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. BIOLOGICAL JOURNAL OF THE LINNEAN SOCIETY vol. 95, pp. 611-619.
GRITTI A, GALLI R, VESCOVI AL (2008) Clonal analyses and
cryopreservation of neural stem cell cultures. METHODS
MOL BIOL. Vol. 438, pp.173-84.
GUASTI L, CROCIANI O, REDAELLI E, PILLOZZI S, POLVANI S, MASSELLI M, MELLO T, GALLI A, AMEDEI A, WYMORE RS, WANKE E, ARCANGELI A (2008) Identification of a
posttranslational mechanism for the regulation of hERG1
K+ channel expression and hERG1 current density in tumor
cells. MOL CELL BIOL. Vol. 28, pp. 5043-60.
HEIDEN ZM, ZAMPELLA G, DE GIOIA L, RAUCHFUSS TB
(2008) [FeFe]-Hydrogenase Models and Hydrogen:
Oxidative Addition of Dihydrogen and Silanes. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION vol. 47, pp. 97569759.
JAVOR S, NATALELLO A, DOGLIA SM, REYMOND JL (2008)
a-Helix Stabilization within a Peptide Dendrimer J. AM.
CHEM. SOC. Vol. 130, pp. 17248-17249.
JI HF, SHEN L, GRANDORI R., MUELLER N (2008) The
effect of heme on the conformational stability of micromyoglobin. THE FEBS JOURNAL. vol. 275, pp. 89-96
JUSTICE AK, DE GIOIA L, NILGES MJ, RAUCHFUSS TB,
WILSON SR, ZAMPELLA G (2008) Redox and structural
properties of mixed-valence models for the active site of the
[FeFe]-hydrogenase: Progress and challenges. INORGANIC
CHEMISTRY vol. 47, pp. 7405-7414.
JUSTICE AK, NILGES MJ, RAUCHFUSS TB, WILSON SR,
DE GIOIA L, ZAMPELLA G (2008) Diiron dithiolato carbonyls
related to the H-ox(CO) state of [FeFe]-hydrogenase. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY vol. 130, pp.
5293-5301.
INVERNIZZI G, ANNONI E, NATALELLO A, DOGLIA SM,
LOTTI M (2008) In vivo aggregation of bovine beta-lactoglobulin is affected by Cys121. PROTEIN EXPRESSION AND
PURIFICATION, vol. 62, pp. 111-115.
LONGHESE MP (2008) DNA damage response at functional
and dysfunctional telomeres. GENES & DEVELOPMENT.
vol. 22, pp. 125-140.
LONGHESE MP, GUERINI I, BALDO V, CLERICI M (2008)
Surveillance mechanisms monitoring chromosome breaks
during mitosis and meiosis. DNA REPAIR. vol. 7, pp. 545557.
MALATESTA M, BIGGIOGERA M, BALDELLI B, BARABINO
S., MARTIN TE, ZANCANARO C (2008) Hibernation as a farreaching program for the modulation of RNA transcription.
MICROSCOPY RESEARCH AND TECHNIQUE, vol. 71, p.
564-572.
MALERBA M, CONTRAN N, TONELLI M, CROSTI P, CERANA R (2008) Role of nitric oxide in actin depolymerization
and programmed cell death induced by fusicoccin in sycamore (Acer pseudoplatanus) cultured cells. PHYSIOLOGIA
PLANTARUM vol. 133, pp. 449-457.
[ 64 ]
P UBLICATIONS
MARTUCCI C, TROVATO AE, COSTA B, BORSANI E,
FRANCHI S, MAGNAGHI V, PANERAI AE, RODELLA LF,
VALSECCHI AE, SACERDOTE P, COLLEONI M (2008) The
purinergic antagonist PPADS reduces pain related behaviours and interleukin-1ß, interleukin-6, iNOS and nNOS
overproduction in central and peripheral nervous system
after peripheral neuropathy in mice. PAIN vol. 137, pp. 81-95.
MATHEWS DJ, SUGARMAN J, BOK H, BLASS DM, COYLE
JT, DUGGAN P, FINKEL J, GREELY HT, HILLIS A, HOKE A,
JOHNSON R, JOHNSTON M, KAHN J, KERR D, KURTZBERG J, LIAO SM, MCDONALD JW, MCKHANN G, NELSON KB, RAO M, REGENBERG A, SIEGEL AW, SMITH K,
SOLTER D, SONG H, VESCOVI A, YOUNG W, GEARHART JD,
FADEN R. (2008) Cell-based interventions for neurologic
conditions: ethical challenges for early human trials. NEUROLOGY vol. 71(4), pp. 288-93.
MEREGHETTI P, GANADU ML, PAPALEO E, FANTUCCI P,
DE GIOIA L (2008) Validation of protein models by a neural
network approach. BMC BIOINFORMATICS 9.
NATALELLO A, PROKOROV VV, TAGLIAVINI F, MORBIN M,
FORLONI G, BEEG M, MANZONI C, COLOMBO L, GOBBI M,
SALMONA M, DOGLIA SM (2008) Conformational Plasticity
of the Gerstmann-Sträussler-Scheinker Disease Peptide
as Indicated by Its Multiple Aggregation Pathways". J MOL
BIOL vol. 381, pp. 1349-1361.
NATALELLO A, SANTARELLA R, DOGLIA SM, DE MARCO A
(2008) Physical and chemical perturbations induce the formation of protein aggregates with different structural features. PROTEIN EXPR PURIF vol. 58, pp. 356-361.
NERI M, MADERNA C, CAVAZZIN C, DEIDDA-VIGORITI V,
POLITI LS, SCOTTI G, MARZOLA P, SBARBATI A, VESCOVI
AL, GRITTI A (2008) Efficient in vitro labeling of human neural precursor cells with superparamagnetic iron oxide particles: relevance for in vivo cell tracking. Stem Cells, vol.
