1 September 2009 ISSN: Volume 3 Special issue Scientifica Acta GIORNATE DELLA FACOLTÀ DI SCIENZE MAT. FIS. NAT. DELL UNIVERSITÀ DI PAVIA ATTI DEL CONVEGNO LA RICERCA SCIENTIFICA IN FACOLTÀ OTTOBRE OBRE 2008 Università degli Studi di Pavia Biblioteca Delle Scienze Scuola di Dottorato in Scienze e Tecnologie Alessandro Volta
2 September 2009 Volume 3 Special issue Scientifica Acta Contenuto Introduzione al Convegno (Lucio Toma) 3 Intervento (Gianluca Introzzi) 7 Biologia 9 Department of Genetics and Microbiology A. Buzzati Traverso of the University of Pavia: recent research activity Alessandra M. Albertini 9 Biochemistry Paolo Iadarola 27 Physiology and Pharmacology Jacopo Magistretti 33 Biologia Cellulare Marco Biggiogera 39 Chimica 51 Chimica Organica Angelo Albini 51 Inorganic Chemistry Antonio Poggi 61 Chimica Analitica Antonella Profumo 70 Chimica Fisica Giorgio Spinolo 81 Fisica 89 Le ricerche nel Dipartimento di Fisica Alessandro Volta Lucio Claudio Andreani 89 Fisica Nucleare e Subnucleare Claudio Conta 101 Matematica 119 Analisi, Algebra, Geometria Maurizio Cornalba 119 Matematica Applicata Giuseppe Toscani Università degli Studi di Pavia
3 2 Scientifica Acta 3, Special issue (2009) Scienze della Natura 135 Zoologia Giuseppe Bogliani 135 Botanica e Micologia Graziano Rossi 143 Geologia e Geologia Ambientale Cesare R. Perotti 158 Mineralogia, Petrologia, Geochimica Riccardo Vannucci 179 Conclusione del Convegno (Alessandro Coda) 191
4 Scientifica Acta 3, Special issue, 0 (2009) Introduzione al Convegno A distanza di più di trent anni dal precedente analogo Convegno, la Facoltà di Scienze Matematiche, Fisiche e Naturali, si è nuovamente riunita nell Aula del 400 per la presentazione della Ricerca Scientifica in Facoltà. Il simbolo scelto per il logo dell evento è stato l icosaedro a simboleggiare la grande pluralità delle tematiche di ricerca scientifica che si svolgono nella Facoltà, tutte altrettanto importanti come ciascuna delle venti facce triangolari di questo poliedro regolare. Come nell icosaedro le facce rappresentano diverse espressioni dello stesso oggetto, i diversi ambiti della ricerca scientifica, che possono sembrare estremamente eterogenei, sono unificati dallo stesso approccio metodologico, dal rigore del metodo scientifico che accomuna discipline così diverse ma così sostanzialmente simili come la matematica e la biologia, la geologia e la fisica, le scienze naturali e la chimica. Il comune linguaggio delle due giornate del Convegno ha infatti confermato come gli steccati di separazione tra le diverse discipline siano sostanzialmente una sovrastruttura del sistema accademico italiano; la conoscenza della realtà di quanto si svolge nei laboratori e dipartimenti in cui operano membri della Facoltà deve essere di stimolo all attivazione di nuove collaborazioni interdisciplinari che sono oramai la sostanza di ogni approccio veramente moderno alle problematiche scientifiche. L interdisciplinarietà, auspicabile e da promuovere all interno della Facoltà, deve essere una linea di indirizzo anche all esterno della Facoltà non soltanto per quel che riguarda l attività scientifica ma anche per quella didattica, visto il continuo emergere di nuove figure professionali che, nella nostra società basata sull interazione delle conoscenze, è sempre più importante contribuire a formare. In maniera del tutto imprevedibile le due giornate del Convegno sono coincise con uno dei momenti più difficili che l intero sistema universitario italiano abbia attraversato negli ultimi anni. Da una parte la forte riduzione, progressivamente sempre più pesante, delle risorse pubbliche stanziate per le Università e dall altra la forte campagna di dequalificazione e di disinformazione sul sistema universitario portata avanti da tutti i mezzi di comunicazione tendenti ad accreditare la realtà universitaria come un mondo di privilegi e di sprechi. I relatori che si sono succeduti in questi due giorni hanno mostrato quanto false e generiche fossero le accuse che il mondo dell informazione e, purtroppo, l intera società civile ci muove. Hanno mostrato quanto l attività scientifica sia vitale, incisiva e ricca di ricadute positive, soprattutto grazie al contributo essenziale che tanti giovani ricercatori strutturati e non strutturati riescono a dare, spesso su basi praticamente volontaristiche. Tuttavia, non è il momento per l autocompiacimento. Piuttosto che ad un autovalutazione, o un autoassoluzione, la nostra Facoltà, l Ateneo pavese e tutto il mondo universitario italiano devono mostrarsi disponibili ad un serio processo di valutazione operato dall esterno che faccia emergere le vere eccellenze e che possa portare, con coraggio, anche a potare qualche ramo secco. Questo fascicolo, che riassume le relazioni svolte nel Convegno, mostra come la Facoltà di Scienze Matematiche, Fisiche e Naturali di Pavia non abbia nulla da temere da un sistema di valutazione veramente serio. Prof. Lucio Toma, Preside della Facoltà di Scienze Matematiche, Fisiche e Naturali
5 4 Scientifica Acta 3, Special issue (2009) Comitato organizzatore del Convegno: Alessandro Coda, Giovanni Desimoni, Adalberto Piazzoli, Francesco Sartori, Giuseppe Toscani, Riccardo Tribuzio. Curatore degli Atti del Convegno: Adalberto Piazzoli. Le relazioni contenute in questo fascicolo non riportano risultati scientifici originali ma costituiscono una rassegna della recenti attività di ricerca dei Dipartimenti afferenti alla Facoltà di Scienze; sono state redatte sulla base delle informazioni fornite dagli autori di queste ricerche. Le relazioni contenute in questo fascicolo non corrispondono del tutto al programma riportato nel fac-simile della locandina allegato: un relatore (M. Livan) ha dovuto farsi sostituire per causa di forza maggiore e alcune discipline (Biologia e Chimica) hanno aggiunto relazioni su ricerche non illustrate oralmente nel convegno per ragioni di tempo disponibile.
