fait pressante de par la disponibilité décroissante des carburants fossiles. Ce besoin a motivé l’émergence du concept de bio-raffinerie dont certains produits obtenus en grande quantité sont des dérivés d’acides gras (Fatty Acid Methyl Ester, FAME). Ces FAME sont principalement produits par hydrolyse des triglycérides et ils ont déjà trouvé de nombreux domaines d’application. Dans le contexte d’utilisation optimale des différents produits par les bio-raffineries, la modification des FAME peut être mise en œuvre via la catalyse, et notamment la métathèse des oléfines, en tirant avantage de la double liaison interne présente dans ces composés. D’importants efforts ont été portés sur l’éthénolyse des FAME [Delta]9 en [Alpha]-oléfines, car ces dernières ont un large éventail d’applications, mais aussi sur la diminution de la chaine carbonée par réaction avec des oléfines de petites tailles (C4). De plus, dans le but de limiter la contamination métallique des produits et de permettre un recyclage des catalyseurs, l’utilisation de catalyseur hétérogène est de grand intérêt. Nous avons hétérogénéisé des catalyseurs commerciaux au ruthénium en deux étapes : modification du catalyseur ou de la silice et greffage sur silice par une substitution d’un ligand. Les réactions étudiées au cours de cette thèse concernent l’homométathèse, l’éthénolyse et diverses métathèses croisées de l’oléate de méthyle ainsi que sur des FAME dérivés de bio-sources, peu étudiés, en utilisant des catalyseurs homogènes de métathèse d’oléfines et leurs analogues greffés sur silice. Malgré l’impossibilité d’un recyclage performant, les catalyseurs hétérogènes ont permis de réaliser nos réactions de façon efficace, certaines des ces réactions étant les premiers exemples de telles transformations par catalyse hétérogène. / Production of materials from renewable raw materials is urgent by the decreasing availability of fossil fuels. This need has motivated the emergence of the concept of bio-refinery that certain products obtained in large quantities are derivatives of fatty acids (Fatty Acid Methyl Ester, FAME). These FAMEs are mainly produced by hydrolysis of triglycerides and have already found many application areas. In the context of optimal use of different products by bio-refineries, FAMEs modifications can be implemented thanks to catalysis, including olefin metathesis, taking advantage of the internal double bond present in these compounds. Significant efforts have been made on the ethenolysis of [Delta]9 FAME into [Alpha]-olefins, as these have a wide range of applications, but also on reducing the carbon chain size by reaction with small olefins (C4). Moreover, in order to limit metal contamination of products and to allow catalyst recycling, the use of heterogeneous catalyst is of high interest. We thus prepared heterogenized commercial ruthenium catalysts. These heterogeneous catalysts were prepared in a two-step procedure: modification of the catalyst or the silica and catalysts grafting by ligand exchange on silica. The reactions studied in this thesis concern self-metathesis, ethenolysis and various cross-metatheses of methyl oleate and FAME bio-derived sources using homogeneous catalysts and their grafted analogs. Despite the impossibility of recycling performance, heterogeneous catalysts have achieved our reactions efficiently and some of these reactions are the first example of such transformations with heterogeneous catalysis.

