Organismi acquatici e ambiente: meccanismi biochimici di interazione, risposta e adattamento

The research is focused on the relationship between some Mg2+-dependent ATPase activities of plasma- and mitochondrial membranes from tissues of cultured marine bivalve molluscs and potentially stressful environmental conditions, such as the exposure to contaminants both of natural origin (ammonia nitrogen, the main contaminant of aquaculture plants) and of anthropic source (alkyltins). The two filter-feeding bivalve species selected colonize different habitats: the common mussel Mytilus galloprovincialis binds to hard substrates and the Philippine clam Tapes philippinarum burrows into sea bottom sandy beds. The choice of typical species of coastal waters, extremely suitable for environmental studies due to their features of poor motility, resistance to transport and great filtering efficiency, may constitute a model to evaluate responses to contaminants of membrane-bound enzyme activities involved in key biochemical mechanisms, namely cell ionic regulation and mitochondrial energy production. In vitro and in vitro approaches have been pursued. In vitro assays were carried out by adding the contaminants (NH4Cl and alkyltins) directly to the ATPase reaction media. In vivo experiments were carried out by exposing mussels to various tributyl tin (TBT) concentrations under controlled conditions in aquaria. ATPase activities were determined spectrophotometrically according to the principles of the method of Fiske and Subbarow (1925). The main results obtained are detailed below. In Tapes philippinarum the interaction of NH4 +, the main form of ammonia nitrogen at physiological and seawater pHs, with the Na,K-ATPase and the ouabaininsensitive Na-ATPase was investigated in vitro on gill and mantle microsomal membranes. The proven replacement by NH4 +of K+ in the activation of the Na,KATPase and of Na+ in the activation of the ouabain-insensitive ATPase displayed similar enzyme affinity for the substituted cation. on the one hand this finding may represent one of the possible mechanisms of ammonia toxicity and, on the other, it supports the hypothesis that NH4 + can be transported across the plasma membrane through the two ATPases. In this case both microsomal ATPases may be involved and co-operate, at least under peculiar circumstances, to nitrogen excretion and ammonia detoxification mechanisms in bivalve molluscs. The two ATPase activities stimulated by NH4 + maintained their typical response to the glycoside ouabain, specific inhibitor of the Na,K-ATPase, being the Na++ NH4 +-activated ATPase even more susceptive to the inhibitor and the ouabain-insensitive ATPase activity activated indifferently by Na+ or NH4 + unaffected by up to 10-2 M ouabain. In vitro assays were carried out to evaluate the response of the two Na-dependent ATPases to organotins in clams and mussels and to investigate the interaction of TBT with mussel mitochondrial oligomycin-sensitive Mg-ATPase. Since no literature data were available, the optimal assay conditions and oligomycin sensitivity of mussel mitochondrial MgATPase were determined. In T. philippinarum the ouabain-insensitive Na-ATPase was found to be refractory to TBT both in the gills and in the mantle, whereas the Na,K-ATPase was progressively inhibited by increasing TBT doses; the enzyme inhibition was more pronounced in the gills than in the mantle. In both tissues of M. galloprovincialis the Na,K-ATPase inhibition by alkyltins decreased in the order TBT>DBT(dibutyltin)>>MBT(monobutyltin)=TeET(tetraethyltin) (no effect). Mussel Na-ATPase confirmed its refractorimess to TBT and derivatives both in the gills and in the mantle. These results indicate that the Na,K-ATPase inhibition decreases as the number of alkyl chains bound to tin decreases; however a certain polarity of the organotin molecule is required to yield Na,K-ATPase inhibition, since no enzyme inhibition occurred in the presence of tetraalkyl-substituted derivatives such as TeET . Assays carried out in the presence of the dithioerythritol (DTE) pointed out that the sulphhydrylic agent is capable to prevent the Na,K-ATPase inhibition by TBT, thus suggesting that the inhibitor may link to -SH groups of the enzyme complex.. Finally, the different effect of alkyltins on the two Na-dependent ATPases may constitute a further tool to differentiate between the two enzyme activities. These results add to the wealth of literature data describing different responses of the two enzyme activities to endogenous and exogenous modulators . Mussel mitochondrial Mg-ATPase was also found to be in vitro inhibited by TBT both in the gills and in the mantle: the enzyme inhibition followed non competitive kinetics. The failed effect of DTE pointed out that in this case the interaction of TBT with the enzyme complex is probably different from that with the Na,K-ATPase. The results are consistent with literature data showing that alkyltin may interact with enzyme structures with different mechanisms. Mussel exposure to different TBT sublethal doses in aquaria was carried out for 120 hours. Two samplings (after 24 and 120 hrs) were performed in order to evaluate a short-term response of gill and mantle Na,K-ATPase, ouabain-insensitive Na-ATPase and Mg-ATPase activities. The in vivo response to the contaminants of the enzyme activities under study was shown to be partially different from that pointed out in the in vitro assays. Mitochondrial Mg-ATPase activity appeared to be activated in TBTexposed mussels with respect to control ones, thus confirming the complexity of evaluating in vivo responses of the enzyme activities to contaminants, due to possible interactions of toxicants with molluscan metabolism. Concluding, the whole of data point out that microsomal and mitochondrial ATPase activities of bivalve molluscs are generally responsive to environmental contaminants and suggest that in some cases membrane-bound enzyme activities may represent the molecular target of their toxicity. Since the Na,K-ATPase, the Na-ATPase and the Mg-ATPase activities are poorly studied in marine bivalves, this research may contribute to enlarge knowledge in this quite unexplored field.

