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annualreport department of biotechnology and biosciences
A R N E N P U O A R L T D E PA R T M E N T O F B I O T E C H N O LO G Y AND BIOSCIENCES INTRODUCTION This report has been prepared in 2010, the international year of biodiversity. The Department of Biotechnology and Biosciences (BtBs) well represents biodiversity both from the research point of view, its activities as well as with its personnel. The multidisciplinary approach to research topics and the different competencies of our scientists are described in the section 2 of this report and resulted in over 100 scientific publications on international journals and in several book chapters and invited conferences at meetings. It is worth mentioning that, besides excellent research performed by the single laboratories, work carried out in collaboration between groups with different expertise keeps growing and several multidisciplinary research projects have been approved during this past year. At the end of 2009, our Department employed 15 Full Professors, 17 Associate Professors and 29 Assistant Professors. Permanent staff included also 13 technicians and 9 members of the Administration team who actively contributed to the scientific activity and the organization of the structure, together with post-docs, fellowship holders, PhD students and Master degree students. Besides our involvement in the Erasmus project we signed an agreement with the University Paris Diderot for a joint degree in Biology and Biotechnology. Moreover, 16 members of the Department spent time working and teaching abroad and we hosted 7 researchers from foreign countries. 2009 has seen the organization of two conventions open to the public and named “Darwin Day – The evolution of a scientific revolution” and “The reality of Industrial Biotechnologies in Italy”, the scientific conference “Sysbio Health Symposium” and one symposium called “The different forms of knowledge exploitation” about technology transfer. We also put our efforts into improving our contacts with external parties, both academic and industrial, to promote technology transfer activities and to fund raising (see section 3). To this end, some colleagues formed a team dedicated to external relations. This translated in improved and strengthened collaborations with the University offices in charge of fund raising and technology transfer, in the participation to fairs and conferences, in the releasing of interviews. Last, but not least, BtBs was also home to 2.000 students of Biology, Biotechnology and Bioinformatics first and second level degrees. In 2009 we graduated 387 students, out of which 90 in the first level degree in Biology and 136 in Biotechnology, 53 students in the second level degree in Biology, 98 in Biotechnology and 10 in Bioinformatics. Cover picture: Matteo Urbano [2] [3] STRUCTURE AND O R G A N I Z AT I O N INDEX 1. STRUCTURE AND ORGANIZATION 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Financial Resources Department Management Structure and Staff Organization and Structure Instrumentation and Facilities Education Advanced Training PhD and Master Theses Seminars 4 4 8 10 14 15 16 24 2. RESEARCH GROUPS 27 3. SCIENTIFIC PUBLICATION INDEX, GRANTS 3.1 Publications 3.2 Book chapters 3.3 Research Grants and Contracts 3.4 Patents 60 65 65 67 [4] [5] 1.1 FINANCIAL RESOURCES 1.2 DEPARTMENT MANAGEMENT STRUCTURE & STAFF FULL PROFESSORS NAME FIELD NAME FIELD NAME FIELD Alberghina Lilia Castagnoli Paola* Fantucci Piercarlo Lotti Marina Lucchini Giovanna BIO/10 MED/04 CHIM/03 BIO/10 BIO/18 Martegani Enzo Nicotra Francesco Ottolenghi Sergio Porro Danilo Tortora Paolo BIO/11 CHIM/06 BIO/18 CHIM/11 BIO/10 Vai Marina Vanoni Marco Wanke Enzo Zaza Antonio Zullini Aldo BIO/11 BIO/10 BIO/09 BIO/09 BIO/05 ASSOCIATE PROFESSORS Research Grants Head of the Department: MIUR (PRIN, FISR, FIRB) 270.778 EU GRANTS 561.685 REGIONE LOMBARDIA GRANTS CARIPLO GRANTS 67.418 1.012.472 Prof. Marina Lotti Chief Financial Officer: Dr. Anastasia Sguera OTHER FUNDING AGENCIES 459.840 FAR (FONDI D’ATENEO PER LA RICERCA) 182.652 RESEARCH SERVICES 133.297 Management Board: OTHER RESOURCES 312.290 Prof. Paolo Tortora Prof. Marina Vai Other Funding Sources Prof. Francesca Granucci DEPARTMENT FUNDING (DOTAZIONE) 122.000 Prof. Maria Pia Longhese FUNDS FOR TEACHING 347.568 Dr. Barbara Costa PHD COURSES FUNDS FOR LARGE INSTRUMENTS 28.000 184.000 Dr. Maurizio Casiraghi Dr. Anastasia Sguera NAME FIELD NAME FIELD NAME FIELD Barabino Silvia Becchetti Andrea Cipolla Laura Crosti Paolo De Gioia Luca Doglia Silvia Maria BIO/11 BIO/09 CHIM/06 BIO/01 CHIM/03 FIS/01 Giagnoni Gabriella Grandori Rita Granucci Francesca Longhese Maria Pia Moro Giorgio Nicolis Silvia BIO/14 BIO/10 MED/04 BIO/18 CHIM/02 BIO/18 Peri Francesco Piatti Simonetta* Polissi Alessandra Ronchi Antonella Vescovi Angelo CHIM/06 BIO/18 BIO/19 BIO/18 BIO/13 ASSISTANT PROFESSORS NAME FIELD NAME FIELD NAME FIELD Ambrosini Roberto Bertini Luca Brambilla Luca Branduardi Paola Brocca Stefania Casiraghi Maurizio Ceriani Michela Chiaradonna F. Clerici Michela Coccetti Paola Colangelo A.M. BIO/07 CHIM/03 CHIM/11 CHIM/11 BIO/10 BIO/05 BIO/11 BIO/10 BIO/18 BIO/10 BIO/10 Colombo Sonia Combi Romina Costa Barbara De Filippis Lidia Foti Maria Fraschini Roberta Frascotti Gianni Fusi Paola Galli Paolo Gelain Fabrizio Labra Massimo BIO/11 BIO/13 BIO/14 BIO/13 (to 10-’09) MED/04 BIO/18 CHIM/11 BIO/10 BIO/07 BIO/13 ( to10-’09) BIO/01 La Ferla Barbara Lecchi Marzia Orlandi Ivan Prosperi Davide Regonesi Elena Rocchetti Marcella Tisi Renata Zampella Giuseppe Zanoni Ivan CHIM/06 BIO/09 BIO/11 BIO/10 BIO/10 BIO/09 BIO/11 CHIM/03 MED/04 TECHNICAL STAFF NAME NAME NAME Accardo Elena Citterio Stefania D’Urzo Annalisa Gullo Francesca Malerba Massimo Marinoni Sara Mostacciuolo Gaspare* Passolunghi Simone * Pedroni Paolo Tonelli Maria Grazia* Urbano Matteo Villa Anna Maria Sacchetti Francesco NAME NAME NAME Bottani Elena Bruno Stefania Bruni Giuseppe (15/09-31/12) Campbell Neil Comi Roberto Gotti Maria Cristina Mormile Bruno Pacecca Simona (1/03 –31/12) Sguera Anastasia Settembre Claudio (1/01-30/08) Smeraldi Carla ADMINISTRATION * leave of absence [6] [7] PhD STUDENTS INTERNATIONAL MOBILITY 2009 NAME NAME NAME Alemanni Matteo Ambrosi Paola Aprile Francesco Aquaro Giovanni Bazzi Marco Barbieri Valentina Bertagnoli Stefano Bigi Alessandra Bodio Caterina Broggi Achille Broggi Serena Busnelli Sara Caccia Roberta Cantù Claudio Cardona Francisco Chisci Riccardo Codazzi Vera Colombo Miriam De Mattia Fabrizio DiDomizio Alessandro Fontana Gabriele Fumagalli Silvia Galati Elena Galimberti Andrea Galliani Paolo Giaccherini Cinzia Groppi Silvia Lancini Cesare Maffezzoli Andrea Manfrini Nicola Marangoni Stefano Mariani Jessica Mazzantini Elisa Mazzucchelli Serena Merlini Laura Orsato Alexandre Ostuni Renato Palmioli Alessandro Pasi Marco Passolunghi Simone Pastori Valentina Piazza Matteo Pozzi Chiara Redaelli Elisa Rossi Giorgia Russo Laura Sansoni Veronica Santambrogio Carlo Shaik Nasrin Spinelli Michela Strona Giovanni Taraballi Francesca Testa Lorenzo Torri Anna Tosetti Valentina Venkatesh Aparna Viganò Matteo Villa Riccardo Vitali Caterina Vivarelli Silvia Zona Cristiano FELLOWSHIP HOLDERS NAME NAME NAME Altomare Claudia Alvarez Reinaldo Barresi Simona Barbuto Michela Binda Elena Bini Davide Bonanomi Marcella Bruni Ilaria Cattaneo Francesca Catusi Ilaria Corti Ambra Damore Gaetana Fragni Martina Frana Anna Maria Greco Claudio Mainoldi Federica Pessina Stefania Ranghetti Varonica Reghellin Veronica Sangalli Elena Verderio Paolo Viggiani Sandra Villa Omar POST-DOCS NAME NAME NAME Airoldi Cristina Amigoni Loredana Aracri Patrizia Baldo Veronica Baldissera Michela Barile Lucio Belotti Fiorella Benzoni Francesca Bonetti Diego Calabrese Valentina Cassinelli Letizia Cirulli Claudia Comelli Francesca Dato Laura Dos Santos Cunha Carla Favaro Rebecca Ferrari Daniela Ferri Anna Lucia Forcella Matilde Fossati Tiziana Gorletta Tatiana Invernizzi Gaetano Lenzken Carolina Leoni Giampaolo Morini Raffaella Mortellaro Alessandra Natalello Antonino Occhipinti Emanuela Paiardi Chiara Papaleo Elena Pontiroli Francesca Redaelli Cristina Rossio Valentina Rota Nodari Laura Samalikova Maria Saracino Gloria Sommaruga Silvia Sperandeo Paola Stefani Fabrizio Tripodi Farida INCOMING FROM GROUP PERIOD Isil Teseli Ankara University, Turkey Nicolis 3 months Carol Yang National Institute for Medical Research, London, UK Nicolis 3 months Pieter Van Neuten Katholieke Universiteit Leuven Belgium Martegani 4 months Aparna Venkatesh Singapore Castagnoli Alexandre Orsato Federal University of Paraná, Brasil Nicotra Nasrin Shaikh University of Pune, India Nicotra Francisco Cardona University of Aveiro, Portugal Nicotra OUTGOING TO GROUP PERIOD Simonetta Piatti Centre de Recherche en Biochimie Macromoléculaire in Montpellier, France Piatti 1 year Farida Tripodi Thomas Jefferson University, Philadelphia, Pennsylvania, USA Alberghina 3 months Roberto Spreafico Singapore Castagnoli Cristina Conforti Andreoni Singapore Castagnoli Ottavio Beretta Singapore Castagnoli Alessandra Mortellaro Singapore Castagnoli Matteo Piazza Iowa University, Iowa City, Iowa USA Peri 4 months Laura Dato VTT Technical Center of Finland, Espoo, Finland Porro 8 months Francesco Peri Ecole Normale Superièure de Lion, France Peri 10 days David Metalli Kimmel Cancer Center, T. Jefferson University, Philadelphia PA, USA Vanoni 8 months Daniela Gaglio MIT Boston, MA, USA Vanoni 3 months Michela Spinelli Kimmel Cancer Center, T. Jefferson University, Philadelphia PA, USA Vanoni 8 months Rita Grandori Joannes Kepler University, Linz Austria Grandori Elena Dossi Leipzig University, Leipzig Germany Wanke 4 months [8] [9] 1.3 ORGANIZATION AND STRUCTURE FACILITIES FOR TEACHING ACTIVITIES 10 laboratories devoted to teaching activities are located in building U3. The organization of each laboratory is supervised by a member of the technical staff. Lab 1028 (Genetics) Stefania Citterio Lab 1011 and 1015 (Chemistry) Francesca Gullo, Anna Maria Villa ADMINISTRATION OFFICE Anastasia Sguera – Chief Financial Officer Stefania Bruno - Foreign payments, VAT related accounting Roberto Comi - Accounts payable (contracts, scholarships, travel reimbursement) Bruno Mormile - Supplier’s accounting Francesco Sacchetti - Property inventory, technical support Claudio Settembre, Giuseppe Bruni – Purchase Orders During 2009 the Department Administration processed over 4.300 purchase orders and 439 travel expense reimbursements STUDENT ADMINISTRATION OFFICE Maria Cristina Gotti, Elena Bottani, Simona Pacecca The student administration office manages all administrative aspects related to the teaching activities of the first level degrees in Biotechnology and Biology and second level degrees in Industrial Biotechnology, Biology and Bioinformatics. The student administrative office assists all students in the bureaucratic aspects of their career; it is responsible for the content of the web pages of the department web site with regard to teaching activities; it organizes the calendar of lessons and exams and manages the data related to all degree courses through the information system called SIFA ON LINE. Lab 2010 and 2013 (Microscopy, Morphology) Massimo Malerba Lab 1026 (Biochemistry) Sara Marinoni Lab 2026 and 2030 (Microbiology, Fermentation, Molecular Biology) Simone Passolunghi Lab 1027 and 1029 (Immunology and Cell Biology) Matteo Urbano TECHNICAL SERVICES The technical staff carries out common services, is responsible for the maintenance of common instruments and collaborates in research activities. In 2009 the staff duties were as follows: Mass Spectrometry: Elena Accardo Cytometry: Stefania Citterio Biacore (Surface Plasmon Resonance): Annalisa D’ Urzo Technical gases, MEA workstation: Francesca Gullo Chemical and Biological Waste Disposal: Sara Marinoni IT support: Paolo Pedroni TECHNOLOGY TRANSFER COMMISSION Biotechnicum: Simone Passolunghi Francesco Peri, Alessandra Polissi, Carla Smeraldi Molecular Immunology, BL2 Laboratory: Matteo Urbano The Technology Transfer Commission acts as a liaison between the Department and the Technology Transfer and Intellectual Property office of the University. It promotes and facilitates contacts between the research groups, small and medium enterprises, incubators, patent experts in order to capitalize and exploit scientific results, promote innovation and fill the gap between the academic world and the socio-economical one. During 2009 the Commission organized a one-day symposium with renowned experts about the different tools available to exploit scientific results. The symposium was open to the entire University and listed among the speakers a representative of the Ministry of Economis. Confocal Microscopy: Anna Maria Villa [ 10 ] 1.4 INSTRUMENTATION AND FACILITIES A number of platform technologies and advanced instrumentations are available both to the research groups working in the Department and to external users. [ 11 ] two different cell types or tissue samples, such as in tissues from healthy and diseased subjects. Gene expression has been successfully used to classify complex diseases, enabling researchers to identify genetic changes that are more likely to be causative and, therefore, better diagnostic indicators and possible therapeutic targets. In addition, since global expression profiling allows for discovery of new pathways disrupted in disease states, researchers can better understand the full complement of changes associated with a particular disease. Arrays are currently available for human samples as well as many biologically relevant model organisms including mouse, rat and plant (Arabidopsis). BIOSAFETY LEVEL 2 FACILITY (BL2) BBC (BICOCCA BIOTECHNICUM CENTER) The Biotechnicum (BCC) is a facility aimed at the development of proprietary industrial strains, fermentation and bioconversion processes for the production of commercially interesting proteins, metabolites and enzymes. BCC is particularly strong in services that require an integrated multidisciplinary approach. The main focuses are on those areas which require a combined know-how of bioprocess technology, microbiology and biochemistry applied to industrial biotechnology processes. In this position BBC is able to assist in the selection, modification and development of microorganisms, “scaling-up” and “scaling down” of production processes. The core of the facility are two 10 lt bioreators. All the operations are carried out in GLP (Good Laboratory Practice) and GMP-like (Good Manufacturing Practice) environment. Among the services offered by BCC, in complete confidentiality, the main ones are: • Strain selection and optimization • Optimization of growth fermentation media • Optimization of production of bioprocesses • Pilot fermentation - Batch, fed-batch and continuous - High cell density fermentation • Process design and scale-up • Scaling down • Bioreactor modelling and simulation • Product purification and analysis • Consultancy services www.bbc.btbs.unimib.it MICROARRAY FACILITY Microarray technology allows for rapid measurement and visualisation of differential expression among genes at the whole genome scale. In DNA microarrays, or DNA chips, probes with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. Microarrays may be used to compare gene expression in This new core facility is located in a dedicated closed laboratory space, with restricted access. The facility consists in a Vector Production room, a Cell Manipulation room (directly connected with a Pass-Through Cabinet) and two general support areas. These laboratories house tissue culture hoods, CO2/O2 incubators, microscopes and small equipment for cellular and molecular biology work. All areas are designed to ensure a high standard of cleanliness and an orderly flow of the entire experimental process. The air is HEPA-filtered, and manufacturing conditions have been further optimized by a system of differential air pressures between individual rooms. The personnel working in this facility receives appropriate training on the safety guidelines required for safe handling of potentially biohazardous materials and the necessary updates to comply with possible procedural or policy changes. This facility is suitable for experiments involving agents of moderate potential hazard to personnel and environment, and, in particular, for safe production and handling of lentiviral and retroviral vectors. The facility has been approved by the Ministry of Health, Department of Sanitary Prevention. MASS SPECTROMETRY The mass spectrometry (MS) facility of our department supports the analysis of small and large molecules, including protein non-covalent complexes. The laboratory is equipped with two instruments with electrospray-ionization (ESI) sample source and one with matrix-assistedlaser-desorption/ionization (MALDI) sample source. The mass analyzers are based on different technologies. A triple-quadrupole instrument (QTRAP, Applied Biosystems) is equipped with an electrospray-ionization (ESI) sample source and it is hyphenated with a micro-HPLC system (Perkin Elmer) for coupling to liquid chromatography (LC). The mass analyzer combines triple quadrupole and linear-ion trap capabilities enabling LC-MS/MS and LC-MS/MS/MS measurements for proteomics or analytical chemistry. The instrument is particularly well suited for the analysis of post-translational modifications of proteins by neutral-loss scan, precursor-ion scan and multiple-reaction monitoring. A hybrid quadrupole-time-of-flight (q-TOF) instrument (QSTAR, Applied Biosystems) is equipped with a regular and a nano-ESI sample source and it is particularly well suited for protein analysis under non-denaturing conditions. This instrumentation enables MS and MS/MS measurements at high sensitivity and high resolution for proteomics studies, as well as for protein conformational studies and for the analysis of protein-protein and protein-ligand non-covalent complexes. [ 12 ] MEA - MULTI ELECTRODE ARRAY WORKSTATION The MEA workstation consists of a complex machine for the acquisition, in real time for days or weeks, and under no invasive conditions, of the stimulated, or spontaneous, activity from networks of excitable cells (from sensory, cardiac or neuronal origin). It has 256 points of observation from which about 400 cells will be simultaneously recorded. The quality and sensitivity of this new recording technique has been recently validated because it has been shown that this recording method allows to detect the decline of synaptic spike/burst ratio induced by neurotoxic stimuli, such as the application of the ß-amyloid protein, a main factor involved in Alzheimer’s disease pathogenesis. [ 13 ] Flo® high speed cell sorter (Cytomation-BeckmanCoulter) equipped with three lasers (354 nm; 488 nm and 635 nm) which enables to perform 9-colours analyses. The MoFlo® has a dedicated operator. Cell Lab Quanta SC (Beckman Coulter) with Mercury arc excitation optimized at 365, 405, and 435 and 488nm laser diode excitation. It has 3 broad range ultra sensitive photomultiplier tubes and a 125µm triangular flow cell. With this instrumentation it is possible to measure simultaneously Electronic Volume, side scatter, time, and 3 colour detections. This flow cytometer is used for analyses only. OPTICAL SPECTROSCOPY AND OPTICAL MICROSCOPY NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROMETRY The Nuclear Magnetic Resonance (NMR) lab is equipped with a Brucker ADVANCE 600 MHz equipped with a high-resolution liquid-state quadruple resonance cryo-probe, a HR-MAS triple resonance probe and a solid-state triple resonance probe and a Varian MERCURY 400 MHz spectrometer. One inverse-detection gradient probe (good sensitivity for 1H and 19F) and one direct-detection probe (good sensitivity for 13C and 31P) are available. This instrument allows the structural and conformational characterization of small-medium size molecules (up to 6 kDa molecular weight), such as low molecular-weight drugs, mono-, di-, tri- and oligosaccharides, oligonucleotides and peptides. A large panel of pulse sequences is available to perform: i) monodimensional experiments (1H, 19F, 13C, 15N, 31P spectra, 1D-TOCSY, 1D-NOESY, 1D-ROESY, T1 and T2 measurements); ii) bidimensional experiments (COSY, 2D-TOCSY, 2D-NOESY, 2D-ROESY, HMBC, HSQC); iii) tridimensional experiments (TOCSY-NOESY, TOCSY-HSQC). Small ligand-receptor (such as inhibitor/activator-protein or substrate-enzyme) interaction studies are performed via DOSY, Saturation Transfer Difference (STD) and transferred-NOESY (trNOESY) experiments. FLOW CYTOMETRY Flow cytometry (abbreviated: FCM) is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to hundreds of particles per second. Each suspended particle that passes through the beam scatters the light in some way. Fluorescent chemicals found in the particle or attached to it may be excited into emitting light at a higher wavelength than the light source. This combination of scattered and fluorescent light is picked up by the detectors. A cell sorter, or flow sorter, is a flow cytometer that uses electrical and/or mechanical means to divert and collect cells (or other small particles) with measured characteristics that fall within a user-selected range of values. In our Department different research groups utilize this type of instrument for many different studies, mainly: analysis and sorting of microorganisms (especially yeasts) for industrial biotechnological applications; analysis of yeasts for studying cell cycle progression and ageing; analysis of mammalian cells for typization and sorting of specific subpopulations. Instrumentation Available Includes: A MoFlo (DakoCytomation) Core Facility which allows automated cytofluorimetric analysis and sterile sorting of specific mammalian cell types. The equipment of our facility consists of a Mo- Laser scanning confocal fluorescence microscope Leica TCS SP2 is a confocal microscope with acoustic optical beam splitter (AOBS), equipped with three lasers for fluorescence excitation: an Argon laser with excitation wavelength at lexc = 458nm, 476nm, 488nm, 496nm, 514nm; and two He-Neon lasers, respectively, with excitation wavelengths at lexc= 543nm and at lexc = 633nm. The scanning head of the system is coupled to an inverted motorized optical microscope Leica DFMIR2, equipped with dry objectives of 10x e 20x magnification, as well as with oil immersion objectives of high magnification 40x and 63x. The Leica TCS SP2 prism spectrometer enables also to measure fluorescence spectra and to set the wavelength band of the collected fluorescence to the real emission spectrum. The easy to use acquisition and processing software for image analysis enables also the three-dimensional reconstruction of the specimen. Inverted motorized microscope Nikon Eclipse E600. Fluorescence microscope with halogen lamp for transmitted light illumination and Xenon lamp for fluorescence excitation. The microscope is coupled to a digital video camera Leica DC 350 F that enables to obtain high image quality at low light intensity. The video camera is equipped with image acquisition software and image analysis algorithms for three-dimensional reconstruction. Circular dichroism spectropolarimeter Jasco J815. This spectropolarimeter works in the ultraviolet and visible ranges from 163 nm and 900 nm. It also allows to measure the fluorescence of the sample in the range 200-800 nm. The temperature of the sample is controlled by a Peltier system operating between -10 °C e + 110 °C. The instrument is equipped with a Stopped-Flow accessory for kinetic and titration studies. Fourier transform infrared spectrometer (FTIR) Varian 670-IR FTIR spectrometer for absorbance measurements in the medium infrared range, with dynamic alignment of the interferometer and MCT detector. The instrument allows measurements in transmission mode and in attenuated total reflection (using a 9 reflection diamond plate) with temperature control. The spectrometer is coupled to the infrared microscope 610IR Varian. Spectrofluorimeter Varian Cary Eclipse Is a highly sensitive spectrometer for fluorescence emission and excitation measurements from 200 nm to 900 nm on minimum sample volumes. It allows the temperature control up to four samples simultaneously. It is equipped with a static anisotropy fluorescence accessory (automatically controlled) and with a microplate reader working in reflecting optics. [ 14 ] [ 15 ] 1.5 EDUCATION BIOTECHNOLOGY (Coordinator Prof. Danilo Porro) www.biotecnologie.unimib.it BACHELOR IN BIOTECHNOLOGY The course is articulated in three years, two of which are common to all students, while the third year is focused on either Industrial Biotechnology, or Molecular Biotechnology, or Medical Biotechnology (in cooperation with the Faculty of Medicine and Surgery). MASTER IN INDUSTRIAL BIOTECHNOLOGY The master course in Industrial Biotechnology is two year long with two specializations: 1) Pharma-genomics and 2) Processes and Products. 