Relazione 2008 - BTBS - University of Milano
Transcript
Relazione 2008 - BTBS - University of Milano
‘08 A N N U A L R E P O R T DEPARTMENT OF BIOTECHNOLOGY AND BIOSCIENCES “ “ This Annual report has been printed in 2009, Darwin commemorative year. The cover wants to thank all the people that contributed their work to the activities of the Department. Cover picture: Maurizio Casiraghi [1] This report provides an overview of the Department of Biotechnology and Biosciences (BtBs) of the State University of Milano-Bicocca, it describes its structure and organization, its permanent, temporary and in-training staff and it also includes financial information and a detailed description of teaching and research activities. At the end of 2008, the Department employed 15 Full Professors, 17 Associate Professors and 29 Assistant Professors, 3 of which joined in 2008 for the areas of Immunology, Genetics and Biochemistry. Permanent staff included also 12 Technicians and 9 members of the Administration team. Together with post-docs, fellowship holders, PhD students and Master degree students, our activity involved about 230 people working on both basic and contract/applied research as well as in teaching in the curricula of Biotechnology, Biological Sciences and Bioinformatics. During 2008 we added to our facilities a BL2 laboratory for the safe production and handling of lentiviral and retroviral vectors and core instrumentation was upgraded with new equipment for circular dichroism, infrared spectroscopy and DNA sequencing. Moreover, a Multi Electrode Array Workstation was acquired that supports the acquisition in real time and under no invasive conditions of the stimulated or spontaneous activity from networks of excitable cells (sensory, cardiac, neuronal). Scientific and academic productivity is well documented in over 100 scientific publications and 15 PhD theses discussed during 2008. BtBs members took part in a large number of national and international research programmes. Funding was provided by the University, regional and national sources, European research programs, research contracts and private foundations. BtBs was home to 2.000 students of Biology, Biotechnology and Bioinformatics first and second level degrees. In 2008 we graduated 377 students, out of which 88 in the first level degree in Biology and 163 in Biotechnology, 52 students in the second level degree in Biology, 60 in Biotechnology and 14 in Bioinformatics. INDEX 1 STRUCTURE AND ORGANIZATION 1.1 1.2 1.3 1.4 1.5 1.6 1.7 Financial Resources Department Management Structure and Staff Organization and Structure Instrumentation and Facilities Education Advanced Training Master and PhD theses 3 4 4 8 11 14 15 19 2 RESERCH GROUPS 25 3 SCIENTIFIC PUBLICATION INDEX, GRANTS 57 3.1 3.2 3.3 3.4 Research grants and contracts Publications Book chapters Patents 58 60 67 67 1 S TRUCTURE AND O R G A N I Z AT I O N [4] 1.2 1.1 DEPARTMENT MANAGEMENT STRUCTURE & STAFF FINANCIAL RESOURCES Director: Research grants MIUR (PRIN, FISR, FIRB) EU GRANTS REGIONE LOMBARDIA GRANTS 937.775 1.107.381 79.062 CARIPLO GRANTS 690.781 OTHER FUNDING AGENCIES 541.878 FAR (FONDI D'ATENEO PER LA RICERCA) 219.445 RESEARCH SERVICES 92.582 OTHER RESOURCES 94.674 Prof. Marina Lotti from October 1, 2008 Prof. Francesco Nicotra Up to September 30, 2008 Management Board: (up to September 30, 2008) Other funding sources DEPARTMENT FUNDING (DOTAZIONE) 116.000 FUNDS FOR TEACHING 319.812 PhD COURSES FUNDS FOR LARGE INSTRUMENTS 70.434 980.000 Prof. Francesco Nicotra, Prof. Antonio Zaza, Prof. Enzo Martegani, Prof. Giorgio Moro, Prof. Luca De Gioia, Dr. Paola Branduardi, Dr. Maurizio Casiraghi, Dr. Anastasia Sguera. Chief Financial Officer: Dr. Anastasia Sguera [5] FULL PROFESSORS NAME Alberghina Lilia Castagnoli Paola Fantucci Piercarlo Lotti Marina Lucchini Giovanna FIELD BIO/10 MED/04 CHIM/03 BIO/10 BIO/18 NAME FIELD NAME FIELD Martegani Enzo Nicotra Francesco Ottolenghi Sergio Porro Danilo Tortora Paolo BIO/11 CHIM/06 BIO/18 CHIM/11 BIO/10 NAME FIELD Giagnoni Gabriella Grandori Rita Granucci Francesca Longhese Mariapia Moro Giorgio Nicolis Silvia BIO/14 BIO/10 MED/04 BIO/18 CHIM/02 BIO/18 NAME FIELD Colombo Sonia BIO/11 Labra Massimo BIO/01 Combi Romina BIO/13 La Ferla Barbara CHIM/06 Costa Barbara BIO/14 Lecchi Marzia BIO/09 De Filippis Lidia BIO/13* Orlandi Ivan BIO/11 Foti Maria MED/04 Prosperi Davide BIO/10 Fraschini Roberta BIO/18 Regonesi Elena BIO/10 Frascotti Gianni CHIM/11 Rocchetti Marcella BIO/09 Fusi Paola BIO/10 Tisi Renata BIO/11 Galli Paolo BIO/07 Zampella Giuseppe CHIM/03 Gelain Fabrizio BIO/13* Zanoni Ivan MED/04 Vai Marina Vanoni Marco Wanke Enzo Zaza Antonio Zullini Aldo BIO/11 BIO/10 BIO/09 BIO/09 BIO/05 ASSOCIATE PROFESSORS NAME Barabino Silvia Becchetti Andrea Cipolla Laura Crosti Paolo De Gioia Luca Doglia Silvia Maria FIELD BIO/11 BIO/09 CHIM/06 BIO/01 CHIM/03 FIS/01 NAME FIELD Peri Francesco CHIM/06 Piatti Simonetta BIO/18 Polissi Alessandra BIO/19 Ronchi Antonella BIO/18 Vescovi Angelo BIO/13 ASSISTANT PROFESSORS NAME Ambrosini Roberto Bertini Luca Brambilla Luca Branduardi Paola Brocca Stefania Casiraghi Maurizio Ceriani Michela Chiaradonna F. Clerici Michela Coccetti Paola Colangelo A.Maria FIELD BIO/07 CHIM/03 CHIM/11 CHIM/11 BIO/10 BIO/05 BIO/11 BIO/10 BIO/18 BIO/10 BIO/10 NAME FIELD TECHNICAL STAFF Accardo Elena Malerba Massimo Tonelli Maria Grazia Citterio Stefania Marinoni Sara Urbano Matteo D’Urzo Annalisa Mostacciuolo Gaspare Villa Anna Maria Gullo Francesca Pedroni Paolo Sacchetti Francesco Bottani Elena Comi Roberto Mormile Bruno Bruno Stefania Delcarro Francesca Sguera Anastasia Campbell Neil Gotti Maria Cristina Smeraldi Carla ADMINISTRATION * temporary positions [6] PhD STUDENTS Adamo Giusy Alemanni Matteo Ambrosi Paola Aquaro Giovanni Araujo Ana Catarina Balestrieri Chiara Bigi Alessandra Bodio Caterina Bonetti Diego Broggi Achille Busti Stefano Caccia Roberta Cantù Claudio Casalgrande Maura Chisci Riccardo Codazzi Vera Colombo Daniele Comelli Francesca Contran Nicla De Mattia Fabrizio DiDomizio Alessandro Di Resta Chiara Falcetta Francesca Fumagalli Silvia Gaglio Daniela Galati Elena Galbusera Elena Galimberti Andrea Galliani Paolo Gotti Laura Gregori Maria Groppi Silvia Guerini Ilaria Lancini Cesare Losio Dario Maffezzoli Andrea Manfrini Nicola Rossio Valentina Marangoni Stefano Russo Laura Mariani Jessica Santambrogio Carlo Mazzantini Elisa Shaik Nasrin Mazzucchelli Serena Strona Giovanni Merlini Laura Metalli David Orsato Alexandre Ostuni Renato Palmioli Alessandro Pasi Marco Passolunghi Simone Taraballi Francesca Torri Anna Tosetti Valentina Tripodi Farida Venkatesh Aparna Venturetti Marianna Pastori Valentina Viganò Matteo Piazza Matteo Villa Riccardo Pozzi Chiara Vitali Caterina Redaelli Elisa Vivarelli Silvia Rossi Giorgia Zona Cristiano FELLOWSHIP HOLDERS Barresi Simona Bruni Ilaria Mezzasalma Valerio Spinosa Valerio Barbuto Michela Della Fiorentina Silvia Moretti Silvia Straka Daniele Bellati Adriana Ferri Emanuele Papagna Angela Tsiarentsyeva Viktoria Bettoni Isabella Fragni Martina Ranghetti Anna Tuana Giacomo Biancolini Donatella Gaviraghi Marco Sommaruga Silvia Vanzin Alessia Binda Elena Kapetis Dimos Sottotetti Elisa Viggiani Sandra Bini Davide Mainoldi Federica Spinelli Michela Villa Omar Airoldi Cristina Cirulli Claudia Gorletta Tatiana Papaleo Elena Altomare Claudia Dato Laura Invernizzi Gaetano Pontiroli Francesca Alvarez Reinaldo De Candia Paola Latorre Elisa Redaelli Cristina Dos Santos Cunha Carla Lenzken Carolina Rota Nodari Laura Favaro Rebecca Morini Raffaella Sacco Elena Ferrari Daniela Mortellaro Alessandra Samalikova Maria Ferri Anna Lucia Natalello Antonino Sperandeo Paola Benzoni Francesca Forcella Matilde Occhipinti Emanuela Stefani Fabrizio Cassinelli Letizia Fossati Tiziana Paiardi Chiara Vitale Elena Chiroli Elena Gatti Lafranconi Pietro Panseri Silvia Zorlezzi Francesca POST-DOCS Amigoni Loredana Aracri Patrizia Baldo Veronica Baldissera Michela Belotti Fiorella [7] INTERNATIONAL MOBILITY 2008 INCOMING from group Vladimir Muronetz Lomonosov University, Moscow, Russia Lotti Jan-March 2008 Jan Van der Goten Katholieke Universiteit Leuven, Belgium Martegani Feb-May 2008 Alexandre Orsato Federal University of Paranà, Curitiba, Brazil Nicotra Jan-Dec 2008 Nasrin Shaikh University of Pune India Nicotra Jan-Dec 2008 Ana Catarina Araujo Silva University of Lisbona, Portugal Nicotra Jan-Feb 2008 Monica Enguita Pamplona University, Spain Nicolis May-July 2008 OUTGOING to group Sara Tettamanti MRC Clinical Science Center, Hammersmith, London, UK Nicolis Feb-July 2008 Danilo Porro Visiting Professor, University of Osaka, Japan Porro Feb 2008 David Metalli Kimmer Cancer Center, Jefferson University Philadelphia, USA Vanoni July-Dec 2008 Silvia Vivarelli University of Bern Switzerland Barabino Oct-Nov 2008 Simona Paro Medical Research Council, Edinburgh, UK Barabino May-Sep 2008 Rita Grandori Johannes Kepler University, Linz, Austria Grandori May 2008 Elena Galbusera University Hospital of Geneva Switzerland Tortora Oct 2007 - Mar 2008 [8] 1.3 ORGANIZATION AND STRUCTURE The Department is organized as follows: a. Managing Director’s Office b. Administration Office c. Students Administration Office d. Facilities for Teaching Activities e. Technical Services f. Software and Hardware Assistance g. Wastes Disposal Service h. Safety and Prevention Service [9] a. MANAGING DIRECTOR OFFICE Director: Francesco Nicotra up to September 30, 2008, Marina Lotti from October 1, 2008 b. ADMINISTRATION OFFICE Anastasia Sguera – Chief Financial Officer; Stefania Bruno - Foreign payments, VAT related accounting; Roberto Comi - Accounts payable (contracts, scholarships, travel reimbursement); Francesca Delcarro - Purchase orders, travel reimbursement, supplier's accounting; Bruno Mormile - Supplier's accounting contractors Francesco Sacchetti - Property inventory, technical support. The Administration Office works together with higher management to comply with all administrative aspects and procedures of the department management: It is responsible for general budget planning and financial reports; it manages the funds of the research groups and those dedicated to teaching activities; it manages relations with suppliers and external contractors. It also arranges the Department and the administrative board meetings. c. STUDENT ADMINISTRATION OFFICE Maria Cristina Gotti, Elena Bottani, Marzia Colombo, Carla Smeraldi 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. d. FACILITIES FOR TEACHING ACTIVITIES The Department hosts the following laboratories devoted to practical courses in Biotechnology (BT) and Biological Sciences (BS) LABORATORY (number and name) PRACTICAL TEACHING ACTIVITIES 1011-1015 Chemistry Lab Lab. of General and Inorganic Chemistry (BT) Lab. of Organic Chemistry (BT) Bio-Organic Chemistry (BT) Purification and Downstream (BT) Lab. of Chemistry (BS) 1026 Biochemistry Lab Lab. of Biochemical Techniques (BT) Lab. of Biomolecular Techniques (BT) Molecular Pharmacology (BT) 1027-1029 Cell Biochemistry and Immunology Lab Cell Biochemistry (BT) Lab. of Immunological Techniques (BT) Lab. of Cell Biochemistry Techniques (BT) 1028 Genetics and Microbiology Lab Fermentation Technology (BT) Lab. of Fermentative Techniques (BT) Lab. of Genetic Techniques (BT) Applied Microbiology (BT) Lab. of Experimental Biology (BS) 2010 Zoology and Comparative Anatomy Lab Lab. of Experimental Biology (BS) Zoology (BS) Techniques of measure (BS) 2013 Microscopy Lab Fundamentals of Biology (BS) Cytology and Histology (BS) Plant Systems (BS) Anatomy I (BS) Anatomy II (BS) [ 10 ] 2029 Multimedia Lab Supramolecular Chemistry (BT) Physical Chemistry of Biological Systems (BT) Bioinformatics: Basics (BT) Bioinformatics: Structure-Function Relationships (BT) Molecular Biology (BT) Numerical Methods for Bioinformatics (BT) Fundamentals of Informatics (BT) Computational Biochemistry (BT) Immunogenomics (BT) e. TECHNICAL SERVICES The technical staff carries out common services, is responsible for the maintainance of common instruments and collaborates in research activities. In 2008 the staff duties were as follows: Elena Accardo Lab 1011-1015, mass spectrometry Stefania Citterio Lab 1028, Cytofluorimetry Annalisa D’Urzo Biacore (plasmon resonance) Massimo Malerba Lab of Morphological Microscopy Sara Marinoni Waste Disposal, Lab 1026 Gaspare Mostacciuolo Francesca Gullo Technical gases (fluids), workshop Paolo Pedroni IT support Maria Grazia Tonelli Lab 1028 Matteo Urbano Lab1027-1029, Liquid Nitrogen Service Cooperation for Laboratory PL2 planning Anna Maria Villa Confocal Microscopy f. SOFTWARE AND HARDWARE ASSISTANCE Paolo Pedroni Department IT services. g. WASTES DISPOSAL SERVICE Sara Marinoni The Service guarantees, in compliance with Italian law, the disposal of chemical and biological wastes produced by the Department of Biotechnologies and Biosciences and by the Department of Geological Sciences and Geotechnologies. h. SAFETY AND PREVENTION SERVICE The service consists in following activities: • maintaining contact with the Protection and Safety Office of the University • collection, evaluation and filing of forms and news about Safety • checking the correct working of security systems • Cooperation for training and communication in safety issues [ 11 ] 1.4 INSTRUMENTATION AND FACILITIES A number of platform technologies and advanced instrumentations are available to the research groups working in the Department, for scientific collaborations and to external users. a. TECHNOLOGICAL PLATFORMS AND SPECIAL LABORATORIES BBC (BICOCCA BIOTECHNICUM CENTER) A structure based on the most advanced scientific and technological platforms required for bioreactor production, downstream and analysis of the product (GMP-like env.). This structure is aimed at implementing technological transfer through the development of biotechnological processes, pilot fermentations and research contracts with companies as well as assistance in business plan preparation and patent writing and defence. SYSTEMS BIOLOGY PLATFORM Equipment and expertise for systems biology. i.e. iterative integration of molecular and computational approaches, aiming to structure genetic, biochemical and genome-wide data (that produce detailed molecular descriptions of physiological and pathological processes) so to foster the ability to comprehend and predict complex cellular functions. TRANSCRIPTOMICS Microarray technology allows for rapid measurement and visualisation of differential expression between 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 two different cell types or tissue samples, such as in tissue from healthy and diseased subject. Arrays are currently available for human samples as well as many biologically relevant model organisms including mouse, rat and plant (Arabidopsis). BL2 FACILITY This new core facility is located in a dedicated closed laboratory space, with restricted access, that houses tissue culture hoods, CO2/O2 incubators, microscopes and small equipment for cellular and molecular biology work. The facility consists in a Vector Production room, a Cell Manipulation room (directly connected with a PassThrough Cabinet) and in two general support areas. 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. BL2 laboratory personnel receive appropriate training on the potential hazards associated with their work and additional training as necessary for procedural or policy changes. This facility is suitable for experiments involving agents of moderate potential hazard to personnel and the 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. [ 12 ] b. ADVANCED INSTRUMENTATION MEA MULTI ELECTRODE ARRAY WORKSTATION The new (2008) 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 can 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 ßamyloid protein, a main factor involved in Alzheimer's disease pathogenesis. tional 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, 2DTOCSY, 2D-NOESY, 2D-ROESY, HMBC, HSQC); iii) tridimensional experiments (TOCSY-NOESY, TOCSY-HSQC). Small ligand-receptor (such as inhibitor/activatorprotein or substrate-enzyme) interaction studies are performed via DOSY, Saturation Transfer Difference (STD) and transferred-NOESY (tr-NOESY) experiments. 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-assisted-laser-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 to 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 nondenaturing 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. The installation of 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 is expected for autumn 2009. NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROMETRY The Nuclear Magnetic Resonance (NMR) lab is equipped with 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 conforma- FLOW CYTOMETRY Cytometry is a process in which physical and/or chemical characteristics of single cells are measured. In flow cytometry, the measurements are made as the cells or particles pass through the measuring apparatus in a fluid stream. 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. At the Department different research groups utilize this technological platform 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 provides instrumentation and expertise for automated cytofluorimetric analysis and sterile sorting of specific mammalian cell types. The equipment of our facility consists of a MoFlo® 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, 2-22mW user adjustable. It has 3 broad range ultra sensitive photomulti- [ 13 ] plier tubes. It has a 125µm triangular Flow Cell and a standard set of Emission Filters 460BP, 525BP, 575BP and 670LP. With this instrumentation it is possible to measure simultaneously Electronic Volume, side scatter, time, and 3 color detection with log/linear and FC/FSD options. This is used for analysis only. OPTICAL SPECTROSCOPY AND OPTICAL MICROSCOPY 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 y with excitation wavelength at _exc = 458nm, 476nm, 488nm, 496nm, 514nm; and two He-Neon lasers, respectively, with excitation wavelengths at y_ =543nm and at y_ = 633nm. The scanning head exc exc 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 enable 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 threedimensional 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 680-IR FTIR spectrometer for absorbance measurements in the medium and far infrared ranges, 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 UMA 500 (Digilab, USA). 