Supramolecular hybrid organic-inorganic multicomponent architectures in solution and on surface

Supramolecular architectures can be built-up from a single molecular component (building block) to obtain a complex of organic or inorganic interactions creating a new emergent condensed phase of matter, such as gels, liquid crystals and solid crystal. Further the generation of multicomponent supramolecular hybrid architecture, a mix of organic and inorganic components, increases the complexity of the condensed aggregate with functional properties useful for important areas of research, like material science, medicine and nanotechnology. One may design a molecule storing a recognition pattern and programming a informed self-organization process enables to grow-up into a hierarchical architecture. From a molecular level to a supramolecular level, in a bottom-up fashion, it is possible to create a new emergent structure-function, where the system, as a whole, is open to its own environment to exchange energy, matter and information. “The emergent property of the whole assembly is superior to the sum of a singles parts”. In this thesis I present new architectures and functional materials built through the selfassembly of guanosine, in the absence or in the presence of a cation, in solution and on the surface. By appropriate manipulation of intermolecular non-covalent interactions the spatial (structural) and temporal (dynamic) features of these supramolecular architectures are controlled. Guanosine G7 (5',3'-di-decanoil-deoxi-guanosine) is able to interconvert reversibly between a supramolecular polymer and a discrete octameric species by dynamic cation binding and release. Guanosine G16 (2',3'-O-Isopropylidene-5'-O-decylguanosine) shows selectivity binding from a mix of different cation's nature. Remarkably, reversibility, selectivity, adaptability and serendipity are mutual features to appreciate the creativity of a molecular self-organization complex system into a multilevelscale hierarchical growth. The creativity - in general sense, the creation of a new thing, a new thinking, a new functionality or a new structure - emerges from a contamination process of different disciplines such as biology, chemistry, physics, architecture, design, philosophy and science of complexity.

Distribution and state of fishery resources in the Northern and Central Adriatic Sea using trawl surveys data

The Ecosystem Approach to Fisheries represents the most recent research line in the international context, showing interest both towards the whole community and toward the identification and protection of all the “critical habitats” in which marine resources complete their life cycles. Using data coming from trawl surveys performed in the Northern and Central Adriatic from 1996 to 2010, this study provides the first attempt to appraise the status of the whole demersal community. It took into account not only fishery target species but also by-catch and discharge species by the use of a suite of biological indicators both at population and multi-specific level, allowing to have a global picture of the status of the demersal system. This study underlined the decline of extremely important species for the Adriatic fishery in recent years; adverse impact on catches is expected for these species in the coming years, since also minimum values of recruits recently were recorded. Both the excessive exploitation and environmental factors affected availability of resources. Moreover both distribution and nursery areas of the most important resources were pinpointed by means of geostatistical methods. The geospatial analysis also confirmed the presence of relevant recruitment areas in the North and Central Adriatic for several commercial species, as reported in the literature. The morphological and oceanographic features, the relevant rivers inflow together with the mosaic pattern of biocenoses with different food availability affected the location of the observed relevant nursery areas.

Jasmonates and abscisic acid influence fruit ripening and plant water use: practical, physiological and morphological aspects

