216 research outputs found
Demand Analysis Models to Support Cultural Tourism Strategy: Application of Conjoint Analysis in North Sardinia (Italy)
This study focuses on an inner area in Northern Sardinia (Italy) currently experiencing depopula-tion and considered 'fragile' due to its lack of typical regional summer tourism. However, this area boasts an intriguing cultural heritage, currently under-recognized and undervalued. The aim of this contribution is to apply to this territory the Conjoint Analysis methodology, which examines target demand and constructs scenarios. Drawing from the regional plan for tourism, an explora-tory phase was employed to support future strategies in cultural and touristic enhancement by stakeholders. In this regard, the experiment, involving a survey of 600 interviews among inhabi-tants (301) and tourists (299), aims to: 1) estimate the economic value of the cultural heritage and tourist consumption preferences using WTP, 2) raise awareness among decision-makers about the varied value perceptions of the territory and its heritage by its users, 3) craft a knowledge fra-mework of this territory to diversification of the tourism offer. The study showcases innovative elements both in terms of internal technical aspects and strategic approach. The collection of pre-ferences, based on a set of 9 itinerary cards, highlights undervalued aspects related to intangible components, community well-being, sustainable consumption habits, sustainable mobility, and microeconomies not strictly tied to traditional tourism type
The Crowdmapping Mirafiori Sud experience (Torino, Italy): an educational methodology through a collaborative and inclusive process
The CrowdMapping Mirafiori Sud (CMMS) pilot project, carried out by the Polytechnic of Turin (Italy), involved the academic world (students and professors), the Mirafiori Onlus Foundation, the local administration and the community in a participatory and inclusive process. The district is a large area of Turin characterized by a high average age of its inhabitants and a high percentage of foreigners. The aim of the project is to identify and report, through the use of ICT, the obstacles that prevent residents - the most vulnerable categories - from using public space. In the start-up phase, it was not born as a PBL approach, but during the application the PBL was considered a development perspective in the educational experience starting from the students' proposals and the exchange of their skills, in the specific declination of the PBL approach known as community engaged learning. For students, this version of PBL has proven to be strategic in the implementation phases of the project, in particular in the mapping of interested parties and the data set and connections between networks of actors
SiForAge Social Innovation Reseach Prize 2014
This prize aims to give recognition to projects that undertake research into social innovation in active and healthy ageing and that make significant contributions to ager communities, their families, and caregivers in order to promote an inclusive society. The three research projects proposals which have won are the Crowdmapping Mirafiori Sud project, the Active aging and intergenerational solidarity: an example in the University context project and the Enred@te project
La symétrisation des protéines : un nouvel outil pour la biologie structurale
Structural determination of proteins at atomic level resolution is crucial for unravelling their function. X-ray crystallography has successfully been used to determine macromolecular structures with sizes ranging from kDa to MDa, and currently remains the most efficient method for the high-resolution structure determination of monomeric proteins within the 40-100 kDa range. However, this method is limited by the ability to grow well diffracting crystals, which is problematic for several targets, such as membrane proteins. Single particle cryo electron microscopy (cryoEM) allows near atomic (3-4Ă
