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Macrofite come bioindicatori : analisi ecologica lungo il bacino del fiume Tevere e valutazione degli indici macrofitici

Wed, 16 Dec 2009 23:00:00 GMT

Macrofite come bioindicatori : analisi ecologica lungo il bacino del fiume Tevere e valutazione degli indici macrofitici Bisceglie, Sara 2009-12-17 Per la corretta valutazione e caratterizzazione di un ecosistema fluviale si ritiene oggi fondamentale utilizzare un approccio di tipo integrato tra metodologie chimico-fisiche e biologiche. La recente emanazione a livello europeo della Water Frame Directive (2000/60/CE) impone un innovativo aggiornamento delle metodiche analitiche e di classificazione dei corpi idrici. La WFD, infatti, attribuisce un’importanza prioritaria agli organismi viventi ritenendo che questi costituiscano gli indicatori più validi per definire lo stato ecologico delle acque superficiali e per valutare realmente lo stato di salute di un corpo idrico. Nell’ambito dell’attuazione della WFD diventa, quindi, prioritaria la messa a punto di nuovi descrittori biologici dei diversi livelli trofici dell’ecosistema. Infatti l’applicazione della WFD prevede l’analisi non solo delle comunità di macroinvertebrati bentonici, già inclusi nella precedente normativa nazionale (152/99), ma anche di quelle diatomiche, ittiche e macrofitiche. Da queste premesse nasce il presente progetto di ricerca riguardante in particolare lo studio ecologico della componente macrofitica che, comprensiva di fanerogame erbacee, macroalghe, briofite e pteridofite rinvenibili sia all’interno che in prossimità di corpi idrici, risulta sicuramente la meno indagata a livello nazionale. L’area oggetto di studio è il bacino del fiume Tevere, il secondo bacino idrografico per estensione in Italia ed identificato dalle istituzioni governative come bacino pilota per studi di tipo ecologici (Bagnini et al., 2005). Lungo il fiume Tevere e i suoi principali affluenti sono state selezionate, ai fini del campionamento macrofitico, 61 stazioni con caratteristiche morfologiche ed idrologiche differenti, per poter avere una maggiore eterogeneità di situazioni ecologiche. Ai fini delle elaborazioni e delle analisi floristiche ed ecologiche, oltre ai dati raccolti, sono stati considerati alcuni rilievi floristico-vegetazionali disponibili in letteratura (Caneva et al., 2005; Ceschin et al., 2008; Ceschin et al., 2009) inerenti ad alcuni corsi d’acqua del bacino oggetto di studio. I rilievi floristici sono stati affiancati da analisi chimico-fisiche delle acque; nello specifico per ogni stazione sono state misurate la temperatura, il pH, la conducibilità, la salinità e la concentrazione di ossigeno disciolto. Attraverso analisi spettrofotometriche si sono registrate, inoltre, le concentrazioni di fosfati, nitrati e ione ammonio, utili per valutare il grado di eutrofizzazione dei corsi d’acqua in esame. Sono stati inoltre analizzati attraverso un’osservazione diretta altri parametri ambientali come la torbidità, la velocità della corrente e la granulometria del substrato. III La raccolta dei dati macrofitici assieme a quelli abiotici sopra menzionati è stata la base di partenza per poter intraprendere indagini più specificatamente ecologiche. I dati sono stati ordinati in una matrice ed elaborati secondo procedure di analisi statistica multivariata utilizzando metodi quali classificazione e ordinamento. Per alcune indagini di tipo esclusivamente ecologico è stato utilizzato anche l’approccio della teoria dei fuzzy set. Queste indagini hanno permesso di definire la nicchia ecologica delle specie macrofitiche e i loro ranges di compatibilità e di ottimizzazione in relazione ai gradienti dei fattori ambientali considerati. Sono state, quindi, elaborate curve di risposta ecologica per ogni specie relative a ciascuna delle variabili prese in esame. Le curve e i risultati ottenuti dalla classificazione e ordinamento dei dati hanno permesso di individuare quali specie possono essere utilizzate come indicatori biologici dello stato qualitativo delle acque. Queste specie possono essere considerate dei bioindicatori a tale scopo, in quanto risultano associate ad una ristretta gamma di valori dei fattori abiotici analizzati. E’ stato possibile osservare, pertanto, in acque fluenti, fresche, ossigenate, meso-oligotrofiche e in genere poco inquinate, la presenza caratteristica di specie come le alghe Lemanea fluviatilis, Nostoc punctiforme e Chamaesiphon conferviculus, le briofite Cratoneuron filicinum, Fissidens viridulus, Plagionium undulatum, Cinclidotus aquaticus, Plathypnidium riparioides e Pellia endiviifolia e infine le fanerogame Hippuris vulgaris, Elodea canadensis, Groelandia densa, Berula erecta f. fluitans, Mentha aquatica f. fluitans, Lemna trisulca e Ranunculus tricophyllus. Nei tratti fluviali, invece, caratterizzati da acque lente, poco ossigenate, eutrofiche ed inquinate si trovano in modo peculiare specie come le alghe Enteromorpha flexuosa, Stigeoclonium sp., Oscillatoria amphibia e le fanerogame Persicaria amphibia, Schoenoplectus lacustris, Eleocharis palustris, Potamogeton natans, Spirodela polyrhiza e Potamogeton pectinatus. Si evidenzia, in quest’ultima tipologia di acque, l’assenza totale di briofite sottolineando la sensibilità che questo gruppo tassonomico nel suo insieme mostra nei confronti di condizioni generali di inquinamento. Infine, sono stati testati sul set di dati raccolti alcuni dei principali indici macrofitici (MTR, GIS e IBMR), elaborati in ambito Centro europeo, al fine di valutarne l’adeguatezza e applicabilità ai corsi d’acqua del bacino in oggetto rientranti in un contesto più propriamente mediterraneo. Dai risultati emerge che questi indici, seppur mostrando una buona strutturazione metodologica, non riescono ad interpretare la vera condizione qualitativa dello stato trofico delle stazioni in oggetto; tali indici, infatti, non arrivano a discriminare in modo sensibile situazioni qualitativamente differenti. Appaiono, quindi, evidenti alcuni limiti di applicabilità di questi indici a contesti differenti da quelli di elaborazione, non solo per la scarsità di specie in comune, come nel caso dell’indice MTR, ma anche per problematiche di tipo tassonomico; si fa presente, infatti, che talvolta questi indici considerano bioindicatori solo a livello di genere, mentre come sottolineano anche i risultati emersi in questa indagine, è spesso necessario arrivare al rango di specie a causa delle diverse risposte che, a volte, possono esistere tra specie del medesimo genere. Inoltre confrontando le risposte autoecologiche ottenute nel presente studio con i coefficienti specie-specifici di sensibilità attribuiti dai diversi indici macrofitici alle specie in comune, si nota che l’informazione autoecologica non è talvolta coincidente, per alcune specie, come ad esempio Ranunculus trichophyllus e Myriophyllum spicatum, elemento questo che sottolinea l’importanza di un adattamento di tali coefficienti per le specie in ambito mediterraneo. In ultimo, analizzando le liste dei bioindicatori relative agli indici macrofitici, è stato possibile evidenziare che queste riportano anche alcune entità che, in questo studio, si sono rivelate ad ampia ecologia e quindi non idonee a tale scopo. I risultati ottenuti hanno permesso di ampliare le conoscenze floristiche relative alla componente macrofitica presente nell’area di studio. In particolare, oltre alle fanerogame, sono stati indagati con particolare attenzione i gruppi tassonomici relativi alle alghe, ai cianobatteri e alle briofite acquatiche di cui risultano, in ambito nazionale, solo poche e frammentarie informazioni. Infine, i risultati ottenuti, hanno permesso di analizzare le relazioni esistenti tra le specie macrofitiche e i principali fattori ambientali, oltre che a creare un modello ecologico che possiede una sua congruità e che testimonia l’importanza dell’uso dei bioindicatori per la stima della qualità delle acque e del grado di alterazione ambientale nel suo complesso. I risultati del presente progetto di ricerca rappresentano la base per future ricerche che avranno come obiettivo la formulazione di un indice macrofitico più idoneo per il contesto italiano ed in particolare per il settore mediterraneo contribuendo in questo all’implementazione delle linee guida di biomonitoraggio per la Water Frame Directive.; For the correct evaluation and characterisation of a river ecosystem it is considered essential nowadays to adopt an integrated approach that takes into account physical, chemical and biological methods. The recent adoption at the European level of the Water Frame Directive (2000/60/EC) requires an innovative update of the analytical and classification methods for water bodies. The WFD in fact, gives top priority importance to living organisms as it sees these as the most valid indicators to define the ecological status of surface waters and to truly assess the health of a watercourse. In the context of the implementation of the WFD it therefore becomes a priority to develop new biological indicators of the different trophic levels of the ecosystem. In fact the implementation of the WFD involves examining not only the communities of benthic macroinvertebrates, that are already included in previous regulation (152/99), but also the diatom, fish and macrophyte communities. These are the premises that underlie the present research project, that pertains in particular to the ecological study of the macrophytic component found both within and in proximity of water bodies, which includes herbaceous phanerogams, macroalgae, bryophytes and pteridophytes, and which is the least explored at national level . The area under study is the basin of the River Tiber, the second basin in Italy by extent and identified by governmental institutions as a basin for pilot ecological studies (Bagnini et al., 2005). For the purpose of the macrophyte survey, 61 sampling stations were selected along the River Tiber and its major tributaries, which had different hydrological and morphological characteristics, in order to have a greater diversity of ecological circumstances. For the purposes of the elaborations and for the floristic and ecological analysis, in addition to the collected data, reference was made to certain floristic and vegetation surveys available in the literature and linked to some of the watercourses in the river basin under study (Caneva et al., 2005; Ceschin et al., 2008; Ceschin et al., 2009). The floristic survey was supported by chemical and physical analysis of the waters, which measured for each station the temperature, pH, conductivity, salinity and dissolved oxygen concentration. Furthermore, the concentrations of phosphates, nitrates and ammonium were measured with spectrophotometric analysis, useful for assessing the degree of eutrophication of waterways under consideration. Whilst through direct observation other environmental parameters were analysed, including turbidity, current velocity and particle size of the substrate. The collection of macrophytic data together with the abiotic data mentioned above was the starting point to carry out the ecological investigations. The data was arranged in an array and processed according to procedures of multivariate statistical analysis using methods such as classification and sorting. For some analysis of an exclusively ecological nature, a fuzzy set theory approach was also applied. These investigations allowed to define the ecological niche of macrophytic species and their ranges of compatibility and optimisation in relation to gradients of the environmental factors considered. Ecological response curves were then elaborate for each species to each of the variables taken into consideration. The response curves and the results obtained from the classification and sorting of data made it possible to identify which species may be used as biological indicators of the qualitative state of the waters. These species can be considered as bioindicators for this purpose because they are associated with a narrow range of values for the abiotic factors analysed. It has been possible to observe therefore, in cool, oxygenated, meso-oligotrophic, flowing waters, with generally little pollution, the presence of distinctive species such as the algae Lemanea fluviatilis, Nostoc punctiforme and Chamaesiphon conferviculus, the bryophytes Cratoneuron filicinum, Fissidens viridulus, Plagionium undulatum, Cinclidotus aquaticus, Plathypnidium riparioides and Pellia endiviifolia, and finally the phanerogams Hippuris vulgaris, Elodea canadensis, Groelandia densa, Berula erecta f. fluitans, Mentha aquatica f. fluitans, Lemna trisulca and Ranunculus trichophyllus. In the stretches of water characterized instead by slow currents, poor oxygenation, eutrophication and pollution, there are distinctive species such as the algae Enteromorpha flexuosa, Stigeoclonium sp., Oscillatoria amphibia and the phanerogams Persicaria amphibia, Schoenoplectus lacustris, Eleocharis palustris, Potamogeton natans, Spirodela polyrhiza and Potamogeton pectinatus. We highlight for the latter type of waters, the total absence of bryophytes, emphasizing the sensitivity that this taxonomic group as a whole shows towards general conditions of pollution. Finally, certain of the principal macrophytic indices elaborated in a central European context (MTR, GIS and IBMR) were tested on the data set collected in order to assess their suitability and applicability to the watercourses of the basin under consideration and falling within a more strictly Mediterranean context. The results show that these indices, although exhibiting a good methodological framework, fail to interpret the true qualitative condition of the trophic state at the stations in question; indeed these indices do not discriminate in appreciable manner qualitatively different situations. There appear to be therefore, some obvious limits of applicability of these indices to contexts that are different from those where they were developed, not only because of the scarcity of species in common, as in the case of the MTR index, but also for taxonomical issues; we highlight in fact that sometimes these indices consider bioindicators only at the genus level, but as has also been outlined in this study, it is often necessary to reach the rank of the species due to different responses that can sometimes exist between species of the same genus. Furthermore by comparing the autoecological responses obtained in this study with the species specific coefficients of sensitivity conferred by the various macrophytic indices to the species in common, we note that the autoecological information does not always coincide, as for species such as Ranunculus trichophyllus and Myriophyllum spicatum; an element which emphasises the importance of adapting these coefficients to species in the Mediterranean context. Lastly, by analysing the lists of bioindicators linked to the macrophytic indices, it has also been possible to highlight that these lists also report entities that in this study have displayed wide ecological amplitude, and therefore are not suitable for this purpose. The results obtained have allowed to increase the floristic knowledge relating to the macrophytic component present in the area of study. In particular, apart from the phanerogams, other taxonomic groups were investigated with particular attention: the algae, cyanobacteria and aquatic bryophytes groups, for which there is, nationally, only modest and fragmentary information. Finally, the information obtained made it possible to analyse the relationships between the macrophytic species and the main environmental factors, as well as to create an ecological model that has its own congruity and demonstrates the importance of the use of bioindicators in assessing the quality of the waters and the degree of environmental alteration as a whole. The results of this research project are the basis for future research that will have as its objective the formulation of a macrophytic index that is more suitable to the Italian context and in particular for the Mediterranean region, thus contributing to the implementation of the guidelines for biomonitoring under the Water Frame Directive 2000/60/EC.

