INDEX
Introduction....
3
.
Staff Members............................................................................................................................. 4
Research Units ............................................................................................................................. 5
Functional Post-Genomics and Protein Engineering ................. 5
Fermentation Biotechnology ................................................................................. 7
Human Genetics .................................................................................................................... 9
Population Genetics ..................................................................................................... 11
Cell Biology ............................................................................................................................. 11
Applied Biology ................................................................................................................. 12
13
Aquaculture and Fishery Ecology................................................................. 14
Ecology ....
.
Relevant Grants 2000-2005 ................................................................................ 17
Relevant Publications 2000-2005 .............................................................. 18
Where we are ........................................................................................................................... 24
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INTRODUCTION
The Department of Biotechnology and Molecular Sciences was founded one year ago, on
September 1st 2004, at the University of Insubria in Varese.
This new institution was established to provide a reference site for the multidisciplinary study
of biological phenomena and processes and their application in the field of biotechnology. In
fact, the research performed in the Department unify in a complementary and synergistic way
different expertises and skills in several disciplines such as Biochemistry, Proteomics, Genetics,
Environmental Sciences, Biomarkers Development, Molecular Biology, Fermentation
Technology and Aquaculture.
This integration of theoretical and applied approaches is further supported by the integration of
dedicated laboratories and facilities, providing the development of innovative and competitive
research projects within our University.
Research themes in the Department include the study of biological systems that span from
prokaryotes to eukaryotes up to human pathologies, the experimental approaches range from cell
biology to genomic and proteomic analysis, the study of protein structure-function relationship,
protein-protein interaction and the study of some important pathologies at the molecular level.
The members of the department are organized in several research groups on the basis of their
scientific interests and expertises in the different fields of biotechnology, molecular sciences and
environmental technology.
In this presentation booklet, a detailed description of the research groups and their activities is
reported.
Currently, the Department staff includes 4 full professors, 7 associate professors, 5 researchers and
6 technical and administrative officers. The research activities of the single units are supported
by a wide range of National and International programs funded by the Italian Ministry of
University and Research, the Italian Ministry of Agriculture, the European union, the University
of Insubria itself (“Progetti di Eccellenza”) and by charities such as Fondazione Cariplo, the
Italian Association for Cancer Research (AIRC) and by research contracts with pharmaceutical
and agro- and food-industry. Reserch funds are also provided by the Provincia di Varese and by
local administration.
The Department academic staff is fully involved in Undergraduate and Graduate teaching
programs, within the curricula of Biotechnology, Biological Sciences and Analysis and
Management of Natural Resources and in research training programs for undergraduate, graduate
students and post-doc fellows partecipating to the Department’s scientific activity. Moreover, the
Department hosts the PhD School in “Analysis, Protection and Management of Biodiversity”.
In conclusion, this new Department represents a dynamic institution working in the field of
biotechnology and molecular sciences, with the goal of interacting with public and private
companies, especially in the Varese territory, to achieve an efficient technology transfer.
Mirella S. Pilone
Head of the Department
Varese, July 26th, 2005
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STAFF MEMBERS
FULL PROFESSORS
GIOVANNI BERNARDINI
MIRELLA PILONE
MARCO SAROGLIA
ROBERTO TARAMELLI
ASSOCIATE PROFESSORS
FRANCESCO ACQUATI
GIORGIO BINELLI
GIUSEPPE CROSA
FLAVIA MARINELLI
LOREDANO POLLEGIONI
MARIANGELA PRATI
CARLO ROSSETTI
RESEARCHERS
PAOLA CAMPOMENOSI
ROSALBA GORNATI
GIANLUCA MOLLA
LUCIANO PIUBELLI
GENCIANA TEROVA
ADMINISTRATIVE STAFF
RAFFAELE AUCIELLO
MOTTA STEFANO
TECHNICIANS
MICAELA ANTONINI
ANGELO BOSELLI
ANNA GIULIA CATTANEO
FEDERICA ROSSI
POST-DOC FELLOWS
RAFFAELLA CINQUETTI
MARILENA MELONI
MONICA MOLTENI
FRANCESCO POMATI
SIMONA RIMOLDI
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SILVIA SACCHI
FABRIZIO STEFANI
SERENA ZACCARA
OTHER FELLOWS
GIORGIA LALUMERA
SIMONA LORENZI
GIORGIA LETIZIA MARCONE
PHD STUDENTS
MARIA GRAZIA BERNASCONI
FABIO BRAMBILLA
LAURA CALDINELLI
SARA CASTIGLIONI
PIETRO CECCUZZI
VALENTINA CHINI
DEBORAH MARABELLA
LAURA MONTI
LAURA MOTTERAN
ELENA PAPIS
DAVIDE PERINI
ELENA ROSINI
SILVIA SALIS
GRADUATE STUDENTS
ILEANA BADI
GIORGIA CARABELLI
ELENA CASTELLI
MONICA CELENTANO
MOIRA CLERICI
FLAVIO GASPARI
FEDERICA PAVAN
MATTIA PEDOTTI
ELISA ZAGHETTO
SIMONA ZANONI
RESEARCH UNITS
FUNCTIONAL POST-GENOMICS AND PROTEIN ENGINEERING
Proteins are involved in almost all aspects of cellular function. The characterization of proteins has
become such a significant part of modern biology that it has inspired a new discipline: proteomics
– the classification of the protein complement expressed by the genome of an organism. Technology
development has driven, and continues to drive, rapid evolution in this field. The next step is
represented by the post-genomic (functional proteomics) approach, i.e. the understanding of the
role at the molecular level of specific proteins, in particular those present under pathophysiological
conditions: this is actually the main topic of the FPGPE lab at the University of Insubria. This
Laboratory includes at present 10 research scientists and 7 students. A number of foreigners PhD
students, post-doc fellowships and well-known researchers have been hosted (even for the sabbatical
period) in this lab during the last years. In particular, the FPGPE Laboratory of the University
of Insubria is a well-known component of the international research network on “Flavins and
Flavoproteins”. The specific competences and expertises of the Laboratory include:
1. Molecular biology and microbiology
2. Protein expression and purification
3. Protein structure and engineering
4. Enzymology
At present the Laboratory is engaged in the characterization of proteins involved in human
pathologies (e.g., schizophrenia), in the investigation of folding process and of catalytic competence
in flavooxidases (depending upon the binding of the flavin coenzyme and/or of other proteins),
and in the application of rational and ‘irrational’ mutagenesis approaches to obtain enzymes with
novel functional properties.
Research Topics
1. PROTEIN ENGINEERING OF ENZYMATIC
ACTIVITIES
Loredano Pollegioni, Gianluca Molla, Elena Rosini,
Simona Lorenzi, Letizia Marcone, Silvia Sacchi,
Mirella S. Pilone
The wide experience acquired in the past years in
resolving, identifying, and characterising proteins,
introduced our research group in “Proteomics”, the
emerging area of research of the post-genomic era that
deals with the global analysis of gene expression, and
provided us with the tools and know-how to design
and engineer “novel” enzymatic activities. In fact,
natural enzymes have not evolved to answer to applied
research requirements.