Reduced-order modelling, circuit-level design and SOI fabrication of microelectromechanical resonators

This thesis deals with two important research aspects concerning radio frequency (RF) microresonators and switches. First, a new approach for compact modeling and simulation of these devices is presented. Then, a combined process flow for their simultaneous fabrication on a SOI substrate is proposed. Compact models for microresonators and switches are extracted by applying mathematical model order reduction (MOR) to the devices finite element (FE) description in ANSYS c° . The behaviour of these devices includes forms of nonlinearities. However, an approximation in the creation of the FE model is introduced, which enables the use of linear model order reduction. Microresonators are modeled with the introduction of transducer elements, which allow for direct coupling of the electrical and mechanical domain. The coupled system element matrices are linearized around an operating point and reduced. The resulting macromodel is valid for small signal analysis around the bias point, such as harmonic pre-stressed analysis. This is extremely useful for characterizing the frequency response of resonators. Compact modelling of switches preserves the nonlinearity of the device behaviour. Nonlinear reduced order models are obtained by reducing the number of nonlinearities in the system and handling them as input to the system. In this way, the system can be reduced using linear MOR techniques and nonlinearities are introduced directly in the reduced order model. The reduction of the number of system nonlinearities implies the approximation of all distributed forces in the model with lumped forces. Both for microresonators and switches, a procedure for matrices extraction has been developed so that reduced order models include the effects of electrical and mechanical pre-stress. The extraction process is fast and can be done automatically from ANSYS binary files. The method has been applied for the simulation of several devices both at devices and circuit level. Simulation results have been compared with full model simulations, and, when available, experimental data. Reduced order models have proven to conserve the accuracy of finite element method and to give a good description of the overall device behaviour, despite the introduced approximations. In addition, simulation is very fast, both at device and circuit level. A combined process-flow for the integrated fabrication of microresonators and switches has been defined. For this purpose, two processes that are optimized for the independent fabrication of these devices are merged. The major advantage of this process is the possibility to create on-chip circuit blocks that include both microresonators and switches. An application is, for example, aswitched filter bank for wireless transceiver. The process for microresonators fabrication is characterized by the use of silicon on insulator (SOI) wafers and on a deep reactive ion etching (DRIE) step for the creation of the vibrating structures in single-crystal silicon and the use of a sacrificial oxide layer for the definition of resonator to electrode distance. The fabrication of switches is characterized by the use of two different conductive layers for the definition of the actuation electrodes and by the use of a photoresist as a sacrificial layer for the creation of the suspended structure. Both processes have a gold electroplating step, for the creation of the resonators electrodes, transmission lines and suspended structures. The combined process flow is designed such that it conserves the basic properties of the original processes. Neither the performance of the resonators nor the performance of the switches results affected by the simultaneous fabrication. Moreover, common fabrication steps are shared, which allows for cheaper and faster fabrication.