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8 Scientifica Acta 3, Special issue, 7 8 (2009) Intervento Pro-Rettore, Preside, Docenti, studenti e illustri ospiti, vi parlo oggi a nome dei Ricercatori della facoltà di Scienze. Ringrazio innanzitutto il Comitato organizzatore che ha concesso questa opportunità. Sono Gianluca Introzzi, del Dipartimento di Fisica Nucleare e Teorica. Vi dirò, in breve, cose a voi in gran parte note e che riecheggeranno - credo - in molte relazioni di questo Convegno. Ma le dirò come ex studente dell Università di Pavia, allievo di un collegio storico, ricercatore di questa Facoltà. Ho trovato qui ottimi docenti, biblioteche ben fornite, laboratori efficienti. Studiosi dediti a ricerche interessanti in settori avanzati, con contatti e scambi internazionali. Di tutto questo son grato all Università di Pavia, e al Collegio di cui sono stato alunno. Vorrei, a mia volta, aver la possibilità di trasmettere questo patrimonio di sapere e d esperienza alle generazioni che verranno. Vorrei poter lasciare ai miei allievi un Università tanto eccellente quanto quella in cui ho studiato. Ma, come sapete, questo sta diventando sempre più difficile, forse impossibile. La contrazione, continua e progressiva, delle risorse destinate alla ricerca ha reso arduo trattenere i giovani, fornire loro prospettive credibili, mantenere laboratori e biblioteche a livello internazionale, recarsi all estero per studi e ricerche, invitare studiosi da altri Paesi. In queste condizioni la ricerca langue, i giovani più promettenti se ne vanno. Non - come giusto e come avvenuto a molti della mia generazione - per un difficile e a volte lungo tirocinio all estero, ma con un biglietto di sola andata. Un emigrazione intellettuale che ci fa perdere i migliori talenti, e il prolungato investimento del Paese nella loro educazione e formazione. La mia generazione rischia di essere l ultima ad aver appreso in Italia come si fa ricerca. L abbiamo imparato, ma non avremo più studenti a cui insegnarlo. Gli effetti di questa situazione appaiono ovvi: l Italia è destinata a perdere talenti, brevetti, competitività internazionale. Impossibilitati a gareggiare con le nazioni emergenti sul piano del costo del lavoro, non saremo in grado di difenderci economicamente nemmeno mediante l alta qualità tecnologica e scientifica dei nostri prodotti. La Legge 133/2008 approvata in agosto dal Parlamento riduce del 20% il ricambio del personale nelle Università per i prossimi anni: ogni cinque studiosi che si pensionano, solo un giovane verrà assunto. Nature, un importante rivista internazionale che si occupa di ricerca scientifica, ha definito questi tagli un macello, nel senso letterale del termine. Che fare? Come porre rimedio a tutto ciò? Nessuno - credo - ha oggi una risposta certa. Ma vi invito, in tempi eccezionali e d emergenza, a fare quello che non si è finora fatto: a distogliervi dai vostri studi, ad uscire da laboratori e biblioteche per riaffermare a tutti i livelli l importanza della conoscenza nel funzionamento di un Paese progredito. Occorre sospendere per qualche tempo le nostre quotidiane occupazioni, per difenderne la legittimità e la centralità; per evitare che scelte miopi ci impediscano per sempre di fare ciò che sappiamo fare, nell interesse e a beneficio di tutti. E necessario far giungere questo messaggio alla società e ai politici, se necessario anche con prese di posizione determinate e inusitate. Se la mediazione e il dialogo si dovessero dimostrare impossibili, si trovi la forza di fare quello che fecero anni fa i direttori dei centri di ricerca in tutta la Francia: per resistere alle pressioni del governo che voleva imporre un controllo politico sulla ricerca, presentarono tutti le dimissioni al Primo Ministro.
9 8 Scientifica Acta 3, Special issue (2009) La situazione in Italia non pare oggi meno grave di quanto fosse quella francese di allora. In questo frangente, coesione e determinazione sono fattori decisivi. Mi auguro che tutte le Università, tutti i Rettori, i Presidi, i Direttori dei dipartimenti si impegnino nella difesa di un patrimonio collettivo che rischiamo di perdere: per noi e per le generazioni future. Prof. Gianluca Introzzi Ricercatore della Facoltà di Scienze MM. FF. NN.