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 simultaneously up to four samples. It is equipped with a static anisotropy fluorescence accessory (automatically controlled) and with a microplate reader working in reflecting optics. [ 14 ] 1.5 E D U CAT I O N BIOTECHNOLOGY (Coordinator Prof. Danilo Porro) BIOLOGY (Coordinator Prof. Antonio Zaza) BACHELOR IN BIOTECHNOLOGY www.biotecnologie.unimib.it BACHELOR IN BIOLOGY www.biologia.unimib.it The course is articulated in three years, two of which are common for 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). The overall number of enrolled students for the academic year 2007/2008 was 965. The course is articulated in a common year and a two-year period devoted to the following areas; Bioecology, Biomolecular and Physio-pathological studies. The overall number of enrolled students for the academic year 2007/2008 was 810. MASTER IN INDUSTRIAL BIOTECHNOLOGY www.biotecnologie.unimib.it MASTER IN BIOLOGY www.biologia.unimib.it The master course in Industrial Biotechnology is two year long with two specializations: 1) Pharma-genomics and 2) Processes and Products. The overall number of enrolled students for the academic year 2007/2008 was 120. 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 2007/2008 was 85. MASTER IN BIOINFORMATICS The Master course in Bioinformatics is carried out over two years in cooperation with the Degree in Informatics. The overall number of enrolled students for the academic year 2007/2008 was 20. ERASMUS PROGRAM FOR INTERNATIONAL MOBILITY Coordinator for Biotechnology: Prof. Maria Pia Longhese Coordinator for Biology: Prof. Silvia Nicolis [ 15 ] 1.6 ADVANCED TRAINING The Department hosts two PhD programs in the frame of the School of Doctorate in Sciences www.scuoladottorato.scienze.unimib.it: the PhD Program in Industrial Biotechnology and the PhD Program in Biology. Moreover, staff members participate in 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 Coordinator: Prof. M. Vanoni PhD PROGRAM IN BIOLOGY Coordinator: Prof. P. Tortora The Doctorate in Industrial Biotechnology is highly multi- and inter-disciplinary, the areas of expertise present within the Board of Professors include several aspects of modern Biotechnology: Biophysics, Biochemistry and Systems Biology, Molecular Biology, Genetics, Industrial Microbiology, Organic and Computational Chemistry. The curriculum is three yearlong and is planned as a full-time occupation, strongly based on the development of a research project. Students are encouraged to conduct a 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-of-the-art topics chosen year-by-year, 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 Biology and Bioscience 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. 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 conducted under the guidance of a member of the board of Doctoral Instructors, with which the student agrees upon a research project theme. 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 native-speaker and specifically planned to allow the students to acquire language skills that are indispensable for biological researchers. [ 16 ] SEMINARS 2008 14.01.2008 Luca Canova Dipartimento di Biologia Animale, Università di Pavia 16.01.2008 Vladimir I. Muronetz Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia Strumenti di valutazione degli impatti ambientali: la Valutazione Ambientale Strategica Influence of molecular and artificial chaperones on protein aggregation 17.01.2008 Luca Canova Dipartimento di Biologia Animale Università di Pavia Strumenti di valutazione degli impatti ambientali: Valutazione di Incidenza 28.01.2008 Jean-Louis Reymond Department of Chemistry and Biochemistry, University of Berne, Switzerland Substrate Arrays for Fluorescence-Based Enzyme Fingerprinting and High-Throughput Screening 05.02.2008 Luca Jovine Department of Biosciences and Nutrition, Karolinska Institutet, Sweden Sex & the molecule: Structural basis of biodiversity generation in metazoa 15.02.2008 Marco Geymonat National Institute for Medical Research MRC, London, UK Ruolo di Lte1 nella regolazione dell'uscita dalla mitosi in S. cerevisiae 26.02.2008 Vladimir I. Muronetz Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia Differential scanning calorimetry and dynamic light scattering principles and use for study of proteins, pro tein-protein interactions and protein aggregation 26.02.2008 Vladimir I. Muronetz Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia Immobilization of proteins and use of protein-protein interactions for affinity chromatography 11.03.2008 Alessandra Fischetti BioTeltec, srl Tecnologie innovative per l'avanzamento della ricerca genomica e post-genomica: Oligo microarray COMBIMATRIX e Reversed-Phase LysateCells Microarray AUSHON 27.03.2008 Anna Villa Istituto Tecnologie Biomediche, CNR- Milano Immunodeficienze primarie determinate da difetti del processo di ricombinazione VDJ del T-cell Receptor 07.04.2008 Aldo Pagano Dip. Oncologia Biologia e Genetica, Università di Genova Malattie associate a trascritti non codificanti della RNA Polimerasi III 11.04.2008 Annarosa Arcangeli Università di Firenze Integrine, canali del potassio e neoplasia 17.04.2008 Manolis Fanto Dulbecco Telethon Institute e D.I.B.T HSR, Milano Drosophila melanogaster: dalla biologia dello sviluppo alla patologia 18.04.2008 Marion Peter Institute of Molecular Genetics, Montpellier France Imaging molecular interactions by FRET and FLIM 21.04.2008 Giovanni Perini Dip. di Biologia Evoluzionistica Sperimentale Università di Bologna Complessità funzionale dell'oncogene N-Myc nella genesi e progressione del neuroblastoma [ 17 ] SEMINARS 2008 29.04.2008 Ario de Marco Consorzio Tecnologie Genomiche (COGENTECH), IFOM-IEO, Milano Recombinant antibodies in basic research, diagnostic, and therapy 08.05.2008 Michele Papa Dipartimento di Medicina Pubblica Clinica e Preventiva Seconda Università di Napoli Azione di NGF in diversi quadri patologici 12.05.2008 Roberto Spagnoli Sanofi Aventis, France Industrial bioconversions - General philosophy and strategy. Selected examples at large and small scale 13.05.2008 Roberto Spagnoli Sanofi Aventis, France Industrial bioconversions - Metabolic engineering of yeast for the production of hydrocortisone. Technical aspects and general learning 14.05.2008 Roberto Spagnoli Sanofi Aventis, France Interest of biomolecules in a large Pharmaceutical company: from research applications to therapeutic proteins and monoclonal antibodies 14.05.2008 Monica Beltrame Dip. di Scienze Biomolecolari e Biotecnologie Università degli Studi di Milano Ruolo del fattore trascrizionale Sox18, associato alla sindrome umana HLT (Hypotrichosis-LymphedemaTelangiectasia), nel differenziamento delle cellule endoteliali: uno studio funzionale in zebrafish 20.05.2008 Jean-Stéphane Joly &Alessandro Alunni Institut de Neurosciences A. Fessard CNRS, Gif-sur-Yvette, France Looking for neural stem cells? Just fish them 23.05.2008 Carmen Valenzuela Instituto de Investigaciones Biomédicas "Alberto Sols" CSIC/UAM Madrid, Spain Interaction between Kv1.5 and Kv_1.3 subunits. Pharmacological consequences 26.05.2008 Susanna Dolci Dipartimento di Sanità Pubblica e Biologia Cellulare Università di Roma Torvergata Roma Dalle cellule germinali alle cellule staminali: un punto di origine in comune?" 28.05.2008 Giuseppe Testa European Institute of Oncology Milano Histone lysine demethylation in stem cell commitment 11.06.2008 Philippe Compain Institut de Chimie Organique et Analytique, CNRS, Orléans, Francia Iminosugars: from synthetic methodologies to therapeutic applications 20.06.2008 Jakob Troppmair University of Medicine, Innsbruck, Austria Survival signaling: RAF kinases and beyond 20.06.2008 Marco Giorgio IFOM-IEO Campus, Milano P66Shc, oxidative stress and apoptosis 20.06.2008 Luca Scorrano Dulbecco-Telethon Institute, Padova The many shapes of mitochondrial death 24.06.2008 Cecilia Garlanda Istituto Clinico Humanitas, Milano Pentrassine: recettori solubili dell'immunità innata [ 18 ] SEMINARS 2008 25.06.2008 Paolo Riccio Dept. of Biology D.B.A.F., University of Basilicata, Potenza Novel biotechnological approaches for the study and treatment of Multiple Sclerosis 25.06.2008 Eero Castren Neuroscience Center, University of Helsinki, Helsinki, Finland Neurotrophic effects of antidepressant drugs 25.06.2008 Joris Winderickx Laboratory of Functional Biology, Catholic University of Leuven, Belgium Yeast models to study fundamentals of Parkinson's disease 25.06.2008 Tommaso Russo Dept. of Biochemistry and Medical Biotechnology, Univ. of Napoli “Federico II”, Napoli Role of Fe65-APP complex in cellular sensitivity to DNA damage: potential implications for the onset of neurodegenerative diseases 25.06.2008 Brian B Rudkin Ecole Normale Superieure de Lyon, Lyon, France The 'ins' and 'outs' of NGF signalling: a dynamic view 27.06.2008 Alessandra Gliozzi Dipartimento di Fisica, Università di Genova Molecular mechanisms of amyloid generation and toxicity 27.06.2008 Josè Luis R. Arrondo Departamento de Bioquimica, Universidad del Pais Vasco, Bilbao, Spagna Infrared Spectroscopy: A tool to study protein conformation 27.06.2008 Josè Luis R. Arrondo Departamento de Bioquimica, Universidad del Pais Vasco, Bilbao, Spagna 2D-IR spectroscopy in protein studies 27.06.2008 Thomas Laurell Dept. Electrical Measurements, Lund University, Sweden Free flow acoustophoresis - A new modality to cells separation 27.06.2008 Thomas Laurell Dept. Electrical Measurements, Lund University, Sweden Microchip platforms for low abundant protein analysis and diagnostics 22.09.2008 Andrea Morrione Kimmel Cancer Center, Thomas Jefferson University Philadelphia, PA, USA Growth Factors signalling in cell growth and transformation 11.12.2008 Thomas J. D. Jørgensen University of Southern Denmark Odense, Denmark Protein hydrogen exchange monitored by mass spectrometry 12.12.2008 Florian Hollfelder University of Cambridge, UK Multiple Catalytic Promiscuity: Mechanism and Evolution [ 19 ] 1.7 MASTER AND PHD THESES INDUSTRIAL BIOTECHNOLOGY Adamo Giusy “Studio di fattori coinvolti nella risposta a stress acido in lieviti e potenziali applicazioni biotecnologiche”. P. Branduardi Bologna Serena “Gene targeting in embryonic stem cells to generate transgenic mice expressing in an inducible manner the Bmi-1 oncoprotein”. F. Granucci Aldeghi Alessia “Ruolo della peptidil-prolil isomerasi PIN1 nella risposta al peptide Aß in cellule ippocampali di ratto: modulazione dello stato di fosforilazione della proteina TAU”. F. Peri Brambilla Elena “Caratterizzazione molecolare di preparati di fattore VIII tramite approccio proteomico MUDPIT”. P. Tortora Antonica Francesco “Generazione e caratterizzazione farmacologica di anticorpi monoclonali anti-hLHr ottenuti mediante immunizzazione genetica di topi con hLHrcDNA”. M. Foti Artesi Flora “Il RasGEF di lievito Cdc25 ha una funzione nucleare Ras-indipendente”. R. Tisi Barranco Giulia “Studi su antitumorali coniugati a ligandi di recettori cellulari”. B. La Ferla Barbieri Valentina "Il legame dello ione Cu(II) alla regione N-terminale del peptide ß-amiloide, Aß42: uno studio di modellistica molecolare classica e quantistica". L. Bertini Bartolacelli Chiara “Fund raising nell'ambito della ricerca italiana in biotecnologie: analisi delle opportunità e dei rischi nella prospettiva di elaborare un modello gestionale”. G. Casucci Bazzi Marco “Ruolo della protein fosfatasi Glc7 di lievito nello spegnimento del checkpoint da danni al DNA”. M.P. Longhese Bertolotti Matteo “Studio dei geni codificanti i fattori trascrizionali SOX2 e DMRT5 in cellule staminali embrionali e neurali murine”. M. Vanoni Broggi Achille “Ruolo delle cellule dendritiche nell'induzione di tolleranza delle cellule T negli organi linfoidi periferici”. F.Granucci Casati Alessio “Studio dello stress acido in lievito”. L. Brambilla Casola Marco “Search of NADH dependant amine dehydrogenases”. F. Peri Castelli Maddalena “La policistina-1 umana regola la polarità durante la migrazione cellulare tramite cdc42 e il citoscheletro dei microtubuli”. S. Piatti Catusi Ilaria “Spindle assembly checkpoint in S. cerevisiae: ruolo nel controllo della separazione degli spindle pole bodies e modulazione della sua attivazione”. S. Piatti Cerbone Pamela “L'amide endogena, palmitoiletanolamide riduce il dolore neuropatico dopo somministrazione sistemica nel topo: coinvolgimento dei recettori PPAR-y, TRPV1 e fattori neutrofici”. B. Costa Cereda Marco. “Sviluppo e applicazioni diagnostiche di un sistema point-of-care basato su Real-Time PCR”. M. Vai Bertolotti Milena “Omeostasi redox durante il differenziamento delle plasmacellule”. M. Foti Corneo Paola Elisa “Consumo di latte crudo: valutazione del livello si esposizione ai principali patogeni attraverso metodiche di microbiologia classica e molecolari”. L. Brambilla Bianco Sergio “Studio sul meccanismo neuroprotettivo di cellule staminali neurali in un modello murino di ischemia cerebrale”. F. Chiaradonna Colaiocovo Moreno “Analisi di network di regolazione per la definizione del fenotipo infiammatorio in cellule dendritiche murine”. M. Foti [ 20 ] Colciago Giorgia “Trasporto tra nucleo e citoplasma della miosina non convenzionale VI”. M. Vai Colombo Emanuela. “Neurotrofine e loro recettori nel muscolo scheletrico umano in condizioni fisiologiche e nelle miopatie infiammatorie”. A.M. Colangelo Corbetta Monica “Ingegnerizzazione di Saccharomyces cerevisiae per lo sviluppo di ceppi ad elevata robustezza”. P. Branduardi Cordani Francesca “Analisi filogenetica e caratterizzazione farmacologica di GPR17, recettore duale per nucleotidi e cisteinil-leucotrieni”. G. Giagnoni Corsiero Elisa Francesca: “Anti-inflammatory activity of vitamin D receptor agonists” F. Granucci Groppi Silvia “L'ingresso di calcio indotto da glucosio in Saccharomyces cerevisiae è mediato da diversi sistemi di trasporto e modula l'attività della calcineurina”. R. Tisi Lenti Elisa “Valutazione del ruolo del fattore trascrizionale Prep-1 nella tumorigenesi nel topo e nell'uomo”. S. Piatti Magni Ruben “Analisi delle differenze di espressione genica in cellule endoteliali fetali umane in gravidanze fisiologiche e in gravidanze complicate da ritardo di crescita intrauterine”. M. Vai Mariani Cinzia “Effetto di ibuprofene e isosorbidedinitrato e loro associazione in un modello animale di distrofia muscolare”. A. M. Colangelo Costantini Gabriele “Templati glicidici per la sintesi di molecule bioattive”. B. La Ferla Manrincola Gabriella “Metabolismo del carbonio e modificazioni post-traduzionali della cromatina nel lievito Saccharomyces cerevisiae”. M. Vai Crespolini Paolo “Sviluppo di sistemi regolati per la produzione di apossidi funzionalizzati mediante l'impiego di geni di Pseudomonas”. G. Bestetti Margariti Nadia “Vettori bicistronici per la generazione di suini transgenici per lo xenotrapianto”. M.L. Lavitrano D'Amore Gaetana “Sintesi di nuovi inibitori della proteina Ras umana: esperimenti di attività selettiva sul mutante oncogenico Ras G13D”. F. Peri Maroso Mattia “Interleuchina-1 beta come nuovo target per il controllo delle convulsioni: meccanismi coinvolti”. B. Costa Della Fiorentina Silvia “Nuovi mimetici del lipide A a struttura monosaccaridica”. F. Peri Meani Licia “Modulazione della produzione di IL1beta e della attività infiammatoria di TLR4 tramite HDACi”. M. Vanoni Della Vedova Sergio “Analisi Microarray: Integrazione di nuovi moduli di processamento dati nel software AMDA”. M. Foti Mingotto Federica “Valutazione sull'adesione, proliferazione e differenziamento di colture di preadipociti e condrociti su scaffold micro strutturati”. M. Foti Di Vona Chiara “Ruolo dell'ubiquitini-ligasi COP1 nella risposta all'irraggiamento ultravioletto”. G. Lucchini Farath Laila “Il glucosio come scaffold per la sintesi di mimetici del fosfatidilinositolo”. F. Nicotra Ferrari Emanuela “Ex vivo generation and expansion of cytotoxic T lymphocytes EBV specific”. M. Foti Fontana Paolo “Sviluppo di un metodo diagnostico di polimorfismi genetici, basato sulla microstampa di sequenze di DNA”. M. Vai Fuga Isabella “Valutazione della localizzazione e del differenziamento delle cellule staminali murine adulte trapiantate all'interno dei ventricoli laterali in un modello murino di SLA mediante metodi alternativi di tracciabilità”. G. Giagnoni Gaviraghi Marco “Caratterizzazione degli effetti dell'AMP ciclico sul pathway di Ras e sull'attività mitocondriale di fibroblasti murini normali e trasformati”. F. Chiaradonna Genovesi Elisabetta “Il recettore GABAA: sintesi e caratterizzazione farmacologica di nuovi potenziali ligandi”. L. Cipolla Oliva Paolo “Messa a punto di metodiche in vitro per lo screening di potenziali molecole antitumorali che inibiscono pathaways cellulari correlati all'ipossia e all'angiogenesi tumorale”. G. Lucchini Ottina Eleonora “Trasportatori mitocondriali del NAD+ in Saccharomyces cerevisiae: effetti di un alterato dosaggio genico”. M. Vai Pessina Stefania “Modificazioni post-traduzionali della cromatina nella risposta ai danni al DNA da raggi ultravioletti in Saccharomyces cerevisiae”. M.Vai Piccinini Sara “La metilazione del DNA: un marker epigenetico per l'identificazione di nuove epimutazioni in Zea mays”. E. Martegani Pietrogrande Giovanni “Ruolo del recettore dell'urochinasi (uPAR) nella modulazione del compartimento staminale epidermico”. G. Lucchini Pinto Alessandro “Systematic evolution of ligands