) resolution structural determination of large, preferably symmetric, assemblies in solution. Biological molecules scatter electrons weakly and, to avoid radiation damage, only low electron doses can be used during imaging. Consequently, raw cryoEM images are extremely noisy. However, averaging many molecular images aligned in the same orientation permits one to increase the signal-to-noise ratio, ultimately allowing the achievement of a 3D density map of the molecule of interest. Nevertheless, as the molecular size and degree of symmetry decrease, the individual images loose adequate features for accurate alignment. Currently, cryoEM analysis is practically impossible for monomeric proteins below ~100 kDa in mass. We propose to circumvent this obstacle by fusing such monomeric target proteins to a homo-oligomeric protein (template), thereby generating a self-assembling particle whose large size and symmetry should facilitate cryoEM analysis. In the present thesis we seek to test and demonstrate the feasibility of this âprotein symmetrization' approach and to evaluate its usefulness for protein structure determination. To set up the pilot study we combined selected targets of known structure with two templates: Glutamine Synthetase (GS), a 12-mer with D6 symmetry and a helical N-terminus, and the E2 subunit of the pyruvate dehydrogenase complex, a 60-mer with icosahedral symmetry and an unstructured N-terminus. After recombinant production in E.coli we identified by negative stain EM a promising dodecameric chimera for structural analysis, comprising maltose binding protein (Mbp) connected to GS by a tri-alanine linker (denoted âMagâ). In order to optimize sample homogeneity we produced a panel of Mag deletion constructs by sequentially truncating the 17 residues between the Mbp and GS domains. A combination of biophysical techniques (thermal shift assay, dynamic light scattering, size exclusion chromatography) and negative stain EM allowed us to select the best candidate for cryoEM analysis, MagÎ5. By enforcing D6 symmetry we obtained a cryoEM map with a resolution of 10Ă
(FSC 0.5 criterion). The density of the symmetrized 40 kDa Mbp presents shape and features corresponding to the known atomic structure. In particular, the catalytic pocket and specific α-helical elements are distinguishable. The cryoEM map is additionally validated by a 7Ă
crystal structure of the MagÎ5 oligomer. The presence of a continuous helical connection between target (Mbp) and template (GS) likely contributed to the conformational homogeneity of MagÎ5. Moreover, comparing MagÎ5 with other chimeras studied in this work suggests that a large buried surface area and favorable interactions between the target and template limit the flexibility of the chimera and improve its resolution by cryoEM. For the symmetrization of a target of unknown structure, we envisage proceeding by a trial and error approach by fusing it to a panel of templates with helical termini and different surface properties, and subsequently selecting the best ones using biophysical assays. In conclusion, the present work establishes the proof-of-concept that protein symmetrization can be used for the structure determination of monomeric proteins below 100 kDa by cryoEM, thereby providing a promising new tool for analyzing targets resistant to conventional structural analysis.La dĂ©termination de la structure des protĂ©ines Ă une rĂ©solution atomique est cruciale pour la comprĂ©hension de leur fonction cellulaire. Actuellement, la cristallographie aux rayons X est la mĂ©thode la plus efficace pour la dĂ©termination Ă haute rĂ©solution de la structure de protĂ©ines monomĂ©riques allant 40 et 100 kDa. Par contre, elle est limitĂ©e par la croissance de cristaux de bonne qualitĂ©, qui est problĂ©matique pour nombreuses cibles. La cryo-microscopie Ă©lectronique (cryoME) permet la