The aim of the present thesis was to better understand the physiological role of the phytohormones jasmonates (JAs) and abscisic acid (ABA) during fruit ripening in prospect of a possible field application of JAs and ABA to improve fruit yield and quality. In particular, the effects of exogenous application of these substances at different fruit developmental stages and under different experimental conditions were evaluated. Some aspects of the water relations upon ABA treatment were also analysed. Three fruit species, peach (Prunus persica L. Batsch), golden (Actinidia chinensis) and green kiwifruit (Actinidia deliciosa), and several of their cvs, were used for the trials. Different experimental models were adopted: fruits in planta, detached fruit, detached branches with fruit, girdled branches and micropropagated plants. The work was structured into four sets of experiments as follows: (i) Pre-harvest methyl jasmonate (MJ) application was performed at S3/S4 transition under field conditions in Redhaven peach; ethylene production, ripening index, fruit quality and shelf-life were assessed showing that MJ-treated fruit were firmer and thus less ripe than controls as confirmed by the Index of Absorbance Difference (IAD), but exhibited a shorter shelf-life due to an increase in ethylene production. Moreover, the time course of the expression of ethylene-, auxin- and other ripening-related genes was determined. Ripening-related ACO1 and ACS1 transcript accumulation was inhibited though transiently by MJ, and gene expression of the ethylene receptor ETR2 and of the ethylene-related transcription factor ERF2 was also altered. The time course of the expression of several auxin-related genes was strongly affected by MJ suggesting an increase in auxin biosynthesis, altered auxin conjugation and release as well as perception and transport; the need for a correct ethylene/auxin balance during ripening was confirmed. (ii) Pre- and post-harvest ABA applications were carried out under field conditions in Flaminia and O’Henry peach and Stark Red Gold nectarine fruit; ethylene production, ripening index, fruit quality and shelf-life were assessed. Results show that pre-harvest ABA applications increase fruit size and skin color intensity. Also post-harvest ABA treatments alter ripening-related parameters; in particular, while ethylene production is impaired in ABA-treated fruit soluble solids concentration (SSC) is enhanced. Following field ABA applications stem water potential was modified since ABA-treated peach trees retain more water. (iii) Pre- and post-harvest ABA and PDJ treatments were carried out in both kiwifruit species under field conditions at different fruit developmental stages and in post-harvest. Ripening index, fruit quality, plant transpiration, photosynthesis and stomatal conductance were assessed. Pre-harvest treatments enhance SSC in the two cvs and flesh color development in golden kiwifruit. Post-harvest applications of either ABA or ABA plus PDJ lead to increased SSC. In addition, ABA reduces gas exchanges in A. deliciosa. (iv) Spray, drench and dipping ABA treatments were performed in micropropagated peach plants and in peach and nectarine detached branches; plant water use and transpiration, biomass production and fruit dehydration were determined. In both plants and branches ABA significantly reduces water use and fruit dehydration. No negative effects on biomass production were detected. The present information, mainly arising from plant growth regulator application in a field environment, where plants have to cope with multiple biotic and abiotic stresses, may implement the perspectives for the use of these substances in the control of fruit ripening.

Stream sediments analysis for geochemical mapping of Romagna Apennines (Northern Italy): monitoring and management tool of environmental resources at various scales

Geochemical mapping is a valuable tool for the control of territory that can be used not only in the identification of mineral resources and geological, agricultural and forestry studies but also in the monitoring of natural resources by giving solutions to environmental and economic problems. Stream sediments are widely used in the sampling campaigns carried out by the world's governments and research groups for their characteristics of broad representativeness of rocks and soils, for ease of sampling and for the possibility to conduct very detailed sampling In this context, the environmental role of stream sediments provides a good basis for the implementation of environmental management measures, in fact the composition of river sediments is an important factor in understanding the complex dynamics that develop within catchment basins therefore they represent a critical environmental compartment: they can persistently incorporate pollutants after a process of contamination and release into the biosphere if the environmental conditions change. It is essential to determine whether the concentrations of certain elements, in particular heavy metals, can be the result of natural erosion of rocks containing high concentrations of specific elements or are generated as residues of human activities related to a certain study area. This PhD thesis aims to extract from an extensive database on stream sediments of the Romagna rivers the widest spectrum of informations. The study involved low and high order stream in the mountain and hilly area, but also the sediments of the floodplain area, where intensive agriculture is active. The geochemical signals recorded by the stream sediments will be interpreted in order to reconstruct the natural variability related to bedrock and soil contribution, the effects of the river dynamics, the anomalous sites, and with the calculation of background values be able to evaluate their level of degradation and predict the environmental risk.

Supporting requirement elicitation and ontology testing in knowledge graph engineering

Knowledge graphs and ontologies are closely related concepts in the field of knowledge representation. In recent years, knowledge graphs have gained increasing popularity and are serving as essential components in many knowledge engineering projects that view them as crucial to their success. The conceptual foundation of the knowledge graph is provided by ontologies. Ontology modeling is an iterative engineering process that consists of steps such as the elicitation and formalization of requirements, the development, testing, refactoring, and release of the ontology. The testing of the ontology is a crucial and occasionally overlooked step of the process due to the lack of integrated tools to support it. As a result of this gap in the state-of-the-art, the testing of the ontology is completed manually, which requires a considerable amount of time and effort from the ontology engineers. The lack of tool support is noticed in the requirement elicitation process as well. In this aspect, the rise in the adoption and accessibility of knowledge graphs allows for the development and use of automated tools to assist with the elicitation of requirements from such a complementary source of data. Therefore, this doctoral research is focused on developing methods and tools that support the requirement elicitation and testing steps of an ontology engineering process. To support the testing of the ontology, we have developed XDTesting, a web application that is integrated with the GitHub platform that serves as an ontology testing manager. Concurrently, to support the elicitation and documentation of competency questions, we have defined and implemented RevOnt, a method to extract competency questions from knowledge graphs. Both methods are evaluated through their implementation and the results are promising.