by exponential enrichment: aptamer selection against PSA”. M. Vanoni Giunti Giulia “Studio dell'attivazione e della migrazione delle cellule NK mediata dalle cellule dendritiche: ruolo nell'attivazione enti-tumorale”. F. Granucci Polenghi Alice “Azione del Calcitonin gene-related peptide (CGRP) su astrociti in coltura: morfologia e distribuzione intracellulare di un componente del recettore del CGRP”. A.M. Colangelo Grimoldi Dario “Preparazione e studio di enzimi immobilizzati mediante formazione di cross-linked enzyme aggregates (CLEA)”. F. Peri Quarello Caterina Federica ”Correlazione dell'attività con il profilo strutturale di eparine a basso peso molecolare”. B. La Ferla [ 21 ] Redaelli Erika “Indagine sul proteoma urinario: l'approccio proteomico MudPIT permette la caratterizzazione di profili urinari di pazienti affetti da diverse patologie oncologiche”. P. Tortora Ripamonti Francesca “Meccanismo di regolazione di ∆Np63a in carcinomi esprimenti epidermal growth factor receptor”. F. Chiaradonna Ronzoni Riccardo “Un modello per lo studio delle patologie da accumulo di proteine”. M. Lotti Rusconi Paolo “Caratterizzazione funzionale di Drago, un gene con potenziale attività oncosoppressiva”. S. Piatti Savoia Paola “Mitocondri e neurodegenerazione: ruolo della m-AAA proteasi nella maturazione proteilitica di OPA1”. S. Colombo Sberna Irene “Espressione eterologa di citocromi in lievito per la produzine di fine e bulk chemicals”. P. Branduardi Scarpellini Milena “Studio funzionale del polimorfismo arg990gly del calcium sensing receptor ed effetto del calciomimetico r-568”. R. Tisi Vanzin Alessia “Progettazione, espressione, purificazione e caratterizzazione di peptidi auto-penetranti che inibiscono la via di Ras”. M. Vanoni Zanella Elisa “Malattie Cistiche renali causate da mutazioni di uromodulina: caratterizzazione di due limee murine transgeniche condizionali” .M.L. Lavitrano Zani Anna: “Caratterizzazione di mutazioni del DNA mitocondriale associate a difetti nel complesso I”. S. Colombo BIOLOGY Aprile Francesco “Studi sul ruolo fisiologico e sugli interattori della atgl umana, una proteina chiave nel metabolismo lipidico intracellulare”. P. Tortora Assandri Roberto “Biogenesi della membrana esterna di Escherichia coli: ruolo della proteina LptC nel trasporto del Lipopolisaccaride”. A. Polissi Bagnaresi Eleonora “Regulation of neural enhancers of the Shh gene by the Sox2 transcription factor”. S. Nicolis Spinelli Michela “Approcci allo sviluppo di inibitori peptidici del pathway di Ras”. M. Vanoni Bellini Martina “Mutazioni nella regione V3 dell'envelope di HIV-1 in pazienti Slow Progressor e Fast Progressor sono in grado di influenzare il legame con i co-recettori CCR5 e CXCR4”. P. Tortora Solinas Marta “Stress in Saccharomyces cerevisiae: effetti di un alterato livello delle modificazioni post-traduzionali degli istoni”. M. Vai Bisighini Cinzia “Analisi della risposta anticorpale mucosale in liquidi seminali di soggetti HIV sieropositivi”. F. Granucci Stracka Daniele “Espressione genica e codice istonico in starvation di azoto in Saccharomyces cerevisiae”. M. Vai Bonanomi Marcella ”Le sialidasi da zebrafish: caratteristiche strutturali e funzionali”. P. Fusi Torella Rubben “Studio di design e dinamica molecolare sulla micro-mioglobina, la minima unità proteica legante l'eme” L. De Gioia Branchini Irene “Effetti di alcuni antiepilettici sull'attività di reti corticali di topo”. E. Wanke Terzi Simona “Identificazione e caratterizzazione di cellule progenitrici glomerulari nel rene umano adulto”. M. Lotti Troina Filippo “Introni come regolatori dell'espressione genica nelle piante”. I. Orlandi Tsiarentsyeva Viktoryia “In Saccharomyces cerevisiae Snf1/AMPK controls entrance into S phase by modulating accumulation of Clb5 protein at a post-transcriptional level. Master thesis in Systems Biology, Goteborg University”. P. Coccetti Bussini Adelaide “Studio di un percorso diagnostico genetico in soggetti con ritardo mentale e/o malformazioni congenite”. S. Ottolenghi Cattaneo Pamela “Utilizzo dell'habitat per camoscio (Rupicapra Rupicapra Rupicapra) e muflone (Ovis [Orientalis] Musimon) in un'area delle Dolomiti di Brenta (Trentino)”. M. Casiraghi Ceccon Monica “Ruolo dei linfociti NKT nel controllo della crescita neoplastica. Studio in modelli murini geneticamente modificati”. F. Granucci Uboldi Sarah “Caratterizzazione autoradiografica del recettore gabaergico in aree cerebrali di un modello murino di epilessia mioclonica progressiva”. G. Giagnoni Cavallucci Elisabetta ”Effects of antiepileptic drugs on neuronal nicotinic receptors containing the a2-I279N subunit, linked to nocturnal epilepsy”. A.Becchetti Vajani Valentina “Analisi di geni coinvolti nello sviluppo del sistema vascolare in Zebrafish”. A.M. Colangelo Cesana Stefania “Studio citofluorimetrico della trasduzione del segnale nella leucemia mielomonocitica giovanile: ipersensibilità al GM_CSF e profili di fosforilazione proteica”. F. Granucci Valle Andrea: “Sviluppo di un trattamento combinato per la cura del diabete autoimmune in un modello animale pre-clinico”. F. Granucci Vanini Roberto “Analisi comparativa della risposta a diversi stress ossidativi nei lieviti”. L. Brambilla Danieli Elena “Ontogenesi di MRP1 e MRP3 in fegato di ratto e loro modulazione in seguito ad esposizione materna a PCB durante la gestazione e allattamento”. A. Colombo [ 22 ] Dossi Elena “Indagine sugli effetti della proteina Aß amiloide sull'attività elettrica di reti neuronali corticali murine in coltura mediante la tecnica del multielectrode array(MEA)”. E. Wanke Pannone Katiuscia “Coinvolgimento delle sialidasi NEU2 e NEU3 nei meccanismi di resistenza all'apoptosi caratteristici delle cellule di leucemia mieloide cronica, K562”. P. Fusi Frana Anna Maria “Studi di fibrillogenesi in vitro di varianti dell'atassina-3: ruolo del contesto proteico nel processo di aggregazione”. M. E. Regonesi Panzarino Claudia “Supporto trsfusionale di pazienti con alloimmunizzazione verso antigeni piastrinici: creazione di un registro di donatori di piastrine tipizzati per HPA”. F.Granucci Franchi Michela “Analisi delle mutazioni del gene WT1: un nuovo marcatore con significato prognostico nella leucemia acuta mieloblastica a cariotipo normale dell'adulto”. A. Ronchi Gara Nicola “Cicli biologici di alcune specie di efemerotteri (Insecta:Ephemeroptera) in torrenti alpini piemontesi” P. Galli Giussani Flavia “Effetto di un estratto di Cannabis sativa in un modello di neuropatia diabetica”. B. Costa Paro Simona “Caratterizzazione dei complessi proteici associati a SRPK2 da linee cellulari di Neuroblastoma”. S. Barabino Pedrini Olga “Studio dell'effetto in vitro su cellule leucemiche di due nuovi composti, inibitori delle chinasi ciclinadipendenti”. A. Vescovi Grande Vito “Role of the transcription factor Sox6 in erythroid development”. A. Ronchi Perego Alberto “Caratterizzazione molecolare di mutazioni nel gene di ADAMTS13 in pazienti affetti da Porpora Trombotica Trombocitopenica”. A. Ronchi Gulizia Carla “DNA barcoding e filogenesi molecolare dei chirotteri italiani”. M. Casiraghi Pezzoli Laura “Indagine molecolare del gene JAG1: analisi di 51 casi con sospetta sindrome di alagille”. S. Nicolis Koukkonis Vaios “Caratterizzazione della trealasi da larve di Chironomus riparius”. P. Fusi La Placa Chiara Maria “Analisi proteomica di tessuti paraffinati da carcinoma gastrico mediante MudPIT per la ricerca di nuovi marcatori”. P. Tortora Lazzaro Mario “DNA barcoding: un mezzo oggettivo per la caratterizzazione di prodotti ittici”. P. Galli Leoni Bianca “Immunogenicità di cellule di leucemia promielocitica Acuta (APL) cells attraverso apoptosi indotta dalle antracicline e dagli altri farmaci inclusi nel protocollo APL”. S. Nicolis Maccabruni Irene “Caratterizzazione funzionale in vitro della proteina PAX5/TEL, identificata in pazienti affetti da leucemia linfoblastica acuta con traslocazione t(9;12)”. S. Barabino Maurizio Eleonora “Diversità dell'azione ß2-adrenergica nella risposta delle cellule dendritiche agli agonisti dei recettori Toll-like”. F. Granucci Menduni Francesca “Ruolo delle Chinasi PH-domaindipendenti nella Modulazione della Contrattilità Cardiaca”. A. Zaza Messina Ornella “Riorganizzazione dei microdomini lipidici di membrana durante la capacitazione di spermatozoi di maiale”. M. Pitto Montano Simone “Monitoraggio del Coral Bleaching nel parco marino di Watamu, Kenya”. P. Galli Ravasi Elena “Ruolo dell'oncogene RET/PTC1, specifico del carcinoma papillare tiroideo, nella regolazione dell'espressione del gene LOX (LISIL OSSIDASI)”. A. Ronchi Riva Chiara “Ridescrizione del genere Protogyrodactylus Johnston e tiegs, 1922 (Monogenoidea: Dactylogrydae)”. P. Galli Rizzi Laura “Effetti di un trattamento a breve termine con GHRELIN e GHS sintetici in un modello animale di cachessia”. G. Giagnoni Ronchetti Monica “Effetto di URB602, un inibitore della monoacilglicerolo lipasi, sul dolore infiammatorio e neuropatico”. G. Giagnoni Sacchetti Andrea “Ecologia e selezione a livello di habitat dei gobidi anfibi (Gobiidae: Oxudercinae) del Fly River, papua Nuova Guinea”. P. Galli Scivittaro Silvia Francesca “Il metabolismo di S. cerevisiae e le variazioni indotte da xenobiotici, in particolare dal permanganato di potassio”. M. Milani Seveso Davide “Indagine sulle conseguenze post-sbiancamento in differenti taxa di madrepore (scleractinia) nel parco marino di Watamu, Kenya”. P. Galli Silva Elisabetta “Analisi delle molecole costimolatorie nella patogenesi e prognosi della Sclerosi Multipla”. M. Foti Morosi Lavinia “Studio del meccanismo di ancoraggio alle membrane della sialidasi umana NEU4”. P. Fusi Spada Roberto “Sviluppo di un saggio di screening per interattori trascrizionali della transizione epitelio-mesenchimale e della transizione mesenchimo-epiteliale: generazione di linee cellulari stabili via sistema di trasduzione lentivirale cromatina indipendente”. S. Nicolis Mulone Ludovica “Role of phosphorylation in PML posttransational modfications regulated by AS2O3 “. P. Fusi Specchio Fabio “Caratterizzazione di cellule ganglionari di retina di topo adulto”. E. Wanke Moretti Cinzia “La teoria delle reti negli studi genomici”. M. Casiraghi [ 23 ] Stocchetti Elisa “Proposta di definizione dello stato ecologico ai sensi della Direttiva 2000/60/EC mediante analisi delle comunità macrobentoniche nei corsi d'acqua superficiali della Provincia di Foggia”. P. Galli Tettamanti Sarah “Studio dei geni codificanti i fattori trascrizionali SOX2 e DMRT5 in cellule staminali embrionali e neurali murine”. S. Nicolis Toffolon Roberta “Può una ridotta fascia tampone rimuovere i nutrienti di origine agricola? Un caso di studio della bassa Pianura Reggiana”. P. Galli Tonoli Diletta “In vivo and in vitro study of two regulatory elements of the Sox2 gene active the developing brain and in neural stem cells”. S. Nicolis Villa Riccardo “Ruolo di LptB nella membrana esterna di Escherichia coli”. A. Polissi Zanini Sara “Diagnosi dei principali virus e batteri atipici in pazienti affetti da broncopneumopatia cronica ostruttiva (BPCO)”. A. Polissi Zonca Manuela “Characterisation of cyclin A2 interactions with its cell cycle regulatory partners during the G2 and M phase”. S. Nicolis Consonni Silvia “Electrophysiological and morphological approaches to the study of the nicotinic cholinergic transmission in the cerebral cortex and the pathogenesis of ADNFLE”. PhD DIMET. Tutor: A. Becchetti Contran Nicla “Plant antioxidant systems in stress responses”. PhD in Biology. Tutor: P.Crosti Di Resta Chiara “Electrophysiological study of human neuronal nicotinic receptors containing a2-I279Nß4 subunit, linked to a sleep-related epilepsy”. PhD DIMET. Tutor: A. Becchetti Ferrara Silvia “Environmentally friendly processes for the production of molecules of industrial interest. PhD in Biology. Tutor: P. Tortora Galbusera Elena “Bacillus subtilis improvement for the development of fermentative processes aimed at producing pyrimidine nucleotides”. PhD in Biology. Tutor: P. Tortora Gregori Maria “Synthesis of biologically relevant carbohydrate analogues with potential pharmacological activity”. PhD in Chemistry. Tutor: L. Cipolla BIOINFORMATICS Amara Flavio “Caratterizzazione del sito catalitico della carbossipeptidasi di Sulfolobus solfataricus mediante uno studio di dinamica molecolare”. G. Moro Milan Chiara “Stabilizing micromyoglobin by molecular design”. R. Grandori, Sensi Cristina “Sviluppo di un sistema bioinformatica integrato per la classificazione di campionature biologiche attraverso l'analisi di mappe di elettroforesi bidimensionale”. M. Vanoni Sana Maria Elena “Structural determination of the second Plant HomeoDomain (PHD) of AutoImmune Regulator (AIRE) by solution NMR spectroscopy and computational methods”. R. Grandori PhD DISSERTATIONS Amigoni Loredana “PH-PxxP domain of RalGPS2 is a dominant negative factor for RalA activation in PC12 cell”. PhD in Industrial Biotechnology, Tutor: E. Martegani Bonetti Diego “Crosstalks between DNA damage checkpoints and telomere metabolism in Saccharomyces cerevisiae”. PhD in Industrial Biotechnology, Tutor: M. P. Longhese Comelli Francesca “The modulation of the endocannabinoid system in order to treat neuropathic pain”. PhD in Biology. Tutor: B. Costa Guerini Ilaria: “Preserving balanced genetic information in gametes: Checkpoint response during meiosis can distinguish between accidental and programmed DNA double-strand breaks”. PhD in Industrial Biotechnology, Tutor: M.P. Longhese Lancini Cesare “Functional roles of the transcription factor Sox2 and its interactions with Emx2 in mouse brain neural stem cells and neuronal differentiation”. PhD DIMET. Tutor: S. Nicolis Panseri Silvia “Central and peripheral nervous system regeneration via nano-structured scaffolds”. PhD in Biology. Tutor: B. Costa Pontiroli Francesca “Late production of type I IFNs by Listeria monocytogenes-infected dendritic cells affects early innate immunity by interfering with dendritic cellNK cell cross talk”. PhD DIMET. Tutor: M. Foti Rossio Valentina “Cellular response to mitotic perturbations: PP2A-mediated control of sister chromatid cohesion and adaptation to spindle assembly checkpoint activation”. PhD in Biology. Tutor: G.Lucchini Venturetti Marianna “Exit from mitosis in Saccharomyces cerevisiae: regulation of the small GTPase Tem1”. PhD in Industrial Biotechnology, Tutor: S. Piatti 2 R ESEARCH G ROUPS [ 26 ] 1 MOLECULAR GENETICS OF DEVELOPMENT AND CELL DIFFERENTIATION IN MOUSE AND MAN Silvia Nicolis, Sergio Ottolenghi, Antonella Ronchi, Rebecca Favaro, Anna Ferri. 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. 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 Sox2-dependent 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. 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). 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). 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. Casalgrandi , 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 Soxfamily 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) 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) [ 27 ] MECHANISMS OF POST-TRANSCRIPTIONAL REGULATION OF MAMMALIAN GENE EXPRESSION AND THEIR ROLE IN HUMAN DISEASE 2 Reinaldo Alvarez, Silvia Barabino, Silvia C. Lenzken, Silvia Vivarelli. 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. Our laboratory studies the molecular mechanisms involved in the processing of pre-messanger RNA transcripts in the neuronal cells. Eukaryotic messenger RNA precursors (pre-mRNAs) 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 shut- tling may reflect 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 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. [ 28 ] CONTROL OF GENETIC INTEGRITY BY THE DNA DAMAGE CHECKPOINT PATHWAYS 3 Maria Pia Longhese, Giovanna Lucchini, Michela Clerici, Veronica Baldo, Diego Bonetti, Ilaria Guerini, Nicola Manfrini and Marco Bazzi. 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. [ 29 ] MITOTIC PROCESSES PREVENTING GENOME INSTABILITY AND ANEUPLOIDY 4 Roberta Fraschini, Elena Chiroli, Valentina Rossio, Marianna Venturetti, Laura Merlini, Elena Galati, Ilaria Catusi, Giovanna Lucchini 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 surveillance mechanisms, or checkpoints, which monitor the completion of critical cell cycle events and delay cell cycle progression until errors have been corrected. Thus, these control mechanisms ensure 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, where 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. Therefore, anaphase must be kept under check and delayed until all chromosomes are bi-oriented, a task carried out by the spindle assembly checkpoint (SAC). 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 already at the G1/S transition, concomitantly with bud emergence and much earlier than bipolar spindle formation. A surveillance mechanism called 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 previously uncharacterized yeast proteins that we named 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. Dma1/2 proteins, as well as Chfr and Rnf8, are ubiquitin ligases with a forkhead-associated domain that is normally implicated in the interaction with phosphorylated proteins, and a Ringfinger domain typical of E3 ligases. 