dĂ©termination structurale Ă rĂ©solution quasi-atomique de larges structures protĂ©iques, de prĂ©fĂ©rence symĂ©trique et en solution. Cependant, les images de cryoME sont trĂšs bruitĂ©es, car une faible dose d'Ă©lectrons est appliquĂ©e de maniĂšre Ă limiter les dommages d'irradiation. En moyennant des dizaines d'images correspondant Ă la mĂȘme orientation molĂ©culaire, le rapport signal sur bruit est amĂ©liorĂ©. La combinaison des images moyennĂ©es de plusieurs orientations permet l'obtention d'une carte de densitĂ© Ă©lectronique 3D de la molĂ©cule d'intĂ©rĂȘt. Si la taille et la symĂ©trie de la molĂ©cule diminuent, l'analyse cryoME devient de moins en moins prĂ©cise, il est alors impossible d'analyser des protĂ©ines monomĂ©riques de taille infĂ©rieure Ă 100 kDa. Le but de ce travail a Ă©tĂ© de dĂ©velopper une nouvelle approche pour rĂ©duire cette limite de poids molĂ©culaire. Elle consiste Ă fusionner la protĂ©ine d'intĂ©rĂȘt (cible) Ă une matrice homo-oligomĂ©rique, gĂ©nĂ©rant une particule symĂ©trique et de taille importante adaptĂ©e Ă l'analyse par cryoME. Dans cette thĂšse, nous avons cherchĂ© Ă tester et dĂ©montrer la faisabilitĂ© de cette approche de symĂ©trisation en utilisant des protĂ©ines cibles de structure connue.Pour mettre en place notre Ă©tude pilote, nous avons choisi diffĂ©rentes combinaisons de cibles et de matrices connectĂ©es par des peptides de liaison (linker) de longueur diffĂ©rentes. Nous avons caractĂ©risĂ© les fusions exprimĂ©es en bactĂ©ries par microscopie Ă©lectronique aprĂšs coloration nĂ©gative et par plusieurs techniques biophysiques. Grace Ă ces techniques, nous avons trouvĂ© que la meilleure combinaison est la fusion entre la protĂ©ine matrice glutamine synthĂ©tase (GS), un 12-mer de symĂ©trie D6 et la cible maltose binding protein (Mbp), connectĂ©es par un linker contenant trois alanines, que nous avons appelĂ©e « Mag ». En jouant sur la longueur du linker nous avons ensuite sĂ©lectionnĂ© la fusion la plus compacte pour l'analyse cryoME: MagÎ5. Nous avons obtenu la carte cryoME Ă 10 Ă
de MagÎ5, qui prĂ©sente une bonne corrĂ©lation avec les modĂšles atomiques de Mbp et GS. Plus particuliĂšrement, le site catalytique et quelques hĂ©lices α sont identifiables. Ces rĂ©sultats sont confirmĂ©s par l'Ă©tude cristallographique que nous avons conduite sur MagÎ5. L'ensemble de ce travail souligne que la prĂ©sence d'une grande interface d'interactions cible-matrice stabilise la fusion et amĂ©liore la rĂ©solution en cryoME. Pour la symĂ©trisation d'une cible inconnue, nous envisageons la mĂȘme procĂ©dure expĂ©rimentale que celle dĂ©veloppĂ©e pour MagÎ5. La matrice et le linker les plus adaptĂ©s devront ĂȘtre identifiĂ©s en utilisant les mĂȘmes mĂ©thodes biophysiques.En conclusion, ce travail Ă©tablit la preuve de concept que la mĂ©thode de symĂ©trisation des protĂ©ines permet la dĂ©termination de la structure de protĂ©ines de poids molĂ©culaire infĂ©rieur Ă 100 kDa par cryoME. Cette mĂ©thode a le potentiel d'ĂȘtre un nouvel outil prometteur, qui faciliterait l'analyse de cibles rĂ©sistantes Ă l'analyse structurale conventionnelle
La symétrisation des protéines : un nouvel outil pour la biologie structurale
Structural determination of proteins at atomic level resolution is crucial for unravelling their function. X-ray crystallography has successfully been used to determine macromolecular structures with sizes ranging from kDa to MDa, and currently remains the most efficient method for the high-resolution structure determination of monomeric proteins within the 40-100 kDa range. However, this method is limited by the ability to grow well diffracting crystals, which is problematic for several targets, such as membrane proteins. Single particle cryo electron microscopy (cryoEM) allows near atomic (3-4Ă