A possible molecular mechanism for parasitic inhibition by lactoferrin

Mon, 14 Dec 2009 23:00:00 GMT

A possible molecular mechanism for parasitic inhibition by lactoferrin Leboffe, Loris 2009-12-15 Transferrins (Tfs) belong to a family of iron-binding glycoproteins possessing similar aminoacid sequence though they have different biological functions and locations. Lactoferrin (Lf) is expressed and secreted from glandular epithelial cells and from mature neutrophiles of mammalian and it is an important component of the aspecific host defence or natural immunity, including resistance to parasitic infections. Serum transferrin (sTf) is synthesized by the liver of mammals and secreted into the blood stream; its primary function is iron transport. Ovotransferrin (Otrf), synthesized by avians, displays both iron transport and protective functions. Parasites synthesize papain-like cysteine proteases that are relevant for the virulence and pathogenicity of parasites, being involved in several aspects of the parasite life cycle, it is therefore possible that the antiparasitic activity of Lf could be due to the inhibition of parasitic papain-like cysteine protease that we have recently observed. In this study we have investigated the thermodynamic parameters of hLf, bLf and Otrf inhibition of the parasitic papain-like type I cysteine proteases from Leishmania infantum, Trypanosoma cruzi and Trypanosoma brucei. bLf, hLf and Otrf, both in the apo- and olo-forms, showed time- and concentration-dependent inhibition of the catalytic activity of papain and of type I proteases from L.infantum, T. cruzi and T. brucei. The KI values observed for bLf and hLf inhibition of L. infantum, T. cruzi and T. brucei proteases were in the nanomolar range (KI = 3.1 nM), lower than KI values observed for papain inhibition (KI = 24 nM). Otrf showed lower inhibition of cysteine proteases (KI = 0.6 µM). On the contrary, sTf did not display any inhibition towards parasitic proteases, according to its different role in mammals. The inhibition of parasitic cysteine proteases by hLf, bLf and Otrf appeared to conform to a competitive mechanism. The observed pH optimum for bLf inhibition of parasitic proteases was around neutrality, while it was acidic for hLf and alkaline for Otrf. The further quantitative analysis of pH dependence of the intrinsic ligand-independent inhibition constant KI allowed the evaluation of pKa values that define the acid-base equilibrium of amino acidic residue(s) modulating the enzyme(s)-inhibitor recognition events. SDS-PAGE showed that hLf, bLf and Otrf were easily degraded by either papain or parasitic type I protease during the assay incubation time (few minutes) and it is likely that one or more protease inhibitory peptides were generated from protein hydrolysis. As a matter of fact, a sequence present near the C-terminus of human (hLf) and bovine (bLf) lactoferrin shows homology with the sequence of the active site of cystatins, which are competitive inhibitors of papain-like cysteine proteases. The same sequence is present, though with lower homology, in Otrf and, with even lower homology, in sTf. Therefore, we have charactherized by MALDI-TOF the profile of Lf cleavage by papain and preliminary data suggest the presence of a cystatin-like peptide in two proteolytic fragments of hLf and in one proteolytic fragment of bLf.; Le transferrine (Tfs) sono una famiglia di glicoproteine in grado di legare reversibilmente il ferro, e presentano un’elevata omologia di sequenza tra tutti i membri di questa famiglia. In particolare la lattoferrina (Lf) è prodotta e secreta nelle cellule ghiandolari epiteliali ed è presente nei granuli dei granulociti neutrofili dei mammiferi. Essa rappresenta un’importante componente della difesa immunitaria aspecifica dell’ospite. La siero transferrina (sTf) invece è sintetizzata nel fegato, ed è coinvolta unicamente nel trasporto del ferro. L’ovotransferrina (Otrf), l’omologa aviaria della Lf , svolge sia una funzione di difesa che di trasporto del ferro. Le proteasi a cisteina papaina-simili da protozoi e metazoi parassiti, secretorie e di membrana, partecipano ai processi di morfogenesi degli organismi patogeni, sono implicate nell’invasione delle cellule e dei tessuti da parte dei parassiti, riducono la risposta immunitaria dell’ospite e costituiscono uno dei fattori più rilevanti nelle patologie associate alle infestazioni da parassiti. Pertanto, a fronte della capacità della Lf di svolgere un’azione antiparassitaria, noi abbiamo ipotizzato che la Lf possa svolgere tale azione grazie all’inattivazione delle suddette proteasi a cisteina. Nel presente lavoro abbiamo studiato le proprietà termodinamiche dell’inibizione delle proteasi a cisteina di alcuni parassiti, quali Leishmania infantum, Tripanosoma cruzi e Trypanosoma brucei. Tale studio è stato esteso anche verso la papaina, capostipite di questa famiglia. Innanzitutto è emerso una maggiore affinità delle transferrine studiate (bLf, hLf e Otrf, in forma apo e olo) verso le proteasi parassitarie (KI = 3.1 nM) rispetto a quella verso la papaina (KI = 24 nM). Otrf risulta essere l’inibitore con minore affinità (KI = 0.6 µM). Inoltre è emerso che la sTf non è in grado di svolgere un’azione inibitoria, coerentemente con la sua funzione nei mammiferi. L’inibizione di tutte le proteasi risulta essere conforme ad un meccanismo di inibizione competitivo. Da un’analisi della dipendenza del pH dei suddetti fenomeni di inibizione si è osservato una maggiore attività della bLf a pH fisiologico mentre la hLf inibisce più efficacemente a valori di pH acidi (pH = 5.0) e la Otrf a valori di pH alcalini (pH = 9.0). La dipendenza dal pH del parametro KI relativo all’interazione degli inibitori studiati con gli enzimi, ha permesso di evidenziare la variazione di diversi valori di pKa, ascrivibili ad eventi di protonazione e deprotonazione di differenti gruppi ionizzabili. Inoltre si è osservato, a seguito di SDS-page, una parziale idrolisi della Lf e della Otrf durante diverse incubazioni con la papaina e con le proteasi parassitarie, e l’insieme di tali dati suggerisce la liberazione di un peptide, che può quindi inibire competitivamente le suddette proteasi a cisteina. A supporto di tale ipotesi, si è osservato in presenza della porzione C-terminale della Lf, una sequenza aminoacidica che presenta un’elevata omologia con il sito attivo delle cistatine, una (super)famiglia di proteina, riconosciute essere gli inibitori reversibili per eccellenza delle proteasi a cisteina papaina-simili. Abbiamo quindi caratterizzato, mediante MALDI TOF, il profilo di idrolisi della Lf ad opera della papaina, ed è stata riscontrata la presenza di frammenti contenenti tale sequenza cistatinosimile, sia nella bLf che nell’hLf.

Regulation of cellular functions by nitric oxide pathway

Mon, 14 Dec 2009 23:00:00 GMT

Regulation of cellular functions by nitric oxide pathway Casadei, Manuela 2009-12-15 Nitric oxide (NO) is a highly diffusible gas that is synthesized by three distinct isoforms of nitric oxide synthases (NOS). Two isoforms are constitutively expressed and generate NO for cell-signalling purposes: neuronal NOS (nNOS) and endothelial NOS (eNOS), and the third member of the family is an inducible isoform (iNOS) which releases NO in larger quantities during inflammatory or immunological defence reactions and is involved in host tissue damage. NO has been shown to act as a multifunctional gaseous modulator in many cellular events. It is an important messenger molecule synthesized in a variety of tissues and involved in various physiological and pathological functions. The regulatory actions of NO are explained both by the physiological intracellular concentrations of NO and by some well known NO-dependent cell signalling and regulatory pathways. During this research project we focused on two of these regulatory pathways: the NO-dependent protein modifications of cysteine residues and the reversible inhibition of mitochondrial cytochrome c oxidase (CcOX), the terminal complex of the mitochondrial respiratory chain. Modulation of thiol-disulfide status of critical cysteines on enzymes, receptors, transport proteins, and transcription factors is recognized as an important mechanism of signal transduction and an important consequence of oxidative/nitrosative stress associated with aging, cardiovascular and neurodegenerative diseases. Within these contexts, a prevalent form of cysteine modification is reversible formation of protein mixed disulfides with glutathione (GSH), the major non-protein thiol compound in cells. Protein S-glutathionylation increases globally during overt oxidative stress, but selective/local generation of reactive oxygen and nitrogen species mediates physiological redox signaling. Moreover, reversible modifications, as S-glutathionylation, have been suggested to have a dual role: protection from cysteine irreversible oxidation and modulation of protein function. Among the wide list of proteins demonstrated to be susceptible to oxidative cysteine modifications, as S-nitrosylation, we focused on Metallothionein (MT) and the transcription factor Sp1, two zinc-binding metalloproteins that play fundamental roles in cellular functions, and whose activity has been shown to be affected upon ageing. Thus we investigated the susceptibility to S-glutathionylation of these proteins. Analysis of the three-dimensional structure of both MT and Sp1 showed the presence of some Cys residues likely targets for S-glutathionylation, both for their solvent accessibility and electrostatics induced reactivity glutathionylable cysteine residues. Western blot and dot blot assays performed after in vitro exposure to GSNO, diamide and H2O2 (oxidant agents acting through different mechanisms) revealed that both MT and Sp1 can be susceptible to S-glutathionylation upon oxidative/nitrosative stress conditions. Moreover, this effect was completely reversed by treatment with the reducing agent DTT, indicating the involvement of protein-mixed disulphides. Together our findings support a potential functional role for S-glutathionylation in protecting these proteins from irreversible oxidation that could occur during oxidative/nitrosative stress into the cell, and may represents an important antioxidant mechanism in the ageing process. NO has been observed to act as an effective signal molecule that regulates mitochondrial events, including oxygen consumption and reactive oxygen species production. Binding of NO with the terminal electron acceptor of the mitochondrial electron transport chain, CcOX, plays crucial roles in mediating the physiological effects of NO. Interaction between NO and CcOX is bidirectional resulting not only in the modulation of the mitochondrial enzyme activity by NO, but also in the regulation of NO concentrations by CcOX. Recently, we reported that mtNOS is physically associated with CcOX, and that this binding is mediated by the PDZ motif of mtNOS. In order to further analyze the role played by NO in CcOX activity modulation, in the present work we explored whether and how the interaction between the mtNOS PDZ motif and the subunit Va of CcOX can be regulated. Through molecular modeling simulations we individuated a potentially critic specific tyrosine residue (Tyr77) in the PDZ of nNOS (whose alpha isoform is identical to mtNOS). Considering that the importance of phosphorylation/dephosphorylation in regulating NOS activity and, more recently, mitochondrial processes has been recognized and several protein kinases and phosphatases have been identified in mitochondria, we investigated whether nNOS PDZ Tyr77 phosphorylation could be implicated in the mtNOS/CcOX interaction modulation. To this purpose we utilized different experimental approaches such as nNOS alpha cloning and mutagenesis, expression of wild type and mutant nNOS in mammalian cells, and, finally, analysis of nNOS/CcOX interaction in wild type and transfected cells by means of confocal microscopy and co-immunoprecipitation assays. Together our results suggest phosphorylation as a likely mechanism of mtNOS/CcOX interaction regulation. Moreover it seems that this modulation is mediated by the action of some src tyrosine kinases.