Lab-on-a-chip technology is a rapidly growing research area. Joining together several disciplines, such as physics, biology and several instances of nanotechnologies. The aim of this research is mainly to produce chips that can do the same types of measurements as large lab equipment and measurement systems, but at a fraction of the size and cost. In this work a multifunctional measuring device have been developed. It can measure optical absorbance and fluorescence while performing a range of potentiometric techniques; including chronoamperometry, linear- and cyclic voltammetry. From all these measurements it is possible to calculate particle concentrations in fluid samples. The aim is to bring simpler and cheaper point of care devices to the public. Without larger losses in accuracy and reliability of the medicinal test. To do this our device is intended to be used with lab-chip, which are capable of amplifying the signals while reducing the sample size. Lab-chips could be used in several areas but the ones being designed with this device are made for biomedical purposes, applying suitable nanostructures and reagents to measure the presence of biomarkers. With these techniques, medicinal diagnostics can be made a few minutes after samples have been collected from patients. Much quicker and more direct than sending the samples to a lab and waiting hours if not days for the results. The measuring device or lab-chip reader will use two different lab-chips in the future. One that is optimised for optical absorbance and the other for fluorescence. Both will work with electrochemical measurements, but at present only the absorbance chip have been available for testing and that without any signal enhancing techniques. Assessment of the reader's capabilities was made with solutions of gold nanoparticles, TMB (tetramethylbenzidine), iron dissolved in PBS (Phosphate-buffed saline) and with a film made of PPV (Poly para-phenylenevinylene). The first two were used to test absorbance; while the iron and PBS have been used to test electrochemical system; and the PPV was coated on a glass substrate and used to test fluorescence. During the optical absorption test, it was found that the reader can distinguish between different concentrations of the various solutions. The results are promising and further removal of signal drifts will improve signals considerably. Fluorescence can be induced and measured with the device. This part of the system is, however, untested in general and future work will show if it is sufficient. The iron solution was tested with three different methods. chronopotentiometry, linear sweep voltammetry and cyclic voltammetry. It was however found that our measurements were distorted in comparison with the expected voltammogram for iron in PBS. Additional peaks were found in the voltammogram and it is believed that these are a result of oxidation of the electrodes on the lab-chip.

The objective of this study was to evaluate the effects of inclusion of Bio-Mos in the growing ration for weaned lambs on growth rate, feed efficiency, and clinical measures of health of the lambs. Mannan oligosaccharides (MOS), when included as a supplement to the diet, have been shown to have a positive effect on immune response in several species and in turn, positively affect the growth of the animal. MOS are commercially available as BioMos®, which is a nutritional supplement manufactured by Alltech, Inc. out of Nicholasville, KY. Forty-seven weaned Suffolk × Hampshire (n=47) lambs were used in this trial. Of the group, twenty (n=20) were ewe lambs and twentyseven (n=27) were wether lambs. The lambs were placed on their assigned diets and remained on the trial for a four week period (d+28). All responses evaluated in this study were influenced by time (p < 0.05) over the 28-d trial. A GENDER × WEEK interaction was observed for ADG and feed conversion (p < 0.05). Control lambs tended (p = 0.10) to have a higher intake over the 28-d period in comparison to Bio-Mos fed lambs. There were no statistically significant differences (p < 0.05) between control and Bio-Mos fed lambs for any of the growth parameters that were measured. There was a tendency (p = 0.10) for GENDER × WEEK to influence intake. Gender also tended to interacted with diet (GENDER × DIET, p = 0.09) to influence intake over the trial period. A GENDER × WEEK interaction was observed (p < 0.05) for feed conversion. Diet influenced fecal pH (p < 0.05). This study indicates Bio-Mos had minimal influence on growth and health.