1.6 ADVANCED TRAINING BIOLOGY (Coordinator Prof. Antonio Zaza, Prof. Paolo Tortora from October 2009) www.biologia.unimib.it BACHELOR IN BIOLOGY The course is articulated in a year common to all students and a two-year period devoted to the following areas; Bioecology, Biomolecular and Physio-pathological studies. MASTER IN BIOLOGY The master course in Biology is two year long and organized into the following areas: 1) Functional and Molecular Biology 2) Bio-Ecology. The overall number of enrolled students for the Academic Year 2008/2009 was 920. MASTER IN BIOINFORMATICS The Master course in Bioinformatics is carried out over two years in cooperation with the Degree in Informatics. ERASMUS PROGRAM FOR INTERNATIONAL MOBILITY Coordinator for Biotechnology: Prof. Maria Pia Longhese The overall number of enrolled students for the Academic Year 2008/2009 was 943. Coordinator for Biology: Prof. Silvia Nicolis The Department hosts two PhD programs of the School of Doctorate in Sciences (www.scuoladottorato.scienze.unimib.it): the PhD Program in Industrial Biotechnology and the PhD Program in Biology. Additionally, department members contribute to the PhDs in Chemistry, Nanostructures and Nanotechnology and in the International PhD Program in Translational and Molecular Medicine offered by other Departments. PhD PROGRAM IN INDUSTRIAL BIOTECHNOLOGY www.scuoladottorato.scienze.unimib.it Coordinator Prof. Marco Vanoni PhD PROGRAM IN BIOLOGY www.scuoladottorato.scienze.unimib.it Coordinator Prof. Paolo Tortora The Doctorate in Industrial Biotechnology is highly multi- and inter-disciplinary, the areas of expertise present within the Board of Professors include virtually all aspects of modern Biotechnology: Biophysics, Biochemistry and Systems Biology, Molecular Biology, Genetics, Industrial Microbiology, Organic and Computational Chemistry. The curriculum is three year-long and is a full-time occupation, strongly based on the development of a research project. Students are encouraged to conduct part of their doctoral project in an international laboratory, to be chosen in agreement with their tutor. Teaching activities include: single seminars on topics relevant to modern Biotechnology given by Italian and foreign visiting researchers, journal clubs, intensive coordinated seminars on state-ofthe-art topics, seminars and courses of general interest organized by the Doctoral School of the Faculty of Science. The Doctorate in Biological Research is run by professors from the Departments of Biotechnology and Biosciences as well as Environmental Sciences. Their areas of expertise include virtually all aspects of modern Biology: Physiology, Biochemistry, Molecular Biology, Genetics, Pharmacology, Microbiology, Ecology, Botany, Plant Genetics and Physiology, Zoology, Cytology and Histology. This makes the Doctorate in Biological Research particularly multi- and inter-disciplinary. Doctoral students have access to developing research projects in all the above areas using state-of-the-art genetic, physiological, biochemistry and morpho-functional technologies. Students are encouraged to conduct a part of their doctoral project abroad at one of the many partner institutions of the program. The doctoral research project is chosen and conducted under the supervision of a member of the board of Doctoral Instructors. Teaching activities include: single seminars on the topics mentioned above held by Italian and foreign visiting researchers, journal clubs, an English course taught by an English nativespeaker and specifically planned to allow the students to acquire language skills Overall number of PhD students: 31 Overall number of PhD students: 23 [ 16 ] [ 17 ] 1.7 PhD AND MASTER THESES Galbusera Elena “Bacillus subtilis improvement for the development of fermentative processes aiAlemanni Matteo “The modulation of SERCA med at producing pyrimidine nucleotides” PhD in pump activity as a tool for management of hearth Biology. Tutor: P. Tortora failure”. PhD in Translational and Molecular Medicine. Tutor: A. Zaza Jessica Mariani “Transcriptional regulation, PhD DISSERTATIONS Anna Torri “Gene expression profiling in host-pathogen interactions and identification of the molecular mechanisms involved in dendrictic cells activation”. PhD in Translational and Molecular Medicine. Tutor: M. Foti target genes and functional roles of the SOX2 transcription factor in mouse neural stem cells maintenance and neuronal differentiation” PhD in Translational and Molecular Medicine. Tutor: S. K. Nicolis Borgogni Erica “Profiling human cell-mediated Metalli David “Sviluppo di derivati del GEF Cdc 25 immune response to pre-pandemic vaccination”. Mm per uso in terapia antiproliferativa”. PhD in PhD in Industrial Biotechnology. Tutor: M. Vanoni Industrial Biotechnology. Tutor: M. Vanoni Busti Stefano “Nutrienti e regolazione del ciclo cellulare in S. cerevisiae: analisi di mutanti con alterazioni nei meccanismi di sensing e di trasporto del glucosio”. PhD in Industrial Biotechnology. Tutor: M. Vanoni Panseri Silvia “Central and peripheral nervous system regeneration via nano-structures scaffolds”. PhD in Biology. Tutor: B. Costa Pasi Marco “A dynamical perspective on coldClaudio Cantù “The Sox6 transcription factor: its adapted enzymes at the molecular level”. PhD in role in human and murine erythroid differentia- Industrial Biotechnology. Tutor: L. De Gioia tion and mechanisms for its regulation”. PhD in Translational and Molecular Medicine. Tutor: S. Passolunghi Simone “Matching biotech needs Ottolenghi and yeast physiology”. PhD in Industrial BiotechContran Nicla Aurora “Plant antioxidant systems nology. Tutor: D. Porro in stress responses”. PhD in Biology. Tutor: P. Piazza Matteo “Sviluppo di nuovi ligandi del reCrosti, R. Cerana cettore TLR4 e caratterizzazione della loro funDi Domizio Alessandro. PhD in Chemistry. Tutor: zione biologica”. PhD in Industrial Biotechnology. P. Fantucci Tutor: F. Peri Ferrara Silvia “Environmentally friendly proces- Comelli Francesca “The modulation of the endoses for the production of molecules of industrial cannabinoid system in order to treat neuropathic interest”. PhD in Biology. Tutor: P. Tortora pain”. PhD in Biology. Tutor: B. Costa Gaglio Daniela “Ruolo dei nutrienti sulla proliferazione ed esecuzione del ciclo cellulare di fibroblasti murini immortalizati e K-Ras trasformati”. PhD in Industrial Biotechnology. Tutor: F. Chiaradonna Pozzi Stefano “Caratterizzazione del trascrittoma di PBMCs di pazienti affetti da aneurisma all’aorta addominale e da ostruzione carotidea”. PhD in Industrial Biotechnology. Tutor: M. Vanoni Rebuzzini Gabriele “Studio del dominio elicasico- Binding per Tanchirasi 1”. F. Peri di NS3 di HCV per l’utilizzo in un saggio diagnosticodi tipo CLIA”. PhD in Industrial Biotechnology. Bevilacqua Sara “Ruolo della protein chinasi Tell di S. cerevisiae nell’omeostasi telomerica: Tutor: D. Porro caratterizzazione funzionale di una variante ipeRossio Valentina “Cellular response to mitotic rattiva”. M.P. Longhese perturbations: PP2A - mediated control of sister chromatid cohesion and adaptation to the sac”. Bolamperti Simona “GH e 17 ß-ESTRADIOLO: Interazioni molecolari nell’osteoblasta umano”. PhD in Biology. Tutor: G. Lucchini B. Costa Tripodi Farida “Protein Kinase CK2: a major regulator of G1/S transition in Saccharomyces ce- Bonifacio Silvia “Attività antiangiogenica della revisiae”. PhD in Industrial Biotechnology. Tutor: trombospondina-1 (TSP-1): caratterizzazione funzionale di un dominio che lega FGF-2”. G. P. Coccetti Giagnoni Zerilli Francesco “Progettazione e sviluppo di saggi isotermi per la rilevazionedi stati di iperme- Borghetti Marco “Caratterizzazione genetico – tilazione del DNA”. PhD in Industrial Biotechnolo- molecolare del gene TPMT in pazienti in trattamento con Azatioprina”. M.L. Lavitrano gy. Tutor: S.M. Doglia Boroni Chiara “Effetti dell’assistenza ventricolare sinistra sullo stato redox e sull’infiammazione Abete Domenico “Cofattori nicotianamidici e in pazienti con scompenso cardiaco avanzato”. ubiquitinazione dell’istone H2B in Saccharomy- F. Granucci ces cerevisiae”. M. Vai Brioschi Elisabetta “Inibitori di AKT: sintesi ed Alfieri Michela “Caratterizzazione biochimica e effetti biologici su miociti ventricolari”. L. Cipolla molecolare di proteine legate all’amido in caBroggi Serena “Studi sulla localizzazione delle riossidi di avena”. E. Martegani proteine Ras attive nel lievito Saccharomyces Aloi Valentina “Caratterizzazione di possibili al- cerevisiae”. S. Colombo terazioni morfologiche e funzionali in astrociti spinali con una mutazione nel gene vps54”. E. Brunialti Electra Athena Salomé “Isolamento, clonaggio e caratterizzazione di enzimi esteroliMartegani tici da batteri psicrofili: adattamento al freddo e Aurilia Dario “Studi sul targeting di antitumora- promiscuità catalitica”. M. Lotti li: progettazione e sintesi di analoghi della BomBuscaino Maria Grazia “Ferrodossina-NADP+ besina”. B. La Ferla riduttasi di Plasmodium falciparum: ruolo della Bargna Anna “Inibitori idrosolubili della proteina Lys249 nel legame sul NADPH e meccanismo di Ras: verso la definizione del meccanismo d’azio- inibizione da parte dell’ebselen”. G. Frascotti ne”. M. Vanoni Busnelli Sara “Ruolo svolto dalla proteina chiBertolini Valentina “Caratterizzazione struttu- nasi Snf1 nella transizione G1/S in Saccharomyrale e biochimica di KdsD di Escherichia coli, un ces cerevisiae”. P. Coccetti enzima coinvolto nella biosintesi del lipopolisacCalcinotto Arianna “Sinergia tra immunoteracaride”. A. Polissi pia, targeting vascolare e chemioterapia per la Bettoni Serena “Sintesi e valutazione di Probes cura del cancro: studio in un modello murino di Fluorescenti per messa a punto di un saggio di melanoma”. F. Granucci MASTER IN INDUSTRIAL BIOTECHNOLOGY [ 18 ] Casatta Nadia “Genetic characterization and Ferranti Benedetta “Espressione di anticorpi risynthetic interaction studies among SFP-, combinanti con tag specifici: potenzialità e proSCH9- and PHO85-signalling in Saccharomyces blematiche”. L. Brambilla cerevisiae”. M. Vai Ferrario Anna “Caratterizzazione neuropatologiCausio Jessica “Sviluppo di funzionalità antimi- ca di topi knock-out per il gene lxrb”. G. Lucchini crobiche di superfici cellulosiche mediante bio/ Ferrera Lorenzo “Studi per lo sviluppo di ceppi nano-tecnologie”. D. Porro di Escherichia coli come potenziali produttori di Cerutti Camilla “Effect of cannabinoids on bre- acido succinico”. P. Branduardi ast cancer”. B. Costa Ferrigno Davide “Esperimenti di catalisi mulColombo Stefania “Sviluppo e ottimizzazione di tienzimatica: utilizzo di enzimi ossidoriduttivi in una piattaforma portatile per analisi quantitative biotrasformazioni sequenziali”. G. Bestetti di acidi nucleici su LAB-ON-CHIP”. M. Vai Frattini Véronique “Identificazione dei precursoConz Cecilia “Mechanistic understanding of lac- ri osteoblastici a caratterizzazione del pathway di tate induction and effect of down-regulation of WNT nel tessuto osseo: effetti dell’invecchiamenLactate Dehydrogenase-A by siRNAs in Chine- to”. B. Costa se Hamster Ovary cells producing recombinant protein” . M. Vanoni Fumagalli Erica “Continuità e discontinuità genealogiche attraverso 2.500 anni della popolazione Corti Ambra “Alterazioni dei livelli intracellulari toscana”. G. Lucchini di NAD+ in Saccharomyces cerevisiae: risposte trascrizionali ed effetti metabolici”. M. Vai Fumagalli Sonia “Sviluppo e formulazione di dispositivi medici a base di Zinco gluconato e Taurina Cozzi Lorenzo “Ciclodestrine Vettorizzate con nel trattamento di patologie del cavo orale”. B. La Glicosaminoglicani per il trasporto di farmaci Ferla antitumorali”. F. Peri Fusco Ileana “Preparazione di FAD chimicamente Cracco Marco “Interaction studies of E6 proteins modificato ed impiego nella ricostituzione dell’olowith the non-muscle myosin H9”. M. Ceriani enzima della fenilacetone-monoossigenasi da Thermobifida fusca . F. Peri D’Orazio Giuseppe “Modulators of Sodium-Glucose contrasporter (SGLT1) and their high-in- Gelain Federica “Analisi del rapporto genotipo feflammatory activity”. B. La Ferla notipo della mutazione aIIbP258S responsabile di un caso familiare di trombastenia di Glanzmann”. De Ceglia Roberta “Studio del pathway di tra- E. Martegani sduzione del segnale del complesso multirecettoriale del lipopolisaccaride in cellule dendriti- Giaccherini Cinzia “L’enzima deubiquitinante che”. F. Granucci UBPy/USP8 promuove la deubiquitinazione del recettore TrkA e blocca il differenziamento neuronale Di Benedetto Davide “DNA Barcoding e Micro- indotto da NGF in cellule PC12”. E. Martegani contact Printing: Strumenti Innovativi per la Diagnostica nel Settore Agroalimentare”. F. Peri Giuliani Luca Davide “Identification of loci that contribute to the high ethanol tolerance of a BraDusi Sabrina “Ruolo delle sirtuine nell’invecchia- zilian bioethanol production strain using genetic mento cerebrale e nella demenza: studi genetici markers”. E. Martegani e di biologia cellulare”. R. Tisi [ 19 ] Granata Elena “Studio di inibitori dell’attività enzimatica dell’eparanasi aventi struttura glicosaminoglicanica o loro mimetici”. B. La Ferla Merlini Luca “Progettazione, sintesi e caratterizzazione biologica di nuove molecole capaci di interagire con il pathway del Toll-like Receptor 4 (TLR4)”. F. Peri Krenn Veronica “Studio funzionale della proteina p31comet implicata nella regolazione del Merlo Silvia “D-arabinosio-5-fosfato isomerasi: caratterizzazione del riconoscimento e dell’incheckpoint mitotico”. F. Chiaradonna terazione dei substrati naturali tramite spettroLacava Michele “Effetto di cellule mesenchimali scopia NMR”. F. Nicotra staminali isolate da midollo osseo in un modello di Meroni Eleonora “Caratterizzazione strutturale danno renale da adriamicina”. B. Costa della proteina MTI-2”. R. Consonni Lavazza Martina “Studi di ossidazione di galattomannani da guar con laccasi fungine”. M. Vanoni Monteleone Nicola “Influenza della componente aminoacidica nella definizione aromatica del Longo Valeria “Acido succinico: dallo “Spirito vino”. P. Branduardi d’Ambra” alla produzione per via microbica”. P. Branduardi Mozzi Alessandra “Studio dell’interazione dei residui distali del sito catalitico della sialidasi Lopa Silvia “Sottopopolazioni di cellule staminali NEU2 con i propri substrati”. P. Fusi mesenchimali isolate da tessuto adiposo umano: caratterizzazione e valutazione del potenzia- Musolino Vincenzo “Characterization of novel le differenziativo”. B. Costa factors influencing alpha-synuclein-induced toxicity in a yeast model for Parkinson’s disease”. Mancini Rosanna “Impatto clinico dei controlli di S. Colombo qualita’ eseguiti secondo standard internazionali nel trapianto di cellule staminali ematopoieti- Ortica Sara “Role of the Notch pathway in hepache” M.L. Lavitrano tic differentiation” . S. Nicolis Mapelli Erika “Espressione della trans-o-idrossibenzilidenepiruvato idratasi-aldolasi di Pseudomonas fluorescens N3: purificazione, caratterizzazione e applicazione in catalisi enzimatica”. G. Bestetti Palorini Roberta “Studio del ruolo della via cAMP-PKA nel funzionamento mitocondriale di cellule tumorali murine ed umane mutate nell’oncogene K-Ras”. F. Chiaradonna Pascolutti Roberta “Regolazione della citochineMarchesi Maria “Role of the Aurora B kinase du- si nel lievito Saccharomyces cerevisiae: coinvolring plasma cell differentiation”. M. Vai gimento della GTPasi Rho1 nella dinamica delle septine”. G. Lucchini Martina Marina “Multiple pathways regulate 3’ overhang generation at Saccharomyces cerevi- Patercoli Simona “Studio della chinasi Aurora B siae telomeres”. M.P. Longhese attraverso un approccio chimico-genetico”. M.P. Longhese Mascheretti Iride “Caratterizzazione di nuovi canali del calcio indotti da stress e nutrienti nel lie- Pendino Vera “Caratterizzazione strutturale delvito Saccharomyces cerevisiae”. R. Tisi la proteina intrinsecamente disordinata Sic1, un inibitore delle chinasi ciclina dipendenti del lieviMazzolini Simone “Ruolo delle tirosino-chinasi to Saccharomyces cerevisiae”. R. Grandori Pyk-2 e Src nell’azione pro-angiogenica dell’ossitocina in cellule endoteliali umane”. G. Gia- Pignatari Chiara “Studio di markers genici nelle cellule del cumulo ooforo e sviluppo di una megnoni [ 20 ] todica molecolare per la valutazione della qualità on the proliferation and differentiation in vitro of ovocitaria durante le procedure di fecondazione human neural stem cells (hNSC) immortalyzed assistita”. M. Vai with v-myc”. A.L. Vescovi Pinton Siria “Modificazione superficiale di poli- Sberna Irene “Espressione eterologa di citocromeri tramite enzimi lipolitici: studi su un siste- mi in lievito per la produzione di fine e bulk chema modello a base di poli-etilene tereftalato”. micals”. P. Branduardi M. Vanoni Scarpellini Chiara “Impiego di laccasi per increPiovan Claudia “Ruolo del microRNA-205 nella mentare le proprietà antimicrobiche e antiossiregolazione dell’espressione del recettore HER3 danti di strutture fenoliche”. G. Bestetti nel carcinoma della mammella”. M. Vai Sironi Erika “Studi sulla caratterizzazione NMR Porrino Lucy “Meccanismi di suscettibilità ge- dei peptidi amiloidi e delle loro interazioni con netica alle infezioni virali: analisi di un polimor- composti anti-amiloidogenici” F. Nicotra fismo del singolo nucleotide (SNP-168) nella regione del promotore di una chinasi cellulare Solinas Nicola “Lieviti produttori di acido ascor(PKR) indotta da interferone”. M. Foti bico imparano dalle piante come riciclarlo”. P. Branduardi Raimondi Chiara “Caratterizzazione di LptC: una proteina coinvolta nella biogenesi del lipopoli- Spinelli Chiara Carmela “Analisi sistematica delle mutazioni germinali del gene BRCA1 in una saccaride in E. coli”. A. Polissi popolazione sudanese affetta da tumore mamRaspelli Erica “Regolazione della chinasi Swe1 mario”. G. Lucchini da parte delle ubiquitina-ligasi Dma1 e Dma2 nel lievito S.cerevisiae”. R. Fraschini Stanco Deborah “L’età del donatore può influenzare l’uso delle cellule staminali mesenchimali Ravani Martino “Possibilità di attribuire un pro- umane in applicazioni di medicina rigenerativa? filo genetico ad un gruppo etnico alla luce dei Studio in vitro del loro potenziale proliferativo, clonogenico e differenziativo”. G. Giagnoni polimorfismi del cromosoma Y”. M. Vai Reghellin Veronica “Studio dell’attività della pro- Stravalaci Matteo “Analisi del processo di aggreteina chinasi CK2 e della fosforilazione del suo gazione della proteina beta-amiloide mediante la substrato Sic1 in funzione delle condizioni nutri- “Risonanza Plasmonica di Superficie”. L. De Gioia zionali in Saccharomyces cerevisiae”. P. Coccetti Testa Lorenzo “Ordine/Disordine strutturale del Rinaldi Anna “Sviluppo e funzione antitumorale dominio inibitorio di Sic1, un inibitore delle chidei linfociti iNKT dopo trapianto di midollo osseo nasi ciclina-dipendenti del lievito Saccharomyces cerevisiae”. R. Grandori, S. Brocca in pazienti leucemici”. F. Granucci Rusconi Damiana “Localizzazione funzionale di Tezza Sara “Analisi quantitativa e qualitativa delCdc25, scambiatore di Ras in Saccharomyces la risposta immunitaria in pazienti affetti da mecerevisiae”. R. Tisi lanoma sottoposti a trattamenti di immunoterapia attiva e specifica con linfociti geneticamente Sambi Ilaria “Studio dell’influenza di un tag in- modificati”. F. Granucci trinsecamente destrutturato su solubilità e struttura del partner di fusione”. M. Lotti Tonin Stefano “Ruolo del camp nel controllo della transizione G1/S nel lievito Saccharomyces Santilli Guido “Effects of oxygen concentration cerevisiae”. E. Martegani [ 21 ] Torella Rubben “Studio di design e dinamica MASTER IN BIOLOGICAL SCIENCES molecolare sulla micro-mioglobina, la minima Al Megrabi Fabio “Caratterizzazione morfologica proteina legante l’eme”. L. De Gioia di mastociti in topi affetti da neuropatia periferiTosca Marta Cecilia “Cellule staminali mesen- ca”. A. Colombo chimali da tessuto adipose nella rigenerazione Alfieri Antonella “Ruolo di antagonisti dei recettissutale”. A.L. Vescovi tori GABAa nell’attività di reti di neuroni corticali Trovesi Camilla “L’uscita dalla mitosi in presen- di topo embrionale e postnatale”. E. Wanke za di danni al DNA richiede le proteine del FEAR Bagnati Marta “Identificazione di nuovi autoantiin FEAR in S.cerevisiae”. M. Clerici geni nel diabete di tipo I”. A. Ronchi Turati Laura “Mannose-Binding Lectin: un nuovo target terapeutico nel danno ischemico cere- Bagnobianchi Alessia “Inibizione dell’asse PI3KAKT-mTOR come strategia per ridurre la prolifebrale”. B. Costa razione di cellule tumorali”. G. Giagnoni Vanini Roberto “Analisi comparativa della risposta a diversi stress ossidativi nei lieviti “. L. Banfi Daniele “Modulazione orexinica delle correnti GABAergiche nel V strato della corteccia Brambilla prefrontale murina”. A. Becchetti Vecchi Mauro “Role of apoptosis-associated pathways in neural stem cell differentiation”. A. Bargna Federica “Il DNA Barcoding entra nel quotidiano: l’autenticazione di prodotti ittici di Colangelo largo consumo”. M. Casiraghi Verga Viola “Studio della protein chinasi GSK3 (Glycogen Synthase Kinase 3) in fibroblasti mu- Bernareggi Davide “Analisi dell’espressione e rini NIH3T3 trasformati dall’oncogene K-ras in della funzione di Oct4 in cellule staminali neurali condizioni di diversa disponibilità di glucosio”. murine”. A. Vescovi M. Vanoni Betta Ardisson Giulia “Ghrelin and des-acyl ghVilla Alessandro “Analisi molecolari e computa- relin as anti atrophic factors in the skeletal muzionali della regolazione intra-dominio di hSOS1, scle- in vitro and in vivo studies”. S. Nicolis il maggior attivatore della proto-oncoproteina Bravi Luca “Studio del profilo di espressione Ras”. M. Vanoni proteica correlato alla risposta ad agenti antiVilla Francesco “Screening genomico di ultra proliferativi in un pannello di linee cellulari del centenari: associazione del gene FOX03A con la tumore della mammella”. P. Tortora longevità in una popolazione del sud dell’Italia”. Carcano Lorenzo “Morfologia e CaratterizzazioG. Lucchini ne di Biofilm (S.cerevisiae)”. M. Milani Zagarrì Elisa “Clonaggio, espressione e purificazione e caratterizzazione enzimologica di Conti Valentina “Ruolo di Necdin nel differenziaisoforme mutate del recettore tirosin-chinasico mento dei mesoangioblasti e nella rigenerazioFGFR2b coinvolte nel carcinoma endometriale”. ne muscolare”. S. Ottolenghi M. Lotti Corradi Sara “Role of Mesd in modulating Wnt Zanini Fabiana “Valorizzazione di scarti agro- signaling in organogenesis and disease”. S. K. forestali mediante l’utilizzo di metodi chimici ed Nicolis enzimatici”. B. La Ferla [ 22 ] Costalunga Christian “Tossicità in vitro da PCB: Lazzati Silvia “Studio dei tumori secernenti valutazione della genotossicità in una linea cel- ACTH”. B. Costa lulare di trota (RTG-2)”. A. Santagostino Leggio Patrizia “Caratterizzazione citogenetiDi Gioia Marco “Studio del ruolo delle cellule ca-molecolare di un cromosoma soprannumedendritiche nella migrazione e nell’attivazione rario: descrizione di un caso e correlazione con delle cellule NK a livello dei linfonodi periferici”. il fenotipo”. S. K. Nicolis F. Granucci Malara Francesca “Qualità dei laghi italiani e Di Venosa Alessandra “Nuove tossine di taranto- definizione delle condizioni di riferimento in rela riconoscono la famiglia dei canali del potassio lazione alle normative europee”. P. Galli hERG”. E. Wanke Mejetta Stefania “The loss of a wild type copy of Federici Silvia “Filogeografia di pelobates fuscus Prep1 gene accelerates tumorigenesis in Eμ-myc (amphibia: pelobatidae) e identificazione mole- mouse model of lymphomas”. S. Ottolenghi colare di un patogeno in popolazioni naturali di Micheli Laura “Esposizione pre-natale e postanfibi”. M. Casiraghi natale di ratti a una miscela ricostituita di PCB: Fragola Giulia “Development of a protocol for cinetica di accumulo e distribuzione nei tessuti producting induced pluripotent stem cells and della madre e della prole”. A. Colombo study of the contribution of Histone H3 Lysine 27 Trimethylation to nuclear reprogramming”. S. Minafra Tommaso “Quantificazione dei livelli di una miscela di pcb ed analisi morfologica della Nicolis gonade maschile in ratti maturi esposti durante la gestazione e l’allattamento”. A. Colombo Frigerio Emanuele “Microglia e cellule staminali neuronali: cross-talk e modulazione dello stato Nobili Paola “Generazione e caratterizzazione infiammatorio”. B. Costa di un modello animale “Double Hit” di epilessia cronica associata a malformazioni dello svilupGalimberti Valentina “Cross-talk tra autofagia e po corticale”. A. Becchetti apoptosi nella morte neuronale”. A. Colangelo Orlando Antonina “Ruolo degli endocannabiGemma Marta “Dosaggio delle catecolamine, dei noidi sulla fisiologia dell’endometrio umano”. loro metaboliti e dell’enolasi neurono-specifica G. Giagnoni nei pazienti pediatrici con sospetto di neuroblastoma”. A. Zaza Pagani Chiara “Effetto renoprotettivo dell’associazione di ACE inibitore e di antagonista recetGrande Vito “Ruolo del fattore trascrizionale toriale di Endotelina-1 in un modello sperimenSox6 nel differenziamento eritroide”. A. Ronchi tale di Diabete di tipo 2”. B. Costa Grataroli Emanuela “Ruolo delle cisteine nella Paro Simona“Caratterizzazione dei complessi fibrillogenesi dell’atassina 3 in Escherichia coli”. proteici associati a SRPK2 da linea cellulare di E. Regonesi neuroblastoma”. S. Barabino Ierardi Maria Angela “Studi per l’identificazione Pema Monika “Un nuovo modello muridei residui coinvolti nel legame al substrato del- no di rene policistico: analisi molecolare della la pnpasi di Escherichia coli”. P. Tortora cascata chinasica di mTOR e suo ruolo nella cistogenesi”. S. Barabino Isella Mariachiara “Biofouling e deposito calcareo su acciaio al carbonio protetto catodicamen- Pezzolato Eleonora “Processi infiammatori indotti da particolato fine su sistemi in vitro”. M. Camatini te in acque marine fotiche”. D. Basso [ 23 ] Rizzetto Riccardo “Risposte adattative all’ipossia MASTER IN BIOINFORMATICS cronica in miociti ventricolari di ratto: variazione della corrente di sodio persistente”. A. Zaza Riccombeni Alessandro “Molecular Evolution of human cancer genes MDS2 and TCL6”. L. De Ronco Barbara “Studio delle modificazioni indot- Gioia te da inquinanti ambientali alle proteine di pollini allergenici”. S. Citterio Bracchi Lorenzo “La tutela del software: analisi della normativa a livello nazionale e comunitaRusso Ilaria “Conseguenze dell’ischemia/riper- rio”. L. Fontanesi fusione miocardica in topi knock-out per il recettore Liver X”. A. Zaza Daminelli Simone “Cluster analysis of MudPIT proteomic data for disease profiling and biomarSangalli Elena “Ruolo della fosforilazione ker discovery”. G. Mauri dell’atassina-3 nella patogenesi dell’atassia spinocerebellare di tipo 3 (SCA3)”. P. Fusi Gianti Eleonora “Exploring Protein Flexibility in Sansanelli Serena “Association study of candi- Drug Design Analysis of Hsp90 Conformations”. date genes for premature ovarian failure”. S. Ot- L. De Gioia tolenghi Mattara Manuela Sonia “Studio della proteina Sansoni Veronica “Studio del coinvolgimento Ras con metodi bioinformatici allo scopo di evidel gene SCN1A nell’Epilessia Generalizzata con denziarne il comportamento in soluzione e il legame a potenziali agenti farmacologici”. L. De Convulsioni Febbrili Plus”. R. Combi Gioia Stella Annalisa “Analisi genetica del polimorfismo Thr325Ile e valutazione biochimica dell’ini- Pagani Francesco “Costruzione di modelli prebitore della fibrinolisi, TAFI, in una popolazione di dittivi di proprietà ADME”. G. Moro centenari italiani”. S. K. Nicolis Lori Francesca “Il Progetto Val Borbera: Analisi Tumminello Leonardo “Studio della composizio- descrittiva della struttura della popolazione dell’ ne di exosomi urinari: ricerca di possibili biomar- isolato genetico