2011 ANNUAL REPORT - Istituto Pasteur

2011 

ANNUAL REPORT

2012 – Istituto Pasteur-Fondazione Cenci Bolognetti 

Edited by Lucia Ugo 

P.le Aldo Moro 5 – 00185 Roma 

pasteurcenci@uniroma1.it 

www.istitutopasteur.it

Contents 

Contents 

• Forward 7 

• Boards and Staff 8 

• Fellowships awarded 9 

• Conferences, Seminars and Bookshop talks 13 

• Scientific Reports 15 

Research Area 1: Molecular biology of microorganism and viruses 

Andrea Bellelli, Structural biology of the thiol-dependent redox systems of Schistosoma 

mansoni and Plasmodium falciparum 17 

Alberto Boffi, Bacterial globins as regulators of thiol redox equilibrium in bacteria 19 

Daniela De Biase, The acid resistance genes of Escherichia coli: does their complex 

transcriptional control hide novel biological roles 21 

Alberto Faggioni, Epstein-Barr virus interactions with cellular microRNAs 23 

Claudio Falcone, Study of RNA damage in yeast as a model for age-related degenerative 

diseases 25 

Paola Londei, From leaderless to leadered mRNAs: mRNA features modulating ribosome 

binding and translation initiation in Archaea 27 

Research Area 2: Pathogenic mechanisms of microbially associated diseases 

Guido Antonelli, Molecular characterization of viruses causing bronchiolitis and study of 

viral and host factors affecting Type I IFN antiviral response induced by respiratory viruses 31 

Maria Lina Bernardini, Lipopolysaccharide and peptidoglycan adaptation to host as an 

immune evasion strategy of gram-negative pathogens 33 

Bianca Colonna, Involvement of sRNA molecules in the complex regulatory circuits of 

virulence gene expression in Shigella flexneri and in enteroinvasive E.coli 35 

Francesca Cutruzzolà, Inhibition of Pseudomonas aeruginosa biofilms: new molecular 

strategies targeting cyclic-di-GMP metabolism 37 

Paolo Sarti, Defense mechanisms against oxidative and nitrosative stress in pathogenic 

protozoa 39 

Vincenzo Vullo, Immunopathogenesis of HIV infection: study of innate immunity and 

dendritic cells 41 

Research Area 3: Molecular genetics of eukaryotes 

Fiorentina Ascenzioni, Assembly and functional analysis of genomic context vectors 

containing the human CFTR locus 45 

Paola Ballario, Light control of filamentous fungi life cycle: from system models to 

applications 47 

Silvia Bonaccorsi, Functional analysis of morgana, a gene involved in the control of 

centrosome duplication 49 

Irene Bozzoni, RNA-RNA and RNA-protein interactions: role of small non-coding RNAs in 

gene expression control 51 

3

Contents 

Emanuele Cacci, Role of metalloproteinases and their tissue inhibitors in the regulation of 

neurogenesis and gliogenesis from neural stem/progenitor cells 53 

Paola Caiafa, Does PARylated PARP-1 introduce an epigenetic mark on chromatin 55 

Giorgio Camilloni, DNA recombination of repeated sequences and genome instability: 

epigenetic implications 57 

Antonella De Jaco, Processing of the Neuroligins proteins and autism-related mutations 59 

Patrizio Dimitri, Functional analysis of CG40218, a Drosophila melanogaster gene 

encoding a BCNT-like protein required for chromosome organization 61 

Maurizio Gatti, Genetic and molecular analysis of the mechanisms of Drosophila telomere 

protection 63 

Alessandro Giacomello, Role of epicardium and EMT/MET processes in cardiac progenitor 

cells generation and differentiation 65 

Alberto Gulino, Hedgehog signaling regulatory networks in brain cancer stem cells 67 

Marco Lucarelli, The interplay between epigenetics, cell cycle and homologous 

recombination in gene therapy by Small Fragment Homologous Replacement (SFHR) 69 

Rossella Maione, Interplay between myogenic factors and cell cycle control: regulation and 

role of the cdk inhibitor p57kip2 71 

María Elena Miranda Banos, Handling and toxicity of mutant serpins underlying human 

disease 73 

Antonio Musarò, Characterization of the muscle-nerve crosstalk in a neuromuscular 

disease 75 

Rodolfo Negri, Role of the COP9 signalosome in transcription modulation and chromatin 

organization in yeast and plants 77 

Sergio Pimpinelli, The biogenesis of piRNAs and their involvement in transposon silencing 

and heterochromatin formation in Drosophila 79 

Sabrina Sabatini, To the root of organ growth: the control of root meristem activity in 

Arabidopsis 81 

Mario Stefanini, Biological characterization and in vitro culture of spermatogonial stem cells 83 

Marco Tripodi, Molecular mechanisms driving liver stem cell fate 85 

Giuseppe Zardo, Profiling the Polycomb/Trythorax target genes in normal and leukemic 

hematopoiesis 87 

Research Area 4: Molecular recognition in biomolecules 

Enzo Agostinelli, Toxic effects induced by polyamine metabolites on melanoma cells: a 

new therapeutic approach 91 

Fabio Altieri, Cellular response to oxidative stress: involvement of protein disulfide 

isomerases 93 

Paolo Bianco, Role of Gsα signalling in the bone marrow hematolymphopoietic 

microenvironment as revealed by novel transgenic models 95 

Maurizio Brunori, The dark side of protein folding: denatured states and misfolded species 

in molecular recognition and pathological processes 97 

Stefano Cacchione, The role of nucleosomes in the stability of human telomeres 99 

Felice Cervone, Plant innate immunity: signalling and recognition of Damage-Associated 

Molecular Patterns (DAMPs) 101 

Ernesto Di Mauro, Spontaneous generation and evolution of genetic information 103 

Cristina Limatola, Molecular and functional approaches to investigate the neuroprotective 

and neuromodulatory roles of chemokines and their receptors in the central nervous 

system 105 

4

Contents 

Paola Paggi, Neurone response to experimental injury and lack of dystrophin: a molecular, 

functional and structural study in autonomic ganglia in vivo and in vitro 107 

Maria Rosaria Torrisi, Molecular mechanisms integrating endocytosis and signalling of 

fibroblast growth factor receptors 109 

Research area 5: Cellular and molecular immunology 

Vincenzo Barnaba, Interplay amongst chronic immune activation, apoptosis, crosspresentation, 

immune-regulation, and autoimmunity 113 

Rossella Paolini, Signalling events negatively regulating FceRI expression and mast cell 

functional responses 115 

Angela Santoni, Anti-tumor effector functions of NK cells in tumor microenviroment 117 

Isabella Screpanti, Dissection of Notch signaling-dependent pathways involved in the 

progression of T cell leukemia 119 

Rosa Sorrentino, Predisposing factors in autoimmune diseases: correlation between 

common genetic variations and function 121 

Loretta Tuosto, CD28 co-stimulatory molecule as a key regulator of NF-κB signalling 

pathway: role of cytoskeleton in coupling CD28 to NF-κB activation 123 

Elio Ziparo, Anti-tumor pathways mediated by innate immune responses 125 

Research area 6: New antimicrobial and antiviral agents 

Donatella Barra, Peptide effectors of innate immunity 129 

Mariangela Biava, New pyrrole derivatives of BM 212: a new class of antimycobacterial 

agents. Design, synthesis, biological evaluation and study of their mode of action 131 

Roberto Di Santo, New azole derivatives as antiprotozoal agents 133 

Romano Silvestri, Drug design and synthesis of non-nucleoside inhibitors of both HIV-1 

wild type and resistant mutant strains reverse transcriptase and Coxsackie B4 virus 135 

Research area 7: Biology of malaria and other vector-borne diseases 

Alessandra Della Torre and Vincenzo Petrarca, Genetic and phenotypic characterization 

of species and “molecular forms” of the Anopheles gambiae complex (Diptera: Culicidae), 

afrotropical malaria vectors 139 

David Modiano, Immune responses to malaria and autoimmune disorders: investigating 

common gene-regulatory networks 141 

Start-up program 

Giuseppe Lupo, Specification and maintenance of retinal stem cells 145 

5

Annual Report 2011 

Forward 

The Istituto Pasteur-Fondazione Cenci Bolognetti is a private non-profit foundation 

established according to the terms of the bequest of Princess Beatrice Fiorenza Cenci 

Bolognetti to Sapienza University of Rome for the purpose of encouraging scientific research. 

The funding of research projects is possible thanks to the income from real estate and, this 

year, thanks also to the support of BNP Paribas BNL, that we gratefully acknowledge. 

The Istituto Pasteur-Fondazione Cenci Bolognetti, the Italian member of the Institut 

Pasteur International Network, is committed to studying the mechanisms that regulate the 

basic processes of life, for the understanding and treatment of severe human pathologies for 

which there are no cures as yet, and for improving the existing ones. 

Researchers in the areas of biology, medicine, medicinal chemistry, bioinformatics and 

biotechnologies are dedicated to the study and development of novel drugs as well as 

cellular and gene therapies for the treatment of emerging, or re-emerging infectious diseases 

(influenza, herpes, tuberculosis, HIV, HCV); vector-borne diseases (malaria, Dengue fever); 

neurodegenerative and neoplastic diseases; disorders of the immune and haematopoietic 

systems (leukemia). Furthermore, the Institute has been active in promoting the study of 

stem cell biology and of the use of cardiac, muscle, hepatic, neuronal and male reproductive 

system stem cells in the repair of damaged tissues. 

Our Institute finances high-level research projects selected through a peer review 

system. We give special attention to the new generation of researchers awarding fellowship 

grants that support their training in highly advanced research centres abroad, upon their 

return to Italy and at the beginning of their career following a PhD degree. Recently, we have 

also activated a three-year Start-Up program for a young scientist during which he may build 

a solid research project and attract independent funding. 

Moreover, we are active in promoting scientific communication. We organize seminars, 

conferences and international meetings, which are important occasions for the comparison of 

results. We also promote a school project involving a series of meetings and practical 

activities with students, so they can learn to appreciate the importance of science. Lessons 

and courses in the fields of genetics, biotechnology, microbiology, genomics, evolution 

biology, ecology and neuroscience are carried out in the laboratories of the Sapienza 

University of Rome or, when possible, are performed directly by teachers in schools. 

Furthermore, we organize cultural and social events, such as bookshop talks, for the wider 

public. 

Last but not least, our financial commitment is devoted to the construction of laboratories 

in which established scientists of any origin may improve our research. 

This Annual Report documents the results obtained during this year thanks to the 

enthusiasm and the effort of the scientists involved. It is our pleasure to invite you to follow 

our activity on our website (www.istitutopasteur.it), where you may find further information. 

Paolo Amati 

President 

Angela Santoni 

Scientific Director 

7


Boards and Staff 

Administrative Board 

The Board of Administration is presided 

over by a President appointed by the 

Rector of Sapienza University of Rome 

chosen from a list of three names selected 

during a joint session of the board and the 

Scientific Council. Members of the board 

include a Scientific Director, four members 

of the Faculties of Pharmacy and Medicine, 

Natural Sciences, and Medicine and 

Dentistry. Other members are a legal 

expert designated by the University of 

Rome Board of Administration, three 

auditors nominated by the University, the 

Ministry of the Treasury, and the Ministry of 

Universities and Scientific Research. 

President 

Paolo Amati (Pharmacy and Medicine) 

Members 

Paolo Costantino (Natural Sciences), 

Alberto Faggioni (Medicine and 

Dentistry), David Modiano (Pharmacy and 

Medicine), Angela Santoni (Pharmacy and 

Medicine), Romano Silvestri (Pharmacy 

and Medicine) 

Secretary 

Emanuela Gloriani 

Administrative Expert 

Giuseppina Capaldo 

Auditors 

Anna Carmela Ferrante, Simona Ranalli, 

Carla Vassallo 

Scientific Council 

The Scientific Council is a board of seven 

scholars in the field of the pasteurian 

sciences. Members are elected to a fouryear 

term by the Faculties of Natural 

Sciences, and Pharmacy and Medicine. 

The Scientific Director, appointed by the 

Scientific Council, is ex-officio a member of 

the Board of Administration. The Scientific 

Council attends to examine and coordinate 

the research programs as well as the 

several scientific activities. 

Scientific Director 

Angela Santoni (Pharmacy and Medicine) 

Members 

Ernesto Di Mauro (Natural Sciences), 

Laura Frontali (Natural Sciences), Anna 

Teresa Palamara (Pharmacy and 

Medicine), Anna Tramontano (Pharmacy 

and Medicine), Marco Tripodi (Pharmacy 

and Medicine), Carlo Turano (Pharmacy 

and Medicine) 

Secretariat 

Sarah Gainsforth, Maria Pia Lorenzoni, 

Lynda Romani, Nicoletta Silvestri, Lucia 

Ugo 

Consultants 

Tommaso De Dominicis (legal affairs); 

Anna Maria Pivetti (architectural 

supervision); Barbara Hell (financial 

affairs) 

8


Fellowships awarded 

Fellowships awarded for two-year 

training in foreign laboratories 

Michele ARDOLINO, from Department of 

Molecular Medicine to Dept. of Molecular 

and Cell Biology, University of California, 

Berkeley, USA 

Valerio BERARDI, from Department of 

Biology and Biotechnologies "C. Darwin" 

to State University of New York, Health 

Science Center, NY, USA 

Sonia CONI, from Department of Molecular 

Medicine to IBDC, Université de Nice 

Sophia-Antipolis, France 

Daniela DIMASTROGIOVANNI, from 

Department of Biochemical Sciences "A. 

Rossi Fanelli" to Department of 

Biochemistry, University of Cambridge, 

UK 

Michela ESPOSITO, from Department of 


to Institut de Génétique et Microbiologie, 

Université Paris XI, France 

Roberto GAETANI, from Department of 

Experimental Medicine to Department of 

Cardiology, University Medical Center, 

Utrecht, The Netherlands 

Francesca GASPARRINI, from 

Department of Molecular Medicine to 

Cancer Research UK, London Research 

Institute, UK 

Marta MORETTI, from Department of 

Experimental Medicine to Division of 

Immunology and Inflammation, Imperial 

College, London, UK 

Tommaso MOSCHETTI, from Department 

of Biochemical Sciences "A. Rossi 

Fanelli" to Department of Biochemistry, 

University of Cambridge, UK 

Giuseppe SCIUMÈ, from Department of 

Experimental Medicine to NIH/NIAMS, 

Bethesda, MD, USA 

Fellowships awarded to students who 

had a two-year experience abroad 

Daniela CECCARELLI, from Université de 

Sherbrooke, Québec, Canada, to 

Department of Biology and 

Biotechnologies "C. Darwin" 

Antonio COLUCCIA, from School of 

Pharmacy, University of Cardiff, UK, to 

Department of Medicinal Chemistry and 

Technologies 

Elvira FORTE, from Burnham Institute for 

Medical Research, La Jolla, CA, USA, to 

Department of Molecular Medicine 

Valentina MANGANO, from MRC Centre 

for Genomics and Global Health, Oxford, 

UK, to Department of Public Health and 

Infectious Diseases 

Fellowships awarded to students who 

have completed their PhD and are 

seeking a position 

Andrea BRENNA, at Department of 


Cristina CAPUANO, at Department of 

Experimental Medicine 

Elisa CESARINI, at Department of Biology 

and Biotechnologies "C. Darwin" 

Isotta CHIMENTI, at Department of 


Laura CURCURÙ, at Department of 


9


Alessandra GALATI, at Department of 


Francesca GASPARRINI, at Department 

of Experimental Medicine 

Emanuela MICHELI, at Department of 


Arianna MONTANARI, at Department of 


Michela MUSCOLINI, at Department of 


Marianna PELLONI, at Department of 


Marialaura PETRONI, at Department of 

Molecular Medicine 

Fabrizio TESTA, at Department of 

Biochemical Sciences "A. Rossi Fanelli" 

Fellows working on the Institute 

research programs 

Georgia ABATE, at Department of Cellular 

Biotechnologies and Hematology 

Angela. A. ALAGIA, at Department of 


Chiara ARDICCIONI, at Department of 


Pamela AVELLINO, at Department of 

Public Health and Infectious Diseases 

Maria Giulia BACALINI, at Department of 

Cellular Biotechnologies and Hematology 

Cecilia BATTISTELI, at Department of 


Orlando BORRÁS HIDALGO, at 

Department of Biology and 

Biotechnologies "C. Darwin" 

Anna BUSANELLO, at Department of 


David CACCHIARELLI, at Department of 


Roberta CALABRESE, at Department of 


Maria Rosaria CARBONE, at Department 

of Cellular Biotechnologies and 

Hematology 

Mariateresa CARCURO, at Department of 


Nicoletta CASTIGLIONE, at Department 


Fanelli" 

Roberta CASTRATARO, at Department of 

Physiology and Pharmacology "Vittorio 

Erspamer" 

Francesca CECERE, at Department of 


Arcangela Anna CERA, at Department of 


Sara CHIARELLA, at Department of 


Isotta CHIMENTI, at Department of 


Fabio CICCARONE, at Department of 


Raffaela CIPRIANI, at Department of 


Erspamer" 

Ivan COLADARCI, at Department of 


Elisabetta DAMIA, at Department of 


Federica DE ANGELIS, at Department of 


Francesca DE VITO, at Department of 


Enea Gino DI DOMENICO, at Department 

of Biology and Biotechnologies "C. 

Darwin" 

Maria Letizia DI MARTINO, at Department 

of Biology and Biotechnologies "C. 

Darwin" 

Antonella FARINA, at Department of 


Manuela FORNARA, at Department of 


10


Fedra FRANCOCCI, at Department of 


Alessandra GALATI, at Department of 


Francesca GARIBALDI, at Department of 


Francesca GASPARRINI, at Department 

of Experimental Medicine 

Leonardo GIUSTINI, at Department of 


Roberta GONNELLA, at Department of 


Gaia GRASSINI, at Department of Medical- 

Surgical Sciences and Biotechnologies 

Alfonso GRIMALDI, at Department of 


Erspamer" 

Maria Luisa IANNITTO, at Department of 


Raffaella LA SCALEIA, at Department of 


Ramona LATTAO, at Department of 


Rosalba LEPORE, at Department of 


Valerio LICURSI, at Department of Biology 


Carmen MARESCA, at Department of 


Federica MARINO, at Department of 


Ramona MARRAPODI, at Department of 


Valentina MENGOLI, at Department of 


Emanuela MICHELI, at Department of 


Arianna MONTANARI, at Department of 


Linda Celeste MONTEMIGLIO, at 

Department of Biochemical Sciences "A. 

Rossi Fanelli" 

Angela MORRONE, at Department of 


Cassandra MOSTOCOTTO, at 

Department of Cellular Biotechnologies 

and Hematology 

Gaelle NOEL, at Department of Biology 


Ida PACIELLO, at Department of Public 

Health and Infectious Diseases 

Francesca PAGANI, at Department of 


Erspamer" 

Valeria Luciana PALUMBO, at 

Department of Biology and Biotechnologies 

"C. Darwin" 

Paola PARISI, at Department of Biology 


Eugenia PENNACCHIETTI, at Department 


Fanelli" 

Serena PERILLI, at Department of Biology 


Francesco PISCITELLI, at Department of 

Medicinal Chemistry and Technologies 

Dorina POLINARI, at Department of 


Biancamaria RICCI, at Department of 


Cinzia RIZZO, at Department of Public 

Health and Infectious Diseases 

Maddalena ROMANELLI, at Department 

of Molecular Medicine 

Valentina ROMANO, at Department of 

Physics 

Emanuele ROSCIOLI, at Department of 


Daniele RUNCI, at Department of 


11


Daniel SAVATIN, at Department of Biology 


Riccardo SILIGATO, at Department of 


Helena STABILE, at Department of 


Carolina UGENTI, at Department of 


12


Conferences 

Seminars 

Istituto Pasteur Science Days 

Abbazia di Sant’Andrea in Flumine 

Novembre 18-19, 2011 

• Molecular mechanisms of cellular 

differentiation 

• Immunity and cellular stress response 

• Molecular genetics and epigenetics 

• Molecular biology of microorganisms viruses 

and parasites 

Institut Pasteur International Network 

European Regional Meeting 

Infection related cancer and neurodegenerative 

disorders 

Rome, October 13-15, 2011 

• Pathogen driven mucosal inflammation and 

cancer of Acute Respiratory Infections 

• Hepatitis viruses, inflammation and cancer 

• Viral infection and neurodegenerative 

disorders 

European Day of Immunology 

Un amico sorprendente: il tuo sistema 

immunitario 

Rome, April 29, 2011 

October 5 • CD28: therapeutic target for 

lethal cytokine storm induced by 

superantigen toxins and influenza virus 

Prof. Raymond Kaempfer 

The Hebrew University, Jerusalem, Israel 

July 15 • How not to become a systems 

biologist 

Prof. Arthur M. Lesk 

Dept. of Biochemistry and Molecular Biology, 

The Pennsylvania State University, USA 

June 30 • Epigenetics: the new 

molecular and medical genetics 

Prof. John C. Lucchesi 

Dept. of Biology and Winship Cancer Institute, 

Emory University Atlanta GA, USA 

June 9 • The Segregation Distorter 

system in Drosophila melanogaster: 

a case study of a meiotic cheater 

Prof. Terrence W. Lyttle 

Dept. Cell and Molecular Biology University of 

Hawaii Manoa Honolulu, USA 

Joint Workshop 

Innovative targets and drug design 

strategies in cancer therapy 

in collaboration with PhD Schools. 

Rome, February 18, 2011 

• Mitosis as a “druggable” target 

• Targeting global regulators of cellular 

processes 

• Targeting protein modifications and 

interactions 

May 16 • The mitochondrial genome in 

life death and disease 

May 17 • Revising the mitochondrial 

genetic code - a shift in our thinking 

May 18 • Can we rescue the defect 

associated with a pathogenic 

mitochondrial tRNA mutation 

Prof. Robert Lightowlers and Prof. Zosia 

Chrzanowska Lightowlers 

University of Newcastle upon Tyne, UK 

13


April 11 • From Aristotele to malaria 

control: a bioinformatic approach 

April 13 • Vector-borne diseases in 

Greece: a journey through time 

Prof. Kitsos Louis 

University of Crete, Greece 

Bookshop talks 

Virus herpes: il nemico nascosto 

by Anna Teresa Palamara 

Noi e i batteri: una lunga storia di 

amicizia e guerra 

by Maria Lina Bernardini and Daniela De 

Biase 

Le energie del futuro: la fotosintesi e le 

biomasse 

by Felice Cervone and Roberto Bassi 

…nunc est bibendum… 

Il divin teatro e la mente 

by Nicola Modugno, Paolo De Vita and 

Giovanni Mirabella. Introduction by Cristina 

Limatola 

…genere di…vino… 

Il genere… influenza tutto 

by Flavia Franconi and Letizia Gabaglio 

..in vino e …in vitro 

Cellule staminali: realtà e illusioni 

by Paolo Bianco and Gilberto Corbellini 

…In vino veritas… 

Il Dr. Semmelweiss e altre terribili storie 

Percorsi per la verità scientifica 

by Marco Tripodi, Angela Santoni and 

Mario Polsinelli 

Come siamo apparsi 

Il senso di…vino della vita 

by Ernesto Di Mauro, Giuditta Perozzi and 

Elio Ziparo 

14

Area 1 

Molecular biology of 

microorganisms and viruses

Area 1: Molecular biology of microorganisms and viruses 

Structural biology of the thiol-dependent redox systems of 

Schistosoma mansoni and Plasmodium falciparum 

Andrea Bellelli 

Department of Biochemical Sciences "A. Rossi Fanelli" 

℡: +39 06 49910955 - @: andrea.bellelli@uniroma1.it 

Schistosomiasis is a widespreadd tropical parasitic disease, caused by three species of 

the blood-fluke Schistosoma. The disease is debilitating and affects 200 million people in 

tropical areas; the death toll is estimated at two hundred thousands people per year. 

Schistosomiasis is currently treated with one drug, Praziquantel, whose precise molecular 

target is unknown. Several other drugs are known to kill the schistosomes in vivo and in vitro, 

but these are seldom employed because of toxicity, high cost, complex administration or 

other reasons. The improvement of known drugs or the development of entirely new ones is 

a desirable goal, in view of the fact that strains of Schistosoma mansoni with reduced 

sensitivity to Praziquantel have appeared. In this project we are exploring known or putative 

macromolecular targets of schistosomicidal drugs; thus we focus on the biochemistry and 

molecular biology of the parasite. The rationale of this approach is that drug design may 

become realistic if the mechanism of action of each drug were known at atomic detail, ideally 

as the 3D structure of the drug in complex with its target. The enzymes involved in the 

detoxification of reactive oxygen species (ROS) and other oxidants are potential drug targets. 

We have already characterized from the structural and functional point of view: Glutathione 

Transferase; Thioredoxin Glutathione Reductase; and Glutathione Peroxidase from either S. 

mansoni or S. haematobium. We have also characterized a Fatty Acid Binding Protein and a 

Cyclophilin, both from S. mansoni, even though these two proteins are not directly related to 

the ROS detoxification pathway. 

Two of the enzymes we characterized are known to be “druggable”, i.e. they can serve as 

the target of known or putative drugs: Thioredoxin Glutathione Reductase (TGR) and 

Glutathione Peroxidase. In the case of Thioredoxin Glutathione Reductase an effective 

inhibitor is available, that is known to kill not only the schistosomes, but also malarial 

parasites: the gold containing drug Auranofin. TGR is a NADPH-dependent flavoreductase 

containing a selenocysteine residue (Sec). During its enzymatic cycle thiolates and 

selenolates that have high affinity for transition metals are generated. Auranofin inhibits TGR 

(and other selenocysteine-containing flavoreductases) more effectively than non Secontaining 

ones (glutathione reductase); this preference was traditionally ascribed to the high 

affinity of selenium for gold. We solved the structure of the gold-TGR complex, and found Au 

combined to the sulfur of Cys residues, rather than (or in addition to) the Se of Sec. Thus our 

results challenge the commonly held view. Kinetic measurements have demonstrated that 

the relative velocity of the reaction rather than the relative affinity, depends on the presence 

of Sec residues, which appear to dictate AF selectivity. Indeed when an external source of 

17


selenium (benzeneselenol) was added to the reaction mixture, auranofin was able to inhibit 

the Se-lacking reductases Glutathione Reductase and Sec->Cys mutated TGR with an 

efficiency similar to Se-containing ones. 

In order to complete the structural characterization of the enzymes belonging to the thiolmediated 

detoxification pathway, we solved the high-resolution crystal structure of S. 

mansoni Thioredoxin (SmTrx) in three states, namely: the wild-type oxidized adult enzyme 

and the oxidized and reduced forms of a juvenile isoform, carrying an N-terminal extension. 

SmTrx shows a typical thioredoxin fold, highly similar to the other components of the 

superfamily. SmTrx presents some functional peculiarities, e.g. the ability to reduce oxidized 

glutathione. Moreover it is one of the few defence proteins expressed in mature eggs and in 

the hatch fluid, thus confirming an important role in the parasite. 

Only one relevant enzyme of the pathway remains to be characterized, namely 

Peroxiredoxin; this protein has been heterologously expressed in E. coli, purified and 

crystallized and its structure is the next target of our project. 

Publications 

Angelucci A, Miele AE, Boumis G, Brunori M, Dimastrogiovanni D, Bellelli A. Macromolecular 

bases of antischistosomal therapy. Curr Top Med Chem 2011, 11: 2012-28. doi: 

10.2174/156802611796575939. 

Boumis G, Angelucci F, Bellelli A, Brunori M, Dimastrogiovanni D, Miele AE. Structural and 

functional characterization of Schistosoma mansoni Thioredoxin. Protein Sci 2011, 20: 

1069-76. doi: 10.1002/pro.634. 

Saccoccia F, Angelucci F, Boumis G, Brunori M, Miele AE, Williams DL, Bellelli A. On the 

mechanism and rate of gold incorporation into thiol-dependent flavoreductases. J Inorg 

Biochem 2011 Epub. doi: 10.1016/j.jinorgbio.2011.11.005. 

Research Group 

Adriana Erica Miele, Francesco Angelucci, 

researchers; Giovanna Boumis, technician, 

Fulvio Saccoccia, PhD student. 

Collaborations 

David L. Williams, Rush University, Chicago, 

IL, USA. 

18


Bacterial globins as regulators of thiol redox equilibrium in bacteria 

Alberto Boffi 


℡: +39 06 49910990 - @: alberto.boffi@uniroma1.it 

The specific targets of the project concern the elucidation of the mechanism of heme 

based thiol oxidation in bacterial truncated globins (trHbs) and the discovery of novel 

pathways in thiol redox homeostasis in bacteria. Spectroscopic techniques have been 

applied in order to characterize the properties of bacterial hemoglobins in solution as a 

function of standard parameters (pH, salt composition, reducing environment etc., see 

methods). In particular, a complete resonance Raman spectroscopic characterization of the 

globins from Thermobifida fusca and Bacillus subtilis has been carried out both on native 

proteins and in selected active-site mutants. The Raman studies brought out that these 

globins display the spectroscopic signature characteristics of heme peroxidases, with a 

strong electron donation from the heme iron to the distal ligand both in their ferrous and ferric 

adducts. The nature of the heme-fluoride complexes has been further explored in 

spectroscopic experiments that allowed for the first time the assignment of the specific ironfluoride 

stretching frequencies in a hemoprotein (Droghetti et al., 2011). In parallel, molecular 

dynamics of fluoride motion inside the heme pocket has been investigated and highlighted a 

novel mechanism of ligand stabilization within the heme pocket, based on hydrogen bonding 

from a conserved tryptophan residue to the iron bound fluoride atom (Nicoletti et al., 2011). A 

set of combinatorial mutants of the distal heme pocket has been analyzed in order to focus 

on the properties of distal heme cavity. The results obtained can be considered as a unique 

data set, because they have been obtained for a complete group of mutants where the three 

key amino acids of Tf-trHb (WG8, YCD1, YB10) are progressively substituted with the nonhydrogen 

bonding phenylalanine. The spectroscopic characterization of the fluoride 

complexes has unveiled a well-defined correlation between ν(Fe–F) vibrational frequencies 

and CT1 electronic transition energies. For the case of Tf-trHb, we have obtained a detailed 

picture of H-bonding in the distal cavity environment. The interpretation of the spectroscopic 

data is strengthened by the close relation with the observed fluoride dissociation kinetics and 

molecular dynamics simulations. All the techniques yield evidence that TrpG8 and TyrCD1 

can form strong H bonds with fluoride, whereas TyrB10 can only interact weakly. The second 

target of the research concerns the identification of partner proteins involved in the complex 

physiological network of thiol homeostasis. In the case of Bacillus subtilis the partner protein 

YjbH has been identified already and paralogues have been found in actinomyces, including 

mycobacteria, and in parasitic microorganisms, including Lehismania. On these basis, it is 

suggested that the globin may be able to catalyze disulfide bonds formation and thus act as a 

heme based peroxiredoxin. The first step towards the identification of a novel thiol redox 

19


pathway in Bacillus subtilis concerns the demonstration of the postulated interaction between 

the globin (yjbI) and the “disulfide isomerase-like” protein (yjbH) located upstream within the 

same operon. In this framework, the whole operon contaning yjbI and yjbH has been 

expressed as a copy of the original operon from B. subtilis. Overexpression of the whole 

operon allowed us to single out that trHb is capable of direct oxidation of the -CxxC- motif of 

YjbH thus regenerating active protein. Thus, trHbs act as redox switches that are capable of 

oxidizing disulfide bonds of the YjbH regulator. In this framework, trHbs would be terminators 

of the redox stress response. The solution of these complex biological functions will be 

addressed in the last year of the project, by examining in detail the nature of the interactions 

of trHbs with their partner proteins. 

Publications 

Droghetti E, Nicoletti FP, Bonamore A, Sciamanna N, Boffi A, Feis A, Smulevich G. The 

optical spectra of fluoride complexes can effectively probe H-bonding interactions in the 

distal cavity of heme proteins. J Inorg Biochem 2011, 105: 1338-43. doi: 10.1016/ 

j.jinorgbio.2011.07.007. 

Nicoletti FP, Droghetti E, Boechi L, Bonamore A, Sciamanna N, Estrin DA, Feis A, Boffi A, 

Smulevich G. Fluoride as a probe for H-bonding interactions in the active site of heme 

proteins: The case of Thermobifida fusca hemoglobin. J Am Chem Soc 2011, 133: 20970- 

80. doi: 10.1021/ja209312k. 


Alessandra Bonamore, Alberto Macone, 

research fellows; Paola Baiocco, post-doc 

fellow; Andrea Ilari, CNR researcher. 


Giulietta Smulevich Dipartimento di Chimica, 

Università di Firenze; Leonardo Boechi 

Departamento de Química Inorgánica, Analítica 

y Química Física, Universidad de Buenos Aires. 

20


The acid resistance genes of Escherichia coli: does their complex 

transcriptional control hide novel biological roles 

Daniela De Biase 

Department of Medical-Surgical Sciences and Biotechnologies 

℡: +39 0773 1757212 - @: daniela.debiase@uniroma1.it 

For successful colonization of the gut, enteric bacteria must overcome the extreme acidic 

stress (pH


Structure-function relationships in GadB 

We performed a biochemical characterization of the GadB site-specific double mutant 

D86N-H465A, which was also provided to Dr G. Capitani (PSI, Villigen, Switzerland) for 

crystallographic analysis. We designed this mutant because D86 in the GadB active form is 

involved in binding of the γ-carboxylate of glutamate. We found that the Asp86Asn mutation, 

in association with the His465Ala mutation, which is necessary to maintain the GadB active 

site open, affects GadB catalytic activity by extending its pH range of activity up to pH 8.0. 

Because the double mutant D86N-His465A is active at pH well above neutral, we expect that 

it might be employed for biotechnological applications. The double mutant has been 

crystallized and the crystal structure is under refinement (manuscript in preparation). 

Role of the gadBC system in Brucella microti 

As part of a newly established collaboration with Dr. A. Occhialini (Montpellier, France), 

we constructed three acid sensitive E. coli mutants of MG1655 K12 strain, i.e. ∆gadC, 

∆gadA-∆gadB and ∆gadA-∆gadBC. These mutants were used for heterologous in trans 

complementations with the B. microti gadBC operon. All mutants recovered glutamatedependent 

acid resistant phenotype, were able to export GABA and to decarboxylate 

glutamate, thereby confirming conserved functions of the gadBC system in both bacterial 

species. 

Publications 

Doucette GJ, Mikulski CM, King KL, Roth PB, Wang Z, Leandro LF, Degrasse SL, White KD, 

De Biase D, Gillett RM, Rolland RM. Endangered North Atlantic right whales (Eubalaena 

glacialis) experience repeated, concurrent exposure to multiple environmental neurotoxins 

produced by marine algae. Environ Res 2011 Epub. doi: 10.1016/j.envres. 2011.09.010. 

Yu Z, Bekker M, Tramonti A, Cook GM, van Ulsen P, Scheffers DJ, de Mattos JT, De Biase 

D, Luirink J. Activators of the glutamate-dependent acid resistance system alleviate 

deleterious effects of YidC depletion in Escherichia coli. J Bacteriol 2011, 193: 1308-16. 

doi: 10.1128/JB.01209-10. 


Francesco Bossa, professor; Angela Tramonti 

CNR researcher; Eugenia Pennacchietti, postdoc 

fellow; Daniela Bastianelli, Gaia Grassini, 

PhD students. 


Guido Capitani, Paul Scherrer Institute, Villigen 

PSI, Switzerland; Maurice CR Franssen 

Wageningen University and Research Centre, 

The Netherlands; Sylvie Rimsky, CNRS-ENS de 

Cachan, France; Alessandra Occhialini, 

Université Montpellier I, CPBS, UMR 5236, 

France; Joen Luirink, Institute of Molecular Cell 

Biology, Amsterdam University, The Netherlands. 

22


Epstein-Barr virus interactions with cellular microRNAs 

Alberto Faggioni 

Department of Experimental Medicine 

℡: +39 06 4461500 - @: alberto.faggioni@uniroma1.it 

The discovery of microRNA (miR) represents a novel paradigm in RNA based regulation 

of gene expression and their dysregulation has become a hallmark of many tumors. In virally 

associated cancers, the host-pathogen interaction could involve alteration in miR expression. 

Interestingly EBV, besides encoding its own miRs, can also influence cellular miRs. The 

initial evidence that this is so came from studies showing that EBV negative and EBV positive 

BLs differ in their miR expression signature. Moreover, among EBV positive BLs, it was 

shown that the miR profile of restricted latency expressing BLs (latency I) and broader 

latency expressors (latency III) is diverse. Given the importance on the one hand, of LMP1 

and EBNA2 in EBV driven transformation and on the other, of the dysregulation of miRs in 

cancer, we asked how these viral proteins influence cellular miR expression. 

We first showed that LMP1 negatively regulates a major oncogene, TCL1, in diffuse large 

B-cell lymphoma (DLBCL) and BL cells. MicroRNA (miR) profiling of LMP1 transfectants 

showed that among others, miR-29b, is upregulated. LMP1 diminished TCL1 by inducing 

miR-29b through C-terminus activation region 1 (CTAR1) and CTAR2. miR-29b locked 

nucleic acid (LNA) antisense oligonucleotide transfection into LMP1-expressing cells reduced 

miR-29b expression and consequently reconstituted TCL1, suggesting that LMP1 negatively 

regulates TCL1 through miR-29b upregulation. The ability of LMP1 to negatively regulate 

TCL1 through miR-29b might underlie its B-cell lymphoma growth antagonistic property. As 

LMP1 is also important for B-cell transformation, we suggested that the functional dichotomy 

of this viral protein may depend on a combination of levels of its expression, lineage and 

differentiation of the target cells and regulation of miRs, which then directs the outcome of 

the cellular response. 

