annualreport department of biotechnology and biosciences

2010
A
R
N
E
N
P
U
O
A
R
L
T
D E PA R T M E N T O F
B I O T E C H N O LO G Y
AND BIOSCIENCES
Francesco Peri / Luce dalla finestra
This annual report represents an overview of the structure, organization and activities of the Department of
Biotechnology and Biosciences (BTBS) in 2010.
Our staff is presently composed by 13 Full Professors,
16 Associate Professors and 28 Assistant Professors.
Permanent staff also includes 12 technicians and 9
members of the administration team who actively contribute to the activity and organization of the
structure. Despite the difficulties experienced by all
Universities in the present time, the year went rather
well for the Department with several research grants
and contract that brought our budget to be over 8 million euros. The scientific activity resulted in about 140
papers on international magazines and to some book
chapters and patents.
As in previous years, we have been heavily involved in educational activities, which include undergraduate courses in Biology and Biotechnology,
Master’s courses in Industrial Biotechnology, Biology
and Bioinformatics and different PhD programs. The
number of students this year was well above 2.000.
Internationalization of education was also pursued.
Besides our involvement in the Erasmus Project,
some of our students prepared their Master’s degree
thesis abroad thanks to the support of the EXTRA
project and newly graduates had the opportunity to
attend a stage in a foreign company or academic
laboratory in the frame of the program Unipharma
Graduates. Moreover, some students of Biology and
Biotechnology were admitted to an international
degree in the frame of an agreement with the
Universities of Paris V and Paris VII.
Our international collaborations were intense as
can be seen from the International Mobility section
and from the projects active. Moreover, during this
year two well renewed scientists from Germany
and Spain spent some months in the Department
to work on common research projects and to participate in the education of young scientists.
Even this year, I would like to thank all staff members,
post-docs, PhD and Master’s students for their excellent work and collaboration. Many thanks are
also due to the colleague who kindly allowed us to
use his paintings for this report’s art work.
INDEX
1. STRUCTURE AND ORGANIZATION
1.1
1.2
1.3
1.4
1.5
Financial Resources
Department Management Structure and Staff
Organization and Structure
Spin-offs
Instrumentation and Facilities
3
4
4
8
9
10
2. RESEARCH GROUPS 15
3. PUBLICATIONS, GRANTS, PhD THESES
49
50
56
56
57
59
3.1
3.2
3.3
3.4
3.5
Publications
Book chapters
Patents
Research Grants and Contracts
PhD Theses
Marina Lotti
[1]
Francesco Peri / Mare Vento
[2]
STRUCTURE AND
ORGANIZATION
1
[3]
1.1
FINANCIAL
RESOURCES
1.2
DEPARTMENT
MANAGEMENT
STRUCTURE &
STAFF
Research Grants
Head of the Department
MIUR (PRIN, FISR, FIRB)
1.723.000
EU GRANTS
451.000
REGIONE LOMBARDIA GRANTS 768.000
CARIPLO AND AIRC GRANTS
509.000
OTHER FUNDING AGENCIES
39.000
FAR (fondi d’ateneo per la ricerca) 306.000
RESEARCH SERVICES
232.000
OTHER RESOURCES
125.000
Other Funding Sources
DEPARTMENT FUNDING
FUNDS FOR TEACHING PhD COURSES
126.000 90.000
27.200
Prof. Marina Lotti
Chief Financial Officer
Dr. Anastasia Sguera
Management Board
Prof. Paolo Tortora
Prof. Marina Vai
Prof. Francesca Granucci
Prof. Maria Pia Longhese
Dr. Barbara Costa
Dr. Maurizio Casiraghi
Dr. Anastasia Sguera
COORDINATORS OF BACHELOR AND
MASTER COURSES
Prof. Danilo Porro (Biotechnology)
www.biotecnologie.unimib.it
Prof. Paolo Tortora (Biology)
www.biologia.unimib.it
COORDINATORS OF PhD COURSES
Prof. Marco Vanoni (Biotechnology)
Prof. Enzo Wanke (Biology)
www.scuoladottorato.scienze.unimib.it
[4]
FULL PROFESSORS
NAME
FIELD
Alberghina Lilia (Until 5.2010)
Castagnoli Paola*(Until 10.2010)
Fantucci Piercarlo
Lotti Marina
Lucchini Giovanna
BIO/10
MED/04
CHIM/03
BIO/10
BIO/18
NAME
Martegani Enzo
Nicotra Francesco
Ottolenghi Sergio
Porro Danilo
Tortora Paolo
FIELD
NAME
FIELD
BIO/11
CHIM/06
BIO/18
CHIM/11
BIO/10
Vai Marina
Vanoni Marco
Wanke Enzo
Zaza Antonio
Zullini Aldo
BIO/11
BIO/10
BIO/09
BIO/09
BIO/05
ASSOCIATE PROFESSORS
NAME
FIELD
Barabino Silvia
Becchetti Andrea
Branduardi Paola
Cipolla Laura
Crosti Paolo
De Gioia Luca
BIO/11
BIO/09
CHIM/11
CHIM/06
BIO/01
CHIM/03
NAME
Doglia Silvia Maria
Giagnoni Gabriella (Until
10.2010)
Grandori Rita
Granucci Francesca
Longhese Maria Pia
Moro Giorgio
FIELD
NAME
FIELD
FIS/01
BIO/14
BIO/10
MED/04
BIO/18
CHIM/02
Nicolis Silvia
Peri Francesco
Piatti Simonetta*
Polissi Alessandra
Ronchi Antonella
Vescovi Angelo
BIO/18
CHIM/06
BIO/18
BIO/19
BIO/18
BIO/13
FIELD
NAME
FIELD
BIO/11
BIO/13
BIO/14
MED/04
BIO/18
CHIM/11
BIO/10
BIO/07
BIO/01
CHIM/06
Lecchi Marzia
Orlandi Ivan
Prosperi Davide
Regonesi Elena
Rocchetti Marcella
Tisi Renata
Zampella Giuseppe
Zanoni Ivan
BIO/09
BIO/11
BIO/10
BIO/10
BIO/09
BIO/11
CHIM/03
MED/04
ASSISTANT PROFESSORS
NAME
FIELD
Ambrosini Roberto
Bertini Luca
Brambilla Luca
Brocca Stefania
Casiraghi Maurizio
Ceriani Michela
Chiaradonna F.
Clerici Michela
Coccetti Paola
Colangelo A.M.
BIO/07
CHIM/03
CHIM/11
BIO/10
BIO/05
BIO/11
BIO/10
BIO/18
BIO/10
BIO/10
NAME
Colombo Sonia
Combi Romina
Costa Barbara
Foti Maria
Fraschini Roberta
Frascotti Gianni
Fusi Paola
Galli Paolo
Labra Massimo
La Ferla Barbara
TECHNICAL STAFF
NAME
Accardo Elena*
Bruni Ilaria (from Oct 2010)
Citterio Stefania
D’Urzo Annalisa
NAME
Gullo Francesca
Malerba Massimo
Marinoni Sara
Mostacciuolo Gaspare
NAME
Tonelli Maria Grazia*
Urbano Matteo
Villa Anna Maria
Sacchetti Francesco
ADMINISTRATION
NAME
NAME
NAME
Bottani Elena
Bruno Stefania
Campbell Neil
Comi Roberto
Gotti Maria Cristina
Mormile Bruno
Pacecca Simona
Sguera Anastasia
Smeraldi Carla
* leave of absence
[5]
PhD STUDENTS
NAME
NAME
NAME
Adamo Giusy Manuela
Ambrosi Paola
Anbalagan Savani
Aprile Francesco
Balestrieri Chiara
Barbieri Valentina
Bazzi Marco
Bertagnoli Stefano
Bigi Alessandra
Broggi Achille
Broggi Serena
Busnelli Sara
Caccia Roberta
Cantù Claudio
Cardona Francisco
Casatta Nadia
Codazzi Vera
Colombo Miriam
Di Gioia Marco
Doizy Anthony
D’ Orazio Giuseppe
Dossi Elena
Falcettoni Marco
Fontana Gabriele
Fumagalli Silvia
Gabrielli Luca
Gaglio Daniela
Galimberti Andrea
Giaccherini Cinzia
Gotti Laura
Groppi Silvia
Hasan Mehedi
Loffreda Alessia
Longo Valeria
Marangoni Stefano
Mariani Jessica
Martina Marina
Marzi Roberta
Merlo Silvia
Palorini Roberta
Pastori Valentina
Raspelli Erica
Rizzetto Riccardo
Rossi Giorgia
Russo Laura
Santambrogio Carlo
Shaik Nasrin
Sironi Erika
Testa Lorenzo
Trovesi Camilla
Venkatesh Aparna
Viganò Matteo
Villa Riccardo
Vivarelli Silvia
Zona Cristiano
NAME
NAME
NAME
Acquaro Giovanni
Alemanni Matteo
Altomare Claudia
Alvarez Reinaldo
Baruffa Chiara
Biancolini Donatella
Bini Davide
Bonanomi Marcella
Busti Stefano
Cattaneo Francesca
Clarelli Ferdinando
Damore Gaetana
Frana Anna Maria
Gaviraghi Marco
Kapetis Dimos
Mazzoleni Elisa
Monestiroli Andrea
Montano Simone
Monza Francesca
Mozzi Alessandra
Papagna Angela
Parravicini Federica
Pessina Stefania
Sala Danna Lara
Sangalli Elena
Seveso Davide
Strona Giovanni
Verderio Paolo
Zacco Elsa
Zalfa Maria Cristina
NAME
NAME
NAME
Airoldi Cristina
Ami Diletta
Amigoni Loredana
Aracri Patrizia
Barbuto Michela
Barile Lucio
Belotti Fiorella
Benzoni Francesca
Berbenni Miluscia
Bodio Caterina
Bonetti Diego
Bruni Ilaria
Calabrese Valentina
Cirulli Claudia
Comelli Francesca
Dato Laura
De Filippis Lidia
De Mattia Fabrizio
Favaro Rebecca
Ferri Anna Lucia
Forcella Matilde
Fossati Tiziana
Gelain Fabrizio
Gorletta Tatiana
Greco Claudio
Invernizzi Gaetano
Lenzken Carolina
Leoni Giampaolo
Lombardi Alessio
Maffezzoli Andrea
Manfrini Nicola
Martorana Alessandra
Mazzucchelli Serena
Natalello Antonino
Paiardi Chiara
Papaleo Elena
Piazza Matteo
Redaelli Cristina
Sacco Elena
Salvadé Agnese
Samalikova Maria
Servettini Ilenio
Sperandeo Paola
Stefani Fabrizio
Tripodi Farida
Torri Anna
FELLOWSHIP HOLDERS
POST-DOCS
[6]
INTERNATIONAL MOBILITY 2010
Our Department participates in programs of exchange of students and professors such as Erasmus and EXTRA. Mobility of researchers is supported either in the frame of institutional projects or on the basis of individual research projects and collaborations.
In the following an overview is given of incoming and outgoing scientists during 2010. Two foreign scientists were hosted in the
Department in 2010 for research and training projects financed by Fondazione Cariplo.
INCOMING
FROM
PERIOD
Barbotin Céline
Cardona Francisco
Colbert Marlène
Doizy Anthony
Ebner Florian
Lin Charles
Orsato Alexandre
Prikrylova Iva Masaryk
Romano Annalisa
Shaikh Nasrin
Venkatesh Aparna
Yan Carol Université Paris VII Denis Diderot, Paris, France University of Coimbra, Portugal Université Paris VII Denis Diderot, Paris, France
Université de Marseille, France Universitaet Wien, Austria Princeton University, USA Federal University of Paraná, Brazil University of Czech Republic Université de Nice Sophia-Antipolis, Nice, France Pune University, India
A-Star Singapore
National Institute for Medical Research, London, UK 3
12
3
2
3
2
12
12
2
12
12
3
OUTGOING
TO
PERIOD
Adamo Giusy
Aprile Francesco
Balestrieri Chiara
Colombo Miriam
Dato Laura
Gaglio Daniela
Pagella Pierfrancesco Peri Francesco
Piazza Matteo
Rizzetto Riccardo
Russo Laura
Servettini Ilenio
Villa Riccardo
University of Goteborg, Sweden University of Cambridge, UK
University of Helsinki, Finland
Philipps University of Marburg, Germany VTT Technical Center of Finland
Massachusetts Institute of Technology (MIT), USA
Ludwig-Maximilians Universitaet, Munich, Germany
Ecole Normale Supérieure (ENS) Lyon, France
University of Iowa, USA
IGH-CNRS Montpellier, France
Imperial College, London, UK
Leipzig University, Germany
Harvard University, Cambridge MA, USA 4
10 7
6
2
1
3
1
4
6
6
1.5 6
months
months
months
months
months
months
months
months
months
months
months
months
months
months
months
months
months
month
months
month
months
months
months
months
months
FONDAZIONE CARIPLO GRANTS
Prof. Peter Illes, Dept. of Pharmacology and Toxicology, Leipzig University (Germany) “Functional regeneration of the mesocorticolimbic dopaminergic system as a model to study novel neuroreparative strategies” collaboration with Prof. E. Wanke
Prof. Jesus Jimenez Barbero, CIB, CSIC Madrid “Development of NMR techniques for tissue engineering studies” collaboration
with Prof. F. Nicotra
COORDINATORS OF INTERNATIONAL MOBILITY
Prof. Maria Pia Longhese (for Biotechnology)
Prof. Silvia Nicolis (for Biology)
[7]
1.3
ORGANIZATION
AND STRUCTURE
ADMINISTRATION OFFICE
Building U3, III floor. [email protected]
Anastasia Sguera / Chief Financial Officer
Stefania Bruno / Foreign payments, VAT related accounting
Roberto Comi / Accounts payable (Contracts, scholarships)
Bruno Mormile / Supplier’s accounting, travel reimbursement
Francesco Sacchetti / Property inventory, technical support
STUDENT ADMINISTRATION OFFICE
Building U3, II floor. [email protected]
Maria Cristina Gotti, Elena Bottani, Simona Pacecca
The student administration office manages all administrative aspects related to the teaching activities of the degrees in
Biotechnology and Biology and assists all students in the bureaucratic aspects of their career; it is responsible for the content of
the web pages of the Department website 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.
TECHNOLOGY TRANSFER COMMISSION
Francesco Peri, Alessandra Polissi, Carla Smeraldi
Contact person: Carla Smeraldi /[email protected]
The Technology Transfer Commission acts as a liaison between the Department and the Technology Transfer and Intellectual Property
office of the University. It also promotes contacts between the research groups, small and medium enterprises, patent experts, in order to
exploit scientific results and promote innovation.
TECHNICAL SERVICES
The technical staff carries out common services, is responsible for the maintenance of common instruments and collaborates in
research activities.
Mass Spectrometry: Annalisa D’ Urzo. Cytometry: Stefania Citterio. Biacore (Plasmon Resonance): Annalisa D’ Urzo. Technical gases,
MEA workstation: Francesca Gullo. Chemical and Biological Wastes Disposal: Sara Marinoni. Biotechnicum: Simone Passolunghi.
Molecular Immunology, BL2 Laboratory: Matteo Urbano. Confocal Microscopy: Anna Maria Villa.
EXTERNAL SERVICES
The Department offers its know-how and services to outside customers. For a complete list of services and contacts see:
www.btbs.unimib.it
[8]
1.4
SPIN-OFFS
BLUEPRINT BIOTECH s.r.l
Established in 2006 with a focus on red biotechnologies and more specifically on developing NGFlike peptides for several clinical applications. It owns
patents related to new NGF-like peptides for neurodegenerative disorders, patents related to the use of
NGF for ophthalmic applications and the know-how
related to the production of recombinant NGF.
For more information tel +39.02.6448.3515
FEM2 – AMBIENTE
Established in January 2010, Fem2-Ambiente’s
mission is to provide customers with products and
services for environmental education, natural resources and biodiversity preservation. The company is
promoting a series of easy-to-use kits to test water
physical and chemical characteristics that can be
used at home or in the classroom. It also provides
molecular diagnostic services that, for example,
allow to identify animal or vegetal foods or contaminated food; to determine the sex of birds from
the DNA of a feather; to identify parasites using the
DNA barcoding technology; to determine the
microbial load of a sample etc.
http://www.fem2ambiente.com
[9]
1.5
INSTRUMENTATION
AND FACILITIES
A number of platform technologies and advanced instrumentations are available both to the research groups working in the Department and to external users.
BBC (BICOCCA BIOTECHNICUM CENTER)
The Biotechnicum (BCC) is a facility aimed at the development of proprietary industrial strains,
fermentation and bioconversion processes for the production of commercially interesting
proteins, metabolites and enzymes. BCC is particularly strong in services that require
an integrated multidisciplinary approach. The main focuses are on those areas which require a combined know-how of bioprocess technology, microbiology and biochemistry applied to
industrial biotechnology processes. In this position BBC is able to assist in the selection,
modification and development of microorganism, “scaling-up” and “scaling down” of production
processes. The core of the facility are two 10 lt bioreators. All the operations are carried out in
GLP (Good Laboratory Practice) and GMP-like (Good Manufacturing Practice) environment.
For more information www.bbc.btbs.unimib.it
MICROARRAY FACILITY
Microarray technology allows for rapid measurement and visualisation of differential expression
among genes at the whole genome scale. In DNA microarrays, also called 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 tissues from healthy and
diseased subjects. Gene expression has been successfully used to understand complex
diseases, design diagnostic tests and possible therapeutic targets. Arrays are currently
available for human samples as well as many biologically relevant model organisms including
mouse, rat and plant (Arabidopsis).
BIOSAFETY LEVEL 2 FACILITY (BL2)
This new core facility is located in a dedicated closed laboratory space, with restricted access.
The facility consists in a Vector Production room, a Cell Manipulation room (directly connected
with a Pass-Through Cabinet) and two general support areas. These laboratories house tissue
culture hoods, CO2/CO2 incubators, microscopes and small equipment for cellular and mole-
[ 10 ]
cular biology work.
All areas are designed to ensure a high standard of cleanliness and an orderly flow of the
entire experimental process. The air is HEPA-filtered, and manufacturing conditions have been
further optimized by a system of differential air pressures between individual rooms. This facility is suitable for experiments involving agents of moderate potential hazard to personnel
and environment, and, in particular, for safe production and handling of lentiviral and retroviral
vectors. The facility has been approved by the Ministry of Health.
MASS SPECTROMETRY
The mass spectrometry (MS) facility supports the analysis of small and large molecules, including
protein non-covalent complexes. The laboratory is equipped with two instruments, one with
electrospray-ionization sample source (ESI) and one with matrix-assisted-laser-desorption/
ionization sample source (MALDI). The two mass analyzers are based on different technologies and
are connected respectively with a micro-HPLC system for liquid chromatography (LC) and a
nano-ESI sample source. Together they enable LC-MS/MS and LC-MS/MS/MS measurements for
proteomics or analytical chemistry, analysis of post-translational modifications of proteins
by neutral-loss scan, precursor-ion scan and multiple-reaction monitoring and protein
analysis under non-denaturing conditions. We can therefore conduct proteomics studies,
protein conformational studies and analyses of protein-protein and protein-ligand non-covalent
complexes.
MEA - MULTI ELECTRODE ARRAY WORKSTATION
The MEA workstation consists of a complex machine for the acquisition of the stimulated, or
spontaneous, activity of networks of excitable cells (from sensory, cardiac or neuronal origin)
in real time (for days or weeks), and under non-invasive conditions. 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 ß-amyloid protein, a main factor involved in Alzheimer’s
disease pathogenesis.
NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROMETRY
The Nuclear Magnetic Resonance (NMR) lab is equipped with a Brucker ADVANCE 600 MHz
equipped with a high-resolution liquid-state quadruple resonance cryo-probe, a HR-MAS triple
resonance probe and a solid-state triple resonance probe and a Varian MERCURY 400 MHz
spectrometer. One inverse-detection gradient probe (good sensitivity for 1H and 19F) and one
direct-detection probe (good sensitivity for 13C and 31P) are available. This instrument allows the
structural and conformational characterization of small-medium size molecules (up to 6 kDa
molecular weight), such as low molecular-weight drugs, mono-, di-, tri- and oligosaccharides,
oligonucleotides and peptides. A large panel of pulse sequences can be performed, i.e monobi- and tri-dimensional experiments. Small ligand-receptor interaction studies (such as inhibitor/activator-protein or substrate-enzyme) are performed via DOSY, Saturation Transfer
Difference (STD) and transferred-NOESY (tr-NOESY) experiments.
[ 11 ]
FLOW CYTOMETRY
Flow cytometry (FCM) allows to count and examine microscopic particles, such as cells and
chromosomes, suspending them in a stream of fluid and capturing their fluorescence with an
electronic detection apparatus, after they have been hit by a laser beam. Each suspended
particle that passes through the beam scatters the light in some way. Fluorescent chemicals
found in the particle or attached to it may be excited to emit light at a higher wavelength than
the light source. This combination of scattered and fluorescent light is picked up by the detectors
allowing simultaneous multiparametric analysis of physical and/or chemical characteristics of
up to hundreds of particles per second.
A cell sorter, or flow sorter, is a flow cytometer that uses electrical and/or mechanical means to
divert the analyzed particles with characteristics that fall within a user-selected range of values.
Several research groups use this type of instrument for the analysis and sorting of microorganisms (especially yeasts) for industrial biotechnological applications; analysis of cell cycle progression and ageing of yeast subpopulations; analysis of mammalian cells for typization and sorting of
specific subpopulations. Flow cytometers present in our department include:
A MoFlo® high speed cell sorter (Cytomation-BeckmanCoulter) equipped with three lasers
(354 nm; 488 nm and 635 nm) which enables to perform 6-colour analyses. The MoFlo® has a
dedicated operator.
Cell Lab Quanta SC (Beckman Coulter) with Mercury arc excitation optimized at 365, 405, and
435 and 488nm laser diode excitation. It has 3 broad range ultra sensitive photomultiplier tubes
and a 125µm triangular flow cell. With this instrumentation it is possible to measure simultaneously electronic volume, side scatter, time, and 3 colour detections. This flow cytometer is
used for analyses only.
A FacScan® (Becton & Dickinson) that allows to perform 3-colour analyses
OPTICAL SPECTROSCOPY AND OPTICAL MICROSCOPY
The laser scanning confocal fluorescence microscope Leica TCS SP2 is a confocal microscope with an acoustic optical beam splitter, equipped with three lasers for fluorescence
excitation (an Argon laser and two He-Neon lasers). The scanning head of the system is coupled to an inverted motorized optical microscope Leica DFMIR2, equipped with dry objectives
of 10x e 20x magnification, as well as with oil immersion objectives of high magnification
40x and 63x. The Leica TCS SP2 prism spectrometer enables also to measure fluorescence
spectra and to set the wavelength band of the collected fluorescence to the real emission
spectrum. The easy-to-use acquisition software for image analysis enables also the threedimensional reconstruction of the specimen.
