Poster Session I (Topics 1-12 & 15)

Wednesday, 23 September, 17:30-21:00


1. Bioinformatics, genomics and proteomics

Total number of abstracts in this session: 10

 

P1.1

Structural and biochemical investigations on human kynurenine aminotransferase II: a promising target for the treatment of schizophrenia
Valentina Casazza1, Valeria Montalbano1, Franca Rossi1, Yasushii Kajii2, Menico Rizzi1
1DisCAFF, University of Piemonte Orientale, Novara, Italy
2Mitsubishi-Tanabe Pharma Corporation, Yokohama, Japan

Kynurenic acid (KYNA) is an endogenous neuroactive compound, whose unbalancing is involved in the etiogenesis of several pathological conditions. In particular, high KYNA levels have been associated with schizophrenia and cognitive impairment. KYNA production in human brain is sustained by two aminotransferases: hKAT I and hKAT II whose 3D structures have been solved by our group. Despite the fact that both KAT enzymes catalyze the same reaction, they display important structural differences. This feature could be usefully exploited in order to identify a selective inhibitor for hKAT II, that is responsible for the majority of KYNA synthesis in human brain. A selective inhibitor for hKATII, BFF-122, has recently been found in an in vitro screening. We solved the 3D structure of the hKATII-BFF122 complex at 2.3 Å, revealing inhbitor mode of binding. Intriguingly, human and rat KATII reveal a significant different inhibitory pattern. We used site directed mutagenesis, structural biology and kinetics analysis to dissect such a behaviour providing data of high relevance for the pharmaceutical exploitation of this target.

 


P1.2

Heterologous expression and biochemical characterization of UvrD2 helicase from Mycobacterium tuberculosis
C. Cassani, K. Kazarian, M. Rizzi
DiSCAFF, University of Piemonte Orientale, Novara, Italy


Tuberculosis (TB) still remains one of the major lethal diseases in the world killing around 2 million people a year. The efficiency of M. tuberculosis as a parasite is its ability to persist in host tissues in latent state. According to the WHO data, about one third of world’s population carries latent TB infection. M. tuberculosis survives and replicates in host macrophages where is exposed to highly reactive nitrogen and oxygen radicals potent in damaging lipids, proteins and DNA.
Helicases appear to be important for genome stability of dormant Mycobacterium tuberculosis responsible for latent tuberculosis infection and big proportion of active disease cases caused by reactivation. It was demonstrated that in both M. tuberculosis and Mycobacterium smegmatis, a helicase termed UvrD2 is essential for bacterial growth making it a promising target to fight latent tuberculosis. We report here, for the first time, the expression purification and biochemical characterization of UvrD2 helicase from M. tuberculosis. Interestingly, the enzyme shows a significant ATP-ase activity in the absence of DNA, in contrast with what was reported for its paralog UvrD.

 


P1.3

A new member of the discoidin family: in silico analysis of Paracentrotus lividus nectin based on its cDNA sequence
C. Costa, F. Zito, V. Matranga
Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy”, Consiglio Nazionale delle Ricerche, Palermo, Italy

We obtained the cDNA sequence coding for Pl-nectin, an extracellular matrix protein purified from the sea urchin, composed of two 105 kDa equivalent subunits. The protein, synthesized as a 984aa precursor, containing a 23aa signal peptide, is a new member of the galactose-binding protein superfamily. In fact, it consists of six 151-156aa-long tandemly-repeated domains, which are homologous to the discoidin-like domains, also known as F5/8 Type C domains. Phylogenetic analysis with discoidin domains from other known proteins confirmed the importance of their biological role during evolution. In addition, we describe the three-dimensional structure for each Pl-nectin domain, obtained on the basis of the homology modelling with crystal structures of the human coagulation factor V and VIII C2 domains and neuropilin-1 b1 domain. A further two-domains modelling and the subsequent association of the three two-domains modelled dimers produced the assembled Pl-nectin subunit. The in silico quaternary structure of the two subunits, composed of two 105kDa C-shaped monomers linked by a disulfide bridge accounts for the whole 210 kDa Pl-nectin molecule. The model presented here is consistent with earlier biochemical reports on the molecule and extends previous conclusions concerning its function. In addition, the in silico analysis offers a basis for predictions to be experimentally tested.

 


P1.4

Transcriptional changes related to maturation and mating in male and female medfly, Ceratitis capitata
L.M. Gomulski1, G. Dimopoulos2, Z. Xi2, P. Gabrieli1, F. Scolari1, P. Siciliano1, A.R. Malacrida1, G. Gasperi1
1Dept Animal Biology, Univ. Pavia, Pavia, Italy
2Johns Hopkins Bloomberg School of Public Health, Baltimore, USA


The medfly is an invasive agricultural pest that has become a model insect for the development of biological control strategies. These strategies depend on knowledge of the behaviour, physiology and molecular genetics of reproduction. The recent availability of medfly ESTs has permitted the development of microarrays for mass-gene expression profiling.
A microarray-based approach was used to compare the adult head transcriptomes of each sex at differential physiological stages, sexually immature and mature virgin and mated individuals. Particular attention was placed on transcripts involved in reproduction, behaviour, olfaction, and the immune system. Compared to the massive transcriptional changes during the maturation of the female, post-mating changes were modest, suggesting that mating does not trigger extensive transcriptional changes. Mating in the medfly does not appear to trigger changes in gene expression to the extend seen in Drosophila.
Apart from increasing our understanding of the molecular machinery behind these biological processes, the genes implicated may represent important targets for control programmes aimed at controlling populations of this pest species.

 


P1.5

Prediction of the biological effect of polymorphisms falling within micro-RNA binding sites
D. Landi 1, R. Barale1, F. Gemignani1 , S. Landi1
1Dept of Biology, Univ. of Pisa, Pisa, Italy

miRNAs are negative gene regulators acting at the 3’UTR level, modulating the translation of cancer-related genes. SNPs within the 3’UTRs could impact the miRNA-dependent gene regulation either weakening or reinforcing the binding sites. The alteration of the normal regulation of a given gene could affect the individual’s risk of cancer. It is helpful to develop a tool enabling the researchers to predict which of the many SNPs could really impact the regulation of a target gene. There are several available databases and algorithms able to predict potential miRNAs-binding sites. Each algorithm gives different predictions and none of them gives, for each SNP, a direct measurement of the biological impact. We propose an approach allowing the assignment to each SNP of a ranking of its biological impact. The method is based on a simple elaboration of predictions from pre-existing well-established algorithms. We selected 140 genes candidate for colorectal cancer. These genes were identified following a genome-wide sequencing of 20,857 transcripts from 18,191 genes in 11 colorectal cancer specimens and found somatically mutated and thought to be crucial for the development of cancer.

 


P1.6

Identifcation of genetic variants using pooled multiplexed sequencing
F. Marroni1, S. Pinosio1,2, G. Zaina2, N. Felice2, F. Cattonaro1, M. Morgante1,2
1Institute of Applied Genomic, Udine, Italy
2Dept of Agricultural and Environmental Sciences, Univ. of Udine, Udine, Italy

In poplar, cinnamyl alcohol dehydrogenase (CAD4) is involved in the biosynthesis of lignin, a major component of plant cell wall which negatively impacts paper pulp processing and biomass fermentation to ethanol.
In the framework of EnergyPoplar project, we set out to survey the natural genetic variation of CAD4 in 1152 poplar trees. After Sanger sequencing of 384 samples, we analysed the additional 768 by Illumina next generation sequencing in multiplexed runs, and obtained complete CAD4 sequences for 12 pools of 64 subjects each; two of them were analysed also by Sanger sequencing. Considering as positives the SNPs found by Sanger sequencing in the selected pools, we evaluated accuracy of Illumina in the two pools (100% sensitivity, 97% specificity, allele frequency correlation r=0.99) and in the whole sample, in which pooling caused a slight decrease in accuracy (84% sensitivity and 96% specificity, r=0.84). In the whole sample, we identified 113 SNPs; of them 33 had already been identified using Sanger sequencing. Our results suggest that next generation sequencing is a fast, reliable and cost-effective technique to study genetic variation in a large number of subjects.

 


P1.7

A potent 3-isopropylmalate dehydrogenase inhibitor shows anti-mycobacterial activity: structural, enzymatic and microbiological investigations
S. Martignon1, P. Sander2, M. Rizzi1
1 DiSCAFF, Piemonte Orientale Univ., Novara, Italy
2 Institut für Medizinische Mikrobiologie, Zürich Univ., Switzerland

Many pathogens become starved for certain essential amino acids during infection. Attenuated M. tuberculosis strains mutated in genes encoding for key enzymes in the synthesis of essential amino acids, have been reported. Leucine auxotrophy makes M. tuberculosis incapable of replicating in macrophages in vitro. Unlike humans M. tuberculosis can synthesise leucine through a pathway characterised by three specific enzymes that include 3-isopropylmalate dehydrogenase (3-IPMDH), a protein reported to be essential for the bacterium growth. Here we report a structural and enzymatic analysis unravelling the mechanism of action of O-isobutenyloxalylhydroxamate (O-IbOHA), a potent bacterial 3-IPMDHs inhibitor. Determination of enzyme kinetics revealed a competitive inhibition with a Ki of 2.5 microM and the 1.7 A resolution crystal structure showed the mode of binding of O-IbOHA to the active site. Moreover, we observed that O-IbOHA kills M. tuberculosis and other mycobacteria with MICs in the range of 30-50 microM. Our data will be used to drive the identification of a novel class on anti-tubercular agents interfering with leucine synthesis.

 


P1.8

Tomato genome sequencing
M. Pietrella1, G. Falcone1, E. Fantini1, M. Gonzalez1, M.R. Ercolano2, A. Barone2, M.L. Chiusano2, N. D’Agostino2, A. Traini2, L. Frusciante2, S. Grandillo3, G. Perrotta4, A. Vezzi5, G. Valle5, G. Giuliano1
1Dept BAS, ENEA, Casaccia Research Center, Roma, Italy
2Dept of Soil, Plant, Environmental and Animal Production Sciences, Univ. of Naples "Federico II", Portici, Italy
3CNR-IGV, Institute Of Plant Genetics, Portici
4ENEA, Trisaia Research Center, Rotondella (MT), Italy
5CRIBI Biotechnology Centre and Depnt of Biology, Univ. of Padova, Padova, Italy

Tomato (Solanum lycopersicum) is an economically and nutritionally valuable crop and constitutes a model plant for the Solanaceae family. Its genome encodes approx. 35.000 genes, which are largely grouped in contiguous euchromatic regions corresponding to approx. 25% of the 950 Mb genome. An international project is under way to sequence the euchromatic DNA on a BAC-by-BAC strategy (http://sgn.cornell.edu/about/tomato_sequencing.pl). Italy is sequencing chromosome 12 and providing mapping and bioinformatic tools to the international effort. Seed BACs are selected for the presence of genetic markers and validated via genetic (Introgression Line) and cytogenetic (FISH) mapping. Each sequenced seed BAC is then extended into a minimum tiling path. Approx 41% of the euchromatic portion is publicly available (43% on Chromosome 12). A bioinformatic platform has been built to provide a preliminary annotation of the genome. An additional effort, to obtain a draft WGS sequence with Next Generation sequencing, is under way.

 


P1.9

Molecular characterization and expression profiles of putative Pheromone-Binding Protein genes from the Mediterranean fruit fly, Ceratitis capitata
P. Siciliano1, F. Scolari1, P. Gabrieli1, L.M. Gomulski1, C.R. Guglielmino2, M. Falchetto1, A. Bonomi1, A.R. Malacrida1, G. Gasperi1
1Dept Animal Biology, Univ. of Pavia, Pavia, Italy
2Dept Genetics and Microbiology, Univ. of Pavia, Pavia, Italy

Insect Pheromone Binding Proteins (PBPs) play an important role in intra- and inter-sex communication. Identification of genes encoding the PBPs of Ceratitis capitata (medfly), a worldwide fruits and crops pest, remains unaddressed. Here we report the cloning, characterization and expression profiles of 7 putative PBP Genes (CcPBP1 to CcPBP7) isolated from two cDNA libraries (head and embryos). Genomic analyses revealed a very high similarity between C. capitata and D. melanogaster gene structures, except for longer introns length in the medfly. RT-PCR assays aimed at identifying sex- age- and tissue-mediated differential expression showed higher transcript amounts in the heads in respect to the bodies for 6 PBP genes. In inter-sex comparison, significant sex-mediated differential expression was identified in only one of the genes. Additional quantitative RT-PCR analyses revealed differential expression in relation to sexual maturation for 3 of the genes. The expression profiles of these 7 genes, together with molecular and phylogenetic analyses, provide the first step towards the understanding of medfly pheromonal communication.

 


P1.10

Centaurin-α2 interacts with tubulin beta through microtubules’ anchoring
M. Stroppi1, M. Crippa1, D. Cartelli2, G. Cappelletti2, M. Venturin1, E. Battaglioli1, P. Riva1
1Dept of Biology and Genetics for Medical Sciences, Univ. of Milan, Milan, Italy
2Dept of Biology, Univ. of Milan, Milan, Italy


Centaurin-α2 is characterized by an Arf-GAP zinc binding domain and two PH domains. It promotes the inactivation of Arf-6, involved in intracellular vesicular trafficking and in cytoskeletal rearrangement. In stimulated cells centaurin-α2 localizes at plasma membrane through PIP2/PIP3 binding. Its expression pattern and function have not yet been investigated. In situ hybridization studies on mouse embryos show that Centa2 mRNA is expressed in early developmental stages of encephalon and heart. We carried out an yeast two-hybrid assay to identify interactors of centaurin-α2. Tubulin beta and NUP53 have been identified as possible interactors. The interaction with the C-ter tubulin beta, probably mediated by centaurin-α2 PH domain, has been validated by co-immunoprecipitation. Confocal microscopy and western blotting experiments allowed us to co-localize centaurin-α2 and microtubules. Our findings allow us to hypothesize that centaurin-α2 can move to plasma membrane through microtubules anchoring. Experiments aimed at investigating whether centaurin-α2 binds specific microtubule organization/s, such as mitotic spindle, and at validating NUP53 interaction are in progress.

 


2. Cell cycle

Total number of abstracts in this session: 1

 

P2.1

RNA interference of MAD2 and BubR1 causes mitotic spindle alterations, aneuploidy and cell cycle arrest
D. Piscitello, L. Lentini, T.Schillaci, V. Barra, A.Di Leonardo

MAD2 and BubR1 are crucial components of the spindle assembly checkpoint, that delays the onset of anaphase until the kinetochores of each duplicated chromosomes pair have successfully established bipolar attachment to the microtubules and are under tension. We investigated the effects of simultaneous reduction of MAD2 and BubR1 expression by small interfering RNA-mediated silencing. Transient reduction of MAD2 (40%) and BubR1 (70%) affected both cell survival and growth, in comparison to MAD2 and BubR1 alone silenced cells. Real time RT-PCR analysis showed p21 overexpression that might account for the partial G1/S arrest observed in MAD2/BubR1 co-depleted cells.After MAD2/BubR1 post-trascriptional silencing cells showed spindle alterations observed by β tubulin immunostaining, and the presence of lagging chromosomes revealed by time-lapse video microscopy . These findings suggest that co-depletion of MAD2 and BubR1 could affect the correct assembly of the mitotic spindle causing aneuploidy and chromosomal alterations.

 


3. Cellular stress, apoptosis and autophagy

Total number of abstracts in this session: 12

 

P3.1

Cellular Prion protein (PrPC) silencing induces autophagic cell death in human glial tumor cells
G. Barbieri1, S. Palumbo1, E. Sbalchiero1, N. Marchesi1, C. Muzzini1, F. Calderaro1, A. Azzalin2, S. Comincini1
1Dept of Genetics and Microbiology, Univ. of Pavia, Pavia, Italy
2Istituto di Genetica Molecolare-Consiglio Nazionale delle Ricerche, Pavia, Italy

A neuro- and cyto-protective function has been recently suggested for cellular Prion protein (PrPC). In order to determine if PrPC is involved in the resistance of glial tumors to cell death, three different phosphorothioate oligonucleotides (ODNs) targeting the Prion protein gene transcript (PRNP-ODN1/2/3) were designed and transfected into human glioma cells (IPDDC-A2, T98G, U87-MG, U373-MG, D384-MG, PRT-HU2), control healthy human primary fibroblasts, and rat adult astrocytes. While control cells were not affected, treatment with PRNP-ODN3 induced profound morphological changes and a highly significant cellular mortality in the majority of glioma cells. Typical apoptotic markers such as BAX, caspase 3/7, p53 and PARP-1 were not affected. On the contrary, acridine orange staining, electron microscopy analysis, altered expression of autophagic markers (i.e. Beclin1 and BCL2), and the accumulation of GFP-LC3II in autophagosomal vescicles, indicated a predominant activation of autophagic cell death as a consequence of PrPC silencing. These results suggest a possible role for cellular Prion protein as a susceptibility modulator of cancer cell death.

 


P3.2

Tumour biology of melanoma: a novel role of acid sphingomyelinase
L. Bizzozero1, C. Verdelli1, G. Milani2, E. Clementi1,3, C. Perrotta1
1Dept of Preclinical Sciences, Univ. of Milano, Milano, Italy
2H. San Raffaele Scientific Institute, Milano, Italy
3E. Medea Scientific Institute, Bosisio Parini, Italy

Recent evidence indicates that Acid Sphingomyelinase (A-SMase), plays important role in tumour biology, including tumour response to chemotherapeutic drugs. The effects of A-SMase were shown in vivo to result from a combination of effects of the enzyme expressed by the tumour cells themselves, and the surrounding microvascular endothelial and imnflammatory cells. We investigate the specific role of A-SMase expressed by melanoma B16 cells which show different tumorigenic and metastatic properties dependent on their melanin content. In order to investigate a possible correlation between A-SMase expression and B16 cells phenotype we isolated several B16 clones on the basis of pigmentation, indicating the pigmented clones as “black”, and the not-pigmented as “white” and we observed a higher expression and activity of A-SMase in the white B16 clones. in vivo experiments showed that the white and the black clones differ in terms of growth rate, invasiveness and histological characteristics. These data suggest a strong relationship between A-SMase expression and tumour behaviour. These studies might help refine therapeutic strategies against tumour based on regulation of A-SMase activity/expression.

