Generazione di fotorecettori a partire da cellule pluripotenti della calotta animale di Xenopus laevis tramite sovraespressione di noggin e Xotx5.

La retina dei Vertebrati è l'organo di senso deputato alla trasduzione del segnale luminoso. Istologicamente, la retina presenta una tipica organizzazione trilaminare: lo strato nucleare esterno, contenente i fotorecettori, lo strato nucleare interno, contenente cellule bipolari, orizzontali, amacrine e cellule della glia di Müller, e lo strato di cellule gangliari, che con i loro assoni costituiscono il nervo ottico. Le malattie degenerative della retina, come la Retinite pigmentosa, portano alla graduale perdita della vista mediante l'attuazione di un programma multifasico di morte cellulare e di perdita dell'organizzazione trilaminare retinica. Tale neurodegenerazione potrebbe essere efficacemente contrastata con una terapia basata sulla “sostituzione cellulare”, cioè sul trapianto in organo di cellule staminali o progenitori capaci di sostituire le cellule in degenerazione dell'ospite. Tale approccio richiede anzitutto una fase di differenziamento in vitro di cellule staminali multipotenti, al fine di ottenere cellule indirizzate verso un destino retinico, capaci di integrarsi nel tessuto ospite e differenziarsi in tutti i sottotipi cellulari affetti dalla degenerazione. Xenopus laevis rappresenta un buon modello per lo studio della funzione genica e dello sviluppo dell'occhio. I suoi embrioni possono essere microiniettati durante le prime fasi di sviluppo con RNA messaggeri trascritti in vitro. Successivamente, allo stadio di blastula, gli emisferi animali (animal cap), ai quali compete un destino epidermico se non diversamente trattati, possono essere asportati e coltivati in soluzione salina. Ciò consente di utilizzare queste cellule come materiale biologico di partenza per saggi di differenziamento, dove la sovraespressione di geni coinvolti nella specificazione di tipi cellulari retinici può istruire le cellule dell’animal cap e indirizzarle verso un particolare destino. Nel laboratorio dove ho svolto il mio internato di tesi da molti anni si studiano i meccanismi molecolari del differenziamento retinico nell'anfibio Xenopus laevis. Diversi fattori di trascrizione di tipo homeobox, come Xotx5, Xotx2 e Xvsx1, sono importanti per il differenziamento di specifici neuroni retinici. La trascrizione e la traduzione dei geni sopraelencati vengono finemente regolate nel tempo e nello spazio all’interno della retina di Xenopus. Esperimenti di trasfezione in vivo hanno dimostrato che Xotx5 promuove il destino di fotorecettori, mentre Xotx2 e Xvsx1 promuovono entrambi il destino bipolare. Più recentemente è stato evidenziato il ruolo di noggin nell'indurre un destino retinico in cellule embrionali multipotenti, quali le cellule staminali embrionali della calotta animale (ACES). L’iniezione di mRNA di noggin determina l’espressione di marcatori terminali di differenziamento retinico in cellule animali (animal cap cells) della blastula di Xenopus, coltivate in vitro. In seguito a trapianto in vivo, cellule della calotta animale esprimenti alte dosi di noggin formano occhi ben sviluppati, sia quando trapiantate nella regione presuntiva dell’occhio sia quando inserite in posizione ectopica posteriore, anche se negli occhi derivanti dai trapianti posteriori non si ha mai la formazione del cristallino. Lo scopo del mio lavoro di tesi è stato duplice: · Innanzitutto mi sono occupato di verificare la funzionalità degli occhi derivati da trapianto di animal cap iniettati con noggin mRNA. Questo è stato realizzato con tecniche di elettrofisiologia, quali patch-clamp e registrazione di risposta alla luce. In particolare, ho effettuato trapianti di animal cap in vivo, rimuovendo metà del campo morfogenetico dell’occhio di embrioni di Xenopus a stadio di neurula (st. 15) e sostituendolo con animal cap iniettati con noggin mRNA. Ho quindi allevato i girini, sia wild type (WT) di controllo che animali sottoposti a trapianto, fino a stadi tardivi e recuperato gli occhi a stadio di sviluppo 46/47 tramite dissezione sotto luce infrarossa o rossa; ho poi ripulito gli occhi da epitelio pigmentato e cristallino, e ho infine ridotto il materiale in frammenti adatti all’applicazione di tecniche di elettrofisiologia, dissociando o meno le cellule con opportuni enzimi a seconda delle esigenze. Questo lavoro è stato svolto in collaborazione con il Prof. Gian Carlo Demontis (Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa), che ha effettuato le registrazioni elettrofisiologiche. La tecnica del patch-clamp perforato è servita a verificare la presenza di canali voltaggio-dipendenti caratterizzanti i fotorecettori, mentre la suzione del segmento esterno è stata il punto di partenza per la registrazione di risposte a lampi di luce con lunghezza d’onda di 520 nm (adatta alla stimolazione dei bastoncelli). · Inoltre, nel mio lavoro di tesi ho anche indagato sulla potenzialità di Xotx5 nell’indurre un differenziamento fotorecettoriale. Senza la compresenza di altri fattori implicati nel differenziamento retinico, l’azione della sola proteina Noggin induce un differenziamento retinico generale, dove la calotta animale risulta arricchita di popolazioni cellulari corrispondenti ai vari tipi retinici dell’occhio WT. Mi sono occupato quindi di analizzare se la microiniezione di noggin + Xotx5 fosse in grado di indirizzare le cellule della calotta animale non più verso destini retinici generali bensì verso lo specifico destino di fotorecettore. Tecniche quali ibridazione in situ, immunoistochimica e PCR quantitativa Real Time hanno consentito di analizzare il differenziamento cellulare avvenuto all’interno degli animal cap. Per quanto riguarda la prima parte del mio lavoro, l’analisi dei risultati ha mostrato notevole somiglianza di funzionamento e differenziamento tra fotorecettori WT e fotorecettori derivanti da occhi trapiantati: in entrambi i casi viene espresso in membrana lo stesso set di canali ionici e si riscontrano risposte alla luce equivalenti. Inoltre la circuiteria retinica è perfettamente funzionante nel recepire e trasdurre, attraverso neuroni di secondo ordine, lo stimolo luminoso captato in primis dai fotorecettori. I risultati ottenuti durante la seconda parte del mio internato di tesi, invece, hanno mostrato come la coiniezione di Xotx5 con alte dosi di noggin determini un aumento di espressione del gene opsina, marcatore di fotorecettori terminalmente differenziati, oltre a un aumento percentuale di cellule opsina-positive e alla facilitazione dell’organizzazione di tali cellule in strati ordinati. Ciò candida Xotx5 come possibile fattore di differenziamento da impiegare in associazione a noggin per l’ottenimento di fotorecettori in vitro, da utilizzarsi in protocolli di terapia cellulare sostitutiva

