PRESSO L’UNIVERSITA’ DEGLI STUDI DI ROMA LA SAPIENZA
Convenzioni 12.6 All. 73
CONVENZIONE QUADRO PER IL CENTRO LIFE-NANOSCIENCE (CLNS@Sapienza) DELL’ISTITUTO ITALIANO DI TECNOLOGIA
PRESSO L’UNIVERSITA’ DEGLI STUDI DI ROMA LA SAPIENZA
TRA
La Fondazione Istituto Italiano di Tecnologia, con sede legale in Xxxxxx, Xxx Xxxxxx 00, C.F. 97329350587, rappresentata dal Direttore Scientifico, xxxx. Xxxxxxx Xxxxxxxxx (di seguito anche indicata come “la Fondazione” o “IIT”)
L’Università degli Studi di Roma la Sapienza, con sede legale in Xxxx, X.xx Xxxx Xxxx 0, C.F.
n. 80209930587, PI n. 02133771002, rappresentata dal Rettore Xxxx. Xxxxxxx Xxxxxx (di seguito anche indicata come “Sapienza” o “l’Università”)
di seguito congiuntamente denominate “le Parti”
d) Xxxxxxxx si prefigge di valorizzare il rapporto tra formazione e ricerca scientifica nonché la collaborazione interdisciplinare tra i settori scientifico-disciplinari in essa rappresentati, anche allo scopo di favorire la sua migliore interazione con l’esterno e per il raggiungimento dei suoi fini istituzionali;
e) a tale scopo l’Università, come centro di ricerca scientifica nazionale ed internazionale, promuove e attiva forme di collaborazione con altri atenei, centri di ricerca, enti pubblici locali, nazionali e internazionali, con istituzioni scientifiche, culturali ed economiche, pubbliche e private;
f) presso l’Università è operante il Complesso ex Regina Xxxxx sito in Roma, Viale Regina Xxxxx, di proprietà demaniale e concesso in uso perpetuo a titolo gratuito all’Università, presso il quale si realizzano attività di ricerca e formazione ai più elevati standard di qualità;
g) Sapienza e IIT, ravvisando l’opportunità di proseguire l’attività di ricerca congiunta utilizzando sinergicamente le reciproche risorse e valorizzando lo scambio di conoscenze e professionalità, hanno manifestato il comune interesse di collaborare: 1) per la prosecuzione delle attività di ricerca congiunta già proficuamente avviate mediante la sottoscrizione, in data 3 giugno 2011, della Convenzione Quadro per la realizzazione del Centro Life-Nanoscience dell’Istituto Italiano di Tecnologia con l’Università di Roma La Sapienza prorogata sino a tutto il 2 giugno 2017 con delibera del Senato Accademico n. 35/17 e del Consiglio di Amministrazione n. 79/17; 2) per la realizzazione di un programma scientifico in ambito life-nanoscience (Allegato 1), come previsto dalla presente Convenzione e dai relativi Allegati.
Tutto ciò premesso, le Parti convengono e stipulano quanto segue:
Le Premesse e gli Allegati formano parte integrante e sostanziale della presente Convenzione.
2.1 Sapienza ed IIT, nell’ambito dei fini previsti dai rispettivi ordinamenti e statuti, si impegnano reciprocamente a consolidare i rapporti di collaborazione istituzionale e scientifica secondo le modalità di cui alla presente Convenzione.
f) pari a circa 1.596 mq, comprensiva di studi, laboratori e spazi comuni. Per i suddetti spazi è previsto un rimborso dei costi connessi al funzionamento come da successivo art. 4.
Sapienza garantisce che gli spazi sono in regola con tutte le autorizzazioni e normative vigenti e a tal fine dichiara e garantisce a IIT di essere in possesso di:
• Certificato di agibilità dei locali;
• Certificati di conformità degli impianti;
• Certificato di verifica degli impianti di messa a terra;
• Certificato di verifica dell’impianto per le scariche atmosferiche;
• Certificato di autorizzazione agli scarichi idrici;
• Certificato di autorizzazione alle emissioni in atmosfera;
• Certificato di Prevenzione Incendi.
3.2 Con convenzioni attuative da concordarsi con i dipartimenti interessati, si potrà prevedere la temporanea dislocazione di strumentazione scientifica di proprietà di IIT presso i dipartimenti stessi in quanto, e se, finalizzata alla migliore esecuzione dei programmi di ricerca. Analogamente potrà regolamentarsi l’accesso e l’utilizzo della strumentazione al personale di IIT ed ai dottorandi affiliati ad IIT ed impegnati nelle attività oggetto della presente convenzione.
4.1 La Fondazione IIT, fermo restando quanto previsto ai successivi artt. 10, 11 e 12, si impegna a:
a) allocarvi esclusivamente i laboratori e/o studi/uffici di cui al presente accordo;
b) osservare tutte le prescrizioni di leggi in tema di tutela ambientale;
Art. 5
Coordinatore del Centro di Ricerca
- il Coordinatore del Centro di Ricerca, in qualità di Presidente;
- un membro nominato da IIT entro tre mesi dalla firma della presente Convenzione;
- due membri nominati da Sapienza entro tre mesi dalla firma della presente Convenzione.
6.3 Il Comitato delibera a maggioranza degli aventi diritto.
6.5 Il Comitato bilaterale predispone annualmente una relazione che renda conto di tutte le attività svolte congiuntamente. La relazione è trasmessa al Rettore della Sapienza e al Direttore scientifico di IIT.
7.4 Gli overhead prodotti dai progetti di ricerca competitivi, regionali, nazionali o internazionali, saranno impiegati da Sapienza in attività di ricerca di tipo generale.
Ciascun Accordo di cui al precedente articolo 7.3 dovrà indicare:
- gli obiettivi da conseguire, le specifiche attività da espletare e gli impegni da assumere;
Sicurezza - Responsabilità - Assicurazioni
9.3 Fermo restando quanto previsto dai precedenti commi 1 e 2, i datori di lavoro Sapienza e IIT, ai sensi e per gli effetti del D. Lgs n. 81/08, si impegnano comunque a promuovere la cooperazione ed il coordinamento allo scopo di garantire la tutela della salute e la sicurezza dei lavoratori che saranno occupati nelle attività oggetto della presente Convenzione. In questo senso, l’Università e IIT si impegnano a comunicarsi vicendevolmente, con cadenza annuale, per mano dei rispettivi Servizi di Prevenzione e Protezione, l’elenco nominativo dei soggetti individuati ai sensi dell’art. 2 comma 4, del D. M. 5 agosto 1998, n. 363, cui competono gli obblighi previsti dal D. Lgs. n. 81/08.
9.4 Sapienza si impegna a garantire la rispondenza dei locali concessi all’IIT, nonché degli spazi di uso comune (quali connettivo e servizi), alle vigenti normative in materia di sicurezza nei luoghi di lavoro. Un documento generale di valutazione dei rischi, redatto dal Servizio Prevenzione e Protezione dell’Università, verrà consegnato a IIT.
9.5 IIT si impegna per suo conto ad assicurare, per le attività svolte all’interno dei locali medesimi, l’applicazione delle misure generali e specifiche per la protezione della salute dei lavoratori. Il datore di lavoro di IIT si impegna altresì ad individuare e valutare i rischi cui sono esposti i propri lavoratori per effetto dell’attività svolta, nonché a trasmettere formalmente all’Università copia del documento di cui all’art. 17, comma 1, lettera a, del D. Lgs n. 81/08. Ogni qual volta si dovessero verificare modifiche delle attività tali da richiedere un aggiornamento del documento di valutazione dei rischi, IIT provvederà a trasmetterne una copia all’Università.
Pubblicazioni e Proprietà intellettuale
12.1 Lo sviluppo del CLNS@Sapienza e l’esecuzione del relativo programma di ricerca saranno oggetto di valutazione da parte del Comitato Tecnico-Scientifico di IIT integrato da Sapienza con un membro nominato dal Rettore. Nell’ipotesi in cui, sulla base della valutazione, tale Comitato dovesse ritenere gravemente insufficiente lo sviluppo del Centro di Ricerca e/o l’esecuzione del relativo programma scientifico, IIT avrà la facoltà di interrompere ogni attività del medesimo nonché il relativo programma scientifico.
12.2 Per quanto riguarda i progetti comuni disciplinati da accordi attuativi di cui agli articoli 7 e 8 della presente Convenzione, le Parti concordano di valutare la progressione di tali progetti, riservandosi la facoltà di proseguirli, emendarli o interromperli sulla base dei risultati scientifici conseguiti congiuntamente.
ad un livello di diligenza atta a prevenire usi non autorizzati, divulgazioni interne o esterne indebite.
17.1 Le Parti si impegnano ad improntare i loro rapporti ad un principio di leale collaborazione evitando qualsiasi comportamento od azione che possano risultare dannosi ad una delle parti stesse.
18.1. Per ogni eventuale controversia che dovesse sorgere tra l’IIT e l’Università in merito all’applicazione, interpretazione, esecuzione, risoluzione della presente convenzione, le Parti si impegnano ad esperire tentativo per comporre la controversia tra di esse insorta.
18.2 In caso di mancato raggiungimento di un accordo xxxxxxx, per la composizione amichevole della controversia, sarà competente il Foro di Roma.
Art. 19 Registrazione
19.1 La presente Convenzione viene redatta in triplice originale ed è soggetta a registrazione in caso d'uso. Le spese di registrazione saranno a carico della Parte richiedente.
Fondazione Istituto Italiano di Tecnologia Xxx Xxxxxx, 00x
Università degli Studi di Roma la Sapienza P.le Xxxx Xxxx, 5
pec: xxxxxxxxxxxxxxxxxx@xxxx.xxxxxxx0.xx
Roma,
Università degli Studi di Roma Fondazione Istituto Italiano di Tecnologia La Sapienza
IL RETTORE IL DIRETTORE SCIENTIFICO
Xxxx. Xxxxxxx Xxxxxx Xxxx. Xxxxxxx Xxxxxxxxx
Allegato 1: Progetto Scientifico Allegato 2: Accordo di affiliazione
Allegato 3: Accordo per la protezione e la valorizzazione della proprietà intellettuale Allegato 4: Disciplina in materia ambientale
ALLEGATO 1 - PROGETTO SCIENTIFICO
IIT-CLNS@Sapienza Center for Life NanoScience
Research Project 2017-2020
Index
Lab Organization and Research Activities 2
Lab Organization and Research Activities
The laboratory revolves around two very relevant activities of biomedical interest where technological innovation is key to reach the goals.
The first biomedical area of interest cover neurodegenerative disorders, hereditary and sporadic conditions characterized by progressive nervous system dysfunction. The project study the molecular, cellular and tissutal processes underlying nervous system homeostasis and differentiation and their misregulation (project A1).
The other area on which the efforts laboratory focus is brain tumours, the most life-threatening diseases of adulthood and childhood. Primary aims here is the understanding of the interplay between Cancer Stem Cells (CSC) and neoangiogenesis, the dynamics of the CSC population and the set-up of pharmacological screenings of available small drug and natural products libraries as well as of innovative immunotherapeutic strategies. At the same time the project employees and further develop in vivo molecular imaging technologies to improve tumour detection (Project A2).
The detailed A1 and A2 project plans can be found at the end of this document.