Next, we studied how the expression of EBNA2, a protein indispensable for the capacity 

of the virus to transform B cells in vitro, might affect cellular miRs. Extensive miR profiling of 

the virus infected and EBNA2 transfected B lymphoma cells revealed that oncomiR miR-21 is 

positively regulated by this viral protein. Conversely, Burkitt lymphoma (BL) cell lines infected 

with EBNA2 lacking P3HR1 strain did not show any increase in miR-21. EBNA2 increased 

phosphorylation of AKT and this was directly correlated with increased miR-21.The primary 

miR-21 transcripts were unaltered by EBNA2 and this suggests that the increase in mature 

miR-21 is due to higher post-transcriptional processing. Interestingly, miR-146a, whose 

expression is known to be induced by LMP1, was severely downregulated by EBNA2 in 

U2932 DLBCL cells. The primary miR-146a transcription and the promoter activity was 

hampered by EBNA2 whereas LMP1 had no such effect. Low miR-146a expression generally 

23


correlates with high IRAK1 and consequently higher type I interferon level. Indeed an 

elevated level of IRAK1 and type I interferon was found in EBNA2 transfectants. 

Further studies will be required to investigate the precise molecular mechanisms how 

EBNA2 affects miR expression and in particular miR-21 and miR-146a. However the data 

suggest that EBNA2 might contribute to EBV induced B cell transformation by a positive 

regulation of oncomiR miR-21 and by interfering with the innate immunity through regulating 

a key regulator of it, namely miR-146a (Rosato et al., submitted). Additionally, this miR has 

been attributed with oncosuppressive functions. Our data thus suggest that EBNA2 may 

promote B cell transformation by simultaneously increasing an oncomiR and reducing a miR 

with known tumor suppressing functions. 

Publications 

Imig J, Motsch N, Zhu JY, Barth S, Okoniewski M, Reineke T, Tinguely M, Faggioni A, 

Trivedi P, Meister G, Renner C, Grässer FA. MicroRNA profiling in EBV-associated B-cell 

lymphomas. Nucleic Acid Res 2011, 39: 1880-93. doi: 10.1093/nar/gkg1043. 


Antonio Angeloni, Pankaj Trivedi, professors; 

Roberta Santarelli, Mara Cirone, Antonella 

Farina, Roberta Gonnella, researchers; Marisa 

Granato, Eleni Anastasiadou, post-doc 

fellows; Francesco Boccellato, Paola Rosato, 

PhD students. 

24


Study of RNA damage in yeast as a model for age-related 

degenerative diseases 

Claudio Falcone 

Department of Biology and Biotechnologies "Charles Darwin" 

℡: +39 06 49912278 - @: claudio.falcone@uniroma1.it 

The aim of the present project is to elucidate how cells can handle and respond to the 

accumulation of oxidized RNAs. 

Recently, a growing body of studies suggests that RNA oxidation is an early event in a 

wide variety of neurological diseases, including Alzheimer's disease (AD) and amyotrophic 

lateral sclerosis (ALS), and in the progressive loss of muscle mass and strength which occur 

during aging (sarcopenia). Uncovering on the consequences and cellular handling of the 

oxidatively damaged RNA may provide significant insights into the pathogenesis of 

neurodegenerative diseases. Due to the complexity of studying these processes in 

mammalian model organisms, yeast could represent, because of its easy handling and the 

high conservation of fundamental cell pathways, a convenient model to trace the basal root 

for determining the effects of RNA damage on cell physiology. We previously demonstrated 

that mutants in mRNA degradation, beside the accumulation of mRNAs, show premature 

aging, high level of intracellular ROS and apoptotic cell death features. These mutants 

represent a convenient tool to study the effects of mRNA modifications during aging. 

In preliminary experiments, we observed that in mRNA degradation mutants (Kllsm4∆1) 

the reversion rate of mutation hugely increased with aging, compared to the wild type. In fact, 

we found that after 14 days of growth, when cell viability dropped to 0,004%, about 30% of 

survivals were Trp+ revertants while, in the same conditions, the revertants in the wild type 

population were only 0,008%. 

High reversion rate, as that measured in mRNA degradation mutants, was also observed 

in extremely aged wild type cells after 20 days of cultivation, suggesting that such 

phenomenon is physiologically related to aging. 

Interestingly, we found that most of the reversions observed in both wild type and 

Kllsm4∆1 mutant, were not due to adaptive DNA mutations since cells, after repeated 

cultivation, showed again the tryptophan auxotrophy. 

This fact suggests that such mutations were not at the DNA level but, rather, at the RNA 

level (mRNA and/or tRNA). We hypothesize that the mechanism underlying the suppression 

of the auxotrophic mutations under selective pressure might result from translational errors 

induced by oxidized mRNAs. 

According to this, our data concerning the mutation rate might indicate that, in yeast cells, 

the capability to handle oxidized RNAs drops with aging. In this respect, the Kllsm4∆1 

mutant, which accumulates mRNAs and ages much faster that the wild type strain, 

25


represents a useful tool for deciphering the molecular mechanisms of cell response to mRNA 

oxidation. 

We observed that the use of a lower glucose concentration, 0,2% instead of 2%, and the 

over-expression of HIR1 and PGK1 genes extended lifespan of the Kllsm4∆1 mutant. We are 

currently measuring the mutation rate in these conditions. 

Hydroxyl radicals, produced within cells, can easily modify RNA because they are highly 

reactive and cannot diffuse from their sites of formation. The most prevalent oxidized base in 

RNA is 8-hydroxyguanosine (8-OHG), which can be detect/quantified through Highperformance 

liquid chromatography-electrochemical detection (HPLC-ECD). To make a 

correlation between mutation frequency and RNA oxidation, we are developing a protocol to 

measure the oxidized fraction of total RNAs from wild type and Kllsm4Ä1 mutant cells during 

aging. 

Publications 

Mazzoni C, Falcone C. mRNA stability and control of cell proliferation. Biochem Soc Trans 

2011, 39: 1461-5. doi: 10.1042/BST0391461. 

Palermo V, Pieri L, Silvestri R, La Regina G, Falcone C, Mazzoni C. Drug-induced inhibition 

of tubulin polymerization induces mitochondrion-mediated apoptosis in yeast. Cell Cycle 

2011, 10: 3208-09. doi: 10.4161/cc.10.18.16514. 


Cristina Mazzoni, researcher; Vanessa 

Palermo, post-doc fellow; Michele Saliola, 

graduated technician. 


Frank Madeo, Institute of Molecular Biosciences, 

University of Graz, Austria. 

26


From leaderless to leadered mRNAs: mRNA features modulating 

ribosome binding and translation initiation in Archaea 

Paola Londei 

Department of Cellular Biotechnologies and Hematology 

℡: +39 06 4940463 - @: londei@bce.uniroma1.it 

Background and aims 

Initiation of protein synthesis is the rate-limiting step of translation and the main target of 

translation regulation. However, the mechanism and molecular machinery for initiation have 

diverged in the primary domains of life: Bacteria, Archaea and Eukarya. In Bacteria, 

translation initiation is relatively simple, while in Eukarya is complex and requires more 

components. The Archaea, despite their prokaryotic phenotype, have an unexpectedly 

elaborated initiation process, requiring at least six initiation factors. Archaeal mRNAs differ 

widely in the structure of their translation initiation regions (TIR). Only a minority of TIRs are 

endowed with Shine-Dalgarno (SD) sequences for ribosome binding; in a majority of cases 

there is no SD motif, and very often there is no 5’ untranslated region (5’UTR) at all. In the 

latter case the mRNA is called leaderless. Leaderless mRNAs can constitute up to 50% of all 

transcripts in certain archaeal species. Archaeal SD-less and leaderless mRNAs have been 

proposed to be translated using initiation mechanisms alternative to those employed for 

canonical mRNAs having SD motifs, but experimental data in support of this hypothesis are 

still scarce. Using the thermophilic archaeon Sulfolobus solfataricus as the model system, we 

will study the features of mRNA/ribosome interaction in Archaea, analyzing different types of 

model mRNAs endowed with different TIRs for their translational capacity, efficiency of 

interaction with the ribosomes and formation of 30S and 70S initiation complexes. 

Results and perspectives 

To investigate the mRNA features involved in the control of translational efficiency, we 

prepared a number of model constructs to be translated in vitro and in vivo. The genes of 

choice were a natural leaderless mRNA (lacS), a natural “quasi leaderless” mRNA (aSUI-1), 

whose start codon is preceded by 5 nucleotides, and an artificial leaderless mRNA (104∆L). 

Preliminarily, the predicted 5’ termini of aSUI1 and lacS were validated experimentally by 

primer extension assays. The artificial leaderless mRNA (104∆L) was derived from a 

leadered mRNA by removing its 5’UTR and cloning the truncated gene in the plasmid (pBS) 

SK+ under the control of the viral promoter T7. The aSUI1 gene and the lacS gene were also 

cloned in (pBS) SK+, the former from a PCR amplificate, the second by subcloning from the 

construct pCmalLacS (a kind gift of Sonja Albers, Marburg ). 

The in vitro transcripts were incubated in our well-established cell-free system for protein 

synthesis. The translation products were revealed by radioactive labelling, or, in the case of 

the lacS protein, by colorimetric detection of the products of enzymatic activity. All the 

27


mRNAs were translated with sufficient efficiency. Then, we modified the TIR of each of the 

above three constructs. Specifically, 5’-UTR of monotonous sequence (oligo-A) and different 

lengths were added to the leaderless genes, while the 5-nucleotide-5’UTR of the quasileaderless 

aSUI1 gene was shortened progressively to 4, 3 and one nucleotide. These 

experiments are aimed at unravelling the features of a translatable SD-less 5’-UTR in 

thermophilic archaea. In particular, the use of 5’-UTRs of monotonous sequence, and, later, 

of 5’-UTRs capable of forming secondary structures, should enable us to discriminate 

between the effects of length, sequence and folding. So far, we have found that the addition 

of short oligo-A tracts to mRNA 104∆L drastically reduces its translational efficiency. Further 

experiments are in progress. 

In parallel, we set up to develop a system for in vivo translational analysis. In vitro 

translation has several limitations, for instance that it is impossible to obtain perfectly 

leaderless mRNAs by transcription with T7 polymerase. In vivo analysis is carried out using 

the PBL2025 strain of Sulfolobus solfataricus, which is uracil auxotrophic. This strain was 

transformed with the plasmid pCMalLacS which contains the selectable marker pyrEF and 

the leaderless lacS gene. A number of positive transformants have been selected on uracildeficient 

plates and lacS expression has been detected by the colorimetric β-galactosidase 

assay. The starting construct has been engineered by adding to its 5’ terminus monotonous 

oligo-A sequences of varying length. Translational analysis is in progress. 

Once completed the in vitro and in vivo analysis of TIR features, our next intent will be to 

determine whether an artificially increased intracellular concentration of certain translation 

initiation factors results in a selective advantage for the translation of leaderless mRNAs. To 

this end S. solfataricus PBL2025 will be co-transformed with the pCmal-IF, which carries the 

IF gene of interest, and the plasmid pCMalLacS encoding the leaderless lacS gene. 

Publications 

Benelli D, Londei P. Translation initiation in Archaea: conserved and domain-specific 

features. Biochem Soc Trans 2011, 3: 89-93. 


Dario Benelli, researcher; Michela Pinzaglia, 

PhD student; Dorina Polinari, graduated yellow. 


Anna La Teana, Università Politecnica delle 

Marche; Sonja-Verena Albers, Max Plank 

Institute for Terrestrial Microbiology, Marburg, 

Germany. 

28

Area 2 

Pathogenic mechanisms of microbially 

associated diseases

Area 2: Pathogenetic mechanisms of microbially associated diseases 

Molecular characterization of viruses causing bronchiolitis and 

study of viral and host factors affecting Type I IFN antiviral 

response induced by respiratory viruses 

Guido Antonelli 


℡: +39 06 4474122 - @: guido.antonelli@uniroma1.it 

Bronchiolitis is the principal cause of hospitalization for infants. Several risk factors are 

associated with the severity of bronchiolitis, such as premature birth, immunodeficiency, and 

chronic disease, but the majority of the infected infants has no obvious risk factors. It is 

tempting to speculate that virus features (e.g. different and specific strains, viral loads, etc.), 

or host factors associated to an effective immune response might be also important in 

determining the disease severity. 

Hence, we proposed to characterize respiratory viruses determinants of virulence and 

study the role of innate immunity in antiviral response, either in patients or in cell lines. 

First, we characterized viral infections, with a comprehensive panel of PCR-based 

reactions, in respiratory samples from patients seeking medical care in Emergency 

Departments: no respiratory signs/symptoms could differentiate or were useful to distinguish 

respiratory viruses (either children or adults). Hence, a comprehensive virological diagnosis 

is needed to differentiate respiratory agents and their role in specific respiratory syndromes. 

During the first winter circulation of pandemic Influenza A H1N1 (InfA-pH1N1), 

respiratory syncytial virus (RSV) was the pathogen with the highest severity of infection in 

children, with highest intensive care rates, even though total days of hospitalization of 

children were independent on the type of virus found in their specimens. RSV was the major 

pathogen in bronchiolitis, followed by rhinovirus (RV) while metapneumovirus (hMPV) and 

pH1N1 were found only in few cases. 

Nasal washings from children positive to RSV, RV, pH1N1, Bocavirus (hBoV), and hMPV 

were inoculated onto tubes of different cell cultures; with the exception of the not-yetcultivated 

hBoV, viral stocks were obtained to characterize in vitro type I Interferon (IFN) 

antiviral activity. InfA pH1N1 resulted partially resistant to type I IFN with respect to a 

seasonal InfA and hMPV was more sensitive to IFN beta than to leukocyte IFN. 

We then evaluated the association between RSV load and disease severity or between 

different biochemical/clinical parameters and mRNA-levels of the interferon stimulated gene 

(ISG) 56 in respiratory samples from 132 infants hospitalized for bronchiolitis and infected 

with RSV as a single (90%), or as a dual infection with other respiratory viruses (10%). In the 

same context we also evaluated how viral load varied during the course of RSV infection. 

Results indicated that viral load was positively related to the clinical severity of 

bronchiolitis, the length of hospital stay, the levels of glycaemia and the number of ISG56- 

mRNA copies, whereas an inverse correlation was observed with levels of haemoglobin. We 

also found that the RSV load significantly decreased between the first and second sample in 

31


most single and RSV coinfections. Indeed, infants with high RSV load on hospital admission 

are more likely to have both more severe bronchiolitis and a higher airway activation of 

antiviral immune response. 

To clarify the important issue of RV proposed association with development of paediatric 

asthma, the rate of recurrent wheezing was analyzed 12 months after hospitalization for 

bronchiolitis: multivariate analysis identified as major independent risk factors for recurrent 

wheezing, rhinovirus infection and a positive family history for asthma. 

To tentatively relate a clinical phenotype to a RV strain, we performed RV genomic region 

5’ UTR and VP4/VP2 sequencing and a phylogenetic tree was constructed. We then 

analyzed in field isolates the close 5’ proximity of polyprotein initiation site (AUG at nt 611), a 

conserved 24 amino acids (aa) motif exposed during the so-called capsid breathing, the 

Met67-Ser68 cleavage site at the VP4-VP2 junction, the position of a putative internal cisacting 

replication element (cre) located within viral protein 2 (VP2). The 5’ proximity of the 

initiator AUG and the VP4-VP2 cleavage sites were highly conserved in patient strains of all 

RV species, the cre position was characteristic and conserved among RV C strains, and the 

24 aa motif was conserved in RV A and B (21/24 aa) but not in RV C isolates (only 9 aa out 

of 24). Patient data were then analyzed: age distribution and clinical data of RV A and C 

were not significantly different, even though there was a tendency of RV C strains to be 

associated with hospitalization of older children and with wheezing. 

Publications 

Pierangeli A, Scagnolari C, Selvaggi C, Verzaro S, Spina MT, Bresciani E, Antonelli G, 

Bertazzoni G. Rhinovirus frequently detected in elderly adults attending an emergency 

department. J Med Virol 2011, 83: 2043-7. doi: 10.1002/jmv.22205. 

Scagnolari C, Trombetti S, Selvaggi C, Carbone T, Monteleone K, Spano L, Di Marco P, 

Pierangeli A, Maggi F, Riva E, Antonelli G. In vitro sensitivity of human metapneumovirus 

to type I interferons. Viral Immunol 2011, 24: 159-64. doi: 10.1089/vim.2010.0073. 

Scagnolari C, Trombetti S, Soldà A, Selvaggi C, Monteleone K, Spano L, Pierangeli A, 

Clementi M, Turriziani O, Antonelli G. Pandemic 2009 H1N1 influenza virus is resistant to 

the antiviral activity of several interferon alpha subtypes. J Interferon Cytokine Res 2011, 

31: 475-9. doi: 10.1089/jir.2010.0125. 


Ombretta Turriziani, professor; Laura 

Antonelli, Fabio Midulla, Paola Papoff, 

Alessandra Pierangeli, Carolina Scagnolari, 

researchers; Carla Selvaggi, research fellow; 

Katia Monteleone, PhD student. 


Stefano Chiavelli, Carlo Concato, Ospedale 

Bambin Gesù, Roma; Monica Ferreri, Patrizia 

Bagnarelli, Ospedale Generale di Torrette, 

Ancona; Christina Christodoulou, Jan Richter, 

Molecular Virology, Cyprus Institute of Neurology 

& Genetics. 

32


Lipopolysaccharide and peptidoglycan adaptation to host as an 

immune evasion strategy of gram-negative pathogens 

Maria Lina Bernardini 


℡: +39 06 49917850 - @: marialina.bernardini@uniroma1.it 

Pathogens have evolved sophisticated strategies to finely tune the host innate immune 

responses and the inflammatory reaction. In fact, symptoms of infectious diseases are often 

based on the inflammatory reaction induced by pathogens in the site of infection. 

Shigella is an enteroinvasive pathogen provoking an acute and severe inflammation of 

colonic and rectal mucosa. Shigella is able to drive the immune sensing and response mainly 

by manipulating the recognition of PAMPs (Pathogens Associated Molecular Patterns) by the 

PRRs (Pattern Recognition Response) and by subverting the host cell-death machinery. 

Pseudomonas aeruginosa (PA) is an opportunistic pathogen causing acute and chronic 

infection in the airways of Cystic Fibrosis (CF) patients. Strains establishing life-long chronic 

infection are usually pathogenic variants distinguished from the initially acquired strains, often 

differing for the presence/absence of virulence factors and harboring PAMPs modifications, 

e.g. in the lypopolysaccharide (LPS) structures or in flagellin. 

Our research was focused on the impact of PAMPs on the host response during the 

invasive process of Shigella and lung colonization of PA. We also evaluated the ability of 

these two pathogens to hijack the host cell machinery controlling the death processes. 

Our findings show that Shigella proliferating within the epithelial cells is able to trigger 

both apoptosis and necrosis of the infected cells, depending on several parameters, including 

the number of intracellular bacteria and the time of infection. Apoptosis is dependent on 

mitochondrial dysfunction and caspase-9 activation. The stress-response molecule 

GADD45alfa is involved in this process. Likewise, Shigella kills dendritic cells through various 

mechanisms, including apoptosis and pyroptosis. 

PA clinical strains, isolated from a CF patient at sequential stages of lung infection, 

induce a pyroptotic cell death of macrophages and dendritic cells likely using different 

PAMPs and PRRs as elicitors. PAMPs of these PA clinical isolates display modified 

structures and immunopotential. LPS, Peptidoglycan (PGN) and flagellin are deeply different 

in the clonal strains isolated at various times of the disease. The structural modifications are 

reflected by the different ways of these structures to interact with their respective PRRs, 

leading the PA strains isolated from the chronic stage of the infection to be able of escaping 

from the immune system recognition. However, the PAMPs modifications make these 

“chronic” strains better elicitors of the inflammasome. 

Similarly, PAMPs of Shigella vary during infection and exhibit a dramatic influence on 

host cell responses. PGN is remodeled through a set of enzymes including the plasmid 

encoded gene SfpgdA, which acts as a PGN deacetylase producing a PGN more resistant to 

33


the lisozyme activity. PGN shedding is also a mechanism by which this pathogen dampens 

the immune system, especially through the sensing of the intracellular PRRs Nod1 and 

Nod2. Moreover, Shigella modifies LPS during infection, in this way modulating the response 

of different cell populations including macrophages, neutrophils and dendritic cells. The 

majority of the inflammatory cytokines are influenced by the LPS changes as well as the 

ability of neutrophils to produce factors needed to Shigella eradication. 

Finally, our findings suggest that, despite the different style of life, these two pathogens 

exploit the same strategies to establish a survival niche into the host. 

Publications 

Cigana C, Lorè NI, Bernardini ML, Bragonzi A. Dampening host sensing and avoiding 

recognition in Pseudomonas aeruginosa pneumonia. J Biomed Biotechnol 2011, 852513. 

doi: 10.1155/2011/852513. 

Lembo Fazio L, Nigro G, Noël G, Rossi G, Chiara F, Tsilingiri K, Rescigno M, Rasola A, 

Bernardini ML. Gadd45α activity is the principal effector of Shigella mitochondriadependent 

epithelial cell death in vitro and ex vivo. Cell Death Dis 2011, 2:e122. doi: 

10.1038/cddis.2011.4. 


Laura Curcurù, Luigi Lembo Fazio, post-doc 

fellows; Valeria Ciancarella, Gaëlle Noël, PhD 

students. 


Abdel Allaoui, Lab. Bactériologie Moléculaire, 

Université Libre de Bruxelles, Belgium; 

Alessandra Bragonzi, Divisione di Immunologia, 

Trapianti e Malattie Infettive, Istituto Scientifico 

San Raffaele, Milano; Antonio Molinaro, 

Dipartimento di Chimica Organica e Biochimica, 

Università di Napoli Federico II; Maria Rescigno, 

Dipartimento di Oncologia Sperimentale, Istituto 

Europeo di Oncologia, Milano. 

34


Involvement of sRNA molecules in the complex regulatory circuits 

of virulence gene expression in Shigella flexneri and in 

enteroinvasive E. coli 

Bianca Colonna 


℡: +39 06 49917582 - @: bianca.colonna@uniroma1.it 

Recently small noncoding RNAs (sRNA) have emerged as crucial elements in bacterial 

cell regulation. In particular, it is becoming increasingly evident that sRNAs, besides acting in 

gene regulation in general, can also play a key role in controlling the expression of virulence 

genes or affect adaptive stress responses which are crucial for bacteria to survive within the 

host. 

In several human bacterial pathogens sRNAs appear to be important for integrating 

environmental stimuli into outputs that are relevant for the pathogenicity process. Therefore 

we have analyzed the potential involvement of sRNA transcripts in the modulation of 

virulence gene expression in Shigella, a human pathogen that causes a severe enteric 

syndrome mainly in the developing countries. The cellular pathogenesis and the clinical 

presentation of shigellosis result from the complex action of a large number of bacterial 

virulence factors mainly located on a large virulence plasmid (pINV). By analyzing the large 

intergenic region separating the virA and icsA genes, we have identified the first pINV 

encoded sRNA of Shigella flexneri, RnaG. 

RnaG acts as antisense on the transcript of the icsA gene, which encodes the protein 

required for the intra- and intercellular spread of the bacterium inside the host cell. A detailed 

in vivo and in vitro characterization of the RnaG molecule and of its promoter has allowed us 

to understand the strategy adopted by this molecule to control icsA. As opposed to other 

sRNAs, which affect the ability of the target mRNA to be translated, we have found that 

RnaG is capable to downregulate icsA transcription by means of two, not mutually exclusive 

mechanisms. As for the first mechanism we have shown that silencing of the RNAG promoter 

results in higher activity of the convergent icsA promoter. On the other hand, RnaG can also 

cause a direct repression of icsA transcription by acting as antisense RNA, provoking a 

premature termination of the icsA messenger. In fact, binding of RnaG to the nascent icsA 

transcript induces the formation of a stem-loop motif at the 5’end of the mRNA, mimicking an 

intrinsic terminator and resulting in the synthesis of a truncated icsA transcript. 

Over the years evidence has built up indicating that the regulation of icsA depends on the 

nucleoid protein H-NS which acts as a repressor, and on the AraC-like protein VirF, the major 

activator of the Shigella invasivity regulon. Since the molecular details of this regulatory 

mechanism are still not fully understood, we have analysed the synergistic and antagonistic 

interplay among the regulatory proteins and the RnaG molecule in order to evaluate their 

contribution to the expression of the icsA gene. The results obtained show that both proteins, 

H-NS and VirF, recognize the icsA and RnaG promoter regions in vivo and in vitro and that 

35


the temperature-dependent expression of icsA depends on a delicate balance among H-NS 

repression, VirF activation and RnaG transcription. 

All together our data evidence the complexity of the circuits regulating virulence in 

Shigella and give insight into the strategy adopted by pathogenic bacteria to obtain a 

coordinated and efficient expression of their key virulence determinants inside the host. 

Publications 

Barbagallo M, Martino ML, Marcocci L, Pietrangeli P, Carolis ED, Casalino M, Colonna B, 

Prosseda G. A new piece of the Shigella pathogenicity puzzle: spermidine accumulation by 

silencing of the speG gene. PLoS One 2011, 6:e27226. doi: 10.1371/journal.pone. 

0027226. 

De Carolis E, Posteraro B, Florio AR, Colonna B, Prosseda G, Bugli F, Lorenzetti SR, 

Fiscarelli E, Inzitari R, Iavarone F, Castagnola M, Fadda G, Sanguinetti M. Analysis of 

heat-induced changes in protein expression of Stenotrophomonas maltophilia K279a 

reveals a role for GroEL in the host-temperature adaptation. Int J Med Microbiol 2011, 301: 

273-81. doi: 10.1016/j.ijmm.2010.10.001. 

Nicoletti M, Iacobino A, Prosseda G, Fiscarelli E, Zarilli R, De Carolis E, Petrucca A, Colonna 

B, Casalino M. Stenotrophomonas maltophilia isolated from cystic fibrosis patients: 

genotyping analysis and molecular characterization of virulence determinants. Int J Med 

Microbiol 2011, 301: 34-43. doi: 10.1016/j.ijmm.2010.07.003. 

Tran CN, Giangrossi M, Prosseda G, Brandi A, Di Martino ML, Colonna B, Falconi M. A 

multifactor regulatory circuit involving H-NS, VirF and an antisense RNA modulates 

transcription of the virulence gene icsA of Shigella flexneri. Nucleic Acids Res 2011, 39: 

8122-34. doi: 10.1093/nar/gkr521. 


Gianni Prosseda, researcher; Maria Letizia Di 

Martino, post-doc fellow; Rosaria Campilongo, 

PhD student. 


Gioacchino Micheli, Istituto di Biologia e 

Patologia Molecolari, CNR, Roma; Maurizio 

Falconi, Dipartimento di Biologia Molecolare, 

Cellulare e Animale, Università di Camerino; 

Mariassunta Casalino, Dipartimento di Biologia, 

Università di Roma Tre. 

36


Inhibition of Pseudomonas aeruginosa biofilms: new molecular 

strategies targeting cyclic-di-GMP metabolism 

Francesca Cutruzzolà 


℡: +39 06 49910713 - @: francesca.cutruzzola@uniroma1.it 

Biofilms formed by bacterial pathogens are responsible of more than 70% of all infections 

in developed countries and are less sensitive to treatments with antimicrobial agents. The 

ubiquitous second messenger 3', 5'-cyclic diguanylic acid (c-di-GMP) is used in most bacteria 

to control the switch to the biofilm lifestyle; c-di-GMP has no counterpart in eukaryotic cells 

and thus it is an ideal target to develop effective anti-biofilm strategies. 

The aim of this proposal is to study the metabolism of c-di-GMP in the opportunistic 

human pathogen Pseudomonas aeruginosa in order to find new targets for effective antibiofilm 

drugs. We plan to characterize strategic P. aeruginosa proteins involved in c-di-GMP 

metabolism (diguanylate cyclases-DGCs and phosphodiesterases-PDEs) and to study the 

connection between the c-diGMP pathway and other metabolic pathways relevant for 

pathogenesis. 

We developed the enzymatic and c-di-GMP binding assays using the reference DGC 

PleD from Caulobacter crescentus. We have measured the DGC activity of purified PleD and 

confirmed, by titration of PleD with c-di-GMP (employing Isothermal Titration Calorimetry- 

ITC), that PleD binds c-di-GMP (2 mol c-di-GMP/mol protein; Kd 0,9 µM) in agreement with 

literature data. We have tested putative DGC inhibitors, starting from selected compounds, 

previously identified in microbiological screening of a chemical library by Landini and 

coworkers. These compounds do not significantly inhibit PleD in vitro (up to 500 µM) 

suggesting that they do not inhibit c-di-GMP biosynthesis through direct interaction with DGC 

proteins, but likely affect the availability of nucleotide substrate(s). 

We have characterized putative P.aeruginosa DGCs or PDEs, focusing our attention on 

selected targets, i.e. the DGC PA1120 and the PDEs PA4781 and PA4108. The PA1120 

protein (TpbB) is a DGC linking Las-induced quorum sensing (QS) to the formation of matrix 

exopolysaccharide (EPS) and extracellular DNA, necessary for biofilm development and 

coordinated group response in P. aeruginosa. We have produced the isolated catalytic 

domain of TpbB and measured its catalytic activity (0,1 nmol/min/mg); PA1120 is not 

significantly inhibited by c-di-GMP at low GTP concentrations. Identification and 

characterization of inhibitors are in progress. 

Few biochemical data are available on PDEs containing the HD-GYP domain, despite 

their importance in pathogenesis and their role in controlling biofilm formation. While EALtype 

PDEs hydrolyze c-di-GMP to linear diguanylate (pGpG), HD-GYPs completely hydrolyze 

c-di-GMP to GMP in a single reaction. In P.aeruginosa, 2 HD-GYP proteins are found, which 

are able to decrease c-di-GMP levels in vivo; these proteins contain the PDE catalytic 

37


domain and a (putative) regulatory domain, either of the CheY-type (PA4781) or unknown 

(PA4108). Both proteins were purified and tested for their PDE activity in vitro, monitoring c- 

di-GMP hydrolysis to GMP: while the PA4108 enzyme shows c-di-GMP hydrolytic activity, 

PA4781 is inactive. In order to bypass a possible activation step, a truncated version of 

PA4781 was also analyzed (PA4781-G) and found to be inactive in vitro, but active in cell 

extracts or in intact E. coli cells. These results suggest that the activity of PA4781 may 

depend on the interaction with an intracellular partner, as previously proposed for other HD- 

GYP PDEs. 

In parallel, we have also studied Aromatic L-Aminoacid decarboxylase (DOPA 

decarboxylase-DDC), the human enzyme responsible for a key step in the synthesis of 

adrenaline, one of the eukaryotic signal molecules sensed by Gram-negative pathogens. 

These molecules bind to bacterial membrane receptors and activate signaling pathways 

involving, among others, c-diGMP, thus leading to modulation of biofilm formation. Analysis 

of the three-dimensional structure coupled to a kinetic study allows to identify the structural 

determinants of the open/close conformational change occurring upon PLP binding and 

thereby propose a model for the stability of Group II decarboxylases in vivo (Giardina et al., 

2011). 

Publications 

Giardina G, Montioli R, Gianni S, Cellini B, Paiardini A, Voltattorni CB, Cutruzzolà F. Open 

conformation of human DOPA decarboxylase reveals the mechanism of PLP addition to 

Group II decarboxylases. Proc Natl Acad Sci USA 2011, 108: 20514-9. doi: 10.1073/ 

pnas.1111456108. 


Nicoletta Castiglione, Giorgio Giardina, 

Serena Rinaldo, post-doc fellows; Valentina 

Stelitano, PhD student; Manuela Caruso, 

technician. 


Davide Antoniani, Paolo Landini, Università 

di Milano; Livia Leoni, Giordano Rampioni, 

Università Roma Tre; Loredana Cappellacci, 

Università di Camerino. 

38


Defense mechanisms against oxidative and nitrosative stress in 

pathogenic protozoa 

Paolo Sarti 


℡: +39 06 49910944 - @: paolo.sarti@uniroma1.it 

Giardiasis is a well-known human infectious disease caused by the amitochondriate 

protozoan parasite Giardia intestinalis, whose genome has been sequenced. Giardia is an 

O 2 -sensitive microorganism lacking some conventional ROS-scavenging enzymes, such as 

catalase and superoxide dismutase. Despite that, Giardia preferentially colonizes the fairly 

oxygenated small intestine. Our research has been focusing on the structural and functional 

characterization of the enzymes playing a key role in the antioxidant defense system of this 

microbial pathogen. 

We have shown that Giardia cells express a flavodiiron protein (FDP), a superoxide 

reductase (SOR) and, under nitrosative stress conditions, a flavohemoglobin (FlavoHb). This 

is surprising because FDPs and SORs are typically absent in eukaryotes and FlavoHbs are 

very rarely found in protists. The three proteins were expressed in E. coli and purified. The 

FDP is endowed with a high H 2 O-producing O 2 -reductase activity (TN > 40 s -1 , K M,O2 ≤ 2 µM, 

T = 20 °C), that led us to propose that the primary function of the FDP is to protect Giardia 

from O 2 toxicity, a function originally attributed to NADH-oxidase. The recombinant Giardia 

FlavoHb aerobically metabolizes nitric oxide (NO) with high efficacy (TN = 116 ± 10 s -1 at 1 

µM NO, K M,O2 = 22 ± 7 µM, T = 37 °C). Consistently, we found that the NO-induced 

expression of FlavoHb in Giardia cells endows the parasite with the ability to degrade NO in 

the presence of O 2 , which likely represents a strategy to evade the host immune response 

during infection. In collaboration with M.Teixeira and L.M.Saraiva (New University of Lisbon, 

Portugal) and D.E.Cabelli (Brookhaven National Laboratory, New York, USA), more recently 

the recombinant Giardia SOR was also characterized. The protein, whose expression in the 

trophozoite was assessed by immunoblotting, shows negligible superoxide dismutase 

activity, but in the reduced state is highly reactive with superoxide anion, as shown by pulse 

radiolysis and stopped-flow spectrophotometry. 

These results suggest that these antioxidant enzymes favor parasite survival in the host. 

Publications 

Borisov VB, Forte E, Sarti P, Giuffrè A. Catalytic intermediates of cytochrome bd terminal 

oxidase at steady-state: ferryl and oxy-ferrous species dominate. Biochim Biophys Acta 

2011, 1807: 503-9. doi: 10.1016/j.bbabio.2011.02.007. 

39


Giuffrè A, Borisov VB, Mastronicola D, Sarti P, Forte E. Cytochrome bd oxidase and nitric 

oxide: from reaction mechanisms to bacterial physiology. FEBS Lett. 2011 Epub. doi: 

10.1016/j.febslet.2011.07.035. 

Mastronicola D, Giuffrè A, Testa F, Mura A, Forte E, Bordi E, Pucillo LP, Fiori PL, Sarti P. 

Giardia intestinalis escapes oxidative stress by colonizing the small intestine: a molecular 

hypothesis. IUBMB Life 2011, 63: 21-5. doi: 10.1002/iub.409. 

Sarti P, Forte E, Mastronicola D, Giuffrè A, Arese M. Cytochrome c oxidase and nitric oxide 

in action: molecular mechanisms and pathophysiological implications. Biochim Biophys 

Acta 2011 Epub. doi: 10.1016/j.bbabio.2011.09.002. 

Testa F, Mastronicola D, Cabelli DE, Bordi E, Pucillo LP, Sarti P, Saraiva LM, Giuffrè A, 

Teixeira M. The superoxide reductase from the early diverging eukaryote Giardia 

intestinalis. Free Radic Biol Med 2011, 51: 1567-74. doi: 10.1016/j.freeradbiomed. 

2011.07.017. 


Elena Forte, researcher, Daniela Mastronicola, 

Fabrizio Testa, post doc fellows, Alessandro 

Giuffrè, CNR researcher. 


Diane E. Cabelli, Brookhaven National 

Laboratory, New York, USA; Pier Luigi Fiori, 

Università di Sassari; Brajendra K. Singh, 

Department of Chemistry, University of New 

Delhi, India; Miguel Teixera, Instituto de 

Tecnologia Química e Biológica, Universidade 

Nova de Lisboa, Portugal; João B. Vicente, 

Universidade de Lisboa, Portugal. 

40


Immunopathogenesis of HIV infection: study of innate immunity 

and dendritic cells 

Vincenzo Vullo 

Department of Public Health and Infectious Diseases 

℡: +39 06 49970313 - @: vincenzo.vullo@uniroma1.it 

The study of dendritic cells (DC) as a link among innate and specify immunity is very 

important for pathogenesis, clinical and therapeutic approaches in HIV infection. The 

chemotactic recruitment of APC at the site of infection is critical for the initiation of 

appropriate immune responses. There are several data suggesting that chemokine receptor 

CCR5 is involved in both positive and negative regulation of APC system by the modulation 

of leukocyte trafficking, cellular activation and cytokine expression. What are the in vitro and 

in vivo immunologic consequences of pharmacologic inhibition of CCR5 function remain to 

be investigated. Recently, compounds targeting CCR5 have been introduced into clinical 

practice for the treatment of HIV infection. In this first year of our projects study, we have 

investigated the direct in vitro effect of anti-HIV CCR5 antagonist maraviroc (a new 

antiretroviral drugs) on chemotactic activity of DCs and innate immune cells. In particular, we 

evaluated the in vitro effect of different concentrations of CCR5 antagonist maraviroc (MVC) 

on cell migration of monocytes, macrophages (MO) and monocyte-derived dendritic cells 

(MDC) towards peptide formyl-methionyl-leucyl-phenylalanine (fMLP) and chemokines 

regulated upon activation normal T cell expressed and secreted (RANTES) and 

CCL4/macrophage inflammatory protein-1 (MIP-1beta) and CCL2/monocyte chemotactic 

protein-1 (MCP-1). Results off low cytometric analysis showed that monocytes treated in vitro 

with MVC exhibited a significant dose-dependent reduction of chemotaxis towards MIP-1beta 

and MCP-1. fMLP-induced chemotactic activity decreased only at higher concentration (1 

mM and 10 mM of MVC). In addition, all concentrations of MVC (0·1, 1 and 10 mM) induced 

in vitro a significant inhibition of chemotaxis of MO and MDC in response to all tested 

chemoattractants. In another set of experiments, cell viability and phenotype (CD14 for 

monocytes, MO and CD1a for MDC) and expression of chemoattractant receptors CCR1, 

CCR4, CCR5 and FPR expression were investigated. We found no alteration in viability and 

phenotype in cells treated with MVC. Moreover, treatment with different concentrations of 

MVC did not modulate CCR1, CCR4, CCR5 and FPR expression in monocytes, MO and 

MDC. In summary our findings suggest that CCR5 antagonist MVC may have the in vitro 

ability of inhibiting the migration of innate immune cells by mechanism which could be 

independent from the pure anti-HIV effect. The drug might have a potential role in the downregulation 

of HIV-associated chronic inflammation by blocking the recirculation and trafficking 

of MO and MDC. Considering the increasing use of MVC in patients with HIV infection, 

further studies should be encouraged to understand the immunological consequences of 

41


CCR5 blockade in innate immune cells. Future studies are needed to understand more 

clearly the mechanism underlying this inhibitory phenomenon exerted in vitro by maraviroc. 