 


P3.3

Mitochondrial DNA variability (mtDNA) modulates the expression of SIRT3 gene in oxidative stress by Sp-1 and AP-2 factors: evidence from cybrid cell lines
P. D'Aquila, F. Di Cianni, G. Passarino, G. Rose, D. Bellizzi
Dept Cell Biology, Univ. of Calabria, Rende, Italy

Several evidences indicate that multiple mitochondria/nucleus signaling pathways in response to oxidative stress are regulated by mtDNA variability. Considering the emerging role of sirtuins as sensor of cellular energy and oxidative stress, we investigated in cybrid cells if mtDNA variability is able to modulate the expression profiles of human sirtuin genes and molecular mechanisms underlying the stress response.
By RT-PCR we found that only SIRT3 gene was down-expressed under oxidative stress and this expression was influenced by mtDNA variability. Transient transfections of the SIRT3 promoter reporter construct revealed that such regulation was correlated to a different activity of this promoter in cybrid cells. Two transcription factors, Sp-1 and AP-2, which bioinformatics analysis demonstrated to recognize binding sites on SIRT3 promoter, are differently activated upon stress in cybrid cells.
On the whole, our study provides the first experimental evidence that mtDNA variability modulates the SIRT3 gene expression in oxidative stress and that Sp-1 and AP-2 factors could be the main players of this modulation, by acting as mediators of the mitochondria/nucleus interaction.

 


P3.4

Selective increase of mtDNA and of proteins controlling mitochondrial biogenesis in fibroblast from Leber's patients grown in the presence of a respiratory substrate
F. Fanelli1, P. Loguercio Polosa1, A. Ghelli2, L. Iommarini3, A. Maresca3, M. Roberti1, V. Carelli3, P. Cantatore1
1Dip. di Biochimica e Biologia Molecolare "Ernesto Quagliariello", Univ. degli Studi di Bari, Bari, Italy
2Dip. di Biologia Evoluzionistica Sperimentale, Univ. of Bologna, Bologna, Italy
3Dip. di Scienze Neurologiche, Univ. of Bologna, Bologna, Italy

Leber's hereditary optic neuropathy is a genetically determined degeneration of retinal ganglion cells, associated with mtDNA mutations in complex I subunit genes. The pathogenic mechanism underlying the disease is not completely understood; points to be clarified include the tissue specificity, the incomplete penetrance of the disease and the male prevalence. To obtain information on some of these aspects we determined the mtDNA copy number and the content of some mitochondrial proteins in fibroblast cell lines deriving from affected patients as well as from subjects that although presenting the pathogenic mutation did not develop the disease (unaffected carriers). We found that changing the carbon source from glucose to galactose caused an increase in the content of mtDNA and of the proteins TFAM, mtSSB, NRF1 and PGC1α in the carrier's fibroblasts. No substantial changes were found in fibroblasts from control and affected patients. These data suggest that a mechanism responsible for the different penetrance of the disease may be associated with an increase of mitochondrial biogenesis that takes place selectively in the carriers but not in the affected patients.

 


P3.5

Expression analysis of proapoptotic genes in skull base chordoma
L. Ferrari1, M. Longoni1, M. Stroppi1, N. Boari2, P. Castellazzi2, P. Mortini2, P. Riva1
1Dept of Biology and Genetics for Medical Sciences, Univ. of Milan, Milan, Italy
2Dept of Neurosurgery, Vita-Salute S.Raffaele Univ. , Milan, Italy

Chordoma is a rare tumor arising from remnants of the notochord, characterized by local invasiveness and variable tendency for recurrence. Loss of heterozigosity of 1p36 occurs in 80% of tumors.
Given the implication of apoptosis in notochord regression we studied the expression by RT-PCR of 8 proapoptotic genes mapping in 1p36 in 27 chordomas. TNFRSF8 and TNFRSF9 are differently expressed compared to control in 48% tumors, while DFFA, DFFB, CASP9, TNFRSF1B, TNFRSF14 and TP73 showed occasionally a different pattern of expression from control. We also studied the expression of miR-34a, mapping in 1p36, of its target SIRT1 and its transcriptional factor p53 by Real-Time PCR in 21 chordomas. We detected a miR-34a overexpression, probably caused by the p53 overexpression as reported in chordomas. SIRT1 protein expression is under study.
As FAS-FASL complex is involved in notochord regression, we studied their expression in 34 tumors and in 3 chordoma cell lines. Since most chordomas expressed FAS but not FASL, we treated 1 chordoma cell line with FASL and observed that apoptosis is induced after its administration. This evidence may suggest a pharmacological approach for chemotherapy.

 


P3.6

TRIM50 Williams Beuren syndrome gene is a novel component of aggresomes that allows a link between autophagy and proteasome
Carmela Fusco1, Mikhail Egorov 2, Lucia Micale1, Maria Monti3, Maria Giuseppina Turturo1, Bartolomeo Augello1, Ester D’Addetta1, Roman S. Polishchuk2, Piero Pucci3, Flora Cozzolino3, Giuseppe Merla1

1Laboratory of Medical Genetics, IRCCS Casa Sollievo Della Sofferenza Hospital, San Giovanni Rotondo, Italy
2Unit of Membrane Sorting and Biogenesis Department of Cell Biology and Oncology, "Mario Negri Sud Consortium" Santa Maria Imbaro, Italy
3CEINGE Advanced Biotechnology and Dept of Organic Chemistry and Biochemistry, Federico II Univ., Napoli, Italy

We showed that TRIM50 encodes an E3-ubiqutin ligase. TRIM50 is hemizygous in the Williams Beuren syndrome (WBS), a developmental genomic disorder, caused by a 1.5 Mb deletion at 7q11.23 including 25 genes. The contributions of TRIM50, to the WBS defects are still undetermined.We founded that TRIM50 protein localizes in mobile and dynamic cytoplasmic bodies. Electron microscopy showed that it localizes in the multi-vesicular aggregate structures. Further, proteasomal inhibition cause relocation of TRIM50 to the aggresomes together with HDAC6, an aggresomes marker. Using biochemical approaches we showed that TRIM50 interacts with HDAC6 and P62/SQSTM1, an essential protein involved in transport of polyubiquitinated proteins to the Proteasome or Autophagy. Together these experimental evidences strongly suggested that TRIM50 is part of a multimeric and dynamic protein complex that govern the cargo and shuttle of polyubiquitinated misfolded protein for both proteasome and autophagy clearance. We anticipated that the haploinsufficiency of TRIM50 could account for a consistent part of WBS phenotypes through the accumulation of its substrates.

 


P3.7

Cell damage induced by expanded ataxin-1 involves mechanical instability of nuclear membrane
S. Averaimo1, C. Canale2,3, D. Pesci4, V. Fortunati4, D. Paulis1, A. Gliozzi2,5, A. Relini2,5, M. Mazzanti1, C. Jodice4,5
1Dept Biomolecular Sciences and Biotechnology, Univ. Milan, Italy
2Dept Physics, Univ. Genoa, Italy
3Italian Institute of Technology (IIT), Genoa, Italy
4DeptBiology, Univ. “Tor Vergata” Rome, Italy.
5Inter-University Research Centre On The Molecular Basis Of Neurodegenerative Diseases (CIMN), Italy

SCA1 is an autosomal dominant late onset progressive ataxia. Normal SCA1 alleles contain 6-39 CAG repeats, encoding a polyGln stretch. The polyGln in the expanded (>40 units) pathological alleles is always uninterrupted. Rare non-pathological expanded alleles have been observed, but they show the polyGln interrupted by two blocks of histidines.
Analysis by confocal microscope of eukaryotic cells (COS1), transfected either by wild type proteins or by variants containing specific destabilizing or stabilizing mutations previously described, have highlighted that “expanded pathological” and “expanded normal” proteins produce mature aggregates at comparable degree, supporting the hypothesis that they are not toxic to the cell or at least do not trigger the neuropathology.
Homogeneous populations of oligomers of these proteins have been investigated also for their ability to permeabilize synthetic lipidic membranes; the membrane mechanical resistance has been tested by atomic force microscopy. These analyses have provided evidence that prefibrillar aggregates of expanded pathologic polyGln could damage the nuclear membrane eventually causing cell death.

 


P3.8

Effect of cadmium and manganese on gene expression and “in vitro” proliferative and invasive behaviour of MDA-MB231 human breast cancer cells
A. Longo1, R. Rizza1, C. Lourenço2, R. Sirchia1, C. Luparello1
1Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Palermo, Italia
2Erasmus student, University of Salford (UK)

It is known that cell response to cadmium (Cd) intoxication may not only involve protective reactions against the toxicity but also cell addressing to death, and that in some model systems manganese (Mn) has been proven to counteract Cd effects. We have exposed MDA-MB231 cells to 5 μM CdCl2 for 96 h, corresponding to the IC50 at this time period, and examined the expression patterns of apoptosis-related and stress response genes, also extending the analysis to the “differential display”-PCR identification of other genes whose expression was affected by the treatment. These latter experiments revealed AEG-1 and PLP2 as Cd-dependent genes whose involvement in cell response to the metal deserves further study. We also exposed cells to 1-100 μM MnCl2 or to CdCl2/MnCl2 co-treatment for 96 h and examined their proliferative and invasive behaviour. The results obtained indicate that 5 and 100 μM MnCl2 induced an approx. 50% increase and 10% decrease of tumor cell number, respectively, and, that MnCl2 at every concentration tested was unable to counteract Cd-triggered reduction of MDA-MB231 cell number. Boyden chamber analysis of MDA-MB231 cell motility and invasion show that, in addition to the effect on cell number, as expected 5 μM CdCl2 induced also a decrease of cell invasive ability, whilst 5 and 100 μM MnCl2 exerted a promoting and restraining effect, respectively, on cell invasion, both alone and in combination with CdCl2.

 


P3.9

Des-acyl ghrelin protects skeletal muscle from atrophy
Paolo E. Porporato1, Nicoletta Filigheddu1, Viola F. Gnocchi1, Simone Reano1, Giulia Bettas Ardisson1, Roy G. Smith2, Michele Fornaroo 3, Stefano Geuna3, Andrea Graziani1
1Univ.of Piemonte Orientale, Dip.Clin and Exp.Medicine
2The Scripps Research Institute, Scripps Florida
3Univ.of Torino

Skeletal muscle atrophy is a debilitating process associated to several diseases, fasting, or disuse, resulting in a massive loss of muscle mass and functionality. Des-acyl ghrelin is a peptide produced by the ghrelin gene which undergoes acylation to generate ghrelin.Ghrelin stimulates GH release and positive energy balance through binding to its receptor GHSR-1a. Des-acyl ghrelin, which does not bind GHSR-1a, nevertheless shares with ghrelin the ability to stimulate skeletal myoblasts differentiation and to inhibit apoptosis.
Here we show that des-acyl ghrelin and ghrelin inhibit skeletal muscle atrophy in vitro, by activating the Akt/mTOR pathway. In vivo up-regulation of circulating D-GHR, obtained by either pharmacological treatment or in transgenic mice, inhibits skeletal muscle atrophy induced by either fasting or denervation, through a mechanism which does not involve GHSR-1a-mediated activation of GH/IGF-1 axis.
Moreover, by showing that both des-acyl ghrelin and ghrelin activate Akt and mTOR in the skeletal muscle of GHSR-1a deficient mice, we provide the first genetic evidence that ghrelin and des-acyl ghrelin activate anti-atrophic signaling independently of GHSR-1a. Altogether these results, besides unveiling a novel direct anti-atrophic activity of des-acyl ghrelin, offer also new therapeutic strategies, alternative to IGF-1, for the treatment of skeletal muscle atrophy and cachexia.

 


P3.10

Autophagy during development of sea urchin embryos
M. Agnello, R. Chiarelli, L. Bosco, M.C.Roccheri
Dip. di Biologia Cellulare e dello Sviluppo, A. Monroy, Univ. degli Studi di Palermo, Italy

Autophagy is a highly regulated mechanism that enhances cell eukaryotic survival under various environmental and cellular stresses, by breakdown and recycling of macromolecules and organelles.
Here we report that in Paracentrotus lividus embryos autophagic process occur, at a lesser extend during physiological development and at greater levels after a cadmium treatment.
By Acridine Orange staining, we found that embryonic cells exposed to cadmium display green fluorescence in cytoplasm and nucleus, and show considerable red fluorescent dots in cytoplasm. This evidence suggests formation of acidic auto-phagolysosomal vacuoles. By Neutral Red vital staining, specific for lysosomes and cellular acid compartments, we obtained analogous results. These data have been sustained by anti-LC3 antibody detection (specific marker of autophagy). By Western blot we found a peak of physiological autophagy at late gastrula stage, while in treated embryos autophagy seem to anticipate massive apoptosis, probably for providing the energy for the removal of apoptotic cells or, alternately, as a less deleterious defence strategy for the safeguard of development program.

 


P3.11

Effects of H2O2 on mesoangioblast stem cells: survival and cell death
G. Turturici, C. Geraci, M.M. Barreca, A. Mingrino, G. Sconzo
Dept of Developmental and Cellular Biology, Univ. of Palermo

An important property of mesoangioblast stem cells is their ability to cross the endothelial barrier when injected into the mouse circulation, localizing to experimentally damaged tissues. They have the capacity to reduce the severity of experimental muscular dystrophy and to ameliorate myocardial damage in infarcted mouse hearts. When these stem cells are close to the damaged/inflamed tissues they are subjected to cellular stress due to inflammatory mediators and to reactive oxygen species, ROS. To determine whether ROS affect these stem cells, we subjected them at different H2O2 concentrations close to those who may be accumulated in inflamed tissues. We estimated the amount of cell death in response to the H2O2 treatments and analyzed the cell cycle, the activity of caspase 8-9 and 3 and the cytosolic LC3 ratio during the treatment and the recovery of 72 h. Preliminary data showed that a number of cells died (25-50%) depending on the treatment (conc./time), that cell growth is stopped and start again at the end of recovery, that the caspase 3 reaches maximum activity soon after treatment and LC3 is peaking during the first part of recovery to return then to normal value.

 


P3.12

Role of the prolyl-isomerase Pin1 in regulating the transcription-independent apoptotic activity of p53
M. Mioni1, G. Sorrentino1, F. Mantovani1,2, P. Pinton3, G. Del Sal1,2
1Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB), Area Science Park, Padriciano, Trieste
2Dipartimento di Scienze della Vita, Univ. di Trieste, Trieste
3Dept of Experimental and Diagnostic Medicine, Univ. of Ferrara, Ferrara

The major tumor suppressive activity of p53 is the induction of apoptosis in response to stress, relying on both regulation of transcription and on direct roles at the mitochondria. In the nucleus p53 is able to induce the expression of key proapoptotic genes in response to genotoxic stress. A key regulator of this pathway is the prolyl-isomerase Pin1, which is able to transduce phosphorylation of p53 into conformational changes in order to trigger dissociation of p53 from the E3-ubiquitin ligase MDM2 and the inhibitor iASPP with a consequent increase of proapoptotic transcriptional activity of p53. At the mitochondria p53 induces outer membrane permeabilization. We hypothesize that Pin1 might regulate also the mitochondrial apoptotic activity of p53, given the fact that the prolyl-isomerase has been previously shown to regulate other apoptotic proteins acting at the mitochondria, such as BIMEL, Bcl2 and p66 Shc. Here we show that Pin1 is necessary to p53-mediated transcription-independent apoptosis. Furthermore, we demonstrate that Pin1 is necessary for efficient localization of p53 to mitochondria and that it modulates the ability of p53 to interact with BclXL.

 


4. Chromosome biology

Total number of abstracts in this session: 7

 

P4.1

Human common fragile sites FRA2H and FRA7B
N. Bosco, F. Pelliccia , C. Viscomi , S. Graziano , A. Rocchi
Dept Genetics and Molecular Biology, Univ. Sapienza, Rome, Italy

The chromosomes of all analysed individuals show gaps or breaks in specific regions, the common fragile sites (CFS) (n ≤ 100), when the cells are exposed to replication stress or to some DNA-binding compounds. So, they are “normal” features of human genome, but the frequency of their expression is different in different individuals. CFSs cause genetic instability and are frequently involved in mutations in cancer cells. The causes of their fragility are still under investigation. The analysis of the twenty CFSs until now molecularly characterized revealed same shared features: the AT bases richness, the high DNA flexibility and the DNA late replication.
In this work we determined the DNA sequence of the CFS FRA2H (2q32.1-q32.2) and of the telomeric fragile site FRA7B (7p22.3-p21.3) using BAC clones and fluorescent in situ hybridization (FISH). The expression of both CFSs is induced by aphidicolin. FRA7B is also inducible by DAPI (4’,6-diamidino-2-phenylindole). We analysed the molecular composition of these two sequences and searched for the presence of DNA helix high flexibility regions.
The chromosome bands 2q32.1 and 7p22 are recurrent breakpoints in chromosome abnormalities in different types of neoplasms.

 


P4.2

Silencing of miniwhite reporter gene induced by functional domains of constitutive heterochromatin
P. Dimitri, Ilaria Passacantilli, Maria Carmela Accardo
Dip. di Genetica e Biologia Molecolare, Univ. La Sapienza, Roma

Position effect variegation (PEV) exhibited by a P element-derived reporter gene is thought to reflect the silenced state of a given heterochromatin region where the P element is inserted. It is now clear however that constitutive heterochromatin in Drosophila melanogaster contains a large variety of active genes. In the present work, we asked the following question: are P element reporter genes subjected to PEV when inserted into functional heterochromatin domains? To answer this question we examined 25 KV insertion lines mapping to the heterochromatin of the right arm of chromosome 2. Each lines carries a single SUPor-P transposon with a miniwhite reporter gene flanked by Su(Hw) boundary elements and under the control of specific enhancer. Although an analysis of the flanking regions showed that in all tested KV lines, the SUPor-P elements are inserted into, near or in between annotated genes, all lines exhibited a strongly white variegated eye phenotype and only 4 were sensitive to the suppression by Su(var)205 e Su(var)101. In addition, there appears to be no relationship between location or nature of flanking sequences and sensitivity to suppression. Notably, we found lines with strong silencing of the miniwhite, where the SUPor-P is inserted into the widely expressed heterochromatin genes. Our data show that position effect variegation can be induced by functional heterochromatin. The molecular bases underlying this phenomenon will be discussed.