Inverted motorized microscope Nikon Eclipse E600. Fluorescence microscope with halogen lamp for transmitted light illumination and Xenon lamp for fluorescence excitation. The
microscope is coupled to a digital video camera Leica DC 350 F that enables to obtain high
image quality at low light intensity. The video camera is equipped with image acquisition
software and image analysis algorithms for three-dimensional reconstruction. Circular dichroism spectropolarimeter Jasco J815. This spectropolarimeter works in the ultraviolet and visible ranges from 163 nm and 900 nm. It also allows to measure the fluorescence of
the sample in the range 200-800 nm. The temperature of the sample is controlled by a Peltier
system operating between -10 °C e + 110 °C. The instrument is equipped with a Stopped-Flow
accessory for kinetic and titration studies.
[ 12 ]
Fourier transform infrared spectrometer (FTIR) Varian 670-IR
This spectrometer is used for absorbance measurements in the medium infrared range, with
dynamic alignment of the interferometer and MCT detector. The instrument allows measurements in transmission mode and in attenuated total reflection (using a 9 reflection diamond
plate) with temperature control. The spectrometer is coupled to the infrared microscope
610IR Varian.
Spectrofluorimeter Varian Cary Eclipse
This is a highly sensitive spectrometer for fluorescence emission and excitation measurements
from 200 nm to 900 nm on minimum sample volumes. It allows for temperature control up to
four samples simultaneously. It is equipped with a static anisotropy fluorescence accessory
(automatically controlled) and with a microplate reader working in reflecting optics
Genetic Analyzer ABI prism 3130
The Genetic Analyzer ABI prism 3130 (Applied Biosystem) is a versatile tool, able to perform
analysis of DNA fragments and regions ranging in size from a few hundred bases to about
1000 bp. It is a 4 capillary electrophoresis system that uses fluorescently labeled dyes for
detection of DNA. The sequence is displayed as a series of peaks, one for each nucleotide,
represented in different colors: green for A, blue for C, black for G and red for T.
The present system has several preparation kits for the analysis of sequence and for fragment
analysis (microsatellites, AFLP, SNP).
Real time PCR System 7500 FAST
The real time PCR System 7500 (Applied Biosystem) is equipped with a sophisticated system
of detection of fluorescence emitted by dyes that bind double-stranded DNA, or by hybridization probes that are used in the amplification reaction. This detection system allows to monitor the performance of real-time PCR reaction eliminating non-specific signals and allowing
to quantify the PCR amplification products compared to a calibration curve.
Applications of this technological resource range from the detection of a gene or a specific
marker to the quantification of gene expression.
[ 13 ]
Francesco Peri / Opus Quadratum
[ 14 ]
RESEARCH
GROUPS
2
[ 15 ]
1
Silvia Nicolis,
Sergio Ottolenghi,
Antonella Ronchi,
Rebecca Favaro,
Anna Ferri,
Jessica Mariani,
Roberta Caccia,
Claudio Cantù.
Molecular genetics of
stem/progenitor cells in
development and differentiation;
studies in the nervous and
hematopoietic systems
The Group of Molecular Genetics is headed by
three scientists who are interested in the molecular control of tissue-specific development and
differentiation at the stem/progenitor cell level.
S. Nicolis is interested mainly in the nervous system, A. Ronchi and S. Ottolenghi in the hematopoietic system. Some research on solid tumors
and leukemia is also carried out.
S. Nicolis focuses on the transcription factor
Sox2. Using conditional gene ablation in mouse,
the group showed that Sox2 is essential for the
normal genesis of the dentate gyrus of the hippocampus, via a mechanism controlling the expression of the Sonic hedgehog cytokine. By deleting
Sox2 at appropriate developmental stages, they
are investigating region-specific roles of Sox2 in
the cerebellum, ventral brain and cortex. Studies
in neural stem cells in vitro allow to investigate
Sox2 molecular mechanisms of action; in particular, a collaboration with the Genome Institute
of Singapore (Drs. Chia-lin Wei, Paul Robson) is
looking at long range effects of Sox2 in the control of chromatin organization. As Sox2 is also
expressed in non-nervous stem/progenitor cells,
the group has been investigating, in collaborative studies, other systems (osteoblasts, melanocytes, germ cells). Finally, Sox2 is expressed
in putative stem cells in various tumors, such as
glioblastoma, medulloblastoma, etc.. Conditional
ablation of Sox2, in vitro or in vivo, might affect
the development/maintenance of these cells;
these hypotheses are currently being explored.
[ 16 ]
A. Ronchi looked at differentially expressed genes during the development of the hematopoietic
system in mouse. Among several differentiallyexpressed genes, they selected the transcription factor Sox6 for further studies, showing that
overexpression of Sox6 strongly stimulates the in
vitro growth arrest and differentiation of primary
human and mouse hematopoietic progenitors,
and of cell lines (including some leukemic lines).
This effect is, in part, mediated by the stimulation of the expression of SOCS3 ( that appears to
be a direct Sox6 target), a factor that inhibits the
activities of several cytokine signalling systems
(erythropoietin, IGF1). An important effect of Sox6
overexpression is on the globin genes; these genes are all stimulated by Sox6, but the embryonic- and fetal-globin (epsilon and gamma) genes
are much less activated than the adult (beta)-globin genes. This effect, and its mechanisms, are
of great potential interest for the development of
therapies of inherited diseases (thalassemia, Sickle Cell Disease), in which replacement of betaby gamma-globin would be beneficial.
S. Ottolenghi is interested in the c-Kit gene, a
membrane receptor of Stem Cell Factor, important in hematopoietic stem/progenitor cells and
other stem cell types. Using reagents such as
c-Kit/GFP constructs and trasgenic mouse lines
expressing GFP under the control of regulatory
elements of the c-Kit locus, the group is studying
stem/progenitor cells in the hematopoietic, cardiac and germ cell systems, in collaboration with
several research groups.
Mechanisms of post-transcriptional
regulation of mammalian gene
expression and their role in
human disease
The research interests of our laboratory is in 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 premRNA, with major emphasis on the role of the
arginine-serine (SR) family of proteins, and their
kinases, in the regulation of alternative splicing. To
complement our biochemical studies we use a cell
biological approach and look at the intracellular distribution of these factors by fluorescence and confocal microscopy.
The main lines of research in the laboratory are:
1.Multiple roles of SR proteins in RNA processing
2.RNA processing and signal transduction
3’ END PROCESSING AND TRANSPORT OF mRNAs
We have characterized the intracellular localization
of the 3’ end processing factor CF Im and we have
shown that it shuttles continuously between the nucleus and the cytoplasm in association to mRNA.
Nucleo-cytoplasmic shuttling may reflect the association of CF Im with mature mRNPs and participate
in coupling mRNA processing to later events in the
life of mRNA. We have shown that CF Im plays a direct role in nuclear export of mRNAs.
AMYOTROPHIC LATERAL SCHLEROSIS 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. We
identified a common set of genes involved in neuritogenesis, axon growth and guidance, and synaptogenesis that are deregulated at the transcription
and alternative splicing level in both models of mitochondrial stress.
DNA DAMAGE AND RNA SPLICING
We have investigated the effect of genotoxic treatments on the subcellular localization and the activity
of SRPK2. SRPK2 is a kinase the SR family of splicing regulatory proteins. SRPK2 is normally localized in the cytoplasm where it phosphorylates spliceosomal SR proteins that can thus be re-imported
into the nucleus. We have shown that the activation
of the DNA damage response leads to the accumulation of SRPK2 in the cell nucleus and to increased phosphorylation of SR proteins. Relocalization
of SRPK2 correlates with changes in the alternative splicing pattern of the E1A splicing reporter. In
addition, we have identified a set of mRNAs whose
splicing is modulated by genotoxic stress. These
observations thus identify SRPK2 as a novel kinase
involved in the cellular response to DNA damage.
2
Silvia Barabino,
Reinaldo Alvarez,
Gabriele Fontana,
Silvia C. Lenzken,
Alessia Loffreda,
Silvia Vivarelli.
[ 17 ]
3
Mechanisms controlling
genome integrity
Maria Pia Longhese,
Giovanna Lucchini,
Michela Clerici,
Diego Bonetti,
Nicola Manfrini
Marco Bazzi,
Camilla Trovesi,
Marco Falcettoni,
Savani Anbalagan,
Marina Martina.
[ 18 ]
Myriad genetic and epigenetic alterations are
required to drive normal cells toward malignant
transformation. Cancer cell genomes are highly
rearranged and are characterized by complex
translocations and regional copy number alterations that target loci harboring cancer-relevant
genes. Therefore, those mechanisms that maintain genome stability, in principle, protect from
cancer. DNA double-strand breaks threat genome integrity, because failure to repair these
lesions can lead to rearrangements and/or loss
of genetic information. A broken chromosome
can be repaired by either nonhomologous endjoining or homology-directed recombination, allowing cells to continue their divisions with an
intact genome. Furthermore, the exposed DNA
ends at a double-strand break activate the DNA
damage checkpoint, which arrests cell division
cycle and can induce cell death. Central components of this pathway are highly conserved protein kinases such as human ATR and ATM and
their S. cerevisiae orthologs Mec1 and Tel1.
Although genomic instability is thought to drive
tumorigenesis, the biological basis of selection
for cells with an aberrant DNA damage response in cancer remain poorly understood. Efforts
to uncover the mechanisms underlying genome
instability in cancer cells have revealed a prominent role for telomeres, specialized structures at
the end of eukaryotic chromosomes. Telomeres
are needed to ensure that the entire chromosome is faithfully replicated and that chromosome
ends are not mistakenly treated as DNA doublestrand breaks. Loss of telomeric sequences or
failure of telomeres to mask themselves from
recognition as double-strand breaks activates
a DNA damage checkpoint response that inhibits cell proliferation and contribute to ageing.
Attempts to “repair” these dysfunctional telomeres would have devastating consequences
for genome integrity and such telomere-initiated genetic instability can lead to carcinogenesis. Thus, normally functioning telomeres need
to be protected from DNA repair activities and
checkpoint activation.
Our research activity aims to elucidate the molecular mechanisms that i) control the cellular
response to double-strand breaks and ii) protect
telomeres from being recognized as DNA damage. In particular, we are using different approaches in order to study how cells sense, process
and repair double-strand breaks. Furthermore,
we are studying the mechanisms that ensure telomere homeostasis and inhibit DNA repair activities at telomeres. Finally, we are investigating
how the above mechanisms are coupled to cell
cycle progression and are interconnected with
each other.
As many aspects of telomeres and DNA damage
response are remarkably conserved throughout
evolution, the organism chosen to tackle these
issues is Saccharomyces cerevisiae. Because
both the DNA damage response and telomere
protection are necessary to maintain genetic stability, our research activity could contribute to the
understanding of the molecular processes that
are critical for preventing cancer development.
Role of evolutionarily
conserved factors
in preventing aneuploidy
Our research aims to shed light on molecular processes controlling progression of mitosis and cytokinesis in order to prevent the formation of cells
with abnormal chromosome content (aneuploidy), a
hallmark of cancer cell. In order to avoid unbalanced chromosome segregation, the events leading to
mitotic cell division are tightly controlled in all eukaryotic cells by multiple mechanisms. Mutations impairing a number of genes involved in these controls
have been implicated in tumorigenesis. As aneuploidy is the most common characteristic of human
solid tumor cells and likely contributes to tumor
development. a deep knowledge of these controls
is crucial not only for our understanding of tumorigenesis, but also for the development of targeted
strategies for cancer diagnosis and therapy.
In this context, we are using the budding yeast
Saccharomyces cerevisiae, which is widely recognized as a very suitable model system for these
studies, to unravel the still poorly understood roles
and targets of evolutionarily conserved mitotic regulators.
In particular, we are characterizing the role of the
functionally redondant and evolutionarily conserved
ubiquitin ligases Dma1 and Dma2 in controlling the
mitotic cell cycle. Our recent results indicate that the
Dma proteins contribute to block entry into mitosis
in response to DNA replication stress by inhibiting
the degradation of the protein kinase Swe1 (Raspelli
et al., 2011, MBC, in press). Also Swe1 is evolutionarily conserved and plays an important role in cell
cycle control, as it can block entry into mitosis through inhibitory phosphorylation of the catalytic subunit
of cyclin-dependent kinase. Timely Swe1 down-re-
gulation is critical for proper cell cycle progression,
and we showed that the Dma proteins participate in
this regulation likely by controlling Swe1 ubiquitylation. We are now further investigating the molecular
details of this control.
We are also investigating the role of Dma1 and Dma2
in controlling cytokinesis, the final step of the cell
cycle that allows physical separation of the mother
from the daughter cell. The events leading to cytokinesis must be tightly controlled and coordinated
with nuclear division in order for a single eukaryotic
cell to generate two identical daughter cells at the
end of the mitotic cell cycle, thus preserving genetic stability in proliferating cell populations. These
events are basically conserved in all eukaryotes,
but several molecular details of their regulation
still need to be elucidated. Despite the apparent differences in cytokinesis modes among species, the
formation of a septin ring and the contraction of an
actomyosin ring are essential for cytokinesis in both
fungal and animal cells. In yeast, the septin ring
serves as a scaffold for recruiting other proteins at
the bud neck, among which the type II myosin heavy
chain Myo1 that forms a ring that is coincident with
a ring of F-actin. The resulting actomyosin ring contracts, thus accomplishing formation of the septum,
which is the physical barrier between mother and
daughter cell.
Our data indicate that Dma proteins inhibit cytokinesis in a step following septin ring deposition and
actomyosin ring formation, and we are now investigating the Dma target(s) in this regulation.11
4
Roberta Fraschini,
Giovanna Lucchini,
Erica Raspelli.
[ 19 ]
5
Lilia Alberghina,
Marco Vanoni,
Elena Sacco,
Stefano Busti,
Laura Gotti,
Mehedi Hasan,
Elisa Mazzoleni,
Stefano Lamperti.
1_A model of DNA replication
initiation (from Brummer et
al. 2010)
2_A Ras Grf1-derived
peptide inhibits growth of
transformed cells (from
Sacco et al. 2011)
[ 20 ]
Systems biology and cellular
proliferation in lower and higher
eukaryotes
Our research groups are developing a modular
systems biology approach to the study of cell
cycle (most notably of the G1/S transition) in the
model organism, Saccharomyces cerevisiae, as
well as in normal and transformed mammalian
cells. The approach involves both wet experiments as well as computer modelling and simulation. Experimental data are used to extract
information on network topology leading to mathematical models and to estimate parameter
values. In order to understand this complex phenomenon, it is mandatory not only to study the
core machinery driving the cell cycle, but also its
modulation by genetic and enviromental conditions, including nutrient and growth factor availability, as well as the interconnections with differentiation, signal transduction and cell death
pathways. Ultimately, these approaches should
lead to a more rational and more efficient drug
discovery process.
Nutritional modulation of cell cycle
progression in yeast
Combining genetic, physiological, biochemical
and post-genomic techniques we are studying
nutritional modulation of growth/cell cycle coordination with the aim to characterize the connection of pathways sensing nutrients (notably
glucose) with cell cycle execution.
Modelling of cell cycle and signal
transduction pathways
A mathematical model of the G1/S network
(Barberis et al, 2007) has been studied using the
circuit metaphor. This modelling approach has
been extended to the the G1/S network in mammalian fibroblasts (in collaboration with E. Klipp
(Berlin) and G. Milanesi (CNR, MI) (Alfieri et al,
2009). A cellular model of the entire yeast cell
cycle is under construction.
In mammalian cells Sos1 is the main activator
of Ras proteins that coordinate signalling pathways leading to cell proliferation, differentiation,
senescence, survival and motility. In collaboration with M. Farina and D. Liberati (Politecnico,
MI) we developed a mathematical model that
describes functional inter-domains rearrangements regulating the Sos1 activity and predicts
the effect of clinically relevant mutations on Sos
activity (Sacco, Farina et al., 2011 in the press).
Controlling the onset of DNA replication
in yeast and mammalian cells
Eukaryotic genomes are duplicated starting
from hundreds of thousands replication origins,
through tightly regulated multi-step processes.
Failure in regulation of such processes can originate aberrant DNA structures and incorrect
chromosomal duplication, that in mammalian
cells correlate with genome instability and tumorigenesis. A mathematical model of the
network governing the assembly of the yeast
replication machinery, generated in collaboration with T. Hofer (Heidelberg), highlighted a
major role for protein multisite phosphorylation
in DNA replication initiation. These studies are
being extended to mammalian cells.
Design, development and characterization of RasGRF1-derived Ras inhibitors
Mutations of Ras proteins and their regulators
are critical events in the pathogenesis of human
tumors and developmental syndromes. Starting
from a Ras activator, we developed cell-penetrating, Ras-inhibitory peptides (Sacco, Metalli et
al., 2011 in the press). Using computational and
molecular methods we are using these peptides
and sugar-derived Ras inhibitors (provided by F.
Peri, this Department; Sacco, Abraham, et al,
2011) as models for Ras-inhibitory drugs, and
as tools to improve molecular understanding of
the Ras activation cycle.
Real time analysis of protein-protein
Interaction
The BIAcore technology is being used to analyze
protein/protein and protein/ligand interactions
in real time. The technique is being applied mostly to interaction of proteins of potential pharmaceutical interest, including the Ras oncoprotein, prion-derived peptides, cell cycle inhibitors
and ataxin.
Molecular analysis of cancer
cells metabolic alterations
by transcriptomics and
metabolomics
Several decades ago Otto Warburg first described
that tumors exhibit glycolytic metabolism with a reduced rate of oxidative phosphorylation, despite the
availability of adequate oxygen. This phenomenon,
known as “Warburg effect,” has been proposed to be
a key driver of tumor progression (Warburg, 1956).
Since then, several researchers have observed altered glucose metabolism associated with a high
rate of lactate secretion in cancer cells and tumor
tissues. Furthermore, aerobic glycolysis represents
a robust hallmark of cancer that is employed for
tumor detection by positron emission tomography
analysis with the 2-[18F]fluoro-2-deoxy-D-glucose
tracer. However, we and others have recently provided evidences on an important anabolic role of
glutamine in tumor cell proliferation (DeBerardinis
et al, 2007; Gaglio et al, 2009). Indeed, by using different experimental approaches, it has been shown
that glycolytic cancer cells consume more glutamine as compared to their normal counterparts to
synthesize proteins, nucleotides and fatty acids and
to produce energy (DeBerardinis et al, 2007; Kovacevic
& McGivan, 1983).
Several reports suggest that nutrient uptake
changes and metabolic alterations are both under direct control of ras or myc oncogenes (Chiaradonna et al, 2006b; Vander Heiden et al, 2009).
In particular, oncogenic Ras proteins, identified
in 25% of human cancers (Bos, 1989), correlate
with metabolic alterations, including increased
rate of glucose and glutamine consumption, lactic acid accumulation, altered expression of mitochondrial genes, increased ROS production, reduced mitochondrial activity (Chiaradonna et al,
2006a; Vizan et al, 2005; Weinberg et al, 2010; Yun
et al, 2009). A result of this metabolic reprogramming is the dependence of K-Ras transformed
cells on glucose and glutamine availability, since
their withdrawal induces apoptosis and cell cycle
arrest, respectively (Chiaradonna et al, 2005; Ramanathan et al, 2005; Telang et al, 2006; Yun et
al, 2009). However, the precise metabolic effects
downstream of oncogenic Ras signaling as well
as the mechanisms controlling both survival and
apoptosis in cancer cells have not been completely elucidated.
Therefore, to better understand the regulation of
cancer cell metabolism and to identify key metabolic routes altered in K-Ras transformed cells, we are
currently applying a systems-level approach based
on the integration of molecular, metabolic and transcriptional analyses. In particular metabolic fluxes
have been analyzed by using 13C-labeled glucose
and glutamine as well as [ 15N]glutamine tracers.
Such approach has shown that K-ras oncogene expression enhances glucose uptake but decreases
its utilization in the tricarboxylic acid (TCA) cycle
and associated anabolic pathways. Furthermore,
we have shown that while K-Ras transformation decreases overall flux through TCA cycle, it increases
utilization of carbon backbone and nitrogen moiety
of glutamine either through TCA cycle or transamination activities, in order to sustain biosynthetic
reactions including amino acid, nucleotide and
glutathione synthesis. Further demonstration of
the main role of glutamine in cancer cells proliferation, has been obtained by inhibition of key
enzymes along glutamine pathway as well as by
analysis of transcriptional data.
Since cancer cells frequently encompass mitochondrial dysfunctions, currently we are analyzing also
the role of these dysfunctions in cancer cells metabolic reprogramming. In particular we are examining
the possible alterations of the cAMP/PKA pathway in
K-ras transformed murine NIH3T3 fibroblasts and
human breast cancer MDA-MB-231 cells and their
impact on cell growth and energy metabolism. Our
unpublished results indicate that exogenous stimulation of PKA pathway in cancer cells, stimulates
complex I activity and aerobic ATP production, induces mitochondrial fusion and depresses apoptosis,
upon glucose deprivation, resulting in promotion of
cell growth.
In order to identify the molecular mechanisms involved in cancer cells apoptosis, upon glucose deprivation, we are also evaluating the role of the Unfolded
Protein Response process (UPR). In fact, both transcriptomic and proteomic analyses, performed on
NIH3T3 and K-Ras-NIH3T3 cells grown in low glucose, has indicated that UPR maybe an important
determinant in the ability of the cells, upon nutrient
stress, to survive or to die.
6
Ferdinando Chiaradonna,
Daniela Gaglio,
Roberta Palorini,
Andrea Monestiroli,
Chiara Balestrieri,
Lara Sala Danna,
Marco Gaviraghi.
1_Forskolin treatment
increases mitochondrial
interconnections in
transformed cells
2_Metabolic pathways altered
in cancer cells. Schematic
representation of principal
alterations identified in
glycolyisis, glutamine
utilization and fatty acid
metabolism of cancer cells.
[ 21 ]
7
Paola Coccetti,
Farida Tripodi,
Claudia Cirulli,
Stefania Pessina,
Sara Busnelli.
Snf1 is involved in control of
MBF G1/S specific transcription
factor. The Swi6 subunit of
MBF transcription factor
recruits the α-catalytic
subunit of Snf1 to MBFdependent promoter. Mbp1
and Swi6 form the MBF
factor which binds the MBFdependent promoters found
upstream of genes involved
in the G1/S transition.