In a second line of investigation, we assessed also the immunologic response to 

tuberculosis by assessing interferon-γ release assays (IGRAs). The study of this new 

immunologic assay is important to understand the dynamic of immune response during 

tuberculosis, which is known to be an important and increasing opportunistic infections 

especially in HIV-infected patients. In a longitudinal study we demonstrated that the classic 

tuberculin skin test (TST) does not influence the outcome of subsequent IGRAs testing in 

individuals with negative TST results, but it can boost the IFN-g response in subjects 

sensitized to TB antigens and not detected by IGRA. The evaluation of the possibility that the 

TST administration might affect the outcome of subsequent QFT-GIT testing is important to 

support the adoption of the assay in immunosuppressed populations in an attempt to predict 

the risk of developing TB disease. 

Publications 

Rossi R, Lichtner M, De Rosa A, Sauzullo I, Mengoni F, Massetti AP, Mastroianni CM, Vullo 

V. In vitro effect of anti-human immunodeficiency virus CCR5 antagonist maraviroc on 

chemotactic activity of monocytes, macrophages and dendritic cells. Clin Exp Immunol 

2011, 166: 184-90. doi: 10.1111/j.1365-2249.2011.04409.x. 

Sauzullo I, Massetti AP, Mengoni F, Rossi R, Lichtner M, Ajassa C, Vullo V, Mastroianni CM. 

Influence of previous tuberculin skin test on serial IFN-γ release assays. Tuberculosis 

(Edinb) 2011, 91: 322-26. doi: 10.1016/j.tube.2011.05.004. 


Claudio M. Mastroianni, professor; Miriam 

Lichtner, researcher; Raffaella Rossi, Ilaria 

Sauzullo, post-doc fellows; Gabriella d’Ettorre, 

Claudia D’Agostino, hospital physicians; Fabio 

Mengoni, graduated technician. 

42

Area 3 

Molecular genetics 

of eukaryotes

Area 3: Molecular genetics of eukaryotes 

Assembly and functional analysis of genomic context vectors 

containing the human CFTR locus 

Fiorentina Ascenzioni 


℡: +39 06 49917577 - @: fiorentina.ascenzioni@uniroma1.it 

We have very recently published BAC vectors containing the wt CFTR allele 

(polymorphisms: (GATT) 7 in intron 6a; (TG) 11 T 7 in intron 8; V470 at position 470) and 

including the upstream and downstream regions. Functional activity of BAC-encoded CFTR 

was demonstrated in model FRT cells by different approaches including RT-PCR, 

quantitative real-time PCR, Western blotting, indirect immunofluorescence, and 

electrophysiological methods. Finally, and supporting the hypothesis that CFTR functions as 

a receptor for Pseudomonas aeruginosa, we demonstrated that CFTR-expressing cells 

internalized more bacteria than parental cells that do not express CFTR. 

The main objectives of the project are: 

1) Functional caracterization of the BAC in CF cells 

2) CFTR-BAC engineering in order to improve handling of the molecule and to add 

sequences that may be relevant for CF genetic research, such as specific mutations. 

CF defects complementation by cCFTRD12 

We have recently shown, by different functional assays, that the BAC vector cCFTRD12, 

when transfected into rat cells, encodes a functional CFTR protein (Auriche et al., 2010]. To 

extend this analysis to cell models relevant to CF gene therapy cCFTRD12 we need to 

transfer this vector to human epithelial cells from lung origin and having mutation in the 

CFTR gene. Due to the lack of efficient methods to transfer such large molecules, we 

performed preliminary work to select the most useful transfection reagents for bronchial 

epithelial cells. This was motivated by the fact that lipofectamine, which was successfully 

used to transfer the BAC into FRT (fisher rat thyroid) cells, didn’t work well with the human 

bronchial cell lines (CFBE and 16HBE). Nanoparticle-mediated gene delivery may represent 

a valid alternative to un-structured transfection reagent. Thereby, we are testing chitosanbased 

nanoparticles to deliver DNA to different cell lines, including 16HBE and CFBE cells. 

Preliminary results obtained with HEK cells transfected with EGFP-plasmids showed that the 

fraction of GFP positive cells, as determined by FACS analyses, reaches about 30% 4-5 

days after transfection and, keeping the cells in culture, it declines slowly over 15-20 days. Of 

note, cells treated with control reagents (EscortV from SIGMA or Lipofectamine from 

Invitrogen) showed similar efficiency but the fraction of GFP+ cells picks 2 day after 

transfection and rapidly decrease down to 0% 4-5 days after transfection. As previously 

reported by other, these results suggest that chitosan nanoparticles mediate a slow release 

of intracellular DNA and protect it from rapid intracellular degradation. We are currently 

45


optimizing the chitosan-nanoparticle mediated transfection protocol and extend this method 

to BAC vectors. 

CFTR-BAC engineering 

The most used approaches to engineer inserts cloned into BAC vectors make use of 

homologous recombination or site specific homologous recombination in bacterial cells. In 

both cases the target region remains signed by the marker gene or by the site used by the 

recombinase. Consequently, the engineered region would contain sequences unrelated to its 

function. To overcome this problem, we are setting a new procedure based on yeast 

homologous recombination to insert/exchange DNA fragments without using selectable 

markers for the targeted region. Preliminary experiments have been done with the BAC 

cCFTRD12 which has three selectable markers, herein identified as marker 1, 2 and 3, in the 

vector backbone. The BAC has been co-transfected into yeast cells with a fragment having 

complementary ends to either marker 1 and 2; the transformants have been selected for 

marker 3. All the transformants recovered have been analyzed for the presence of the unselected 

marker, which was detected in 10% of the clones. Phenotypic and molecular 

analyses of these clones are undergoing. In the same time we have designed a series of 

experiments in order to highlight possible preferential sites of recombination that would limit 

the use of this procedure. 

Publications 

Del Porto P, Cifani N, Guarnieri S, Di Domenico EG, Mariggiò MA, Spadaro F, Guglietta S, 

Anile M, Venuta F, Quattrucci S, Ascenzioni F Dysfunctional CFTR alters the bactericidal 

activity of human macrophages against Pseudomonas aeruginosa. PLoS One 2011, 

6:e19970. doi: 10.1371/journal.pone.0019970. 

Mattarocci S, D'Ambrosio E, Tafaro L, Somma V, Zannino G, Marigliano V, Ascenzioni F, 

Cimino-Reale G. Erosion of telomeric 3'-overhangs in white blood cells of aged subjects 

with high frequency of very short telomeres. Mech Ageing Dev 2011, 132: 27-32. 

http://dx.doi.org/10.1016/j.bbr.2011.03.031. 


Enea Gino Di Domenico, post-doc fellow, 

Cifani Noemi, PhD student; Paola, Parisi, 

graduated student; Barbara Pompili, student. 


Dr. Cleofe Palocci Dipartimento di Chimica, 

Sapienza Università di Roma. 

46


Light control of filamentous fungi life cycle: from system models to 

applications 

Paola Ballario 


℡: +39 06 49912392 - @: paola.ballario@uniroma1.it 

The study of blue light transduction pathways in filamentous fungi is the main field of 

interest of our group. This project persecutes two short term goals. The first is to clarify the 

mechanism controlling the switch ON and OFF of light regulated genes. The second is the 

characterization of the blue light sensor, the LOV domain. Genetically encoded protein 

photosensors as LOV are promising tools for engineering optical control of cellular behaviour; 

we are only beginning to understand how to couple these light detectors to effectors of 

choice (D. Strickland et al., Nat Methods 2010, 7: 623-26). 

The first fungal blue light BL photoreceptor, White collar-1 (WC-1), was isolated in 

Neurospora (by us and Prof Macino group) in 1996 and identified as a phototropin-like 

protein previously described only in plants. The blind wc-1 mutant is a phenocopy of white 

collar mutant wc-2. This coincidence of phenotype suggested that the two proteins (WC-1 

and WC-2) were in the same signal transduction pathways and that both were necessary for 

light responses in Neurospora. In fact WC-1 and WC-2 constitute the WC Complex (WCC) 

that now we know is the most diffuse type of photoreceptor among fungi. In Neurospora 

crassa light induced gene expression is dependent from the presence of the heterodimeric 

WC-1WC-2 complex and the acetylation NGF-1-dependent (Neurospora Gcn Five)of histone 

H3 residue K16 at the LRR (light responsive region) of specific promoters. The acetylation of 

histones is light dependent and transient (Grimaldi et al., 2006). 

Structural and functional aspects of WC-1 suggest that it can be considered as a primitive 

form of Nuclear Receptor (pNR), whose incoming signals are photons. Vertebrate nuclear 

receptors activate the transcription of their controlled genes through the action of a 

Coactivator. We already demonstrated the role of NGF-1 an Histone Acetyltransferase HAT 

(the ortologue of S. cerevisiae Gcn5p) as coactivator for light dependent switch on of 

trascription in Neurospora. During this first year of the project we demonstrated that NGF-1 

binds to WC-1 in D and L conditions of growth, and, only upon irradiation, it becomes active 

in the acetylation of histone H3 in the promoter region of light-genes. The conserved LXXLL 

sequences, involved in vertebrate NR-coactivators interactions have been identified in WC-1 

and NGF-1 and their relevance tested by mutagenesis. Our analyses revealed also a stable 

acetylation of WC-1 that is probably necessary in order to stabilize the photoreceptor, this 

acetylation however is independent either by light and by NGF-1. These data (reported in a 

manuscript just submitted) suggest a model based on a stable pNR-Coactivator interaction 

that becomes enzymatically active, as chromatin modifier, only after blue light illumination. 

47


The other aspect we have developed is the characterization of the light sensor domain 

(LOV for Light Oxygen and Voltage) of Tuber borchii and Tuber melanosporum. The LOV 

domain is a region of around 150 amino acids deriving from the less specialized proteinprotein 

interaction domain called PAS (stand for PER ARNT and SIM). LOV is the light 

sensor domain of WC-1 and of many other proteins. It is a sensor domain that shares the 

mixed α/β fold common to the Per-ARNT-Sim class of environmental sensors and carry a 

flavin chromophore (FMN), that broadly absorbs blue light (see the figure). While these 

cofactors are non-covalently associated with the surrounding LOV domain in the dark, 

photoexcitation generates a covalent adduct between the flavin C4a carbon atom and an 

invariant cysteine residue. There is now strong interest in characterizing LOVs from different 

sources or mutagenized version in order to utilize such “new” LOV domain fused to effectors 

domain to obtain active molecules under dark/light control. With the aim of contributing to this 

“collection” of LOV domains we are characterizing Tuber LOV that we postulate to have 

particular properties in the response to blue light deriving from its life cycle spent in the dark. 

We constructed a Neurospora strain 

containing a chimeric WC-1 with its 

LOV domain substituted by TBLOV 

under a Neurospora promoter. We 

are characterizing the intensity and 

timing of bleu light photoresponses 

(i.e. carotenoids biosynthesis) comparing 

the chimeric strain to a Wild 

NcWC1 LOV 

TbWC1 LOV 

Type Neurospora and to a wc-1 KO 

mutant. 

PHY3 

(Crosson and Moffat, 2001) 

Fig. 1 - NcWC-1 and TbWC-1 LOV domains 

structures obtained by modellization on the 

experimentally resolved structure of PHY3. 

Note the loops of random coils indicated by 

the arrows, this variable region should be 

responsible of the individual specificity of the 

different LOVs. 


Patrizia Filetici, CNR researcher; Cristina De 

Luca, research fellow; Andrea Brenna, postdoc 

fellow; Raffaele Gerace, PhD student. 


Simone Ottonello, Dipartimento di Biochimica, 

Università di Parma; Leonardo Baciarelli 

Falini, Dipartimento di Biologia Vegetale e 

Biotecnologie Agroambientali e Zootecniche, 

Università di Perugia; Giuseppe Briganti, 

Dipartimento di Fisica, Università Sapienza 

Roma. 

48


Functional analysis of morgana, a gene involved in the control of 

centrosome duplication 

Silvia Bonaccorsi 


℡: +39 06 49912473 - @: silvia.bonaccorsi@uniroma1.it 

We have recently demonstrated that Morgana/chp1, a conserved CHORD (cysteine and 

histidine rich domains) containing protein, is required for proper centrosome duplication and 

genome stability in both Drosophila and mouse. Null mutations in Drosophila morgana (mora) 

cause lethality and elicit a strong mitotic phenotype. Larval brains of mora mutants exhibit a 

high mitotic index with cells arrested in a prophase/prometaphase-like state and a high 

frequency of polyploid cells. In addition, most diploid cells of mora mutants display multiple 

centrosomes. Notably, the fly mitotic phenotype is fully rescued by the human orthologue of 

mora, highlighting a conserved function of this protein. morgana -/- mice die at the 

perimplantation stage, while morgana +/- primary cells and mice display a high frequency of 

supernumerary centrosomes and increased susceptibility to neoplastic transformation. In 

addition, tumor tissue array histochemical analysis revealed that Morgana is underexpressed 

in a large fraction of breast and lung human cancers. Thus, Morgana/chp-1 appears to 

prevent both centrosome amplification and tumorigenesis. 

Our research programme is aimed at understanding the mechanism of centrosome 

duplication, by elucidating the role of Morgana (Mora) in the Drosophila model system. Given 

the striking evolutionary conservation of the Mora function, these studies will provide 

information that can potentially be applied to humans, specifically in the realm of cancer 

biology. 

By combining a proteomic approach with the sophisticated Drosophila genetics, we are 

pursuing the identification and functional characterization of Mora-interacting proteins. To this 

end, in the course of the first year of the project we have generated transgenic flies carrying 

either a mora or a mora-GFP transgene under the PUASp GAL4-responsive promoter. This 

promoter responds very well to the V32 GAL4 driver, causing very high overexpression levels 

in the female germline. Flies carrying V34 GAL4 maternal driver and the PUASp-mora 

transgene will be then used to overexpress wild type and GFP-tagged Mora to isolate Morainteracting 

proteins by co-IP experiments. 

We have also carried out a functional analysis of mora in the Drosophila embryo, using 

video time-lapse microscopy. We have performed injection experiments in wild type embryos 

using an Alexa 633-labeled anti-Mora antibody. In both S2 tissue culture cells and larval 

brains immunostained with an anti-Mora antibody, the protein had previously been shown to 

accumulate in the cytoplasm of dividing cells, with no specific localization. Conversely, in the 

microinjection experiments carried out on embryos Mora exhibits a specific enrichment at the 

centrosome and at the spindle microtubules (Fig.1A). In addition, injection of the antibody 

49


caused a deformation of the embryonic spindles, strongly suggesting a role of Mora in the 

control of spindle structure and dynamics (Fig. 1B). We now plan to confirm the centrosomal 

localization of Mora also in larval brains, using flies overexpressing GFP-tagged Mora that 

we have recently constructed (see above). 

We have also generated transgenic flies carrying a rok-GFP transgene under the PUASp 

GAL4-responsive promoter. We are currently using these flies in immunoprecipitation 

experiments with anti-GFP antibodies to verify whether Rok interacts with Mora. In addition, 

we will determine whether V32-induced Rok overexpression leads to centrosome 

amplification in the embryo. 

Fig. 1 - Selected frames from time 

lapse sequences of a buffer-injected 

embryo (control) and embryos injected 

with an Alexa 633-labeled anti-Mora 

antibody. (A) Prometaphase cells from 

an anti-Mora injectd embryo, showing 

a clear accumulation of Mora at the 

centrosomes (insets). (B) Metaphase 

cells from two subsequent cycles of 

division. Note, at cycle 11, the anti- 

Mora staining of spindle microtubules 

and the presence of a few aberrant 

spindles (inset). As a result of spindle 

degeneration, at the next cycle (cycle 

12), the embryo contains fewer cells 

than the control and many dramatically 

defective spindles (inset). 

Publications 

Mottier-Pavie V, Cenci G, Vernì F, Gatti M, Bonaccorsi S. Phenotypic analysis of misato 

function reveals roles of noncentrosomal microtubules in Drosophila spindle formation. J Cell 

Sci 2011, 124: 706-717. doi: 10.1242/jcs.072348. 


Maria Grazia Giansanti, CNR researcher; 

Valeria Palumbo, postd-doc fellow; Ramona 

Lattao, PhD student. 


James Wakefield, College of Life and 

Environmental Sciences, University of Exeter, 

UK. 

50


RNA-RNA and RNA-protein interactions: role of small non-coding 

RNAs in gene expression control 

Irene Bozzoni 


℡: +39 06 49912202 - @: irene.bozzoni@uniroma1.it 

It is becoming largely accepted that the non-coding portion of the genome rather than its 

coding counterpart is likely to account for the greater complexity of higher eukaryotes. High 

throughput transcriptome analysis of the last years has disclosed that the mammalian 

genome is pervasively transcribed into many different complex families of RNAs: besides a 

large number of alternative transcriptional start sites, termination and splicing patterns, a 

complex collection of new antisense, intronic and intergenic transcripts was found. Moreover, 

almost half of the polyadenylated species resulted to be non-protein-coding RNAs. Although 

many studies have helped unveiling the function of many small non-coding RNAs, very little 

is known about the long non-coding (lncRNA) counterpart of the transcriptome. 

So far, a large range of functions has been attributed to lncRNAs: it is clear that ncRNA 

molecules associate with chromatin structures to regulate transcription, others exert their 

control at the post-transcriptional level and still others perform key structural or catalytic 

functions, hence approximating the hitherto conventional roles of proteins. Therefore, it is 

evident that most, if not all, cellular pathways and developmental routes are intricate 

networks comprising a multitude of RNA and protein molecules acting in concert to 

accurately convey biological signals and respond to environmental cues. 

In the last year we have defined the role of several small non coding RNAs (microRNA) in 

the translation control of target mRNAs with a relevant function in muscle differentiation and 

identified several new circuitries of gene expression control involving miRNA as central 

players. 

More recently, we have shown the identification of a long non-coding RNA (linc-MD1) that 

cross-talks with specific mRNAs by competing for miRNA binding via their miRNA recognition 

motifs (MRE), thereby imposing an additional level of post-transcriptional regulation. These 

are features described as competing endogenous RNA (ceRNA). 

We showed that linc-MD1 controls muscle differentiation both in mouse and human 

myoblasts through its ability to bind specific miRNAs and in turn to affect the translation of 

the corresponding targets. Downregulation or overexpression of linc-MD1 correlated with 

retardation or anticipation of the muscle differentiation program, respectively. We showed 

that linc-MD1 “sponges” miR-133 and miR-135 to regulate the expression MAML1 and 

MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we 

demonstrated that linc-MD1 exerted the same control over differentiation timing in human 

myoblasts, and that linc-MD1 levels are strongly reduced in the Duchenne muscle cells. 

51


Publications 

Cacchiarelli D, Incitti T, Martone J, Cesana M, Cazzella V, Santini T, Sthandier O, Bozzoni I. 

miR-31 modulates dystrophin expression: novel implications in Duchenne muscular 

dystrophy therapy. EMBO Rep 2011, 12: 136-41. doi: 10.1038/embor.2010.208. 

Cacchiarelli D, Legnini I, Martone J, D’Amico A, Bertini E, Bozzoni I. miRNAs as serum 

biomarkers for Duchenne muscular dystrophy. EMBO Mol Med 2011, 5: 258-65. doi: 

10.1002/emmm.201100133. 

Cesana M, Cacchiarelli D, Legnini I, Santini T, Sthandier O, Chinappi M, Tramontano A, 

Bozzoni I. Key ceRNA role for the long non-coding RNA linc-MD1 in the control of muscle 

differentiation. Cell 2011, 147: 358-69. doi: 10.1016/j.cell.2011.09.028. 

Ragno R, Gioia U, Laneve P, Bozzoni I, Mai A, Caffarelli E. Identification of small-molecule 

inhibitors of the XendoU endoribonucleases family. ChemMedChem 2011, 6: 1797-805. 

doi: 10.1002/cmdc.201100281. 

Salvatori B, Iosue I, Djodji Damas N, Mangiavacchi A, Chiaretti S, Messina M, Padula F, 

Guarini A, Bozzoni I, Fazi F, Fatica A. Critical role of c-Myc in acute myeloid Leukemia 

involving direct regulation of miR-26a and histone methyltransferase EZH2. Genes Cancer 

2011, 2: 585-92. doi: 10.1177/1947601911416357. 


Presutti Carlo, professor; Fatica Alessandro, 

researcher; Elisa Caffarelli, CNR researcher; 

Julie Martone, Tiziana Santini, Ubaldo Gioia, 

Beatrice Salvatori, research fellows; Davide 

Cacchiarelli, post-doc fellow; Cesana Marcella, 

PhD student; Mariangela Morlando, graduated 

technician. 


Alberto. Gulino, Antonio. Musarò, Sapienza 

Università di Roma; Alberto Auricchio, 

TIGEM, Napoli. 

52


Role of metalloproteinases and their tissue inhibitors in the 

regulation of neurogenesis and gliogenesis from neural 

stem/progenitor cells 

Emanuele Cacci 


℡: +39 06 49912206 - 06 4991 2371 - @: emanuele.cacci@uniroma1.it 

Neural progenitor (NP) cells continuously generate new neurons and glia in the adult 

brain. Regulation of adult neurogenesis involves a wide spectrum of signals from the 

microenvironment, the so-called neurogenic niche. The identification of intrinsic and extrinsic 

factors implicated in the modulation of neurogenesis is crucial to define strategies to 

successfully manipulate the neurogenic and gliogenic potential of NPs. 

Microglial cells are crucial components of the niche and they exert either beneficial or 

detrimental effects on neurogenesis, depending on their activation state and ranges of 

mediators produced (Cacci et al., 2005; Cacci et al., 2008; Ajmone Cat et al., 2010). Acute 

exposure to the prototypical activating agent lipopolysaccharide confers antineurogenic 

properties upon microglial cells. We have recently shown that docosahexaenoic acid (DHA), 

a long-chain polyunsatured fatty acid (L-PUFA) with potent immunomodulatory properties, 

exerts, through the modulation of microglial function, a proneurogenic effect (Ajmone Cat et 

al., 2011). 

Among extrinsic factors, matrix metalloproteinases (MMPs) and their natural tissue 

inhibitors (TIMPs) are emerging as contributors to neurogenesis modulation. In addition, 

MMPs are multifaceted contributors to neuroinflammation. Particularly MMP-2 and MMP-9 

have been extensively studied in the brain, where they are found expressed at low levels 

under physiological conditions and up-regulated in various pathological conditions. MMPs 

and TIMPs are expressed in NPs and other brain cell types, including microglia. Noteworthy, 

MMPs and TIMPs regulate cytokine production in microglia in autocrine/paracrine manner 

and, vice versa, cytokines appear to be potent regulators of microglial MMP production. 

We explored whether MMPs modulate NP properties. We found that treatment of NPs 

with the clinically tested broad-spectrum MMP inhibitor Marimastat profoundly affected NP 

differentiation fate. Marimastat treatment allowed for enrichment in neuronal cells, inducing 

NPs to generate a higher percentage of neurons and a lower percentage of astrocytes, 

possibly affecting NP commitment. Consistently with its pro-neurogenic effect, Marimastat 

early down-regulated the expression of Notch target genes, such as HES1 and HES5. 

Interestingly, a potent inhibitor of the Notch signalling (DAPT), increased NP differentiation 

into neurons with an efficiency comparable to that of Marimastat and its administration in 

combination with Marimastat increased slightly, although significantly, neuronal 

differentiation. These data suggest that the early Marimastat-mediated inhibition of Notch 

signalling supports NP differentiation. 

53


Since MMP-2 and MMP-9 are the main MMPs in the brain, we speculated that the MMP 

inhibitor effects on NPs were mediated through MMP-2 and/or MMP-9 inhibition. To clarify 

this point we investigated the expression profile of MMP-2 and MMP-9 in NPs. MMP-2 and 

MMP-9 profiling on proliferating and differentiating NPs revealed that MMP-9 was not 

expressed, whereas MMP-2 increased in the medium as proMMP-2 during differentiation, 

although its active form was not detectable. MMP-2 silencing or administration of 

recombinant active MMP-2 demonstrated that MMP-2 does not affect NP neuronal 

differentiation, nor it is involved in Marimastat pro-neurogenic effect. We also found that 

TIMP-2 is expressed in NPs and increases during late differentiation, mainly as a 

consequence of astrocyte generation. Endogenous TIMP-2 did not modulate NP neurogenic 

potential; however the pro-neurogenic action of Marimastat was at least partially mediated by 

TIMP-2, as demonstrated by silencing experiments. Our data exclude a major involvement of 

MMP-2 and MMP-9 in the regulation of basal NP differentiation, but highlight the ability of 

TIMP-2 to act as a key effector of the pro-neurogenic response to an inducing stimulus, such 

as Marimastat. 

We also addressed the molecular mechanisms involved in the regulation of NP 

properties. We focused our attention on the RE1-silencing transcription factor (REST), a 

master regulator of neuronal genes that can induce neuronal differentiation in NPs. 

To clarify which genes are transcriptionally repressed by REST we have used an 

adenovirus carrying the dominant negative of REST (DN:REST), or shRNA against REST. 

Analysis of the gene expression profile, including the whole spectrum of MMPs and TIMPs 

were performed by microarray. Combination of microarray and Chip sequencing analysis 

identified, among many other genes, two MMPs either directly (MMP-24) or indirectly 

(ADAM19ts) regulated by REST. We propose these MMPs as candidate for regulating NP 

differentiation, possibly cooperating with TIMP-2 in mediating the pro-neurogenic effects of 

Marimastat. 

Publications 

Ajmone-Cat M, Salvatori M, De Simone R, Mancini M, Biagioni S, Bernardo A, Cacci E, 

Minghetti L. Docosahexaenoic acid modulates inflammatory and antineurogenic functions 

of activated microglial cells. J Neurosci Res 2011 Epub. doi: 10.1002/jnr.22783. 


Stefano Biagioni, professor; Tonino Anelli, 

research fellow; Pasquale Caramanica, PhD 

student. 


Ferdinando Mannello, Gaetana A. Tonti, 

Università Carlo Bo, Urbino; Luisa Minghetti, 

Maria Antonietta Ajmone-Cat, Istituto 

Superiore di Sanità, Roma; Bukley Noel, 

King’s College, London, UK. 

54


Does PARylated PARP-1 introduce an epigenetic mark on 

chromatin 

Paola Caiafa 


℡: +39 06 49976530 - @: caiafa@bce.uniroma1.it 

Poly(ADP-ribosyl)ation (PARylation), a post-translational modification catalyzed by 

enzymes of PARP family, leads to the covalent introduction of ADP-ribose units onto 

acceptor chromatin proteins and even onto PARPs themselves. Poly(ADP-ribose) 

glycohydrolase (PARG) degrades ADP-ribose polymers (PARs) reverting the modification by 

its exo and endoglycosydase activities (Hassa and Hottiger, Front Biosci 2008; Krishnakumar 

and Kraus, Mol Cell 2010). PARs, present on covalently PARylated proteins and 

heterogeneous with respect to length and the presence of branches, are themselves able to 

interact non covalently with other proteins that bring a 20-amino-acid PAR-binding motif 

(Malanga and Althaus, Biochem Cell Biol 2005) as well as well-known “macro domain” 

(Krishnakumar and Kraus, Mol Cell 2010). PARP1 orchestrates chromatin dynamics 

modulating transcription and genome organization with different mechanisms. PARylation 

leads to chromatin decondensation modifying PARP1 itself as well as core histones or the 

linker histone H1 (Altmeyer et al., Nucleic Acids Res 2009; Krishnakumar and Kraus, Mol 

Cell 2010, Messner et al., Nucleic Acids Res 2010) while the block of PARylation induces 

chromatin condensation and the presence on some nucleosomes of macro H2A, whose NHD 

domain recruits PARP1, mediates heterochromatic silencing (Nusinow et al., J Biol Chem 

2007). Concerning gene expression PARylation is also important for the maintaining of DNA 

methylation patterns on genome (Caiafa et al., FASEB J 2009). In fact, PARylated PARP1 

and Dnmt1 interact. In this complex PARs, present on PARP1 molecule, interact non 

covalently with Dnmt1 preventing its access to DNA and thus its DNA methyltransferase 

activity (Reale et al., Oncogene 2005). As a consequence, PAR depletion - due to treatment 

of cells with a competitive inhibitor of PARP activity or ectopic over-expression of PARG - 

leads to the introduction of new anomalous methyl groups onto DNA. The multifunctional 

factor CTCF has been identified as an important player in the mechanism that sees together 

PARylation and DNA methylation (Guastafierro et al., J Biol Chem 2008). In fact, CTCF is by 

itself able to activate PARylation of PARP1 also in absence of DNA. This, together with the 

fact that CTCF binds only non methylated sequences on DNA and that, as shown by in silico 

data, its binding sites on genome often coincide with those of PARP1, suggests that CTCF 

marks on the genome those regions that must be maintained non methylated. 

Our aim has been to verify this hypothesis and to establish if PARylated Parp1, by itself 

and/or following PARylation of CTCF and/or other transcription factors, introduces an 

epigenetic code onto chromatin by marking those DNA sequences that must be maintained 

in an unmethylated state in normal cells thus preventing Dnmt1 access to these sequences. 

55


The maintaining of DNA methylation patterns in mammals is required for correct 

development, genome stability, and transcriptional regulation. The mechanism(s) by which 

DNA sequences rich in CpG dinucleotides are maintained unmethylated has still to be 

defined. We demonstrated this mechanism for the DMR1 region present in the mouse 

Igf2/H19 imprinted locus where the binding of Ctcf, Parp1, Dnmt1 and PARs within the 

unmethylated DMR1 depends on PARs. In cells depleted of PARs new methyl groups are 

introduced onto this control region and the Ctcf-Parp1 complex is released from Ctcf target 

sequence (Zampieri et al., Biochem J 2012). Globally, our results, together with those 

reported for the control of p16 gene (Witcher and Emerson, Molecular Cell 2009), support a 

mechanism by which Parp1 tethered and activated at specific DNA target sites by Ctcf or 

other transcription factors to identify, preserves their methylation-free status. 

Publications 

Bacalini MG, Di Lonardo D, Catizone A, Ciccarone F, Bruno T, Zampieri M, Guastafierro T, 

Calabrese R, Fanciulli M, Passananti C, Caiafa P, Reale A. Poly(ADP-ribosyl)ation affects 

stabilization of Che-1 protein in response to DNA damage. DNA Repair (Amst) 2011, 10: 

380-9. doi:10.1016/j.dnarep.2011.01.002. 

Bacalini MG, Tavolaro S, Peragine N, Marinelli M, Santangelo S, Del Giudice I, Mauro FR, Di 

Maio V, Ricciardi MR, Caiafa P, Chiaretti S, Foà R, Guarini A, Reale A. A subset of chronic 

lymphocytic leukemia patients display reduced levels of PARP1 expression coupled with a 

defective irradiation-induced apoptosis. Exp Hematol 2011 Epub. doi: 10.1016/j. 

exphem.2011.11.005. 

Calabrese R, Zampieri M, Mechelli R, Annibali V, Guastafierro T, Ciccarone F, Coarelli G, 

Umeton R, Salvetti M, Caiafa P. Methylation-dependent PAD2 upregulation in multiple 

sclerosis peripheral blood. Mult Scler 2011 Epub. doi: 10.1177/1352458511421055. 


Anna Reale, professor, Michele Zampieri, 

researcher, Maria Giulia Bacalini, Tiziana 

Guastafierro, post-doc fellows, Fabio 

Ciccarone, PhD student, Roberta Calabrese, 

graduated student. 

56


DNA recombination of repeated sequences and genome instability: 

epigenetic implications 

Giorgio Camilloni 


℡: +39 06 49912808 - @: giorgio.camilloni@uniroma1.it 

This project is centered on the simple model organism Saccharomyces cerevisiae, in 

order to gain information concerning basic mechanisms of genome stability to be translated 

to mammal model systems. We focus our attention on the ribosomal DNA (rDNA) locus, a 

repeated region where transcription, replication and recombination meet, both physically and 

functionally. These basic DNA transactions must be tightly regulated to avoid rDNA instability 

and common regulatory mechanisms should be active. 

We intend to explore the role of epigenetics in connecting replication, recombination and 

transcription. In fact, this aspect has not been deeply investigated yet, and its detailed 

characterization could make epigenetics a further element to manipulate in diseases due to 

genome instability. 

During this first year of activity we identified 2 fundamental processes affecting genome 

stability at rDNA: 

i) the histone content and the nucleosome number 

ii) the hyperacetylation of the H4K16 residue 

Regulation of the histone content and nucleosome number 

The basic unit of genome packaging is the nucleosome, and nucleosomes have long 

been proposed to restrict DNA accessibility both to damage and to transcription. Nucleosome 

number in cells was considered fixed, but recently aging yeast and mammalian cells were 

shown to contain fewer nucleosomes. We have shown that mammalian cells lacking High 

Mobility Group Box 1 protein (HMGB1) contain a reduced amount of core, linker, and variant 

histones, and a correspondingly reduced number of nucleosomes, possibly because HMGB1 

facilitates nucleosome assembly. Yeast nhp6 mutants lacking Nhp6a and -b proteins, which 

are related to HMGB1, also have a reduced amount of histones and fewer nucleosomes. 

Nucleosome limitation in both mammalian and yeast cells increases the sensitivity of DNA to 

damage, increases transcription globally, and affects the relative expression of about 10% of 

genes. In yeast nhp6 cells, the loss of more than one nucleosome in four does not affect the 

location of nucleosomes and their spacing, but nucleosomal occupancy. The decrease in 

nucleosomal occupancy is non-uniform and can be modelled assuming that different 

nucleosomal sites compete for available histones. Sites with a high propensity to occupation 

are almost always packaged into nucleosomes both in wild type and nucleosome-depleted 

cells; nucleosomes on sites with low propensity to occupation are disproportionately lost in 

nucleosome-depleted cells. We suggest that variation in nucleosome number, by affecting 

57


nucleosomal occupancy both genomewide and gene-specifically, constitutes a novel layer of 

epigenetic regulation. 

Hyperacetylation of the H4K16 residue 

Transcription-associated recombination is an important process involved in several 

aspects of cell physiology. In the rDNA of S. cerevisiae, RNA polymerase II transcription 

dependent recombination among the repeated units has been demonstrated. In our 

experimental results, we showed the existence of mechanisms controlling this process at the 

chromatin level. On the basis of a small biased screening, we found that mutants of histone 

deacetylases and chromatin architectural proteins alter both the amount of Pol II-dependent 

non coding transcripts and recombination products at rDNA, in a coordinate manner. More 

interestingly, ChIP analyses revealed, in the above mutants, a correspondent variation of the 

histone H4 acetylation along the rDNA repeat, particularly at the lysine 16. We provided 

evidence that a single, rapid and reversible post-translational modification, the acetylation of 

the H4K16 residue, represents the epigenetic basis of the coordination of transcription and 

recombination at rDNA. 

Publications 

Celona B, Weiner A, Di Felice F, Mancuso FM, Cesarini E, Rossi RL, Gregory L, Baban D, 

Rossetti G, Grianti P, Pagani M, Bonaldi T, Ragoussis J, Friedman N, Camilloni G, Bianchi 

ME, Agresti A. Substantial histone reduction modulates genomewide nucleosomal 

occupancy and global transcriptional output. PLoS Biol 2011, 9:e1001086. doi: 

10.1371/journal.pbio.1001086. 

Orecchia A, Scarponi C, Di Felice F, Cesarini E, Avitabile S, Mai A, Mauro ML, Sirri V, 

Zambruno G, Albanesi C, Camilloni G, Failla CM. Sirtinol treatment reduces inflammation 

in human dermal microvascular endothelial cells. PLoS One 2011, 6:e24307. doi: 

10.1371/journal.pone.0024307. 


Elisa Cesarini, Francesca Di Felice, post-doc 

fellows; Anna D’Alfonso, Davide Gaglio, PhD 

students. 


Marco Bianchi, Università Vita-Salute San 

Raffaele, Milano; Bastia Deepak, Department 

of Biochemistry & Molecular Biology; University 

of Charleston, USA. 

