Conference Programme & Abstract Book

Faculty of Sciences, University of Lisboa, Portugal

18th July 2013

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CONTENTS

Pag.

Conference Sponsors 5

Welcome FCUL/EMBL Director 7

Conference Programme 9

Poster Titles & Authors 11

Poster Abstracts 17

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Institutional Sponsors

Other Sponsors

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Welcome Message

The Faculty of Sciences, University of Lisboa (FCUL) and the European Molecular Biology

Laboratory (EMBL) have organised this event dedicated to address latest developments in

Molecular Biology in Portugal and at EMBL, mostly targeted for an audience of young

researchers.

The event aims firstly to inform life scientists at all levels in Portugal (in academia and

industry) about the latest molecular biology developments in Portuguese labs and about the

excellent science, facilities, training opportunities and services offered by EMBL. Secondly,

the event is an excellent opportunity to further strengthen scientific links within the

Portuguese community and with EMBL.

EMBL and Portugal have a longstanding and a successful partnership in molecular biology

and we are certain that your contribution will represent an important step towards even

closer collaborations amongst Portuguese scientists as well as between Portugal and EMBL.

We hope that you will enjoy this original initiative!

José Pinto Paixão Iain Mattaj Director Director General Faculty of Sciences Lisbon University EMBL

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Molecular Biology in Portugal and EMBL (and EMBL Alumni) Faculty of Sciences, University of Lisboa, Lisbon, 18 July 2013

Anf. 3.2.14

Programme 8.00 – 9.00 Registration of participants & poster set-up

9.00 - 9.30 Welcome address

Prof. Pinto Paixão, Director of the Faculty of Sciences, University of Lisboa

Prof. Miguel Seabra, FCT President

Prof. Claudio Sunkel, EMBL Council Chair, EMBO Member

9.30 – 11.00 Scientific talks I.

Prof. Margarida Amaral, Faculty of Sciences, University of Lisbon, EMBL Alumni

Using Functional Genomics to Tackle Cystic Fibrosis

Prof. Maria Carmo Fonseca, IMM Director, EMBO Member, EMBL Alumni

Imaging transcription and splicing in real time

Dr. Luis Teixeira, Gulbenkian Institute, EMBL Alumni, Faculty of Sciences’ Alumni

Symbiont modulation of host-pathogen interactions

11.00 – 11.30 Coffee break

11.30 – 13.15 Scientific talks II.

Prof. Claudina Rodrigues-Pousada, ITQB, EMBO Member

Activation of the Yap8-target, ACR2 gene, requires the function of the Mediator complex in S. cerevisiae cells exposed to arsenate

Dr. Rainer Pepperkok, Head of ALMF Core Facilities, EMBL

Prof. Iain Mattaj, Director General EMBL:

EMBL – research, services, training

Dr. Gerlind Wallon, EMBO Deputy Director:

EMBO – what is in it for you

13.15 – 15.15 Lunch and poster session

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15.15 – 16.45 Scientific talks III

Dr. Lars Steinmetz, Joint Head of Unit, EMBL:

System Genetics

Dr. Paulo Matos, Young Investigator, Faculty of Sciences, University of Lisbon

Rac1 signalling in health: not too little... not too much... just enough.

Dr. Reto Gassmann, IBMC, EMBO IGs

16.45 – 17.00 Poster prize and closing remarks

Dr. Gerlind Wallon, EMBO Deputy Director: delivery of a Poster prize

Prof. Pinto Paixão, Director of the Faculty of Sciences, University of Lisbon – Closing remarks

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Poster Titles & Authors

P.1 The role of mycobacteriophage Ms6 LysB in mycobacteria lysis Adriano Gigante, Francisco Martins, Filipa Gil and Madalena Pimentel

P.2 Internal ribosome entry site-mediated translational regulation of mammalian target of rapamycin (mTOR) in stress conditions Marques-Ramos A, Lacerda R, Teixeira A, Romão L

P.3 miR-34c-5p at the crossroads of human naive CD4 T-cell activation and HIV infection Ana Margarida Matos, Russell B Foxall, Andreia J Amaral, Jorge Andrade, Paula Matoso, Mariana Santa-Marta, Rita Tendeiro, Ana Serra-Caetano, Rui S Soares, João Gonçalves, Margarida Gama-Carvalho, Ana E Sousa

P.4 RefirMAR, a neuromodulator bioactive ingredient derived from a deep sea microorganism: applications for daily skincare Martins A, Andrade G, Rodrigues I, Almeida A, Rodrigues C, Pinheiro R, Calado P, Vieira H.

P.5 Post-transcriptional regulation of SMN2 expression by hnRNP G and LARP Family proteins Ana Miguel Fernandes, Ana Luísa Gomes, Mariana Oliveira, Gonçalo Costa, Isabel Peixeiro, Ana Margarida Matos, Carlos Cordeiro and Margarida Gama-Carvalho

P.6 An approach to study glia reactivity in Amyotrophic Lateral Sclerosis Gomes C, Correia AM, Gomes R, Vaz AR, Brites D

P.7 Aberrant CFTR mRNA Processing: Which Are Consequences? Anabela S Ramalho, Verónica Felício, Margarida D Amaral

P.8 Amyloid-ß peptide dysregulates TrkB receptors and BDNF function by a calpain-dependent mechanism André Jerónimo-Santos, Sandra Henriques Vaz, Sara Parreira, Sofia Rapaz-Lérias, António P. Caetano, Valérie Buée-Scherrer, Eero Castrén, Claudia A. Valente, David Blum, Ana Maria Sebastião, Maria José Diógenes.

P.9 An automated high-throughput screening platform to study the role of C.elegans Kinesin-5 André F. Maia, Daphne Lelieveld, Matilde Galli, David A. Egan, Marvin E.Tanenbaum, Claudio Sunkel, Sander van den Heuvel and René H. Medema

P.10 Deep-sequencing profile of small ncRNAs in human CD4+ T cells upon TCR-mediated activation and its modulation by HIV infection Andreia J Amaral, Jorge Andrade, Ana Margarida Matos, Paula Matoso, Rui Soares, Russel Foxall, Rita Tendeiro, Margarida Gama-Carvalho, João Gonçalves, Ana Espada Sousa

P.11 Changing laminin isoforms during skeletal muscle development: different players or a backup system? Andreia M. Nunes, André Gonçalves, Patrícia Ybot-Gonzalez, Marianne Deries & Sólveig Thorsteinsdóttir

P.12 Deciphering grapevine resistance to Plasmopara viticola: hints from proteomics Andreia Figueiredo, Joana Martins, Mónica Sebastiana, Filipa Monteiro, Ana Coelho

P.13 m7GDP mononucleotide is not a substrate for DcpS enzyme but might regulate its activity in vitro Anna Wypijewska, Elzbieta Bojarska, Maciej Lukaszewicz, Janusz Stepinski, Jacek Jemielity, Richard E Davis and Edward Darzynkiewicz

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P.14 Comparative Genomics of Bacillus spp.: can core genome be the key for new therapeutics? Assessment of the core genome of Bacillus spp. and its stability

Carlos Alonso, Francisco M Couto & Ricardo Dias

P.15 Revertants, Low temperature and Correctors Provide Clues to Mechanism of F508del-CFTR Rescue by VX-809 and Suggest Multiple Agents for Full Correction Carlos M Farinha, John King-Underwood, Bárbara J Henriques, Ana Raquel Correia,Marisa Sousa, Jonathan Williams, Simon Hirst, Cláudio Gomes, Margarida D Amaral

P.16 A novel in vitro model to study hypoxia-induced effects in retinal pigment epithelium Inês P. Rodrigues, Sara Maia, Cristiana F. Pires, Martim D. Portal,Carolina Thieleke-Matos Olaf Strauss, Miguel C. Seabra José S.Ramalho

P.17 Posttranscriptional regulation of bacterial virulence Christian G Ramos, André M Grilo, Joana R. Feliciano, Paulo JP da Costa, Jorge H Leitão

P.18 Translational regulation of human hemojuvelin expression via upstream open reading frames Cláudia Onofre, Cristina Barbosa, Luísa Romão

P.19 Translation of the human erythropoietin transcript is regulated by an upstream open reading frame in response to hypoxia Cristina Barbosa, Luísa Romão

P.20 Matching Biomedical Ontologies using External Resources Daniel Faria

P.21 Dynamic expression of Notch pathway components in the ovary during the mouse estrous cycle Daniel Murta, Marta Batista, Alexandre Trindade, Elisabete Silva, Domingos Henrique, António Duarte, and Luís Lopes-da-Costa

P.22 Monitoring anthracnose emergence and pathogen population dynamics in the expanding cashew tree culture in Guinea Bissau Batista D, Romeiras MM, Talhinhas P, Silva DN, Vieira A, Loureiro A, Duarte MC, Catarino L

P.23 Regulation of Drug Metabolizing Enzymes and Transporters in Mouse Liver by Primaquine Gonçalves E, Marques C, Nogueira F, Rosário V, RibeiroV

P.24 The role of lipid membrane in amyloid fibril formation and toxicity in Alzheimer’s disease. Eric Finot

P.25 The role of Arabidopsis thaliana diacylglycerol kinase 4, in pollen tube growth and fertilization a preliminary approach." Fernando Vaz Dias, Susana Serrazina and Rui Malhó

P.26 BDNF modulates type 1 cannabinoid receptor (CB1R)-stimulation of neurogenesis in murine dentate gyrus stem/progenitor cell cultures Ferreira F, Ribeiro FF, Sebastião AM, Xapelli S

P.27 Evaluation of 99mTc-TMEOP as Probe for Functional Monitoring of Multidrug Resistance Filipa Mendes, Lurdes Gano, Célia Fernandes, António Paulo e Isabel Santos

P.28 Uncovering basal defense response in grapevine through gene expression analysis Filipa Monteiro, Mónica Sebastiana, Andreia Figueiredo

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P.29 A roadmap for tissue specific RNA-seq analysis of Drosophila shRNA models Francisco Brito, Andreia Amaral, Takakazu Yokokura, David van Vactor and Margarida Gama-Carvalho

P.30 Ms6 Gp1 behaves as a chaperone assisting LysA delivery to its substrate Francisco Martins, Adriano Gigante, Maria João Catalão, José Moniz-Pereira and Madalena Pimentel