The laboratory include different technological platforms (Genomics, Bioinformatics, Bioprinting, Flow Cytometry, Microscopy and cell Culture) that contain state-of-the-art commercial equipment and serve the technological needs of the projects. The platforms also provide support for the development of new techniques and instrumentation.
There are numerous research collaborations with Sapienza researchers and with other institute of the Rome area. Sapienza personnel is allowed to the use of the technological platforms free of charge and on the basis of scientific agreements.
The technological platforms are (or will be) also involved in “service activities”, through specific commercial agreement with private companies or other research institute.
The laboratory also have a tight collaboration with a small company, CREST-Optics, a highly dynamic SME present at international level. On the basis of a formal CLNS-CREST joint lab, the partner are developing new instrumentation in the fields of microscopy and diagnostic. Particularly relevant is the activity focused on the development of a specific “Bessel” microscope for the early diagnoses of the Alzheimer Disease.
A number of the activities described in the two specific projects A1-A2 can very much benefit from the establishment of common technological platforms that can effectively support the biomedical applications and their research teams. In order to exploit the synergy among the various aspects of research and provide relevant added value to their development, the Laboratory has established the following platforms:
• Genomics
• Bioinformatics
• Bioprinting
• Flow Cytometry
• Microscopy
• Cell Culture
The technological platforms occupy about 500 m2 on the ground floor of Edificio B. The area is composed by lab spaces to be equipped with state of the art commercial instrumentation and will provide the technological services. The remaining part of the ground floor of Edificio B will be occupied by the technological R&D laboratories, as described in the A1-A2 projects.
Whenever reasonable and possible, agreements have been sought with instrument providers in order to have “open versions” of the instruments that can be further developed by assembling and/or integrating them with novel instruments that can be i) used for the research activities carried out in the boiling pot and ii) exploited for the construction of new instrumentation of potential commercial interest. These activities will be carried out in agreement with the instrument providers.
Access to the technological platform is granted to the IIT personnel, to the IIT associated personnel working in lab. Access is also granted - on the basis of IIT-Sapienza collaborative short term projects - to Sapienza researchers.
A more detailed explanation of the specific activities for each platform is given below.
The Integrative Genomic and Epigenomic Platform (IGEP) collaborates with scientists at the IIT-Sapienza Life- NanoScience Laboratory to identify and characterize patterns of genetic variation and gene expression. These patterns can yield a deeper understanding of how genetic factors influence disease risk and treatment outcomes in a wide range of human diseases, with a particular focus to neurodegenerative diseases and brain tumours.
IGEP provide a wide range of genomic technologies and platforms, including genotyping, chromatin dynamics, gene expression and directed re-sequencing, and routinely apply these technologies to studies of both human and non-human samples. In addition, the IGEP platform will work to test out emerging genetic technologies, develop informatics tools and infrastructure to enhance scientific research and coordinate projects involving the IIT- Sapienza Life-NanoScience Laboratory.
The platform's major technological activities include:
High-throughput sequencing. IGEP NGS activity will produce the massive quantities of genomic data needed to investigate the whole genome structure, genome wide genetic associations (GWAS) of SNPs, genome wide protein/chromatin associations (ChIPSeq), the epigenome (histone modifications) and the DNA methylome, xxXXX and sncRNAs genome wide profiling and the complete transcriptome (RNASeq). In some cases, the rough drafts of genome wide information are sufficient to achieve the research and clinical objectives but directed activities will be set up when appropriate.
Libraries preparations. IGEP will coordinate with users to design the “best fit” experiments and to provide the appropriate sample processing technologies in order to maximize the effort to gathered information ratio and thus minimize overall the costs (“sustainable genomics”). Genome wide technologies are rapidly evolving and a major focus of the IGEP will be to track/test/adapt/evolve new technologies as well as develop new applications.
Sequencing informatics (in collaboration with the bioinformatics section). These activities will provide the infrastructure and tools necessary to process, store, analyze and track the millions of samples handled by the platform.
Altogether these integrated activities will generate a large collection of genomic data sets amenable to further cross referencing with similar or complementary data sets produced elsewhere. Ultimately these efforts will advance discovery in biology and medicine and will hopefully shorten the lag time to the application of new knowledge to diagnosis and cure.
We are well aware that the development of tools for biological data analysis is a moving target as new technologies appear continuously. Each will require novel approaches and will deliver new data types that must be integrated with available ones.
- Illumina Hi-Seq 1500 (soon a 500) NGS platform or higher
- Covaris S2 (DNA shearing instrument for NGS fragment library preparation)
- Caliper Lab ChIP XT (Automated nuclei acids fractionation apparatus for NGS)
- Agilent Microarray Station
- AB 7900 HT Fast (real-time PCR thermocycler)
- Production Computer cluster with disk space for storing laboratory data
- Development Computer cluster
- Phosphoimager, chemidoc and luminometer
- High-capcity radiographic processor Genomics platform set-up
The advent of next-generation technologies has fuelled an explosion in the quantity of raw DNA sequence that can be generated. The chips (flow-cells) that are currently utilized for the GAIIx have eight channels or lanes, allowing eight sample libraries to be simultaneously analysed. Additional samples can be analysed using the so called multiplexing or indexing to mix different samples in a single lane of the chip; these samples can be subsequently separated in software using their unique sequence barcodes. Typically, all eight lanes of a 100-cycle run generate about 30 Gb of sequence in paired-end mode (sequencing sequentially 100 bases, e.g., from each end of the molecules).
The IGEP platform will both operate as a project-focused service for the IIT-Sapienza Life- NanoScience Laboratory A1 and A2 projects and will independently develop datasets and tools. The facility will assist users all along the research pipeline workflow from the experiments design to their actual performance to end with data analysis (in concert with the bioinformatics section of this platform) and, eventually, to results validation by providing an integrated access to high throughput PCR validation assays.
Running as a facility requires a great amount of support and integration in three critical areas:
a) library preparation, b) maintenance and c) sequencing informatics. All these activities require the presence of qualified dedicated personnel.
The IGEP platform will hire one to two full time technicians devoted to the maintenance and running of the facility as well as one senior post-doc to coordinate projects design and management and one to two post-docs / PhD students to provide coordination/advice and eventually assist users in the pre-sequencing (project oriented) phase as well as in supervising/performing the NGS workflow. The actual data analysis will be performed in concert with the bioinformatics platform. Expert assistance for the NGS results validation phase will be provided according to needs / request.
The facility will be initially centered around one Illumina GAIIx apparatus. The acquisition of a second NGS apparatus is planned to year 2/3 of the project in order to best accommodate the growth curve of users demand and the consequent increase in the facility workload as well as to cope with the continuous development of both the technology and the hardware. The facility will also include support technology for sample preparation and fragments length selection (i.e. Covaris and Caliper LabChip) as well as an Agilent Array platform and a Bead Express for results validation phase. The Illumina high throughput sequencing techniques and microarrays- based techniques will be used to generate mRNAs and miRNAs profiles, to study chromatin dynamics and epigenetic histone modifications (ChIP-Seq) and assess DNA methylation (me- DIP). Standard datasets analysis will be integrated with the refined clustering and classification algorithms for gene expression and chromatin immuno- precipitation (ChIP) analysis generated to identify robust interaction and regulatory patterns. The facility will also provide assistance for the extensive validation of new mid-to large size molecular signatures using the DASL (cDNA-mediated Annealing, Selection, Extension, and Ligation) multiplex expression profile protocol that allows robust quantitative analysis from small quantities of input RNA from both frozen tissues or paraffinated sections.
Research & Development
Besides performing the genomic analysis described in projects A1 and A2, IGEP will develop independent R&D mainly in the field of pre-NGS procedures (target selection, libraies preparation).
The R&D activity will focus on the invention of novel and improved protocols to take better advantage of this new technology. This will be mainly applied to the pre-NGS procedures (target selection, libraries preparation). In spite of the high throughput of NGS it is not feasible to sequence large numbers of complex genomes in their entirety, because the cost and time taken are still too great. In addition to the burden in terms of actual time and funding the primary analysis, where the image files captured during the sequencing reaction are converted into nucleotide sequences, a huge burden would fall on the informatics infrastructure, for storage of the resulting sequence information. One way to cope with these limitations is to perform a 'target enrichment', where unwanted genomic regions are selectively depleted from a DNA sample prior to sequencing, as part of the sample preparation. IGEP will actively focus on the development of application oriented protocols aiming to ideally perform “personalized” pulldown target enrichments. Another area of development will be to invent/design/optimize/test new multiplexing procedures adapted to specific sequencing projects where the maximum high throughput of NGS runs would represent a massive excess. By combining barcoding and pooling of up to a hundred samples to be sequenced as a single sequencing library will make NGS more and more attractive in planning powerful NGS-based clinical investigation studies. To increase the barcoding design and reading capacity represent an additional area of possible R&D.
The projects of our scientific centre are challenging and employ cutting edge technologies producing not only a vast amount of data, but also data of different types, from sequencing to imaging, to functional results. All of them need to be stored, retrieved, analysed and, especially, integrated. This implies that the computational aspects of the projects are challenging as well and require extensive research and development activities that are performed in collaboration with the computational biology scientists in the A1 and A2 projects.
Bioinformatics platform set-up
The bioinformatics facility was set up, with one Post Doc responsible for ensuring the correct operation and maintenance.
The facility is supported by a dedicated High Performance Computing platform, with an architecture specifically suited to biological data analysis: a cluster for parallel computing, a SAN storage area, a supporting backup system.
The activities of the facility have been selected to meet the specific needs of the laboratory:
• storage and calculating capacity are provided for diverse research projects in the laboratory;
• the bioinformatics platform is directly connected to the genomics laboratory
instruments, converting raw sequencing data into standard formats, providing data storage and backup;
• the data produced in the laboratory are made accessible to many scientists to perform “in silico” simulations and hypothesis testing;
• the most used NGS data analysis software is available for analyse RNA-seq, ChIP- seq, Whole exome- seq , Whole genome-seq, miR-seq data;
• specific experimental designs and computational pipelines to analyse diverse/novel sequencing data types are designed and implemented, in tight collaboration between bioinformaticians and experimental researchers;
• customized bioinformatics tools are developed to be easily usable by scientists, including clinical researchers;
• the bioinformatics facility also offers a number of fee-based services to satisfy commercial requests including RNA-seq, miR-seq, ChIP-seq, whole exome and genome sequencing (WES, WGS) in a standard and/or an advanced level of analysis.
Research & Development
The facility currently participates in multiple IIT internal and external projects and collaborations. Since the ever growing amount of data and the continuous innovations in technology, it is essential to keep the expertise up-to- date at all times on new procedures as they are developed.
In particular, we are exploring novel solutions to substantially speed up the analysis of NGS data with the aim of processing them as fast as they are produced, by testing different hardware and software possibilities. This is done in collaboration with CINECA, former CASPUR (Consorzio di Supercalcolo per Universita’ e Ricerca), which is already exploring the possibility of using GPUs and that provides us (through an official agreement) with additional power for data storage and handling.
Our main interest is the lack of tools to extract knowledge from integration of heterogeneous and large omics data, through both data discovery and data exploitation. The plan here is to develop more and more sophisticated data integration strategies, which will be designed in collaboration with the scientists of the laboratory, in order to make them effective and suitable to their needs.
Since all members of the laboratory should acquire the necessary knowledge to interpret their results correctly, we want to provide short hand-on courses, especially for students and young post-docs, to make the most out of their data and to be able to objectively evaluate the effectiveness of newly appearing methods for their specific task.