26(2), 505-16.
NICOTRA F, CIPOLLA L, PERI F, LA FERLA B, REDAELLI C
(2008) Chemoselective neoglycosylation. ADVANCES IN
CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, vol.
61, pp. 353-397.
OLSEN MT, BRUSCHI M, DE GIOIA L, RAUCHFUSS TB,
WILSON SR (2008) Nitrosyl derivatives of diiron(I) dithiolates mimic the structure and Lewis acidity of the [FeFe]hydrogenase active site. JOURNAL OF THE AMERICAN
CHEMICAL SOCIETY vol. 130, pp. 12021-12030.
OLUFSEN M, PAPALEO E, BTANDSDALl B, SMALAS A
(2008) Pairs and their role in modulating stability of coldand warm-active uracil DNA glycosylase. PROTEINS, vol.
71, pp. 1219-1230.
PANSERI S, CUNHA C, LOWERY J, DEL CARRO U, TARABALLI F, AMADIO S, VESCOVI A, GELAIN F (2008)
Electrospun micro- and nanofiber tubes for functional nervous regeneration in sciatic nerve transections. BMC BIOTECHNOL. 2008 Apr 11;8:39.
PAOLETTI E, CONTRAN N, MANNING WJ, TAGLIAFERRO
F, RANIERI A, CASTAGNA A (2008) Protection of ash
(Fraxinus excelsior) trees from ozone injury by ethylenediurea (EDU): Roles of biochemical changes and decreased
stomatal conductance in enhancement of growth. ENVIRONMENTAL POLLUTION vol. 155, pp. 464-472.
PAPALEO E, PASI M, RICCARDI L, SAMBI I, FANTUCCI P,
DE GIOIA L (2008) Protein flexibility in psychrophilic and
mesophilic trypsins. Evidence of evolutionary conservation
of protein dynamics in trypsin-like serine-proteases. FEBS
LETTERS vol. 582, pp. 1008-1018.
PEREIRA MBP, TISI R, FIETTO LG, CARDOSO AS, FRANÇA
MM, CARVALHO FM, TROPIA MJM, MARTEGANI E,
CASTRO IM, BRANDÃO RL. (2008) Carbonyl cyanide mchlorophenylhydrazone induced calcium signaling and activation of plasma membrane H+-ATPase in the yeast
Saccharomyces cerevisiae. FEMS YEAST RES. Vol. 8, pp.
622-630.
PIAZZA M, ROSSINI C, DELLA FIORENTINA S, POZZI C,
COMELLI F, BETTONI I, FUSI P, COSTA B, PERI F (2008)
Glycolipids and benzylammonium lipids as novel anti-sepsis agents: synthesis and biological characterization". J
MED CHEM vol. 52 (4), pp. 1209–1213.
PESSIA M, SERVETTINI I, PANICHI R, GUASTI S, GRASSI S,
ARCANGELI A, WANKE E, PETTOROSSI VE (2008) ERG voltage-gated K+ channels regulate excitability and discharge
dynamics of the medial vestibular nucleus neurons. J PHYSIOL. Vol. 586, pp. 4877-90.
PORRO D, VAI M, VANONI M, ALBERGHINA L AND HATZIS
C (2008) Analysis and modeling of growing budding yeast
populations at the single cell level. CYTOMETRY vol. 75A (2),
pp. 114-120.
POZZI C, VALTORTA M, TEDESCHI G, GALBUSERA E,
PASTORI V, BIGI A, NONNIS S, GRASSI E, FUSI P. (2008)
Study of subcellular localization and proteolysis of ataxin-3.
NEUROBIOLOGY OF DISEASE vol. 30 (2), pp. 190-200.
[ 65 ]
P UBLICATIONS
QUERIN L, SANVITO R, MAGNI F, BUSTI S, VAN DORSSELAER A, ALBERGHINA L, VANONI M (2008) Proteomic
Analysis of a Nutritional Shift-up in Saccharomyces cerevisiae Identifies Gvp36 as a BAR-containing Protein Involved
in Vesicular Traffic and Nutritional Adaptation. J BIOL
CHEM. Vol. 283, pp. 4730-43.
RAUCCI A, CUGUSI S, ANTONELLI A, BARABINO S., MONTI
L, BIERHAUS A, REISS K, SAFTIG P, BIANCHI ME (2008) A
soluble form of the receptor for advanced glycation endproducts (RAGE) is produced by proteolytic cleavage of the
membrane-bound form by the sheddase a disintegrin and
metalloprotease 10 (ADAM10). FASEB JOURNAL, vol. 22,
pp. 3716-3727.
ROCCHETTI M, ALEMANNI M, MOSTACCIUOLO G ET AL.
(2008) Modulation of sarcoplasmic reticulum function by
PST2744 [istaroxime; (E,Z)-3-((2-aminoethoxy)imino)
androstane-6,17-dione hydrochloride)] in a pressure-overload heart failure model. J PHARMACOL EXP THER. vol.
326, pp. 957-965.
ROMANO M, CAPRIOLI M, AMBROSINI R, RUBOLINI D,
FASOLA M, SAINO N (2008) Maternal allocation strategies
and differential effects of yolk carotenoids on the phenotype and viability of yellow-legged gull (Larus michahellis)
chicks in relation to sex and laying order. JOURNAL OF
EVOLUTIONARY BIOLOGY vol 21, pp 1626-1640.
SAINO N, AMBROSINI R (2008) Climatic connectivity between Africa and Europe may serve as a basis for phenotypic adjustment of migration schedule of trans-Saharan
migratory birds. GLOBAL CHANGE BIOLOGY vol 14, pp 250263.
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