Studio archeometrico-tecnologico e conservazione dei mosaici romani del sito archeologico di Suasa

Una breve introduzione sulla storia del mosaico: dalle origine alle sue evoluzione tipologiche e tecnologiche nel tempo, di come si organizzavano le antiche botteghe del mosaico e le suddivisione dei compiti tra il pictor imaginarius, pictor parietarius e il musivarius (la gerarchia) all’interno di essi; la tecniche esecutiva per la messa in opera dei mosaici pavimentali romani. Visto che i mosaici si trovano a Suasa, quindi è stata riassunta la storia della città romana di Suasa con le sua varie fase edilizie, con maggior approfondimenti per gli edifici che presentano pavimentazione a mosaico: in primo luogo è la domus dei Coiedii contenente più di diciotto pavimenti in opus tessellatum. Il secondo è quello del così detto Edificio 4 (ancora inedito e di incerta natura e destinazione) portato in luce solo parzialmente con due settore a mosaico. Successivamente è stato effettuato in maniera dettagliata lo studio dello stato di conservazione dei vani musivi che sono state oggetto in senso stretto dei varie interventi conservativi, sia nella domus dei Coiedii (vano AU, oecus G e vano BB) che in Edificio 4 (vano A e vano D), evidenziando così le diverse morfologie di degrado in base “Normativa UNI 11176/2006 con l’aiuto della documentazione grafica ed fotografica. Un ampio e complessivo studio archeometrico-tecnologico dei materiali impiegati per la realizzazione dei musaici a Suasa (tessere e malte) presso i laboratori del CNR di Faenza.. Sono stati prelevati complessivamente 90 campione da tredici vani musivi di Suasa, di cui 28 campione di malte, comprese tra allettamento e di sottofondo,42 tessere lapidee e 20 tessere vitree; questi ultimi appartengono a sette vani della domus. Durante l’operazione del prelevo, è stato presso in considerazione le varie tipologie dei materiali musivi, la cromia ed le morfologie di degrado che erano presente. Tale studio ha lo scopo di individuare la composizione chimico-mineropetrografico, le caratteristiche tessiturali dei materiali e fornire precisa informazione sia per fine archeometrici in senso stretto (tecnologie di produzione, provenienza, datazione ecc.), che come supporto ai interventi di conservazione e restauro. Infine si è potuto costruire una vasta banca dati analitici per i materiali musivi di Suasa, che può essere consultata, aggiornata e confrontata in futuro con altri materiali proveniente dalla stessa province e/o regione. Applicazione dei interventi conservativi: di manutenzione, pronto intervento e di restauro eseguiti sui vani mosaicati di Suasa che presentavano un pessimo stato di conservazione e necessitavano l’intervento conservativo, con la documentazione grafica e fotografica dei varie fase dell’intervento. In particolare lo studio dei pregiatissimi materiali marmorei impiegati per la realizzazione dell’opus sectile centrale (sala G) nella domus dei Coiedii, ha portato alla classificazione e alla schedatura di sedici tipi di marmi impiegati; studio esteso poi al tessellato che lo circonda: studio del andamento, tipologie dei materiali, dei colore, dimensione delle tessere, interstizio ecc., ha permesso con l’utilizzo delle tavole tematiche di ottenere una chiara lettura per l’intero tessellato, di evidenziare così, tutti gli interventi antiche e moderni di risarciture, eseguiti dal II sec. d.C. fio ad oggi. L’aspetto didattico (teorico e pratico) ha accompagnato tutto il lavoro di ricerca Il lavoro si qualifica in conclusione come un esempio assai significativo di ricerca storicoiconografiche e archeometriche, con risultati rilevanti sulle tecnologie antiche e sui criteri di conservazione più idonei.

Evidenze molecolari del ruolo antiproliferativo e pro-apoptotico della clusterina in cellule epiteliali prostatiche umane

The Clusterin (CLU) gene produces different forms of protein products which vary in their biological properties and distribution within the cell. Both the extra- and intracellular CLU forms regulate cell proliferation and apoptosis. Dis-regulation of CLU expression occurs in many cancer types, including prostate cancer. The role that CLU plays in tumorigenesis is still unclear. We found that CLU over-expression inhibited cell proliferation and induced apoptosis in prostate cancer cells. Here we show that depletion of CLU affects the growth of PC-3 prostate cancer cells. Following siRNA, all protein products quickly disappeared, inducing cell cycle progression and higher expression of specific proliferation markers (i.e. H3 mRNA, PCNA and cyclins A, B1 and D) as detected by RT-qPCR and Western blot. Quite surprisingly, we also found that the turnover of CLU protein is very rapid and tightly regulated by ubiquitin–proteasome mediated degradation. Inhibition of protein synthesis by cycloheximide showed that CLU half-life is less than 2 hours. All CLU protein products were found poly-ubiquitinated by co-immuniprecipitation. Proteasome inhibition by MG132 caused stabilization and accumulation of all CLU protein products, strongly inducing the nuclear form of CLU (nCLU) and committing cells to caspase-dependent death. In conclusion, proteasome inhibition may induce prostate cancer cell death through accumulation of nCLU, a potential tumour suppressor factor.