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. [ 30 ] 5 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, David Metalli, Daniele Colombo, Chiara Balestrieri, 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 in the model organism, Saccharomyces cerevisiae, as well as in normal and transformed mammalian cells. The approach involves both “wet” experiments and 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 CONTROL OF THE G1 TO S TRANSITION: NETWORK IDENTIFICATION AND PARAMETER ESTIMATION Paola Coccetti, Farida Tripodi, Claudia Cirulli, Marco Vanoni, Lilia Alberghina. CK2 is a highly conserved enzyme ubiquitously distributed among eukaryotes that phosphorylates a wide range of substrates. Genetic studies indicate that CK2 activity is required for cell cycle progression in both mammals and yeast. Recent results newly indicate a major involvement of CK2 in the regulation of cell cycle progression in budding yeast since several targets of CK2 have been found to serve essential functions. Specifically, we found that CK2 promote ubiquitin-conjugating activity of the E2 ubiquitinconjugating enzyme Cdc34 -required for the G1/S transition- by phosphorylating Ser130 and Ser167 within the catalytic domain. Based on our work on CK2-mediated phosphorylation of Sic1 and Cdc34 (Coccetti et al 2006, Coccetti et al 2008) and on available literature data, the goal of our research is to elucidate the role of CK2 phosphorylation on the G1/S transition in budding yeast studying by mass spectrometry the phosphorylation state of its relevant substrates as a function of growth conditions and cell cycle position in exponential and perturbed growth. NUTRITIONAL MODULATION OF CELL CYCLE PROGRESSION IN SACCHAROMYCES CEREVISIAE STUDIED BY BIOCHEMICAL AND POST-GENOMIC TECHNIQUES Marco Vanoni, Paola Coccetti, Stefano Busti, Farida Tripodi, Laura Gotti, Viktoria Tsiarentsyeva, Lilia Alberghina Cell proliferation requires an exquisite coordination between continuous events of the growth cycle and discontinuous events of the nuclear division cycle which results in nutritionally-modulated cell mass homeostasis and correct duplication and segregation of the genetic material. Combining genetic, physiological, biochemical and postgenomic 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 transition. We could show that the Gpr1/Gpa2, but not the Snf3/Rgt2, pathway plays a direct role in setting cell mass. Snf1 deletion affects translation, but not transcription of Clb5. These results are being used to move towards a more complete network identification of the G1 to S transition. MODELLING OF CELL CYCLE AND SIGNAL TRANSDUCTION PATHWAYS Lilia Alberghina, Ferdinando Chiaradonna, Daniela Gaglio, Stefano Busti, Elena Sacco, Marco Vanoni We have incorporated finding regarding interaction of Cln3 with the Cki Far1 in a nutritionally modulated threshold controlling the G1/S transition (Alberghina et al, 2004) into a mathematical model of the G1 to S network (Barberis et al, 2007) that newly shows that Ps is an emergent property of network strongly dependent on growth rate. A mathematical model of the entire cell cycle is now under construction. Time-course analysis of key players in the G1/S transition of normal mammalian fibroblasts have been collected and have been integrated with literature data to develop a model for the G1/S transition of normal mammalian fibroblasts (in collaboration with E. Klipp (Berlin) and G. Milanesi (CNR, MI). assuming a conservation during evolution of the basic structure of this network. 1 [ 31 ] 2 Cdc34 1_Mitochondrial morphology (green staining) and cytoskeleton (red staining) NIH3T3 cells 2_A schematic representation of Cdc34 functional domains 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. We functionally expressed such signalling module in budding yeast (Busti et al., 2008). In collaboration with M. Farina and D. Liberati (Politecnico di Milano) we developed a mathematical model descibing 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 NEUROPROTECTION BY NGF Anna Maria Colangelo, Daniele Colombo, Sandra Viggiani, Martina Fragni, Lilia Alberghina Apoptosis is the main form of neuronal death during neurodegenerative diseases. Global analysis of neuronal apoptosis in Alzheimer Disease (AD) has led to a modular molecular model where mitochondrial function is modulated by molecules regulating survival/differentiation in response to Nerve Growth Factor (NGF) (Alberghina & Colangelo, 2006). We are using NGF-differentiated PC12 cells to dissect molecular mechanisms of neuronal apoptosis following NGF deprivation by analyzing the temporal correlation between activation of cell cycle regulatory proteins, mitochondrial dysfunction, increased ROS production and the final apoptotic steps. In addition, we are studying mechanisms of neuroprotection by NGF both in vitro and in vivo (in animal models of peripheral nerve injury, in collaboration with Prof. M Papa (UniNA). The different pathways of cell death and their relation to longevity have also been analyzed (Salvioli et al, 2008). RHNGF AND NGF-LIKE PEPTIDES FOR THE THERAPY OF NEUROPATHIES Lilia Alberghina, Anna Maria Colangelo, Sandra Viggiani, Daniele Colombo, Enzo Martegani The role of decreased availability of NGF in development and progression of neurodegenerative processes (peripheral neuropathies and AD) involving NGF-dependet neurons is well established. Based on our previous work on production of recombinant human (rhNGF) (Colangelo et al., 2005), we are currently working in collaboration with Primm and Blueprint Biotech, on projects aiming at: i) developing bioassays for development of rhNGF; ii) developing and analyzing NGF-like molecules that might be characterized by better pharmacological properties (Colangelo et al., 2008). CANCER AND METABOLISM: ROLE OF ONCOGENIC K-RAS PROTEIN IN METABOLIC REPROGRAMMING OF CANCER CELL Ferdinando Chiaradonna, Daniela Gaglio, Marco Gaviraghi, Elisa Sottotetti, Marco Vanoni, Lilia Alberghina The relation between alterations of metabolism and transformed phenotype has recently received increased attention. We showed that selective growth advantage of rastransformed fibroblasts is lost upon growth in sub-optimal glucose concentration (Chiaradonna et al., 2006a, 2006b). Such dependence of transformed cells on glucose availability correlates with a reduced activity of Complex I and ensuing reduced oxidative phosphorylation (in collaboration with G. Lenaz, Unibo). We could identify an essential role of the PKA pathway in the control of mitochondria activity. Bioinformatic analysis of the PKA pathway in 60 different cancer cell lines showed that in transformed cells the PKA pathway is repressed as compared to normal counterpart possibly due to oncogenic activation of Ras pathway (Chiaradonna et al., 2008, Balestrieri et al., 2009). In addition, we showed that glutamine shortage strongly reduces proliferation ability of transformed cells, without inducing apoptosis and with no effect on overall protein synthesis or ATP level. Fragility of ras-transformed cells to glutamine depletion is largely due to a reduced supply of DNA replication precursors in the presence of active signalling inputs leading to execution of the G1/S transition (Gaglio et al., 2009). DESIGN, DEVELOPMENT AND MOLECULAR CHARACTERIZATION OF RASGRF1-DERIVED RAS INHIBITORS E. Sacco, D. Metalli, M. Spinelli, A. Vanzin, M. Vanoni Mutations of Ras proteins and their regulators are critical events in the pathogenesis of human tumors and developmental syndromes. We are using molecules derived from Ras-sequestering peptides (Sacco et al., 2005) and small sugar-derived Ras inhibitors (provided by F. Peri, this Department) 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 M. Vanoni, A. D'urzo, E. Sacco The BIAcore technology is being used to analyze protein/protein interaction of proteins of potential pharmaceutical interest in real time. These proteins include: Ras, prion-derived peptides, cell cycle inhibitors and ataxin. [ 32 ] 6 SIGNAL TRANSDUCTION IN EUKARYOTIC CELLS Enzo Martegani, Sonia Colombo, Renata Tisi, Michela Ceriani, Fiorella Belotti, Chiara Paiardi, Loredana Amigoni, Silvia Groppi SIGNAL TRANSDUCTION IN YEAST: Enzo Martegani, Sonia Colombo, Renata Tisi, Chiara Paiardi, Fiorella Belotti In Saccharomyces cerevisiae one of the main signalling transduction pathways is the Ras/cAMP/adenylate cyclase pathway, finely tuning pKA activity in the cell. The Ras-GEF Cdc25 is essential for viability of yeast cells. Beside this essential function related to its GEF activity, this protein is revealing additional functions. Cell membrane fractionation allowed to localize the Cdc25 protein in the internal membranes, but nuclei purification reveals that Cdc25 is also physiologically imported and efficiently retained in the nucleus. Forcing nuclear export of Cdc25 causes a growth defect on glycerol as a carbon source, suggesting an involvement of Cdc25 nuclear localization in derepression from glucose. Moreover, PKA hyper-activation induces complete Cdc25 export from the nucleus, suggesting a regulation on Cdc25 localization by phosphorylation. We also investigated the localization of active Ras2 in vivo through the use of a trimeric Ras binding domain of Raf1 (RBD3), which binds selectively Ras2-GTP, fused with GFP. Our results show that the localization of the probe is dependent on the abundance and quality of the carbon sources. In fact, in a wild type strain growing on medium containing 2% glucose, the RBD3-GFP probe accumulates mostly at the plasma membrane and into the nucleus, in 2% galactose it accumulates also in internal membranes and mitochondria, while in starved cells it accumulates only in internal membranes and mitochondria. Finally, upon addition of glucose to starved wild type cells, a rapid recruitment of the probe back to the plasma membrane and into the nucleus was observed. We initiated also a study on the role of the cAMP-PKA pathway in the coordination between cell growth and cell cycle and in the modulation of expression of key cell cycle proteins involved in the G1/S transition. We used a cyr1∆, pde2∆, msn2∆, msn4∆ strain. Deletion of PDE2 allows this strain to use cAMP added to the medium, bypassing lethality caused by deletion of CYR1; moreover, deletion of genes encoding the transcriptional factors Msn2 and Msn4 allow this strain to grow even in the absence of cAMP. Our preliminary results show that the addition of cAMP to the cyr1∆, pde2∆, msn2∆, msn4∆ strain strain influences cell growth parameters and the level of expression of Sic1. CALCIUM SIGNALING IN YEAST Enzo Martegani, Renata Tisi, Fiorella Belotti, Silvia Groppi Collaboration with: Rogelio Brandão The addition of glucose to glucose-deprived cells of Saccharomyces cerevisiae triggers a quick and transient flux of calcium incoming from the extracellular environment. This involves at least three different carrier systems: the Mid1 Cch1 system, only functioning during growth on a minimal medium; the low affinity system, functioning mainly during growth on a rich medium; and another calcium transport system, not yet identified, that can substitute the two known systems when they are inactivated. The unknown calcium transporter is also activated by hypotonic shock or by high calcium concentration in the medium, and is poorly sensitive to magnesium. Calcineurin, a phosphatase with serine and threonine specificity, is involved in the regulation of calcium homeostasis. For the first time we have shown that calcineurin can also be activated by nutrients: by using a calcineurin responsive CDRE-LacZ reporter, the activation of calcineurin was observed in derepressed cells after addition of glucose in the presence of 1 mM extracellular calcium. SIGNAL TRANSDUCTION MECHANISMS IN NGF-MEDIATED DIFFERENTIATION. Enzo Martegani, Michela Ceriani, Loredana Amigoni Collaboration with: Stefano Morara, Giovanna Berruti Ligand-activated receptors tyrosine kinase (RTK) endocytosis and endosomes-mediated transport to lysosomes for degradation are crucial to downregulate the cell proliferation signals. Receptors ubiquitination is a sorting signal for this trafficking. 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. To evaluate TrkA-USP8 interaction in vivo, a coimmunoprecipitation assay was performed in PC12-TrkA cells transiently transfected with hUBPy stimulated for different time with NGF. TrkA and UBPy interaction seems to depend on stimulation. The coimmunoprecipitation assay performed on untransfected PC12 cells verified that the interaction is physiological and occurs at the endogenous level. We next analyzed the subcellular localization of UBPy in PC12 cells and the effect of the overexpression of this deubiquitinating enzyme on PC12 differentiation. UBPy localize in cytosol. Cells transfected with UBPy-GFP fusion construct did not differentiate also after 72h from stimulation while cells transfected with UBPyC748A, a catalytically inactive mutant, presents a high degree of differentiation. These data let us suppose that the overexpression of UBPy blocks differentiation probably promoting TrkA degradation. [ 33 ] YEAST AS A MODEL SYSTEM FOR STUDYING AGING AND STRESS-RELATED PROCESSES 7 Marina Vai, Ivan Orlandi, Gabriella Marincola, Matteo Viganò, Domenico Abete, Nadia Casatta, Ambra Corti, Pietro Giani HISTONE MODIFICATIONS AND AGING IN SACCHAROMYCES CEREVISIAE - Marina Vai, Ivan Orlandi, Gabriella Marincola, Domenico Abete, Ambra Corti, Pietro Giani DNA of eukaryotes is wrapped around nucleosomes and packaged into chromatin. The details of this packaging are crucial for many cellular processes including aging. Changes in chromatin are mediated by histones modifications that include acetylation, methylation and ubiquitination. The Sir2 family (Sirtuins), comprises the unique class of NAD+ -dependent deacetylases. Sirtuins are phylogenetically conserved and beyond silencing, they promote longevity. In yeast, proper association of Sir2 to silent chromatin requires the deubiquitinating enzyme, Ubp10 that regulates the levels of H2B monoubiquitination. We are focusing on i) the role of Ubp10 in the regulation of the chromatin state, studying histones modifications in different experimental conditions related to aging; ii) the role of Sir2 as a metabolic sensor that links calorie intake to transcriptional gene expression. Therefore, processes such as glycolysis, respiration and NAD synthesis, that influence the pool of nicotinamide metabolites, have a profound effect on Sirtuins activity. In this context, a molecular characterization of yeast strains that have altered mitochondrial NADH/NAD+ ratios is underway (in collaboration with L. Palmieri, Università di Bari, Italy). emphasis has been directed towards a family of glucanosyltranferases that play an important role in cell wall biogenesis and in the response to cell wall stress. A functional characterization of these enzymes 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 changes (in collaboration with L. Alberghina, this Department). YEAST IN SPACE THE FUNGAL CELL WALL AS A TARGET FOR ANTIFUNGAL DRUGS - Marina Vai, Ivan Orlandi Marina Vai, Ivan Orlandi Opportunistic fungal infections have increased in recent years as a result of increased immunosuppression associated with AIDS, organ transplants, aggressive treatment of cancer and autoimmune disorders. 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 regarded as a target for new antifungals due to its essential biological role in fungal cells and its absence in mammalian ones. Special Last year, a suitable experimental equipment containing yeast cells has been sent in one of the spaceflights organized by ESA. In this context, yeast has been used as a model system for studying the effects of lack of gravity. On yeast cells, returned from the space, experiments testing the activation of pathways involved in the stress response have been performed and spaceflight simulations on the ground are underway (in collaboration with S. Bradamante, C.N.R., Milano, Italy). [ 34 ] 8 PROTEIN MASS SPECTROMETRY Rita Grandori, Maria Samalikova, Carlo Santambrogio, Elena Accardo Mass spectrometry (MS) is employed on one side as an analytical tool for proteomics. The focus is on the phosphorylation states and intracellular partners of regulators of the yeast cell cycle. On the other side, mass spectrometry is applied to the direct investigation of noncovalent interactions and intact protein structures for conformational studies and binding analysis. This information is complemented by data obtained by other biophysical methods and bioinformatics. SIC1 CONFORMATIONAL PROPERTIES Internal collaborators: Maria Samalikova, Stefania Brocca, Marina Lotti, Lilia Alberghina, Marco Vanoni. External collaborators: Vladimir Uversky, (Indianapolis, IN). 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, structural characterization of this protein has been progressing very slowly. 