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Synthesis, characterization and applications of highly stable pyrazolate-based metal-organic frameworks.
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Patterns of population structure and adaptive genetic variation in alpine populations of Picea abies (L.) Karst.
No full textForest trees dominate many alpine landscapes and are currently exposed to changing climate. Picea abies (L.) Karst (Norway spruce) is one of the most important conifer species of the Italian Alps due to its ecological and economical relevance. Natural populations of this species are found across steep environmental gradients with large differences in temperature and moisture availability. Steep environmental gradients represent interesting models to study the interaction between natural selection and gene flow, especially when aiming to understand adaptation processes under global change. The present work aims to understand adaptive responses to changing climate by determining and quantifying patterns of genetic diversity in natural population of P.abies. a wide array of potential candidate genes was tested, by means of Single Nucleotide Polymorphisms (SNPs), for correlation with climatic and environmental parameters at different spatial scales: i) a geographical scale corresponding to the natural distribution of P.abies across the Italian Alps and ii) at a regional scale on the Eastern Italian Alps. Weak population structure was revealed at the geographical scale with only one population clearly divergent from the unique major genetic cluster identified. At the regional scale, hierarchical analyses of molecular variance revealed that most of the genetic variability was found within populations (ca. 99%), and small but significant variation was also found due landscape features (ca. 0.38%). In order to detect potentially adaptive markers, classical FST outlier approaches were first applied and five outlier loci were revealed at broad scale, while contrasting results were obtained at the regional scale according to the model used. Subsequently, environmental association analysis were performed: at the geographical scale temperature and precipitation were found to influence allelic variation at seven polymorphic loci, while at the regional scale, the Alpine topography resulted a potential adaptive determinants at 19 polymorphic loci, thus considered of ecological relevance. The results obtained in this study may provide relevant information for forestry management and genetic conservation, to understand and quantify the effect of climate change on conifer species as well as their adaptive potential
Validazione di un innovativo sistema di telemonitoraggio e creazione di percorsi diagnostico-terapeutici per l'identificazione dei meccanismi di presentazione ed evoluzione della fibrillazione atriale.
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Bio-materials and surgical options for the treatment of lung cancer invading the superior vena cava.
No full textBackground. The oncologic value of superior vena cava (SVC) resection for lung malignancies remains controversial. The aim of this study was to review postoperative outcome and long-term oncologic results in the surgical treatment of locally advanced lung cancer (NSCLC) invading the SVC system.
Methods. The clinical data of 88 consecutive patients who underwent surgery for locally advanced NSCLC invading SVC system from 1998 to november 2011 were retrospectively reviewed. Patients were grouped according to the type of surgical treatment performed. The study endpoints were overall survival, post-operative morbidity, mortality and lenght of postoperative stay.
Results. Forty-seven (72.4%) patient underwent partial SVC resection. Replacement of the SVC system by prosthesis was performed in 18 patients (27.6%). In 20 patients (30.7%) SVC resection was associated with carina resection (SVC-CR). In 23 patients a replacement of the SVC was performed by a biomaterial: polytetrafluoroethylene graft (13 patients), biological custom-made bovine pericardial tube (14 patients) or autologus pericardium patch (5 patients). Twenty-three patients underwent explorative thoracotomy (ET). In the resected group overall postoperative major morbidity and mortality rates were 17% (minor 28%, major 16%) and 9,2% respectively. In multivariate analysis, advanced age (≥60 years) and advanced stage (III-IV) are independent predictors of survival. Mortality was not influenced by any factor. Five-year survival probability was 25% for overall SVC resection, and 20% when resection of SVC was associated with CR. In SVC resection patients, lymph node status was the most important prognostic factor of survival (5-year survival in N0 patients 44%; 22% in N+). In ET group 5-year survival was 12%.
Conclusions. Pursuing extended resection in locally advanced NSCLC invading SVC may achieve permanent cure in selected patients who would otherwise be defined inoperable with very poor prognosis. Satisfactory long-term results in NSCLC infiltrating the SVC system can be achieved only when pathologic N2 disease is excluded
Amino acid oxidases in red biotechnologies: a target and a tool.