10 Scientifica Acta 3, Special issue, 9 26 (2009) Biologia Department of Genetics and Microbiology A. Buzzati Traverso of the University of Pavia: recent research activity Dipartimento di Genetica e Microbiologia Adriano Buzzati-Traverso, Università di Pavia, Via Ferrata 1, Pavia, Italy Presenta: Alessandra M. Albertini, I temi di ricerca qui riassunti ed illustrati nel corso del Convegno della Facoltà di Scienze del Ottobre 2008, sono parte delle ricerche svolte da gruppi che afferiscono e svolgono le loro attività di ricerca nel Dipartimento di Genetica e Microbiologia fondato il 26 Ottobre Le attività presentate appartengono ad ambiti di ricerca apparentemente eterogenei, quali biologia strutturale, genetica evolutiva di popolazioni, genetica molecolare e genomica umana ed animale, immunogenetica, biologia e biotecnologie vegetali, microbiologia molecolare, genetica e biotecnologie microbiche. Diversi aspetti culturali e metodologici accomunano queste linee di ricerca: fondamentale è l approccio che si propone di studiare, a diversi livelli (macromolecolare, cellulare, di organismo e di popolazione) la complessità della struttura, della funzione e dell evoluzione del materiale genetico e dei meccanismi per la sua espressione, sino alle macromolecole ed alle loro funzioni, nell individuo ed in insiemi di individui. Le metodologie e le tecniche sperimentali utilizzate sono interdisciplinari: genomica e genetica, biologia molecolare e cellulare, chimica e biochimica, enzimologia, biometria e bioinformatica. The research activities hereafter introduced, and illustrated during the Sciences Faculty Meeting of October 2008, are part of the underway projects of the groups working at the Dipartimento di Genetica e Microbiologia founded in 1982, October 26. These researches belong to fields apparently heterogeneous such as structural biology, evolutionary and population genetics, molecular genetics, human and animal genetics and genomics, immunogenetics, plant biology and biotechnology, molecular microbiology, microbial genetics and biotechnology. On the contrary, these research lines share a number of cultural and methodological approaches. Essential is the multilevel (macromolecules, cells, organism and populations) approach to the study of the complexity of the structure, the function and the evolution of the genetic material, and of its expression mechanisms, up to the study of the macromolecular functions in the individual or in the whole of the organisms of a population. The experimental methods an techniques used are interdisciplinary: genomics and genetics, molecular and cellular biology, chemistry and biochemistry, enzimology, biometry and bioinformatics. 1 Introduction The Department of Genetics and Microbiology DGM was created in 1982 from the merging of the Institute of Genetics founded in 1948, the Institute of Microbiology and Plant Physiology (1969) and the Institute of Cristallography (1971). Till 1991, when the new and present DGM building was ready in the Cravino Campus of the University of Pavia, the DGM has been scattered among various institutions for years, with a main core hosted at the Botanical Gardens of Pavia. In the new place the DGM recreated the historical and scientific connection with the Institute of Molecular Genetics of the National Council of Research, IGM-CNR: actually the library of the Department is physically linking the two buildings. The research activity performed by people working at the DGM covers many scientific areas: microbial genetics, biotechnology, biochemical and cellular genetics, animal genetics, human genetics, evolutionary and population genetics, immunogenetics, tumor genetics, microbiology, plant molecular biology and biochemistry, biocristallography and structural biology. The DGM, in strict collaboration with the IGM-CNR, hosts the didactic and research Programmes of the Doctoral School in Genetic and Biomolecular Sciences of the Univeristy of Pavia, founded 25 years ago,
11 10 Scientifica Acta 3, Special issue (2009) and participates to the programmes of the Doctoral School in Biomolecular Sciences and Biotechnologies of the Istituto Universitario di Studi Superiori, IUSS (Institute for Advanced Study of Pavia), hosting seminars, courses and research activities of many of the PhD candidates. With a budget of about 1.3Me/year (mean of the last six years) the staff of the DGM (10 full professors, 6 associate professor, 6 researchers, 15 technicians, 13 Post-Docs and fellows, 44 PhD students) carries out research activities of high scientific level. In the last four years the researchers of DGM have published a mean of 52 ISI full paper/year, reaching an average IF of 5.5 (16.9, for the top five). In the last two years at the DGM have been awarded twelve PhDs/year, and more than fifty masters and first degrees in Biological Sciences or Biotechnology/year. 2 Structural Biology The structural biology laboratory at the Dept. of Genetics and Microbiology of the University of Pavia has long-standing interest in a range of biochemical questions encompassing the structural basis of enzyme catalysis and the dynamic properties of proteins. A common theme to the laboratory s research projects is the investigation of medically relevant enzymes with interesting chemical properties, such as complex multifunctional systems and proteins performing unusual catalytic functions[1-7]. The core of the research activity is represented by X-ray crystallography, employed to study protein three-dimensional structures. This is complemented by other approaches such as site-directed mutagenesis, analysis of enzyme kinetics and computational chemistry. Current research is focusing on enzymes of the neurotransmitter metabolism, on a protein complex involved in chromatin re-modeling, on an enzymatic system for the biosynthesis of a class of membrane phospholipids, on the structural genomics of viral replicative enzymes, and on the reaction of flavoenzymes with oxygen. 2.1 Bayer - Villiger Monoxygenases At the end of the 19th century, Baeyer and Villiger discovered that cyclic ketones react with oxidants such as peroxymonosulphuric acid to yield lactones. Baeyer-Villiger reactions are of enormous value in synthetic organic chemistry and the number of their applications is countless. Several microorganisms produce enzymes capable to catalyse Baeyer-Villiger reactions. These proteins are extensively studied for their exploitation in biocatalytic applications. This interest follows the problems related to the toxicity and instability of the oxidising reactants that are currently being used in chemical processes. In addition, enzymatic reactions exhibit a superior degree of enantioand regio-selectivity. Baeyer-Villiger monooxygenases are FAD-dependent proteins that use NADPH and molecular oxygen to insert an oxygen atom into their substrate. Fig. 1: The Bayer-Villiger reaction Kinetic and spectroscopic data have shown that the mechanism underlying the enzyme reaction is conceptually identical to that of the equivalent non-enzymatic processes. The key feature is the reaction of the NADPH-reduced flavin with molecular oxygen to generate a stable flavin-peroxide intermediate, which attacks the carbonyl carbon of the substrate. Rearrangement of the resulting Criegee intermediate yields