) resolution structural determination of large, preferably symmetric, assemblies in solution. Biological molecules scatter electrons weakly and, to avoid radiation damage, only low electron doses can be used during imaging. Consequently, raw cryoEM images are extremely noisy. However, averaging many molecular images aligned in the same orientation permits one to increase the signal-to-noise ratio, ultimately allowing the achievement of a 3D density map of the molecule of interest. Nevertheless, as the molecular size and degree of symmetry decrease, the individual images loose adequate features for accurate alignment. Currently, cryoEM analysis is practically impossible for monomeric proteins below ~100 kDa in mass. We propose to circumvent this obstacle by fusing such monomeric target proteins to a homo-oligomeric protein (template), thereby generating a self-assembling particle whose large size and symmetry should facilitate cryoEM analysis. In the present thesis we seek to test and demonstrate the feasibility of this âprotein symmetrization' approach and to evaluate its usefulness for protein structure determination. To set up the pilot study we combined selected targets of known structure with two templates: Glutamine Synthetase (GS), a 12-mer with D6 symmetry and a helical N-terminus, and the E2 subunit of the pyruvate dehydrogenase complex, a 60-mer with icosahedral symmetry and an unstructured N-terminus. After recombinant production in E.coli we identified by negative stain EM a promising dodecameric chimera for structural analysis, comprising maltose binding protein (Mbp) connected to GS by a tri-alanine linker (denoted âMagâ). In order to optimize sample homogeneity we produced a panel of Mag deletion constructs by sequentially truncating the 17 residues between the Mbp and GS domains. A combination of biophysical techniques (thermal shift assay, dynamic light scattering, size exclusion chromatography) and negative stain EM allowed us to select the best candidate for cryoEM analysis, MagÎ5. By enforcing D6 symmetry we obtained a cryoEM map with a resolution of 10Ă
(FSC 0.5 criterion). The density of the symmetrized 40 kDa Mbp presents shape and features corresponding to the known atomic structure. In particular, the catalytic pocket and specific α-helical elements are distinguishable. The cryoEM map is additionally validated by a 7Ă
crystal structure of the MagÎ5 oligomer. The presence of a continuous helical connection between target (Mbp) and template (GS) likely contributed to the conformational homogeneity of MagÎ5. Moreover, comparing MagÎ5 with other chimeras studied in this work suggests that a large buried surface area and favorable interactions between the target and template limit the flexibility of the chimera and improve its resolution by cryoEM. For the symmetrization of a target of unknown structure, we envisage proceeding by a trial and error approach by fusing it to a panel of templates with helical termini and different surface properties, and subsequently selecting the best ones using biophysical assays. In conclusion, the present work establishes the proof-of-concept that protein symmetrization can be used for the structure determination of monomeric proteins below 100 kDa by cryoEM, thereby providing a promising new tool for analyzing targets resistant to conventional structural analysis.La dĂ©termination de la structure des protĂ©ines Ă une rĂ©solution atomique est cruciale pour la comprĂ©hension de leur fonction cellulaire. Actuellement, la cristallographie aux rayons X est la mĂ©thode la plus efficace pour la dĂ©termination Ă haute rĂ©solution de la structure de protĂ©ines monomĂ©riques allant 40 et 100 kDa. Par contre, elle est limitĂ©e par la croissance de cristaux de bonne qualitĂ©, qui est problĂ©matique pour nombreuses cibles. La cryo-microscopie Ă©lectronique (cryoME) permet la dĂ©termination structurale Ă rĂ©solution quasi-atomique de larges structures protĂ©iques, de prĂ©fĂ©rence symĂ©trique et en solution. Cependant, les images de cryoME sont trĂšs bruitĂ©es, car une faible dose d'Ă©lectrons est