Identification of Drosophila heart-specific Cis-Regulatory Modules under Hox control

Cardiac morphogenesis is a complex process governed by evolutionarily conserved transcription factors and signaling molecules. The Drosophila cardiac tube is linear, made of 52 pairs of cardiomyocytes (CMs), which express specific transcription factor genes that have human homologues implicated in Congenital Heart Diseases (CHDs) (NKX2-5, GATA4 and TBX5). The Drosophila cardiac tube is linear and composed of a rostral portion named aorta and a caudal one called heart, distinguished by morphological and functional differences controlled by Hox genes, key regulators of axial patterning. Overexpression and inactivation of the Hox gene abdominal-A (abd-A), which is expressed exclusively in the heart, revealed that abd-A controls heart identity. The aim of our work is to isolate the heart-specific cisregulatory sequences of abd-A direct target genes, the realizator genes granting heart identity. In each segment of the heart, four pairs of cardiomyocytes (CMs) express tinman (tin), homologous to NKX2-5, and acquire strong contractile and automatic rhythmic activities. By tyramide amplified FISH, we found that seven genes, encoding ion channels, pumps or transporters, are specifically expressed in the Tin-CMs of the heart. We initially used online available tools to identify their heart-specific cisregutatory modules by looking for Conserved Non-coding Sequences containing clusters of binding sites for various cardiac transcription factors, including Hox proteins. Based on these data we generated several reporter gene constructs and transgenic embryos, but none of them showed reporter gene expression in the heart. In order to identify additional abd-A target genes, we performed microarray experiments comparing the transcriptomes of aorta versus heart and identified 144 genes overexpressed in the heart. In order to find the heart-specific cis-regulatory regions of these target genes we developed a new bioinformatic approach where prediction is based on pattern matching and ordered statistics. We first retrieved Conserved Noncoding Sequences from the alignment between the D.melanogaster and D.pseudobscura genomes. We scored for combinations of conserved occurrences of ABD-A, ABD-B, TIN, PNR, dMEF2, MADS box, T-box and E-box sites and we ranked these results based on two independent strategies. On one hand we ranked the putative cis-regulatory sequences according to best scored ABD-A biding sites, on the other hand we scored according to conservation of binding sites. We integrated and ranked again the two lists obtained independently to produce a final rank. We generated nGFP reporter construct flies for in vivo validation. We identified three 1kblong heart-specific enhancers. By in vivo and in vitro experiments we are determining whether they are direct abd-A targets, demonstrating the role of a Hox gene in the realization of heart identity. The identified abd-A direct target genes may be targets also of the NKX2-5, GATA4 and/or TBX5 homologues tin, pannier and Doc genes, respectively. The identification of sequences coregulated by a Hox protein and the homologues of transcription factors causing CHDs, will provide a mean to test whether these factors function as Hox cofactors granting cardiac specificity to Hox proteins, increasing our knowledge on the molecular mechanisms underlying CHDs. Finally, it may be investigated whether these Hox targets are involved in CHDs.

Studio del comportamento meccanico di smalti porcellanati per substrati metallici

Composite porcelain enamels are inorganic coatings for metallic components based on a special ceramic-vitreous matrix in which specific additives are randomly dispersed. The ceramic-vitreous matrix is made by a mixture of various raw materials and elements and in particular it is based on boron-silicate glass added with metal oxides(1) of titanium, zinc, tin, zirconia, alumina, ecc. These additions are often used to improve and enhance some important performances such as corrosion(2) and wear resistance, mechanical strength, fracture toughness and also aesthetic functions. The coating process, called enamelling, depends on the nature of the surface, but also on the kind of the used porcelain enamel. For metal sheets coatings two industrial processes are actually used: one based on a wet porcelain enamel and another based on a dry-silicone porcelain enamel. During the firing process, that is performed at about 870°C in the case of a steel substrate, the enamel raw material melts and interacts with the metal substrate so enabling the formation of a continuous varying structure. The interface domain between the substrate and the external layer is made of a complex material system where the ceramic vitreous and the metal constituents are mixed. In particular four main regions can be identified, (i) the pure metal region, (ii) the region where the metal constituents are dominant compared with the ceramic vitreous components, (iii) the region where the ceramic vitreous constituents are dominant compared with the metal ones, and the fourth region (iv) composed by the pure ceramic vitreous material. It has also to be noticed the presence of metallic dendrites that hinder the substrate and the external layer passing through the interphase region. Each region of the final composite structure plays a specific role: the metal substrate has mainly the structural function, the interphase region and the embedded dendrites guarantee the adhesion of the external vitreous layer to the substrate and the external vitreous layer is characterized by an high tribological, corrosion and thermal shock resistance. Such material, due to its internal composition, functionalization and architecture can be considered as a functionally graded composite material. The knowledge of the mechanical, tribological and chemical behavior of such composites is not well established and the research is still in progress. In particular the mechanical performances data about the composite coating are not jet established. In the present work the Residual Stresses, the Young modulus and the First Crack Failure of the composite porcelain enamel coating are studied. Due to the differences of the porcelain composite enamel and steel thermal properties the enamelled steel sheets have residual stresses: compressive residual stress acts on the coating and tensile residual stress acts on the steel sheet. The residual stresses estimation has been performed by measuring the curvature of rectangular one-side coated specimens. The Young modulus and the First Crack Failure (FCF) of the coating have been estimated by four point bending tests (3-7) monitored by means of the Acoustic Emission (AE) technique(5,6). In particular the AE information has been used to identify, during the bending tests, the displacement domain over which no coating failure occurs (Free Failure Zone, FFZ). In the FFZ domain, the Young modulus has been estimated according to ASTM D6272-02. The FCF has been calculated as the ratio between the displacement at the first crack of the coating and the coating thickness on the cracked side. The mechanical performances of the tested coated specimens have also been related and discussed to respective microstructure and surface characteristics by double entry charts.