12 Scientifica Acta 3, Special issue (2009) 11 the oxygenated product and a hydroxy-flavin adduct, which must be hydrolyzed to regenerate the oxidized flavin. Recently the Structural Biology group identified a novel microbial Baeyer-Villiger enzyme, phenylacetone monooxygenase, which offers several unique and attractive features (Fig. 2): (1) it is thermostable and tolerant towards organic solvents, (2) it catalyzes enantioselective Baeyer-Villiger oxidations and sulfoxidations, and (3) it represents the only Baeyer-Villiger monooxygenase for which a crystal structure is available (solved in the Structural Biology team laboratory) [1, 2, 7]. Fig. 2: Three dimensional structure of a bacterial Baeyer-Villiger flavin monoxoygenase complexed with FAD and NADP. By enzyme redesign in the OXYGREEN EU Project, phenylacetone monooxygenase mutants will be created that combine exquisite operational stability with tailor-made substrate specificities and regioand/or enantioselectivities. Particular emphasis will be put on the development of a suite of enantiocomplementary Baeyer-Villiger monooxygenases, displaying diverse regioselectivities to the conventional chemical oxidation process. 2.2 Epigenetics and cancer Cancer cells display a multitude of epigenetic abnormalities. Such abnormalities can be viewed as a counterpart of the role that epigenetic modifications of DNA and histones play in the development and differentiation of normal cells. In the case of solid tumors, specific epigenetic marks have been linked to increased risk of cancer recurrence, suggesting an active involvement of chromatin alterations in cancer establishment and progression. Methylation of lysine residues is a well-characterized histone mark, whose existence has been known since the early days of chromatin research. Histone methylation has been long thought to be a low-turnover epigenetic mark. Consistently, the enzymes capable of reverting such modification have been the object of conjectures but have remained elusive until recently. At the end of 2004/beginning of 2005, the Structural Biology group reported the discovery of LSD1 [3-5]. The group demonstrated that the protein, formerly known as KIAA0601 or BHC110, is a histone demethylase that specifically acts on mono- and di-methylated Lys4 of H3 (Fig.3). The protein was shown to catalyze the demethylation reaction through an oxidative process involving an essential flavin cofactor. It appears that LSD1 is involved in a surprisingly broad range of both gene activation and gene repression events, from repression of neuronal genes in non-neuronal cells, to differentiation of hematopoietic cell lines and pituitary development. The discovery and characterization of the biochemical function of LSD1 together with the growing understanding of its crucial role in the regulation of gene expression, cell development, and tumor pathogenesis provide the foundation for a research project that aims atr the exploitation on LSD1 as potential drug target.
13 12 Scientifica Acta 3, Special issue (2009) Fig. 3: LSD1, associated with the transcriptional corepressor CoREST and histone deacetylase HDAC, catalyzes the demethylation of Lys4 of histone H3, through a flavin-dependent oxidative reaction. Components of the research group: Sandro Coda, Andrea Mattevi, Claudia Binda, Stefano Rovida, Daniele Bonivento, Federico Forneris, Marcello Tortorici, Arystotele Karytinos, Roberto Orrù, Elena Carpanelli, Silvia Speroni, Simone Nenci, Kittisan Thotsapon. Grants: PRIN; FAR; European Community; Contracts with Private Industry. 3 Mammalian Genome Structure and Instability Three research units of the Dept. of Genetics and Microbiology are involved in the analysis of mammalian genomes through different molecular and cytogenetic approaches. This analysis is mainly focused on genome instability and plasticity. Several aspects are investigated by means of complementary techniques in a collaborative effort aimed at elucidating mechanisms involved in: 1) the occurrence of genomic alterations, including large chromosome rearrangements, associated with cancer; 2) the evolution of mammalian karyotypes; 3) the remodeling of mammalian chromosomes. 3.1 Molecular and Cellular Biology Mammalian chromosomes are linear DNA molecules containing peculiar structures at their termini, called telomeres, which are essential for the maintenance of chromosome integrity. Genomic instability associated with telomere disfunction can lead to tumorigenesis. Telomeric DNA is composed of tandem repetitions of the TTAGGG hexamer, synthesized by the specialized enzyme telomerase. In addition to their location at terminal positions (Fig. 4), telomeric-like repeats are also present at internal sites (ITSs). Using molecular, cytogenetic and bioinformatic approaches has been addressed the question as to how such sequences arise and showed that they were inserted in the genomes during the repair of DNA breaks that occurred in the germ-line during evolution [8, 9]. This analysis strongly suggested that ITSs were inserted at break sites by telomerase, indicating that this enzyme, besides its role in telomere maintenance, may be involved in DNA repair . Recently this group, in collaboration with a Swiss group, described another new function of telomeric DNA; telomeres, that were thought to be silent regions of the genome, are actively transcribed producing a heterogeneous fraction of RNA molecules involved in the regulation of telomere stability . Besides telomeres, the main structure required for chromosome stability is the centromere. The group of Molecular and Cellular Biology discovered that, in the genus Equus (horses, donkeys and zebras) centromere repositioning occurred surprisingly often during the last 3 million years; therefore, this otherwise rare phenomenon played an important role in the rapid evolution of these animals . Another manifestation of the genomic instability is gene amplification (copy number increase of a DNA sequence), a key mechanism for oncogene activation and drug resistance in tumour cells which involves DNA breakage and repair. To study the molecular mechanisms generating amplified DNA, was measured the amplification ability in tumour cells in which the expression of genes involved in the repair of DNA