appliquĂ©e de maniĂšre Ă limiter les dommages d'irradiation. En moyennant des dizaines d'images correspondant Ă la mĂȘme orientation molĂ©culaire, le rapport signal sur bruit est amĂ©liorĂ©. La combinaison des images moyennĂ©es de plusieurs orientations permet l'obtention d'une carte de densitĂ© Ă©lectronique 3D de la molĂ©cule d'intĂ©rĂȘt. Si la taille et la symĂ©trie de la molĂ©cule diminuent, l'analyse cryoME devient de moins en moins prĂ©cise, il est alors impossible d'analyser des protĂ©ines monomĂ©riques de taille infĂ©rieure Ă 100 kDa. Le but de ce travail a Ă©tĂ© de dĂ©velopper une nouvelle approche pour rĂ©duire cette limite de poids molĂ©culaire. Elle consiste Ă fusionner la protĂ©ine d'intĂ©rĂȘt (cible) Ă une matrice homo-oligomĂ©rique, gĂ©nĂ©rant une particule symĂ©trique et de taille importante adaptĂ©e Ă l'analyse par cryoME. Dans cette thĂšse, nous avons cherchĂ© Ă tester et dĂ©montrer la faisabilitĂ© de cette approche de symĂ©trisation en utilisant des protĂ©ines cibles de structure connue.Pour mettre en place notre Ă©tude pilote, nous avons choisi diffĂ©rentes combinaisons de cibles et de matrices connectĂ©es par des peptides de liaison (linker) de longueur diffĂ©rentes. Nous avons caractĂ©risĂ© les fusions exprimĂ©es en bactĂ©ries par microscopie Ă©lectronique aprĂšs coloration nĂ©gative et par plusieurs techniques biophysiques. Grace Ă ces techniques, nous avons trouvĂ© que la meilleure combinaison est la fusion entre la protĂ©ine matrice glutamine synthĂ©tase (GS), un 12-mer de symĂ©trie D6 et la cible maltose binding protein (Mbp), connectĂ©es par un linker contenant trois alanines, que nous avons appelĂ©e « Mag ». En jouant sur la longueur du linker nous avons ensuite sĂ©lectionnĂ© la fusion la plus compacte pour l'analyse cryoME: MagÎ5. Nous avons obtenu la carte cryoME Ă 10 Ă
de MagÎ5, qui prĂ©sente une bonne corrĂ©lation avec les modĂšles atomiques de Mbp et GS. Plus particuliĂšrement, le site catalytique et quelques hĂ©lices α sont identifiables. Ces rĂ©sultats sont confirmĂ©s par l'Ă©tude cristallographique que nous avons conduite sur MagÎ5. L'ensemble de ce travail souligne que la prĂ©sence d'une grande interface d'interactions cible-matrice stabilise la fusion et amĂ©liore la rĂ©solution en cryoME. Pour la symĂ©trisation d'une cible inconnue, nous envisageons la mĂȘme procĂ©dure expĂ©rimentale que celle dĂ©veloppĂ©e pour MagÎ5. La matrice et le linker les plus adaptĂ©s devront ĂȘtre identifiĂ©s en utilisant les mĂȘmes mĂ©thodes biophysiques.En conclusion, ce travail Ă©tablit la preuve de concept que la mĂ©thode de symĂ©trisation des protĂ©ines permet la dĂ©termination de la structure de protĂ©ines de poids molĂ©culaire infĂ©rieur Ă 100 kDa par cryoME. Cette mĂ©thode a le potentiel d'ĂȘtre un nouvel outil prometteur, qui faciliterait l'analyse de cibles rĂ©sistantes Ă l'analyse structurale conventionnelle
Digital Participatory Platforms for Urban Regeneration: A Survey of Italian Case Studies
Despite the increase in the globally connected population, there is still a high percentage of European citizens who do not have basic digital skills. In the era of smart cities, the Digital Divide affects the possibility for citizens to participate in public life through the use of ICT tools. To deal with this issue,
the European Union promotes strategies to develop e-government tools, such as digital participatory platforms (DPPs), in order to connect citizens with the public administration. The research proposes a survey of Italian DPPs, investigated through a questionnaire, to bring out which strategies have
been adopted in relation to participation, social inclusion and digital illiteracy, transparency of data, processes, and user-friendliness of the platform. With regard to these issues, certain elements of success of the DPPs presented are highlighted
CAR-modified Cellular Therapies in Chronic Lymphocytic Leukemia: Is the Uphill Road Getting Less Steep?