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

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

Inibizione selettiva del gene MYCN mediante PNA (acidi peptido nucleici) anti-gene nel rabdomiosarcoma umano

MYCN oncogene amplification/expression is a feature of many childhood tumors, and some adult tumors, and it is associated with poor prognosis. While MYC expression is ubiquitary, MYCN has a restricted expression after birth and it is an ideal target for an effective therapy. PNAs belong to the latest class of nucleic acid-based therapeutics, and they can bind chromosomal DNA and block gene transcription (anti-gene activity). We have developed an anti-gene PNA that targets specifically the MYCN gene to block its transcription. We report for the first time MYCN targeted inhibition in Rhabdomyosarcoma (RMS) by the anti-MYCN-PNA in RMS cell lines (four ARMS and four ERMS) and in a xenograft RMS mouse model. Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, comprising two main subgroups [Alveolar (ARMS) and Embryonal (ERMS)]. ARMS is associated with a poorer prognosis. MYCN amplification is a feature of both the ERMS and ARMS, but the MYCN amplification and expression levels shows a significant correlation and are greater in ARMS, in which they are associated with adverse outcome. We found that MYCN mRNA and protein levels were higher in the four ARMS (RH30, RH4, RH28 and RMZ-RC2) than in the four ERMS (RH36, SMS-CTR, CCA and RD) cell lines. The potent inhibition of MYCN transcription was highly specific, it did not affect the MYC expression, it was followed by cell-growth inhibition in the RMS cell lines which correlated with the MYCN expression rate, and it led to complete cell-growth inhibition in ARMS cells. We used a mutated- PNA as control. MYCN silencing induced apoptosis. Global gene expression analysis (Affymetrix microarrays) in ARMS cells treated with the anti-MYCN-PNA revealed genes specifically induced or repressed, with both genes previously described as targets of N-myc or Myc, and new genes undescribed as targets of N-myc or Myc (mainly involved in cell cycle, apoptosis, cell motility, metastasis, angiogenesis and muscle development). The changes in the expression of the most relevant genes were confirmed by Real-Time PCR and western blot, and their expression after the MYCN silencing was evaluated in the other RMS cell lines. The in vivo study, using an ARMS xenograft murine model evaluated by micro-PET, showed a complete elimination of the metabolic tumor signal in most of the cases (70%) after anti-MYCN-PNA treatment (without toxicity), whereas treatment with the mutated-PNA had no effect. Our results strongly support the development of MYCN anti-gene therapy for the treatment of RMS, particularly for poor prognosis ARMS, and of other MYCN-expressing tumors.

L'inerbimento nell'ecosistema vigneto: confronto di metodologie di misura del consumo idrico del prato e risposta allo sfalcio

Water resources management will become increasingly important in agriculture as global warming takes place. Cover crop is largely used in viticultural areas based on the many positive agronomic and environmental benefits it provides. However, it is not clear what effect the cover crop can have on water use in the vineyard. This study is designed to develop a further understanding of the role cover crops play in total water use in the vineyard and develop our understanding of the potential use of cover crops as a water management tool. Two techniques were used to measure cover crop water use, the mini-lysimeters and a portable open chamber and data from both was compared to reference evapotranspiration (ETo) (FAO guidelines). While the mini-lysimeters seemed to be limited in their ability to accurately represent the water use of the surrounding soil, the open chamber method is a reliable and suitable instrument to be used for the accurate measurement of evapotranspiration. Further, the relationship between vineyard grass water use and the contributing environmental factors thought to influence water use were analyzed. A strong relationship between total available radiation and cover crop evapotranspiration was found suggesting the possibility of an indirect method of evapotranspiration measurement in a vineyard grass cover crop. Mowing the cover crop was determined to significantly effect transpiration as shown by both the mini-lysimeter and open chamber, however, the reduction was largely dependent on the growth rate of the grass.