58


Processing of the Neuroligins proteins and autism-related 

mutations 

Antonella De Jaco 


℡: +39 06 49912310 - @: antonella.dejaco@uniroma1.it 

The α/β-hydrolase fold family is one of the largest families of proteins presenting 

significant structural homology with divergent functions: from catalytic hydrolysis to 

heterophilic cell adhesive interactions and hormone production. All the proteins of the family 

share a common three-dimensional core structure, containing the α/β-hydrolase fold domain 

that is crucial for proper protein localization and function. Mutations affecting the integrity of 

the α/β-hydrolase domain in conserved residues within of neuroligins, butyrylcholinesterase 

and thyroglobulin have been found associated with congenital disorders (De Jaco et al., 

Protein Pept Lett 2011). Characterization of the mutations found in the α/β-hydrolase fold 

domain shows that they mainly disrupt the architecture of the common structural domain, 

impairing trafficking along the secretory pathway and causing retention of the mutant protein 

in the endoplasmic reticulum (ER). This is indicating that the structural integrity of the α/βhydrolase 

fold domain is required for the export of the protein from the ER (De Jaco et al., 

Protein Pept Lett 2011, De Jaco et al., submitted). The α/β-hydrolase fold domain of the 

postsynaptic adhesion molecules neuroligins (NLGNs) interacts directly with presynaptic 

ligands belonging to the neurexins (NRXNs) family. In the last years, studying processing of 

the NLGNs proteins has been particularly interesting in relation to autism spectrum disorders 

(ASDs). Molecular genetic studies on ASDs are focusing on alterations in the functioning of 

synapses with the synaptic proteins playing a central role. The NLGNs-NRXNs synaptic cell 

adhesion complex is the best-characterized pathway implicated in ASDs. Genetic alterations 

in the NLGNs genes have been identified in autistic patients, suggesting that impairments in 

synaptic adhesion might lead to deficits in brain development (Betancur et al., Trends 

Neurosci 2009). We have studied an autism-associated mutation, R451C in NLGN3. We 

have shown that the mutation induces local protein misfolding of the α/β-hydrolase fold 

domain of NLGN3 that ultimately results mostly retained in the ER. The mutation slows down 

protein processing in the HEK-293 cell line and trafficking in neurons with a small fraction still 

reaching the cell surface (De Jaco et al., JBC 2010). More recently we have been 

investigating whether the retention of the NLGN3 R451C mutant protein in the ER is 

determining the activation of an ER stress response. The Unfolded Protein Response (UPR) 

is the most important ER signaling pathway that regulates protein folding and processing 

capacity of the ER (Kaufman, Genes Dev 1999). UPR is initiated by three transmembrane 

ER proteins, PERK (PKR-like ER kinase), IRE1 (inositol requiring kinase 1), and ATF6 

(activating transcription factor 6) that sense protein-misfolding and activate an ER-to-nucleus 

59


signaling cascades in order to maintain homeostasis in the ER (Marciniak and Ron, Physiol 

Rev 2006). UPR activation has been detected in experimental models of neurodegenerative 

diseases (Matus et al., Curr Opin Cell Biol 2011) but a UPR signaling pathway in ASDs has 

never been described. To evaluate whether the ER retention of the misfolded R451C NLGN3 

mutant protein was determining the activation of the UPR, we have studied the ATF6 

signaling pathway in the HEK-293 cell line. We have overexpressed in transient the NLGNs 

constructs together with a vector containing binding sites for ATF6, upstream of the 

luciferase reporter gene. Since these sequences are found in the promoters of most UPR 

target genes, the luciferase signal would indicate the activation of ATF6 and therefore and 

ER stress condition. Luciferase activity, measured at 24 and 48 hours after transfection 

shows that ATF6 activation is significantly higher for the mutant R451C NLGN3 compared to 

the wild type protein. We also studied IRE1 activation in response to accumulation of the 

R451C NLGN3 protein in the ER. Under ER stress condition, IRE1 activates the 

unconventional splicing of the mRNA of a transcriptional factor named XBP-1. We have 

studied the splicing of XBP1 mRNA by semiquantitative RT-PCR by and Real Time RT-PCR 

analysis, detecting both unspliced and spliced isoforms for XBP1. We have found that XBP1 

splicing is enhanced in the presence of the R451C mutant compared to the wild type protein 

(manuscript in preparation). 

From our results it is emerging that the autism-associated R451C mutation in NLGN3 is 

causing retention of the protein in the ER and this is determining an ER stress condition that 

results with the activation of ATF-6 and XBP-1, two of the UPR signaling pathways. 

Publications 

De Jaco A, Comoletti D, Dubi N, Camp S, Taylor P. Processing of cholinesterase-like 

alpha/beta-hydrolase fold proteins: alterations associated with congenital disorders. Protein 

Pept Lett 2011 Epub. 


Federica De Angelis, Lisa Ulbrich, post-doc 

fellows ; Flores Lietta Favaloro, student. 


Davide Comoletti, Palmer Taylor, , University 

of California, San Diego, USA; Michael Lin, 

Stanford University, San Francisco, USA, 

Stefan Marciniak, University of Cambridge, 

UK. 

60


Functional analysis of CG40218, a Drosophila melanogaster gene 

encoding a BCNT-like protein required for chromosome 

organization 

Patrizio Dimitri 


℡: +39 06 49917948 - @: patrizio.dimitri@uniroma1.it 

Aim of the project 

The GC40218 protein, also called YETI, is a member of the evolutionarily conserved 

family of BCNT proteins, whose function is still unclear. Using genetic and molecular 

analyses, as well as reverse genetics tools, we plan to delucidate the function played by 

YETI and CFDP1, the human ortholog of YETI. 

Transgenic lines expressing the YETI-tagged protein 

We tagged the YETI protein with GFP and 3xFLAG. Expression of the YETI-GFP and 

3xFLAG-YETI transgenes was induced in salivary glands and brains by the elav-GAL4 driver. 

Immunostaining of salivary gland chromosomes in Yeti-GFP, elav-GAL4 larvae using specific 

anti-GFP antibodies clearly show that the YETI-GFP fusion protein localizes in all nuclei and 

is recruited at hundreds of sites along the chromosome arms. Notably, the banding pattern 

produced by anti-GFP overlaps well with that produced by DAPI-staining. Although YETI- 

GFP is able to bind chromosomes, its expression driven by tub-gal4 transgene apparently 

does not rescue or mitigate the lethal phenotype of larvae homozygous for the LP1 null 

allele. We also made several transgenic lines carrying different deletions of the Yeti coding 

region (DYETI) tagged with GFP or 3xFLAG. The expression of such DYETI constructs 

under control of elavGAL4 driver will allow us to map in vivo the YETI region required for 

binding to chromosome. 

Identification of YETI interactors 

In order to identify the proteins that interact with YETI, we decided to perform 

Immunoprecipitation (IP) experiments in Drosophila S2 cells. For this purpose, a first step is 

to construct a pjZ4-3xFLAG-Yeti plasmid that will be transfected into S2 cells. The 3X-FLAG 

epitope coding region was inserted in pJZ4 and the obtained pJZ4-3XFLAG plasmid was 

used to clone the Yeti cDNA in frame with the epitope. Expression of such contruct in S2 

cells will give rise the YETI protein tagged at the N-terminal portion with the FLAG epitope. IP 

assay will be then performed on S2 cells containing pjZ4-3xFLAG-Yeti plasmids using the α- 

FLAG antibody. 

61


Expression of the CFDP1 human protein in wild-type flies 

In order to investigate the effect of CFDP1 protein expression in the fly, we constructed 

Drosophila melanogaster transgenic lines carrying the CFDP1 human c-DNA. Expression of 

the CFDP1 protein in Drosophila melanogaster was performed by crossing two trangenic 

UAS-CFDP1 lines (15 and 16) to lines carrying ubiquitously active or tissue-specific GAL4 

drivers. By Western blot analysis using anti-CFDP1 monoclonal antibodies, we detected the 

presence of CFDP1 in protein extracts from w/w, elav-GAL4; UAS-CFDP1/+ larvae carrying 

the UAS-CFDP1 transgene under control of the elev-GAL4 driver. By immunostaing with anti- 

CFDP1, a diffuse signal was detected on polytene chromosomes and nuclei from w/w, elav- 

GAL4; UAS-CFDP1/+ larvae, while no staining was observed in the polytene chromosome of 

w/Y; UAS-CFDP1/+ control larvae. However, expression of UAS-CFDP1 transgene in wildtype 

flies results in lethality and phenotypic abnormalities, depending on the driver used. With 

elav-GAL4, lethality mainly occurs at third instar larvae, which often show reduced brains, 

sporadic melanotic masses in the larval hemocoel and chromosome defects; most notably, in 

elav-GAL4, UAS-CFDP1 larvae abnormally condensed polytene chromosomes were found. 

All the defects observed are clearly reminiscent of those found in Yeti mutants. In addition, 

when expressed simultaneously under control of elav-GAL4 driver, YETI-GFP and CFDP1 

protein mainly colocalize in the same polytene nuclei and their citological pattern remain 

unaffected. It is conceivable that the CFDP1 protein behaves as a dominant-negative and 

disrupts the normal function of YETI. 

Together, the results of our work provide evidence in favour of a conserved role of YETI 

and CFDP1 proteins in chromatin organization, which is extremely interesting in the light of 

the involvement of CFDP1 in complex developmental syndromes. 


Francesca Romana Mariotti, post-doc fellow, 

Elisabetta Damia, Giovanni Messina, PhD 

students, Emanuele Celauro, student. 


Ruggiero Caizzi, Università di Bari; Ennio 

Giordano, Università di Napoli “Federico II”. 

62


Genetic and molecular analysis of the mechanisms of Drosophila 

telomere protection 

Maurizio Gatti 


℡: +39 06 49912842 - @: maurizio.gatti@uniroma1.it 

Telomeres are nucleoprotein complexes that protect chromosome ends from incomplete 

replication, degradation and fusion. In most organisms, telomeres are extended by 

telomerase and contain GC-rich repeats. Drosophila telomeres are elongated by 

transposition of 3 specialized retroelements rather than telomerase activity, and are 

assembled independently of the terminal DNA sequence. In organisms with telomerase, 

telomeres are protected by the CST and shelterin complexes, which specifically bind the 

telomeric DNA sequences. We have recently identified a Drosophila telomere-capping 

complex, terminin, which binds telomeric DNA in a sequence independent fashion and 

shares functional analogies with CST and shelterin. Terminin includes 4 telomere-specific 

proteins, HOAP, HipHop, Modigliani (Moi) and Verrocchio (Ver); Ver is structurally similar to 

Stn1, while HOAP, HipHop and Moi do not exhibit obvious homologies with known telomere 

proteins. Our research is aimed at the structural and functional characterization of terminin. 

We have 3 principal aims: 1) analyze the molecular interactions and the DNA binding 

properties of the terminin subunits using both biochemical methods and atomic force 

microscopy; 2) predict the three-dimensional structures of terminin subunits by bionformatic 

analysis, so as to design mutant proteins that will be tested for their functional properties; 3) 

perform genome-wide searches to identify additional proteins required for Drosophila 

telomere protection. 

In collaboration with Dr. Domenico Raimondo, we have performed a bioinformatic 

analysis of Moi and Ver and elaborated a structural model for Ver and a preliminary model for 

Moi. Based on these models we prepared several tagged (with GST or 6His) Moi and Ver 

truncations. We had already in hand tagged HOAP truncations fused with GST. Using these 

protein fragments we performed several pairwise pulldown experiments and defined the 

interaction domains within the terminin complex. These analyses also showed that the C- 

terminus of Ver contains an alpha-helix domain that mediates both DNA binding activity and 

formation of Ver homomultimers. These results are currently being used to produce mutant 

forms of HOAP, Moi and Ver, which will allow a variety of functional analyses. 

Our GST pulldown experiments revealed an unexpected and interesting molecular 

interaction. We found that Moi physically interacts with the Drosophila TAT-like (Dtl) protein, 

a trimethyl guanosine synthase homologous to yeast Tgs1 and human PIMT/hTGS1. Tgs1 is 

known to catalyze the conversion of the 7-monomethylguanosine caps of snRNAs, snoRNAs 

63


and telomerase TLC1 RNA to 2,2,7-trimethylguanosine caps; Tgs1 has been implicated in 

nucleolar assembly, splicing of meiotic pre-mRNAs, and regulation of telomerase activity. Dtl 

is encoded by the bicistronic CG31241 locus, which produces a single transcript that 

contains 2 ORFs: One encodes Dtl and the other the Moi protein. These findings suggested 

a possible involvement of Dtl in regulation of transposition of the 3 Drosophila telomerespecific 

retrotransposons (HeT-A, TART and TAHRE). Preliminary results using real time RT- 

PCR showed that dtl mutants exhibit a 9-fold increase of TART transcripts compared to wild 

type. We are now exploring the possible mechanisms underlying this increase in TART 

transcript level. 

In humans, TGS1 is associated with and functionally dependent on the Survival of Motor 

Neuron (SMN) complex, which plays an essential role in spliceosomal snRNP assembly. 

Defects in human SMN1 cause spinal muscular atrophy autosomal recessive type 1 (SMA1), 

a neuromuscular disorder characterized by degeneration of the anterior horn cells of the 

spinal cord. We found that Dtl physically interacts with the Drosophila homologue of SMN1, 

and that hypomorphic mutations in dtl result in neurological problems in the fly. These data 

establish an unexpected link between the telomere and motoneuron maintenance, which will 

be explored in the future. 

Publications 

Mottier-Pavie V, Cenci G, Vernì F, Gatti M, Bonaccorsi S. Phenotypic analysis of misato 

function reveals roles of noncentrosomal microtubules in Drosophila spindle formation. J 

Cell Sci 2011, 124: 706-717. doi: 10.1242/jcs.072348. 

Raffa GD, Capponi L, Cenci G, Gatti M. Terminin: a protein complex that mediatesepigenetic 

maintenance of Drosophila telomeres. Nucleus 2011, 2: 383-91. doi: http://dx.doi.org/ 

10.4161/ nucl.2.5.17873. 


Laura Ciapponi; professor; Grazia Daniela 

Raffa, Fiammetta Vernì, researchers; Maria 

Patrizia Somma, CNR researcher. 


Stefano Cacchione, Dipartimento di Biologia e 

Biotecnologie “Charles Darwin”. Sapienza 

Università di Roma; Giovanni Cenci, 

Dipartimento di Biologia di Base ed Applicata, 

Università dell’Aquila; Domenico Raimondo, 

Dipartimento di Scienze Biochimiche “A. Rossi 

Fanelli”, Sapienza Università di Roma. 

64


Role of epicardium and EMT/MET processes in cardiac progenitor 

cells generation and differentiation 

Alessandro Giacomello 


℡: +39 06 4461481 - @: alessandro.giacomello@uniroma1.it 

Aim of the study. Cardiac progenitor cells (CPCs) isolated as Cardiospheres (CSps), 

represent a promising candidate for autologous cardiac cell therapy. CSps can be easily 

obtained from cells spontaneously migrating out of primary cardiac explants (explant-derived 

cells, EDCs) and re-create in vitro a niche-like microtissue, which seems to favor the 

maintenance of a “stemness” status and confers resistance to oxidative stress, thus 

enhancing in vivo engraftment (Messina et al., 2004, Li et al., 2011). CSp-derived cells 

(CDCs) can be expanded in monolayers and retain the ability to form secondary 

cardiospheres (IICSps) when plated back in the appropriate conditions. Thus, this method 

yields a significant number of adult autologous CPCs suitable for clinical application, which 

can contribute in vivo to all the three main cell lineages of the heart. A better understanding 

of the mechanism underlying CSps formation and differentiative potential is required to 

control their fate in vivo and potentiate a cardiogenic outcome rather than a fibrogenic one, 

even in the hostile ischemic environment of an infarcted heart. Epithelial-to-mesenchymal 

transitions (EMT) play a central role in embryogenesis. Four distinct waves of EMT occur at 

different stages of heart morphogenesis. EMT has also been associated with the acquisition 

of stem cells properties both in adult tissues and cancer and with cardiac post-ischemic 

remodeling. The aim of this study was to analyze the possible role of EMT in CSp generation 

and development, by means of gene expression analysis at different culture stages and in 

vitro treatments with TGFβ, which is a key EMT inducer, and its antagonist SB431452. 

Results. Gene expression analysis, of the four different culture stages (EDCs, CSps, 

CDCs, IICSps), conducted using Taqman microfluidic array cards, and immunostainings 

reveal the involvement of EMT in CSps formation. In fact, EMT associated genes (Snail, 

FGFR, Slug, Twist, alk2/5, n-cadherin and smad2/3), mesenchymal markers (SMA, CX43) 

and mobility-associated genes (β-catenin, MMP2) are upregulated at all culture stages 

compared to EDCs, especially in CSps and IICSps. WT1, marker of the activated epicardium 

and EMT mediator, is significantly upregulated at all culture stages, following CSps 

formation. WT1 expression can be reactivated in adult epicardium in response to specific 

signals, including growth factors and ischemia. Since CSps are 3D structures, a hypoxic 

gradient is conceivably present from the periphery to the center, and hypoxia-mediated 

signals could be responsible for this re-activation. Another mediator of EMT, directly 

activated both by HIF1α and BMP/TGF pathway, is Notch1 involved in CPCs proliferation 

and maintenance. In our system it is significantly upregulated in CSps and IICSps compared 

65


to EDCs and CDCs, similarly to Notch3 (known to control self-renewal and hypoxia survival 

in human mammospheres). In vitro treatment with TGFβ speeds up CSps formation and 

significantly increases overall CSps yield. Treatment with SB431542 induces a significant 

downregulation of genes related to the TGFβ pathways, and of Notch3 and 1, while BMP4 is 

significantly upregulated; such treatment results in a drastic inhibition of CSps formation, 

while it causes the spreading of pre-formed CSps. 

Future prospectives. In conclusion, Notch-Wt1 mediated EMT has a crucial role in CSps 

formation and may be involved in the acquisition of stem-cell like properties by cardiacexplant 

derived cells, just as observed in other adult tissues (Mani et al., 2008; Caja et al., 

2011). In order to discern if Wt1 upregulation is the result of an enrichment for pre-existing 

Wt1+ progenitors or rather of the de-differentiation of EPDCs, transgenic Wt1-cre mice are 

going to be used. Based on these insights into molecular pathways involved in CSps 

formation and maintenance, small molecules previously screened on embryonic stem cells 

are going to be tested in order to enhance the in vivo cardiogenic potential of CSps. 

Publications 

Barile L, Cerisoli F, Frati G, Gaetani R, Chimenti I, Forte E, Cassinelli L, Spinardi L, Altomare 

C, Kizana E, Giacomello A, Messina E, Ottolenghi S, Magli MC. Bone marrow-derived cells 

can acquire cardiac stem cells properties in damaged heart. J Cell Mol Med 2011, 15: 63- 

71. doi: 10.1111/j.1582-4934.2009.00968.x. 

Chimenti I, Rizzitelli G, Gaetani R, Angelini F, Ionta V, Forte E, Frati G, Schussler O, 

Barbetta A, Messina E, Dentini M, Giacomello A. Human cardiosphere-seeded gelatin or 

collagen scaffolds as cardiogenic engineered bioconstructs. Biomaterials 2011, 32 : 9271- 

81. doi: 10.1016/j.biomaterials.2011.08.049. 

Fabrizi C, Angelini F, Chimenti I, Pompili E, Somma F, Gaetani R, Messina E, Fumagalli L, 

Giacomello A, Frati G. Thrombin and thrombin-derived peptides promote proliferation of 

cardiac progenitor cells in the form of cardiospheres without affecting their differentiation 

potential. J Biol Regul Homeost Agents 2011, 25: S43-51. 

Forte E, Chimenti I, Barile L, Gaetani R, Angelini F, Ionta V, Messina E, Giacomello A. 

Cardiac cell therapy: the next (re)generation. Stem Cell Rev 2011, 7: 1018-30. doi: 

10.1371/journal.pone. 0025669. 


Elisa Messina, medical manager; Francesco 

Angelini, research fellow; Isotta Chimenti, 

Elvira Forte, Roberto Gaetani, post-doc 

fellows; Vittoria Ionta, PhD student. 


Luigi Aurisicchio, Takis Srl, Biogem, Rome; 

Mark Mercola, Sanford-Burnham Medical 

Research Institute, San Diego, USA; Pilar 

Ruiz-Lozano, Stanford School of medicine, 

Stanford, CA, USA. 

66


Hedgehog signaling regulatory networks in brain cancer stem cells 

Alberto Gulino 


℡: +39 06 4464021 - @: alberto.gulino@uniroma1.it 

Hedgehog pathway plays a pivotal role in development and tumorigenesis, processes 

sustained by stem cells. The transcription factor Nanog controls stemness acting as a key 

determinant of both embryonic stem cell self-renewal and differentiated somatic cells reprogramming 

to pluripotency, in concert with the loss of the oncosuppressor p53. We have 

shown that Nanog is highly expressed in stem cells from postnatal cerebellum and 

medulloblastoma, and acts as a critical mediator of Hedgehog-driven self-renewal through 

Gli1 and 2-dependent and p53-independent transcriptional regulation. Our data reveal a 

mechanism for the role of Hedgehog in the control of stemness that represents a crucial 

component of an integrated circuitry determining cell fate decision and involved in the 

maintenance of cancer stem cells. 

We have also observed that acetylation of Gli1 and 2 proteins (human Gli1 K518 and Gli2 

K757) is a transcriptional checkpoint of Hedgehog signaling, having acetylated Gli reduced 

transcriptional function while it is enhanced by HDAC1 and 2-dependent deacetylation. 

HDAC1 degradation is in turn controlled by the tumor suppressor REN KCTD11 , that is 

monoallelically deleted and silenced by hypermethylation in medulloblastoma. REN forms a 

tetrameric complex with Cul3 E3 ligase and HDAC1, leading to HDAC degradation. REN is a 

member of a novel family of Cul3 adaptors that includes two additional members (KCASH2 

and KCASH3 - KCTD Cullin3 Adaptors and Suppressors of Hedgehog). They also form 

homo- and heteromers of KCASH/Cul3 complexes, ubiquitinate and degrade HDAC1 and 

suppress in this way Hedgehog signaling. They are all downregulated in medulloblastoma 

and decrease tumor growth. Therefore, Hedgehog and KCASH/REN work in an opposite and 

coordinated fashion, by regulating HDAC1 levels and, ultimately, Gli1 acetylation, suggesting 

that HDAC inhibitors may be a promising and novel therapeutic strategy for 

medulloblastoma. 

Ubiquitination represents an additional Heddehog regulatory mechanism, in which we 

have shown that Numb activates the catalytic activity of Itch, releasing it from an inhibitory 

intramolecular conformation, that results in a Gli1 PPXYs and a phospho-serine/proline 

degron-dependent ubiquitination and degradation. Itch and Numb-insensitive Gli1 mutant 

determines enhanced Gli1-dependent medulloblastoma growth, migration and invasion 

abilities, as well as in vitro transforming activity. Our data reveal a novel mechanism of 

regulation of Gli1 stability and function, which influences Hedgehog/Gli1 oncogenic potential. 

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Publications 

Correale S, Pirone L, Di Marcotullio L, De Smaele E, Greco A, Mazzà D, Moretti M, Alterio V, 

Vitagliano L, Di Gaetano S, Gulino A, Pedone EM. Molecular organization of the cullin E3 

ligase adaptor KCTD11. Biochimie 2011, 93: 715-24. http://dx.doi.org/10.1016/j.biochi. 

2010.12.014. 

De Smaele E, Di Marcotullio L, Moretti M, Pelloni M, Occhione MA, Infante P, Cucchi D, 

Greco A, Pietrosanti L, Todorovic J, Coni S, Canettieri G, Ferretti E, Bei R, Maroder M, 

Screpanti I, Gulino A. Identification and characterization of KCASH2 and KCASH3, 2 novel 

Cullin3 adaptors suppressing histone deacetylase and Hedgehog activity in 

medulloblastoma. Neoplasia 2011, 13: 374-85. doi: 10.1593/neo.101630. 

Di Marcotullio L, Canettieri G, Infante P, Greco A, Gulino A. Protected from the inside: 

endogenous histone deacetylase inhibitors and the road to cancer. Biochim Biophys Acta 

Rev Cancer 2011, 1815: 241-52. http://dx.doi.org/10.1016/j.bbcan.2011.01.002. 

Di Marcotullio L, Greco A, Mazzà D, Canettieri G, Pietrosanti L, Infante P, Coni S, Moretti M, 

De Smaele E, Ferretti E, Screpanti I, Gulino A. Numb activates the E3 ligase Itch to control 

Gli1 function through a novel degradation signal. Oncogene 2011, 30: 65-76. 

doi:10.1038/onc.2010.394. 


Gianluca Canettieri, Elisabetta Ferretti 

professors, Enrico De Smaele, Lucia Di 

Marcotullio, researchers, Agnese Po, Evelina 

Miele, Marta Moretti, Sonia Coni, Azzura 

Greco, research fellows. 


Alesse Edoardo, Università dell’Aquila, 

Pedone Carlo, Università di Napoli, Schininà 

Eugenia, Sapienza Università di Roma. 

68


The interplay between epigenetics, cell cycle and homologous 

recombination in gene therapy by Small Fragment Homologous 

Replacement (SFHR) 

Marco Lucarelli 


℡: +39 06 4451784 - @: marco.lucarelli@uniroma1.it 

The aim of this project is to clarify the molecular mechanisms underlying an in situ gene 

targeting approach called Small Fragment Homologous Replacement (SFHR). A small DNA 

fragment (SDF) is used as a corrector to stably modify a genomic sequence, restoring the 

wild-type gene function. Although DNA repair pathways, in particular homologous 

recombination (HR), seem to be involved, the molecular mechanisms of SFHR are poorly 

understood, with its potential currently limited by a low and variable frequency of correction. 

This project focuses on the scarcely studied relationships between SFHR, DNA methylation 

and repair, chromatin structure and cell cycle. 

Mouse embryonic fibroblast (MEF) clones were stably modified with a genomic 

integration of the wild type enhanced green fluorescent protein (wtEGFP clones) sequence, 

in which a non sense mutation was also introduced (mEGFP clones). In this reporter system, 

a corrector SDF homologous to the wtEGFP sequence can integrate in mEGFP clones and 

restore the fluorescence of the cells. Several molecular and cellular analyses of MEF clones 

confirmed the genomic correction, its persistence over time, and the absence of detectable 

random integration of SDF. The efficiency of SFHR is evaluated at DNA and RNA levels, as 

well as functionally quantified by cytofluorimetric analysis. 

The best experimental characteristics of corrector SDF and the best transfection 

conditions, in term of correction efficiency and cell viability, were selected in unsynchronized 

MEF clones. To determine the cell cycle influence, we evaluated gene targeting in G0/G1, 

G1/S and G2/M phases. In G2/M the SFHR resulted favoured, reaching 0.5% of correction, 

probably because of the absence of nuclear envelope, the tetraploid status of the cells and 

the higher activity of HR. Artificially methylated and plasmid-excised SDFs showed a lower 

correction efficiency than unmethylated PCR synthesized SDF, probably because of the 

creation of complexes between methylated SDFs and nuclear proteins that inhibit the 

integration. After sorting, corrected MEF gradually lost EGFP fluorescence concomitantly 

showing an increased site-specific methylation pattern at EGFP locus, possibly influenced by 

the correction event. A cause / effect relationship was evidenced by the reversion of DNA 

methylation and EGFP expression after treatment with the demethylating agent 5-aza-2’- 

deoxycytidine (aza-dC). An increment of SFHR efficiency to 2.5% was evidenced by the 

simultaneous inhibition of DNA methylation and poly-(ADP-ribose) polymerase, highlighting 

synergistic effects of these two pathways on SFHR. 

The quantitative expression of 84 genes involved in the response to several kinds of DNA 

damage, using real time expression arrays, was also investigated. Their expression levels in 

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MEF were heterogeneous and most of the genes affected by SFHR resulted to be low 

expressed genes. After an early (24h after treatment with SDF) upregulation of 70 DNA 

damage repair genes, there was a late (72h after treatment) downregulation with 51 genes 

reaching a lower expression level than untreated control. The genes with statistically 

significant expression differences between treatment and control resulted to be 33. On the 

contrary to current opinions, these genes belong to different DNA repair pathways, and not 

only to HR pathway. Their early upregulation may constitute the molecular basis for the 

correction, whereas their late downregulation may be responsible for the reduced correction 

efficiency, possibly as an adaptive response to cell invasion by exogenous DNA. 

These results contribute to the comprehension of the molecular mechanisms underlying 

cell invasion by exogenous DNA and its genomic integration. The selection of specific 

molecular targets to manipulate provides suggestions for increasing gene repair efficiency to 

achieve an higher correction for a practical SFHR application to ex vivo therapeutic 

approaches. 


Roberto Strom, professor; Fabrizio Ceci, 

Giampiero Ferraguti, researchers; Silvia 

Pierandrei, PhD student; Sabina Maria Bruno, 

graduated student. 


Paola Borgiani, Annalisa Botta, Andrea 

Luchetti, Arianna Malgieri, Giuseppe Novelli, 

Federica Sangiuolo, Dip. di Biopatologia e 

Diagnostica per Immagini, Università di Roma 

Tor Vergata. 

70


Interplay between myogenic factors and cell cycle control: 

regulation and role of the cdk inhibitor p57kip2 

Rossella Maione 


℡: +39 06 4457737 - @: maione@bce.uniroma1.it 

Cell proliferation and differentiation are highly coordinated processes during 

development, homeostasis and regeneration of multicellular organisms. Cell cycle exit is not 

only critical for the formation of appropriate cell numbers, but also is a prerequisite for 

terminal differentiation in many cell types. 

The interdependence of cell proliferation and differentiation has been extensively 

investigated in the skeletal muscle system, in which the whole differentiation program can be 

initiated by the MyoD family of muscle-specific transcription factors, even when ectopically 

expressed in non muscle cell types. MyoD directs a complex program of gene expression 

through the cooperation with other transcription factors and chromatin remodeling enzymes. 

In addition to regulate muscle-specific genes, MyoD is also involved in coupling the onset of 

differentiation with cell cycle withdrawal. The regulated expression of cdk inhibitors (CKIs) 

plays a fundamental role in muscle differentiation. 

The function and the regulation of the CKI p57 kip2 (p57) are quite peculiar. p57 is the only 

CKI which deficiency causes severe developmental defects both in humans and mice. 

Moreover, compared to other CKIs, p57 shows a more restricted tissue and cell-type 

distribution. Finally, p57 belongs to a network of imprinted genes involved in growth 

regulation and is subject to a complex epigenetic control. Despite the importance of this CKI, 

the specific functions of p57 and the molecular mechanisms regulating its expression during 

differentiation processes are still poorly understood. 

Our research project is focused on two related topics. One concerns the specific roles 

that p57 plays in the establishment and in the maintenance of terminal differentiation in 

muscle cells. The other one concerns the transcriptional mechanisms controlling p57 

expression during myogenesis. 

During the last year, we mainly focused on the second topic. The results obtained 

allowed us to highlight a novel epigenetic mechanism by which MyoD regulates gene 

expression during myogenesis. We have shown that the induction of p57 requires MyoD 

binding to a long-distance element located within the imprinting control region KvDMR1 and 

the consequent release of a repressive chromatin loop involving p57 promoter. We have also 

provided evidence that differentiation-dependent regulation of p57, while involving a region 

implicated in the imprinting process, is distinct and hierarchically subordinated to the 

imprinting control. These findings highlight a novel mechanism, involving the modification of 

higher order chromatin structures, by which MyoD regulates gene expression. Our results 

also suggest that a repressive chromatin loop mediated by KvDMR1 could account for the 

71


highly restricted expression of p57 during normal development and, possibly, for its aberrant 

silencing in some pathologies. 

The results are detailed in the following submitted manuscripts: Busanello et al., MyoD 

regulates p57 kip2 expression by interacting with a distant cis-element and modifying a higherorder 

chromatin structure and Battistelli et al., Functional interaction between MyoD and 

CTCF in the regulation of long-range chromatin interactions during muscle differentiation. 


Anna Busanello, Rosaria Carbone, post-doc 

fellows; Cecilia Battistelli, Agnese Ciotti, 

Cassandra Mostocotto, PhD students. 


Maurizia Caruso, Istituto di Neurobiologia e 

Medicina Molecolare, CNR, Roma. 

72


Handling and toxicity of mutant serpins underlying human disease 

María Elena Miranda Banos 


℡: +39 06 49912310 - @: mariaelena.mirandabanos@uniroma1.it 

Research summary 

The serpinopathies are human pathologies caused by mutations that promote 

polymerisation and intracellular deposition of proteins of the serpin (serin protease inhibitors) 

superfamily, leading to toxicity and cell death. The dementia FENIB is caused by 

polymerisation of the neuronal serpin neuroserpin (NS). The identification of the molecular 

bases of polymer accumulation and toxicity using in vitro model systems is an important 

route for understanding these conditions and developing therapeutic interventions. 

Aims 

Our aim is to understand how NS polymers accumulate within the endoplasmic reticulum 

(ER) and what is the mechanism of their cellular toxicity. More specifically, we aim to: 

- Define the role of N-linked glycosylation on the ER handling of NS. 

- Characterise the cell toxicity of NS polymers in a new neuronal model of FENIB. 

First year results 

1. Role of N-linked glycosylation in the ER handling of NS. NS has three consensus 

motifs for N-linked glycosylation (N157, N321 and N401). We have shown that only two of 

them are glycosylated on wild type NS, while the fastest polymerising mutant (Gly392Glu NS) 

presents as two populations, with two and three glycan chains respectively. To understand 

the role of the extra glycan, we used direct mutagenesis to eliminate each of the three 

glycans in our pcDNA3.1 plasmids carrying wild type (WT) and Gly392Glu (G392E) NS. We 

have studied these mutants, along with the control WT and G392E plasmids, by transient 

transfection in COS-7 cells. Cells and culture media were analysed by SDS and nondenaturing 

PAGE followed by western blot for NS, luciferase (transfection efficiency control) 

and GAPDH (protein loading control). Our results show that: i) the N401 glycosylation site is 

not used in wild type NS and is additionally used in Gly392Glu NS; ii) removing the N321 site 

causes polymerisation of wild type NS, demonstrating that glycosylation is important for the 

structural stability of NS; and iii) removing the N401 glycosylation site in Gly392Glu NS 

causes a higher degree of mutant protein accumulation within the cells, probably by 

interfering with mutant protein degradation by the proteasome. 

2. Cell toxicity of NS polymers in a new model of FENIB. We have prepared expression 

constructs for constitutive, stable expression of wild type, Ser52Arg (medium degree of 

73


polymerisation), Gly392Glu (high degree of polymerisation) and delta (a truncated version 

that does not polymerise) NS, by cloning these cDNAs into the pTP6 vector, suitable for high 

expression in neural progenitors. This vector also has an antibiotic selection marker for 

efficient selection of transfected cells with stable transgene integration and robust expression 

levels of the gene of interest. Cell lines of neural progenitors from embryonic and adult 

mouse brain cortex are already available for transfection through our collaboration with Dr. 

Giuseppe Lupo and Dr. Emanuele Cacci. 

Future work 

1. Role of N-linked glycosylation on the ER handling of NS. We will: i) assess the 

amounts of intra- and extracellular NS quantitatively, by using two complementary sandwich 

ELISA specific for total and polymerised NS respectively; ii) study the degradation of 

Asn401Gln-Gly392Glu NS at steady state, by using proteasome inhibitors and comparing the 

amounts of the glycosylation mutant with the control plasmid (Gly392Glu NS); iii) in 

collaboration with Prof. Lomas (Cambridge, UK), we will study the intracellular processing of 

our glycosylation mutants in more detail by pulse-chase analysis in COS-7 cells. 

2. Cell toxicity of NS polymers in a new model of FENIB. We will transfect our new 

constructs into neural progenitor cell lines and will generate stably transfected cells by 

antibiotic selection. We will assess the expression levels of each NS variant by SDS-PAGE 

and western blot, and their monomeric vs. polymeric state by non-denaturing PAGE and 

western blot. We will use sandwich ELISA to quantify intracellular vs extracellular presence 

of each type of neuroserpin, and immunocytochemistry to study the intracellular localisation 

of retained NS. After the initial evaluation, we will proceed with long-term cultures to assess 

the toxicity of intracellular polymers of Ser52Arg and Gly392Glu NS in this system, by looking 

at activation of NFkB, oxidative stress and apoptosis. This will be done in terminally 

differentiated neural progenitor cells, using protocols available in our lab that allow efficient 

terminal differentiation of neural progenitors into neurons, astrocytes or oligodendrocytes. 


Claudia Moriconi, post-doc fellow; Valentina 

Timpano, student. 


Emanuele Cacci, Nicoletta Carucci, 

Antonella De Jaco, Giuseppe Lupo, Sapienza 

Università di Roma; David A. Lomas, Stefan 

Marciniak, University of Cambridge, UK; Juan 

Perez, University of Malaga, Spain; Nigel 

Birch, University of Auckland, New Zealand; 

Martino Bolognesi, Stefano Ricagno, 

Università di Milano, Mauro Manno, Vincenzo 

Martorana, Rosetta Noto, CNR, Istituto di 

Biofisica, Palermo; Luisa Schiaffonatti, Anna 

Fra, Università di Brescia. 

74


Characterization of the muscle-nerve crosstalk in a neuromuscular 

disease 

Antonio Musarò 

Department of Anatomical, Histological, Forensic & Orthopaedic Sciences 

℡: +39 06 49766956 - @: antonio.musaro@uniroma1.it 

One of the crucial systems severely affected in several neuromuscular diseases, 

including Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy (SMA) and aging, is 

the loss of effective connection between muscle and nerve, leading to a pathological noncommunication 

between the two tissues. 

In a previous work (Dobrowolny et al., JCB 2005), we demonstrated that muscle 

expression of a local IGF-1 isoform protects motor neurons in an ALS mouse model. More 

recently, we showed that mIGF-1 improves muscle mass and extends the survival of severe 

SMA mice independent of SMN induction (Bosch-Marcé et al., Hum Mol Genet 2011). Our 

studies suggests that muscle fibers provide appropriate factors, such as mIGF-1, for the 

maintenance of a functional muscle and neuron survival and that skeletal muscle is a primary 

target for the dominant action of inherited SOD1 mutation. This hypothesis has been 

validated by our recent studies, demonstrating that muscle-restricted expression of mutant 

SOD1 G93A was sufficient to recapitulate some of the pre-symptomatic sign of ALS 

(Dobrowolny et al., Cell Met 2008). The goals of this project is to define the molecular 

signature of the retrograde muscle-nerve interaction. 