P.31 Identification of CFTR traffic regulators as a source of novel cystic fibrosis therapeutic targets Hugo M. Botelho, Shehrazade Dahimène, Inna Uliyakina, Beate Neumann, Christian Tischer, Rainer Pepperkok, Margarida D Amaral

P.32 Splicing factors in CD5 mRNA alternative polyadenylation Inês Lago-Baldaia, Nair Lopes, Inês Boal-Calvalho, Rita G Domingues, Danica Drpic, Telmo Henriques, Vânia Glória, Isabel Pereira Castro, Alexandre M Carmo, Alexandra Moreira

P.33 Hydrogen Peroxide sensor in an anaerobic bacterium: Desulfovibrio vulgaris Hildenborough PerR Vasco Ribeiro, Isabel Silva, David Sardinha, Alice Pereira, Pedro Tavares and Cristina Timóteo

P.34 Regulation of mRNA metabolism by U2AF65 splicing factor - novel mechanisms for the coordination of gene expression? Isabel Peixeiro, Samuel Casaca, Margarida Gama‐Carvalho

P.35 Studying the interaction of dengue virus capsid protein inhibitor pep14-23 with membranes André F Faustino, Gabriela M Guerra, Axel Hollmann, Miguel ARB Castanho, Fábio CL Almeida, Andrea T Da Poian, Nuno C. Santos, Ivo C. Martins

P.36 AUG-Proximal CFTR nonsense mutations are NMD resistant and induce translation re-initiation João P. Amorim, Margarida D Amaral and Anabela S. Ramalho

P.37 Deciphering the genetic architecture of haemolysis in sickle cell anaemia Andreia Coelho, Alexandra Dias, Anabela Morais, Emanuel Ferreira, Isabel Picanço, Baltazar Nunes, Paula Faustino, João Lavinha

P.38 Semantic Similarity in the Biomedical Domain João D. Ferreira & Francisco M. Couto

P.39 Nucleic acid delivery vectors derived from viral proteins: Cell Penetrating Peptides vs. Supercharged Proteins João Miguel Freire, Ana Salomé Veiga, Inês Rego de Figueiredo, Beatriz G de la Torre, Nuno C Santos, David Andreu, Andrea T Da Poian and Miguel ARB Castanho

P.40 Molecular regulation of wood formation in Eucalyptus Jorge Paiva

P.41 CRiSPy evolution of venom Kartik Sunagar, Warren E Johnson, Stephen J O’Brien, Vitor Vasconcelos and Agostinho Antunes

P.42 Towards understanding the role of PTEN genes in P. patens Laura Saavedra, Rita Catarino, Tobias Heinz, Ingo Heilmann, Magdalena Bezanilla and Rui Malhó

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P.43 Disclosing the role of DNA methylation in salt tolerance/response mechanisms in rice Liliana Ferreira, João Maroco, Mª Margarida Oliveira, Ana Paula Santos

P.44 Effect of the intron 22 inversion mutation of the f8 gene in hemophilia A on the structure and function of the FVIII protein. Linda MEDDAH

P.45 The impact of rpiA overexpression on plasmid biopharmaceutical production by Escherichia coli GALG20, a pgi-gene knockout strain. Luís AM Carreira, Geisa AL Gonçalves, Duarte MF Prazeres and Gabriel A Monteiro

P.46 Dynamics of Akt activation during mouse embryo development: distinct subcellular patterns distinguish proliferating versus differentiating cells. Luís Marques and Sólveig Thorsteinsdóttir

P.47 Tauroursodeoxycholic acid prevents MPTP-induced dopaminergic cell death in a mouse model of Parkinson's disease M Castro-Caldas, A Neves Carvalho, E Rodrigues, CJ Henderson, CR Wolf, CMP Rodrigues, MJ Gama

P.48 Integrating survey and molecular approaches to better understand the origin of endemic flora in the Cape Verde Islands Romeiras MM, Monteiro F, Fernandes C, Duarte MC

P.49 Neuronal Protection in Parkinson's Disease: is there a role for Gst pi? Andreia Neves Carvalho, Margarida Castro-Caldas, Carla Marques, Elsa Rodrigues,Colin J Henderson, C Roland Wolf, Paulo Pereira, Maria João Gama

P.50 Yeast Two-Hybrid System: TCTEX1D4 interactome, a protein is known by the company it keeps Maria João Freitas, Luís Korrodi-Gregório, Margarida Fardilha

P.51 Exploring a link between mycobacteria structure and virulence C Silva, J Perdigao, E Alverca, AP Matos, PA Carvalho, I Portugal, L Jordao

P.52 Measuring transcript abundance of candidates from selected gene families: a first glimpse on the molecular basis of postharvest biocontrol in pears Maria Manuel Cristóvão, Luis F Goulao and Claudia Sánchez

P.53 P.53 NGSOnto - keeping track of the NGS pipeline process using an ontological approach and its application on molecular epidemiology Mickael Silva, Catia Vaz, Alexandre Francisco, Mário Ramirez, Francisco M Couto, João André Carriço

P.54 Proteomic analysis of Quercus suber – Pisolithus tinctorius ectomycorrhizal symbiosis Mónica Sebastiana, Joana Martins, Andreia Figueiredo, Filipa Monteiro, Catarina Franco, Ana Coelho

P.55 Effect of peanut extraction protocols on yield of crude protein, quantities of allergens Ara h 1 and 2, and detection in subsequent experiments Nicole E Walczyk, Euan Tovey, Pen CSmith, and Thomas H Roberts

P.56 Reserpine Inhibits the CFTR Chloride Channel in Caco-2 Monolayers Nikhil T Awatade, Margarida Ramos, Margarida D Amaral and John P Winpenny

P.57 Role of 2CysPrx and PrxIIB in H2O2 antioxidant system under heat stress Patrícia Vidigal, Ana Montserrat Martin-Hernandez, Cèlia Guiu-Aragonés, Sara Amâncio, Luísa Carvalho

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P.58 The role of fibronectin matrix in regulating segmentation in the vertebrate embryo Patrícia Gomes Almeida, Ana Lina Rodrigues, Isabel Palmeirim and Sólveig Thorsteinsdóttir

P.59 Cortical microtubule and ß-tubulin organization in developing maize (Zea mays L.) endosperm transfer cells and starchy endosperm cells Sara Rocha, Artur da Câmara Machado, Paula Sampaio, Roberto Salema, Paulo Monjardino

P.60 Bioinformatics awareness through training provision in Portugal Pedro L. Fernandes, Cath Brooksbank

P.61 Centromere-independent accumulation of cohesin at ectopic heterochromatin sites induces chromosome stretching during anaphase Oliveira R, Kotadia S, Mirkovic M, Bowlin K, Eichinger C, Nasmyth K, and Sullivan W

P.62 Intra-strain phenotypic and genomic variability of the commercial Saccharomyces cerevisiae strains Zymaflore VL1 recovered from vineyard environments Ricardo Franco-Duarte, Laura Carreto, Inês Mendes, Brigitte Cambon, Sylvie Dequin, Manuel AS Santos, Margarida Casal, Dorit Schuller

P.63 Glycine uptake in brain nerve terminals is decreased by BDNF through a reduction in GlyT2 membrane insertion Rita I Aroeira, Ana M Sebastião and Cláudia A Valente

P.64 Towards understanding the role of PTEN genes in P. patens Laura Saavedra, Rita Catarino, Tobias Heinz, Ingo Heilmann, Magdalena Bezanilla and Rui Malhó

P.65 Quaking A - a post-transcriptional regulator of early patterning in zebrafish Fernandes SF, Fior R, Gama-Carvalho M and Saúde L

P.66 Potential involvement of Smurf2 in altered regulation of the TGF-beta signalling pathway in Cystic Fibrosis disease Sara Canato, Margarida Telhada, Carlos Farinha, Margarida Amaral, Luka Clarke

P.67 A derivative of the natural compound kakuol affects DNA relaxation of topoisomerase IB Silvia Castelli, Sara Vieira, Ilda D’Annessa, Prafulla Katkar, Loana Musso, Sabrina Dallavalle and Alessandro Desideri

P.68 Cross talks between amyloid-forming proteins in neurodegeneration Sofia B. Carvalho, Sónia S. Leal, Isabel Cardoso, Hugo M Botelho, Günter Fritz and Cláudio M. Gomes

P.69 Microbial Stress to Metals and ROS Cláudia Nóbrega, Iris L. Ziober, Célia Silveira, Bart Devreese, Manolis Matzapetakis, Sofia R. Pauleta

P.70 A ClpB chaperone knockout mutant of Mesorhizobium ciceri shows a delay in the root nodulation of chickpea plants Clarisse Brígido, Marta Robledo, Esther Menéndez, Pedro F Mateos, Solange Oliveira

P.71 Chestnut transcriptome after Phytophthora cinnamomi challenge Susana Serrazina, Carmen Santos, Helena Machado, Cátia Pesquita, Rita Costa, Maria Salomé Pais and Mónica Sebastiana

P.72 Analysis of the effect of the two in cis CFTR splice variants c.2562T>G and c.2657+5G>A Susana Igreja, Teresa Casals, Margarida D Amaral, Anabela S Ramalho

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P.73 Protein misfolding at the mitochondria: lessons from fatty acid oxidation diseases Bárbara J Henriques, Tânia G Lucas, Cláudio M Gomes

P.74 Magnetic labeling and detection of cfDNA for cancer diagnosis Tomás Dias

P.75 Alternative splicing of tumour-related Rac1b is regulated by upstream signalling pathways Vânia Gonçalves and Peter Jordan

P.76 An Hydrogen Peroxide sensor in an anaerobic bacterium: Desulfovibrio vulgaris Hildenborough PerR Vasco Ribeiro, Isabel Silva, David Sardinha, Alice Pereira, Pedro Tavares and Cristina Timóteo

P.77 Using an mRNA-based approach to detect rare CFTR mutations Verónica Felicio, Margarida D Amaral and Anabela Ramalho

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P.1

The role of mycobacteriophage Ms6 LysB in mycobacteria lysis

Adriano Gigante, Francisco Martins, Filipa Gil and Madalena Pimentel

Centro de Patogénese Molecular, Unidade dos Retrovirus e Infecções Associadas, Faculdade de

Farmácia, Universidade de Lisboa

Nowadays bacterial resistance to antibiotics is a disturbing reality and casts a dark veil over the future combat to infectious diseases. It is urgent to find and develop new molecules that can be used as alternative therapeutics. In this sense, bacteriophages have been explored due to their ability to destroy essential structures of bacteria. We have been studying the lysis properties of mycobacteriophage Ms6, a phage that infects Mycobacterium smegmatis, a non-pathogenic strain used as a model to study mycobacteria, which includes M. tuberculosis, the causal agent of tuberculosis, one of the leader causes of death by a single pathogen. Ms6 encodes a lytic enzyme, named LysB, capable of hydrolyzing important structures of the mycobacterial cell envelope with a high lipidic content. In this work we show that using a new method, named Bacteriophage Recombineering of Electroporated DNA (BRED) we efficiently constructed an Ms6 mutant deleted in gene lysB. We show that LysB has an important role on host lysis. One-step growth curve assays revealed that lysis is less efficient in absence of LysB, resulting in a delay of the timing of lysis, as a consequence of a deficient release of the phage particles which are trapped in cell debris. Understanding the bacteriophage-host interactions at the molecular level, will contribute to the discovery of new antibacterial therapeutic targets and for the design of phage-based products with medical and biotechnological applications.