della Val Borbera tramite i policatori del carcinoma renale”. M. Pitto morfismi del DNA mitocondriale e del Cromosoma Y”. G. Mauri Vatri Roberta “Studio del processo di domesticazione di Vitis vinifera mediante approcci molecoTraglia Michela “Progetto Val Borbera: Carattelari”. M. Labra rizzazione di tratti fenotipici in un’ampia popolazione geneticamente isolata per l’identificazione Venturati Sara “Valutazione degli effetti cito-genotossici indotti dal BDE-47 e dal BDE-100 sul di varianti genotipiche di malattie complesse”. bivalve Dreissena polymorpha mediante l’appli- F. Stella cazione di una batteria di biomarkers”. A Santagostino . Rogora Alessandro “Clustering relazionale e GO enrichment: un metodo per l’analisi di dati biologici e loro validazione”. V. Messina Zalfa Cristina “Caratterizzazione in vitro ed in vivo di linee cellulari staminali neurali umane hNSC, immortalizzate e non, tramite trapianto in Volpato Viola “Data Mining su dati microarray: un modello animale di demielizzazione focale”. classificazione dello stato di attivazione di cellule dendritiche”. F. Stella A. Vescovi [ 24 ] [ 25 ] 1.8 SEMINARS 26.05.2009 Roberto Spagnoli Sanofi Aventis, France Industrial Specificities of Bioconversions Part 1: Traditional and cutting-edge approaches 27.05.2009 Roberto Spagnoli Sanofi Aventis, France Industrial Specificities of Bioconversions Part 2: Traditional and cutting-edge approaches 28.05.2009 Roberto Spagnoli Sanofi Aventis, France Industrial Specificities of Biotechnology. Production of Proteins 28.05.2009 Meng Li Imperial College London, UK Midbrain dopaminergic neuron differentiation from pluripotent stem cells 3.06.2009 Ted Weinert University of Arizona, Tucson, USA Genome instability using yeast: its getting simpler, interesting and intriguing 5.06.2009 Manuela Battaglia San Raffaele Telethon Institute for Gene Therapy, Milano Terapie avanzate per ristabilire la tolleranza immunologica in patologie autoimmuni e/o dopo trapianto 6.06.2009 Paolo Malatesta Università degli Studi di Genova Progressione tumorale e dipendenza dall’oncogene in un modello di gliomagenesi 15.06.2009 Giovanni Cazzaniga Università di Milano-Bicocca, Milano Genetica molecolare della leucemia pediatrica 22.01.2009 Luca Canova Università di Pavia Strumenti di valutazione degli impatti ambientali: la Valutazione Ambientale Strategica e la VIA 23.01.2009 Luca Canova Università di Pavia Strumenti di valutazione degli impatti ambientali: Valutazione di Incidenza 26.01.2009 Davide Sassera Università degli Studi di Milano Midichloria: simbiosi tra un batterio intracellulare e le zecche ixodidae 28.01.2009 Jannette Carey Princeton University, USA Structure and function of WrbA, a novel flavoprotein 17.02.2009 Elly M. Hol Netherlands Institute for Neuroscience, The Netherlands Neurogenic astrocytes in the adult human brain: the role of GFAP-delta 24.02.2009 Peter Illes Rudolf-Boehm-Institute of Pharmacology and Toxicology, Leipzig, Germany Cross-talk between neurons and astrocytes in the brain by gliotransmitters and the Cariplo project 25.02.2009 Giampiero Corradin Université de Lausanne, Switzerland Exploiting stable protein domains for the identification of new malaria vaccine candidates 16.06.2009 Gioacchino Natoli IFOM IEO, Milano L’epigenoma e la regolazione trascrizionale dell’infiammazione 5.03.2009 Giorgio Colombo CNR, Milano Drug design to dynamics and function in macromolecular machines: what can simulations tell us 17.06.2009 Simone Fattorini Università di Roma la Sapienza Rarità e biogeografia della conservazione 9.03.2009 Artur Silva University of Aveiro, Portougal Styrylchromones: synthesis, transformations and evaluation of their antioxidant activity 22.06.2009 Ullrich Wuellner Universtatklinikum Bonn, Germany Pathogenesis of Polyglutamine disorders: SCA3 19.03.2009 Otto Holst Research Centre Brostel, Germany Structure and function of the bacterial cell envelope 25.06.2009 Luca Mollica Istituto Dulbecco Telethon c/o S. Raffaele, Milano Structural characterization of the interactions between HMGB1 and inhibitors of its pro-inflammatory activity 26.03.2009 Michael Krützen University of Zurich, CH The mother of all cultures - the vertical skill transmission syndrome applied to wild tool-using bottlenose dolphins 8.07.2009 Gennaro Agrimi Università di Bari Identificazione di carriers mitocondriali in S. cerevisiae e loro ruolo metabolico 26.03.2009 Richard Lavery Institut de Biologie et Chimie des Protéines, Lyon, France A mechanical look at protein structure 16.07.2009 Benjamin G. Davis Oxford University, UK Sugars and Proteins 15.04.2009 Giorgio Dieci Università di Parma Promoter demarcation by a subtelomeric protein in yeast 2.10.2009 François Lefoulon Servier, Neuilly sur Seine Cedex, France Upscaling synthesis of antiinflammatory glycomimetics 28.04.2009 Vincent L. Pecoraro University of Michigan, Ann Arbor, USA The Adventures of Metalloprotein Design 14.10.2009 Vadim Gaponenko University of Illinois at Chicago, USA The tale of Ras tails: K-Ras4B tail modulates proteinlipid and protein-protein interactions 8.05.2009 Olexandr Holovachov University of California, Riverside, USA Systematics of bacterivorous Cephaloboidea - a challenge of a morphologically diverse though genetically uniform group of nematodes 26.10.2009 Rolf Kinne Max Planck, Dortmund, Germany Dynamics of the Sodium-D-Glucose Cotransporter SGLT1: Cellular and Molecular Aspects 11.05.2009 Giorgio Bertorelle Università di Ferrara E’ possibile discriminare gli effetti della selezione, della demografia e delle reintroduzioni sulla variabilità genetica? Il caso del camoscio alpino 3.11.2009 Iros Barozzi IFOM- IEO, Milano Bioinformatics plays a central role in understanding the epigenome 26.11.2009 Michèle Studer Université de Nice Sophia Antipolis, France Area identity and cell-type specification in the mammalian brain 30.11.2009 A. Gräslund Stockholm University, Sweden The amyloid ß peptide involved in Alzheimer´s disease: NMR studies of molecular interactions and secondary structure conversions 2.12.2009 Miguel A. Valvano University of Western Ontario, Canada Assembly and export of lipopolysaccharide O antigen cell precursors across the bacterial cel membrane 18.12.2009 Andrè Junqueira Zaharenko Universidade de São Paulo, Brazil Diversity and complexity of sea anemone toxins targeting ion channels 18.12.2009 Giuseppe Legname Scuola Internazionale Superiore di Studi Avanzati (SISSA) Basovizza (TS) Prion Biology and Disease 15.05.2009 Ulrich Schueller Ludwig-Maximilians-University, Munich, Germany The origin of medulloblastoma – developmental biology meets oncology 18.05.2009 Alessandro Guffanti Genomnia srl - Lainate, Milano Strategie per l’annotazione funzionale di dati da sequenziamento massivo 21.05.2009 Giovanna Musco Istituto Dulbecco-Telethon, S. Raffaele, Milano Solution NMR: a useful tool to study protein-ligand interactions? 25.05.2009 Camilla Bellone Université de Geneve, Switzerland Reward dependence and addiction 26.05.2009 Laura Feltri DIBIT, Ospedale San Raffaele, Milano Laminin receptors and signals in peripheral nerve development [ 26 ] [ 27 ] Photo: Matteo Urbano RESEARCH GROUPS [ 28 ] 1 [ 29 ] MOLECULAR GENETICS OF DEVELOPMENT AND CELL DIFFERENTIATION IN MOUSE AND MAN MECHANISMS OF POST-TRANSCRIPTIONAL REGULATION OF MAMMALIAN GENE EXPRESSION AND THEIR ROLE IN HUMAN DISEASE Silvia Nicolis, Sergio Ottolenghi, Antonella Ronchi, Rebecca Favaro, Anna Ferri Reinaldo Alvarez, Silvia Barabino, Silvia. C. Lenzken, Gabriele Fontana, Silvia Vivarelli The development of complex organs and tissues, such as brain and the hematopoietic system, requires the ordered expression of key transcription factors controlling cell typeand tissue-specific gene expression. Stem cells represent the self renewing compartment of rapidly replicating cell types, as in the hematopoietic system, but are present, in small numbers, also in adult brain, heart and other organs which do not show active cell replication in adults. The group uses a common set of approaches (conditional and standard targeted mutagenesis in mouse, cell culture and gene transduction, chromatin studies, etc.) to investigate the role of key transcription factors in the development, maintenance and differentiation of a variety of stem cells. THE ROLE OF SOX2 IN STEM CELLS S.Nicolis, R.Favaro, A.L.M. Ferri, C. Lancini, J. Mariani, R.Caccia, V.Tosetti, Sox2 is a transcription factor critically involved in multipotency. Using Cre-mediated conditional ablation of Sox2, in vivo and in vitro, we are investigating the mechanisms of Sox2dependent regulation of the development of Embryonic and Neural Stem cells, and of their neuronal differentiation. Our results indicate an important requirement for Sox2 in the expansion of hippocampal neural stem cells early after birth (in mouse) and in the long-term maintenance of neural stem cells grown in vitro, as well as in early stages of neuronal differentiation (Favaro et al., 2009). Targets of Sox2, required for these activities, have been identified by genomic and proteomic studies, and by Chromatin Immunoprecipitation. The functional role of some of these targets has already been validated by in vitro lentiviral overexpression of the identified genes and in vivo functional rescue. Studies have also been started, based on the notion of the importance of Sox2 for neural stem cell biology, to assess a potential role of Sox2 overexpression in neural tumour development or maintenance (glioblastoma and medulloblastoma). Finally, the transcriptional repression of Sox2 by the homeobox gene Emx2 has been investigated by the study of transgenic and knock-out mice, and by in vitro transfection and protein/protein and protein/DNA interaction studies. Collaborations: A.Smith (Cambridge, UK) (neural stem cells and ES cells); F. Watt (Cambridge, UK) (Sox2 and skin stem cells); C.-L. Wei, P. Robson (Genome Institute of Singapore)(functional genomics of neural stem cells); S. Goldman (Rochester, NY) (human neural stem cells); D. Epstein (Pennsylvania University) (Sox2 in hypothalamus); C. Basilico (osteogenic stem cells). Work on tumours is being carried out with P. Malatesta and G. Corte (Genova). The role of Emx2 on the control of Sox2 expression is being studied with A.Okuda, (Saitama Medical School, Japan) and V. Zappavigna (Università di Modena). 2 MOLECULAR REGULATION OF THE c-KIT GENE IN HEMATOPOIETIC AND CARDIAC PROGENITORS. S. Ottolenghi, L.Cassinelli, M.Baldissera Using transgenic constructs, we identified a subset of c-Kit genomic sequences which drive expression of a reporter gene in Primordial Germ Cells and some of their descendants, as well as in Hematopoietic Stem Cells and early progenitors, and in Cardiac Stem Cells. Using Lentiviral transduction of transcription factors which might affect the regulation of c-Kit, Chromatin Immunoprecipitation and the Chromatin Conformation Capture Assay (3C assay), we are trying to define transcription factors interacting with the main regulatory areas of the gene in hematopoietic progenitors and in cardiac stem cell-like cells grown in vitro. THE ROLE OF SOX AND OTHER TRANSCRIPTION FACTORS IN HEMATOPOIETIC DEVELOPMENT AND GLOBIN REGULATION. A. Ronchi, S.Ottolenghi, C. Cantu’, M. Baldissera We previously identified a set of genes which are differentially expressed during the develoment of the mouse hematopoietic system and its initial differentiation (in fetal liver). We are now focusing on a number of differentially expressed transcription factors, among which some Sox-family factors. By in vitro transfection, in vitro protein interaction studies, Chromatin Immunoprecipitation assays, proteomic analysis and lentiviral transduction in primary mouse and human hematopoietic cells, we are trying to identify relevant functional targets of these transcription factors and to assess their effects on proliferation, differentiation and regulation of embryonic, fetal and adult globin synthesis. In particular, we showed that Sox6 greatly stimulates the differentiation to erythroid cells of human neonatal CD34 hematopoietic progenitors, and we identified some of its transcriptional targets. Collaborations: G. Ferrari (TIGET-HSR Institute, Milano), MD. Cappellini (University of Milano), I. Dianzani (Università del Piemonte Orientale). Another gene whose expression changes during erythroid differentiation is Gelsolin, an actin-severing protein involved in actin filaments remodelling. We detected several abnormalities in red blood cell maturation in the late hepatic phase of fetal development of gelsolin null mice. Collaborations: L. Spinardi (Fondazione Policlinico Milano), W.Witke (EMBL Monterotondo) and E. Reali (INMG, Milano). The research interests of our laboratory focus on the field of molecular neurobiology. By integrating the disciplines of protein biochemistry, cell biology, and molecular biology, we hope to gain a better understanding of the cellular and molecular processes underlying neuronal differentiation in normal and pathophysiological disease states. flect the association of CF Im with mature mRNPs and participate in coupling mRNA processing to later events in the life of mRNA. We are currently testing with different in vitro and in vivo approaches if CF Im plays a direct role in mRNA transport. CELL STRESS AND RNA SPLICING Our laboratory studies the molecular mechanisms involved in the processing of pre-messanger RNA transcripts in the neuronal cells. Eukaryotic messenger RNA precursors (premRNAs) are synthesized and processed in the nucleus prior to their export to the cytoplasm, where they serve as templates for protein synthesis. Transcription is coupled spatially and temporally to capping of the pre-mRNA at the 5’end, to splicing of introns and to 3’end polyadenylation. In the nervous system, alternatively pre-mRNA splicing plays a crucial role in the synthesis of specific protein isoforms that participate functions such as learning and memory, neuronal cell recognition, neurotransmission, ion channel function, and receptor specificity. We are studying the processing of eukaryotic pre-mRNA, with major emphasis on the role of the arginine-serine (SR) family of proteins, and their kinases, in the regulation of alternative splicing. To complement our biochemical studies we use a cell biological approach and look at the intracellular distribution of these factors by fluorescence and confocal microscopy. The main lines of research in the laboratory are: 1. Multiple roles of SR proteins in RNA processing 2. RNA processing and signal transduction 3’ END PROCESSING AND TRANSPORT OF mRNAS We have characterized the intracellular localization of the 3’ end processing factor CF Im and we have shown that it shuttles continuously between the nucleus and the cytoplasm in association to mRNA. Nucleo-cytoplasmic shuttling may re- Coupling of pre-mRNA splicing to extracellular signals is crucial for altering splicing patterns according to the physiological state of cells. Since protein phosphorylation is often the response of cells to external signals, our working hypothesis is that alternative splicing pathways will be ultimately regulated by phosphorylation-dependent signal transduction cascades. We have recently established a cellular model that will allow us to elucidate the molecular changes in the alternative splicing machinery induced by the oxidative stress response. Oxidative stress arising from mitochondrial dysfunction has been proposed as concurring to the pathogenesis of many neurodegenerative diseases, including Parkinson Disease and Amyotrophic Lateral Sclerosis (ALS). Defects in the splicing of individual mRNAs have also been observed in the affected tissues of ALS patients. Based on these observations we are investigating in our cellular model whether oxidative stress can induce aberrant alternative mRNA processing thus contributing to the development and the progression of ALS. To better define the molecular mechanisms underlying the response to oxidative stress caused by mitochondrial insufficiency on a genome-wide scale we profiled at the same time SH-SY5Y neuroblastoma cell line upon treatment with a mitochondrial complex 1 inhibitor, and the same cell line stably transfected with wild type or mutant SOD1(G93A), found in some of the cases of familial ALS. To resolve the response into transcription and exon-level regulation we used Exon 1.0 ST GeneChips (Exon GeneChips, Affymetrix), which allow the definition of both transcription patterns and alternative pre-mRNA maturation events. Results are currently being evaluated. [ 30 ] 3 [ 31 ] MECHANISM CONTROLLING GENOME INTEGRITY MITOTIC PROCESSES PREVENTING GENOME INSTABILITY AND ANEUPLOIDY Maria Pia Longhese, Giovanna Lucchini, Michela Clerici, Veronica Baldo, Diego Bonetti, Nicola Manfrini, Marco Bazzi, Camilla Trovesi and Marina Martina The genome of living organisms can suffer both spontaneous and induced DNA damage. DNA double-strand breaks (DSBs) are among the most deleterious types of damage that can occur in the genome of eukaryotic cells, because failure to repair these lesions can lead to genetic instability. Eukaryotic cells have to cope with three different types of DSBs: accidental DSBs, programmed DSBs and natural DSBs. Accidental DSBs can arise during both mitosis and meiosis of eukaryotic cells either by DNA replication problems or by exposure to environmental factors, whereas site-specific DSBs are introduced into the genome in a programmed manner to initiate meiotic recombination in germ cells. Finally, eukaryotic cells contain natural DSBs that are represented by the ends of their linear chromosomes. The cellular response to either accidental or programmed DSBs requires highly conserved surveillance pathways, called DNA damage checkpoint and recombination checkpoint, respectively, which delay mitotic and meiotic cell cycle progression until DSBs are repaired. Mechanistically, the DNA damage checkpoint is related to the recombination checkpoint. In fact, highly conserved protein kinases, including mammalian ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and RAD3-related (ATR), as well as their S. cerevisiae orthologs Tel1 and Mec1, are necessary to activate both the DNA damage and the recombination checkpoint. Not surprisingly, defects in these networks result in a variety of diseases ranging from severe genetic disorders to cancer predisposition and accelerated aging. In contrast to accidental and programmed DSBs, the physical ends of eukaryotic chromosomes are protected from checkpoints and other events that normally promote DSB repair. This differentiation is thought to be the consequence of a unique organization of chromosomal ends into specialized nucleoprotein complexes called telomeres. When chromosome end protection fails, dysfunctional telomeres are targeted by the DNA repair and recombination pathways. The outcomes of such events at telomeres range from the generation of chromosomal abnormalities, general hallmarks for cancer cells in humans, to permanent cell cycle arrest and cell death. Given the different fates of DSBs and telomeres, it is remarkable that Tel1/ATM and Mec1/ATR are telomere-associated and are involved in ensuring telomere length and identity, implying that the difference between a DNA break and a telomere is less pronounced than previously assumed. Our research project aims to elucidate the molecular mechanisms protecting telomeric ends and controlling the cellular response to DSBs during both the mitotic and meiotic cell cycles. In particular, we are using different approaches in order to provide new insights into the roles of ATM/Tel1 and ATR/Mec1 checkpoint kinases in sensing, processing and signalling mitotic and meiotic DSBs and telomeres. Moreover, we are searching for new molecular targets of these kinases and we are studying how these mechanisms are coupled to cell cycle progression and interconnected with each other. Valentina Rossio, Laura Merlini, Elena Galati, Ilaria 4 Catusi, Roberta Pascolutti, Erica Raspelli, Giovanna Lucchini, Roberta Fraschini and Simonetta Piatti Genetic instability involves gain or loss of genetic information and is thought to be one of the major causes of cancer development. An altered chromosome number, referred to as aneuploidy, is a hallmark of cancer cells. Mistakes during mitosis may be responsible for the abnormal karyotypes of many human tumour cells and have an important role in oncogenesis. The integrity of the genome depends upon checkpoints that delay cell cycle progression until errors have been corrected, thus ensuring the genetic stability of a cell’s lineage. Checkpoint defects can pave the road to chromosome alterations and, ultimately, to cancer. Similarly, recent findings indicate that human cells undergoing a faulty cytokinesis accumulate numerical and structural chromosome aberrations, presumably due to the formation of multipolar spindles. Thus, cytokinesis needs to be tightly regulated in order to avoid aneuploidy. Our group studies these issues using the budding yeast S. cerevisiae as model organism. In particular, we are focusing on three main research topics: REGULATION OF MITOTIC PROGRESSION BY THE SPINDLE ASSEMBLY CHECKPOINT Once mitotic chromosomes are duplicated into two sister chromatids, their segregation is mediated by a bipolar microtubule spindle, to which they attach via their kinetochores. When the sister kinetochores of each chromatid pair are captured by microtubules emanating from opposite spindle poles, the chromosome becomes bi-oriented. Finally, the onset of anaphase, when sister chromatids split and migrate to the spindle poles, is one of the major points of no return in the cell cycle, and unbalanced chromosome segregation at this stage will inevitably result in the production of aneuploid cells. The spindle assembly checkpoint (SAC) keeps anaphase under check until all chromosomes are bi-oriented. In case of errors, the SAC sends an inhibitory signal that delays the separation of sister chromatids and mitotic exit until bipolar attachment is achieved. The target of the SAC is the Cdc20/APC ubiquitin ligase, which is normally required for sister chromatid separation and mitotic exit. We study some aspects of SAC activation and switch-off in yeast. CONTROL OF MITOTIC EXIT AND CYTOKINESIS BY THE SPINDLE POSITION CHECKPOINT In most eukaryotic cells the site where cytokinesis takes place is dictated by the position of the mitotic spindle. In budding yeast, conversely, the site of cell division, the bud neck, is established at the G1/S transition, concomitantly with bud emergence and much earlier than bipolar spindle formation. The spindle position checkpoint delays cytokinesis in the presence of misoriented spindles. The spindle position checkpoint operates through down regulation of the small GTPase Tem1, acting at the top of the mitotic exit network (MEN), a signal transduction cascade that drives inactivation of mitotic cyclin-dependent kinases and is strictly necessary for mitotic exit and cytokinesis. We are investigating the molecular mechanisms of this process. REGULATION OF CYTOKINESIS BY Dma1/2 PROTEINS We implicated two ubiquitin ligases (Dma1 and Dma2) in the control of cytokinesis. We showed that they are required, together with the PAK kinase Cla4, for deposition of the septin ring at the bud neck, which is in turn essential for proper spindle positioning and subsequent cytokinesis. In addition, Dma1 and Dma2 participate to the spindle position checkpoint. Therefore, Dma1 and Dma2 are likely to be crucial for preserving genome stability. Dma1/2 proteins are functionally redundant and they share the same structural organization as S. pombe Dma1 and human Chfr and Rnf8, which are all involved in checkpoint mechanisms. We hypothesised that Dma1/2 may ubiquitinate protein(s) that regulate septin ring assembly or function and we are trying to identify their possible targets through genetic screens and biochemical analysis of candidate proteins. [ 32 ] 5 [ 33 ] SYSTEMS BIOLOGY OF CELL PROLIFERATION AND DIFFERENTIATION Lilia Alberghina, Marco Vanoni, Paola Coccetti, Ferdinando Chiaradonna, Anna Maria Colangelo, Elena Sacco, Claudia Cirulli, Paola DeCandia, Daniela Gaglio, Lara Sala Danna, Marco Gaviraghi, Roberta Palorini, Chiara Balestrieri, David Metalli, Daniele Colombo, Farida Tripodi, Laura Gotti, Sandra Viggiani, Martina Fragni, Viktoria Tsiarentsyeva, Annalisa D’Urzo The research groups of L. Alberghina and M. Vanoni are developing a modular systems biology approach to the study of cell cycle (most notably of the G1/S transition) in the model organism, Saccharomyces cerevisiae, as well as in normal and transformed mammalian cells. The approach involves both “wet” experiments as well as computer modelling and simulation. Experimental data are used to extract information on network topology leading to mathematical models and to estimate parameter values. In order to understand this complex phenomenon, it is mandatory not only to study the core machinery driving the cell cycle, but also its modulation by genetic and enviromental conditions, including nutrient and growth factor availability, as well as the interconnections with differentiation, signal transduction and cell death pathways. Ultimately, these approaches should lead to a more rational and more efficient drug discovery process. CK2 ACTIVITY CORRELATES WITH GROWTH RATE IN BUDDING YEAST Paola Coccetti, Farida Tripodi, Claudia Cirulli, Veronica Reghellin, Marco Vanoni, Lilia Alberghina CK2 is a highly conserved protein kinase involved in different cellular processes, that shows a higher activity in actively proliferating mammalian cells and in various types of cancer and cancer cell lines. A clear demonstration of the relationship between CK2 activity and growth rate has not been provided so far. We used Saccharomyces cerevisiae as a model organism to investigate this question, since yeast growth rate can be strictly modulated by changing nutritional conditions in batch or by changing dilution rates in chemostat cultures. We found that although levels and localizations of both catalytic and regulatory subunits were not affected by the carbon source, CK2 activity was significantly lower in ethanol-growing cells than in glucose-growing ones. In chemostat-grown cells, CK2 activity was higher at higher dilution rates, while no difference was observed in cells grown at the same dilution rate in media supplemented with either ethanol or