 


P4.3

Radial nuclear location of human chromosome 7 loci is evolutionary conserved in Primate cell nuclei
C. Federico, R. Picciotto, S. Motta, S. Saccone
Dept Biologia Animale “M. La Greca”, Univ. Catania, Italy

Human chromosome territories, in the cell nucleus, are radial organized, and this spatial organization seems to be strictly related to replication/expression pattern of large chromosomal regions. DNA sequences from human chromosome 7 were hybridized on human and other Primate nuclei, to define the radial nuclear location (RNL) of specific loci in cells from different species. Loci were selected taking into account their evolutional relocation along the mitotic chromosomes, to evaluate the effects of evolutionary translocation/inversion events on the nuclear DNA organization. Our results showed a conserved RNL of the investigated loci, in spite of their relocation along the homologous chromosomes from the different Primate species, arguing that RNL of specific loci is preserved during Primate chromosomal evolution. The evolutionary conservation of RNL indicates that contiguity of specific DNA sequences, in cell nucleus, could be maintained even if they changed the position along mitotic chromosomes, then consenting preservation, during evolution, of pre-existing functional interactions among loci.

 


P4.4

Peripheral nuclear location of chromosomal loci is an important feature for evolutionary new-centromere formation
C. Federico, R. Picciotto, M. Galvagno, S. Motta, S. Saccone
Dept Biologia Animale “M. La Greca”, Univ. Catania, Italy

Evolutionary New-Centromeres (ENC) are centromeres that emerged, during evolution, in new sites along the chromosome, with inactivation of the pre-existing centromere. In human chromosomes, new-centromeres were occasionally detected on bands corresponding to ENC sites in homologous chromosomes from other Primates, and these new-centromeres are generally not associated to any clinical features. To date, no consensus-sequence was identified, and epigenetic mechanisms have been proposed to explain the emergence of new centromeres in non-canonical chromosomal loci. During extensive analysis of the human genome organization in cell nuclei, we focused our attention on the radial nuclear location of four ENC-related human chromosomal bands. Our results showed that these bands, and their homologous regions on other Primate chromosomes, other than low GC-level, late replication timing, and low gene-density, are endowed by a very peripheral nuclear location. Thus, we can conclude that the peripheral nuclear location of a locus could be considered one of the main parameter “predisposing” a chromosomal region to become an ENC.

 


P4.5

The length of the telomeric 3’overhang in senescent cells
V. Somma1,2, S. Mattarocci2,3, G. Cimino Reale2, E. Pascale4, E. G. Di Domenico5, F. Ascensioni5, E. D’Ambrosio2
1Dipartimento di Istologia ed Embriologia Medica, Università “Sapienza”, Roma, Italia
2Istituto di Neurobiologia e Medicina Molecolare, CNR, Roma, Italia
3Dipartimento per lo sviluppo di programmi terapeutici, Istituto dei Tumori Regina Elena, Roma, Italia
4Dipartimento di Medicina Sperimentale, Università “Sapienza”, Roma, Italia
5 Dipartimento di Biologia Cellulare e dello Sviluppo, Università “Sapienza”, Roma, Italia

The length of telomeric 3’overhang is crucial for an efficient capping but it is unclear its specific role to trigger the senescence. We analyzed cultures of HUVEC until they reached the senescence by a specific method (t-OLA). The 3’overhang did not undergo to progressively shorten and constantly resulted as a distribution of length ranging 50-300nt (m.v.90nt). In senescent cultures the longer tails disappear and the modal value drops to around 50nt. A drop of the length distribution occurs whenever a cell culture enters a replicative quiescence even in very early passages. Thus the 3’overhang erosion occurring in senescent cell, it would be due to the high number of non-cycling cells. We also performed a “life long” analyzes of the 3’overhang in leukocytes of old subjects (91-106 years) and compared it with general population (10-85 years). We found that the length of the 3’overhang do not change during the life span remaining around 90nt, but 3 out of the old subjects had highly eroded 3’overhang (m.v.48nt) and the highest frequency of very short telomeres. These data support the hypothesis that cell senescence is triggered by a few very short telomeres with a poor 3’overhang.

 


P4.6

Evolutionary novel centromeres in the genus equus
M.F. Piras, S.G. Nergadze, E. Magnani, L. Bertoni, C. Attolini, E. Raimondi, E. Giulotto
Dipartimento di Genetica e Microbiologia, Univ. di Pavia, Italy

We showed previously that neocentromere formation through centromere repositioning (movement of centromeric function without chromosome rearrangement) is exceptionally frequent in the genus Equus (horses, asses and zebras) (Carbone et al, Genomics 2006). No satellite sequences were detectable at all Equus evolutionary neocentromeres, following FISH experiments. We then demonstrated, at the sequence level, that the neocentromere of horse chromosome 11 is totally devoid of satellite repeats. This observation strongly supports the hypothesis that this centromere was formed recently during the evolution of the horse lineage and, in spite of being functional and stable in all horses, did not acquire all the marks typical of mammalian centromeres, probably representing the first example of an evolutionary “immature” centromere. The presence of so many apparently-satellite-free neocentromeres suggests that, at least in this genus, there is no adaptive requirement for the acquisition of centromeric satellite DNA once neocentromeres are formed.

 


P4.7

Ectopic expression of human telomerase allows the immortalization of primary cells from equus species
P. Vidale1, E. Magnani1, S.G. Nergadze1, Mondello2, E. Giulotto1
1Dipartimento di Genetica e Microbiologia, Univ. di Pavia, Italy
2Ist, di Genetica molecolare CNR, Pavia, Italy

Genetic studies with primary mammalian cells are limited by their short-term proliferative capacity. It was shown that primary cells from several mammalian species can acquire unlimited proliferative potential upon ectopic expression of the human telomerase protein component (TERT). The aim of this work was to obtain immortal cell lines from three Equus species (donkey, Grevy’s zebra and Burchelli’s zebra) as a general method to immortalize primary cells from endangered mammals. However, when we transfected fibroblasts of these species with human TERT, we were unable to prolong their proliferative capacity. We then introduced both the protein and the RNA component of human telomerase in the same cell lines and were able to obtain immortal cell lines. We showed that in these cells telomerase is active and telomeres are greatly elongated. While Burchellì’s zebra immortalized fibroblasts maintained a diploid karyotype, the cells from the other two species became aneuploid.. Ectopic expression of the two subunits of human telomerase can then be viewed as a general method to immortalize primary cells from endangered species.

 


5. Development and differentiation

Total number of abstracts in this session: 10

 

P5.1

Noggin elicits retinal fates in Xenopus animal cap embryonic stem cells
L. Lan1, A. Vitobello1,2, M. Bertacchi1, M. Andreazzoli1, F. Cremisi1, R. Vignali1, G.C. Demontis3, G. Barsacchi1, S. Casarosa1,4
1Dept of Biology, Univ. of Pisa, Italy
2Present address: Fredrich Miescher Institute for Biomedical Research, Basel, Switzerland
3Dept of Psychiatry, Neurobiology, Pharmacology and Biotechnology, Univ. of Pisa, Italy
4Centre for Integrative Biology, Univ. of Trento, Italy

Driving specific differentiation pathways in stem cells is a main goal of cell therapy. We have exploited the differentiating potential of Xenopus animal cap embryonic stem cells (ACES) to investigate the factors necessary to drive stem cells toward retinal fates, as ACES are multipotent. We have found that a secreted molecule, Noggin, is sufficient to elicit retinal fates in ACES. High Noggin doses support terminal differentiation of retinal neurons in ACES cells in vitro, as seen by the activation of terminal differentiation genes. Following in vivo transplant, high Noggin-expressing ACES rescue eye development when grafted to replace one of the eye fields. These eyes are functionally equivalent to normal eyes, as they respond to a light stimulus. Moreover, high Noggin-expressing ACES grafted in a posterior ectopic location still induce the formation of an eye-like structure.
Our data show that in Xenopus embryos, proper doses of a single secreted molecule, Noggin, can drive ACES toward retinal differentiation without additional cues. This makes ACES a suitable model to direct differentiation of stem cells toward retinal fates and encourages further studies on the role of Noggin in the retinal differentiation of mammalian stem cells.

 


P5.2

MiRNAs couple cell cycle, cell fate and developmental timing in retina
S. Decembrini1*, D. Bressan2,3*, R. Vignali1, L. Pitto2, S.Mariotti1, G. Rainaldi2, X. Wang4, M. Evangelista2, G. Barsacchi1, F. Cremisi1,2,3,5
1Dip. di Biologia, Univ. di Pisa, Pisa, Italy
2Istituto di Fisiologia Clinica, CNR, Pisa, Italy
3Scuola Normale Superiore, Pisa, Italy
4 Center for Brain and Cognitive Sciences, Institute of Biophysics, Beijing, China
*These authors contributed equally to this work

Cell identity is acquired in different brain structures following a stereotyped timing schedule, by accommodating the proliferation of multipotent progenitor cells and the generation of distinct types of mature nerve cells at precise times. However, the molecular mechanisms coupling the identity of a specific neuron and its birth date are poorly understood. In the neural retina, only late progenitor cells that divide slowly can become bipolar neurons, by the activation of otx2 and vsx1 genes. In Xenopus, we found that Xotx2 and Xvsx1 translation is inhibited in early progenitor cells that divide rapidly by a set of cell cycle related microRNAs (miRNAs). Through expression and functional screenings, we selected four miRNAs, mir-129, mir-155, mir-214 and mir-222, which are highly expressed at early developmental stages in the embryonic retina and bind to the 3\' UTR of Xotx2 and Xvsx1 mRNAs inhibiting their translation. The functional inactivation of these miRNAs in vivo releases the inhibition, supporting the generation of extra bipolar cells. We propose a model in which the proliferation rate and the age of a retinal progenitor are linked to each other and determine the progenitor fate through a set of miRNAs.

 


P5.3

Raver1 expression and alternative splicing events during skeletal muscle differentiation
E. Diani, C. Morandi, M.G. Romanelli

Raver1 is a dual compartment protein that interacts in the nucleus with polypyrimidine tract-binding protein (PTB/hnRNPI), acting as a co-repressor in the regulation of alternative splicing, and in the cytoplasm, with vinculin, directing mRNA to focal adhesions. It has been demonstrated that PTB expression is related to alternative splicing events during myoblasts differentiation of specific genes, such as calpain 3 (CAPN3) and myotubularin-related protein 1 (MTMR1). CAPN3 is a non-lysosomal cysteine protease involved in disassembly of the sarcomere and muscle cytoskeleton, whereas MTMR1 plays a role in muscle formation and represents a novel target for abnormal mRNA splicing in myotonic dystrophy.
The aim of this study is to investigate the expression pattern of Raver1 in myogenesis and its role in alternative splicing events that occur in CAPN3 and MTMR1 transcripts. Alternative exons switch during C2C12 myoblast cells differentiation has been analyzed by Real Time-PCR and Raver1 over-expression in transfected cells. The results will highlight the individual contribution of Raver1 in alternative splicing events regulated by trans-acting regulatory proteins of the hnRNP family.

 


P5.4

Proteins participating to the post transcriptional regulation of the mitochondrial cytochrome c oxidase subunit IV via elements located in the 3’UTR
G. Cannino, E. Ferruggia, A.M. Rinaldi

In higher eukaryotes, nucleus encodes all mitochondrial proteins with the exception of 13 subunits of the respiratory complexes. These subunits, together with 2 rRNAs and 22 tRNAs are coded by mitochondrial genome. Biogenesis and assembly of cytochrome c oxidase complex thus require fine interplay between the two compartments. In order to shed light on some aspects of the cross-talk between nucleus and mitochondria, we studied the regulation of COXIV (nucleus-encoded) subunit.
In developing rat brain we demonstrated that the COXIV expression is also regulated at post-transcriptional level and we identified two 3’UTR-COXIV RNA-binding factors. We isolated from brain of newborn rats two proteins of about 86 and 42kDa, whose putative identities are respectively Hsp90 and actin. These proteins maintain the RNA-binding ability and specificity for COXIV messenger and interacting with the 3’UTR-COXIV inhibit the translation. We also evaluated the content of Hsp90 and actin during postnatal brain development. In addition, we demonstrate that in just born rat brain, when the COXIV protein appears at low levels, Hsp90 is not phosphorylated hence able to partecipate to the formation of a complex with the mRNA. Vice versa in the adult tissue, when COXIV level increased, the Hsp90 is phosphorylated in serine residues causing the release of the mRNA, which therefore is ready for translation and consequently the protein can be accumulated.

 


P5.5

Sex and the single embryo: early development in the medfly, Ceratitis capitata
P. Gabrieli1, A. Falaguerra1, P. Siciliano1, L.M. Gomulski1, F. Scolari1, A. Bonomi1, A.R. Malacrida1, G. Gasperi1
1Dept of Animal Biology, Univ. of Pavia, Pavia, Italy

In embryos the maternal-to-zygotic transition (MTZ) integrates post-transcriptional regulation of maternal transcripts with transcriptional activation of the zygotic genome. Although the molecular mechanisms underlying this event are about to be clarified in Drosophila melanogaster, little is know about the MTZ in other insect species, like the global pest species Ceratitis capitata (medfly). Using a novel PCR-based sexing method, which takes advantage of a putative LTR retrotransposon MITE insertion on the medfly Y chromosome, the expression of the sex determination and cellular blastoderm formation genes was analysed in individual early male and female embryos. This study allowed us to clarify i) when the MTZ occurs during the medfly embryogenesis, ii) when and how the maternal information of “female-development” is reprogrammed in the embryos and iii) what are the similarities and the differences in the regulation of gene expression in C. capitata and D. melanogaster. The slow rate of early development in the medfly makes it a suitable candidate to improve our understanding of the molecular mechanisms of the MTZ transition in Diptera.

 


P5.6

A new role for β-dystrobrevin in neuronal differentiation
B. Artegiani1,3, A. Sferra1, C. Labbaye2, M.T. Quaranta2, P. Torreri1, G. Macchia1, M. Ceccarini1, T.C. Petrucci1, P. Macioce1
1Dept Cell Biol & Neurosci, Istituto Superiore di Sanità, Roma, Italy
2Dept Haematol Oncol & Mol Med, Istituto Superiore di Sanità, Roma, Italy
3Present address: Center for Regenerative Therapies Dresden, Germany

α And β dystrobrevins are cytoplasmic components of the Dystrophin-associated Protein Complex we have reported to be involved as scaffold proteins in intracellular transport and signal transduction. Recently, we have identified iBRAF/HMG20a, an HMG (High-Mobility Group)-protein with close sequence and structural homology to BRAF35/HMG20b, as a specific partner of β-dystrobrevin. HMG proteins are non-histone proteins that bind to DNA and chromatin, acting as architectural elements that modulate chromatin structure and controlling the expression of a number of genes. We also found that β-dystrobrevin directly interacts with BRAF35, in vitro. BRAF35 is required to maintain repression of neuronal specific genes, while iBRAF expression induces the neuronal differentiation programme. Our results corroborate the role of dystrobrevin as a multifunctional scaffold protein, and suggest that β-dystrobrevin might be involved in neuronal differentiation as a component of co-activator/co-repressor complexes required for regulation of neuronal gene expression.

 


P5.7

Organizational principles of the NuMA/LGN/Gαi complex: opening the switch to orient the mitotic spindle during asymmetric divisions
Andrea Alfieri1, Vaibhav Jadhav1, Marina Mapelli1
1European Institute of Oncology, IFOM-IEO-Campus, Milan, Italy

The asymmetric outcome of a cell division requires the correct placement of the mitotic spindle within the cell, a process coordinated by cortical force generators. Force generators are macromolecular machines able to localize at spatially-restricted cortical sites in conjunction with polarity cues and to generate microtubule pulling forces. In recent years the NuMA/LGN/Gαi complex has emerged as the core module of force generators in several systems. Topologically, LGN constitutes the molecular link between Gαi subunits anchored at the plasma membrane and the microtubule associated protein NuMA. To unveil the force generators\' organizational principles, we characterized the biochemical properties of the reconstituted NuMA/LGN/Gαi complex.
Data from our in vitro analyses are consistent with the \'conformational switch\' model, according to which during interphase LGN is kept in an inhibited state by head-to-tail intramolecular interactions, while in mitosis NuMA and Gαi synergize to prime the trimeric complex assembly. Interestingly, we found that the association of Gai with the four C-terminal GoLoco motifs of LGN is strongly cooperative, and suggest this might favor cortical clustering of force generators. Interaction studies also revealed that the LGN homologue AGS3 is endowed with very similar properties: it directly binds NuMA and exhibits a cooperative behavior toward Gαi.

 


P5.8

Role of KGFR/FGFR2b in the control of human keratinocyte differentiation
V. Purpura1, F. Belleudi2, M.R. Torrisi2
1Dipartimento di Medicina Sperimentale,“Sapienza” Università di Roma
2Dipartimento di Medicina Clinica e Molecolare,“Sapienza” Università di Roma

The keratinocyte growth factor (KGF/FGF-7) is a member of the FGF family which is secreted by mesenchimal cells and binds specifically to the tyrosine kinase KGF receptor (KGFR/FGFR2b) expressed exclusively on epithelial cells. KGF is a potent mitogen in vitro and promotes the early differentiation program of human cultured keratinocytes. Here we studied the role of KGFR in the epithelial differentiation process analyzing the effects in human HaCaT keratinocytes of the expression by transfection of KGFR WT, a kinase negative KGFR mutant or a KGFR generated with a Y769F mutation affecting the proliferative signaling. The differentiation program in subconfluent cells was induced by treatment with Thapsigargin, an inhibitor of endoplasmic reticulum Ca-ATPase pump family. Our results demostrated that the overexpression of KGFR WT and KGFR Y769F, but not of KGFR Kin-, induces an increased expression of the early differentiation marker K1 and a decreased expression of the late differentiation marker filaggrin, suggesting that: i) KGFR triggers early but inhibits late stages of differentiation in keratinocytes and ii) the Y769F mutation does not alter the KGFR-induced differentiation.