The microRNA miR-21 Is a Mediator of FGF8 Action on Cortical COUP-TFI Translation

The morphogen FGF8 plays a pivotal role in neocortical area patterning through its inhibitory effect on COUP-TFI/Nr2f1 anterior expression, but its mechanism of action is poorly understood. We established an in vitro model of mouse embryonic stem cell corticogenesis in which COUP-TFI protein expression is inhibited by the activation of FGF8 in a time window corresponding to cortical area patterning. Interestingly, overexpression of the COUP-TFI 3′UTR reduces the inhibitory effect of FGF8 on COUP-TFI translation. FGF8 induces the expression of few miRNAs targeting COUP-TFI 3′UTR in silico. We found that the functional inhibition of miR-21 can effectively counteract the inhibitory effect of FGF8 in vitro and regulate COUP-TFI protein levels in vivo. Accordingly, miR-21 expression is complementary to COUP-TFI expression during corticogenesis. These data support a translational control of COUP-TFI gradient expression by FGF8 via miR-21 and contribute to our understanding of how regionalized expression is established during neocortical area mapping

SACHER Project: A Cloud Platform and Integrated Services for Cultural Heritage and for Restoration

The SACHER project provides a distributed, open source and federated cloud platform able to support the life-cycle management of various kinds of data concerning tangible Cultural Heritage. The paper describes the SACHER platform and, in particular, among the various integrated service prototypes, the most important ones to support restoration processes and cultural asset management: (i) 3D Life Cycle Management for Cultural Heritage (SACHER 3D CH), based on 3D digital models of architecture and dedicated to the management of Cultural Heritage and to the storage of the numerous data generated by the team of professionals involved in the restoration process; (ii) Multidimensional Search Engine for Cultural Heritage (SACHER MuSE CH), an advanced multi-level search system designed to manage Heritage data from heterogeneous sources