Derivati polimerici organometallici ancorati su matrice inorganica per applicazioni in chimica fine

Organotin compounds have found in the last few decades a wide variety of applications. Indeed, they are used successfully as antifouling paints, PVC stabilizers and ion carriers, as well as homogeneous catalysts. In this context, it has been proved that the Lewis acidity of the metal centre allows these compounds to promote the reaction between alcohol and ester. However their use is now limited by their well-known toxicity, moreover they are hardly removable from the reaction mixture. This problem can be overcome by grafting the organotin derivative onto a polymeric cross-linked support. In this way the obtained heterogeneous catalyst can be easily filtered off from the reaction mixture, thus creating the so-called "clean organotin reagents", avoiding the presence of toxic organotin residues in solution and the tin release in the environment. In the last few years several insoluble polystyrene resins containing triorganotin carboxylate moieties have been synthesized with the aim of improving their catalytic activity: in particular we have investigated and opportunely modified their chemical structure in order to optimize the accessibility to the metal centre and its Lewis acidity. Recently, we replaced the polymeric matrix with an inorganic one, in order to dispose of a relatively cheaper and easily available support. For this purpose an ordered mesoporous silica, characterized by 2D-hexagonal pores, named MCM-41, and an amorphous silica have been selected. In the present work two kinds of MCM-41 silica containing the triorganotin carboxylate moiety have been synthesized starting from a commercial Cab-O-Sil M5 silica. These catalysts have two different spacers between the core and the tin-carboxylate moiety, namely a polyaliphatic chain (compound FT29) or a poliethereal one (compound FT6), with the aim to improve the interaction between catalyst and reacting ester. Three catalysts supported onto an amorphous silica have been also synthesized: the structure is the same as silica FT29, i.e. a compound having a polialiphatic chain, and they have different percentage of organotin derivative grafted on the silica surface (10, 30, 50% respectively for silica MB9, SU27 and SU28). The performances of the above silica as heterogeneous catalysts in transesterification reactions have been tested in a model reaction between ethyl acetate and 1-octanol, a primary alcohol sensitive to the reaction conditions. The alcohol conversion was assessed by gas-chromatography, determining the relative amount of transesterified product and starting alcohol after established time intervals.

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

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

Sviluppo dei materiali innovativi e studio della loro interazione con sistemi biologici e biomimetici

