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.

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.

Synthetic and mechanistic investigation of new radical processes: reaction of organic azides with group-XIII Lewis acids

Dichloroindium hydride revealed to be a valid alternative to tributyltin hydride for radical reduction of organic (alkyl, aryl, acyl, solfonyl) azides. The new approach entails mild reaction conditions and provides high yields of the corresponding amines and amides, also showing high degrees of selectivity. The system dichloroindium hydride / azides can be utilised in fivemembered ring closures of g-azidonitriles, as a new source of aminyl radicals for the attractive synthesis of interesting amidine compounds in the absence of both toxic reagents and tedious purification procedures. Allylindium dichloride seems a good substitute for dichloroindium hydride for generation of indium centred radicals under photolytic conditions, since it allows allylation of electrophilic azides (e.g. phenylsulfonyl azide) and halogen or ester δ-substituted azides, the latter through a 1,5-H transfer rearrangement mechanism. Evidences of the radical nature of the reactions mechanism were provided by ESR spectroscopy, furthermore the same technique, allowed to discover that the reaction of azides with indium trichloride and other group XIII Lewis acids, in particular gallium trichloride, gives rise to strongly coloured, persistent paramagnetic species, whose structure is consistent with the radical cation of the head-to-tail dimer of the aniline corresponding to the starting azide.

The EU and Dispute Settlement within the WTO: a strategy for the protection of the Union’s autonomy in the domestic regulation of goods?

Oggetto della ricerca è l’accertamento dell’esistenza, nonché la definizione, della strategia dall’UE in materia di controversie commerciali aventi ad oggetto l’interpretazione e l’applicazione di norme facenti capo agli Accordi OMC in materia di misure sanitarie e di barriere tecniche al commercio. Nella prima parte della tesi, si ricostruiscono gli obbiettivi perseguiti dall’UE in materia di controversie SPS e TBT. In questo contesto, un’importanza di primo piano è attribuita alla difesa dell’autonomia regolamentare dell’Unione. Ad essa si riconduce la prassi UE finalizzata a prevenire il sorgere di controversie sul piano bilaterale attraverso la conclusione di accordi di mutuo riconoscimento, la cui portata ella sottolinea essere tuttavia limitata. L’analisi di cinque controversie sorte in ambito OMC di cui l’Unione è o è stata parte convenuta e che si fondano su presunte o accertate violazioni delle norme facenti capo ai due accordi menzionati consente di classificare gli argomenti giuridici avanzati dall’Unione nel contesto di tali controversie. Nella seconda parte della ricerca, la candidata identifica i mezzi a servizio della strategia UE, in primo luogo, attraverso l’analisi del quadro giuridico relativo alla partecipazione dell’Unione e degli Stati Membri al sistema OMC di risoluzione delle controversie; in secondo luogo, attraverso lo studio, da un lato, dello status delle norme OMC nell’ordinamento UE e, dall’altro, degli effetti delle pronunce dell’Organo di Risoluzione delle Controversie e della questione della responsabilità dell’Unione per violazione del diritto OMC. Sulla base del lavoro di ricerca svolto, si conclude che una strategia dell’UE esiste nella misura in cui l’Unione persegue l’obbiettivo di preservare la propria autonomia regolamentare attraverso, anche se non esclusivamente, gli strumenti afferenti all’ordine giuridico interno analizzati nella seconda parte. La candidata conclude altresì che la riforma del diritto delle relazioni esterne operata dal Trattato di Lisbona può indurre un cambiamento di tale strategia.

Optimization of purification procedures in organic semiconductor synthesis for improving the performances of organic field-effect transistors

The aim of the research activity focused on the investigation of the correlation between the degree of purity in terms of chemical dopants in organic small molecule semiconductors and their electrical and optoelectronic performances once introduced as active material in devices. The first step of the work was addressed to the study of the electrical performances variation of two commercial organic semiconductors after being processed by means of thermal sublimation process. In particular, the p-type 2,2′′′-Dihexyl-2,2′:5′,2′′:5′′,2′′′-quaterthiophene (DH4T) semiconductor and the n-type 2,2′′′- Perfluoro-Dihexyl-2,2′:5′,2′′:5′′,2′′′-quaterthiophene (DFH4T) semiconductor underwent several sublimation cycles, with consequent improvement of the electrical performances in terms of charge mobility and threshold voltage, highlighting the benefits brought by this treatment to the electric properties of the discussed semiconductors in OFET devices by the removal of residual impurities. The second step consisted in the provision of a metal-free synthesis of DH4T, which was successfully prepared without organometallic reagents or catalysts in collaboration with Dr. Manuela Melucci from ISOF-CNR Institute in Bologna. Indeed the experimental work demonstrated that those compounds are responsible for the electrical degradation by intentionally doping the semiconductor obtained by metal-free method by Tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) and Tributyltin chloride (Bu3SnCl), as well as with an organic impurity, like 5-hexyl-2,2':5',2''-terthiophene (HexT3) at, in different concentrations (1, 5 and 10% w/w). After completing the entire evaluation process loop, from fabricating OFET devices by vacuum sublimation with implemented intentionally-doped batches to the final electrical characterization in inherent-atmosphere conditions, commercial DH4T, metal-free DH4T and the intentionally-doped DH4T were systematically compared. Indeed, the fabrication of OFET based on doped DH4T clearly pointed out that the vacuum sublimation is still an inherent and efficient purification method for crude semiconductors, but also a reliable way to fabricate high performing devices.