Monitoring of coastal dunes habitats in Tuscany through the “MONITO-RARE” project

“MONITO-RARE” is a project involving Tuscany Region and Universities of Firenze, Siena and Pisa, aimed to improve the knowledge and develop monitoring methodologies for species and habitats included in Natura 2000 Network and considered of community interest, according to Art. 11 and Art. 17 of the Directive 92/43/EEC. Among the habitat of interest (Annex I), coastal sand dune is one of the most threatened by multiple human pressures (1, 2), such as pollution, coastal erosion, effects of global warming, farming practices, urban development, and pressure from tourism (3). As part of this project, was carried out a first year of monitoring of dunal habitats (Natura 200 code 2110, 2120, 2210, 2230, 2240, 2250* and 2260) in five SACs (Special Areas of Conservation) in order to cover different latitudes of Tuscany coast (“Dune litoranee di Torre del Lago/Selva Pisana”, “Tombolo da Castiglion della Pescaia a Marina di Grosseto”, “Dune costiere del Parco dell’Uccellina” and “Duna del Lago di Burano”). The coastal areas to the north and south of Arno basin are very different by climatic, geomorphologic (erosion) and anthropic factors. The northern coast is twice rainy as the southern one. Nevertheless, sand dune habitats are distributed in a fine scale mosaic, not distinguishable through aerial photos and maps. For this reason, we opted for a stratified sampling in three EUNIS habitat types (B1.3, shifting coastal dunes; B1.4, coastal stable dune grassland; B1.6, coastal dune scrub) for psammophilous vegetation. According to Sperandii et al. (4), we recorded these communities by 262 random plots of 2 x 2 m surface in which were surveyed pressures and threats, floristic composition and an estimate of abundance using a percentage cover scale ranging from 1 to 10. Our sampling is congruent with RanVegDunes (GIVD ID EU-IT-020), the first Italian database gathering standardized, randomly-sampled vegetation data in coastal dune environments. We detected significative differences in abundance and species composition between EUNIS habitat types, between SACs, and between habitat types within SACs. In particular, our data shows particular differences between communities of north and south of Tuscany: the northern SACs to Serchio river are heavily impacted by the presence of mass tourism, with bathing establishments, roads and human trampling. From these evidences can be deduced that trails installed within the DUNETOSCA Life Projects and aimed to reduction of tourism impact may be insufficient to reduce anthropic pressures in those contexts and adequate conservation strategies are required. 1) EEA (1999) State and pressures of the marine and coastal Mediterranean Environment. Environmental Issues Series, No. 5, European Environment Agency, Copenhagen 2) Barcelona Convention, Athens (2012) UNEP/MAP 2012. State of the Mediterranean Marine and Coastal Environment https://wedocs.unep.org/bitstream/handle/20.500.11822/364/sommcer_eng.pdf?sequence=4&isAllowed=y 3) Ciccarelli D. (2014) Environmental Management 54, 194–204 4) Sperandii M.G., Prisco I., Stanisci A., Acosta A.T.R. (2017) Phytocoenologia 47, 231–23

Enough Is Enough? Searching for the Optimal Sample Size to Monitor European Habitats: A Case Study from Coastal Sand Dunes

A robust survey method that samples the main characteristics of plant assemblages is needed to assess the conservation status of European habitat in the Natura 2000 network. A measure of variability, called pseudo-multivariate dissimilarity-based standard error (MultSE), was recently proposed for assessing sample-size adequacy in ecological communities. Here, we used it on coastal sand dune systems in three Special Areas of Conservation (SACs) in Tuscany. Our aim was to assess the minimum number of replicates necessary to adequately characterize sand dune environments in terms of differences between habitats and SACs, after a preliminary baseline assessment of plant diversity. Analysis of \u3b1 and \u3b2 diversity indicated that especially between habitats the three SACs protect different plant communities. The study of the MultSE profiles showed that the minimum number of replicates was related to habitat features and varied between 10 and 25 plots. Two-way PERMANOVA and SIMPER analysis on the full and reduced datasets confirmed that SACs and habitats host different plant communities, and that the contribution of the target species remained unchanged even with a reduced sample size. The proposed methodological approach can be used to develop cost-effective monitoring programs and it can be useful for plant ecologists and biodiversity managers for assessing ecosystem health and changes