No full textD-Amino acid oxidase (DAAO; EC 1.4.3.3) has been proposed to play a main role in the degradation of D-serine, an allosteric activator of the N-methyl-D-aspartate-type glutamate receptor in the human brain, associated with the onset of schizophrenia. To prevent excessive D-serine degradation, novel drugs for schizophrenia treatment based on DAAO inhibition were designed and tested on rats. The properties of rat DAAO (rDAAO) are unknown and various in vivo trials reported on the effects of DAAO inhibitors on D-serine concentration in rats. rDAAO was efficiently expressed in Escherichia coli. The recombinant enzyme was purified as an active, 40 kDa monomeric flavoenzyme showing the basic properties of the dehydrogenase-oxidase class of flavoproteins. rDAAO differs significantly from the human enzyme because it: 1)) possesses a different substrate specificity; 2) shows a lower kinetic efficiency (because of a low substrate affinity); 3) differs in affinity for binding of classical inhibitors; 4) is a stable monomer; 5) interacts with the mammalian protein modulator pLG72 yielding a 100 kDa complex in addition to the 200 kDa, as formed by the human DAAO. Interestingly, the concentration of endogenous D-serine in U87 glioblastoma cells was not affected by transfection with rDAAO whereas it was significantly decreased when expressing the human homologue. These results raise doubt on the use of rat as model system for testing new drugs against schizophrenia and indicate a different physiological function of DAAO in rodents and humans.
During past years, a number of variants of D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) with altered substrate specificity (e.g., active on acidic, or hydrophobic, or on all D-amino acids) both by rational design and directed evolution methods have been produced in our laboratory. RgDAAO is the most suitable biotechnological tool for the detection of D-amino acids and in this work we evaluated the capability of some mutant forms of this flavoenzyme previously produced in our laboratory in order to determine D-amino acid content in different biological samples. The kinetic constants for a number of natural and unnatural D-amino acids have been investigated. This information constitutes the basis for considering potential analytical applications of these variants of RgDAAO.
Glycine is implicated in several physiological functions, e.g. as a biosynthetic precursor or neurotransmitter in the central nervous system. Glycine is an important coagonist of NMDA receptor and it is putatively involved in schizophrenia susceptibility and other neurological diseases, such as congenital nonketotic hyperglycinemia. With the final aim to produce an optimized enzyme that can be employed in a specific biosensor for glycine detection, glycine oxidase from Bacillus subtilis (GO) was engineered to improve its kinetic efficiency on this small amino acid. Based on in silico analysis, site saturation mutagenesis was independently performed at positions Met49, Gly51, Ala54, Met95, Tyr241, His244, Tyr246, Met261, Arg302, Arg329 and Asn330. The GO variants were screened by employing a rapid colorimetric assay on 96 well-plates based on the determination of hydrogen peroxide produced on glycine as substrate: seven GO variants were selected. Significant alteration of kinetic parameters was observed for H244K and H244R GO variants: kcat increased about twice and Km decreased 3-5-fold, yielding a 7-12-fold higher kinetic efficiency on glycine, as compared to the wild-type GO. Screening of GO variants at position 49 also identified an improved enzyme (M49I) showing a 1.4-fold decreased Km. Combination of information gathered from the site saturation mutagenesis approach could be useful to obtain an evolved GO variant suitable for biotechnological applications
Screening and heterologous expression of chitinases from environmental metagenomic libraries.