Effetti del chitosano su composti polifenolici in colture cellulari di vite: aspetti molecolari e metabolici

Polyphenols, including flavonoids and stilbenes, are an essential part of human diet and constitute one of the most abundant and ubiquitous group of plant secondary metabolites. The level of these compounds is inducible by stress or fungal attack, so attempts are being made to identify likely biotic and abiotic elicitors and to better understand the underlying mechanism. Resveratrol (3,5,4’-trihydroxystilbene), which belongs to the stilbene family, is a naturally occurring polyphenol, found in several fruits, vegetables and beverages including red wine. It is one of the most important plant polyphenols with proved benefic activity on animal health. In the last two decades, the potential protective effects of resveratrol against cardiovascular and neurodegenerative diseases, as well as the chemopreventive properties against cancer, have been largely investigated. The most important source of polyphenols and in particular resveratrol for human diet is grape (Vitis vinifera). Since stilbenes and flavonoids play a very important role in plant defence responses and enviromental interactions, and their effects on human health seem promising, the aim of the research of this Thesis was to study at different levels the activation and the regulation of their biosynthetic pathways after chitosan treatment. Moreover, the polyphenol production in grape cells and the optimisation of cultural conditions bioreactor scale-up, were also investigated. Cell suspensions were obtained from cv. Barbera (Vitis vinifera L.) petioles and were treated with a biotic elicitor, chitosan (50 μg/mL, dissolved in acetic acid) to promote phenylpropanoid metabolism. Chitosan is a D-glucosamine polymer from fungi cell wall and therefore mimes fungal pathogen attack. Liquid cultures have been monitored for 15 days, measuring cell number, cell viability, pH and grams of fresh weight. The endogenous and released amounts of 7 stilbenes (trans and cis isomers of resveratrol, piceid and resveratroloside, and piceatannol), gallic acid, 6 hydroxycinnamic acids (trans-cinnamic, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acids), 5 catechines (catechin, epicatechin, epigallocatechin-gallate (EGCG), epigallocatechin and epicatechin-gallate) and other 5 flavonoids (chalcon, naringenin, kaempferol, quercetin and rutin) in cells and cultural medium, were measured by HPLC-DAD analysis and total anthocyanins were quantified by spectrophotometric analysis. Chitosan was effective in stimulating trans-resveratrol endogenous accumulation with a sharp peak at day 4 (exceeding acetic acid and water controls by 36% and 63%, respectively), while it did not influence the production of the cis-isomer. Compared to both water and acetic acid controls, chitosan decreased the release of both trans- and cis-resveratrol respect to controls. No effect was shown on the accumulation of single resveratrol mono-glucoside isomers, but considering their total amount, normalized for the relative water control, it was possible to evidence an increase in both accumulation and release of those compounds, in chitosan-treated cells, throughout the culture period and particularly during the second week. Many of the analysed flavonoids and hydroxycinnamic acids were not present or detectable in trace amounts. Catechin, epicatechin and epigallocatechin-gallate (EGCG) were detectable both inside the cells and in the culture media, but chitosan did not affect their amounts. On the contrary, total anthocyanins have been stimulated by chitosan and their level, from day 4 to 14, was about 2-fold higher than in both controls, confirming macroscopic observations that treated suspensions showed an intense brown-red color, from day 3 onwards. These elicitation results suggest that chitosan selectively up-regulates specific biosynthetic pathways, without modifying the general accumulation pattern of other flavonoids. Proteins have been extracted from cells at day 4 of culture (corresponding to the production peak of trans-resveratrol) and separated by bidimensional electrophoresis. The 73 proteins that showed a consistently changed amount between untreated, chitosan and acetic acid (chitosan solvent) treated cells, have been identified by mass spectrometry. Chitosan induced an increase in stilbene synthase (STS, the resveratrol biosynthetic enzyme), chalcone-flavanone isomerase (CHI, that switches the pathway from chalcones to flavones and anthocyanins), pathogenesis-related proteins 10 (PRs10, a large family of defence proteins), and a decrease in many proteins belonging to primary metabolisms. A train of six distinct spots of STS encoded by the same gene and increased by chitosan, was detected on the 2-D gels, and related to the different phosphorylation degree of STS spots. Northern blot analyses have been performed on RNA extracted from cells treated with chitosan and relative