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La nullita’ del provvedimento amministrativo per mancanza degli elementi essenziali.
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Stress ossidativo ed infiammazione contribuiscono alla disfunzione vascolare in un modello murino di sindrome metabolica.
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Rapid structural characterization of human antibody-antigen complexes through experimentally validated computational docking.
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Synthesis of fused-ring heterocycles from intramolecular reactions on alkene derivatives.
No full textHeterocycles form by far the largest of the classical divisions of organic chemistry and are of immense importance biologically, industrially, and indeed to the functioning of any developed human society. The majority of pharmaceuticals and biologically active agrochemicals are heterocyclic, as are countless additives and modifiers used in industries as varied as cosmetics, reprography, information storage, and plastics.
The work accomplished in this thesis concerns three different projects to achieve fused-ring heteropolicyclic systems having potential pharmacological properties. The synthetic procedures employed to reach this goal involve well known methodologies based on the functionalization of carbon-carbon double bonds.
The first project deals with diversity-oriented synthesis to access complex structures containing a piperidine unit starting from enantiopure 2-allyl-piperidine.
The second one concerns the study of a synthetic protocol for a new class of enantiopure tetracyclic 1,4-benzodiazepin-5-ones, containing an imidazole and a pyrazole ring.
Finally, third one is related to the obtainment of bicyclic oxazolidinone derivatives by Pd(II)-CuCl2 catalyzed intramolecular reactions on alkoxycarbonyl-protected amino alkenes
Conservation of Biodiversity in Alps: mountain hare (Lepus timidus) as model system.
No full textKnowledge about mountain hare (Lepus timidus) is still scarce, especially in alpine ecosystem. We collected information about mountain hare behaviour and distribution to asses if this species can be a useful indicator of environmental changes across the Alps.
First, we tested two di_erent live capture methods for mountain hare in Scotland and we verified if sex or age composition and body condition of hares di_ered between methods.
Individuals in poor condition were more frequently taken with traps, a tendency which may reflect increased risk taking of individuals in poorer body condition and less aversion to entering traps to bene_t from eating bait. We then used data of body mass of recaptured hares in two study areas in Italy. Here we did not _nd di_erence in body mass between _rst capture and recaptured hares, supporting the hypothesis that di_erences in capture methods, at least partly, depend on the habitat type where they have been applied.
The main part of the _eldwork aimed to investigate space and habitat use of mountain hare in relation to habitat structure and composition. We analysed the space-use patterns of two mountain hare populations from the Central Alps using radiotracking.
We monitored 34 hares, estimating home range size, overlap and site _delity, and compared our data with space use in Scottish and North-European populations. Home ranges of mountain hares did not di_er between two study sites with di_erent habitat types.
Subadult animals used larger ranges than adults and both age groups reduced home range size in autumn, a period that might be critical for hares due to changes in diet and/or high energy expenditure during the previous breeding season. Younger hares probably made exploratory movements to gather information about surrounding habitats. Home ranges in alpine 'edge' populations were smaller than in Scandinavian populations, but within the range of populations in di_erent habitat types in Scotland. Seasonal home ranges overlapped considerably, but di_ered among the sexes: intersexual overlap in males (thus with females) was larger than spatial overlap among other sexes. Seasonal shifts of home ranges were small and site _delity remained constant over the di_erent seasons, suggesting that resource distribution remained constant throughout the year and that the knowledge of an intensively frequented area is an important element of habitat quality.
From data collected with radiotracking we also analysed habitat selection of mountain hares. Hares selected closed habitat and avoided open habitat with poor productivity.
We found the same patterns at di_erent temporal scales: day and night ranges, seasonal ranges and annual ranges. The most preferred habitat type was dwarf mountain-pine, important for hares during winter, being the only available food resource, and still fundamental during the rest of year because it o_ers resting places. Dwarf mountain-pine present the highest High Heating Value (HHV) among the available food resources. For conservation reasons, we also analysed mountain hare interaction with cattle on alpine pastures. Presence of cattle on pastures did not result in a shift in habitat selection by hares.