In the first part of the research activity, we extended previous study with the aim to define 

the potential signal transduction pathways activated in response to SOD1 G93A , expressed 

either locally or ubiquitously, that cause muscle atrophy. 

Several studies have indicated the activation of apoptotic events in pathological 

conditions associated with muscle atrophy. The central component and the executor of the 

apoptotic machinery are a family of cysteine proteases called caspases. We reported that 

muscle atrophy in MLC/SOD1 G93A mice does not involve the activation of caspases 

(Dobrowolny et al., Skelet Muscle 2011). Of note, the expression and activity of caspases 

increased in the muscle of the classical animal models of ALS, which express the SOD1 G93A 

gene ubiquitously, only at paralysis stage. 

Thus, it seems likely that muscle atrophy is a primarily events associated with the toxic 

effects of SOD1 G93A mutant protein, whereas motor neuron degeneration is a later event that 

contributes to exacerbate the atrophic phenotype, promoting paralysis and muscle wasting. 

The present work has provided new insights concerning the control of muscle fiber size and 

the mechanisms of atrophy in animal models of ALS. Moreover we started monitoring the 

intracellular organelles and neuromuscular junctions (NMJ). Preliminary experiments 

revealed significant alterations in mitochondria morphology and function and in NMJ. 

75


Mitochondria were often clustered, abnormally shaped, and presented a translucent 

appearance, edematous internal matrix with abnormal and/or missing internal cristae. 

This initial morpho-functional alteration of skeletal muscle might be followed by NMJ 

destruction, distal axonopathy, astrocytosis in the spinal cord, and finally motor neuron loss. 

This might lead to a late exacerbation of muscle atrophy caused by caspases activation. 

Further work will define if and how skeletal muscle impacts the nervous system. 

Publications 

Bosch-Marcé M, Wee CD, Martinez TL, Lipkes CE, Choe DW, Kong L, Van Meerbeke JP, 

Musarò A, Sumner CJ. Increased IGF-1 in muscle modulates the phenotype of severe 

SMA mice. Hum Mol Genet 2011, 20: 1844-53. doi: 10.1093/hmg/ddr067. 

Carosio S, Berardinelli MG, Aucello M, Musarò A. Impact of ageing on muscle cell 

regeneration. Ageing Res Rev 2011, 10: 35-42. http://dx.doi.org/10.1016/j.arr.2009.08.001. 

Dobrowolny G, Aucello M, Musarò A. Muscle atrophy induced by SOD1G93A expression 

does not involve the activation of caspase in the absence of denervation. Skelet Muscle 

2011, 1: 3. doi: 10.1186/2044-5040-1-3. 

Forte G, Pietronave S, Nardone G, Zamperone A, Magnani E, Pagliari S, Pagliari F, Giacinti 

C, Nicoletti C, Musaró A, Rinaldi M, Ribezzo M, Comoglio C, Traversa E, Okano T, Minieri 

M, Prat M, Di Nardo P. Human cardiac progenitor cell grafts as unrestricted source of 

supernumerary cardiac cells in healthy murine hearts. Stem Cells 2011, 29: 2051-61. doi: 

10.1002/stem.763. 


Gabriella Dobrowolny, Emanuele Rizzato, 

post-doc fellows; Carmine Nicoletti, technician. 


Marco Sandri, Dipartimento Scienze 

Biomediche- VIMM, Università di Padova; 

Feliciano Protasi, Ce.S.I., Università degli 

Studi G. d’Annunzio, Chieti; Rüdiger Rudolf, 

Univ. of the State of Baden-Wuerttemberg and 

National Laboratory of the Helmholtz 

Association- Germany; Charlotte Sumner, 

Department of Neurology, Johns Hopkins 

Bloomberg School of Public Health, USA. 

76


Role of the COP9 signalosome in transcription modulation and 

chromatin organization in yeast and plants 

Rodolfo Negri 


℡: +39 06 49917790 - @: rodolfo.negri@uniroma1.it 

Accurate regulation of transcription in time and space is critical for the establishment and 

maintenance of gene expression patterns in eukaryotes. In the past years, several 

components of the ubiquitin/proteasome system (UPS) have been shown to be necessary for 

the tight regulation of gene expression with possible important implications for cellular 

homeostasis. A key component of the UPS is the COP9 signalosome (CSN), a protein 

complex conserved in all eukaryotes. CSN removes the small peptide NEDD8 (an ubiquitin 

like modifier) from the cullin-RING family of E3 ubiquitin ligases. E3 ligases are the enzymes 

responsible for poly-ubiquitination of specific substrates, which can be subsequently 

recognized and degraded by the proteasome. The reaction catalyzed by CSN is necessary 

for the tight regulation of the assembly/disassembly cycles of these ligases and is essential 

for all higher eukaryotes, since null CSN mutants from different organisms ranging from 

plants to mammals are lethal at very early developmental stages. 

At the cellular level, CSN mutants from different organisms display de-repression and, 

more in general, miss-regulation of several sets of genes. This could be due simply to the 

fact that CSN is necessary for the proper degradation of several transcription factors. 

However, recent evidence suggests that CSN might perform specific tasks directly on the 

genomic chromatin. 

The CSN from budding yeast S. cerevisiae has been characterized and, like in higher 

eukaryotes, is responsible for the de-neddylation of cullin-RING ubiquitin ligases. However, 

while the CSN in most higher eukaryotes is composed of 8 subunits and is a paralog of the 

lid of the proteasome, the CSN from budding yeast consists of six subunits: Csn9, Csn10, 

Csn11/Pci8, Csn12, Csn5/Rri1 and Csi1. In addition, in contrast to other eukaryotes, all the 

CSN subunits from S. cerevisiae are non-essential. The non essentiality of CSN components 

and the availability of powerful genetic tools make S. cerevisiae a very promising model 

system to elucidate some aspects of the nuclear role of this complex. 

We performed a transcriptomic analysis of a S. cerevisiae strain deleted in CSN5 (the deneddylating 

subunit of the Cop9 complex), as compared with its isogenic wild type strain. 

Data suggest that Csn5 is involved in the modulation of several genes controlling zinc 

uptake and metabolism and ergosterol biosynthesis. All the modulations were confirmed by 

real-time RT-PCR and most of them were observed in some of the other CSN deletion 

mutants. We also tested the various CSN components deleted strains for phenotypic features 

related to defects in zinc uptake and ergosterol biosynthesis and found a good correlation 

with the transcriptomic pattern. ChIP on chip analysis suggests that Csn5 associates with 

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some of the modulated genes, suggesting a direct mode of transcriptional regulation. This 

mode of regulation is likely to consist in a decrease of the turnover of a transcriptional 

activator or in an increase of the turnover of a transcriptional repressor (Fig.1). We are now 

trying to identify putative targets. A promising candidate is Mot3/Rox7 which represses genes 

involved both in ergosterol biosynthesis and in zinc import and metabolism. 

Fig. 1 - Csn5 could act by increasing 

the turnover of a repressor or 

decreasing the turnover of an 

activator 

Publications 

Bosio MC, Negri R, Dieci G. Promoter architectures in the yeast ribosomal expression 

program. Transcription 2011, 2: 71-77. doi: 10.4161/ trns.2.2.14486. 

Piccinni E, Chelstowska A, Hanus J, Widlak P, Loreti S, Tata AM, Augusti-Tocco G, Bianchi 

MM, Negri R. Direct interaction of Gas41 and Myc encoded by amplified genes in nervous 

system tumours. Acta Biochim Pol 2011, 58: 529-34. 

Romagnoli G, Cundari E, Negri R, Crescenzi M, Farina L, Giuliani A, Bianchi MM. 

Synchronous protein cycling in batch cultures of the yeast Saccharomyces cerevisiae at log 

growth phase Exp Cell Res 2011, 317: 2958-68. doi: 10.1016/j.yexcr.2011.09.007. 


Giovanna Serino, professor; Teresa Rinaldi, 

researcher; Valerio Licursi, post-doc fellow; 

Angela Alagia, Chiara Salvi, PhD students. 


Gianni Balliano, Università degli Studi di 

Torino; Elah Pick, Haifa University, Israel. 

78


The biogenesis of piRNAs and their involvement in transposon 

silencing and heterochromatin formation in Drosophila 

Sergio Pimpinelli 


℡: +39 06 49912876 - @: sergio.pimpinelli@uniroma1.it 

The "canalization" concept (Waddington, 1959) describes the resistance of a 

developmental process to phenotypic variation, regardless of genetic and environmental 

perturbations, owing to the existence of buffering mechanisms. Severe perturbations, which 

overcome such buffering mechanisms, produce altered phenotypes that can be heritable and 

can themselves be canalized by a genetic "assimilation" process. 

Recent studies on Hsp90, a protein involved in several cellular processes and 

development pathways, seem to indicate possible molecular mechanism for canalization and 

genetic assimilation. In both flies and plants, mutations in the Hsp90-encoding gene induce a 

wide range of phenotypic abnormalities which have been interpreted as an increased 

sensitivity of different developmental pathways to hidden genetic variability (Rutherford and 

Lindquist, 1998; Queitsch, 2002). From these data, it has been proposed that Hsp90 is a 

capacitor of morphological evolution and buffers a pre-existing genetic variation that is not 

expressed and accumulates in neutral conditions. Thus, Hsp90 chaperone machinery may 

be an evolutionarily conserved buffering mechanism of phenotypic variance, which provides 

the genetic material for natural selection. 

Our recent study (Specchia et al., 2010) suggests an additional, perhaps alternative, 

explanation for proposals of a concrete mechanism underlying canalization. We show that, in 

Drosophila, functional alterations of Hsp90 affect the Piwi interacting RNA (piRNA; a class of 

germ-line-specific small RNAs) silencing mechanism leading to transposon activation and the 

induction of morphological mutants. This indicates that Hsp90 mutations can generate new 

variation by transposon-mediated ‘canonical’ mutagenesis. 

We propose that, in general, stress causes the activation of transposons that induce de 

novo gene mutations affecting development pathways. The transposon induced mutations 

can be expressed and fixed across subsequent generations by a process consisting in a coselection 

of two independent somatic and germinal events giving the same phenotype. 

The research activity during 2011 has been mainly focused on the effect of different type 

of stress on transposons mobilization and a screening to identify genes that are involved in 

heterochromatin formation and/or piRNA biogenesis in Drosophila melanogaster. 

1. to study the effect of stress on transposon activity, we applied discrete of continuous 

heat shocks to larvae and adults and analyzed, in their germ and somatic cells, the 

transcription and mobilization of different classes of transposons also assessing the 

presence of their corresponding piRNAs or endo-siRNAs. The results sofar obtained, strongly 

79


suggest that stress activates transposons trancription and mobilization. To obtain a global 

view of transposons genomic redistribution, we are performing an analysis by New 

Generation Sequencing methodology. We are also performing a global analysis of piRNAs by 

cloning and deep sequencing. 

2. we have identified several mutations that affect piRNAs biogenesis. Their analysis is 

still ongoing. 

3. we are performing a proteomic analysis of Hsp90 complexes before and after Heat 

Shock. Preliminary results have indicated that Hsp90 changes some cofactors in the two 

different conditions. 

Part of these data are discussed in a paper in preparation. 


Laura Fanti, professor; Lucia Piacentini, 

researcher; Enzo Marchetti, research fellow; 

Marcella Marchetti, post-doc fellow. 


Maria Pia Bozzetti, Università di Lecce; Maria 

Berloco; Università di Bari. 

80


To the root of organ growth: the control of root meristem activity in 

Arabidopsis 

Sabrina Sabatini 


℡: +39 06 4991 7916 - @: sabrina.sabatini@uniroma1.it 

A crucial aspect of development, in animals as well as plants, is how the balance 

between cell division and cell differentiation is maintained to ensure coherent growth of the 

organs and determine their correct shape and size. 

In plant roots, meristems ensure growth by constantly supplying fresh cells thanks to the 

division of stem cells; as in other developmental systems, including animal (e.g. the 

mammalian bone growth plate, and gut crypt), stem cell division and terminal differentiation 

take place in distinct and physically separated tissutal districts. 

We had previously shown that in the root meristem the balance between cell division and 

cell differentiation is maintained by the interaction between the plant hormone auxin, 

promoting cell division and the plant hormone cytokinin, promoting cell differentiation. 

In particular, we have shown that cytokinin perception at the root meristem transition 

zone, mediated by the histidine kinase AHK3, activates two type-B primary ARABIDOPSIS 

RESPONSE REGULATORS, ARR1 and ARR12 which act as transcription factors and 

mediate the cytokinin-dependent cell diffentiation input. The transcription factor ARR1 

activates the gene SHY2, a repressor of auxin signalling that negatively regulates the PIN 

genes that encode auxin transport facilitators. Thus, cytokinin controls the distribution of 

auxin, prompting cell differentiation. Conversely, auxin mediates degradation of the SHY2 

protein, sustaining the activity of the PIN genes and prompting cell division. 

Our work has therefore shown that the size of the Arabidopsis root meristem is 

established by a balance between the antagonistic effects of cytokinin and auxin and has 

provided a genetic and molecular framework - the ARR1/SHY2/PIN circuit - for this 

interaction. 

Nevertheless, major unanswered questions remained: as cells divide in the stem cell 

niche while cell differentiation takes place in the transition zone at the opposite end of the 

meristem, how the activities of these two regions of the meristem are coordinated with each 

other at a distance And which of the regulatory components that ensure stem cell niche 

maintenance mediate the coordination with the circuit active in the transition zone at the 

opposite end of the root meristem 

By means of the construction and analysis of relevant single and multiple mutants, the 

use of diverse reporter gene fusions, pharmacological experiments, chromatinimmunoprecipitation 

and quantitative RT-PCR, we provide mechanistic evidence - at the 

genetic and molecular level - of how the spatial coordination between cell division in the 

stem cell niche and cell differentiation at the meristem transition zone is achieved. 

81


We have shown that the SCR transcription factor maintains the activity of stem cell niche 

by suppressing cytokinin signaling in the QC, and that it presides over the spatial 

coordination of stem cell division with cell differentiation in transition zone at the opposite end 

of the root meristem. SCR achieves this coordination by positioning the cytokinin response 

regulator ARR1. SCR binds directly to the promoter of ARR1 in the QC, repressing its 

expression and allowing stem cell division. In the transition zone, SCR controls expression of 

ARR1 via the DELLA protein RGA, thus controlling the rate of cell differentiation. This 

modulation of the cytokinin input by SCR allows a fine tuning of auxin concentration along the 

meristem, acting both on its synthesis via auxin biosynthesis gene ASB1, and on signalling 

and transport via SHY2, thus ensuring coherent root growth. 


Laila Moubayidin, Serena Perilli, post-doc 

fellows; Riccardo Di Mambro, Phd student. 


Renze Heidstra, Faculty of Science, Dept of 

Biology, section Molecular Genetics, Utrecht 

University, The Netherlands. 

82


Biological characterization and in vitro culture of spermatogonial 

stem cells 

Mario Stefanini 


℡: +39 06 49766570 - @: mario.stefanini@uniroma1.it 

The adult human testis produces millions of sperm each day trough spermatogenesis. 

This highly efficient process, finely tuned by hormone and paracrine balance, relies on the 

biological activity of spermatogonial stem cells (SSC), the stem cells of germline. Because of 

the ability to transmit genes from one generation to the next, SSCs represent the only 

replicating, potentially totipotent, population of cells in the adult body. 

Surface antigenic profile of SSCs, for their prospective isolation, has been partially 

defined. Moreover, data from our and other laboratories support the idea that SSCs are 

heterogeneous in term of gene expression profile. SSC subsets may be readout of niche 

signals or they could represent hierarchically distinct stem/progenitor cell populations. 

Interestingly, we found that spermatogonial stem cell displaying SP phenotype may be 

generated very early during embryonic testis development (Scaldaferri et al., 2011). The 

anatomical location and molecular components of the SSC niche are largely unknown. The 

molecular dissection of germline niche, will certainly shed light on SSCs maintenance and 

differentiation. 

To gain insight into lineage relationship among subsets of SSC we have developed an 

animal model expressing the receptor GFRA1 tagged with the enhanced green fluorescent 

protein (EGFP) to isolate and enrich SSCs from mice at different stage of testis development. 

We confirmed that expression of tagged protein is restricted to early spermatogonia. We 

are at the present testing the stem cell activity of sorted cell populations that have been 

selected based on EGFP-expression level by means of germ cell transplantation. Moreover, 

by video-time laps analysis we are testing the migratory activity of GFP-expressing early 

spermatogonia. 

We have also analyzed the role of the niche-derived factor GDNF upon the gene 

expression of SSCs. At the present GDNF is the best characterized niche-derived factor, 

produced by Sertoli cells the nurse cells of male germ cells. A large body of evidences show 

that GDNF is essential for the maintenance of SSCs both in vivo and in vitro. By in vitro 

short-term culture of intact seminiferous tubules and qRT-PCR we have recently found that 

GDNF regulates the expression of genes known to be involved in spermatogonial 

differentiation such as Tex14, Sohlh1 and Kit. Our data indicate that GDNF, besides its 

crucial role in the self-renewal of stem cells also functions in the differentiation of chained 

undifferentiated spermatogonia (Grasso et al., 2011). 

Finally we were able to establish human and mouse SSC cultures. Development of SSC 

culture is highly relevant to the study of molecular mechanisms regulating early step of 

83


spermatogensis, for future applications on male infertility, and to unveil molecular 

mechanisms underlying pluripotency. 

Publications 

Grasso M, Fuso A, Dovere L, de Rooij D, Stefanini M, Boitani C, Vicini E. Distribution of 

GFRA1-expressing spermatogonia in adult mouse testis. Reproduction 2011 Epub. doi: 

10.1530/REP-11-0385. 

Scaldaferri ML, Fera S, Grisanti L, Sanchez M, Stefanini Mario, De Felici M, Vicini E. 

Identification of side population cells in mouse primordial germ cells and prenatal testis. Int 

J Dev Biol 2011, 55: 209-14. doi: 10.1387/ijdb.092977ms. 


Carla Boitani, professor; Elena Vicini, 

researcher; Lisa Dovere, Barbara Muciaccia, 

post-doc fellows; Dante Lamberti, PhD student; 

Stefania Fera, Tiziana Menna, technicians. 


Dirk de Rooij, Department of Endocrinology, 

Utrecht University, The Netherlands; Ans Van 

Pelt, Center for Reproductive Medicine, 

Academic Medical Center, University of 

Amsterdam, The Netherlands. 

84


Molecular mechanisms driving liver stem cell fate 

Marco Tripodi 


℡: +39 06 4461387 - @: tripodi@bce.uniroma1.it 

Our work focuses on liver stem cell plasticity and differentiation mechanisms. To these 

aims we dispose of a number of stem cell lines named RLSCs (from Resident Liver Stem 

Cells) we previously stabilized and characterized. These cells spontaneously differentiate 

into periportal hepatocytes that, in turn, are able to switch into perivenular hepatocytes. This 

post-differentiative switch is triggered by Wnt signaling and converges on the HNF4a-driven 

transcription (Conigliaro et al., CDD 2008; Colletti et al., Gastroenterology 2009). RLSCs 

show a metastable molecular phenotype typical of embryonic and adult stem cells and 

characterized by co-expression of epithelial and mesenchymal markers as well as of a 

variety of chromatin remodeling genes. This highly dynamic cell state, thought to be the 

major determinant of stem cell fate, may be considered as the result of continue oscillations 

between EMT/METs. 

Result 1. In frame of the study of molecular mechanisms controlling EMT/MET in hepatic 

cells, we published a work describing the pivotal role of HNF4α in the induction and 

maintenance of hepatocyte identity. HNF4α was over-expressed in different hepatocyte cell 

lines and the resulting gene expression profile determined by qRT-PCR. HNF4α recruitment 

on promoters of both mesenchymal and EMT regulator genes was determined by EMSA and 

ChIP assays. The effect of HNF4α depletion was assessed in silenced cells and in the 

context of the whole liver of HNF4-KO animals. By these approaches, we identified key EMT 

regulators and mesenchymal genes as new targets of HNF4α. In particular HNF4α directly 

inhibits transcription of the EMT master gene Snail (Santangelo et al., 2011). Our findings 

indicate that HNF4α transcriptional activation and repression of critical EMT/MET genes is 

pivotal for the maintenance of a stable epithelial phenotype as well as for the regulation of 

the dynamic process of MET. Previously, we demonstrated as Snail causes repression of 

Hnf4α transcription through direct promoter binding (Cicchini et al., 2006). Thus, we propose 

a simple cross-regulatory circuit between Snail and HNF4α in which each factor’s expression 

is mutually exclusive to the other due to the presence of repressor elements in each 

promoter. HNF4α /Snail reciprocal control may provide the molecular rationale for feed-back 

and reversible differentiative processes and explain, at least in part, the coherence and the 

reversibility of the molecular events underlying EMT/MET. 

Result 2. In Garibaldi et al., we described a molecular circuitry controlling RLSCs 

plasticity. Starting from the finding that Snail is expressed in RLSCs, we demonstrated its 

85


positive role in stemness markers expression. This observation, unexpected considering that 

the transcriptional repression is the only function so far attributed to Snail, prompted us to 

investigate on other players integrating/mediating Snail activity. Mirror observations made in 

RLSCs and their derived differentiated hepatocytes RLSCdH allowed us to conclude that 1) 

in RLSC Snail inhibits the hepato-specific program through direct repression of HNF4 gene 

and of the epithelial microRNAs-200c and -34a, 2) in RLSCdH HNF4, together with a direct 

repression of Snail gene, directly up-regulates microRNA-200 family members (200a, b, c) 

and microRNA-34a transcription, thus further stabilizing the hepatocytic phenotype. 

Altogether these data unveiled Snail, HNF4 and microRNAs-200a, b, c and -34a as epistatic 

elements controlling hepatic stem cell maintenance/differentiation. (Garibaldi et al., Cell 

Death and Differentiation 2011). 

Publications 

Garibaldi F, Cicchini C, Conigliaro A, Santangelo L, Cozzolino AM, Grassi G, Marchetti A, 

Tripodi M, Amicone L. An epistatic mini-circuitry between the transcription factors Snail and 

HNF4α controls liver stem cell and hepatocyte features exhorting opposite regulation on 

stemness-inhibiting microRNAs. Cell Death Differ 2011 Epub. doi: 10.1038/cdd.2011.175. 

Mancone C, Steindler C, Santangelo L, Simonte G, Vlassi C, Longo MA, D'Offizi G, Di 

Giacomo C, Pucillo LP, Amicone L, Tripodi M, Alonzi T. Hepatitis C virus production 

requires apolipoprotein A-I and affects its association with nascent low-density lipoproteins. 

Gut 2011, 60: 378-86. doi:10.1136/gut.2010.211292. 

Santangelo L, Marchetti A, Cicchini C, Conigliaro A, Conti B, Mancone C, Bonzo JA, 

Gonzalez FJ, Alonzi T, Amicone L, Tripodi M. The stable repression of mesenchymal 

program is required for hepatocyte identity: a novel role for hepatocyte nuclear factor 4α. 

Hepatology 2011, 53: 2063-74. doi: 10.1002/hep.24280. 


Laura Amicone, Carla Cicchini, Alessandra 

Marchetti, researchers, Carmine Mancone, 

Alice Conigliaro, post-doc fellows; Laura 

Santangelo, Angela Cozzolino, AIRC fellows; 

Valeria De Nonno, Marco De Santis Puzzonia, 

Viviana Costa, PhD students; Claudio 

Cavallari, technician. 

86


Profiling the Polycomb/Trythorax target genes in normal and 

leukemic hematopoiesis 

Giuseppe Zardo 


℡: +39 06 4469843 - @: zardo@bce.uniroma1.it 

Research aims 

Several studies describe the functional effects of the modifications of PcG/TrxG activities 

on hematopoietic stem/progenitor cell self renewal and proliferation. However, insufficient 

information is available about the genes, undergoing PcG/TrxG regulation in normal 

hematopoiesis, and deregulated during terminal differentiation, thus permitting the activation 

of a leukemic program. Our research aims to identify these genes and to investigate the 

existence of functional connections between different epigenetic mechanisms of gene 

regulation during normal and leukemic hematopoiesis. 

Results 

Recent findings have identified NFI-A gene as a regulator of hematopoiesis. NFI-A is a 

post-transcriptional miR-223 target directing human hematopoietic progenitor lineage 

decision. Our research demonstrates that NFI-A down-regulation, which allows normal 

granulopoiesis, is guaranteed also by other epigenetic events affecting NFI-A gene promoter 

activity and chromatin structure, including the resolution of chromatin “bivalent domains” by a 

modification of PcG/TrxG activities; DNA hypermethylation, recruitment of PcG-RNAi 

complex and by miR-223 promoter targeting activity. We found that miR-223 settles on these 

epigenetic events. Confocal microscopy and ChIP analyses revealed that in myeloid cells, 

induced to granulopoiesis by retinoic acid treatment, miR-223 localizes in the nucleus, colocalizes 

with PcG and RNAi proteins, and targets the NFI-A promoter region containing 

PcGs binding sites and miR-223 complementary DNA sequences. The knockdown of PcG 

and RNAi proteins, such as Suz12, Dicer1 and Ago1, and promoter activity assays, showed 

that both the integrity of the PcG-RNAi complex and DNA sequences, matching the seed 

region of miR-223, are required to induce NFI-A transcriptional silencing. PcG and RNAi 

Suz12, Dicer1 and Ago1 knockdown impaired the NFI-A down-regulation and granulocytic 

differentiation potential of HL60 cells treated with retinoic acid, as shown by NFI-A mRNA 

levels, myeloid cell marker CD11b levels and TGaseII expression. Ectopic miR-223 

expression caused heterochromatic repression of NFI-A gene and channeled granulopoiesis, 

while its stable knockdown produced opposite effects. Our findings indicate that endogenous 

miRs can affect gene expression also at the transcriptional level, functioning in a critical 

interface between chromatin remodelling complexes and the genome to direct fate lineage 

determination of hematopoietic progenitors. These results also identify PcG/TrxG functions 

as regulator of hematopoiesis, likely deregulated during leukemic transformation. Polycomb 

87


protein Ezh2, which in association with Suz12 is responsible for establishment of repressive 

chromatin mark H3K27me3, directs DNA methylation by the recruitment of DNA 

methyltransferases on target genes. The presence of H3K27me3 chromatin mark at gene 

promoter regions in human embryonic stem cells (HESC) makes these genes sensitive to 

aberrant DNA methylation during tumour transformation. By RLGS we acquired the DNA 

methylation profiles of several human leukemic cell lines, AML patient samples and of 

CD34+HSC/HPCs and mature CD34- cells as control samples. We selected genes 

hypermethylated and derived, from literature data, the presence at their promoter region of 

H3K27me3 and/or H3K4me3 chromatin marks in HESC. ChIP assays showed that although 

the majority of these hypermethylated genes were H3K27me3 labelled in HESC, 

CD34+HSC/HPCs, and CD34- cells, a relevant number presented the H3K4me3 activating 

mark. In leukemic samples, where these genes appeared consistently hypermethylated, the 

H3K4me3 mark was replaced by H3K27me3 mark, thus proving that leukemic transformation 

is also associated with an anomalous PcG/TrxG activity. 

Perspectives 

The results of this research enable a more comprehensive knowledge of epigenetic 

mechanisms regulating the activity of hematopoietic master regulators and unveil new targets 

for leukaemia therapy. 


Alberto Ciolfi, post-doc fellow; Carmen 

Maresca, technician. 


Clara Nervi, Dip. di Scienze e Biotecnologie 

Medico-Chirurgiche, Sapienza Università di 

Roma; Robin Foa’, Sabina Chiaretti, Dip. di 

Biotecnologie Cellulari ed Ematologia, 

Sapienza Università di Roma; Joseph F. 

Costello, Comprehensive Cancer Center, 

University of California, San Francisco, USA. 

88

Area 4 

Molecular recognition 

in biomolecules

Area 4: Molecular recognition in biomolecules 

Toxic effects induced by polyamine metabolites on melanoma cells: 

a new therapeutic approach 

Enzo Agostinelli 


℡: +39 06 49910838 - @: enzo.agostinelli@uniroma1.it 

The purpose of this research is to develop a new anticancer therapy against melanoma 

(M14), taking advantage of the high polyamine content of tumor cells, since such polycations 

may become a source of cytotoxic metabolites. For their generation an enzyme, bovine 

serum amine oxidase (BSAO) will be employed. It is expected that by delivering BSAO into 

cancer cells, toxic enzymatic oxidation products could be produced intracellularly for 

selective killing in situ. 

Several studies have been performed to overcome the drug-resistant phenotype and to 

develop innovative chemotherapeutic strategies effective against multi-drug resistant (MDR) 

tumours. Our studies demonstrate that BSAO and spermine (SPM) addition to human M14 

cells induces cell growth inhibition and overcomes the MDR phenotype (ADR2). The 

oxidative reaction produces polyamine metabolites (H 2 O 2 and aldehydes) which induce 

oxidative stress. Cytotoxicity induced by SPM metabolites was greater in MDR cells than in 

wild-type ones (WT), due to an increased mitochondrial activity. To increase the induction of 

cell death by toxic polyamine metabolites, we are currently investigating several drug 

combinations. Clonogenic experiments showed that the cytotoxicity induced by BSAO/SPM 

addition was enhanced by the pre-treatment of tumour cells with lysosomotropic compounds, 

the drug chloroquine (CQ) or MDL 72527, in both WT and MDR M14 cells. 

The pre-treatment of M14 cells with CQ or MDL 72527 sensitized both WT and ADR2 

cells to the subsequent exposure to SPM metabolites. Noteworthy, the apoptotic effect was 

higher in ADR2 cells than in their WT counterparts. The cytotoxic effect induced by the 

combined treatment was characterized by cytoplasmic vacuolization and mitochondrial 

damage, as revealed by transmission electron microscopy (TEM) (Fig. 1). We hypothesize 

that the release of lysosomal enzymes produces oxidative stress and apoptosis, indicating a 

contribution of the lysosomotropic properties of CQ to the sensitization of M14 cells to H 2 O 2 

and aldehyde(s). The findings suggest that the association of BSAO/SPM with CQ or MDL 

72527, and mitochondrial alterations induced by SPM oxidation products, allow the design of 

a new therapeutic strategy based on the use of these combinations in human neoplasms, 

making this approach mainly attractive in treating MDR cancer patients. 

From a therapeutic point of view the improvement of the efficacy of in situ formation of 

cytotoxic polyamines metabolites is essential. Since in the present model H 2 O 2 and 

aldehydes are produced outside the cells, the main challenge will be to deliver BSAO into 

cancer cells in order to induce their selective killing by the cytotoxic factors produced 

intracellularly from endogenous polyamines. Therefore, strategies could be developed to find 

91


how the enzyme could be delivered in vivo. To this aim BSAO incorporates into liposomal 

vesicles and dedicated nano-scale drug delivery systems will be designed. Thus, 

endogenous polyamines present in high concentration in tumor cells could be targeted and 

oxidized by the enzyme. 

Fig. 1- Ultrastructural analysis of M14 WT ( top 

panels) and M14 ADR2 (lower panels) cells 

performed by TEM. (a and b) Untreated melanoma 

cells; (c and d) Cells exposed to 300 µM MDL 72527 

for 24 h, at 37˚C; (e and f) Cells treated for 60 min 

with 6.5x10-3 IU/ml BSAO and6 µM SPM at 37˚C; (g 

and h) Cells were first exposed for 24 h to 300 µM 

MDL 72527, and then treated for 60 min with 

6.5x10-3 IU/ml BSAO and 6 µM SPM at 37˚C. 

Publications 

Agostinelli E. Role of polyamines, their analogs and transglutaminases in biological and 

clinical perspectives. Amino Acids 2011 Epub. doi: 10.1007/s00726-011-1129-2 

Agostinelli E, Toninello A, Vianello F, Stevanato R. Do mammalian amine oxidases and the 

mitochondrial polyamine transporter have similar protein structures Amino Acids 2011 

Epub. doi: 10.1007/s00726-011-0988-x. 

Stevanato R, Cardillo S, Braga M, De Iuliis A, Battaglia V, Toninello A, Agostinelli E, Vianello 

F. Preliminary kinetic characterization of a copper amine oxidase from rat liver 

mitochondria matrix. Amino Acids 2011, 40: 713–20. doi: 10.1007/s00726-010-0708-y. 

Valente S, Tomassi S, Tempera G, Saccoccio S, Agostinelli E, Mai A. Novel Reversible 

Monoamine Oxidase A Inhibitors: Highly Potent and Selective 3-(1H-Pyrrol-3-yl)-2- 

oxazolidinones. J Med Chem 2011, 54: 8228-32. Epub 2011 Nov 7. doi: 10.1021/ 

jm201011x. 


Paola Turini, professor; Giampiero Tempera, 

Nikenza Viceconte, Stefania Saccoccio, postdoc 

fellows. 


Annarica Calcabrini, Istituto Superiore di 

Sanità, Roma; Taichi Ueshima, Wakunaga 

Pharmaceutical Co., Hiroshima, Japan. 

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Cellular response to oxidative stress: involvement of protein 

disulfide isomerases 

Fabio Altieri 


℡: +39 06 49910880 - @: fabio.altieri@uniroma1.it 

The aim of this research project is to elucidate the involvement of ERp57/PDIA3 in the 

cellular response to oxidative stress. Different thyroid carcinoma cell lines were subjected to 

oxidative stress and analyzed during the following 24 hours. We compared cell vitality, the 

intracellular levels of reduced and oxidized glutathione and the cellular distribution of ERp57 

and Ref1, STAT3 and Ku70/86, previously identified ERp57 interactors. ARO cells, which 

better survived to oxidative stress, showed the highest levels of ERp57 but the lowest 

glutathione content. A nuclear translocation of ERp57, Ref1 and Ku70/86 as a consequence 

of the stress treatment has been also observed, suggesting the involvement of ERp57, 

beside its known functions within ER, in regulatory processes at the nuclear level. 

We showed that in M14 cells, where STAT3 is constitutively activated, ERp57 binds with 

STAT3 to a subset of well-characterized STAT3-associated promoters/enhancers. The 

presence of ERp57 in these sites appears to be essential for the regulatory activity of STAT3 

on transcription, since inhibition of ERp57 expression by RNA interference is accompanied 

by the decrease of expression of several STAT3 dependent genes. Moreover, the addition of 

vancomycin and phenyl-arsenoxide, two inhibitors of ERp57 redox activity, inhibited the in 

vitro binding of STAT3 to DNA. 

ERp57-calreticulin interaction has been studied by surface plasmon resonance (SPR). 

ERp57 was covalently bound to the sensor surface and its binding with calreticulin (CRT) 

was analyzed. The estimated K D was 2.6 µM, in agreement with the observation of other 

authors. However, the kinetic analysis of obtained binding curves better fits with a two-step 

binding model where the initial protein complex undergoes to conformational changes that 

increase its stability. This result is in agreement with a kind of plasticity in the structure of 

ERp57 that allows, through specific conformational changes, to handle the interaction with so 

many different protein partners. SPR analysis showed that vancomycin can inhibit ERp57- 

CRT interaction and the K D obtained for vancomycin binding to ERp57 was similar to that 

observed for CRT. Vancomycin has also effect on CRT-ERp57 interaction in vivo. It has been 

demonstrated that cell membrane localization of calreticulin is dependent on the interaction 

with ERp57. Vancomycin administration to HeLa cells prevents the ERp57-mediated 

translocation of calreticulin to the plasma membrane. This result confirms a role for ERp57 in 

the trafficking of proteins from endoplasmic reticulum to plasma membrane. 

A joint project was started to analyze the involvement of ERp57 in EGFR activation and 

internalization. Breast cancer cells, where ERp57 was silenced by RNA interfering, were 

subjected to EGF stimulation. Preliminary studies suggest that ERp57 can interact with 

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EGFR and that in EGF stimulated cells the silencing of ERp57 affects internalization of the 

receptor and its autophosphorylation, thus leading to changes in the activation of 

downstream pathways. 

The in vitro binding of ERp57 with known interactors was further analyzed. Using a thiol 

probe able to modify the electrophoretic mobility of reduced and oxidized forms of a protein, 

we confirmed the reducing activity of ERp57 on oxidized Ref1. Solid-phase binding assay 

and SPR experiments verified the close interaction between the two proteins with a K D in the 

micromolar range. Finally, we started to analyze the binding of several natural substances to 

ERp57 and their effects on protein activities, to find molecules able to specifically inhibit the 

binding between ERp57 and selected interactors and modulate its biological functions. We 

focused the attention on green tea catechins (GTCs) and tested their binding with ERp57 by 

fluorescence quenching measurements and SPR analysis. Data analysis showed that 

galloylated forms of GTCs better bind to ERp57 and quench the fluorescence of tryptophan 

residues with epigallocatechin-gallate the most active. Quenching analysis was extended to 

isolated redox domains of ERp57. Data analysis confirmed the binding of catechins near the 

ERp57 redox-active sites and this interaction requires the presence of the galloyl moiety. 

This structural feature is also responsible for the inhibition of both ERp57 reductase activity 

and DNA binding capability. 

Publications 

Frasconi M, Chichiarelli S, Gaucci E, Mazzei F, Grillo C, Chinazzi A, Altieri F. Interaction of 

ERp57 with calreticulin: Analysis of complex formation and effects of vancomycin. Biophys 

Chem 2011 Epub. doi: 10.1016/j.bpc.2011.09.003. 

Turano C, Gaucci E, Grillo C, Chichiarelli S. ERp57/GRP58: a protein with multiple functions. 