The evolution of a new enzymatic function can be
obtained using two different approaches: the “rational
design” approach based on the detailed knowledge of the
structure-function relationships of the enzyme under
investigation, and the “directed evolution approach”
based on the introduction of random mutations. We
developed both experimental approaches. E.g., the
substrate specificity of yeast D-amino acid oxidase a flavoenzyme presenting relevant characteristics for
exploitation in biotechnological and industrial work
(i.e. the high catalytic efficiency and strong coenzyme
Model of the active site of evolved M213R mutant of yeast DAAO
(active on acidic D-amino acids) with bound D-aspartate.
binding) – was modified to isolate an enzyme active
on acidic D-amino acids and an enzyme active on
all D-amino acids. Analogously, we evolved by a
combined approach a cephalosporin C acylase which
is able to directly convert cephalosporin C into 7aminocephalosporanic acid.
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2. CHARACTERIZATION OF
FLAVOPROTEIN OXIDASES:
CHOLESTEROL OXIDASE, D-AMINO
ACID OXIDASE AND GLYCINE OXIDASE
Luciano Piubelli, Angelo Boselli, Laura Motteran,
Gianluca Molla, Silvia Sacchi, Loredano Pollegioni,
Mirella S. Pilone
The flavoenzyme D-amino acid oxidase (DAAO)
is a model of the dehydrogenase-oxidase class
of flavoproteins. The enzyme from the yeast R.
gracilis was the object of an impressive amount of
investigations, allowing to define at the molecular
presence has been first reported in 1998 when the genome
of Bacillus subtilis was fully sequenced. This enzyme
is active on various amines and shares the substrate
specificity with both DAAO and sarcosine oxidase.
In fact, GO is able to oxidise sarcosine and glycine
as well as D-amino acids. Interestingly, GO has been
recently proposed to be a component of the thiamine
pyrophosphate biosynthesis pathway in B. subtilis.
We cloned the cDNA for GO and overexpressed
the recombinant protein in E. coli. Its physicochemical, spectral and kinetic characterisation has
been carried out, and the 3D-structure has been
determined. The main goals of this project are: a) the
investigation of the structure-function relationships
of GO to understand the modulation of the substrate
specificity in enzymes catalysing similar reactions; b)
the identification of its physiological role, specifically
regarding the thiamine pyrophosphate biosynthesis
pathway. Collaborations: Sandro Ghisla - Universitat
Konstanz, Germany; Wolfram Welte -Universitat
Konstanz, Germany; Alice Vrielink – University of
California – San Diego, U.S.A
3. MOLECULAR MECHANISMS OF
SUSCEPTIBILITY TO SCHIZOPHRENIA
Gianluca Molla, Silvia Sacchi, Mariagrazia
Bernasconi, Loredano Pollegioni, Mirella S. Pilone
Schizophrenia is a psychosis that affects nearly 1% of
people around the world and accounts for about 2.5%
of health-care costs. In the last two years several genes
linked to susceptibility to schizophrenia have been
discovered. One of the most intriguing finding has
been a new human gene, G72. The protein encoded by
Tetrameric structure of GO.
level its structure-function relationships.
Cholesterol oxidase (CO) is a FAD containing
flavoenzyme which catalyses the oxidation and the
isomerization of cholesterol to give 4-cholesten-3one. This enzyme exhibits a wide range of clinical
and industrial applications. We are investigating two
different cholesterol oxidases from microorganisms:
from
non-pathogenic
microorganisms
(e.g.,
Brevibacterium sterolicum) having the coenzyme
covalently bound to the protein moiety, and from
pathogenic microorganisms (e.g., R. equi) possessing
a non-covalently bound FAD molecule. In the
recent past, detailed studies on the mechanism of
cholesterol oxidase have been carried out in order to
determine structure-function relationships of this
class of enzymes since the crystal structure of both
cholesterol oxidases has been solved. The aim of the
ongoing studies is to understand the significance of
the covalent link of the coenzyme, and to identify the
structural determinants of its reactivity.
Glycine oxidase (GO) is a novel flavoenzyme, whose
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Schematic representation of some of the inferred schizophreniasusceptibility genes.
this gene, pLG72, interacts with the enzyme D-amino
acid oxidase (DAAO) which role in mammalian brain
is the modulation of the level of D-serine.We are
investigating the structural and functional properties of
the pLG72-DAAO complex and interaction network.
The final goal is to understand how the two genes,
the encoded proteins, and the SNP associated with
increased susceptibility to schizophrenia contribute to
the molecular mechanisms that ultimately lead to the
pathology. We will work at the same time at several
levels: the functional and structural characterization
of isolated proteins, the identification and analysis of
structural determinants involved in the interaction,
the functional characterization and the regulation of
the pLG72-DAAO complex in cultured neuronal cells.
The characterization of this complex, both in vitro and
in cell cultures, will provide an unprecedented insight
into complex brain networks involved
in disorders like schizophrenia.
Collaborations: Kiyoshi Fukui –
University of Tokushima, Japan ; JeanPierre Mothet – CNRS Gif sur Yvette,
France
are often multi-subunit proteins constituted either
by identical or by different polypeptide chains. The
manner in which cofactors affect the folding pathway
remains poorly understood, because kinetic folding
experiments are frequently conducted in the absence of
potential ligands. To have insights on the relationships
between cofactor uptake, stable interaction between
identical subunits (and on its significance), folding,
and intracellular trafficking, we undertook a study of
the stability of structural elements in the homodimeric
peroxisomal flavoenzyme D-amino acid oxidase from
the yeast Rhodotorula gracilis and in the monomeric
flavoprotein cholesterol oxidase from B. sterolicum
that contains a molecule of covalently bound FAD.
Collaborations: Francesco Bonomi, Stefania Iametti
– DISMA University of Milan, Italy; Dimitrios Fessas
– DISTAM University of Milan, Italy
4. INVESTIGATION
OF THE FOLDING/
UNFOLDING PROCESS OF
FLAVOPROTEINS
Loredano Pollegioni, Laura Caldinelli,
Gianluca Molla, Mirella S. Pilone
Protein folding/unfolding is a highly
cooperative process. It has been shown
that the folding/unfolding of small
globular proteins occurs via a two-state
process, whereas the folding/unfolding
of larger proteins (>100 amino acids)
is complex and often involves the
formation of intermediate(s). It is of
interest to extend studies to larger,
more complex, and therefore more
typical proteins. Flavoproteins have
attracted our attention, since they
Proposed conformation of yeast DAAO unfolding intermediate.
FERMENTATION BIOTECHNOLOGY
Fermentation Biotechnology Unit has been recently started up following the joining of Prof.
Flavia Marinelli since January 2005. The Fermentation and Microbiology labs are being installed
and three new fermentors of 3 liter working volume are operative, flanking the 15 liter one
already in use in the PROTEIN ENGINEERING LAB for protein production. The aim of
the unit is to develop research projects mainly devoted to the biotechnological exploitation of
uncommon and /or newly isolated and /or industrially relevant microrganisms, with a particular
focus on actinomycetes. In parallel the research unit is involved in the optimization and process
development for protein production by native or heterologous hosts in synergy with the research
topics of PROTEIN ENGINEERING LAB.