The role of mitotic spindle alterations in the induction of apoptosis

Thu, 17 Dec 2009 23:00:00 GMT

The role of mitotic spindle alterations in the induction of apoptosis Cenciarelli, Chiara 2009-12-18 Microtubules, together with actin and intermediate filaments are the major components of the cytoskeleton of eukaryotic cells and play a critical role in many cellular processes, including cell division, cell motility, intracellular trafficking, and cell shape maintenance. The principal Microtubule Organizing Center (MTOC) in mammalian cells is the centrosome that is composed of a pair of centrioles surrounded by an osmiophilic matrix termed pericentriolar material (PCM). As the microtubule network is highly involved in cell proliferation, it appeared to be a preferential target for cancer therapy. Microtubule Damaging Agents (MDA) represent an important class of anticancer drugs. Although some of this agents promote microtubules stabilization and other induce microtubules disruption, all MDA share the common property of suppressing microtubules dynamics, and thereby microtubules function, leading to the disruption of the mitotic spindle and the blockade of cell cycle progression at the transition from metaphase to anaphase that leads to apoptosis or mitotic catastrophe commitment. Mitotic catastrophe has been described as cell death that occurs from metaphase of mitosis in response to agents that cause mitotic-spindle damage. Recently we focused our attention on the antineoplastic spindle poison agent combretastatin-A4 (CA-4). CA-4 is an MDA isolated from the South African tree Combretum Caffrum that is not recognized by the multi drug resistance pump, a cellular pump which rapidly ejects foreing molecules including many anticancer drugs. MDAs also promote several effects on the centrosome including abnormal centrioles structure, centrosome fragmentation and inappropriate centrosome duplication. CA-4 is able to arrest cells at mitosis in a dose depending manner accompanied by an increasing disorganization of chromosomal arrangement, led to formation of particular structure named 'star-like'. This structures contain pericentrosomal matrix components like γ-tubulin, pericentrin and ninein. Confocal and immunofluorescence microscopy revealed that 'star like' structure are spherical structure formed by residual fragments of microtubules connected to kinetochore. At the centre of this structure are present some component of the pericentriolar matrix like γ-tubulin, pericentrin and ninein. Treatment with CA-4, which produced high frequency of 'star-like' mitosis, was accompanied by mitotic catastrophe commitment characterized by activation of caspases-3/-9 and DNA fragmentation. The mechanisms that lead from microtubules depolymerization to cell death are still not completely clear. Recently it has been demonstrated that Bim, a proapoptotic Bcl-2 protein, is associated to the dynein motor complex and sequestered by microtubules. MDA, through the alteration of microtubules structure, might lead to the release of Bim from cytoskeleton and to its translocation to the mitochondria. Moreover, recent data show that further mechanisms might be at the base of Bim-induced apoptosis such as regulation of Bim transcriptional levels. In a similar way, p53 another well known pro-apoptotic molecule, is associated with cellular microtubules and is transported to the nucleus by dynein. Based on these data, we have investigated whether upon tubulin depolymerization, Bim and p53 could be involved, and eventually in which manner, in the first steps of apoptosis. CA-4 treatment induce the release of Bim from microtubules and its increase at transcriptional level. In addition, Bim silencing strongly reduced apoptotic commitment. It has recently reported that p53 can also induce apoptosis independently of new protein synthesis however the functional importance of p53 in MDA-induced apoptosis of human cancer cells remain poorly understood. Concerning the role of p53 in CA-4-induced apoptosis, we found that treatment determined its release from cytoskeleton and re-localization to mitochondria. Data obtained from this study showed that Combretastatin-treated H460 cells are killed via a mitotic catastrophe mechanism strictly dependent on the achievement of mitotic block, caspases activation and Bim and p53 translocation to mitochondria.; I microtubuli, insieme con l'actina e i filamenti intermedi sono i principali componenti del citoscheletro delle cellule eucariotiche e svolgono un ruolo fondamentale in molti processi cellulari: divisione, motilità, 'trafficking' e mantenimento della forma cellulare. Il principale Centro Organizzatore dei Microtubuli (MTOC) nelle cellule di mammifero è il centrosoma che è composto da una coppia di centrioli circondata da una matrice elettron-densa chiamata materiale pericentriolare (PCM). Essendo i microtubuli fortemente coinvolti nei processi di divisione cellulare, rappresentano un target preferenziale nella terapia del cancro. Gli agenti che danneggiano i microtubuli (MDA) rappresentano una importante classe di farmaci antitumorali e benchè alcune di queste sostanze promuovano la stabilizzazione dei microtubuli e altre inducano la depolimerizzazione, tutti gli MDA presentano la caratteristica comune di sopprimere la dinamica e quindi la funzione dei microtubuli. In questo modo gli MDA causano la distruzione del fuso mitotico e un blocco nella progressione del ciclo cellulare nella transizione metafase-anafase che puo. portare all.attivazione dei pathways apoptotici e alla catastrofe mitotica. Quest'ultima è stata descritta recentemente come una morte cellulare che avviene durante la mitosi in seguito all.utilizzo di agenti che causano alterazioni del fuso mitotico. Recentemente abbiamo focalizzato la nostra attenzione sulla Combetastatina (CA-4), un MDA isolato dal Combretum Caffrum, un albero sud Africano che ha la caratteristica di non essere riconosciuta dalla pompa Multi Drug Resistance, una pompa cellulare che elimina rapidamente sostanze estranee inclusi molti farmaci antitumorali. Gli MDA agiscono inoltre anche sul centrosoma causando alterazioni strutturali, frammentazione e inappropiata duplicazione del centrosoma. La CA-4 è in grado di arrestare le cellule in mitosi causando, in maniera dipendente dalla concentrazione, un aumento della disorganizzazione dell.arrangiamento cromosomico e la formazione di particolari strutture chiamate 'star-like'. L'analisi in microscopia a fluorescenza e confocale ha evidenziato che le 'star-like' sono strutture di forma sferica formate da piccoli fasci di microtubuli connessi al cinetocore. Al centro di queste strutture sono presenti alcuni componenti della matrice pericentriolare come la γ-tubulina, la pericentrina e la nineina. Il trattamento con la CA-4, oltre a determinare alterazioni di tipo strutturale, causa morte cellulare per catastrofe mitotica caratterizzata dall.attivazione delle caspasi-3/-9 e dalla frammentazione del DNA. Il meccanismo che porta dalla depolimerizzazione dei microtubuli alla morte cellulare non è ancora completamente chiarito. Recentemente è stato dimostrato che Bim, una proteina pro-apoptotica appartenente alla famiglia Bcl-2, è associata con il motore proteico dineina e sequestrata quindi dai microtubuli. Gli MDA, attraverso l'alterazione della struttura dei microtubuli, potrebbero causare il rilascio di Bim dal citoscheletro e la sua traslocazione sul mitocondrio. Inoltre dati recenti mostrano che esistono altri meccanismi che regolano Bim come la regolazione trascrizionale. Allo stesso modo, p53, una proteina pro-apoptotica molto conosciuta è associata con i microtubuli ed utilizza il motore proteico dineina per essere trasportata nel nucleo ed agire come fattore di trascrizione. Basandoci su questi dati abbiamo valutato il coinvolgimento di Bim e di p53 nell.apoptosi indotta in seguito al trattamento con CA-4. Il trattamento con la CA-4 determina sia il rilascio dai microtubuli sia un aumento del livello trascrizionale di Bim. Inoltre il silenziamento di Bim riduce fortemente l.attivazione della caspasi-3, una delle caspasi effettrici del pathway apoptotico mitocondriale. Inoltre, recentemente è stato dimostrato che p53 puo. indurre apoptosi non solo agendo come fattore di trascrizione ma anche attraverso una azione indipendente dalla trascrizione grazie alla capacità di interagire direttamente con proteine della famiglia Bcl-2 presenti sul mitocondrio come Bcl-2 e Bcl-Xl. Comunque l'importanza dell'attività indipendente dalla trascrizione di p53 dopo utilizzo degli MDA su cellule tumorali rimane poco compresa e studiata. Nei nostri esperimenti abbiamo verificato che il trattamento con la combretastatina causa una variazione nel legame di p53 con la dineina e parallelamente determina la presenza di tale proteina sul mitocondrio. Inoltre esperimenti di coimmunoprecipitazione rivelano che Bim e p53 interagiscono sul mitocondrio e sono probabilmente responsabili dell.attivazione del pathway mitocondriale apoptotico. I nostri dati indicano quindi che cellule H460 trattate con la combretastatina sono uccise attraverso il meccanimo della catastrofe mitotica dipendente da un prolungato arresto mitotico, attivazione delle caspasi e traslocazione mitocondriale di Bim e p53.

Sviluppo di metodi per la predizione massiva della struttura e funzione di proteine attraverso l'utilizzo di infrastrutture di calcolo condiviso = Development of methods for high through-put proteins structure and function prediction through the use of GRID computing infrastructures

Mon, 14 Dec 2009 23:00:00 GMT

Sviluppo di metodi per la predizione massiva della struttura e funzione di proteine attraverso l'utilizzo di infrastrutture di calcolo condiviso = Development of methods for high through-put proteins structure and function prediction through the use of GRID computing infrastructures Minervini, Giovanni 2009-12-15 The number of natural proteins represents a small fraction of all the possible protein sequences and there is an enormous number of proteins never sampled by nature, the so called “never born proteins” (NBPs). A fundamental question in this regard is if the ensemble of natural proteins possesses peculiar chemical and physical properties or if it is just the product of contingency coupled to functional selection. A key feature of natural proteins is their ability to form a well defined 3D structure. Thus, the structural study of NBPs can help to understand if natural protein sequences were selected for their peculiar properties or if they are just one of the possible stable and functional ensembles. The structural characterization of a huge number of random proteins cannot be approached experimentally, thus the problem has been tackled using a computational approach. A large random protein sequences library (2×104 sequences) was generated, discarding amino acid sequences with significant similarity to natural proteins, and the corresponding structures were predicted using Rosetta. Given the highly computational demanding problem, Rosetta was ported in grid and a user friendly job submission environment was developed within the GENIUS Grid Portal. Protein structures generated were analysed in terms of net charge, secondary structure content, surface/volume ratio, hydrophobic core composition, etc.. The vast majority of NBPs, according to the Rosetta model, are characterized by a compact three-dimensional structure with a high secondary structure content. Structure compactness and surface polarity are comparable to those of natural proteins, suggesting similar stability and solubility. Deviations are observed in α helix-β sheet relative content and in hydrophobic core composition, as NBPs appear to be richer in helical structure and aromatic amino acids with respect to natural proteins. The results obtained suggest that the ability to form a compact, ordered and water-soluble structure is an intrinsic property of polypeptides. The tendency of random sequences to adopt α helical folds indicate that all-α proteins may have emerged early in prebiotic evolution. Further, the lower percentage of aromatic residues observed in natural proteins has important evolutionary implications as far as tolerance to mutations is concerned.