Cell Mol Biol Lett 2011, 16: 539-63. doi: 10.2478/s11658-011-0022-z. 


Margherita Eufemi, professor; Caterina Grillo, 

post-doc fellow; Rossana Cocchiola, PhD 

student. 


Silvia Chichiarelli, Daniela Ricci, Elisa 

Gaucci, Dipartimento di Scienze Biochimiche 

“A. Rossi Fanelli”, Sapienza Università di 

Roma. 

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Role of Gsα signalling in the bone marrow hematolymphopoietic 

microenvironment as revealed by novel transgenic models 

Paolo Bianco 


℡: +39 06 4441049 - @: paolo.bianco@uniroma1.it 

Background 

Cells of osteogenic lineage maintain, organize and transfer the hematopoietic 

microenvironment/niche in the bone marrow. In search for the specific cell type conveying 

these crucial physiological effects, attention over the past few years has been directed first to 

osteoblast proper (i.e., the mature bone forming compartment in the osteogenic lineage) and 

later to osteogenic progenitors (aka “mesenchymal stem cells”). Studies using osteoblasttargeted 

knockout have implicated Gsα signaling within cells of osteogenic lineage in the 

regulation of the HME/niche, particularly with respect to B lymphopoiesis. We have 

generated mice in which an activating missense mutation of Gsα (resulting in a severe 

skeletal disease in humans, Fibrous Dyplasia of bone) is either targeted to osteoblasts 

through the COL1A1 2.3kb promoter, or expressed widely in the bone environment as 

directed by costitutive promoters (PGK or EF1α). The latter model has been established both 

in an FVB and in a B6 background. 

Aim 

The general aim is to elucidate a) the changes in the organization of the HME/niche b) 

the changes in the hemato-lymphopoietic progenitor compartments brought about by 

overactivity of Gsα signaling. 

Results 2011 

1) Functional effects of mutated Gsa in different osteogenic cell compartments. Using a 

well standardized, timed system for monitoring osteogenic differentiation of BM-derived 

skeletal progenitor cells (“mesenchymal stem cells”) in vitro, we have established the 

differential effects of mutated Gsα on the progenitor and mature compartments of the 

osteogenic lineage. In the mature compartment, mutated Gsα potently downregulates SOST, 

a negative modulator of Wnt signaling, leading to an inappropriate excess Wnt lignaling (as 

monitored through Axin2 and LEF1). The latter data directly explain the high bone mass 

phenotype of COL1A1-GsaR201C mice, which indeed is made by these data a phenocopy of 

two human high bone mass disorders, Van Buchem disease and Sclerosteosis. When 

expressed in the progenitor compartment, mutated Gsα induces a complete replica of the 

human FIbrous Dysplasia phenotype. This is linked to specific cellular responses that are not 

not duplicated in the mature compartment, including a potent upregulation of RANKL 

95


expression, a marked blunting of Wnt signaling, and a prolonged delay of osteogenic 

differentiation (2-3 weeks). In vivo, this compartment corresponds to an intraosseous, 

perivascular population of stromal cells, noted for high expression of the Adrb2 (IHC in vivo; 

qPCR ex vivo), and high functional responses to adrb2 agonists ex vivo. This population is at 

the same time both perivascular and endosteal, i.e. is located at the two main sites where the 

HSC niche is thought to be. 

2) Functional effects of mutated Gsα expressed in the osteogenic lineage on hematolymphoid 

compartments. This analysis has so far revealed that detectable changes in 

hematolymphoid compartments do occur in mice as a result of Gsα overactivity in the 

osteogenic lineage. Of note, these changes do unfold in an age-dependent manner, 

matching the development of the bone/bone marrow structural changes. While we have not 

been able to detect so far clear-cut changes in the size of the HSC compartment proper 

(CD34- LSK), or of B, pre-B or pro-B compartments, we do detect changes in the pool of 

progenitors. More importantly, we do detect an age dependent activation of DNA synthesis 

and cell replication (BrdU; Flk-2 subset of LSK) in B6 mice with constitutive expression of 

mutated Gs in osteoprogenitors, so that pools of replicating HSCs approach in size, in old 

mutant mice, those seen in young wt. 

Next directions 

One interesting lead for developing these data further comes from combining three key 

observations: 1) the identification of an intraosseous, perivascular osteoprogenitor as the 

prime mediator of effects of mutated Gsα in vivo (leading to subversion of the HME/niche as 

seen in Fibrous Dysplasia); 2) the evidence that this progenitor compartment is 

physiologically primarily responsive to beta2-adrenergic stimulation, which in turn is a known 

regulator of circadian mobilization of HSCs; 3) the evidence that in conjunction with the 

development of FD lesions, HSCs are activated to divide. While the analysis of 

hematolymphoid compartments will continue in the next year, we will also pursue the 

hypothesis that HSCs are mobilized in mice with constitutively active Gsα in the 

osteoprogenitor compartment. 


Mara Riminucci, Isabella Saggio, professors; 

Alessandro Corsi, Benedetto Sacchetti, 

researchers; Stefania Cersosimo, Rossella 

Costa, Cristina Remoli, post-doc fellows; 

Paola Comite, Letizia Astrologo, PhD 

students; Emanuela Spica, technician. 


Ana Cumano, Pasteur Institut, Paris 

96


The dark side of protein folding: denatured states and misfolded 

species in molecular recognition and pathological processes 

Maurizio Brunori 


℡: +39 06 4991 0579 - 06 4940543 - @: maurizio.brunori@uniroma1.it 

The variety of chemical reactions taking place in the cell demands recognition events to 

be highly specific. Since specificity is achieved through complementarity between interacting 

species, folding is crucial to protein function. We have studied the folding and binding of 

selected protein systems, that are key components of important cellular processes. Particular 

attention has been devoted to elusive states, such as the denatured state, and to intradomain 

allostery, a sophisticated mechanism to modulate specificity. The two model systems 

are: (i) the PDZ domains, small globular domains involved in a variety of cellular processes 

(Model System I), and (ii) a pair of designed proteins with an extraordinarily high degree of 

sequence identity but different 3D structure and function (Model System II). 

Results 

Model System I. Protein-protein interactions mediated by modular protein domains are 

critical for cell’s life. However, given the vast number of ligands competing for binding to a 

limited number of domain families, it is unclear how specificity can be achieved. Such 

specificity may be modulated by intradomain allostery, whereby remote residues are 

energetically connected to the functional binding site. Whereas energetic pathways in 

modular domains have been predicted, there is a paucity of experimental data to validate 

their existence and roles. We have identified these functional energetic networks in one of 

the most common protein-protein interaction modules, the PDZ domain. We exploited the 

use of double mutant cycles, in conjunction with kinetics to capture the fine energetic details 

of the networks involved in peptide recognition. The analysis revealed that the allosteric 

pathways are dictated by the amino acid sequence rather than topology. Model System II. 

We have addressed the folding mechanism of two heteromorphic proteins G A 88 and G B 88, 

with 88% sequence identity but different structure (Fig. 1). This system offered the unique 

opportunity to unveil the mechanism whereby a few key residues commit the polypeptide 

chain to its characteristic and functionally competent native topology. 

An extensive characterization of the folding mechanisms of G A 88 and G B 88 by 

experiments and molecular dynamics (MD) simulations allowed to identify the presence of a 

residual structure in the denatured state of G B 88, not observed for G A 88. A signature of such 

a difference is the different denaturant dependence of the (un)folding rate constants. 

Consistently, MD simulations show that for G B 88 the non-polar solvent-accessible surface 

area decreases at low pH. This unexpected result suggests that the denatured state has a 

primary role in the folding process. Indeed, protein topology appears to be committed very 

97


early along the folding pathway, being “imprinted” in the residual structure of the denatured 

state; this weak, loosely defined topology is sufficient to dictate the pathway of folding. 

Fig. 1 - G A 88 and G B 88 structures. Sequence 

alignment and secondary structure are shown. 

The 7 residues that differ between the two 

proteins are shown in different colors. It may be 

seen that the only a-helix in G B 88 is longer 

compared to the topologically homologous α2 of 

G A 88; this is the physico-chemical feature 

responsible for the residual interaction in the 

denatured state of G B 88 which dictates the 

stabilization of the β-sheet fold. 

We have undertaken an extensive characterization of the folding mechanism of the 

parental protein displaying the structure of G B 88, namely GB1. We demonstrated the 

presence of an on-pathway folding intermediate that previously escaped detection and 

characterization. Elucidation of the folding mechanism of GB1 is critical for future work on the 

different variants with increased sequence identity with respect to the G A proteins. 

Publications 

Gianni S, Haq SR, Montemiglio LC, Jürgens MC, Engström Å, Chi CN, Brunori M, Jemth P. 

Sequence-specific long range networks in PSD-95/discs large/ZO-1 (PDZ) domains tune 

their binding selectivity. J Biol Chem 2011, 286: 27167-75. doi: 10.1074/jbc.M111.239541. 

Morrone A, McCully ME, Bryan PN, Brunori M, Daggett V, Gianni S, Travaglini-Allocatelli C. 

The denatured state dictates the topology of two proteins with almost identical sequence 

but different native structure and function. J Biol Chem 2011, 286; 3863-72. doi: 

10.1074/jbc.M110.155911. 

Morrone A, Giri R, Toofanny RD, Travaglini-Allocatelli C, Brunori M, Daggett V, Gianni S. 

GB1 is not a two-state folder: identification and characterization of an on-pathway 

intermediate”. Biophys. J 2011, 101; 2053-60. doi: 10.1016/j.bpj.2011.09.013. 


Luca Federici, Carlo Travaglini Allocatelli, 

professors; Adele Di Matteo, Stefano Gianni, 

CNR researchers; Rajanish Giri, Angela 

Morrone, PhD students. 


Per Jemth, University of Uppsala, Sweden; 

Valerie Daggett, University of Washington, 

Seattle, Washington, USA. 

98


The role of nucleosomes in the stability of human telomeres 

Stefano Cacchione 


℡: +39 06 49912240 - @: stefano.cacchione@uniroma1.it 

Telomeres are the special nucleoprotein structures that protect chromosome ends. In 

humans telomeric DNA consists of TTAGGG repeats about 10 kbp long, ending in a 3’ G-rich 

overhang of 150-200 nucleotides. Most of telomeric DNA is organized in tightly packed 

nucleosomes. Two proteins, TRF1 and TRF2, compete with the histone octamer for binding 

to double-stranded telomeric DNA and organize a six-protein complex named shelterin, 

essential for telomere stability. Both telomere length maintenance and the establishment of a 

correct capping structure are essential for cell life and survival. Whereas the roles played by 

telomeric proteins in telomere protection have been widely investigated, a satisfactory 

description of nucleosome contribution to telomere structure and function is still lacking. 

Little is known also on the interplay between telomeric proteins and nucleosomes. It is not 

known if nucleosomes and TRF proteins occupy different domains on the telomere or 

whether they cooperate in telomere capping; moreover, it is still unclear if telomere 

nucleosomal organization is affected by abnormal expression of TRF proteins. 

Our goal is to characterize the nucleosomal organization of human telomeres and to 

elucidate the role played by nucleosomes in telomere capping structure. We addressed these 

issues by means of theoretical analyses, in vitro model systems and analyses on cultured 

cells. 

Theoretical analysis of the higher-order organization of telomeric chromatin 

Telomeric chromatin is characterized by a nucleosome repeat length about 40 bp shorter 

than bulk nuclear chromatin. Due to their peculiar DNA sequence features, telomeric 

nucleosomes occupy isoenergetic positions having the periodicity of the telomere repeat. 

The systematic search of the possible nucleosome packing revealed that for nucleosome 

repeat length of 156, 162 and 168 bp (multiples of the vertebrate telomeric repeat) different 

and unique structures are allowed (De Santis and Scipioni, in preparation). 

Nucleosome density at telomeres is regulated by TRF2 

To analyze whether TRF2 affects nucleosomal organization at telomeres, we altered 

TRF2 expression in human cells by transient transfection. We found that the density of 

telomeric nucleosomes was inversely proportional to the dosage of TRF2 at telomeres. This 

effect coincides with the end of S phase of the cell cycle. Moreover, we showed that 

nucleosome spacing at telomeres increased upon TRF2 overexpression. These results were 

confirmed by an in vitro nucleosome assembly on a telomeric DNA in the presence of purified 

99


TRF2 (Galati et al., in press). These findings raise the intriguing possibility that telomere 

protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome 

organization. 

Nucleosomes affect differently TRF1 and TRF2 accessibility to telomeric sequences 

By means of an in vitro model that mimics the binding of TRF proteins to telomeric DNA 

in a nucleosomal context, we found that the presence of nucleosomes close to the binding 

site disfavors TRF2 interaction whereas TRF1 recognizes its binding sequence with higher 

affinity. Our data indicate that this different behavior is mediated by histone tails and suggest 

a different role of TRF1 and TRF2 in the folding of telomeric chromatin (Galati et al., in 

preparation). 

Perspectives 

In the next future our aim is to map telomeric chromatin at a higher resolution than that 

present in literature. To this end, we realized a cell line containing an engineered telomere in 

which a strong nucleosome positioning sequence – the 601 DNA - is placed directly 

upstream of telomeric DNA. This positioned nucleosome will allow us to analyze whether the 

regular and tight nucleosome spacing is a feature of the entire telomere or whether the 

proximal and the distal part of the telomere are differently organized. Induction of telomere 

shortening by inhibition of telomerase activity will allow us to map telomeres over their entire 

length and to study changes of telomere chromatin organization as a function of telomere 

length. The possible molecular structures of telomeric chromatin will be screened adopting 

the theoretical model developed in our lab. 

Publications 

Scipioni A, De Santis P. Predicting nucleosome positioning in genomes: Physical and 

bioinformatic approaches. Biophys Chem 155: 53-64. doi: 10.1016/j.bpc.2011.03.006. 


Stefano Morosetti, professor; Anita Scipioni, 

researcher; Alessandra Galati, Emanuela 

Micheli, Pasqualina Punzi, post-doc fellows. 


Pasquale De Santis, Armandodoriano 

Bianco, Dipartimento di Chimica, Sapienza 

Università di Roma; Daniela Rhodes, Medical 

Research Council, Laboratory of Molecular 

Biology, Cambridge, UK; Eric Gilson, Marie- 

Josephe Giraud-Panis, Institute for Research 

on Cancer and Aging, University of Nice, 

France. 

100


Plant innate immunity: signalling and recognition of Damage- 

Associated Molecular Patterns (DAMPs) 

Felice Cervone 


℡: +39 06 49912517 - @: felice.cervone@uniroma1.it 

The cell wall-derived oligolacturonides (OGs) are typical damage-associated molecular 

patterns (DAMPs) formed during microbial infections. The molecular basis of the plant innate 

immunity elicited by OGs is the focus of this project. In Arabidopsis the accumulation of OGs 

takes place during the degradation of pectin by microbial polygalacturonases (PGs) and is 

favoured when PGs interact with specific plant inhibitors (PGIPs). OGs are recognized by a 

Wall-Associated Kinase (WAK1). We have shown that WAK1 and chimeric WAK1-derived 

receptors may be used to engineer resistance in crop plants by activating a signal 

transduction pathway that has been partially elucidated during this project (De Lorenzo et 


MPK3 and MPK6 are Arabidopsis protein kinases activated by OGs. Analysis of single 

mapk mutants revealed that lack of MPK3 increases basal susceptibility to the fungal 

pathogen Botrytis cinerea but does not significantly affect elicitor-induced resistance. Instead, 

lack of MPK6 has no effect on basal resistance but suppresses OG-induced resistance to B. 

cinerea. Over-expression of the AP2C1 phosphatase leads to impaired OG-induced 

phosphorylation of both MPK3 and MPK6 (Galletti et al., 2011). 

OGs inhibit adventitious root formation induced by auxin in Arabidopsis leaf explants as 

well as the expression of several auxin-responsive genes. The inhibition of auxin responses 

by OGs does not require ethylene, jasmonic acid and salicylic acid signalling and is 

independent of RBOHD-mediated reactive oxygen species production. OG-auxin antagonism 

does not involve a stabilization of auxin-response repressors or decreased levels of auxin 

receptor transcripts through the action of microRNAs (Savatin et al., 2011). 

The ability of bacterial or fungal necrotrophs to produce enzymes capable of degrading 

pectin and producing OGs is often related to a successful initiation of the infective process. 

Pectin is synthesized in a methylesterified form and is subsequently de-esterified in muro by 

pectin methylesterase. Deesterification makes pectin more susceptible to the degradation by 

pectic enzymes such as PGs and pectate lyases secreted by pathogens. Pectobacterium 

carotovorum and B. cinerea induce in Arabidopsis a rapid expression of AtPME3 that acts as 

a susceptibility factor and is required for the initial colonization of the host tissue (Raiola et 


The inhibitors PGIPs modulate the accumulation of OGs by interacting with microbial 

PGs. We have obtained a low-resolution structure of the complex formed by PG from 

Fusarium phyllophilum and PGIP from Phaseolus vulgaris as determined by small-angle x- 

ray scattering analysis. The inhibitor engages its concave surface of the leucine-rich repeat 

101


domain with the enzyme. Both sides of the enzyme active site cleft interact with the inhibitor. 

The structure is in agreement with previous site-directed mutagenesis data and has been 

further validated with structure-guided mutations and subsequent assay of the inhibitory 

activity (Benedetti et al., 2011). 

Publications 

Benedetti M, Leggio C, Federici L, De Lorenzo G, Pavel NV, Cervone F. Structural resolution 

of the complex between a fungal polygalacturonase and a plant polygalacturonaseinhibiting 

protein by Small-Angle X-Ray Scattering. Plant Physiol 2011, 157: 599-607. doi: 

http://dx.doi.org/10.1104/pp.111.181057. 

De Lorenzo G, Brutus A, Savatin DV, Sicilia F, Cervone F. Engineering plant resistance by 

constructing chimeric receptors that recognize damage-associated molecular patterns 

(DAMPs). FEBS Letters 2011, 585: 1521-28. doi: 10.1016/j.febslet.2011.04.043. 

Galletti R, Ferrari S, De Lorenzo G. Arabidopsis MPK3 and MPK6 play different Roles in 

basal and oligogalacturonide- or flagellin-induced resistance against Botrytis cinerea. Plant 

Physiol 2011, 157: 804-14. doi: 10.1104/pp.111.174003. 

Raiola A, Lionetti V, Elmaghraby I, Immerzeel P, Mellerowicz EJ, Salvi G, Cervone F, 

Bellincampi D. Pectin methylesterase is induced in Arabidopsis upon infection and is 

necessary for a successful colonization by necrotrophic pathogens. Mol Plant-Microbe 

Interact 2011, 24: 432-40. doi: http://dx.doi.org/10.1094/MPMI-07-10-0157. 

Savatin DV, Ferrari S, Sicilia F, De Lorenzo G. Oligogalacturonide-auxin antagonism does 

not require posttranscriptional gene silencing or stabilization of auxin response repressors 

in Arabidopsis. Plant Physiol 2011, 157: 1163-74. doi: http://dx.doi.org/ 10.1104/ pp.111. 

184663. 


Giulia De Lorenzo, Daniela Bellincampi, 

professors; Simone Ferrari, Benedetta Mattei, 

researchers; Giovanni Salvi, Daniela 

Pontiggia, Isabel Santori, research fellows; 

Manuel Benedetti, Claudia Fabbri, Fedra 

Francocci, Vincenzo Lionetti, Daniel Savatin, 

Francesca Sicilia, Francesco Spinelli, postdoc 

fellows; Federico Andreani, Elisa 

Bastianelli, Nora Gigli Bisceglia, Giovanna 

Gramegna, Matteo Gravino, Vanessa 

Modesti, Chiara Paparella, PhD students. 

102


Spontaneous generation and evolution of genetic information 

Ernesto Di Mauro 


℡: +39 06 49912880 - @: ernesto.dimauro@uniroma1.it 

The aim of the project is to reconstruct the initial reactions that lead to the generation of 

genetic information. Starting from the consideration that it will not be possible to know how 

life started but that it is nevertheless possible to approximate its first steps, we have 

extended the observations that have brought us to establish three principles: 1) all the 

nucleic bases necessary for building extant nucleic acids can be synthesized nonenzymatically, 

from simple components, with high efficiency and in plausible prebiotic 

conditions. 2) Nucleic bases evolve, within the same prebiotic physico-chemical frame, into 

nucleosides and nucleotides. 3) Polymerization mechanisms exist leading from 

spontaneously activated precursors to long nucleic polymers. 

We have explored the conditions that extend the mechanisms identified so far, passing 

from the abiotic non-enzymatic synthesis of homogenous polymers to the generation of 

complex sequences. The questions asked are: 1) is it possible to spontaneously generate 

RNA sequence complexity, based on intrinsic properties of the RNA structure 2) Is it 

possible to correlate the generation of RNA complexity to the origin of the protein coding 

process 

In addition, somewhat unexpected results (see below) allow us to ask the key question: is 

it possible to correlate the development of (proto)genetic systems with the development of 

(proto)metabolic systems 

The data obtained provide the following answers: 

It is actually possible to generate sequence complementarity-driven nonenzymatic 

ligation of RNA. We have reported two reactions of RNA G:C sequences occurring nonenzymatically 

in water in the absence of any added cofactor or metal ion: (a) sequence 

complementarity-driven terminal ligation; (b) complementary sequence adaptor-driven 

multiple tandemization. The two abiotic reactions increase the chemical complexity of the 

resulting pool of RNA molecules and change the Shannon information of the initial population 

of sequences. 

The reaction of nonenzymatic terminal ligation of homogeneous polyA sequences 

previously discovered (Pino et al., J Biol Chem 2008, 283: 36494-503) has been analyzed by 

Atomic Force Microscopy. The products of ligation reaction of a 24 nucleotides-long PolyA 

RNA adsorbed on mica were analyzed by atomic force microscopy. In that study the 

occurrence of oligonucleotides at different degrees of polymerization was quantitatively 

studied before and after ligation reaction. The microscopy images at the nanoscale showed 

103


that nonenzymatic ligation of pristine RNA monomers resulted in the formation of 

supramolecular aggregates, with prevalence of dimers and tetramers. Analytical conditions 

were defined allowing the identification, the quantitative evaluation, and their distribution after 

ligation reaction, also providing an estimate of the degree of hydration of the objects. Such 

investigation provides the simplest yet model system for direct investigation of RNA reactions 

by advanced microscopy. 

Taken together, these studies show how spontaneously generated short oligonucleotides 

may progress along the path of generation of longer sequences. The possibility of introducing 

“errors” in the abiotic replication of RNA sequences has been shown, suggesting evolutionary 

mechanisms in the absence of complex phenotypes. 

The previously reported polymerization of oligonucleotides from cyclic nucleotides has 

been further studied and the conditions for improvement of the synthetic reactions in part 

published. 

Relevant results pertain the identification of the products of prebiotic synthesis of organic 

compounds from formamide in the presence of specific classes of catalysts like boroncontaining 

minerals and minerals from the Murchison meteorite. In addition to nucleic bases, 

complex population of carboxylic acids were observed, showing the possibility that in the 

same test-tube the precursors formed of both RNA and key metabolic cycles. The rational of 

these studies has been published. 

Publications 

Pino S, Costanzo G, Giorgi A, Di Mauro E. Sequence complementarity-driven nonenzymatic 

ligation of RNA. Biochemistry 2011, 50: 2994-3003. doi: 10.1021/bi101981z. 

Saladino R, Crestini C, Pino S, Costanzo G, Di Mauro E. Formamide and the origin of life. 

Phys Life Rev 2011 Epub. 2011. doi: 10.1016/j.plrev.2011.12.002. 


Giovanna Costanzo, CNR researcher, 

Samanta Pino, post-doc fellow; Silvia Lopizzo, 

technician. 


Raffaele Saladino, Dip. ABAC, Università della 

Tuscia, Viterbo; Claudia Crestini, Università 

Tor Vergata, Roma; Edward N. Trifonov, 

University of Haifa, Israel. 

104


Molecular and functional approaches to investigate the 

neuroprotective and neuromodulatory roles of chemokines and 

their receptors in the central nervous system 

Cristina Limatola 

Department of Physiology and Pharmacology "Vittorio Erspamer" 

℡: +39 06 49690243 - @: cristina.limatola@uniroma1.it 

This research project want to investigate the action mechanism of trans-membrane 

chemokine fractalkine CX3CL1 in the CNS. In particular we are interested in evaluating the 

role of glial cells as modulators of the neuroprotective and neuromodulatory effects of this 

chemokine also with attention to the signaling pathways activated by adenosine receptors 

(ARs). In the last few years we described that microglia and the A1R are involved as 

mediators of the neuroprotective and neuromodulatory effects of CX3CL1. We recently 

extended this study to an in vivo model of cerebral ischemia in rodents and to the effects of 

CX3CL1 on hippocampal synaptic plasticity induced by environmental stimuli. 

In 2011 we completed the study of the neuroprotective activity of CX3CL1 in models of 

permanent cerebral ischemia in rats and mice. In particular, we demonstrated that CX3CL1 is 

neuroprotective also in vivo, reducing the cell death of cortical neurons induced by 

permanent mean cerebral artery (pMCAO) occlusion. The protective effect of CX3CL1 has 

been demonstrated: 1) by a reduction of the ischemic brain area measured with a specific 

vital dye, 24 hours after injury induction; 2) by an improvement of performances in sensory 

motor tests to evaluate the brain damage from the functional point of view. In addition, we 

observed that these same and other parameters (evidenced by NMR analysis) are 

maintained up to 50 days after pMCAO confirming and extending the importance of our 

results. As concern the mechanisms, we prove that in vivo the protective effect of CX3CL1 is 

mediated by the indirect activation of adenosine receptor type 1 (A1R). This result has been 

reached with two different experimental approaches: we used both a specific A1R antagonist, 

DPCPX, which gave a complete inhibition of the neuroprotective effect of CX3CL1 and we 

performed pMCAO in A1R-/- mice, treated for icv injection of CX3CL1 observing no 

protection from ischemic damage (Cipriani et al., 2011). We also investigated another aspect 

of the activity of CX3CL1 in the central nervous system: in particular we have been interested 

in the effects on hippocampal plasticity of mice exposure for a long period to an environment 

“enriched” with sensory, social, motor cues, with particular attention to the changes induced 

in mice lacking CX3CL1 receptor (CX3CR1 GFP/GFP mice). The results demonstrated that in 

CX3CR1 GFP/GFP mice, hippocampal plasticity phenomena like learning in the “Water Maze” 

test and synaptic plasticity modulation like long-term potentiation (LTP) are increased in 

comparison with control wild-type (wt) mice. In contrast, while wt mice in enriched 

environment potentiated LTP and learning and memory ability, CX3CR1 GFP/GFP mice did not 

improve their performances upon “enrichment”. We also analyzed hippocampal neurogenesis 

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in the dentate gyrus observing that environmental enrichment is equally effective in wt and 

CX3CR1 GFP/GFP mice in increasing the number of newborn cells, with no significant 

differences in the two genotypes. In basal conditions, in the absence of enrichment, 

CX3CR1 GFP/GFP mice have a reduced neurogenesis (Maggi et al., 2011). 

Publications 

Di Angelantonio S, Piccioni A, Moriconi C, Trettel F, Cristalli G, Grassi F, Limatola C. 

Adenosine A2A receptor induces protein kinase A-dependent functional modulation of 

human 3{beta}4 nicotinic receptor. J Physiol 2011, 589: 2755-66. doi: 

10.1113/jphysiol.2011.207282. 

Bernareggi A, Grilli M, Marchi M, Limatola C, Ruzzier F, Eusebi F. Characterization of 

GABA(A) receptors expressed in glial cell membranes of adult mouse neocortex using a 

Xenopus oocyte microtransplantation expression system. J Neurosci Methods 2011, 198: 

77-83. http://dx.doi.org/10.1016/j.jneumeth.2011.03.011. 

Maggi L, Scianni M, Branchi I, D'Andrea I, Lauro C, Limatola C. CX3CR1 deficiency alters 

hippocampal-dependent plasticity phenomena blunting the effects of enriched environment. 

Front Cell Neurosci 2011, 5-22: 1-9. doi: 10.3389/fncel.2011.00022. 

Cipriani R., Villa P, Chece G, Lauro C, Paladini P, Micotti E, Perego C, De Simoni MG, 

Fredholm BB, Eusebi F, Limatola C. CX3CL1 is neuroprotective in permanent focal 

cerebral ischemia in rodents. J Neurosci 2011, 31: 16327–35. doi: 

10.1523/JNEUROSCI.3611-11.2011. 

Palma E, Inghilleri M, Conti L, Deflorio C, Frasca V, Manteca A, Pichiorri F, Roseti C, Torchia 

G, Limatola C, Grassi F, Miledi R Physiological characterization of human muscle 

acetylcholine receptors from ALS patients. Proc Natl Acad Sci U S A 2011, 108: 20184-8. 

doi: 10.1073/pnas.1117975108. 


Francesca Grassi, Sergio Fucile, Eleonora 

Palma, Davide Ragozzino, professors; Myriam 

Catalano, Flavia Trettel, researchers; Raffaela 

Cipriani, Clotilde Lauro, post-doc fellows; 

Grimaldi Alfonso, PhD student; Giuseppina 

Chece, technician. 


Maria Grazia De Simoni, Pia Villa, Istituto 

Mario Negri, Milano; Letizia Antonilli, 

Valentina Brusadin, Dipartimento di Fisiologia 

e Farmacologia, Sapienza Università di Roma; 

Bertil Fredholm, Karolinska Institute, 

Stockholm, Sweden; Angelo Spinedi, 

Università di Roma Tor Vergata. 

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Neurone response to experimental injury and lack of dystrophin: a 

molecular, functional and structural study in autonomic ganglia in 

vivo and in vitro 

Paola Paggi 


℡: +39 06 49912323 - @: paola.paggi@uniroma1.it 

Aim of our project is the characterisation in rodent superior cervical ganglion (SCG) of the 

molecular mechanisms and structural changes involved in the establishment, maintenance 

and plasticity of the reciprocal interactions between pre- and post-ganglionic neurones and 

between post-ganglionic neurones and their target organs. Damage of the ganglionic 

connectivity, consequent to pre- and post-ganglionic nerve crush or to the lack of dystrophin 

(Dp427), is used as a tool to perform our investigation. In the year 2011 we investigated the 

relevant pathological changes produced by lack of Dp427, a protein of the cortical 

cytoskeleton, in mdx mice, an animal model for Duchenne muscular dystrophy, investigating 

the following aspects: i) analysis of nicotine-evoked currents in dissociated neurones of mdx 

mouse SCG, in which the nicotinic acetylcholine receptors containing the α3 subunit 

(α3nAChRs) are drastically reduced compared with the wild-type; ii) large-scale analysis of 

gene expression in mdx mouse SCG to search for genes affected by the spontaneous 

mutation in the dystrophin gene and possibly contributing to SCG neuronal death, occurring 

between postnatal day 5 (P5) and 15 (P15), and to the altered reaction to axotomy and axon 

growth of SCG neurones, previously observed. 

i) In the sympathetic superior cervical ganglion (SCG), nicotinic acetylcholine receptors 

(nAChRs) mediate fast synaptic transmission. We previously demonstrated that in SCG 

neurons of mdx mice, lack of dystrophin causes a decrease, compared to the wild-type, in 

post-synaptic nAChRs containing the α3 subunit associated with β2 and/or β4 (α3β2/β4- 

nAChRs), but not of those containing the α7 subunit (α7-nAChRs). We showed, by whole cell 

patch-clamp recordings from cultured SCG neurons, that both nicotine- and acetylcholineevoked 

currents through α3β2/β4-nAChRs are significantly reduced in mdx mice compared 

to wild-type, while those through α7-nAChR are unaffected. This reduction associates with 

that of the protein levels of α3, β2 and β4 subunits. We suggest that, in mdx mouse SCG 

neurons, lack of dystrophin, by specifically affecting membrane stabilization of α3β2/β4- 

nAChRs, could determine an increase in receptor internalization and degradation, with 

consequent reduction in the fast intraganglionic cholinergic transmission. 

ii) By DNA microarray, we looked for changes in gene expression in SCG of P5, P10 and 

6-7 week-old mdx mice to verify whether the lack of Dp427 may affect transcript levels of 

specific genes, possibly giving an insight into the mechanisms involved in the structural and 

functional alterations we previously observed. Ontological Analysis of more than 500 

modulated genes showed significant differences in genetic class enrichment at each post- 

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natal date. Up-regulated genes mainly fell in the categories of vesicular trafficking, 

cytoskeletal and synaptic organization, while those down-regulated were associated to axon 

development, growth factors, intracellular signal transduction, gene expression regulation, 

synapse morphogenesis and nicotinic receptor clustering. In particular, downregulation of 

genes related to trophic factor signaling pathways and involved in axon growth and 

differentiation suggested that lack of Dp427 could affect trophic factor sensitivity and axon 

growth dynamics. 

Publications 

Di Angelantonio S, De Stefano ME, Piccioni A, Lombardi L, Gotti C, Paggi P. Lack of 

dystrophin functionally affects α3β2/β4-nicotinic acethylcholine receptors in sympathetic 

neurons of dystrophic mdx mice. Neurobiol Dis 41: 528-537. doi:10.1016/j.nbd.2010. 

10.024. 


Maria Egle De Stefano, professor, Silvia Di 

Angelantonio, researcher, Valerio Licursi, 

Loredana Lombardi, post-doc fellows. 


Tamara Petrucci, Istituto Superiore di Sanità, 

Laboratorio di Biologia Cellulare, Roma; Cecilia 

Gotti, CNR, Istituto di Neuroscienze, Centro di 

Farmacologia Cellulare e Molecolare, Dip. di 

Farmacologia Medica, Università di Milano; 

Irene Bozzoni, Ernesto Di Mauro, Andrea 

Mele, Rodolfo Negri, Alberto Oliverio, 

Dipartimento di Biologia e Biotecnologie “C. 

Darwin”, Sapienza Università di Roma; Carla 

Perrone Capano, Dipartimento di Scienze 

Biologiche, Università di Napoli “Federico II”; 

Casper Hoogenraad, Erasmus Medical 

Center, Department of Neuroscience, 

Rotterdam, The Netherlands; Falk Schroedl, 

Paracelsus University of Salzsburg, Austria. 

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Molecular mechanisms integrating endocytosis and signalling of 

fibroblast growth factor receptors 

Maria Rosaria Torrisi 

Department of Clinical and Molecular Medicine 

℡: +39 06 33775502 - @: mara.torrisi@uniroma1.it 

Endocytosis of receptor tyrosine kinases (RTKs) represents one of the main mechanisms 

of attenuation of the signal transduction, through re-localization by internalization and 

subsequent degradation of signaling molecules at the plasma membrane. The endocytic 

machinery and the RTK signal transduction appear to be intimately linked. In contrast to most 

of the receptor tyrosine kinases (RTKs), the keratinocyte growth factor receptor 

(KGFR/FGFR2b) appears to play an unique and unusual role in epithelial tissues, controlling 

proliferation and differentiation and exerting a tumor suppressive function in vitro and in vivo. 

KGFR endocytosis, upon binding of its paracrine ligands KGF and FGF10, is clathrindependent, 

but once internalized the receptors may follow two distinct endocytic pathways 

targeting to lysosomal degradation or recycling to the plasma membrane, as a consequence 

of a distinct phosphorylation and ubiquitination. Therefore, FGFR2b/KGFR represent a model 

system to shed light on the molecular mechanisms which control the endocytic trafficking and 

the receptor signaling. In addition, we have proposed that the expression of E5 protein 

encoded by high-risk human papillomavirus (HPV) type 16 can be used as a tool to analyze 

the endocytic indirect recycling pathway (Belleudi et al., Oncogene 2011). 

Since it is known that cell migration requires endocytic trafficking and polarization of 

adhesion molecules and RTKs to the leading edge, during this first year of the research 

project we investigated the role of KGFR endocytosis in the possible polarization of the 

receptors and their signaling and trafficking at the leading edge of migrating cells. We 

demonstrated the polarized localization of KGFR, upon ligand stimulation, regulated by Src. 

In addition, we found that the increased expression of the receptor and its polarization were 

able to enhance cell migration. Interestingly, both Src and the actin-binding protein cortactin 

appear to play a key role in the KGFR endocytosis and polarization, supporting the crucial 

involvement of RTK trafficking in cell motility (Belleudi et al., PLoS One 2011a). Future work 

will be focused to characterize the recycling pathways responsible for KGFR polarization 

during migration. In particular, based on the present observations that KGF and FGF10 have 

different ability to induce Src tyrosine phosphorylation, it would be interesting to verify if the 

two KGFR ligands might be able to mediate the receptor polarization through its targeting to 

different endocytic recycling pathways. During the first year of the project we investigated 

also the contribution of the KGFR expression, ligand-induced activation and receptor 

signaling in regulating the phagocytic process. To this aim we analyzed the uptake in vitro of 

fluorescent latex beads and we found that KGFR ligands trigger phagocytosis. Moreover, our 

results obtained by KGFR depletion or overexpression of wild type or defective mutants 

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demonstrated the promoting effect exerted by the activated receptor on the phagocytic 

ingestion and indicated the crucial role of the PLCγ signaling pathway in this KGFR-mediated 

mechanism of uptake. In addition, while the block of the receptor kinase activity and signaling 

inhibits both phagocytosis and receptor endocytosis, the block of the receptor clathrinmediated 

endocytosis by RNA interference does not impair the KGF activity in promoting the 

phagocytosis, unequivocally demonstrating that the receptor signaling and activation of the 

PLCγ downstream pathway occurs from the plasma membrane (Belleudi et al., FASEB J 

2011). Finally, to identify the possible receptor-mediated signaling pathways involved in the 

earlier induction of differentiation, we modulated in vitro the receptor expression and we 

induced a synchronous wave of differentiation, demonstrating that KGFR overexpression, 

activation and signalling increase the early differentiation, while receptor depletion reduces it. 