glucose. These results strongly indicate that the growth rate, and not the carbon source, is the major factor controlling CK2 activity in yeast. NUTRITIONAL MODULATION OF CELL CYCLE PROGRESSION IN SACCHAROMYCES CEREVISIAE STUDIED BY BIOCHEMICAL AND POST-GENOMIC TECHNIQUES Marco Vanoni, Paola Coccetti, Stefano Busti, Laura Gotti, Lilia Alberghina Combining genetic, physiological, biochemical and post-genomic techniques we are studying nutritional modulation of the cell cycle with the final aim to characterize the connection of nutrient-sensing signalling pathways (i.e, Tor, Zinzalla et al, 2007) with proteins involved in the G1/S. We could show so far that at least short- and medium-term effects of glucose addition on cell cycle progression are independent of gluco- se uptake and that the Gpr1/Gpa2 pathway, but not the Snf3/ Rgt2 pathway. plays a direct role in setting cell mass. A postgenomic analysis of the effect of altering the FAR1 gene dosage showed unexpected links with metabolism and signaling pathways. Sn+1/AMPK PROMOTES S-PHASE ENTRANCE BY CONTROLLING CLB5 TRANSCRIPTION IN BUDDING YEAST Paola Coccetti, Stefania Pessina, Sara Busnelli, Marco Vanoni, Lilia Alberghina The Saccharomyces cerevisiae Snf1 protein kinase has been reported to be required for adaptation to glucose limitation and for growth on non-fermentable carbon sources. We present novel findings indicating that Snf1, the key regulator of cellular energy, is also involved in yeast cell cycle control. The lack of Snf1 a-catalytic subunit down-regulates the growth rate and CLB5 expression, delaying Sld2 phosphorylation and G1/S transition, in cells grown in 2%, but not in 5% glucose. A non-phosphorylatable Snf1 rescues the slow growth phenotype, whereas a wild type or a phosphomimetic mutant is required to rescue growth rate and the G1/S delay. Using either Snf1 or Swi6 as a bait, a specific interaction of Snf1 with Swi6, the regulatory subunit of MBF, was detected. In conclusion, we describe a previously unrecognized role for Snf1 in transcriptional modulation of the G1 to S transition, differing from the reported AMPK role in controlling the G1/S transition in multicellular eukaryotes. MODELLING OF CELL CYCLE AND SIGNAL TRANSDUCTION PATHWAYS Lilia Alberghina, Paola, Coccetti, Ferdinando Chiaradonna, Daniela Gaglio, Matteo Barberis, Stefano Busti, Elena Sacco, Marco Vanoni Within a systems biology approach to the study of yeast cell cycle (Barberis et al., 2007), a detailed network structure of a second relevant regulatory step of the cell cycle, the exit from mitosis, derived from extensive data mining, has been constructed. Allowed to sketch a plan of the regulatory circuits governing the G1/S transition of normal mammalian fibroblasts and to develop a computational model for it (in collaboration with E. Klipp (Berlin) and G. Milanesi (CNR, MI) (Alfieri et al, 2009). The Epidermal Growth Factor Receptor (EGFR) signalling module is a major pathway regulating proliferation, differentiation, survival and motility in mammalian cells by activating Ras through Sos1. In collaboration with M. Farina and D. Liberati (Politecnico di Milano) we developed a mathematical model that describes functional inter-domains rearrangements regulating the Sos1 activity. The model is being tested and validated by simulation and used to predict the effect on catalytic activity of Sos mutants of clinical relevance. MECHANISMS OF NEURONAL APOPTOSIS AND NEUROPRO- 1 2 1_Mitochondrial morphology (green staining) and cytoskeleton (red staining) in NIH3T3 cells 2_A model of the regulatory role of CK2 phosphorylation in the G1/S transition. TECTION BY NGF Anna Maria Colangelo, Giampaolo Leoni, Sandra Viggiani, Martina Fragni, Lilia Alberghina Despite the heterogeneity of their anatomo-pathological features, apoptosis is regarded as the main form of neuronal death in neurodegenerative diseases, as well as in infectious (HIV dementia) and acute neurological disorders, such as trauma and ischemic stroke. The decrease of neurotrophic factors plays a key role in neuronal dysfunction and apoptosis. In our molecular model of neuronal apoptosis, mitochondrial function is modulated by molecules regulating survival/differentiation in response to Nerve Growth Factor (NGF) (Alberghina & Colangelo, 2006). Starting from this model, we are using NGF-differentiated PC12 cells and primary neurons to dissect mechanisms of neuronal apoptosis following oxidative stress and/or NGF deprivation, and investigate the correlation between mitochondrial dysfunction and cell cycle re-entry. Furthermore, besides its direct neuronal activity, our recent studies in collaboration with M. Papa (Second University of Napoli) indicate that neuroprotection by NGF also involves modulation of glial activation and neuro-glial network. In animal models of reactive astrocytosis induced by peripheral nerve injury (CCI), we have recently published that intrathecal administration of NGF is able to i) restore GFAP levels (marker of glial activation), ii) reduce expression levels of Substance P, IB4 and Cb (markers of sensitive fibers) and iii) modify the structure of gangliar fibers by mechanisms involving iv) modification of NGF receptors TrkA and p75. Further studies are in progress for a thorough characterization of this process both at glial and neuronal levels to implement our initial model of neuronal apoptosis and the role of NGF in neuroprotection. PRODUCTION OF RECOMBINANT HUMAN NGF (rhNGF) Anna Maria Colangelo, Sandra Viggiani, Giampaolo Leoni, Lilia Alberghina Based on our previous work about production of rhNGF (Colangelo et al., 2005), we are currently working, in collaboration with Primm and Blueprint Biotech, on rhNGF production for evaluating its efficacy in animal models of corneal ulcers, glaucoma and Alzheimer. CANCER AND METABOLISM: ROLE OF ONCOGENIC k-RAS PROTEIN IN METABOLIC REPROGRAMMING OF CANCER CELL Ferdinando Chiaradonna, Daniela Gaglio, Chiara Balestrieri, Lara Sala Danna, Marco Gaviraghi, Roberta Palorini, Marco Vanoni, Lilia Alberghina The relation between alterations of metabolism and transformed phenotype has recently received increased attention. By studying the role of the ras oncogene in cancer cell metabolic reprogramming, we showed that selective growth advantage of ras-transformed fibroblasts is lost upon growth in sub-optimal glucose concentration (Chiaradonna et al., 2006a, 2006b). In addition, we showed that such dependence of transformed cells on glucose availability correlates with a reduced mitochondrial Complex I activity ensuing reduced oxidative phosphorylation. Moreover, we showed, that glutamine shortage, strongly reduces of the proliferation ability of transformed cells, effect restored by adding the four deoxyribonucleotdes. These results indicated that fragility of ras-transformed cells to glutamine depletion is largely due to a reduced supply of DNA replication precursors in the presence of active signaling inputs leading to execution of the G1/S transition. Complex I dysfunction can be partially recovered by exogenous stimulation of PKA activity. Indeed, Forskolin treatment, of normal and transformed cells grown in 25 and 1mM glucose, protect transformed cells from apoptosis induced by glucose deprivation by enhancement of Complex I activity and by decrease of intracellular ROS accumulation. In order to identify and characterize specific metabolic alterations, able to promote growth of K-ras transformed tumor cells as compared to normal counterpart, we have generated transcriptomic and metabolomic data, upon alterations in nutrient availability. Preliminary results indicate that transformed cells rely on glycolysis more than TCA cycle for energetic processes. To further detail these metabolic alterations, we are currently performing a transcriptional comparative analysis, by using bioinformatics several tools, between NCI60 cancer cell collection and our model of K-ras dependent transformation. Preliminary results of a such comparative analysis show that our cellular mouse model of transformation has a good degree of similarity with human cancer cell lines encompassing Ras mutations and that all transformed cell lines (human and mouse) have common metabolic alterations as identified by transcriptional analysis. DESIGN, DEVELOPMENT AND MOLECULAR CHARACTERIZATION OF RASGRF1-DERIVED Ras INHIBITORS Elena Sacco, David Metalli, Michela Spinelli, Anna Bargna, Annalisa D’Urzo, Marco Vanoni Mutations of Ras proteins and their regulators are critical events in the pathogenesis of human tumors and developmental syndromes. In collaboration with S. Traversa (Creabilis Therapeutics spa) using Ras-sequestering peptides derived from the Ras activator RasGRF1 (Sacco et al., 2005) we obtained down-sized, auto-penetrating peptides that downregulate the Ras pathway. In collaboration with V. Gaponenko (University of Illinois) the inhibitory peptides and small sugarderived Ras inhibitors (provided by F. Peri, this Department) are being used as models for Ras-inhibitory drugs and as tools for improving molecular understanding of the Ras activation cycle. REAL TIME ANALYSIS OF PROTEIN-PROTEIN INTERACTION Annalisa D’Urzo, Elena Sacco, Marco Vanoni The BIAcore technology is being used as an effective tool to analyze protein/protein interaction and protein/ligand interaction in real time. The technique is being applied mostly to interaction of proteins of potential pharmaceutical interest, including the Ras oncoprotein, prion-derived peptides, cell cycle inhibitors and ataxin. [ 34 ] [ 35 ] YEAST AS A MODEL SYSTEM FOR STUDYING AGING AND STRESS-RELATED PROCESSES SIGNAL TRANSDUCTION IN EUKARYOTIC CELLS 6 Enzo Martegani, Sonia Colombo, Renata Tisi, Michela Ceriani, Fiorella Belotti, Loredana Amigoni, Silvia Groppi, Serena Broggi, Cinzia Giaccherini RAS/cAMP SIGNALLING IN YEAST Enzo Martegani, Sonia Colombo, Renata Tisi, Loredana Amigoni, Fiorella Belotti, Serena Broggi To investigate the localization of active Ras2 in vivo, we made a probe consisting of a GFP fusion with a trimeric Ras Binding Domain of Raf1 (eGFP-RBD3), which binds selectively Ras2GTP. The eGFP-RDB3 probe localizes at the plasma membrane and into the nucleus of cells growing on fermentable carbon sources, while in starved cells it accumulates only in internal membranes and mitochondria. Glucose starvation causes a delocalization of the probe, which rapidly relocalizes at the plasma membrane and into the nucleus after glucose addition. A similar pattern was observed in many mutants: cyr1Δ, gpr1Δ, sch9Δ, Ras2Val19, but not in gpa2Δ. In particular, in the in gpa2Δ strain, the probe accumulates in the mitochondria, both when cells are starved or growing on medium containing 2% glucose. Apparently Gpa2, but not Gpr1, is required for the recruitment of Ras-GTP at the plasma membrane and into the nucleus. We have recently found that the RasGEF Cdc25 is mainly accumulated in the nuclear compartment. PKA activity can inhibit Cdc25 nuclear import by phosphorylating two Serine residues nearby the NLS in position 806. Moreover, we found that after starvation for nutrients Cdc25 moves from the internal membranes to the plasma membrane, likely in order to yield a rapid activation of Ras in response to glucose addition. To study the role of the cAMP-PKA pathway in the coordination between cell growth and cell cycle we used a cyr1Δ pde2Δ msn2Δ msn4Δ strain. In this strain the PKA activity is not required for growth, but PKA can be activated by exogenous cAMP. We used this model to investigate the effect of PKA on cell growth parameters and on the expression of the main regulators of the cell cycle. Our results show that cAMP addition transitorily slows the increase in cell numbers, brings a massive increase in cell size and determines a transient decrease in the percentage of budding cells followed by an increase to very high value. Only with CLN1 deletion, a change in the growth parameters is observed compared with the control strain, indicating Cln1 as one of the target of PKA. CALCIUM SIGNALING IN YEAST Enzo Martegani, Renata Tisi, Fiorella Belotti, Silvia Groppi. Collaboration with: Rogelio Brandão Different stimuli induce a rapid increase in free intracellular Ca2+ concentration that is a signal for different signals transduction cascades. On the plasma membrane two calcium influx systems are present, the high affinity (HACS) and the low affinity (LACS) system. HACS is constituted by Mid1 and Cch1 proteins that appear to function as an unique Ca2+ influx system. By analysing the calcium response after glucose addition (in presence of 1 mM CaCl2) to nutrient-starved cells, Marina Vai, Ivan Orlandi, Matteo Viganò, Ambra Corti, Domenico Abete, Nadia Casatta, Cristina Mazzone we also identified a new uncharacterized Ca2+ influx system (transporter X) on the plasma membrane of Saccharomyces cerevisiae, which, differently from HACS system, is almost insensitive to nickel. Calcium is involved in an extremely wide range of cellular responses and calcineurin is one of the effectors activated by calcium. Calcineurin is involved in the regulation of calcium homeostasis and in many other cellular phenomena such as tolerance to high concentrations of Na+ and Li+, the response to pheromones and genes transcription regulation. We found that calcineurin can also be activated by nutrients, suggesting a cross-talk between the calcium signalling and PKA signalling. SIGNAL TRANSDUCTION MECHANISMS IN NGF-MEDIATED DIFFERENTIATION: Enzo Martegani, Michela Ceriani, Cinzia Giaccherini and Loredana Amigoni. Collaboration with Giovanna Berruti Ligand-activated receptors tyrosine kinase (RTK) endocytosis and endosomes-mediated transport to lysosomes are crucial to downregulate the cell proliferation signals. Receptors deubiquitination is a sorting signal for their trafficking to lysosome. UBPy/USP8 is a key regulator of cargo sorting and membrane traffic at early endosomes: it can deubiquitinate monoubiquitinated RTKs, like EGFR regulating their internalization. We study the involvement of the deubiquitinating enzyme UBPy in the internalization and stability of the TrkA receptor. Initially we have demonstrated that UBPy and TrkA interacts and that this interaction is NGF dependent. In un-stimulated PC12 cells UBPy localizes in cytosol while after NGF stimulus UBPy re-localizes to a perinuclear region that partially correspond to endosome system. Subsequently we have performed differentiation experiments on PC12 cells to investigate UBPy role in this pathway; cells transfected with UBPy-GFP fusion construct did not differentiate also after 72h from stimulation while cells transfected with UBPyC748A, a catalytically inactive mutant, present a high degree of differentiation. These data suggest that the overexpression of UBPy blocks differentiation probably promoting TrkA degradation. Using an antibody that recognize the extracellular domain of TrkA we have shown that UBPy overexpression alters the cellular residence of the receptor. In particular in unstimulated cells that overexpress UBPy or its catalitically inactive mutant (C748A) the receptor is almost completly sequestrated in the cytoplasm; after 15 minutes of stimulus with NGF it seems that in cells that overexpress UBPy the receptor disappear while in cells that overexpress the catalitically inactive mutant the cytoplasmatic localization of the receptor persists. These data suggest that the overexpression of UBPy could promote TrkA degradation. In future we will use siRNA to knock-down UBPy in PC12 cells to study the role of UBPy in the turnover and internalization of TrkA receptor. HISTONE MODIFICATIONS AND AGING IN SACCHAROMYCES CEREVISIAE Marina Vai, Ivan Orlandi, Domenico Abete, Ambra Corti, Cristina Mazzone In eukaryotic cells the genetic material is organized into chromatin, a complex structure composed of DNA and specialized proteins, histones. The dynamic organization of chromatin structure influences many cellular processes including aging. Changes in chromatin are mediated by histones modifications that include acetylation, methylation and ubiquitination. Among the enzymes involved in this regulation, the Sir2 family (Sirtuins) comprises the unique class of NAD+ -dependent deacetylases. Sirtuins are phylogenetically conserved and beyond silencing, they promote longevity. In yeast, Sir2 down-regulates histone acetylation at silent chromatin and recently it was suggested that this enzyme can influence also the carbon metabolism through a regulation of non-histone substrates. Due to the relevance of both metabolism and chromatin regulation on aging, we focused on the link between Sir2 activity and processes such as glycolysis, respiration and NAD synthesis. In this context, we also performed a molecular characterization of yeast strains that have altered mitochondrial NAD content. In collaboration with L. Palmieri, Università di Bari, Italy. THE FUNGAL CELL WALL AS A TARGET FOR ANTIFUNGAL DRUGS Marina Vai, Ivan Orlandi Opportunistic fungal infections are common and in immunodepressed patients are even life threatening. Clinically important fungal pathogens display varying degrees of tolerance to the widely used antifungals principally linked to their lack of fungicidal activity. The cell wall is an essential structure for fungal cells with no mammalian counterpart and consequently is an attractive target for antifungal drugs. We focused on a family of glucanosyltranferases that play an important role in the cell wall biogenesis and in the pa- 7 thobiology of several fungi. In particular, a functional characterization of three enzymes expressed by the pathogenic form of Paracoccidioides brasiliensis has been performed (in collaboration with C.M. de Almeida Soares, Universidade Federal de Goiás, Brazil). This fungus is the etiologic agent of one of the most prevalent human systemic mycosis in Latin America. RIBOSOME BIOGENESIS AND CELL SIZE CONTROL Marina Vai, Matteo Viganò, Nadia Casatta Sfp1 is a zinc-finger protein that promotes the transcription of many genes involved in ribosome biogenesis in response to nutrients and stress. Moreover, Sfp1 functions as a dose-dependent cell size regulator and its activity is modulated by the TOR and PKA pathways. Ongoing analyses aim to better define the alteration of regulatory circuits detected after SFP1 inactivation. Particular attention is devoted to characterize the pattern of expression of specific proteins that regulate growth and cell cycle progression in response to environmental stress conditions. In collaboration with L. Alberghina, this Department. SEAWATER ENVIRONMENTAL CHANGES AND BLEACHING OF CORAL REEFS Marina Vai, Ivan Orlandi During the past two decades the frequency of coral bleaching events has increased dramatically as a consequence of climatic changes and human activity in tropical oceans worldwide. Elevated temperatures of sea surface, chemical contamination and habitat destruction leave coral reefs with diminished resistance to additional perturbation resulting in reduced ecological integrity. Based on our experience on yeast, we are investigating the effects of thermal and high salinity stressess on reef coral species of Maldives atolls (in collaboration with P.Galli, this Department). Among the different mechanisms of cytoprotection we are focusing on the role of some heat shock proteins. [ 36 ] 8 [ 37 ] Rita Grandori, Maria Šamalikova, Carlo Santambrogio, Lorenzo Testa Mass spectrometry (MS) is employed, on one side, as an analytical tool for proteomics. On the other side, MS of intact proteins under non denaturing conditions is applied to conformational studies and binding analysis. This information is complemented by data obtained by other biophysical methods and bioinformatics. SIC1 CONFORMATIONAL PROPERTIES Lorenzo Testa, Maria Šamalikova, Rita Grandori The cyclin-dependent protein kinase inhibitor Sic1 is the key regulator of cell cycle progression and its coordination with cell growth. Despite intensive functional studies, Sic1 structural characterization has been undertaken only recently. We have applied complementary biophysical methods to conformational studies of pure Sic1 in solution. It can be concluded that the whole molecule exists in a highly disordered state and can, therefore, be classified as an intrinsically disordered protein (IDP). At the same time, the polypeptidic chain is endowed of some intrinsic structure, making it possible to recognize two distinct “structural domains”. The corresponding protein fragments have been expressed and characterized. It is found that the isolated kinase-inhibitory domain transiently visits compact states. Intrinsic structure and order propensity of IDPs is a very important feature that can mediate recognition of intracellular partners. The future aims of this project will include further structural characterization of order seeds within the Sic1 molecule and the investigation of their role in molecular recognition. In collaboration with Stefania Brocca, Marina Lotti, Lilia Alberghina, Marco Vanoni, Antonino Natalello, Silvia Maria Doglia ( this Departmen) t, Vladimir Uversky (Indiana University, IN), Frank Sobott (University of Antwerp, Belgium) STRUCTURAL AND FUNCTIONAL PROPERTIES OF BACTERIAL LIPASES Rita Grandori PROTEIN ENGINEERING AND INDUSTRIAL ENZYMOLOGY PROTEIN MASS SPECTROMETRY Bacterial lipases are one of the most valuable classes of industrial enzymes. We have investigated deactivation of the lipase from Burkholderia glumae (BGL) induced by temperature, pH and organic solvents, testing protein activity and conformational properties. The results show that, under most considered conditions, inactivation precedes denaturation. This conclusion fits well in the view that inactivation can occur without global unfolding under several physical–chemical conditions. Furthermore, role and accessibility of the calcium ion contained in BGL native structure have been investigated experimentally and by molecular-dynamics simulations. In the native protein, calcium is not accessible unless specific flexible loops are displaced. As a consequence of metal depletion the protein unfolds irreversibly and undergoes aggregation. The absence of the metal ion causes major structural transitions and leads to an increase in beta structure, particularly in regions that are largely unstructured in the native state and that contain the calcium coordination residues. In collaboration with Gaetano Invernizzi, Marina Lotti, Elena Papaleo, Luca De Gioia, this Department. CONFORMATIONAL AND OLIGOMERIC INTERMEDIATES OF AMYLOID PROTEINS Carlo Santambrogio, Rita Grandori Protein folding and unfolding transitions can be monitored by the charge-state distributions (CSDs) obtained by electrospray ionization mass spectrometry (ESI-MS). Since charge reports on structure and mass reports on association, combined information on folding and binding can be retrieved. Furthermore, ESI-MS allows direct detection of the distinct components populating heterogenous samples. These features make nano-ESI-MS a particularly valuable tool for the study of highly complex and dynamic systems such as amyloid proteins. In order to understand the molecular mechanisms that promote fibrillation, it is of central importance to identify the conformational and oligomeric intermediates of the aggregation process, and the environmental variables that affect their properties. We have undertaken this kind of studies on three different systems: a-synuclein (Parkinson’s disease), ß2 microglobulin (Dialysis Related Amyloidosis) and ataxin 3 (spinocerebellar ataxia type 3). Partially folded forms and oligomeric species that accumulate under fibrillation conditions have been identified and characterized. In collaboration with Paolo Tortora, Gaetano Invernizzi, Maria Elena Regonesi, Anna Maria Frana, Antonino Natalello, Silvia Maria Doglia (this Department), Stefano Ricagno, Martino Bolognesi (University of Milan, Italy), Giuseppe Legname (SISSA Triest, Italy) Marina Lotti, Stefania Brocca, Giusy Manuela Adamo, Electra Brunialti Pietro Gatti-Lafranconi, Ilaria Sambi We study enzymes employed in biocatalysis, model proteins and instrinsically disordered proteins (IDPs) by mutagenesis (both directed evolution and site directed mutagenesis) and biochemical and biophysical methods with the goal to highlight the molecular bases of stability, specificity and interactions and, whenever appropriate, to improve these properties. CONFORMATION, STABILITY AND BIOLOGICAL ACTIVITY Marina Lotti, Stefania Brocca, Pietro Gatti-Lafranconi, Ilaria Sambi, Electra Brunialti Different model proteins are used to investigate how function and conformation are related and affected by the experimental or physiological environment. A major focus are cold- adapted enzymes that we investigate both for understanding the molecular bases of temperature dependence and for obtaining novel biocatalysts for processes at low temperature. A lipase produced by the psychrophilic Pseudomonas fragi was randomly mutagenised to produce variants with higher kinetic stability and novel cold-active proteolytic and esterolytic enzymes were cloned from bacterial strains. We have also analyzed in detail the robustness of a lipase applied in biocatalysis to challenging reactions conditions, showing that loss of activity and denaturation are uncoupled and suggesting possible routes towards increasing the operational stability of this enzyme. The newest target of our research are proteins involved in the yeast cell cycle and characterized by the lack of a defined 3D structure in the absence of partner proteins (Instrinsically Disordered Proteins). Among IDPs, the kinase inhibitor Sic1 was produced as a recombinant protein and deeply investigated as for its intrinsic conformational properties. Notwithstanding its largely disordered scaffold, some degree of compactness was recognised and related to the presence of functional organisation in “disordered domains”. The effects on IDPs conformation of posttranslational modifications (i.e. glycosylation, phoshorylation) and of the interaction with non-physiological globular ordered proteins is also investigated. In collaboration with R.Grandori, S.M. Doglia, and L. Alberghina, L. De Gioia from this Department and S. Longhi at the CNRS of Marseille, France 9 STRESS RESPONSES AND AGGREGATION IN BACTERIAL CELLS Pietro Gatti-Lafranconi, Marina Lotti When expressed in a bacterial cell, recombinant proteins might aggregate