Analysis of Vγ9Vδ2 T lymphocytes response to human glioma cell lines: possible implications for therapy

Sun, 15 Feb 2009 23:00:00 GMT

Analysis of Vγ9Vδ2 T lymphocytes response to human glioma cell lines: possible implications for therapy Cimini, Eleonora 2009-02-16 T cells link innate and acquired immunity. In humans 90% of circulating T cells express the V9V2 TCR rearrangement and recognize non peptidic antigens in a MHC-unrestricted manner. After antigen recognition, activated V9V2 T cells rapidly proliferate, produce high levels of cytokines and chemokines and can differentiate in cytotoxic effector cells. Specifically, V9V2 T cells recognize unprocessed non-peptidic compounds such as isopentenyl pyrophosphate (IPP), which are produced through the isoprenoid biosynthesis pathway. Moreover, V9V2 T cells can also be activated by aminobisphosphonates drugs through an indirect mechanism: they inhibit farnesyl pyrophosphate synthase, an enzyme of cholesterol biosynthesis, acting downstream of IPP synthesis; this inhibition, in turn, leads to the accumulation of endogenous IPP, directly recognized by V9V2 T cells. A specific feature of V2 T cell biology is their ability to recognize tumor cells presenting a dis-regulation in mevalonate pathway, resulting in an increase of phosphorilated metabolites such as isopentenyl-pyrophosphate (IPP). Thus, the increased isoprenoid metabolism in cancer cells induces V2 T cell activation through cellular IPP accumulation. Mevalonate cycle is present in all eucaryotic cells and produce cholesterol and prenyl-compounds. The main two enzymes (HMG-CoA reductase and FPP synthase) in the mevalonate pathway are carefully regulated and can be farmacologically modulated by different drugs (mevastatin and aminobisphosphonates respectively). Several studies show that V2 T cells recognize and kill several cancer cells, such as lymphoma, colon-, lung-, renal, breast carcinoma, and glioma. In renal cancer patients, a V2- based immunotherapy with a synthetic phosphorilated compound is in course with promising results. Similarly, aminobisphosphonates (Zoledronic Acid) is currently used for bone metastases in prostate cancer patients. In this context, the possibility to massively activate and expand in vitro a relatively large number of cells opens new interesting prospects in the immunotherapy of cancer disease. Gliomas are tumors arising from glia or their precursors within the central nervous system. Unfortunately, the majority of patients with glioma tumors die in less then of one year; in these patients, new treatment strategies are therefore hardly needed. Aim of this study was to analyse the activity of human V2 T cells against glioma cancer cells and to verify the possibility to target these innate cells in new immunotherapeutical strategies. In a first set of experiments, we set up an in vitro protocol able to expand human V2 T cells by using IPP and IL-2. After 12 days the expanded V2 T cell lines (80-95% of purity) were analysed for their differentiation phenotype, (as expression of CD27 and CD45RA markers of T, B, NK cells), cytokines production (IFN and TNF) and natural cytotoxicity capability (Perforin). Results showed that in vitro expanded V2 T cell lines present an effector memory phenotype and have high functionality both in terms of cytokines production and Perforin release. We then studied three different glioma cell lines: T70, U373 and U251 by analyzing GFAP expression on cell surface by direct immunofluorescence. Resulted showed that all glioma cells was positive for GFAP. In a second set of experiments, V2 T cell lines were co-cultured with glioma cells in order to analyse the activation of V2 T cells and the effects on the viability of glioma cells. In our system, V2 T cell lines were able to recognized glioma cells (T70, U373, U251) by specifically differentiate in effector memory cells, and release Perforin. In contrast, they did not produce cytokines. In order to verify the cytotoxic effect of V2 T cells on glioma cells, we performed a viability test on glioma cells in the absence and in the presence of V2 T cell lines. Briefly, glioma cells were labelled with Annexin/Propidium Iodide and were analysed by flow cytometry. Interestingly, V2 T cells were able to kill glioma cells through an apoptotic mechanism, demonstrating their antitumoral activity. We then decided to study if Zoledronic Acid (ZOL) treatment of glioma cells could improve V2 T lines response. Glioma cells were treated with ZOL in vitro for two hours, and co-cultured with V2 T cell lines, analyzing V2 T cells response by flow cytometry. Results showed that V2 T cell lines were able to recognize glioma cells by releasing high amount of IFN and TNF. V2 T cells activation was mediated by ZOL-induced IPP accumulation, since the incubation with mevastatin was able to completely block this biological effect. Finally, we studied the direct effect of different concentrations of ZOL on glioma cells viability before and after the co-culture with V2 T lines. We observed that treatment with ZOL induced necrosis on glioma cells, but only the co-culture with V2 T lines together to the treatment with ZOL increased both the apoptosis and necrosis of glioma cells. Altogether, our results suggest that the induction of a strong antitumoral response of V2 T cells by using aminobisphosphonates could represent a new interesting immunotherapy approach for glioma care.

Meccanismi alla base degli effetti protettivi degli ormoni estrogeni in differenti contesti cellulari

Thu, 17 Dec 2009 23:00:00 GMT

Meccanismi alla base degli effetti protettivi degli ormoni estrogeni in differenti contesti cellulari Galluzzo, Paola 2009-12-18

Analisi etnobotanica della costiera amalfitana e valutazione dei risultati da un punto di vista scientifico ed economico

Wed, 16 Dec 2009 23:00:00 GMT

Analisi etnobotanica della costiera amalfitana e valutazione dei risultati da un punto di vista scientifico ed economico Savo, Valentina 2009-12-17

Development of novel methodologies for the optimization of production processes of biopharmaceuticals

Tue, 10 Feb 2009 23:00:00 GMT

Development of novel methodologies for the optimization of production processes of biopharmaceuticals Barba, Marco 2009-02-11 Process Analytical Technology (PAT) is defined by the FDA as a "System for designing, analyzing and controlling manufacturing through timely measurements of critical quality and performance attributes of raw and in-process materials and processes, with the goal of ensuring final product quality". The goal of implementing PAT is defined therein as enhancing the understanding and the control of a production process. This broad definition encompasses testing of raw material for batch consistency as well as online sensors that provide feedback for the process control. In this context, rapid methods placed at-line, i.e. close to the process, can be considered to be PAT applications since they will both contribute to the understanding how individual steps impact on product quality and will accelerate and facilitate process development and optimization decisions. There are many tools available that enable process understanding for scientific, pharmaceutical development. These tools can provide effective and efficient means for acquiring information to facilitate process understanding and continuous improvement. From a physical, chemical and biological perspective, pharmaceutical products and processes are complex multi-factorial systems. Methodological experiments based on multivariate statistical principles provide useful means for the identification and study the effect and interaction of product and process variables. Traditional one-factor-at-time experiments cannot address these kinds of interactions. These tools enable the identification and evaluation of product and processes variables that may be critical to product quality and performance. Thanks to the PAT initiative, spectroscopic sensors systems have gained interest for bioprocess monitoring because they allow rapid and non-destructive monitoring of product quality attributes. The improvements in spectrometers, detectors and optics have led to interesting applications related to PAT. The main goal of the thesis is the development of novel methodologies based on spectroscopic techniques coupled with multivariate data analysis for the optimization of production process of biopharmaceuticals. One of the approaches described here combines the strengths of various spectroscopic techniques, such as Circular Dichroism (CD), Infrared (IR), Raman, Fluorescence and UV-Visible-NIR measurements, to provide a more comprehensive description of a substance, the so-called "fingerprint". This, in combination with Principal Component Analysis (PCA) may be use to establish and define quality, equivalence, and comparability of substances while also providing a means to monitor processes and provide relevant information about molecular changes in product. Moreover, it can highlight the relationships between different properties, for example that between structure and aggregation, and a better understanding of the nature of a product. The protein used in this study is a homo-dimeric Fc-fusion protein. In particular, for the first part of the project, focused on PCA of multispectroscopic data, were used ten batches of drug substance produced with the current process called "process C". This bulk material has a concentration of "not less than" 160 mg/ml. Within this set, the batches differ with respect to hydrolysates that were used as feed during the fermentation process. In addition to these difference, three additional batches form the new "process D", after some minor optimisations with respect to "process C", were analysed. In order to generate a wider diversity of samples, with aspects of deterioration, solutions were diluted with water (instead of using a buffer) and then stored at room temperature for several weeks before spectroscopic analysis. The major consequence of this treatment is a change in buffer/additive concentrations, a change in pH, and a deterioration of solutions through aggregation, structural and chemical decomposition. A comprehensive set of spectra of eleven batches thus were acquired, using a variety of techniques. Some twelve variants of five spectroscopies have been employed, covering the complete wavelength range from far-ultra violet to infrared and involving phenomena including absorption, fluorescence, Raman scattering, Rayleigh scattering and circular dichroism. Both concentrated stock and deliberately deteriorated dilute solutions were investigated. All of the techniques employed have yielded useful data of some forms in terms of identifying variance in the batches and are potentially complementary and cross-supporting. Each set of spectra were subjected to multivariate data analysis, primarily PCA, to highlight patterns and differences between batches. Such analysis highlighted an apparent connection between the spectra and the history of batches regarding production date and hydrolysate type used. In particular, a series of anomalous absorptions in the visible wavelength region, together with potentially related fluorescent species, were identified. These may derive from contaminants, post-translational modifications (PTM) dependent on production conditions. Another aim of the thesis was to assess the feasibility of obtaining quantitative data about degradation products of a therapeutic protein when employing Circular Dichroism and infrared spectroscopy in combination with multivariate data analysis, primarily Partial Least Squares (PLS) regression, and an extension of PLS, Orthogonal Partial Least Squares (O-PLS). This is a novel approach since the classical applications for CD and IR spectroscopy are the determination of secondary structure content of proteins. Also the use of multivariate statistical methods for the determination of secondary structure content is reported. Nevertheless, the present approach is to our knowledge the first one that seeks to exploit PLS in order to correlate CD and IR spectral data with quantitative data of common protein degradation forms. In order to generate a suitable calibration matrix, a set of samples containing pre-defined levels of aggregates, oxidized forms, and free Fc, was generated. In order to ensure non-correlation of the degradation levels within the calibration matrix, the target concentrations therein were chosen according to an approach described by Brereton (2000). All the samples generated were then analyzed separately for each of the three degradation forms employing dedicated chromatographic QC assays in order to obtain accurate degradant levels. Furthermore, both CD (near and far UV) and IR spectra were measured. Both the QC and the spectroscopic data form the basis for the generation of various PLS/O- PLS models, i.e. based respectively CD or IR spectra alone, as well as CD and IR data combined. The feasibility of employing PLS/O-PLS analysis to extract quantitative data for common protein degradation forms was successfully demonstrated for an Fc fusion protein. Both CD and IR spectra contained the relevant information, nevertheless, CD-based O-PLS models achieved a higher accuracy compared to that of IR-based models for predicting aggregate and oxidation levels, while the accuracy for free Fc levels could be equally well predicted. Combining CD and IR data improved the accuracy of the prediction for all degradation forms. In addition, we demonstrated that O-PLS models yielded to a better accuracy compared to that obtained with PLS models. The last part of the thesis is based on the "protein design" methodologies. Aim of the present thesis is to study the scaffold stability of contryphan-Vn, a small peptide isolated from the venom of Conus ventricosus formed by only 9 residues and characterized by the presence of a single disulfide bridge, after substitution of 4 of 9 amino acids of its sequence. Contryphans are bio-active peptides, isolated from the venom of marine snails of the genus Conus, which are characterized by the short length of the polypeptide chain and the high degree of unusual post-translational modifications. The cyclization of the polypeptide chain through a single disulphide bond, the presence of two conserved Pro residues and the epimerization of a Trp/Leu residue confer to Contryphans a stable and well defined structure in solution, conserved in all members of the family. The potential of Contryphans as scaffolds for the design of redox- active (macro)molecules was tested by engineering a copper binding site on two different variants of the natural peptide Contryphan-Vn, named Cupryphan and Arg-Cupryphan through the introduction of four His residues. The binding site was designed by computational modelling and the redesigned peptides were synthesized and characterized by optical, fluorescence, electron spin resonance and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupryphan and Arg-Cupryphan bind Cu2+ ions with a 1:1 stoichiometry and a Kd = 1.3(± 0.2) x 10-7 M and 1.0(± 0.4) x 10-7 M, respectively. Other divalent metals (e.g. Zn2+ and Mg2+) are bound with much lower affinity. In addition, Cupryphans catalyze the dismutation of superoxide anions with an activity comparable to other non-peptidic superoxide dismutase mimicks. We tested the potential of conopeptides as scaffolds for the engineering of novel, metal based, biocatalysts starting from the simplest prototype of disulphide constrained conopeptides: the Contryphans. The results of the present work indicate that indeed this class of peptides could be successfully exploited to engineer novel, stable and redox active macromolecules.