In the very last months, the bioinformatics facility started to offer a number of fee-based services to satisfy commercial requests. The plan is to extend this opportunity, providing sophisticated and integrated bioinformatics strategies of analysis on request.
The bioprinting facility is meant to establish new and more relevant in vitro 3D cellular model using cell encapsulation technologies and robotic dispensing systems. Lately, the culture of cells in bio-mimetic 3D environments is becoming an essential requirement for high-impact studies, and a useful intermediate step between in vitro and in vivo tests for both drug discovery and disease modelling.
The development of new cellular models starts from the collaboration with researcher inside and outside of the institute. The intersection between the technology and the existing projects of the institute is vast and mostly to be explored.
The bioprinter:
The bioprinting machine present at CLNS is a custom-made fiber-based 3D bioprinter that allows for the encapsulation of living cells inside bio-active extracellular matrixes (the bio-ink).
The machine is totally “open” for technical implementation, and consists of a computer numerically controlled 3D movement assembly and a fiber-extrusion system.
The time of the instrumentation dedicated to R&D (~ 40%) will focus on the determination of deposition protocols suitable for different types of ECM and cell types.
The remaining machine-time will be dedicated to the support of internal (~40%) and external (~20%) projects.
Research and Development:
The R&D aspects regarding the technology comprehend new microfluidic extruders as well as new chemical systems that allows for the biocompatible deposition of solid bio-inks composed of bioactive, gel-forming macromolecules and living cells.
Thus, new and existing collaboration will regard not only the use of the technology for the production and later use of 3D cellular models, but also the development of the fluidic and chemical aspects of deposition. Specifically, external support may regard micro-fabrication technologies and chemical synthesis for the ECM components.
Organically, the products of the work of the bioprinting laboratory will consist in the production of multicellular model that recapitulate the structure and function of complex, organized tissues, and the technology for their production.
Actual collaborations focus on the creation of 3D cellular models for the study of tumors (e.g. leukaemia, medulloblastoma, multiple myeloma), genetic diseases (e.g. spinal muscular atrophy, SMA, amyotrophic lateral sclerosis, ALS) and viral diseases (HBV).
Future collaboration may include regerative medicine applications, organs-on-chip integration of the 3D living constructs and human-derived drug testing platforms.
This platform at CLNS-IIT@Sapienza provides basic support to the projects in the field of flow cytometry, as well as help in the development of novel protocols and methodology. The laboratory technology supporting the A1-A2 project, as well as the new foreseen development, has the main purpose to characterize and isolate a variety of cell population both of human and mouse origin.
Flow Cytometry platform setup
The flow cytometry laboratory is equipped with two laser-based instruments which technology allows simultaneous multiparametric analysis of thousands of cells for second for rapid analysis and/or isolation of complex cell populations. This is achieved by the use of different labeled antibodies or dyes to target specific molecules express at the surface or in the cytoplasm of the cell, giving a screening able to describe in detail a complex sample or to identify low frequency subset of cells.
Through these instruments, a variety of fluorochromes attached to different antibodies can screen the molecular components of a cell first to characterize it (flow cytometer) and then to specifically select for isolation (cell sorter). The following machinery have been purchased:
• The BD LSRFortessa cell analyzer is a 5 laser cytometer (488nm, UV 355nm, V 405nm, 561nm and 640nm) and 18 fluorescence detectors running a BD FACSDiva software on XP. The main feature of the cell analyzer is to characterize a given cell population.
• The BD FACSAriaIII is a 4 laser cell sorter (488nm, Near UV 375nm, 561 nm and 640nm) and 10 fluorescence detectors running a BD FACSDiva software on XP. The main feature of the cell sorter is to isolate a specific population.
The instrument time is dedicated to the following activities:
- support to the A1-A2 project (approx 60% of the total instrument time)
- R&D for implementation and new protocols (approx 20%)
- Open to collaborative research projects from the Sapienza community (approx 20%)
The time-slot allocation is defined by the scientist responsible for the laboratory. Development of new flow cytometry protocols
The platform supports scientists involved in the A1-A2 project aiming to exploit innovative flow cytometry protocols and improve the one already existing.
Specific focus of the newly developed techniques is oriented not only towards refine cell characterization and isolation but also to improve complexity in multiparametric FACS analysis. The final aim is to gain a detailed screen and isolation of complex and/or rare cell populations for further functional studies.
In the field of nanobiomedicine, the recent technology of nanoparticles is used as effective device to apply for the delivery of drugs, proteins, peptides and nucleic acids into the cells. The improvement of flow cytometry protocols to better investigate this cellular uptake is an important part of the development process in the laboratory.
In details:
‐Development of protocols to characterize fluorescently labeled nanoparticles uptake by different tumor cell lines using BD LSR cell analyzer
‐Development and improvement of protocols for isolation of mouse microglial cells from hippocampus using BD FACSAriaIII instrument
‐Development of specific protocols for isolation of motoneurons derived from mouse embrionic stem cells (mESCs) using BD FACSAriaIII instrument
‐Development of specific protocols for isolation of motoneurons derived from human induced Pluripotent Stem Cells (iPSCs) using BD FACSAriaIII instrument
‐Development and improvement of single cell sorting isolation using BD FACSAriaIII instrument
This platform provides basic support to the projects in the field of bio-oriented microscopy, as well as help in the development of novel instruments and methodology. The microscopy techniques requested by the A1-A2 project, as well as the new foreseen development, are
mainly oriented to the study of intracellular distribution and of traffic of specific macromolecules (i.e. proteins and/or xxXXX).
On one hand, the platform hosts commercial state-of-the art instrumentation, which – according to specific agreement established with the provider – has been left “open” for technical implementations and new protocol definitions. On the other side, it provides the natural environment for the development of new microscopy techniques, as detailed below.
Present microscopy platform set-up
The platform was populated with commercial state-of-the-art instrumentation using the “Start Up” budget during the first years of operation of the project. Specifically, the following instruments have been purchased or developed in-house:
• Single and multi-photon laser scanning confocal microscopy with FRET and FRAP capabilities
• Spinningdisk confocal microscopy, with FRET capability
• Confocal microscopy combined to AFM systems for nanoRaman imaging
• Non-linear microscopy for CARS imaging
• Structured light microscopy with high spatial resolution capabilities
• An optogenetics station for neural network analysis
• IR illumination coupled to AFM microscopy for nanoIR imaging
• Multi-channel elettrophysiology microscopy
The instrument time is dedicated to three activities:
i) support to the A1-A2 project (approx. 50% of the total instrument time)
ii) R&D for implementation and new protocols (approx 20%)
iii) open to collaborative research project from the Sapienza community (approx. 30%)
The time-slot allocation is defined by the scientists responsible for the Laboratory on a case- by-case evaluation.
Development of new microscopy techniques
This part of the platform supports scientists involved in the A1-A2 project aiming to exploit innovative technologies and/or physical processes as yet unexplored. The platform will share the space for the laboratories, allowing the CLNS staff to create the proper synergy between existing commercial materials and new instrumentation.
Specific focus of the newly developed techniques is oriented towards single molecule imaging aiming at investigating macromolecular distribution and traffic at the intracellular level. In a nutshell (the details can be found within the A1-A2 project text) the foreseen activities are:
- New scanning techniques for ”standard” confocal and/or multiphotons microscopy.
- Development of fluorescence crosscorrelation spectroscopy for accurate in-vitro colocalization analysis of proteins or microRNA strands.
- Development of integrated AFM spectromicroscopy based on photonic and plasmonic nanostructures for single molecules in both the intra and extra cellular context.
- Development of integrated spectro-photometric systems for multi spectra analysis (also on chip)
- Development of new nano-phosphorous labels to go beyond the blinking and bleaching limits associated with fluorescent and nano-dots dyes.
- Development of techniques (software and hardware) for tracking and image reconstruction.
Here the computational methodologies will be based on ab-initio methods, DFT, micro-nano fluidic modelization, FTDT for electromagnetic radiation and its interaction with matter.
The Cell Culture Facility, is a small multi-user laboratory, current users mainly consist of principal investigators from the Biology unit and their collaborators.
The purpose of this facility is to provide researchers a shared resource necessary to carry out cell/ tissue culture based experiments, and to maintain and store experimental cell lines. The facility is overseen by a research technician who ensures a safe and clean environment.
It is divided in two adjoining but separate laboratories with mammalian and human cell culture and primary cell culture of Biosafety level 1 and 2; hence all work must be performed following BSL 1 and 2 guidelines.
The shared facility consists of shared laboratory equipment, and provides basic laboratory consumables, users are expected to bring their own cell culture and project-specific materials to the lab.
Access to the facility is granted upon proof of training, knowledge of potential risks, and proficiency in standard and BSL2 special microbiological practices before working with BSL-2 agents.
Lab 8 is designated for biological containment level 1 work and has the following equipment for shared use:
Two Biosafety Cabinets, Class II, Type A2 and vacuum aspirators Two CO2 incubators
37°C water bath Fridge/freezers (-20°C) Benchtop centrifuge
CO2 /O2 gas line , CO2 gas line
Basic lab supplies (hand soap, paper towels, Xxx xxxxx
Biosafety cabinets cleaning supplies (absorbent towels, disinfectants) Biohazardous waste handling supplies (autoclave bags and tape) Biohazard spill kits
Inverted microscope to observe cell morphology and count cells (Zeiss Primo Vert )
Provided by the User:
Personal Protective Equipment (PPE): lab coat, safety glasses, gloves Pipettes and pipette tips
Flasks, vials, tubes, etc.
Cell cultures, media, antibiotics, additives, etc. Any other miscellaneous supplies needed
Lab 29 meets all the requirements of biological containment Level 2, dedicated for cell culture and Induced Pluripotent Stem cells work only and has the following equipment for shared use:
4 Biosafety Cabinets, Class II, Type A2 1 IVF workstation
Four CO2 incubators 37°C water bath Fridge/freezers (-20°C) Benchtop centrifuge CO2 gas tanks
Basic lab supplies (hand soap, paper towels, Xxx xxxxx)
Biosafety cabinets cleaning supplies (absorbent towels, disinfectants) Biohazardous waste handling supplies (autoclave bags and tape) Biohazard spill kits
Invereted microscope to observe cell morphology and count cells ( Zeiss AxioVert ) Cell counter
The following cell culture services can be provided:
Free short training on the biosafety procedures and equipment introduction for working in the facility. This training must be taken by everyone who is new to the facility.
Basic cell culture technique training including sterile skills, cell passage, cell counting and plating, thawing and freezing cells.
Maintain cell lines
Expand and create frozen cell stocks Mammalian cell transfection Mycoplasma contamination test
Project A1
Novel Nanotech-Based Approaches for the Study and Treatment of Neurodegenerative diseases
Background and rationale
Neurodegenerative disorders are defined as hereditary and sporadic conditions which are characterized by progressive nervous system dysfunction. These diseases, that account altogether for more than 600 different species, are often associated with atrophy of the affected central or peripheral structures of the nervous system.
In the framework of this project we want to exploit and further develop the study of the molecular, cellular and tissutal processes underlying nervous system homeostasis and differentiation and at understanding their misregulation occurring in pathological conditions, building upon new discoveries in the field of molecular neurobiology and new opportunities arising from improvement in nano- and bio-technology applications.