 


P5.9

TBX5 modulates the miR-218-1 expression: a possible cardiac circuit
L. Verduci1,2, A. Mercatanti2, L. Dolfi3, F. Cremisi4, L. Pitto2
1Scuola Superiore Sant\'Anna, Pisa, Italia
2Istituto di Fisiologia Clinica CNR, Pisa, Italia
3Univ. di Pisa, Italia
4Scuola Normale Superiore, Pisa, Italia

Transcription factors (TFs) are important components of gene regulatory networks. Like TFs, miRNAs are trans-acting factors that interact with many cis-regulatory elements. It is not surprising that TFs and miRNAs are embedded in common regulatory circuitries. TBX5 is a T-box transcription factor required for embryonic development of heart and forelimbs. An inverse correlation between TBX5 dosage and abnormal cardiac morphogenesis it has been demonstrated. Up to now there are no reports about the impact of TBX5 dosage alteration on miRNA expression. For that, we developed a software to interface gene expression data obtained by cDNA-microarray analysis of cells over-expressing TBX5 with database of annotated miRNAs. This allowed to identify several miRNAs that are candidate TBX5 targets. We focused on miR-218. We demonstrated that miR-218 is upregulated in TBX5 overexpressing C2C12 cells. Moreover, we showed a defined spatial/temporal expression of miR-218 in Xenopus laevis heart by in situ hybridization. Our data demonstrate the usefulness of the developed software in searching miRNAs controlled by TBX5 and suggest the existence of a cardiac circuit involving TBX5 and miR-218.

 


P5.10

Nitric oxide: new prospecs in skeletal muscle embryonic development
D. Cazzato1, S. Falcone2, C. Perrotta2, S. Brunelli3, G. Cossu3, E. Clementi4
1E. Medea Scientific Institute, Bosisio Parini, Lecco, Italy
2Unit of Clinical Pharmacology, Dept of Preclinical Sciences, Univ. Hospital "Luigi Sacco", Univ. degli Studi di Milano
3Division of Regenerative Medicine, San Raffaele Scientific Institute, Milan, Italy
4University of Milan

In skeletal muscle, nitric oxide (NO), synthesized by the neuronal and the endothelial isoforms of Nitric Oxide Synthase (NOS) has been previously suggested to have a role in physiology and regeneration. Using chicken embryos as model, we focused our attention on the possible role of NO in the early stages of muscle embryonic development. To this end, after 24 hours of development, embryos were treated for 48 h with the NOS inhibitor, L-NG-monomethyl Arginine citrate (L-NMMA) or with the NO donor(z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). L-NMMA-treated embryos showed morphological abnormalities as well as a reduced expression of the myogenic markers Pax7, Mef2c, Myogenin and Myosin, while an opposite effect was observed in DETA-NO-treated embryos. Morphological analysis was performed in 8 days-old embryos treated for 48 h and no difference between untreated and treated embryos was found, indicating that NO is essential in the early stage of embryonic development but not in the late phases. These results indicate that NO has a key and time-selective role in muscle development.

 


6. DNA damage and mutagenesis

Total number of abstracts in this session: 7

 

P6.1

Arabidopsis thaliana: a new model for the study of the tolerance to the oxidative stress
A.Amoroso1, L.Concia2, R.Cella2, G.Maga1

As obligatory phototropic organisms, plants are continuously exposed to elevated levels of endogenous genotoxic and environmental stresses, that threatens integrity of their genome. One of the more frequent modifications that affect DNA is the oxidation of bases induced by reactive oxygen species (ROS) that can to form adducts creating 7,8-diidro-8-oxo-guanina (8oxoG) as product. In our laboratory we have been studied, from the enzymatic and molecular point of view, the translesion DNA synthesis reaction (TLS) - a specific repair mechanism that takes part during the nuclear replication - in the bypass of 8oxoG in A.thaliana, a well established model for studying plant DNA repair. We have cloned and expressed three A.thaliana proteins (DNA pol λ, PCNA1 and PCNA2), whose mammalian homologs play key roles in TLS over oxidative DNA damage in mammalian cells. As in the case of mammalian DNA pol λ, we have shown that the Arabidopsis enzyme, which is the only member of the X family of DNA pol present in plants, is very efficient in performing error-free translesion synthesis past 8oxoG. Moreover, its fidelity and efficiency, as tested in vitro using a recombinant Arabidopsis enzyme, is greatly enhanced by the presence of auxiliary proteins PCNA and RP-A.

 


P6.2

Interaction between AID and spliceosome-associated CTNNBL1
S.G. Conticello1, K. Ganesh2, K. Xue2, M. Lu2, C. Rada2, M.S. Neuberger2
1Core Research Laboratory - Istituto Toscano Tumori, Firenze, Italy
2Medical Research Council - Laboratory of Molecular Biology, Cambridge, UK

Activation Induced Deaminase (AID) triggers the Somatic Hypermutation (SHM) and the Class Switch Recombination (CSR) processes in B-cells by deaminating deoxycytidine residues in the antibody genes. While not exclusively, under physiological conditions the primary target of AID is a specific region in the immunoglobulin gene locus, thus suggesting the existence of a targeting machinery.
We have used a cytoplasmic yeast-2-hybrid approach and co-immunoprecipitation assays to identify CTNNBL1 as a specific AID interactor. We have selected mutants of AID that, while retaining deoxycytidine deaminase activity in vitro, are not able to reconstitute neither CSR in B-cells from AID-deficient mice, nor SHM in DT40, a chicken lymphoma cell line. Furthermore, mutants of the DT40 cell line carrying a CTNNBL1 gene disruption exhibit a greatly diminished IgV diversification as well.
These results identify residues in AID involved in its in vivo targeting and suggest that they might act through interaction with CTNNBL1. The observation that CTNNBL1, is associated with the Prp19 complex of the spliceosome through its CDC5L-component, gives possible insight into the linkage between AID recruitment and target-gene transcription.

 


P6.3

DNA ligase I deficient cells: a model to study the impact of DNA damage on cytoskeleton organization
V. Leva, C. Galeone, G. Biamonti, A. Montecucco

DNA ligase I (LigI) is required for maturation of the Okazaki fragments. A LigI-deficiency was described in a patient with immunodeficiency and sun sensitivity that died with lymphoma. LigI mutant mice showed an increased incidence of spontaneous epithelial tumors. We have shown the occurrence of endogenous single stranded and double stranded DNA breaks in M10 cells expressing a phosphomimetic mutant of LigI with a dominant negative effect. This is accompanied by phosphorylation of the H2AX histone variant and the formation of γH2AX foci that mark damaged DNA. Single cell analysis demonstrates that the number of γH2AX foci in LigI defective cells fluctuates during the cell cycle: they form in S-phase, persist in mitosis and eventually diminish in G1-phase. Unexpectedly, replication-dependent DNA damage in M10 cells only moderately delays cell cycle progression and does not activate the S-phase specific ATR/Chk1 checkpoint pathway that also monitors the execution of mitosis. In contrast, the ATM/Chk2 pathway is activated. Notably, the expression of the phosphomimetic mutant drastically affects cell morphology and the organization of the cytoskeleton unveiling an unexpected link between endogenous DNA damage and the structural organization of the cell.

 


P6.4

Multiple pathways regulate 3' overhang generation at S. cerevisiae telomeres
D. Bonetti, M. Martina, M. Clerici, G. Lucchini, M.P. Longhese
Dept Biotecnologie e Bioscienze, Milano-Bicocca Univ., Milan, Italy

Generation of 3\' G-strand overhangs at telomere ends may play a role in regulating telomerase action and occurs by still unclear mechanisms. We show by an inducible short telomere assay that Sae2 and the Sgs1 RecQ helicase control two distinct but partially complementary pathways for nucleolytic processing of S. cerevisiae telomeres, with Sae2 function requiring its serine 267 phosphorylation. No processing activity is detectable in sae2Δ sgs1Δ cells, while the Exo1 exonuclease contributes to telomere end processing and elongation in both sae2Δ and sgs1Δ cells, suggesting that Exo1 telomeric function requires either Sgs1 or Sae2 action. Moreover, also Dna2 might support Sgs1 activity, as it acts redundantly with Exo1, but not with Sgs1. Finally, both length maintenance and G-strand overhang generation at native telomeres are affected in sae2Δ sgs1Δ cells, further supporting the notion that Sae2 and Sgs1 combined activities control telomere length by regulating telomere processing.

 


P6.5

The replication dynamics at common fragile site FRA3B and its possible involvement in the occurrence of genomic instability
E. Tosoni, E. Palumbo, L. Matricardi, A. Russo
Dept Biology, Univ. Padova

Common fragile sites (CFSs) are expressed as chromosome breaks or gaps after partial inhibition of DNA replication, which contribution to CFSs instability is still controversial. We investigated the replication dynamics at FRA3B in human primary lymphocytes and in the lymphoblastoid TK6 cell line. By FISH on TK6 nuclei, late replication was confirmed for FRA3B both in control cells and in a late S-phase enriched (LSE) subpopulation, in which an enhanced frequency of replicated alleles was observed (CTR: 18%, LSE: 43%, P < 0.001); the early replicating sequence LAMIN B2 (LMNB2) was used as control. On the same TK6 cell samples and on primary lymphocytes, the whole genome replication pattern was evaluated by molecular combing, and comparable replication rates and interorigin distances (R = 0.96) were found. On primary lymphocytes, similar mean fork rates were found at LMNB2 (1.17±0.15 kb/min) and at FRA3B (FRA3B 1.40±0.26 kb/min), while unidirectional forks seemed overrepresented at the CFS (LMNB2 19%, FRA3B 31%). The analysis on the LSE TK6 subpopulation is in progress. Moreover, data collected by molecular combing suggest that FRA3B is affected by high instability in its sequence.

 


P6.6

RAD51 SNPs: correlation with clinical radiation sensitivity and protein expression
S. Sterpone1,2,3, L. Vaslin2,3, M. Fernet2,3, D. Landi4 , S. Landi4, J. Hall2,3, R. Cozzi1
1Dept Biology, Univ. degli Studi “Roma TRE”, Italy
2Unité 612-INSERM, Centre Universitaire,, Orsay , France
3Institut Curie, Centre Universitaire, Orsay, France
4Dept Biology, Univ. di Pisa, Italy

It is well known from clinical observations that a considerable inter-patient heterogeneity in normal tissue reactions can be seen in cancer patients following radiotherapy. Part of these differences can be explained by treatment protocols and other predisposing factors but part of the variations are also due to genetically determined intrinsic differences in the cellular responses to damage. Among the possible biological endpoints, the detection and repair of induced-DNA damage are among the most relevant for different clinical side effects. Certain SNPs can alter the expression and functional properties of the corresponding enzymes and the increased risk of adverse reaction to RT. We investigated whether two SNPs in the DNA repair gene RAD51, ex10-52T>C located in a potential mir34 miRNA binding site and ex1-96G>C, a gene modifier for breast cancer risk in BRCA2 mutation carriers, are associated with an increased risk of developing an adverse reaction to RT in a cohort of BC patients. The two SNPs are genotyped by sequencing and RFLP-PCR and these genotyping studies are complemented by investigating the levels of mir34 and the Rad51 mRNA and protein in lymphoblastoid cell lines.

 


P6.7

The zebrafish as model system for the study of ROS-mediated DNA-damage in vivo
G. Deflorian1,2, C. Santoriello2, R. di Micco2, F. Pezzimenti1,2, F. d’Adda di Fagagna2, M. Mione2
1Cogentech, Consortium for Genomic Technologies, Milan, Italy
2IFOM-FIRC, Institute for Molecular Oncology Foundation, Milan, Italy

Our understanding of the DNA-damage (DD) repair system has been gained primarily from cell culture models, which cannot recapitulate dynamic in vivo processes such as temporally and spatially regulated patterns of gene expression. To investigate the interplay between oncogene expression and cancer development in vertebrates and to verify the role of reactive oxygen species (ROS)-mediated DD in this system, we have generated a zebrafish transgenic line, Tg(HSP70::eGFP-H-RASV12), in which the expression of the human activated oncogene H-RASV12 (tagged with the reporter gene eGFP) can be activated at will through heat shock. Activation of HRASV12 expression in zebrafish leads to a proliferative burst associated with hyper-production of ROS. Markers of DD responses are increased and in most cases this leads to arrest in proliferation and increased apoptosis in a few days. The abnormal phenotype of HRASV12 transgenic embryos after HS, and the consequent ROS hyper-production, was partially rescued by L-NAC treatment. In summary, our studies demostrate that oncogene induced DD seems to be caused at least in part by an increase in ROS production and benefit from ROS scavenger treatment.

 


7. Environmental microbiology and microbial ecology

Total number of abstracts in this session: 6

 

P7.1

Antibacterial evaluation of a new textile fabric to make hospital gowns
I. Anacarso, S. de Niederhausern, R. Iseppi, C. Sabia, G. Manicardi, P.Messi, M. Bondi
Dept of Biomedical Sciences, Univ. of Modena and Reggio Emilia, Modena

Since hospital-acquired infections have a significant impact in terms of human lives, the use of professional clothing that can minimise the microorganism transmission would be recognized as an important instrument of prevention. For this purpose in collaboration with the SIGGI GROUP SPA we compared the antibacterial activity of an experimental hospital uniform added with antimicrobial substances, with the traditional gown. For the “in vitro” study textile samples were artificially contaminated with four bacteria (E.coli ATCC 25922, P.aeruginosa ATCC 27853, S.aureus ATCC 6538, E.faecalis ATCC 29212), stored at 37°C and evaluated at different times for bacterial killing activity. Subsequently the study was performed on both types of uniforms worn by hospital medical staff, monitoring the viable count at the beginning and at the end of duty period.
The in vitro results showed a remarkable bactericidal action of the experimental uniforms compared to those traditional (reduction >99.9 of the viable counts after few hours).
The test performed on the gowns worn, although with less evident results, showed the ability of the experimental uniforms to reduce at the end of duty period the increase of viable counts in particular for Gram negatives.

 


P7.2

Human safety and health inside manned space modules: microbial contamination and biofilm development
F. Canganella, G. Bianconi, E. di Mattia

Most of biological space research has been usually devoted to medical issues but current biological research is mainly focused on advanced life support and biosafety: 1) in situ production of fresh food; 2) novel foods and food supplements for astronauts health; 3) biocontrol of microorganisms and biocorrosion inside space modules.
We have carried out research activities concerning biofilm development and metabolic activities of some reference bacteria on materials commonly used for aerospace industry and currently examined for space greenhouses. It was evaluated the effect on these materials of a mixture of emulsifiers produced by Pseudomonas strain AD1. Results showed a diverse affinity of materials for bacterial biofilm formation and occasionally sessile colonization was rejected. Pre-conditioning with the emulsifying extract led in some cases to a diminish of biofilm dehydrogenase activity and development compared to untreated materials, taking into account both concentrations and experimental conditions. These results are also related to the relationship between the physical traits of materials and the level of bacterial biofilm developed under the experimental conditions. Data may be useful to select appropriate material to be used for life support hardware to decrease the risk of surface biocontamination and health problems inside space modules, a great challenge for both biological and medical research.

 


P7.3

Predominance of Alcanivorax genus during simulation of oil spill bioremediation in microcosms
S. Cappello1, F. Smedile2, M. Genovese1, R. Denaro1, M. Hassanshanian3, G. Emtiazi3, M.M. Yakimov1
1Istituto per l'Ambiente Marino Costiero (IAMC), CNR of Messina, Italy
2Dept Biologia Animale ed Ecologia Microbica, Univ. of Messina, Italy
3Dept of Biological Sciences, Univ. of Isfahan, Iran

Microcosm experiments simulating an oil spill event were performed in natural seawater in order to evaluate the response of the natural microbial community following the accidental load of petroleum. Three different series of experiments were carried out. Those experiments were indicated as “Oil", “Oil+IN" and “Oil+Bio" microcosm. All microcosms were supplemented with crude oil, the "Oil+Bio" microcosm was also supplemented with a biosurfactant and the "Oil+IN" microcosm with inorganic nutrients.
Measures on the bacterial abundance, microbial community (16S crDNA) composition and analysis of hydrocarbons (GC-MS) were carried out during all the experimental period (15 days).
Data obtained shown how addition of inorganic nutrients and biosurfactant induces an increase in bacterial abundance and led to a progressive predominance, inside the microbial population, of bacteria related to Alcanivorax genus.
Data obtained in microcosm studies improve our understanding of the natural processes occurring during oil spills and put in evidence how the bacteria related to genus of Alcanivorax are the most important “players” in the bioremediation of oil-contaminated marine environments.

 


P7.4

Biochemical characterization of a recombinant Anopheles gambiae tryptophan 2,3-dioxygenase
A. Cavagnino1, A. Paglino1, F. Lombardo2, B. Arcà2,3, C. Mowat4, S. Chapman4, M. Rizzi1, F. Rossi1
1DiSCAFF, Univ. of Piemonte Orientale “A. Avogadro”, Novara, Italy
2Dept of Public Health, Parasitology Section, Univ. “La Sapienza”, Roma, Italy
3Dept of Structural and Functional Biology, Univ. “Federico II”, Napoli, Italy
4School of Chemistry, Univ. of Edinburgh, Scotland

Malaria is caused by protozoan parasites of the genus Plasmodium, which are horizontally spread throughout human population by the bites of infected Anopheles mosquitoes.
In insects, the metabolites produced along the Kynurenine Pathway (KP) for tryptophan oxidation are implicated in a variety of biological functions. Of great interest is the finding that, in Plasmodium-infected Anopheles, the KP metabolite Xanthurenic Acid acts as an essential trigger of the sexual development of the malaria parasite. Thus, the biochemical analysis of enzymes playing a key role in the KP of the mosquito could inspire the development of novel insecticides and transmission-blocking drugs.
In Anopheles, the heme-dependent Tryptophan 2,3-dioxygenase (TDO) is the only enzyme able to initiate tryptophan degradation through the KP, by catalyzing the first and rate-limiting step of the catabolic cascade. We report here the biochemical characterization of a recombinant form of Anopheles gambiae TDO, which has been highly expressed in and easily purified from E. coli. The enzyme was characterized by spectroscopic analysis and subsequently used to determining kinetics parameters, binding affinity constants for substrates and redox state.
Our data could be exploited for the identification/selection of TDO inhibitors to be used to alter Tryptophan homeostasis in the malaria vector.