controls, using probes for STS, PAL (phenylalanine ammonia lyase, the first enzyme of the biosynthetic pathway), CHS (chalcone synthase, that shares with STS the same precursors), CHI and PR-10. The up-regulation of PAL, CHS and CHI transcript expression levels correlated with the accumulation of anthocyanins. The strong increase of different molecular weight PR-10 mRNAs, correlated with the 11 PR-10 protein spots identified in proteomic analyses. The sudden decrease in trans-resveratrol endogenous accumulation after day 4 of culture, could be simply explained by the diminished resveratrol biosynthetic activity due to the lower amount of biosynthetic enzymes. This might be indirectly demonstrated by northern blot expression analyses, that showed lower levels of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) mRNAs starting from day 4. Other possible explanations could be a resveratrol oxidation process and/or the formation of other different mono-, di-glucosides and resveratrol oligomers such as viniferins. Immunolocalisation experiments performed on grape protoplasts and the subsequent analyses by confocal microscope, showed that STS, and therefore the resveratrol synthetic site, is mostly associated to intracellular membranes close to the cytosolic side of plasma membrane and in a smaller amount is localized in the cytosol. STS seemed not to be present inside vacuole and nucleus. There were no differences in the STS intracellular localisation between the different treatments. Since it was shown that stilbenes are largely released in the culture medium and that STS is a soluble protein, a possible interaction of STS with a plasma membrane transporter responsible for the extrusion of stilbenes in the culture medium, might be hypothesized. Proteomic analyses performed on subcellular fractions identified in the microsomial fraction 5 proteins taking part in channel complexes or associated with channels, that significantly changed their amount after chitosan treatment. In soluble and membrane fractions respectively 3 and 4 STS and 6 and 3 PR-10 have been identified. Proteomic results obtained from subcellular fractions substantially confirmed previous result obtained from total cell protein extracts and added more information about protein localisation and co-localisation. The interesting results obtained on Barbera cell cultures with the aim to increase polyphenol (especially stilbenes) production, have encouraged scale up tests in 1 litre bioreactors. The first trial fermentation was performed in parallel with a normal time-course in 20 mL flasks, showing that the scale-up (bigger volume and different conditions) process influenced in a very relevant way stilbenes production. In order to optimise culture parameters such as medium sucrose amount, fermentation length and inoculum cell concentration, few other fermentations were performed. Chitosan treatments were also performed. The modification of each parameter brought relevant variations in stilbenes and catechins levels, so that the production of a certain compound (or class of compounds) could be hypothetically promoted by modulating one or more culture parameters. For example the catechin yield could be improved by increasing sucrose content and the time of fermentation. The best results in stilbene yield were obtained in a 800 mL fermentation inoculated with 10.8 grams of cells and supplemented with chitosan. The culture was fed with MS medium added with 30 g/L sucrose, 25 μg/mL rifampicin and 50 μg/mL of chitosan, and was maintained at 24°C, stirred by marine impeller at 100 rpm and supplied of air at 0.16 L/min rate. Resveratroloside was the stilbene present in the larger amount, 3-5 times more than resveratrol. Because resveratrol glucosides are similarly active and more stable than free resveratrol, their production using a bioreactor could be a great advantage in an hypothetical industrial process. In my bioreactor tests, stilbenes were mainly released in the culture medium (60-80% of the total) and this fact could be another advantage for industrial applications, because it allows recovering the products directly from the culture medium without stopping the fermentation and/or killing the cells. In my best cultural conditions, it was possible to obtain 3.95 mg/L of stilbenes at day 4 (maximum resveratrol accumulation) and 5.13 mg/L at day 14 (maximum resveratroloside production). In conclusion, chitosan effect in inducing Vitis vinifera defense mechanisms can be related to its ability to increase the intracellular content of a large spectrum of antioxidants, and in particular of resveratrol, its derivates and anthocyanins. Its effect can be observed at transcriptional, proteomic (variation of soluble and membrane protein amounts) and metabolic (polyphenols production) level. The chitosan ability to elicit specific plant matabolisms can be useful to produce large quantities of antioxidant compounds from cell culture in bioreactor.