No full textMy PhD thesis has been developed in the frame of the EU-funded Metaexplore project. This project, conceived in collaboration with eighteen research groups all over Europe, concerns the development of new metagenomic techniques to detect enzymes involved in the biodegradation of recalcitrant natural molecules such as chitin and lignin. The final goal is the heterologous expression and characterization of the newly discovered chitinases and ligninases to exploit them in industrial bioprocesses. My thesis deals with chitinases that include a group of enzymes acting in a synergic and consecutive way, responsible for the complete hydrolysis of chitin into the constitutive monomers of N-acetylglucosamine. An alternative pathway involves chitin deacetylases for the deacetylation of chitin into chitosan, which is then further hydrolyzed by chitosanases in glucosamine units. Chitin derivatives have a multiplicity of industrial applications, which make chitinases attractive at industrial level. University of Insubria participates at the Metaexplore project in different work-packages. In the first part, my work is devoted to the validation of the functional screening methods to detect chitin degrading activities from metagenomic libraries. The following task regards the preliminary analysis of the most promising clones and the heterologous expression of the genes of interest in conventional and alternative hosts. At the moment, protein expression is the major bottleneck for the biotechnological success of the metagenomic approach to discover new enzymes. The optimization of the heterologous expression of metagenome-sourced genes will allow a complete biochemical characterization of the new enzymes and their production at industrial level.
The following chapters report Material and Methods, Results and Discussion related to the experimental work performed for the Metaexplore project, during the three years of my PhD course.
The first chapter, Screening for chitinase activities in actinomycetes, consist in an exploration of the University of Insubria’s actinomycete culture collection for chitin degrading activities. This culture collection comprises microorganisms belonging to Streptomyces genus, but also to other genera of uncommon actinomycetes named “rare”, since difficult to be isolated, cultivated, and manipulated. All the assayed microorganisms are degrading chitin when it is used as a sole carbon and nitrogen source in plate assay, but only some of them seem to possess chitosan and chitin deacetylase activities. Induction and repression of the chitinolytic system has been investigated in representative strains. Enzymatic assays to detect the extracellular and intracellular levels of chitin depolymerase, chitobiosidase, N-acetylglucosaminidase activities have been developed, validated and transferred to the Metaexplore partners to be applied at the screening of the metagenomic libraries. Interestingly, the rare actinomycete Nonomuraea sp. ATCC 39727, previously never explored for its chitin degrading activity, results positive in the chitin deacetylase plate assay and produces chitosan. To my knowledge, this is the first report on chitin deacetylase enzyme discovered from actinomycetes.
The second paper, Bacterial chitinase with biocontrol capacity from suppressive soil revealed by functional metagenomics, presents the research work performed in collaboration with the School of Life Sciences of Södertörn University (Huddinge, Sweden), on the heterologous expression of the first chitinase isolated from a metagenomic library within the Metaexplore consortium. The metagenomic library originates from a soil considered suppressive for clubroot disease of cabbage. Genetic screening using degenerate primers for the conserved central catalytic domain of family 18 chitinases has selected a putative chitobiosidase gene, called chi18H8. The gene is cloned in BL21(DE3) E. coli cells, in transcriptional fusion with the gluthathione-S-transferase tag. In this host, most of the protein accumulates in the insoluble fraction as inclusion bodies, so expression optimization in E. coli soluble fractions is required for protein purification by affinity chromatography. On the pure enzyme, recovered at very low concentration, preliminary analysis confirms its chitinolytic nature and its antifungal properties against common plant phytopathogens.
In the third chapter, entitled Expression of a metagenome-sourced chitinase in Streptomyces lividans, this actinomycete is presented as alternative candidate to E. coli for the heterologous expression of the chi18H8 chitinase. Streptomyces spp. are typical degraders of biomasses in soil habitats. They possess a developed secretory pathway for the extracellular release of their hydrolytic enzymes. As described in the first paper on screening, they usually produce different chitinases, whose activity can be detected also when cultivated in rich media not containing chitin as carbon source. To use S. lividans as a host for heterologous chitinase expression, the chitinolytic system is metabolically repressed by adding glucose to the culture media, and the chi18H8 gene is cloned in a multicopy plasmid under the control of a heterologus constitutive promoter which is not repressed by the presence of glucose. The transformation of S. lividans TK 24 occurs by E. coli - S. lividans integeneric conjugation after a DNA demethylation step. Enzymatic activity assay and zymogram analysis confirm that the recombinant protein is secreted in the extracellular broth with an estimated volumetric productivity of 66 U/L which represents a good starting point for the further attempts of protein production and purification of Chi18H8 in S. lividans host
Un'analisi del modello di sviluppo brasiliano: cambiamento strutturale, specializzazione commerciale e le politiche per l'innovazione.