La labioschisi con o senza palatoschisi non-sindromica (NSCL/P) è tra le più frequenti alterazioni dello sviluppo embrionale, causata dall’interazione di fattori genetici e ambientali, moti dei quali ancora ignoti. L'obiettivo del mio progetto di Dottorato consiste nell’identificazione di fattori di rischio genetico in un processo a due stadi che prevede la selezione di geni candidati e la verifica del loro coinvolgimento nella determinazione della malformazione mediante studi di associazione. Ho analizzato alcuni polimorfismi a singolo nucleotide (SNPs) dei geni RFC1 e DHFR, appartenenti alla via metabolica dell’acido folico, evidenziando una debole associazione tra alcuni degli SNPs indagati e la NSCL/P nella popolazione italiana. Presso il laboratorio della Dott.ssa Mangold dell’Università di Bonn, ho valutato il ruolo di 15 diverse regioni cromosomiche nel determinare la suscettibilità alla malattia, evidenziando una significativa associazione per i marcatori localizzati in 8q24 e 1p22. Ho quindi rivolto la mia attenzione al ruolo del complesso Polycomb nell’insorgenza della schisi. Nell’uomo i due complessi Polycomb, PRC1 e PRC2, rimodellano la cromatina agendo da regolatori dei meccanismi trascrizionali alla base della differenziazione cellulare e dello sviluppo embrionale. Ho ipotizzato che mutazioni a carico di geni appartenenti a PRC2 possano essere considerati potenziali fattori di rischio genetico nel determinare la NSCL/P. Il razionale consiste nel fatto che JARID2, una proteina che interagisce con PRC2, è associata all’insorgenza della NSCL/P ed espressa a livello delle cellule epiteliali delle lamine palatine che si approssimano alla fusione. L’indagine condotta analizzando i geni di elementi o partner dei due complessi Polycomb, ha evidenziato un’associazione significativa con alcuni polimorfismi dei geni indagati, associazione ulteriormente confermata dall’analisi degli aplotipi. Le analisi condotte sui geni candidati mi hanno permesso di raccogliere dati interessanti sull’eziologia della malformazione. Studi indipendenti saranno necessari per poter validare l'associazione tra le varianti genetiche di questi geni candidati e la NSCL/P. / Non-syndromic cleft lip with or without cleft palate (NSCL/P) is the most frequent alterations of embryonic development; it is caused by an interaction of genetic and environmental factors. The aim of this study is identifying of genetic risk factors, selecting candidate genes and testing their involvement in the determination of the malformation through association studies. I analyzed some single nucleotide polymorphisms (SNPs) of RFC1 and DHFR genes, belonging to the folic acid metabolic cascade. I highlighted a low association for some of the SNPs investigated and NSCL/P in the Italian population. In the laboratory of Dr. Mangold of the University of Bonn, I evaluated the role of 15 different chromosomal regions in determining susceptibility to disease. I showed a significant association for the variants at 8q24 and 1p22. Attention has been given to the role of the Polycomb complex in determining NSCL/P susceptibility. In mammals, there are two main Polycomb complexes: PRC1 and 2 PRC2. Both are chromatin-remodeling multiprotein complexes. These complexes control the transcriptional mechanisms implicated in cell differentiation and in embryonic development. I hypothesized that mutations in genes belonging to PRC2 complex can be considered as potential genetic risk factors for NSCL/P. The rationale is that JARID2, protein interacts with PRC2, is associated with the onset of NSCL/P and it is expressed in epithelial cells of the palatine laminae that approach to fusion. Analysis of the polymorphisms of genes encoding elements or partners of the two Polycomb complexes, showed a significant association between some of the SNPs investigated and pathology, further confirmed by the analysis of haplotypes. The analyzes of candidate genes allowed me to collect interesting data sulla'eziologia the malformation. Independent studies will be needed to confirm the association between genetic variants of these candidate genes and NSCL/P.

DNA elongation is performed by Pol III α subunit in E. coli, stimulated by the association with ε and θ subunits. These three subunits define the DNA Pol III catalytic core. There is controversy about the DNA Pol III assembly for the simultaneous control of lagging and leading strands replication, since some Authors propose a dimeric model with two cores, whereas others have assembled in vitro a trimeric DNA Pol III with a third catalytic core, which increases the efficiency of DNA replication. Moreover, the function of the PHP domain, located at the N-terminus of α subunit, is still unknown. Previous studies hypothesized a possible pyrophosphatase activity, not confirmed yet. The present Thesis highlights by the first time the production in vivo of a trimeric E. coli DNA Pol III by co-expressing α, τ, ε and θ subunits. This trimeric complex has been enzymatically characterized and a molecular model has been proposed, with 2 α subunits sustaining the lagging-strand replication whereas the third core replicates the leading strand. In addition, the pyrophosphatase activity of the PHP domain has been confirmed. This activity involves, at least, the H12 and the D19 residues, whereas the D201 regulates phosphate release. On the other hand, an artificial polymerase (HoLaMa), designed by deleting the exonuclease domain of Klenow Fragment, has been expressed, purified and characterized for a better understanding of bacterial polymerases mechanism. The absence of exonuclease domain impaired enzyme processivity, since this domain is involved in DNA binding. Finally, Klenow enzyme, HoLaMa, α subunit and DNA Pol III αεθ have been characterized at the single-molecule level by FRET analysis, combining ALEX and TIRF microscopy. Fluorescently-labeled DNA molecules were immobilized, and changes in FRET efficiency enabled us to study polymerase binding and DNA polymerization.