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P.2

Internal ribosome entry site-mediated translational regulation of mammalian target of rapamycin (Mtor) in stress conditions

Marques-Ramos A1,3, Lacerda R1,3, Teixeira A1,2, Romão L1,3

Departamento de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa

Regulation of translation is a key mechanism by which cells and organisms can rapidly change their gene expression patterns in response to extra- and intracellular stimuli. Translational control can occur on a global basis by modifications of the basic translation machinery, or selectively target defined subsets of mRNAs to maintain synthesis of certain proteins required either for stress responses or to aid recovery from the stress condition. These pathways are evolutionary conserved and have been shown to significantly impact translation in organisms as diverse as yeast and humans. In many cases, features in the 5’ untranslated regions (5’UTRs) of the corresponding mRNAs, such as internal ribosome entry sites (IRESs) are important for them to evade global repression of translation. IRES-mediated translation is an alternative to the cap-dependent mechanism of translation initiation that involves direct recruitment of the ribosome to the vicinity of the initiation codon and may require several IRES trans-acting proteins. Mammalian target of rapamycin (Mtor) is a highly conserved kinase that is responsive to several cellular stimuli. Deregulation of Mtor 18erreira18 is implicated in major diseases, mainly due to its role in regulating protein synthesis. The main Mtor targets are proteins responsible for ribosome recruitment to the Mrna, thus, a specific inhibitor of Mtor, for example rapamycin, leads to global inhibition of translation. Knowing that in stress conditions such as hypoxia, overall protein synthesis is reduced, but synthesis of Mtor protein is not inhibited, we hypothesized that Mtor 5’UTR harbours an IRES allowing cap-independent synthesis of Mtor protein in stress conditions. By using dicistronic reporter plasmids we have tested and confirmed this hypothesis. Our findings provide a framework for understanding how translational reprogramming of Mtor Mrna is regulated in response to cellular stress conditions

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P.3

miR-34c-5p at the crossroads of human © CD4 T-cell activation and HIV infection

Ana Margarida Matos1*, Russell B Foxall2*, Andreia J Amaral1,2, Jorge Andrade1, Paula Matoso2, Mariana Santa-Marta3, Rita Tendeiro2, Ana Serra-Caetano2, Rui S Soares2, João Gonçalves3, Margarida

Gama-Carvalho1, Ana E Sousa2

1BioFIG- Center for Functional and Integrative Genomics, Faculdade de Ciências 2Instituto de Medicina Molecular, Faculdade de Medicina 3URIA- Centro de Patogénese Molecular, Faculdade de

Farmácia, Universidade de Lisboa, Portugal *Co-first authors

A delicate balance of cell proliferation, differentiation and death occurs upon TCR stimulation of

naïve CD4 T cells. These processes are likely to involve microRNAs, which target the 3‟UTR of specific

mRNAs and represent one of the most important mechanisms of posttranscriptional regulation.

Activated CD4 T cells are the main targets of HIV infection, which critically depends of the state of

cellular activation for effective integration and productive viral replication. We hypothesized that HIV

may subvert the microRNA profile induced by TCR stimulation. Based on a massive parallel

sequencing study of the total microRnome of human naïve CD4 T cells upon cell activation and HIV

infection, we identified miR-34c-5p as a novel candidate in the regulation of T cell activation.

Moreover, we found this miR down-regulated during HIV infection. Independent RT-Qpcr validation

of miR-34c-5p expression levels in CD4 T cells confirmed its up-regulation after TCR stimulation, and

down-regulation following HIV infection of activated CD4 T cells. Expression kinetic studies revealed

that miR-34c-5p levels, measured by RT-Qpcr, started to increase later upon activation, suggesting a

role in cell survival/apoptosis. Additionally, our in silico analysis predicted miR-34c-5p to regulate

several target genes that are relevant for T cell function and HIV infection. In order to further

elucidate this, stable Jurkat T cell lines constitutively over-expressing miR-34c-5p were generated. In

addition to changes in cell size and proliferation, miR-34c-5p over-expression resulted in the down-

regulation of Bcl-2 and CXCR4, with potential implications for HIV replication and latency. Moreover,

over-expression of miR-34c-5p was found to inhibit viral replication in stable Jurkat clones,

suggesting an important role for this miR in the process of HIV infection.

Acknowledgements: This project is supported by a grant from the Merck Investigators Study

Program, funded by the Merck Sharp & Dome Corp.

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P.4

RefirMAR, a neuromodulator bioactive ingredient derived from a deep sea microorganism: applications for daily skincare

Martins, A. (1), Andrade, G. (2), Rodrigues, I. (3), Almeida, A. (4), Rodrigues, C. (5), Pinheiro, R. (6), Calado, P.

(7), Vieira, H. (8)

(P) BIOALVO S.A., TEC LABS-CENTRO DE INOVAÇÃO- Campus da FCUL, Campo Grande, 1748-016 Lisboa, Portugal, ana.martins@bioalvo.com; (2) BIOALVO S.A., TEC LABS-CENTRO DE INOVAÇÃO- Campus da FCUL, Campo Grande, 1748-016 Lisboa, Portugal, goncalo.andrade@bioalvo.com; (3) BIOALVO S.A., TEC LABS-CENTRO DE INOVAÇÃO-

Campus da FCUL, Campo Grande, 1748-016 Lisboa, Portugal, ines.rodrigues@bioalvo.com; (4) ) BIOALVO S.A., TEC LABS-CENTRO DE INOVAÇÃO- Campus da FCUL, Campo Grande, 1748-016 Lisboa, Portugal,

ana.almeida@bioalvo.com; (5) BIOALVO S.A., Edifício ICAT – Campus da FCUL, Campo Grande, 1748-016 Lisboa, Portugal, catia.rodrigues@bioalvo.com; (6) BIOALVO S.A., Edifício ICAT – Campus da FCUL, Campo Grande, 1748-016 Lisboa, Portugal, ricardo.pinheiro@bioalvo.com; (7) BIOALVO S.A., Edifício ICAT – Campus da FCUL, Campo Grande, 1748-016 Lisboa, Portugal, patricia.calado@bioalvo.com; (8) BIOALVO S.A., Edifício ICAT – Campus da

FCUL, Campo Grande, 1748-016 Lisboa, Portugal, helena.vieira@bioalvo.com.

Screening of phylogenetically diverse and unique microorganisms from rare or extreme ecosystems has increased tremendously in the last decade with relevant bioactivities being discovered. Nevertheless, marine sources deliverables, in particular, are still far from the expectations and so new extreme sources of microbes should be explored. In this work, a marine prokaryotic collection, isolated from four deep sea hydrothermal vents in the Mid-Atlantic Ridge near Azores islands, Portugal, was created, characterized and tested for daily skincare applications. This library, called PharmaBUG, comprises 139 psychrotolerant aerobic and facultative anaerobic prokaryotes that have been extensively characterized and adapted to sustainable growth conditions in the laboratory and industrial scale, allowing a steady and convenient resupply of marine natural ingredients. RefirMAR ingredient was identified as a novel marine microorganism derived bioactive that inhibits the release of acetylcholine at the synapse of the motor neuron. Interestingly, and in contrast to the well known botulinum toxin (BOTOX®), RefirMAR does not have a permanent action in the motor neuron but instead inhibits the release of acetylcholine vesicles in a transient way (dosage range of 1 mg/ml to 10 mg/ml) making it highly advantageous. This novel bioactive ingredient was isolated from a deep sea crustacean sample collected from the Rainbow hydrothermal vent field, at 2296 m depth, where the local temperature is 3.7 ºC. It is a crude aqueous protein extract highly soluble in water, safe, stable and non-cytotoxic. In addition, RefirMAR shows dermal penetration capacity and thus, potential for topical application, avoiding the inconvenience of administration by injection and medical supervision which offers limitless applications for cosmetics. A 28-day clinical study in 12 volunteers was performed to assess RefirMAR’s efficacy in wrinkle smoothing and line reduction, upon daily usage of a 1% formulation. Interestingly this trial has shown an up to 23% wrinkle depth decrease and an up to 64% hydration increase. RefirMAR is thus the first success story in BIOALVO’s path towards marine microorganism-derived added value products and should be released to the market in the short term.