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Research Topics
1. NOVEL ANTIBIOTICS FROM
UNCOMMON MICROORGANISMS
Flavia Marinelli in collaboration with Fabrizio
Beltrametti, Mara Brunati, Luciano Gastaldo, Daniele
Losi and Enrico Selva at Vicuron Pharmaceuticals labs
Microbial products are still unsurpassed in their
ability to provide novel and unpredictable molecular
diversity able to face the concomitant evolution
of pathogen resistant strategies and complexity of
biological targets. On the other hand it is commonly
agreed that less than 1% of microbial species living on
those described in the target pathogens, i.e. enterococci
and staphylococci. For other recently discovered
antibacterials not only the chemical structure, but also
the mechanism of action seems novel and promising
for generating novel drugs.
2. NOVEL BIOCONVERSIONS FROM
ACTINOMYCETES
Flavia Marinelli in collaboration with Francesco
Molinari, DISTAM, Univerisity of Milan, Raffaella
Gandolfi, Facoltà di Farmacia, University of Milan,
and Srdjan Jovetic, Vicuron Pharmaceuticals
Novel species of actinomycetes have been
recently isolated and employed to perform
specific bioconversion of industrial interest
such as the enantioselective hydrolysis of 1,2O-isopropylidene glycerol esters to obtain the
solketal, an important chiral building block for
the synthesis of many optically active compounds,
such
as
glycerophospholipids,
β-blockers,
prostaglandins and leukotrienes. Bioconversion
processes for regio-stereo specificity modification
of complex antibiotic molecules such as lipoglicopeptides and depsipeptides have been optimized
from the lab scale to the pilot plant reactors.
Actinomycetes can be used as free or immobilized
cells, metabolically active or resting, or as a
source of partially purified extracts, in batch or
continuous reactors. In particular the removal of
one mannose unit from ramoplanin is carried out
by a novel specie of Streptomyces isolated form soil,
and the deacylation of teicoplanin-like molecules
is performed by a group of actinomycetes selected
among the producers of the same chemical class
of antibiotics.
Aerial mycelium of an actinomycete strain producing a novel
peptide antibiotic.
the Earth have been identified till now and probably
much less can be easily cultivated and maintained in
culture collections. By accessing to peculiar habitats
(from Antarctic lake mats to Israeli desert sands)
and by employing selective isolation protocols, we
have isolated novel bacterial species belonging to
biotechnologically relevant microbial groups, first
of all actinomycetes, followed by myxobacteria
and cyanobacteria. Novel metabolic pathways and
products are been discovering in some of these
organisms and their elucidation together with the
development of cultivation methods and fermentation
process represents a main interest of the lab. For
two lipoglycopepetide antibiotics produced by two
different actinomycetes, we are studying the selfresistance mechanisms, condition sine qua non for
the antibiotic production and for its improvement.
Interestingly the resistance genetic determinants and
the phenotypes resemble and probably originated
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Applikon 3 liter fermentation unit.
HUMAN GENETICS
The Research Unit is headed by Prof. Roberto Taramelli and is involved in the study of the genetic
bases of human complex diseseas such as cancer and congenital heart disease. More specifically, the
Unit’s research goals are the identification and characterization of tumor suppressor genes from the
6q27 region on human chromosome 6 and the definition of the molecular pathway underlying the
pathogenesis of Total Anomalous Pulmonary Venous Return, a congenital developmental defect
affecting the heart. A further field of investigation deals with the development of murine transgenic
models in order to evaluate the contribution of the vascular compartment to the pathogenesis of
Alzheimer disease. Several recombinant DNA, molecular and cellular biology techniques are
routinely used by the Unit in the above mentioned projects, which are all carried out within the
frame of a long standing collaboration with several research groups in Italy, Europe and USA. The
Unit provides research opportunities for undergraduate students, PhD and Post-doc fellows in the
fields of human molecular genetics.
Research Topics
A
1. CHARACTERIZATION OF RNASET2,
A RIBONUCLEASE WITH PUTATIVE
ONCOSUPPRESSIVE FUNCTION.
Roberto Taramelli, Francesco Acquati,
Paola Campomenosi, Laura Monti, Silvia Salis,
Davide Mariani, Moira Clerici
The 6q27 region of human chromosome 6 is frequently
involved in chromosomal alterations associated with
both solid and hematological cancer. Therefore, it has
long been suggested that this region harbours one or
more putative tumor suppressor genes. Our group has
been involved in the last years in the identification
of candidate genes within the 6q27 region. Within
this frame, an extensive effort has been devoted to
the characterization of RNASET2, a gene encoding
a ribonuclease which belongs to the Rh/T2/S family.
The RNASET2 protein is secreted, but full length
or proteolytic forms are also present intracellularly,
suggesting that the protein may have different roles
inside or outside the cell. Recently, we have gathered
experimental evidences supporting the involvement
of RNASET2 in the control of the tumorigenic and
metastatic behaviour of two cancer types, namely
ovarian cancer and melanoma. We have, at first,
observed that both RNASET2 transcript and protein
expression levels are extremely low in a panel of
ovarian cancer cell lines and fresh tumor specimens.
Moreover, introduction of RNASET2 cDNA in
various histologically different tumor cell lines
having both tumorigenic and metastatic properties
was sufficient to dramatically reduce the ability of
those cell lines to form primary tumors in vivo in
nude mice. Intriguingly, the RNASET2 catalytic
activity seems to be dispensable for the observed
oncosuppressive activity. Indeed, a catalitically
inactive mutant allele was shown to be as much
B
C
Tumor suppression by the RNASET2 gene. In vivo tumor growth
kinetic of different HEY3MET2 ovarian cancer cell line clones
transfected with control or RNASET2-encoding expression vectors.
(A). H&E staining of tumor sections from control clones (B) or
RNASET2-expressing clones (C). Note the highly differentiated
structure within the RNASET2-expressing tumor.
effective (and possibly more) as the wild-type protein
in suppressing the tumorigenic and metastatic
phenotype in vivo. Strikingly, hystological analysis
showed that RNASET2-expressing tumors have an
higher differentation grade than controls. Further
molecular and functional characterization of this
intriguing gene is ongoing in our lab.
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2. MOLECULAR BASIS OF TOTAL
ANOMALOUS PULMONARY VENOUS
RETURN (TAPVR): STRUCTURAL AND
FUNCTIONAL CHARACTERIZATION OF
CANDIDATE GENES.
Roberto Taramelli, Francesco Acquati,
Raffaella Cinquetti, Ileana Badi
RT-PCR assays on total RNA from lymphoblastoid
cell lines and found CARP mRNA overexpression
in the 10;21 translocation carrier when compared to
his healthy parents and brother. Finally, we recently
identified a second sporadic TAPVR case bearing
a missense mutation (T116M) within the CARP
coding region. The polymorphic nature of this
mutation was ruled out by analyzing more than 100
alleles from healthy subjects. Taken together, these
results suggest a role for CARP in the pathogenesis
of TAPVR, and further work is ongoing in our lab
to validate this hypothesis.
Total Anomalous Pulmonary Venous Return
(TAPVR) is a cardiac congenital defect characterized
by the failure of the pulmonary veins to enter the
left atrium coupled to their drainage
into the right atrium, resulting in
early postnatal circulatory distress.