Specific interest will be devoted to the study of neuromuscular diseases as model systems where to analyze the processes underlying neuronal degeneration as well as muscle function. One of the most severe neurodegenerative disorders is Amyotrophic Latheral Sclerosis (ALS), a progressive and fatal disease in which motor neurons undergo degeneration, causing muscle atrophy, weakness and, ultimately, muscle paralysis. The disease has adult- onset with a typical course of 1 to 5 years. Most forms of ALS are sporadic, but ~10% of patients have inherited familial forms of the disease and a clear family history. Understanding of ALS pathogenesis began with the landmark discovery of dominant causative mutations in the gene encoding copper/zinc superoxide dismutase 1 (SOD1) in ~20% of familial ALS cases and
~1% of sporadic cases. More recently, our view of ALS has been improved by the recent discovery of two new mutations in the DNA/RNA-binding proteins TDP-43 and FUS, which have triggered new interest in ALS pathogenesis (Xxxxxx-Tourenne and Cleveland, 2009). Even in these cases, where a well defined mutation has been linked to the disease, a clear correlation between the genetic defect and the physiopathology of the disease has not yet been disclosed.
The objective of this project will be to identify, in the genetic context of the above mutations, the deregulated molecular processes relevant for ALS initiation and progression and to understand how they affect neuromuscular junctions (NMJ) and motor neurons (MN) survival and activity. The integration of the molecular data with the morpho-functional alterations will eventually enable the identification of the molecular circuitries and cellular functions primarily involved in the disease.
As a next step, we plan to test whether the recovery of the correct expression or function of the identified molecular players in affected animals could confer any beneficial effect to the physiopathology of the disease. Initial tests will be performed in vitro, taking advantage of the cellular model systems described below, where we can test whether any of the identified genes is able to rescue a wild type-like MN phenotype/function. Subsequently, we want to set up conditions for MN-specific gene delivery in mice model systems by utilizing the in vivo transducing features of the Adeno-Associated Virus (AAV). This system has provided very good performances in transducing spinal MNs after a single unilateral intramuscular injection
in muscles of adult mice and monkeys (Xxxxxxx et al. 2006; Xxxxx et al., 2010). Recovery of correct expression of the ALS-associated genes in the affected MN of the mouse models should allow to reach proofs of concept of whether any of them can have therapeutic value. In parallel, we intend to exploit the utilization of ad hoc functionalized nanoparticles for the delivery of selected nucleic acid molecules as an easy tool for in vitro manipulation of cells as well as vehicles for in vivo cell-type specific delivery.
A further aspect of ALS pathogenesis is that, as a consequence of nerve degeneration, muscles become severely compromised. Nevertheless, this is not a one way process, since adult muscle fibers are a source of signals that may influence neuron survival, axonal growth and maintenance of synaptic connections. Therefore, this project will also aim at identifying the molecular/metabolic/physiological alterations taking place in ALS muscles and to define how the progression of a pathological condition in skeletal muscle will eventually affect NMJ and motor neurons survival and activity.
Along this line, we will further deepen our knowledge on the molecular mechanisms controlling proper muscle differentiation as well as tissue homeostasis and integrity.
To achieve all these goals, the project will benefit of integrated and multidisciplinary actions including basic research and technologic development, genetic and bioinformatics approaches, as well as cell biology and functional assessments.
Therefore the aims of this proposal are:
- Produce suitable in vitro and in vivo models for the ALS pathology.
- Characterize the molecular circuitries controlled by ALS-associated proteins and identify crucial targets misregulated in ALS pathology.
- Set-up appropriate bioinformatics tools to study the complex networks of gene expression regulation in normal and pathological neurogenesis.
- Set-up and optimize sensitive and innovative methodologies for the in situ detection of specific macromolecules (RNA/proteins) along the MN axon or at the NMJ in in vitro ALS- reconstituted systems as well as for the study of body wide distribution of viruses or nanoparticles in mice model systems.
- Set-up appropriate electrophysiological tests and bio-mechanical measurements to study motoneurons activity and their impact on muscle activity.
- Optimize strategies of gene delivery into muscles and/or in MN cells
The previously reported experimental plan is composed of 6 Workpackages:
Workpackage 1. Cellular and animal model systems
Produce appropriate cellular model systems, and in particular reprogrammed patient-derived fibroblasts, where to study the activity of FUS and TDP-43 mutations and where to test the ability of identified molecular targets to rescue a functional MN phenotype.
In parallel, transgenic mice for FUS and TDP-43 mutations will allow to carry out morphological, molecular and functional analyses both in muscles and spinal cord and to test whether any of the identified molecular targets of ALS-associated proteins can be exploited for possible ALS therapeutic interventions.
Workpackage 2. Identification of alterations induced by ALS-associated mutant proteins
The combined analysis of the morphological, biochemical and molecular pathways affected by the mutated ALS- associated proteins should allow to identify specific targets that in turn can be tested in reconstituted experiments in order to validate their role in the pathology and to test their possible use as therapeutic molecules.
Reconstruction of the networks implied by the pSILAC data in combination with functional and genomic data in neuronal and muscles cells will allow to elucidate important molecular circuitries responsible for correct tissue homeostasis and their correlation with disease onset and progression.
Further objective will be to obtain a rapid, cheap and reliable system for measuring different types of RNA molecules as biomarkers of the ALS pathology and for testing its severity.
Workpackage 3. Computational and system biology
Reconstruction of the network implied by the data delivered by the project Identification of the key elements of the network and their functional assignment
Novel classification algorithms for gene expression/ protein abundance analysis and design of validation experiments
Simulation of the metabolic network
Prediction of the structure of the identified proteins Construction of the database of Raman spectra of known protein
Implementation of methods for identifying proteins from their Raman spectrum
Workpackage 4. New microscopy technologies
Deliver a table top, ready-to-use, plasmonic antenna combined with Raman analysis for intracellular single molecule detection.
Deliver new generation of sub-diffraction microscopy techniques and protocols, either in labeled (development of new fluorophores, of new fast scan techniques, of Bessel function based structured illumination) or label-free (digital holographic microscopy, mid Infrared chemical sensitive absorption microscopy) samples.
Use the previous techniques to follow the macromolecules (RNA/proteins) traffic along the MN axon or at the NMJ in different in vitro ALS-reconstituted systems to follow muscle regeneration/degeneration.
Workpackage 5. Functional aspects
Derive the most suitable electro-physiological tests and Ca2+ imaging measurements in order to calibrate MN as well as NMJ activities in wild type versus ALS conditions. The set-up of standardized measurements should allow to analyze in reconstituted in vitro systems as well as in mice models the correlation with disease onset and progression and to evaluate the beneficial effect of putative therapeutic treatments.
In line with this, the setting up of experimental procedures and instrumentation to study the biomechanical performances of in vitro reconstituted NMJ (co-culture of the ex-vivo Muscle Engineered Tissue “X-MET” with motoneurons) should provide new strategies for implementing the types of outcome measurements.
Workpackage 6. Strategies of gene delivery into muscle and/or MN cells
Set-up appropriate strategies enabling the delivery of specific molecular constructs to MN or muscle cells. The comparison between viral vectors and nanocarriers will allow to define the most effective strategy for tissue- specific delivery. Moreover, the development of 19F- labelling and MRI analysis will allow to track in vivo the body distribution of the different macromolecular compounds under analysis.
Specific effort will be devoted to the development of nanocarriers for RNA delivery to cells and to their functionalization by selector peptides or modular protein domains in order to provide cell-type specific delivery.
References
- Barbato C, Xxxxxxx F, Xxxxxx C. (2009) X. Biomed. Biotechnol., 2009:871313.
- Xxxx D, et al. (2008) Stem Cells. 26:2564. Chiancone E, Xxxx P. Front Biosci. (2010) 15:122-31
Xxxxxxxxxxxx L, Xxxxxxxx I, Xxxxxxx C, Xxxxxxxx I, Xxxxx MV “2009 Xxxxxxxx 25 11940– 11946
Xxxxxxx E, Xxxxxxxx XX, Xxxxxxxx XX, (2007) J Gene Med. 9:265-74. Xxxxx C, Xxxxxxxxxxx A (2008) Nature Phys. 4:794.
De Angelis et al. (2008) Nano Letter, 8:2321. De Angelis et al. (2009) Nature Nanotech. 5:67.
Dobrowolny G, Xxxxxxxx X, Xxxxxx L, Xxxxxxxxx C, Xxxx X, Xxxxxxx L, Xxxxxxxx M, Xxxxxxxxx N, Xxxxxx A. (2005) X. Cell. Biol. 168:193.
Dobrowolny G, Xxxxxxx M, Xxxxxxxx M, Xxxxxx A. (2008a) Neurol. Res. 30:131
Dobrowolny G, Xxxxxxx M, Xxxxxxx E, Xxxxxxxxx S, Xxxxxxxxx C, Xxxxxxxxxxx S, Xxxxx S, Xxxxxxxx, X Xxxxxxxxx, Xxx Xxxxx Z, Xxxxxxxxx N, Xxxxxxxx M, Xxxxxxx F, Xxxx G, Xxxxxx M, and Xxxxxx A. (2008b) Cell Metabolism 8:425.
Xxxxx RA, Xxxxxxx GJ, Xxxx CH. (2006) Am J Physiol Regul Integr Comp Physiol. 290:R773-84.
Xxxxx R, Xxxxx S, Xxxxxxxx T, Xxxxx K, Xxxxxxxxx A, Xxxxxxxxx T, Xxxxxxxxx T, Xxxxx G G, and Xxxxxx X. (2005) Curr. Biol. 15:587.
Xxxxxxx XX, Xxx KP, Xxxxxxx G, Xxxxxxxxxxx T, Xxxxxxxxx B, Xxxxx N, Xxxxxxxxxxx R. (2004) Nature, 432:235.
Xxxxxxxxx E, Xxxxxxx N (2006) Xxx Xxxxx 38(Supplement): S20–S24. Kalir S, Xxxxxx S, Xxxx U (2005) Mol Syst Biol. 1, 2005;
Karumbayaram S, Xxxxxxx BG, Xxxxxxxxx M, Xxxxxx JA, Xxxxxxx L, Xxxxxxxx A, Xxxxxx AE, Xxxxx AT, Xxxxxxx SA, Wiedau-Xxxxx M, Xxxxxxxx HI, Xxxxx WE. (2009) Stem Cells, 27:806.
Xxxxx X, Xxxxxxxx K (1999) Nature 399, 134.
Xxxxxx-Xxxxxxxx and Xxxxxxxxx (2009) Cell, 136:1001. Xxx Y, Xxx VN. (2007) Methods Enzymol., 427:89.
Xxx KZ et al. (2006) Brithish Journal of Haematology 136, pp. 713-722. Xxxxxxx G, Xxxxx F et al. (2008) Mol. Pharm. 0 000-000
- Xxxxxxx G et al. (2005) X. Med. Chem. 00 0000-0000
Xxxxxx S, Xxxx U (2003) Proc Natl Acad Sci U S A 100: 11980–11985 Xxxxxxxx F et al. (2009)submitted to Applied Physics Letters, arXiv:0907.4586.