 


P7.5

Correlating gene expression and enzymatic activities data: a case study of nitrification assessment in Zinc contaminated soils
E. Puglisi, S. Vasileiadis, D. Coppolecchia, R. E. Hamon, M. Trevisan
Istituto di Chimica Agraria ed Ambientale, Univ. Cattolica del Sacro Cuore, Piacenza

Soil enzymatic activities are commonly used to assess the ecological status of soils. Most methods used are based on the measurement of product released after soil incubation with the enzyme substrate. On the other hand, advances in molecular biology allow now the quantification of genes involved in soil enzymatic processes.
Aim of the present work is to correlate results obtained by the substrate induced nitrification (SIN) method with RT-PCR quantification of the amount and level of expression of the ammonia monoxygenase (amo) gene. Analyses were carried out on a case study with soil samples spiked in the lab with Zinc concentrations up to 5000 mg kg-1. RT-PCR analyses were carried out on the amount (DNA) and level of expression (RNA) of the amo gene of both β-proteobacteria and Archaea. 16S RT-PCR analyses were also carried out to quantify the abundance of the two microbial groups
Results showed a decrease in both amo and 16S genes, thus suggesting that Zinc toxicity involve a reduction of both living cells and gene expression levels. A good correlation between SIN and RT-PCR data was also found. Advantages and drawbacks of both approaches will be discussed.

 


P7.6

Effect of inoculum carrier on fungal remediation of polycyclic aromatic hydrocarbons-contaminated matrices
S. Covino1, E. Galli2, M. Petruccioli1, A. D’Annibale1, Z. Kresinova3, P. Erbanova3, C. Novotny3, T. Cajthaml3
1Dip. di Agrobiologia e Agrochimica, Univ. of Tuscia, Viterbo, Italy
2IBAF-CNR, Area di Montelibretti, Roma, Italy
3
Experimental Mycology Laboratory, Inst. of Microbiology, Academy of Sciences of Czech Republic, Prague, Czech Republic

Main objective of this study was to select effective fungal strains for PAH degradation in several aged-contaminated matrices, such as a contaminated soil from a wood treatment plant (in Sobeslav, Bohemia) and shavings of creosote-contaminated hardwood sleepers arising from the same plant. An additional aim was to identify the most suitable inoculum carrier among a variety of lignocellulosic residues to favour contaminated matrices colonization. A preliminary screening procedure utilizing industrial dyes as model compounds, led to the selection of four white rot strains (Irpex lacteus 617/93, Dichomitus squalens, Pleurotus ostreatus N3 and 3004) and one brown rot (Coprinus comatus). Three different lignocellulosic substrates as inoculum carriers and, among them, granulated straw resulted to be the most suitable one, while chopped wheat straw was the least effective. Among the strains under study, I. lacteus was the most robust fungus for polluted soil/woods colonization. Furthermore, a 52% degradation of PAHs in the creosote-treated wood by I. lacteus was the highest, using both granulated straw and corn cobs as growth substrates. P. ostreatus 3004, previously grown on pelleted straw, was the most efficient PAH degrader leading to a 80% removal from the contaminated soil.

 


8. Epigenetics

Total number of abstracts in this session: 4

 

P8.1

A ncRNA regulates the epigenetic switch at the basis of facioscapulohumeral muscular dystrophy (FSHD)
D.S. Cabianca1, M.V. Neguembor1, D. Gabellini1,2
1Division of Regenerative Medicine, San Raffaele Scientific Institute, Milano, Italy
2Dulbecco Telethon Institute, Milan, Italy

FSHD, the third most common myopathy, is an autosomal dominant disease whose locus maps at 4q35.
In FSHD, reduction in the copy number of 3.3 kb repeats called D4Z4 causes an epigenetic switch leading to de-repression of 4q35 genes.
In healthy subjects, D4Z4 copy number ranges from 11 to 100 repeats, while FSHD patients carry less than 11 units. Importantly, at least one copy of D4Z4 is required to cause FSHD suggesting for a gain of function mechanism.
We found that a chromatin-bound non coding RNA (ncRNA) is transcribed proximally to D4Z4 selectively in FSHD patients. Interestingly, in several experimental settings production of the ncRNA correlates with de-repression of 4q35 genes. Notably, knockdown of the ncRNA prevents 4q35 gene de-repression.
We analyzed the recruitment of Polycomb (PcG) and Trithorax (TrxG) proteins (and related histone marks) to 4q35. Interestingly, we found that recruitment of the TrxG Ash1 and H3K4me3 correlate with production of the ncRNA and de-repression of 4q35 genes. Notably, Ash1 knockdown prevents 4q35 gene de-repression.
We propose that the ncRNA recruits Ash1 to activates the epigenetic cascade culminating with 4q35 gene de-repression in FSHD.

 


P8.2

A novel mammalian flavin-dependent histone demethylase
A. Karytinos1, F. Forneris1, A. Profumo2,
G. Ciossani1, E. Battaglioli3, C. Binda1, A. Mattevi1
1Dip. di Genetica e Microbiologia, Univ. di Pavia, Pavia, Italy
2Dip. di Chimica Generale, Univ. di Pavia, Pavia, Italy
3Dip. di Biologia e Genetica per le Scienze Mediche, Univ. di Milano, Milano, Italy

Methylation of Lys residues on histone proteins is a well characterized epigenetic mark, and the discovery of lysine-specific demethylase 1 (LSD1) demonstrated that lysine methylation can be dynamically controlled.
Among the histone demethylases so far identified, only LSD1 works through a flavin-dependent amine oxidation reaction. Database analyses reveal that mammalian genomes contain a gene homologous to the LSD1-coding gene; in our work we show that the protein encoded by this gene, LSD2, represents a second flavin-dependent histone demethylase.
LSD2 is specific for mono and dimethylated Lys4 of histone H3 N-terminal tail, senses the presence of additional epigenetic marks on the substrate, and is inhibited by tranylcypromine. As opposed to LSD1, LSD2 does not form a stable complex with the C-terminal domain of the corepressor CoREST. Furthermore, LSD2 contains a CW-type zinc finger motif with potential zinc-binding sites not present in LSD1.
We conclude that LSD2 represents a new flavin dependent H3-Lys4 demethylase that features substrate specificity properties highly similar to those of LSD1 but is very likely to be part of different chromatin-remodeling complexes.

 


P8.3

Epigenetic regulation of hypoxia-induced PlGF expression
L. Tudisco, MR. Matarazzo, F. Della Ragione, M. D’Esposito, S. De Falco
CNR, Institute of genetics and Biophysics, "ABT"

Placental Growth Factor (PlGF) is a member of Vascular Endothelial Growth Factor (VEGF) family. It has been reported that hypoxia regulate PlGF expression but the direct involvement of hypoxia-inducible factor-1α (HIF-1α) has not been demonstrated: therefore the mechanism underlying hypoxia-induced PlGF expression is still unclear. We report that PlGF expression is induced by hypoxia in primary endothelial cells as demonstrated both by mRNA and protein quantification. We investigated whether the hypoxia-induced PlGF expression is mediated by epigenetic regulation. Bisulfite sequencing show that methylation status of the PlGF promoter is unchanged. We also analyzed the acetylation status of histones H3 and H4 by ChIP assay: the acetylated isoforms of histones H3 and H4 were enriched in the PlGF intron-2. Sequence analyses of this region revealed the presence of several putative binding sites for HIF-1α and we were able to detect the binding of HIF-1α to this region. These results suggest that the PlGF intron-2 may have a chromatin structure that undergoes change upon being stimulated by hypoxia and we provide new elements in favor of HIF involvement in PlGF hypoxic regulation.

 


P8.4

Global repression of cancer gene expression in a zebrafish model of melanoma is linked to epigenetic regulation
V. Anelli1, C. Santoriello1, M. Distel3, R.W. Köster3, F.D. Ciccarelli2, M. Mione1
1IFOM, The Firc Institute of Molecular Biology, Milan, Italy
2IEO, Institute of European Oncology, Milan, Italy
3Helmholtz Center Munich, Institute of Developmental Genetics, Munich, Germany

We have established a model of melanoma progression in zebrafish through the generation of transgenic lines specifically expressing oncogenic human HRAS in the melanocitic lineage. In these tumors we have carried out quantitative analysis of expression of several potential cancer genes, from known1 and predicted cancer gene lists. In particular we analysed 39 out of 101 novel cancer genes identified with a bioinformatics approach2 and selected for the low frequency of duplication and the high connectivity in protein networks. Data obtained by real time-PCR analysis from zebrafish melanoma tissue show that the expression of many cancer genes is down-regulated in zebrafish melanomas whereas only cell cycle genes are up-regulated. In order to understand whether this trend is due to global repression of genes expression associated to a repressive chromatin state, we investigated whether changes of histone methylation were detectable in our melanoma model. We found substantial differences in the levels of H3K9me3, H4K20me2, H3K27me3, H3K4me3 and H3R2me2a immunostaining in melanoma tissue as compared with normal skin. Thus our analysis suggests that in our model, like in human melanoma, important changes occur to the methylation status of histones, leading to a global repression of gene expression through epigenetic regulation. 1. Futreal PA, Coin L, Marshall M, et al. A census of human cancer genes. Nat Rev Cancer 2004; 4:177-83. 2. Rambaldi D, Giorgi FM, Capuani F, Ciliberto A, Ciccarelli FD. Low duplicability and network fragility of cancer genes. Trends Genet 2008; 24:427-30.

 


9. Evolution

Total number of abstracts in this session: 11

 

P9.1

The genetic structure of the endangered species Patella ferruginea (Mollusca: Gastropoda) suggests possible incipient speciation in Sardinia
G. Dedola1,2, T. Lai1, D. Sanna1, M. Fais1, B. Cristo1, I. Vanetti2, G. Binelli2, M. Curini-Galletti1, M. Casu1
1Dip. di Zoologia e Genetica Evoluzionistica, Univ. di Sassari, Italy
2Dip. di Biotecnologie e Scienze Molecolari, Univ. degli Studi dell’Insubria, Italy

The ferruginean limpet Patella ferruginea, formerly distributed in Western Mediterranean, is now limited to a few restricted areas, due to intense human exploitation. Its distribution in Northern Sardinia is particularly fragmented, the larger populations being encompassed by Marine Protected Areas (MPAs). In order to plan conservation strategies for this species, we studied the levels of genetic variability within and among populations by means of ISSRs, SSRs and, for phylogeographic purposes, of COI sequences.
ISSRs evidenced a low level of gene flow (FST < 0.02 n.s.) between populations from North-East Sardinia (where two relatively close MPAs are present), whereas populations from North-West Sardinia (with two far apart MPAs) show strong differentiation (FST > 0.31 ***), suggesting that gene flow is relevant only at very small geographic scale. The use of heterologous SSRs developed in the congener P. rustica and P. depressa hints at the possibility that at least some populations are tetraploids. Since in Northern Africa diploid populations only are known, this finding suggests a possible phenomenon of incipient speciation. The issue is being investigated by RT-PCR and karyotype determination after cell culture.

 


P9.2

The novel mtDNA haplogroup R: the contribution of Italian aurochsen to modern cattle
S. Bonfiglio1, A. Achilli1,2, A. Malusà1, A. Olivieri1, M. Pala1, B. Hooshiar Kashani1, U. A. Perego1,3, P. Ajmone-Marsan4, R. Negrini4, M. Pellecchia4, L. Colli4, O. Semino1, A. Torroni1, L. Ferretti1
1Dept Genetics and Microbiology, Pavia Univ., Pavia, Italy
2Dept Cellular and Environmental Biology, Perugia Univ., Perugia, Italy
3Sorenson Molecular Genealogy Foundation, Salt Lake City, Utah, USA
4Institute of Zootechnics, Catholic Univ. of the Sacred Heart, Piacenza, Italy

A Neolithic domestication of taurine cattle in the Fertile Crescent from local Bos primigenius is generally accepted, but a genetic contribution from European aurochsen is still under debate. We surveyed about 1000 taurine cattle mitochondrial DNA (mtDNA) from 26 European breeds confirming the overall clustering within haplogroups of Near Eastern ancestry (T1, T2 and T3), but also identifying 21 mtDNAs (2.2%) not clustering within haplogroup T. Whole mitochondrial genome sequencing showed that nine mtDNAs formed a novel haplogroup R which represents a very early split in the mtDNA phylogeny of B. primigenius. The remaining 12 samples belonged to the recently discovered haplogroup Q. Phylogeographic data indicate that R mtDNAs might derive from female aurochsen of the Italian Peninsula sporadically included in domestic herds, whereas Q and T subclades were most likely involved in the same event of Neolithic domestication in the Near East. Since the analysis of the maternally transmitted mtDNA underestimates the extent of gene flow from European aurochsen, the detection of the R mtDNAs in Italian breeds reveals an unexpected reservoir of genetic variation that should be
carefully preserved.

 


P9.3

Simulating genetic and cultural factors in human evolution by a multi-agent system
A. Colosimo
Dept Physiology and Pharmacology, Sapienza Univ., Rome, Italy

Even for people with good informatic background modeling the relative influence of genetic and cultural factors in human evolution was difficult – among other things – because of the problematic choice of appropriate hardware [parallel\\sequential] and software [continuous\\ discontinuos algorithms] computational strategies. A significant improvement in the field was introduced by a new generation of programming languages, primarily Lisp [http://it.wikipedia.org/wiki/Lisp] and Logo, and the ensuing availability of software tools specifically designed to simulate the collective, often unpredictable, behaviour of large sets of independent agents each endowed with relatively simple properties. We used the unique friendliness and flexibility of one of such tools, Netlogo [http://ccl.northwestern.edu/netlogo/] , to simulate the behaviour of populations whose members can take advantage of both genetic and cultural mechanisms to adapt to a changing environment. The results were found in qualitative agreement with data from real populations of different economic level and technological sophistication.

 


P9.4

Evolution and conservation of the 3'Regulatory Region of the Ig heavy chain: apes introduced a new SP1 consensus in the HS1.2 enhancer
S. Lolli1, P. D'Addabbo2, C. Martinez-Labarga3, M. Scascitelli4, V. Giambra5, F. Pandolfi1, M. Rocchi2, D. Frezza3
1Dip. di medicina interna, Univ. cattolica del sacro Cuore, Roma
2Dip. di Genetica e Microbiologia, Univ. degli studi di Bari
3Dip. di Biologia E.Calef, Univ. degli studi di TorVergata, Roma
4Dept of Botany, Univ. of British Columbia, Vancouver, Canada
5Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada

The 3\'Regulatory Region of the immunoglobulin heavy chain cluster is conserved between mouse and human with 3 enhancers, HS3, HS1.2 and HS4, organized in same way. Here we describe the status of variation and conservation of these three enhancers in 18 different species: 1 bird and 17 mammals. In order HS3 and HS1.2 are more conserved in respect to HS4, the most downstream enhancer of cluster. In particular, the polymorphic enhancer HS1.2 shows a structure with different conserved elements. It consists of a 38bp element repeted from 1 to 4 copies preceded by a structure alternatively of a 31bp or 17bp . In Macaca is present one allele with 7 copies of the 38mer with the 31mer at the 5\'. In Pan troglodyte was found one allele with 12 copies of the 38mer preceded by the 31mer element, while the allele with 1 copy of the 38mer is preceded by the 17mer as in humans. These data show that HS1.2 is present from 80 mya. The differences observed and the main structures conserved in the enhancers suggest also the conservation of specific features. The 31mer structure in silico shows a consensus for USF for insulator functions, and the 17mer specific for SP1 factor.

 


P9.5

On the origin and evolution of nitrogen fixation genes
M. Fondi, L. Presta, I. Maida, E. Perrin, M. C. Papaleo, B. Tarabella, R. Fani
Dept of Evolutionary Biology, University of Florence

The building up of nitrogen fixation represented a metabolic innovation that is not only crucial for the extant life, but played a key role in the early stages of evolution as the prebiotic supply of all nitrogen sources decreased. The ancestral nif pathway might have originated in the early stages evolution and the entire process might have been carried out by a limited number of genes coding for multifunctional, unspecific enzymes that could react with a wide range of chemically related substrates. These primordial enzymes were responsible for the interconnection of nitrogen fixation to other metabolic routes, such as bacterial photosynthesis and biosynthesis of leucine/lysine. Gene and operon duplications, gene recruitment and elongation events and an extensive horizontal transfer of nif genes played a crucial role in shaping the entire pathway that was completely assembled before the appearance of the Last Universal Common Ancestor. We report a detailed comparative analysis, based on a computational biology approach, of the available sequences of proteins involved in nitrogen fixation and propose a model for the major evolutionary steps of nitrogen fixation process.

 


P9.6

Ty1-copia retrotransposon-based S-SAP marker development in Myrtus communis L.
I. Kafantaris , P. Woodrow , G. Pontecorvo
Dept of Life Science, II Univ. of Naples, Caserta, Italy

Retrotransposons have proved useful for molecular marker studies and play an important role in genome evolution among different species. The most popular retrotransposon-based molecular marker system for the identification of closely related species, from a defined geographical location, is the sequence-specific amplified polymorphism (S-SAP).
In this study we isolated a new Ty1-copia like retrotransposon in the genome of the plant of Myrtus communis L. and our results also provided evidence that it is present in a high copy numbers. The LTR regions were used successfully to develop S-SAP marker system in order to distinguish four different populations of Myrtus communis L. In fact the clear S-SAP patterns revealed high insertion polymorphisms.
Our finding demonstrate that S-SAP is able to detect retrotransposon polymorphisms and is an efficient way for accession identification and discrimination in Myrtus communis L. The variety/species identification is an important link between the conservation and utilization of a commercially important plant such as Myrtus communis L.