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P.5

Post-transcriptional regulation of SMN2 expression by hnRNP G and LARP Family proteins

Ana Miguel Fernandes1, Ana Luísa Gomes1, Mariana Oliveira1, Gonçalo Costa2, Isabel Peixeiro1, Ana Margarida Matos1, Carlos Cordeiro2,3 and Margarida Gama-Carvalho 1,3

1Gene Expression and Bioinformatics Unit, BioFIG – Center for Functional and Integrative Genomics,

Faculty of Sciences, University of Lisbon, Portugal 2Center for Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, Portugal

3Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, Portugal

Spinal Muscular Atrophy (SMA) is a recessive autosomal neurodegenerative disease caused by the loss of Survival Motor Neuron 1 gene (SMN1). Previous studies have implicated SMN in RNA metabolism, namely in the biosynthesis of small nuclear ribonucleoprotein complexes, and there is evidence that SMN is involved in axonal Mrna transport. Furthermore, in humans there is an almost identical genomic copy of SMN1, the SMN2 gene. SMN2 contains a silent mutation in exon 7 that greatly impairs splicing, leading to the predominant production of a truncated SMN protein that is thought to be rapidly degraded. Interestingly, SMA severity is inversely correlated to the number of SMN2 copies present in the genome. Therefore, SMA would be a strong target for the development of new therapies regarding Mrna stability and translation control. It is widely accepted that Mrna stability and translation are greatly influenced by the action of RNA binding proteins and microRNAs that bind untranslated regions of mRNAs. Hence, identification of proteins binding to SMN2 Mrna would provide a greater insight on mechanisms underlying its expression and ultimately uncover novel targets that favor Mrna stabilization and/or increased translation. In this work we have used in vitro transcribed biotin-labeled probes covering the 3’ UTR of SMN2 Mrna to perform pull-down assays to identify proteins binding its 3’UTR. Several proteins were identified by peptide-mass fingerprinting, among which were proteins described as having a role in Mrna metabolism, namely hnRNP G (hnG) and members of the La-Related Protein (LARP) family, including a novel LARP1 isoform. As LARP family members remain poorly studied, we characterized LARP isoform expression in the cell lines used in this work by RT-Qpcr, confirming the existence of the novel isoform identified by mass-spec. Overexpression of hnG and LARPs resulted in increased expression of both a luciferase reporter containing the SMN 3’UTR sequence and of the endogenous SMN protein. Characterization of SMN Mrna levels identified different mechanisms of action for these proteins at the level of Mrna stability and translation. In order to further clarify the effect of hnG and LARPs on SMN2 expression, hnG and LARPs were co-transfected with various SMN2-3’UTR deletion mutants fused to firefly luciferase. Our data supports a role for hnG and LARPs as positive modulators of SMN Mrna expression.

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P.6

An approach to study glia reactivity in Amyotrophic Lateral Sclerosis

Gomes C1, Correia AM3,4, Gomes R3,5, Vaz AR1, Brites D1,2

1Neuron Glia Biology in Health & Disease, iMed.UL, Faculty of Pharmacy, University of Lisbon, Portugal,

2Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Portugal

3 Unit of Neurosciences, Institute of Molecular Medicine, University of Lisbon, Portugal 4Museu Nacional de História Natural e da Ciência, University of Lisbon, Portugal

5Faculty of Science, University of Lisbon, Portugal

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective loss of motoneurons (MNs), commonly linked to mutations in Cu-Zn superoxide dismutase (SOD1), particularly in familial cases. Glia activation seems to be required for ALS progression. Astrocytes expressing mutant SOD1 were characterized as being dysfunctional, with a higher proliferative and undifferentiated state, while microglia expressing mutant SOD1 evidenced to switch from their normal surveying state to an activated one. Here, we aimed to investigate the phenotypes and the activation stages of these glial cells as potential contributors to ALS progression. Astrocyte and microglia cultures were obtained from brain of 7-days SJL (WT) mice or from those carrying a human SOD1 mutation (TgSOD1-G93A) and maintained for 10 and 23 days in vitro, respectively. The SOD1 expression was measured in both cell types. Astrocytes were quantified based on glial fibrillary acidic protein (GFAP)+ labeling and using Ki-67 staining to assess proliferation. Microglia was stained for CD11b to evaluate activation and for Iba-1 to determine morphology and the number (%) of Iba-1+ cells. SOD1 expression was identified in ~100% of the cells from TgSOD1-G93A, in contrast with ~10% from WT mice, each SOD1+ cell exhibited higher expression levels than WT (4-fold for astrocytes and 3-fold for microglia, p<0.05). In astrocytes from TgSOD1-G93A we observed a decrease in GFAP+ and an increase in Ki-67+ cells when compared to WT, suggesting aberrant phenotypic features. In microglia from TgSOD1-G93A, the number of Iba-1+ cells was slightly higher than in WT, with a prevalence of amoeboid shaped cells, together with higher CD11b levels, suggesting an increase of activated microglia. Altogether, our results show that increased levels of dysfunctional astrocytes and microglia are originated in TgSOD1-G93A mice, early before the disease onset. Exploring the molecular pathways underlying this glial activation will contribute to dissect the role of these cells in ALS onset and progression and to identify key targets that may be restrained in the future. Funded by FEDER (COMPETE Programme) and by National funds (FCT-projects PTDC/SAU-FAR/118787/2010 to DB and Pest-OE/SAU/UI4013/2011/2012).

23

P.7

Aberrant CFTR mRNA Processing: Which Are Consequences?

Anabela S Ramalho1, Verónica Felício1, Margarida D Amaral1,

1 University of Lisbon, Faculty of Sciences, Centre for Biodiversity, Functional and Integrative Genomics (BioFIG), Lisboa, Portugal.

Cystic Fibrosis (CF) is the most common, lethal monogenic disorder in Caucasians. CF is estimated to affect 30,000 individuals in Europe (~900 in Portugal) and is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. Alterations in mRNA processing efficiency are known to have an impact on CF disease manifestation. Thus, correct characterization of CFTR mutations becomes crucial to decide on the adequate therapeutic strategy. Our goal here is to understand the functional impact of CFTR mutations regarding mRNA processing, stability and translatability. CFTR transcripts were analysed by semi-quantitative RT-PCR upon extraction from native tissues of CF patients with F508del in one allele and one the following in the other: 711+1G>T, 1584+18672A>G, 1717-1G>A, 1812-1G>A, 3120+1G>A. Comparing the transcripts relative levels from both alleles (reference-F508del levels), our data show that 1717-1G>A, 1812-1G>A, 3120+1G>A and 1716+18672A>G PTC induce transcripts degradation (63-80%) probably due PTC resulting from the alternative splicing triggered by these mutations. Notwithstanding for the 711+1G>T mutation no transcript decay was observed (ex5 skipping is in-frame). Minigenes with IVS4art+IVS5 introns in full-length CFTR cDNA were constructed to further characterize abnormal mRNA processing. HEK cell expressing the wt and 711+1G>T, 711+3A>G, 711+5G>A (IVS5) mutant mini-genes were. By RT-PCR and Western blot analysis, correct processing of the wt CFTR transcripts and protein were observed confirmed. All mutants CFTR minigenes originated alternative transcripts resulting in complete absence or misprocessed protein. Compounds like Kinetin and EGCG that were proven to correct splicing for other human genes (e.g. neurofibromatosis type 1 –NF1- gene) are being tested using these CFTR minigenes. These data may be of high relevance for CF personalized medicine. Work supported by Pest-OE/BIA/UI4046/2011 BioFig centre grant (POCTI/FCT/PIDDAC, Portugal) and Ciência2008 fellowship (FCT, Portugal) to ASR.

24

P.8

Amyloid-ß Peptide dysregulates TrkB receptors and BDNF function by a calpain-dependent mechanism

André Jerónimo-Santos, Sandra Henriques Vaz, Sara Parreira, Sofia Rapaz-Lérias, António P. Caetano, Valérie Buée-Scherrer, Eero Castrén, Claudia A. Valente, David Blum, Ana Maria Sebastião, Maria José

Diógenes.

Instituto de Medicina Molecular The brain-derived neurotrophic factor (BDNF), through the activation of its full length receptor (TrkB-FL), plays a central role in the nervous system by providing trophic support to neurons and by regulating synaptic plasticity and memory. BDNF 24erreira24 is drastically affected in Alzheimer’s disease (AD), a disease involving the accumulation of amyloid-? (A?) peptide. Previously, we demonstrated that, A? induces a decrease in TrkB-FL and an increase in truncated TrkB-Tc receptors, negative modulators of TrkB-FL 24erreira24, on primary neuronal cultures, in a similar way of what is observed in AD brains, suggesting a direct correlation between Aâ and TrkB receptors alterations. The present work was designed to reveal the mechanisms behind TrkB changes induced by A? and the functional impact of this dysregulation. This work shows that exposure of cortical cultures to A? (25µM, 24h) increases Mrna levels of TrkB-T1 and T2 isoforms by 45±19% and 58±17%, respectively, (n=8, p<0.05) without affecting TrkB-FL Mrna levels. Moreover, we found that A? promotes a calpain-mediated cleavage of TrkB-FL, downstream Shc binding site, generating a new truncated receptor (TrkB-T’) and a stable intracellular domain fragment (TrkB-ICD) which was also detected in human brain samples. Functionally, we found that A? impairs BDNF actions upon neurotransmitter (GABA and glutamate) release on synaptosomes prepared form adult hippocampal slices and impairs hippocampal long-term potentiation (LTP), in a calpain-dependent way. This work shows that A? 1) increases Mrna levels of TrkB-Tc isoforms without affecting TrkB-FL Mrna, 2) induces a calpain-mediated cleavage of TrkB-FL, originating a new truncated receptor and an intracellular fragment and 3) impairs BDNF effects, in a calpain-dependent way, on neurotransmitters release as well as in LTP.

25

P.9

An automated high-throughput screening platform to study the role of C.elegans Kinesin-5

André F. Maia1,2, Daphne Lelieveld3, Matilde Galli4, David A. Egan3, Marvin E.Tanenbaum2, Claudio Sunkel1, Sander van den Heuvel4 and René H. Medema2,5

1 Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal

2 Department of Medical Oncology, Laboratory of Experimental Oncology, University Medical Center Utrecht, Utrecht, The Netherlands

3 Department of Cell Biology, Cell Microscopy Center, University Medical Center Utrecht, Utrecht, The Netherlands

4 Department of Biology, Utrecht University, Utrecht, The Netherlands 5 Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands

Accurate chromosome segregation during mitosis is crucial for the maintenance of genomic stability and prevention of tumorigenesis. The assembly of a bipolar spindle is fundamental in this process. A highly conserved motor protein, Eg5/BMK-1 (Kinesin-5), is essential for bipolar spindle formation in several organisms, including humans, but not in the nematode Caenorhabditis elegans. Therefore, this organism is attractive for identification of redundantly acting genes in a synthetic lethal screen. Accordingly, we assembled an automated high-throughput screening platform and performed a genome-wide RNAi screen in the C.elegans bmk-1 null mutant. For the read-out of the screen we determined the animals’ reproductive fitness, upon ingestion of the double-stranded RNA, and compared to the wild type strain. We were able to successfully screen 82% of the C.elegans genome and found several genes that further decrease the animals’ reproductive fitness when silenced in the bmk-1 null mutant. Amongst these genes are genes that might be involved in the bipolar spindle assembly, as well as genes that may play other functions during the worm development unrelated to the mitotic spindle. We are currently analyzing the function of the identified genes and the relevance of the genetic interaction with kinesin-5, by looking at the early C.elegans embryo mitotic divisions.