Xxxx X, Xxxx-Xxx S, Xxxxxxxxx S, Xxxxxxx N, Xxxxxxxxxx D, et al. (2002) Science 298: 824– 827,
Xxxxxxx M, Xxxxxxxx S, Xxxxxxx M, Xxxx CB, Xxxxxxx A, Xxxxxxxx E. (2010) Mol. Cell. Neurosci., 43:287.
Xxxxxxx C, Xxxxxxx A, Xxxxx F, Xxxxxx F, Xxxxxxx G 2008 Nanotechnology 5 19-25 Xxxxxxxx C et al. (2009) Nature Photonics 3, 178.
Xxxxxxx M, Xxxxxxxxx A, Xxxxxxx G, Xxxx G, Xxxxxxxx MC, Xxxxxxxxx A, Xxxxxxx EI. (2006) X. Clin. Invest., 116: 202-8.
Porcari P. et al. (2008) Physics in Medicine and Biology 53:6979-6989
Prastaro A, Xxxx P, Xxxxx A, Xxxxxxx G, Xxxxxx S. (2010) Tetrahedron Lett. 51 2550–2552. Xxxx-Xxx SS, Xxxx X, Xxxxxx S, Xxxx U (2002) Xxx Xxxxx 31: 64–68
Xxxxx Xxx X, Xxxxxx T, Xxxxx L, Xxxx X, Xxxxxxxxxx M, Xxxxxxx SA. (2005) Exp. Neurol., 196:224.
Sot B et al. (2010) The EMBO Journal, 1–10.
Xxxxxxxxx, X., Xxxxxx, X., Xxxxxx, M., Xxxxxx, X., Xxxxxxxx, X., Xxxxxx, X., and Xxxxxxxx,
S. (2007) Cell 131, 861–872.
Xxxxx C, Xxxxxxxxx BL, Xxxxxx D, Xxxxxxx XX Xx, Xxxxxxxxx P. (2010) Gene Ther., 17:141.
Xxxxxx L, Xxxxx PD, Xxxxxxx T, Xxx YH, Xx H, Xxx F, Xxxxx W, Xxxxxx PK, Xxxxx ZD, Xxxxxxxx A, Xxxxx GQ, Xxxxx AS, Xxxxxxx JJ, Xxxxx C, Xxxxxxxxx TM, Xxxxx DJ. (2010)
Cell Stem Cell. 7:618-30.
Project A2
Novel strategies for the imaging and treatment of brain tumours through targeting cancer stem cell-specific signalling pathways.
Background and rationale
Brain tumors are the most life-threatening diseases of adulthood and childhood. Most of brain tumors arise from aberrant development of stem/progenitor cells belonging to two cell lineages (glial and neuronal) leading to I to IV grade gliomas (up to glioblastoma multiforme, GBM) or medulloblastoma (MB). GBM is mainly occurring in adulthood when it is the most frequent and most aggressive primary brain malignancy with a very poor survival rate (5 year survival less than 3%). Conversely, MB is the most frequent aggressive and poor prognosis brain malignancy of childhood, arising from oncogenic transformation of cerebellar neural progenitors. MB multimodal treatments (surgical resection, chemotherapy, and/or radiotherapy) have improved survival, nevertheless it is still incurable in about a third of cases and survivors commonly have severe treatment-induced long-term side-effects, such as developmental, cognitive and endocrinological defects, due to the young age of patients. MB prognosis is determined by patient classification in two risk classes being average risk the patients aged more than 3 years at diagnosis, non-metastatic and totally or nearly totally resected, while patients not fulfilling these criteria are regarded as high risk. Similarly, the extent of surgical removal is the most significant prognostic factor influencing progression free and overall survival in GBM patients, despite combined chemotherapy and radiotherapy regimens. In addition, GBM and MB are composed of an heterogeneous family of distinct tumours based on different genetic/epigenetic features, whose impact on prognosis and therapy sensitivity is poorly understood.
Improving technologies addressing the major risk factor of GBM and MB by achieving nearly complete surgical tumor resection through more accurate intraoperative imaging delineation of the tumor and better genetic/epigenetic characterization of selected tumor cell populations are thus needed. Furthermore, novel and risk-adapted therapeutic strategies are also needed based on specific targeting of post-surgical residual tumor cells without toxic side affects on normal cells.
Cancer stem cells and the concept of the “niche”. To this regard, an important issue deals with the presence of cancer stem cells (CSC) populating brain tumors, which represent the reservoir of tumor initiating cells sustaining tumor maintenance and progression. These CSCs are also believed to represent the cells of origin of the tumor from untrasformed tissue stem/progenitor cells where a number of genetic and epigenetic oncogenic hits is reponsible for their aberrant proliferation and differentiation leading to malignancy. CSCs are resistant to conventional chemo/radiotherapy and may remain “dormant” untill their reactivation in order to re-initiate tumor. These cells need to be unmasked and specifically eradicated by therapies targeting their stemness properties, in order to prevent tumor recurrence. To this end, signals that regulate both individual CSCs and their assembly as a cell population together with their cell progeny, have to be identified, in order to envisage innovative and targeted therapeutic strategies aimed at CSC eradication and progression towards the growth of bulk tumor cell population. Inded, CSCs reside in a tissue “niche” assuring stemness maintenance, where stem cells i) sustain and are sustained by endothelial cells and tumoral
vasculogenesis, ii) exchange intercellular signals with neighboring cells to maintain the “niche” architecture and iii) eventually generate a progeny of differentiated cells composing the bulk tumor cell population.
Cancer stem cells and the “neoangiogenic niche”. GBM CSCs have been described to closely interact with the vascular niche which plays a critical role for their maintenance (Xxxxxxxxx et al 2007; Xxxxxxxxxx & Xxxx, 2007). The connection between CSCs and the endothelial compartment appears critical in several cancers: indeed, in GBM, neuroblastoma and lymphoma tumor vasculature has been suggested to derive from cancer cells (Xxx et al 2006; Xxxxxxx et al 2007; Xxxxxxx et al 2009). Importantly, normal neural stem cells and more recently GBM CSCs have been shown to be able to differentiate into functional endothelial cells and promote angiogenesis through the release of VEGF and stromal-derived factor 1, in in vitro culture of GSCs and in in vivo orthotopic transplants, indicating the neoplastic origin of a portion of the vascular endothelium (Wurmster et al 2004; Ricci- Vitiani et al 2010). Therefore, the ability of cancer stem-like cells, endowed with stem cell plasticity, to directly contribute to the tumor vasculature by endothelial cell differentiation, represents a new mechanism of angiogenesis that may have considerable therapeutic implications, since they may be targeted by drugs hitting the same genomic alteration as cancer cells, in addition to conventional anti-angiogenic treatments (e.g. VEGF/VEGFR therapy). On the other hand, the targeting of the process of GBM CSCs differentiation into endothelial cells might offer new therapeutic options for cancer treatment. Therefore, the identification of the signals involved in this process represents a very important challenge to be investigated.
Such a challenge deals with i) the features of the cell of origin driving the process and ii) the underlying mechanistic signals. The proponents of this project have undertaken this endeavor in the past few years, obtaining evidence for a general view of the nature of “mesenchymal stem cells” based on an identical set of expressed antigens (first and foremost CD146), with ability to self-renew and the capacity to form clonal colonies in vitro, and an in situ identity as subendothelial microvascular cells (Cossu & Xxxxxx, 2003; Xxxxxx et al. 20038; Xxxxxxxxx et al. 2007). Of note, all of these populations share the newly recognized property of directing the formation of functional blood vessels in which they integrate as subendothelial mural cells.
Stem cells and neoangiogenetic niche signals. Among the cellular signals, Xxxxx and Hedgehog pathways play a critical role in stem cells and neoangiogenic response. Both of them sustain stem cells and brain CSCs and neoangiogenesis (see below). Most importantly, crucial roles of Notch in the neurovascular unit has been described, where the four Notch receptor paralogues and their ligands are expressed in endothelial cells (EC), in vascular smooth muscle cells and pericytes, with a role for Notch signaling in maintaining the integrity and homeostasis of vessels and in the remodeling of the primordial vascular plexus (Xxxxxxx & Iruela-Xxxxxx, 2007; Xxxxxxx et al, 2005; Xxxxx & Xxxxxxxxx, 2009). Most interestingly, the accumulated knowledge regarding the biology of the Notch3 receptor emphasizes its central role in the vasculature (e.g. CADASIL disease, where a key feature is the degeneration of vascular smooth muscle cells thus resembling common forms of small vessel disease). To this regard, The proponents of this project have unraveled several mechanisms of Notch signaling, specifically including Notch3 and provided novel insights through the development of Notch transgenic mouse models (reviewed in Xxxxxx et al, 2008; Xxxxxxxx et al. 2008) as well as the study of the role of Notch3 in the interactions between tumor and endothelial cells (Xxxxxxxxxx et al, 2009).
Targeting pathogenic signaling pathways. A number of signaling pathways has been reported to be responsible for subversion of developmental processes of stem/progenitor cells
leading to malignancy and for sustaining stemness properties of CSCs. Among them, the Hedgehog (Hh) and Notch pathways are the most crucial players during development and their deregulation is a leading cause of a wide variety of tumors, including MB and GBM, where Hh and Notch promote several tumorigenic steps including cell proliferation, maintenance of CSCs, survival, tissue spreading and metastatic potential, angiogenesis.
The PI and other proponents of this project have identified cancer related genes underlying a number of novel Hh regulatory mechanisns, involving ubiquitin-, acetylation- and xxXXX- dependent control as well as the tumorigenic role of Notch through the development of transgenic mouse tumor models (Xx Xxxxxxxxxxx et al, 2004; Xx Xxxxxxxxxxx et al, 2006; Xxxxxxxx et al, 2008; Xxxxxxxxxx et al, 2010; Xx et al, 2010; Xxxxxx et al. 2008). The PI is also using and generating several genetically modified mice which provide useful brain tumor model systems, suitable to study the behavior of CSCs. Xxxxxxxxxx, the unique ability of the proponents of this project have and their prior success in numerical simulation and modeling of complex biological events (Xxxxxxx et al 2008, Xxxxxxxxx et al 2008; Xxxxxxx et al. 2010), will allow to approach numerical simulation of the complex events underlying the nature and range of action of signals involved in dictating rules of the behavior of CSCs as a whole population. The chemists participating to this proposal have also developed a number of molecules that target Hh pathway and may by exploited as drugs specifically affecting CSCs. They include a series of histone deacetylase inhibitors as well as novel lead compounds obtained from screening of chemical libraries from natural extracts derived from medicinal plants.
Indeed, in the last few years Hh and Notch pathways has been identified as druggable therapeutic targets in cancer, providing the proof-of-principle of the efficacy of their antagonism for in vitro or in vivo suppression of the growth of MB and GBM and of brain CSCs, in preclinical models and in clinical trials. The rationale for the development of drugs targeting the multiple levels of regulation of Hh and Notch pathways is based on i) the heterogeneity of molecular defects sustaining the pathway activation in cancer (see above) as well as on ii) the need to overcome the reported resistance occurring during single-agent therapy. Although the heterogeneity of the above described regulatory mechanisms provides a rationale for the search of additional molecules specifically targeting Hh and Notch signaling at various misregulated levels, this issue is still largely not understood and unexplored. Furthermore, the role of targeting Hh and Notch pathways in the control of CSCs is also not understood. Therefore, screening of small molecule libraries are required to search for novel Hh and Notch inhibitors acting upon bulk tumor cell population or specifically CSCs.