TAPVR is often described as an
isolated anomaly, but it can be found in
association with other syndromes. The
non-syndromic form is thought to result
from multifactorial causes, but heritable
mechanisms
for
non-syndromic
TAPVR have also been suggested. We
have recently characterized a male
nonsyndromic TAPVR patient carrying
a de novo balanced translocation
involving
chromosomes
10
and
21, t(10;21)(q23.1;q11.2). The same
rearrangement was not detected in the
other healthy members of the child’s
nuclear family. A detailed molecular
characterization of the breakpoints
was carried out and both junction
fragments were amplified by Long
range PCR, cloned and sequenced in
order to define the genomic region
to be sought for TAPVR candidate
genes. The Cardiac Ankyrin Repeat
Protein (CARP) gene, mapping 100
kb proximal to the chromosome 10
breakpoint, was considered a good
TAPVR candidate. In support of its
involvement in TAPVR pathogenesis,
in situ hybridization experiments on
mouse embryos showed an expression
pattern for CARP which is clearly
compatible with its putative role in the
pathogenesis of TAPVR. Furthermore,
In situ hybridization of a CARP cDNA
sectioned (right) E13 mouse embryos.
we performed quantitative RealTime
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riboprobe on whole-mount (left) and
POPULATION GENETICS
The Research Unit deals with Population Genetics of several plant and animal species, approaching
basic problems in the fields of Conservation Genetics and of Phylogeography. The use of molecular
genetic markers represents a unifying trait for this kind of studies. Besides, numerical taxonomy
techniques are used to solve phylogenetic problems by means of the analysis of DNA sequence.
This approach is being applied also to problems specific of other organisms, such as viruses.
Research Topics
1. POPULATION GENETICS OF PLANTS
Giorgio Binelli, Marilena Meloni, Davide Perini,
Giulia Mameli
Population Genetics of forest species:Neutral genetic
molecular markers are utilised to conduct large-scale
surveys about the distribution of the genetic variability
among different Italian populations of Norway
spruce and Juniperus phoenicea to answer also basic
phylogeographic questions.
Conservation Genetics of endemic plant species:
Molecular genetic markers are being both produced and
used for the assessment of the degree and distribution
of genetic variability in rare endemic species (Primula,
Centaurea).
Conservation Genetics in animal species: molecular
markers and sequence analysis for the assessment
of genetic variability and demographic studies in
freshwater fish species.
2. PHYLOGENY OF VIRAL GENOTYPES
Giorgio Binelli, Marilena Meloni, Davide Perini
Utilisation of numerical taxonomy and phylogenetic
methods for the analysis of the nucleotide sequences
of different genotypes of the Hepatitis B and C viruses
(HBV and HCV). The main purpose of the research is
to assess whether a correlation exists between different
viral genotypes and the degree of the hepatitis-cirrhosishepatocellular carcinoma progression. The results obtained
are also applied at the identification of epidemic events.
CELL BIOLOGY
The Research Unit is involved in the search of molecular biomarkers of expression in vertebrates.
Mammalian cell cultures, Xenopus laevis embryos and fish of economical interest, such as seabass,
pearch and tuna, are the models that we are currently studying.
Research Topics
1. FROG EMBRYO TERATOGENESIS
ASSAY-XENOPUS (FETAX)
Giovanni Bernardini, Mariangela Prati,
Rosalba Gornati, Federica Rossi, Simona Rimoldi,
Elena Papis, Valentina Chini
We use Xenopus laevis embryos in the Frog Embryo
Teratogenesis Assay-Xenopus (FETAX), a powerful
and flexible bioassay for developmental toxicants.
FETAX, thanks to its three endpoints (i.e., mortality,
malformation, and growth inhibition), can also detect
the xenobiotics that affect embryonic development, a
weak link in the life cycle of an organism. The FETAX
protocol, however, is amenable to modification in
several ways and to being integrated with molecular
biology techniques that considerably increase the
capability of the test. As exposure to xenobiotics may
alter gene expression and therefore mRNA and protein
patterns, transcriptome and proteome modifications
can be studied with the aim to obtain new insights on
the mechanisms of embryotoxicity and teratogenesis
or simply to obtain molecular markers of exposure
useful in the early diagnosis of environmental stress.
With this model organism, we have evaluated several
molecules till now and we are currently studying the
fungicide triadimefon and the potent developmental
toxicant TCDD on the expression of several genes
involved in the development.
2. MOLECULAR BIOMARKERS IN
ACQUACULTURE
Giovanni Bernardini, Mariangela Prati,
Rosalba Gornati, Federica Rossi, Simona Rimoldi,
Elena Papis, Valentina Chini
In the field of aquaculture, we are looking for
molecular biomarkers capable to describe fish
welfare both searching among those genes whose
expression could reasonably be modified by the
different farming conditions (heat shock proteins
70 and 90, metallothioneins, cytochrome P4501A,
etc.) and by differential display, a molecular biology
technique that allows the comparison of two or more
mRNA pools. With this last technique, we have
obtained some genes whose expression was modified
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by high population density; one of those resulted
to be 3-hydroxil-3-methyl-glutaryl coenzime A
reductase (HMGR), a key enzyme of the cholesterol
synthesis. These genes can be used as biomarkers to
rapidly detect by qualitative and quantitative PCR
the occurred exposure of the fish to the stressor. By
performing these experiments, we become aware
of the scarsity of the genomic resources for some
fish species, in spite of their economical interest.
Therefore, we decided that an effort should have
been made to reduce, as far as it concerns genomics
resources, the gap that separates farming species from
“model organisms” and we started EST projects on
seabass, pearch and tuna.
APPLIED BIOLOGY
The Research Unit is headed by Prof. Carlo Rossetti and is involved in two main topics:
1) Characterization of Lipopolysaccharides (LPS) extracted from cyanobacteria;
2) Ecological effects of therapeutic drugs in the environment.
Research Topics
1. CHARACTERIZATION OF LPS
EXTRACTED FROM CYANOBACTERIA
Carlo Rossetti, Monica Molteni, Deborah Marabella
Cyanobacteria, also termed blue-green algae, represent
one of oldest forms of life on Earth. They are a large
group of photosynthetic oxygenic prokariots with a
high degree of biological adaptation. They effectively
competed in nature and reached a worldwide
distribution in ecologically diverse habitats, often
capable of surviving in extreme environments.
Earlier works have shown that cyanobacteria are
rich sources of novel natural products and promise a
diverse array of potential pharmaceutical compounds.
Specifically, various bioactive metabolites isolated from
cyanobacteria have been found to display cytotoxic,
anti-fungal, anti-viral and anti-inflammatory
properties.
Our labs are focused on LPS extracted from the
freshwater cyanobacterium Oscillatoria Planktothrix
sp. FP1. We investigated the biological effects of
cyanobacterial LPS on different in vitro models and
compared the effects with LPS obtained from Gram
negative bacteria. The studies on human monocytederived dendritic cells were performed in collaboration
with Dr. Federica Sallusto, Institute for Research in
Biomedicine, Bellinzona. Our results showed that
cyanobacterial LPS is a potent antagonist of E. coli
LPS. Preliminary in vivo results showed protection of
mice against lethal endotoxic shock. These results open
promising perspectives for the use of the compound
as a therapeutic agent able to control innate immune
responses. Other in vitro cell models employed human
tumor cells.
2. ECOLOGICAL EFFECTS OF
THERAPEUTIC DRUGS IN THE
ENVIRONMENT
Carlo Rossetti, Francesco Pomati, Sara Castiglioni
Aim of this research is to investigate the presence and
to study the ecological effects of therapeutics drugs in
the environment.