Budding yeast as a model
system for studying the relevant
mechanisms of cell cycle
progression
SNF1/AMPK PROMOTES S-PHASE ENTRANCE
BY CONTROLLING CLB5 TRANSCRIPTION IN
BUDDING YEAST
Stefania Pessina, Sara Busnelli, Lilia Alberghina, Paola Coccetti
The Saccharomyces cerevisiae Snf1 protein kinase has been reported to be required for adaptation to glucose limitation and for growth on
non-fermentable carbon sources.
We showed that Snf1 is also involved in yeast cell
cycle control. The lack of Snf1 alpha-catalytic
subunit downregulates the growth rate and
CLB5 expression, delaying Sld2 phosphorylation
and G1/S transition. Using either Snf1 or Swi6
as a bait, a specific interaction of Snf1 with Swi6,
the regulatory subunit of MBF, was detected.
We describes a previously unrecognized role for
Snf1 in transcriptional modulation of the G 1 to
S transition in budding yeast.
SYNTHESIS AND BIOLOGICAL EVALUATION OF
COMBRETASTATIN ANALOGS AS CELL CYCLE
INHIBITORS OF THE G1 TO S TRANSITION IN
SACCHAROMYCES CEREVISIAE
Farida Tripodi, Paola Coccetti
[ 22 ]
The Combretastatins were originally isolated
from the South African tree Combretum caffrum. Combretastatin A-4 (CA4) is a natural
cis-stilbene used in traditional medicine for
the treatment of hepatitis and malaria. CA4 is
a microtubule-destabilizing agent that inhibits
microtubule assembly by binding to tubulin, similar to the well-known microtubule-targeted
agent colchicine. CA4P, the water soluble prodrug, is now collectively classified as vascular
disrupting agents (VDAs), since it causes rapid
shut down of the established tumor vasculature. CA4P is being evaluated in Phase III clinical
trials for cancer treatment. Further investigation identifies CA4 as an activator of AMPactivated protein kinase (AMPK), which is a key
regulator of energy balance involved in response
to cellular stress in mammalian cells. In colla-
boration with Dr. Roberto Pagliarin (University
of Milano) a series of Z and E combretastatin
A-4 analogs were synthesized. These derivatives were analysed by monitoring their ability to
inhibit cell growth in Saccharomyces cerevisiae.
Some of these compounds were found to inhibit
yeast growth by inducing a specific G1 arrest by
affecting the synthesis of Clb5 protein, the principal S-phase cyclin. The G1 arrest is coincident
with the activation of the stress activated kinase
AMPK/Snf1.
CK2 ACTIVITY IS MODULATED BY GROWTH
RATE IN SACCHAROMYCES CEREVSIAE
Farida Tripodi, Claudia Cirulli, Lilia Alberghina, Paola Coccetti
Protein kinase CK2 is a highly conserved, essential protein kinase, which phosphorylates more
than 300 substrates, involved in transcription,
translation, signal transduction and cell cycle.
Genetic studies in yeast demonstrated that CK2
is essential for cell viability. CK2 is a constitutively active enzyme, independent of second messengers. CK2 activity is higher after hormone or
growth factor stimulation. Besides, abnormally
elevated CK2 activity is observed in various types
of cancer and cancer cell lines. In accordance with the emerging view of CK2 as a cancer
marker and a putative new therapeutic target,
a positive correlation between CK2 activity and
cellular proliferation rate has been suggested.
We provided the first evidence of an in vivo modulation of CK2 activity, dependent on growth
rate, in Saccharomyces cerevisiae. In fact, CK2
activity, assayed on nuclear extracts, is shown to
increase in exponential growing batch cultures
at faster growth rate. In collaboration with Dr.
Luca Brambilla (this Department) we also showed that in chemostat cultures nuclear CK2 activity is higher in faster growing cells providing
the first unequivocal demonstration that growth
rate itself can affect CK2 activity in a eukaryotic
organism.
Mechanisms of neuronal
apoptosis and neuroprotection
by ngf
8
Anna Maria Colangelo,
Miluscia Berbenni.
Modulation of neuronal and
glial markers by NGF in the
lumbar spinal cord following
peripheral nerve injury
Our research group is involved in studies regarding neurodegenerative diseases in order to gain
a better insight into mechanisms underlying neuronal degeneration and develop novel therapeutic
strategies for neuroprotection. To this purpose,
we are focusing on the role of neurotrophic factors, in particular Nerve Growth Factor (NGF). It
is well-known that members of the neurotrophin
family play a key role in development and function
of the brain. For instance, age-related decrease
of NGF levels plays a key role in triggering neuronal dysfunction and apoptosis (the main form of
neuronal death) in neurodegenerative disorders,
such as Alzheimer’s disease (AD) (Colangelo and
Alberghina, 2010). Moreover, alteration of the
ratio between the precursor form (proNGF) and
the mature active molecule (NGF) seems to be
relevant to neuronal death (Figure 1) in several
neurodegenerative conditions, including Amyotrophic Lateral Sclerosis (ALS).
Starting from our previous modular molecular
model of neuronal apoptosis in AD (Alberghina &
Colangelo, 2006), we are using two in vitro neuronal systems (NGF-differentiated PC12 cells and
primary cortical neurons) to dissect mechanisms of neuronal apoptosis following NGF deprivation and/or oxidative stress. We have recently
demonstrated that exposure of NGF-dependent
neuronal PC12 to NGF deprivation determines
mitochondrial dysfunction and activation of an
abortive cell cycle (Bianco et al., 2011). Interestingly, NGF was also able to prevent mitochondrial
dysfunction and neuronal death following oxidative stress to an extent similar to that obtained with
sodium selenite (Sel). Since Sel is a component of
selenoproteins, such as glutathione peroxidases,
which represent the main neuronal scavenger of
Reactive Oxygen Species (ROS), our data strongly support the hypothesis that effective neuroprotection under neurodegenerative conditions
might be achieved by administration of NGF and
antioxidant molecules.
Development of a NGF-based therapy is difficult
to obtain because of its poor pharmacokinetic
properties (instability and low permeability through the blood-brain barrier). To this purpose, in
collaboration with PRIMM srl and Blueprint Biotech, we developed a NGF-like molecule (BB14)
that behaves as agonist of TrkA, the specific NGF
receptor (Colangelo et al., 2008). In collaboration
with Prof M. Papa (Second University of Napoli)
we have demonstrated that neuroprotection by
NGF also involves modulation of reactive gliosis, a
mechanism underlying neuroinflammation in almost all neurodegenerative conditions. In animal
models of reactive gliosis induced by peripheral
nerve injury (CCI and SNI models), we succeeded
in demonstrating that intrathecal administration
of NGF or BB14 is able to i) reduce reactive gliosis and the sprouting of nociceptive fibers (Figure
2), ii) modify the structure of gangliar fibers and
NGF receptors expression (Cirillo et al., 2010),
iii) restore synaptic homeostasis and GSH levels
(Cirillo et al., 2011). Studies are in progress for
further characterization of this process both at
glial and neuronal levels to implement our initial
model of neuronal apoptosis and the role of NGF
in neuroprotection.
[ 23 ]
9
Signal transduction in
eukaryotic cells
Enzo Martegani,
Sonia Colombo,
Renata Tisi,
Michela Ceriani,
Fiorella Belotti,
Loredana Amigoni,
Silvia Groppi,
Serena Broggi,
Cinzia Giaccherini.
RAS SIGNALLING IN YEAST
Enzo Martegani, Renata Tisi, Sonia Colombo, Fiorella Belotti,
Silvia Groppi, Serena Broggi, Loredana Amigoni
1
In Saccharomyces cerevisiae cAMP/pKA pathway
plays a major role in metabolism, stress resistance and proliferation control. cAMP is produced by
adenylate cyclase, activated by Gpr1/Gpa2 system
and Ras proteins with Cdc25/Sdc25 guanine exchange factors and Ira GTPase activator proteins.
The subcellular localization of Ras complex proteins was investigated both by fluorescent tagging
and by biochemical cell membrane fractionation.
During exponential growth on glucose Cdc25 appears to localize mainly on ER membranes, while
Ira2 and Cyr1 are also significantly present on mitochondria. Besides Cdc25, only Ira1 is efficiently
imported in the nucleus, suggesting an additive
role for Ras signalling in the nucleus specifically
involving Ira1.
Although the cAMP/PKA pathway has been extensively studied, data on the spatiotemporal variation
of cAMP in single cells are still lacking. We used a
FRET (Fluorescence Resonance Energy Transfer)
sensor to monitor the changes in cAMP level in a
single yeast cell. To avoid any interference with the
cAMP/PKA signalling we used a sensor based on
the mammalian protein EPAC. The relative FRET
efficiency was determined from the CFP/YFP fluorescence ratio. Preliminary data indicate that the
sensor is able to detect changes in cAMP level in
yeast living cells.
CALCIUM SIGNALING IN YEAST
Renata Tisi, Fiorella Belotti, Silvia Groppi, Enzo Martegani
Collaboration with: Rogelio Brandão
[ 24 ]
In the yeast Saccharomyces cerevisiae, like in other
eukaryotes, calcium intracellular concentration is
maintained at submicromolar level. Different Ca2+
transport systems situated on yeast plasma membrane and on internal membranes take part to this
mechanism. Different stimuli induce a rapid increase in free intracellular Ca2+ concentration. On the
plasma membrane two calcium influx systems independently regulated are present, the high affini-
ty (HACS) and the low affinity (LACS) system.
By analysing the calcium response after 100 mM
glucose addition (in presence of 1 mM CaCl2) to
nutrient-starved cells, we found a second highaffinity (apparent KM 43.8±10.3 μM) Ca2+ influx system on the plasma membrane of Saccharomyces
cerevisiae, which, differently from HACS system, is
almost insensitive to nickel and to verapamil.
Calcineurin, a Ser/Thr phosphatase activated by
calcium level inside the cytoplasm, is involved
in the regulation of calcium homeostasis and in
many other cellular phenomena. We found that
calcineurin can also be activated by nutrients, and
is responsible for GIC transporter full activity in
rich media.
SIGNAL TRANSDUCTION MECHANISMS IN NGFMEDIATED DIFFERENTIATION
Michela Ceriani, Cinzia Giaccherini, Enzo Martegani
Collaboration with: Giovanna Berruti
We have studied the TrkA receptor internalization
and the role of the deubiquitinating enzyme UBPy
in this process in PC12 cells. We found that UBPy
overexpression alters the cellular residence of the
receptor. In unstimulated cells that overexpress
UBPy or its catalitically inactive mutant (C748A)
the receptor is almost completely sequesterred in
the cytoplasm; after a 15 minutes-stimulus with
NGF the receptor disappeared in cells that overexpressed UBPy while in cells that overexpressed
the catalitically inactive mutant the receptor persisted. These data suggest that UBPy could promote
TrkA degradation.
Since the retrograde trasport of signalling endosomes is essential for signaling and NGF-induced
differentiation, we investigated the role played by
GARP (Golgi-associated retrograde protein) complex, essentially constituted by Vps52, Vps53 and
Vps54 proteins. Preliminary data demonstrated
that Vps54 in HEK 293 cells localizes in endosomes and in the Golgi apparatus and that this protein doesn’t interact and co-localize with UBPy in
this cells. Now we are studying endogenous Vps54
in PC12 cells and in particular we are analyzing its
role in TrkA receptor internalization.
Yeast as a model system
for studying aging and stressrelated processes
SIR2: A REGULATOR OF METABOLISM AND AGING
IN SACCHAROMYCES CEREVISIAE
Marina Vai, Ivan Orlandi, Nadia Casatta
10
Marina Vai,
Ivan Orlandi,
Matteo Viganò,
Nadia Casatta.
size control. Ongoing analyses aim to better define
the alteration of regulatory circuits detected after
SFP1 inactivation with particular attention devoted
to MAPK cascade activation (in collaboration with L.
Alberghina, this Department).
Sirtuins are NAD+-dependent protein deacetylases
conserved from bacteria to humans. They regulate
the activity of a wide range of substrates, including
histones, transcriptional factors and mitochondrial
proteins, influencing many important biological processes, such as gene transcription, stress response and aging. Due to their absolute requirement of
NAD, sirtuins act as sensor of nutritional stimuli,
providing a direct linkage between cellular metabolic status and aging.
In yeast, two metabolites, ethanol and acetate,
have been indicated as pro-aging factors. Ethanol accumulates in the yeast culture media during
fermentative metabolism on glucose and then it is
transformed into acetate by oxidation. The exposure to these metabolites shortens the lifespan of yeast cells in stationary phase (chronological aging).
Recently, it was observed that SIR2 inactivation by
increasing ethanol catabolism extends life span of
yeast chronologically aged cultures. Aimed to elucidate this new relationship among Sir2, metabolism
and aging, we are studying the role of Sir2 in the
metabolic pathways involved in ethanol production/
utilization, such as glycolysis, gluconeogenesis and
respiration.
Opportunistic fungal infections are common and in
immunodepressed patients are frequently serious
and even life threatening. Clinically important fungal pathogens display varying degrees of tolerance
to the widely used antifungals principally linked to
their lack of fungicidal activity. The cell wall is an
essential structure in fungi with no mammalian
counterpart and consequently is an attractive target
for new antifungal drugs. We focused on a family of
glucanosyltranferases that are involved in cell wall
biogenesis and are required in many species for cell
wall remodelling during the infection of the host.
In particular, a functional characterization of three
members of this family in 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.
STRESS RESPONSE, RIBOSOME BIOGENESIS AND
CELL SIZE CONTROL
SEAWATER ENVIRONMENTAL CHANGES AND
BLEACHING OF CORAL REEFS.
Marina Vai, Matteo Viganò
Marina Vai, Ivan Orlandi
Every cell has developed mechanisms to respond to
changes in its environment and to adapt its growth
and metabolism to unfavorable conditions. Eukaryotic cells transduce different cellular stimuli by
multiple mitogen-activated protein kinase (MAPK)
cascades. Yeast cells use six MAPKs, which respond
to different conditions such as pheromone signals,
osmolarity, cell wall stress and nutritional status.
Their activation results in the generation of a set of
adaptive responses that leads to the modulation of
several aspects of cell physiology essential for cell
survival. Sfp1 is a key transcriptional regulator of
ribosome biogenesis in response to nutrients and
stress which also plays an important role in the cell
During the past two decades the frequency of coral
bleaching events has increased dramatically as a
consequence of climatic changes and human activity in tropical oceans worldwide. Elevated temperatures of sea surface, chemical contamination and
habitat destruction leave coral reefs with diminished
resistance to additional perturbation and result in
reduced ecological integrity. Based on our experience in yeast, we are investigating the effects of different stresses on reef coral species inside the lagoon
of Maghoodhoo Island (Maldives) (in collaboration
with P.Galli, this Department). Among the different
mechanisms of cytoprotection we are focusing on
the role of heat shock proteins.
THE FUNGAL CELL WALL AS A TARGET FOR ANTIFUNGAL DRUGS
Marina Vai, Ivan Orlandi
[ 25 ]
11
Protein mass spectrometry
Rita Grandori,
Maria Šamalikova,
Carlo Santambrogio,
Lorenzo Testa.
Cartoon representation of
ß2m tertiary structure.
Residues relevant to this
study are shown in sticks.
Oligomerization of ß2m seen
by nano-ESI-MS:
(•) monomer; ( ) dimer, (Δ)
trimer; ( ) tetramer.
Electrospray-ionization mass spectrometry (ESIMS) has recently developed into a central tool
of structural biology, allowing investigation of
structure and dynamics of proteins and protein
complexes. The ESI process produces multiply
charged, gas-phase protein ions preserving the
non-covalent interactions responsible for protein
conformation and protein-protein association.
Protein complexes are identified by their mass,
while different protein conformations coexisting in
the original liquid sample can be distinguished by
their final charge in the gas phase. Thus, combined information about folding and binding can be
retrieved. Furthermore, the different components
of heterogeneous mixtures can be analyzed individually. These features make this technique particularly useful for the investigation of complex molecular systems. ESI-MS is used in our laboratory
to study two major classes of proteins: intrinsically
disordered proteins (IDPs) and amyloid proteins.
IDPs lack ordered three-dimensional structure in
their free state and undergo folding upon binding
to specific interactors. They play key regulatory
roles in biological systems but are difficult to study because of their inherent dynamics. We have
performed conformational analysis of IDPs by
ESI-MS. It has been shown that even IDPs known
to be disordered in their whole length, like yeast
Sic1 and human -synuclein, display transient
tertiary structure that can be denatured by acids
and organic solvents. These studies allow describing the effects of environmental conditions on
protein conformation and provide useful input for
molecular-dynamics simulations for the development of structural models of these highly dynamic
systems.
[ 26 ]
Amyloid proteins are involved in several human
diseases. The pathological state arises by protein
misfolding and aggregation into insoluble amyloid
fibrils. Particular interest is focused on the first
steps of the aggregation process, in order to identify the amyloidogenic conformers and the early
oligomeric species that trigger fibril formation.
Soluble oligomers are also thought to be the toxic
species and represent the most interesting target
for novel pharmaceutical strategies. We study the
protein ß2-microglobulin (Figure 1), in collaboration with Prof. Martino Bolognesi (University of Milan, Italy). This protein is the causing agent of the
dialysis related amyloidosis that affects patients
with kidney failure treated by long-term dialysis.
Conformation, stability and aggregation of the protein have been investigated by different biophysical methods. ESI-MS analysis reveals an intrinsic
propensity of the protein in a native-like state to
form oligomers without obligate stoichiometry
(Figure 2), in a concentration-dependent manner.
Comparison of wild-type and mutant protein suggests that this behavior might be related to protein
amyloidogenicity.
A related computational project has been carried out in collaboration with Prof. Simone Raugei
(Sissa, Triest, Italy), in order to develop atomistic
structural models of gas-phase ions produced by
ESI of peptides or small proteins. A novel protocol
for energetic analysis of all the possible protomers
of a given peptide has been implemented. The results indicate that complex networks of intramolecular interactions persist upon desolvation. It is
also found that zwitterionic species can be favored,
particularly at low charge states, because the stabilization due to intramolecular hydrogen bonding
and salt-bridges can compensate for the thermodynamic penalty deriving from charge separation
in the gas phase. This study poses the basis for
atomistic modeling and energetic analysis of larger desolvated systems like proteins.
Protein engineering
and industrial enzymology
12
Marina Lotti,
Stefania Brocca,
Giusy Manuela Adamo,
Anthony Doizy,
Federica Parravicini.
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. A major goal of our research is understanding the molecular bases of stability, function and
interactions and to modulate these properties.
Notwithstanding its largely disordered scaffold,
Sic1 turned out to be organized in “disordered
domains”, whose compactness seem to obey to
statistical and physical rules. The functional meaning of such an organization might be clarified
by studying Sic1 in complex with its physiological
partners. Always in the field of IDPs we are studying the properties of disordered viral proteins
in collaboration with a group of Marseille.
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.
Research on cold-adapted enzymes is focused on
a lipase and on an acylaminoacyl peptidase both
obtained from psychrophylic bacteria. The first
enzyme was targeted by directed evolution to generate a lipase active in the cold but still robust
towards temperature. Laboratory evolution induced also a broadening of the range of substrates
accepted by this enzyme, a result that introduced
issues related to enzyme promiscuity in our research. The acylaminoacyl peptidase is of interest
not only because of cold adaptation but also for
its ability to catalyze two different reactions being
active as a protease on small peptides and as a
lipase on fatty acid esters. Our studies on the natural enzyme and on its variants aim at defining
key elements involved in structural stability and
substrate specificity.
Proteins characterized by the lack of a defined 3D
structure in the absence of partner proteins (Instrinsically Disordered Proteins or IDPs) are a relatively new and challenging field for biochemical
and biophysical studies. Our work-horse in this
topic is Sic1, a protein involved in yeast cell cycle.
In collaboration with R .Grandori, S.M. Doglia, L. DeGioia
and L. Alberghina from this Department and S. Longhi at
the CNRS of Marseille, France
MOLECULAR BASES OF YEASTS ADAPTATION TO
HEAVY METALS
Some metal ions, like copper ions, are essential
for life since they are the cofactors of key cell enzymes. However, when present in excess, they may
elicit cytotoxic effects that are related also to severe human pathologies. Cells of Saccharomyces
cerevisiae and other related yeast species, exposed to increasing concentration of copper are our
model to study the physiology and the molecular
events occurring during the exposition to metals.
This choice is supported by the high degree of
conservation of cellular and molecular processes
between higher eukaryotes and Saccharomyces
cerevisiae, that is considered a valuable system
to study basic mechanisms behind devastating
illnesses such as cancer and neurodegenerative
disorders. This research is aimed at unravelling
the complex process of adaptation to heavy metala and to characterize the cellular and molecular
effects thereof.
IMPROVEMENT OF INDUSTRIAL CATALYSTS
We have recently started a new research funded by
the Regione Lombardia and focussed on the improvement of the production of biodiesel with biotechnological tools.
[ 27 ]
13
Molecular and cellular
biophysics
Silvia Maria Doglia,
Antonino Natalello,
Diletta Ami,
Anna Maria Villa.
PROTEIN SECONDARY STRUCTURE, STABILITY
AND AGGREGATION
S.M. Doglia, A. Natalello, D.Ami
[ 28 ]
Structural properties and aggregation of different
proteins and peptides relevant for biotechnology
and biomedicine have been studied by complementary biophysical and biochemical approaches.
In vitro studies of amyloid proteins and peptides
We investigated by Fourier Transform Infrared
Spectroscopy (FTIR) the role of glutamine side
chains in the formation of SDS insoluble fibrils
of the polyQ protein ataxin 3 in collaboration with
the group of P. Tortora (BTBS) (Natalello et al.
PlosOne 2011).
In collaboration with V. Bellotti of the University
of Pavia (I) and his coworkers, we characterized
the aggregate structural properties for several
mutants of a fragment of the apolipoprotein A-I
(Raimondi et al. JMB 2011).
In collaboration with the group of F. Nicotra (BTBS)
we investigated by FTIR spectroscopy the interaction of A-beta peptides with tetracycline (Airoldi et
al.Org. Biomol.Chem. 2011).
The formation of partially folded intermediates of
-synuclein induced by alcohols and copper have
been also studied in collaboration with R. Grandori (BTBS) and G. Legname of SISSA, Trieste-I
(Natalello et al . Proteins 2011).
In collaboration with the group of A. Vescovi (BTBS)
and F.Gelain we investigated the assembling of
functionalized peptides into stable nanostructures as scaffold for biomedical applications (Gelain
et al. ACS nano 2011; Taraballi et al. Frontiers in
Neuroeng. 2010).