14 Scientifica Acta 3, Special issue (2009) 13 breaks was inhibited by RNA interference. These cells are hypersensitive to ionizing radiations and more prone to gene amplification than normal cells, indicating that the cellular genetic background can affect the response to therapy and the probability of tumour progression . Taking advantage of the background in gene amplification the research group set up an efficient method for the construction of mammalian cell lines over-expressing exogenous proteins; joint projects with pharmaceutical companies are under way aimed at the production of recombinant therapeutic proteins. Components of the research group: Elena Giulotto, Carmen Attolini, Livia Bertoni, Lela Khoriauli, Solomon G. Nergadze, Francesca Piras, Marco Santagostino, Alexandra Smirnova, Pamela Vidale. Grants: PRIN; FAR; European Community; Contracts with private industry. Fig. 4: Metaphase chromosomes hybridized in situ with a probe for the telomeric repeats. 3.2 Molecular Cytogenetics It is well documented that certain regions in the human genome contain breakpoints that have been repeatedly used during mammalian evolution. These breakage reuse regions are enriched in segmental duplications and tend to cluster in centromeric and telomeric position. In vertebrates, highly conserved homologous synteny blocks (HSBs) exist. HSBs are chromosomal segments containing genes whose sequence, distance and order, are highly conserved in the course of evolution; despite the extensive reshuffling of HSBs occurring in different genomes, the internal integrity of each block is maintained. The structural stability of HSBs and their ability to move intra and inter genomes, are guaranteed by the existence of recombinogenic regions flanking them; these structurally unstable regions have been demonstrated to be breakage reuse sites . Two different aspects of the topic briefly outlined above are studied. The first one consists in the analysis of a particular region of the human genome, chromosome 9 pericentromeic region, which has been demonstrated to have a redundant architecture and to be rearrangement prone . The data of the Molecular Cytogenetic group suggest that this chromosomal region plays a crucial role in driving structural chromosome rearrangements in both germinal and somatic cells. Moreover, it has been demonstrated that is directly involved in primate karyotype evolution. These results also indicate that a conserved segmental duplication, mapping in the region of interest, may coincide with an evolutionary breakpoint reused in mammalian species [16, 17]. The attention is also focused on another genomic region, mapping in the q21-22 segment of human chromosome 5, where the Adenomatous Polyposis Coli (APC) tumor suppressor gene is localized. By means of FISH on combed human genomic DNA, was built a map of the APC locus. This reference map was used to analyze constitutive large deletions of the APC locus in patients affected with familial adenomatous polyposis (Fig. 5A). The ultimate goal of this analysis is to search for common breakpoints and to characterize their molecular arrangement . State-of-the-art knowledge: it might be hypothesized
15 14 Scientifica Acta 3, Special issue (2009) that recurrent breakpoints have a structural organization resembling that of the breakage reuse regions contained in segmental duplication enriched chromosome districts . Components of the research group: Elena Raimondi, Mirella Bensi, Elisa Belloni, Valeria Biscaro, Francesca Coperchini, Valeria De Pascali, Chiara Marcialis, Francesco Vella. Grants: MIUR - PRIN; FAR. Fig. 5: Identification of partial APC gene deletion in a FAP patient by means of: A) molecular combing; B) multiplex ligation-dependent probe amplification. 3.3 Human Genetics: Cancer Genetics Familial adenomatous polyposis, a phenotypically heterogeneous disease highly predisposing to colorectal cancer, is associated with either APC tumor suppressor gene, or MUTYH DNA base excision repair gene. Selected polyposis patients that have proven to be negative for the most common APC germline mutations  are currently investigated with complementary molecular methods (Fig. 5B) that are suitable to detect different types of genetic lesions, including partial and whole-gene deletions. It has been recently demonstrated that: the molecular heterogeneity of APC deletions does not indicate the presence of hot spot breakpoints in polyposis patients; the APC locus rearrangements are mediated by repeated elements (SINE; LINE) interspersed along intronic and intragenic DNA sequences; the homologous recombination between repeated sequences is likely to represent the mechanism that underlies genetic rearrangements. We found that polyposis patients that are MUTYH mutations carriers, frequently show genetic variants the functional significance of which remains uncertain [21-23]. These researches aim at functionally characterize potentially pathogenetic MUTYH mutations by: analyzing Mutyh-/- mouse defective cell lines that, following transfection, express the human MUTYH mutated proteins; in vitro testing the human mutated proteins purified from bacterial cells transformed with the appropriate expression vectors. Colorectal cancer progress through different genetic lesions, tumor progression and metastasis are considered as the results of early changes that determine the metastasic capacity. We are currently investigating the possible alterations of cancer-related genes in colorectal cancer specimens, with and without associated metastases [24, 25]. These alterations include: mutations of KRAS, BRAF, TGFBR1, and BAT26 loci; rearrangements, such as amplifications and deletions, of 40 different genes encoding for proteins that have a role in cell cycle regulation, signal transduction, motility and angiogenesis. Aim of this study are: to search for a possible molecular signature that is specific for the metastatic capacity; to investigate the extent and the nature of genomic rearrangements underlying tumor progression; to evaluate the instability of cancer cell genome.