Based on culled animals, we also analysed reproductive parameters of mountain and brown hare (Lepus europaes): litter size, number of litters per year and number of leverets per litter. Reproductive parameters of mountain hare were similar to those reported for other subspecies across its distribution range. In contrast, those registered in this study for brown hare suggested adaptation of brown hares to colder environments.
Finally, we explored population-genetic parameters, using mtDNA and nuclear microsatellites analysis. Two animals trapped in one of our study areas were hybrids between mountain and brown hare, supporting the fact that hybridisation processes are present also on the Alps as already reported for other populations living in the Northern parts of mountain hare distribution range.
Data collected support the hypothesis that alpine mountain hare populations undergo pressure related to environmental changes. This tendency becomes evident only taking into account di_erent aspects of its ecology, genetics and distribution
Anomalie cromosomiche strutturali come causa di alterata funzionalità genica, piastrinopenia e conseguente suscettibilità allo sviluppo di mielodispasie e/o leucemie.
No full textIl presente studio è stato effettuato su quattro diversi pazienti portatori di anomalie cromosomiche strutturali sia di natura acquisita che costituzionale. Tutti i riarrangiamenti alterano l'espressione di specifici geni e possono essere causativi delle forme di piastrinopenia da cui i nostri pazienti sono affetti. Ciascuna anomalia è stata indagata con metodi di citogenetica standard, di ibridazione in situ fluorescente (FISH), di comparative genomic hybridization su array (a-CGH) e, laddove necessario, sono state utilizzate tecniche di PCR real-time quantitativa e/o Multipainting. In dettaglio vengono presentati i seguenti casi:
CASO 1: paziente maschio affetto da anemia aplastica grave con scarsa presenza a livello midollare della componente granulopoietica e la completa assenza di megacariociti. E’ stata evidenziata nel midollo un’ anomalia cromosomica clonale a carico di un cromosoma n° 21. L'analisi di FISH effettuata con la sonda informativa CTD-2235K24 ha evidenziato la delezione degli esoni 2-8 di RUNX1, mentre le sonde BAC RP11-88N2 e RP11-625E21 hanno mostrato una duplicazione invertita delle bande q22.2 e q22.3, confermata poi anche dall'analisi di a-CGH. È stato dimostrato con esperimenti di real-time PCR quantitativa che l’evento di delezione a carico degli esoni 2-8 del gene RUNX1 comporta una significativa riduzione nell’espressione del gene stesso. Tale ipoespressione spiega le condizioni ematologiche del paziente, in particolare la completa assenza di megacariociti, vista la nota importanza di RUNX1 nella maturazione degli stessi e nella produzione delle piastrine. In prelievi successivi il paziente è andato incontro a una riduzione del clone con il cromosoma der(21) ma ha acquisito un secondo clone caratterizzato da una delezione interstiziale delle braccia lunghe di un cromosoma 13: in particolare la regione interessata (13q13.3-q21.31) risulta da letteratura essere spesso associata a sindromi mielodisplastiche (MDS) e pazienti affetti da MDS con del(13q) hanno una prognosi negativa per l’evoluzione in leucemia.
Questi due cloni sono stati monitorati nel tempo tramite FISH al fine di mantenere sotto controllo la disfunzione midollare ed eventuali sviluppi mielodisplastici/leucemici.