In collaboration with J-L. Reymond of the University of Bern (CH), we studied by otpical spectroscopies the interaction of glycopeptide dendrimers
with vitamin B12 (Uhlich et al. Chembiochem. 2010)
We also collaborated with the group of D. Prosperi (BTBS) to investigate by FTIR spectroscopy the
structural properties of antibodies after conjugation
to nanoparticles (Occhipinti et al . Nanoscale 2011)
In situ studies of a beta peptides in transgenic
C. elegans
We studied the expression of the A beta peptides in
transgenic C. elegans nematode and the effect of tetracycline in counteracting their aggregation in collaboration with the group of Mario Salmona (Istituto di
Ricerche Farmacologiche “Mario Negri”, Milano ).
In particular, single intact nematode specimens
were investigated by FTIR microspectroscopy, collecting the absorption spectrum from the nematode
pharynx where the highest expression of peptides
was found (Diomede et al. Neurobiol. Dis. 2010).
Inclusion bodies of recombinant proteins in bacteria
In collaboration with the group of M. Lotti (BTBS)
we continued to study the aggregation of recombinant proteins in form of inclusion bodies in
bacterial cells, with particular interest for their
structural properties (Gatti-Lafranconi et al.
FEBS J. 2011)
OPTICAL MICROSCOPIES OF INTACT CELLS
S.M.Doglia, D.Ami , A.M.Villa
Maturation of murine oocytes by FTIR microspectroscopy
In collaboration with C. A. Redi (Università di Pavia-I) we studied by infrared microspectroscopy
the developmental stages of two types of murine
oocytes, characterized by a different chromatin
organization and competence in the embryonic
development. A different infrared response was
found for the two oocyte types, related to their lipid content, DNA methylation and polyadenylation
(Ami et al. BBA-Mol.Cell.Res. 2011)
EB as a marker of replicating mtDNA in living cells
In collaboration with the group of P. Fusi (BTBS) we
investigated by laser scanning confocal microscopy
the fluorescence response of ethidium bromide (EB)
in mitochondria and the replicative status of mtDNA
in human neuroblastoma SHSY-5Y cells.
Using BrdU immunofluorescence and ligation mediated real time PCR, we demonstrated a strong
correlation between the intensity of EB fluorescence
in mitochondria nucleoids and the replication status of mtDNA.
Proteins: structure,
functions, pathogenicity
and conjugation to
nanoparticles
STRUCTURAL STUDIES ON PROTEINS CONTAINING GLUTAMINE REPEATS RESPONSIBLE FOR
NEURODEGENERATIVE DISORDERS
Maria Elena Regonesi, Paolo Tortora, Gaetano Invernizzi,
Alessio Lombardi, Francesco Aprile, Marcella Bonanomi,
Anna Maria Frana.
Some neurodegenerative disorders result from the
expansion of glutamine repeats (poly-Q diseases)
in a set of proteins. Their misfolding and aggregation are likely to be involved in these disorders.
The aim of this investigation is to gain insight into
the molecular mechanism(s) by which expanded
poly-Q stretches in ataxin-3 lead to the MachadoJoseph neurodegenerative disease. We are focusing on two major issues related to the molecular
mechanism of the pathogenesis, i.e., the understanding of the protein’s physiological role, and the
mechanisms by which ataxin-3 generates amyloid
fibrils. These studies are performed on both purified molecules and cellular systems. As regards
the investigations on the protein’s physiological
role, our findings point to an involvement of ataxin-3 in sorting aggregated protein to aggresomes
via microtubules. Furthermore, our studies on the
mechanisms of amyloidogenesis, led to the structural characterization of normal and expanded
variants by taking advantage of different analytical
methods, notably FT-IR, circular dichroism and
ThT fluorimetry, which highlighted major differences between the two. Our findings pave the way to
a deeper understanding of the protein aggregation
process and to development of new antiamyloidogenic compounds.
14
Paolo Tortora,
Davide Prosperi,
Maria Elena Regonesi,
Gaetano Invernizzi,
Serena Mazzucchelli,
Alessio Lombardi,
Agnese Salvadé,
Francesco Aprile,
Miriam Colombo,
Marcella Bonanomi,
Anna Maria Frana,
Paolo Verderio.
DEVELOPMENT OF HYBRID NANOPARTICLES
FOR BIOMEDICAL APPLICATIONS
Davide Prosperi, Serena Mazzucchelli, Agnese Salvadé, Miriam
Colombo, Paolo Verderio.
Nanomaterials within 1-100 nm hold tremendous
potential in biomedical research thanks to a unique
interaction with biological molecular systems. The
production of high quality hybrid (bio)organic/inorganic nanoparticles endowed with inherent optical
and magnetic properties represents a promising
new road to the development of a novel generation
of diagnostic and therapeutic agents for biosensing, preclinical investigations and clinical use. In
particular, magnetic nanoparticles (MNP) appear
as a very promising contrast agent for magnetic
resonance imaging (MRI) clinical diagnostics. Indeed, MNP functionalized with cancer-specific
targeting ligands can be used for early detection
of tumors and of peripheral metastases. Thus, the
aim of this project is to develop a small library of
hybrid MNP consisting of a magnetic core and a
protein shell responsible for cell receptor targeting. Fusion proteins are produced in order to obtain an optimal control on the number and orientation of proteins conjugated to the nanoparticle
surface. Furthermore, we are developing hybrid
MNP for biosensing, protein purification and enzyme recycling. The preparation of nickel(II) nitriloacetic acid (NTA)-modified Fe3O4 MNP enables
a one-step protein purification through binding to
His-tagged proteins. By combining spectroscopy
investigations and bioactivity assays we aim at determining the effects of bioconjugation on protein
structure and biofunctionality.
[ 29 ]
15
Characterization of proteins
of biomedical relevance
Paola Fusi,
Matilde Forcella,
Valentina Pastori,
Alessandra Bigi,
Alessandra Mozzi,
Elena Sangalli.
STUDIES ON ATAXIN-3 PHYSIOLOGICAL ROLE
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, 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. Moreover, in collaboration with Dr Coccetti
(University of Milan-Bicocca) and Prof. Tedeschi
(University of Milan), we showed that Ataxin-3
phosphorylation by casein kinase 2 (CK2) and glycogen synthase kinase 3 (GSK3) is essential for
nuclear localization and pathogenesis. A study
of protein nitration during Sca3 pathogenesis is
currently under way.
[ 30 ]
CHARACTERIZATION OF HUMAN SIALIDASES
Sialidases are widely distributed glycohydrolytic
enzymes removing sialic acid residues from glycoconjugates. A characterization of membrane
bound human sialidase NEU4 carried out in our
laboratory had shown that this sialidase is an extrinsic membrane protein, anchored to the membrane though interactions with other protein(s).
Primary structure analysis of this protein showed
the presence of a proline-rich region which is
unique to NEU4. We hypothesized a role for this
region in interactions with signaling pathways
components. Since Akt and Erk1 kinase motifs
are found in NEU4 proline-rich region, activation
of both PI3K/Akt and MAPK signaling pathways
were studied in stably transfected SK-N-BE clones, overexpressing NEU4. The results showed
that NEU4 is located downstream in these signaling pathways and that it interacts with Akt kinase, through the proline-rich region. Finally, our
data showed that NEU4 is required for retinoic
acid induced neuronal differentiation.
INVESTIGATION OF THE ROLE OF NEU3 IN
COLORECTAL CARCINOGENESIS
Colorectal cancer (CRC) is the second leading cause
of cancer-related death in the Western countries.
Newer therapeutic options for treating advanced
CRC include targeted biologic therapies, such as
the epidermal growth factor receptor (EGFR) antagonists. However, it is now emerging that genetic
alterations of EGFR and its downstream signaling
effectors may predict the efficacy of EGFR-targeted drugs. Recent data pointed out that inhibition
of EGFR-tyrosine phosphorylation is highest in the
presence of deregulation of the plasma membrane
bound sialidase NEU3, raising the possibility that
NEU3 may largely promote EGFR phosphorylation.
In our laboratory we are currently investigating the
role of NEU3 in colorectal carcinogenesis, in collaboration with Dr. Milo Frattini (Istituto Cantonale di
Patologia in Locarno). Preliminary data showed a
correlation between EGFR and NEU3 deregulation.
Investigation of the alterations occurring in EGFR
downstream pathways is currently underway.
CLONING AND EXPRESSION OF A TREHALASE
FROM Chyronomus riparius TO BE EXPLOITED AS A TARGET FOR BIOINSECTICIDES
Trehalase inhibitors have a great potential as human
safe bioinsecticides, this enzyme playing a key role
in insect metabolism. A trehalase has been purified
in our laboratory from the Diptera Chironomus riparius, showing a different specificity towards many
insecticides, compared to mammalian enzymes.
Molecular cloning of its cDNA has been achieved, as
well as expression of the recombinant protein in E.
coli. The recombinant protein has been purified to
homogeneity and is now being tested as a target for
new synthetic bioinsecticides, in collaboration with
Prof. Parenti and Prof. Cipolla (Universiy of Milan-Bicocca), as well as Dr. Francesca Cardona (University
of Florence). Some imminosugars have been shown
to selectively inhibit C. riparius trehalase.
New therapeutic approaches
for chronic pain
16
Gabriella Giagnoni,
Barbara Simona Costa,
Francesca Comelli.
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. As a defense mechanism, nociceptive
pain is essential for survival. By contrast, chronic
pain has not defensive or helpful function. Acute
pain is produced by the physiological functioning
of the normal nervous system, whereas chronic
pain is a reflection of pathologically altered nervous system. Among the most debilitating types
of chronic pain is peripheral neuropathic pain.
Neuropathic pain typically develops when peripheral nerves are damaged. These injuries can
be caused by tumors compressing peripheral
nerves, chemotherapy, metabolic (diabetes) or
viral diseases, severe ischemic insults, trauma
and disc herniation that stretches, compresses
or inflames a nerve root. It is characterized by the
presence of an exaggerated response to painful
stimuli (hyperalgesia), pain response to normal
innocuous stimuli (allodynia) and spontaneous
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, affecting millions of people
worldwide, can be classified as an incurable disease. Our group is involved in the research of new
pharmacological targets and we believe that one
possibility to successfully treat chronic pain is to
develop drugs that are not aimed to suppress the
neuronal activity but that target important modulators of chronic pain instead of neurons. In this
context during this year we first characterized
the role of microglial cells, and in particular of
Toll like receptors expressed by microglial cells
within the spinal cord, in the genesis and maintenance of neuropathic pain. After assessing this
involvement in an animal model that mimics neuropathic pain development after a nerve injury,
we focused the attention to the role of the same
microglial cells in other pathological conditions
leading to neuropathic pain, such as diabetes,
HIV-infection and antiretroviral treatment. The
aim is clearly to find a pharmacological target
suitable for neuropathic pain independently from
the leading cause. Particularly, HIV-associated
sensory neuropathy is the most common neurological complication of HIV infection, symptomatically affecting up to 50% of patients with HIV.
Unfortunately, this problem is particularly severe
just in the era of the highly active antiretroviral
therapy (HAART). There are in fact two clinically
similar settings in which painful HIV neuropathy
occurs: first, a disease–related neuropathic pain
associated with HIV-infection per se; secondly,
a drug-induced neuropathy associated with the
use of nucleoside reverse transcriptase inhibitors
(NRTI), such as zalcitabine (ddC) or didanosine,
normally part of HAART. The use of HAART has
markedly increased patient survival making neuropathic pain an important source of morbidity.
Particularly, during this year, we set up the animal model of HIV-associated neuropathy through
the exposition of mice sciatic nerve to 200 ng of
gp120. For systemic anti-retroviral treatment, the
nucleoside analogue zalcitibine (ddC) has been
used (50 mg/kg in saline, three times a week for
3 weeks). The same strain of mice has been also
used to induce sciatic nerve chronic constriction
neuropathy for comparison. We set up the animal model in terms of development of painful
symptoms: hyperalgesia and allodynia and we
are now evaluated whether the pain hypersensitivity is related to microglia activation and Toll like
receptor stimulation.
Microglia activation
in the spinal cord
[ 31 ]
17
Angelo Luigi Vescovi,
Lidia De Filippis,
Chiara Paiardi,
Fabrizio Gelain,
Maria Cristina Zalfa;
collaborators from
A.O. Niguarda,
collaborators from
Stemgen S.p.A.
A
B
C
1_ Effect of RADA16-I functionalizations. RADA16-I functionalized
with different bio-active motifs,
respectively with: (A) BMHP1 (B)
ALK (C) SDE. Each peptide shows
a different stability in cross-ß
structure after 1 ns of MD simulation in implicit solvent.
2_Functionalized self assembling
peptides and Neural stem cells.
(A) 0G-BMHP1, (B) 2G-BMHP1,
(C) 4G-BMHP1. (D) Positive and
(E) negative controls (F) show
significant differences for all
possible coupled experimental
groups except for (*) 0G-BMHP1
vs negative control, and (**) 2GBMHP1 vs 4G-BMHP1. Values are
reported as means ± standard
error of the mean.
[ 32 ]
Regulation of neural stem cells in
physiology and experimental therapy
CHARACTERIZATION OF GMP-GRADE HUMAN
NEURAL STEM CELL LINES IN VITRO AND IN VIVO
IN SOD1 G93A RAT MODEL OF AMYOTROPHIC LATERAL SCLEROSIS.
A.L. Vescovi, Lidia De Filippis , Laura Rota Nodari , Daniela Ferrari,
Cristina Zalfa, Maurizio Gelati.
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by
progressive degeneration of motor neurons which
leads to definitive muscular paralysis and finally to
respiratory failure. Many authors have demonstrated that is possible to succeed in delaying the onset
of the disease in different animal models through
the transplantation of neural stem cells into lumbar tract of the spinal cord. Moreover, cell therapy
is reaching the stage of clinical application, with the
first few clinical trials already underway in some
post traumatic, post-ischemic or neurodegenerative disorders. A critical element is represented by
the cells to be used in neural transplantation and,
in this view, standardized, clinical grade source of
normal human CNS cells (hNSCs), combining the
plasticity of foetal tissue with extensive proliferative
capacity and functional stability would be of paramount importance in this field. Here, we describe
the establishment of continuous and stable hNSC lines from the foetal CNS and their ability to integrate
in the spinal cord of ALS animal model after multisite injection into the ventral horns. In particular, we
present the analysis of some of the NSC-mediated
therapeutic effects, such as trophic support to host
cells and immunomodulation of the inflammatory
environment. Parallel transplantation into the brain
of nude mice has shown that hNSC lines are not tumorigenic under our culture conditions. hNSC lines
were grown according rules of Good Manufacturing
Practices (GMP) in medium containing EGF and bFGF according to the neurosphere assay paradigm
and they have been recently approved by the Italian
Agency of Pharmacology (AIFA) for clinical trial phase I on ALS patients.
IDENTIFICATION OF NOVEL EFFECTORS REGULATING THE INVASIVENESS OF HUMAN GLIOBLASTOMA MULTIFORME BY EXPLOITATION OF A CANCER
STEM CELL-BASED in vitro/in vivo MODEL
Angelo L. Vescovi, Elena Binda,Alberto Visioli, Fabrizio Giani
The field of brain cancer stem cells, which we have
seen to be born around 2002, has been the most active areas in neuro-oncology and cancer research in
general. As every newborn scientific areas in their
infancy, there have been dramatic changes regarding central tenets and basic paradigms that have
made it one of the most productive and exciting, yet
controversial fields in tumor biology. The overall
objective of our proposed investigation can be summarized as the study of cancer stem cells, perhaps
now better defined as tumor-initiating stem-like cells from human glioblastomas (hGTICs), aimed at i)
characterizing them thoroughly and, thus, providing
a better understanding of these cells’ basic physiology; ii) identifying specific regulatory mechanisms
which, in turn, would lead to iii) the identification of
candidate novel markers and, more important, to
novel therapeutics for these incurable cancers.
NERVOUS REGENERATION VIA NANO-STRUCTURED SCAFFOLDS
Fabrizio Gelain, Francesca Taraballi, Diego Silva, Omar Villa, Daniela Cigognini, Andrea Caprini, Gloria Anna Ada Saracino, Angelo
L. Vescovi
The nervous system is vulnerable to various disorders and for its intrinsic features, even limited damages may strongly affect important neurological and
physiological functions. Our research is focused on
the development of regenerative therapies specific
for nervous injuries by using the potentials offered
by electrospun bio-protheses and nanostructured
scaffolds. Electrospun tubes are polymeric scaffolds
with high porosity and surface/volume relation. Selfassembling peptides are made from natural amino
acids, they undergo self-assembly into nanofibers
forming a scaffold, they can be mixed with growth
factors and cells before the assembling process takes place upon exposure to physiological conditions
of pH and temperature. Over the past year, we developed novel functionalized biomaterials in order to
promote survival and differentiation of neural stem
cells (NSC) in vitro and to regenerate nervous tissue in rodents suffering from spinal cord injury. We
studied the peptides sequence in relation with their
structure and biological functionality (Taraballi et
al, 2009; Taraballi et al, 2010). Thus we used these
self-assembling peptides in combination with electrospun tubes and we showed these prostheses are
permissive micro-environments for regenerating
nervous tissue in rat models of chronic and acute
spinal cord injuries. Our approach is going to be further ameliorated via complementary strategies like
scaffold loading with neurotrophic factors for drug
delivery or seeding with neural stem cells for cell
therapies. The results achieved by our group during
the past year demonstrate that a strategy making
joint use of stem cell technology, tissue engineering
and nanotechnology could foster innovative solutions for regenerative therapies of nervous injuries.
Regulation of innate
immune responses and their
influence on adaptive immunity
The research group has a long record of achievements and expertise in the field of DC biology and
a solid expertise in in vivo systems and in mouse
models of human diseases. The group has pioneered systems biology approaches to study complex dynamic processes, such as host-pathogen
interactions, the process of DC maturation and
the role of DC in activating and controlling NK
cell functions. Moreover, the group has been interested in the study of peripheral mechanisms
of maintenance of T cell tolerance and has produced different transgenic and knock out mouse
models to approach this problem.
SIGNAL TRANSDUCTON PATHWAYS INDUCED
BY LIPOPOLYSACCHARIDE IN DENDRITIC CELLS AND NEUTROPHILS
Microorganism invasions are perceived by special
receptors of the innate immune system including
Toll-like receptors (TLR) expressed on various
immune cells such as macrophages, dendritic
cells (DC), lymphocytes (B and Natural Killer (NK)
cells), and neutrophils. By recruiting different
combinations of adapter proteins, individual TLR
turn on signal transduction pathways that activate different transcription factors, such as nuclear
factor (NF)-kB, activation protein (AP)-1, and interferon regulatory factors (IRF). In a recent study
the group has found that an additional pathway
that leads to the activation of a fourth class of
transcription factors, specifically NFAT proteins,
is activated in dendritic cells and neutrophils following LPS encounter. The group has determined
that the role of this transcription factor is to regulate DC and neutrophil life cycle following activation. Additional roles are under investigation.
DENDRITIC CELLS AND NATURAL KILLER CELLS
Natural Killer (NK) cells exert a direct anti-tumor
and anti-microbial effect and can influence the
development of adaptive T cell responses. Activation of NK cells is regulated by accessory cells
such as dendritic cells (DC). Following activation,
NK cells accumulate at the lymph nodes draining
the site of infection, the key place in which DC
and NK cell interactions occur. Taking advantage
of the two-photon intravital microscopy technology the capacity of activated NK cells to reach
the draining lymph nodes is investigated together
with the DC-derived signals necessary for NK cell
priming in inflammatory conditions induced by lipopolysaccharides.
DENDRITIC CELLS AND REGULATION OF IMMUNE TOLERANCE
The immune system of vertebrate animals has
the capacity to respond to perturbations (invading pathogens, stress signals) limiting selftissue 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.
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. Using genome-wide
approaches we aim to develop genetic signatures
of inflammation and autoimmune diseases.
18
Francesca Granucci,
Maria Foti, Ivan Zanoni,
Caterina Bodio,
Tatiana Gorletta,
Achille Broggi,
Marco Di Gioia,
Roberta Marzi,
Aparna Venkatesh,
Matteo Urbano,
Anna Torri, Silvia
Fumagalli,
Donatella Biancolini,
Angela Papagna,
Dimos Kapetis,
Ferdinando Clarelli.
In collaboration with Dr Davide
Prosperi (UNIMIB) and Dr Raffaele
Allevi (UNIMI)
[ 33 ]
19
Neurophysiology
Enzo Wanke,
Marzia Lecchi,
Francesca Gullo,
Elisa Redaelli,
Andrea Maffezzoli,
Ilenio Servettini,
Elena Dossi.
[ 34 ]
SPATIOTEMPORAL EVOLUTION OF NEURONAL
NETWORKS INVESTIGATED WITH MULTIELECTRODE ARRAYS (MEA)
With the acquisition of a novel multielectrode
array (MEA) electrophysiological system, we aim
at studying neuronal networks (~9 mm2, ~10.000
neurons, ) by recordings from 252 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) Short latency cross-and autocorrelation functions to identify neurons, 2) the
role of protein Aß 1-42 on the activity of cortical
networks.
PHYSIOLOGY AND PATHOLOGY OF THE VISUAL
SYSTEM
In the retina, ganglion cells convey the images
processed from photoreceptors, horizontal and
amacrine cells to the brain. Many different types
of ganglion cells have been identified in vertebrates and humans but their characteristics and
physiological functions are still to be defined. Moreover, visual dysfunctions are a consequence of
several pathologies and the first symptoms that
appear in patients affected by neurodegenerative
diseases.
By using flat-mounted retina of adult rodents as
a model, we investigate ganglion cell electrophysiological properties in normal and pathological
conditions. A project on diabetic retinopathy is
performed with the collaboration of Prof. G. Cavaletti, Università Milano-Bicocca, Monza, and Dr.
Marina Figliuzzi, Istituto Mario Negri, Bergamo.
Nicotinic acetylcholine
receptors and voltage-gated k+
channels in physiology and
pathology
NICOTINIC ACETYLCHOLINE RECEPTORS IN
THE CEREBRAL CORTEX. PHYSIOLOGY AND IMPLICATIONS FOR THE PATHOGENESIS OF SLEEP-RELATED EPILEPSY
The cholinergic fibers ascending from the basal
forebrain and mesopontine nuclei regulate cortical arousal and the sleep-waking cycle. ACh
release is also involved in the control of synaptic
plasticity and, consequently, of memory and learning. Moreover, the mendelian forms of nocturnal frontal lobe epilepsy are often caused by
mutations on subunits of the neuronal nicotinic
receptor. We combine electrophysiological, neuroanatomical and biomolecular approaches to
determine the cholinergic and peptidergic modulation of transmitter release in the neocortex and
thalamus of normal mice and murine models of
sleep-related epilepsy.