in inclusion bodies. Aggregation impacts on the process of production of these proteins and further provides an interesting tool for the study of the molecular and physiological determinants of in vivo aggregation, due to the similarity with the process of deposition of amyloid fibrils. Besides, the ability to control the structure of proteins within the aggregates is of importance in biotechnology since it allows avoiding aggregation or obtaining “relaxed” inclusion bodies from which proteins can be recovered by mild treatments. We are also interested in the stress responses induced in the cells by the expression of recombinant proteins. In this field, we have characterized relevant changes in the cell envelope that depend on the protein expressed and on its state of solubility or aggregation In collaboration with S.M. Doglia, this Department MOLECULAR BASES OF YEASTS ADAPTATION TO HEAVY METALS Giusy Manuela Adamo, Stefania Brocca, Marina Lotti Metal ions, like copper ions, are essential for life being the cofactors of several key enzymes. However, when present in excess they might exert cytotoxic effects that have been also related to severe human pathologies. Cells of Saccharomyces cerevisiae and other related yeast species, grown in the presence of high copper concentration are our model to study the physiology and the molecular events occurring during the exposition to metals. This choice is supported by the high conservation of cellular and molecular processes between higher eukaryotes and the yeast Saccharomyces cerevisiae, that is therefore a valuable system to study basic mechanisms behind devastating illnesses such as cancer and neurodegenerative disorders. This research is aimed at studying the complex process of adaptation to heavy metal and to characterize the effects thereof, through a multidisciplinary approach that employs classical techniques of growth and viability assessment, flow citometry, biochemistry and biophysics. [ 38 ] [ 39 ] PROTEINS: STRUCTURE, FUNCTIONS, PATHOGENICITY AND CONJUGATION TO NANOPARTICLES 10 Paolo Tortora, Davide Prosperi, Maria Elena Regonesi, Gaetano Invernizzi, Serena Mazzucchelli, Francesco Aprile, Anna Maria Frana, Miriam Colombo, Paolo Verderio 1 2 STRUCTURAL AND FUNCTIONAL STUDIES ON EUKARYOTIC PROTEINS Paola Fusi, Chiara Pozzi, Valentina Pastori, Elena Sangalli, Alessandra Bigi, Matilde Forcella. STUDIES ON ATAXIN-3 PHYSIOLOGICAL ROLE Valentina Pastori, Elena Sangalli and Paola Fusi STRUCTURAL STUDIES ON PROTEINS CONTAINING GLUTAMINE REPEATS RESPONSIBLE FOR NEURODEGENERATIVE DISORDERS Maria Elena Regonesi, Paolo Tortora, Gaetano Invernizzi, Serena Mazzucchelli, Francesco Aprile, Anna Maria Frana Some neurodegenerative disorders result from the expansion of glutamine repeats (poly-Q diseases) in a set of proteins. Their misfolding and aggregation are likely to be involved in these disorders. The aim of this investigation is to gain insight into the molecular mechanism(s) by which expanded poly-Q stretches in ataxin-3 lead to the MachadoJoseph neurodegenerative disease. We are focusing on two major issues related to the molecular mechanism of the pathogenesis, i.e., the understanding of the protein’s physiological role, and the mechanisms by which ataxin-3 generates amyloid fibrils. These studies are performed on both purified molecules and cellular systems. As regards the investigations on the protein’s physiological role, our findings point to an involvement of ataxin-3 in sorting aggregated protein to aggresomes via microtubules. Furthermore, our studies on the mechanisms of amyloidogenesis, led to the structural characterization of normal and expanded variants by taking advantage of different analytical methods, notably FT-IR, circular dichroism and ThT fluorimetry, which highlighted major differences between the two. Our findings pave the way to a deeper understanding of the protein aggregation process and to development of new antiamyloidogenic compounds. DEVELOPMENT OF HYBRID NANOPARTICLES FOR BIOMEDICAL APPLICATIONS Davide Prosperi, Miriam Colombo, Paolo Verderio Nanomaterials within 1-100 nm hold tremendous potential in biomedical research thanks to a unique interaction with biological molecular systems. The production of high quality hybrid (bio)organic/inorganic nanoparticles endowed with inherent optical and magnetic properties represents a promising new road to the development of a novel generation of diagnostic and therapeutic agents for biosensing, preclinical investigations and clinical use. In particular, magnetic nanoparticles (MNP) appear as a very promising contrast agent for magnetic resonance imaging (MRI) clinical diagnostics. Indeed, MNP functionalized with cancer-specific targeting ligands can be used for early detection of tumors and of peripheral metastases. Thus, the aim of this project is to develop a small library of hybrid MNP consisting of a magnetic core and a protein shell responsible for cell receptor targeting. Furthermore, we are developing hybrid MNP for biosensing, protein purification and enzyme recycling. The preparation of nickel(II) nitriloacetic acid (NTA)-modified Fe3O4 MNP enables a one-step protein purification through binding to Histagged proteins. By combining spectroscopy investigations and bioactivity assays we aim at determining the effects of bioconjugation on protein structure and biofunctionality. In the effort to understand spinocerebellar ataxia type 3 (Sca3) pathogenesis, subcellular localization and proteolysis of ataxin-3, has been studied in our laboratory, using ataxins-3 with different polyQ lengths. Results showed a mainly cytosolic localization for both pathological and non pathological ataxins-3, but also showed that ataxin-3 is found in mitochondria. Our results also showed that ataxin-3 is extensively proteolyzed, while the pathological form is more resistant to proteolysis. The role of Ataxin-3 phosphorylation by casein kinase 2 (CK2) and glycogen synthase kinase 3 (GSK3) is also being investigated in our laboratory, in collaboration with Dr Coccetti (This Department) and Prof. Tedeschi (University of Milan). A series of phosphorylated sites have been identified and subjected to site-directed mutagenesis. Characterization of these mutants and their phenotypes, through confocal microscopy, subcellular fractionation and mass spectrometry analysis of phosphorylated residues, is currently under way. CHARACTERIZATION OF HUMAN SIALIDASES Alessandra Bigi, Alessandra Mozzi and Paola Fusi Sialidases or neuraminidases are widely distributed glycohydrolytic enzymes removing sialic acid residues from glycoproteins and glycolipids. The structure of the soluble human sialidase NEU2 was elucidated by our group in collaboration with Prof. Soichi Wakatsuki (Head of KEK Structural Biology Group, Tzukuba, Ibaraki, Japan). More recently a further characterization of natural substrates binding to ancillary sites has been obtained, through molecular dynamic studies in collaboration with Prof. De Gioia and Dr. Zampella (this Department), with the aim of designing more selective inhibitors towards viral sialidases, to be used as antiviral agents. Characterization of membrane bound human sialidase NEU4 is also being carried out in our laboratory. Solubilization studies showed that NEU4 is an extrinsic membrane protein, anchored to the membra- 11 ne though interaction with other protein(s). Cross-linking studies are currently carried our to identify these proteins. Moreover, NEU4 involvement in signalling pathways is currently being investigated in our laboratory. STUDY OF THE MECHANISM OF CROSS-PRESENTATION OF TUMOR ANTIGENS FROM BACTERIA-INFECTED MELANOMA CELLS Chiara Pozzi and Paola Fusi In Dr. Rescigno’s laboratory, at the European Institute of Oncology (IEO) in Milan, a new immunotherapy protocol for metastatic melanoma patients has been developed, based on the vaccination of patients against Salmonella followed by the intratumoral injection of a non-virulent, but invasive, strain of S. typhimurium. Our group, in collaboration with Dr. Rescigno, aims at understanding the basis of the observed systemic anti-tumor response and at elucidating the bacterial determinants responsible for this phenomenon. Results suggest that bacteria facilitate processing of tumor antigens within the tumor cell and that these antigens are transferred to the dendritic cells (DCs) via gap junctions without the need of phagocytosis. Our data also strongly suggest that upregulation of connexin 43 and TLRs engagement are involved. CLONINING AND EXPRESSION OF A TREHALASE FROM CHYRONOMUS RIPARIUS TO BE EXPLOITED AS A TARGET FOR BIOINSECTICIDES Matilde Forcella, Raffaella Schirone and Paola Fusi Trehalase inhibitors have a great potential as human safe bioinsecticides, this enzyme playing a key role in insect metabolism. A trehalase has been purified in our laboratory from the Diptera Chironomus riparius, showing a different specificity towards many insecticides, compared to mammalian enzymes. Molecular cloning of its gene has been achieved and expression of recombinant protein is currently underway in our laboratory, as well as testing of new synthetic bioinsecticides (imminosugars), in collaboration with Prof. Parenti and Prof. Cipolla (University of Milano Bicocca). [ 40 ] 12 [ 41 ] MOLECULAR AND CELLULAR BIOPHYSICS PROTEIN SECONDARY STRUCTURE, STABILITY AND AGGREGATION S.M. Doglia, A. Natalello Structural properties and aggregation of different proteins relevant for biotechnology and biomedicine have been studied by complementary biophysical and biochemical approaches: in vitro for the amyloid protein ataxin3 and for proteins in the presence of betaine, as well as in vivo in E.coli cells expressing the GFP-GST fusion protein. In collaboration with the group of Prof. P. Tortora (BtBs) we studied the aggregation process of ataxin-3 (AT3), a polyQ-containing protein, and of a number of its variants to understand the relevance of the different protein regions in the assembly process. In particular, we examined by FTIR spectroscopy the role of the polyQ tract in the variants AT3Q24 and AT3Q55, respectively expanded below and above the pathological threshold. Indeed, in the case of the Q55 variant - but not in that of the Q24 - we observed, in addition to the intermolecular ß-sheet bands characteristic of protein aggregates and fibrils, two new infrared bands of high intensity at 1656 cm-1 and 1604 cm-1. By H/D exchange experiments we demonstrated that these bands were respectively due to the C=O and NH2 vibrational modes of glutamine side chains. The peak position of these bands indicated that the glutamine side chains are involved in strong H bond interactions, forming an ordered network of bonds. This network of additional hydrogen bonds, which is observed only for the variant with poly Q tract expanded over the pathological threshold, lead to a further stability of AT3Q55 fibrils that become SDS insoluble. This glutamine side chain rearrangement into an ordered hydrogen bond array points to a structural plasticity of AT3 aggregates. Interestingly, a structural reorganization within aggregates seems to be a common feature of protein aggregates of different origins, such as proteins in amyloid structures and in bacterial inclusion bodies, where a molecular reorganization of already formed aggregates was found to occur without any disaggregation of the aggregate precursors We completed the study of the effect of the osmolyte betaine on protein misfolding and aggregation in vitro, in collaboration with the research group of Dr. A. de Marco (IFOM-IEO, Milan). Interestingly, we found that betaine can induce protein misfolding and aggregation depending on its concentration, and is also able to disrupt preformed insoluble aggregate into soluble oligomers. In collaboration with the group of Prof. Marina Lotti (BtBs) and with that of Dr. A. de Marco (IFOM-IEO, Milano) we also investigated in vivo the expression of recombinant GST-GFP fusion NEW THERAPEUTIC APPROACHES FOR CHRONIC PAIN Silvia Maria Doglia, Antonino Natalello, Anna Maria Villa Gabriella Giagnoni, Barbara Simona Costa, Francesca Comelli 13 protein in native, misfolded and aggregated form in E. coli. By several complementary biophysical and biochemical techniques we found that protein misfolding and aggregation lead to a significant reorganization of the cell membrane - with reduction of permeability and fluidity - and of the host protein expression, sharing several features with other stress responses. CONFOCAL FLUORESCENCE MICROSCOPY OF INTACT CELLS AND SURFACES S.M. Doglia, A.M. Villa, A. Natalello By laser scanning confocal microscopy (LSCFM) with photon counting detection we completed the study of mitochondria in human carcinoma cells under living conditions, employing two vital fluorescent probes, ethidium bromide (EB) and rhodamine 123 (R123), in order to visualize respectively their mtDNA and membrane potential. In all the examined cell lines (MCF-7, MCF-7/DX, and A549), two mitochondria populations were observed, characterized by a different intracellular localization (in central cytoplasm and in the cell periphery), membrane potential and morphology. Interestingly, the population of peripheral mitochondria has been found to be a peculiar signature only of transformed cells. Important differences were also observed in the EB fluorescence of the two populations, reflecting a different accessibility of EB to mtDNA, in turn related to the mtDNA transcription and replication activity. To quantitatively correlate the level of EB fluorescence in mitochondria to their mtDNA status in living cells, in collaboration with the group of Dr. P. Fusi (BtBs) we investigated the interaction of EB with mitochondria in human neuroblastoma cells SHSY-5Y, where mtDNA replication can be modulated inducing cell differentiation by retinoic acid. In collaboration with the group of Prof. Claudia Riccardi (Physics Dept, Unimib) and with that of Prof. Marina Lotti (BtBs), we studied the adsorption of model proteins on polymer surfaces functionalized by plasma treatments. Examining the intrinsically fluorescent protein GFP and a fluorescent labelled fibrinogen, we evaluated by LSCFM the fluorescence of the adsorbed proteins on the treated surfaces. In collaboration with Dr. Diletta Ami (IRCCS Policlinico San Matteo, Pavia) and with Prof. Carlo Alberto Redi (IRCCS Policlinico San Matteo and Università di Pavia) we started to study by infrared microspectroscopy the developmental stages of two types of murine oocytes (Surrounded Nucleolus e Not Surrounded Nucleolus). Pain is a sensory system that under normal conditions is protective and adaptive, serving as a warning signal for the body. When dysfunctional pain signalling occurs, pathological pain ensues. Despite over fifty years of research there are not yet effective treatments for chronic pain, and pharmacological or physical attempts to control pathological pain give results not lasting over time. In fact, majority of these treatments have a limited effectiveness or produce unwanted side effects. This unsatisfactory therapy can drastically decrease the quality of life. Thus, the search for new drug molecules to alleviate this intractable pain is priority nowadays. Our group believe that one possibility to successfully treat chronic pain is to develop drugs that are not aimed to suppress the neuronal activity but that target important modulators of chronic pain instead of neurons. The further research performed this year aimed to support such hypothesis. hyperalgesia development during chronic inflammatory conditions. Concerning the involvement of TLR2 and TLR4 in the development of acute inflammatory painful conditions, the data showed that these receptors did not contribute to this type of pain. In fact, carrageenan-induced thermal hyperalgesia was still present in knockout mice in the same degree of wild-type ones. The explanation could be related to the fact that this inflammatory model produced mild to moderate glial activation when compared to a chronic inflammation or a peripheral nerve injury which produce an intense glial activation, so suggesting that glia responses depend on the type of stimulus. The blockade of TLR2 and TLR4 signalling pathway in concert with the blockade of other pathways could lead to develop multi-targets pharmacological strategies for the treatment of chronic pain syndromes. THE ROLE OF TOLL-LIKE RECEPTORS IN CHRONIC PAIN ONSET AND DEVELOPMENT Increasing evidence suggest a pivotal role of mast cell-deriving factors in the onset of chronic pain, particularly neuropathic pain. Mast cell mediators which can sensitise nociceptors include histamine, tumor necrosis factor a (TNFa) and nerve growth factor (NGF). Notably, mast cells also express the trkA receptors and thus NGF binding may cause mast cell degranulation leading to a further release of NGF and many other proinflammatory and pronociceptive mediators, finally leading to peripheral sensitization and hyperalgesia. We have employed the chronic constriction injury of the sciatic nerve, CCI, and the administration of palmitoylethanolamide, PEA, known to behave as a local autacoid capable of downregulating mast cell activation. The findings obtained after PEA treatment clearly indicate that this compound is able to significantly delay the mast cell recruitment in the early phase of nerve injury-induced neuropathic pain and to significantly inhibit mast cell degranulation during the subsequent phase. Importantly, there is a good correlation between the timecourse of PEA effect upon neuropathic pain showed in our previous work (Costa et al., 2008) and its action upon mast cell activity shown herein, supporting the idea that such nonneuronal cells may be an important and valuable pharmacological target to treat neuropathic pain since, unfortunately, the current neuronal-direct drugs are still unsatisfactory for many patients. By using TLR2 and TLR4 knockout mice in different series of acute, persistent and chronic pain assays, we demonstrated that TLR2 and TLR4 are required for chronic pain responses, in particular for neuropathic pain states. Our results, showing the striking reduction of thermal hyperalgesia and mechanical allodynia after sciatic nerve injury in tlr2-/-and tlr4-/- mice, provide compelling evidence for a pivotal role of TLR2 and TLR4 in driving chronic neuropathic pain hypersensitivity. This result was consistent with the localization of TLR2 and TLR4 on microglia. Increasing evidence indicates that glial cell activation in the spinal cord plays a critical role in the initiation and/or maintenance of pathological pain with various aetiologies. Upon activation, microglia are characterized by an increased expression of cell surface markers and receptors, including TLR2 and TLR4, whose activation by endogenous ligands (such as molecules released from damaged cells or extracellular matrix breakdown products) lead to hyperalgesia and allodynia by releasing algesic substances, such as cytokines, prostaglandins, excitatory amino acids. TLR2 and TLR4 loss determined an attenuated pain responses by avoiding pro-inflammatory and pro-nociceptive mediator release. Moreover, our study provided for the first time evidence for a crucial role of TLR2 and TLR4 in thermal MAST CELLS MODULATORS TO TREAT CHRONIC PAIN [ 42 ] 14 [ 43 ] REGULATION OF NEURAL STEM CELLS IN PHYSIOLOGY AND EXPERIMENTAL THERAPY A B C 1_ Effect of RADA16-I functionalizations. RADA16-I functionalized with different bio-active motifs, respectively with: (A) BMHP1 (B) ALK (C) SDE. Each peptide shows a different stability in cross-b structure after 1 ns of MD simulation in implicit solvent. Angelo L. Vescovi, Elena Binda, Fabrizio Gelain, Francesca Taraballi, Carla Cunha, Stefania Antonini, Omar Villa, Daniela Ferrari, Cristina Zalfa, Laura Rota Nodari, Lidia De Filippis IDENTIFICATION OF NOVEL EFFECTORS REGULATING THE INVASIVENESS OF HUMAN GLIOBLASTOMA MULTIFORME BY EXPLOITATION OF A CANCER STEM CELL-BASED IN VITRO/ IN VIVO MODEL Angelo L. Vescovi, Elena Binda The invasive properties of malignant tumors of the central nervous system (CNS), and in particular of glioblastoma multiforme (GBM), are of great relevance, since they dramatically contribute to the poor prognosis of these neoplastic diseases. The handling of GBM represents a daunting challenge to clinicians, also considering the few therapeutic options available, none of which can significantly alter the inevitable lethal outcome of these tumors. Hence, the development of effective therapies would greatly benefit from the availability of GBM models that can reliably mimic the characteristics of malignant cells and the features of the human disease. The presence of tumor stem cells within some cancers has been suggested recently and our group has provided the first demonstration that adult human GBMs contain tumor neural stem cells (TNSCs) endowed with tumor-founding capabilities and identified the first TNSC-tailored strategy to inhibit their proliferation both in vitro and in vivo abolishing their tumorigenic potential. Notably, GBM-derived TNSCs, isolated from human glioblastomas, display all of the defining features of stem cells – namely extensive self-renewal capacity, ex vivo multipotentiality – and possess the ability to establish and expand GBM-like tumors in the brain of immune-compromised animals. Cell lines can thus be established that are extremely stable in culture and that can produce infiltrating tumors which resemble GBMs to a much greater extent than any of the cell lines previously available. We have collected human GBM specimens, from which we have established histopatological preparations, assessed chromosomal aberrations, and isolated the TNSCs. Together with the collection of the clinical parameters that have been gathered all throughout the disease development, these data and sample provided the background information regarding the patient and its tumor. By taking advantage of the availability of GBM-TNSCs and of GBM specimens, even from peritumoral areas, each single line has been subjected to a comprehensive characterization to assess its molecular and antigenic pattern, in order to identify antigens/ genes involved in the regulation of the invasive behaviour of the GBM itself. Continuous TNSC lines have been then employed to screen and assess their pattern of sentitivity to classical and new therapeutic drugs and agents. The same lines have been used to define the conditions leading to the establishment and validation of bona-fide models of GBMs following intracerebral transplantation in immune-compromised mice. We have used resonance magnetic imaging (MRI) to study the development of these tumors in living animals and correlate them with their progressive acquisition of neurological impairment, assessed by behavioural testing. MRI and behavioural scores shall be obtained at progressive stages of tumor development and have been compared to the pattern of expression of specific histological features and of candidate molecular and antigenic markers, at the same experimental times as MRI. Our strategy should enable us to define a heretofore unavailable radiographic, anatomical, behavioural, histopatological and molecular/antigenic correlate, describing a reliable GBM animal model. This have provided the opportunity to collect information regrding the molecular, cellular and histological changes associated with the various stages of GBM development, comprised from early tumorigenesis and the establishment of full blown tumors. This have also permitted the identification of new tumor markers thanks to the investigation of the antigenic molecular profile of cells from tumors by molecular analysis or FACS. Matching these findings with the clinical, genetic, chromosomal and histopatological data from the primary specimen from which a given TNSC line has been generated may lead to the establishment of a predictive system that helps to define patient-specific drug-responsiveness and lead to protocols that may help to devise patent-selective therapies, such as patient-tailored chemotherapy regimen, antibody-guided, radionuclide-targeted and differentiation therapies. NERVOUS REGENERATION VIA NANO-STRUCTURED SCAFFOLDS Fabrizio Gelain, Francesca Taraballi, Carla Cunha, Stefania Antonini, Omar Villa, Angelo L. Vescovi The nervous system is vulnerable to various disorders and for its intrinsic features, even limited damages may strongly affect important neurological and physiological functions. Our research is focused on the development of regenerative therapies specific for nervous injuries by using 2_Functionalized self assembling peptides and Neural stem cells. (A) 0G-BMHP1, (B) 2G-BMHP1, (C) 4G-BMHP1. (D) Positive and (E) negative controls (F) show significant differences for all possible coupled experimental groups except for (*) 0G-BMHP1 vs negative control, and (**) 2G-BMHP1 vs 4G-BMHP1. Values are reported as means ± standard error of the mean. the potentials offered by electrospun bio-protheses and nanostructured scaffolds. Electrospun tubes are polymeric scaffolds with high porosity and surface/volume relation. Self-assembling peptides are made from natural amino acids, they undergo self-assembly into nanofibers forming a scaffold, they can be mixed with growth factors and cells before the assembling process takes place upon exposure to physiological conditions of pH and temperature. Over the past year, we developed novel functionalized biomaterials in order to promote survival and differentiation of neural stem cells (NSC) in vitro and to regenerate nervous tissue in rodents suffering from spinal cord injury. We studied the peptides sequence in relation with their structure and biological functionality (Taraballi et al, 2009; Taraballi et al, 2010). Thus we used these self-assembling peptides in combination with electrospun tubes and we showed these prostheses are permissive micro-environments for regenerating nervous tissue in rat models of chronic and acute spinal cord injuries. Our approach is going to be further ameliorated via complementary strategies like scaffold loading with neurotrophic factors for drug delivery or seeding with neural stem cells for cell therapies. The results achieved by our group during the past year demonstrate that a strategy making joint use of stem cell technology, tissue engineering and nanotechnology could foster innovative solutions for regenerative