Targeting and drug delivery into tumor cells and CSCs. Although reducing the side toxic effects on normal cells, therapeutic strategies targeting specific molecular events misregulated in cancer does not rule out unwanted consequences. For instance, inhibition of Hh patwahy, while efficiently killing brain cancer cells, is also impairing skeletal formation in developing young animals, because of the general role of Hh signaling in tissue development and in normal stem cell population. Therefore, selective targeting of cancer cells is needed, through development of innovative and efficient delivery methods. An efficient drug delivery system to be employed for in vivo brain targeting must show: i) ability to circulate in the bloodstream for a prolonged period of time; ii) ability to overcome the blood-brain-barrier (BBB) iii) ability to overcome intracellular membrane barriers such as the endosomal membrane and the nuclear membrane via step-wise membrane. To this regard, functionalized nanoparticles could provide precision detection, targeted treatment, and real- time tracking that conventional technology lacks. There are several approaches using different non-invasive delivery systems, that represent promising although challenging
strategies, such as liposomes and niosomes, polymeric nanoparticles and N- linked peptido-resorcarenes, which have been all developed by chemists and physicists participating to this proposal.
Immunotherapeutic strategies against brain tumors. Glioma development and progression is influenced by intrinsic properties of the glioma cells, as well as by microenvironmental factors and a variety of leukocytes subsets. A number of experimental evidences support a role of the innate and adaptive immune system in the control of glial and neuronal brain tumor development and progression (Xxxxxx et al 2004; Xxxx et al 2007).
Adoptive transfer of cytotoxic lymphocytes such as CD8+ T cells and NK cells alone or in combination with conventional therapeutic agents is under investigation in patients with CNS tumors, but well-defined protocols for the in vitro selection and expansion of cytotoxic lymphocyte populations with efficient anti-tumor activity and preferential ability to migrate to the CNS, the main site of tumor growth, are still largely undefined. The proponents of this project have a long-standing experience in studying the molecular mechanisms controlling the functions and trafficking of cytotoxic effector cells under patho-physiological conditions (Xxxxxxxxxxx et al., 2004; Xxxxxx 2007). In addition, the proponents of this project have recently identified the chemokine CX3CL1, one of the principal chemoattractant for the NK cells, as a potent inhibitor of glioma cell invasiveness (Sciumè et al 2010). This is of particular interest since recent evidence indicates that activated CXCR4 is expressed by both tumor cells and vascular endothelial cells in all grades of astrocytoma (Xxxxxxx et al., 2005), that Hh may regulate CXCR4 gene expression (Xxxxxxxx et al 2005) and that CXCR4- dependent glioma growth was inhibited in vivo by systemic administration of CXCR4 antagonist AMD3100 (Xxxxx et al., 2003).
Imaging brain tumors. Nuclear Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) are well established techniques for brain tumors anatomical and functional imaging. However, there is space for technological improvements, that can crucially increase the performances of these techniques. Ultra-low field MRI (i.e. MRI using precession fields of a few µT) is emerging as a promising development, fully compatible with intraoperative applications and other diagnostic tools. It can acquire images with enhanced contrast, that offer better tumor delineation. PET imaging is complementary to MRI, offering easy functional contrast. Current PET spatial resolution is however poor, due to both the energy and temporal resolution of the sensors. Including information about the time elapsed between the emission and the detection of the photon allows better spatial resolution and reduced background noise.
On the other hand, both techniques can exploit functionalized contrast agents; in this regard, magnetonanoparticles (MNP) can play an important role, because are easily matched to subcellular sizes and properties, an can be easily functionalized. Functionalization with biomolecules allows interaction with biological structures, potentially specific to a target or a function. MNP are inherently good MRI contrats agents, because of their usually superparamagnetic properties. Depending on the particle size, from hundreds to a single cell can be imaged by MRI. Electronic/magnetic properties of nanostructured systems depend on structure and aggregation, thus low level structural characterization of these materials is needed in order to gain full understanding about their action.
In order to fully exploit the molecular imaging power, the imaging technique and a smart contrast agent are obviously essential. However, acting on the biological system can enhance specific features of the signal. The action of strong pulsed electric fields can be used to trigger
and control the delivery of substances from artificial systems (micelles and/or liposomes), or to induce specific responses directly on the living cells. This procedure can be fundamental in enhancing the ability of imaging of biological systems acting in a controlled manner.
Therefore the aims of this proposal are:
1. Providing either in vitro cell culture and in vivo mouse models of brain tumors (MB, GBM) as well as target molecules featuring bulk tumor and CSCs, suitable for being exploited in imaging technologies and/or therapeutic targeting.
2. Understanding the interplay between CSCs and neoangiogenesis in the “tumor stem cell niche” and bulk tumor cell population and involved signaling molecules, suitable for being exploited in imaging technologies and/or therapeutic targeting (supported by the “Integrative Genomic & Bioinformatic” Platform and “Lab-on-chip” Laboratory).
3. Understanding the dynamics of CSC population and cells of their progeny as well as unraveling the underlying intercellular signals through design and validation of mathematical models (supported by the “Integrative Genomic & Bioinformatic” and “Imaging & Microscopy” Platforms).
4. Pharmacological screening of available small drug and natural products libraries to identify drugs able to suppress the growth of MB and/or GBM stem cells, mainly focusing to the Hh and Notch pathways and neoangiogenesis (supported by the “Nanotechnologies for drug delivery” Laboratory).
5. Developing molecular imaging technologies (MRI and PET) in vivo to improve tumor detection during intraoperative brain tumor delineation (to achieve nearly complete tumor resection) and in diagnostic and follow-up procedures, with specific emphasis to the imaging of CSCs based on multifunctional nanoparticle-dependent targeting of specific markers (supported by the “Imaging & Microscopy” Platform).
6. Developing multifunctional therapeutic nanoparticle-based delivery systems targeting CSC-specific signaling pathways and neoangiogenesis to provide proof-of-principle evidence of tumor treatment in vivo (supported by the “Nanotechnologies for drug delivery” Laboratory).
7. Development of innovative immunotherapeutic strategies against glial and neuronal brain tumors based on the infusion of cytotoxic effector cells and haematopoietic progenitor cells (supported by the “Imaging & Microscopy” Platform).
The previously reported experimental plan is composed of 4 Workpackages:
1. Cancer stem cells and in vivo mouse models of brain tumors.
2. Molecular Imaging of brain tumors.
3. Design and production of multifunctional nanoparticles for targeted drug delivery. (These studies will be carried out with the support and collaboration of “Nanotechnologies for drug delivery” Laboratory).
4. Development of innovative immunotherapeutic strategies against glial and neuronal brain tumors based on the infusion of cytotoxic effector cells and haematopoietic progenitor cells.
References
Accapezzato D, Xxxxxxxxxxx V, Xxxxxx M, Xxxxxxxx M, Xxxxxx LV, Xxxxxxxx A, Xxxxxxxxx S, Xxxxxxxx MU, Xxxxxxx V. Hepatic expansion of a virus-specific regulatory CD8(+) T cell population in chronic hepatitis C virus infection. J Clin Invest. 2004; 113:963-72.
Xxx, X. et al. Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor.
Cancer Res 66, 7843-8 (2006).
Xxxxxxxx D, Xxxxxxxxx S, Campese AF, Felli MP, Xxxxxx A, Screpanti I. Notch3: from subtle structural differences to functional diversity. Oncogene. 2008 Sep 1;27(38):5092-8.
Xxxxxxxxxx G, Xxxxxx G, Xxxxxxx D, Xxxxxxx S, Xxxxxxx S, Xxxxxxx A. CCL3 and CXCL12 regulate trafficking of mouse bone marrow NK cell subsets. Blood 2008; 111:3626-34.
Xxxxxx P, Xxxxx PG, Xxxxxxx PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell. 2008; 2:313-9.
Xxxxxxxxx SW, Xxxxxxx D, Xxxxxx E, Xxxxxx T, Xxxx M, Xxxxxxxxxx J, xxx Xxxxxxxxx O, xxx Xxxxxxxx M. Bmi1 controls tumor development in an Ink4a/Arf-independent manner in a mouse model for glioma. Cancer Cell. 2007; 12:328-41.
Xxxxxxxxx, X. et al. A perivascular niche for brain tumor stem cells. Cancer Cell 11, 69-82 (2007).
Canettieri Di Marcotullio L, Xxxxx A, Xxxx S, Xxxxxxxxx L, Xxxxxxx P, Xxxxxxxxxxx L, Xx Xxxxxx E, Xxxxxxxx E, Xxxxx E, Xxxxxxx M, Xx Xxxxxx X, Xxxxxx EM, Xxxxxxxxx P, Xxxxxx A, Xxxxxxxxxxx C, Xxxxxxxxxx L, Xxxxxx C, Xxxxxxx ME, Xxxxxxxxx I, Xxxxxx A. Histone deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation. Nat Cell Biol. 2010, 12:132-42
Xxxxxxx TR et al., Proc Natl Acad Sci U S A 102, 9884 (2005).
Cossu G, Bianco P. Mesoangioblasts—vascular progenitors for extravascular mesodermal tissues. Curr Opin Xxxxx Dev. 2003; 13:537-42.
xx Xxxxx NA, Xxxxxxxxx SW, Xxxxxxx D, xx Xxxxx HI, Xxxxxxxxxx J, Xxxxxx T, Xxxxxx BC, Xxxxxxxx WP, Xxxxxxx JH, xxx Xxxxxxxx M, Xxxxx AJ, xxx Xxxxxxxxx O. Rapid and robust transgenic high-grade glioma mouse models for therapy intervention studies. Clin Cancer Res. 2010; 16:3431-41.
Di Xxxxxxxxxxx Xxxxxxxx E, Xx Xxxxxx E, Xxxxxxx B, Xxxxxxxx C, Xxxxxxxxx F, Xxxxx R, Xxxxxxxx L, Xxxxxxxxxx M, Xxxxxxx M, Xxxxxxx A, Xxxxxxxxxxx F, Xxxxxxxxx I, Xxxxxx E, Xxxxxx A. REN(KCTD11) is a suppressor of Hedgehog signaling and is deleted in human medulloblastoma. Proc Natl Acad Sci U S A. 2004, 101:10833-8
Di Xxxxxxxxxxx Xxxxxxxx E, Xxxxx A, Xx Xxxxxx E, Xx A, Xxxx MA, Xxxxxxxx M, Xxxxxxxx G, Xxxxxxx M, Xxxxxxxxx I, Xxxxxx A. Numb is a suppressor of Hedgehog signalling and targets Gli1 for Itch-dependent ubiquitination. Nat Cell Biol. 2006, 8:1415-23
Xxxx GP, Xxxx IF, Xxxxx WT. Focus on TILs: Prognostic significance of tumor infiltrating lymphocytes in human glioma. Cancer Immun 2007; 7:12.
Ferretti De Smaele E, Xxxxx E, Xxxxxx P, Xx A, Xxxxxxx M, Xxxxxxxxx A, Di Marcotullio L, Xxxxxxxxxx E, Xxxxxxxxx I, Xxxxxxx I, Xxxxxx A. Concerted microRNA control of Hedgehog signalling in cerebellar neuronal progenitor and tumour cells. EMBO J. 2008, 27:2616-27
Xxxxxxx, C. et al. Glioma tumor stem-like cells promote tumor angiogenesis and vasculogenesis via vascular endothelial growth factor and stromal-derived factor 1. Cancer Res 69, 7243-51 (2009).