Filamentous cyanobacterium belonging to the Order Oscillatoriales: Planktothrix sp. FP1 isolated from the Varese Lake.
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The most recent objectives of the research, in
collaboration with the Institute for Pharmacological
Research Mario Negri, Milano, comprise:
- implement environmental concentrations data of
the distribution and occurrence of pharmaceuticals in
surface waters across the Italian peninsula
- define concentrations of pharmaceuticals and their
metabolites in a model sewage treatment plant, and in
the surface waters receiving its effluents
- evaluate the removal efficiency of different wastewater
treatment plants in diverse Italian geographic
and climatic regions and study the variations of
pharmaceuticals emission
- the obtained data of environmental occurrence,
transport and fate of therapeutic agents will then be
used to perform a risk assessment of pharmaceuticals
and their metabolites by refining the PECs (predicted
environmental concentrations)
- study the effects of pharmaceutical mixtures on
animal cells: identify specific effects on cell signalling
and detoxification; identify specific effects on gene
expression (DNA microarrays, real-time PCR)
- identify biomarkers of pharmaceuticals exposure
- study and implement the removal efficiency of
improved tertiary technologies, such as ozonation,
in degrading therapeutic drugs in wastewater
treatment plans (joint project with SOGEIVA Varese
Ambiente).
- induction of antibiotic resistance in environmental
microorganisms in consequence to pharmaceutical
contamination (joint project with the Cyanobacteriology and Astrobiology Laboratory at UNSW).
ECOLOGY
The Unit is headed by Prof. Giuseppe Crosa and staffed by Dr. Serena Zaccara (Molecular Ecology
and Freshwater Ecology), Dr. Giorgia Lalumera (Chemical and Biological Monitoring), Dr. Fabrizio
Stefani (Fish Biology and Molecular Ecology). The research goals of the Unit are: to understand
the structures and functioning of freshwater ecosystems; to assess and monitor the process of
recovery and rehabilitation of stream and lakes; and to promote environmentally-sustainable socioeconomic development in remote countries. The Unit provides research opportunities in the fields
of restoration ecology, environmental science, and aquatic resource management for undergraduate
and graduate students. Current areas of study include river monitoring in West Africa and Central
Asia, development of ecologically based operational schemes for reservoir management in Central
Europe and Asia, eutrophication of shallow lakes and molecular studies of aquatic species. The
Unit collaborates with the local authorities for the scientific coordination of research programmes
on the quality of the Varese Lake.
Research Topics
1. INVESTIGATION OF INNOVATIVE
POLLUTION CLEAN-UP AND
AVOIDANCE STRATEGIES
FOR SURFACE WATER AND
GROUNDWATER RESOURCES AT THE
“DISASTER ZONE” OF THE AMUDARYA LOWERS (UZBEKISTAN)
Giuseppe Crosa, Fabrizio Stefani, Giorgia Lalumera
The overall aim of the project is to achieve an
improvement of the socio-economical conditions
by an increased usability of polluted groundwater
resources at the lower Amu Darya river and to avoid
or minimise future risks (sustainable development).
By extreme pesticide concentrations there no more
groundwater resources are left, that can be used as
drinking water. Child mortality is at a peak level at
Central Asia and cancer diseases are incomparably
widespread. The planned use of the Kaparas reservoir
for drinking water supply leads in turn to a total
modification of the actual water pathways. There is
a considerable lack of surface water for irrigation and
a higher demand of usable groundwater to expect.
The Dam on the Amu Darya river 500 km upstream the Aral Sea
shoreline (Uzbekistan).
Actual purification strategies (e.g. use of activated
coal, ultrasonic or osmosis technologies) are too
expensive, demand unavailable amount of energy or
are not feasible at a scale of 1,2 million hectares. The
specific research objectives of this project are:
- development of site specific risk assessment and
monitoring ecosystems including human health;
- to investigate the feasibility of pesticide wash
DBSM
13
out by waste water re-use (complicated by the
very low energy level) and the required technical
optimisations;
- development of strategies and optimization models
for reducing of agricultural pollution loads,
optimisation of irrigation and drinking water supply,
and minimization of water losses.
2. PHYLOGEOGRAPHY OF AQUATIC
SPECIES
Giuseppe Crosa, Serena Zaccara, Fabrizio Stefani
Phylogeographic studies, based on nucleotide sequence
analysis of the mitochondrial large ribosomal subunit
DNA, are focused on freshwater species, especially
fishes and crayfish following their ecological role in the
rivers ecosystems. The freshwater species considered are
endemic and representative of the historical evolution
of Italian watersheds and biological districts. Specific
researches address the phylogeography of the threatened
crayfish Austropotamobius (Decapoda; Astacidae).
Main objective is to identify the different management
units (MUs) that can be used in conservation plans,
Austropotamobius pallipes, an engaged crayfish species.
preventing any contamination/alteration of the natural
white-clawed crayfish population in Northern Italy.
A second research theme has been recently activated
on the phylogeography of the Italian populations of
Telestes muticellus with the objective of determining
the phylogenetic structure of congener species and the
patterns of genetic differentiation within the Italian
peninsula.
AQUACULTURE AND FISHERY ECOLOGY
Fish production has had an extraordinary growth worldwide, during last half a century, accounting
now over 100 million tonnes, versus 27 million tonnes of 50 years ago. Aquaculture contribute, that
was 1.5% of total fish capture on 1950, represents now the 30% of the total world capture (FAO,
2002 data). On the same time, the average pro-capite fish consumption followed the same growth,
reaching 16.3 kg/year on a world average base. The pro-capite consumption in the 25 European
countries changes with the different cultures and habits, resulting in a 26,3 kg/year average, while
the Italian figure accounts for more than 24 kg/year.
While fishery is not expected to growth because of the already exploited resources, aquaculture is
facing the increasing demand of fish products. Farming technologies are more and more sophisticated
and aquaculture is suggested not only as a source of good quality food for human population, but
for the management of coastal resources and wetlands management as well.
The most important challenges of such an
Animal Biotechnology, are now represented
by the product quality, the minimization of
environmental impacts, the welfare of farmed
animals.
In order to face such goals, the Aquaculture and
Fishery Ecology (AFE) Working Group, has
been established on 2001 at the University of
Insubria in Varese and it is part of DBSM.
Functional Genomics and Bio-Engeneering of
the Aquaculture Environment are the master
lines for the research activities of this Research
Unit, which specific competences include:
• Biology of Aquaculture
• Technologies and husbandry in mariculture
and inland water aquaculture
Sea bass harvesting at a commercial land-based fish farm in
Tuscany at Il Padule, Castiglione della Pescaia, GR, Italy.
• Aquaculture and environment
14
DBSM
• Molecular biology applications in aquaculture
• Fish nutrition
• Fish welfare
Facilities include a water recirculation pilot farm, a molecular biology laboratory, a laboratory for
fish biology studies, water and microbiological analyses. Contacts and collaborations are active
with commercial mariculture farms as well as inland water fish farms, including hatcheries, where
undergraduated and PhD students may do research for their thesis.
Research Topics
1. FUNCTIONAL GENOMICS
IN AQUACULTURE
Genciana Terova, Marco Saroglia, Simona Rimoldi,
Micaela Antonini, Pietro Ceccuzzi
The research goal is the identification, with a functional
genomic approach, of molecular markers that describe
fish response to environmental conditions, nutritional
and feeding strategies.