Biomimetic Materials for Biomedical Applications

Objects with complex shape and functions have always attracted attention and interest. The morphological diversity and complexity of naturally occurring forms and patterns have been a motivation for humans to copy and adopt ideas from Nature to achieve functional, aesthetic and social value. Biomimetics is addressed to the design and development of new synthetic materials using strategies adopted by living organisms to produce biological materials. In particular, biomineralized tissues are often sophisticate composite materials, in which the components and the interfaces between them have been defined and optimized, and that present unusual and optimal chemical-physical, morphological and mechanical properties. Moreover, biominerals are generally produced by easily traceable raw materials, in aqueous media and at room pressure and temperature, that is through cheap process and materials. Thus, it is not surprising that the idea to mimic those strategies proper of Nature has been employed in several areas of applied sciences, such as for the preparation of liquid crystals, ceramic thin films computer switches and many other advanced materials. On this basis, this PhD thesis is focused on the investigation of the interaction of biologically active ions and molecules with calcium phosphates with the aim to develop new materials for the substitution and repair of skeletal tissue, according to the following lines: I. Modified calcium phosphates. A relevant part of this PhD thesis has been addressed to study the interaction of Strontium with calcium phosphates. It was demonstrated that strontium ion can substitute for calcium into hydroxyapatite, causing appreciable structural and morphological modifications. The detailed structural analysis carried out on the nanocrystals at different strontium content provided new insight into its interaction with the structure of hydroxyapatite. At variance with the behaviour of Sr towards HA, it was found that this ion inhibits the synthesis of octacalcium phosphate. However, it can substitute for calcium in this structure up to 15 atom %, in agreement with the increase of the cell parameters observed on increasing ion concentration. A similar behaviour was found for Magnesium ion, whereas Manganese inhibits the synthesis of octacalcium phosphate and it promotes the precipitation of dicalcium phosphate dehydrate. It was also found that Strontium affects the kinetics of the reaction of hydrolysis of α-TCP. It inhibits the conversion from α-TCP to hydroxyapatite. However, the resulting apatitic phase contains significant amounts of Sr2+ suggesting that the addition of Sr2+ to the composition of α-TCP bone cements could be successfully exploited for its local delivery in bone defects. The hydrolysis of α-TCP has been investigated also in the presence of increasing amounts of gelatin: the results indicated that this biopolymer accelerates the hydrolysis reaction and promotes the conversion of α-TCP into OCP, suggesting that its addition in the composition of calcium phosphate cements can be employed to modulate the OCP/HA ratio, and as a consequence the solubility, of the set cement. II. Deposition of modified calcium phosphates on metallic substrates. Coating with a thin film of calcium phosphates is frequently applied on the surface of metallic implants in order to combine the high mechanical strength of the metal with the excellent bioactivity of the calcium phosphates surface layers. During this PhD thesis, thank to the collaboration with prof. I.N. Mihailescu, head of the Laser-Surface-Plasma Interactions Laboratory (National Institute for Lasers, Plasma and Radiation Physics – Laser Department, Bucharest) Pulsed Laser Deposition has been successfully applied to deposit thin films of Sr substituted HA on Titanium substrates. The synthesized coatings displayed a uniform Sr distribution, a granular surface and a good degree of crystallinity which slightly decreased on increasing Sr content. The results of in vitro tests carried out on osteoblast-like and osteoclast cells suggested that the presence of Sr in HA thin films can enhance the positive effect of HA coatings on osteointegration and bone regeneration, and prevent undesirable bone resorption. The possibility to introduce an active molecule in the implant site was explored using Matrix Assisted Pulsed Laser Evaporation to deposit hydroxyapatite nanocrystals at different content of alendronate, a bisphosphonate widely employed in the treatments of pathological diseases associated to bone loss. The coatings displayed a good degree of crystallinity, and the results of in vitro tests indicated that alendronate promotes proliferation and differentiation of osteoblasts even when incorporated into hydroxyapatite. III. Synthesis of drug carriers with a delayed release modulated by a calcium phosphate coating. A core-shell system for modulated drug delivery and release has been developed through optimization of the experimental conditions to cover gelatin microspheres with a uniform layer of calcium phosphate. The kinetics of the release from uncoated and coated microspheres was investigated using aspirin as a model drug. It was shown that the presence of the calcium phosphate shell delays the release of aspirin and allows to modulate its action.