Business model analysis of low-cost carriers on the exaple of Ryanair

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An overview of the Italian forest biodiversity and its conservation level, based on the first outcomes of the 4th Habitat Report ex-Art. 17

In 2019 the 4th Report ex-Art. 17 on the conservation status (CS) of Annex I Habitats of the 92/43/EEC Directive was expected by every EU/28 country, with reference to the period 2013-18. In Italy, the process was in charge to the Italian Institute for Environmental Protection and Research (ISPRA), on behalf of the Ministry for Environment, Land and Sea Protection (MATTM), with the scientific support of the Italian Botanical Society (SBI). A large group of thematic and territorial experts elaborated the available data concerning the 124 types of terrestrial and inland water Habitats present in Italy, 39 of which are represented by Forest Habitats (Group 9),. The main aim of the work was the evaluation of the overall CS of each Habitat by Biogeographic Region (Mediterranean, Continental and Alpine), for a total amount of 294 assessments. A high proportion of these (92, corresponding to 31% of the total) referred to Forest Habitats, including 20 marginal types for which the CS was not requested. The analysis was carried out at different scales: a) administrative territory, through the data contained in the ISPRA database, whose compilation was in charge to the Regions and Autonomous Provinces; b) Natura 2000 site, with the latest updates available (Standard Data Forms updated to 2018); c) national scale, implementing the distribution maps for each Habitat based on the European grid ETRS89-LAEA5210 (10x10 km2 mesh); d) Biogeographic Region, scale of the final assessment. Cartographic outcomes, associated databases and additional data used for the assessments will be available online on the ISPRA Portal as soon as the validation process by the European Commission will be completed. A dedicated archive named "HAB_IT" has been created in the national database "VegItaly" (1), managed by the Italian Society of Vegetation Science, where the phytosociological relevés representative of the various Annex I Habitats in Italy will be archived and freely accessible. An overview of the results regarding the Forest habitats is here provided, including a comparison with the outcomes of the former reporting cycle, the 3rd Report ex-Art. 17 (2). In several cases (e.g. 9120, 91L0), the distribution maps have been remarkably improved due to better knowledge and more fitful interpretation. The conservation status resulted as Favourable (FV) for 6,7%, Inadequate (U1) for 58,7% and Bad (U1) for 32,0% of the 72 assessed forest Habitat types. In no case there was an improvement of the conservation status, while in 6 cases a worsening of the conditions resulted from the data analysis, pointing out the Habitats types with a higher need of action. Similarly to other projects carried out as a team by the network of Annex I Habitat experts of the Italian Botanical Society and the Italian Society for Vegetation Science (e.g. 3, 4), this is another step in the direction of supporting the implementation of the 92/43/EEC "Habitat" Directive in Italy and Europe. On this ground, the high biodiversity of the Italian forest Habitats could be emphasized, however results pointed out that some rare or endemic types (e.g. Alnus cordata or Betula aetnensis-dominated forests) are still scarcely acknowledged by the most prominent EU conservation tools such as the Annex I to the "Habitat" Directive. 1) F. Landucci et al. (2012) Plant Biosyst., 146(4), 756-763 2) P. Genovesi et al. (2014) ISPRA, Serie Rapporti, 194/2014 3) E. Biondi et al. (2009) Società Botanica Italiana, MATTM, D.P.N., http://vnr.unipg.it/habitat/ 4) D. Gigante et al. (2016) Plant Sociology, 53(2), 77-8

Shedding light on typical species : implications for habitat monitoring

Habitat monitoring in Europe is regulated by Article 17 of the Habitats Directive, which suggests the use of typical species to assess habitat conservation status. Yet, the Directive uses the term “typical” species but does not provide a definition, either for its use in reporting or for its use in impact assessments. To address the issue, an online workshop was organized by the Italian Society for Vegetation Science (SISV) to shed light on the diversity of perspectives regarding the different concepts of typical species, and to discuss the possible implications for habitat monitoring. To this aim, we inquired 73 people with a very different degree of expertise in the field of vegetation science by means of a tailored survey composed of six questions. We analysed the data using Pearson's Chi-squared test to verify that the answers diverged from a random distribution and checked the effect of the degree of experience of the surveyees on the results. We found that most of the surveyees agreed on the use of the phytosociological method for habitat monitoring and of the diagnostic and characteristic species to evaluate the structural and functional conservation status of habitats. With this contribution, we shed light on the meaning of “typical” species in the context of habitat monitoring