therapies of nervous injuries. CHARACTERIZATION OF IMMORTALIZED AND NON-IMMORTALIZED HUMAN NEURAL STEM CELL LINES IN A FOCAL DEMYELINATION MODEL Daniela Ferrari, Cristina Zalfa, Laura Rota Nodari, Angelo L. Vescovi, Lidia De Filippis Human neural stem cells (hNSC) represent an optimal tool for the therapy of neurodegenerative diseases, since their ability to differentiate into neurons, astrocytes and oligodendrocytes. In the experimental setting the slow proliferation rate of hNSC represent a limit that can be overcome by the use of immortalized hNSC lines, such as v-myc (v-IhNSC) or c-myc T58A (T-IhNSC) transduced hNSC. We previously showed that, compared with hNSC and v-IhNSC, T-IhNSC rise high percentages of oligodendrocytes soon after removal of mitogens and are prone to a precocious differentiation. Given the differential in vitro oligodendrogenic potential we analyzed the progeny of hNSC, T-IhNSC and v-IhNSC in an animal model of focal demyelination induced by lysolecithine, to verify if local environmental cues inducing endogenous remyelination could address their integration and differentiation. The three lines were all able to survive after transplantation in the subventricular zone (SVZ) and to migrate along the ventricular wall. In particular, after 15 days from transplantation, hNSC and T-IhNSC were able to reach the striatum and the corpus callosum differentiating into O4+ and MBP+ oligodendrocytes, with T-IhNSC colonizing the lesioned area, whereas v-IhNSC remained mainly confined in the SVZ. A significant reduction of Iba1+ microglia activation was also observed in transplanted animals with respect to controls together with a partial switch of the globular macrophagic phenotype to the stellate morphology typical of resident microglia, suggesting an immunomodulatory effect of hNSC on the acute inflammatory reaction. These results support T-IhNSC line as a reliable cell model to study therapeutic applications of hNSC for demyelination disorders and show a differential tropism in vivo of hNSC depending on their intrinsic proliferation potential. We recently derived new non-immortalized hNSC lines from human fetal brain under GMP conditions to evaluate and compare the basal stem cell properties of different cultures derived from different brains. Preliminary data have shown that the different hNSC lines retain a stable kariotype over passages and comparable basal stem properties such as proliferation and multipotency. Recent experiments of transplantation into the brain of nude mice has shown that these lines are not tumorigenic, but in the next future their characterization in SOD rats (Lateral amyotrophic sclerosis animal models) will be aimed to determine if hNSC from different donors display different therapeutic potentials. [ 44 ] 15 [ 45 ] DENDRITIC CELLS IN INNATE AND ADAPTIVE IMMUNITY NEUROPHYSIOLOGY Paola Castagnoli, Francesca Granucci, Maria Foti, Ivan Zanoni, Tatiana Gorletta, Matteo Urbano, Federica Mainoldi, Anna Ranghetti, Anna Torri, Silvia Fumagalli, Francesca Pontiroli, Caterina Vitali, Caterina Bodio, Renato Ostuni, Simona Barresi, Achille Broggi, Aparna Venkatesh. Dendritic cells (DC) are a special type of leukocytes able to alert the immune system for the presence of infections. They are extremely versatile antigen presenting cells involved in the initiation of both innate and adaptive immunity, but also in the differentiation of regulatory T cells required for the maintenance of self-tolerance. Multiple animal models of infections and autoimmunity are used to investigate how DC can mediate all these diverse and almost contradictory functions. DENDRITIC CELLS BIOLOGY AND MOLECULAR MEDICINE Development of innate and adaptive immune response during the course of a microbial infection is dependent upon early interactions between incoming microorganisms with immature dendritic cells (iDCs) which are the first immune cells interacting with the microbial agents. The recent improvements of sequencing technologies, and in particular the publication of the initial version of the human and mouse genome sequences, have opened the field of large-scale functional approaches of biological systems. We employ high-throughput technologies to investigate fundamental aspects of the immune system and their roles in health and disease. In order to identify key cellular genes involved in these processes, we use a transcriptomic approach in which modifications of cellular transcriptome are analysed at several times post-infection. DENDRITIC CELLS AND NATURAL KILLER CELLS Natural Killer (NK) cells exert a direct anti-tumor and anti-microbial effect and can influence the development of adaptive T cell responses. Activation of NK cells is regulated by accessory cells such as dendritic cells (DC). Following activation, NK cells accumulate at the lymph nodes draining the site of infection, the key place in which DC and NK cell interactions occur. Taking advantage of the two-photon intravital microscopy technology the capacity of activated NK cells to reach the draining lymph nodes is investigated together with the DC-derived signals necessary for NK cell priming in inflammatory conditions induced by lipopolysaccharides. DENDRITIC CELLS AND REGULATION OF IMMUNE TOLERANCE The immune system of vertebrate animals has the capacity to respond to perturbations (invading pathogens, stress signals) limiting self-tissue damage. Tolerance to tissue antigens is achieved through a combination of thymic and peripheral events that eliminate or inactivate potentially dangerous T cells. Several mechanisms have been proposed to explain the induction of tolerance in peripheral autoreactive T cells. Taking advantage of different transgenic and knock out mouse models the mechanisms through which dendritic cells induce T cell tolerance in peripheral lymphoid organs are investigated. 16 Enzo Wanke, Marzia Lecchi, Elisa Redaelli, Francesca Gullo, Andrea Maffezzoli PHYSIOLOGY AND PATHOLOGY OF THE VISUAL SYSTEM FUNCTIONAL REGENERATION OF THE MESOCORTICOLIMBIC DOPAMINERGIC SYSTEM AS A MODEL TO STUDY NOVEL NEU- In the retina, ganglion cells convey the images processed ROREPARATIVE STRATEGIES from photoreceptors, horizontal and amacrine cells to the brain. Many different types of ganglion cells have been identi- We characterize the developmental and regeneration features of fied in vertebrates and humans but their characteristics and a brain circuitry involved in working memory and reward proces- physiological functions are still to be defined. Moreover, vi- sing, from the ventral tegmental area-substantia nigra [VTA-SN] sual dysfunctions are a consequence of several pathologies fibers projecting to the prefrontal cortex [PFC] and the comple- and the first symptoms that appear in patients affected by mentary glutamatergic pathways. By utilizing a co-culture sy- neurodegenerative diseases. stem adapted to multi electrode platforms (MEAs), we simul- By using flat-mounted retina of adult rodents as a model, taneously record from 60 electrodes, at brief (5-7 days in-vitro, we investigate ganglion cell electrophysiological properties div) and long-term (15-25 div) conditions, both spikes (bandwith in normal and pathological conditions. A project on diabetic 250-5000 Hz) and local field potentials (LFP, bandwith 1-200 Hz) retinopathy is performed with the collaboration of Prof. G. activity. After few days the co-cultures show a spontaneous acti- Cavaletti, Università Milano-Bicocca, Monza, and Dr. Marina vity in the form of bursts, trains of action potentials, respectively. Figliuzzi, Istituto Mario Negri, Bergamo. Surprisingly, the activity increase in parallel with the growth of new projections from one slice to the other and there was no need of evoking activity in VTA to observe activity in PFC. We will exploit the ability of endogenous stem/precursor cells of brain parenchyma to sustain the regeneration-remodeling of damaged circuitries and to possibly differentiate to new-born neurons and glia able to replace irreversibly damaged cells. [ 46 ] [ 47 ] NICOTINIC ACETYLCHOLINE RECEPTORS AND VOLTAGE-GATED K+ CHANNELS IN PHYSIOLOGY AND PATHOLOGY 17 RESEARCH IN CARDIAC CELL PHYSIOLOGY Antonio Zaza, Marcella Rocchetti, Claudia Altomare, Lucio Barile, Matteo Alemanni, Riccardo Chisci, Stefano Marangoni, Riccardo Rizzetto, Daniele Re Andrea Becchetti, Patrizia Aracri, Raffaella Morini,Paola Ambrosi The research of the cardiac cell physiology group is centered on the ontogenesis and modulation of myocardial excitationcontraction coupling. The research activity in 2009 was articulated in the following projects. EVALUATION OF FUNCTIONAL DIFFERENTIATION IN BETA-SARCOGLYCAN KO MESOANGIOBLASTS NICOTINIC RECEPTORS IN THE CEREBRAL CORTEX. PHYSIOLOGY AND IMPLICATIONS FOR THE PATHOGENESIS OF SLEEPRELATED EPILEPSY The cholinergic fibers ascending from the basal forebrain and mesopontine nuclei regulate cortical arousal and the sleepwaking cycle. ACh release is also involved in the control of synaptic plasticity and, consequently, of memory and learning. In general, the underlying mechanisms are poorly understood. We study the cholinergic and peptidergic modulation of transmitter release and its contribution to the regulation of the neocortical functions in normal and pathological conditions. We carry out patch-clamp recording in murine brain slices and couple the electrophysiological approach with neuroanatomical and molecular biological methods. In addition, we study the properties of mutant subunits of the human nicotinic receptor, linked to mendelian nocturnal frontal lobe epilepsy. Normal and mutant channels are expressed in cell lines and their properties studied in patch-clamp. We are also addressing the nicotinic modulation of the thalamocortical function in murine models of these pathologies. NICOTINIC RECEPTORS IN LUNG CANCER CELLS Nicotinic receptors are also expressed in non-neuronal tissues, including neoplastic cells such as small and non-small cell lung cancer cells. In these, they regulate cell proliferation, apoptosis 18 and angiogenesis, which is suggestive considering that smoking is an established risk factor for lung cancer. The mechanisms of these effects are still debated. Nicotinic receptors may modulate the release of growth factors and other molecules that exert autocrine and paracrine effects on lung cancer cells. We have observed that cancer cells express nicotinic receptors with distinct functional properties that may regulate processes occurring at different time scales, such as transmitter release and cell migration. We are also studying the effect of tobacco-derived carcinogenic nitrosamines on these ion channels. MOLECULAR COMPLEXES AND SIGNALING BETWEEN INTEGRIN RECEPTORS AND ION CHANNELS By mediating cell adhesion to the extracellular matrix, integrins regulate many developmental processes in the broadest sense (from cell choice between differentiation and proliferation, to tissue remodeling and organogenesis). Increasing evidence shows that considerable cross-talk occurs between integrins and ion channels, mediated by direct (i.e. formation of macromolecular complexes) or indirect interaction (e.g. through G proteins). In addition, ion channel stimulation frequently controls integrin activation or expression. We study the channel-integrin interplay in different cell types (from cortical neurons to leukemia cells). Alteration of these mechanisms has clear implications for pathogenetic processes, such as tumour invasiveness and several neurological diseases. The beta-sarcoglycan null transgenic mouse (BSG-/-), which is an animal model of dystrophic cardiomyopathy, was used to evaluate the in vitro amplification and differentiation of adult cardiac stem cells, named cardiac mesoangioblasts (cMabs). cMabs were isolated and cloned from the BSG-/- mice by the M. Sampaolesi group (University of Pavia), and whereas they examined the molecular aspect, the aim of our group was to investigate the functional differentiation of selected cell population. We analysed the functional differentiation of BSG-/cMabs and evidenced a potential correlation between the aberrant cardiac phenotype and development of the cardiomyopathy. The functional evaluation of this aberrant differentiation process, was carried out by studying BSG-/- cMabs in parallel with the skeletal muscle (C2C12 cell line) and neonatal cardiomyocytes (CM) as positive control cells. To discriminate between cardiac and skeletal muscle differentiation we analysed the excitation-contraction (EC) coupling characteristics: calcium dependency (cardiac property) and the nicotine response (skeletal property). As expected, whereas the ventricular CM immediately stopped their contraction after Ca2+ removing, the C2C12 cells mantained the contractile activity for a long time; BSG-/- cMabs showed a behaviour more similar to the C2C12 skeletal myoblasts one. Moreover, whereas C2C12 cells and BSG-/- cMabs contracted under nicotine (100 µM) superfusion, CMs resulted in a strong decrease of twitches or a lack of effects. Therefore, molecular and functional results showed an abnormal differentiation in BSG-/- cMabs, which is probably associated to the dystrophic cardiomyopathy of this mouse model. EFFECTS OF TWO INOTROPIC AGENTS WITH DIFFERENT TOXICITY ON SUBCELLUAR Ca2+ DISTRIBUTION Despite well established efficacy of anti-remodelling agents in the management of heart failure, inotropic support may still be required both in the acute and chronic stages of the disease. Such a need is unmet by the drugs currently available, which fail to improve the long term clinical outcome because of an increased risk of life threatening arrhythmias. This prompts the search for positive inotropic agents with a more favourable balance between inotropic and pro-arrhythmic effects (safety profile). Istaroxime is a novel inotropic agent that has a substantially better safety profile than digoxin. As digoxin, istaroxime inhibits the Na+/K+ pump, but, unlike digoxin, it also stimulates Ca2+ uptake by the sarcoplasmic reticulum (SR), an effect fully preserved in the failing heart and potentially accounting for the favourable safety profile. The present study aims to assess the role of direct RyR channel modulation in determining the difference in toxicity between istaroxime and digoxin. The major finding of this work is that the two drugs does not modulate RyR activity directly, but they differently modulate cytosolic Ca2+, which in turn affects RyR activity. EFFECTS OF CHRONIC HYPOXIA ON MYOCARDIAL ELECTRICAL ACTIVITY Chronic hypoxia (CH) is common in respiratory diseases, a condition in which secondary myocardial involvement is common. Moreover, CH results from uncompensated heart failure and might contribute to its evolution. The aim of this project is to study the effects of chronic exposure of rats to hypoxia (10% O2) on the physiology of cardiac myocytes. Myocytes isolated from the right ventricle (mechanically overloaded by pulmonary hypertension), were compared to those of the left ventricle (not mechanically overloaded). The study focused on the expression of 1) the “late Na+ current” (INaL), which is enhanced by acute hypoxia and may contribute to both electrical and contractile derangements, and 2) the outward potassium current (Ito), which is usually depressed in cardiac hypertrophy models. The results obtained so far indicate that the hypoxia protocol used caused marked right ventricular hypertrophy without clear-cut derangements on left ventricular function. [ 48 ] 19 [ 49 ] ECOLOGY OF MARINE AND MIGRANT BIRDS Maternal effects comprise a class of phenotypic effects where the genotype of a mother is expressed in the phenotype of her offspring, unaltered by paternal genetic influence. We are currently studying maternal effects mediated by nutritional constraints of the yellow-legged gull (Larus michahellis). Migratory connectivity describes the extent of the connection between the areas where populations of migratory animals spend different phases of their annual life- cycle. We have developed a novel method for quantifying migratory connectivity and delimiting highly connected sub-populations. This method may have important spin-offs in the assessment of effective conservation plans for migrators. Roberto Ambrosini Significant temporal changes in the timing (phenology) of bird migration are probably linked to recent climate change albeit the ecological mechanisms linking climatic conditions to migration phenology are still debated. We have first proved that long-distance migrants may be able to predict meteorological conditions in their breeding areas while they are still in Africa and adjust their migration schedule consequently. Since 1999 we are monitoring a large number of breeding colonies of Barn Swallow (Hirundo rustica) a small passerine bird that migrate each year between Europe and Africa and whose population suffered sharp declines in recent years. IDENTIFICATION AND ANALYSIS OF THE MOLECULAR BASIS AND PREDISPOSING FACTORS OF NEUROLOGICAL DISEASES, MAINLY IDIOPATHIC EPILEPSIES 20 Neurological diseases are frequently characterised by a complex inheritance with several genes and environmental factors acting together in determining the observed pathological phenotypes. In the majority of these disorders the genetic background and the molecular mechanisms underlying the clinical phenotype are not fully characterized yet. Among these disorders, idiopathic epilepsies are the most epidemiologically relevant representing a common and devastating neurological phenotype which is assumed to have a strong genetic component, being monogenic or oligo/polygenic with different recurrence risks in the same family. Even in monogenic epilepsy, ethiology, phenotypic manifestations and prognosis are indeed highly heterogeneous. Several loci associated with epilepsies have been mapped by means of linkage analysis, and mutations have been detected in genes encoding ion-channels, leading to hyperexcitability of cortical neurons through alterations in the channel function, as Romina Combi, Veronica Sansoni well as in genes not belonging to the channel family. However, these gene discoveries have been in the tiny fraction of epilepsies characterised by Mendelian inheritance. Moreover, even in these rare forms (of both adult and paediatric age), the identified mutations frequently account for a minority of patients suggesting therefore the existence of additional loci. To address the issue of the molecular and cellular basis of genetic neurological disorders, we analyse large cohorts of patients by means of an integrated clinical and molecular approach (comprising genetic counselling, DNA analysis, DNA sequencing, linkage analysis, CGH and SNPs microarrays). In particular, we search for new genes and new mutations involved in the pathogenesis of each disease performing also functional in vitro studies to evaluate the effect of the identified mutations. Moreover, we check the involvement of candidate predisposing factors in increasing the population risk for the disease. ZOOPLANTLAB - INTEGRATED RESEARCHES IN ANIMAL AND PLANT BIOLOGY Maurizio Casiraghi, Massimo Labra, Aldo Zullini, Michela Barbuto, Fabrizio De Mattia, Emanuele Ferri, Ilaria Bruni, Andrea Galimberti ZooPlantLab (ZPL) links applied and basic researches in the zoological, botanical and agronomic fields. Main projects are based on a molecular approach, but the integration of these data with other biological information is common and essential. For these reasons, ZPL has several collaborations with national and international teams. NEW METHOD FOR THE ANALYSIS OF BIODIVERSITY: APPLICATION OF PYROSEQUENCING TO THE STUDY OF SOIL ORGANISMS The study of biodiversity is becoming more and more central in the international scientific community. Our project meets this interest and proposes an innovative approach to the biodiversity investigation: the pyrosequencing on a massive environmental scale. Pyrosequencing allows to analyse a high number of samples in a short period of time, factors really useful in an environmental study on a large scale. This approach could be considered a pivotal project for biodiversity researches. Recently, most of the molecular environmental studies follow a metagenomic approach, however, in these kind of projects the huge amount of data, makes really complex and expensive the data management, with often the loss of taxonomic details. Our method, due to its concentration on only a fraction of the gene sequences obtainable, permits to achieve a high level of efficiency (comparable to that of a metagenomic approach) coupled with a higher taxonomic accuracy. The results of our large scale screening will be the base to develop a geographic information system of the soil biodiversity in Italy. This is a preliminary result, but plays a key role, being a proposal that could be followed by other researches on different taxonomic groups/kinds of soil/sampling areas. DNA BARCODING: A LINK BETWEEN BASIC AND APPLICATIVE SCIENCES TOWARDS AN INTEGRATED APPROACH TO TAXONOMY We are involved in the generation of a tool for the study of biodiversity based on an integrated approach to taxonomy, in which we propose the interaction of different level of taxonomic information. Taxonomy is an essential tool for biological studies, however this discipline is sometimes perceived as old-fashioned and descriptive. Taxonomy must face this perception and to renew itself, the main challenges are computerisation and a “reasoned” molecular approach. The framework provided by the DNA Barcoding initiative is the key of this renewed approach. Given these considerations, the goal of our projects is to de- 21 velop an identification system for different organisms. These following are our running projects on DNA Barcoding: 1) Food tracking: in particular on fish (both fresh and processed). 2) Parasitic nematodes: discrimination of filarial nematodes and their endosymbionts (Wolbachia). 3) Free-living nematodes: analysing natural population of free-living nematodes hosted in different habitats (i.e. water, moss, soil). 4) Birds: studying populations of non-autochthon species of birds. 5) Bats: studying national populations of bats species. 6) Aromatic plant species: setting DNA plant barcoding sequences to univocally identify each plant species. FROM GENES TO ECOSYSTEMS: DNA BARCODING AS A SYSTEM TO PROTECT BIODIVERSITY The main idea of the project is that to protect is essential to know organisms and environments to be protected. In this project we will couple the molecular identification system provided by the DNA barcoding approach with the analysis of the connectivity of protected areas in a fragmented environment. In the first part of the project we are working in collaboration with the Milan Natural History Museum, to create a reference database of regional organisms. In the second part of the project we are moving from the consideration that Lombardy region has several protected areas, but they are usually separated by highly civilizated parts, in which wild organisms are usually not allowed. In the project we will analyse the level of genetic connection among splitted areas, to detect, understand and (hopefully) protect ecological corridors. SCIENTIFIC EDUCATION: FROM THE UNIVERSITIES TO THE SOCIETY The main idea of the project is to use our scientific knowledges to produce systems for science education in collaboration with Italian associations (i.e. Lega Ambiente, WWF, Fondazione Idra, protected areas, etc). In the course of this project ZPL developed an educational kit for the water analysis, to be used by young kids, or in the school classroom. The kit has been pivotally tested with success on few dozens classrooms, but it will be implemented and directed to a vast majority of schools. [ 50 ] 22 [ 51 ] FRESHWATER AND MARINE ECOLOGY PROGRAMMED CELL DEATH (PCD) IN PLANTS Paolo Crosti, Massimo Malerba Paolo Galli, Fabrizio Stefani, Franscesca Benzoni, Giovanni Strona, Giovanni Aquaro, Simone Montano, Davide Seveso A HOST-PARASITE MODEL FOR THE DISPERSAL OF LESSEPSIAN SPECIES IN THE MEDITERRANEAN The 1869 opening of the Suez Canal created a direct link between the Mediterranean and the Red Sea, allowing the entry into the Levantine aquatic system of non-native species, particularly from the Erythrean basin, process that has accelerated in the recent years concurrently to the warming trend of the seawater. Among fishes Siganus luridus has proven to be extremely successful in colonizing a large part of the Eastern Mediterranean coasts up to Linosa Island, that constitutes the western boundary of the species distribution. The aim of the work is to provide a theoretical framework, through a metapopulation model, to explore alternative assumptions on the Lessepsian invasion by using information on the presence of fish parasite as fingerprint of the adult host arrival time. In the model, host populations are divided into identical interconnected sub-populations that are linked by dispersal and well-mixed with respect to parasite transmission. HEAD GLANDS OF MONOGENOIDEA: CANDIDATES FOR INDUSTRIAL PRODUCTION OF SURGERY BIOADHESIVES Surgical interventions and bleeding control rely on methods for the prevention of excessive blood loss. A number of haemostatic agents, both mechanical and based on biological compounds, are currently available. However, most of them show major drawbacks, like low efficacy, dependence on the coagulation status of the patient and a dry field requirement, which makes them unfit in emergency situations. Moreover some of them are not safe for the patients. The aim of this project is the production of a bioadhesive material produced by some plateminth fish parasites, belonging to the class of Monogenoids. These parasites are able to attach quickly and reversibly to the fish branchial epithelium, the attachment being mediated by two proteins which interact to yield an unsoluble adhesive complex. Parasite detachment is performed by a third, still uncharacterized, protein. The ability to bind reversibly to living tissues in an aqueous environment is a unique feature which renders this adhesive material most suitable to applications in the surgical field. BIODIVERSITY AND BIODIVERSITY PATTERNS OF SCLERACTINIA (CNIDARIA) IN THE GULF OF ADEN, YEMEN Coral reefs are known to be the most diverse marine ecosystem worldwide, and, as such, are receiving increasing attention both on account of their very high