 


P9.7

Methylated genes and non methylated transposons in the honey bee Apis mellifera
M. Mandrioli1, C. Tepedino2
1Dept of Animal Biology, Univ. of Modena and Reggio Emilia, Modena, Italy
1Dept of Paleobiology Museum and Botanical Garden,Univ. of Modena and Reggio Emilia, Modena, Italy

The availability of Apis mellifera genome project prompted us to study the roles played by DNA methylation in honey bees. At this regard, methylated sequences have been isolated by chromatin immuno-precipitation (ChIP) and the occurrence of transposable elements (TEs) in the precipitate methylated fraction of the honey bee genome verified by PCR. Strikingly, no TE has been observed within the methylated fraction of the genome. Interestingly the same TEs have been immuno-precipitated with an anti-monomethylated-K9 histone H3. Successively, 50 immuno-precipitated DNA fragments have been cloned and sequenced and they corresponded to internal portions of transcribed genes and never corresponding to regions located upstream to genes or between genes (intergenic spacer). Intriguing new scenarios can be therefore suggested in insects where the occurrence of a cross-talk between histone and DNA methylation should be carefully verified since this interaction seem to be absent in insects. Furthermore, the existence of mechanisms for TEs control at a transcriptional level in mammals and plants could be due to convergence since they are absent in insects.

 


P9.8

Evolutionary analysis of the LCR9 segmental duplication in hominoids
F. Vella, E. Belloni, M. Bensi, V. Biscaro, F. Coperchini, V. De Pascali, C. Marcialis, E. Raimondi.
Dept Genetics and Microbiology, A. Buzzati Traverso Univ., Pavia, Italy

Segmental duplications (SDs) are euchromatic portions of genomic DNA (≥ 1 kb) that occur at more than one site within the genome, and typically share a high level of sequence identity (>90%). In a previous work we reported the detailed investigation of LCR9 duplications (Paulis et al., Genomics 87-2006, 747-757). Moreover we demonstrated that LCR9 duplicons play a crucial role in driving structural chromosome rearrangements in germinal and somatic cells. We also studied the chromosomal distribution of LCR9 paralogs in hominoids, thus observing that paralogous copies of the duplication are located at evolutionary breakpoints. Here we investigate the dynamic nature of LCR9 during hominoid evolution. We identified the highest LCR9 copy number in human. Our analysis of LCR9 duplicon evolution revealed that SDs contributed to inter-species variation. Furthermore, we could elucidate the link between LCR9 scattering and the phylogeny of human chromosomes 1, 2, 7, 9, 15, 16 and 22.

 


P9.9

GLO-like and DEF-like MADS-box genes in orchids: copy number and evolutionary implications
C. Cantone, L. Gaudio, S. Aceto
Dept of Biological Sciences, Section of Genetics and Molecular biology, Univ. of Naples Federico II, Naples, Italy

DEF- and GLO-like loci encode MADS-box transcription factors involved in flower development. The “orchid code” attributes the high degree of flower diversity of the Orchidaceae to duplication and sub/neo-functionalization of DEF-like genes. Four copies of DEF-like were described in orchids, whereas GLO-like genes seem to be single copy loci, excluding HrGLO1 and 2 of Habenaria (Orchidoideae). We identified four different DEF-like cDNAs in Orchis (Orchidoideae). Phylogenetic analysis confirmed the existance of four DEF-like clades reproducing the systematic relationships reported for orchids. Surprisingly, we found a new GLO-like cDNA (OrcPI2), different from the known OrcPI gene. OrcPI and OrcPI2 nucleotide sequences are similar to HrGLO1 and HrGLO2, respectively. Phylogenetic analysis showed the presence of a cluster including almost all the orchid GLO-like cDNAs (comprising HrGLO2 and OrcPI2) and a second cluster including OrcPI and HrGLO1. The position of the latter cluster suggests the duplication of the orchid GLO-like genes took place after the divergence of the Cypripedioideae and Vanilloideae, with differential gain and loss of duplicate genes during the orchid evolution.

 


P9.10

When migration mimics population contraction in genetic variation data: a simulation study
G. Bertorelle, A. Benazzo, S. Mona
Dipartimento di Biologia ed Evoluzione, Univ. di Ferrara, Ferrara, Italy

The effective number of individuals, Ne, in a population or a species is a fundamental parameter in population genetics and evolution. The relative role of genetic drift and natural selection during the evolution of a species is strictly related to Ne. Statistical methods based on the coalescent theory allow the estimation of this parameter and the reconstruction of its changes through time. However, this inference relies on the assumption that the population under investigation evolved in isolation. In this study we investigated if and how different levels of migration affect the statistical reconstruction of the population size dynamic based on molecular data. We simulated genetic data sets assuming a model with migration, and estimated the population size dynamic in a single population using the Bayesian Skyline plot algorithm, a bayesian coalescent method which assumes complete isolation. We showed that migration can frequently mimic the effect of a population decline in a population with constant size. In other words, DNA data can erroneously support a bottleneck effect in a population receiving immigrants. This result should be considered when evolutionary processes producing a population contraction are inferred from the genetic variation data.

 


P9.11

In silico comparative analysis of antibiotics RND-efflux pumps in Burkholderia genus
E. Perrin1, M. Fondi1, M.C. Papaleo1, I. Maida1, B. Tarabella1, S. Buroni2, M.R. Pasca2, G. Riccardi2, R. Fani1
1Laboratory of Molecular and Microbial Evolution, Dept of Evolutionary Biology, Univ. of Florence, Florence, Italy
2Dept of Genetics and Microbiology, Univ. of Pavia, Pavia, Italy

The genus Burkholderia includes a variety of species inhabiting different ecological niches, with opportunistic human pathogenic strains, whose increasing global resistance to antibiotics has become a public health problem. In this context a major role is played by multidrug efflux pumps belonging to RND family, that allow bacterial cells to extrude a wide range of different substrate, including antibiotics. We performed an extensive phylogenetic analysis of these proteins in the 21 completely sequenced Burkholderia genomes, and we found the occurrence of several independent events of gene loss and duplication across the different lineages, leading to notable differences in the number of paralogs between different genomes. A putative substrate (antibiotics/heavy-metal) was also assigned to the majority of these proteins. Interestingly, we found no correlation between the ecological niche and the lifestyle of Burkholderia strains and the number/type of efflux pumps they possessed, while a relation can be found with genome size and taxonomy. Taken together these data might represent an important reservoir of targets for further experimental validations.

 


10. Genetics of microorganisms

Total number of abstracts in this session: 13

 

P10.1

Involvement of a sRNA in the regulation of plasmid virulence genes of Shigella flexneri
M. Giangrossi 1, G. Prosseda 2, C. N. Tran 1, A. Brandi 1, B. Colonna 2, M. Falconi1
1Dip. di Biologia MCA, Università di Camerino, Camerino (MC)
2Istituto Pasteur-Fondazione Cenci Bolognetti, Dip. di Biologia Cellulare e Sviluppo, Sapienza Univ. di Roma, Italy

Like in other life-threatening human pathogens also in Shigella and enteroinvasive E.coli, the etiological agents of bacillary dysentery, regulation of virulence gene occurs at diverse levels and is modulated by the host environmental stimuli. Shigella is a highly adapted human pathogen that causes bacillary dissentery, a severe enteric syndrome, mainly in the developing world. Recently small noncoding RNAs (sRNA or ncRNA) 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 important for bacteria to survive within the host. We have recently identified a sRNA transcribed from the pINV plasmid of S. flexneri. Data obtained indicates that it acts as antisense and interferes at transcriptional level with the expression of the target gene. This opens new and interesting perspectives in deciphering the expression of invasivity genes in Shigella.

 


P10.2

Pyrosequencing analysis of the D1-D2 region of the 28S ribosomal DNA discriminates between clinical and environmental isolates of Sporothrix schenckii and reveals two well-conserved nucleotide polymorphisms
B. Costanzo1, O. Romeo1, F. Scordino1, G. Criseo1
1Dept of Life Sciences section of Microbiological, Genetic and Molecular Sciences, Univ. of Messina, Italy

The authors reported a genetic characterisation of environmental and clinical isolates of Sporothrix schenckii, the aetiological agent of a chronic granulomatous mycosis in humans and animals called sporotrichosis.
The environmental strains used were isolated from commercial amended and garden soils in a previous study whereas the Italian clinical isolates were: one from nasal sporotrichosis in a dog and one from human. Amplification and sequencing of the of the D1-D2 region of the 28S rDNA were performed by using existing universal fungal primers NL-1/NL-4.
Nucleotide sequence and phylogenetic analysis of the large-subunit ribosomal DNA gene revealed a degree of difference sufficient to justify the separation of the examined isolates in two principal groups (environmental and clinical). Such separation in two groups is further supported by two well-conserved nucleotide polymorphisms, caused by single-base transitions, in the D1-D2 domain of rDNA. These nucleotide changes seems to be related to virulence and could be used as potential genetic marker for distinguishing pathogenic and non-pathogenic strains within S. schenckii clade.

 


P10.3

Transcriptome analysis of Neisseria meningitidis during growth in human whole blood reveals new survival strategies and virulence factors
E. Del Tordello1, H. Echenique-Rivera1, A. Muzzi1, K. Seib1, I. Delany1, B. Aricò1, R. Rappuoli1, M. Pizza1, D. Serruto1
1Research Center, Novartis Vaccines and Diagnostics, Siena, Italy

Neisseria meningitidis (Nm) is a Gram-negative exclusively human pathogen that represents the major cause of septicemia and meningitis. During the infection, Nm adapts to changing environments and host factors, but the study of Nm pathogenesis in vivo has been limited by the lack of animal models and the species-specificity. To understand how Nm adapts to grown in blood, we performed a whole-genome transcriptome analysis using an ex vivo model of human whole blood infection. We observed changes in the expression of transcriptional regulators, energy metabolism, transport and binding factors and several virulence genes were up-regulated. From the list of the genes up-regulated, we selected genes coding for proteins involved in nutrient uptake, host-cell interaction and transcriptional regulation. Knock-out mutant strains were generated and analysed for the survival in the ex-vivo human blood model. The results indicate that KO strains of nalP, fur, tbp2, lctP and nlpD are more sensitive to killing by human whole blood. Although the screening of other mutants is still ongoing, this approach has the potential to identify new virulence factors involved in the survival of Nm in human blood.

 


P10.4

Analysis of NhhA expression variability in Neisseria meningitidis B and its role in pathogenesis
H. Echenique-Rivera, M. Scarselli, P. Montanari, M. Pizza, D. Serruto, B. Aricò
Novartis Vaccines, Siena, Italy

Neisseria meningitidis is a leading cause of meningitis and septicemia worldwide. NhhA (Neisseria hia/hsf homologue) is a neisserial outer membrane protein, belonging to the family of trimeric autotransporter adhesins. The NhhA mediates bacterial attachment to heparin sulfate and laminin of extracellular matrix and to host epithelial cells, facilitating meningococcal colonization of the nasopharyngeal mucosa. Here we reported the differential expression of NhhA in a panel of N. meningitidis B strains and investigated its relevance in meningococcus pathogenesis. The expression variability seems to be an effect of two mechanisms: i) transcriptional regulation that affects the level of nhhA mRNA, and ii) a single aminoacid substitution in the translocator domain that affects trimerization and surface localization of the passenger domain. Furthermore, to study the effect of NhhA in the host interaction, we evaluated in vitro the capacity of N. meningitidis B strains expressing different levels of NhhA to adhere and invade epithelial cells, or traverse epithelial barrier. The contribution of NhhA on the capacity of N. meningtidis B strains to attach, enter and/or traverse the epithelial barrier could help understand the importance of NhhA in the evolutionary process of colonizing, invasive and hyperinvasive strains.

 


P10.5

SYM1, the yeast ortholog of the MPV17 human disease gene, is required for TCA function, mtDNA stability and mitochondrial morphology in stress conditions
C. Dallabona1, R. M. Marsano2, P. Mancini3, D. Ghezzi4, M. Zeviani4, I. Ferrero1, C. Donnini1
1Dept of Genetics, Biology of Microorganisms, Anthropology, Evolution, Univ. of Parma, Italy
2Dept of Genetics and Microbiology, Univ. of Bari
3Dept of Experimental Medicine and Pathology, Univ. of Rome "La Sapienza”
4Pierfranco and Luisa Mariani Center for Mitochondrial Disease, Division of Molecular Neurogenetics, National Neurological Institute ‘C. Besta’, Milano, Italy


A peculiar form of hepatocerebral mtDNA depletion syndrome is caused by mutations in the MPV17 gene. Although the function of Mpv17 is unknown, its yeast ortholog, termed Sym1, is a heat-induced gene product, with a role in ethanol metabolism and tolerance.
In an attempt to define the molecular role of Mpv17 we took advantage of S. cerevisiae as a model system. From the analysis of phenotypic alterations of sym1D mutant it emerged that SYM1 gene was essential for maintenance of a correct OXPHOS phenotype, mitochondrial morphology and mtDNA stability in stressing conditions.
To get insight into the molecular basis of the phenotypic alterations of the sym1∆ strain we identified and characterized multicopy and metabolic suppressors. All together the results obtained suggest for Sym1 a critical role on OXPHOS mitochondrial functions: Krebs cycle and electron transport chain. As for the altered mitochondrial morphology, our results suggest that Sym1 is crucial for the maintenance of mitochondrial cristae. The effect of Sym1 ablation on organellar morphology suggests destabilization of the inner mitochondrial membrane that could impinge on the determination of the mtDNA instability.

 


P10.6

A yeast model for mitochondrial diseases: a study of pathogenetic human equivalent base substitutions and search for correcting genes
L. Frontali1, C. De Luca1, A. Montanari1, G. Ercoli1, M. Bolotin-Fukuhara2, S. Francisci1
1Dept Cell and Developmental Biology, Pasteur Institute-Cenci Bolognetti Foundation, Univ. Sapienza, Rome, Italy
2Institut de Génétique et Microbiologie, IGM. Bat. 400, Univ. Paris Sud, Orsay, France

S. cerevisiae can be used as a model to study the molecular mechanisms of the mt tRNA mutations equivalent to those causing mt diseases and to identify correcting genes. Phenotypic analyses of the mutants reveal that also in the “homoplasmic” yeast the severity of phenotypes varies in different nuclear contexts, which corresponds to the variable penetrance and tissue specificity of defects observed in humans. We took advantage of this peculiarity to choose the strain in which to perform the experiments necessary to study the molecular mechanisms of the mutations.
The defective phenotypes could be suppressed by tRNA interactors such as the mt elongation factor EF-Tu and the aa-tRNA synthetases. The rescue of the respiratory defect of tRNAVal and tRNALeu mutants (human equivalent: 1624C>T and 3256C>T respectively) are of special interest. We found that the defect could be suppressed not only by EF-Tu and by the cognate synthetase, as expected, but also by the non cognate synthetase. We could hypothesize that the rescue mechanism of these suppressions is based on a chaperon-like stabilising effect of interactors on the mutated tRNAs.

 


P10.7

Virulence factors of Stenotrophomonas maltophilia: an opportunistic pathogen
Angelo Iacobino1, Bianca Colonna2, Mauro Nicoletti3, Gianni Prosseda2, Mariassunta Casalino1
1Dept of Biology, Univ. “Roma Tre”, Rome
2Dept of Cell and Developmental Biology, Univ. “La Sapienza”, Rome
3Dept of Biomedical Science, Univ. “G. D’Annunzio”, Chieti

Stenotrophomonas maltophilia is emerging as one of the most frequently isolated bacteria from cystic fibrosis (CF) patients. The role of S. maltophilia in the pathophysiology of the CF lung diseases has not been fully elucidated. A progressive deterioration of pulmonary functions has been observed during S. maltophilia colonization. As far as potential virulence associated factors are concerned, we have screened 41 S. maltophilia strains, isolated from respiratory secretions of CF patients at the Hospital “Bambino Gesù“ of Rome, and two clinical reference strains: K279a and ATCC 13637. The strains were screened for the expression of the major and minor extracellular proteases (StmPr1 and StmPr2), for fimbriae of type 1 (SMF-1 fimbriae) and for esterase (EstM) by using in vitro techniques and detection of correlated genes by PCR.
The virulence of representative S. maltophilia CF strains presenting different phenotypes regarding protease, fimbriae and esterase production were assayed by in vivo tests by using Galleria mellonella larvae as an infection model. Results obtained suggest that extracellular protease StmPr1, could play a role in the infective process of S. maltophilia.

 


P10.8

Construction and validation of a yeast model system for studying in vivo the susceptibility to stavudine of Polg allelic variants
E. Baruffini, I. Ferrero, T. Lodi
Dip. di Genetica, Biologia dei microrganismi, Antropologia, Evoluzione, Università degli Studi di Parma, Parma

The use of nucleoside reverse transcriptase inhibitors (NRTIs) in the highly active antiretroviral therapy (HAART) has increased significantly the life expectancy of HIV-infected subjects. However, mitochondrial dysfunctions have been observed in subjects treated with NRTIs, in particular with stavudine (D4T), as they interfere with mitochondrial DNA replication through direct inhibition of DNA polymerase gamma (Polg) by D4T-TP, incorporation of D4T-MP in the nascent strand and/or inefficient excision by exonuclease activity of Polg. Recently, D4T induced mitochondrial dysfunction was associated to a non pathological mutation (R964C) and to a SNP (E1143G) in the POLG gene. We constructed a yeast model system to evaluate in vivo the association between D4T toxicity and mutations in MIP1 gene, the yeast ortholog of POLG. We found that: (1) D4T treatment increased mtDNA instability in yeast; (2) strains carrying mutations equivalent to R964C and E1143G were more susceptible to D4T toxicity; (3) these mutations, which are recessive, behave as dominant concerning D4T toxicity. This model system will be useful to assess potential association between POLG polymorphisms and D4T toxicity.