26

P.10

Deep-sequencing profile of small ncRNAs in human CD4+ T cells upon TCR-mediated activation and its modulation by HIV infection

Andreia J Amaral1,3, Jorge Andrade1, Ana Margarida Matos1, Paula Matoso3, Rui Soares3, Russel

Foxall3, Rita Tendeiro3, Margarida Gama-Carvalho1, João Gonçalves2,3, Ana Espada Sousa3

1BioFIG- Center for Functional and Integrative Genomics, Faculty of Sciences, University of Lisbon, Portugal; 2URIACentro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, Portugal;

3Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal CD4+T-cells are the main orchestrators of immune responses. They are produced in the thymus and egress as so-called “naïve” T-cells that patrol the body. Naïve T-cells will be activated through their specific T-cell receptor (TCR) upon encounter with their cognate antigen and this activation is essential for their clonal expansion and differentiation into memory/effector cells critical for the mounting of immune responses. CD4+ T–cell activation is a tightly controlled process in order to prevent disruptions of T-cell homeostasis, requiring a delicate balance of cell proliferation, differentiation and cell death. Furthermore, activated CD4+ T-cells are the main targets for HIV infection, which depends upon the cell-activated state for effective viral integration into the host genome and replication. In recent years, miRNAs have been identified in cells of the innate and adaptive immune systems, which are modulated by HIV infection in T-cells. Moreover, other types of small non-coding RNAs (ncRNAs) have been identified in mammalian cells that have a role in the regulation of cell proliferation and differentiation. We have investigated the changes in the profile of small ncRNAs upon TCR-mediated activation of purified naïve CD4+ T-cells and after infection with HIV. Small RNA molecules were isolated from total RNA and sequenced using sequencing by synthesis (Illumina), generating the largest profile ever reported of ncRNAs in human CD4+T-cells. Similarly to previous studies, about 50% of the small RNA molecules identified corresponded to known miRNAs. Additionally, we identified different types of ncRNAs that were differentially expressed upon cell activation and modulated by HIV infection, which have not been reported before. Our results provide novel insights into the gene expression networks involved in the activation of CD4+ T-cells, which can be explored to identify key regulators of T-cell activation and differentiation, as well as of the intrinsic cell response to HIV infection. Acknowledgements: This project is supported by a grant from the Merck Investigators Study Program, funded by the Merck Sharp & Dome Corp.

27

P.11

Changing laminin isoforms during skeletal muscle development: different players or a backup system?

Andreia M. Nunes1, André Gonçalves1, Patrícia Ybot-Gonzalez2, Marianne Deries1 & Sólveig

Thorsteinsdóttir1,3

1Centro de Biologia Ambiental/Departamento de Biologia Animal, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal; 2 Unidad de Gestion de Pediatria, Hospital Infantil Virgen del Rocio, Instituto de Biomedicina de Sevilla (IBIS), Sevilla, Spain; 3 Instituto Gulbenkian de Ciência,

Oeiras, Portugal. Skeletal muscle development is a complex process which starts early in embryogenesis, and progressively builds up skeletal muscles. Laminins are glycoproteins which compose the extracellular matrix that engage muscle cells and are essential for muscle development and function. Laminins 111 and 511 are thought to be essential for early stages of myogenesis, while laminins 211/221 are crucial for later stages concomitantly with myotube formation. When alpha1-laminins are absent, as in Dmrt2 mutants, early myogenesis is perturbed, but recovers later on. However, when alpha2-laminins are lacking, as in Merosin (laminin alpha2)-deficient congenital muscular dystrophy type1A (MDC1A) in humans, crippling muscle weakness is evident from birth. We are addressing the question of when and how the transition between these two laminin niches occurs during development, whether they overlap in time and/or complement each other. Surprisingly, our data show that in wild type embryos laminin alpha2 is present in myotomes already at E9.5, suggesting a role in early stages of myogenesis. Laminin alpha1 and alpha5 are also present at E9.5 and laminin alpha5 persists in muscle masses at least until E12.5. Thus there is a temporal overlap of laminins 111, 511 and 211/221 suggesting they may be able to compensate for each other. To address this issue, we are currently studying myogenesis in Dmrt2 mutants, which lack laminin-111 (and possibly also laminin-511) during early stages of myogenesis to determine whether (and, if so, how) laminins 211/221 are able to rescue the muscle in these mutants. This work was financed by FCT project PTDC/SAU-BID/120130/2010.

28

P.12

Deciphering grapevine resistance to Plasmopara viticola: hints from proteomics

Andreia Figueiredo1, Joana Martins2, Mónica Sebastiana1, Filipa Monteiro1, Ana Coelho2

1 Plant Systems Biology Lab, FCUL, Center for Biodiversity, Functional and Integrative Genomics, Portugal

Grapevine (Vitis vinifera L.) is one of the major cultivated crops as well as an emerging model species for fruit crop biology, reflecting the recent completion of its draft genome sequence. The cultivated 28erreira28 is highly susceptible to several diseases including downy mildew caused by the oomycete Plasmopara viticola (Berk and Curt.) Berl. And de Toni. Disease control currently requires the repeated application of chemical fungicides that cause extensive damage to the environment. The introgression of specific genetic traits, such as resistance to pathogens in traditional crops by breeding programs is one of the most promising methods to avoid the massive use of pesticides in each growing season. In the last 20 years, parallel to the development of technologies for high throughput analyses, grapevine-P. viticola interaction has captured the interest of researchers and several studies have been conducted in order to characterize grapevine specific defense mechanisms. Despite all the knowledge gathered on this interaction, recent reports documented the emergence of P. viticola resistance-breaking isolates in Europe, support the need to continue identifying candidate resistance genes and to pursue breeding strategies that perform knowledge-based pyramiding of resistance. Previously we have used high-throughput approaches as Cdna microarrays and 1H NMR to characterize the differences between resistant and susceptible grapevine genotypes prior (constitutive) and after inoculation with P. viticola. We have selected several candidates that are putatively associated to pathogen recognition, 28erreira28 and defense. In order to complement the previously used approaches and to integrated the knowledge on this interaction, a difference gel electrophoresis (DIGE) approach is being conducted. So far several differentially expressed proteins when comparing a resistant (Vitis vinifera cv Regent) and a susceptible (V. vinifera cv Trincadeira) grapevine at 0h, 6, 12 and 24 hpi, were identified. This systems biology approach will allow the identification of several targets that could potentially be exploited to develop new protection strategies. Acknowledgments: Project funding: FCT-PTDC/AGR-GPL/119753/2010. Fellowship: FCT-SFRH/BPD/63641/2009 and #Pest-OE/EQB/LA0004/2011.

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P.13

m7GDP mononucleotide is not a substrate for DcpS enzyme but might regulate its activity in vitro

Anna Wypijewska1, Elzbieta Bojarska1, Maciej Lukaszewicz1, Janusz Stepinski1, Jacek Jemielity1,2,

Richard E. Davis3 and Edward Darzynkiewicz1

1 Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland

2 Centre of New Technologies, University of Warsaw, Warsaw, Poland 3 Department of Biochemistry and Molecular Genetics, University of Colorado, School of Medicine,

Aurora, Colorado, United States

DcpS is a pyrophosphatase that functions in the 3’→5’ Mrna decay to catalyze the hydrolysis of the 5’ Mrna cap structure within dinucleotide species (m7GpppG) remaining after the exosome-mediated turnover of the Mrna chain. In some reports, DcpS was also assigned as the hydrolase responsible for the degradation of 7-methylguanosine diphosphate (m7GDP), a product of the Dcp2-mediated decapping in the 5’→3’ Mrna decay. Examining DcpS activity from four species: Homo sapiens, Caenorhabditis elegans (nematode; model organism), Ascaris suum (parasitic nematode) and Saccharomyces cerevisiae (yeasts; model organism) we evidenced by HPLC chromatography that DcpS has no ability to digest m7GDP in vitro. Interestingly, by means of fluorescence titrations we

-1) and is a competitive inhibitor of DcpS enzymes (shown in HPLC assays), regardless of their origin. Concluding, DcpS enzyme is likely not involved in the direct turnover of m7GDP in the 5’→3’ Mrna decay. However, m7GDP might interact with DcpS and regulate its activity. Thus, we proposed an amended model of Mrna degradation.

30

P.14

Comparative Genomics of Bacillus spp.: can core genome be the key for new therapeutics? Assessment of the core genome of Bacillus spp. And its stability

Carlos Alonso1,2, Francisco M. Couto3& Ricardo Dias1

1LMB-BioFIG Laboratory for Microbiology and Biotechnology – Center for Biodiversity, Functional &

Integrative Genomics 2Laboratório de Bromatologia e Defesa Biológica, Exército Português

3LASIGE, Department of Informatics, Faculty of Sciences, University of Lisbon

Anthrax is a disease caused by the bacteria Bacillus anthracis which primarily 30erreir ruminants, but may occasionaly infect human beings. This disease has been nearly 30erreira3030 in western countries in the last decades, but has been brought back to light following its use in the terrorist attacks in 2001 in the USA. Given this bacteria’s ability to survive in the environment and its easy dissemination thanks to its spore forming ability, it is an election agent to be used in a bioterrorist attack. In addition to the bacteria’s innate pathogenic caracteristics, taking in account the current state of molecular biology, there is a chance that a B. anthracis strain used in a bioterrorist attack has been genetically modified to gain antimicrobial resistance mechanisms. With the advent of synthetic biology there is also the risk of B. anthracis pathogenic charateristics being introduced in a different Bacillus species. This would create a new pathogen with the same pathogenic characteristics as B. anthracis, but which would not be identified by the detection methods for B. anthracis and might need a different clinical treatment. The objective of this work is to identify the core genome and essential genes of the genus Bacillus. The present work aims in its first phase to identify the core genome of the genus Bacillus. Within the scope of this work, the core genome is considered as the genetic information which is transversally present in most or all strains of the same genus.The fact that this genetic material remains conserved in divergent species suggests that it is essential for survival. In order to identify the core genome of the genus Bacillus, all the completely sequenced genomes of the genus Bacillus available in NCBI will be used, along with a set of controls, and a clustering 30erreira will be 30erreira to identify sets of orthologous genes conserved in all or most strains of the genus Bacillus. The second phase of the project will consist in the identification of essential genes in Bacillus spp. Essential genes are genes that are indispensable to support cellular life. These genes constitute a minimal gene set required for a living cell. The identification of essential genes will be made through comparison with known essential genes, the assessment of several parameters which may reflect essentiality – such as function, Ka/Ks ratio, 30erreira localization, proteic and aminoacidic parameters – and machine learning methods. The knowledge of the set of core and essential genes in the Bacillus genus would make way for the subsequent task of selecting a subset of target genes, which would be used to develop molecules with therapeutic or prophylactic action in B. anthracis. Seeing as these molecules would interact with essential, conserved proteins, it would be more difficult for someone to engineer a resistant strain, or for the bacteria to naturally develop resistance mechanisms.In a preliminary run with 80 proteomes (57 Bacillus spp. Proteomes and 23 from other bacterial genus as control), 30erreira 289405 proteins, it was observed that 210153 proteins grouped in 27272 clusters, from which 20791 were found in the Bacillus genus. 18468 proteins were assigned to clusters (n = 324) including all Bacillus proteomes analyzed. Analysis of the main functional classes will be presented.