CASO 2: paziente femmina affetta da piastrinopenia cronica. Su prelievi di midollo, sangue periferico e fibroblasti cutanei è stata riscontrata un’anomalia cromosomica costituzionale a carico di un cromosoma n° 21 caratterizzata da un evento di inversione pericentrica. Analisi di FISH hanno dimostrato rottura del gene RUNX1 tra il 4° e il 5° esone. L'analisi di a-CGH ha confermato i risultati di FISH e mostrato anche una duplicazione degli esoni 2-3-4 del medesimo gene, una duplicazione della banda 21q22.2 e una grossa regione di delezione terminale di 2,35 Mb. La FISH effettuata con la sonda fosmidica WI2-1915K14 ha confermato la duplicazione degli esoni 2-3-4 di RUNX1, che sono ritenuti sul braccio lungo del cromosoma invertito, e, con la sonda BAC RP11-88N2, la delezione terminale. La Real-time quantitativa ha mostrato una riduzione nell'espressione relativa del gene RUNX1. Anche in questo caso gli eventi di rottura e duplicazione, di natura costituzionale, spiegano la piastrinopenia di cui la nostra paziente è affetta.
CASO 3: paziente maschio con fenotipo clinico suggestivo per trombocitopenia amegacariocitica congenita (CAMT) in assenza, però, delle mutazioni del gene MPL riconosciute come causa diretta della patologia. La CAMT, come le altre sindromi da insufficienza midollare, potrebbe essere uno stadio pre-leucemico, anche se i casi riportati in letteratura sono assai rari. E’ stata evidenziata un’anomalia cromosomica acquisita a carico di un cromosoma n° 1, caratterizzata da un evento di inversione paracentrica con punti di rottura in p13 e p36. Le analisi di FISH effettuate con sonde BAC informative hanno confermato l'evento di inversione e la presenza di due regioni di delezione prossime ai punti rottura, evidenziate prima solo con l'a-CGH. La Real-time quantitativa ha evidenziato una ridotta espressione del gene MPL, il quale non risulta rotto dall'evento di inversione ma solo traslocato in una regione più centromerica dello stesso cromosoma: l’ipotesi di un effetto di posizione sul gene MPL potrebbe spiegare il fenotipo simil-CAMT riscontrato nel paziente.
CASO 4: paziente femmina con quadro morfologico del midollo e delle piastrine compatibile con Sindrome di Paris-Trousseau: si tratta di una forma costituzionale di piastrinopenia con inclusione di piastrine caratterizzate da granuli α giganti e causata da delezione terminale di un cromosoma n° 11, in una regione dove è mappato il gene FLI1 (11q24), causativo della patologia. Da analisi su midollo è stato evidenziato un cariotipo complesso caratterizzato dalla presenza di un cromosoma derivativo 11 da traslocazione t(3;11)(q21;q25), di un cromosoma derivativo 2 e da monosomia 3. Analisi di MultiFISH a a-CGH hanno meglio chiarito la natura del riarrangiamento. La regione monosomica del cromosoma 11, confermata con FISH con la sonda BAC RP11-744N12, dimostra la perdita clonale di una copia del gene FLI1: questo spiega il quadro di piastrinopenia della paziente.
CONCLUSIONI
La rottura del gene RUNX1 (CASO 1 e 2), la perdita di una copia del gene FLI1 (CASO 4) e l'effetto di posizione del gene MPL (CASO 3) comportano disfunzione midollare. Nei casi 1 e 2 la rottura del gene è causa diretta della piastrinopenia di cui soffrono, poiché questo gene è coinvolto nella maturazione dei megacariociti e nella produzione delle piastrine. La Real-time quantitativa ha dimostrato una riduzione nell'espressione relativa di RUNX1. Nel CASO l'effetto di posizione a carico del gene MPL è probabilmente causa del fenotipo simil-CAMT del paziente: la Real-time quantitativa ha confemato una riduzione nell'espressione del gene. Nel CASO 4 un complesso riarrangiamento cromosomico strutturale comporta la perdita clonale di una copia del gene FLI1, che è la causa della piastrinopenia della paziente
Versatilità del palladio in chimica organica: attività catalitica di nuovi complessi e reazioni di carbonilazione, amminazione e alcossilazione per la sintesi di sistemi eterociclici.