heritage value and as potentially major genetic reservoirs. Such a rapidly rising need for a better understanding of coral reefs overall biodiversity applies in particular to their fundamental component: the reef corals (Scleractinia). The objectives of the project are to capitalize on the existing scientific information and data, extend the study area from Balhaf to the Bir Ali and Mukallah areas and to develop them into a definitive and authoritative work that would represent a benchmark and a reference at the local and regional level, for reef coral biodiversity and its distribution patterns in the Gulf of Aden, Yemen. Therefore, the objectives are: To develop a reference collections of Scleractinia skeletons, digital in vivo images, and ethanol preserved voucher specimens from different sites along the Yemeni coast of the Gulf of Aden. To analyse the collected material and identify it at species level both by means of morphological and molecular means, in order to quantify Scleractinia diversity in the area of study. To evaluate Scleractinia biodiversity patterns along the Yemeni coast of the Gulf of Aden and investigate the relationships between such patterns and different environmental factors (e.g. the Arabian Sea upwelling). PROGRAMMED CELL DEATH (PCD) and its most studied form in animals, apoptosis, are genetically controlled processes present in all living organisms, aimed to eliminate unwanted or detrimental cells. In plants PCD plays a pivotal role in several developmental processes (formation of tracheary elements, sex determination, senescence) and it is involved in the responses to environmental stresses and in defence mechanisms (hypersensitive response, HR). Researches to elucidate the basic mechanisms of PCD in plants are in rapid expansion and at least three different forms of PCD based on the cell organelle first involved have been reported: a “nuclear” (apoptotic-like) form, typical of the defense response against pathogen attack, a “chloroplastic” form, typical of the foliar senescence, and a “vacuolar” form, typical of the maturation of the vascular elements. During the last years we showed that in sycamore (Acer pseudoplatanus L.) cultured cells fusicoccin (FC), a well known phytotoxin acting as a 14-3-3 protein-mediated activator of the plasma membrane H+-ATPase, induces a set of stressrelated responses (including the production of ethylene, H2O2 and NO), and PCD which only in a fraction of dead cells shows the typical hallmarks of apoptosis (cell shrinkage, chromatin condensation, nucleus and DNA fragmentation and release of cytochrome c from the mitochondrion). This suggests that FC can trigger different cell death programs. While the dependence of the stress responses on H2O2 and NO production induced by FC has been extensively investigated (Malerba et al., 2003; 2005; 2008), the possible role of ethylene as signal- 23 ling molecule for these responses is still unknown. Ethylene is an important gaseous plant hormone that is involved in many physiological and developmental processes of plants. It is well known that ethylene, which biosynthesis is stimulated by a variety of abiotic stresses as well as by pathogens or pathogen-derived elicitors, is involved in stress responses and it is assumed to play a role in the development of disease resistance. Several reports also indicate that, in addition to its role in physiological and morphological processes as well as in senescence-associated cell death, ethylene participates as a regulator in other developmental cell death, such as aerenchyma formation in hypoxic roots and endosperm cell death in cereals. Thus, in the last year, by means of Co2+, a well known specific inhibitor of ethylene biosynthesis, we investigated the possible involvement of ethylene in the stress responses induced by FC in sycamore cells: i) accumulation of dead cells and of cells with fragmented DNA in the culture; ii) production of H2O2 and NO; iii) accumulation of regulative 14-3-3 proteins in the cytosol and of molecular chaperone Binding Protein (BiP) in the endoplasmic reticulum; iv) release of cytochrome c from the mitochondrion. In addition, we compared the effect of FC on these parameters with that of the ethylene-releasing compound ethephon (2-chloroethane phosphonic acid). The results suggest that ethylene is involved in several of the stress responses above reported, including a form of cell death that does not show apoptotic features and possibly involves NO as signalling molecule. [ 52 ] [ 53 ] CONFORMATIONAL INVESTIGATION OF STRUCTURE-ACTIVITY RELATIONSHIPS IN PROTEINS AND BIOMIMETIC COMPLEXES 24 DESIGN, SYNTHESIS AND MOLECULAR RECOGNITION STUDIES ON BIOACTIVE COMPOUNDS AND BIOMATERIALS Piercarlo Fantucci, Luca De Gioia, Luca Bertini, Giuseppe Zampella, Elena Papaleo, Claudio Greco, Marco Pasi, Valentina Barbieri, Alessandro Di Domizio Francesco Nicotra, Laura Cipolla, Barbara La Ferla, Cristina Airoldi, Cristina Redaelli,Paolo Galliani, Alexander Orsato, Davide Bini, Cristiano Zona, Francisco Cardona, Nasrin Shaikh, Laura Russo DESIGN, SYNTHESIS AND MOLECULAR RECOGNITION STUDIES ON POTENTIAL DRUGS Francesco Nicotra, Laura Cipolla, Barbara La Ferla, Cristina Airoldi, Cristina Redaelli,Paolo Galliani, Alexander Orsato, Davide Bini, Cristiano Zona, Francisco Cardona DFT INVESTIGATIONS OF METALLO PROTEINS AND RELATED BIOMIMETIC COMPOUNDS Claudio Greco, Luca Bertini, Giuseppe Zampella, Piercarlo Fantucci, Luca De Gioia The project is aimed at elucidating the relationship between the three-dimensional structure of proteins containing metal ions and their function. In particular, recent studies have been focussed on hydrogenases, which are a family of enzymes able to convert, with a particularly high catalytic efficiency, protons and electrons into molecular hydrogen. The elucidation of the catalytic mechanism of hydrogenases is fundamental not only to understand the structure-function relationship in this enzyme family, but also for the design of novel synthetic compounds characterized by high catalytic activity. Hydrogenases and related synthetic compounds are studied in our laboratory using different quantum chemical techniques, ranging from Density Functional Theory calculations to QM/MM models. BIOINFORMATICS TOOLS AND MOLECULAR DOCKING TO STUDY PROTEIN LIGAND INTERACTIONS Giuseppe Zampella, Luca De Gioia, Piercarlo Fantucci Computed-aided drug design is a powerful tool that can nicely complement experimental studies aimed at the identification of novel compounds characterized by potential pharmacological activity. In our laboratory, molecular modelling and docking methods are applied to study protein-ligand interaction in several families of potential targets. Recently, molecular modelling and docking studies have contributed to the identification of watersoluble Ras inhibitors, and to the structural characterization of enzymes involved in lipopolysaccharide biosynthesis. COMPUTATIONAL INVESTIGATIONS OF PROTEIN DYNAMICS Elena Papaleo, Marco Pasi, Piercarlo Fantucci, Luca De Gioia Molecular dynamics simulations and homology modelling are used as main techniques with the aim of investigating structure-function relationship in enzymes and proteins. In particular, long and multiple simulations of biomolecular systems has allowed obtaining insights into biomolecular processes at the atomic level, which are often hardly accessible to experimental methods. Recent studies carried out in our laboratory have been focused in studying the effect of temperature on protein stability, as well as the interaction between enzymes and their cofactor or inhibitors. 25 The area of investigation of the research group ranges in the field of design, synthesis and biological evaluation of bioactive compounds. Particular attention is devoted to the generation of inhibitors, agonists and antagonists not only as new lead compounds in drug research, but also as tools to understand unknown biological pathways (chemical genetic studies). Furthermore, in a large integrated FP7 project, nanoparticles for diagnosis and therapy of Alzheimer Disease are developed. Synthetic targets focused in 2009 are: • Ligands of Abeta peptides and their conjugation to nanoparticles for diagnosis and therapy of Alzheimer Disease • Inhibitors of bacterial LPS biosynthesis as potential antibacterial agents • Regulators of SGLT1 as anti-inflammatory and antitumor agents • Drugs fused into glycidic structures, in particular in order to modulate the pharmacokinetic and the conformational properties • Glycidic scaffolds and their use for the synthesis of Gastrin Releasing Peptide (GRP) receptor agonists/antagonists NMR studies are performed for: • Structure elucidation • Conformational analysis • Epitope mapping studies (ligand-receptor interactions studies at atomic level) • Adhesion kinetic studies GENERATION OF SMART BIOMATERIALS Francesco Nicotra, Laura Cipolla, Nasrin Shaikh, Laura Russo The generation of smart biomaterials for tissue engineering requires mimicking natural ECMs that regulate complex morphogenetic processes in tissue formation and regeneration. Their functionality should be adjustable to a particular biological environment to obtain cell- and tissue-specificity. Ideally, one would create them from an array of biocompatible scaffolds decorated with an array of ligands inducing cell adhesion and/or proliferation and/or differentiation. Ideally, the biomaterial should include cell-adhesive ligands (such as integrin-binding peptides of the prototypical RGD family or carbohydrates such as hialuronic acid), binding sites for growth factor (GF) proteins, domains with susceptibility to degradation by cell-secreted or cell-activated proteases to facilitate bidirectional cell-matrix interactions, but also domains with structural function (such as the elastin-derived peptide sequence VPGVG). Synthetic networks can be obtained by cross linking of these biofunctional components (from an entire array of building blocks) by distinct linkage schemes. The use of such synthetic approaches in “bioactive” material design may allow matrices to be tailor-made for a specific cell or tissue. In collaboration with research groups expert in material science and in stem cells, a research project devoted to the functionalization of solid and gel scaffolds with properly selected peptides and sugars is under developement. [ 54 ] 26 [ 55 ] BIOORGANIC AND MEDICINAL CHEMISTRY MOLECULAR MODELLING AND COMPUTATIONAL CHEMISTRY Francesco Peri, Alessandro Palmioli, Matteo Piazza, Valentina Calabrese, Gaetana Damore Giorgio Moro, Gloria Saracino 27 1_Structure of the activated (TLR4-MD-2-endotoxin) 2 complex 2_Synthetic inhibitor bound to Ras INVESTIGATING THE LPS/TLR4 SIGNALING WITH A CHEMICAL GENETIC APPROACH: NEW MOLECULES TARGETING SELECTIVELY THE CD14 AND MD-2 RECEPTORS This is the main project of our group. In 2009 we projected and synthesized new molecules derived from natural sugars and from aromatic compounds that are active in modulating the LPS-mediated signaling. These compounds target selectively the CD14 and the MD-2 receptors and are lead compounds for the development of anti-sepsis, anti-inflammatory agents as well as vaccine adjuvants and stand-alone immunotherapeutics. The development of such specific chemical tools for the study of the endotoxin recognition process has been possible through an interdisciplinary research program that merged the expertise in organic and medicinal chemistry of the research unit of University of Milano (Italy) with that of immunologists and biochemists at the University of Iowa (USA). Positively charged monosaccharides with lipid chains have been developed by our group, that inhibit LPS and lipid A-induced cytokine production in innate immunity cells. These molecules are also active in vivo in contrasting septic shock and other syndromes, such as neuropathic pain, caused by TLR4 activation in microglia. Biochemical studies (2009) indicated that these monosaccharides inhibit the TLR4 pathway by selectively antagonizing the endotoxin binding to the CD14 receptor. In 2009 we also discovered new lipodisaccharides containing negatively charged sulfate groups, active as selective and mild agonists of TLR4. These molecules selectively target the MD-2 receptor and are promising leads for the development of nontoxic vaccine adjuvant. SUGAR-DERIVED RAS PATHWAY INHIBITORS We are involved in an ongoing project on the synthesis of novel molecules that are able to interfere with the signal transduction pathway of the Ras proteins. In previous years, since 2005, we have developed small molecules that are able to bind human Ras and inhibit the guanine nucleotide exchange that is the essential step for Ras activation. As constitutively active Ras mutants are responsible of the generation and growth of about the 30% of human tumor (in particular prostatic and colorectal cancers), small organic molecules that bind and deactivate Ras are potential highly selective antitumor drugs. The new compounds developed in 2009 include a panel of sugar-containing small molecules that were found to be active in inhibiting oncogenic Ras activation in vitro. These compounds made possible, in collaboration with the University of Chicago (USA), the determination at an atomic resolution of the Ras-inhibitor binding interface by means of NMR experiments. The interaction between our compounds and Ras was also detected by Isothermal Calorimetry (ITC). We are currently trying to co-crystallize our inhibitors with oncogenic Ras variants in collaboration with Dr. Nicolas Nassar (Stoney Brook Univeristy, New York, USA). NEW ANTI-INFLAMMATORY COMPOUNDS TO TREAT THE INFLAMMATORY BOWEL DISEASE (IBD) In 2009 our group started a research project aimed at finding new synthetic molecules as drugs to treat Chron disease and other inflammatory intestinal diseases. The project is currently funded by the AMICI association. Computational approaches based on Molecular Dynamics simulations, Quantum Mechanical methods and 3D Quantitative Structure-Activity Relationships are employed to study biological processes at the molecular level. The computational approach taken in our research on biological processes focuses mainly on three methodological areas. One includes a variety of methods based on Molecular Mechanics (MM) and Molecular Dynamics (MD). The second is an approach based on advanced Quantum Mechanical (QM) methods applied to model systems. The third is an approach aimed at obtaining statistical models through an analysis of data inferring relative Quantitative Structure-Activity Relationships (QSAR). As is well known, approaches based on MD theories are the only ones presently available to study complex systems like proteins in solution. The approach to the problem of protein structure at the classical level is even more acute when there is the modelling of interaction between proteins themselves, between protein and DNA fragments or between protein and substrates (as in drug discovery, toxicology studies or virtual enzyme engineering). However, MD methods are not completely free of difficulties, which are generated just by the very high number of degrees of freedom (about 105). In practice it is impossible to sample the phase space exhaustively due to the limitations in reliability of the final results. Given our awareness of the difficulties involved, we took great care when applying the MD to maximize the degree of phase space sampling, using the repeated trajectory technique, the essential dynamics technique extensively, in order to extract the low frequency motions of biological relevance, and the replca exchange technique to overcome the potential energy holes problem. In collaboration with Ugo Cosentino, this University Specific topics of interest are: - properties of prion protein peptides (collaborations with dott. Alessandra Villa - Karolinska Institutet, Stoccolma, Svezia; dott. Mario Salmona – Istituto Mario Negri – Milano) - thermal stability of the Sulfolobus solfataricus Carboxypeptidase active site (collaborations with prof. Paolo Tortora - Dipartimento Biotecnologie e Bioscienze) - characterization of a new contrast agent for selective targeting in Magentic Resonance Molecular Imaging (collaborations with prof. Francesco Nicotra and prof. Laura Cipolla – Dipartimento Biotecnologie e Bioscienze) - interaction of the HIV-1 viral protein R with the adenine nucleotide translocator protein [ 56 ] 28 [ 57 ] OUTER MEMBRANE BIOGENESIS IN ESCHERICHIA COLI Alessandra Polissi, Paola Sperandeo, Silvia Sommaruga, Riccardo Villa The cell envelope of Gram-negative bacteria represents an effective permeability barrier against external noxious agents and cell envelope components are primarily involved in host colonization or infection. However many aspects of cell envelope biogenesis remain still obscure. A peculiar structure of Gramnegative envelope is the outer membrane an asymmetric lipid bilayer with phospholipids and LPS forming the inner and outer leaflet, respectively. LPS is a complex essential molecule relevant to initial bacterial attachment, evasion of host defenses, and establishment of infection. Despite structure and composition of the OM have long since been known, many aspects of its biogenesis still remain obscure. My laboratory has recently identified new proteins required for transport of LPS to the outer membrane. The research of the group focuses on two main interconnected objectives: MOLECULAR MECHANISMS OF LPS TRANSPORT TO THE OM Alessandra Polissi, Paola Sperandeo, Riccardo Villa Genetic and biochemical approaches are being used to identify new proteins implicated in the LPS biogenetic pathway and to study how these proteins interact. By dissecting the mechanisms of LPS transport, identifying new components involved and understanding how the protein machinery is assembled we aim at obtaining a deeper knowledge of outer membrane biogenesis, a fundamental process for bacterial cell life and pathogenicity. This not only will allow a better understanding of the mechanisms that control bacteria-host interactions but is also a prerequisite and a significant step forward to the second objective of this research. THE LPS BIOGENETIC PATHWAY AS TARGET FOR THE DESIGN AND SYNTHESIS OF NOVEL ANTIBACTERIALS Alessandra Polissi, Silvia Sommaruga, Paola Sperandeo Structural and functional studies of target proteins known to play key roles in the biogenesis of LPS are currently ongoing. As LPS is a strategic structure for bacterial virulence and an important modulator of the innate immune response LPS biogenesis is an important topic to be explored in order to shed light on pathogen-host interaction, being also a largely unexplored target for drug development. Target proteins under study are being purified from both, Escherichia coli and Pseudomonas aeruginosa, an opportunistic pathogen that causes a wide variety of infections in compromised patients, given that intrinsic and acquired resistance of the pathogen to most conventional drugs makes the treatment of such infections very difficult. Structural information will be used to design and synthesize novel lead compounds that inhibit the LPS biogenetic pathways in the hope to develop new therapeutic strategies against infectious diseases. INDUSTRIAL BIOTECHNOLOGY: ADAPTATION OF THE MICROBIAL CELL FACTORY TO TECHNICAL CONSTRAINTS 29 Danilo Porro, Luca Brambilla, Paola Branduardi, Gianni Frascotti, Tiziana Fossati, Laura Dato, Stefano Bertagnoli, Vera Codazzi, Simone Passolunghi, Giorgia Rossi Evolution has produced a huge variety of organisms living in radically different environments. Some of these organisms have evolved metabolic pathways leading to the synthesis of potentially useful compounds that are difficult to produce by the chemical industry or that are environmentally harmful to manufacture. It has to be reminded that the fundamental basis of evolution is the need to survive and reproduce, not to produce potentially important and commercially valuable products. Indeed, interesting proteins and metabolites are very often produced by wild type organisms in such low concentrations that biotechnological exploitation is today still impractical. rDNA platforms allow, sometimes in a quite simple way, the development of new micro-organisms leading to the production of new products. The existing rDNA applications for eukaryotic microbial hosts are the results of less than three decades of global experience developing processes for the production of heterologous proteins, fine chemicals, vitamins, nutraceuticals, biofuels and animal nutritional aids such as amino acids. Unfortunately, the majority of the rDNA engineering processes, besides the challenges encountered during the research and development phase, fail during the scale-up phase. Indeed, in an industrial process, the microorganism used as a mean of production, is exposed to several stresses that can lead to lower production, lower productivity and lower yield of the product. A stress is typically caused by stressors (or stimuli), i.e. agents of physical, chemical or biological nature that represent a change in the usual intracellular or extracellular conditions. It is therefore highly desirable to consider strategies for minimizing stress. In this respect, our laboratory has developed (i) a series of cell factories producing heterologous compounds, like proteins, enzymes, organic acids, biofuels and nutraceuticals (ii) a series of yeast strains with improved resistance to specific constraints imposed by the process itself and (iii) a study and a model of the correlation between the size of the single yeast cell and its cellular metabolism. MICROBIAL CELL FACTORIES AND MAIN PRODUCTS For twentyfive years our group has been involved in the production of homologous and heterologous proteins in a variety of yeast hosts, from the conventional S.cerevisiae, to the nonconventional Kluyveromyces lactis, Torulaspora delbrueckii, Zygosaccharomyces bailii applying different fermentativetechnologies (batch, continuous and fed-batch). As an example, we developed yeast strains capable of producing organic acids from glucose (i.e. lactic and ascorbic acid). More recently, our attention is also focused on the production of biofuels. IMPROVING RESISTANCE IN MICROBIAL CELL FACTORIES In order to develop an effective process of production, cell factories not only have to produce the molecule of interest, but they also have to face the constraints often imposed by the process itself. We proved that yeast cells engineered to produce ascorbic acid acquire an increased robustness in respect to different limiting conditions such as low pH, oxidative stress and the presence of high concentrations of organic acids. In addition, said resistance can be achieved also by modulating other key elements. PHYSIOLOGICAL AND MODELLING STUDIES OF THE CELL FACTORIES The control of both metabolism and cell cycle progression by modulating the cellular environment has a key role in the regulation of growth and cell proliferation and production in all organisms. Specific attention has been devoted to study and to model the correlation between the size of the single yeast cell, its metabolism, redox unbalance and cofactors alterations. Redox cofactors are involved in hundreds of metabolic reactions and cells devoid large amounts of nutrients and energy to maintain their redox balance. Changes in NADH/NAD ratio as well as in cofactor localization can deeply affect either cellular growth and accumulation of biotechnological relevant metabolites. Alterations of redox homeostasis in yeast is studied coupling genetic and physiological techniques. [ 58 ] [ 59 ] SCIENTIFIC PUBLICATION INDEX, GRANTS [ 60 ] [ 61 ] [3.1] PUBLICATIONS ALBERGHINA L (2009) Systems Biology for biotechnological innovation. J. BIOTECHNOL. vol. 144; p. 165-166. ALBERGHINA L, COCCETTI P, ORLANDI I (2009) Systems biology of the cell cycle of Saccharomyces cerevisiae: from network mining to system-level properties. BIOTECH ADVANCES vol 27; p. 960-978. ALBERGHINA L, HOFER T, VANONI M. (2009) Molecular networks and system-level properties. J. BIOTECHNOL. vol. 144; p. 224-233. ALFIERI R, BARBERIS M, CHIARADONNA F, GAGLIO D, MILANESI L, VANONI M., KLIPP E, ALBERGHINA L (2009) Towards a systems biology approach to mammalian cell cycle: modeling the entrance into S phase of quiescent fibroblasts after serum stimulation. BMC BIOINFORMATICS, vol. 10; p. S16. AMBROSINI R., MøLLER AP, SAINO N (2009) A Quantitative measure of migratory connectivity. JOURNAL OF THEORETICAL BIOLOGY, vol. 257; p. 203-211. AMI D, NATALELLO A, SCHULTZ T, GATTI-LAFRANCONI P, LOTTI M, DOGLIA SM, DE MARCO A (2009) Effects of recombinant protein misfolding and aggregation on bacterial membranes. BIOCHIMICA ET BIOPHYSICA ACTA - PROTEINS AND PROTEOMICS vol. 1794; p. 263-269. AQUARO G, RIVA C, GALLI P (2009) Monogenoids from the gills of Acanthopagrus bifasciatus (Forsskål, 1775) (Perciformes: Sparidae) of the Red Sea, Egypt with the description of Lamellodiscus donatellae sp. n. (Diplectanidae). COMPARATIVE PARASITOLOGY vol. 76; p. 51–57. ARCANGELI A, CROCIANI O, LASTRAIOLI E, MASI A PILLOZZI S, BECCHETTI A (2009) Targeting ion channels in cancer: a novel frontier in antineoplastic therapy. CURR MED CHEM vol. 16, pp. 66-93. ARINGHIERI C, RUEPP MD, VIVARELLI S, CARDINALE S, PARO S, SCHÜMPERLI D, BARABINO S (2009) Mammalian 3’ end processing factor CF Im68 functions in mRNA export. MOL. BIOL. CELL vol. 20; p. 5211-5223. BALESTRIERI C, ALBERGHINA L, VANONI M., CHIARADONNA F (2009). Data recovery and integration from public databases uncovers transformation-specific transcriptional downregulation of cAMP-PKA pathway-encoding genes. BMC BIOINFORMATICS, vol. 10; p. S1-. BÁNYÁSZ T, HORVÁTH B, VIRÁG L, BÁRÁNDI L, SZENTANDRÁSSY N, HARMATI G, MAGYAR J, MARANGONI S, ZAZA A, VARR” A, NÁNÁSI PP (2009) Reverse rate dependency is an intrinsic property of canine cardiac preparations. CARDIOVASC RES vol. 84(2); p. 237-244. [3.1] PUBLICATIONS BARACCA A, CHIARADONNA F., SGARBI G, SOLAINI G, ALBERGHINA L, LENAZ G (2009) Mitochondrial Complex I decrease is responsible for bioenergetic dysfunction in K-ras transformed cells. BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1797; p. 314-323. BARILE L, CERISOLI F, FRATI G, GAETANI R, CHIMENTI I, FORTE E, CASSINELLI L, SPINARDI L, ALTOMARE C, KIZANA E, GIACOMELLO A, MESSINA E, OTTOLENGHI S, MAGLI MC (2009) Bone marrow-derived cells can acquire cardiac stem