31

P.15

Revertants, Low temperature and Correctors Provide Clues to Mechanism of F508del-CFTR Rescue by VX-809 and Suggest Multiple Agents for Full Correction

Carlos M Farinha1, John King-Underwood2, Bárbara J Henriques3, Ana Raquel Correia3,Marisa Sousa1,

Jonathan Williams2, Simon Hirst2, Cláudio Gomes3, Margarida D Amaral1

1BioFIG – Center for Biodiversity, Functional and Integrative Genomics, Faculty of Sciences,University of Lisboa, Portugal; 2Sygnature Discovery, Nottingham, UK; 3ITQB, Oeiras, Portugal

The most common disease-causing mutation in the CFTR gene, F508del, causes Cystic Fibrosis through impairment of protein from exiting the endoplasmic reticulum due to misfolding. The investigational drug VX-809 is a small-molecule that rescues F508del-CFTR localization which recently went into clinical trial but with still unknown mechanism of action (MoA). Herein, we assessed if VX-809 is additive or synergistic with genetic revertants of F508del-CFTR, other correctors (VRT-325 and Corr-4a) and low temperature to attempt determining its MoA. We explored and integrated those various agents in combined treatments, showing how they add to each other, to identify their complementary MoA and the full scope for F508del-CFTR correction. Results from biochemical, functional and 31erreira approaches pointed to major insights into its MoA point to major insights into its MoA: 1) VX-809 is additive to both VRT-325 and Corr-4a, suggesting that each compound operates by a different MoA. 2) VX-809 is additive to low temperature rescue of the mutant almost to wt-CFTR levels. 3) VX-809, VRT-325 and Corr-4a show variable additive effects with the genetic revertants tested (4RK, G550E and R1070W) thus providing clues for their possible action being exerted at specific protein binding pockets: VX-809 at the NBD1:TM2 interface (and VRT-325 at NBD1:NBD2) or acting unspecificallly (Corr-4a). 4) VX-809 does not rescue the diacidic-code traffic mutant (D565D567/AA) in contrast to low temperature which seems to act at trafficking surveillance checkpoints. Besides suggesting a MoA for VX-809, our data also indicate the scope for further synergistic F508del-CFTR correction by other compounds at distinct conformational sites/cellular checkpoints. Altogether, these data provide a plausible explanation for the limited success of VX-809 in clinical trials and suggest that combination therapies may thus be required to achieve full F508del-CFTR correction, so as to reach the functional CFTR threshold necessary to avoid CF. These findings should be exploited for therapeutic strategies of Cystic Fibrosis. Work supported by CFF-Cystic Fibrosis Foundation, USA (Ref 7207534) and PTDC/SAU-GMG/122299/2010, PTDC/BIA-BCM/112635/2009 and BioFig (Pest-OE/BIA/UI4046/2011) grants from FCT/POCTI. Authors thank CFF-USA for correctors and antibody.

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P.16

A novel in vitro model to study hypoxia-induced effects in retinal pigment epithelium

Inês P. Rodrigues1, Sara Maia1, Cristiana F. Pires1, Martim D. Portal1, Carolina Thieleke-Matos1,2, Olaf Strauss3, Miguel C. Seabra1,4, José S. Ramalho1

1CEDOC, NOVA Medical School, 1169-056 Lisboa, Portugal. 2Instituto Gulbenkian de Ciência, 2780-

156 Oeiras, Portugal. 3Charite University Medicine Berlin, Berlin, Germany. 4Molecular Medicine Section,

National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United Kingdom. The retinal pigment epithelium (RPE) cells constitute the blood-retinal barrier and integrate essential functions for maintenance of visual homeostasis. Aging and retinal degenerative disorders involve deregulation of homeostatic processes, namely the secretion of growth factors, like vascular endothelial growth factor (VEGF). Here, we show that B6-RPE07 mouse cell line displays important retinal epithelial features similar to primary RPE cells. Expression of epithelial markers was assessed by immunofluorescence for ZO1 and Na+/K+ ATPase proteins, and we concluded that both are expressed in cultured RPE cells. Transepithelial resistance measurements showed similar values between B6-RPE07 and primary cells. Regarding VEGF secretion, we showed that B6-RPE07 secretes VEGF under unstimulated conditions, reinforcing the idea that this cell line is a reliable model to study RPE biology. We further tested whether cobalt chloride (CoCl2) could be used, in these cells, as a hypoxia-mimicking agent to potentiate VEGF secretion. We determined the optimal concentration of CoCl2 to use with minimal cytotoxicity and proved that B6-RPE07 secretes higher levels of soluble VEGF in response to presence of CoCl2. We propose, therefore, to use B6-RPE07 as a new mouse model to study VEGF biology in the RPE under hypoxia, an existing condition of most retinal disorders.”

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P.17

Posttranscriptional regulation of bacterial virulence

Christian G. Ramos1, André M. Grilo1, Joana R. Feliciano1, Paulo J.P da Costa1, Jorge H. Leitão1,2

1IBB – Institute for Biotechnology and Bioengineering, and 2Department of Bioengineering, Instituto Superior Técnico, Universidade Técnica de Lisboa, Portugal.Email: 33erreira33.ramos@ist.utl.pt

Bacterial virulence is a multifactorial phenomenon, requiring a wide array of distinct effectors (1), which have been increasingly shown to be regulated at the post-transcriptional level by sRNAs, mediated by Hfq-like RNA chaperones (2). Burkholderia cepacia complex (Bcc) bacteria are opportunistic multidrug resistant human pathogens, able to cause life-threatening infections especially, but not restricted, to cystic fibrosis and immunocompromised patients. SRNAs generally require the action of the ubiquitous RNA chaperone Hfq. In the case of Bcc bacteria, two distinct RNA chaperones, Hfq and Hfq2, are required for virulence (3). We have recently used a RNOmics-based strategy to identify Bcc sRNAs that interact with Hfq or Hfq2 (4). A total of 24 sRNAs were identified by co-precipitation with Hfq. These sRNAs are unevenly distributed within the genome: 13 in the chromosome1, 9 in chromosome 2, and 2 in chromosome 3 (5). While the novel Srna h2Cr was found to regulate the ability of the bacterium to persist within the digestive tract of the nematode Caenorhabditis elegans, used as an infection model. H2Cr overexpression reduces the survival of the nematodes, despite a lower number of CFU’s thriving inside C. elegans (6), indicating an important role played by h2Cr in persistence, but not in killing the nematode. H2Cr negatively regulates the hfq2 Mrna through binding to part of its 5’-UTR, resulting in accelerated decay and reduced protein levels in exponentially growing cells. Details on the functional and molecular characterization of h2Cr will be presented, and the relevance of sRNAs on B. cenocepacia virulence will be discussed. References

P. Leitão JH et al (2010). Appl Microbiol Biotechnol 87, 31-40. 2. Ramos CG et al (2010) Small non-coding RNAs in Prokaryotes, LAP, pp. 75. 3. Ramos CG et al (2011). J Bacteriol 193, 1515-1526. 4. Ramos CG et al (2011). ENBENG 2011, 1-4. 5. Ramos CG et al (2013). Genomics 101 : 139-148 6. Ramos CG et al (2012). PloS ONE 7: e47896.

Acknowledgements This work was partially supported by Fundação Ciência e a Tecnologia, Portugal (contract PTDC/BIA-MIC/119091/2010), post-doc grant (GCR) and PhD grants (AMG and JRF).

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P.18

Translational regulation of human hemojuvelin expression via upstream open reading frames

Cláudia Onofre, Cristina Barbosa, Luísa Romão

Departamento de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo Jorge, Av. Padre Cruz,

1649-016 Lisboa, PORTUGAL

Hemojuvelin (HJV) is a glycosylphosphatidylinositol (GPI)-linked membrane protein shown to be a co-receptor for class of ligands called bone morphogenetic proteins (BMPs). The HJV is involved on iron homeostasis through regulation of hepcidin transcription levels. Mutations on the hepcidin gene or in the HJV gene cause an early-onset inherited disorder associated to iron overload named juvenile hemochromatosis. A better knowledge of the mechanisms implicated in HJV gene expression is crucial to understand its role in the iron homeostasis. The 5’ leader sequence of the human HJV Mrna has two upstream open reading frames (Uorf) with 28 and 19 codons created by two upstream AUGs (uAUGs) that share the same stop codon. Reporter constructs containing several HJV 5’ leader sequences fused to the Firefly luciferase cistron were tested in HeLa and HepG2 cells to evaluate the effect of these uORFs in the translational regulation of HJV Mrna. Luciferase activity was measured by luminometry and the corresponding Mrna levels, quantified by real-time RT-PCR. The results revealed that the HJV uORFs decrease the translational efficiency of the main ORF in about 6-fold. Furthermore, we have observed that the production of HJV protein is mainly due to translation reinitiation. Thus, HJV Mrna has a low leaky scanning ability that contributes to the translational repression of the main ORF. We also observed that the amino acid sequence of the Uorf2-encoded peptide seems to cause ribosomal stalling, which also impedes translation of the downstream main ORF. In addition, our preliminary results show that in HepG2 cells submitted to Eif2? Phosphorylation or iron overload, the HJV uORFs-mediated translational repression is released. These results suggest that these uORFs play a role in regulating HJV expression levels in response to iron overload.