No full textLe reazioni catalizzate da palladio sono diventate strumenti indispensabili in chimica organica. I complessi di palladio mostrano un comportamento diverso a seconda dello stato di ossidazione in cui si trovano, e la molto facile interconversione tra essi è la causa principale della versatilità del palladio in chimica organica. Questa tesi di dottorato si prefiggeva di promuovere processi catalizzati dal palladio nei vari stati di ossidazione, dando origine sia a legami C-C che C-N che C-O per la sintesi di una serie di sistemi eterociclici.
All’interno dei risultati ottenuti, vanno sottolineati alcuni aspetti:
1. Con il lavoro descritto nella prima parte della tesi si è studiata l’attività catalitica di una serie abbastanza ampia di complessi di σ-alchilpalladio, intermedi della reazione di Heck, per la prima volta isolati e caratterizzati sistematicamente. Questi palladacicli sono risultati efficaci precatalizzatori in una serie di processi di formazione di legami C-C come le reazioni di Heck, Suzuki e Stille. Questi prodotti sono una valida alternativa ai tradizionali catalizzatori a base di palladio per la loro eccellente stabilità.
2. Si è sviluppato una nuova procedura per la sintesi diretta di diidroisochinolinoni portanti una porzione metossicarbonil-metilica in posizione 4 tramite una ciclizzazione/carbonilazione palladio catalizzata. Si è trovato che la pressione di monossido di carbonio e la temperatura sono fattori cruciali per l’ottimizzazione della resa di isolamento dei prodotti desiderati.
3. Si sono sviluppati processi catalizzati da palladio per reazioni intramolecolari di carboaminazione e idroaminazione di indolilallenammidi che danno composti eteropoliciclici ad anelli fusi portanti rispettivamente una porzione stirenica o vinilica. In particolare un risultato rilevante è stata la reazione di idroaminazione catalizzata da palladio in condizioni blande tramite irraggiamento alle microonde.
4. Si sono ottenuti processi di ciclizzazioni di N-allil-N-idrossietilammidi, dando prodotti diidrossazinici, tramite formazione di legami C-O in presenza di una quantità catalitica di Pd(II) e di solo ossigeno molecolare come ossidante. Il processo è particolarmente interessante dal punto di vista ambientale in quanto l’ossidante utilizzato non genera waste. Questo processo è stato esteso ad altri amminoalcoli ed ad altri gruppi come sostituenti all’azoto. Si sono inoltre ottenuti processi domino in condizioni carbonilative e in presenza di vari nucleofili dando origine ad una serie di composti ossazinici completamente saturi diversamente sostituiti
Development and application of a marine biotic index for the evaluation of the influence of aquaculture activities on the benthic ecosystem in Mediterranean coastal areas.
No full textThe project was oriented to the development of an innovative tool for a more complete evaluation of the effect of marine aquaculture on the benthic ecosystem. Due to their high sensitivity, the attention was focused on the macroinvertebrates communities and on their use as descriptor of the marine soft bottom “health status”. Therefore, the AMBI, an existing marine biotic index already applied to detect impacts along European Coasts but never for the evaluation of the effects of aquaculture in the Mediterranean Sea, was developed and tested in four cases study: two off-shore fish farms, one in Sardinia and another one in Cyprus and two land-based fish farms in Tuscany. Assigning the macrobenthic species to one of the five Ecological Groups defined by this index, related to species sensitivity to disturbance, the AMBI gives back a classification of the site based on the benthic community “health status”. Thus, for each fish farm, samples of sediment were collected and chemical (total nitrogen, total carbon, organic matter, water content), physical (granulometry) and biological analysis (quantitative and qualitative indices calculation) were carried out. Concerning the biological analysis, all the organisms collected were counted and identified to the lowest possible taxon. The obtained data were used to calculate biological indices, including AMBI. For each case study, new species were added to the AMBI database, and this allowed the correct calculation of the index. The obtained results underlined the potentiality of AMBI in detecting effects of aquaculture on the benthic ecosystem and the development of this index database extended the possibility to use it also for the Mediterranean Sea
Coastal fish assemblages associated with rocky substrates: differences between a marine reserve (Pianosa island) and an unprotected area (Elba island).