Nell’ambito della Chimica Sostenibile e dell’applicazione dei suoi principi per la salvaguardia dell’ambiente, il progetto di dottorato ha riguardato lo sviluppo di materiali innovativi e lo studio della loro interazione con sistemi biologici e biomimetici. In particolare l’attività si è focalizzata sulla sintesi di liquidi ionici ed indagini delle interazioni con membrane cellulari e sull’utilizzo ed isolamento di molecole da fonti rinnovabili. I liquidi ionici sono sali organici liquidi a temperature inferiori ai 100 °C; sono considerati promettenti solventi a ridotta tossicità, ma vanno chiarite a pieno le modalità di interazione con i sistemi biologici ed i meccanismi di tossicità. A questo scopo è stata impiegata una batteria di test bio-chimici, con saggi di fluorescenza e colorimetrici, che hanno permesso di discriminare le diverse tipologie di interazioni con varie strutture di membrana. Le informazioni raccolte sono servite per progettare sostanze meno dannose per le strutture cellulari, al fine di scegliere le funzionalità molecolari che consentano ai liquidi ionici di mantenere la loro attività ma di essere meno dannosi per l’ambiente. Per quanto riguarda l’utilizzo ed isolamento di molecole da fonte rinnovabili, si è utilizzata la tecnica della pirolisi per l’ottenimento di starting materials ed il loro impiego nella sintesi di chemicals in alternativa a composti derivanti da fonti fossili. La pirolisi tradizionale della cellulosa fornisce una molecola interessante, per semplicità denominata LAC, in quantità insufficienti ad un uso applicativo. Nell’ambito delle ricerche svolte è stato scoperto che la pirolisi condotta in presenza di catalizzatori meso-strutturati (MCM-41) drogati con metalli di transizione, fornisce buone quantità di LAC. LAC si è dimostrato promettente sia per la produzione di nuove molecole con possibili applicazioni nella chimica fine e farmaceutica, che come monomero per nuovi polimeri (copolimero ed omopolimero).

Effects of Resources Exploitation on Water Quality: case studies in Salt Water Intrusion and Acid Mine Drainage

Throughout the world, pressures on water resources are increasing, mainly as a result of human activity. Because of their accessibility, groundwater and surface water are the most used reservoirs. The evaluation of the water quality requires the identification of the interconnections among the water reservoirs, natural landscape features, human activities and aquatic health. This study focuses on the estimation of the water pollution linked to two different environmental issues: salt water intrusion and acid mine drainage related to the exploitation of natural resources. Effects of salt water intrusion occurring in the shallow aquifer north of Ravenna (Italy) was analysed through the study of ion- exchange occurring in the area and its variance throughout the year, applying a depth-specific sampling method. In the study area were identified ion exchange, calcite and dolomite precipitation, and gypsum dissolution and sulphate reduction as the main processes controlling the groundwater composition. High concentrations of arsenic detected only at specific depth indicate its connexion with the organic matter. Acid mine drainage effects related to the tin extraction in the Bolivian Altiplano was studied, on water and sediment matrix. Water contamination results strictly dependent on the seasonal variation, on pH and redox conditions. During the dry season the strong evaporation and scarce water flow lead to low pH values, high concentrations of heavy metals in surface waters and precipitation of secondary minerals along the river, which could be released in oxidizing conditions as demonstrated through the sequential extraction analysis. The increase of the water flow during the wet season lead to an increase of pH values and a decrease in heavy metal concentrations, due to dilution effect and, as e.g. for the iron, to precipitation.

Synthesis and applications of N-Metallo ketene imines

N-metallo ketene imines are attractive for the preparation of a wide range of organic compounds. Our research group has been engaged in the preparation and application of the N-metallo imines (SKIs). In this frame we have studied the uncatalyzed reaction of SKIs with isocyanates to give the corresponding malonamides with good yields. It has been demonstrated that the use of SKIs, instead of simple lithium anion of nitriles, is essential for the success of the reaction. A possible explanation assumes that this new reaction proceeds via a silatropism. In the course of our studies, reported in this thesis, the synthesis and the reactivity of N-silyl ketene imines in the preparation of 2,2-diaryl-3,4- dihydroxy- alcanonitrile in an uncatalyzed adol-type reaction has been performed. Our conception has been to use a chiral aldehyde to introduce asymmetric induction at the β-position and at the α-quaternary stereogenic center in the new forming diols. To achieve this goal, we used diarylacetonitrile as the substrate to form the corresponding N-trimethylsylilketene-imines to be reacted with (S)–lactic aldehyde with different protecting groups on the hydroxyl functionality. A number of 2,2-diaryl-3,4-dihydroxy-pentanenitrile were prepared with good to excellent stereo-control and satisfactory yields. Extension of this protocol to other metallo-ketene imines was performed. Accordingly, the preparation of tin ketene imines was attempted in analogy of the corresponding silyl ketene imine. The reaction of tin ketene imines with aldehydes was tested as a new tool for the synthesis of beta-hydroxynitriles starting from carbonyl compounds (aldehydes and/or ketones). Dialkyl(aryl)silyl nitriles and dialkyl(aryl)tin nitriles presents different reactivity. Finally, N-aluminium-ketene imines, as nucleophilic partner in the opening reaction of epoxides were studied. Preliminary positive results foster us to continue our studies in enlightening the scope and the limitations of this new reaction.