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 reveals a surprising degree of compactness, and can be “denatured” to a completely unfolded state. Intrinsic structure and order propensity of IDPs is a very important feature that can mediate recognition of intracellular partners. Interestingly, the most structured region of the molecule seems to include part of the kinase-inhibitory domain. The future aims of this project will include further structural characterization of the order seeds within the Sic1 molecule and the investigation of their role in molecular recognition. THE TANFORD TRANSITION IN BETA-LACTOGLOBULIN Internal collaborators: Carlo Santambrogio Protein folding and unfolding transitions can be monitored by the charge state distributions (CSDs) obtained by ESIMS. However, minor conformational changes, such as displacement of secondary-structure elements, typically do not alter protein CSDs and require more sensitive tools for structural studies. The fluorescent dye 1-anilinonaphthalene-8-sulfonate (ANS) is used to monitor protein conformational changes by the increased fluorescence of the dye upon binding to hydrophobic structures. ANS binding to exposed hydrophobic patches can be considered to be quite conformation-specific. However, ANS also binds to proteins by rather unspecific electrostatic interactions. To which category the complexes detectable by MS belong is still subject of debate. The Tanford transition in beta-lactoglobulin (BLG) was exploited as an experimental device to dissect hydrophobically- from electrostatically-driven binding. The results indicate stronger binding to the “open” protein conformation at pH 8 than to the “closed” structure at pH 6 supporting the hypothesis of conformation-specific binding. Control experiments show that ANS binding inside protein cavities is detectable by ESI-MS only in well folded structures, like the BLG calyx and the apoMb heme pocket, while ANS interactions with highly dynamic structures or molten globules, although detectable in solution, are easily lost in the gas phase. PROTEIN-LIGAND INTERACTION Internal collaborators: Antonino Natalello, Silvia Doglia. External collaborators: Jannette Carey (Princeton, NJ), Norbert Mueller (Linz, Austria), HongFang Ji and Liang Shen (Shandong, China), Iva Kuta Smatanova and Ruediger Ettrich (Nove Hrady, Czech Republic). We are currently studying two protein-ligand systems. The oligomeric flavodoxin-like WrbA, which binds flavin mononucleotide (FMN), and micromyoglobin (µMb), a minimal fragment derived from whale myoglobin, which binds heme. We have solved the crystal structure of the apo and holo forms of WrbA. The comparison reveals the bases of the effect of FMN on protein oligomerization and stability previously revealed by MS and IR spectroscopy. Molecular-dynamics simulations have been carried out in order to analyze the effect of heme on the conformational stability of µMb. The simulations reproduce the experimental evidence that the fragment in the absence of heme does not maintain a native-like conformation. The results indicate that, besides specific protein-ligand interactions, a shielding effect of heme on long-range electrostatic interactions contribute to conformational stability of holo-µMb. [ 35 ] PROTEIN ENGINEERING AND INDUSTRIAL ENZYMOLOGY 9 Marina Lotti, Stefania Brocca, Pietro Gatti-Lafranconi, Giusy Manuela Adamo, Riccardo Villa Enzymes employed in biocatalysis, model proteins and instrinsically disordered proteins (IDPs) are studied by a combined approach of mutagenesis (both directed evolution and site directed mutagenesis) and biochemical and biophysical characterization. Major goals of our research are to highlight the molecular bases of stability, function and interactions and to modulate these properties. Cold adapted enzymes are studied both to understand the structural determinants of temperature adaptation and to develop low-temperature biocatalysts. Aggregation is studied in vitro and in vivo, in particular in bacterial inclusion bodies. Moreover, novel biocatalysts are isolated from non commercial sources or produced by protein engineering. CONFORMATION, STABILITY AND BIOLOGICAL ACTIVITY Different model proteins are used to investigate how function and conformation are related and affected by the experimental or physiological environment. In the following a few relevant examples are quoted to illustrate our general approach. The Burkholderia glumae lipase is an enzyme of wide use in biocatalysis whose robustness is of importance for the feasibility of the process. It was exposed to high temperature, extreme pH and organic solvents and residual activity was monitored in parallel with the maintenance of the native structure as determined by ESI-MS and circular dichroism. This study revealed that the loss of activity precedes denaturation and is probably due to minor movements in the polypeptide architecture, thus suggesting some strategies of stabilization. Another lipase, produced by the psychrophile Pseudomonas fragi was randomly mutagenised and variants showing improved stability extensively characterized from the structural and conformational point of view, to highlight the fine detail of temperature adaptation in this protein. 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). The effect on protein conformation of post-translational modifications (i.e. glycosylation, phoshorylation) is also investigated. This work is performed in collaboration with the labs of S.M. Doglia, R. Grandori and L. Alberghina from this Department and S. Longhi at the CNRS of Marseille, France. PROTEIN AGGREGATION AND STRESS RESPONSES Several recombinant proteins when expressed in bacterial cells aggregate in inclusion bodies. This phenomenon has practical implications for the production of recombinant proteins but is also of interest for the study of general mechanisms of aggregation that can be translated to pathological aggregation in eukaryotic cells. During this year we have studied the deposition of inclusion bodies composed by proteins differing in sequence, stability and for the presence of disulfide bonds, showing how the specific protein sequence and the fermentation conditions influence the structure of the protein within aggregates and, as a consequence, its residual biological activity. The production of recombinant proteins can be also considered as a cause of stress for the producing cells. Accordingly we have characterized stress responses affecting the cell membrane and dependent on the aggregation state of the recombinant protein. A new field of investigation concerns the stress produced in yeast cells by the exposition to heavy metals in the environment and the molecular mechanisms of adaptation. These studies are performed in collaboration with the labs of S.M. Doglia and P. Tortora from this Department and Ario De Marco IFOM, Milano. [ 36 ] 10 STRUCTURE FUNCTION-PATHOGENICITY RELATIONSHIPS IN PROTEINS Paolo Tortora, Maria Elena Regonesi, Gaetano Invernizzi, Elena Galbusera, Serena Mazzucchelli, Elisa Mazzantini, Emanuela Occhipinti Amyloid fibrils generated by human ataxin-3, as shown by atomic force microscopy. The arrows highlight regularly spaced ridges. Major topics in protein chemistry are the understanding of the structure-function relationship and of the mechanisms by which some proteins are capable of triggering specific diseases. We address these issues by studying structural and functional properties of the proteins under investigation, as well as their intracellular localization and interactors. As far as enzyme proteins are concerned, their catalytic behavior and sensitivity to inhibitors and activators are characterized. Structural features, in particular the aggregation state, are explored by FT-infrared spectroscopy and atomic force microscopy. Intracellular interactors are identified by advanced mass spectrometry techniques. These approaches are matched with the development and characterization of mutated forms of the proteins under investigation, which helps clarify the structural properties associated with function and pathogenicity. ical role, and the mechanisms by which ataxin-3 generates amyloid fibrils. These studies are performed on both purified molecule and cellular systems. STRUCTURAL STUDIES ON PROTEINS CONTAINING GLUTAMINE REPEATS RESPONSIBLE FOR NEURODEGENERATIVE DISORDERS INVESTIGATIONS ON STRUCTURE, STABILITY AND FUNCTIONS OF PROTEINS FROM THE ARCHAEON SULFOLOBUS SOLFATARICUS Maria Elena Regonesi, Paolo Tortora, Gaetano Invernizzi, Serena Mazzucchelli Paolo Tortora, Emanuela Occhipinti 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 Machado-Joseph 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 physiolog- ROLE OF POLYNUCLEOTIDE PHOSPHORYLASE IN MATURATION OF PROKARYOTIC TRANSCRIPTS Paolo Tortora, Elisa Mazzantini Polynucleotide phosphorylase (PNPase) is a prokaryotic enzyme that degrades RNAs phosphorolytically. It plays a major role in regulation of their stability, degradation and maturation. This project is aimed at providing a better insight into the role of PNPase in the aforementioned degradative mechanisms and the factors which control its activity. To this end, we take advantage of a set of mutants, which are being characterized in terms of physiological behavior, enzymatic properties and aggregation state. S. solfataricus carboxypeptidase (CPSso) is a thermostable metalloenzyme endowed with broad substrate specificity and the ability to withstand extreme chemical-physical conditions, such as temperatures up to 85°C and high concentrations of organic solvent. A process aimed at synthesizing N-blocked amino acids in organic medium is being developed by taking advantage of the properties of CPSso. Also, by combining mass spectrometry, molecular modeling, and sitedirected mutagenesis we could identify key structural features responsible for its thermostability. [ 37 ] STRUCTURAL AND FUNCTIONAL STUDIES ON PROTEINS 11 Paola Fusi, Matilde Forcella, Chiara Pozzi, Valentina Pastori, Alessandra Bigi, Lavinia Morosi STUDIES ON ATAXIN-3 PHYSIOLOGICAL ROLE Valentina Pastori and Paola Fusi 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 (University of Milan-Bicocca) and Prof. Tedeschi (University of Milan). A series of phosphorylated sites have been identified and subjected to sitedirected 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, Lavinia Morosi, Chiara Pozzi 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 Strcuctural Biology Group, Tzukuba, Ibaraki, Japan). Mutants have been produced to validate NEU2 crystallographic structure and verify the proposed catalytic mechanism. More recently, molecular dynamic studies have been undertaken, in collaboration with Prof. De Gioia and Dr. Zampella (University of MilanBicocca), to elucidate binding to ancillary substrate sites, 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 membrane though interaction with other protein(s). Cross-linking studies are currently carried our to identify these proteins. Moreover, NEU4 membrane anchoring mechanism is investigated, through site-directed mutagenesis. 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, aim 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. Upregulation of connexin 43 and TLRs engagement are currently investigated on bacteria activated melanoma cells. CLONINING AND EXPRESSION OF A TREHALASE FROM CHYRONOMUS RIPARIUS TO BE EXPLOITED AS A TARGET FOR BIOINSECTICIDES Matilde Forcella 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 and characterized: results show that it has a different specificity towards commercially available insecticides, compared to mammalian enzymes. Molecular cloning of its gene is currently underway in our laboratory, as well as testing of new synthetic bioinsecticides (imminosugars), in collaboration with Prof. Parenti and Prof. Cipolla (Universiy of Milan-Bicocca). [ 38 ] 1 12 1_ EB in peripheral mitochondria of MCF-7 cells. 2_Enlarged view and image analysis of the inset. MOLECULAR AND CELLULAR BIOPHYSICS Silvia Maria Doglia, Antonino Natalello, Anna Maria Villa 2 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 in vitro and in vivo by complementary biophysical and biochemical approaches. Particular attention has been addressed to the study of amyloid proteins. In collaboration with the group of Dr M. Salmona (Istituto di Ricerche Farmacologiche “Mario Negri”, Milan) we studied the kinetics of aggregation of the human prion peptide PrP82-146 and the structural properties of its oligomers and fibrils. The PrP82-146 peptide was found to undergo several aggregation pathways, with end products displaying different secondary structures and intermolecular interactions. These findings underline the high plasticity of the prion peptide, which is a crucial feature of prion proteins to overcome species barriers (Natalello et al. J.Mol.Biol. 2008). The effect of several chemical and physical effectors, and of the osmolyte betaine on protein misfolding and aggregation has been investigated in vitro, in collaboration with the research group of Dr.A. de Marco (IFOM, Milan). Interestingly, we found that betaine not only can induce protein misfolding and aggregation, but depending on its concentration - is able to disrupt preformed insoluble aggregate into soluble oligomers (Natalello et al. Protein Expr. Purif. 2008; Natalello et al. Proteins 2009). In collaboration with the group of Prof. J-L Reymond (University of Berne, Swiss) we studied the. stability and aggregation of peptide dendrimers. Dendritic branching of helix-forming peptide was found to induce a more stable a-helical structure than the corresponding linear peptide toward pH-induced unfolding and thermal aggregation (Javor et al. J. Am. Chem. Soc. 2008) . We recently proposed a new FT-IR method to study in vivo the aggregation of recombinant proteins in bacterial cells in the form of inclusion bodies (IB) (Doglia et al. Biotechnol. J. 2008). By this approach we studied, in col- laboration with the group of Prof. M. Lotti of this Department, the role of Cys 121 in the in vivo aggregation of bovine ß-lactoglobulin. We found that in E. coli the mutant C121S is more prone to aggregation than the wild type protein, an effect that depends on the oxidation of disulfide bonds (Invernizzi et al. Protein Expr. Purif. 2008). CONFOCAL FLUORESCENCE MICROSCOPY OF INTACT CELLS AND SURFACES . S.M. Doglia, A.M. Villa, A. Natalello In collaboration with Prof. Claudia Riccardi (Physics Department of the University of Milano Bicocca) we characterized the adsorption of model proteins on polymer surfaces functionalized by plasma treatments through laser scanning confocal fluorescence microscopy. Taking advantage of photon counting detection to evaluate the protein fluorescence on the treated surfaces, we found that plasma treatments of short duration reduce the protein adsorption down to the 30 % of that of the untreated surface. Longer plasma treatments lead to an increased protein adsorption, reflecting a damaging of the surface. Our fluorescence approach enabled to optimize the treatment conditions to obtain polymer surfaces useful for biomedical applications . The interaction of ethidium bromide (EB) with mitochondria was investigated in human carcinoma cells under living conditions. Two coexisting mitochondria populations with distinct localization, membrane potential and morphology were observed in MCF-7, MCF-7/DX, and A549 cells. Differences were also found in the EB fluorescence of the two pools of mitochondria, indicating a different EB accessibility to mtDNA that could be related to its transcription and replication activity. To establish whether the level of EB fluorescence was correlated with the mtDNA status, in collaboration with the group of Dr P. Fusi we studied mitochondria in neuroblastoma cells. By modulating the mtDNA replication in these cells, the EB fluorescence intensity was found to correlate with the percentage of mtDNA nascent strands detected by real time PCR. [ 39 ] THERAPEUTICAL STRATEGIES FOR CHRONIC PAIN 13 Gabriella Giagnoni, Barbara Simona Costa, Francesca Comelli, Isabella Bettoni A great paradox of pain is that acute, nociceptive pain is a necessary defense mechanism that warns against existing or imminent damage to the body, whereas chronic pain is only deleterious. Among the most debilitating types of chronic pain is peripheral neuropathic pain. Despite over fifty years of research there are not yet effective treatments, and pharmacological or physical attempts to control neuropathic pain give results not lasting over time. Therefore, neuropathic pain can be classified as an incurable disease. Our research aimed to find new pharmacological targets in order to develop new effective drugs against neuropathic pain. degranulating mast cells were increased. MCP-I positive granules were scattered throughout the endoneurium at the site of the injury already 48h post-injury and their number was significantly correlated to the distance from injury site. The administration of PEA protects mast cells against degranulation: the number of intact mast cells was significantly higher in PEA-treated mice than in vehicle-treated animals even if the PEA treatment did not allow a complete preservation of intact mast cells. These results suggest that mast cell modulation represent an important tool to counteract the development of hyperalgesia in neuropathic pain states. A. TARGETING MAST CELLS IN NEUROPATHIC PAIN WITH THE ENDOGENOUS MODULATOR PALMITOYLETHANOLAMIDE. The endogenous lipid palmitoylethanolamide (PEA) behaves as a local autacoid capable of downregulating mast cell activation. We have shown that PEA induces relief of neuropathic pain probably through both an action upon receptors located on the nociceptive pathway and a more direct action on mast cells. During this year we aimed to better characterize the role of mast cells during neuropathic pain and to relate the anti-hyperalgesic effects of PEA to its capability to inhibit mast cell degranulation. The chronic constriction injury (CCI) model in mice was employed and PEA was administered i.p. to CCI mice once a day for one week starting the day after the lesion. After assessing PEA-induced relief of pain, mice were sacrificed and sciatic nerves were submitted to different preparations and inclusions to evaluate the axon morphology (indicative of Wallerian degeneration) and the number of intact or degranulated mast cells, through both toluidine staining and immunostaining employing the polyclonal antibody anti-mouse mast cell protease I (MCP-I). The histological analysis of sciatic nerve sections showed a marked degeneration of myelinated fibers in CCI mice, which was substantially reduced after repeated administration of PEA, suggesting that the compound may favour myelin repair. The immunostaining revealed that the number of intact mast cells was dramatically reduced following the nerve injury, whereas activated B. CANNABINOID RECEPTOR ANTAGONISTS AS A THERAPEUTIC APPROACH FOR DIABETIC NEUROPATHIC PAIN. Diabetes is one of the leading causes of painful neuropathy and to date, besides a tight glycemic control, a viable treatment for this complication is not available. Rimonabant is a selective cannabinoid CB1 receptor antagonist that produced a significant increase in insulin sensitivity and a reduction of HbA1c in diabetic patients. In this study we showed that the repeated treatment with rimonabant evoked a significant attenuation of mechanical allodynia in diabetic mice that is dose- and timedependent. This effect occurred without alteration of hyperglycemia but it was associated with significant effects on many key players in the pathogenesis of diabetic neuropathy. Metabolic changes induced by hyperglycemia lead to oxidative stress, dysregulation of cytokine control and reduced production and transport of nerve growth factor (NGF), and all these factors contribute to the nerve degeneration and consequently to neuropathic pain. Rimonabant treatment evoked a reduction of oxidative stress in peripheral nerve as revealed by the ability of compound to counteract the reduced glutathione (GSH) depletion. In addition rimonabant elicited an inhibition of TNF_ overproduction in the spinal cord and an increase in the NGF support. These findings support the hypothesis that CB1 antagonists would represent a new opportunity for diabetic patients in which the CB1 receptor blockade has been already shown to increase insulin sensitivity and to reduce HbA1c. [ 40 ] REGULATION OF NEURAL STEM CELLS IN PHYSIOLOGY AND EXPERIMENTAL THERAPY FOR CANCER AND NEURODEGENERATIVE DISORDERS. 