 


P10.9

A Systems Biology approach to investigate the interplay between peroxisomes and mitochondria in yeast
E. Marchi, I. Stefanini, D. Cavalieri

The way Saccharomyces cerevisiae uses carbon sources has long been a landmark for studies related to cellular responses to environmental conditions. In S. cerevisiae only peroxisomal β-oxidation occurs, but mitochondria play a fundamental role both for the completion of fatty acids degradation and for the well established continuous interplay between the two organelles relative to metabolic intermediates.
Mitochondria are fundamental for FA metabolism and several mitochondrial defects affected fitness on oleate more deeply then peroxisomal ones. We investigated some aspects of mitochondrial activity in BY4741 wt strain compared to Δsco2, Δoaf1 and Δfox2 mutants. While Δoaf1 and Δfox2 mutants were defective in oleic acid utilization, Δsco2 mutation didn’t affect fitness. We studied respiratory chain cytochromes absorption spectra, O2 consumption, mitochondrial membrane potential activation and ROS accumulation in the selected strains undergoing metabolic shift from YPD to YP added with oleic acid 0.1% and 5%. Δfox2 mutation elicited the more intense effects in oleate, resulting in sharp ROS accumulation, suggesting ROS detoxification as a central process in oleate utilization.

 


P10.10

Yeast model of human disease: functional study of a novel SDHB germline missense mutation diagnosed in a patient affected by a glomus tumor
F. Meloni1, E. Panizza1, T. Ercolino2, M. Mannelli2, I. Ferrero1, P. Goffrini1
1Dept of Genetics, Biology of Microrganisms, Anthropology and Evolution, Univ. of Parma, Parma, Italy
2Dept of Clinical Pathophysiology, Univ. of Florence, Florence, Italy

We report the functional study in Saccharomyces cerevisiae of a novel SDHB germline missense mutation (C191Y) diagnosed in a patient affected by an apparently sporadic glomus tumor. The missense mutation hits an amino acid residue conserved from mammals to the yeast S. cerevisiae. To validate the pathogenic significance of the human mutation, we introduced into a yeast null SDH2 mutant (sdh2), characterized by an OXPHOS negative phenotype and by the absence of SDH activity, the mutant allele. SDH2C184Y, equivalent to human SDHBC191Y, did restore neither the OXPHOS phenotype nor the SDH activity. Moreover, SDH2C184Y mutant, similarly to the null mutant, displayed a reduction of mitochondrial cytochrome content, particularly of the peak corresponding to complex IV. Accordingly, respiratory activity was reduced by 45%. The mutants displayed enhanced sensitivity to menadione compared to wild type strain, suggesting that the null and pathological mutations determined an increase of ROS. In addition, SDH2C184Y mutation caused an increase of mtDNA mutability approximately 6/7 fold the frequency detected in wild type parental strain.

 


P10.11

Poly-gamma-glutamate production in Bacillus subtilis
C. Osera, C. Calvio, A. Galizzi
Dept Genetics and Microbiology, Pavia Univ., Italy

Poly-gamma-glutamic acid (gamma-PGA) is an anionic polymer of increasing industrial interest, composed of thousands of glutamic acid residues linked by gamma-glutamyl bonds. Secretion of the polymer into the medium confers a mucoid colony morphology to the Bacillus producer strains grown on LB agar plates. Although Bacillus subtilis possesses the functional biosynthetic pgs operon, containing four genes, laboratory strains do not have the ability to produce the polymer because pgs transcription is not active. We dissected the genetic elements involved in the conversion of laboratory non-producer strains into gamma-PGA producers and established that the synergic action of two gene products is required. The co-presence of the wild-type swrAA allele, a gene involved in swarming motility, and the hyperphosphorylated form of the transcriptional factor degU, belonging to the two-component system degS/degU, is sufficient to drive pgs transcription and gamma-PGA production.

 


P10.12

Regulation of glycopeptide antibiotic A40926 biosynthesis in Nonomuraea sp. ATCC 39727
R. Alduina1, M. Bibb2, A. M. Puglia1
1Dipartimento di Biologia Cellulare e dello Sviluppo, Univ. di Palermo
2John Innes Centre, Norwich Research Park, Norwich, UK

Glycopeptides are an important class of antibiotics, interfering with the bacterial cell wall synthesis. Nonomuraea dbv gene cluster for the glycopeptide antibiotic A40926 biosynthesis includes 37 open reading frames participating in antibiotic biosynthesis, regulation, resistance, and export. Specifically, the cluster encodes the regulator Dbv4. dbv4 transcription is negatively influenced by phosphate and a recombinant His-Dbv4 binds the upstream regions of two dbv operons, inducing the transcription of 13 out of 37 dbv genes. However, it is not currently known whether Dbv4 is essential for antibiotic production. We are adopting a genetic approach to determine the role of Dbv4 and a biochemical approach to investigate the hierarchy of regulatory events triggering dbv4 transcription. The genetic approach consists of inactivating dbv4 and assessing the subsequent effect on dbv gene expression and antibiotic yield. The biochemical approach consists of isolating the proteins binding to the upstream region of dbv4 by using a series of chromatographic steps and monitoring binding activity by gel retardation experiments. Investigation of regulatory mechanisms of antibiotic production is of great interest, as these studies provide a potential platform for manipulating industrially important strains to increase production of their secondary metabolites.

 


P10.13

Candida albicans hyphal wall protein 1 (hwp1): one gene, many possible alleles
S. Strangio1, O. Romeo1, G. Criseo1
1Dept of Life Sciences, Univ. of Messina

Gene hwp1 encodes a Candida albicans hyphae cell wall protein which is a substrate of mammalian transglutaminases, promoting the cross-link of the fungus to epithelial cells.
Recently, our group showed that atypical chlamydospore-negative C. albicans isolates, called C. africana, have a new hwp1 allele (Genbank: EU477610) that has been found to be different from that of typical C. albicans strains and from homologous gene of C. dubliniensis.
The occurrence of considerable allelic variability at the hwp1 locus, led us to study also the hwp1 gene of Candida stellatoidea type I that although it is now regarded as synonymous with C. albicans differs from the latter in several major genetic characteristics.
The hwp1 gene was amplified by PCR and sequenced. The sequences were assembled and deposited in GenBank database (FJ715440, FJ715441 and FJ769073).
The results showed that a considerable allelic variability occurred at the hwp1 locus.
The data suggest that the analysis of genetic polymorphisms of the hwp1 gene could be an useful tool in order to study gene function, expression, evolutionary relatedness among C. albicans isolates and/or used for epidemiological identification purposes.

 


11. Human genetic diversity

Total number of abstracts in this session: 14

 

P11.1

Toward a greater understanding of the Native American mtDNA phylogeny: a detailed analysis of the poorly characterized pan-American C1d clade
U.A. Perego1,2, N. Angerhofer1, M. Pala2, J. E. Gomez-Palmieri1, A. Olivieri2, K. H. Ritchie1, B. Hooshiar Kashani2, V. Carossa2, H. Lancioni3, N. M. Myres1, O. Semino2, W. Parson4, A. Salas5, S. R. Woodward1, A. Torroni2, A. Achilli2,3
1 Sorenson Molecular Genealogy Foundation, Salt Lake City, Utah, USA
2Dept of Genetics and Microbiology, Univ. of Pavia, Pavia, Italy
3Dept of Cellular and Environmental Biology, University of Perugia, Perugia, Italy
4Institute of Legal Medicine, Innsbruck Medical Univ., Innsbruck, Austria
5Institute of Legal Medicine, Univ. of Santiago de Compostela, Santiago de Compostela, Galicia, Spain

The genetic history of Early Americans has begun to unfold more fully through the contribution of recent studies based on complete mitochondrial DNA (mtDNA) sequences. However, phylogeographic analyses of Native American haplogroups still suffer from two major problems: an overall underrepresentation of complete mtDNA sequences, especially when compared to haplogroups from other regions of the world, and a very poor definition, if any, of the internal sub-clades.
One example is represented by haplogroup C1d that – according to recent studies – could be one of the founding Paleo-Indian lineages along with the other pan-American haplogroups A2, B2, C1b, C1c, D1 and D4h3a. Nevertheless, the “founder role” of C1d is based on the analysis of only nine complete (or almost complete) sequences currently available in public databases.
To expand on the current knowledge about the events that characterized the peopling of America, we have sequenced more than sixty novel mtDNA genomes belonging to haplogroup C1d, which were carefully selected on the basis of both control-region variation and geographic/ethnic origin. These data allowed an accurate evaluation of the expansion time of C1d in the Americas, and provided a detailed picture of its current distribution in both general mixed and indigenous populations.

 


P11.2

No evidences of association between genetic polymorphisms within the region 8q24 and the risk of thyroid cancer. The Italian study on the cancer of thyroid (ITASCaT) experience
L. Cancemi1, D. Landi1, R. Elisei2, C. Romei2, I. Spitaleri3, M. Cipollini1, F. Gnudi1, A. Cecchini1, F. Gemignani1, S. Landi1
1Dip. di Biologia, Genetica, Univ. di Pisa Italy
2Dip. di Endocrinologia e Metabolismo, Ortopedia, Traumatologia, Medicina del Lavoro, Ospedale Cisanello, Pisa, Italy
3Ospedale Meyer, Firenze, Italy

Differentiated thyroid carcinoma (DTC), namely papillary (PTC) and follicular (FTC) thyroid carcinomas, represents the most frequent histotype among thyroid tumors. Only a few is known on the genetic predisposing factors to DTC. Recently, several polymorphic variants within the 8q24 region have been associated with the risk of developing several types of cancer. In this manuscript we investigate the possible involvement of polymorphic variants in 8q24 region in relation to individual genetic susceptibility to DTC through a case-control association study on a Caucasian population (the ITASCaT study, the Italian study on the Cancer of Thyroid). We genotyped 1000 cases and 1000 healthy controls for the single nucleotide polymorphisms (SNPs) rs6983267 and rs7000448, both associated with different types of cancer, following genome-wide association studies (GWAS). A further analysis was carried out examining the haplotype and diplotype combinations between the considered polymorphisms. Data were analysed with a multivariate logistic regression. Our study did not support evidences for a significant association between the considered allelic variants and the risk of DTC.

 


P11.3

Efficacy of cholinesterase inhibitors for Alzheimer's disease: a pharmacogenetic study
C. Chianella1, P. Maisano Delser1, D. Gragnaniello2, M.R. Tola2, G. Barbujani1, S. Fuselli1
1Dept Biology and Evolution, Univ. of Ferrara
2Dept Neuroscience, Azienda Ospedale-Univ. S. Anna, Ferrara

Pharmacogenetics aims at identifying the right drug and dose for each patient. Individual differences in drug response can result from a number of different factors, including genetics, that may influence both the efficacy of a drug and the likelihood of an adverse reaction.
To test if a correlation exists between the efficacy of three different anti-cholinesterase drugs and genetic variation affecting drug disposition and drug targets, we sampled 142 Alzheimer’s patients (AD) treated by anti-cholinesterase drug Donepezil, Galantamine, or Rivastigmine. Response was quantified by a method for grading the cognitive state of patients, the Mini-Mental State Examination, performed after 6 and 12 months from the beginning of treatment.
For each individual involved in this study we typed and analysed genetic variation at two polymorphic loci: CYP2D6 involved in drugs metabolism, and BCHE coding for the drugs target. To control for sample stratification, a locus associated with risk of AD and inefficacy of specific AD treatments, APOE, was genotyped in a subset of patients. No association was detected between APOE genotypes and drug response. Our preliminary results show that a clear relationship between genetic variation at drug metabolizing and target genes and anti-cholinesterase response in AD is not evident, suggesting that other factors concur in determining such a complex phenotype.

 


P11.4

Mitochondrial variation patterns in Trentino (BIOSTRE project)
V. Coia1, S. Grimaldi1, I. Boschi2, F. Salis2, F. Trombetta2, G. Destro-Bisol 3,4, A. Pedrotti1
1Dip. di Filosofia, Storia e Beni Culturali, Univ. degli Studi di Trento, Trento, Italia
2Istituto di Medicina Legale, Univ. Cattolica di Roma, Roma, Italia
3Dip. di Biologia Animale e dell’Uomo, Sapienza Univ. di Roma, Roma, Italia 4Istituto Italiano di Antropologia, Roma, Italia

With the project “Biodiversity and history of the populations from Trentino (BIOSTRE) we intend to investigate the genetic variation (mtDNA, Y-chromosome and autosomal loci) of Trentine populations, with the final aim to assess how cultural and geographic factors have contributed to the present patterns of genetic variation. The results obtained are being used to reconstruct the peopling processes of this area, by analyzing the genetic structure of Trentine populations and comparing it with that of other European groups with a focus on populations from the alpine area.
We present some preliminary results on the mtDNA variation in 396 samples collected from the principal valleys in Trentino previously selected in order to cover the linguistic and the geographic diversity. Samples were analyzed for 17 SNPs and the hypervariable region 1 and 2. A preliminary intra and inter-population analysis showed some noteworthy differences in the mtDNA variation patterns among the distinct geographic and linguistic groups. The final results are discussed in a historical and cultural context with the collaboration of an interdisciplinary team (Archaeologists, Historical and Linguists).

 


P11.5

Effects of genetic variants of mitochondrial UnCoupling Protein (UCP) genes on longevity
P. Crocco, F. Tallaro, P. D’Aquila, F. De Rango, D. Bellizzi, G. Passarino, G. Rose
Dept of Cell Biology, Univ. of Calabria, Rende, Italy

UnCoupling Proteins (UCPs) are mitochondrial membrane transporters that uncouple respiration from ATP production. Human UCP1, -2, -3 affect the energy balance and the Reactive Oxygen Species (ROS) production. Energy metabolism and ROS-induced damages are among the major contributors to the aging process. Therefore, it has been proposed that UCPs may contribute to slow down aging and extend lifespan by controlling the body metabolic rate and reducing ROS production. This hypothesis has been named “uncoupling-to-survival”. We set up a multivariate logistic regression model, including gender as confounding variable, to evaluate the effects of six selected SNPs in the UCP1,-2,-3 genes on the probability to attain longevity. Two different age groups were analyzed: 313 subjects aged from 64 to 89 years (controls) and 308 subjects aged from 90 to 105 years (cases).We found that the UCP1-rs12502572 (OR=1.44), UCP2-rs660339 (OR=1.53), and UCP3-rs1800849 (OR=1.94) affect significantly (p<0.05) survival at very old age. No clear gene-gene interaction was identified. Our data suggest a significant relationship between the uncoupling function and survival at old age.

 


P11.6

Insights into Pygmy demographic history from complete mitochondrial genomes
C. Batini1,2, J. Lopes3, F. Calafell1, L. Quintana-Murci4, L. van der Veen5, L. Jorde6, J. Bertranpetit1, G. Spedini2,7, D. Behar8, G. Destro-Bisol2,7, D. Comas1
1Unitat de Biologia Evolutiva, Facultat de Ciències de la Salut i de la Vida, Univ. Pompeu Fabra, Barcelona, Spain
2Dipartimento di Biologia Animale e dell’Uomo, Univ. La Sapienza, Roma, Italy
3School of Biological Sciences, Univ. of Reading, Reading, UK
4Human Evolutionary Genetics, CNRS URA3012, Institut Pasteur, Paris, France
5UMR 5596, Institut des Sciences de l'Homme, Lyon, France
6Dept of Human Genetics, University of Utah Health Sciences Center, Salt Lake City, USA
7Istituto Italiano di Antropologia, Rome, Italy
8Molecular Medicine Laboratory, Rambam Health Care Campus, Haifa, Israel

In this study we present a population based analysis of whole mitochondrial genomes in a total of 205 individuals from eight Pygmy populations (Western cluster: Babinga, two Baka, Bakola, Biaka, Mbenzele; Eastern Cluster: 2 distinct Mbuti group) and two Bantu-speaking groups from Central Africa. Genetic structure and population differentiation has been studied. A reanalysis of recently published phylogenetic reconstructions has been also performed, in order to test their robustness and to reassess the contribution of Pygmies to the human mitochondrial variation in sub-Saharan Africa. Finally, ABC (Approximate Bayesian Computation) simulations have been carried out to test three different evolutionary scenarios, built on the basis of external evidence (mainly archaeological and paleoclimatological), as well as estimating specific demographic parameters. The results obtained will be discussed in the light of the current debate on the origin of Pygmies and their genetic and cultural relations with Bantu speakers. Comparison with autosomal data will be shown, with the aim of detecting possible sex-biased admixture processes.

 


P11.7

Inferring genealogical processes from patterns of bronze-age and modern DNA variation in Sardinia
S. Ghirotto1, S. Mona1, A. Benazzo1, F. Paparazzo 1,2, D. Caramelli3, G. Barbujani1
1 Dip. di Biologia ed Evoluzione, Univ. di Ferrara, Ferrara, Italy
2Dip. di Biologia, Univ. di Milano, Milano, Italy
3Dip. di Biologia Evoluzionistica, Laboratorio di Antropologia, Univ. di Firenze, Firenze, Italy

The ancient inhabitants of a region are often regarded as ancestral to the modern dwellers (for instance, in studies of admixture), but so far this assumption has not been tested empirically using ancient DNA data. We studied mitochondrial DNA variation in Sardinia, across a time span of 2,500 years, comparing 23 Bronze-Age (nuragic) mitochondrial DNA sequences with those of 254 modern individuals from two regions, Ogliastra (a likely genetic isolate) and Gallura, and considering the possible impact of gene flow from mainland Italy. To understand the genealogical relationships between past and present populations we developed seven explicit demographic models and tested whether these models can account for the levels and patterns of genetic diversity in the data, and which one does it best. Simulation based on a serial coalescent algorithm allowed us to compare the posterior probability of each model and estimate the relevant evolutionary and demographic parameters, by Approximate Bayesian Computations. We show that a direct genealogical continuity between Bronze-Age Sardinians and the current people of Ogliastra, but not Gallura, has a much higher probability than any alternative scenarios, and that genetic diversity in Gallura evolved largely independently, owing in part to gene flow from the mainland.