We found also that the PI3K/Akt signaling pathway is involved in the control of KGFRmediated 

keratinocyte differentiation. This in vitro experimental model indicates that 

FGFR2b/KGFR expression represents a key event regulating keratinocyte early 

differentiation during the switch from undifferentiated to differentiating cells (Belleudi et al., 

PLoS One 2001b) 

Publications 

Belleudi F, Purpura V, Scrofani C, Persechino F, Leone L, Torrisi MR. Expression and 

signaling of the tyrosine kinase FGFR2b/KGFR regulates phagocytosis and melanosome 

uptake in human keratinocytes. FASEB J 2011, 25: 170-81. doi: 10.1096/fj.10-162156. 

Belleudi F, Leone L, Purpura V, Cannella F, Scrofani C, Torrisi MR. HPV16 E5 affects the 

KGFR/FGFR2b-mediated epithelial growth through alteration of the receptor expression, 

signaling and endocytic traffic. Oncogene 2011, 30: 4963-76. doi: 10.1038/onc.2011.203. 

Belleudi F, Scrofani C, Torrisi MR, Mancini P. Polarized endocytosis of the keratinocyte 

growth factor receptor in migrating cells: role of Src-signaling and cortactin. PLoS One 

2011, 6:e29159. doi: 10.1371/journal.pone.0029159. 

Belleudi F, Purpura V, Torrisi MR. The receptor tyrosine kinase FGFR2b/KGFR controls 

early differentiation of human keratinocytes. PLoS One 2011, 6:e24194. doi: 

10.1371/journal.pone.0024194. 


Patrizia Mancini, Maurizio Alimandi, Lavinia 

Lotti, Vincenzo Visco, professors, Francesca 

Belleudi, Salvatore Raffa, researchers; Valeria 

Purpura, Danilo Ranieri, PhD students; 

Antonio Sabatucci, technician. 

110

Area 5 

Cellular and molecular 

immunology

Area 5: Cellular and molecular immunology 

Interplay amongst chronic immune activation, apoptosis, crosspresentation, 

immune-regulation, and autoimmunity 

Vincenzo Barnaba 

Department of Internal Medicine and Medical Specialties 

℡: +39 06 491268 - @: vincenzo.barnaba@uniroma1.it 

In previous studies, we found that the proteome of apoptotic T cells includes prominent 

caspase-cleaved cellular proteins and that a high proportion of distinct epitopes in these 

fragments (apoptotic epitopes) can be cross-presented by DCs to autoreactive CD8 + T cells 

(Moroni-Rawson et al., Nat Med 2007). In chronic HIV infection, these autoreactive CD8 + T 

cells correlate with the proportion of apoptotic CD4 + T cells in vivo and are involved in 

establishing polyclonal T cell activation that in the long run results in generalized T cell 

dysfunction/depletion. In addition, our previous report showed that apoptotic cells derived 

from activated T cells (in contrast to those derived from resting T cells or from non-lymphoid 

cells) retain the expression of CD40 ligand (L) and can then condition CD40 + DCs to acquire 

high capacities to prime or cross-prime autoreactive T cells (Propato et al., Nat Med 2001). 

This mechanism is consistent with the evidence that the signals provided by CD40L + 

apoptotic cells and not those provided by conventional apoptotic cells facilitate the 

emergence of autoreactive T cell responses to apoptotic self-antigens. 

Here we used the hepatitis C virus (HCV) infection as a human model of acute infection 

that generally undergoes chronic progression to verify whether CD8 + T cells that are specific 

for apoptotic self-epitopes have a distinct effector type-1, -2, or -17 phenotype, to distinguish 

which of them may participate in determining the fate of a viral infection (recovery versus 

chronicity), and to ascertain the mechanisms whereby these responses are induced and 

maintained. We demonstrated for the first time that the emergence of mixed polyfunctional 

(type-1, -2, -17) CD8 + T EM cell responses to apoptotic self-epitopes is related to the chronic 

evolution of acute HCV infection. The responses were directly correlated with the plasma 

viral load or the serum ALT levels, and were then sustained over time in relation to the viral 

persistence. These results suggest that, in conditions in which HCV has been able to evade 

the virus-specific immunity, strong CD8 + T cell responses against apoptotic self-epitopes are 

maintained and may contribute to the liver immunopathology through the production of high 

levels of inflammatory cytokines. This hypothesis is further emphasized by our parallel study 

indicating that similar autoreactive CD8 + T cell responses in chronically infected patients are 

recruited in the inflamed livers, are related with the signs of hepatic damage, and decrease in 

relation with the decline or the disappearance of the viral load upon antiviral therapy 

(interferon plus ribavirin @ ). 

We also demonstrated that cross-presentation of apoptotic T cells by DCs promptly 

activates CD8 + T EM cells specific for caspase-cleaved apoptotic self-epitopes ex vivo 

indicating that this mechanism might be operative in the induction of the resulting 

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polyfunctional autoreactive responses in patients with acute HCV infection who are 

experiencing chronic progression. The strong production of IFN-γ and IL-17 may favor the 

triggering of recruitment of inflammatory cells to the liver, which contribute to liver pathology. 

An important facet of our findings is that they demonstrate a link between the TCR avidity 

of autoreactive CD8 + T cells and the difference in the responsiveness of apoptotic epitopespecific 

CD8 + T cells exhibited by patients experiencing chronic infection and those 

undergoing infection resolution. Our results suggest that autoreactive CD8 + T cells with 

higher avidity for apoptotic self-epitopes are sustained over time and correlate with the 

progression toward chronic infection. The selection of the autoreactive CD8 + T cells with 

higher avidity likely occurs because of a sustained stimulation by apoptotic antigens. By 

contrast, lower avidity CD8 + T cells in the presence of weaker stimuli would undergo rapid 

contraction, as seen in the peripheral blood of patients with self-limited HCV infection. 

Publications 

Oliviero B, Cerino A, Varchetta S, Paudice E, Pai S, Ludovisi S, Zaramella M, Michelone G, 

Pugnale P, Negro F, Barnaba V, Mondelli MU. Enhanced B-cell differentiation and reduced 

proliferative capacity in chronic hepatitis C and chronic hepatitis B virus infections. J 

Hepatol 2011, 55: 53-60. doi.org/10.1016/j.jhep.2010.10.016. 

Spadaro F, Lapenta C, Donati S, Abalsamo L, Barnaba V, Belardelli F, Santini SM, Ferrantini 

M. Interferon-alpha enhances cross-presentation in human dendritic cells by modulating 

antigen survival, endocytic routing and processing. Blood 2011 Epub. doi: 10.1182/blood- 

2011-06-363564. 


Daniele Accapezzato, Silvia Piconese, 

researcher; Alessandra Citro, Helen Martini, 

Valeria Schinzari, post-doc fellows; Carmela 

Martire, Alessandra Proia, Eleonora Timperi, 

PhD students. 

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Signalling events negatively regulating FcεRI expression and mast 

cell functional responses 

Rossella Paolini 


℡: +39 06 4468448 - @: rossella.paolini@uniroma1.it 

Mast cells are key effectors in allergic diseases: upon ligation of the high affinity receptor 

for IgE (FcεRI) they release preformed and newly synthesized pro-inflammatory mediators. 

FcεRI is composed of an IgE-binding α chain, and the ITAM-containing β and γ subunits. 

Upon FcεRI cross-linking, the β chain-associated Src family PTK Lyn, phosphorylates β and 

γ-chain ITAMs allowing the recruitment and consequent activation of Syk, responsible for 

signal propagation (1). Notably, the full activation of FcεRI requires the recruitment of 

aggregated receptors into lipid rafts, where engaged receptors are concentrated (2) and can 

more easily interact with signaling molecules, such as active Lyn (3), favouring 

phosphorylation events. 

Besides these positive signals, FcεRI engagement has recently been understood to 

generate negative intracellular signals capable of limiting the rate and the extent of mast cell 

functional responses (4). Relevant to this, others and we have demonstrated that the Cbl 

family proteins control the amplitude of FcεRI-generated signals by specific ubiquitin 

modification of activated receptor subunits and associated protein tyrosine kinases (5,6). 

Concurrently, engaged receptors trigger their own endocytosis to extinguish signaling 

through removal of activated FcεRI complexes from the cell surface and delivery to 

lysosomes for degradation (7). 

The present study was aimed at identifying the molecular mechanisms ensuring the 

clearance of antigen-stimulated FcεRI complexes from the cell surface, thus contributing to 

the termination of mast cell functional program. 

We initially demonstrated that FcεRI subunits are monoubiquitinated by c-Cbl at multiple 

sites upon stimulation, and provided evidence for a role of ubiquitin as a signal regulating the 

initial step of internalization: endocytosis of engaged FcεRI complexes is dramatically 

affected in conditions of impaired receptor ubiquitination and requires the integrity of lipid 

rafts (8). 

We have further investigated the involvement of adaptor proteins harboring ubiquitin 

interacting motifs, namely Eps15, Eps15R, epsin and Hrs, in coupling ubiquitinated FcεRI to 

the endocytic machinery. Eps15, Eps15R and Epsin display similar functions: they control the 

early steps of the endocytic route coupling ubiquitinated receptors with components of the 

budding vesicles (9,10). The key role of Hrs is, instead, the delivery of ubiquitinated proteins 

to the outer membrane of the late endosomes and the sorting of the cargo into internal 

vesicles of multivesicular bodies for lysosomal degradation (11). 

Although we failed to observe a significant decrease of FcεRI entry in early endosomes 

upon individual depletion of Eps15, Eps15R or Epsin, the simultaneous depletion of all of 

115


them impaired ligand-induced receptor endocytosis, suggesting a partial overlapping function 

of these adapters in ubiquitinated FcεRI uptake. Notably, Hrs depletion retains ubiquitinated 

receptors into early endosomes and partially prevents their sorting into lysosomes supporting 

a critical role for Hrs in controlling the fate of internalized receptor complexes (8). 

In summary, our data demonstrate a key role for the ubiquitin pathway to ensure proper 

endocytic trafficking of antigen-stimulated FcεRI to a lysosomal compartment where 

degradation of the complexes can take place (8,12). 

The outcome of our proposal would be a better comprehension of the molecular 

mechanisms that functionally regulate the endocytic adaptor Hrs. In this respect, our 

preliminary results demonstrate that Hrs is subjected to antigen-dependent tyrosine 

phosphorylation and monoubiquitination, and that Syk is the main kinase regulating both Hrs 

covalent modifications (Gasparrini F. et al., unpublished observations). At present we are 

investigating whether and how Syk-induced modifications of Hrs impact on its endosomal 

localization, thus regulating Hrs function. 

References 

1) Nadler MJ, Matthews SA, Turner H, Kinet JP. Adv Immunol, 76:325-355, 2000. 

2) Field KA, Holowka D, Baird B. J Biol Chem, 272:4276-4280, 1997. 

3) Sheets ED, Holowka D, Baird B. J Cell Biol, 145:877-887,1999. 

4) Molfetta R, Peruzzi G, Santoni A, Paolini R. Arch Immunol Ther Exp, 55:219-229, 2007. 

5) Paolini R, Molfetta R, Beitz LO, Zhang J, Scharenberg AM, Piccoli M, Frati L, Siraganian R, 

Santoni A. J Biol Chem, 277:36940-36947, 2002. 

6) Kyo S, Sada K, Qu X, Maeno K, Miah SM, Kawauchi-Kamata K, Yamamura H. Genes Cells, 

8:825-836, 2003. 

7) Molfetta R, Belleudi F, Peruzzi G, Morrone S, Leone L, Dikic I, Piccoli M, Frati L, Torrisi M R, 

Santoni A, Paolini R. J Immunol, 175: 4208-4216, 2005. 

8) Molfetta R, Gasparrini F, Peruzzi G, Vian L, Piccoli M, Frati L, Santoni A, Paolini R. PLoS One, 

4(5):e5604, 2009. 

9) Carbone R, Fré S, Cannolo G, Belleudi F, Mancini P, Pelicci PG, Torrisi M, Di Fiore PP. Cancer 

Res, 57:5498-5504, 1997. 

10) Chen H, Fre S, Slepnev VI, Capua MR, Takei K, Butler MH, Di Fiore PP, De Camilli P. Nature 

394:793-797, 1998. 

11) Bache K., Brech A, Mehlum A, Stenmark H. J Cell Biol, 162:435-442, 2003. 

12) Molfetta R, Gasparrini F, Santoni A, Paolini R. Mol Immunol, 47:2427-2434, 2010. 

Publications 

Gasparrini F, Molfetta R, Santoni A, Paolini R. Cbl family proteins: balancing FcεRI-mediated 

mast cell and basophil activation. Int Arch Allergy Immunol 2011; 156: 16-26. doi: 

10.1159/000322236. 


Rosa Molfetta, researcher; Alessandra Porzia; 

post-doc fellow; Linda Quatrini, PhD student. 

116


Anti-tumor effector functions of NK cells in tumor microenviroment 

Angela Santoni 


℡: +39 06 44340632 - @: angela.santoni@uniroma1.it 

Our research proposal is aimed at identifying the molecular mechanisms underlying the 

NK cell recognition of tumor cells, and the defects of NK cell maturation and functional 

competence promoted by tumor microenvironment. To this end, a tumor type particularly 

suitable is the multiple myeloma (MM), a clonal B cell malignancy characterized by an excess 

of mature plasma cells mainly in the BM where NK cell maturation occurs. We have 

previously shown that treatment of MM cells with low doses of drugs largely used in the 

therapy of MM, such as doxorubicin, melphalan and bortezomib, up-regulate DNAM-1 and 

NKG2D ligands and is associated with increased NK cell degranulation. Drug-induced 

DNAM-1 and NKG2D ligand expression was abolished following treatment with the 

pharmacological inhibitors (caffeine and KU-55933) of the DNA Damage Response (DDR)- 

initiating kinases ATM/ATR, and was preferentially observed on senescent cells arrested in 

the G2 phase of the cell cycle (Soriani et al., Blood 2009). 

More recently, we have investigated the signaling pathways underlying NK cell 

recognition of drug-induced senescent MM cells with particular attention given to the role 

played by changes in the cellular redox state. We initially found that low doses of doxorubicin 

and melphalan can trigger the DNA damage response as indicated by chk1/2 checkpoint 

kinase activation, phosphorylation of histone H2AX and p53 serine 15 phosphorylation in MM 

cells. Thus, we tested the effect of pharmacological inhibitors targeting chk1/chk2 

(SB218078, UCN-01) and p53 (pifitrin-a) on the expression of NKG2D and DNAM-1 ligands. 

We found that MICA, MICB and PVR up-regulation was dependent on chk1/chk2 activation, 

whereas it was not affected upon treatment with pifitrin-a, thus excluding the involvement of 

p53 in this event. To evaluate the role of changes in the cellular redox state in ligand upregulation 

by doxorubicin and melphalan known to induce ROS generation, we performed 

experiments in the presence NAC (N-acetyl-L-cysteine), a potent scavenger of ROS in SKO- 

007(J3) MM cell line or in MM patients’ CD138 + malignant PCs. We observed that druginduced 

up-regulation of NKG2D and DNAM-1 ligand protein and mRNA expression was 

reduced in the presence of NAC. NAC-mediated inhibition of ligand up-regulation was 

associated with impaired ATM and chk1/chk2 activation and senescent cell associated-cell 

cycle G2/M arrest. 

We have also analyzed whether drug treatment of MM cell lines could also result in MICA 

and MICB release. Our preliminary findings show that basal levels of soluble MICB were 

strongly up-regulated after 24h-treatment and peaked at 72h, whereas soluble MICA was 

barely detected. In an effort to elucidate the molecular mechanisms underlying MICB 

117


shedding, we analyzed the ability of different inhibitors to affect MICB shedding on drugtreated 

cells. Our results indicate that the metalloproteinase inhibitor Marimastat dramatically 

decreased the shedding of MICB from drug-treated cells and this decrease was 

accompanied by a concomitant increase of cell surface MICB. Similar results were obtained 

using the dithiobisnitrobenzoic acid that inhibits protein disulfide isomerase activity. Finally, 

we demonstrated that cell surface MICB but not MICA is associated with the membrane raft 

compartment, further suggesting that different mechanisms control MICA and MICB shedding 

in MM cell lines in response to therapeutic agents. 

Overall, our findings show that low doses of chemotherapeutic drugs up-regulate NKG2D 

and DNAM-1 ligands on MM cells in a ATM/Chk1/2- dependent and p53-independent 

manner, and this event is triggered by ROS-dependent activation of DNA Damage 

Response. In addition, we suggest a model in which the senescence program promotes 

tumor cell recognition and elimination by NK cells. 

Publications 

La Regina G, Bai R, Rensen W, Coluccia A, Piscitelli F, Gatti V, Bolognesi A, Lavecchia A, 

Granata I, Porta A, Maresca B, Soriani A, Iannitto ML, Mariani M, Santoni A, Brancale A, 

Ferlini C, Dondio G, Varasi M, Mercurio C, Hamel E, Lavia P, Novellino E, Silvestri R. 

Design and Synthesis of 2-Heterocyclyl-3-arylthio-1H-indoles as Potent Tubulin 

Polymerization and Cell Growth Inhibitors with Improved Metabolic Stability. J Med Chem 

2011, 54: 8394-406. doi: 10.1021/jm2012886. 

Gasparrini F, Molfetta R, Santoni A, Paolini R. Cbl family proteins: balancing FcεRI-mediated 

mast cell and basophil activation. Int Arch Allergy Immunol 2011, 15: 16-26. doi: 

10.1159/000322236. 

Ardolino M, Zingoni A, Cerboni C, Cecere F, Soriani A, Iannitto ML, Santoni A. DNAM-1 

ligand expression on Ag-stimulated T lymphocytes is mediated by ROS-dependent 

activation of DNA-damage response: relevance for NK-T cell interaction. Blood 2011;117: 

4778-86. doi: 10.1182/blood-2010-08-300954 

Sciumè G, De Angelis G, Benigni G, Ponzetta A, Morrone S, Santoni A, Bernardini G. 

CX3CR1 expression defines 2 KLRG1+ mouse NK-cell subsets with distinct functional 

properties and positioning in the bone marrow. Blood 2011, 117: 4467-75. doi: 

10.1182/blood-2010-07-297101. 


Marco Cippitelli, Angela Gismondi, 

professors; Giovanni Bernardini, Cristina 

Cerboni, Cinzia Fionda, Alessandra Soriani, 

Helena Stabile, Alessandra Zingani, 

researchers; Maria Luisa Iannitto, PhD student. 

118


Dissection of Notch signaling-dependent pathways involved in the 

progression of T cell leukemia 

Isabella Screpanti 


℡: +39 06 44700816 - @: isabella.screpanti@uniroma1.it 

The role of Notch signaling in the development of T cell leukemia is well established, 

whereas its role in promoting and sustaining leukemia progression remains undefined. We 

previously showed that the triggering of NF-kB canonical pathway, sustained by the 

constitutive expression of preTCR, inducing in turn the activation of several anti-apoptotic 

and pro-proliferative signals (Bcl2-A1, IL2, Cyclin D1), appears to exert an essential role 

during the expansion phase of the disease. 

However, although it is widely known the role of Notch intracellular domain as a 

transcriptional activator, a lot remains to be clarified on the mechanisms regulating the 

switch-on/off of the signalling. This is particularly important with respect to the possibility to 

pharmacologically intervene on the constitutive activation of Notch signaling in T cell 

leukemia. We recently identified a specific novel property of Notch3, known to be 

overexpressed in T-ALL, that is acetylated and deacetylated by p300 and HDAC1, 

respectively. Notch3 acetylation primes ubiquitination and proteasomal-mediated degradation 

of the protein. As a consequence, its transcriptional activity is decreased, thus resulting in the 

impairment of downstream signaling as well as in vitro T cell proliferation and in vivo growth 

of Notch3-induced T cell leukemia in transgenic mice. 

Although the acetylatable lysine residues in the RAM domain are evolutionarily 

conserved among almost all the Notch proteins from various species, whether the 

mechanism, as well as the functional consequences, we described might be extended to 

other members of the Notch family and to different cell contexts remain to be further 

investigated. Indeed, the Sirt1 loss of function-induced Notch1 acetylation was shown to 

sustain its stabilization and to play a positive role in endothelial cells and in in vivo models of 

vascular maturation/differentiation, while in our study HDAC inhibition-induced Notch3 

acetylation, resulting in its degradation, is inhibitory in human and mouse T-ALL cell lines and 

in a in vivo mouse model of T-ALL. 

Overall this observations, together with ours, representing the first evidence highlighting 

the involvement of a reversible acetylation mechanism in Notch3 protein stability and 

function, suggest two possible scenarios: either the cell context determines the acetylationdependent 

output of the Notch signaling or the overall Notch signaling is mediated by the 

integration of the differential roles of Notch1 and Notch3 acetylation. 

We previously reported that, besides its oncogenic/leukemogenic role, Notch3, in 

cooperation with the pTalpha/preTCR pathway, positively regulates the generation and 

function of naturally occurring regulatory T cells, which have been suggested to facilitate 

119


tumor development by suppressing protective antitumor immune responses. More recently, 

we showed that Notch3 triggers the trans-activation of Foxp3 promoter depending on the T 

cell developmental stage. Moreover, we discovered a novel CSL/NF-kB overlaping binding 

site within the FoxP3 promoter and demonstrate that the activation of NF-kB, mainly 

represented by p65-dependent canonical pathway, plays a positive role in Notch3-dependent 

regulation of Foxp3 transcription. Accordingly, the deletion of PKCtheta, which mediates 

canonical NF-kB activation, markedly reduces regulatory T cell number and per cell Foxp3 

expression in transgenic mice with a constitutive activation of Notch3 signalling. 

Collectively, our data indicate that the cooperation among Notch3, PKCtheta and p65/NFkB 

subunit modulates Foxp3 expression, adding new insights in the understanding of the 

molecular mechanisms involved in regulatory T cell homeostasis and function. 

Publications 

Palermo R, Checquolo S, Giovenco A, Grazioli P, Kumar V, Campese AF, Giorgi A, 

Napolitano M, Canettieri G, Ferrara G, Schininà ME, Maroder M, Frati L, Gulino A, Vacca 

A, Screpanti I. Acetylation controls Notch3 stability and function in T-cell leukemia. 

Oncogene 2011 Epub. doi: 10.1038/onc.2011.533. 

Barbarulo A, Grazioli P, Campese AF, Bellavia D, Di Mario G, Pelullo M, Ciuffetta A, 

Colantoni S, Vacca A, Frati L, Gulino A, Felli MP, Screpanti I. Notch3 and canonical NFkappaB 

signaling pathways cooperatively regulate Foxp3 transcription. J Immunol 2011, 

186: 6199-206. doi: 10.4049/jimmunol.1002136. 

De Smaele E, Di Marcotullio L, Moretti M, Pelloni M, Occhione MA, Infante P, Cucchi D, 

Greco A, Pietrosanti L, Todorovic J, Coni S, Canettieri G, Ferretti E, Bei R, Maroder M, 

Screpanti I, Gulino A. Identification and characterization of KCASH2 and KCASH3, 2 novel 

Cullin3 adaptors suppressing histone deacetylase and Hedgehog activity in 

medulloblastoma. Neoplasia 2011, 13: 374-85. doi: 10.1593/neo.101630. 


Maria Pia Felli, professor; Diana Bellavia, 

Antonio F. Campese, researchers; Paola 

Grazioli, Gaia Scafetta, post-doc fellows; Maria 

Pelullo, Roberta Quaranta, PhD students. 


Guido Franzoso, Department of Immunology, 

Imperial College of London, UK; Antony J. 

Capobianco, Molecular Oncology Research 

Program, Division of Surgical Oncology, 

University of Miami, USA. 

120


Predisposing factors in autoimmune diseases: correlation between 

common genetic variations and function 

Rosa Sorrentino 


℡: +39 06 49917706 - @: rosa.sorrentino@uniroma1.it 

Many complex disorders share a common ground with the autoimmune diseases that is 

the dysregulation of the network controlling the inflammatory response. The main interest of 

our research group is the correlation between common genetic polymorphisms and their 

functional outcome in models of inflammatory diseases, with a particular emphasis on the 

role played by the HLA-B27 molecules in conferring susceptibility to Ankylosing Spondylitis 

(AS). 

In order to establish the effect of the polymorphisms in the HLA-B27 molecules on the 

antigen presentation properties, much work has been done by us and much is still ongoing. 

In particular, our research has been firstly focused on the functional differences between two 

HLA-B27 alleles differing for a single amino acid (D116H) in the antigen presenting groove 

but differentially associated with AS. In this context, a further association between AS and a 

gene mapping in the HLA-region has been pinpointed by us. This SNP, rs 1264457, is a 

common polymorphism in the HLA-E gene mapping close to the HLA-B locus. This prompted 

us to investigate in more details how the expression of the HLA-E molecules is modulated. 

These molecules, present on the surface of many cell types, are the only known ligands for 

NKG2A and NKG2C that are inhibitory and activating receptors, respectively, present on 

Natural Killer and cytotoxic T cells. The engagement of one or the other receptor induces 

opposite signals that halt or trigger the killing by these cells. We have firstly analyzed the 

expression of the HLA-E molecules in cells of the immune system such as B cells and 

monocytes. We have found that their expression is modulated along with cell differentiation. 

We are now exploring whether this might have an impact on the defense of differentiated 

cells from the killing by cytotoxic T cells. 

Meanwhile, along this line of research, we have taken part to a project carried out by 

many groups around the world showing that the reported association of a common 

polymorphism in the ERAP1, a gene involved in antigen presentation, with the AS, occurs 

only in the presence of HLA-B27 since patients who are HLA-B27 negative do not show this 

association (Evans et al., Nat Genet 2011). 

Monocytes and the plasticity of their effector functions plays a major role in tuning the 

inflammatory response. These aspects have become the focus of a leading interest in our 

laboratory as also shown by a recent publication (Paladini et al., Toxicol Sci 2011). We 

pointed our attention on the Vasoactive Intestinal Peptide and its receptors as modulators of 

the inflammatory response in monocytes. We have first analyzed the distribution of 

121


polymorphisms mapping in this gene and found, in agreement with other groups, an 

association with several diseases such as Rheumatoid Arthritis, Ankylosing Spondylitis, 

Idiopathic Achalasia and, finally, type II diabetes (Paladini et al., Gene Epub 2011). 

Moreover, we have identified a microRNA, 525-5p, as negative regulator of the expression of 

this receptor (Cocco et al., PLoS One 2010). Interestingly, this microRNA is upregulated by 

LPS and, in turn, down-regulates the expression of VPAC1, tuning down its 

counterinflammatory functions when monocytes are exposed to bacterial infections. Work is 

in progress to establish the effect of SNPs mapping in the VPAC1 3’UTR region and in the 

microRNA 525-5p on the expression of this receptor that plays a major role in the 

neuroinflammatory network and is considered an interesting therapeutic target in several 

complex diseases. 

Publications 

Paladini F, Adinolfi V, Cocco E, Ciociola E, Tamburrano G, Cascino I, Lucantoni F, Morano 

S, Sorrentino R. Gender-dependent association of type 2 diabetes with the vasoactive 

intestinal peptide receptor 1. Gene 2011 Epub. doi: 10.1016/j.bbr.2011.03.031. 

Paladini F, Cocco E, Potolicchio I, Fazekasova H, Lombardi G, Fiorillo MT, Sorrentino R 

Divergent effect of cobalt and Beryllium salts on the fate of peripheral blood monocytes and 

T lymphocytes. Toxicol Sci 2011, 119: 257-69. doi: 10.1093/toxsci/kfq328. 


Maria Teresa Fiorillo, researcher; Fabiana 

Paladini, post-doc fellow; Giorgio Camilli, 

Elisa Cocco, Sinem Tuncer, PhD student, 

Valentina Tedeschi, student. 


Isabella Cascino, CNR, Roma; Alessandro 

Mathieu, Università di Cagliari; Andreas 

Ziegler, Charité, Freie Universität, Berlin, 

Germany; Giovanna Lombardi, King’s 

College, London, UK; Rainer A. Bockmann, 

University of Erlangen-Nurnberg, Erlangen, 

Germany. 

122


CD28 co-stimulatory molecule as a key regulator of NF-κB 

signalling pathway: role of cytoskeleton in coupling CD28 to NF-κB 

activation 

Loretta Tuosto 

Department of Biology and Biotechnology "Charles Darwin" 

℡: +39 06 49917595 - @: loretta.tuosto@uniroma1.it 

CD28 is an important co-stimulatory receptor for T lymphocytes that following 

engagement by its natural ligand B7 may act as a TCR-independent signalling units and 

activate a non-canonical NF-κB2-like cascade and the selective transcription of both proinflammatory 

and survival genes. We recently found that the actin binding protein filamin A 

(FLNa), a large cytoplasmic protein that crosslinks cortical actin, constitutively binds NIK, a 

MAP-3 kinase involved in the activation of IKKα and NF-κB, and that both FLNa and NIK are 

recruited to the the C-terminal proline rich motif (Y 206 QP 208 YAPP) of CD28, an important 

domain also involved in the recruitment of Vav-1. 

Starting from these data the aim of the present project was to identify: 

1) The molecular basis of FLNa/NIK association and its role in the activation of IKKα and 

NF-κB 

2) The mechanisms and molecules involved in the recruitment of Vav-1 to the C-terminal 

Y 206 QP 208 YAPP motif and their role in CD28 signalling 

Role of FLNa/NIK association in the activation of IKKα and NF-κB in CD28 stimulated cells 

The activation of IKKα and the non-canonical NF-κB pathway requires NIK. We found 

that in both Jurkat T cell lines and primary T cells NIK associated with IKKα and CD28 

engagement by B7 significantly induced the kinase activity of NIK-bound IKKα. Mutation of 

proline residues in the C-terminal Y 206 QP 208 YAPP motif of CD28 (CD28-3A) failed to induce 

NIK-associated IKKα kinase activity. 

Altogether, these data evidenced that the C-terminal proline-rich motif of CD28 regulates 

the recruitment of both FLNa/NIK complexes, thus leading to NIK/IKKα activation and to the 

induction of the non-canonical NF-κB2 pathway. 

Identification of a novel interaction between Vav-1 and the type I phosphatidylinositol-4- 

phosphate 5-kinase (PIP5KI) 

In searching for specific molecules coupling CD28 to Vav-1, we identify PIP5KIα as a 

novel binding partner of Vav-1. PIP5KIα belongs to a family of phosphatidylinositol 

phosphate kinases, which phosphorylate phosphatidylinositol-4-phosphate in the D5 position, 

thus generating phosphatidylinositol 4,5-biphosphate (PIP2) a key lipid that serves as a 

substrate of PLC-γ and regulates both the Ca 2+ -calcineurin/NF-AT and PKCθ/NF- κB 

123


signalling cascades. Vav-1 constitutively associated with PIP5KIα in un-stimulated cells and 

also PIP5KIα was recruited to the C- 

terminal Y 206 QP 208 YAPP of CD28 

following stimulation with B7 (Fig. 1). We 

also found that CD28 stimulation by both 

B7 and anti-CD28 antibodies induces 

PIP5KIα kinase activity. 

Fig. 1 - Confocal Analysis of PIP5KIa and actin 

recruitment in Jurkat cells (JCH7C17) expressing 

CD28WT or CD28Y 191 F or CD28-3A mutants. 

The future prospectives will be aimed to characterize the nature of Vav-1/PIP5KIα 

interaction and identify the functional role of this association in the context of CD28-mediated 

regulation of both Ca 2+ -calcineurin/NF-AT and PKCθ/NF-κB signalling cascades. 

Publications 

Muscolini M, Montagni E, Palermo V, Di Agostino S, Gu W, Abdelmoula-Soussi S, Mazzoni 

C, Blandino G, Tuosto L. The cancer-associated K351N mutation affects the ubiquitination 

and the translocation to mitochondria of p53 protein. J Biol Chem 2011, 286: 39693-702. 

doi: 10.1074/jbc.M111.279539. 

Muscolini M, Sajeva A, Caristi S, Tuosto L. A novel association between Filamin A and NFkB 

Inducing Kinase couples CD28 to Inhibitor of NF-kB kinase a and NF-κB activation. 

Immunol Lett 2011,136: 203-12. doi:10.1016/j.imlet.2011.01.011. 

Tuosto L. NF-κB family of transcription factors: Biochemical players of CD28 co-stimulation. 

Immunol Lett 2011, 135: 1-9. doi:10.1016/j.imlet.2010.09.005. 


Cristina Camperio, research yellow; Michela 

Muscolini, post-doc fellow; Laura Muzi, PhD 

student; Petra Tomassini, graduate student; 

Silvana Caristi, technician. 


Balbino Alarcón, Centro de Biología Molecular 

Severo Ochoa, Universidad Autónoma de 

Madrid, Spain; Giovanni Blandino, Laboratorio 

di Oncogenomica Traslazionale, Roma; 

Ricciarda Galandrini, Dipartimento di 

Medicina Sperimentale, Sapienza Università di 

Roma. 

124


Anti-tumor pathways mediated by innate immune responses 

Elio Ziparo 


℡: +39 06 49766586 - @: elio.ziparo@uniroma1.it 

Toll-like receptors (TLRs), membrane-bound pattern recognition receptors (PRRs) that 

play a central role in responses to pathogens, have been recently implied in the control of 

tumor progression. 

Our previous data demonstrated that the synthetic TLR-3 agonist poly(I:C) induces 

apoptosis (Paone et al., 2008), accompanied by NF-κB activation and parallel induction of 

cytokines and chemokines (Galli et al., 2010), in the prostate cancer cell line LNCaP. 

Poly(I:C), by exerting both a direct pro-apoptotic effect on LNCaP cells and an immunemediated 

effect due to the recruitment of NK and cytotoxic CD8 cells, might therefore be a 

potentially valid therapeutic agent in prostate cancer. However, we observed that TLR3 

activation provokes anti-tumour signaling leading to apoptosis of prostate cancer cells 

LNCaP and PC3 with much more efficiency in the former than in the second more aggressive 

line (Paone et al., 2008). Since PC3 cells represent an androgen-independent cell line, it 

would be crucial to render it sensitive to cytotoxic agents. A family of cytosolic PRRs, such as 

RNA helicases (retinoic acid inducible gene protein 1, RIG-1 and melanoma differentiationassociated 

gene-5, MDA5) (Inohara et al., 2005) and the RNA-dependent protein kinase 

PKR, use the mitochondrial-bound MAVS as adapter (Kawai, 2005) and downstream activate 

the same protein kinases and transcription factors as TLR-3. It has been shown that the 

complex of poly(I:C) and cationic liposome (PIC-liposome) induces tumor growth inhibition 

(Fujimura, 2006) or apoptosis (Besch, 2009) and the anti-tumoral effect is mediated by RIG-1 

and MDA-5. 

An aim of this project is to increase the apoptotic rate of poly (I:C) in the more aggressive 

cell line PC3. To this purpose, we transfected poly(I:C) by means of lipofectamine reagent 

into PC3 cells to stimulate cytosolic PRR and then evaluated apoptosis rate and the 

cytoplasmic molecules involved. We found that the transfection of poly (I:C) at 2 µg/ml in PC3 

cells for 24 h induced a cell viability inhibition higher than 80%, (evaluated by MTT assay) 

respect to the untreated cells and a parallel increase of PC3 apoptosis up to 40% after 

poly(I:C) transfection compared to 10% of apoptosis after extracellularly delivered poly(I:C) 

(evaluated by PI staining). Moreover, cleaved form of caspase-3 was clearly upregulated and 

caspase-3 inhibitor completely reverted apoptosis caused by transfection of poly(I:C). To 

clarify the nature of apoptosis, we used specific inhibitors of caspase-9 and caspase-8, the 

mediators of intrinsic and extrinsic apoptosis pathways, respectively. Both inhibitors blocked 

PC3 apoptosis, suggesting the involvement of the two apoptotic pathways that now we are 

further investigating. 

125


Thus, we analysed the expression of cytosolic PRR by RT-PCR and Western blot. 

Although the constitutive expression of MDA-5 was relatively low in PC3 cells, it is 

significantly up-regulated when exposed to PIC-liposome, whereas RIG-1 was absent and 

the functional phosphorylated form of PKR was similarly expressed in PC3 cells treated with 

poly(I:C)/liposome or not. These data strongly suggest that MDA-5 could play a key role in 

anti-tumor effect of poly(I:C)/liposome in PC3 cells and we are performing siRNA-mediated 

ablation of MDA-5 to definitely determine its function. To test the involvement of TLR3, we 

are going to investigate this issue by the analysis of apoptosis and signalling molecules after 

transfection of poly(I:C) in a stable TLR-3 dominant negative PC3 cell line. 

Publications 

D'Alessio A, Esposito B, Giampietri C, Ziparo E, Pober JS, Filippini A. Plasma membrane 

micro domains regulate TACE-dependent TNFR1 shedding in human endothelial cells. J 

Cell Mol Med 2011 Epub. doi: 10.1111/j.1582-4934.2011.01353.x. 

Esposito B, Gambara G, Lewis AM, Palombi F, D'Alessio A, Taylor LX, Genazzani AA, 

Ziparo E, Galione A, Churchill GC, Filippini A. NAADP links histamine H1 receptors to 

secretion of von Willebrand factor in human endothelial cells. Blood 2011, 117: 4968-77. 

doi: 10.1182/blood-2010-02-266338. 


Antonio Filippini, professor; Anna Riccioli, 

researcher; Claudia Giampietri, post-doc 

fellow; Sara Palchetti, PdD student; Fabrizio 

Padula, Simonetta Petrungaro, Donatella 

Starace, technicians. 


Paola De Cesaris, Dipartimento di Medicina 

Sperimentale, Università de L’Aquila. 