35

P.19

Translation of the human erythropoietin transcript is regulated by an upstream open reading frame in response to hypoxia

Cristina Barbosa, Luísa Romão

Departamento de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo Jorge, Av. Padre Cruz,

1649-016 Lisboa, PORTUGAL

In mammalian cells, most translational regulation is exerted at the cap-translation initiation step, where the AUG codon is identified and decoded. However, when translation is inhibited by cell stress, alternative mechanisms of translation initiation act to maintain the synthesis of certain proteins required either for the stress response or to aid recovery from the stress stimulus. These pathways are evolutionarily conserved and have been shown to dramatically impact translation. In many cases, features in the 5’ untranslated region (UTR) of these mRNAs are important for translational control. These include small structural elements, internal ribosome entry sites and regulatory upstream open reading frames (uORFs). UORFs modulate translation of the main ORF by decreasing the number and/or efficiency of scanning ribosomes to reinitiate at the start codon of the main ORF. In its classical hormonal role, human erythropoietin (EPO) is a glycoprotein synthesized and released mainly from the kidney, which has a key role in hematopoiesis. However, recent studies have revealed that EPO is a multifunctional molecule produced and utilized by many tissues that rapidly responds to different cell stress stimuli and tissue injuries. Thus, it has the potential to be used as a therapeutic target/strategy for the treatment of several human disorders. Understanding the EPO translational control mechanisms will be valuable in the determination of these therapies. Knowing that human EPO transcript presents a 5’UTR with 181 nucleotides containing a 14-codon-Uorf, conserved among different species, which might indicate its role in translational regulation, we aimed to prove this hypothesis. To explore the role of the EPO Uorf in controlling translation, we cloned the EPO 5’UTR, with the intact or disrupted Uorf, fused to the firefly luciferase reporter cistron. HepG2, HEK293 and REPC cells were transiently transfected with these constructs. Luciferase activity was measured by luminometry and the corresponding Mrna levels were quantified by real-time RT-PCR to obtain translation efficiencies. Results have shown that the Uorf can decrease the main ORF translation efficiency in about 3-fold. In addition, results support the conclusion that reinitiation, and in less extent leaky scanning, are responsible for the main ORF translation. Furthermore, this repression is released under hypoxia, specifically in REPC renal cells, via Eif2? Phosphorylation. These findings provide a framework for understanding that production of high levels of EPO induced by hypoxia is also regulated at the translational level.

36

P.20

Matching Biomedical Ontologies using External Resources

Daniel Faria

Faculty of Sciences, University of Lisbon

Ontology matching is a task that has gained particular relevance in the biomedical domain in recent years. On the one hand, ontologies and structured controlled vocabularies have become the preferred media for knowledge representation and storage in this domain. On the other hand, there is a significant overlap between the existing biomedical ontologies, so integrating them is paramount to ensure interoperability and facilitate knowledge sharing. Matching biomedical ontologies is a difficult task, due to the complexity and ambiguity of biomedical terminology. However, the availability of biomedical ontologies with overlapping domains can facilitate the matching process, as it enables the use of external ontologies as background knowledge for matching two ontologies. One of the main goals of the SOMER project is precisely to exploit the availability of ontologies in the biomedical domain for ontology matching. In this context, we have developed the AgreementMakerLight ontology matching system, which features a methodology for automatically selecting the external ontologies that are more useful as background knowledge in a given ontology matching problem. Additionally AgreementMakerLight includes three different strategies for using the external ontologies: cross-reference matching, lexical matching and lexical extension. These features have enabled AgreementMakerLight to surpass the results of top ontology matching systems in the benchmark biomedical ontology matching tasks of the Ontology Alignment Evaluation Initiative (OAEI).

37

P.21

Dynamic expression of Notch pathway components in the ovary during the mouse estrous cycle

Daniel Murta,a* Marta Batista,a Alexandre Trindade,a,b Elisabete Silva,a Domingos Henrique,c António

Duarte,a,b and Luís Lopes-da-Costa a

Reproduction and Obstetrics, CIISA, Faculty of Veterinary Medicine, Technical University of Lisbon, Lisbon, PORTUGAL; b. Gulbenkian Institute of Science, Oeiras, PORTUGAL; c. Institute of Molecular

Medicine, Faculty of Medicine, University of Lisbon, Lisbon, PORTUGAL

* Presenting author:addanielmurta@fmv.utl.pt Faculdade de Medicina Veterinária, Avenida da Universidade Técnica, 1300-477 Lisboa, PORTUGAL

Tel.: (+ 351) 21 365 2825 Following puberty and during the estrous cycle throughout life, only a small proportion of the ovarian pool of primordial follicles escape their arrested state and resume growth and development, the major proportion becoming atretic. As development continues, the follicle reaches maturity and ovulation occurs, originating the corpus luteum (CL). This cyclic transient endocrine gland, which main function is progesterone (P4) production, plays a central role on the regulation of the estrous cycle and on pregnancy establishment and maintenance. The ovarian dynamics are tightly regulated, involving extensive cell-to-cell communication. Notch pathway is an evolutionarily well-conserved cell signaling pathway that has been implicated in cell fate decisions in several tissues. Here, we evaluated the presence of Notch proteins during estrous cycle follicle and CL development. Mature CD1 female mice were monitored through vaginal cytology, and at each of the four stages of the estrous cycle (proestrus, estrus, metaestrus and diestrus) five females were euthanized. Blood samples were collected for measurement of P4 concentrations and ovaries were processed for immunohistochemistry, to evaluate the expression patterns of Notch receptors (1, 2 and 3) and ligands (Jagged1, Delta-like1 (Dll1) and Dll4) in follicle and CL developmental stages. During follicle development (stages: primordial, primary, secondary or pre-antral and tertiary or antral), Notch2 and Dll4 are expressed at all stages, whereas Notch1 is not expressed at any stage. Notch3 and Dll1 are expressed respectively from the primary and secondary stage onwards, whereas Jagged1 is expressed from primordial to pre-antral follicles. Dll1 is specifically immunolocalized in granulosa cells completely surrounding the oocyte zonae pellucidae. Notch receptors (1, 2 and 3) are expressed during all CL developmental stages. However, while Notch2 staining is at the highest intensity during diestrus (when P4 concentrations peak), Notch3 staining is at the highest intensity during estrus (at the lowest P4 concentrations). Regarding Notch ligands, Dll1 is only expressed in small luteal cells during metaestus and diestrus, whereas Dll4 and Jagged1 are expressed at all stages of CL development. These results prompt for a functional role of Notch signaling in the regulation of follicle and CL development during the estrous cycle. We suggest that the specific dynamic combination of different Notch pathway components is involved in the orchestration of cyclic follicle and CL developmental events.

38

P.22

Monitoring anthracnose emergence and pathogen population dynamics in the expanding cashew tree culture in Guinea Bissau

Batista, D.1, Romeiras, M.M.2, Talhinhas, P.1, Silva, D.N.1, Vieira, A.1, Loureiro, A.1, Duarte, M.C.2,

Catarino, L.2

IICT, Instituto de Investigação Científica Tropical – 1BIOTROP Oeiras, Quinta do Marquês, 2784-505 Oeiras, Portugal, 2 JBT, Trav. Conde da Ribeira, nº 9, 1300-142 Lisboa, Portugal

In the last decades, agriculture in Guinea Bissau (GB) has been following a strong trend of moving on from traditional farming systems towards commercial production, mainly due to cashew expansion. Cashew is by far the most important cash crop grown in GB and its main export product. It is mostly produced by small farmers in monospecific orchards, involving in some way more than 85% of the rural population. Given the near optimal conditions provided for this crop in GB, nuts of superior quality with virtually no agro-chemical inputs are produced. In spite of no plant genetic improvement or suitable husbandry practices have been implemented, yields appear relatively acceptable and comparable to those in India and Brazil, with a tendency of quickly getting higher. As the area of cashew orchards increases, negative impacts are expected to occur, namely pest and disease emergence driven either by adaptation to local conditions or inadvertent importation. Some worrying signs of localized dying trees due to anthracnose (Colletotrichum gloeosporioides) are coming to light but the extent of its widespread and the severity of damages are largely unknown. Given this scenario and the experience in other cashew producing countries, an early action is of the utmost importance in controlling the disease and limiting production losses. A research work was recently initiated to address this question by conducting a biogeographical survey of anthracnose incidence in GB and assessing pathogen genetic diversity and differentiation as to trace patterns of migration/dispersal, gene flow and phylogeographical structure. A comprehensive knowledge on the spatial organization of pathogen genetic variation and on how population dynamics is being shaped is needed to provide reasonable guidelines for disease control strategies. Funded by FCT (project PTDC/AFR/117785/2010)

39

P.23

Regulation of Drug Metabolizing Enzymes and Transporters in Mouse Liver by Primaquine

Gonçalves,E1, Marques,C1, Nogueira,F2, Rosário,V2, Ribeiro,V1

1Centre of Molecular and Structural Biomedicine (CBME) / Institute of Biotechnology and Bioengineering (IBB), University of Algarve, Faro, Portugal 2Center of Malaria and other Tropical

Diseases, IHMT-UNL, Lisboa, Portugal

Malaria is one of the greatest of all infectious diseases, afflicting more than 500 million people and causing around 2 million deaths each year. Although many classes of drugs have been used in the treatment and prophylaxis of malaria, therapeutic failure is an increasingly important problem either through parasite resistance or variability in the host’s metabolism. Drug metabolizing enzymes (mainly Cytochromes P450) and transporters are often involved in therapeutic efficacy as well as in clinically significant drug-drug interactions. An additional source of variability is the regulation of those genes, which is mediated by a group of nuclear receptors. Although the genes involved in mammalian drug metabolism and transport have been object of many studies, their response to antimalarial drugs is poorly understood. In this study we evaluated the effect of antimalarial drugs in the hepatic expression levels of these genes using a rodent malaria model. Expression patterns were evaluated by RT-PCR in three strains of mice infected with Plasmodium chabaudi and subject to a treatment with primaquine. The analysed genes included Cytochromes P450 (from families Cyp1, Cyp2 and Cyp3), ABC transporters (abcb1) and nuclear receptors (PXR and CAR). The observed changes in gene expression triggered by primaquine exposure may be relevant for the understanding of the mechanisms underlying drug failure and drug-drug interactions, two of the most serious problems in malaria therapeutics.

40

P.24

The role of lipid membrane in amyloid fibril formation and toxicity in Alzheimer’s disease.