The aim of this study is to investigate on some molecular mechanisms contributing to the pathogenesis of osteoarthritis (OA) and in particular to the senescence of articular chondrocytes. It is focused on understanding molecular events downstream GSK3β inactivation or dependent on the activity of IKKα, a kinase that does not belong to the phenotype of healthy articular chondrocytes. Moreover, the potential of some nutraceuticals on scavenging ROS thus reducing oxidative stress, DNA damage, and chondrocyte senescence has been evaluated in vitro. The in vitro LiCl-mediated GSK3β inactivation resulted in increased mitochondrial ROS production, that impacted on cellular proliferation, with S-phase transient arrest, increased SA-β gal and PAS staining, cell size and granularity. ROS are also responsible for the of increased expression of two major oxidative lesions, i.e. 1) double strand breaks, tagged by γH2AX, that associates with activation of GADD45β and p21, and 2) 8-oxo-dG adducts, that associate with increased IKKα and MMP-10 expression. The pattern observed in vitro was confirmed on cartilage from OA patients. IKKa dramatically affects the intensity of the DNA damage response induced by oxidative stress (H2O2 exposure) in chondrocytes, as evidenced by silencing strategies. At early time point an higher percentage of γH2AX positive cells and more foci in IKKa-KD cells are observed, but IKKa KD cells proved to almost completely recover after 24 hours respect to their controls. Telomere attrition is also reduced in IKKaKD. Finally MSH6 and MLH1 genes are up-regulated in IKKαKD cells but not in control cells. Hydroxytyrosol and Spermidine have a great ROS scavenging capacity in vitro. Both treatments revert the H2O2 dependent increase of cell death and γH2AX-foci formation and senescence, suggesting the ability of increasing cell homeostasis. These data indicate that nutraceuticals represent a great challenge in OA management, for both therapeutical and preventive purposes.

The impact of a chromium smelter on pollution was evaluated by determining Cr(VI) in topsoil, grass and tree bark by electhrothermal atomic absorption spectrometry (ETAAS). It was found that bark reflected the levels of air pollution better than soil and grass due to its high accumulative ability of Cr(VI). The tree bark was contaminated with Cr(VI) by a factor of 9 than in soil. It is therefore suggested that the bark be used as an indicator of air pollution for long-term exposure. The concentration of Cr(VI) in the bark was always a fraction of the total concentration of Cr and ranges between 1.6 and 3%. The method used in the preparation of samples was validated by the analysis of certified reference materials.

Obiettivo Analisi di consumi e costi degli antibiotici sistemici negli ospedali dell’Emilia-Romagna dal 2004 al 2011, con attenzione alla variabilità interaziendale e al significato, in termini di resistenza batterica, dell’aumento di alcuni gruppi terapeutici; Sottoanalisi nei reparti pediatrici, individuando i gruppi terapeutici critici, e valutazione delle reazioni avverse pediatriche da antibiotici segnalate, per il periodo in esame. Metodi I dati di consumo e spesa degli antibiotici sistemici per il periodo 2004-2011 sono stati ottenuti dal database regionale AFO e le giornate di degenza per ogni reparto dal database regionale di dimissione ospedaliera SDO. Le segnalazioni di sospette reazioni avverse da antibiotici tra gennaio 2004 e dicembre 2011 sono state estratte dal database nazionale VigiSegn. Risultati Negli otto anni, il consumo di antibiotici negli ospedali dell’Emilia-Romagna è aumentato del 27% e la spesa del 3%. Il consumo è apparso nettamente superiore nei reparti chirurgici che medici. La prima classe per consumo e spesa sono le penicilline/inibitori delle beta lattamasi. Nei reparti pediatrici, sono stati utilizzati 65 principi attivi diversi e amoxicillina/acido clavulanico è stato il più usato (26% del totale del 2011). Tra gli antibiotici critici, le cefalosporine di terza generazione sono state le più consumate in tutti i reparti pediatrici nel 2011. Tra le molecole il cui uso ospedaliero è vincolato, spiccano il linezolid e la teicoplanina che, comunque, hanno inciso più di tutte nella spesa del 2011 (18% e 15%, rispettivamente). Per la farmacovigilanza, i bambini (3-13 anni) sono stati coinvolti in 23 casi, mentre gli infanti (≤2 anni) solo in 4. L’associazione amoxicillina/acido clavulanico è stata