NICOTINIC RECEPTORS AND CANCER
Nicotinic receptors are also expressed in nonneuronal tissues, including tumors. In these,
they regulate cell proliferation, apoptosis and angiogenesis, which is suggestive considering that
smoking is an established risk factor for cancer,
particularly in the lung. The mechanisms of these
effects are still debated. We found that lung cancer cells express distinct nicotinic receptor subtypes that may regulate processes occurring at
different time scales, such as transmitter/growth
factor release and cell migration. In addition, we
20
Andrea Becchetti,
Patrizia Aracri,
Paola Ambrosi.
observed that some tobacco-derived carcinogenic
nitrosamines are partial agonists with high affinity for human neuronal nicotinic receptors. Such
information is crucial to understand the pathologic effects. It suggests that these compounds can
act as stimulators or inhibitors depending on the
concentration of the full agonist (acetylcholine or
nicotine).
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). Growing
evidence indicates that considerable cross-talk
occurs between integrins and ion channels, mediated by direct (i.e. formation of macromolecular
complexes) or indirect interaction (e.g. through G
proteins). In addition, ion channel stimulation frequently controls integrin activation or expression.
We study the channel-integrin interplay in different cell types (from cortical neurons to leukemia
cells). Alteration of these mechanisms has clear
implications for pathogenetic processes such as
tumour invasiveness and neurodevelopmental alterations
[ 35 ]
21
Research in cardiac cell
physiology
Antonio Zaza,
Marcella Rocchetti,
Lucio Barile,
Claudia Altomare,
Riccardo Rizzetto,
Stefano Marangoni,
Matteo Alemanni.
The research of the cardiac cell physiology group
is centered on the ontogenesis and modulation of
myocardial excitation-contraction coupling. The
research activity in 2010 was articulated in the following projects.
New Projects
Functional characterization of a Brugada syndrome mutation (NaV1.5 S216L) and
its modulation by a common polymorphism
(H558R).
The Brugada syndrome (BrS) is an arrhythmogenic
syndrome that has been associated to mutations of
several cardiac ion channels, including Na+ ones.
Within the collaboration established by the NEDD
network, the Genetic unit of San Raffaele hospital
identified a mutation (S216L) and a polymorphism
(H558R) in a patient with a BrS ECG phenotype. We
expressed the mutation, alone and in association
with the polymorphism, in HEK cells and evaluated
its electrophysiological phenotype by standard and
dynamic V-clamp studies. The mutation caused a
decrease in channel expression, resulting from abnormal protein folding, and subtler changes in gating properties, whose functional relevance was fully
disclosed by dynamic clamp experiments. The mutation phenotype was attenuated by coexpression of
the polymorphism. The study, currently in press on
Cardiovascular Research, has been funded by the
NEDD network.
[ 36 ]
Effects of INaL inhibition in a model of pulmonary hypertension
In the previous years the group evaluated the effects of pulmonary hypertension induced by chronic
hypoxia on myocardial cell function. These studies
disclosed an enhancement of the late Na+ current
(INaL), a change potentially contributing to the development of right ventricular (RV) hypertrophy. To
discriminate whether INaL enhancement depended
on hemodynamic overload or hypoxia itself, we
developed a an hypoxia-independent model of RV
hypertrophy based on administration of monocro-
talin, a toxin causing pulmonary vasoconstriction.
Furthermore, to test whether INaL contributed to
hypertrophy development, we tested the effect of INaL
inhibition throughout the study period. The results
showed induction of marked INaL enhancement,
thus suggesting mechanical load as its cause, and
reversal of many, but not all aspects of hypertrophy,
by INaL blockade. The study is close to termination.
The project has been supported by grants from CVTherapeutics (Palo Alto, CA).
Continuing Projects
Evaluation of functional differentiation
in beta-sarcoglycan KO mesoangioblasts.
The study, ongoing from previous years, has been
completed and currently under submission for publication. The findings provide proof that transgenic beta-sarcoglycan knock out (BSG-/-) switches
functional differentiation of cardiac mesangioblast
precursors (cMabs) from cardiogenic (as previously
published) to skeletal muscle. This study was initially funded by a Cariplo 2007 grant
Effects of two inotropic agents with different toxicity on subcelluar Ca2+ distribution
The study, initiated in previous years, has been completed and published on the Journal of Molecular
and Cellular Cardiology. The outcome of this study is
that concurrent stimulation Ca2+ compartmentation
by the sarcoplasmic reticulum through SERCA2 stimulation may substantially limit the arrhythmogenic effect of cell Ca2+ overload resulting from Na+/K+
pump inhibition and improve the contractile response (positive lusitropy). The results are relevant to the
development of new inotropic agents. This study
was funded by private grants from Prassis SigmaTau, Debiopharm (Lausanne); student exchanges
with the university of Montpellier within this project
were supported by the MIUR Interlink program.
Ecology of migrant birds
22
Roberto Ambrosini
Rapidly ongoing climate change is influencing the
ecology of bird migration, particularly the timing
of migration, the choice of wintering areas, and
winter survival. In addition, several populations of
migratory birds are suffering steep demographic
declines.
To evaluate the critical events that influence the
population dynamic of migratory birds, a detailed
knowledge of the timing and route of migration
is required. Until now, the amount of information
available on migration journeys has been limited
by the impossibility to equip small-sized birds,
which represent the large majority of migrants,
with instruments able to record the position of
individuals during the annual life-cycle. New
miniaturized data-loggers may bridge this fundamental gap in our knowledge of the ecology of
bird migration.
Our research group is currently involved in two
studies aiming at applying miniaturized (< 1 g)
data-loggers (light-level geolocators), on different
populations of Barn Swallow (Hirundo rustica),
a small migrant passerine bird, whose populations have suffered steep demographic declines
in the last years. These studies will evaluate the
efficiency of miniaturized light-level geolocators
for the study of migration of small-sized birds. In
addition, these instruments will record detailed
information on the wintering grounds and on the
migration timing and routes of these birds. It is
the first time that the migration strategy of a large number (~ 50) of small passerine birds will be
known in details.
The group also continues a long-term monitoring project of Barn Swallow populations in the
Adda Sud Regional Park started in 1999. This
long-term study indicated that Barn Swallow populations have steeply declined (-60%) in the last
decade. This dramatic decline prompted the Regional Administration to start a research project,
which will be concluded in the next year under the
scientific coordination of our research group, for
assessing the actual consistency of Barn Swallow
populations in Lombardy.
The integration of information on the population
dynamics in the breeding quarters, the wintering
areas and the timing and route of migration of
individuals from the same population may allow
for a precise identification of the causes of the
observed population declines, and will help planning proper conservation strategies for migrant
birds.
[ 37 ]
23
Romina Combi,
Veronica Sansoni.
[ 38 ]
Identification and analysis
of the molecular basis
and predisposing factors of
neurological diseases
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 characterised yet.
Our projects are mainly focused on the study of two
groups of neurological disorders: ASDs (autism
spectrum disorders) and idiopathic epilepsies. These are the most epidemiologically relevant neurological disorders of childhood representing common
and devastating phenotypes which are assumed to
have a strong and complex genetic component. Even
in monogenic epilepsy, ethiology, phenotypic manifestations and prognosis are indeed highly heterogeneous.
Several loci associated with epilepsies have been
mapped by means of linkage analysis, and mutations have been detected in genes encoding ionchannels, leading to hyperexcitability of cortical neurons through alterations in the channel function, as
well as in genes not belonging to the channel family.
However, these gene discoveries have been in the
tiny fraction of epilepsies characterised by Mendelian inheritance. Moreover, even in these rare forms,
the identified mutations frequently account for a minority of patients suggesting therefore the existence
of additional loci.
Several ASDs susceptibility loci have also been mapped and an overlapping between these genes and
those associated with epilepsy has been observed.
To address the issue of the molecular and cellular
basis of these neurological disorders, we analyse
large cohorts of patients by means of an integrated
clinical and molecular approach (comprising genetic
counselling, DNA analysis, DNA sequencing, linkage analysis, CGH and SNPs microarrays, NGS techniques). 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 these diseases
Integrated researches
in animal and plant biology
24
Maurizio Casiraghi,
Massimo Labra,
Aldo Zullini,
Michela Barbuto,
Ilaria Bruni,
Fabrizio De Mattia,
Andrea Galimberti,
Francesca Cattaneo,
Emanuele Ferri.
ZooPlantLab (ZPL) links applied and basic researches in the zoological, botanical and agronomic
fields. Main projects are based on a molecular approach, but the integration of these data with other
biological information is common and essential. For
these reasons, ZPL has several collaborations with
national and international teams.
NEW METHOD FOR THE ANALYSIS OF BIODIVERSITY: USE OF PYROSEQUENCING
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. The method allows to analyse
a high number of samples in a short period of time,
factors really useful in an environmental study on a
large scale.
We are using pyrosequencing to investigate different
matrices ranging from environmental ones (soil,
water) to organic (gut content of different animals
like arthropods or mammals).
DNA BARCODING: A LINK BETWEEN BASIC AND
APPLICATIVE SCIENCES TOWARDS AN INTEGRATED APPROACH TO TAXONOMY
We are involved in the generation of a tool for the study of biodiversity based on an integrated approach
to taxonomy, in which we propose the interaction of
different level of taxonomic information. Taxonomy is
an essential tool for biological studies, however this
discipline is sometimes perceived as old-fashioned
and descriptive. Taxonomy must face this perception
and to renew it, the main challenges are computerisation and a “reasoned” molecular approach. The
framework provided by the DNA Barcoding initiative
is the key of this renewed approach.
Given these considerations, the goal of our projects
is to develop an identification system for different organisms. These following are our running projects
on DNA Barcoding:
1) Food tracking: in particular on fish (both fresh and
processed).
2) Parasitic nematodes: discrimination of filarial nematodes and their endosymbionts (Wolbachia).
3) Free-living nematodes: analysing natural population of free-living nematodes hosted in different
habitats (i.e. water, moss, and soil).
4) Birds: studying populations of non-autochthon
species of birds.
5) Bats: studying national populations of bats species.
6) Aromatic plant species: setting DNA plant barcoding sequences to univocally identify each plant
species.
FROM GENES TO ECOSYSTEMS: DNA BARCODING
AS A SYSTEM TO PROTECT BIODIVERSITY
The main idea of the project is that to protect is essential to know organisms and environments to be
protected. In this project we will couple the molecular identification system provided by the DNA barcoding approach with the analysis of the connectivity of
protected areas in a fragmented environment.
In the first part of the project we are working in collaboration with the Milan Natural History Museum,
to create a reference database of regional organisms. In the second part of the project we are moving from the consideration that Lombardy region
has several protected areas, but they are usually
separated by highly civilizated parts, in which wild
organisms are usually not allowed. In the project we
will analyse the level of genetic connection among
splitted areas, to detect, understand and (hopefully)
protect ecological corridors.
SCIENTIFIC EDUCATION: FROM THE UNIVERSITIES TO THE SOCIETY
The main idea of the project is to use our scientific
knowledge to produce systems for science education in collaboration with Italian associations (i.e.
Legambiente, WWF, Fondazione Idra, protected areas, etc). In the course of this project ZPL developed
an educational kit for the water analysis, to be used
by young kids, or in the school classroom.
The kit has been pivotally tested with success in few
dozens classrooms, but it will be implemented and
directed to a vast majority of schools.
[ 39 ]
25
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 nonnative species, particularly from Erythrean basin,
process that has accelerated in the recent years
concurrently to the warming trend of the seawater.
“Among fishes Siganus luridus”, has proven to be
extremely successful in colonizing a large part of
Eastern Mediterranean coasts until Linosa Island,
that constitutes the western boundary of the species distribution. Our aim 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.
[ 40 ]
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 platyhelminths
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 insoluble adhesive complex.
Parasite detachment is performed by a third, still
uncharacterized, protein. The ability to bind reversibly to living tissues in an aqueous environment is a
unique feature which renders this adhesive material
most suitable to applications in the surgical field.
CORAL DISEASES
Coral diseases have increased in number, species
affected, and geographic extent in the last two decades, but little is known about coral diseases in the
Indian Ocean, especially in the Republic of Maldives.
The objective of this study was to document the distribution and prevalence of coral diseases in the
Maldives at the genus level. Surveys for lesions in
scleractinians were conducted at eight sites around
Magoodhoo Island, Faafu Atoll. We documented five
different coral diseases. The white syndrome, skeleton eroding band disease, and Porites dark discoloration syndrome were the most common diseases
on the reefs of this island. A first estimate of prevalence of each of these coral diseases was calculated
based on 25 x 1 m belt transects. Significant differences were found between shallow and deep sites
only for Porites dark discoloration syndrome. These
data represent the first report of coral diseases for
the Republic of Maldives and confirm the wide
spread of a new threat to reef community structure
and biodiversity.
Programmed cell death
(pcd) in plants
26
Paolo Crosti,
Massimo Malerba.
Programmed cell death (PCD) and its most studied
focused on chitosan (CHT). CHT is a natural, non-
form in animals, apoptosis, are genetically control-
toxic and inexpensive compound obtained by partial
led processes present in all living organisms, aimed
alkaline deacetylation of chitin, the main component
to eliminate unwanted or detrimental cells. In plants
of the exoskeleton of crustaceans and other arthro-
PCD plays a pivotal role in several developmental
pods. Although the exact mode of action of CHT is
processes (formation of tracheary elements, sex
still unknown, in agriculture it has been shown to
determination, senescence) and it is involved in the
be a versatile compound that controls numerous pre
responses to environmental stresses and in defence
and postharvest diseases on various horticultural
mechanisms (hypersensitive response, HR). Rese-
commodities. In sycamore (Acer pseudoplatanus L.)
arches to elucidate the basic mechanisms of PCD in
cultured cells CHT rapidly induces a set of defense/
plants are in rapid expansion and at least three dif-
stress responses: accumulation of dead cells and of
ferent forms of PCD based on the cell organelle first
cells with fragmented DNA, production of H2O2and
involved have been reported: a “nuclear” (apoptotic-
nitric oxide (NO), accumulation of regulative 14-3-3
like) form, typical of the defense response against
proteins in the cytosol and of HSP70 molecular cha-
pathogen attack, a “chloroplastic” form, typical of
perone Binding Protein (BiP) in the endoplasmic
the foliar senescence, and a “vacuolar” form, typical
reticulum, release of cytochrome c from the mito-
of the maturation of the vascular elements.
chondrion. These findings, in particular the produc-
During the last years we utilized different plant cell
tion of signalling molecules like H2O2 and NO open
lines and several PCD inducers to investigate the
the possibility to investigate the signalling pathways
plant PCD process. Recently our attention has been
leading to the CHT-induced responses.
[ 41 ]
27
Piercarlo Fantucci,
Luca De Gioia,
Giuseppe Zampella,
Luca Bertini,
Elena Papaleo,
Claudio Greco.
[ 42 ]
Computational
investigations of proteins
and related biomimetic
compounds
Our work is aimed at elucidating the relationship
between the three-dimensional structure of proteins and their function. In particular, recent studies
have been focussed on hydrogenases, which are a
family of enzymes able to convert, with a particularly
high catalytic efficiency, protons and electrons into
molecular hydrogen. The elucidation of the catalytic
mechanism of hydrogenases is fundamental not
only to understand the structure-function relationship in this enzyme family, but also for the design
of novel synthetic compounds characterized by high
catalytic activity. Hydrogenases and related synthetic compounds are studied in our laboratory using
different quantum chemical techniques, ranging
from Density Functional Theory calculations to QM/
MM models.
In our laboratory, molecular modelling and docking
methods are also used, to study protein-ligand interaction in several families of potential targets.
Recently, molecular modelling and docking studies
have contributed to the identification of water-soluble Ras inhibitors, and to the structural characterization of enzymes involved in lipopolysaccharide
biosynthesis.
Finally, molecular dynamics simulations and homology modelling are used with the aim of investigating
structure-function relationship in enzymes and proteins. In particular, simulations of biomolecular systems has allowed obtaining insights into biomolecular processes at the atomic level, which are often
hardly accessible to experimental methods. Recent
studies carried out in our laboratory have been focused in studying the effect of temperature on protein
stability, as well as the interaction between enzymes
and their cofactors or inhibitors.
Molecular modelling
and computational chemistry
28
Giorgio Moro
Computational approaches based on Molecular Dynamics simulations, Quantum Mechanical methods
and 3D Quantitative Structure-Activity Relationships
are employed to study biological processes at the
molecular level. The computational approach taken in our research on biological processes focuses
mainly on three methodological areas. One includes
a variety of methods based on Molecular Mechanics
(MM) and Molecular Dynamics (MD). The second is
an approach based on advanced Quantum Mechanical (QM) methods applied to model systems. The
third is an approach aimed at obtaining statistical
models through an analysis of data inferring relative Quantitative Structure-Activity Relationships
(QSAR).
As is well known, approaches based on MD theories
are the only ones presently available to study complex systems like proteins in solution. The approach
to the problem of protein structure at the classical
level is even more acute when there is the modelling
of interaction between proteins themselves, between protein and DNA fragments or between protein
and substrates (as in drug discovery, toxicology studies or virtual enzyme engineering).
However, MD methods are not completely free of
difficulties, which are generated just by the very
high number of degrees of freedom. 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 A. Villa - Karolinska Institutet, Stoccolma, Svezia; M. Salmona – Istituto Mario Negri – Milano)
- characterization of a new contrast agent for selective targeting in Magentic Resonance Molecular
Imaging (collaborations with F. Nicotra and L. Cipolla – Dipartimento Biotecnologie e Bioscienze)
- mechanism of the adenine nucleotide translocator
protein
- interaction between mono- and multivalent ligands and human galectins (collaboration with F. Peri,
Dipartimento Biotecnologie e Bioscienze)
[ 43 ]
29
Francesco Nicotra,
Laura Cipolla,
Barbara La Ferla,
Cristina Airoldi,
Alexander Orsato,
Cristiano Zona,
Nasrin Shaikh,
Francisco Cardona,
Laura Russo,
Erika Sironi,
Silvia Merlo,
Giuseppe D’Orazio,
Luca Gabrielli,
Davide Bini
Design, synthesis and
molecular recognition studies
on bioactive compounds and
biomaterials
Design synthesis and biological evaluation
of bioactive compounds
Particular attention is devoted to the generation of
inhibitors, agonists and antagonists not only as new
lead compounds in drug research, but also as tools
to understand unknown biological pathways (chemical genetic studies). Furthermore, in a large integrated FP7 project, nanoparticles for diagnosis and
therapy of Alzheimer Disease are developed.
Synthetic targets focused in 2010 are:
• Ligands of Abeta peptides and their conjugation to nanoparticles for diagnosis and therapy
of Alzheimer Disease; synthesis of radiolabelled
ligands for in vitro-vivo blood brain barrier passage studies.
•Inhibitors of bacterial LPS biosynthesis as
potential antibacterial agents
•Inhibitors of insect trehalase as potential
“green” pesticides
•Synthesis of paramagnetic sialic acid
conjugates as MRI contrast agents
•Glycomimetics as regulators of SGLT1 as
anti-inflammatory and antitumor agents
•Drugs fused into glycidic structures, in order to modulate the fisico-chemical, pharmacokinetic and conformational properties
•Glycidic scaffolds and their use for the synthesis of Gastrin Releasing Peptide (GRP) receptor
agonists/antagonists, as potential antitumor
agents and/or antitumor drug targeting
compounds
NMR studies are performed for:
•Structure elucidation
• Conformational analysis
• Epitope mapping studies (ligand-receptor
interactions studies at atomic level)
• Adhesion kinetic studies
[ 44 ]
BIOMATERIALS
The generation of smart biomaterials for tissue en-
gineering requires mimicking natural ECMs that
regulate complex morphogenetic processes in tissue formation and regeneration. Their functionality
should be adjustable to a particular biological environment to obtain cell- and tissue-specificity. Ideally, one would create them from an array of biocompatible scaffolds decorated with an array of ligands
inducing cell adhesion and/or proliferation and/or
differentiation.
Several issues are taken into accounts for the design
of bioactive and biocompatible materials:
a)nature of the material (collagen, composites with synthetic organic polymers or inorganic materials such as hydroxyapatite);
b)molecular cues for eliciting the desired
biological activity: small peptidic or glycidic epitopes are synthesised and tested for their biological activity in solution and linked to
the material
c) “biodecoration” step: chemoselective
methods are optimised in order to covalently functionalise materials with different
features and chemical nature
Ideally, the biomaterial should include cell-adhesive
ligands (such as integrin-binding peptides of the
prototypical RGD family or carbohydrates such as
hialuronic acid), binding sites for growth factor (GF)
proteins, domains with susceptibility to degradation by cell-secreted or cell-activated proteases to
facilitate bidirectional cell-matrix interactions, but
also domains with structural function (such as the
elastin-derived peptide sequence VPGVG). The use
of such synthetic approaches in “bioactive” material
design may allow matrices to be tailor-made for a
specific cell or tissue.
In collaboration with research groups expert in material science and in stem cells, a research devoted
to the functionalization biomaterial scaffolds with
properly selected peptides and sugars is developed.
Organic, bioorganic
and medicinal chemistry
30
Francesco Peri,
Matteo Piazza,
Valentina Calabrese,
Roberto Cighetti,
Chiara Baruffa,
Francesca Monza,
Gaetana Damore.
The research interests of Peri Lab span the disciplines of organic, bioorganic and medicinal chemistry with an emphasis of study of the interactions
between small molecules and their biological (pharmacological) targets. In 2010, innovative hit and lead
compounds for drug discovery were projected and
synthesized. These molecules were also used as tools for “chemical genetics” to study, in collaboration
with biologists and immunologists, some important
signal pathways in humans.
The main ongoing projects in medicinal chemistry
focus on:
1) Development of new chemical entities (mainly
small molecules derived from natural sugars) that
are active on the innate immunity receptors of bacterial endotoxins.
These compounds target the human Toll-like Receptor 4 (TLR4), and other proteins (CD4, MD-2,
LBP) involved in the process of detection of bacterial
lipopolysaccharides (LPS) and subsequent trigger of
innate immune response in animals and humans.
This project is funded by NIH (project “Regulation of
MD-2 function and expression”) and is developed in
collaboration with J. Weiss and T. Gioannini (University of Iowa, USA).
2) New sugar-based small molecules targeting the
human Ras protein, whose oncogenic variants are
responsible for the insurgence and maintenance of
about 30% human tumors. Ras plays a pivotal role
in the pancreatic tumor that still lack efficient pharmacological treatment.