Molecular responses to stress in fish
In addition to several indicators that have been
proposed for assessing fish welfare, such as cortisol,
haematocrit, blood glucose or total haemoglobin,
molecular biomarkers directly indicating gene activity
may have the characteristics for being very sensitive
and reliable indicators. In this context, we have first
determined the sequence of a glucocorticoid receptor
(GR), a stress-related gene in a teleost (Dicentrarchus
labrax) of high commercial interest for Mediterranean
aquaculture, for which only little is known at present
about its genomics. GR acts as a ligand-dependent
transcription factor to control and regulate gene
expression. We have then investigate the impact
of long-term exposure to crowding stress on GR
expression levels in the liver of sea bass quantified by
real-time RT-PCR. Our study clearly demonstrated
that chronic stress by high rearing density affects GR
expression, whose levels in the liver decrease inversely
with blood cortisol levels. Therefore, GR may be a valid
bio-indicator capable of providing useful information
about fish welfare.
Hypothetical model of DNA–binding domain of glucocorticoid
receptor
Studies on the “compensatory growth” in Sea bass.
The exceptional fast growth that farmed fish
experience after a feed deprivation period has been
called “compensatory growth” or “catch up “ growth.
In intensive aquaculture this phenomenon has been
studied in order to find a possibility of enhancing
feed conversion efficiency. In this context cloning and
sequencing of the complete coding sequences of sea bass
(Dicentrarchus labrax, L) insulin-like growth factor I
(IGF-I) and IGF-II, myostatin and fibroblast growth
factor 6 (FGF6), all genetic determinants of skeletal
muscle growth, have been carried out. The complete
coding sequences of sea bass IGF-I , IGF-II , FGF6
and myostatin were deposited in the GenBank. Then,
the expression profile of IGF-I, IGF-II, myostatin
and FGF6 in liver and myotomal muscle, in response
to different feeding regimens, has been studied. The
expression levels of all aforementioned genes has been
quantified by real-time RT-PCR. Nutritional status
influenced significantly the IGF-I and II expression
levels in liver as well as in muscle, inducing a downregulation during fasting and an up-regulation during
the recovery from fasting. The myostatin expression
levels, confirmed the involvement of this gene in
the compensatory growth induced by re-feeding. A
novel finding was the increase in myostatin mRNA
abundance in the muscle of fasted sea bass, indicating
that this autocrine/paracrine growth factor has a role
in modulating muscle growth in response to different
feeding regimens. Unlike the three aforementioned
genes, the FGF6 expression pattern did not provide us
any clue to its function, as its muscular mRNA levels
were not affected whatever the feeding status of the
animals was.
Oxygen-regulated gene expression in Sea bass
Low oxygen is prevalent in many aquatic habitats
and one response of fish to hypoxia is compensatory
changes in gene expression. In mammals this
phenomenon is mediated, in part, by the HypoxiaInducible Factor (HIF). This transcription factor
accumulates at low oxygen, binds to hypoxia response
elements of a variety of target genes, and ultimately
increases gene expression. In fish, homologues of HIF
are present. However the targets of HIF regulation
remain largely unknown. In efforts to characterize
mechanisms of hypoxia adaptation in marine fish we
have isolated the complete coding sequence of HIF1α in sea bass (Dicentrarchus labrax), which has been
deposited in the GenBank. To study the expression
patterns of HIF-1α, fish were exposed to severe acute
DBSM
15
and chronic hypoxia conditions and the mRNA
abundance levels were measured by real time PCR
in liver and kidney. Upon exposure to acute hypoxia
for 3 hours transcription of HIF-1α was significantly
upregulated in both tissues, indicating that its mode of
regulation is similar to its mammalian homologues.
the production, more than 30 are utilized to improve
water quality (15 at the intake and 15 at the outfall),
while more that 20 ha are still unexploited and the
intent is to utilize it to improve the bio-depuration
with algae and other organisms that may be utilized as
by-product goods. A number of monitoring campaign,
2. BIO-INGENEERING AND
AQUACULTURE ENVIRONMENT
Marco Saroglia, Fabio Brambilla, Pietro
Ceccuzzi, Micaela Antonini, Genciana Terova
Closed water re-circulation systems
The Unit’s laboratories are equipped with a pilot
closed water re-circulation system, utilized
for fish rearing, experimental challenges
and experimentation of new technological
equipments, studies on the biofilters, including
their microbiological aspects.
Impact assessment and improvement of water
quality inside and at the outlet of a mild
intensive euryhaline fish farm in Tuscany.
Modelling benthonic faecal pellets diffusion at a 1200 tonnes production
pen cage fish farm, located in mid Thyrrenian coast (Gaeta gulf). Different
Intensive aquaculture in coastal shallow water blue tones indicate the water deepness, while the pink colours rate the
lagoons is strongly affected by the quality of faecal benthonic deposition in g/m 2/y
water intake; so, the fish stile strongly depends
from the so called “internal impacts”, due to the including nictemeral cycles, have been carried on at
released catabolites and fish faecal pellets as well as the site, during which chemical and physical water
the quality of water at the intake. An important study characteristics were measured. Results indicate the
is in progress to assess the impacts originated from possibility to re-circulate a high percentage of the
a fish farm located in a shallow water coastal lagoon water, while important benefits to the surronding
and an evaluation of an adequate phyto-depuration lagoon are provided by the hydrodinamism
technology. The farm (Azienda Ittica Il Padule) transferred from the farm. Important elements for the
insists on the Diaccia Botrona Natural Wetland Park productive management of coastal water aquaculture
at Castiglione della Pescaia (Grosseto, Italy). Natural have already been developed and a model for coastal
hydrodinamism in the lagoon is almost absent, due wetlands exploitation is the expected final product of
to the small tide (15-20 cm), so the risk of naturally this research.
induced anoxic events is elevated. To ensure an
adequate oxygen partial pressure to the water, in order Environmental Impact Assessment of Mariculture
to sustain the yearly yield of 400 tons high quality Modern Mediterranean mariculture is mainly developing
Sea bass, liquid oxygen is utilized. Moreover, of the with pen cages technologies, with farming facilities
70 ha surface owned by the farm, 15 are dedicated to installed along the coasts. Water currents providing
oxygen supply and a diffusion of fish
catabolites, the environmental impacts are
generally not relevant. Nevertheless, when
a forecast EIA is due, available mathematic
models for a forecast of faecal pellets
distribution on the bottom and dissolved
catabolites dispersion may be calibrated at
the site. Recommendations for the farming
and monitoring protocols may than be
produced.
The DOPAMOD model for a forecast
dispersion of faecal and unutilized feed
pellets, developed for salmon farms
in North Europe, is now applied for
Mediterranean Sea bass and Sea bream
farms. Our Working Group is involved
in some forecast evaluations in the
Pen cages mariculture in Sicily
Thyrrenian Sea.