Xxxxxx MA, Xxxxxxx A, Xxxxxx M. The innate immune response in the central nervous system and its role in glioma immune surveillance. Onkologie 2004; 27:487-91.
Xxxxxxxxxx, X. X. & Xxxx, J. N. Making a tumour’s bed: glioblastoma stem cells and the vascular niche. Nat Rev Cancer 7, 733-6 (2007).
Xxxxxxx XX, M. L. Xxxxxx-Xxxxxx, Circ Res 100, 1556 (2007).
Indraccolo S, Xxxxxxx S, Xxxxxxx M, Xxxxxxxx I, Xxxxxxx L, Xxxxxxx L, Xxxxxxx A, Xxxxxx E, Xxxxxxxxx M, Xx Xxxxx X, Xxxxxxxxx I, Xxxxxxx M, Xxxxxxxx C, Xxxxxxx A. Cross-talk between tumor and endothelial cells involving the Notch3-Dll4 interaction marks escape from tumor dormancy. Cancer Res. 2009; 69:1314-23.
Xxxxxxxx F, Xxxxxxx A, Xxxxxxxxx R, Xxxxxxxxx J, Xxxxxxxx A, Xxxxxxx M, Xxxxx L, Xxxxxxx A. RAC1/P38 MAPK signaling pathway controls beta1 integrin-induced interleukin-8 production in human natural killer cells. Immunity. 2000; 12:7-16.
Xxxxxxx, X. et al. Tumor origin of endothelial cells in human neuroblastoma. J Clin Oncol 25, 376-83 (2007).
Po Ferretti E, Xxxxx E, De Smaele E, Xxxxxxxxx A, Xxxxxxxxxx G, Xxxx S, Di Marcotullio L, Xxxxxxx M, Xxxxxxx L, Di Xxxxx C, Xxxxxxxxx I, Xxxxxx A. Hedgehog controls neural stem cells through p53-independent regulation of Nanog. EMBO J. 2010, 29:2646-58
Xxxxxx PM, Xxxxxxx C, Xxxxxxx M, Xxxxxxx L, Xxxxxxxxxxxx D, Xxxxxx X, Xxxxxxxx L, Xxxxxxx A, Xxxxxx M, Xxxxxx F, Xxxxxxxxxxx CM, d’Ettorre G, Xxxxxx X, Xxxxx A, Xxxxxxx X. Cross- presentation of caspase-cleaved apoptotic self antigens in HIV infection. Nat Med. 2007;13:1431-9.
Ricci-Xxxxxxx L, Xxxxxxx R, Xxxxxxx M, Xxxxxx M, Xxxxxxxxx G, Xxxxx T, Xxxxx G, Xxxxxx EA, Xxxxxx G, Xxxxxxx LM, Xx Xxxxx X. Tumor vascularization via endothelial differentiation of glioblastoma stem-like cells Nature 2010 in press.
Xxxxx JB, Xxxx AL, Xxxxx RS, Xxxx JA, Xxx Y, Xxxxxxx K, Xxxxxx MW, Xxxxxx AD, Xxxxx RA. A small- molecule antagonist of CXCR4 inhibits intracranial growth of primary brain tumors. Proc Natl Acad Sci U S A 2003; 100:13513-18.
Xxxxxxxxx B, Xxxxxx A, Xxxxxxxxx S, Xx Xxxxxx S, Xxxxxxxxx S, Xxxxxx I, Xxxxxxxxxx E, Xxxxxxx S, Xxxxx PG, Xxxxxxxxx M, Xxxxxx P. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell. 2007; 131:324-36.
Xxxxxxxx U, Xxxx A, Xxxxxxxx W, Xxxxx ML, Xxxxxxx CG, Xxxx A, Xxxxxxxx N, Xxxxxxxx OD, Xxxxxxx T. Subtype-specific expression and genetic alterations of the chemokinereceptor gene CXCR4 in medulloblastomas. Int J Cancer 2005;117:82-9.
Sciumè G, Xxxxxxx A, Xxxxxxx M, Xxxxx L, Xxxxxxx A, Xxxxxxxxxx X. CX3CR1/CX3CL1 axis negatively controls glioma cell invasion and is modulated by transforming growth factor- beta1. Neuro-Oncology 2010, 12:701- 10.
Stabile H, Xxxxxxx C, Xxxxx C, Xxxxxxx S, Xxxxxxx S, Xxxxxxxxxxx LD, Santoni A, Xxxxxxxx
A. Impaired NK-cell migration in WAS/XLT patients: role of Cdc42/WASp pathway in the control of chemokine-induced beta2 integrin high-affinity state. Blood. 2010; 115:2818-26.
Xxxxxxxx, X. et al. Lymphoma-specific genetic aberrations in microvascular endothelial cells in B-cell lymphomas.
N Engl J Med 351, 250-9 (2004).
Xxxxx MR, B. M. Xxxxxxxxx, Circ Res 104, 576 (2009).
Xxxxxxxxx G, Xxxx O, Xxxxx R, Eyüpoglu IY, Xxxxxxxx AM, Xxxxxxxxxx J, Xxxxxx J, Xxxxxxx I, Xxxxxx M, Xxxx W. Lessons from the bone marrow: how malignant glioma cells attract adult haematopoietic progenitor cells. Brain. 2005; 128:2200-11.
Talora C, Campese AF, Xxxxxxxx D, Xxxxx MP, Xxxxx A, Xxxxxx A, Screpanti I. Notch signaling and diseases: an evolutionary journey from a simple beginning to complex outcomes. Biochim Biophys Acta. 2008; 1782:489- 97.
Xxxxxxx BM, Xxxxxxxxxx NM, Xxxx AL, Xxxxx A, Xxxxx XX. Widespread CXCR4 activation in astrocytomas revealed by phospho-CXCR4-specific antibodies. Cancer Res 2005; 65:11392-99.
Xxxxxxx, A. E. et al. Cell fusion-independent differentiation of neural stem cells to the endothelial lineage.
Nature 430, 350-6 (2004).
ALLEGATO 2 ACCORDO DI AFFILIAZIONE
L’Università degli Studi di Roma la Sapienza, con sede legale in Xxxx, X.xx Xxxx Xxxx 0, C.F. 80209930587, in persona del Rettore Xxxx. Xxxxxxx Xxxxxx, domiciliato per la carica presso la sede legale, debitamente autorizzato alla firma del presente atto, nel prosieguo (l’ “Università”)
E
La Fondazione Istituto Italiano di Tecnologia, con sede legale in Xxxxxx, Xxx Xxxxxx x. 00, codice fiscale 97329350587, in persona del Direttore Scientifico, xxxx. Xxxxxxx Xxxxxxxxx, domiciliato per la carica presso la sede legale (di seguito anche indicata come “la Fondazione” o “IIT”)
di seguito congiuntamente denominate “le Parti”
PREMESSO CHE
convengono e stipulano quanto segue:
Le Premesse formano parte integrante e sostanziale del presente Accordo.
Con il presente atto, nell’ambito dei fini previsti dai rispettivi ordinamenti e statuti, le parti intendono disciplinare le modalità di affiliazione del personale dipendente, collaboratore e in formazione, di cui all’art. 4.1 lettere c) e d) della Convenzione Quadro, dell’Università alle attività del Centro di Ricerca IIT.
Art. 3 Modalità di affiliazione
3.1 Il personale dell’Università interessato a partecipare all’esecuzione del programma di ricerca del Centro di Ricerca IIT sarà individuato dal Comitato Bilaterale di cui all’Art. 6 della Convenzione Quadro.
3.2 L’individuazione di tali soggetti deve essere compiuta da parte del Comitato Bilaterale secondo criteri trasparenti, riferiti esclusivamente alle doti intellettuali e pratiche manifestate, al
3.3 Il Comitato Bilaterale inviterà il soggetto ad affiliarsi al Centro. L’affiliazione sarà predisposta per programmi scientifici specifici e per un periodo determinato comunque non superiore alla durata del programma del Centro stesso.
Status, diritti e doveri dell’affiliato
4.1 La qualità di affiliato non implica un cambiamento di status o l’insorgere di alcun vincolo contrattuale con IIT. Ai fini dell’affiliazione, è onere dei soggetti individuati richiedere alle proprie strutture di appartenenza la relativa autorizzazione.
Pubblicazioni e Proprietà intellettuale
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7.2 Tale obbligo riguarda anche le pubblicazioni dei soggetti affiliati relativamente all’attività scientifica svolta su programmi del Centro stesso.
7.3 Resta inteso che in base alla normativa vigente, nell’ambito dei processi di valutazione dei risultati della ricerca, i prodotti dei ricercatori:
- Sapienza saranno considerati attribuiti all’Ateneo;
- IIT saranno considerati attribuiti alla Fondazione.
7.4 I prodotti dei ricercatori Sapienza dovranno comunque essere inseriti nel Catalogo della Ricerca di Ateneo IRIS.
7.5 Le modalità di gestione della proprietà intellettuale sviluppata durante la collaborazione sono regolate dall’Allegato 3 alla presente Convenzione
Resta inteso tra le Parti che, per quanto qui non espressamente previsto e/o richiamato, restano ferme le previsioni contenute nella Convenzione Quadro, nessuna esclusa e/o eccettuata, e che, laddove non diversamente precisato, i termini utilizzati nel presente Allegato hanno lo stesso significato attribuito loro nella Convenzione Quadro.
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ALLEGATO 3
ACCORDO PER LA PROTEZIONE E LA VALORIZZAZIONE DELLA PROPRIETÀ INTELLETTUALE
L’Università degli Studi di Roma “La Sapienza”, con sede legale in X.xx Xxxx Xxxx, 0 xxx 00000 Xxxx (XX) C.F. 80209930587 in persona del Rettore e legale rappresentante Xxxx. Xxxxxxx Xxxxxx, domiciliato per la carica presso la sede legale, debitamente autorizzato alla firma del presente atto, nel prosieguo (“Sapienza”)
E
La Fondazione Istituto Italiano di Tecnologia, con sede legale in Xxxxxx, Xxx Xxxxxx x. 00, codice fiscale 97329350587, in persona del Direttore Scientifico, xxxx. Xxxxxxx Xxxxxxxxx, domiciliato per la carica presso la sede legale (di seguito anche indicata come “la Fondazione” o “IIT”)
PREMESSO CHE
convengono e stipulano quanto segue:
Definizioni
a) Per “Affiliati” si intende il personale della Sapienza che, debitamente autorizzato, partecipa all'esecuzione del Programma di Ricerca IIT di cui all’Allegato 1) della Convenzione.
b) Per “Personale della Sapienza” si intende i lavoratori subordinati di ogni genere, a tempo indeterminato o determinato, nonché studenti, studenti di PhD, borsisti, assegnisti, contrattisti e collaboratori di ogni genere, appartenenti alla Sapienza.
c) Per “Personale IIT” si intende il personale dipendente nonché il personale a contratto di IIT, come definiti nel “Regolamento IIT sulla Proprietà Industriale” approvato in data 23 novembre 2010.
d) Per “Invenzione” si intende ogni risultato utile della ricerca scientifica che abbia un valore patrimoniale e/o sia suscettibile di un diritto di esclusiva, come le invenzioni industriali, il software, i procedimenti o i prodotti microbiologici, i disegni e modelli industriali, il know-how, i marchi.