126

Area 6 

New antimicrobial and 

antiviral agents

Area 6: New antimicrobial and antiviral agents 

Peptide effectors of innate immunity 

Donatella Barra 


℡: +39 06 4456663 - @: donatella.barra@uniroma1.it 

Peptides with diverse biological activities are particularly abundant in skin secretions of 

amphibians. Many of these compounds are pivotal components of the innate immune 

response, while others are related to mammalian hormones or neurotransmitters. 

We have focused the attention on the peptide components of amphibian skin secretions, 

discovering a number of molecules displaying antimicrobial activity, as well as small proteins 

with different properties, among which the most interesting is Bv8. Mammalian homologues 

of Bv8 have been described also in humans (prokineticin 1/EG-VEGF and prokineticin 

2/human Bv8), and found to display chemokine-like activities. These two classes of 

molecules can be considered as the effectors of the evolutionary ancient immune system, 

having overlapping functions: inactivate invading bacterial, fungal, or viral pathogens and 

recruit and activate leukocytes. 

Referring to the first issue, we have studied the mechanism of synergism between 

temporins, a property described in a previous publication (Mangoni et al., J Biol Chem 2008). 

Using NMR and fluorescence spectroscopy, the structures and interactions of temporins in 

LPS micelles were investigated providing the first structural insight into the mode of action 

and synergism of antimicrobial peptides at the level of the LPS-outer membrane. 

On temporin A, to obtain a peptide with a better therapeutic index, we used alanine 

scanning analogs to elucidate the contribution of the side chains of each amino acid residue 

to the peptide's antimicrobial and hemolytic activity, thus providing the basis for optimizing 

the design of temporin-based lead structures for the production of new anti-infective agents. 

On temporin L, which displays the strongest antimicrobial activity, but also a high toxicity 

on human erythrocytes, we studied the structure-activity relationships of a library of temporin 

L derivatives, focusing on the correlation between the α-helix content of the peptides, the 

nature of their cationic residues, and their antibacterial/antiyeast/hemolytic activities. 

Studies on bombinins, specifically the H2/H4 pair, revealed interesting relationships 

between the presence of a single D-amino acid in the sequence of an antimicrobial peptide 

and its target microbial cell selectivity/membrane-perturbing activity. 

Dr. Mangoni has been invited to act as guest Editor for an issue of Cellular and Molecular 

Life Science on “Host-defense peptides: from biology to therapeutic strategies”. 

Concerning Bv8/prokineticins, we have studied their selective interaction with the G 

protein-coupled receptors, PKR1 and PKR2. Defects in the signalling pathways lead to 

various pathologies, in particular the Kallmann syndrome and carcinogenesis. We have 

demonstrated the dimerization of the PK2 receptor through in vivo experiments on a modified 

yeast cells strain that does not express PK receptors. 

129


Publications 

Bhunia A, Saravanan R, Mohanram H, Mangoni ML, Bhattacharjya S. NMR structures and 

interactions of temporin-1Tl and temporin-1Tb with lipopolysaccharide micelles: 

mechanistic insights into outer membrane permeabilization and synergistic activity. J Biol 

Chem 2011, 286: 24394-406. doi: 10.1074/jbc.M110.189662. 

Coccia C, Rinaldi AC, Luca V, Barra D, Bozzi A, Di Giulio A, Veerman EC, Mangoni ML. 

Membrane interaction and antibacterial properties of two mildly cationic peptide 

diastereomers, bombinins H2 and H4, isolated from Bombina skin. Eur Biophys J 2011, 40: 

577-88. doi: 10.1007/s00249-011-0681-8. 

Grieco P, Luca V, Auriemma L, Carotenuto A, Saviello MR, Campiglia P, Barra D, Novellino 

E, Mangoni ML. Alanine scanning analysis and structure-function relationships of the frogskin 

antimicrobial peptide temporin-1Ta. J Pept Sci 2011, 17: 358-65. doi: 10.1002/ 

psc.1350. 

Mangoni ML, Carotenuto A, Auriemma L, Saviello MR, Campiglia P, Gomez-Monterrey I, 

Malfi S, Marcellini L, Barra D, Novellino E, Grieco P. Structure-activity relationship, 

conformational and biological studies of temporin L analogues. J Med Chem 2011, 54: 

1298-307. doi: 10.1021/jm1012853. 

Mangoni ML, Shai Y. Short native antimicrobial peptides and engineered ultrashort 

lipopeptides: similarities and differences in cell specificities and modes of action. Cell Mol 

Life Sci 2011, 68: 2267-80. doi: 10.1007/s00018-011-0718-2. 

Marsango S, Bonaccorsi di Patti MC, Barra D, Miele R. Evidence that prokineticin receptor 2 

exists as a dimer in vivo. Cell Mol Life Sci 2011, 68: 2919-29. doi: 10.1007/s00018-010- 

0601-6, doi: 10.1007/s00018-010-0601-6. 


Maurizio Simmaco, Giuseppina Mignogna, 

professors; M. Luisa Mangoni, Rossella Miele, 

Alessandro Paiardini, researchers; Sara 

Marsango, Vincenzo Luca, PhD students; 

Alessandra Franco, Alessandra Giorgi, 

technicians. 


Günther Kreil, Institute of Molecular Biology, 

Austrian Academy od Sciences, Salzburg, 

Austria; Yechiel Shai, Department of Biological 

Chemistry, Weizmann Institute, Rehovot, 

Israele; Lorenzo Stella, Dipartimento di 

Scienze e Tecnologie Chimiche, Università di 

Roma Tor Vergata; Lucia Negri, Dipartimento 

di Fisiologia e Farmacologia “V. Erspamer”, 

Sapienza Università di Roma; Paolo Grieco, 

Dipartimento di Chimica Farmaceutica e 

Tossicologica, Università di Napoli “Federico II”. 

130


New pyrrole derivatives of BM 212: a new class of antimycobacterial 

agents. Design, synthesis, biological evaluation and study of their 

mode of action 

Mariangela Biava 

Department of Medicinal Chemistry and Technologies 

℡: +39 06 49913812 - @: mariangela.biava@uniroma1.it 

In 2010, the World Health Organization estimated that one-third of the global population 

has been infected with M. tuberculosis (MTB) 1 , thus Tuberculosis still represents a major 

challenge toward which research efforts are needed. 

We identified a new chemical class of 1,5-diphenyl pyrrole derivatives endowed with potent 

antimycobacterial activity whose hit compounds are BM212 (MIC=0.7µg/mL; CC 50 =4µg/mL; 

PI(CC 50 /MIC)=5.6) and BM521 (MIC=0.25µg/mL; CC 50 =64µg/mL; PI=256µg/mL). This class of 

small molecules exhibited a very good biological profile, better than that shown by streptomycin 

and rifampin 2 . 

This project was devoted to the synthesis and characterization of new derivatives, 

starting from the chemical structures of BM212 and BM521, in the attempt to further improve 

both their biological and ADME profile. The syntheses of three chemical classes of BM 

derivatives were planned, following both literature and molecular modelling suggestions. 

Noteworthy, these 1,5-diphenyl pyrrole analogues were synthesized employing a very easy 

synthetic pathway comprised of three steps, that were optimized by us, allowing us to obtain 

them quickly and in good yields. 

Compounds synthesized till now and belonging to the first and second classes were 

tested as previously done for BM 212 and BM 521 and many of them proved to be very 

active against MTB. 

In particular, BM579, exhibited a very good activity with a MIC= 0.125µg/mL, CC 50 = 

>128µg/mL, PI= >1000, higher than that of BM212, BM521 and rifampin, and comparable to 

that of Isoniazid. 

N NCH 3 

N 

S 

N 

S 

N 

N 

N 

Cl 

H 3 C 

H 3 CS 

Cl 

BM212 BM 521 

F 

F 

BM 579 

The last year of the project has been focused on the synthesis of the third class of 

compounds, designed according to the results previously obtained. Among them, several 

derivatives proved to be very active. Data-sets are, however, still incomplete. In particular 

131


BM708 (MIC=0.016µg/mL, CC 50 =18.89µg/mL, PI=1180.62) and BM720 (MIC=0.125µg/mL, 

CC 50 = 179.26µg/mL, PI=1434.08) proved to be the best ones. 

Pursuing the hit-to-lead optimization process, the best compounds of the three classes 

were further evaluated for better defining their microbiological profile as well as their ADME 

profile. In particular, the LORA (low-oxygen recovery assay) test, running in oxygen deprived 

conditions simulating the not-replicating persistent –TB phenotype environment (NRP-TB), 

has been used for evaluating the activity of the selected compounds towards persistent 

strains, that represent one of the most important problem with respect to current therapies. 

All selected derivatives showed good activity with a MIC ranging from 5.7 µg/ml to 7.7 µg/ml 3 . 

They also showed both good microsomal stability and good activity against intramacrophagic 

mycobacteria 4 . Pharmacokinetic studies for the best derivatives have been planned, and they 

are still in progress. Finally, during this last year of project the molecular target for BM212 

has been identified 5 , and different approaches for shedding light on the direct interaction of 

BM212 with the molecular receptor as well as for elucidating the mechanism of action have 

been planned. 

In conclusion, we have currently reached all our preset goals on our way toward the 

development of a promising class of antimycobacterial compounds. 

References 

1) WHO Report 2011. 

2) Biava M. et al., J Med Chem. 2008, 51: 3644-3648,. 

3) Biava M. et al., manuscript in preparation. 

4) a) Biava M. et al., Eur. J. Med. Chem. 2009, 44: 4734–4738; b) Biava M. et al. Bioorg. Med. 

Chem. 2010, 18: 8076–8084; c) Biava M. et al. ChemMedChem 2011, 6: 593-599. 

5) La Rosa V. et al., Antimicrob. Agents Chemother. 2012, 56: 324–331. 

Publications 

Biava M, Porretta GC, Poce G, Battilocchio C, Alfonso S, de Logu A, Manetti F, Botta M. 

Developing pyrrole-derived antimycobacterial agents: a rational lead optimization 

approach. ChemMedChem 2011, 6: 593-9. doi: 10.1002/cmdc.201000526. 


Giulio Cesare Porretta, professor, Giovanna 

Poce, post-doc fellow; Claudio Battilocchio, 

Salvatore Alfonso, PhD students, Roberto 

Torri, Technician. 


Raffaello Pompei, Alessandro De Logu, Dip. 

di Scienze e Tecnologie Biomediche, Università 

di Cagliari; Maurizio Botta, Fabrizio Manetti, 

Dip. Farmaco Chimico Tecnologico, Università 

di Siena; Edda De Rossi, Dip. di Genetica e 

Microbiologia, Università di Pavia; Scott G. 

Franzblau, Institute for Tuberculosis Research, 

College of Pharmacy, University of Illinois at 

Chicago, USA; Eric Rubin, Dep. of 

Immunology and Infectious Diseases, Harvard 

School of Public Health, Boston, USA. 

132


New azole derivatives as antiprotozoal agents 

Roberto Di Santo 

Department of Medicinal Chemistry and Technologies 

℡: +39 06 49913150 - @: roberto.disanto@uniroma1.it 

Chronic infection with Trypanosoma cruzi is a major cause of morbidity and mortality in 

Latin America with as many as 200000 new cases of infection (Chagas disease) occurring 

per year. Parasites of the genus Leishmania give rise to a number of different clinical 

manifestations. The most severe is visceral leishmaniasis (VL), which is almost always fatal 

unless treated. Estimated numbers of new VL cases are 500,000 per annum, with 

approximately 50,000 deaths each year, although VL is often not recognized or reported. 

Limitations of current drugs useful against Chagas and VL include significant toxicity, high 

cost and long treatment courses as well as a lack of efficacy due to the insorgence of drug 

resistance. Moreover, no vaccines are available against the above pathogens, up to day. 

A series of compounds that were determined to be highly potent inhibitors of the T. cruzi 

lanosterol 14α-demethylase (L14DM) enzyme are in preclinical development for Chagas 

disease. The same target is also present in Leishmania donovani and has potential to be 

exploited for treatment of VLs. 

Fig. 1 - Hit compound found as antiprotozoal agent. 

A few years ago we started studies on new antifungal agents characterized by imidazole 

and pyrrole moieties, exemplified by the structure of RDS 2394 (Fig. 1), that displayed EC 50 s 

5-10 nM against C. albicans and Candida spp. The target of these azole antifungal agents is 

the L14DM of fungi. Thus, taking account of the presence of L14DMs in protozoa and our 

availability of an in house library of azole antifungal compounds, we decided to test RDS 

2394 and a few derivatives against a panel of protozoa. RDS 2394 resulted the best hit and 

have been proven to bind L14DM of T. cruzi by monitoring the spectral shift of the heme-iron 

Soret band. 

Main results. During the first year of the project about the research on new azole 

derivatives as anti-protozoal agents, we have designed and synthesized a series of RDS 

2394 analogues. The first round of compounds was designed based on information coming 

form the binding mode of RDS 2394 with T. cruzi L14DM, that was found by the means of 

133


molecular modelling. As a parallel approach we decided to try to validate the binding mode of 

the RDS 2394, by the means of the design of further azole derivative characterized by the 

lack of one or more features depicted by the above binding mode. In general, the newly 

synthesized compounds were good antiprotozoal agents. The potency of these compounds 

is high against T. cruzi, good against P. falciparum, medium against L. donovani, low against 

T. brucei. A number of compounds were more potent than the reference compounds, and a 

few derivatives were active at nanomolar concentrations. 

Nome 

T. b. rhod. T. cruzi L. don. axen. P. falciparum 

IC 50 (µg/mL) IC 50 (µg/mL) IC 50 (µg/mL) IC 50 (µg/mL) 

RC 184 5.56 5.59 5.03 2.12 

RC 190 13.9 0.068 6.65 0.046 

RC 195 15.3 0.090 6.77 0.026 

RC 201 4.03 0.013 1.35 0.133 

RC 230 5.98 0.007 2.46 0.131 

RC 247 6.89 0.020 2.02 0.136 

RDS 416 6.98 0.014 1.52 0.064 

RDS 2037 14.9 0.002 2.15 0.072 

RDS 2060 34.8 0.153 3.72 0.076 

RDS 2087 13.6 0.027 0.89 0.035 

RDS 2056 2.72 0.001 1.11 0.289 

RDS 2128 15.8 0.001 0.88 0.258 

RDS 2137 15.3 0.001 2.18 0.539 

RDS 2394 8.92 0.026 0.20 0.119 

Melarsoprol 0.03 

Benznidazole 0.435 

Miltefosine 0.149 

Chloroquine 0.105 

Table 1 - In vitro potency of the newly synthesized azoles of RDS and RC series 

against T. cruzi Tulahuen C4 amastigotes, L. donovani MHOM-ET-67/L82 

amastigotes, as well as other protozoa like T. brucei rhodiesiense STIB 900 

trypomastygotes and Plasmodium falciparum K1 IEF, Reference compounds are 

melarsoprol, benznidazole, miltefosine and RDS 2394. 

Perspective. The activity showed by these new azole derivatives led us to hypothesize 

that further targets in addition to L14DM could be involved in inhibition of the growth of 

protozoa. Efforts will be devoted to determine if farnesyl transferase enzyme is involved as a 

further target. Thus the main perspective of our work will be the development of structureactivity 

relationships in these series of compounds to better elucidate information useful to 

define the mechanism of action of these azoles as well as to further improve the potency 

against protozoa of this compounds. 


Roberta Costi, professor; Giuliana Cuzzucoli 

Crucitti, Luca Pescatori, Gaetano Miele, 

Alberto Iacovo, post-doc fellows; Federica 

Rosi, PhD student. 


Luciana Marinelli, Ettore Novellino, 

Università di Napoli “Federico II”; Michael 

Gelb, Christophe Verlinde, Washington 

University, Seattle, USA. 

134


Drug design and synthesis of non-nucleoside inhibitors of both 

HIV-1 wild type and resistant mutant strains reverse transcriptase, 

and Coxsackie B4 virus 

Romano Silvestri 

Department of Drug Chemistry and Technologies 

℡: +39 06 49913800 - @: romano.silvestri@uniroma1.it 

Acquired immunodeficiency syndrome (AIDS) causes over 5700 deaths every year, and 

at the same time over 6800 people become infected with human immunodeficiency virus 

(HIV). We obtained new potent non-nucleoside inhibitors of the HIV-1 reverse transcriptase 

(NNRTIs) having the indolylarylsulfone (IAS) scaffold by coupling the indolo-2-carboxamide 

functionality with natural and unnatural aminoacids and introducing different electronwithdrawing 

substituents at position 4 and 5 of the indole nucleus. The new IASs inhibited the 

HIV-1 replication in human T-lymphocyte (CEM) cells at low/subnanomolar concentration and 

were weakly cytostatic. Against the mutant L100I, K103N, and Y181C RT HIV-1 strains in 

CEM cells, these sulfones were comparable to EFV. Most importantly, these new IASs were 

inhibitors to Coxsackie B4 virus at low micromolar concentration, that is 25 times lower than 

required for ribavirin. The compounds were evaluated against Coxsackie B4 virus in Vero cell 

cultures. The antiviral potency was in general less pronounced in Vero than in HeLa cell 

cultures. Five compounds showed anti-Coxsackie B4 virus activity in both HeLa and Vero cell 

cultures, and for two derivatives the inhibitory concentrations against respiratory syncytial 

virus were ~4 µM. The activity against Coxsackie B4 virus seemed to be positively affected 

by the presence of the bromine atom at position 5 of the indole and an amino acid unit 

bearing a methyl group. The antivirally active compounds showed selectivity index against 

Coxsackie B4 that was several orders of magnitude lower than the selectivity index for HIV. 

We questioned whether the observed antiviral activity is due to a direct anti-Coxsackie virus 

effect or caused by the cytotoxic activity of the compounds. Several substituted 

benzimidazole-based structures have been identified in the past to be endowed with anti- 

Coxsackie virus activity in cell culture. One of these compounds (TBZE-029) was, like the 

present structures, initially discovered as an NNRTI active against HIV- 1. TBZE-029 is active 

at an EC 50 value of 1.2 µg/mL, a concentration close to the EC 50 of the most inhibitory IAS 

derivatives. The TBZE-029 compound affects viral RNA synthesis by targeting the 

nonstructural protein 2C. The most active indole derivatives are under investigation for their 

ability to target virus-specific protein to reveal a potential viral target for this novel class of 

compounds. Therefore, we believe that this study allowed disclosure of a new lead class of 

Coxsackie B4 virus and respiratory syncytial virus inhibitors. Coxsackie B virus may be the 

causative agent of viral polymyositis and rhabdomyolysis, a pathological condition that may 

be associated with immunodepressed conditions.Such agents may provide an useful starting 

point for the development of new effective inhibitors of both HIV-1 RT and Coxsackie B4 

virus. 

135


Recently, a new concept of binding interaction with the HIV-1 drug-resistant mutant 

strains emerged: the “flexibility hypothesis”. According to this idea, a powerful inhibition of the 

mutant variants of the HIV-1 should be correlated to the ability of the molecule to adopt 

variable binding conformations which would be not affected by the interchanges of the amino 

acids residues into the NNBS of the mutant RT. Having in mind the newer “horseshoe-like” 

model, we synthesized new IAS derivatives bearing cyclic substituents at the indole-2- 

carboxamide linked through a methylene ethylene spacer. The new IASs were potent 

inhibitors of HIV-1 WT replication in CEM cells and showed inhibitory concentrations in the 

low nanomolar range. Against the mutant L100I and K103N RT HIV-1 strains, these 

compounds showed antiviral potency superior to that of NVP and EFV and were equipotent 

to ETV. The compounds proved to inhibit effectively different HIV clades in PBMC with EC 50 

values in the higher picomolar or lower nanomolar range of concentrations. These IASs may 

be useful in EFV-based HIV-1 therapies that show the emergence of the L100I and K103N 

mutations. 

Publications 

La Regina G, Gatti V, Piscitelli F, Silvestri R. Open vessel and cooling while heating 

microwave-assisted synthesis of pyridinyl N-aryl hydrazones. ACS Comb Sci 2011, 13: 2-6. 

doi: 10.1021/co100015b. 

La Regina G, Coluccia A, Brancale A, Piscitelli F, Gatti V, Maga G, Samuele A, 

Pannecouque C, Schols D, Balzarini J, Novellino E, Silvestri R. Indolylarylsulfones as HIV-1 

non-nucleoside reverse transcriptase inhibitors. New cyclic substituents at the indole-2- 

carboxamide. J Med Chem 2011, 54, 1587-1598. doi: 10.1021/jm101614j. 

Samuele A, Bisi S, Kataropoulou A, La Regina G, Piscitelli F, Gatti V, Silvestri R, Maga G. 

Mechanism of interaction of novel indolyl arylsulfone derivatives with K103N and Y181I 

mutant HIV-1 reverse transcriptase in complex with its substrates. Antiviral Chem Chemoth 

2011, 22, 107-118, doi: 10.3851/IMP1855. 


Giuseppe La Regina, researcher; Cesare 

Giordano, CNR researcher; Antonio Coluccia, 

post-doc fellow; Valerio Gatti, PhD student. 


Giovanni Maga, CNR, Istituto di Genetica 

Molecolare, Pavia; Jan Balzarini, Rega 

Institute for Medical Research, K.U. Leuven, 

Belgium; Andrea Brancale, Welsh School of 

Pharmacy, Cardiff University, UK; José A. 

Esté, IrsiCaixa, Hospital Universitari Germans 

Trias i Pujol, Universitat Autonoma de 

Barcelona, Badalona, Spain. 

136

Area 7 

Biology of malaria and other 

vector-borne diseases

Area 7: Biology of malaria and other vector-borne diseases 

Genetic and phenotypic characterization of species and “molecular 

forms” of the Anopheles gambiae complex (Diptera: Culicidae), 

afrotropical malaria vectors 

della Torre Alessandra - Vincenzo Petrarca 

Dept of Public Health and Infectious Diseases - Dept of Biology and Biotechnologies “C. Darwin” 

℡: +39 06 49694268 - +39 06 49914932 

@: ale.dellatorre@uniroma1.it - vincenzo.petrarca@uniroma1.it 

In 2011, we focused on 2 major lines of research, as follows: 

1. Analysis of the genetic and ecological differentiation of Anopheles gambiae M and S 

molecular forms in areas of high hybridization. M and S populations from the western edge 

of the species, where hybridization rates have been show to be much higher than in eastern 

regions (>20%), offer the opportunity to clarify several aspects of the speciation process 

ongoing within the major Afrotropical malaria vector species. We applied recently developed 

diagnostic assays for detection of M- and S-specific pericentromeric SNPs to samples 

collected in The Gambia and in Guinea Bissau, and revealed a level of admixture not 

observed in the rest of the range (Caputo et al., 2011). In particular, we found: i) 

heterozygous genotypes at each marker, although at frequencies lower than expected under 

panmixia; ii) virtually all possible genotypic combinations between markers on different 

chromosomes, although genetic association was nevertheless detected; iii) discordant M and 

S genotypes at two X-linked markers close to the centromere, suggestive of introgression 

and inter-locus recombination. We hypothesized that this geographic area may represent a 

secondary contact zone between M and S. 

2. Analysis of differentiation of genes involved in post-mating behaviour in the An. 

gambiae complex. Proteins involved in reproduction evolve rapidly due to positive selection 

resulting from intersexual interaction and sexual conflict. In Drosophila, fast evolution driven 

by positive selection has been detected in proteins expressed both in the male accessory 

glands (MAGs) and in the female lower reproductive tract (LRT). In A. gambiae, MAGproducts 

are transferred to females as a solid mating plug that induces a series of 

physiological post-mating responses in females. We have analyzed the molecular evolution 

of two clusters of 3 MAG- and 3 LRT- specific genes potentially involved in post-mating 

mechanisms in five members of the A. gambiae complex. Two MAG-specific paralog genes 

were shown to be highly conserved among species, although one replacement fixed along 

the whole geographical distribution of A. arabiensis was found. Moreover, we identified at 

third gene clustering with the former two, which is not annotated in the A. gambiae genome, 

and showed a high homology with one of the previous genes. Intriguingly, this gene was 

found to be highly differentiated among A. melas, A. merus and A. quadriannulatus, due to 

positive selective pressure and purifying selection maintaining lineage-specific products 

(Mancini et al., 2011a).The three LRT-specific genes analyzed encode serine-proteases that 

are down-regulated after mating, two of which are expressed in the atrium and interact with 

the mating plug and one in the spermatheca. The analysis of polymorphisms revealed a high 

139


level of replacement polymorphisms consistent with relaxed evolutionary constraints of 

duplicated genes, allowing to rapidly fix novel replacements to perform new or more specific 

functions. Adaptive evolution was detected in several codons of the 3 genes and evidences 

of episodic selection were also found. In addition, the structural modelling of these proteases 

highlighted some important differences in their substrate specificity, and provided evidence 

that a number of sites evolving under selective pressures lie relatively close to the catalytic 

triad and/or on the edge of the specificity pocket, known to be involved in substrate 

recognition or binding. The observed patterns suggest that these proteases may interact with 

factors transferred by males during mating and that differently evolved in independent A. 

gambiae lineages (Mancini et al., 2011b). 

Publications 

Caputo B, Santolamazza F, Vicente JL, Nwakanma DC, Jawara M, Palsson K, Jaenson T, 

White BJ, Mancini E, Petrarca V, Conway DJ, Besansky NJ, Pinto J, della Torre A. The 

“Far-West” of Anopheles gambiae molecular forms. PLoS One 2011, 6:e16415. doi: 

10.1371/journal. pone.0016415. 

Mancini E, Baldini F, Tammaro F, Calzetta M, Serrao A, George P, Morlais I, Masiga D, 

Sharakhov IV, Rogers DW, Catteruccia F, della Torre A. Molecular characterization and 

evolution of a gene family encoding for male-specific reproductive proteins in the African 

malaria vector Anopheles gambiae. BMC Evol Biol 2011a, 11: 292. doi: 10.1186/1471- 

2148-11-292. 

Mancini E, Tammaro F, Baldini F, Via A, Raimondo D, George P, Audisio P, Sharakhov IV, 

Tramontano A, Catteruccia F, della Torre A. Molecular evolution of a gene cluster of serine 

proteases expressed in the Anopheles gambiae female reproductive tract. BMC Evol Biol 

2011b, 11: 72. doi: 10.1186/1471-2148-11-72. 


Marco Pombi, researcher, Beniamino Caputo, 

Emiliano Mancini, Federica Santolamazza, 

post-doc fellows, Maria Calzetta, research fellow, 

Federica Tammaro, PhD student. 


Nora J Besansky, University of Notre Dame, 

USA; Flaminia Catteruccia, Imperial College, 

London, UK; David Conway, MRC, Banjul, The 

Gambia; Elena Levashina, CNRS, Strasburg, 

France; Paolo Pelosi, Università di Pisa; Joao 

Pinto, Universidad de Nova Lisboa, Portugal; 

Hilary Ranson, Liverpool School of Tropical 

Medicine, UK; N’Fale Sagnon, CNRFP, 

Ouagadougou, Burkina Faso; Igor Sharakhov, 

Virginia Tech, Blacksburg, USA; Fred Simard, 

IRD, Montpellier, France; Anna Tramontano, 

Sapienza Università di Roma. 

140


Immune responses to malaria and autoimmune disorders: 

investigating common gene-regulatory networks 

David Modiano 

Department of Public Health and Infectious Diseases 

℡: +39 06 49914933 - @: david.modiano@uniroma1.it 

Background 

The Fulani mount stronger immune responses to P. falciparum and are less susceptible 

to malaria than sympatric populations (Modiano et al., PNAS 1996). The Fulani also show 

higher responses to other pathogens, and both their Th1 and Th2 responses are enhanced, 

suggesting that their resistance to malaria could result from a generally stronger immune 

activation. Indeed, key genes related to T reg cell function are down-regulated in the Fulani 

(Torcia et al., PNAS 2008). This disorder of immune homeostasis could be driven by genetic 

factors positively selected by P. falciparum and may underlie the higher susceptibility of the 

Fulani to diseases with autoimmune pathogenesis. To investigate this hypothesis, we are 

conducting a large-scale epidemiological and immunogenetic study supported by the Malaria 

Genomic Epidemiology Network (MalariaGEN, Nature 2008). 

Genotype data 

We genotyped 363 SNPs on 2186 DNA samples from Fulani, Mossi and Rimaibé using 

the Sequenom MassArray System. SNPs included polymorphisms involved in susceptibility 

and immune response to malaria, as well as SNPs at autoimmunity loci. 

Population genetics analysis 

Principal component analysis revealed that Mossi and Rimaibé are not genetically distinct 

among themselves, whereas the Fulani are a distinct group, in agreement with data on HLA 

class I and class II alleles (Modiano et al., Tissue Antigens 2001; Lulli et al., Hum Immunol 

2009) 

Genetic association analysis 

We conducted association analysis with P. falciparum infection as phenotype using 

logistic regression adjusted for age, gender and village, both within each ethnic group and in 

the all population stratifying by ethnicity. When conducting the analysis in Fulani and Non- 

Fulani separately the first observation was that in both populations the strongest signals are 

shown by the 5q31 region, which has been linked to P. falciparum blood infection levels 

(Rihet et al., AJHG 1998) and actually lie in the IRF1 gene, encoding Interferon Regulatory 

Factor-1. These results confirm previous evidence that IRF1 polymorphisms are associated 

with the control of P. falciparum infection (Mangano et al., Genes Immun 2008). Among 

genes involved in resistance to severe malaria, we noticed association with the ABO locus, 

141


encoding for the blood group system ABO. Children with blood group O have been reported 

to have a lower risk of severe malaria (Fry et al., Hum Mol Genet 2008). Also the DDC locus 

shows signals of association, among the Non-Fulani only. The DDC locus encodes for dopa 

decarboxylase, which is involved in dopamine and serotonin synthesis and has been recently 

associated with severe malaria in a GWAS in The Gambia (Jallow et al., Nat Genet 2009) as 

well as linked to malaria refractoriness in mosquitoes (Romans et al., Am J Trop Med 1999). 

Among genes related to T regulatory cell function and/or involved in autoimmunity the most 

interesting association signals were shown by IL10, CD25 and TGFBR3 SNPs. IL10 

polymorphisms have been associated with SLE and IBD (Franke et al., Nat Genet 2008; 

Nath et al., Hum Genet 2005) while CD25 polymorphisms have been associated with AIT, 

MS, T1D (Xavier et al., Nat Rev Immunol 2008). On the other hand, it has been shown that 

the expression of TGFBR3 is lower in T regulatory cells from Fulani than Non-Fulani (Torcia 

et al., PNAS 2008). Most of the protective factors against P. falciparum infection that we 

identified have a higher frequency in the Fulani, similar to that of Europeans, who have a 

higher prevalance of autoimmune diseases. 

Publications 

Paganotti GM, Gallo BC, Verra F, Sirima BS, Nebié I, Diarra A, Coluzzi M, Modiano D. 

Human genetic variation is associated with Plasmodium falciparum drug resistance. J 

Infect Dis 2011, 20411: 1772-8. doi: 10.1093/infdis/jir629. 

Rizzo C, Ronca R, Fiorentino G, Mangano VD, Sirima SB, Nèbiè I, Petrarca V, Modiano D, 

Arcà B. Wide cross-reactivity between Anopheles gambiae and Anopheles funestus SG6 

salivary proteins supports exploitation of gSG6 as a marker of human exposure to major 

malaria vectors in tropical Africa. Malar J 2011, 10: 206. doi: 10.1186/1475-2875-10-206. 

Rizzo C, Ronca R, Fiorentino G, Verra F, Mangano V, Poinsignon A, Sirima SB, Nèbiè I, 

Lombardo F, Remoue F, Coluzzi M, Petrarca V, Modiano D, Arcà B. Humoral response to 

the Anopheles gambiae salivary protein gSG6: a serological indicator of exposure to 

Afrotropical malaria vectors. PLoS One 2011, 6:e17980. doi: 10.1371/journal.pone. 

0017980. 


Pamela Avellino, Valentina Mangano, Cinzia 

Rizzo, post-doc fellows. 


Dominic Kwiatkowski, Wellcome Trust Center 

for Human Genetics, Oxford, UK; Eleanor 

Riley, London School of Tropical Medicine and 

Hygiene, London, UK; Bienvenu Sirima, 

Centre National de Recherche et Formation sur 

le Paludisme, Ouagadougou, Burkina Faso. 

142

Start up program 

Specification and maintenance of 

retinal stem cells

Start-up Program 

Specification and maintenance of retinal stem cells 

Giuseppe Lupo 

Department of Biology and Biotechnology "Charles Darwin" 

℡: +39 06 49912206 - @: giuseppe.lupo@uniroma1.it 

Our work is focused on the molecular mechanisms controlling specification and 

maintenance of regional identity in progenitor/stem cells of the mammalian central nervous 

system (CNS), with particular interest in retinal and forebrain cell fates. We are using 

embryonic stem cells (ESCs) to model the extrinsic signalling systems directing the early 

patterning of the developing CNS, and neural stem cells (NSCs) isolated from specific 

regions of the foetal CNS to model the intrinsic regulatory systems that maintain positional 

identities of neural progenitors following their initial specification. 

While several studies have described the production of various neural cell types from 

ESCs, there have been few systematic studies of how different regional identities are 

established. Using chemically-defined culture conditions, we have found that ESCs can be 

efficiently converted into neuroectoderm by Nodal pathway inhibition along with either Bone 

Morphogenetic Protein (BMP) inhibition or treatment with Fibroblast Growth Factor (FGF). 

However, in the presence of Nodal inhibition, BMP inhibition allows specification towards 

anterior neural fates (forebrain/midbrain), while FGF promotes predominantly posterior fates 

(hindbrain/spinal cord). Upregulation of endogenous Wnt signalling during differentiation also 

contributes to neuroectoderm posteriorization. Based on these results, we have obtained 

efficient forebrain specification by inhibition of Nodal, BMP and Wnt pathways and 

minimization FGF signalling. Notably, these condition are not permissive for the specification 

of retinal progenitor fates. We are currently addressing the signals needed to stir early 

forebrain progenitors to retinal fates and our available evidences point to a requirement for 

functional Nodal and BMP pathways. 

We have also found that NSCs derived from different foetal CNS regions maintain in vitro 

transcriptional profiles consistent with their region of derivation. For example, NSCs derived 

from the foetal cortex and striatum retain expression of forebrain, but not spinal cord 

markers, while the opposite pattern is detectable in spinal cord-derived NSCs. To address 

whether these regional identities are irreversibly specified or can be influenced by molecular 

cues involved in CNS patterning, region-specific NSCs were treated with Retinoic Acid (RA) 

or FGF. We have found that both pathways were functional and elicited transcriptional 

responses in different NSC lines. Remarkably, however, each NSC line showed a unique 

transcriptional output coherent with its positional character. These results indicate that the 

regional identity of NSCs is specified at an early phase of neural development through 

mechanisms that can be maintained in vitro even after exposure to exogenous patterning 

signals. Experiments are underway in order to elucidate the intrinsic mechanisms controlling 

the differential response of region-specific NSCs to RA and FGF. 

145

Start-up Program 

In collaboration with Prof. Ohta at Kumamoto University, Japan, we have also 

investigated the signals supporting proliferation of RSCs located at the periphery of the 

mouse retina. Our work has shown a crucial role for the secreted molecule Tsukuchi (TSK), 

belonging to the small leucine-rich proteoglycan (SLRP) family, in regulating RSC 

proliferation. TSK is expressed in the peripheral retina from embryonic stages to adulthood 

and TSK knock-out mice show increased proliferation in this region. Activation of the Wnt 

pathway also stimulates RSC proliferation and we found that TSK inhibits Wnt signalling by 

competing for Wnt ligands to receptor binding. These results suggest that RSC proliferation 

in the peripheral retina is modulated by the antagonistic interaction between TSK and Wnt 

ligands and they also raise the interesting possibility that TSK activity may be responsible for 

the limited or absent RSC self-renewal in the adult peripheral retina. In order to gain insight 

into the molecular mechanisms of TSK action of cell proliferation, we are studying its 

expression pattern and function in NSCs derived from the foetal mouse brain, where TSK is 

also expressed in vivo. Preliminary real time PCR analyses showed that TSK is transcribed 

in NSCs from both the embryonic cortex and striatum and that its expression levels 

significantly increase during cell cycle exit and differentiation induced by growth factor 

withdrawal, in agreement with TSK role in inhibiting progenitor proliferation. We are currently 

analysing expression of TSK protein in proliferating and differentiating NSCs. Furthermore, 

we are establishing stable NSC lines where TSK expression is abrogated or enhanced, in 

order to determine whether it is necessary and/or sufficient to restrain proliferation of neural 

progenitors. 

Publications 

Ohta K, Ito A, Kuriyama S, Lupo G, Kosaka M, Ohnuma S, Nakagawa S, Tanaka H. 

Tsukushi functions as a Wnt signaling inhibitor by competing with Wnt2b for binding to 

transmembrane protein Frizzled4. Proc Natl Acad Sci USA 2011, 108: 14962-7. doi: 

10.1073/pnas.1100513108. 


Nicoletta Carucci, post-doc fellow; Valentina 

Taschetta, undergraduate student. 


Emanuele Cacci, Maria Elena Miranda 

Banos, Dipartimento di Biologia e 

Biotecnologie “C. Darwin”, Sapienza Università 

di Roma; William Harris, Department of 

Physiology, Development and Neuroscience, 

Univ. of Cambridge, UK; Roger Pedersen, 

Anne McLaren Laboratory for Regenerative 

Medicine, Univ. ofCambridge, UK; Kunimasa 

Ohta, Dept. of Developmental Neurobiology, 

Kumamoto University, Japan. 

146

Istituto Pasteur – Fondazione Cenci Bolognetti 

P.le Aldo Moro 5, 00185 Roma - Tel: +39 06 49255625/6/7/8 - Fax: +39 06 49255629 

www.istitutopasteur.it - pasteurcenci@uniroma1.it

2011 ANNUAL REPORT - Istituto Pasteur

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