16
DBSM
RELEVANT GRANTS 2000-2005
• Crosa G. Guidelines for reservoirs operations
compatible with the protection of local fish
populations – 2005 Regione Lombardia
• Pilone M.S. Role of molecular interactions in the
acquisition of the functional structure of model
proteins – 2005-2006 MURST-PRIN
• Rossetti C. Farmaci nell' ambiente - 2005-2006
MURST-COFIN
• Rossetti C. Sviluppo di un biosensore - 2005
Fondazione Comunitaria del Varesotto
• Gornati R. Acquacultura e benessere animale – 2004
Fondazione Amici dell’Università Onlus
• Pollegioni L. – Molecular mechanisms of susceptibility to schizophrenia - 2004 Fondazione Cariplo
• Terova Saroglia G. Animal welfare and qualità
in intensive fish farming. - 2004 Ministero per le
Politiche Agricole e Forestali MIPA
• Rossetti C. Off-Flavours negli allevamenti ittici 2004-2005 ARSIA-Regione Toscana
• Prati M. Effetti embriotossici del fungicida triadimefon in Xenopus laevis - 2003 MURSTCOFIN
• Crosa G. Investigation of innovative pollution
clean-up and avoidance strategies for surface water
and groundwater resources at the „Disaster Zone“
of the Amu-Darya lowers (Uzbekistan) - 2003 EU
INTAS
• Bernardini G. Provision of freshly isolated neuronal and glial cells - 2003, 2004, 2005 ECVAM
• Bernardini G. Creazione di una libreria di espressione di gonadi di tonno rosso. - 2004 Ministero per
le Politiche Agricole e Forestali MIPAF
• Bernardini G. Gangliosides as tumor biomarker. 2004 Consorzio Interuniversitario di Biotecnologie
• Bernardini G. – 2004 Sequenziamento automatico
DNA. Fondazione Cariplo
• Pilone S.M. Directed evolution of binding properties and molecular recognition in proteins 2003-2004 MURST-PRIN
• Rossetti C. Progetto cianobatteri - 2003-2005 Fondazione Cariplo
• Saroglia M. Welfare of teleost fish farmed with
intensive technologies: sustainable technologies and
search for diagnostic molecular markers in sea bass
(Dicentrarchus labrax) - 2003 MURST-COFIN
• Taramelli R. Functional characterization by transgenic and gene knock-out technologies of a gene
(DRAP/BACE2) involved in both neurodegenerative
and neoplastic diseases - 2003 MURST COFIN
• Acquati F. Analisi genetico-molecolare di una
cardiopatia congenita umana - 2003 Fondazione
delle Comunità del Varesotto Onlus
• Crosa G. Development of integrated water management tools for the Tuyamuyn reservoir complex
for the improvement of the drinking water supply
and public health in the disaster zone of the lower
Amu-Darya – Uzbekistan and Turkmenistan
(Central Asia) - 2002 EU-INTAS
• Pilone M.S. Struttura e funzione nella scienza delle
proteine Progetto di Eccellenza -2002 Università
degli Studi dell’Insubria
• Pilone M.S. Directed engineering of specific functions, binding properties and thermal stability of
proteins 2001-2002 MURST-PRIN
• Pollegioni L., Pilone M.S. Produzione di enzimi
per intermedi di antibiotici β-lattamici -2002-2005
Antibioticos S.p.A
• Saroglia M. Study for an “open sky” depuration
and recirculation of water effluents from a mild
intensive euryhaline fish farming in Tuscany.- 2002
ARSIA/Toscana
• Taramelli R. Identificazione e caratterizzazione di
geni con proprietà antioncogeniche localizzati sul
cromosoma 6 umano -2002 Fondazione Carialo
• Marinelli F. Actapharm QLK3-CT-2001-01783
2001-2004 Comunità europea
• Marinelli F. Micromat BIO4-90-0040- 2001 Comunità europea
• Binelli G. Scelta e validazione di geni candidati che
influenzano nelle piante caratteri di interesse agroforestale – 2001 MURST-COFIN
• Crosa G. Genetic heterogeneity of A. Pallipes
populations for restocking plans – 2001 Life
Natura
• Taramelli R. Identificazione di pathways molecolari implicate in patologie neurodegenerative dell’uomo – 2001 FIRB
• Binelli G. Conservazione di Salmo marmoratus e
Rutilus pigus nel Fiume Ticino -2000 Life Natura
DBSM
17
RELEVANT PUBLICATIONS & PATENTS
2000-2005
2005
Acquati, F., Possati, L., Ferrante, L., Campomenosi, P.,
Talevi, S., Bardelli, S., Margiotta, C., Russo, A., Bortoletto,
E., Rocchetti, R., Calza, R., Cinquetti, R., Monti, L., Salis,
S., Barbanti-Brodano, G. and Taramelli, R (2005) Tumor
and metastasis suppression by the human RNASET2 gene.
Int J Oncol 26:1159-68.
Brunati, M., Bava, A., Marinelli, F. and Lancini, G. (2005)
Influence of leucine and valine on ramoplanin production
by Actinoplanes sp-ATCC 33076. In press, J Antibiotics.
Caldinelli, L., Iametti, S., Barbiroli, A., Bonomi, F., Fessas,
D., Molla, G., Pilone, M.S., Pollegioni, L.(2005) Dissecting
the structural determinants of the stability of cholesterol
oxidase containing covalently bound flavin. J Biol Chem
280 (24): 22572-22581.
Crosa G., Froebrich J., Nikolayenko V., Stefani F. Spatial
and seasonal variations in the water quality of the Amu
Darya River (Central Asia). In press, Water Research.
Crosa G. and Zaccara S. Key aspects of the water quality
variability in the Amu Darya River upstream the Tuyamun
hydroengineering complex. In press, Water Resources.
Fratini, S., Zaccara, S., Barbaresi, S., Grandjean, F.,
Souty-Grosset, C., Crosa, G. and Gherardi, F. (2005)
Phylogeography of the threatened crayfish (genus
Austropotamobius) in Italy: implications for its taxonomy
and conservation. Heredity 94 :108–118.
Gaspari, F., Paitan, M., Mainini, D., Losi, D., Ron, E.Z.
and Marinelli, F. (2005) Myxobacteria isolated in Israel as
potential source of new anti-infectives. J Appl Microb 98:
429-439.
Gornati, R., Gualdoni, S., Cavaliere, R., Terova, G., Saroglia,
M. and Bernardini, G. (2005) Molecular biology and fish
welfare: a winning combination. Aquaculture Int 13: 5155.
Gornati, R., Papis, E., Rimoldi, S., Chini, V., Terova, G.,
Prati, M., Saroglia, M., Bernardini, G. (2005). Molecular
markers for animal biotechnology: sea bass (Dicentrarchus
labrax, L.) HMG-CoA reductase mRNA. Gene 344: 299-305.
Jovetic, S., Marinelli, F. and Tramper, J. (2005) Continuous
biotransformation of glycopeptide antibiotic A40926 in
a cascade of three airlift bioreactors using immobilized
Actinoplanes teichomyceticus cells. In press, Enz Microb
Tech .
Lazzarini, A., Gastaldo, L., Candiani, P., Ciciliato, I., Losi,
D., Marinelli, F., Selva, E. and Parenti, F. (2005) Antibiotics
107,891, its factor A1 and A2, pharmaceutically acceptable salts
and compositions, and use thereof. WO 2005/014628 A1
Molinari, F., Romano, D., Gandolfi, R., Kroppenstedt,
R.M. and Marinelli, F. (2005) Newly isolated Streptomyces
spp. as enantioselective biocatalysts: hydrolysis of 1,2-Oisopropylidene glycerol racemic esters. In press, J App
Microb.