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e) Per “Protezione dell’Invenzione” si intende la tutela della Proprietà Intellettuale, realizzabile in diversi modi quali, tra gli altri, i brevetti per invenzione, le registrazioni di disegni e modelli, i marchi. Per brevetti si intendono, inoltre, quelli previsti da convenzioni internazionali, dal diritto comunitario, dalla legislazione nazionale o di ogni altro stato.
Articolo 1 - Oggetto -
1.1 Il presente Accordo ha ad oggetto la disciplina della Proprietà Intellettuale delle Invenzioni che possano derivare dalle seguenti attività:
A. esecuzione di progetti svolti congiuntamente da IIT e Sapienza, cofinanziati dalle Parti sia in misura paritetica che in diversa proporzione;
B. esecuzione di progetti svolti congiuntamente da IIT e Sapienza, finanziati da soggetti terzi;
C. esecuzione del programma di ricerca IIT presso IIT@Sapienza, di cui indicato all'Allegato
1) della Convenzione, con la partecipazione di soggetti Affiliati appartenenti alla Sapienza.
Articolo 2 - Titolarità dei diritti sulle Invenzioni -
2.1. Fermo restando il diritto delle Parti di utilizzare in modo gratuito per le proprie attività di ricerca scientifica la Proprietà Industriale e le Opere protette dal Diritto d’Autore frutto della ricerca, le Parti convengono che la quota di titolarità dei diritti sulle Invenzioni è stabilita come segue:
i. per le attività di cui all’art. 1.1 (A), la quota di titolarità sarà ripartita tra le Parti in ragione del numero degli inventori di ciascuna Parte, al loro contributo inventivo e all'ammontare del cofinanziamento apportato da ciascuna Parte;
ii. per le attività di cui all’art. 1.1 (B), la proprietà delle Invenzioni realizzate in comune saranno disciplinate dagli specifici accordi con le terze parti finanziatrici;
iii. per le attività di cui all’art. 1.1 (C), ossia inerenti il programma di ricerca IIT presso il Centro di Ricerca IIT e svolte con la collaborazione di soggetti Affiliati, la quota di titolarità dei diritti sulle Invenzioni sarà ripartita nella misura del 65 % (sessantacinque per cento) a favore di IIT e del 35 % (trentacinque per cento) a favore della Sapienza.
2.2 In tutti i casi, agli inventori spettano i diritti morali sulle proprie Invenzioni, i quali non sono alienabili.
Articolo 3 - Modalità operative -
3.1 Le Parti, di comune accordo, definiranno per iscritto la Parte che sarà responsabile della gestione operativa delle fasi di Protezione e sfruttamento di ciascuna Invenzione (nel seguito “Parte Operativa”).
3.2 La Parte Operativa sarà la Parte che possiede la maggiore quota di proprietà dell'Invenzione, secondo quanto disposto dal precedente art. 2, o, in via subordinata e nel caso di quote di proprietà paritetiche, la Parte che verrà designata di comune accordo.
3.3 La Parte Operativa potrà in ogni momento rimettere il mandato, comunicando la sua decisione per iscritto all’altra Parte con un preavviso di 60 giorni.
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3.4 La Parte Operativa, per la gestione delle attività di protezione e trasferimento tecnologico delle Invenzioni di cui agli artt. 5.1, 6.2 e 7.2, tratterrà una somma pari al 10% dei ricavi generati dallo sfruttamento delle Invenzioni, al netto delle spese sostenute dalle Parti per la Protezione dell’Invenzione.
Articolo 4 - Interesse alla Protezione e allo sfruttamento dell’Invenzione -
4.1 Le Parti si impegnano, entro un tempo ragionevole e comunque non superiore a 30 (trenta) giorni a decorrere dalla comunicazione di concepimento dell'Invenzione, a comunicarsi reciprocamente il proprio interesse alla Protezione dell’Invenzione e ad individuare la Parte Operativa.
4.2 Nel caso in cui una Parte non abbia interesse alla Protezione dell’Invenzione, l’altra avrà ogni diritto su tale Invenzione e sarà libera di procedere alla sua Protezione ed al relativo sfruttamento senza nulla dovere all'altra Parte, fatto salvo il diritto morale degli inventori ad esserne riconosciuti autori.
Articolo 5 - Disciplina dei diritti di Proprietà Intellettuale a titolarità congiunta -
5.1 La Parte Operativa avrà competenza sulla predisposizione delle domande di brevetto, o di altra forma di privativa industriale, concernenti le Invenzioni di cui è congiunta la titolarità, al loro deposito e prosecuzione, sulla scelta dell'ufficio cui affidare la gestione della procedura di brevettazione nonché sulla proposta dei Paesi e/o le Organizzazioni presso i quali depositare le domande di brevetto in questione. Tale ultima proposta dovrà essere comunicata tempestivamente all’altra Parte la quale, entro il termine massimo di 30 giorni dal ricevimento della sopra citata comunicazione, comunicherà a sua volta la propria decisione sulla proposta della Parte Operativa.
5.2 Le Parti parteciperanno agli oneri che si riferiscono al deposito della domanda di brevetto o di altra forma di privativa industriale, al mantenimento del medesimo, alla sua eventuale estensione internazionale e alle eventuali spese dirette legate alle procedure di valorizzazione dell’Invenzione in relazione alle rispettive quota di titolarità.
5.3 Qualora una Parte decidesse di rinunciare alla partecipazione agli oneri relativi al mantenimento del brevetto o altra forma di privativa industriale e/o all'estensione internazionale, dovrà informare tempestivamente l'altra Parte entro un termine ragionevole, comunque non inferiore a 30 (trenta) giorni precedenti al decorrere dell'atto previsto dalla procedura brevettuale e al relativo impegno di pagamento. In caso di mancata tempestiva comunicazione, la Parte rinunciataria sarà comunque tenuta al rimborso della sua quota di pagamento. La Parte ricevente la comunicazione avrà un diritto di opzione sulla concessione, a titolo gratuito, della piena titolarità del brevetto o altra forma di privativa industriale in quei Paesi non di interesse, o non più di interesse, della Parte rinunciataria. Resta inteso che la Parte rinunciataria non potrà vantare alcun diritto patrimoniale sullo sfruttamento delle privative industriali in quei Paesi nei quali abbia rinunciato.
5.4 Ciascuna Parte s'impegna a distribuire gli eventuali utili e premi inventivi spettanti ai propri inventori in ottemperanza a quanto previsto dai propri Regolamenti interni vigenti in materia. Ciascuna Parte terrà indenne l'altra da eventuali pretese dei propri dipendenti, collaboratori, consulenti o diversi soggetti comunque da essa impiegati per l’esecuzione dei progetti regolati dalla Convenzione e dal presente Accordo, per i compensi concernenti eventuali attività inventive
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ai sensi delle norme vigenti.
Articolo 6 - Concessione di Licenze d’uso sulle Invenzioni a titolarità congiunta -
6.1 Ciascuna Parte potrà condurre, anche autonomamente, le attività che verranno ritenute da essa opportune per la promozione delle Invenzioni. In tale caso, ciascuna Parte si impegna a tenere informata con tempestività e con diligenza l’altra Parte sulle azioni di promozione che intraprende e sui risultati da essa raggiunti.
6.2 Le Parti stabiliscono sin da ora che la Parte Operativa avrà competenza esclusiva riguardo alle attività negoziali e alla gestione delle licenze d’uso sulle Invenzioni.
6.3 La Parte non Operativa s'impegna sin da ora a sottoscrivere i contratti di licenza d'uso sulle Invenzioni di cui è congiunta la titolarità, alle condizioni concordate dalla Parte Operativa con il licenziatario e, comunque, informata la Parte non Operativa; la Parte Operativa dovrà gestire tale sua competenza esclusiva secondo le regole di buon comportamento in uso nel settore di riferimento.
6.4 Le Parti stabiliscono sin da ora che tutti i proventi derivanti dalle licenze d'uso delle Invenzioni a titolarità congiunta, al netto delle spese sostenute per la Protezione dell’Invenzione e per le attività di gestione effettuate dalla Parte Operativa di cui agli artt. 5.1, 6.2 e 7.2, saranno suddivisi tra le Parti in proporzione delle rispettive quote di titolarità.
Articolo 7- Riservatezza -
7.1 Le Parti si danno atto che qualunque informazione di carattere tecnico-scientifico comunicata da una delle Parti all’altra e relativa alle Invenzioni ha carattere confidenziale; pertanto, si impegnano a non utilizzarle né comunicarle a terzi, né in tutto né in parte, né direttamente né indirettamente, per fini diversi dall'esecuzione di quanto previsto dal presente Accordo.
7.2 Le Parti s’impegnano, altresì, a sottoscrivere appositi accordi di riservatezza nel caso in cui sottopongano le Invenzioni a terzi possibili licenziatari prima della Protezione delle Invenzioni medesime.
Articolo 8 - Uso del Nome e del Marchio -
8.1 Nessun contenuto di quest'Accordo conferisce alcun diritto di usare per scopi pubblicitari, o per qualsiasi altra attività promozionale, alcun nome, marchio, o altra designazione di entrambe le Parti, incluse abbreviazioni. L'uso del nome è obbligatorio in ambienti scientifici e in documentazioni tecniche, divulgazioni scientifiche e articoli stampa.
Articolo 9 - Integrazioni e Conservazioni degli Effetti -
9.1 Qualsiasi modifica o integrazione del presente Accordo verrà redatta esclusivamente in forma scritta e sarà valida se sottoscritta da entrambe le Parti.
9.2 Le Parti stabiliscono sin d'ora che, nel caso in cui alcune condizioni concordate in questo Accordo vengano ritenute non valide, illegali, o inapplicabili in alcuni aspetti, ciò non influenzerà le altre condizioni dell’Accordo, che verrà interpretato come se le condizioni non valide, illegali o inapplicabili non fossero mai state pattuite.
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10.1 Resta inteso tra le Parti che, per quanto qui non espressamente previsto e/o richiamato, restano ferme le previsioni contenute nella Convenzione, nessuna esclusa e/o eccettuata, e che, laddove non diversamente precisato, i termini utilizzati nel presente Allegato hanno lo stesso significato attribuito loro nella Convenzione.
10.2 Restano altresì ferme le previsioni contenute nel Regolamento Brevetti di Sapienza e nel Regolamento IIT sulla Proprietà Industriale.
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ALLEGATO 4
DISCIPLINA IN MATERIA AMBIENTALE--
Sapienza e IIT nel prosieguo indicate anche come “Parti” o, singolarmente, come “Parte”.
Tutto ciò premesso, Xxxxxxxx e XXX convengono e stipulano quanto segue:
Articolo 1 Acque reflue dei laboratori
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Articolo 2 Emissioni in atmosfera
Articolo 3 Gestione rifiuti prodotti nelle attività di ricerca
Per garantire ciò, entrambe le parti, ciascuna per gli spazi di propria competenza, si faranno carico, dal momento del loro conferimento negli appositi contenitori posti nei locali all’uopo destinati, della loro gestione, assumendo la qualità di Produttore dei Rifiuti e, quindi, assumendo in toto la titolarità dei Rifiuti prodotti nei propri spazi.
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Articolo 4 Disposizioni generali
Il presente Allegato integra il contenuto della Convenzione in epigrafe richiamata.
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