16 Scientifica Acta 3, Special issue (2009) 15 Components of the research group: Guglielmina Nadia Ranzani, Vito D Agostino, Giulia Garbin, Giuseppe Malpasso, Agnese Loda. Grants: FAR; Fondazione Banca del Monte di Lombardia; Fondazione Cariplo (Nobel Project); AIRC. 4 Evolutionary studies of human and animal populations These studies of human and animal evolution have two main focuses: (i) the analysis of biodemographic and environmental factors which influence human life-history traits such as fertility, age at reproduction, reproductive longevity; (ii) molecular and phylogeographic analyses of two genetic systems mitochondrial DNA (mtdna) and the male-specific portion of the Y chromosome (MSY) that are characterized by a strict uniparental transmission. For the sake of clarity, and space reasons, separate results and conclusions for the human life-history traits, the mtdna and the Y chromosome are provided in the following. 4.1 Biodemographic and Environmental Determinants of Human Life-History Traits Life-history traits are influenced by environmental factors and, when genetic components underlie the relations, by micro-evolutionary forces [26-31]. Sardinian population, which is characterized by historicgeographical isolation and peculiar genetic structure, represents an ideal context for studies on local differentiations of the determinants of life-history traits, as the maternal aptitude to reproduce later and successfully. Through the spatial analysis of all Sardinian birth data from the Italian Central Institute of Statistics we identified some central inland areas, qualified by higher incidence of late maternities at lower cost for perinatal mortality . These reproductive longevity areas correspond fairly well to high consanguinity and high longevity areas; with respect to the neighbouring areas, the inbreeding coefficient is 3.7 fold higher, suggesting possible population homozygosity in genetic factors affecting the trait, and the mortality in women aged 80 years and over is lower, suggesting the role of similar factors on the extension of both the life and the reproductive period. Further and deeper investigations on the relative role of genetic, biological and environmental determinants could focus on these target areas. 4.2 Uniparental Genetic Systems At fertilization, half of the DNA of the mother and half of that of the father are brought together in the zygote. The sperm contribution differs from that of the egg in two complementary ways: it does not contribute viable mitochondria, which harbour their own circular genomes ( 17,000 base pairs), thus the mtdna is maternally-transmitted; only the sperm contributes a Y chromosome in half of the fertilizations (giving rise to male embryos), thus the Y chromosome is of holoandric-inheritance. The mtdna and MSY do not recombine at meiosis, and this means that the first is a molecular record of the history of females who transmitted it through the generations, while the latter recapitulates the evolutionary history of males. Thus, these two uniparentally-transmitted systems provide complementary genetic information, which can be compared to each other, and to that provided by the autosomes and the X chromosome (Fig. 6) Y-Chromosome Studies. The Male Specific region of Y-Chromosome represents the largest portion of the genome ( 60 Mbase pairs) uniparentally transmitted but, differently from mtdna, only less than 1% of it has been studied so far. Although for the width of global human population diversity much more information is yet to be revealed, the study of the MSY variation has immediately proved to be an important tool in investigating human past both at macro- and micro-evolutionary scale . Similarly to that already observed for mitochondrial markers, a comprehensive picture of European human peopling according to the male perspective , confirmed that the European genetic variability originated from a reduced number of lineages and that the present European population derived by two major different events. The first involved the merging
17 16 Scientifica Acta 3, Special issue (2009) Fig. 6: Schematic pedigree illustrating the transmission of the uniparental genetic systems (mitochondrial DNA and Y chromosome) relative to autosomes. of local Mesolithic groups from different refugia after the last glacial maximum, and the second account for the spread of Neolithic farmers from the Near East. The studies carried out in the last years contributed to clarify the modality of the European peopling [35, 36] and the relative contribution of demic and cultural diffusion of agriculture. At this regard, a deep analysis of more that 1000 subjects belonging to 17 different Southeast European population groups revealed that cultural diffusion played an important role in the transition to farming in the Balkans : although has been detected distinctive genetic evidence of the first farming colonists arriving from the Middle East, this analysis has shown for the first time with biological markers, that they were fundamental not so much in shaping the genetic landscape of South East Europe, as in spreading the concept and practice of farming to local indigenous Mesolithic foragers. They then vigorously adopted it, giving rise to their own demic expansion Mitochondrial DNA studies In the last few years, human mtdna studies have entered a new phase: the blossoming of complete genome analyses. Of course, sequencing complete mtdnas is much more expensive and requires much more labour than restriction analysis or simply sequencing the control region of the molecule. However, the efforts are paying off, as the phylogenetic resolution of the mtdna tree has been greatly improved, meaning that, in turn, phylogeographic interpretations can be given correspondingly greater precision in terms of the timing and direction of human dispersals. Therefore, despite the fact that mtdna makes up only a tiny fraction of our total genome, the deciphering of its evolution is profoundly changing our perception about how modern humans spread across our planet. This applies to relatively ancient migrations such as the initial dispersal out of Africa , which occurred about 70,000 years ago, and the Early Upper Palaeolithic back-migration from western Asia to North Africa , but also to more recent events such as expansions due to the major climate change that, after the Last Glacial Maximum, led to the peopling of the Americas  and the human recolonization of Europe . However, mtdna is also an extremely powerful tool for