Modelling coupled physical-biogeochemical processes in ice-covered oceans

The last decades have seen a large effort of the scientific community to study and understand the physics of sea ice. We currently have a wide - even though still not exhaustive - knowledge of the sea ice dynamics and thermodynamics and of their temporal and spatial variability. Sea ice biogeochemistry is instead largely unknown. Sea ice algae production may account for up to 25% of overall primary production in ice-covered waters of the Southern Ocean. However, the influence of physical factors, such as the location of ice formation, the role of snow cover and light availability on sea ice primary production is poorly understood. There are only sparse localized observations and little knowledge of the functioning of sea ice biogeochemistry at larger scales. Modelling becomes then an auxiliary tool to help qualifying and quantifying the role of sea ice biogeochemistry in the ocean dynamics. In this thesis, a novel approach is used for the modelling and coupling of sea ice biogeochemistry - and in particular its primary production - to sea ice physics. Previous attempts were based on the coupling of rather complex sea ice physical models to empirical or relatively simple biological or biogeochemical models. The focus is moved here to a more biologically-oriented point of view. A simple, however comprehensive, physical model of the sea ice thermodynamics (ESIM) was developed and coupled to a novel sea ice implementation (BFM-SI) of the Biogeochemical Flux Model (BFM). The BFM is a comprehensive model, largely used and validated in the open ocean environment and in regional seas. The physical model has been developed having in mind the biogeochemical properties of sea ice and the physical inputs required to model sea ice biogeochemistry. The central concept of the coupling is the modelling of the Biologically-Active-Layer (BAL), which is the time-varying fraction of sea ice that is continuously connected to the ocean via brines pockets and channels and it acts as rich habitat for many microorganisms. The physical model provides the key physical properties of the BAL (e.g., brines volume, temperature and salinity), and the BFM-SI simulates the physiological and ecological response of the biological community to the physical enviroment. The new biogeochemical model is also coupled to the pelagic BFM through the exchange of organic and inorganic matter at the boundaries between the two systems . This is done by computing the entrapment of matter and gases when sea ice grows and release to the ocean when sea ice melts to ensure mass conservation. The model was tested in different ice-covered regions of the world ocean to test the generality of the parameterizations. The focus was particularly on the regions of landfast ice, where primary production is generally large. The implementation of the BFM in sea ice and the coupling structure in General Circulation Models will add a new component to the latters (and in general to Earth System Models), which will be able to provide adequate estimate of the role and importance of sea ice biogeochemistry in the global carbon cycle.

Il coniglio come modello sperimentale per lo studio degli effetti neurologici indotti da Cobalto

Sono stati studiati gli effetti tossici dell’esposizione cronica a cobalto e cromo. In passato, questa tossicità, che colpiva lavoratori esposti per ragioni occupazionali, è stata un problema molto sentito. Tuttavia, recenti pubblicazioni hanno descritto una specifica tossicità mediata da elevati livelli di cobalto e cromo, anche in pazienti portatori di protesi metalliche, quali gli impianti d’anca. Anche se sintomi clinici tra cui, cecità, sordità e neuropatia periferica, suggeriscono uno specifico neurotropismo, ancora poco è conosciuto delle basi neuropatologiche di questo processo ed oltretutto non ne è ancora stata apportata un’evidenza sperimentale. In questo progetto di ricerca, quindi, si è voluto approfondire il meccanismo patogenetico da cui scaturiscono tali sintomi neurologici, utilizzando come modello sperimentale il coniglio. Conigli New Zealand White sono stati trattati con dosi endovenose ripetute di cobalto e cromo, inoculati singolarmente od in associazione tra loro. Nessuna evidente alterazione clinica o patologica è stata associata alla somministrazione di solo cromo, nonostante gli elevati livelli in sangue e tessuti, mentre i trattati con cobalto-cromo o solo cobalto hanno mostrato segni clinici gravanti sul sistema vestibolo-cocleare; il cobalto, quindi, è stato identificato come il maggiore elemento scatenante neurotossicità. Inoltre all’esame istopatologico gli animali hanno mostrato severa deplezione delle cellule gangliari retiniche e cocleari, assieme a danno al nervo ottico e perdita di cellule sensitive capellute dell’orecchio. È risultato infine evidente che la gravità delle alterazioni è stata correlata al dosaggio ed al tempo di esposizione; dati questi che confermano, quindi, le precedenti osservazioni fatte su pazienti umani esposti a rilascio abnorme di cobalto e cromo da usura di protesi d’anca. È stato ipotizzato che il cobalto agisca sui mitocondri provocando l’incremento di produzione di specie reattive dell’ossigeno e il rilascio di fattori proapoptotici, causando sulle cellule neuronali un danno proporzionale al loro fabbisogno energetico e grado di mielinizzazione.