14 Angelo L. Vescovi, Lidia De Filippis, Fabrizio Gelain, Elena Binda, Daniela Ferrari, Carla Cunha, Silvia Panseri, Laura Rota Nodari, Omar Villa, Sara Piccirillo,Francesca Taraballi. SEM image of a sectioned cell embedded in a 3D self-assembled scaffold. Cell nucleus and cytoskeleton are visible. The self-assembled scaffold completely wraps cell body and membrane. 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, Sara Piccirillo, Elena Binda Glioblastoma multiforme (GBM) represents the most aggressive brain tumor, characterized by the presence of a well-vascularized diffuse infiltrative tissue pattern, which eventually leads to the extensive dissemination of the tumor cells within the brain and to the inability to achieve a complete surgical resection. Therefore, disease recurrence occurs in the majority of the patients and life expectancy is dramatically short. We have recently reported that long-term proliferating tumor stem cells (TSCs) can be identified and isolated from post-surgery specimens of human GBMs. These TSCs display all the cardinal features of bona fide stem cells, and, of note, following intracranial implantation, also satisfy the essential requirement for a cancer stem cell, i.e. the capability to generate new tumors, which infiltrate the surrounding brain parenchyma, migrating along commissural fibers across the corpus callosum. We have collected human GBM neoplastic tissues to establish histopatological preparations and isolate new different TSC lines. Each single line has been subjected to a comprehensive characterization to assess its molecular and antigenic pattern, in order also to identify genes whose expression strictly correlate with the GBM TSCs invasive and infiltrative behaviour. As TSCs from different specimens retain their distinctive, linespecific stable properties identical to those of their parenteral bulk cultures, together with the analysis of the clinical parameters, these data gave us the background information regarding the patient and its tumor. The same TSCs have been employed to define the conditions leading to the establishment of bona fide models of GBMs following intracerebral transplantation in immune-compromised mice. By taking advantage of resonance magnetic imaging (MRI) at progressive stages of tumor development, we also studied the progression of these tumors in living animals and correlated them with progressive acquisition of neurological impairment. This combined in vitro and in vivo strategy may allow the identification of genes and antigens not only as promising diagnostic/prognostic markers but also as possibly novel therapeutic patient-tailored targets in order to successfully interfere with the characteristic migratory and infiltrative process of this pathology. [ 41 ] NERVOUS REGENERATION VIA NANO-STRUCTURED SCAFFOLDS Fabrizio Gelain, Silvia Panseri, Francesca Taraballi, Carla Cunha, Omar Villa, Angelo L. Vescovi A traumatic injury to adult nervous system often leads to persistent deficits, due to the inability of mature axons to regenerate after damage, which results on a significant impact on quality of life and life expectancy for the patients. Our project focuses on traumatic injury both in central and peripheral nervous system. In order to enhance nervous regeneration our approach make joint use of diverse nanotechnology derived biomaterials: electrospun micro- and nanofiber channels and selfassembling peptides. Both are bio-reabsorbable and have been shown not to elicit marked immune response, nor inflammatory reactions in animals. Electrospun tubes are flexible tubular scaffolds showing 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 before the self-assembling takes place upon exposure to neutral pH solutions. In the last year we demonstrated how electrospun tubes filled with self-assembling peptides are permissive micro-enivornments for regenerating nervous tissue. Our work provided evidence that electrospun nanofiber channels are promising bioabsorbable scaffolds for stimulating and guiding spinal cord regeneration in rat models of chronic spinal cord injury. 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 therapy. SEM image of a sectioned cell embedded in a 3D selfassembled scaffold. Cell nucleus and cytoskeleton are visible. The self-assembled scaffold completely wraps cell body and membrane. ESTABLISHMENT OF A NOVEL HUMAN NEURAL STEM CELL LINE IMMORTALIZED WITH C-MYC T58A Lidia De Filippis, Daniela Ferrari, Laura Rota Nodari, Angelo L. Vescovi Human neural stem cells (hNSC) represent an essential source of renewable brain cells for both experimental studies and cell replacement therapies. Their relatively slow rate of proliferation and physiological senescence in culture make their use cumbersome under some experimental and pre-clinical settings. The immortalization of hNSC with the v-myc gene (vIhNSC) has been shown to generate stem cells endowed with enhanced proliferative capacity, which greatly facilitates the study of hNSCs, both in vitro and in vivo. Despite the excellent safety properties displayed by v-IhNSCs – which do not transform in vitro and are not tumorigenic in vivo – the vmyc gene contains several mutations and recombination elements, whose role(s) and effects remains to be elucidated, yielding unresolved safety concerns. To address this issue, we used a c-myc T58A retroviral vector to establish an immortal cell line (T-IhNSC) from the same hNSCs used to generate the original v-IhNSCs and compared their characteristics with the latter, with hNSC and with hNSC immortalized using c-myc wt (c-IhNSC). T-IhNSCs displayed enhanced self-renewal ability, with their proliferative capacity and clonogenic potential being remarkably comparable to those of v-IhNSC and higher than wild type hNSCs and c-IhNSCs. Upon growth factors removal, T-IhNSC promptly gave rise to well-differentiated neurons, astrocytes and most importantly, to a heretofore undocumented high percentage of human oligodendrocytes (up to 23%). Persistent growth-factor dependence, steady functional properties, lack of ability to generate colonies in soft-agar colony-forming assay and to establish tumors upon orthotopic transplantation, point to the fact that immortalization by c-myc T58A does not bring about tumorigenicity in hNSCs. Hence, this work describes a novel and continuous cell line of immortalized human multipotent neural stem cells, in which the immortalizing agent is represented by a single gene which, in turn, carries a single and well characterized mutation. From a different perspective, these data report on a safe approach to increase human neural stem cells propagation in culture, without altering their basic properties. This T-IhNSC line provides a versatile model for the elucidation of the mechanisms involved in human neural stem cells expansion and for development of high throughput assays for both basic and translational research on human neural cell development. The improved proclivity of T-IhNSC to generate human oligodendrocytes propose T-IhNSC as a feasible candidate for the design of experimental and, perhaps, therapeutic approaches in demyelinating diseases. [ 42 ] 1 15 DENDRITIC CELLS IN INNATE AND ADAPTIVE IMMUNITY 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. 2 1_Dendritic cells and Bacteria Interactions 2_Dendritic cell-T cell crosstalk. 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 DCderived 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. [ 43 ] NEUROPHYSIOLOGY 16 Enzo Wanke, Marzia Lecchi, Elisa Redealli, Francesca Gullo, Andrea Maffezzoli FUNCTIONAL STUDIES ON Na+ CHANNEL MUTATIONS IN FEBBRILE EPILEPSY AND GENERALIZED EPILEPSY WITH FEBBRILE SEIZURE (GEFS+) Enzo Wanke, Marzia Lecchi, Elisa Redaelli Febrile seizures (FS) affect 5-12 % of infants up to six years of age. Familial epilepsies are often caused by mutations of voltage-gated Na+ channels, but correlation genotype-phenotype is not clear yet. We have found that a missense mutation (M145T) on SCN1A (the alpha subunit of the voltage-gated Na+ channel) cosegregates in a large italian family affected by simple FS. Overall, the M145T substitution appears to determine a “loss-of-function” phenotype, suggesting a putative expression of mutated channels in inhibitory neurons capable to produce a network hyperexcitability that selectively causes FS. We also studied Nav1.1 Na+ channel alpha subunit M1841T mutation, found in an epileptic family characterized by a particularly large phenotypic spectrum. The mutant resulted to be a loss of function because is resulted to be “trafficking-defective”. In collaboration with R. Combi, this Department. SPATIOTEMPORAL EVOLUTION OF NEURONAL NETWORKS INVESTIGATED WITH MULTIELECTRODE ARRAYS (MEA) Enzo Wanke, Marzia Lecchi, Francesca Gullo, Andrea Maffezzoli With the acquisition of a novel multielectrode array (MEA) electrophysiological system we aim at studying neuronal networks (~3 mm2, ~3000 neurons, ) by recordings from 260 electrodes, in parallel and in real time. Excitable activity is produced by the balanced interaction of excitatory and inhibitory neurons connected by synapses (~106), therefore it has intrinsic properties characterized by well defined statistical properties: mean discharge frequency, correlation between neighbouring neurons, stimulation-dependent local field potentials, etc. We investigated the following problems: 1) the properties of the cortical spreading depression in KI mice which mimics the human channelopaty of Ca2+ channels (FHM-1), 2) the epileptic seizures present in KO mice for the K+ channel Kv1.1. In 2008, the group participated to the MEA Meeting (Reutlingen, Germany), to the Molecular Neuroscience Meeting (Milano, Italy) and to the Neuroscience Meeting (Washington, USA) with a poster entitled “Functional pharmacology in long-term cultured neocortical networks”. [ 44 ] 17 NICOTINIC RECEPTORS AND VOLTAGE-GATED K + CHANNELS IN PHYSIOLOGY AND PATHOLOGY Andrea Becchetti, Patrizia Aracri, Raffaella Morini, Chiara Di Resta, Paola Ambrosi NICOTINIC MODULATION OF THE THALAMOCORTICAL SYSTEM. In the mammalian brain, the cholinergic fibres ascending from the basal forebrain and mesopontine nuclei contribute to regulate the transitions between states with different level of vigilance, including the transition between the non rapid-eye-movement and the rapid-eye-movement phases of sleep. ACh release is also involved in the control of synaptic plasticity and, consequently, of memory and learning. The mechanisms by which the cortical cholinergic transmission brings about its functions are poorly understood. We are devoting particular attention to the cholinergic modulation of transmitter release and its contribution to the regulation of the cortical functions in normal and pathological conditions (such as sleeprelated epilepsy). We carry out patch-clamp recording in murine brain slices and couple the electrophysiological approach with neuroanatomical and molecular biological methods. NEURONAL NICOTINIC RECEPTORS AND SLEEPRELATED EPILEPSY. We study the properties of mutant subunits of the human neuronal nicotinic receptors, linked to certain forms of nocturnal epilepsy. Normal and mutant channels are expressed in cell lines and their biophysical and pharmacological properties studied in patch-clamp. In addition, we will address the nicotinic modulation of the thalamocortical function in murine models of these pathologies, by applying the approaches outlined in the previous paragraph. 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). Ion channels would appear instead to be better suited for rapid cellular signalatory tasks. Increasing evidence shows however 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. The study of channel-integrin interplay has important mechanistic implications for understanding how the extracellular matrix regulates as disparate processes as muscle excitability, synaptic plasticity and lymphocyte activation, just to mention a few. The derangement of these processes has clear implications for pathogenetic processes, such as tumour invasivity and neurology. [ 45 ] CARDIAC CELL PHYSIOLOGY 18 Antonio Zaza, Marcella Rocchetti, Claudia Altomare, Matteo Alemanni, Riccardo Chisci, Stefano Marangoni. The research of the cardiac cell physiology group is centered on the ontogenesis and modulation of myocardial excitation-contraction coupling. The research activity in 2008 was articulated in the following projects. Italy). Funding was provided by an extension of the FP6 project “SC&CR”, coordinated by Dr. M.Capogrossi (IDI, Rome); this project is in collaboration with the laboratory of E. Messina, Università La Sapienza (Roma, Italy). EVALUATION OF FUNCTIONAL DIFFERENTIATION IN STEM-CELL DERIVED CARDIOMYOCYTES This is a continuation of the activities started in 2005 and funded by the 6th Framework Program of the EU. We tested the possibility to obtain information on the functional differentiation of precursors by imaging methodologies which could be applied to cell populations. The need of such an approach is dictated by the inefficiency of identifying a low frequency event (cell differentiation) by labor-intensive techniques, such as patch-clamp, addressing one cell at a time. Moreover, at variance with previous ones, this approach can provide objective estimates of the frequency of differentiation. The strategy tested thus far is the search of muscle-specific Ca2+ signaling, triggered by suitable agonists (caffeine, ATP etc), in populations of precursor-derived cells. This was implemented, through the use of Ca2+ -sensitive fluorescent dyes, by counting the number of cells responding to agonist challenge in wide-field confocal images. We developed an image-analysis software to automatically count the responsive cells and study the time course of the Ca2+ response in individual cells. The frequency of Ca2+ responding cells and the pattern of Ca2+ responses in a population was then matched with the expression of molecular markers of muscle differentiation in the same population. This analysis disclosed that the ratio between caffeine and ATP triggered Ca2+ -signals markedly differs between early stage precursors and cells cultivated in differentiating conditions; this is accompanied by a change in cell expression and localization of molecular markers. These findings suggest that the approach developed in this investigation may be suitable to identify early functional differentiation toward muscle phenotype and will be applied to test the differentiation of specific cell populations. Preliminary results have been presented at the ISHR meeting (Bologna, MODULATION OF MYOCARDIAL EC-COUPLING BY THE PI3K/AKT PATHWAY Recent observations indicate that the PI3K/Akt signaling pathway is deeply involved in controlling the phosphorylation state of central components of the cardiac EC-coupling machinery, such as Ca2+ channels and phospholamban (PLB). Moreover, changes in the extent and mode of activation of the PI3K/Akt pathway occur during myocardial remodeling and may account for contractile dysfunction of heart failure. We have tested the possibility to modulate the PI3K/Akt pathway by molecules specifically designed to bind the pleckstrin-homology domain (PH-domain) of Akt protein, thus obstructing Akt recruitment and activation. This is an innovative approach with still undefined functional consequences. For this purpose we have used two chemically unrelated PH-domain antagonists, obtained through a collaboration with the organic chemistry group of this Department (Prof. Cipolla and Nicotra) and with a company (Nerviano Medical Science, Dr. Venturi and Castoldi). The effects of these compounds on myocyte contractility and Ca2+ handling were compared to those of highly selective silencing of Akt1 protein translation by the RNA-interference technique. The results obtained show that the compounds exert significant effects on contractility by cooperatively interacting with ß-adrenergic receptor stimulation and that these effects are indeed mediated by Akt modulation. Also, proteins of the sarcoplasmic reticulum have been identified as the target of Akt phosphorylation and the ultimate effectors of functional changes. This study, now almost completed, identifies a novel target for pharmacological modulation of cardiac function. Funding was partly provided by a grant from Nerviano Medical Science, which covered a 1 year post-doc fellowship. Preliminary results have been presented at the EWGCCE Meeting (Manchester, UK). [ 46 ] 19 ECOLOGY OF MARINE AND MIGRANT BIRDS Roberto Ambrosini, Massimiliano Mori 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 carotenoids content in the eggs 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. 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 IDIOPATHIC EPILEPSIES 20 Romina Combi 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 and they are considered channelopathies owing to the identification of mutations in genes encoding ion-channels. However, the detected mutations 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). 