 


P11.8

Ancient Umbria: the mitochondrial DNA perspective
H. Lancioni1, L. Milani2, G. Peruzzi1, M. Lari2, U. A. Perego3,4, N. Babudri1, D. Caramelli2, F. Panara1, A. Achilli1,3
1Dept of Cellular and Environmental Biology, Univ. of Perugia, Perugia, Italy
2Dept of Evolutionary Biology, Univ. of Florence, Florence, Italy
3Dept of Genetics and Microbiology, Univ. of Pavia, Pavia, Italy
4Sorenson Molecular Genealogy Foundation, Salt Lake City, Utah, USA

Located in the heart of the Italian peninsula, Umbria since prehistoric times has been a nodal point for the trans-Apennine routes between the Tyrrhenian regions and the Adriatic coast. The Umbrian civilization is testified by various urban remains and by important necropolises. A series of pre-Roman hill forts around the ancient Plestinam Paludem (now Colfiorito) are interpreted not as mere temporary refuges, but as foci of regular occupation, trade and administration. The inhabitants of this area gradually became a formal community since the 10th-9th century BC, with the emergence of a local aristocracy that eventually came in contact with the Etruscans thanks to the Iron Road trade. However, up to the present day, ancient Umbria clearly played a minor role when compared to Etruria with its much better documented history.
In order to fill this gap, we analyzed the mitochondrial DNA variation of more than 250 unrelated Umbrian subjects, mostly living in the Colfiorito plateau and surrounding hills. In addition to modern DNA samples, the molecular analysis of a number of human skeletal remains, excavated at Colfiorito by the Superintendence of Archaeological Heritage of Umbria, are also providing a rare opportunity to take a glimpse into the past of this poorly studied region of Italy

 


P11.9

Survival advantages of parents and sblings of calabrian nonagenarians
V. Latorre1, A. Montesanto1, C. Martino1, F. De Rango1, F. Domma2, G. Passarino1
1Dept Cell Biology, Univ. of Calabria
2Dept Statistics and Economics, Univ. of Calabria


During the last decade an impressive and coherent series of epidemiological studies provided evidence of a strong familial and genetic component of human longevity. These studies showed that parents, siblings and the offspring of long-lived subjects have a significant survival advantage and an higher probability than the general population to attain longevity.
In the present study, we report a population-based study of 100 families of long-lived subjects (>90 years) recruited from a genetically and socially homogeneous population of southern Italy (Calabria). This region has often been the focus of genetic studies as its population exhibits a number of unique features, such as highest rate of long-lived subjects; male-biased female-to-male sex ratio among nonagenarians and centenarians. We then compared the survivorship functions of the long-lived subjects’ parents and siblings with their corresponding birth cohorts.
We found that parents of long-lived subjects lived longer than the subjects of the same birth cohorts, and that to have a nonagenarian sib decreases significantly the instantaneous mortality rate throughout lifetime.

 


P11.10

Haptoglobin genetic polymorphism and human longevity
V. Napolioni1, P. Giannì1, F. Concetti1, N. Lucarini1
1Laboratory of Human Genetics, Dept Molecular, Cellular and Animal Biology, Univ. of Camerino, Camerino, Italy

Ageing is a complex, challenging phenomenon arising from a combination of oxidative, immunological and metabolic factors. Haptoglobin (HP), given its relevant role in oxidative stress and in immunological responses, can be considered an excellent candidate gene for human longevity.
Haptoglobin (HP) is an α2-sialoglycoprotein with hemoglobin (Hb)-binding capacity and is characterised by a molecular heterogeneity with three major genotypes: HP 1*/1*, HP 1*/2* and HP 2*/2*.
991 individuals was recruited from the same geographical area of Central Italy. Whole population studied was divided into three cohort: younger group (8-79yrs., 188 females and 188 males), octogenarians (80-89yrs., 210 females and 182 males), nona/centenarians (90-106yrs., 135 females and 88 males). HP genotyping was performed by allele-specific PCR using DNA extracted from whole blood samples.
We observed a significant increase of HP 1*/1* individuals aged over 79 in both sexes (age >79 vs. age <80: males 79% vs 21%; females 77% vs. 23%). A three-way contingency table analysis by log-linear model shows a very significant interaction between HP genotype and age [HP-age-(sex): G=24,754; df=4; p= 5,64 x 10-5].
Our findings are supported by functional and biochemical evidences since HP 1-1 molecule shows a greater anti-oxidative capacity and anti-inflammatory action compared to other haptoglobin molecules.

 


P11.11

A collaborative project on genetic diversity within and among Italian Isolates
D. Pettener1, C. Battaggia2, A. Boattini1, C.M. Calò3, C. Capelli4, L. Castrì1, V. Coia5, L. Corrias3, G. Destro-Bisol2, D. Luiselli1, E. Sanna3, S. Tofanelli6, G. Vona3, G. Paoli6
1Dept Evol. Exper. Biology, Univ. of Bologna, Bologna, Italy
2Dept Animal and Human Biology, Univ. of Rome “La Sapienza”, Rome, Italy, and Italian Institute of Anthropology, Rome, Italy
3Dept Experimental Biology, Anthropological Sciences Section, Univ. of Cagliari, Monserrato, Italy
4Dept Zoology, Univ. of Oxford, Oxford, United Kingdom
5Dept Philosphy, History and Cultural Heritage, Univ. of Trento, Trento, Italy
6Dept of Biology, Univ. of Pisa, Pisa, Italy

The collaborative project “Isolating the Isolates: geographic and cultural factors of human genetic variation” (MIUR, PRIN projects 2007-2009; Universities of Bologna, Cagliari, Pisa and Rome “La Sapienza”) is aimed at evaluating the degree of isolation in geographic and linguistic isolates settled in the Italian territory through the analysis of demographic data and genetic structure, while assessing their genetic continuity with the ancestral populations, through the analysis of demographic data and genetic data (mtDNA and Y-chromosome). The ultimate goal of the project is to shed light on the micro-evolutionary processes and cultural factors shaping human genetic diversity which may be relevant to gene-culture co-evolution processes.
Here we present a survey of the main genetic and demographic evidence so far obtained in the following case studies: Carloforte and Benetutti (Sardinia), Sappada (Veneto) and Sauris (Friuli), Apuan Alps (Tuscany) and Arbereshe and Calabrian communites of the Pollino area (Calabria). Preliminary results move towards the interpretation of isolates\' genetic structure as a floating balance between culture (language) and geography.

 


P11.12

The genotype of PV-92 locus studied in a population of 2720 high school students from Latium
V. Somma1, F. Boccellato2, K. Ikeda2, F. Gabanella1, A. Salvatore1, E. D’Ambrosio1,2
1Istituto di Neurobiologia e Medicina Molecolare, CNR , Roma, Italia
2Fondazione Avanzamento Ricerche in Medicina Molecolare, Roma, Italia

The PV92 is a polymorphic locus present on chromosome 16. The polymorphism is due to the insertion of an element of the Alu family. The locus is not coding and no recognizable phenotype is associated. Genotypization of this locus is widely used for didactic purpose in several country and data on allele frequencies in several population have been reported (1). In the last three years 2720 high school students of Latium (Italy) had their first approach with molecular biology techniques and learned the basis of population genetics by genotyping their own DNA for PV92 polymorphism. Under tutor supervision, each student carried out DNA extraction from oral cells, PCR and electrophoresis analysis. In our population it was found that the frequency of the Alu inserted allele was 20,5% while the heterozygote’s were 32,65%. The study group that can be considered representative of Italian population, being the presence of other nationality episodic, is in Hardy Weinberg equilibrium (p value <0,5). Students can analyze their results compared with groups of population from all over the world so they can appreciate the sense of genetic diversity. (1)Batzer et al. PNAS (1994) vol 91, p.12288-92

 


P11.13

Insulin/IGF-1 Signalling pathway and longevity in humans: preliminary results of a multilocus approach
F. Tallaro, F. Di Cianni, P. Crocco, A. Montesanto, D. Bellizzi, G. Passarino, G. Rose
Dept of Cell Biology, Univ. of Calabria, Rende, Italy

Evidence has accumulated that mutations in genes involved in the Insulin/IGF-1 Signalling (IIS) pathway affect lifespan in a variety of model organisms. However, there are few evidences that variations in human homologues of genes in this pathway are associated with a long-lived phenotype. We performed a multilocus association analysis to find out if, and to what extent, human longevity is affected by the genetic variability of fifteen genes of the IIS pathway. Using the APEX technology (Arrayed Primer EXtention) we genotyped a sample of 77 centenarians and 69 younger controls. We first performed an univariate feature selection; then, the genotypes resulted to be significantly associated with longevity were used as independent variables in a multivariate regression logistic model. We found that five genes IRS-1, PI3K, GRB2, IGF1 and GAP are significantly associated with human longevity. Moreover, using the Area Under the Curve (AUC) as a measure of the classification performance, we found that the predictivity of the model is about 70%. On the whole, our data support the view that the Insulin/IGF-1 Signalling pathway play a key role also in human longevity.

 


P11.14

Malagasy admixture: the tale of a recent encounter between deep-rooted lineages
S. Tofanelli1, S. Bertoncini1, L. Castrì2, D. Luiselli2, F. Calafell3, G. Donati4, G. Paoli1
1Dept Biology, Univ. of Pisa, Pisa, Italy
2Dept Experimental Evolutionary Biology, Univ. of Bologna, Bologna, Italy
3Dept Experimental Sciences and Health (CEXS), Pompeu Fabra Univ., Barcelona, Spain
4Dept Anthropology and Geography, Oxford Brookes Univ., Oxford, UK

We fit the Malagasy admixture history in a highly resolved phylo-geographic framework by typing a large set of uni-parentally transmitted markers in unrelated individuals from inland and coastal ethnic groups. The uniqueness of Malagasy was confirmed to be due to a recent encounter between gene pools (Southeast Asian and sub-Saharan African) that have been shaped by at least 60,000 years of independent evolution. The distribution of the two ancestral components was ethnic and sex biased, with the Asian ancestry appearing more conserved in the female than in the male gene pool and in inland than in coastal groups. The focus about the origin of Malagasy lineages was enlarged in space and pushed back in time. Complex underlying demography after the admixture event could make the search of univocal ancestries inconclusive and the close link between Malagasy and Bornean (Maanyan) vocabulary misleading. The pattern of diffusion was compatible with a primary admixture of proto-Malay people with Bantu speakers bearing a western-like pool of haplotypes, followed by a secondary flow of Southeastern Bantu speakers unpaired for gender and geography.

 


12. Immunology

Total number of abstracts in this session: 1

 

P12.1

Constitutively active STAT3 triggers the development of autoimmune myocarditis
A. Camporeale1,2, F. Marino1,2, A. Brero3, R. Chiarle4, O. Jensen5, R. Levi3, V. Poli1,2
1Molecular Biotechnology Center, Univ. of Turin, Turin, Italy
2Dept of Genetics, Biology and Biochemistry, Univ. of Turin, Turin, Italy
3Dept of Animal and Human Biology, Univ. of Turin, Turin, Italy
4Dept of Pathology and Center for Experimental Research and Medical Studies (CeRMS), Univ. of Turin, Turin, Italy
5Dept of Biochemistry and Molecular Biology, Univ. of Southern Denmark, Odense, Denmark

Stat3 is a pleiotropic transcription factor displaying different functions. It is required for the differentiation of IL-17 secreting cells (Th17), which have been correlated with harmful inflammatory effects in autoimmune disorders. Thus Stat3 could be involved in the development and regulation of autoimmune diseases, but no model investigating Stat3 pathogenic functions in autoimmunity was so far available. We have recently generated knock/in mice expressing physiological levels of the constitutively active mutant form Stat3C (Stat3C/C mice). These mice develop a lethal form of autoimmune myocarditis, characterized by myeloid infiltration of the heart, production of anti-alpha myosin autoantibodies and high mRNA levels of proinflammatory cytokines. In addition, the development of Th17 cells appears to be enhanced by Stat3C expression as well as the circulating levels of the complement component C3. Thus, Stat3C/C mice appear to be a suitable model to study the role of chronically activated Stat3 in the development of autoimmunity. Further characterization of this model may lead to identify potential new strategies for therapeutic intervention in autoimmune diseases.

 


15. Neurobiology

Total number of abstracts in this session: 4

 

P15.1

Proliferation of local progenitors in the cerebellar cortex of peripuberal rabbits: involvement of Bergmann glia
P. Crociara, G. Ponti, L. Bonfanti
Dept of Veterinary Morphophysiology, Univ. of Turin, Grugliasco, Italy

Adult neurogenesis in mammals is confined within restricted sites (hippocampus and SVZ) being sustained by stem cells located in brain neurogenic niches. As a consequence, most mammalian CNS tissue is physiologically non-neurogenic. Recent studies showed that dividing cell progenitors also persist in the mature parenchyma, yet not granting constitutive neurogenesis (Nishiyama, Nat Rev Neurosci 2009). In the mammalian cerebellum, neurogenesis is limited to early postnatal periods, until the exhaustion of granule cell genesis. We previously showed that in rabbits many cells continue to be generated on the cerebellar surface (peripuberally; Ponti et al. Dev Biol 2006) and in the cortex (even in adults; Ponti et al. PLoS ONE, 2008). Our preliminary results suggest that some dividing Sox2+ cells are Bergmann glia. Here we studied in vivo cell proliferation within the Purkinje cell layer of peripuberal rabbits, wherein the cell bodies of Bergmann glia are contained. The occurrence of BrdU+ nuclei in BLBP+ cells with the morphology of Bergmann glia indicates that in lagomorphs this radial glia-derived cell type continues to undergo division at very late postnatal stages.

 


P15.2

Investigations of GABAergic mechanisms in SSADH deficiency
P. Blasi1, I. Vardya2, E.J. Hager3, K. Jensen2, K.M. Gibson3, P. Malaspina1
1Dept of Biology, Tor Vergata Univ., Rome, Italy
2Dept of Physiology and Biophysics, Aarhus Univ., Denmark
3Dept of Biological Sciences, Michigan Technological Univ., Houghton, MI, USA


Succinic semialdehyde dehydrogenase (SSADH) deficiency (4-HBA) is an autosomal recessive disorder of the GABA catabolism. The phenotype is variable and associated with neurodevelopmental delays and seizures. Identification of patients is facilitated by detection of increased urinary gamma-hydroxybutyric acid (GHB).
The pathophysiology of 4-HBA has been recently modeled in SSADH KO mice, which display similar symptoms. Previous studies have shown significant elevations of GHB and GABA in newborn KO mice. Based upon these accumulations, ain view of the seizure phenotype of KO mice and the imaging abnormalities observed in human patients, we hypothesized that GABA might be significantly elevated also in embryonic KO mice.
To evaluate the molecular and neurophysiological effects of excessive GABA and GHB levels in KO mice, we addressed the RGS (regulators of G-protein signaling) as part of the signal pathway downstream to GABA and GHB binding. We report on RGS mRNA levels in selected brain regions. Since several receptors are G-protein coupled, such knowledge may point to new therapeutic targets for the treatment of 4-HBA, and systems in which GHB accumulates due to illicit consumption.

 


P15.3

Cholesterol homeostasis and prion misfolding: a comparative study in mouse neuroblastoma cells and in sheep brains and skin fibroblasts
M.D. Cannas, C.D. Orru’, S. Vascellari, C. Abete, F. Angius, P.L. Cocco, C. Norfo, P. La Colla, S. Dessi’, A. Pani
Dept Biomedical Science & Technology, Univ. of Cagliari, Italy

Background. Abnormal accumulation of cholesterol esters were observed in N2a cells infected with mouse-adapted strains of scrapie as well as in ex vivo skin fibroblasts and brain biopsies from scrapie-affected sheep indicating a relationship between susceptibility to prion infection/replication and altered cholesterol homeostasis.
Results. We now show that accumulation of cholesterol esters in brains and cultured skin fibroblasts from scrapie-susceptible sheep, with or without scrapie, was accompanied by altered expression levels of ACAT1 (up-regulated) and Cav-1 (down-regulated), involved in cholesterol esterification and trafficking, and of PrP itself (up-regulated). Moreover, Oil Red-O, Nile Red and filipin staining of prion-infected N2a cells revealed a general derangement of intracellular lipids (i.e.cholesterol esters, phospholipids, and free cholesterol). Synergic anti-prion effects by drug combinations able to restore lipids content and distribution similar to uninfected cells, support the idea that derangement of intracellular lipids may favour and assist prion misfolding by affecting plasma membrane functional integrity.
Conclusions. Cholesterol ester accumulation in peripheral cells might serve to identify a distinctive lipid metabolic profile associated with increased susceptibility to develop prion disease.

 


P15.4

Expression of dysbindin in the retina is reduced in diabetic rats
L. Gaddini, A. Matteucci, G. Macchia, P. Macioce, M. Villa, T.C. Petrucci, F. Malchiodi-Albedi, F. Pricci, M. Ceccarini
Dept of Cell Biology and Neuroscience, Istituto Superiore di Sanitá, Rome, Italy

Dysbindin is a dystrobrevin-binding protein expressed in several tissues, including brain and nervous system. Although its functional role has not yet been elucidated, it is believed to participate in intracellular trafficking and biogenesis of organelles and vesicles, and it has been suggested that it plays a role in the exocytosis of synaptic vesicles through the association with molecules in presynaptic machinery. Recent data suggest that reductions in presynaptic protein and mRNA levels in the retina may be a mechanism for establishing the neuronal dysfunction, including impaired synaptic function and loss of neurotransmission, at the basis of visual deficits found as consequences of diabetes. We examined the expression of dysbindin in rat retina and found it to be localized to the ganglion cell layer, as well as in the inner plexiform layer. Treatment of rats with streptozotocin induced an experimental diabetes that resulted in heavily reduction of dysbindin, as evidenced by Western blot experiments. This variation suggests an involvement of dysbindin in the impaired neural function observed in diabetic retinopathy.

 


Deadlines

  • Abstract
    submissions
    17 July 2009
  • Grant
    requests
    17 July 2009
  • Registration
    17 July 2009
  • Payment
    17 July 2009

Important dates

  • Abstract
    submissions
    open
    22 June
  • Accepted Posters available on website
    3 August