18 Scientifica Acta 3, Special issue (2009) 17 reconstructing very recent migrations; a paradigmatic example is represented by the study that has revealed that the ancestral homeland of the Etruscans is the Near East . As for animal studies, the same whole genome approach was employed to bovine mtdna. The recent analysis of 108 mitochondrial genomes from modern bovines has revealed that extinct wild aurochsen, most likely from two geographically distinct populations of Europe, occasionally transmitted their mitochondrial genomes to domesticated taurine breeds. These two genomes belong to different haplogroups (P and Q) whose ancestral geographic distributions, according to ancient DNA data, were rather different. One (P) is from an aurochs breed that lived in northern and central Europe, while the other (Q), which we found in an indigenous and endangered Italian breed (the Cabannina breed of Liguria), is from a population of aurochsen that might have ranged only south of the Alps . Overall, these findings support a single Neolithic domestication event for B. taurus in the Near East, followed by their spread throughout the Old World accompanying human migrations. However, the study demonstrates that, despite the selection performed by farmers and pastoralists against admixture between domesticated stocks and local aurochsen, a certain amount of gene flow between domesticated breeds and at least two wild European populations of B. primigenius occurred . However, mtdna is not only a string of DNA with peculiar features useful for evolutionary studies; it contains genes involved in the generation of cellular energy through the process of oxidative phosphorylation. Thus, the high mutation rate of the mitochondrial genome has resulted not only in the accumulation of the broad spectrum of sequence polymorphisms that distinguish haplogroups and sub-haplogroups, but also in a large number of disease-causing mutations. These human diseases generally manifest in organs and tissues that rely heavily on mitochondrial energy production and a paradigmatic example is represented by Leber hereditary optic neuropathy (LHON). In addition to the search for disease-causing mutations, the sequence variation of the human mitochondrial genome has been the focus of studies investigating its role as a contributing factor in the pathogenesis of neurodegenerative diseases. Recent studies carried on patients with LHON and Parkinson disease provide important clues about haplogroup effects . Moreover, they revealed that mtdna association studies are probably affected by a major specific problem: the resolution of the mtdna haplogroup structure is generally too low, and the definition of sub-haplogroups is necessary. In conclusion, for the small mtdna molecule the final level of molecular resolution that of complete sequences has been reached for both humans and cattle, and soon it will be reached for the Y chromosome, but the task of determining and interpreting worldwide variation has only just begun. Components of the research group: Antonio Torroni, Ornella Semino, Luca Ferretti, Silvana Santachiara, Paola Astolfi, Laura Zonta, Rosalba C. Guglielmino, Nadia Al-Zahery, Maria Pala, Vincenza Battaglia, Silvia Bonfiglio, Viola Grugni, Baharak H. Kashani, Arianna Malusà, Anna Olivieri, Ugo A. Perego, Cristina Uboldi, Carmela Nici Grants: PRIN, FIRB, FAR, Fondazione Cariplo, European Community 5 Functional Oncogenomics In the last years this laboratory was involved in studies aimed at the set up of new molecular biology tools to be used, at the diagnostic and therapeutic level, in the field of human malignant gliomas [45-50]. Several intriguing lines of evidence have recently emerged indicating that the cellular prion protein (PrP C ) may exert a neuro- and cyto-protective function: PrP C overexpression protects not only cultured neurons but also tumor cell lines from various pro-apoptotic stimuli. 5.1 The cellular Prion protein as a novel molecular target in human glial tumors Malignant gliomas are the most common intracranial tumors and are considered incurable. In order to determine if PrP C is involved in the resistance of glial tumors to cell death, the research Unit of Functional Oncogenomic investigated the effects of the down-regulation of cellular prion protein in human malignant glioma cell lines, by means of an antisense approach . Different phosphorothioate oligonucleotides
19 18 Scientifica Acta 3, Special issue (2009) (s-odn) targeting the PrP C transcript were designed. Prion-specific and negative control s-odns were transfected into different human glioma, murine glioma, human non-glial tumor cell lines as well as in healthy human primary fibroblasts and rat adult astrocytes. Treatment with s-odn, induced profound morphological changes, marked retarded cell grown and a highly significant cellular mortality in most of the glioma cells. Importantly, human primary healthy fibroblasts and rat adult astrocytes were not affected by s-odn treatment. Deeper molecular and cellular investigations indicated that PrP C silencing mediated by s-odn antisense oligonucleotide was accomplished by significant reduction of BCL2 and Beclin 1 expression, while typical apoptotic markers such as caspase 3/7, p53 and PARP-1 were not affected. Acridine orange staining, electron microscopy analysis and an accumulation of GFP-LC3II in autophagosomal membranes in glioma cells transfected with GFP-LC3 indicated a predominant activation of autophagic cell death. In