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. [ 47 ] ZOOPLANTLAB - INTEGRATED RESEARCHES IN ANIMAL AND PLANT BIOLOGY 21 Maurizio Casiraghi, Massimo Labra, Aldo Zullini, Michela Barbuto, Fabrizio De Mattia, Emanuele Ferri, Ilaria Bruni, Andrea Galimberti ZooPlantLab (ZPL) links applied and basic sciences in the zoological, botanical and agronomic fields. Main projects are based on a molecular approach, but the integration with all the biological information is the rule. 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. Our project put a bridge between metagenomic approaches and DNA Barcoding initiative, achieving a high level of processivity 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. 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. We are working on different topics: 1) Food traceability: 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 identify each plant species. FROM GENES TO ECOSYSTEMS: DNA BARCODING HAS A SYSTEM TO PROTECT BIODIVERSITY In the project we will couple the molecular identification systems approach with the analysis of the connectivity of protected areas in fragmented environments. The project is in collaboration with the Milan Natural History Museum, and aims to create a reference database of regional organisms. In the second part of the project we will analyse the level of genetic connection among non contiguous areas, to detect, understand and (hopefully) protect ecological corridors. SCIENTIFIC EDUCATION: FROM THE UNIVERSITIES TO THE SOCIETY The aim 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, National and Regional Parks, etc). ZPL developed an educational kit for the water analysis, to be used by young kids, or in the school classrooms. 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. [ 48 ] 22 FRESHWATER AND MARINE ECOLOGY Paolo Galli, Fabrizio Stefani, Francesca Benzoni, Giovanni Strona, Giovanni Aquaro, Simone Montano, Davide Seveso A HOST-PARASITE MODEL FOR THE DISPERSAL OF LESSEPSIAN SPECIES IN MEDITERRANEAN. The 1869 opening of the Suez Canal created a direct link between Mediterranean and Red Sea, allowing the entry into the Levantine aquatic system of non-native species, particularly from Erythrean basin, process that has accelerated in the recent years concurrently to the warming trend of the seawater. Among fish Siganus luridus, has proven to be extremely successful in colonizing a large part of Eastern Mediterranean coasts until 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 tis- sues 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 Biodiversity and its conservation are one of the major environmental challenges. 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. The push for a detailed appraisal of their overall biodiversity is therefore very strong as exemplified, for instance, by the establishment in the recent years of an « International Coral Reef Initiative » (I.C.R.I.) aimed at developing worldwide relevant management and conservation strategies for coral reefs for the benefit of future generations. 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 morphologic 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). [ 49 ] PROGRAMMED CELL DEATH (PCD) IN PLANTS 23 Paolo Crosti, Massimo Malerba, Nicla Contran 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 stress-related responses and PCD which only in a fraction of dead cells shows the typical morphological 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. In recent years the actin cytoskeleton has been identified as a major target and effector of signalling cascades including PCD in both animal and plant cells. In fact, clear evidence shows that alteration of actin filaments dynamics can initiate or modulate the apoptotic signalling cascade in yeast and animal cells. Besides, stomatal opening by FC accompanied by depolymerization of actin filaments has been reported in guard cells of Commelina communis. Thus, in the last year, we investigated whether FC can induce changes in actin filaments dynamics of sycamore cells and we demonstrated that the phytotoxin induces a massive depolymerization of actin filaments that is prevented by scavengers of nitric oxide (NO). The same scavengers strongly reduce the FC-induced PCD. The addition of actin-depolymerizing drugs induces PCD in control cells and overcomes the inhibiting effect of NO scavengers on FC-induced cell death. Vice versa, the addition of actin-stabilizing drugs to FC-treated cells partially inhibits the phytotoxin-induced PCD. These results suggest that changes in actin cytoskeleton mediated by NO are involved in FC-induced PCD. [ 50 ] COMPUTATIONAL INVESTIGATION OF STRUCTURE-ACTIVITY RELATIONSHIPS IN PROTEINS AND BIOMIMETIC COMPLEXES 24 Piercarlo Fantucci, Luca De Gioia, Giuseppe Zampella, Luca Bertini, Elena Papaleo, Marco Pasi, Alessandro Di Domizio, Francesca Falcetta DFT INVESTIGATIONS OF METALLO PROTEINS AND BIOMIMETIC METAL COMPLEXES Luca Bertini, Luca De Gioia, Piercarlo Fantucci, Giuseppe Zampella The project is aimed at elucidating both the activity mechanism and the stereo-electronic properties of some active sites in metalloenzymes, as well of the key regions of proteins, involved in their biological role. Effort is put in determining the chemical features which characterize a transition metal when bound to the polypeptide. Ab initio Density Functional Theory (DFT) approaches are used in order to compute the electronic structures and perform a detailed analysis of models employed to simulate the biosystems under study. COMPUTATIONAL INVESTIGATIONS OF STRUCTUREACTIVITY RELATIONSHIPS IN PROTEINS Luca De Gioia, Piercarlo Fantucci, Elena Papaleo, Marco Pasi, Giuseppe Zampella Molecular dynamics simulations and homology modelling are used as main techniques with the aim of investigating structure-function relationship in enzymes and proteins. In fact, long and multiple simulations of biomolecular systems can allow obtaining insights into biomolecular processes at the atomic level, which are often hardly accessible to experimental methods. Attention is addressed to the effect of the temperature on protein stability and the interaction between enzymes and their cofactor or some inhibitors. DEVELOPMENT OF BIOINFORMATICS TOOLS FOR ANALYSIS OF PROTEINS AND THEIR POST-TRANSLATION MODIFICATIONS AND FOR MOLECULAR DOCKING Luca Bertini, Francesca Falcetta, Alessandro Di Domizio, Piercarlo Fantucci In order to overcome the limitations of proteomic techniques to determine post-translational modifications (PTM), computer programs have been developed to analyze amino acid sequences for PTMs and compute modifications of molecular mass and isoeletric point. New methodologies for molecular docking have been developed and applied to ligand-protein interaction in the framework of computed-aided drug design. [ 51 ] DESIGN, SYNTHESIS AND MOLECULAR RECOGNITION STUDIES ON BIOACTIVE COMPOUNDS 25 Francesco Nicotra, Laura Cipolla, Barbara La Ferla, Cristina Airoldi, Cristina Redaelli, Maria Gregori, Cristiano Zona, Nasrin Shaikh, Alexandre Orsato, Ana Catarina Araujo, Paolo Galliani, Laura Russo, Valerio Spinosa, Davide Bini DESIGN, SYNTHESIS AND MOLECULAR RECOGNITION STUDIES ON POTENTIAL DRUGS Francesco Nicotra, Laura Cipolla, Barbara La Ferla, Cristina Airoldi, Cristina Redaelli, Ana Catarina Araujo, Paolo Galliani, Alexander Orsato, Davide Bini 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). Synthetic targets focused in 2008 are: Inhibitors of bacterial LPS biosynthesis as potential antibacterial agents Inhibitors of Protein Kinase B as potential antitumor agents and cardiac modulators Inhibitors of glycosidases as potential antiviral agents and metabolic diseases regulators Drugs fused into glycidic structures, in particular glycobenzodiazepines and GABA-receptor ligands, in order to modulate the pharmacokinetic and the conformational properties NMR studies are performed for: Structure elucidation Conformational analysis Epitope mapping studies (ligand-receptor interactions studies at atomic level) Adhesion kinetic studies [ 52 ] 26 BIOORGANIC AND MEDICINAL CHEMISTRY Francesco Peri, Alessandro Palmioli, Matteo Piazza, Silvia della Fiorentina Synthesis of pharmacologically active molecules and investigation of their interaction with biological systems. 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 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 2008 include the first totally water-soluble Ras inhibitor. With this compound, in collaboration with the University of Chicago (USA), it was possible to determine the Rasinhibitor binding interface by NMR. The binding between our compounds and Ras was also detected by Isotermal Calorimetry measurements in collaboration with the University of Montana (USA). These experimental data constitute a big step forward in the perspective to clarify at a molecular level the mode of action of such inhibitors. We also performed experiments to investigate the activity of our Ras inhibitors on human colorectal cancer cells HCT-116 and DLD-1, in collaboration with the research group of prof. Alberto Bardelli (IRCC, Candiolo, TO). In 2008 the following collaborations were active on Ras project: Prof. Marco Vanoni, Prof. Enzo Martegani, Prof. Luca de Gioia (our department), Dr. Vadim Gaponenko (University of Illinois at Chicago, USA), Dr. C. Thomas (University of Montana, Missoula, USA), Prof. A. Bardelli (IRCC, Candiolo, TO). NOVEL GLYCOLIPIDS AND BENZYLAMMONIUM LIPID INHIBITORS OF THE TLR4 PATHWAY We are developing a new class of compounds constituted by glycolipids and benzylammonium lipids that are able to inhibit the activation of the TLR-4 receptor and the signal pathway associated to this receptor. Bacterial lipopolysac- Structure of the Ras-GDP-inhibitor complex. charides (LPS) and their bioactive portion, the lipid A, bind to TLR-4 initiating the signal cascade that causes cytokine production. Several syndromes ranging from the simple inflammation, to the neuropathic pain and acute sepsis and septic shock, depend on the activation (or abnormal activation) of the TLR-4 receptor. Our compounds have been patented as hits for the development of innovative anti-inflammatory and anti-sepsis drugs. In 2008 we investigated the molecular details of the action of our compounds and demonstrated their activity in cells and in animals. Some of these molecules showed a potency in contrasting LPS-induced septic shock in mice similar to that of the most potent anti-sepsis agents currently in clinical phase of development. In collaboration with Dr. T. Gioannini and Dr. J. Weiss (University of Iowa, USA), we applied for an NIH grant on the structure determination of some receptors of the TLR4 pathway. This grant was financed for 5 years and ranked in the top 0.6% of 2008 NIH projects in the USA. In 2008 the following collaborations were active on TLR4 project: Prof. Francesca Granucci, Dr. Barbara Costa, Dr. Paola Fusi (our department), Dr. Theresa Gioannini, Dr. Jerrold Weiss (University of Iowa, USA). NEW DENDRIMERIC MOLECULES FOR MULTIPLE ANTIGEN PRESENTATION AND SIGNAL AMPLIFICATION We are developing in collaboration with Diasorin S.p.A. (Nerviano, MI) new molecules that present several copies of clinically important antigens and of luminescent molecules (isoluminol derivatives). These compounds, with a tree-like shape (dendrimers) will be used as components of kits for the detection of antigens in the body fluids of patients. The multiple presentation to immobilized antibodies of the antigen and the presence of several signalgenerating units in the molecules, should ensure signal amplification that is very important for the sensitivity and reliability of immunochemistry tests. In 2008 this project was developed in collaboration with the immunology and immunochemistry unit of Diasorin S.p.A. (Nerviano, MI). [ 53 ] MOLECULAR MODELLING AND COMPUTATIONAL CHEMISTRY 27 Giorgio Moro, Gloria Saracino, Flavio Amara. MOLECULAR MODELLING AND COMPUTATIONAL CHEMISTRY Giorgio Moro, Gloria Saracino, Flavio Amara 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 num- ber of degrees of freedom. 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. Specific topics of interest are: properties of prion protein peptides (collaborations with dott. Alessandra Villa - Max-Planck-Institute for Polymer Research - Mainz - Germany; 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 [ 54 ] 28 OUTER MEMBRANE BIOGENESIS IN ESCHERICHIA COLI 1 Alessandra Polissi, Paola Sperandeo, Silvia Sommaruga, Riccardo Villa 2 1_Escherichia coli Rod-shaped Bacterium with Multiple Flagella 2_Escherichia coli strains undergoing conjugation 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 Gram-negative 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 NOVELS 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. Target proteins under study are being purified from both Escherichia coli and Pseudomonas aeruginosa an important pathogen that causes a wide variety of infections in immune-compromised hosts. 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. [ 55 ] INDUSTRIAL BIOTECHNOLOGY: ADAPTATION OF THE MICROBIAL CELL FACTORY TO TECHNICAL CONSTRAINTS 29 Danilo Porro, Luca Brambilla, Paola Branduardi, Gianni Frascotti, Simone Passolunghi, Vera Codazzi, Laura Dato, Dario Losio, Tiziana Fossati, Valerio Mezzasalma, Giorgia Rossi, Elena Vitale. 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 micro-organism 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 con- straints 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. (I) 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 non-conventional Kluyveromyces lactis, Torulaspora delbrueckii, Zygosaccharomyces bailii applying different fermentative technologies (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. (II) 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. (III) 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 and its metabolism. 3 SCIENTIFIC PUBLICATION INDEX , GRANTS [ 58 ] RESEARCH GRANTS AND CONTRACTS 3.1 ALBERGHINA L. ITALBIONET. Rete Italiana di Bioinformatica. FIRB, MIUR CASTAGNOLI P. Microarrays a DNA per lo studio della variabilità genetica. FIRB, MIUR ALBERGHINA L. Yeast Systems Biology Network"( YSBN) FP6 Coordination Action, 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 2007, MIUR ALBERGHINA L. Eukaryotic unicellular organism biology – systems biology of the control of cell growth and proliferation. FP7, European Commission BARABINO S. Genomica e proteomica del processamento degli RNA mesaggeri nella sclerosi laterale amiotrofica. Fondazione Cariplo 2006 COSTA B. Ruolo spinale e sovra spinale di citochine e BDNF nel dolore neuropatico e loro modulazione dopo trapianto di cellule staminali mesenchimali umane nella corteccia agranulare insulare. PRIN 2007, MIUR BARABINO S. A role for the pre-mRNA processing factor CF Im in quality control of mRNA function. PRIN 2006, MIUR DOGLIA SM. Processi di funzionalizzazione dei polimeri per la modifica della biocompatibilità dell' adesione di proteine. Fondazione Cariplo BRANDUARDI P. Systems Biology as a Driver for Industrial Biotechnology. FP7, European Commission FOTI M. Generation of a coronavirus-based multigene AIDS vaccine and evaluation in a preclinical SIV model. FP6, European Commission BECCHETTI A. Recettori nicotinici cerebrali e patologie epilettiche. PRIN MIUR BECCHETTI A. Recettori nicotinici cerebrali e patologie epilettiche. BML Foundation CASIRAGHI M. Nuova metodica per l'analisi della biodiversità: un'applicazione del pirosequenziamento allo studio degli organismi del suolo. PRIN 2007, MIUR. CASIRAGHI M. Qualità delle acque superficiali: studio degli effetti di xenobiotici ambientali di origine farmaceutica sulla produttività agricola. Fondazione Banca del Monte di Lombardia. FOTI M. Identificazione dei meccanismi molecolari indotti in cellule dendritiche da batteri commensali e patogeni importanti nella polarizzazione di linfociti T. PRIN 2007, MIUR FUSI P. Sialidasi umane: biologia strutturale, biochimica funzionale e implicazioni patologiche. PRIN, MIUR GALLI P. BioInspired Adhesives for Surgery. Fondazione Cariplo GALLI P. BENZONI F. Biodiversity and Biodiversity patterns of Scleractinia (Cnidaria) in the Gulf of Aden, Yemen. Creocean CASIRAGHI M. E LABRA M. Connessione ecologica e rinaturazione nel sistema delle aree protette del nord Milanese. Fondazione Cariplo GRANUCCI F. Dendritic cells for novel immunotherapies (DC THERA), European Commission CASIRAGHI M. E LABRA M. Dai geni all'ecosistema: il DNA barcoding come supporto innovativo per la protezione della biodiversità e l'analisi della funzionalità delle reti ecologiche. Fondazione Cariplo GRANUCCI F. Meccanismi d' induzione di tolleranza in cellule T autoreattive coinvolte nella risposta autoimmune presente nella cheratite erpetica stremale. Fondazione Cariplo CASIRAGHI M. Sviluppo di un sistema di identificazione molecolare di organismi target della fauna nel suolo. PRIN 2007, MIUR CASTAGNOLI P. Integrated functional genomics in mutant mouse models as tools to investigate the complexity of human immunological disease (CE MUGEN). European Commission CASTAGNOLI P. Microbial Action on immune survival. European Commission GRANUCCI F. Key regulators of DC-primed anti tumor NK cell functions. Associazione Italiana per la Ricerca sul Cancro GRANUCCI F. Ruolo delle cellule dendritiche nell' attivazione delle funzioni anti-tumorali delle cellule NK: meccanismi cellulari e molecolari. PRIN 2007, MIUR GRANUCCI F. Normalization of immune reactivity in old age – from basic mechanisms to clinical application, European Commission [ 59 ] GRANUCCI F. European Network for cell imaging and tracking expertise, European Commission LABRA M. Tutela della biodiversità con azioni di riqualificazione e valorizzazione di praterie su suolo calcareo (Fetuco brometalia) nei SIC Monte Sangiano e Monti della Valcuvia. Fondazione Cariplo NICOTRA F. Essential proteins of Pseudomonas aeruginosa outer membrane biogenesis as novel targets for new anti-microbial drugs design and synthesis. Fondazione per la Ricerca sulla Fibrosi Cistica PERI F. Synthesis of novel multivalent chemical entities for enhancing luminescent properties of probes for immunologicals assays. DIASORIN S.p.A (Nerviano, MI) LABRA M. Acqua in brocca. Fondazione Cariplo LONGHESE MP. Genetic integrity maintenance: interrelationships between DNA damage checkpoints and telomere metabolism. Associazione Italiana per la Ricerca sul Cancro LONGHESE MP. Initial Training Networks: High Resolution Microscopy in the DNA damage Response. 