Ricerche di campo per lo sviluppo della tecnologia del maschio sterile nella lotta ad Aedes albopictus (Skuse)

Aedes albopictus (Skuse), comunemente detta Zanzara Tigre, ha invaso, negli ultimi anni, molti paesi, soprattutto in modo passivo attraverso il commercio di pneumatici usati. Questa specie è particolarmente adatta all'applicazione della tecnica dell'insetto sterile (SIT), basata su allevamento massale, sterilizzazione e rilascio in campo di un gran numero di maschi della specie vettrice. I maschi sterili rilasciati devono essere in grado di volare, di disperdersi sul territorio, di sopravvivere, di essere sessualmente attivi abbastanza a lungo per coprire il tempo tra una fase di rilascio e la successiva, di individuare le femmine vergini selvatiche e competere con successo per l'accoppiamento con i maschi selvatici. La dispersione e la sopravvivenza dei maschi di Ae. albopictus allevati in laboratorio, sono state studiate mediante tecniche di marcatura, rilascio e ricattura. Le catture sono state eseguite da tecnici specializzati, in un raggio di 350 m dal sito di rilascio. Gli esperimenti condotti hanno dimostrato che i maschi sono in grado di disperdersi, dal sito di rilascio, per circa 200 m ma la loro longevità in campo è fortemente dipendente dalle condizioni climatiche. In studi di semi-campo e di campo è stato valutato uno speciale dispositivo progettato per essere incluso nella stazione di rilascio dei maschi in grado di fornire loro fonti energetiche per migliorarne le prestazioni. I risultati ottenuti sono stati positivi. Studi di competitività sono stati condotti in tunnel costruiti in un ambiente naturale al fine di validare un protocollo per studi sulla competitività dei maschi in questo modello sperimentale. Maschi irraggiati mediante l'applicazione di raggi gamma alla dose di 30 Gy sono stati messi in competizione con maschi fertili per l'accoppiamento con femmine vergini con diversi rapporti. I risultati ottenuti hanno dimostrato le buone prestazioni e l'affidabilità di questo modello sperimentale rimanendo però irrisolto il problema dell’elevata variabilità.

Sindrome da Asfissia Perinatale nel puledro neonato: protocolli diagnostico-terapeutici

La Sindrome da Asfissia Perinatale (PAS) è una delle più comuni patologie che colpiscono il puledro neonato nelle prime 72 h di vita. È una patologia difficile da diagnosticare in quanto non esistono parametri o segni clinici specifici, la sintomatologia è molto variabile in base alla durata e all’intensità dell’insulto ipossico ischemico e al tipo di organo maggiormente colpito. Lo scopo di questo studio è la ricerca e la valutazione di alcuni parametri biochimico-clinici e di alcuni biomarkers per la diagnosi precoce e il corretto trattamento dei puledri affetti da PAS. Nei puledri neonati che presentano questa patologia è stata riscontrata un’ipermagnesiemia al momento del ricovero associata a prognosi infausta, probabilmente causata da un grave danno cellulare con rilascio in circolo del magnesio intracellulare. La PAS potrebbe essere un’ulteriore causa di Euthyroid Sick Syndrome, in quanto abbiamo riscontrato una diminuzione delle concentrazioni di T3 e T4 nei puledri malati rispetto ai sani della stessa età, come avviene in altre malattie sistemiche. Lo studio del profilo proteomico ha permesso di separare le più importanti frazioni proteiche del liquido amniotico di cavalla, mettendo in evidenza similitudini e differenze qualitative e quantitative nei ferogrammi dei puledri sani e di quelli affetti da PAS ed una maggiore variabilità è stata riscontrata nei profili dei liquidi amniotici dei puledri malati. Il glutatione è risultato poco espresso nel puledro neonato, i puledri sani presentano concentrazioni più basse sia rispetto ai malati della stessa età sia agli adulti ma con una tendenza all’aumento nelle prime 24 ore di vita per i sani ed un calo nei malati. La somministrazione della terapia antiradicalica non influisce sulle concentrazioni di glutatione totale ed i puledri deceduti presentano concentrazioni più alte.