This project is in collaboration with V. Gaponenko
(University of Chicago, IL, USA), and M. Vanoni.
3) Innovative, non-classical, Chemoselective Glycosylation (CG) methods for the convergent synthesis
of neoglycoconjugates.
4) Nanoparticles functionalized with sugars, LPS,
other organic molecules for biotechnology and nanomedicine applications (in collaboration with D.
Prosperi).
5) New anticancer drugs targeting human transglutaminase-2 (TG-2) protein (in collaboration with K.
Mehta, University of Houston, Texas, USA).
6) New anti-hepatitis C virus (HCV) agents based on
the discovery of new molecular targets (in collaboration with R. De Francesco, INBM, Milano).
[ 45 ]
31
Outer membrane biogenesis in
Gram-negative bacteria
Alessandra Polissi,
Paola Sperandeo,
Alessandra Martorana,
Riccardo Villa.
Escherichia coli
Rod-shaped Bacterium
with Multiple Flagella
(SEM) image
Pseudomonas aeruginosa
Scanning Electron
Microscopy
[ 46 ]
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 outer membrane have long
since been known, many aspects of its biogenesis
still remain obscure.
My laboratory has recently identified new essential
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 OUTER MEMBRANE
Genetic and biochemical approaches are being used
to characterize the proteins implicated in the LPS
biogenetic pathway and to study how these proteins
interact to assemble the functional protein machinery devoted to LPS transport to the cell surface. 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 namely to target outer membrane
biogenesis for the development of new generation of
antibacterial drugs.
These studies are being carried out using both
Escherichia coli, a Gram-negative model organism
and a pathogen implicated in severe gastrointestinal
and extraintestinal diseases, and Pseudomonas aeruginosa, an opportunistic pathogen that causes a
wide variety of infections in compromised patients,
indeed the treatment of such infections is currently
very difficult given the intrinsic and acquired resistance of the pathogen to most conventional drugs.
THE LPS BIOGENETIC PATHWAY AS TARGET FOR
THE DESIGN AND SYNTHESIS OF NOVELS ANTIBACTERIALS
Structural and functional studies of target proteins
known to play key roles in the biogenesis of LPS are
currently ongoing. As LPS is a strategic structure for
bacterial virulence and an important modulator of
the innate immune response LPS biogenesis represents a very attractive, but also largely unexplored
target for drug development. Our approach for novel
antibacterial development is based on rational drug
design, a focused approach that exploits information about the 3D-structure of a target protein and/
or its natural ligands that has proved successful for
the development of effective drugs. For the success of this approach, it is crucial to know the 3D
structure of the target protein and to understand its
structure-function relationships. For this purpose a
multidisciplinary team comprising microbiologists,
structural biologists, bioinformatics and medicinal
chemists has been built. Target proteins under study are being purified from both, Escherichia coli and
Pseudomonas aeruginosa. Structural information is
used to design and synthesize novel lead compounds that inhibit the LPS biogenetic pathways in the
hope to develop new therapeutic strategies against
infectious diseases.
Industrial biotechnology:
adaptation of the microbial
cell factory to technical
constraints
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.
Micro-organisms in general represent on one hand
a still unlimited source of said proteins and products and on the other the best resource for recruiting highly efficient and safe cell factory. rDNA
platforms allow, sometimes in a quite simple way,
the development of new micro-organisms leading
to the production of new products (or process) such
as heterologous proteins, fine and bulk chemicals,
vitamins, nutraceuticals, biofuels and animal
nutritionals (i.e. amino acids). However, this apparently simple statement implies that for rendering a
lab-scale project really feasible at industrial scale,
the whole process has to be deeply examined, understood, developed and optimised. The cell factories has to be tested for their potential efficiency
and easy of manipulation, while at the same time
non-conventional ones can be investigated as a
source of determinants important for the transferring of peculiar physiological traits. In this respect
it has to be underlined that 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. Studying how to improve the cell factory robustness as well as a deep
understanding of its innate potentiality can help in
overcoming this challenging bottleneck. In this respect, our laboratory has developed (i) a series of
cell factories producing heterologous compounds,
like proteins, enzymes, organic acids, biofuels and
nutraceuticals (ii) a series of yeast strains with improved resistance to specific constraints imposed by
the process itself and (iii) a study and a model of the
correlation between the size of the single yeast cell
and its cellular metabolism. Finally (iv), a University
facility unit dedicated to the first scale-up of the labscale project is extensively and routinely utilised.
32
Danilo Porro,
Paola Branduardi,
Luca Brambilla,
Gianni Frascotti,
Simone Passolunghi,
Laura Dato,
Tiziana Fossati,
Vera Codazzi,
Giorgia Rossi,
Stefano Bertagnoli,
Valeria Longo.
MICROBIAL CELL FACTORIES AND MAIN PRODUCTS
For twenty five 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 and 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 and not only
from yeasts but also from bacteria, naturally producing the product of interest or engineered for it.
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, deriving either from the yeast itself or fishing said elements
from other organisms. Single cell analysis by flow
cytometer is a potent technique for monitoring at
different levels said robustness.
PHYSIOLOGICAL AND MODELLING STUDIES OF
THE CELL FACTORIES
The control of both metabolism and cell cycle progression by modulating the cellular environment
has a key role in the regulation of growth and cell
proliferation and production in all organisms. Specific attention has been devoted to study and to model
the correlation between the size of the single yeast
cell, its metabolism, redox unbalance and cofactors
alterations.
[ 47 ]
Francesco Peri / Sassi
[ 48 ]
PUBLICATIONS,
GRANTS,
PhD THESES
3
[ 49 ]
[3.1] PUBLICATIONS
AIROLDI C, MERLO S, NICOTRA F (2010) Synthesis of 3-DeoxyD-threopentofuranose 5-Phosphate, a Substrate of Arabinose
5-Phosphate Isomerase. J CARBOHYDR CHEM vol. 29; p. 30–38.
plexes [HFe2(SR)2(PR3)(x)(CO)(6-x)]+ (x = 2, 3, 4) relevant to the
active site models for the [FeFe]-hydrogenases. DALTON TRANS
vol. 39 (12), p. 3011-3019.
AIROLDI C, SOMMARUGA S, MERLO S, SPERANDEO P, CIPOLLA
L, POLISSI A, NICOTRA F (2010) Targeting Bacterial Membranes:
NMR Characterization of Substrate Recognition and Binding Requirements of D-arabinose 5P Isomerase, a key enzyme in the
biosynthesis of LPS. CHEMISTRY EUR J vol. 16; p. 1897–1902.
BASU-ROY U, AMBROSETTI D, FAVARO R, NICOLIS SK, MANSUKHANI A, BASILICO C (2010) The transcription factor Sox2 is
required for osteoblast self-renewal. CELL DEATH AND DIFFERENTIATION vol. 17, p. 1345-1353.
ALBERGHINA L, CIRULLI C (2010) Proteomics and systems biology to tackle biological complexity: Yeast as a case study. PROTEOMICS vol. 10(24); p.4337-4341.
ALTOMARE C, BARILE L, MARANGONI S, ROCCHETTI M, ALEMANNI M, MOSTACCIUOLO G, GIACOMELLO A, MESSINA E, ZAZA
A (2010) Caffeine-induced Ca(2+) signaling as an index of cardiac
progenitor cells differentiation. Basic Res Cardiol vol.105; p.
737-749.
AMBROSINI R, SAINO N (2010) Environmental effects at two nested spatial scales on habitat choice and breeding performance of
barn swallow. EVOLUTIONARY ECOLOGY vol. 24; p. 491-508.
AMI D, NATALELLO A, MEREGHETTI P, NERI T, ZANONI M, MONTI M, DOGLIA SM, REDI CA (2010) FT-IR spectroscopy supported
by PCA-LDA analysis for the study of embryonic stem cell differentiation. SPECTROSCOPY- AN INTERNATIONAL JOURNAL vol.
24; p. 89-97.
ARACRI P, CONSONNI S, MORINI R, PERRELLA M, RODIGHIERO
S, AMADEO A, BECCHETTI A (2010) Tonic modulation of GABA
release by nicotinic acetylcholine receptors, in layer V of the murine prefrontal cortex. CEREB CORTEX vol. 20; p. 1539-1555.
ARCANGELI A, BECCHETTI A (2010) New trends in cancer therapy: targeting ion channels and transporters. PHARMACEUTICALS vol. 3; p. 1202-1224.
BARACCA A, CHIARADONNA F, SGARBI G, SOLAINI G, ALBERGHINA L, LENAZ G (2010) Mitochondrial Complex I decrease is
responsible for bioenergetic dysfunction in K-ras transformed
cells. BIOCHIM BIOPHYS ACTA vol.1797(2); p. 314-23.
BARANDI L, VIRAG L, JOST N, HORVATH Z, KONCZ I, PAPP R,
HARMATI G, HORVATH B, SZENTANDRASSY N, BANYASZ T,
MAGYAR J, ZAZA A, VARRO A, NANASI PP (2010) Reverse ratedependent changes are determined by baseline action potential
duration in mammalian and human ventricular preparations. BASIC RES CARDIOL vol. 105; p. 315-323.
BARBUTO M, GALIMBERTI A, FERRI E, LABRA M, MALANDRA R,
GALLI P, CASIRAGHI M (2010) DNA barcoding reveals fraudulent
substitutions in shark seafood products: The Italian case of “palombo” (Mustelus spp.). FOOD RESEARCH INTERNATIONAL vol.
43; p. 376-381.
BARTON BE, ZAMPELLA G, JUSTICE AK, DE GIOIA L, RAUCHFUSS TB, WILSON SR (2010) Isomerization of the hydride com-
[ 50 ]
BAVARO T, UBIALI D, BROCCA S, ROCCHIETTI S, NIETO S, PREGNOLATO M, LOTTI M, TERRENI M (2010) Recombinant lipase
from Candida rugosa for regioselective hydrolysis of peracetylated nucleosides. A comparison with commercial non recombinant
lipases. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY vol. 28;
p. 108-116.
BAZZI M, MANTIERO D, TROVESI C, LUCCHINI G, LONGHESE MP
(2010) Dephosphorylation of H2A by Glc7/protein phosphatase 1
promotes recovery from inhibition of DNA replication. MOL CELL
BIOL. Vol. 30, p. 131-45.
BECCHETTI A, ARCANGELI A (2010) Integrins and ion channels
in cell migration: implications for neuronal development, wound
healing and metastatic spread. ADV EXP MED BIOL vol. 674; p.
107-123.
BECCHETTI A, PILLOZZI S, MORINI R, NESTI E, ARCANGELI A
(2010) New insights into the regulation of ion channels by integrins. INT REV CELL MOL BIOL vol. 279; p. 135-190.
BECCHETTI A (2010) Hippocampal formation and the classical
art of memory. PROC NATL ACAD SCI (USA) vol. 107; p. E104.
BENNINGHOFF J, GRITTI A, RIZZI M, LAMORTE G, SCHLOESSR
RJ, SCHMITT A, ROBEL S, GENIUS J, MOESSNER R, RIEDERER
P, MANJI HK, GRUNZE H, RUJESCU D, MOELLER HJ, LESCH KP,
VESCOVI AL (2010) Serotonin depletion hampers survival and
proliferation in neurospheres derived from adult neural stem cells. NEUROPSYCHOPHARMACOLOGY vol.35, p. 893-903.
BENZONI F, GALLI P, PICHON M (2010) Pink spots on Porites:
not always a coral disease. CORAL REEFS vol. 29, p. 153.
BENZONI F, STEFANI F, PICHON M, GALLI P (2010) The name
game: morpho-molecular species boundaries in the genus
Psammocora (Cnidaria, Scleractinia). ZOOLOGICAL JOURNAL
OF THE LINNEAN SOCIETY vol. 160, p. 521-456.
BERRUTI G, RIPOLONE M, CERIANI M (2010) USP8, a regulator
of endosomal sorting, is involved in mouse acrosome biogenesis through interaction with the spermatid ESCRT-0 complex and
microtubules. BIOL REPROD vol. 82; p. 930-939.
BERTINI L, GRECO C, BRUSCHI M, FANTUCCI P, DE GIOIA L
(2010) CO Affinity and Bonding Properties of [FeFe] Hydrogenase
Active Site Models. A DFT Study ORGANOMETALLICS vol. 29 (9);
p. 2013-2025.
BIGI A, MOROSI L, POZZI C, FORCELLA M, TETTAMANTI G, VE-
NERANDO B, MONTI E, FUSI P (2010) Human sialidase NEU4
long and short are extrinsic proteins bound to outer mitochondrial membrane and the endoplasmic reticulum, respectively.
GLYCOBIOLOGY vol. 20 (2); p. 148-157.
CASIRAGHI M, LABRA M, FERRI E, GALIMBERTI A, DE MATTIA
F (2010) DNA barcoding: a six-question tour to improve users’
awareness about the method. BRIEFINGS IN BIOINFORMATICS
vol. 11; p. 440-453.
BOLOGNINI D, COSTA B, MAIONE S, COMELLI F, MARINI P, DI
MARZO V, PAROLARO D, ROSS RA, GAUSON LA, CASCIO MG, PERTWEE RG (2010) The plant cannabinoid Δ9-tetrahydrocannabivarin
can decrease signs of inflammation and inflammatory pain in
mice. BR J PHARMACOL vol.160; p. 677-687.
CAVALIERI D, RIVERO D, BELTRAME L, BUSCHOW SI, CALURA E, RIZZETTO L, GESSANI S, GAUZZI MC, REITH W, BAUR A,
BONAIUTI R, BRANDIZI M, DE FILIPPO C, D’ORO U, DRAGHICI
S, DUNAND-SAUTHIER I, GATTI E, GRANUCCI F, GÜNDEL M,
KRAMER M, KUKA M, LANYI A, MELIEF CJ, VAN MONTFOORT
N, OSTUNI R, PIERRE P, POPOVICI R, RAJNAVOLGYI E, SCHIERER S, SCHULER G, SOUMELIS V, SPLENDIANI A, STEFANINI I,
TORCIA MG, ZANONI I, ZOLLINGER R, FIGDOR CG, AUSTYN JM
(2010) DC-ATLAS: a systems biology resource to dissect receptor
specific signal transduction in dendritic cells. IMMUNOME RES
vol. 19; p. 6-10.
BONETTI D, CLERICI M, MANFRINI N, LUCCHINI G, LONGHESE
MP (2010) The MRX complex plays multiple functions in resection
of Yku- and Rif2-protected DNA ends. PLoS One 5: e14142.
BONETTI D, CLERICI M, ANBALAGAN S, MARTINA M, LUCCHINI
G, LONGHESE MP (2010) Shelterin-like proteins and Yku inhibit
nucleolytic processing of Saccharomyces cerevisiae telomeres.
PLoS Genet. 6: e1000966.
BRADAMANTE S, VILLA A, VERSARI S, BARENGHI L, ORLANDI I, VAI M (2010) Oxidative stress and alterations in actin cytoskeleton trigger glutathione efflux in Saccharomyces cerevisiae.
BIOCHIMICA BIOPHYSICA ACTA (MOL CELL RES) vol. 1803; p.
1376-1385.
BRÜMMER A, SALAZAR C, ZINZALLA V, ALBERGHINA L, HÖFER
T (2010) Mathematical modelling of DNA replication reveals a
trade-off between coherence of origin activation and robustness
against rereplication. PLOS COMPUT BIOL vol.6(5); p. e1000783.
BRUNI I, DE MATTIA F, GALIMBERTI A, GALASSO G, BANFI E,
CASIRAGHI M, LABRA M (2010) Identification of poisonous plants
by DNA barcoding approach. INTERNATIONAL JOURNAL OF LEGAL MEDICINE vol. 124; p. 595-603.
CERIANI M, AMIGONI L, SCANDIUZZI C, BERRUTI G, MARTEGANI E (2010) The PH-PxxP domain of RalGPS2 promotes PC12 cells differentiation acting as a dominant negative for RalA GTPase
activation. NEUROSCIENCE RES vol. 66; p. 290-298.
CHAVAS L, KATO R, SUZUKI N, VON ITZSTEIN M, MANN M,
THOMSON R, DYASON J, MCKIMM-BRESCHKIN J, FUSI P,
TRINGALI C, VENERANDO B, TETTAMANTI G, MONTI E, WAKATSUKI S (2010) Complexity in Influenza Virus Targeted Drug
Design: Interaction with Human Sialidases. J Med Chem vol. 53
(7); p. 2998-3002.
CIPOLLA L, ARAÚJO AC, BINI D, GABRIELLI L, RUSSO L, SHAIKH
N (2010) Discovery and design of carbohydrate-based therapeutics. EXP OPIN DRUG DISCOV vol. 5(8); p. 721-737.
CIPOLLA L, GABRIELLI L, BINI D, RUSSO L, SHAIKH N (2010)
Kdo: a critical monosaccharide for bacteria viability. NAT PROD
REP vol. 27; p. 1618–1629.
BRUSCHI M, GRECO C, BERTINI L, FANTUCCI P, RYDE U, DE
GIOIA L (2010) Functionally Relevant Interplay between the Fe4S4
Cluster and CN- Ligands in the Active Site of [FeFe]-Hydrogenases. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY vol. 132
(14); p. 4992-4993.
CIPOLLA L, LA FERLA B, AIROLDI C, ZONA C, ORSATO A, SHAIKH N, RUSSO L, NICOTRA F (2010) Carbohydrate Mimetics and
Scaffolds: Sweet Spots in Medicinal Chemistry. FUTURE MEDICINAL CHEMISTRY vol. 2; p.587-599.
BUSTI S, COCCETTI P, ALBERGHINA L AND VANONI M (2010)
Glucose Signaling-Mediated Coordination of Cell Growth and Cell
Cycle in Saccharomyces cerevisiae. SENSORS vol. 10(6), p. 61956240.
CIPOLLA L, REDAELLI C, GRANUCCI F, ZAMPELLA G, ZAZA A,
CHISCI R, NICOTRA F. (2010) Straightforward synthesis of novel
Akt inhibitors based on a glucose scaffold. CARBOHYDR RES vol.
345; p.1291-1298.
CANTU’ C, GRANDE V, ALBORELLI I, CASSINELLI L, CANTÙ I,
COLZANI MT, IERARDI R, RONZONI L, CAPPELLINI MD, FERRARI G, OTTOLENGHI S, RONCHI A (2010) A highly conserved
Sox6 double binding site mediates Sox6 gene downregulation in
erythroid cells. NUCLEIC ACIDS RESEARCH vol. 39; p. 486-501.
CIRILLO G, CAVALIERE C, BIANCO MR, DE SIMONE A, COLANGELO AM, SELLITTI S, ALBERGHINA L, PAPA M (2010) Intrathecal NGF administration reduces reactive astrocytosis and changes neurotrophin receptors expression pattern in a rat model of
neuropathic pain. CELL MOL NEUROBIOL vol. 30(1), p. 51-62.
CARDONA F, GOTI A, PARMEGGIANI C, PARENTI P, FORCELLA M, FUSI P, CIPOLLA L, ROBERTS SM, DAVIES GJ, GLOSTER
TM (2010) Casuarine-6-O- -D-glucoside and its analogues are
tight binding inhibitors of insect and bacterial trehalases. CHEM
COMM vol.46(15); p. 2629-2631.
COCCETTI P, MONTANO G, LOMBARDO A, TRIPODI F, ORSINI F,
PAGLIARIN R (2010) Synthesis and biological evaluation of combretastatin analogs as cell cycle inhibitors of the G1 to S transition in Saccharomyces cerevisiae. BIOORG MED CHEM LETT vol.
20(9), p. 2780-2784.
[ 51 ]
[3.1] PUBLICATIONS
COLOMBO M, CORSI F, FOSCHI D, MAZZANTINI E, MAZZUCCHELLI S, MORASSO C, OCCHIPINTI E, POLITO L, PROSPERI D,
RONCHI S, VERDERIO P (2010) HER2 targeting as a two-sided
strategy for breast cancer diagnosis and treatment: Outlook and
recent implications in nanomedical approaches. PHARMACOL
RES vol. 62(2); p. 150-165.
COLOMBO S, PALMIOLI A, AIROLDI C, TISI R, FANTINATO S, OLIVIERI S, DE GIOIA L, MARTEGANI E, PERI F (2010) Structure-Activity Studies on Arylamides and Arysulfonamides Ras Inhibitors.
CURRENT CANCER DRUG TARGETS vol. 10 (2); p. 192-199.
COMBI R, REDAELLI S, BEGHI M, CLERICI M, CORNAGGIA CM,
DALPRÀ L (2010) Clinical and genetic evaluation of a family showing
both autism and epilepsy. BRAIN RES BULL vol. 82; p. 25-28.
COMELLI F, BETTONI I, COLOMBO A, FUMAGALLI P, GIAGNONI
G, COSTA B, RIMONABANT A (2010) Cannabinoid CB(1) receptor
antagonist, attenuates mechanical allodynia and counteracts oxidative stress and nerve growth factor deficit in diabetic mice. EUR
J PHARMACOL vol. 637; p. 62-69.
CORSI F, PROSPERI D (2010) Molecular nanoclinics: Dream or
reality? PHARMACOL RES vol. 62(2); p. 55-56.
COSTA B, BETTONI I, PETROSINO S, COMELLI F, GIAGNONI G, DI
MARZO V (2010) The dual fatty acid amide hydrolase/TRPV1 blocker, N-arachidonoyl-serotonin, relieves carrageenan-induced
inflammation and hyperalgesia in mice. PHARMACOL RES vol.
61; p. 537-546.
DATO L, BRANDUARDI P, PASSOLUNGHI S, CATTANEO D, RIBOLDI L, FRASCOTTI G, VALLI M, PORRO D (2010) Advances in
molecular tools for the use of Zygosaccharomyces bailii as host
for biotechnological productions and construction of the first auxotrophic mutant. FEMS YEAST RESEARCH vol. 10; p. 894-908.
DI RESTA C, AMBROSI P, CURIA G, BECCHETTI A (2010) Effect of
carbamazepine and oxcarbazepine on wild type and mutant neuronal nicotinic receptors linked to nocturnal frontal lobe epilepsy.
EUR J PHARMACOL vol. 643; p. 13-20.
DIOMEDE L, CASSATA G, FIORDALISO F, SALIO M, AMI D, NATALELLO A, DOGLIA SM, DE LUIGI A, SALMONA M (2010) Tetracycline and its analogues protect Caenorhabditis elegans from
ß-amyloid-induced toxicity by targeting oligomers. NEUROBIOL
DIS vol. 40; p. 424-431.