The clinical development of chimeric antigen receptor (CAR) T-cell therapy has been more challenging for chronic lymphocytic leukemia (CLL) compared to other settings. One of the main reasons is the CLL-associated state of immune dysfunction that specifically involves patient-derived T cells. Here, we provide an overview of the clinical results obtained with CAR T-cell therapy in CLL, describing the identified immunologic reasons for the inferior efficacy. Novel CAR T-cell formulations, such as lisocabtagene maraleucel, administered alone or in combination with the Bruton tyrosine kinase inhibitor ibrutinib, are currently under investigation. These approaches are based on the rationale that improving the quality of the T-cell source and of the CAR T-cell product may deliver a more functional therapeutic weapon. Further strategies to boost the efficacy of CAR T cells should rely not only on the production of CAR T cells with an improved cellular composition but also on additional changes. Such alterations could include (1) the coadministration of immunomodulatory agents capable of counteracting CLL-related immunological alterations, (2) the design of improved CAR constructs (such as third- and fourth-generation CARs), (3) the incorporation into the manufacturing process of immunomodulatory compounds overcoming the T-cell defects, and (4) the use of allogeneic CAR T cells or alternative CAR-modified cellular vectors. These strategies may allow to develop more effective CAR-modified cellular therapies capable of counteracting the more aggressive and still incurable forms of CLL
Piattaforme collaborative per progetti di innovazione sociale. Il caso Miramap a Torino - Collaborative platforms for social innovation projects. The Miramap case in Turin
MiraMap Ăš un progetto di innovazione sociale del Dipartimento DAD del Politecnico di Torino che si avvale di una piattaforma digitale collaborativa per favorire la comunicazione, la partecipazione e l'interazione tra cittadini e amministratori. MiraMap coniuga modalitĂ on line e off-line e consiste in una mappa interattiva georiferita, utilizzata dai cittadini per riportare problemi e proposte riguardo lo spazio pubblico, e in un sistema di back office a disposizione del personale tecnico-amministrativo per gestire le segnalazioni degli utenti. In questo modo si rafforza il ruolo di partecipazione attiva dei cittadini e di governance del territorio della PA, che diventa soggetto in grado di gestire contributi di natura diversa, in unâottica di co-progettazione e co-produzione di servizi
Cryo-EM structure of the full-length Lon protease from Thermus thermophilus.
In bacteria, Lon is a large hexameric ATP-dependent protease that targets misfolded and also folded substrates, some of which are involved in cell division and survival of cellular stress. The N-terminal domain of Lon facilitates substrate recognition, but how the domains confer such activity has remained unclear. Here, we report the full-length structure of Lon protease from Thermusâthermophilus at 3.9âĂ
resolution in a substrate-engaged state. The six N-terminal domains are arranged in three pairs, stabilized by coiled-coil segments and forming an additional channel for substrate sensing and entry into the AAA+ ring. Sequence conservation analysis and proteolysis assays confirm that this architecture is required for the degradation of both folded and unfolded substrates in bacteria
Immunotherapeutic strategies in chronic lymphocytic leukemia: advances and challenges
Immune-based therapeutic strategies have drastically changed the landscape of hematological disorders, as they have introduced the concept of boosting immune responses against tumor cells. Anti-CD20 monoclonal antibodies have been the first form of immunotherapy successfully applied in the treatment of CLL, in the context of chemoimmunotherapy regimens. Since then, several immunotherapeutic approaches have been studied in CLL settings, with the aim of exploiting or eliciting anti-tumor immune responses against leukemia cells. Unfortunately, despite initial promising data, results from pilot clinical studies have not shown optimal results in terms of disease control - especially when immunotherapy was used individually - largely due to CLL-related immune dysfunctions hampering the achievement of effective anti-tumor responses. The growing understanding of the complex interactions between immune cells and the tumor cells has paved the way for the development of new combined approaches that rely on the synergism between novel agents and immunotherapy. In this review, we provide an overview of the most successful and promising immunotherapeutic modalities in CLL, including both antibody-based therapy (i.e. monoclonal antibodies, bispecific antibodies, bi- or tri- specific killer engagers) and adoptive cellular therapy (i.e. CAR T cells and NK cells). We also provide examples of successful new combination strategies and some insights on future perspectives
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