Metabolismo telomerico in risposta al danno al DNA indotto da radiazioni ionizzanti di diversa qualità

Sun, 15 Feb 2009 23:00:00 GMT

Metabolismo telomerico in risposta al danno al DNA indotto da radiazioni ionizzanti di diversa qualità Berardinelli, Francesco 2009-02-16 Ionizing radiations are a well known genotoxic agents, widely studied for the great impact of their applications (i.e., radiotherapy and hadrontherapy) and effects (i.e., exposure risk for astronauts in space missions). Exposure to ionising radiations (IR) can result in the deposition of energy to DNA molecules, thus leading to DNA damage. IR-induced DNA damage is localized, and the level of localization is believed to increase with increasing linear energy transfer (LET) values of the radiation. Because LET is a measure of the energy released to an object along the path of the radiation, high-LET radiation can deposit more energy than low-LET one. Condensed or concentrated energy deposition results in cluster of ionization events. When the target is the DNA, the site of such lesions is termed "clustered DNA damage" or "locally multiply damaged site", which consists in two or more lesions localized in close proximity on the DNA duplex. In order to study the biological effects of high-LET radiations, several endpoints have been evaluated both in rodent- and in human-irradiated cells, including chromosomal aberrations, micronuclei (MN), chromosomal non-disjunction, mutations, DNA fragmentation, clonogenic survival, and cell cycle effects. However, aspects related to telomere length modulation and telomere metabolism have been so far poorly investigated both in primary and in immortalized cells exposed to low- and high-LET radiations. The aim of the first part of the study was to analyze the DNA-damage and the genotoxic effects induced by graded doses (0, 25-2 Gy) of low-energy protons (high-LET radiation), and X-rays (low- LET radiation) in human primary fibroblasts. DSB induction and repair as mesured by scoring for -H2AX foci indicated that 3MeV protons, with respect to X-rays, yielded a lower number of DSBs per Gy, which showed a slower kinetics of disappearance in the first hours from irradiations. Furthermore, irrespective of dose delivered, a higher fraction of unrejoined DSBs persisted in sample harvested 24 hours from exposure to protons. The higher clastogenic effect of protons was in agreement with the extent of micronuclei (MN) induction in binucleated cells up to 1, 5 Gy. Our results support the notion that DNA DNA damage produced by 28.5 keV/µm protons appears less amenable to be repaired and could be transformed in cytogenetic damage in the form of MN in the first cell cycle from irradiation . After confirming the greater biological effectiveness of high-LET radiations compared to low-LET ones, we focused our attention on studying telomere metabolism within 24 hours from the exposure to both types of radiations. Interestingly, data obtained showed a different kinetics of telomere length modulation in cells exposed to low- or high-LET radiations. Moreover, the phenomenon observed appeared to be conserved both in primary and in immortalized cell lines. Interestingly, exposure of human primary fibroblasts to 4Gy high-LET radiation determined a telomere elongation respect to untreated cells, whereas no telomere length modulation was observed in low- LET treated fibroblasts. In order to investigate the molecular mechanism underlying the observed elongation, the expression levels of the telomerase (i.e., hTERT) and its enzymatic activity were evaluated. Results obtained excluded the involvement of the telomerase in the observed telomere lengthening induced by high-LET radiation, thus supporting the activation of a telomerase- independent mechanism. Some mammalian cells lacking in any telomerase activity are able to maintain the length of their telomeres for many population doublings (PDs). This indicated the existence of one or more non-telomerase mechanism(s) for telomere maintenance, further termed Alternative Lengthening of Telomeres (ALT). To date, clear evidences of the existence of an ALT activity has been demonstrated only in human tumours and immortalized cell lines, and in telomerase-null mouse cell lines. To analyze whether a recombinational mechanism could be responsible for the high-LET-induced telomere lengthening observed in human primary fibroblasts, two types of experiments were performed. On one side, the incidence of recombinational events at telomeres (T-SCE) was measured, and on the other side the colocalization of telomeres and PML bodies (that are considered as an hallmark of cells with activated ALT pathway), was analyzed. Strikingly, our results indicated that the DNA damage induced by high-LET radiation is somehow able to induce telomere lengthening through the transient activation of an ALT recombinational pathway. Recent reports demonstrated that NBS1 is essential for the correct functioning of the ALT pathway. NBS1 gene, mutated in the NBS human chromosome instability disorder, encodes for the NBS1 protein, a central player in the response to the ionizing radiation-induced DNA damage, as well as in the homologous recombination repair. In order to confirm the high-LET-induced recombinational ALT pathway, telomere length was evaluated in Lymphoblastoid Cell Lines (LCLs) heterozygous (NBS1+/- ) and homozygous (NBS1-/- ) for a mutation of the NBS1 gene, as well as in normal cells (NBS1+/+ ) exposed to 4 Gy of carbon ions (39keV/m). Remarkably, a telomere elongation was observed in NBS1+/+ and NBS1+/- cells, but not in NBS1-/- ones. These data evidenced that the process of telomere lengthening induced by high-LET radiation is NBS1- dependent, thus supporting the hypothesis that telomere elongation is mediated by recombinational mechanisms. Beside the analysis performed at 24 hours, telomere length modulation was followed up to 15 days from the irradiation of both human primary fibroblasts and LCLs. Dynamics of telomere lengths modulation appeared to be different after low- and high-LET irradiation. Our data showed that the telomere lengthening observed in high-LET-treated cells seems to be maintained at 3-4 days, as well as 15 days after exposure. Interestingly, the time-course of the low-LET radiation-induced telomere length modulation appeared to be more complex than the high-LET one. In fact, after 3-4 days telomere erosion was reported, whereas after 15 days from the exposure a telomere lengthening was observed in primary as well as in immortalized cell lines. To explain the time course of low-LET-induced telomere length modulation we have hypothesized that a direct correlation between telomere length and radioresistance/radiosensitivity could account for this phenomenon. To test our hypothesis, we decided to perform experiments in TK6 lymphoblast cells, since they represent a good and widely used radiobiological cellular model. Data obtained brought us to suggest a model: the radioresistance of cells with longer telomeres drives a selection process that led to an increased telomere length in clones survived to low-LET radiation exposure. A direct correlation between telomere length and radisensitivity/radiresistance has already been proposed in some published reports and imply that telomeres length measurement could be potentially used as a tool to predict clinical radiation response in radiotherapy.

Studio delle proprietà funzionali di una libreria peptidica a sequenza casuale : implicazioni per lo studio dell'origine della vita

Tue, 10 Feb 2009 23:00:00 GMT

Studio delle proprietà funzionali di una libreria peptidica a sequenza casuale : implicazioni per lo studio dell'origine della vita De Lucrezia, Davide 2009-02-11 La scienza assume che la vita sulla Terra abbia avuto origine attraverso un processo spontaneo di autorganizzazione ed aumento di complessità a partire dalla materia inanimata. Diverse teorie sono state proposte per spiegare questo spontaneo aumento di complessità. Wächtershäuser identifica cicli metabolici privi di enzimi come il cardine alla base dell’ origine della vita; Kauffmann propone cicli di peptide auto-catalitici come il principale motore, mentre Cech individuò in molecole auto replicanti di RNA la struttura fondamentale del primo sistema con proprietà “viventi”. Al contrario, Luisi identifica nella struttura autopoietica dei sistemi viventi il principio primo, mentre Lancet enfatizza l’ereditarietà composizionale come caratteristica saliente dei sistemi viventi. Nonostante le differenze, tutte le teorie devono affrontare la stessa questione fondamentale: è il percorso di transizione alla vita univocamente determinato dalla legge della fisica e della chimica? Oppure è piuttosto il risultato della simultanea interazione di diversi fattori contingenti? La controversia tra determinismo e contingenza emerge con forza se si considera l'emergere di biopolimeri funzionale nel quadro di origine della vita. Infatti, l'intera architettura della vita si basa su biopolimeri così che l'eziologia dei biopolimeri rappresenta un problema cruciale nel settore della ricerca sull’origine della vita. La questione principale è come biopolimeri funzionale siano stati selezionati in condizioni pre- o protobiotiche. Infatti, il numero di sequenze teoricamente possibile cresce esponenzialmente al crescere della lunghezza raggiungendo rapidamente cifre astronomiche così che non è ragionevole assumere che l’intero spazio delle sequenze sia stato esplorato durante l’evoluzione prebiotica. Da questa osservazione nasce l’interrogativo di come siano stati selezionati biopolimeri funzionali in un contesto prebiotico. Vi sono forse principi chimico-fisici particolari che sottendono alla selezione di biopolimeri funzionali? O piuttosto l’evoluzione molecolare è il risultato dell’interazione di fattori contingenti? Nel tentativo di rispondere a queste domande una libreria di sequenze peptide completamente casuale è stata sintetizzata e studiata per le sue proprietà funzionali al fine di verificare la possibilità di isolare nuove sequenze funzionali che non presentassero omologie con quelle presenti in natura. La libreria è stata progettata senza vincoli strutturali o sequenza in modo che possa essere ragionevolmente considerata come omologa ad una popolazione di peptidi prodotta in condizioni prebiotiche. La strategia sperimentale adottata ha utilizzato la tecnica dell’evoluzione in vitro che permette di testare simultaneamente un numero consistente di sequenze diverse al fine di individuare quelle che soddisfano un particolare criterio di selezione. L’evoluzione in vitro mima l’evoluzione naturale tramite cicli iterativi di mutazione-selezione-amplificazione. Ci sono due requisiti fondamentali per applicare l’evoluzione in vitro: il primo è la possibilità di creare un legame diretto tra genotipo e fenotipo. Il secondo si basa sulla disponibilità di una idonea procedura