EL-BOUBBOU K, ZHU DC, VASILEIOU C, BORHAN B, PROSPERI D, LI W, HUANG X (2010) Magnetic glyco-nanoparticles: a tool
to detect, differentiate, and unlock the glyco-codes of cancer
via magnetic resonance imaging. J AM CHEM SOC vol. 132(12);
p.4490-4499.
FAN X, KHAKI L, ZHU TS, SOULES ME, TALSMA CE, GUL N, KOH C,
ZHANG J, LI YM, MACIACZYK J, NIKKHAH G, DIMECO F, PICCIRILLO S, VESCOVI AL, EBERHART CG (2010) NOTCH pathway blockade
depletes CD133-positive glioblastoma cells and inhibits growth of
tumor neurospheres and xenografts. STEM CELLS vol. 28, p. 5-16.
[ 52 ]
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Unit2D.6.
FERRARIO F, CALCINAI B, ERPENBECK D, GALLI P, WORHEIDE G (2010) Two Pione species (Hadromerida, Clionaidae) from
the Red Sea: a taxonomical challenge. ORGANISMS DIVERSITY &
EVOLUTION vol. 10; p. 275-285.
FERRI E, NOVATI S, CASIRAGHI M, SAMBRI V, GENCO F, GULMINETTI R, BANDI C (2010) Plasma levels of bacterial DNA in HIV
infection: the limits of quantitative polymerase chain reaction. J
INFECT DIS vol. 202; p. 176-177.
FICETOLA GF, CROTTINI A, CASIRAGHI M, PADOA-SCHIOPPA
E (2010) New data on amphibians and reptiles of the Northern
Areas of Pakistan: distribution, genetic variability and conservation
issues. NORTH-WESTERN JOURNAL OF ZOOLOGY vol. 6; p. 1-12.
FONTANETO D, AMBROSINI R (2010) Spatial niche partitioning in
epibiont rotifers on the waterlouse Asellus aquaticus. LIMNOLOGY AND OCEANOGRAPHY vol. 255; p. 1327-1337.
FORCELLA M, CARDONA F, GOTI A, PARMEGGIANI C, CIPOLLA L, GREGORI M, SCHIRONE R, FUSI P, PARENTI P (2010) A
membrane-bound trehalase from Chironomus riparius larvae:
purification and sensitivity to inhibition. GLYCOBIOLOGY vol. 20;
p. 1186-1195.
GATTI-LAFRANCONI P, NATALELLO A, REHM S, DOGLIA SM,
PLEISS J, LOTTI M (2010) Evolution of stability in a cold-active
enzyme elicits specificity relaxation and highlights substrate-related effects on temperature adaptation. JOURNAL OF MOLECULAR BIOLOGY vol. 395; p. 155-166.
GOURLAY LJ, SOMMARUGA S, NARDINI M, SPERANDEO P,
DEHÒ G, POLISSI A, BOLOGNESI M (2010) Probing the active site
of the sugar isomerase domain from E. coli arabinose-5-phosphate isomerase via X-ray crystallography. PROTEIN vol. 19; p.
2430-2439.
GRECO C, FANTUCCI P, DE GIOIA L, SUAREZ-BERTOA R, BRUSCHI M, TALARMIN J, SCHOLLHAMMER P (2010) Electrocatalytic dihydrogen evolution mechanism of [Fe2(CO)4(kappa(2)Ph2PCH2CH2PPh2)(mu-S(CH2)3S)] and related models of the
[FeFe]-hydrogenases active site: a DFT investigation. DALTON
TRANS vol. 39 (31), p. 7320-7329.
GUGLIELMETTI S, TAVERNITI V, MINUZZO M, ARIOLI S, ZANONI
I, STUKNYTE M, GRANUCCI F, KARP M, MORA D (2010) A dairy
bacterium displays in vitro probiotic properties for the pharyngeal
mucosa by antagonizing group A streptococci and modulating the
immune response. INFECT IMMUN vol. 78(11); p. 4734-4743.
GULLO F, MAZZETTI S, MAFFEZZOLI A, DOSSI E, LECCHI M,
AMADEO A, KRAJEWSKI J, WANKE E (2010) Orchestration of
“presto” and “largo” synchrony in up-down activity of cortical networks. FRONTIERS IN NEURAL CIRCUITS vol. 4; p. 1-16.
HAYNES RK, CHAN WC, WONG HN, LI KY, WU WK, FAN KM, SUNG
HH, WILLIAMS ID, PROSPERI D, MELATO S, COGHI P, MONTI D
(2010) Facile oxidation of leucomethylene blue and dihydroflavins
by artemisinins: relationship with flavoenzyme function and antimalarial mechanism of action. CHEM MED CHEM vol. 5(8), p.
1282-1299.
HE J, LIU Y, XIE X, ZHU T, SOULES M, DIMECO F, VESCOVI AL, FAN
X, LUBMAN DM (2010) Identification of cell surface glycoprotein
markers for glioblastoma-derived stem-like cells using a lectin microarray and LC-MS/MS approach. J PROTEOME RES vol. 9(5), p.
2565-2572.
KRITSKY D, AQUARO G, GALLI P (2010) Microncocotyle bicoccae
n. gen., n. sp. (Monogenoidea: Dactylogyridae) from the Gills of the
Longtail Silverbiddy, Gerres longirostris (Teleostei: Gerreidae), in the
Red Sea, Egypt. COMPARATIVE PARASITOLOGY vol. 77, 137-144.
LA FERLA B, CERVI G, NICOTRA F, PAPEO G, FELDER ER (2010)
Synthesis of a ß-carboline scaffold properly functionalysed for the
generation of libraries of bioactive compounds. SYNTHESIS vol.4;
p. 601-604.
LA FERLA B, SPINOSA V, D’ORAZIO G, PALAZZO M, BALSARI A,
FOPPOLI AA, RUMIO C, NICOTRA F (2010) Dansyl C-Glucoside as
a Novel Agent Against Endotoxic Shock. CHEM MED CHEM vol. 5;
p. 1677–1680.
LABRA M, DE MATTIA F, BERNASCONI M, BERTACCHI D, GRASSI
F, BRUNI I, CITTERIO S (2010) The Combined Toxic and Genotoxic
Effects of Chromium and Volatile Organic Contaminants to Pseudokirchneriella subcapitata. WATER AIR SOIL POLLUT vol. 213; p.
57-70.
LONGHESE MP, BONETTI D, MANFRINI N, CLERICI M (2010) Mechanisms and regulation of DNA end resection. EMBO J. 29: 2864-74.
MACIAS A, MORENO C, MORAL-SANZ J, COGOLLUDO A, DAVID
M, ALEMANNI M, PEREZ-VIZCAINO F, ZAZA A, VALENZUELA
C, GONZALEZ T (2010) Celecoxib blocks cardiac Kv1.5, Kv4.3 and
Kv7.1 (KCNQ1) channels: effects on cardiac action potentials. J MOL
CELL CARDIOL vol. 49; p. 984-992.
MALERBA M, CROSTI P, CERANA R (2010) Effect of heat stress
on actin cytoskeleton and endoplasmic reticulum of tobacco BY-2
cultured cells and its inhibition by Co2+. PROTOPLASMA vol. 239; p.
23–30.
MALERBA M, CROSTI P, CERANA R (2010) Ethylene is involved in
stress responses induced by fusicoccin in sycamore cultured cells.
JOURNAL OF PLANT PHYSIOLOGY vol. 167, p. 1442–1447.
MALFATTO G, ROCCHETTI M, ZAZA A (2010) The role of the autonomic system in rate-dependent repolarization changes. HEART
RHYTHM vol. 7; p. 1700-1703.
MANFRINI N, GUERINI I, CITTERIO A, LUCCHINI G, LONGHESE
MP (2010) Processing of meiotic DNA double strand breaks requires cyclin-dependent kinase and multiple nucleases. J BIOL
CHEM. 285: 11628-37.
MARCHESE R, GRANDORI R, CARLONI P, RAUGEI S (2010) On
the zwitterionic nature of gas-phase peptides and protein ions
PLOS COMPUTATIONAL BIOLOGY vol. 6; p. e1000775.
MAZZUCCHELLI S, COLOMBO M, DE PALMA C, SALVADÈ A,
VERDERIO P, COGHI MD, CLEMENTI E, TORTORA P, CORSI F,
PROSPERI D (2010) Single-domain protein A-engineered magnetic nanoparticles: toward a universal strategy to site-specific
labeling of antibodies for targeted detection of tumor cells. ACS
NANO vol. 4(10), p. 5693-5702.
MEREGHETTI P, RICCARDI L, BRANDSDAL BO, FANTUCCI P, DE
GIOIA L, PAPALEO E (2010) Near native-state conformational landscape of psychrophilic and mesophilic enzymes: probing the folding funnel model. J PHYS CHEM B vol. 114 (22); p. 7609-7619.
METALLI D, LOVAT F, TRIPODI F, GENUA M, XU SQ, SPINELLI
M, ALBERGHINA L, VANONI M, BAFFA R, GOMELLA LG, IOZZO
RV, MORRIONE A (2010) The insulin-like growth factor receptor
I promotes motility and invasion of bladder cancer cells through
Akt- and mitogen-activated protein kinase-dependent activation
of paxillin. AM J PATHOL vol. 176(6), p. 2997-3006.
MIDDELDORP J, BOER K, SLUIJS JA, DE FILIPPIS L, ENCHARAZAVI F, VESCOVI AL, SWAAB DF, ARONICA E, HOL EM (2010)
GFAPdelta in radial glia and subventricular zone progenitors in the
developing human cortex. DEVELOPMENT vol.137(2), p. 313-21.
MONTANO S, SEVESO D, GALLI P, OBURA D (2010) Assessing
coral bleaching and recovery with a colour reference card in Watamu Marine Park, Kenya. HYDROBIOLOGIA vol. 655; p. 99-108.
MORASSO C, COLOMBO M, RONCHI S, POLITO L, MAZZUCCHELLI S, MONTI D, BUSCAGLIA M, BELLINI T, PROSPERI D (2010)
Towards a universal method for the stable and clean functionalization of inert perfluoropolymer nanoparticles: exploiting photopolymerizable amphiphilic diacetylenes. ADV FUNCT MATER vol.
20; p. 3932-3940.
NÉMETI B, REGONESI ME, TORTORA P, GREGUS Z (2010) Polynucleotide phosphorylase and mitochondrial ATP synthase mediate reduction of arsenate to the more toxic arsenite by forming
arsenylated analogues of ADP and ATP. TOXICOL SCI vol. 117(2),
p. 270-281.
NERI M, MADERNA C, FERRARI D, CAVAZZIN C, VESCOVI AL,
GRITTI A (2010) Robust generation of oligodendrocyte progenitors
from human neural stem cells and engraftment in experimental
demyelination models in mice. PLOS ONE vol. 5(4); p. e10145.
OCCHIPINTI E, VERDERIO P, NATALELLO A, GALBIATI E, COLOMBO M, MAZZUCCHELLI S, SALVADÈ A, TORTORA P, DOGLIA
SM, PROSPERI D (2010) Investigating the structural biofunctionality of antibodies conjugated to magnetic nanoparticles. NANOSCALE vol. 3; p. 387-390.
ORAIN PY, CAPON JF, GLOAGUEN F, PETILLON FY, SCHOLL-
[ 53 ]
[3.1] PUBLICATIONS
HAMMER P, TALARMIN J, ZAMPELLA G, DE GIOIA L, ROISNEL T
(2010) Investigation on the Protonation of a Trisubstituted [Fe-2(CO)(3)(PPh3)(kappa(2)-phen)(mu-pdt)] Complex: Rotated versus
Unrotated Intermediate Pathways. INORGANIC CHEMISTRY vol.
49 (11); p. 5003-5008.
ORLANDI I, BETTIGA M, ALBERGHINA L, NYSTRÖM T, VAI M (2010)
Sir2-dependent asymmetric segregation of damaged proteins in
ubp10 null mutants is independent of genomic silencing. BIOCHIMICA BIOPHYSICA ACTA (MOL CELL RES) vol. 1803; p. 630-638.
OSTUNI R, ZANONI I, GRANUCCI F (2010) Deciphering the complexity of Toll-like receptor signaling. CELL MOL LIFE SCI vol.
67(24), 4109-4134.
PALUMBO P, MAVELLI G, FARINA L, ALBERGHINA L (2010)
Networks and circuits in cell regulation. BIOCHEM BIOPHYS RES
COMMUN vol. 396(4), p. 881-886.
PAPALEO E, RUSSO L, SHAIKH N, CIPOLLA L, FANTUCCI P, DE
GIOIA L (2010) Molecular dynamics investigation of cyclic natriuretic peptides: Dynamic properties reflect peptide activity.
JOURNAL OF MOLECULAR GRAPHICS & MODELLING vol. 28 (8);
p. 834-841.
PASSOLUNGHI S, RIBOLDI L, DATO L, PORRO D, BRANDUARDI P
(2010) Cloning of the Zygosaccharomyces bailii GAS1 homologue
and effect of cell wall engineering on protein secretory phenotype.
MICROBIAL CELL FACTORIES vol. 9; p. 4512-4513.
PASTORI V, SANGALLI E, COCCETTI P, POZZI C, NONNIS S, TEDESCHI G, FUSI P (2010) CK2 and GSK3 phosphorylation on S29
controls wild-type ATXN3 nuclear uptake. BIOCHIMICA BIOPHYSICA ACTA vol. 1802(7-8); p. 583-592.
PERI F, PIAZZA M, CALABRESE V, DAMORE G, CIGHETTI R (2010)
Exploring the LPS/TLR4 signal pathway with small molecules.
BIOCHEM SOC TRANS vol. 38(5), p. 1390-1395.
PESSINA S, TSIARENTSYEVA V, BUSNELLI S, VANONI M, ALBERGHINA L, COCCETTI P (2010) Snf1/AMPK promotes S phase entrance by controlling CLB5 transcription in budding yeast.
CELL CYCLE vol. 9(11); p. 2189-2200.
PIAZZA M, CALABRESE V, BARUFFA C, GIOANNINI T, WEISS J,
PERI F (2010) The cationic amphiphile 3,4-bis(tetradecyloxy)benzylamine inhibits LPS signaling by competing with endotoxin for
CD14 binding. BIOCHEM PHARMACOL vol. 80; p. 2050-2056.
PIAZZA M, COLOMBO M, ZANONI I, GRANUCCI F, TORTORA P,
WEISS J, GIOANNINI T, PROSPERI D, PERI F (2010) Uniform
Lipopolysaccharide (LPS)-Loaded Magnetic Nanoparticles for
the Investigation of LPS-TLR4 Signaling. ANGEW CHEM INT ED
ENGL 50(3):622-626
RE F, AIROLDI C, ZONA C, MASSERINI M, LA FERLA B, QUATTROCCHI N, NICOTRA F (2010) Beta Amyloid Aggregation Inhibitors: Small Molecules as Candidate Drugs for Therapy of Alzheimer’s Disease. CURR MED CHEM vol. 17, p. 2990-3006.
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REDAELLI E, RESTANO-CASSULINI R, FUENTES-SILVA D, CLEMENT H, SCHIAVON E, ZAMUDIO FZ, ODELL G, ARCANGELI A,
CLARE JC, ALAGON A, RODRÍGUEZ DE LA VEGA RC, POSSANI
LD, WANKE E (2010) Target promiscuity and heterogeneous effects of tarantula venom peptides affecting ion channels. J BIOL
CHEM vol. 285; p. 4130-4142.
ROSSI G, SAUER M, PORRO D, BRANDUARDI P (2010) Effect of
HXT1 and HXT7 hexose transporter overexpression on wild-type
and lactic acid producing Saccharomyces cerevisiae cells. MICROBIAL CELL FACTORIES vol. 9; p. 15.
ROTA NODARI L, FERRARI D, GIANI F, BOSSI M, RODRIGUEZMENENDEZ V, TREDICI G, DELIA D, VESCOVI AL, DE FILIPPIS
L (2010) Long-term survival of human neural stem cells in the
ischemic rat brain upon transient immunosuppression. PLOS
ONE vol. 5(11); p. e14035.
RUEPP M-D, VIVARELLI S, PILLAI RS, KLEINSCHMIDT N, AZZOUZ TN, BARABINO SML, SCHÜMPERLI D (2010) Association
of the 68 kDa subunit of mammalian cleavage factor I with the
U7 snRNP: possible role in 3’ end processing of animal histone
mRNAs. NUCL ACIDS RES vol. 38; p.7637-7650.
RYDE U, GRECO C, DE GIOIA L (2010) Quantum Refinement of
[FeFe] Hydrogenase Indicates a Dithiomethylamine Ligand.
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY vol. 132 (13);
p. 4512-4513.
SACCHERI F, POZZI C, MITTAL D, AVOGADRI F, BAROZZI S, FARETTA M, FUSI P, RESCIGNO M (2010) Bacteria induced gap junctions in tumors favor antigen cross-presentation and anti-tumor
immunity. SCIENCE TRANSLATIONAL MEDICINE vol. 11, 2(44);
p. 44-57.
SAINO N, ROMANO M, CAPRIOLI M, AMBROSINI R, RUBOLINI
D, FASOLA M (2010) Sex allocation in yellow-legged gulls (Larus
michahellis) depends on nutritional constraints on production of
large last eggs. PROCEEDINGS OF THE ROYAL SOCIETY OF LONDON - SERIES B vol. 277; p.1203-1208.
SAINO N, ROMANO M, RUBOLINI D, CAPRIOLI M, AMBROSINI
R, FASOLA M (2010) Food supplementation affects egg albumen
content and body size asymmetry among yellow-legged gull siblings. BEHAVIORAL ECOLOGY AND SOCIOBIOLOGY vol. 64; p.
1813-1821.
SAINO N, RUBOLINI D, SERRA L, CAPRIOLI M, MORGANTI M,
AMBROSINI R, SPINA F (2010) Sex-related variation in migration
phenology in relation to sexual dimorphism: a test of competing
hypotheses for the evolution of protandry. JOURNAL OF EVOLUTIONARY BIOLOGY vol. 23; p. 2054-2065.
SAINO N, RUBOLINI D, VON HARDENBERG J, AMBROSINI R,
PROVENZALE A, ROMANO M, SPINA F (2010) Spring migration
decision in relation to weather are predicted by wing morphology among trans-Mediterranean migratory birds. FUNCTIONAL
ECOLOGY vol. 24; p. 658-669.
SALAZAR C, BRÜMMER A, ALBERGHINA L, HÖFER T (2010) Timing control in regulatory networks by multisite protein modifications. TRENDS CELL BIOL vol. 20(11); p. 634-641.
SAMBI I, GATTI-LAFRANCONI P, LONGHI S, LOTTI M (2010) How
disorder influences order and viceversa: mutual effects in fusion
proteins containing an intrinsically disordered and a globular protein. FEBS J vol. 277; p. 4438-4451.
SANTAMBROGIO C, RICAGNO S, COLOMBO M, BARBIROLI A, BONOMI F, BELLOTTI V, BOLOGNESI M, GRANDORI R (2010) DE-loop
mutations affect ß2 microglobulin stability, oligomerization and the
low-pH unfolded form. PROTEIN SCIENCE vol. 19; p. 1386-1394.
SANTILLI G, LAMORTE G, CARLESSI L, FERRARI D, ROTA NODARI L, BINDA E, DELIA D, VESCOVI AL, DE FILIPPIS L (2010)
Mild hypoxia enhances proliferation and multipotency of human
neural stem cells. PLOS ONE vol. 5; p. e8575.
SAUER M, PORRO D, BRANDUARDI P (2010) 16 years research on lactic acid production with yeast – ready for the market? BIOTECHNOLOGY & GENETIC ENGINEERING REVIEWS
vol. 27; p. 1-28.
SCHIAVON E, STEVENS M, ZAHARENKO A, KONNO K, TYTGAT J,
WANKE E (2010) Voltage-gated Sodium Channels Isoform-specific Effects of Pompilidotoxins. FEBS J vol. 277; p. 918-30.
SHAIKH N, RUSSO L, PAPALEO E, GIANNONI P, DE GIOIA L, NICOTRA F, QUARTO R, CIPOLLA L (2010) C-Type Natriuretic Peptide: Structural Studies, Fragment Synthesis, and Preliminary
Biological Evaluation in Human Osteosarcoma Cell Lines. BIOPOLYMERS vol. 94 (2); p. 213-219.
STRONA G, STEFANI F, GALLI P (2010) Monogenoidean parasites
of Italian marine fish: an updated checklist. ITALIAN JOURNAL
OF ZOOLOGY vol. 77; p. 419-437.
TARABALLI F, NATALELLO A, CAMPIONE M, VILLA O, DOGLIA
SM, PALEARI A, GELAIN F (2010) Glycine-spacers influence
functional motifs exposure and self-assembling propensity of
functionalized substrates tailored for neural stem cell cultures.
FRONT NEUROENGINEERING vol. 3:1.
TORRI A, BERETTA O, RANGHETTI A, GRANUCCI F, RICCIARDICASTAGNOLI P, FOTI M (2010) Gene expression profiles identify
inflammatory signatures in dendritic cells. PLOS ONE vol. 5(2);
p. e9404.
TRIPODI F, CIRULLI C, REGHELLIN V, MARIN O, BRAMBILLA
L, SCHIAPPELLI MP, PORRO D, VANONI M, ALBERGHINA L,
COCCETTI P (2010) CK2 activity is modulated by growth rate in
Saccharomyces cerevisiae. BIOCHEM BIOPHYS RES COMMUN
vol. 398(1); p. 44-50.
UHLICH NA, NATALELLO A, KADAM RU, DOGLIA SM, REYMOND
JL, DARBRE T (2010) Structure and binding of peptide-dendrimer
ligands to vitamin B12. CHEMBIOCHEM vol. 11; p. 358-365.
VALSECCHI E, CORKERON PJ, GALLI P, SHERWIN W, BERTORELLE G (2010) Genetic evidence for sex-specific migratory
behaviour in western South Pacific humpback whales. MARINE
ECOLOGY PROGRESS SERIES vol. 398; p. 275–286.
VILLA CE, CACCIA M, SIRONI L, D’ALFONSO L, COLLINI M, RIVOLTA I, MISEROCCHI G, GORLETTA T, ZANONI I, GRANUCCI
F, CHIRICO G (2010) Accumulative difference image protocol for
particle tracking in fluorescence microscopy tested in mouse
lymphonodes. PLOS ONE vol. 5(8); p. e12216.
WANG S, CHANDLER-MILITELLO D, LU G, ROY NS, ZIELKE A,
STANWOOD N, NICOLIS SK, GESCHWIND D, COPPOLA G, SIM
FJ, GOLDMAN SA (2010) Prospective identification, isolation, and
profiling of a telomerase expressing subpopulation of human
neural stem and progenitor cells, using Sox2 enhancer-directed
FACS. JOURNAL OF NEUROSCIENCE vol. 30; p. 14635-14648.