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, MIUR. LUCCHINI G. Fattori di checkpoint coinvolti nell'omeostasi telomerica nel lievito S. cerevisiae. PRIN 2007, MIUR MARTEGANI E. Valutazione attività NGF umano ricombinante. PRIMM-Blueprint NICOLIS SK. A genetic toolkit for analysis of mouse neural stem cells. Fondazione Cariplo, Progetto NOBEL NICOLIS SK. Ruolo funzionale e meccanismi molecolari d' azione del fattore trascrizionale Sox2 nelle cellule staminali neurali e nella differenziazione dei neuroni: studio mediante mutagenesi condizionale nel topo. PRIN 2007, MIUR NICOLIS SK. Genetic approaches to the study of the role of the Sox2 transcription factor in cancer neural stem cells. Associazione Italiana per la Ricerca sul Cancro NICOTRA F. Materiali innovativi per lo sviluppo di bio-protesi articolari. FIRB, MIUR NICOTRA F. NAD, Nanoparticles for therapy and diagnosis of Alzheimer Disease. FP7, European Commission 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). Metadistretti, Regione Lombardia PIATTI S. Molecular mechanism preventing the occurrence of aneuploidia hallmark of cancer cells. Associazione Italiana per la Ricerca sul Cancro POLISSI A. Essential proteins of Pseudomonas aeruginosa outer membrane biogenesis as novel targets for new anti-microbial drugs design and synthesis. Fondazione per la Ricerca sulla Fibrosi Cistica PORRO D. Novel high performance enzymes and microorganisms for conversion of lignocellulosic biomass to bioethanol. FP7, European Commission RONCHI A. Genomica funzionale della transizione embrionico-adulta nell'ematopoiesi. PRIN 2007, MIUR TORTORA P. Genoproteomics of Age Related Disorders (GUARD). Fondazione Cariplo (N.O.B.E.L. project) TORTORA P. Network tecnologico integrato per lo studio proteomico e transcrittomico di malattie neurodegenerative correlate a deposizione di amiloidi. Ministero della Salute/Regione Lombardia TORTORA P. Caratterizzazione biofisica e biochimica dei processi di aggregazione in vitro e in vivo di varianti di proteine ricombinanti che contengono poliglutammine. PRIN 2007, MIUR VANONI M. Sensing extracellulare e intracellulare dei nutrienti e progressione del ciclo cellulare nel lievito Saccharomyces cerevisiae. PRIN 2006, MIUR VANONI M. Sviluppo di inibitori peptidici di Ras. Creabilis. VESCOVI AL. Cis-regulatory logic of the transcriptional control in neural stem cells (CISSTEM). FP7, European Commission VESCOVI A.L. PLURIGENES Pluripotency associated genes to de-differentiate neural cells into pluripotent cells. European Commission VESCOVI A.L. Functional genomics of the retina in health and disease (EVI_GENORET). European Commission [ 60 ] R ESEARCH GRANTS AND CONTRACTS VESCOVI A.L. "Cellule staminali neurali umane e ingegneria dei tessuti biologici per la rigenerazione di lesioni al sistema nervoso centrale e periferico" e "Cellule staminali neurali umane e biomateriali nanostrutturati per la medicina rigenerativa". Fondazione Cariplo 2005 e 2007 VESCOVI A.L. Tumor neural stem cells in the in vitro and in vivo modeling and studying of the adult human glioblastomas. Associazione Italiana per la Ricerca sul Cancro VESCOVI A.L. Utilizzo di cellule staminali neurali tumorali come modello di studio in vitro e in vivo di glioblastoma adulto umano. PRIN 2006, MIUR VESCOVI A.L. Towards the neuronal Machine (NEURO). European Commission WANKE E. Molecular bases of channelopathies and functional characterization in single neurons and in neuronal networks. PRIN 2007, MIUR WANKE E. Studio delle proprietà biofisiche di specifici sottotipi del canale del sodio in modelli in vitro. NewronMilano Ricerche WANKE E. Studio di ricerche funzionali su proteine di membrana. – Axxam-Milano Ricerche ZAZA A. Terapia genica in cellule staminali cardiache in vitro per la correzione di una cardiomiopatia ereditaria. Fondazione Cariplo ZAZA A. Ruolo della corrente di Na+ persistente nel danno miocardico indotto da ipossia cronica. PRIN 2007, MIUR ZAZA A. Modulation of SR function by Istaroxime. Debiopharm, Lausanne (CH) ZAZA A. Effect of chronic hypoxia on myocardial function. CVT Therapeutics, Palo Alto (CA) USA ZULLINI A. Nematodi a vita libera come mezzo di valutazione delle qualità ambientale e di fertilità dei suoli. PRIN 2007, MIUR PUBLICATIONS 3.2 AMI D, NERI T, NATALELLO A, MEREGHETTI P, DOGLIA SM, ZANONI M, ZUCCOTTI M, GARAGNA S, REDI CA (2008) Embryonic stem cell differentiation studied by FT-IR spectroscopy. BBA-MOL. CELL RES. 1783, 98-106. ANDRIETTI F, CASIRAGHI M., MARTINOLI A, POLIDORI C, MONTRESOR C (2008). Nesting habits of two spider wasps: Anoplius infuscatus and Episyron sp. (Hymenoptera: Pompilidae), with a review of the literature. ANNALES DE LA SOCIÉTÉ ENTOMOLOGIQUE DE FRANCE, vol. 44, pp. 93-111. AQUARO G, RIVA C, GALLI P. (2008) 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 (in press). ARAUJO AC, NICOTRA F, AIROLDI C, COSTA B, GIAGNONI G, FUMAGALLI P, CIPOLLA L. (2008). Synthesis and biolo- gical evaluation of novel rigid 1,4-benzodiazepine-2,5-dione chimeric scaffolds. EUROPEAN JOURNAL OF ORGANIC CHEMISTRY vol. 2008, pp. 635-639. ARAUJO AC, NICOTRA F, COSTA B, GIAGNONI G, CIPOLLA L. (2008) Fructose-fused gamma butyrolactones and lactams, synthesis and biological evaluation as GABA receptor ligands. CARBOHYDRATE RESEARCH vol. 343, pp. 1840-1848. BAIN O, CASIRAGHI M., MARTIN C, UNI S. (2008). The Nematoda Filarioidea: critical analysis linking molecular and traditional approaches. PARASITE, vol. 15, pp. 342-348. BALDO V, TESTONI V, LUCCHINI G, LONGHESE M.P. (2008) Dominant TEL1-hy mutations compensate for Mec1 lack of functions in the DNA damage response. MOLECULAR AND CELLULAR BIOLOGY. vol. 28, pp. 358-375. [ 61 ] P UBLICATIONS BECCHETTI A, ARCANGELI A. (2008) A comment on ion channels as pharmacological targets in oncology. J GEN PHYSIOL Vol 132, pp. 313-314. cerevisiae Rad53 checkpoint kinase in signaling doublestrand breaks during the meiotic cell cycle. MOLECULAR AND CELLULAR BIOLOGY. vol. 28, pp. 4480-4493. BENZONI F, CALCINAI B, EISINGER M, KLAUS R. (2008) Coral disease mimic: sponge attacks Porites lutea in Yemen. CORAL REEF vol. 27, pp. 695-695. CAVALLARO M, MARIANI J, LANCINI G, LATORRE E, CACCIA R, GULLO F, VALOTTA M, DEBIASI S, SPINARDI L, RONCHI A, WANKE E, BRUNELLI S, FAVARO R, OTTOLENGHI S, NICOLIS SK (2008) Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants. DEVELOPMENT vol. 135, pp. 541-57. BETTONI I, COMELLI F, ROSSINI C, GRANUCCI F, GIAGNONI G, PERI F, COSTA B (2008) Glial TLR4 receptor as new target to treat neuropathic pain: efficacy of a new receptor antagonist in a model of peripheral nerve injury in mice. GLIA vol. 56, pp. 1312-1319. BRANDUARDI P, SMERALDI C, PORRO D (2008) Metabolically engineered yeasts: 'potential' industrial applications. J MOL MICROBIOL BIOTECHNOL. Vol 15(1), pp. 31-40. BRUSCHI M, DE GIOIA L, MITRIC R, BONACIC-KOUTECKY V, FANTUCCI P (2008) A DFT study of EPR parameters in Cu(II) complexes of the octarepeat region of the prion protein. PHYSICAL CHEMISTRY CHEMICAL PHYSICS vol. 10, pp. 4573-4583. BRUSCHI M, GRECO C, FANTUCCI P, DE GIOIA L (2008) Structural and electronic properties of the [FeFe] hydrogenase H-cluster in different redox and protonation states. A DFT investigation. INORGANIC CHEMISTRY vol. 47, pp. 6056-6071. CAZZANIGA P, PESCINI D, BESOZZI D, MAURI G, COLOMBO S, MARTEGANI E. (2008). Modeling and stochastic simulation of the Ras/cAMP/PKA pathway in the yeast Saccharomyces cerevisiae evidences a key regulatory function for intracellular guanine nucleotides pools". J BIOTECHNOL vol. 133, pp. 377-385. CHIARADONNA F, BALESTRIERI C, GAGLIO D, VANONI M (2008) RAS and PKA pathways in cancer: new insight from transcriptional analysis. FRONTIERS IN BIOSCIENCE. vol. 13, pp. 5257-5278. CIPOLLA L, AIROLDI C, GALLIANI P, POLISSI A, NICOTRA F (2008) Re LPS biogenetic pathway: enzyme characterisation and synthetic efforts towards inhibitors. CURRENT ORGANIC CHEMISTRY. vol. 12, pp. 576-600. CIPOLLA L, PERI F, AIROLDI C (2008) Glycoconjugates in cancer therapy. ANTI-CANCER AGENTS IN MEDICINAL CHEMISTRY, vol. 8 (1), pp. 92-121. BRUSCHI M, GRECO C, ZAMPELLA G, RYDE U, PICKETT CJ, DE GIOIA L (2008) A DFT investigation on structural and redox properties of a synthetic Fe6S6 assembly closely related to the [FeFe]-hydrogenases active site. COMPTES RENDUS CHIMIE vol. 11 (8), pp.834-841. CIPOLLINA C, VAN DEN BRINK J, DARAN-LAPUJADE P, PRONK JT, PORRO D, DE WINDE JH (2008) Saccharomyces cerevisiae SFP1: at the crossroads of central metabolism and ribosome biogenesis. MICROBIOLOGY vol. 154(6), pp.1686-99. BUSTI S, SACCO E, MARTEGANI E, VANONI M (2008) Functional coupling of the mammalian EGF receptor to the Ras/cAMP pathway in the yeast Saccharomyces cerevisiae. CURRENT GENETICS vol. 53, pp. 153-62. CIPOLLINA C, VAN DEN BRINK J, DARAN-LAPUJADE P, PRONK JT, VAI M, DE WINDE JH (2008) Rivisiting the role of yeast Sfp1 in ribosome biogenesis and cell size control: a chemostat study. Microbiology vol. 154, pp. 337-346. CACCIA M, SIRONI L, COLLINI M, CHIRICO G, ZANONI I, GRANUCCI F. (2008) Image filtering for two-photon deep imaging of lymphonodes. EUR BIOPHYS J vol. 37, pp.979987. CLERICI C, MANTIERO D, GUERINI I, LUCCHINI G, LONGHESE MP (2008). The Yku70-Yku80 complex contributes to regulate double-strand break processing and checkpoint activation during the cell cycle. EMBO REPORTS. vol. 9, pp. 810-818. CARDONA F, LA FERLA B (2008) Synthesis of CGlycoconjugates from readily available Unprotected C-Allyl Glycosides by Chemoselective Ligation. J CARBOHYDR CHEM vol. 27(4), pp. 203-213. CARTAGENA-LIROLA H, GUERINI I, MANFRINI N, LUCCHINI G, LONGHESE MP (2008) Role of the Saccharomyces COCCETTI P, TRIPODI F, TEDESCHI G, NONNIS S, MARIN O, FANTINATO S, VANONI M AND ALBERGHINA L (2008) A novel mechanism for stimulation of Cdc34 activity through phosphorylation of its catalytic domain by CK2 in Saccharomyces cerevisiae. CELL CYCLE. vol. 7, pp. 1391-1401. [ 62 ] P UBLICATIONS COLANGELO AM, BIANCO MR, VITAGLIANO L, CAVALIERE C, CIRILLO G, DE GIOIA L, DIANA D, COLOMBO D, REDAELLI C, ZACCARO L, MORELLI G, PAPA M, SARMIENTOS P, ALBERGHINA L, MARTEGANI E (2008) A new nerve growth factor-mimetic peptide active on neuropathic pain in rats. JOURNAL OF NEUROSCIENCE vol. 28, pp. 2698-2709. COMBI R, FERINI-STRAMBI L, TENCHINI ML (2008) Compound heterozygosity with dominance in the CRH promoter in a case of nocturnal frontal lobe epilepsy. SLEEP RESEARCH, vol. 17; p. 361-362. COMBI R, SALA E, VILLA N, CROSTI F, BECCARIA L, COGLIARDI A, TENCHINI ML, DALPRA' L. (2008) Maternal -/iso-disomy and paternal supernumerary ring of chromosome 7 in a child with Silver-Russell Syndrome. CLINICAL DYSMORPHOLOGY. vol. 17, pp. 35-39. COMELLI F, GIAGNONI G, BETTONI I, COLLEONI M, COSTA B (2008) Antihyperalgesic effect of a Cannabis sativa extract in a rat model of neuropathic pain: mechanisms involved. PHYTOTHERAPY RESEARCH vol. 22, pp. 1017-1024. COSENTINO U, PITEA D, MORO G, SARACINO GAA, CARIA P, VARI RM, COLOMBO L, FORLONI G, TAGLIAVINI F, SALMONA M (2008) The anti-fibrillogenic activity of tetracyclines on PrP 106-126: a 3D-QSAR study. J MOLECULAR MODELING, vol. 14, pp. 987-994. COSTA B, COMELLI F, BETTONI I, COLLEONI M, GIAGNONI G (2008) The endogenous fatty acid amide, palmitoylethanolamide, has anti-allodynic and anti-hyperalgesic effects in a murine model of neuropathic pain: involvement of CB1, TRPV1 and PPARgamma receptors and neurotrophic factors. PAIN vol. 139, pp. 541-550. CROTTINI A, MAROTTA R, BARBUTO M, CASIRAGHI M, FERRAGUTI M (2008) The world in a river? A preliminary analysis of the 16S rDNA variability of Tubifex species (Clitellata: Tubificidae) from the Lambro River. MOLECULAR PHYLOGENETICS AND EVOLUTION, vol. 48, pp. 11891203. DATO L, SAUER M, PASSOLUNGHI S, PORRO D, BRANDUARDI P (2008) Investigating the multibudded and binucleate phenotype of the yeast Zygosaccharomyces bailii growing on minimal medium. FEMS Yeast Res. Vol. 8(6), pp. 906-15. DE FILIPPIS L, FERRARI D, ROTA NODARI L, AMATI B, SNYDER E, VESCOVI AL (2008) Immortalization of human neural stem cells with the c-myc mutant T58A. PLoS ONE. 2008 Oct 2;3(10):e3310. DE MATTIA F, IMAZIO S, GRASSI F, LABRA M (2008) Chloroplast and nuclear DNA markers to characterize cultivated and spontaneous ribes accessions. PLANT BIOSYSTEMS vol. 142, pp. 204-212. DE MATTIA F, IMATIO S, GRASSI F, DOULATY BANEH H, SCIENZA A, LABRA M (2008) Study of genetic relationships between wild and domesticated grapevine distributed from Middle East Regions to European Countries. RENDICONTI LINCEI vol. 19, pp. 223-240. DEL FAVERO M, MAZZANTINI E, BRIANI F, ZANGROSSI S, TORTORA P, DEH" G (2008) Regulation of Escherichia coli polynucleotide phosphorylase by ATP. J BIOL CHEM vol. 283, pp. 27355-27359. DELLASEGA D, FACIBENI A, DI FONZO F, BOGANA M, POLISSI A, CONTI C, DUCATI C, CASARI CS, LI BASSI A, BOTTANI CE (2008) Nanostructured Ag4O4 films with enhanced antibacterial activity. NANOTECHNOLOGY. vol. 19, pp. 475602-475608. DOGLIA SM, AMI D, NATALELLO A, GATTI-LAFRANCONI P, LOTTI M (2008) Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies. BIOTECHNOL. J. vol. 3, pp. 193-201. FRANSIOLI J, BAILEY B, GUDE NA, COTTAGE CT, MURASKI JA, EMMANUEL G, WU W, ALVAREZ R, RUBIO M, OTTOLENGHI S.,SCHAEFER E, SUSSMAN MA (2008) Evolution of the c-kit-positive cell response to pathological challenge in the myocardium. STEM CELLS, vol. 26, p. 1315-1324. FRASCHINI R, VENTURETTI M, CHIROLI E, PIATTI S. (2008) The spindle position checkpoint: how to deal with spindle misalignment during asymmetric cell division in budding yeast. BIOCHEM SOC TRANS vol 36, pp. 416-20. GALLI P, KRITSKY DC (2008) Three New Species of Protogyrodactylus (Monogenoidea, Dactylogyridae) from the Gills of the Longtail Silverbiddy, Gerres longirostris (Teleostei: Gerreidae) in the Red Sea. SYSTEMATIC PARASITOLOGY vol. 69, pp. 221-231. GALLI R, GRITTI A, VESCOVI AL. (2008) Adult neural stem cells. METHODS MOL BIOL vol. 438, pp. 67-84. GALLINA E, STRONA G, GALLI P (2008) Thaparocleidus siluri, monogenoidean parasite of Silurus glanis: a new record from Italy". PARASSITOLOGIA. (in press) [ 63 ] P UBLICATIONS GASSER B, SALOHEIMO M, RINAS U, DRAGOSITS M, RODRIGUEZ-CARMONA E, BAUMANN K, GIULIANI M, PARRILLI E, BRANDUARDI P, LANG C, PORRO D, FERRER P, TUTINO ML, MATTANOVICH D, VILLAVERDE A (2008) Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. MICROB CELL FACT. Vol. 7(1):11. GATTI-LAFRANCONI P, CALDARAZZO S.M, VILLA A, LOTTI M (2008) Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase. FEBS LETTERS. vol. 582, pp. 2313-2318. GIVOGRI MI, BOTTAI D, ZHU HL, FASANO S, LAMORTE G, BRAMBILLA R, VESCOVI A, WRABETZ L, BONGARZONE E (2008) Multipotential neural precursors transplanted into the metachromatic leukodystrophy brain fail to generate oligodendrocytes but contribute to limit brain dysfunction. DEV NEUROSCI. Vol. 30(5), pp. 340-57. GONZALEZ-MONTALBAN N, NATALELLO A, GARCÍAFRUITOS E, VILLAVERDE A, DOGLIA SM (2008) In situ protein folding and activation in bacterial inclusion bodies. BIOTECHNOL. BIOENG. vol. 100, pp. 797-802. GRANUCCI F, ZANONI I (2008) Role of Toll like receptoractivated dendritic cells in the development of autoimmunity. Front Biosci. Vol. 13, pp. 4817-4826. GRANUCCI F, ZANONI I, RICCIARDI-CASTAGNOLI P (2008) Central role of dendritic cells in the regulation and deregulation of immune responses. CELL MOL LIFE SCI. vol. 65, pp.1683-1697. GRASSI F, DE MATTIA F, ZECCA G, SALA F, LABRA M (2008) Historical isolation and Quaternary range expansion of divergent lineages in wild grapevine. 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Editors: A. Becchetti, A. Arcangeli. LANDES BIOSCIENCE.www.landesbioscience.com/curie/chapter/4009). MORINI R, BECCHETTI A (2008) INTEGRIN RECEPTORS AND LIGAND-GATED CHANNELS: In : Integrins and Ion Channels: Molecular Complexes and Signaling. Editors: A. Becchetti, A. Arcangeli. LANDES BIOSCIENCE.www.landesbioscience.com/curie/chapter/4014). PATENTS 3.4 ALBERGHINA L, COLANGELO AM, MARTEGANI E. "Method for the production of biologically active rhNGF". USP 2008/0214464A1 Published on Sept. 4, 2008. RUMIO C, PALAZZO M, BALSARI A, NICOTRA F, LA FERLA B "Composti a struttura glicosidica attivi nella terapia di stati infiammatori locali e sistemici." n. MI2008A846, 09.05. 2008. PORRO D, BRANDUARDI P, VALLI M, ALBERGHINA L. "Process for expression and secretion of proteins by the non-conventional yeast Zygoasaccharomyces bailii" EP1558722 B123/04/08, AT393210T15/05/08 SAUER M, PORRO D, BRANDUARDI P, MATTANOVICH D, VALLI M. "Improved strains for the production of organic acids" EP1929009 A2 11/06/08, WO2007038130 A815/05/08 BRANDUARDI P, PORRO D, SAUER M, MATTANOVICH D. "Ascorbic acid production from D-glucose in yeast" EP1874947 09/01/08; CN101171340 30/04/08, JP2008536497 11/09/08 PORRO D, DATO L, BRANDUARDI P. "Method for improving acid and low pH tolerance in yeast" WO2008153890 (A1) 18/12/08 PORRO D, MATTANOVICH D, SAUER M, BRANDUARDI P. "Method for improving stress tolerance during fermentation" CA2561408 13/04/08 PORRO D, SAUER M, BRANDUARDI P. "Improved yeast strains for organic acid production" EU patent application Filing date: 28/05/08. [ 68 ] Dipartimento di Biotecnologie e Bioscienze - Università degli Studi di Milano Bicocca Piazza della Scienza 2, 20126 Milano - Tel. ++39 02 6448 3330 - Fax ++39 02 6448 3569 [email protected] - www.btbs.unimib.it graphic project okio_design