No full textAlong the coastline of Italy, there are many areas of high environmental value.
Nevertheless, only for few of these the protection proposed in 1982 by low has been really enforced. Understanding if marine reserves work, requires two levels of analysis.
The first requires that particular reserves be evaluated in the context of the goals inherent in their establishment. The second level of analysis should be a broad comparison across reserves in different ecosystems to understand if reserves in general have a local or regional effect. Fish assemblage inside the MPAs, usually include many species targeted by fishing, so that they are primarily expected to benefit from protection, especially those having no-take reserve. The evaluation of the benefits, in terms of increase in density and size of target fishes can be useful to assess the ecological effectiveness of reserves. Up to now, there are very few long-term series of density estimates in and around MPAs before and after protection to verify these assumptions in the context of protected populations.
Fish fauna distributions patterns can be useful for testing the effectiveness of habitat protection in marine reserves. Most target fishes are high-levels predator and their functional extinction may cause community wide changes. The impact of fishing has consequent top-down perturbations in marine communities structures and organization.
Benthic communities in the shallow Mediterranean rocky sublittoral have 2 extreme alternative succession endpoints: coralline barrens and complex macroalgal beds. In the Mediterranean, Paracentrotus lividus is a key species that a high densities influences the dynamics of the phytobenthos, by eliminating erect algae and seagrasses, and inducing the formations of coralline barrens. Despite ecologists’ increasing interest in fish fauna living in marine protected areas, fish assemblage of some Italian marine reserves are still completely unknown.
Pianosa represents the ideal area where to verify the hypothesis that are the subject of this PhD. The PhD thesis is aimed at: 1- determine the structure of the infalittoral fish assemblage inhabiting the protected (Pianosa Island) and adjacent (Elba Island) non protected areas of Tuscan Archipelago, in terms of species composition, diversity and relative density, assessing differences in the community parameters in different areas; 2- to estimate the fish community responses to the trophic cascades in shallow rocky shores as possible results that indicate the reserve effect.
In infralittoral area, is possible to assess that structure of rocky fish assemblage testing the effectiveness of habitat protection in MPAs? Underwater Visual Census (UVC) is choised as the more appropriate method to conduct study on infralittoral fish community in Pianosa and Elba islands. Abundance data were collected by using a scale based on a geometric progression factor which was approximately 2. Fish density was calculated by considering the mid point of each abundant class. The size of individual fish was assigned to one of four size classes.
Extensions of barren cover, was visually estimated as relative percentage by means of square of 1 m-2 located on the bottom. P. lividus was chosen as indicator liable to possible shift from macroalgal cover to barren.
During the survey the degree of exploitation on fish species at Elba island, was evaluated on the basis of direct observation of fishing landings in Marina di Campo harbour.
A one way ANOVA was performed to test for differences among the areas in the assemblage parameters. Permanova was performed to test differences in analysis fish community using locality as fixed factor. The reserve effect (response ratio, R), was calculated for trophic categories and exploitation levels, as ln of ratio between value of response variable (density) inside the protected area and in fished areas.
During this study, 46 fish species were identified. Labridae and Sparidae were the most relevant families in relation to species richness. Some large sized specimens, belonging to species of high commercial value (i.e. Seriola dumerilili, Dentex dentex, and Ephinephelus marginatus), were also observed.