cells properties in damaged heart. J CELL MOL MED Nov 13. PMID: 19912439 BERTINI L, GRECO C, DE GIOIA L, FANTUCCI P (2009) DFT/ TDDFT Exploration of the Potential Energy Surfaces of the Ground State and Excited States of Fe-2(S2C3H6)(CO)(6): A Simple Functional Model of the [FeFe] Hydrogenase Active Site. JOURNAL OF PHYSICAL CHEMISTRY A. vol. 113, pp.5657-5670. BONETTI D, MARTINA M, CLERICI M, LUCCHINI G, LONGHESE MP (2009) Multiple pathways regulate 3’ overhang generation at S. cerevisiae telomeres. MOLECULAR CELL, vol. 35; p. 70-81. BONFANTI P, COLOMBO AE, VILLA S, COMELLI F, COSTA B, SANTAGOSTINO A (2009) The effects of accumulation of an environmentally relevant polychlorinated biphenyl mixture on cytochrome P450 and P-glycoprotein expressions in fetuses and pregnant rats. CHEMOSPHERE vol. 75; p. 572-579. BROCCA S, SAMALIKOVA M, UVERSKY VN, LOTTI M, VANONI M., ALBERGHINA L, GRANDORI R (2009) Order propensity of an intrinsically disordered protein, the cyclin-dependentkinase inhibitor Sic1. PROTEINS, vol. 76; p. 731-746. BRUSCHI M, GRECO C, KAUKONEN M, FANTUCCI P, RYDE U, DE GIOIA L (2009) Influence of the [2Fe](H) Subcluster Environment on the Properties of Key Intermediates in the Catalytic Cycle of [FeFe] Hydrogenases: Hints for the Rational Design of Synthetic Catalysts. ANGEWANDTE CHEMIEINTERNATIONAL EDITION vol. 48, pp. 3503-3506. CARLESSI L, DE FILIPPIS L, LECIS D, VESCOVI AL, DELIA D (2009) DNA-damage response, survival and differentiation in vitro of a human neural stem cell line in relation to ATM expression. CELL DEATH DIFFER vol. 16; p. 795-806. CASTRO NS, DE CASTRO KP, ORLANDI I, FEITOSA LS, ROSA E, SILVA LK, VAINSTEIN MH, BÁO SN, VAI M, SOARES CM (2009) Characterization and functional analysis of the beta1,3-glucanosyltransferase 3 of the human pathogenic fungus Paracoccidioides brasiliensis. FEMS YEAST RESEARCH vol. 9; p. 103-114. CELLOT G, CILIA E, CIPOLLONE S, RANCIC V, SUCAPANE A, GIORDANI S, GAMBAZZI L, MARKRAM H, MICAELA GRAN- DOLFO, SCAINI D, GELAIN F, CASALIS L, PRATO M, GIUGLIANO M, BALLERINI L (2009) Carbon nanotubes direct interactions with neuronal membranes ignite post spike excitability. NATURE NANOTECHNOLOGY vol. 4(2); p. 126-133. COMELLI F, BETTONI I, COLLEONI M, GIAGNONI G, COSTA B (2009) Beneficial effects of a Cannabis sativa extract treatment on diabetes-induced neuropathy and oxidative stress. PHYTOTHERAPY RESEARCH vol. 12; p. 1678-1684. CERISOLI F, CASSINELLI L, LAMORTE G, CITTERIO S, BERTOLOTTI F, MAGLI MC, OTTOLENGHI S (2009) Green fluorescent protein transgene driven by Kit regulatory sequences is expressed in hematopoietic stem cells. HAEMATOLOGICA vol. 94(3); p. 318-325. CONSONNI S, LEONE S, BECCHETTI A, AMADEO A (2009) Developmental and neurochemical features of cholinergic neurons in the murine cerebral cortex. BMC NEUROSCI vol. 10 (18), pp. 1-9. CHIROLI E, RANCATI G, CATUSI I, LUCCHINI G, PIATTI S (2009). Cdc14 inhibition by the spindle assembly checkpoint prevents unscheduled centrosome separation in budding yeast. MOL BIOL CELL vol. 20; p. 2626-2637. CIPOLLA L, ARAÚJ AC,. AIROLDI C, BINI D (2009) Pyrrolo[2,1-c][1,4]benzodiazepine as a scaffold for the design and synthesis of anti-tumour drugs. ANTI-CANCER AGENTS MED. CHEM. vol. 9; p. 1-31. CIPOLLA L, POLISSI A, AIROLDI C, GALLIANI P, SPERANDEO P, NICOTRA F (2009) The Kdo biosynthetic pathway toward OM biogenesis as target in antibacterial drug design and development. CURRENT DRUG DISCOVERY TECHNOLOGIES Vol. 6; p. 19-33. CIPOLLA L, REDAELLI C, GRANUCCI F, ZAMPELLA G, ZAZA A, CHISCI R, NICOTRA F (2009) Straightforward synthesis of novel Akt inhibitors based on a glucose scaffold. CARBOHYDR RES published on line Dec 16, in press COLOMBO G, RUSCONI F, RUBINO T, CATTANEO A, MARTEGANI E, PAROLARO D, BACHI A, ZIPPEL R (2009) Transcriptomic and proteomic analysis of mouse cerebellum reveals alterations in RasGRF1 expression following in vivo chronic treatment with delta 9-tetrahydrocannabinol. J MOL NEUROSC vol. 37; p. 111-122. COLOMBO M, RONCHI S, MONTI D, CORSI F, TRABUCCHI E, PROSPERI D. (2009) Femtomolar detection of auto-antibodies by magnetic relaxation nanosensors. ANALYTICAL BIOCHEMISTRY, vol. 392; p. 96-102. COMBI R, FERINI-STRAMBI L, TENCHINI ML (2009) CHRNA2 mutations are rare in the NFLE population: evaluation of a large cohort of Italian patients. SLEEP MEDICINE, vol. 10; p. 139-142. COMBI R, GRIONI D, CONTRI M, REDAELLI S, REDAELLI F, BASSI MT, BARISANI D, LAVITRANO M, TREDICI G, TENCHINI ML, BERTOLINI M, DALPRÀ L (2009) Clinical and genetic familial study of a large cohort of Italian children with idiopathic epilepsy. BRAIN RESEARCH BULLETIN vol. 79; p. 89-96. CORSI F, DE PALMA C, COLOMBO M, NEBULONI M, RONCHI S, RIZZI G, ALLEVI R, TOSONI A, TRABUCCHI E, CLEMENTI E, PROSPERI D. (2009) Nanodiagnostics: Small 22/2009. SMALL vol. 5; p. NA. COSENTINO U, PITEA D, MORO G, SARACINO GAA, VILLA A (2009) Conformational behaviour determines the low-relaxivity state of a conditional MRI contrast agent. PHYS. CHEM. CHEM. PHYS vol. 11; p. 3943–3950. DANTAS-TORRES F, LIA RP, BARBUTO M, CASIRAGHI M., CROVACE A, CALIGIANI L, GENCHI C, OTRANTO D (2009) Ocular dirofilariosis by Dirofilaria immitis in a dog: first case report from Europe. J. SMALL ANIMAL PRACTICE vol. 50; p. 667-669. DE MATTIA F, LOVICU G, TARDAGUILA J, GRASSI F, IMAZIO S, SCIENZA A, LABRA M (2009). Genetic relationships between Sardinian and Spanish viticulture: the case of Cannonau and Garnacha. J. HORTICULTURAL SCIENCE AND BIOTECHNOLOGY vol. 84; p. 65-71. DI DOMENICO EG, AURICHE C, VISCARDI V, LONGHESE MP, GILSON E, ASCENZIONI F (2009) The Mec1p and Tel1p checkpoint kinases allow humanized yeast to tolerate chronic telomere dysfunctions by suppressing telomere fusions. DNA REPAIR vol. 8; p. 209-218. DOULATI BANEH H., MOHAMMADI S.A., MAHMOUDZADEH H, DE MATTIA F, LABRA M (2009) Analysis of SSR and AFLP markers to detect genetic diversity among selected clones of grapevine (Vitis vinifera L.) cv. Keshmeshi. SOUTH AFRICAN J. ENOLOGY AND VITICULTURE vol. 30; p. 38-42. FAVARO R, VALOTTA M, FERRI A, LATORRE E, MARIANI J, GIACHINO C, LANCINI C, TOSETTI V, OTTOLENGHI S, TAYLOR V, NICOLIS SK (2009) Hippocampal development and neural stem cell maintenance require Sox2-dependent regulation of Shh. NATURE NEUROSCIENCE vol.12; p. 1248-1256. FERREIRA-MARTINS J, RONDON-CLAVO C, TUGAL D, KORN JA, RIZZI R, PADIN-IRUEGAS ME, OTTOLENGHI S, DE ANGELIS A, URBANEK K, IDE-IWATA N, D’AMARIO D, HOSODA T, LERI A, KAJSTURA J, ANVERSA P, ROTA M (2009) Spontaneous calcium oscillations regulate human cardiac [ 62 ] [ 63 ] [3.1] PUBLICATIONS progenitor cell growth. CIRC RES vol. 105(8); p. 764-774. FERRI E, BARBUTO M, BAIN O, GALIMBERTI A, UNI S, GUERRERO, FERTÉ H, BANDI C, MARTIN C, CASIRAGHI M (2009) Integrated taxonomy: traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda). FRONTIERS IN ZOOLOGY vol. 6; p. 1-. GAGLIO D, SOLDATI C, VANONI M., ALBERGHINA L, CHIARADONNA F (2009) Glutamine deprivation induces abortive S-phase rescued by deoxyribonucleotides in K-ras transformed fibroblasts. PLOS ONE vol. 4; p. e4715/1-e4715/17. GALLI P, STRONA G, GIOVANNONI R, LAVITRANO M (2009) Head glands of monogenoidea: morphology, functionality and potentialities in industrial production of surgery bioadhesives. JOURNAL OF PARASITOLOGY vol. 95; p. 13301341. GALLIA GL, TYLER BM, HANN CL, SIU IM, GIRANDA VL, VESCOVI AL, BREM H, RIGGING GJ (2009) Inhibition of Akt inhibits growth of glioblastoma and glioblastoma stem-like cells. MOL CANCER THER vol. 8; p. 386-393. GILBERT D, LECCHI M, ARNAUDEAU S, BERTRAND D, DEMAUREX N (2009) Local and global calcium signals associated with the opening of neuronal ß7 nicotinic acetylcholine receptors. CELL CALCIUM vol. 45, pp. 198-207. GRANDI E, PASQUALINI FS, PES C, CORSI C, ZAZA A, SEVERI S (2009) Theoretical investigation of action potential duration dependence on extracellular Ca2+ in human cardiomyocytes. J MOL CELL CARDIOL vol. 46(3); p. 332-342. GRANDORI R, SANTAMBROGIO C, BROCCA S, INVERNIZZI G, LOTTI M. (2009) Electrospray-ionization mass spectrometry as a tool for fast screening of protein structural properties. BIOTECHNOLOGY JOURNAL vol. 4; p. 73-87. GRASSI F, MINUTO L, CASAZZA G, LABRA M, SALA F (2009) Haplotype richness in refugial areas: phylogeographical structure of Saxifraga callosa. J. PLANT RESEARCH vol. 122; p. 377-387. GRECO C, BRUSCHI M, FANTUCCI P, DE GIOIA L (2009) Relation between coordination geometry and stereoelectronic properties in DFT models of the CO-inhibited [FeFe]-hydrogenase cofactor. JOURNAL OF ORGANOMETALLIC CHEMISTRY vol. 694 pp. 2846-2853. GULLO F, MAFFEZZOLI A, DOSSI E, WANKE E (2009) Short latency cross-and autocorrelation identify clusters of interacting neurons recorded from muti-electrode arrays. J NEUROSCI METH vol. 181; p.186-198. [3.1] PUBLICATIONS INVERNIZZI G, CASIRAGHI L, GRANDORI R, LOTTI M. (2009) Deactivation and unfolding are uncoupled in a bacterial lipase exposed to heat, low pH and organic solvents. JOURNAL OF BIOTECHNOLOGY, vol. 141; p. 42-46. MORASSO C, BELLINI T, MONTI D, BASSI M, PROSPERI D, RIVA S (2009) Dispersed Phantom Scatterer technique reveals subtle differences in substrate recognition by phospholipase d inactive mutants. CHEMBIOCHEM vol. 10; p. 639-644. INVERNIZZI G, PAPALEO E, GRANDORI R, DE GIOIA L, LOTTI M. (2009) Relevance of metal ions for lipase stability: structural rearrangements induced in the Burkholderia glumae lipase by calcium depletion. JOURNAL OF STRUCTURAL BIOLOGY vol. 168; p. 562-570. MORTELLARO A, URBANO M, CITTERIO S, FOTI M., GRANUCCI F, AND RICCIARDI-CASTAGNOLI P (2009) Generation of mouse growth factor-dependent long-term dendritic cell lines to investigate host-parasite interactions. METHODS IN MOLECULAR BIOLOGY vol. 531; p. 17-27. JUNKER K, BARBUTO M, CASIRAGHI M, MARTIN C, UNI S, BOOMKER J, BAIN O (2009) Litomosa chiropterorum Ortlepp, 1932 (Nematoda: Filarioidea) from a South African miniopterid: redescription, Wolbachia screening and phylogenetic relationships with Litomosoides. PARASITE, vol. 16; p. 43-50. MUELLER C, GOMEZ-ZURITA FRAU MA, BALLINARI D, COLOMBO S, BITTO A, MARTEGANI E, AIROLDI C, VAN NEUREN AS, STEIN M, WEISER J, BATTISTINI C, PERI F (2009). Design, Synthesis and Biological Evaluation of Levoglucosenone-derived Ras Activation Inhibitors. CHEMMEDCHEM vol. 4(4); p. 524-528. LA FERLA B, RUSSO L, AIROLDI C, NICOTRA F (2009) Solid-phase supported mimic of GDP-L-Galactose, TETRAHEDRON ASYMM vol. 20; p. 744–745. 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DNA REPAIR vol. 8; p. 1127-1138. MAZZUCCHELLI S, DE PALMA A, RIVA M, DURZO A, POZZI C, PASTORI V, COMELLI F, FUSI P, VANONI M, TORTORA P., MAURI P, REGONESI ME (2009) Proteomic and biochemical analyses unveil tight interaction of ataxin-3 with tubulin. THE INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY vol. 41; p. 2485-2492. PIAZZA M, ROSSINI C, DELLA FIORENTINA S, POZZI C, COMELLI F, BETTONI I, FUSI P, COSTA B, PERI F (2009) Glycolipids and Benzylammonium Lipids as Novel Antisepsis Agents: Synthesis and Biological Characterization. J MED CHEM vol. 52(4); p. 1209-1213. PIAZZA M, YU L, TEGHANEMT A, GIOANNINI T, WEISS J, PERI F (2009). Evidence of a specific interaction between new synthetic antisepsis agents and CD14. BIOCHEMISTRY vol. 48; p. 12337-12344. PICCIRILLO SG, BINDA E, FIOCCO R, VESCOVI AL, SHAH K (2009) Brain cancer stem cells. J MOL MED vol. 87; p.10871095. NATALELLO A, LIU J, AMI D, DOGLIA SM, DE MARCO A (2009) The osmolyte betaine promotes protein misfolding and disruption of protein aggregates. PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS vol. 75; p. 509-517. PICCIRILLO SGM, COMBI R, CAJOLA L, PATRIZI A, REDAELLI S, BENTIVEGNA A, BARONCHELLI S, MAIRA G, POLLO B, MANGIOLA A, DIMECO F, DALPRÀ L, VESCOVI AL (2009) Distinct pools of cancer stem-like cells coexist within human glioblastomas and display different tumorigenicity and independent genomic evolution. ONCOGENE vol. 28(15); p.18071811. NICOTRA F, CIPOLLA L, LA FERLA B, AIROLDI C, ZONA C, ORSATO A., SHAIKH N, RUSSO L (2009) Carbohyrate scaffolds in chemical genetic studies, J. BIOTECHNOLOGY vol. 144; p. 234–241. POLGAR G, BURRESON EM, STEFANI F, KAMRANI E (2009) Leeches on Mudskippers: Host–Parasite Interaction at the Water’s Edge. JOURNAL OF PARASITOLOGY vol. 95; p. 10211025. PALMIOLI A, SACCO E, ABRAHAM S, THOMAS CJ, DI DOMIZIO A, DE GIOIA L, GAPONENKO V, VANONI M., PERI F (2009) First experimental identification of Ras-inhibitor binding interface using a water-soluble Ras ligand. BIOORGANIC & MEDICINAL CHEMISTRY LETTERS vol. 19; p. 4217-4222. PORRO D, BRANDUARDI P (2009) Yeast cell factory: fishing for the best one or engineering it? MICROBIAL CELL FACTORY vol. 8; p.51. PALMIOLI A, SACCO E, AIROLDI C, DI NICOLANTONIO F, D’ URZO A, SHIRASAWA S, SASAZUKI T, DI DOMIZIO A, DE GIOIA L, MARTEGANI E, BARDELLI A, PERI F, VANONI ME (2009). Selective cytotoxicity of a bicyclic Ras inhibitor in cancer cells expressing K-RasG13D. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS vol. 386; p. 593-597. PAPALEO E, MEREGHETTI P, FANTUCCI P, GRANDORI R, DE GIOIA L (2009) Free-energy landscape, principal component analysis, and structural clustering to identify representative conformations from molecular dynamics simulations: The myoglobin case. 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SUN P, XIA S, LAL B, EBERHART CG, QUINONES-HINOJOSA A, MACIACZYK J, MATSUI W, DIMECO F, PICCIRILLO SG, VESCOVI AL, LATERRA J (2009) DNER, an epigenetically modulated gene, regulates glioblastoma-derived neurosphere cell differentiation and tumor propagation. STEM CELLS Jul 27; p.1473-1486 TARABALLI F, CAMPIONE M, VESCOVI AL, SASSELLA A, PALEARI A, WHANG W, GELAIN F (2009) Effect of Functionalization on the Self-Assembling Propensity of Sheet Forming Peptides. SOFT MATTER vol. 5; p. 660–668. VILLA AM, DOGLIA SM (2009) Ethidium bromide as a vital probe of mitochondrial DNA in carcinoma cells. EUROPEAN JOURNAL OF CANCER vol. 45; p. 2588-2597. VIRÁG L, ACSAI K, HÁLA O, ZAZA A, BITAY M, BOGÁTS G, PAPP JG, VARRÒ (2009) Self-augmentation of the lengthening of repolarization is related to the shape of the cardiac action potential: implications for reverse rate dependency. BR J PHARMACOL vol. 156(7); p. 1076-1084. SAINO N, RUBOLINI D, LEHIKOINEN E, SOKOLOV LV, BONISOLI-ALQUATI A, AMBROSINI R, BONCORAGLIO G, MøLLER AP (2009) Climate change effects on migration phenology may mismatch brood parasitic cuckoos and their hosts. BIOLOGY LETTERS vol. 5; p. 539-541. WANKE E, ZAHARENKO AJ, REDAELLI E, SCHIAVON E (2009) Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms. TOXICON vol. 54; p. 1102-1111. SAINO N, RUBOLINI D, VON HARDENBERG J, AMBROSINI R., PROVENZALE A, ROMANO M, SPINA F (2009) Spring migration decision in relation to weather are predicted by wing morphology among trans-Mediterranean migratory birds. FUNCTIONAL ECOLOGY vol. 2009. WOLFOVA J, SMATANOVA IK, BRYNDA J, MESTERS JR, LAPKOUSKI M, KUTY M, NATALELLO A, CHATTERJEE N, CHERN SY, EBBEL E, RICCI A, GRANDORI R, ETTRICH R, CAREY J (2009) Structural organization of WrbA in apo- and holoprotein crystals. BIOCHIMICA ET BIOPHYSICA ACTA vol. 1794; p. 1288-1298. SEVERI S, CORSI C, ROCCHETTI M, ZAZA A (2009) Mechanisms of beta-adrenergic modulation of I(Ks) in the guineapig ventricle: insights from experimental and model-based analysis. BIOPHYS J. vol. 96(9); p. 3862-3872. SOMMARUGA S, DE GIOIA L, TORTORA P, POLISSI A (2009) Structure prediction and functional analysis of KdsD, an enzyme involved in lipopolysaccharide biosynthesis. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS vol. 388, pp. 222-227. SPERANDEO P, DEHO’ G, POLISSI A (2009). The Lipopolysaccharide transport system of Gram-negative Bacteria. BIOCHIMICA ET BIOPHYSICA ACTA vol. 1791; p. 594-602. ZAMPELLA G, FANTUCCI P, DE GIOIA L (2009) Unveiling How Stereoelectronic Factors Affect Kinetics and Thermodynamics of Protonation Regiochemistry in [FeFe] Hydrogenase Synthetic Models: A DFT Investigation. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. Vol. 131 pp.10909-10917. ZANONI I, OSTUNI R, CAPUANO G, COLLINI M, CACCIA M, RONCHI AE, ROCCHETTI M, MINGOZZI F, FOTI M, CHIRICO G, COSTA B, ZAZA A, RICCIARDI-CASTAGNOLI P, GRANUCCI F (2009) CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation. NATURE vol. 460; p. 264-268. PROSPERI D, POLITO L, MORASSO C, MONTI D (2009) Biofunctionalization of spherical & anisotropic bimetallic nano- materials. In: C. S. S. R. KUMAR. Nanomaterials for the Life Sciences. Vol. 3, p. 197-240, NEW YORK: WILEY-VCH. GELAIN F, WANG X, HORII A, HUCKNALL A, KOUTSOPOULOS S, ZHANG S (2009) Designer self-assembling peptides scaffolds for 3-dimensional tissue cell cultures Methods in Bioengineering, Artech House Press, Boston. 59-81 Book Chapter LOWERY J, PANSERI S, CUNHA C, GELAIN F (2009) Electrospinning for Tissue Engineering Applications Electrospun Nanofibers Research: Recent Developments, NOVA Science Publishers, Hauppauge. 1-18 Book Chapter [3.3] RESEARCH GRANTS AND CONTRACTS (external resources) ALBERGHINA L. Rete italiana di Bioinformatica FIRB, MIUR ALBERGHINA L. Eukaryotic unicellular organism biology – systems biology of the control of cell growth and proliferation FP7, European Commission AMBROSINI R. Effetti dei cambiamenti climatici sulla migrazione degli uccelli. La Rondine come modello di studio scientifico, applicazione conservazionistica e disseminazione della cultura scientifica in materia ambientale. Fondazione Cariplo BARABINO S. Genomic and Proteomic Analysis of Pre-mRNA Processing in Amyotrophic Lateral Sclerosis. Fondazione Cariplo BECCHETTI A. Recettori nicotinici cerebrali e patologie epilettiche. BML Foundation CASIRAGHI M. Le connessioni ecologiche nelle selve castanili nel Parco Regionale Campo dei Fiori: valutazione e sviluppo di sistemi di gestione. Fondazione Cariplo CASIRAGHI M. LABRA M. Bio.Api. Le Api come Bioindicatori. Parco della Grigna Settentrionale. Comunità Montana della Valsassina, Valvarrone, Val d’Esino e Riviera CASTAGNOLI P. Integrated functional genomics in mutant mouse models as tools to investigate the complexity of human immunological disease. European Commission COLANGELO AM. Processo di scale up per la produzione di Nerve Growth Factor ricombinante umano (rhNGF) in cellule di mammifero, purificazione e caratterizzazione molecolare PRIN, MIUR FOTI M. Generation of a coronavirus-based multi gene AIDS vaccine and evaluation in a preclinical SIV model. European Commission FOTI M. A systems biology approach to identify biomarkers and therapeutic targets in Multiple sclerosis. FISM FOTI M. Identificazione dei meccanismi molecolari indotti in cellule dendritiche da batteri commensali e patogeni importanti nella polarizzazione di linfociti T. PRIN, MIUR GALLI P. BioInspired Adhesive for Surgery. Fondazione Cariplo GALLI P. BENZONI F. Biodiversity and Biodiversity patterns of Scleractinia in the Gulf of Aden, Yemen. Creocean GIAGNONI G. Spinal and supraspinal role of cytokines and BDNF during neuropathic pain and their modulation after human mesenchymal stem cells transplatation in the rostral agranular insular cortex. MIUR, PRIN GRANUCCI F. Key regulators of DC-primed anti tumor NK cell functions Associazione Italiana per la Ricerca sul Cancro Investigator Grant 2007 GRANUCCI F. Dentritic cells for novel immunotherapies. European Commission GRANUCCI F. Ruolo delle cellule dendritiche nell’attivazione delle funzioni anti-tumorali delle cellule NK: meccanismi cellulari e molecolari. PRIN, MIUR GRANUCCI F. Normalization of immune reactivity in old age - from basic mechanisms to clinical application. European Commission GRANUCCI F. European network for cell imaging and tracking expertise. European Commission LABRA M. Milano da Bere. Regione Lombardia. LABRA M. Insetti Pronubi: mezzi di connessione e diffusione di specie vegetali rare ed endemiche del Parco Regionale della Grigna Settentrionale. Fondazione Cariplo [ 66 ] [ 67 ] [3.3] RESEARCH GRANTS AND CONTRACTS (external resources) [3.3] RESEARCH GRANTS AND CONTRACTS (external resources) LABRA M. Think Green, persone che hanno cura del territorio. Fondazione Cariplo li nanostrutturati per la medicina rigenerativa. Fondazione Cariplo LABRA M. Tassonomia integrata per lo studio della biodiversità vegetale: DNA barcoding e analisi morfologiche. MIUR, PRIN LABRA M. PERI F. Messa a punto di sistemi innovativi per valutare e migliorare la qualità dell’ambiente lavorativo al fine di proteggere la salute dei lavoratori. Neomed srl - Regione Lombardia LONGHESE MP. Genetic integrity maintenance: inter-relationships between DNA damage checkpoints and telomere metabolism. Associazione Italiana per Ricerca sul Cancro LONGHESE MP. High Resolution Microscopy in the DNA damage Response. Initial Training Networks, European Commission LOTTI M. Valorizzazione delle risorse biologiche. Sviluppo di nuove tecnologie per l’identificazione, caratterizzazione e produzione di molecole di interesse farmaceutico e industriale presenti nelle Brassicacee. Projects for Industrial Research (FAR), MIUR LUCCHINI G. Fattori di checkpoint coinvolti nell’omeostasi telomerica nel lievito S. cerevisiae. MIUR, PRIN NICOTRA F. Piattaforma integrata per la progettazione e la produzione high throughput di enzimi e peptidi ingegnerizzati. Valutazione della loro attività biologica rispetto a specifici” substrati molecolari di interesse farmaceutico. (PANDA). Regione Lombardia, Metadistretti OTTOLENGHI S. Gene therapy in cardiac stem cells in vitro for the correction of an inherited cardiomyopathy. Fondazione Cariplo PERI F. Ligandi mono- e multivalenti per le galectine umane: nuovi sistemi molecolari e nanoparticelle funzionalizzate per la diagnosi e per terapie antitumorali specifiche. MIUR, PRIN PERI F. Regulation of MD-2 function and expression.NIH (USA) PERI F. Sintesi di molecole con potenziale attività antiinfiammatoria. Associazione Malattie Infiammatorie Croniche dell’Intestino PIATTI S. Molecular mechanisms preventing the occurrence of aneuploidy, a hallmark of cancer cells. Associazione Italiana Ricerca sul Cancro MARTEGANI E. Sviluppo di peptidi con attività NGF-like, PRIMM POLISSI A. Essential proteins of Pseudomonas aeruginosa outer membrane biogenesis as novel targets for new antimicrobial drugs design and synthesis. Fondazione per la Ricerca sulla Fibrosi Cistica MARTEGANI E. Valutazione attività NGF umano ricombinante. Blueprint PORRO D. Nuovi processi per la produzione di biofuels con lieviti ingegnerizzati. ENI NICOLIS S. Functional role and molecular mechanisms of action of the Sox2 transcription factor in neural stem cells and in neuronal differentiation: a study by conditional mutagenesis in the mouse. MIUR, PRIN NICOLIS S. NS-toolkit – a genetic toolkit for analysis of mouse neural stem cells. NOBEL Cariplo NICOLIS S. Studio di un modello genetico animale di mutazione condizionale del gene Sox2, implicato nella malattia neurologica umana e nella biologia delle cellule staminali neurali. Fondazione Banca del Monte di Lombardia NICOLIS S. Genetic approaches to the role of the Sox2 transcription factor in cancer neural stem cells. Associazione Italiana Ricerca sul Cancro NICOTRA F. Development of NMR techniques for tissue engineering studies. Fondazione Cariplo NICOTRA F. Materiali innovativi per lo sviluppo di bio-protesi articolari. MIUR, FIRB NICOTRA F. NAD, Nanoparticles for therapy and diagnosis of Alzheimer Disease. European Commission RONCHI A. Genomica funzionale della transizione embrionico-adulta nell’ematopoiesi. MIUR, PRIN TORTORA P. Network Operativo per la Biomedicina di Eccellenza in Lombardia, Fondazione Cariplo TORTORA P. Un approccio multidisciplinare per lo studio dell’aggregazione in vivo e in vitro di proteine contenenti poliglutammine. Ruolo di fattori molecolari e ambientali. MIUR, PRIN TORTORA P. Network Tecnologico integrato per lo studio proteomico e trascrittomico di malattie neurodegenerative correlate a deposizioni di amiloidi. Regione Lombardia, Ministero della Sanità TORTORA P. Ottimizzazione della reazione di amplificazione di una sequenza altamente specifica del gene 5’UTR degli enterovirus. Dia.Pro Diagnostic Bioprobes VANONI M. Sviluppo di inibitori peptidici di Ras. Creabilis VESCOVI A. Cellule staminali neurali umane e biomateria- VESCOVI A. Tumor neural stem cells in the in vitro and in vivo modeling and studying of the adult human glioblastomas. Associazione Italiana per Ricerca sul Cancro VESCOVI A. Cis-regulatory logic of the transcriptional control in neural stem cells. European Commission VESCOVI A. Towards the neuronal Machine. European Commission WANKE E. Functional regeneration of the mesocorticolimbic dopaminergic system as a model to study novel neuroreparative strategies. Fondazione Cariplo ZAZA A. Funzione del reticolo sarcoplasmico e stabilità del deposito di Ca2+ nel muscolo cardiaco. Interlink Montpellier (II04C570GL), France ZAZA A. Modulation of SR function by Istaroxime Debiopharm, Lausanne (CH) ZAZA A. Role of the “late Na+ current” in myocardial and neuronal effects of chronic hypoxia. CV-Therapeutics, Palo Alto CA (USA) ZAZA A. Role of the “late Na+ current” in myocardial damage induced by chronic hypoxia. MIUR, PRIN ZAZA A. Gene therapy in cardiac stem cells in vitro for the correction of an inherited cardiomyopathy. Fondazione Cariplo [3.4] PATENTS RUMIO C, PALAZZO M, BALSARI A, NICOTRA F, LA FERLA B Compounds with glycidic structure active in the therapy of sistemi and local inflammation, PCT/EP2009/003267, 07.05.2009 PORRO D., DATO L., BRANDUARDI P. Method for improving acid and low pH tolerance in yeast. Publication info: US2009081793 (A1) publication date 26_03_2009 PORRO D., BRANDUARDI P., VALLI M. ALBERGHINA L. Process for expression and secretion of proteins by the non- conventional yeast Zygosaccharomyces bailii. Publication info: DE60320573 (T2) publication date 28_05_2009 BRANDUARDI P., PORRO D., SAUER M., MATTANOVICH D. Ascorbic acid porduction from D-glucose in yeast. Publication info: BRPI0606117 (A2) publication date 06_10_2009 PORRO D., BRANDUARDI P., SAUER M. Improved yeast strain for the production of organic acids. Publication info: EP2128262, publication date 02_12_2009 Dipartimento di Biotecnologie e Bioscienze Università degli Studi di Milano Bicocca Piazza della Scienza 2, 20126 Milano - Italia Tel. ++39 02 6448 3330 - Fax ++39 02 6448 3569 [email protected] - www.btbs.unimib.it Finito di stampare nel mese di luglio 2010 graphic project okio_design
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