No full textThe dissertation deals with the Brazilian economic model and the role of the State in managing the economy through industrial and innovation policies.
The first part of the dissertation is dedicated to the analysis of the productive structure and the international competitiveness of the Brazilian economy, while the second part focuses on the industrial and innovation policies implemented by the Brazilian government.
An analysis of the economy‟s structural change is conducted, pointing out which sectors have gained importance in terms of manufacturing value added during the economic reforms of the „90s and later on. A broad analysis of trade specialization is also conducted, and an investigation on the relationships between Brazil and its main trade partners is carried out, specifically focusing on China, currently its main trade partner. The trend in Foreign Direct Investments (FDI) flows is analysed, pointing out the increasing importance of the Brazilian multinationals in the international markets.
In order to evaluate the sustainability of the economic model, a broad evaluation of the government industrial and innovation policies is conducted to assess the consistency of those policies with the structural problems of the economy. In particular, policies to improve the international trade performance are analysed. Furthermore, programmes and policies to strengthen the National Innovation System (NIS) are evaluated, and an in depth analysis of the NIS is also carried out. In addition, one chapter deals with an important agent of the Brazilian NIS: the National Development Bank (BNDES) and its role in the credit market.
The increasing role of the State in managing the economy through industrial policies after several years of just “free markets” policies is positively evaluated, although some structural economic problems still remain
Solution of the 2D Navier-Stokes equations by a new FE fractional step method.
No full textIn this work, a mathematical and numerical approach for the solution of the 2D Navier-Stokes equations for incompressible fluid flow problems is investigated. A new flux conservative technique for the solution of the elliptic part of the equations is formulated.
In the new model, the non linear convective terms of the momentum equations are approximated by means of characteristics and the spatial approximations, of equal order, are obtained by polynomials of degree two. The advancing in time is afforded by a fractional step method combined with a suitable stabilization technique so that the Inf-Sup condition is respected.
In order to keep down the computational cost, the algebraic systems are solved by an iterative solver (Bi-CGSTAB) preconditioned by means of Schwarz additive scalable preconditioners.
The properties of the new method are verified carrying out several numerical tests. At first, some elliptic, parabolic and convective-diffusive problems are solved and discussed, then the results of some time dependent and stationary 2D Navier-Stokes problems (in particular the well known benchmark problem of the natural convection in a square cavity) are discussed and compared to those found in the literature.
Another, potentially very important application of the numerical tools developed, regards the solution of 1D Shallow-Water equations. In fact the use of the fractional steps scheme for advancing in time and the finite elements (of different polynomial degrees) for the spatial approximation, makes the above mentioned approach computationally profitable and convenient for real applications. The efficiency and accuracy of the numerical model have been checked by solving a theoretical test.
Finally, a brief description of the software suitably developed and used in the tests conclude the thesis
Mechanical and functional characterization of the diaphragmatic lymphatic network.
No full textThe development of transplantation procedures and increased clinical problems involving edema and cancer have created a growing need for detailed studies of the lymphatic system due to its role as a component in the circulatory system and as a component in the immunological system.
In the initial phase of the present study, we measured the compliance of the lymphatic vessel wall in vivo and performed ex vivo mechanical tests on diaphragmatic tissue strips.
A finite element model (FEM) was thus developed, using the vessel’s actual dimensions, its compliance Measurement and the diaphragmatic tissue’s elastic module. Visualization of the model’s stress distribution then showed the functional differences between superficial, intermediate and deep lymphatic vessels.
The next step was to delve into the study of the lymphatic network with vertical connections draining from superficial capillaries to deeper collecting vessels though, given their anatomical position, these are rather challenging to assess experimentally.
The experimental data thus obtained was then processed in the lumped parameter model, clarifying significantly the way in which the lymph is drained by the diaphragmatic lymphatic network considering both superficial and deep lymphatic vessels.
In part III of the present thesis, we developed a mathematical model that include ribs diaphragm and lungs to quantify and visualize the stress distribution that are generated on the diaphragm, and therefore on the lymphatic vessels, during a whole breathing cycle.
The result is a simulation of a normal breathing