di screening per selezionare le sequenze che soddisfano i criteri di selezione. In questo progetto di dottorato la connessione tra genotipo e fenotipo è stata ottenuta utilizzando la tecnica del phage display, mentre la capacità di legare un analogo dello stato di transizione (TSA) per la reazione di idrolisi di esteri ed ammidi è stata utilizzata come criterio di selezione. La probabilità di successo in un esperimento di evoluzione in vitro è direttamente correlata alla complessità totale della libreria ed alla robustezza del processo di selezione. Conseguentemente, la prima parte di questo progetto di dottorato è stata dedicata alla costruzione di librerie di DNA codificanti peptidi a sequenza casuale di 20 residui e all'ottimizzazione della tecnica del phage display. Infine si è proceduto allo screening di suddetta libreria per la capacità di legare il TSA vincolante peptidi in diverse condizioni chimiche. La costruzione di librerie peptidiche pone una serie di problemi tecnici che limitano la loro applicabilità, per ovviare a queste limitazioni un nuove vettore fagemidico è stato sviluppato per consentire la costruzione di librerie altamente degeneri e diverse. I risultati ottenuti sono riassunti qui di seguito: Complessità della libreria @ 108 Diversità della libreria > 70% Copertura dello Sequenza delle spazio @ 10-56 Un ulteriore problema inerente la tecnologia del phage display risiede nella eterogeneità della popolazione fagica. Tale eterogeneità limita severamente la porzione di spazio delle sequenze effettivamente esplorabile e riduce drasticamente la possibilità di trovare funzioni rare come quella catalitica. Per ovviare a questi limiti è stata condotta un’ottimizzazione del processo di produzione e purificazione dei fagi che garantisse l’omogeneità del prodotto. I risultati ottenuti sono riassunti qui di seguito: Rapporto tra fagi ricombinante e non ricombinante aumentato di un fattore 10 rispetto agli standard commerciali e di letteratura. Rapporto tra genotipo wild-type e genotipo ricombinante ridotto del 33% La libreria è stata quindi sottosta a screening per il legame al TSA in diverse condizioni chimica ed in particolare a diversi pH (da 4 a 10) e concentrazione di zinco (0 mm a 10 mm). per mezzo di 4 cicli iterativi di selzioneamplificazione (biopanning). Al contempo, le condizioni di selezione, forza ionica e concentrazione di detergente, sono state modulate per minimizzare il binding aspecifico. Infine, i tempi di incubazione ed eluizioni sono stati modificati nei diversi cicli di biopanning per favorire il ricupero di peptidi con costanti di affinità basse. Il sequenziamento di cloni selezionati ha rivelato la presenza di pattern conservati al N- e C-terminale e la presenza di residui acidi a valle della regione N-terminale. L’analisi delle sequenze non hanno evidenziato altri pattern conservati ad eccezion fatta di quelli sopramenzionati. L’allineamento delle sequenze ha evidenziato una distribuzione isotropica delle stesse nello spazio delle sequenze. Inoltre, i risultati mostrano come il legame all’aptene sia favorito a bassi pH e non sia influenzato dalla concentrazione di zinco. Questi risultati mostrano che è possibile recuperare selettivamente peptidi funzionali capaci di legare il TSA in diverse condizioni chimiche anche se emerge chiaramente che alcune condizioni (i.e. pH>4) risultano in una selezione non ottimale. Inoltre, l’analisi delle sequenze mostra una notevole eterogeneità dei peptidi selezionati che suggerisce che vi siano diverse famiglie di sequenze non omologhe capaci di legare il TSA. Nel loro complesso i risultati suggeriscono che biopolimeri funzionali diversi da quelli presenti in natura sono uniformemente distribuiti nello spazio delle sequenze a sostegno della teoria della contingenza.; Science assumes that life on Earth originated from inanimate matter by a gradual and spontaneous increase of molecular complexity. Several different theoretical frameworks have been proposed to account for the spontaneous emergence of life. Wächtershäuser identifies enzyme-free metabolic cycles as the pivotal system underpinning life’s origin; Kauffmann proposes autocatalytic peptide cycles as the primary motor, whereas Cech fostered the idea that RNA was the scaffold of the first living system. Conversely, Luisi emphases the autopoietic nature of life; whereas Lancet proposes composition inheritance as the foundation of life. Despite the differences, all theories must confront the same fundamental question: is the transition to life pathway determined univocally by the law of physic and chemistry? Or it is rather the result of the simultaneous interplay of different contingent factors? The controversy between determinism and contingency emerges forcefully when one considers the emergence of functional biopolymers in the framework of the origin of life. Indeed, the entire architecture of life relies on biopolymers so that the aetiology of biopolymers represents a major issue in the field of origin of life research. The main question is how functional biopolymers have been selected under pre- or protobiotic conditions. Indeed, the number of theoretically possible sequences quickly reaches astronomic figures as the length increases so that the correspondent sequence space could not have been sampled exhaustively during natural evolution even for short biopolymers. A straightforward question arises: how were biopolymers selected? Were these biopolymers the best ones ever possible? Or were they simply the outcome of contingency shaped by natural evolution? To tackle this question, a completely de novo random library of short peptides has been designed and tested for potential catalytic activity. The library has been designed with no sequence or structural constrains so it can be reasonably considered as a mirror image of a peptide population produced under plausible prebiotic conditions. The selection criterion was based on the ability to bind a transition state analogue of the ester and amide bond hydrolysis in order to asses the frequency and distribution in sequence space of functional peptides. The ultimate objective of this doctoral project was to investigate the catalytic properties of a random library of peptides in order to assess whether and to what extend functional biopolymers display catalytic function and how functional peptides are distributed in sequence space. Accordingly, the envisaged experimental strategy had to ensure the screening of a vast library of random peptide in order to explore effectively the sequence space. The choice fell on in vitro evolution which allows the simultaneous screening of a vast library of candidate peptides for a priori defined function without a prior knowledge. In vitro evolution mimicries natural evolution in a test tube by means of iterative cycles of mutation-selection-amplification. There are two fundamental requirements to carry out directed evolution: the first is the availability of physical link between the genotype and the phenotype. The second relies on the availability of a suitable screening procedure to enrich the initial peptide population of those sequences satisfying the selection criteria. In this doctoral project the phage display technique has been employed to ensure a physical link between the genotype and the phenotype, whereas the binding to a transition state analogue (TSA) for the ester and amide bond hydrolysis has been used as selection criterion. The likelihood of success in a in vitro evolution experiment is directly related to the total size library, as evaluating more sequences increases the chances of finding one with the desired properties. Accordingly, the first part of this doctoral project has been devoted to the construction of a DNA library encoding for random 20mer peptides and to the optimization of the screening technique. Subsequently, the designed library and the optimised screening technique have been used to select TSA-binding peptides under different chemical conditions. The selection of catalytic peptides from a random library of sequences has never been attempted before and poses a number of technical challenges due to technical difficulties related to short fragment cloning, short peptide expression and purification. To tackle these problems a novel multipurpose vector has been developed that allowed the cloning of short DNA fragment with >90% efficiency. The results obtained are summarised hereafter: Library complexity @108 Library diversity: >70% Sequence space coverage: 10-56 Phage display relies on the presentation of foreign peptides/protein on M13 phage capside whereas the correspondent gene is encapsulated within. One of the major bottleneck of phage display is the inefficient display of foreign peptides on the virion capside and the encapsulation of wild-type phage genome into the recombinant viral particles. These limitations severely affect the screening efficiency and ultimately reduced the sequence space coverage. To overcome these limitations, the phage display techniques has been optimised in order increase the ratio of phage displaying foreign peptides and minimize the ratio of wild-type genome encapsulation. The results obtained are summarised hereafter: recombinant : non-recombinant phage ratio increased 10-fold wild-type : phagemid ratio decreased 33% The random DNA library encoding for random peptides has been screened for binding to the TSA under different chemical condition with respect to pH (4 to 10) and zinc concentration (0 mM to 10 mM). Phage library has been subjected to 4 selections round of increasing stringency with respect to incubation time, wash condition (ion strength and surfactant concentration) and elution time. Sequencing of selected clones retrieved a highly conserved consensus sequence at the N- and C-terminus of selected peptide, no consensus sequence could be identified except for a conserved acid residues downstream the conserved Nterminal region. Sequences alignment shows that selected peptides are isotropically distributed in sequence space. In addition, recovery yield greatly changed under different chemical condition with highest recovery yield at pH 4 with suboptimal recovery at different pH. Finally, zinc concentration does not seem to affect TSA binding irrespective of the pH. These results show that is possible to selectively recover peptides binding to the transition state analogue (TSA) for the ester and amide bond hydrolysis reaction under different chemical environment although this results in a suboptimal selection under certain conditions. In addition, sequence analysis shows a remarkable heterogeneity of selected peptides that may suggest that multiple sequences are capable to perform binding. Although an enzymatic validation of results is required, results suggest that potentially functional sequences are evenly distributed in sequence space supporting the contingency theory.