conclusion, these results provide evidence that the development of strategies targeting PrP C might modulate cell death pathways and might constitute innovative molecular treatment for malignant glial tumor. Components of the research group: Sergio Comincini, Alberto Azzalin, Elena Sbalchiero, Giulia Barbieri, Silvia Palumbo, Nicoletta Marchesi, Cristina Muzzini, Laura Marongiu, Francesca Loraschi, Elisa Moroni. Grants: FAR; Contracts with private industry 6 Human Immunogenetics These researches encompass the complexity of the field of human Immunogenetics. MHC genes controlling self recognition and autoimmune mechanisms, serum complement proteins (C4, Bf) and CR1, CR2 receptors are studied as well as proteins involved in inflammation controlled by RAGE (Receptor for Advanced Glycation Endproducts) and TNF (Tumour Necrosis Factor) genes [52-54]. Research extended to the complete HLA region in order to define supra-types has demonstrated to be of great interest both for defining gene markers in different human populations and for its applications in the study of HLA-disease associations. The mechanisms of gene regulation, especially in immunoglobulin isotype deficiencies, in antibody involvement in organ transplantation, in the immunobiology of human reproduction and in the anti-tumor effects of lymphocyte-activated killer cells (LAK) and KIR receptors are investigated . Experiments involve the use of in vitro cell cultures, serum analysis, lymphocyte genotyping, and include work at proteins and DNA levels. In particular research lines are carried out in gender studies: the fetal-maternal vertical transmission of hepatitis C virus and the mother-fetus tolerance (micro-chimerism phenomena) are important for the future use of cordonal blood in transplantation (collaboration with the Cordonal Blood Bank of Foundation IRCCS Policlinico S. Matteo, Dott. L. Salvaneschi). The HLA haplotype mendelian segregation distortion seems correlated to newborn weight and the sex disequilibrium in autoimmune disease are of peculiar interest. Serum complement genes (C4A, C4B and Bf), HLA class III new allelic variations are studied for a more precise definition of the region; the molecular mechanisms involved in the lack of response to recombinant anti-hepatits B vaccination are investigated in families with individuals already classified as non responders to vaccine [56, 57]. HLA gene polymorphisms are studied in Mediterranean populations  with the aim of constructing gene maps to be used in origins, migrations and diversification of peoples in association with linguistic and archeological studies; in social health studies to identify potential bone marrow donors; in preventive health studies to identify families with a immune disease susceptibility. Chronic Fatigue Syndrome, a pathological condition of unknown aetiology which is assuming greater relevance in medicine is also investigated. The group is the first in Italy to have developed a biological bank of DNA, RNA, serum and red blood cells of CFS patients. The group is studying MHC class III and cytokine promoter polymorphisms and gene expression in order to define a genetic profile of the subject
20 Scientifica Acta 3, Special issue (2009) 19 and to propose diagnostic markers [59, 60]. The Immunogenetics team is available to exchange material from the bank in collaboration with other groups. Gene polymorphisms that code for inflammatory molecules in CAD (coronary artery disease) and in ALS (Amyotrophic Lateral Sclerosis) are investigated in collaboration with CIRMC (Research Centre of Molecular Cardiology- University of Pavia, Prof. Falcone)  and with IRCCS Foundation C. Mondino (Dott. C. Cereda) . Components of the research group: Mariaclara Cuccia, Enrica Capelli, Chiara Boiocchi, Ilaria Sbarsi, Cristina Capittini, Sara Bozzini. Grants: FAR; Regione Lombardia; PRIN. 7 Plant Molecular Biology and Biotechnology 7.1 Plant Molecular Biology The group involved in this field follows three themes of research: the functional characterization of cellcycle regulators, the role of DNA polymerases involved in translesion DNA synthesis, the set up of new methods for the expression and purification of cell wall degrading enzymes. The E2F family of Arabidopsis comprises eight members: two DPs and six E2Fs (two activators, one repressor and three atypical which lack a transactivation domain and have two DNA-binding domains). Results from current studies suggest a dynamic scenario characterized by an interplay between AtE2Fs in regulating cell-cycle and development in Arabidopsis . One of the consequences of oxidative stress is the accumulation of oxidated derivatives of bases in the DNA. The aim of the project is to characterize Arabidopsis plant translesion DNA polymerases and produce plant cell lines characterized by an altered expression of the relevant genes in order to evaluate their response to oxidative stress. To improve the manufacturing of biofuel by sugar sources recovering from lignocellulosic wastes, the group is involved in the developing of methods for the production of recombinant cell-wall degrading enzymes, Fig These researches are conducted in collaboration with: Istituto di Genetica Molecolare, CNR, Pavia, Italy; Università di Sassari e Ferrara,Italy; Institut de Biotechnologie des Plantes (UMR8618), Université Paris-XI, France; North Carolina State University, Raleigh, USA; ICGEB, New Delhi, India. Components of the research group: Rino Cella, Caterina Maggio, Augusta Cueva, Lorenzo Concia, Paolo Longoni. Grants: PRIN; FAR; Private Foundation. Fig. 7: Plant cell factory. 1: Biotechnological system for cellulolytic enzymes production; 2: Ferricyanide ironstaining of bean wt and lpa mutant.; 3: Programmed cell death in plant cell suspension cultures. 7.2 Plant Biochemistry The goal of one the four projects of this research unit is the increase of cations (particularly iron) and of phosphate bioavailability in grain-derived food and feed to improve their nutritional value and reduce