XIAO ZY, XU FF, LONG L, LIU YQ, ZAMPELLA G, DE GIOIA L, ZENG
XR, LUO QY, LIU XM (2010) Influence of the basicity of internal
bases in diiron model complexes on hydrides formation and their
transformation into protonated diiron hexacarbonyl. JOURNAL
OF ORGANOMETALLIC CHEMISTRY vol. 695 (5); p. 721-729.
YING M, SANG Y, LI Y, GUERRERO-CAZARES H, QUINONESHINOJOSA A, VESCOVI AL, EBERHART CG, XIA S, LATERRA J
(2010) KLF9, A Differentiation-Associated Transcription Factor,
Suppresses Notch1 Signaling and Inhibits Glioblastoma-Initiating
Stem Cells. STEM CELLS [Epub ahead of print]
ZAMPELLA G, FANTUCCI P, DE GIOIA L (2010) DFT characterization of the reaction pathways for terminal- to -hydride isomerisation in synthetic models of the [FeFe]-hydrogenase active site.
CHEM COMMUN (CAMB) vol. 46 (46); p. 8824-8826.
ZANINI S, RICCARDI C, GRIMOLDI E, COLOMBO C, VILLA AM,
NATALELLO A, GATTI-LAFRANCONI P, LOTTI M, DOGLIA SM
(2010) Plasma-induced graft-polymerization of polyethylene glycol acrylate on polypropylene films: chemical characterization
and evaluation of the protein adsorption. JOURNAL OF COLLOID
& INTERFACE SCIENCE vol. 341; p. 53–58.
ZANONI I, GRANUCCI F (2010) Regulation of antigen uptake, migration, and lifespan of dendritic cell by Toll-like receptors. J MOL
MED vol. 88(9), p. 873-880.
ZAZA A (2010) Control of the cardiac action potential: The role of
repolarization dynamics. J MOL CELL CARDIOL vol. 48; p. 106-111.
ZECCA G, DE MATTIA F, LOVICU G, LABRA M, GRASSI F (2010)
Wild grapevine: silvestris, hybrids or cultivar escaped from vineyards? Morphological and molecular evidence in Sardinia. PLANT
BIOLOGY vol.12; p. 558–562.
ZHONG W, TANG Y, ZAMPELLA G, WANG XF, YANG XL, HU B,
WANG JA, XIAO ZY, WEI ZH, CHEN HW, DE GIOIA L, LIU XM
(2010) A rare bond between a soft metal (Fe-l) and a relatively
hard base (RO-, R = phenolic moiety). INORGANIC CHEMISTRY
COMMUNICATIONS vol. 13 (9); p. 1089-1092.
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[3.2] BOOK CHAPTERS
BECCHETTI A, ARCANGELI A (2010) Integrin receptors and Ion
Channels. Molecular Complexes and Signaling. Springer Science
and Landes Bioscience (Eds.).
COLANGELO AM, ALBERGHINA L (2010) Apoptotic Mechanisms
Involved in Neurological Disorders. In: Modern Insights Into Disease
From Molecules to Man: Apoptosis (Preedy VR, Ed.) Science Publishers Inc.
KONNO K, SCHIAVON E, STEVENS M, ZAHARENKO A, TYTGAT J,
WANKE E, KAWAI N (2010) Pompilidotoxin, petides neurotoxins in
solitary wasp venom blocking sodium channel inactivation. Peptide
Science 2009: K. Okamoto (Ed).
LECCHI M, HODA JC, HOGG RC, BERTRAND D (2010) Extracellular
ligand-gated ion channels: Cys-loop receptors - Nicotinic acetylcholine receptors” in Ion channels: Structure and function, edited
by James Kew and Ceri Davies, Oxford University Press.
NATALELLO A, DOGLIA SM (2010) Intrinsically disordered proteins
and induced folding studied by Fourier transform infrared spectroscopy” in Instrumental Analysis of Intrinsically Disordered Proteins:
Assessing Structure and Conformation (Vladimir N. Uversky and
Sonia Longhi, Eds.). In The Wiley Protein and Peptide Science Book
Series (Uversky V.N. series Ed.). John Wiley & Sons, Inc., Hoboken,
New Jersey-USA. pp. 225-252.
ŠAMALIKOVA M, SANTAMBROGIO C, GRANDORI R (2010) Massspectometry tools for the investigation of structural disorder and
conformational transitions in proteins” in “Assessing structures
and conformations of intrinsically disordered proteins – instrumental foundation of experimental unfoldomics” pp 629-652. Uversky V.
and Longhi S. (Eds.), John Wiley and Sons.
[3.3] PATENTS
ANTIMISIARIS S, MOURTAS S, NIARAKIS A, NICOTRA F, LA
FERLA B, ZONA C, MASSERINI M Novel curcumin derivatives with improved physicochemical properties and surfacedecorated Nanoliposomes (with the derivatives) with very
high affinity for Amyloid-β1-42 peptide. Publication info:
202000100563 publication date 30_09_2010
MAGNANI M, BARTOLUCCI E, PORRO D, BRANDUARDI P,
CODAZZI V, BENATTI U, DAMONTE G, SHIPPA G, BIANCHINI
S Sviluppo di una cell factory ricombinante per la produzione
di glucobrassicina. Publication info: RM2010R000142 publi-
[ 56 ]
cation date 24_02_2010
NICOTRA F, AIROLDI C, LA FERLA B, JIMENEZ-BARBERO J,
GIOVANARDI S Nuova metodologia RMN (Risonanza Magnetica Nucleare) per la caratterizzazione di interazioni recettore
di membrana–ligando. Publication info: n. RM2010A000647
publication date 09_12_2010
PORRO D, DATO L, BRANDUARDI P Method for improving acid
and low pH tolerance in yeast. Publication info: EP2162528
publication date 17_03_2010
[3.4] RESEARCH GRANTS AND CONTRACTS
ALBERGHINA L. Eukaryotic unicellular organism biology – systems biology of the control of cell growth and proliferation. FP7,
European Commission
ALBERGHINA L. ITALBIONET - Rete Italiana di Bioinformatica.
FIRB, MIUR
ALBERGHINA L. Yeast Systems Biology Network. European
Commission
BARABINO S. Chromatin-dependent regulation of alternative
pre-mRNA splicing in cellular models of neurodegeneration.
PRIN 2008, MIUR
GALLI P. Bioinspired Adhesives for surgery. Fondazione Cariplo
GRANUCCI F. Dendritic Cells for Novel Immunotherapies. European Commission
GRANUCCI F. European Network for Cell Imaging and Tracking
Expertise. FP7, European Commission
GRANUCCI F. Key regulators of DC-primed anti-tumor NK cellfunctions. Associazione Italiana Ricerca sul Cancro
GRANUCCI F. LIIN Lombard Innate Immunity network. Regione
Lombardia
BARABINO S. Genomica e proteomica del processamento degli
RNA mesaggeri nella sclerosi laterale amiotrofica. Fondazione
Cariplo
GRANUCCI F. Meccanismi d’induzione di tolleranza in cellule T
autoreattive coinvolte nella risposta autoimmune presente nella
Cheratite Erpetica Stremale. Fondazione Cariplo.
BRANDUARDI P. Systems Biology as a Driver for Industrial Biotechnology. FP7, European Commission
GRANUCCI F. Normalisation of immune reactivity in old age –
from basic mechanisms to clinical application. FP7, European
Commission
CASIRAGHI M. Sviluppo di un sistema di identificazione molecolare di organismi target della fauna del suolo. PRIN, MIUR
CASTAGNOLI P. Integrated functional genomics in mutant mouse
models as tools to investigate the complexity of human immunological disease. European Commission
CHIARADONNA F. Transcriptional profiling, proliferation and
metabolic analysis of 3AB-OS cancer stem cells as compared to
MG-63 cells. PRIN 2008, MIUR
CIPOLLA L. Funzionalizzazione di biomateriali nanostrutturati
per il trattamento dei difetti cartilaginei articolari. Fondazione
Cariplo
CIRULLI C. Genomica funzionale e disfunzioni patologiche dei sistemi redox e bioenergetici cellulari. FIRB FUTURO E RICERCA,
MIUR
COLANGELO A. Processo di scale-up per la produzione di Nerve
Growth Factor ricombinante umano (rhNGF) in cellule di mammifero, purificazione e caratterizzazione molecolare. Analisi in vitro
dell’attività biologica di rhNGF in sistemi cellulari. PRIN, MIUR
COSTA B. Ruolo spinale e sovraspinale di citochine e BDNF nel
dolore neuropatico e loro modulazione dopo trapianto di cellule
staminali mesenchimali umane nella corteccia agranulare insulare. PRIN, MIUR
DOGLIA SILVIA MARIA. Processi di funzionalizzazione dei polimeri per la modifica della biocompatibilità d e dell’adesione di
proteine. Fondazione Cariplo
FANTUCCI P. Molecular modelling of the interactions between
copper ions or ligands and proteins involved in angiogenesis processes. PRIN 2008, MIUR
FOTI M. Generation of a coronavirus-based multigene AIDS vaccine and evaluation in a preclinical SIV model. European Commission
FOTI M. Identificazione dei meccanismi molecolari indotti in cellule dendritiche da batteri commensali e patogeni importanti nella polarizzazione di linfociti T. PRIN, MIUR
FRASCHINI R. Identification and functional characterization of
budding yeast genes involved in cytokinesis regulation. PRIN
2008, MIUR
GRANUCCI F. Ruolo delle cellule dendritiche nell’attivazione delle funzioni anti-tumorali delle cellule NK: meccanismi cellulari e
molecolari. PRIN, MIUR
LABRA M. Acqua e compost grandi amici. Fondazione Cariplo
LABRA M. Acqua in BROCCA. Fondazione Cariplo
LABRA M. Connessione ecologica e rinaturazione nel sistema
della aree protette del nord milanese. Fondazione Cariplo
LABRA M. Dai geni dell’ecosistema: il DNA barcoding come supporto innovativo per la protezione della biodiversità e l’analisi della funzionalità delle reti ecologiche. Fondazione Cariplo
LABRA M. Development of DNA barcoding approach to identify
plant species and seed bank samples. PRIN 2008, MIUR
LABRA M. Diagnostica molecolare avanzata per il settore agroalimentare. CORI S.R.L.
LABRA M. Il corridoio ecologico del Lambro: interventi per il consolidamento e l’implementazione della connettività e della biodiversità. Fondazione Cariplo
LABRA M. Insetti Pronubi: mezzi di connessione e diffusione di
specie vegetali rare ed endemiche del Parco regionale della Grigna settentrionale. Fondazione Cariplo
LABRA M. Le connessioni ecologiche nelle selve castanili nel parco regionale Campo dei Fiori. Fondazione Cariplo
LABRA M. Tutela della biodiversità con azioni di riqualificazione
e valorizzazione di praterie su suolo calcareo (Festuco Brometalia) nei SIC Monte Sangiano e Monti della Valcuvia. Fondazione
Cariplo
LONGHESE M.P. Cellular response to accidental and natural DNA
interruptions. PRIN 2008, MIUR
LONGHESE M.P. Cross talks between telomere protection and
DNA damage checkpoints: two endogenous anti-cancer barriers
Associazione Italiana Ricerca sul Cancro
LONGHESE M.P. High Resolution Microscopy in the DNA damage
Response. FP7, PEOPLE-2007, European Commission
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[3.4] RESEARCH GRANTS AND CONTRACTS
LOTTI M. Diesel Biotech. ASTIL, Regione Lombardia
acidi grassi per la produzione di monomeri da fonte rinnovabile
utilizzabili nella sintesi di polimeri biodegradabili. Novamont
LOTTI M. Valorizzazione delle risorse biologiche: sviluppo di nuove
tecnologie per l’identificazione, caratterizzazione e produzione di
molecole di interesse farmaceutico ed industriale presenti nelle
Brassicacee” Fondo per le Agevolazioni alla Ricerca (FAR). MIUR
PORRO D., BRANDUARDI P. Selezione di ceppi di Escherichia coli
ricombinanti, sviluppo delle corrispondenti banche cellulari e fermentazioni. EXPLORA LABORATORIES SA
LUCCHINI G. Fattori di checkpoint coinvolti nel controllo dell’omeostasi telomerica nel lievito S. cerevisiae. PRIN, MIUR
SPERANDEO P. Outer membrane Biogenesis in Gram Negative.
Fondazione Cariplo
MARTEGANI E. Regulation of TrkA receptor, interaction with
UBPy, endosomal sorting and retrograde tran sport. PRIN 2008,
MIUR
TORTORA P. Caratterizzazione biofisica e biochimica dei processi
di aggregazione in vitro e in vivo di varianti di proteine ricombinanti che contengono poliglutammine. PRIN, MIUR
NICOLIS S. A Genetic toolkit for analysis of mouse neural stem
cells (NS-Toolkit). Progetto Nobel Fondazione Cariplo
TORTORA P. Genoproteomics of Age Related Disorders (GUARD).
Progetto Nobel Fondazione Cariplo.
NICOLIS S. Genetic approaches to the study of the role of the Sox2
transcription 11factor in cancer neural stem cells. Associazione
Italiana Ricerca sul Cancro
TORTORA P. Network tecnologico integrato per lo studio proteomico e transcrittomico di malattie neurodegenerative correlate a
deposizione di amiloidi. Ministero della Sanità
NICOLIS S. Ruoli funzionali del fattore trascrizionale Sox2. Fondazione Cariplo
VANONI M. Identificazione di Proteine rilevanti per la trasformazione tumorale e ruolo della loro fosforilazione nell’instaurarsi
del fenotipo tumorale. PRIMM s.r.l.
NICOLIS S. 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, MIUR
NICOLIS S. Ultra High-throughput DNA sequencing. ASTIL, Regione Lombardia
NICOTRA F. Development of NMR techniques for tissue engineering studies. Fondazione Cariplo
NICOTRA F. NEDD. Regione Lombardia
NICOTRA F. New antibacterial agents via overexpression, characterisation and inhibition of enzymes involved in LPS biogenesis.
PRIN 2008, MIUR
NICOTRA F. Piattaforma integrata per la progettazine e produzione high throughput di enzimi e peptidi ingegnerizzati. Valutazione
della loro attività biologica rispetto a specifici substrati molecolari
d’interesse farmaceutico, con riferimento allo screening di prodotti oncologici ed alla produzione di antibiotici e nutraceutici.
Metadistretti, Regione Lombardia
VESCOVI A.L. Cellule staminali neurali umane e biomateriali nanostrutturati per la medicina rigenerativa. Fondazione Cariplo
VESCOVI A.L. Cis-regulatory logic of the transcriptional control in
neural stem cells. FP7, European Commission
VESCOVI A.L. Functional genomics of the retina in health and disease. European Commission
VESCOVI A.L. Pluripotency associated genes to de-differentiate
neural cells into pluripotent cells. European Commission
VESCOVI A.L. Towards the Neuronal Machines. European Commission
VESCOVI A.L. Tumor neural stem cells in the in vitro and in vivo
modeling and studying of the adult human glioblastomas. Associazione Italiana Ricerca sul Cancro
WANKE E. Basi molecolari di canalopatie e analisi funzionale in
singoli neuroni e in reti neuronali. PRIN, MIUR
PERI F. Synthesis of galectin ligands and their assembly on magnetic nanoparticles (MNP). PRIN 2008, MIUR
WANKE E. Functional Regeneration of the Mesocorticolimbic Dopaminergic System as a Modelto Study neuro-reparative startegies. Fondazione Cariplo
PIATTI S. Molecular mechanismpreventing the occurence faneuploidya hallmark of cancer cells. Associazione Italiana Ricerca
sul Cancro
ZANONI I. Role of interleukin 2 and probiotics in modulating cancer immunosurveillance: identification of new therapeutic strategies. Fondazione Cariplo
POLISSI A. Innovazione nei processi di controllo della qualità per
la creazione di valore nell’industria cosmetica. FAR.CO.S. s.r.l.
ZAZA A. Valutazione “in vitro” dell’effetto del COMPOSTO sull’entità e dinamica della contrazione e del rilasciamento di cardiomiociti di cavia isolati. ROSTAQUO S.p.A
POLISSI A. Rational Drugs Design. ASTIL, Regione Lombardia
PORRO D. Novel high performance enzymes and micro-organisms for conversion of lignocellulosic biomass to bioethanol. FP7,
European Commission
ZAZA A. Ruolo della corrente di Na+ persistente nel danno miocardico indotto da ipossia cronica. PRIN, MIUR
ZAZA A. Terapia genica in cellule staminali cardiache in vitro per
la correzione di una cardiomiopatia ereditaria. Fondazione Cariplo
PORRO D. Piattaforma di biotecnologie verdi e di tecniche gestionali per un sistema agricolo ad elevata sostenibilità. Regione
Lombardia
ZAZA A. Valutazione “in vitro” dell’effetto del COMPOSTO. PRASSIS – Istituto di Ricerche Sigma-Tau
PORRO D., BRANDUARDI P. Programma di ricerca nel settore
delle biotecnologie sui microrganismi e della ossidazione degli
ZULLINI A. Nematodi a vita libera come mezzo di valutazione della qualita’ ambientale e di fertilita’ dei suoli. PRIN, MIUR
[ 58 ]
[3.5] PhD THESES
Bigi Alessandra “Characterization of human sialidase neu4:
role of the proline-rich region in signal transduction”. PhD in
Biology. Tutor: P. Fusi
Ostuni Renato “The role of CD14-NFAT pathway in the regulation of innate immune functions” PhD in Translational and
Molecular Medicine. Tutor: F. Granucci
Balestrieri Chiara “High-throughput bioinformatic approaches to study tumorigenesis in mammalian cells”. PhD in Industrial Biotechnology. Tutor: F. Chiaradonna
Pastori Valentina “Role of phosphorylation of ataxin-3 and
oxidative stress in the pathogenesis of spinocerebellar ataxia
type 3 (sca3)”. PhD in Biology. Tutor: P. Fusi
Bodio Caterina “Mechanisms of DC-mediated Nk cell activation” PhD in Translational and Molecular Medicine. Tutor: F.
Granucci
Caccia Roberta “Defects in neuronal differentiation and
axonal connectivity in mice mutant in the Sox2 transcription
factor gene: in vitro and in vivo studies” PhD in Translational
and Molecular Medicine. Tutor: S. Nicolis
Rigamonti Valeria “Development of a quantitative chemiluminescent immunoassay for the hepatitis B antigen detection”
PhD in Industrial Biotechnology. Tutor: F. Peri
Rossi Giorgia “Metabolic opportunities offered by wild-type
and engineered Saccharomyces cerevisiae strains for biofuels
production”. PhD in Industrial Biotechnology. Tutor: D. Porro
Cantù Claudio “Sox6 transcription factor: its role in human
and murine erythroid differentiation and mechanisms for its
regulation”. PhD in Translational and Molecular Medicine. Tutor: A. Ronchi
Santambrogio Carlo “The aggregation mechanism of
amyloid proteins studied by mass spectrometry and other biophysical techniques”. PhD in Physics. Tutor: R. Grandori
Cappellini Daniele “An evaluation of an innovative technological platform for straightforward purification of recombinant proteins” PhD in Biology. Tutor: P. Tortora
Shaikh Nasrin “Design & Synthesis of relevant biomolecules for functionalization of biomaterials in tissue engineering’’.
PhD in Chemistry. Tutor: F. Nicotra
Codazzi Vera “Breaking phylogenetic barriers for fine and bulk
chemical products in engineered Saccharomyces cerevisiae”.
PhD in Industrial Biotechnology. Tutor: P. Branduardi
Matteo Sicolo “Negative effects on a bioindicator by electromagnetic field exposures alone, and in combination with
UVC rays” PhD in Biology. Tutor: A. Santagostino
Galati Elena “Yeast response to prolonged activation of the
spindle assembly checkpoint”. PhD in Industrial Biotechnology. Tutor: S. Piatti
Viganò Matteo “Yeast cell size control: an interplay among
ribosome biogenesis, protein synthesis and MAPK routes”.
PhD in Industrial Biotechnology. Tutor: M.Vai
Galimberti Andrea “DNA barcoding: a link between basic
and applied research” PhD in Biology. Tutor: M. Casiraghi
Gotti Laura “Nutritional modulation of cell size at S-phase
initiation in the budding yeast Saccharomyces cerevisiae: a role
for glucose sensing and the cyclin-dependent kinase inhibitor
FAR1”. PhD in Industrial Biotechnology. Tutor: M. Vanoni
Manfrini Nicola “Maintenance of genome integrity on gametes: coping with accidental and programmed DNA double-strand breaks during meiosis”. PhD in Biology. Tutor: MP
Longhese
Vivarelli Silvia “New roles for the RNA processing factors
CFIm and SRPK2 highlight unexpected links in the control
of mammalian gene expression”. PhD in Translational and
Molecular Medicine. Tutor: S. Barabino
Zona Cristiano “Design and Synthesis of Nanoparticles for
Therapy and Imaging of Alzheimer’s Disease” . PhD in Chemistry.
Tutor: F. Nicotra
Marangoni Stefano ”A Brugada Syndrome mutation (p.S216L)
and its modulation by p.H558R polymorphism: standard and dynamic characterization. “ PhD in Biology. Tutor: A. Zaza
Mazzantini Elisa” Polynuclotide phosphorylase from Escherichia coli: regulation mechanisms and substrate binding. PhD
in Biology. Tutor: P. Tortora
Mazzucchelli Serena ”Studies on the mechanism and
physiological role(s) of the interaction of ataxin-3 with tubulin.”
PhD in Biology. Tutor: P.Tortora
Merlini Laura “Cell cycle regulation of septins: implications
for cytokinesis and the spindle position checkpoint” PhD in Industrial Biotechnology. Tutor: S. Piatti
Missaglia Sara “Molecular genetics of familial tubulopathies: Claudin-16 and Claudin-19 Mutations in Familial Hypomagnasemia, Hypercalciuria,and Nephrocalcinosis” PhD in
Biology. Tutor: P. Tortora
Orsato Alexandre “Studies on new antitumor drug targeting” . PhD in Chemistry. Tutor: F. Nicotra
[ 59 ]
Dipartimento di Biotecnologie e Bioscienze
Università degli Studi di Milano Bicocca
Piazza della Scienza 2, 20126 Milano - Italia
Tel. ++39 02 6448 3330 - Fax ++39 02 6448 3569
[email protected] - www.btbs.unimib.it
Finito di stampare nel mese di agosto 2011
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graphic project okiodesign.com