A study on flavin-containing amine = Studio di ammino ossidasi flaviniche in arabidopsis thaliana

Tue, 10 Feb 2009 23:00:00 GMT

A study on flavin-containing amine = Studio di ammino ossidasi flaviniche in arabidopsis thaliana Spedaletti, Valentina 2009-02-11 Le poliammino ossidasi (PAO) sono enzimi FAD-dipendenti che ossidano le poliammine spermina (Spm) e spermidina (Spd) e/o i loro derivati acetilati a livello del gruppo amminico secondario. L'identità chimica dei prodotti di reazione delle PAO dipende dall'origine dell'enzima e riflette la modalità di ossidazione del substrato. In particolare, le PAO presenti nelle piante monocotiledoni, come la PAO di mais (ZmPAO), ossidano l'atomo di carbonio interno adiacente al gruppo amminico secondario della Spm e della Spd, con produzione di 1, 3-diamminopropano (Dap), perossido di idrogeno e di un'amminoaldeide ed in tale maniera partecipano ad una via catabolica terminale delle poliammine. Le PAO animali e le spermina ossidasi (SMO) ossidano, invece, l'atomo di carbonio esterno adiacente al gruppo amminico secondario della Spd o della Spm (o dei loro derivati acetilati) producendo rispettivamente putrescina (Put) o Spd, un'amminoaldeide e perossido di idrogeno ed in tale maniera sono coinvolte in una via di interconversione delle poliammine. In Arabidopsis thaliana, sono stati identificati cinque geni codificanti per PAO putative: l'At5g13700 (AtPAO1), l'At2g43020 (AtPAO2), l'At3g59050 (AtPAO3), l'At1g65840 (AtPAO4), e l'At4g29720 (AtPAO5). L'AtPAO1 e l'AtPAO5 presentano un'omologia di sequenza con la ZmPAO (la PAO vegetale maggiormente caratterizzata ed avente una localizzazione apoplastica) rispettivamente del 23% e del 25% ed una probabile localizzazione citosolica. L'AtPAO2, l'AtPAO3 e l'AtPAO4 presentano un'omologia con la ZmPAO del 23%, un'omologia fra loro che varia tra il 58% e l'85% ed una localizzazione perossisomale. Recentemente, è stato dimostrato che l'AtPAO1 è in grado di ossidare la Spm, ma non la Spd e che è coinvolta in una via di interconversione delle poliammine in maniera simile a alle PAO animali e alle SMO. Nel presente lavoro di tesi, è stato effettuato uno studio sulle proprietà biochimiche delle proteine ricombinanti AtPAO2 e AtPAO4 in seguito alla loro espressione eterologa in Escherichia coli. Tale studio ha dimostrato che le proteine ricombinanti AtPAO2 e AtPAO4 sono attive nei confronti della Spm e della Spd e che producono Spd dall'ossidazione della Spm e Put dall'ossidazione della Spd. Questi dati indicano quindi che queste due AtPAO hanno una modalità di ossidazione del substrato simile a quella dell'AtPAO1 e delle PAO e SMO animali e che sono coinvolte in una via di interconversione delle poliammine. L'esistenza di una via di interconversione delle poliammine in A. thaliana è stata dimostrata anche in vivo. Infatti, durante l'incubazione di protoplasti ottenuti da foglie di A. thaliana con Spd o Spm radioattiva è stato osservato un aumento della quantità di Put o Spd radioattiva. Tale aumento 2 risulta fortemente inibito in presenza di guazatina, un inibitore specifico delle PAO. Nel presente lavoro, è stato dimostrato anche che le proteine ricombinanti AtPAO1, AtPAO2 e AtPAO4 sono in grado di ossidare le poliammine non comuni termospermina (Termo-Spm) e norspermina (Nor-Spm), che sono state associate alla tolleranza agli stress. In particolare, è stato dimostrato queste poliammine non comuni sono per l'AtPAO1 dei substrati migliori rispetto alla Spm facendo ipotizzare che potrebbero essere i suoi substrati fisiologici. Questo dato è di fondamentale importanza se si considera che recentemente è stato identificato un gene (ACAULIS5) codificante per una proteina capace di sintetizzare la Termo-Spm dalla Spd e che piante di A. thaliana che presentano una mutazione in questo gene (acaulis5) mostrano dei difetti nell'allungamento dello stelo. In A. thaliana sono presenti altri quattro geni: l'At1g62830 (AtLSD1), l'At3g13682 (AtLSD2), l'At3g10390 (AtLSD3) e l'At4g16310 (AtLSD4) che codificano per proteine aventi un dominio ammino ossidasico. Queste proteine presentano anche un dominio SWIRM, che è generalmente presente nei complessi coinvolti nelle modificazioni della cromatina, ed hanno un'omologia di sequenza con la proteina umana HsLSD1 (KIAA0601) che varia dal 26 al 30%. È stato dimostrato che l'HsLSD1, che ha gli stessi domini funzionali delle AtLSD, catalizza la demetilazione ossidativa dell'istone H3 mono o dimetilato sulla lisina 4 e fa parte di complessi multiproteici importanti nella regolazione dell'espressione genica. Nel presente lavoro, in seguito ad espressione eterologa in E. coli è stata effettuata una parziale caratterizzazione biochimica della proteina AtLSD1, scelta come membro rappresentativo di questa famiglia genica, ed è stato dimostrato che questo enzima vegetale ha un'attività iston-demetilasica e presenta la stessa specificità di substrato della corrispondente proteina umana. Inoltre, dall'analisi del modello della struttura tridimensionale dell'AtLSD1, eseguito sulla base del cristallo dell'HsLSD1, è risultato un'elevato grado di conservazione delle strutture secondarie dell'HsLSD1 e dei residui facenti parte del sito catalitico e sono emerse alcune importanti differenze che suggeriscono che i partners molecolari dell'AtLSD1 possano essere differenti da quelli della proteina ortologa umana. Per effettuare un'analisi del profilo di espressione dei geni AtLSD, sono stati condotti degli esperimenti di RT-PCR dai quali è emerso che i livelli di espressione di tali geni risultano simili nei vari organi testati. Inoltre, allo scopo di approfondire l'analisi del profilo di espressione dell'AtLSD1, il promotore di tale gene è stato amplificato tramite PCR dal DNA totale estratto da foglie di A. thaliana ed è stato clonato in un vettore per l'espressione in pianta, mediata da Agrobacterium tumefaciens, a monte della sequenza codificante per la green-fluorescent protein (GFP) in fusione con la -glucuronidasi (GUS). In questo modo è stato ottenuto un costrutto AtLSD1 prom::GFP-GUS. Per individuare i geni regolati dall'AtLSD1 tramite analisi 3 microarray ed esperimenti di immunoprecipitazione della cromatina è stato preparato un costrutto per la sovraespressione dell'AtLSD1 in A. thaliana. In particolare, la regione codificante per l'AtLSD1 è stata amplificata tramite PCR utilizzando primers sequenza-specifici disegnati in modo tale da permetterne il clonaggio in un vettore che guidi la sovraespressione delle proteine in pianta e per aggiungere all'estremità 3' una coda di 6 istidine che faciliti l'individuazione della proteina. Per isolare i complessi nei quali la proteina AtLSD1 è eventualmente coinvolta attraverso cromatografia di affinità, è stato preparato anche un costrutto per la sovraespressione della proteina in fusione con una coda FLAG-HA. Tutti i plasmidi ricombinanti sono stati utilizzati per trasformare il ceppo GV301 di A. tumefaciens ed i batteri trasformati sono stati al loro volta utilizzati per trasformare piante di A. thaliana. Per determinare i ruoli fisiologici svolti dalle AtLSD, mutanti inserzionali per ognuno dei quattro geni (Atlsd1, Atlsd2, Atlsd3 e Atlsd4) sono stati ottenuti da banche di semi di A. thaliana e sono stati analizzati. In particolare, per confermare la presenza dell'inserzione del T-DNA e per identificare le piante mutanti omozigoti per l'inserzione è stata effettuata un'analisi tramite PCR del DNA totale estratto dalle piante mutanti. In seguito, è stata eseguita un'analisi dettagliata del fenotipo dei mutanti Atlsd che ha evidenziato un fenotipo nel mutante Atlsd3 caratterizzato da un ritardo nella fioritura. Polyamine oxidases (PAOs) are FAD-dependent enzymes which oxidize the polyamines spermine (Spm) and spermidine (Spd) and/or their acetylated derivatives at the secondary amino group. The chemical identity of PAO reaction products depends on the enzyme source and reflects the mode of substrate oxidation. In particular, PAOs from monocotyledonous plants, such as maize PAO (ZmPAO), oxidize the carbon on the endo-side of the secondary amino group of Spm and Spd producing 1, 3-diaminopropane (Dap), H2O2 and an aminoaldehyde, and are considered involved in a terminal catabolic pathway of polyamines. Conversely, animal PAOs and spermine oxidases (SMOs) oxidize the carbon on the exo-side of the secondary amino group of Spd or Spm (or their acetylated derivatives) producing putrescine (Put) or Spd, respectively, in addition to an aminoaldehyde and H2O2, and are considered involved in a polyamine back-conversion pathway. In Arabidopsis thaliana, five putative PAO genes have been identified: At5g13700 (AtPAO1), At2g43020 (AtPAO2), At3g59050 (AtPAO3), At1g65840 (AtPAO4), At4g29720 (AtPAO5). AtPAO1 and AtPAO5 have a sequence homology of 45% and 25%, respectively, with ZmPAO (the so far best characterized plant PAO which has an apoplastic localization) and a predicted cytosolic localization. AtPAO2, AtPAO3 and AtPAO4 display an homology of about 23% with ZmPAO, an homology of 58-85% to each other and a 4 peroxisomal localization. Recently, AtPAO1 has been shown to oxidize only Spm and not Spd and to be involved in a polyamine back-conversion pathway similarly to the animal PAOs/SMOs. In the present work, a study on the biochemical properties of recombinant AtPAO2 and AtPAO4 expressed in Escherichia coli was performed. This study demonstrated that recombinant AtPAO2 and AtPAO4 are active towards both Spd and Spm and that produce Spd from Spm and Put from Spd. These data indicate that these two AtPAOs have a mode of substrate oxidation similar to that of AtPAO1 and animal PAOs/SMOs and thus that they are also involved in a polyamine back-conversion pathway. The existence of a polyamine back- conversion pathway in A. thaliana has been demonstrated also in vivo. Indeed, incubation of A. thaliana protoplasts with radiolabelled Spd or Spm resulted in the accumulation of radiolabelled Put or Spd, respectively, which was strongly reduced in the presence of the PAO-specific inhibitor guazatine. In the present work, it was also shown that recombinant AtPAO1, AtPAO2 and AtPAO4 are able to oxidize the stress related uncommon polyamines thermospermine (Thermo-Spm) and norspermine (Nor-Spm). In particular, it was shown that these uncommon polyamines are better substrates than Spm for AtPAO1, suggesting that these polyamines may be the physiological substrates of this enzyme. This is of great importance considering that a gene (ACAULIS5) encoding for a protein able to synthesize Thermo-Spm from Spd has been recently characterized in A. thaliana and the acaulis5 Arabidopsis mutant presents defects in stem elongation. In A. thaliana, four more genes have been also identified: At1g62830 (AtLSD1), At3g13682 (AtLSD2), At3g10390 (AtLSD3), At4g16310 (AtLSD4) encoding for proteins with an amine oxidase domain. These proteins bear also a SWIRM domain, which is usually present in chromatin-modifying complexes, and display a 26-30% sequence homology with human HsLSD1 (KIAA0601). HsLSD1, which has the same functional domains as the four AtLSDs, has been shown to catalyse the oxidative demethylation of mono- or dimethylated lysine 4 of histone H3 and to participate in multiprotein complexes important in the regulation of gene expression. In this work, partial biochemical characterization of AtLSD1, chosen as a representative member of this gene family, following expression in E. coli demonstrated that this plant enzyme has a demethylase activity with the same substrate specificity as the corresponding human protein. Modeling of the AtLSD1 three-dimensional structure, using the HsLSD1 crystal structure, evidenced a high degree of conservation of the HsLSD1 secondary structures and of the residues building up the catalytic site, but also some important differences which suggest that the AtLSD1 molecular partners are probably different from those of the human orthologue. To analyse the expression pattern of AtLSDs, RT-PCR experiments were performed which showed that the AtLSD1, AtLSD2, AtLSD3 and AtLSD4 transcripts are present at similar 5 levels in all organs tested. With the aim to go deeper into the AtLSD1 expression pattern, the AtLSD1 promoter was amplified by PCR from Arabidopsis total DNA and cloned in an Agrobacterium tumefaciens-based plant expression vector upstream of the sequence encoding green-fluorescent protein (GFP) in fusion with -glucuronidase (GUS). In this way, an AtLSD1 prom::GFP-GUS construct was obtained. Furthermore, to identify the genes regulated by AtLSD1 through microarray analysis and chromatin immunoprecipitation experiments, a construct for AtLSD1 overexpression in A. thaliana was prepared. Indeed, the coding region of AtLSD1 was amplified by PCR using sequence specific primers designed in a way to allow AtLSD1 cDNA cloning through the Gateway technology in a vector which guides overexpression of proteins in plant and to add at the 3 terminus of cDNA a sequence encoding for a 6-His tag to facilitate detection. To isolate the complexes in which AtLSD1 is eventually involved through a two-step affinity chromatography, a construct for AtLSD1 overexpression in A. thaliana in fusion with a FLAG-HA tag was also prepared. All the recombinant plasmids were used to transform the A. tumefaciens GV301 strain and the transformed bacteria were in turn used to transform A. thaliana plants. To determine the physiological roles of AtLSDs, insertional knock-out mutants for each one of the four AtLSD genes (Atlsd1, Atlsd2, Atlsd3 and Atlsd4 mutants) were obtained from A. thaliana seed banks and analyzed. In particular, PCR analysis of total DNA from mutant seedlings was performed to confirm the presence of T-DNA insertion and to identify the homozygous mutant plants for this insertion. Detailed phenotypic analysis of the Atlsd mutants was also performed which evidenced a delayed flowering phenotype for Atlsd3 mutant. Dott.ssa Valentina Spedaletti