Taxonomic composition of fish community in Pianosa and Elba islands agrees with other similar northern-western in Mediterranean. The abundance of target species correlates strongly with the reserve effect, and therefore shows that the reserve has been effective in providing protection for such species. Some highly spearfished species such as E. marginatus, S. umbra, or D. dentex, have been censed exclusively within the reserve. Other such as Sparus aurata are far more abundant in the reserve. Species diversity index, and Evenness, show significant differences between sites located in Pianosa. Here, Diplodus vulgaris and Diplodus sargus, showed higher abundance in protected than in unprotected area. The study of four size classes of some target species has confirmed that large individuals were significantly more abundant in the protected area.
The increase of the size of fishes is a phenomenon widely reported in other MPAs and this general pattern has primarily attributed to the lack of fishing impact. Protected area seems to have the potential to restore depleted fish stocks. The response ratio evaluated for exploitation level shows that the reserve has been effective in protection for heavily fished species. Protected area, support greater density of D. sargus and D. vulgaris as predators on sea urchin P. lividus, than unprotected area. At the same time, the cover of barrens was significantly higher in unprotected area than in Pianosa. This study has evidenced a reserve effect or refuge effect characterized by the presence of large individuals. This refuge effect, within the presence of large individuals and species vulnerable to fishing, is one of the characteristics described in the definition of reserve effect. The results of the present study suggest that the recovery of Diplodus species in Pianosa seems to reestablish predatory control upon P. lividus similar to what is observed in other temperate regions and in other Mediterranean protected areas. The results of this study can corroborate the hypothesis of a transition from macroalgal beds, which harbour hundred of species of algae and invertebrates, to barrens through sea urchins grazing.In protected area, the effect of predator density on prey and on its population parameter is density-mediated indirect interactions (DMIIs).
This study, is a further step toward a better knowledge of the rocky reef fish assemblage of this area. At the community level, the present study will provide a useful reference point for planning future investigations on inter-specific relationship such as competition. Pianosa is an protected area where is possible to demonstrate that reserves aimed at restoring whole assemblages and ecological processes should be established as permanent no take area. This studies confirms that besides direct effects on target species, fishing may cause changes in trophic relations and in ecosystem functioning. Despite Pianosa is not a typical MPA since actual permanent presence of penal institute, it represent an important site where to study isolated infralittoral fish community on rocky substrata. It is clear that future experiments testing the reserve effect on fish biodiversity will have to take into account the necessity of being repeated on larger spatial and temporal scales
Phylogeography of Italian barbels (Cyprinidae, Barbus) inferred by mitochondrial and nuclear markers.
No full textSpecies of the genus Barbus, being primary freshwater fishes intolerant of salt water, are of great value for biogeographic studies since their dispersal strictly depends on geological
evolution of the landmasses (i.e. catchments watershed, mountain chains and fluctuations of sea level). In Italian peninsula four specie are formally recognized: B. caninus, B. balcanicus, B. plebejus and B. tyberinus. Their genetic relationships were assessed using both mitochondrial and nuclear markers. The study was carried out as first developing new nuclear primers for the S7 ribosomal protein and the Growth hormone genes (Gh); then performing a SNPs characterization of these loci on 18 populations (264 specimens in total). Results from nuclear sequences were then compared with those from partial sequences of the Cytochrome b mitochondrial gene (733 bp). Recovered phylogenies were congruent with the current morphology-based systematic and taxonomy. Results highlighted the close relationships between species belonging to the fluvio-lacustrine ecological group: B. plebejus and B. tyberinus and the high genetic distance between species belonging to the riverine group: B. caninus and B. balcanicus. Moreover findings were congruent with hypotheses of partial permeability of principal biogeographic barriers (Alpine and the Apennine chains) to freshwater fish fauna.
Successively the influence of different ecological preferences on gene flow was tested for B. caninus and B. tyberinus on 6 and 7 populations respectively. Results pointed out that the riverine B. caninus has higher structured populations than B. tyberinus, probably due to the different dispersion ability and the different habitat colonized. Moreover, for the first time, molecular evidences were shown about hybridization events occurring between B. caninus and B. plebejus, B. tyberinus and B. barbus