Studio elettrofisiologico di modificazioni a lungo termine della forza della trasmissione sinaptica nel sistema nervoso centrale

Il nucleo accumbens (NAc), il maggior componente del sistema mesocorticolimbico, è coinvolto nella mediazione delle proprietà di rinforzo e nella dipendenza da diverse sostanze d’abuso. Le sinapsi glutammatergiche del NAc possono esprimere plasticità, tra cui una forma di depressione a lungo termine (LTD) dipendente dagli endocannabinoidi (eCB). Recenti studi hanno dimostrato un’interazione tra le vie di segnalazione del sistema eCB e quelle di altri sistemi recettoriali, compreso quello serotoninergico (5-HT); la vasta colocalizzazione di recettori serotoninergici e CB1 nel NAc suggerisce la possibilità di un’interazione tra questi due sistemi. In questo studio abbiamo riscontrato che una stimolazione a 4 Hz per 20 minuti (LFS-4Hz) delle afferenze glutammatergiche in fettine cerebrali di ratto, induce una nuova forma di eCB-LTD nel core del NAc, che richiede l’attivazione dei recettori CB1 e 5-HT2 e l’apertura dei canali del Ca2+ voltaggio-dipendenti di tipo L. Inoltre abbiamo valutato che l’applicazione esogena di 5-HT (5 M, 20 min) induce una LTD analoga (5-HT-LTD) a livello delle stesse sinapsi, che richiede l’attivazione dei medesimi recettori e l’apertura degli stessi canali del Ca2+; LFS-4Hz-LTD e 5-HT-LTD sono reciprocamente saturanti. Questi risultati suggeriscono che la LFS-4Hz induce il rilascio di 5-HT, che si lega ai recettori 5-HT2 a livello postsinaptico incrementando l’influsso di Ca2+ attraverso i canali voltaggio-dipendenti di tipo L e la produzione e il rilascio di 2-arachidonoilglicerolo; l’eCB viaggia a ritroso e si lega al recettore CB1 a livello presinaptico, causando una diminuzione duratura del rilascio di glutammato, che risulta in una LTD. Queste osservazioni possono essere utili per comprendere i meccanismi neurofisiologici che sono alla base della dipendenza da sostanze d’abuso, della depressione maggiore e di altre malattie psichiatriche caratterizzate dalla disfunzione della neurotrasmissione di 5-HT nel NAc.

The role of CD8+CCR4+ T-cells in axial spondyloarthritis

Axial spondyloarthritis (AxSpA) is an inflammatory disease affecting the axial skeleton. The infiltrate of T-cells in the structural lesions has been found to contribute to bone remodeling, but consensus relating the functional contribution of different T-cell subsets to pathogenesis has not been reached yet. Aim of the project was to characterize circulating T-cells and their homing markers from axSpA patients in order to identify cellular populations that could migrate to inflamed tissues and be implicated in axSpA. We found an altered proportion of circulating naïve and memory T-cells in axSpA patients, and a skew in favor of CD8+ T-cells expressing the chemokine receptor CCR4. Since CCL17 and CCL22, the two ligands for CCR4, are found to be elevated in the sera of axSpA patients, we investigated in details the role of CD8+CCR4+ T cells in axSpA. Our data showed that circulating CD8+CCR4+ T-cells display an effector memory phenotype and express homing markers for tissues that are target of the disease. Noteworthy, CD8+CCR4+ T cells from axSpA patients were activated, expressed markers of proliferation and acquired a cytotoxic phenotype, as demonstrated by the increased production of granzyme and perforin. CD8+CCR4+ T cells from axSpA patients upregulate the transcription of genes involved in bone mineralization and downregulate genes involved in osteoclast differentiation, indicating their possible involvement in bone remodeling. Furthermore, CD8+CCR4+ T cells stimulated with PMA and ionomycin were able to produce and release TNF and IL-8, two cytokines involved in osteoclastogenesis, indicating that CD8+CCR4+ T-cells after stimulation would be able to promote osteoclasts differentiation and neutrophils recruitment. Taken together our data suggest that CD8+CCR4+ T cells might exert a pathogenic role in axSpA, by releasing mediators of tissue damage, bone remodeling and recruitment of other pro inflammatory cells.