Ubiquitin-proteasome dependent regulation of p73 protein stability

Sun, 15 Feb 2009 23:00:00 GMT

Ubiquitin-proteasome dependent regulation of p73 protein stability Scialpi, Flavia 2009-02-16 Background and aim p73 is a structural and functional homologue of the tumor suppressor transcription factor p53, which binds to canonical p53 DNA-binding sites, activates transcription from p53-responsive promoters and, hence, induces cell cycle arrest and apoptosis (reviewed in Melino et al., 2002). In contrast to p53, p73 exists as several distinct protein isoforms (-) generated by alternative splicing at the C-terminal (De Laurenzi et al, 1998, 1999 and 2000). Additionally, the p73 gene has two distinct promoters; the first promoter (P1) yields proteins possessing an N-terminal transactivation domain (TAD), the transcriptionally active (TA) isoforms. The usage of the alternative internal promoter (P2) gives rise to N-terminally truncated proteins (N isoforms), which lack the TAD and, as a result, act as dominant negative inhibitors of p53 and TAp73 tumor suppressive functions (Kaghad et al., 1997; Ueda et al., 1999). Thus, TA- and Np73 isoforms display antagonistic functions: the TAp73 variants largely mimic p53 suppressive activities, while the Np73 proteins promote cell survival and exhibit oncogenic properties (Yang et al., 2000a; Grob et al., 2001; Sayan et al., 2004). As a result of the opposite activities exerted by TA- and Np73 proteins, the balance between cell death and survival, particularly in cells harboring p53 mutations, will crucially depend on the relative proportions of the two isoforms (Melino et al., 2002). Similarly to p53, p73 expression is maintained at low levels in mammalian cells, and its cellular induction and activation is mainly controlled at the post-translational level. p73 is polyubiquitylated in vivo and degraded via the proteasomal proteolytic system (Bernassola et al., 2004; Maisse et al., 2004). It has been previously reported that p73 ubiquitylation is catalysed by the HECT type E3 ubiquitin ligase (E3) Itch (Rossi et al., 2005). In unstressed cells, Itch targets both TA- and Np73 for protein ubiquitylation, thereby keeping their expression levels low under normal conditions. Following DNA damage treatment, TAp73 protein levels accumulate, while Np73 is rapidly degraded, in an Itch-independent manner. In several tumor cell lines, the induction of TAp73 in response to chemotherapeutic drugs is, at least partially, accomplished through Itch downregulation (Rossi et al., 2005). Our findings imply that different E3 ligases can account for p73 degradation in different conditions. On the basis of these evidences, my PhD project has been focused on ubiquitin-dependent degradation of p73, by one side testing the possible implication of another E3 ligase activity in the regulation of p73 protein level, and by the other analyzing the molecular mechanisms of Itch self- ubiquitylation and investigating its possible involvement in the regulation of Itch protein stability. Results Self-ubiquitylation activity of E3 ligases (E3s) has been previously described for both RING-type and HECT-type E3s (Bruce et al., 2008). It is thought to mainly act as a regulatory mechanism that controls the abundance of E3s by marking them for degradation (Yang et al, 2000b; Fang et al, 2000). It has been previously reported that Itch is capable to undergo self-ubiquitylation although its physiological role has not been clearly elucidated (Gao et al., 2004; Mouchantaf et al., 2006). In the context of the first part of my project, we tested whether Itch self-ubiquitination could affect its protein stability. We demonstrated that Itch generates self-assembled Lysine-63 linked polyubiquitin chains, a signal generally not involved in targeting proteins for proteasome-dependent degradation. Consistently with this, we shown that Itch is a high stable protein, whose levels are not significantly affected by treatment by either proteasome or lysosome inhibitors. Furthermore, we demonstrated that the decay rate of a catalytic inactive Itch mutant, which is devoided of self-ubiquitylating activity, is indistinguishable from the one of the wild-type protein. All these results demonstrate that Itch self-ubiquitylation activity does not regulate its protein stability. As discussed above, the evidence that Itch is responsible for keeping both TAp73 and Np73 levels low under normal condition but not in response to DNA damage suggests the involvement of another pathway to target p73 for degradation (Rossi et al., 2005). Additionally, it has been described that in C. elegans, the regulation of the p53-like protein CEP-1 is controlled by the F-box protein named FSN-1 (Gao et al., 2008). Among 520 genome-predicted F- box proteins, FSN1 is one of the few to be conserved through evolution, and its human ortholog is FBXO45. F-box proteins represent the substrate targeting subunit of a class of RING-type E3 ubiquitin ligases known as Skp1-Cul1-F-box complexes (SCF). Taken together, these data led us to test the involvement of SCFFBXO45 complex in the regulation of p73 stability. We firstly proved the existence of a functional relation between SCF complex and p73 by demonstrating that the expression of the dominant negative of Cul1, but not the other cullins, stabilizes p73. Subsequentially, we found that SCFFBXO45 specifically interacts with p73, both TAp73 and Np73 isoforms, suggesting that, similarly to Itch, FBXO45 is not able to discriminate between these two. Given that the major function of F-box proteins involves the ubiquitination of their target proteins, we sought to determine whether FBXO45 ubiquitinates p73 in mammals cells. We demonstrated indeed that FBXO45 significantly stimulates the ubiquitination of p73, both in vitro and in vivo. Significantly, on one hand the overexpression of FBXO5 induces the proteasome-dependent degradation of p73, and on the other the silencing of FBXO45 through RNA interference results in accumulation of p73. Moreover we found that FBXO45, similarly to Itch, is down-regulated in response to DNA damage, allowing thus p73 levels to increase in response to stress. Conclusions p73, a member of the p53 family, is a transcription factor controlling different biological processes, including cell death, tumorigenesis and neuronal differentiation. Knowledge on the mechanisms regulating p73 levels in basal conditions as well as in response to stress is essential to design new therapies that require p73 induction. Our group has previously demonstrated that the HECT E3 ubiquitin ligase Itch is capable to polyubiquitylate p73 and induce its degradation in a proteasome-dependent manner. The work performed in this PhD project has been focused on ubiquitin-dependent degradation of p73, analyzing the molecular mechanisms of Itch self- ubiquitylation and its possible role in Itch protein stability, and testing the possible implication of an E3 ubiquitin ligase, different from Itch, in the regulation of p73 protein level. In this study, we provide evidences that Itch engages an intermolecular reaction generating Lys63 polyubiquitin chains, and that this auto-modification does not regulate Itch protein stability. Furthermore, our findings demonstrate that the self-polyubiquitin chains generated by Itch do not serve as either proteasome or lysosome targeting. Both mono- and poly-ubiquitylation of protein substrates have been associated with internalization, sorting and changes in their subcellular localization. Hence, ubiquitin conjugation might represent a signal for Itch to translocate to distinct cellular compartments, which ultimately, would modify its accessibility to certain substrate molecules. Itch is predominantly localized to early and late endosomal compartments and lysosomes. Numerous Itch substrates are transcription factors mainly residing in the nuclear compartment. Hence, self-ubiquitylation may represent an auto-regulatory mechanism controlling Itch cytoplasmic-nuclear shuffling. The F-box proteins recruit specific substrates to the E3 ligase SCF complex, thus targeting them to proteasome-dependent degradation. Despite the large number of F-box proteins, only nine human SCF ubiquitin ligases have well-established substrates. Here we show that the F-box protein FBXO45 is recruited into SCFFbx45 complex and is able to bind to p73 promoting its ubiquitylation and its proteasome- dependent degradation. Since FBXO45 depletion sensitizes cell to apoptosis, we elucidate a new, conserved mechanism that could be potentially used to develop new strategies aimed to potentiate the apoptotic response of cancer cells following chemotherapy. p73 è un omologo strutturale e funzionale del soppressore tumorale p53, in grado di legare siti canonici di binding riconosciuti da p53, attivare la trascrizione attraverso promotori responsivi a p53, e quindi indurre arresto del ciclo cellulare e apoptosi (funzioni riassunte in Melino et al., 2002). Al contrario di p53, p73 possiede diverse isoforme (-) generate attraverso splicing alternativo all'espremità C-terminale (De Laurenzi et al, 1998, 1999 and 2000). Inoltre, il gene TP73 presenta due distinti promotori, il primo dei quail (P1) genera delle isoforme che contengono all'N-terminale il dominio di transattivazione (TAD), e che sono dunque in grado di indurre transattivazione (isoforme TA). L'utilizzo di un promotore alternativo interno (P2) produce isoforme tronche all'estremità N- terminale (isoforme N), che sono prive del TAD e dunque agiscono come inibitori dominanti negativi delle funzioni di soppressori tumorali ascrivibili a p53 e a p73 (Kaghad et al., 1997; Ueda et al., 1999). Dunque, le isoforme TAp73 e Np73 mostrano funzioni opposte: le isoforme TAp73 mimano le attività di p53, mentre le isoforme N promuovono la sopravvivenza cellulare e mostrano proprietà oncogeniche (Yang et al., 2000a; Grob et al., 2001; Sayan et al., 2004). Ne risulta che l'equilibrio tra sopravvivenza e morte cellulare, in particolare in cellule che possiedono mutazioni per p53, dipende dal rapporto tra le due isoforme (Melino et al., 2002). Come p53, l'espressione di p73 è manenuta a livelli bassi nelle cellule di mammifero, e la sua induzione e attivazione è prevalentemente controllata a livello post-traduzionale. p73 è poliubiquitinata in vivo e degradata attraverso il proteasoma (Bernassola et al., 2004; Maisse et al., 2004). È stato riportato che l'ubiquitinazione di p73 è catalizzata da Itch, una E3 ubiquitina ligasi (E3) di tipo HECT (Rossi et al., 2005). In condizioni normali. Itch ubiquitina sia TAp73 che Np73, mantenendo dunque bassa la loro abbondanza nelle cellule. A seguito di stress genotossico, i livelli proteici di TAp73 aumentano, mentre Np73 è rapidamente degradato, in maniera Itch- indipendente. In diverse linee tumorali, l'induzione di TAp73 in risposta a chemioterapici è ottenuta almeno parzialmente attraverso la downregolazione di Itch (Rossi et al., 2005). I nostri precedenti risultati suggeriscono dunque che diverse E3 ligasi possano essere responsabili della degradazione ubiquitina-dipendente di p73 in differenti condizioni. Sulla base di ciò, il mio progetto di dottorato ha riguardato la degradazione ubiquitina-dipendente di p73, da una parte testando l'ipotesi dell'esistenza di di un'altra E3 ligasi la cui attività sia implicata nella regolazione dei livelli di p73, e dall'altra analizzando i meccanismi molecolari alla base dell'autoubiquitinazione di Itch e il suo possibile coinvolgimento nella regolazione della sua stabilità proteica. Risultati L'autoubiquitinazione di E3 ligasi è stata precedentemente descritta sia per ligasi di tipo RING che di tipo HECT (Bruce et al., 2008). Si ritiene che agisca prevalentemente come un meccanismo regolatorio che controlla l'abbondanza delle E3 marcandole per la degradazione attraverso il proteasoma (Yang et al, 2000b; Fang et al, 2000). È stato descritto che Itch ha la capacità di catalizzare la sua autoubiquitinazione, sebbene il suo ruolo fisiologico non sia stato chiarito (Gao et al., 2004; Mouchantaf et al., 2006). Nella prima parte del mio progetto di dottorato, ci siamo chiesti se l'autoubiquitinazione di Itch potesse influire sulla sua stabilità proteica. Abbiamo dimostrato che Itch è in grado di generare su un'altra molecola di Itch catene di poliubiquitina costruite attraverso la Lisina 63 presente nell'ubiquitina. Catene di poliubiquitina così assemblate costituiscono un segnale che non è coinvolto nella marcatura delle proteine per la degradazione proteasoma-dipendente. Coerentemente con ciò, abbiamo dimostrato che Itch è una proteina molto stabile, i cui livelli sono sono modificati in maniera significativa da trattamento con inibitori del proteasoma o del lisosoma. Inoltre, abbiamo dimostrato che il tasso di decadimento del mutante di Itch cataliticamente inattivo (ovvero incapace di autoubiquitinazione), è indistinguibile da quello della proteina wild type. Tutti questi risultati dimostrano che l'autoubiquitinazione di Itch non regola la stabilità proteica di questa E3 ubiquitina ligasi. Come già discusso, il fatto che in condizioni normali Itch sia responsabile del mantenimento a bassi livelli sia di TAp73 che di Np73, ma non in seguito a danno al DNA, suggerisce il coinvolgimento di almeno un altro pathway responsabile della degradazione di p73 (Rossi et al., 2005). Inoltre, è stato descritto che, in C. elegans, la regolazione dell'ortologo di p53, CEP-1, è controllata da una proteina F-box denominata FSN1 (Gao et al., 2008). Tra circa 520 F-box protein predette nel genoma di C. elegans, FSN-1 è una delle poche conservate attraverso l'evoluzione, e il suo ortologo umano è FBXO45. Le proteine F-box rappresentano la subunità responsabile del riconoscimento specifico del substrato di una sottofamiglia di E3 di tipo RING nota come complesso Skp1- Cul1-F-box (SCF). Tutti insieme questi dati ci hanno spinto a indagare il possibile coinvolgimento del complesso SCFFBXO45 nella regolazione della stabilità di p73. In primo luogo abbiamo verificato l'esistenza di una relazione funzionale tra il complesso SCF e p73 dimostrando che l'espressione del mutante dominante negativo di uno dei componendi dell'SCF, la cullina Cul1, stabilizza p73, al contrario delle altre culline. Successivamente, abbiamo dimostrato che SCFFBXO45 interagisce in maniera specifica sia con TAp73 che con Np73, suggerendo che, in maniera simile ad Itch, FBXO45 non è in grado di distinguere tra le due isoforme. Poiché la funzione principale delle proteine F-box riguarda l'ubiquitinazione dei loro substrati, abbiamo verificato se FBXO45 ubiquitinasse p73 nelle cellule di mammifero. Abbiamo dimostrato che FBX045 stimola significativamente l'ubiquitinazione di p73, sia in vitro che in vivo. Inoltre, abbiamo osservato che FBXO45, come Itch, è downregolata in risposta a danno al DNA, permettendo che i livelli di p73 aumentino. Conclusioni p73, un membro della famiglia di p53, è un fattore di trascrizione che controlla diversi processi biologici, tra cui la morte cellulare, la tumorigenesi e il differenziamento neuronale. Conoscere i meccanismi che regolano i livelli di p73 in condizioni basali e in risposta allo stress cellulare è essenziale per progettare nuove terapie che richiedano l'induzione di p73. Il nostro gruppo ha precedentemente dimostrato che l'E3 ubiquitina ligasi di tipo HECT denominata Itch è in grado di poliubiquitinare p73 ed indurre la sua degradazione attraverso il proteasoma. Il lavoro riguardante questo progetto di dottorato è stato incentrato da una parte sulla degradazione ubiquitina-dipendente di p73, analizzando i meccanismi molecolari dell'autoubiquitinazione di Itch e il suo possibile ruolo nella stabilità proteica di Itch stesso, e dall'altra testando il possibile coinvolgimento di una E3 ubiquitina ligasi diversa da Itch nella regolazione dei livelli proteici di p73. In questo lavoro, abbiamo dimostrato che Itch è impegnata in una reazione intermolecolare che genera catene di poliubiquitina sintetizzate impiegando la Lys63 dell'ubiquitina stessa, e che questa auto-modificazione non regola la stabilità proteica di Itch. Inoltre, i nostri dati dimostrano che l'auto-poliubiquitinazione di Itch non conduce Itch stessa alla degradazione proteasoma- o lisosoma- dipendente. Sia la mono che la poliubiquitinazione delle proteine è stata associata con internalizzazione, smistamento e con cambiamenti nella localizzazione subcellulare. Per cui l'ubiquitinazione di Itch potrebbe rappresentare un segnale per la sua traslocazione in altri compartimenti cellulari, e dunque modificare il suo accesso ad alcuni suoi substrati. Itch è infatti localizzata in maniera predominante negli endosomi precoci e tardivi e nei lisosomi. Numerosi substrati di Itch sono fattori di trascrizione che si trovano dunque prevalentemente nel compartimento nucleare. Quindi, l'autoubiquitinazione potrebbe rappresentare un meccanismo autoregolatorio che controlla il trasporto di Itch tra il citoplasma e il nucleo. Le proteine F-box reclutano substrati specifici al complesso E3 ligasi Skp1-Cul1-F-box (SCF) ed quindi alla degradazione proteasoma- dipendente. Malgrado il gran numero di proteine F-box esistenti, solo di nove di esse si conoscono approfonditamente i substrati. In questa tesi abbiamo dimostrato che la proteina F-box FBXO45, è parte di un complesso SCF ed è capace di legare p73, di cui promuove l'ubiquitilazione e la degradazione proteasoma- dipendente. Poiché il silenziamento di FBXO45 sensibilizza le cellule all'apoptosi, noi abbiamo descritto un nuovo meccanismo, conservato attraverso l'evoluzione, che può essere potenzialmente oggetto dello sviluppo di nuove strategie per aumentare la risposta apoptotica in cellule cancerose attraverso la chemioterapia.

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