più frequentemente segnalata (n=7), e soltanto 2 casi erano gravi. Conclusioni I risultati mostrano un quadro critico sul massiccio uso delle cefalosporine di terza generazione e sull’incremento del linezolid, da approfondire se per inappropriatezza d’uso oppure per aumento delle resistenze batteriche. / Aim To assess antibiotic consumption and expenditure in all hospitals of Emilia-Romagna from 2004 to 2011, highlighting on variability and meaning, in terms of bacterial resistance, of the rise in some therapeutic groups; To analyze antibiotic use among the pediatric wards, identifying the critical therapeutic groups, and evaluation of antibiotic-associated adverse reactions in pediatric inpatients, in the period considered. Methods Consumption and expenditure data of antibacterial for systemic use in the period 2004-2011 were obtained from the regional database (AFO) and the number of bed-days was available for the single wards from the regional hospital discharge database (SDO). Adverse reaction reports associated to antibiotics and recorded between January 2004 and December 2011 have been extracted from the national database VigiSegn. Results In the eight years analyzed, the overall antibiotic consumption increased by 27% and expenditure by 3% in all hospitals of Emilia-Romagna. Antibiotic consumption appeared markedly higher in the wards of surgical wards than medical ones. Penicillins/beta-lactamase inhibitors ranked first in consumption and expenditure. Sixty-five different antibiotics were used among the pediatric wards and amoxicillin/clavulanic acid was the most used (26% of the total in 2011). Among the critical antibiotics identified, the third-generation cephalosporins were the most used in all pediatric wards in 2011. Among the antibiotics with a defined limited use, the data highlighted that linezolid and teicoplanin affected most of all expenditure in 2011 (18% and 15%, respectively). About pharmacovigilance data, children (3-13 years) were involved in 23 cases, whereas infants (≤2 years) just in 4. Amoxicillin/clavulanic acid was the antibiotic most frequently reported (n = 7), and 2 cases of them were serious. Conclusions Results show a critical picture on the marked use of third-generation cephalosporins and on increase of linezolid consumption, because of inappropriateness of use or rise of bacterial resistance.

As a large and long-lived species with high economic value, restricted spawning areas and short spawning periods, the Atlantic bluefin tuna (BFT; Thunnus thynnus) is particularly susceptible to over-exploitation. Although BFT have been targeted by fisheries in the Mediterranean Sea for thousands of years, it has only been in these last decades that the exploitation rate has reached far beyond sustainable levels. An understanding of the population structure, spatial dynamics, exploitation rates and the environmental variables that affect BFT is crucial for the conservation of the species. The aims of this PhD project were 1) to assess the accuracy of larval identification methods, 2) determine the genetic structure of modern BFT populations, 3) assess the self-recruitment rate in the Gulf of Mexico and Mediterranean spawning areas, 4) estimate the immigration rate of BFT to feeding aggregations from the various spawning areas, and 5) develop tools capable of investigating the temporal stability of population structuring in the Mediterranean Sea. Several weaknesses in modern morphology-based taxonomy including demographic decline of expert taxonomists, flawed identification keys, reluctance of the taxonomic community to embrace advances in digital communications and a general scarcity of modern user-friendly materials are reviewed. Barcoding of scombrid larvae revealed important differences in the accuracy of the taxonomic identifications carried out by different ichthyoplanktologists following morphology-based methods. Using a Genotyping-by-Sequencing a panel of 95 SNPs was developed and used to characterize the population structuring of BFT and composition of adult feeding aggregations. Using novel molecular techniques, DNA was extracted from bluefin tuna vertebrae excavated from late iron age, ancient roman settlements Byzantine-era Constantinople and a 20th century collection. A second panel of 96 SNPs was developed to genotype historical and modern samples in order to elucidate changes in population structuring and allele frequencies of loci associated with selective traits.