Molla, G., Bernasconi, MG., Sacchi, S., Pilone, M.S.,
Pollegioni, L. (2005) Expression in E. coli and in vitro
refolding of the human protein pLG72. In press, Prot.
Expr. Purif.
18
DBSM
Molteni, M., Marabella, D., Orlandi, C. and Rossetii,
C. (2005) Melanoma cell lines are responsive in vitro to
lipopolysaccharide and express TLR-4. In press, Cancer
Lett .
Mothet, J.P., Pollegioni, L., Ouanouno, G., Martineau, M.
Fossier, P. and Baux G. (2005) Glutamate receptor activation
triggers a calcium-dependent and SNARE proteindependent release of the gliotransmitter D-serine. Proc Nat.
Acad Sci U.S.A. 102: 5606-5611.
Pollegioni, L., Pilone, M.S., Molla, G., Cucchetti E., Verga,
R. and Cabri W. (2005) Cephalosporin C acylases. European
Patent 05000102.3.
Pollegioni, L., Lorenzi, S., Rosini, E., Marcone, G.L.,
Molla, G., Verga, R., Cabri, W., Pilone, M.S. (2005)
Evolution of an acylase active on cephalosporin C. In press,
Protein Science.
Rinaldi, L., Basso, P., Tettamanti, G., Grimaldi, A.,
Terova, G., Saroglia, M., DeEguileor, M. (2005). Oxygen
availability causes morphological changes and a different
VEGF/Flk-1/HIF-2 expression pattern in sea bass gills.
Ital. J. Zool. 72, 103-111.
Terova, G., Gornati, R., Rimoldi, S., Bernardini, G. and
Saroglia, M. (2005) Quantification of a glucocorticoid
receptor in sea bass (Dicentrarchus labrax, L) reared at
high stocking densities. In press, Gene. Available on line
29 august 2005 doi:10.1016/j.gene.2005.06.016
Terova, G., Bernardini, G., Binelli, G., Gornati, R. and
Saroglia, M. (2005) cDNA encoding sequences for myostatin
and FGF6 in sea bass (Dicentrarchus labrax, L) and the
effect of fasting and refeeding on their expression levels. In
press, Domestic Animal Endocrinol. Available on line 12
september 2005 doi:10.1016/j.domanied.2005.08.003 .
Terova, G., Bernardini, G., Chini, V., Gornati, R. and
Saroglia, M. (2005) Expression patterns of insulin-like
growth factor I and II in liver and muscle of sea bass
(Dicentrarchus labrax, L) during long term fasting and
refeeding. In press, Gen. Comp. Endocrinol.
Sacchi, S., Boselli, A., Job, V., Pilone, M.S., Pollegioni,
L., Molla, G. (2005) The role of tyrosines 223 and 238
in Rhodotorula gracilis D-amino acid oxidase catalysis:
interpretation of double mutations. In press. Enz. Micr.
Technol.
Zaccara S. and Crosa G. Mitochondrial diversity in the
endangered white-clawed crayfish (Austropotamobius
italicus) from the Po River drainage: genetic knowledge for
its conservation. In press, Freshwater Biology.
2004
Beltrametti, F., Jovetic, S., Feroggio, M., Gastaldo, L., Selva,
E. and Marinelli, F. (2004) Valine influences production and
complex composition of glycopeptide antibiotic A40926 in
fermentation of Nonomuraea sp. ATCC 39727. J Antibiotics
57:37-44.
Boselli, A., Piubelli, L., Molla, G., Sacchi, S., Pilone, M.S.,
Ghisla, S. and Pollegioni L. (2004) On the mechanism of
Rhodotorula gracilis D-amino acid oxidase: role of the
active site Serine 335. Biochim. Biophys. Acta 1702: 19-32.
Levels and Saxitoxin Production in Cylindrospermopsis
raciborskii T3. Microbiology 150:455-61.
Brunati, M., Marinelli, F., Bertolini, C., Gandolfi, R.,
Daffonchio, D. and Molinari, F. (2004) Biotransformations
of cinnamic and ferulic acid with actinomycetes. Enz
Microb Technol 34:3-9.
Ribas, L., Planas, J.V., Barton, B., Monetti, C., Bernardini G.,
Saroglia, M., Tort, L., MacKenzie, S. (2004) A differentially
expressed enolase gene isolated from the gilthead sea
bream (Sparus aurata) under high-density conditions is upregulated in brain after in vivo lipopolysaccharide challenge.
Aquaculture 241: 195-206.
Caldinelli, L., Iametti, S., Barbiroli, A. Bonomi, F., Ferranti,
M., Pilone, M.S. and L. Pollegioni (2004) Unfolding of the
peroxisomal flavoprotein D-amino acid oxidase. J. Biol.
Chem. 279: 28426-28434.
Casiraghi, M.A., De Paschale, M., Romanò, L., Biffi,
R., Assi, A., Binelli, G. and Zanetti, A.R. (2004) Longterm outcome (35 years) of hepatitis C in individuals who
acquired infection through mini transfusions of blood given
at birth. Hepatology 39: 90-96.
Ciciliato, I., Corti, E., Sarubbi, E., Stefanelli, S., Gastaldo,
L., Montanini, N., Kutz, M., Losi, D., Marinelli, F. and
Selva, E. (2004) Antibiotics GE23077, novel inhibitors
of bacterial RNA polymerase I. Taxonomy, Isolation and
Characterization. J Antibiotics 57: 210-221.
Diodovich, C., Bianchi, M.G., Bowe, G., Acquati, F.,
Taramelli, R., Parent-Massin, D., and Gribaldo, L. (2004)
Response of human cord blood cells to styrene exposure:
evaluation of its effects on apoptosis and gene expression by
genomic technology. Toxicology. 200: 145-57.
Gornati, R., Papis, E., Rimoldi, S., Terova, G., Saroglia, M.
and Bernardini, G. (2004). Rearing density influences the
expression of stress-related genes in sea bass (Dicentrarchus
labrax, L.). Gene. 341: 111-118.
Gornati, R., Terova, G., Vigetti, D., Prati, M., Saroglia, M.,
and Bernardini, G. (2004) Effects of population density on
sea bass (Dicentrarchus labrax) gene expression. Aquaculture
230: 229-239.
Sacchi, S., Rosini, E., Molla, G., Pilone, M.S. and Pollegioni
L. (2004) Modulating D-amino acid oxidase substrate
specificity: production of an enzyme for analytical
determination of all D-amino acids by directed evolution.
Protein Engineering Design and Selection 17: 517-525.
Stefani, F., Galli, P., Crosa, G., Zaccara, S. and Calamari,
D. (2004) Alpine and Apennine barriers determining the
differentiation of the rudd (Scardinius erythrophthalmus
L.) in the Italian peninsula. Ecology of Freshwater Fish 13:
168-175.
Stefani, F., Galli, P., Zaccara, S and Crosa, G. (2004) Genetic
variability and phylogeography of the cyprinid Telestes
muticellus within the Italian peninsula as revealed by
mitochondrial DNA. J Syst Evol Res 42: 1-9.
Taramelli, R. and Acquati, F.(2004) The human genome
project and the discovery of genetic determinants of cancer
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