Eric Finot

Université de Bourgogne

Alzheimer’s disease is a progressive neurodegenerative disease associated with amyloid fibril formation in the brain. It is now accepted that the cytotoxicity is a result of the non-specific interaction of toxic soluble amyloid oligomers with the surface of plasma membrane. We used atomic force microscopy (AFM), atomic force spectroscopy (AFS), frequency modulated Kelvin probe microscopy (FM-KPFM), Langmuir-Blodgett monolayer technique and Surface Plasmon Resonance (SPR) to study effect of membrane structure and composition on binding of amyloid-? (1-42) peptide and fibril formation. We show that cholesterol induces electrostatic domains in lipid membrane which creates a target for amyloid binding. Hormone melatonin, which regulates and maintains the body’s circadian rhythm, has been shown to be protective against AD, but molecular mechanism of this protection is not understood. We show that melatonin and cholesterol have the opposite effects of the lipid membrane properties which, in turn, affect amyloid binding to the lipid membrane.

41

P.25

The role of Arabidopsis thaliana diacylglycerol kinase 4, in pollen tube growth and fertilization a preliminary approach

Fernando Vaz Dias1, Susana Serrazina1 and Rui Malhó1.

1Universidade de Lisboa, Faculdade de Ciencias de Lisboa, BioFIG, 1749-016, Lisboa, Portugal. E-

mail:fmdias@fc.ul.pt

Polarized growth depends on an intricate and dynamic link between membrane secretion and the actin cytoskeleton. Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol (DAG), a crucial second messenger of receptor-mediated 41erreira41, to form phosphatidic acid (PA). Regardless of the evidence for the role of PA in tip growth (and production by phospholipase D), little is known about DGKs, their activity and its interacting partners. In other cell types DGK activity is associated with a complex of proteins including Rop/Rac GTPases which regulate cytoskeleton dynamics. The family of DGKs is well conserved among most species, seven isoforms (AtDGK1 to 7) have been identified in Arabidopsis. Genevestigator suggest that diacylglycerol kinase 4 (AtDGK4) is highly expressed in Arabidopsis pollen. Here, we adopted a reverse genetics approach to investigate the function of the Arabidopsis thaliana pollen-expressed gene encoding AtDGK4. Preliminary results show that pollen germination, tube growth, and polarity were not significantly impaired in homozygous mutant plants lacking DGK4. Analysis of homozygous Arabidopsis plants KO for DGK4 showed nevertheless an abnormal phenotype compared to wild type with KO DGK4 plants displaying smaller flowers and silique. Further cellular and genetical analysis suggest a role for DGK4 in pollen development and a working model presented.

42

P.26

BDNF modulates type 1 cannabinoid receptor (CB1R)-stimulation of neurogenesis in murine dentate gyrus stem/progenitor cell cultures

Ferreira F1,2, Ribeiro FF1,2, Sebastião AM1,2, Xapelli S1,2

1Institute of Pharmacology and Neurosciences, Faculty of Medicine, University of Lisbon, 1649-028

Lisboa, Portugal. 2Unit of Neurosciences, Institute of Molecular Medicine, University of Lisbon, 1649-028 Lisboa,

Portugal.

Constitutive neurogenesis takes place in both adult mammalian subventricular zone and, in less extent, in the subgranular zone of the dentate gyrus (DG). Since the endocannabinoid system and BDNF have both been implicated in the modulation of neurogenesis, the aim of this study was to evaluate whether BDNF influences CB1R activation-induced neurogenesis. DG neurospheres were prepared from early postnatal (P1-3) Sprague-Dawley rats in serum-free medium supplemented with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2). Neurospheres with 6 days were then grown in differentiation conditions (withdrawal of EGF and FGF-2) and were seeded onto poly-D-lysine coated coverslips. One day after plating, the medium was renewed in the presence or absence (control) of Win 55,212-2 (CB1R agonist, 100Nm-1?M) and/or TrkB-Fc (2 ?g/ml). We evaluated cell viability by propidium iodide (PI) assay after incubating the cultures for 48h with the drugs and we observed that none of the drugs altered cell viability when comparing with control. Importantly, CB1R agonist treatment increased proliferation (increase in BrdU-positive cells) at 48 hours and neuronal differentiation (increase in NeuN-positive cells) after 7 days in DG cell cultures. Interestingly, the use of Trkb-Fc, which prevents the activation of TrkB by the endogenous BDNF, dramatically suppressed CB1R-induced proliferation and differentiation suggesting that CB1R-induced proliferation and differentiation depends on endogenous BDNF. Our data suggest that a cross-talk between the BDNF and endocannabinoid 42erreira42 pathways is required to stimulate neural stem cells proliferation and differentiation.

43

P.27

Evaluation of 99mTc-TMEOP as Probe for Functional Monitoring of Multidrug Resistance

Filipa Mendes, Lurdes Gano, Célia Fernandes, António Paulo e Isabel Santos

Instituto Superior Técnico, Campus Tecnológico e Nuclear, Estrada Nacional 10, 2695-066 Bobadela LRS, Portugal

Chemotherapy remains an important therapy in many malignant tumours and is used extensively. Resistance to chemotherapeutic agents is a major obstacle in the successful treatment of cancer patients. Therefore, non invasive detection of multidrug resistance (MDR) to chemotherapeutic agents is highly advantageous to define a successful therapeutic regimen. Cationic radiotracers originally developed as myocardial perfusion agents, such as 99mTc-Sestamibi, tend to localize in tumour cells due to the increased negative mitochondrial potentials and have been used for both cancer early detection and non-invasive monitoring of MDR by nuclear imaging. Recently, we developed a new 99mTc complex (99mTc-TMEOP) with potential for myocardial imaging, that presented high initial and persistent heart uptake associated to rapid blood and liver clearance. The goal of this work is to assess the usefulness of 99mTc-TMEOP for functional assessment of MDR. The in vitro uptake and efflux kinetics of 99mTc-TMEOP was evaluated in human cancer cell lines, H69 and MCF-7 (and the corresponding drug-resistant H69 Lx4 and MCF/MDR-1, which overexpress PgP, the most widely studied transporter associated to MDR. The uptake kinetics of 99mTc-TMEOP is comparable with 99mTc-Sestamibi, being significantly reduced in the cells over-expressing PgP and increased in the presence of verapamil, a modulator of PgP. The biodistribution data of 99mTc-TMEOP in rats show that the effect of cyclosporine A, a PgP inhibitor, induces a significant decrease in the washout rate from liver, kidneys and lungs, organs with a high PgP expression. In nude mice bearing MDR-negative and MDR-positive tumour xenografts, the biodistribution of 99mTc-TMEOP was similar in noncancerous organs. However, the tumour uptake was almost 2 times higher in the MCF-7 xenografts compared with the MCF/MDR-1 tumours. In summary, 99mTc-TMEOP is a promising candidate for tumor imaging and functional assessment of MDR mediated drug resistance.

44

P.28

Uncovering basal defense response in grapevine through gene expression analysis

Filipa Monteiro, Mónica Sebastiana, Andreia Figueiredo

Plant Systems Biology Lab, FCUL, Center for Biodiversity, Functional and Integrative Genomics, University of Lisbon, Portugal

Downy mildew caused by the oomycete Plasmopara viticola (Berk and Curt.) Berl. And de Toni is one of the most destructive of grapevine diseases worldwide. Most of the Vitis taxa endemic from North America is resistant to P. viticola due to coevolution with the pathogen. Instead, as P. viticola only entered in Europe in the 19th century, European grapevine cultivars are highly susceptible to this pathogen. Besides the application of fungicides by breeders, hybrids between V. vinifera and American species, including multiple backcrossing with European cultivars, have been made to achieve resistance to downy mildew. So far, several research groups have been devoted to the analysis of post-inoculation responses of resistant and susceptible grapevines. Thus, there is a lack of knowledge regarding the constitutive differences between resistance and susceptibility. From our previous works, we have identified defense related transcripts that are expressed constitutively and could be correlated with P. viticola resistance. These constitutively expressed genes could be acting as an early line of defense (plant immunity). Quantification of the expression of these genes requires normalizing Qpcr data using reference genes with stable expression in the system studied. In this study, a set of twelve genes were evaluated to identify reference genes in different Vitis species and V. vinifera cultivars, known to display different degrees of resistance and susceptibility towards P. viticola. Several comparison groups were used in order to select the best reference genes accounting genotype-dependent response towards P. viticola infection. Reference genes inferred from our study allowed obtaining insights into grapevine immunity in this particular pathosystem. Acknowledgments: Project funding: FCT-PTDC/AGR-GPL/119753/2010. Fellowship: FCT-SFRH/BPD/63641/2009.

45

P.29

A roadmap for tissue specific RNA-seq analysis of Drosophila shRNA models

Francisco Brito1, Andreia Amaral1,2, Takakazu Yokokura3, David van Vactor3,4 and Margarida Gama-

Carvalho 1,5

1Center for Biodiversity, Functional and Integrative Genomics, Faculty of Sciences, University of

Lisbon, Portugal; 2Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa,

Portugal; 3Okinawa Institute of Science and Technology Graduate School, Okinawa, Japan; 4Department of Cell Biology, Harvard Medical School, Boston, MA, USA; 5Department of Chemistry

and Biochemistry, Faculty of Sciences, University of Lisbon, Portugal.

Next generation RNA sequencing (RNA-Seq) has shown to be a powerful tool for studying the transcriptome as it allows characterizing gene expression, alternative splicing events, gene fusion and allelic imbalances. Drosophila melanogaster is considered to be one of the best models for studying neuro-muscular diseases, as their genes are highly conserved when compared to humans and genetic manipulations are cost effective. In this study, we used RNA-Seq to identify tissue specific changes in the transcriptome of a Drosophila melanogaster shRNA knockdown line that models a human neuro-muscular disease. RNA-Seq libraries were generated from pools of ~200 brains from shRNA knockdown and control wt larvae and data analysis was performed with the aim of identifying changes in gene expression and transcript isoform expression. During the development of the analysis pipeline, several unexpected difficulties were encountered emerging from the inherent complexity of the process of preparing tissue specific RNA samples requiring the dissection and pooling of multiple larvae brains, the presence of an shRNA expression vector and possible sequencing batch effects This resulted in intra-treatment variance that needed to be addressed and stabilized. Furthermore, we found that some widely used algorithms for discovery of novel transcript isoforms can perform poorly on Drosophila, requiring the selection of alternative approaches. Here, we present some key concepts and considerations that are critical for RNA-Seq data analysis in Drosophila shRNA models providing a complete workflow with example data from the quality control procedures to the quantification of differential gene expression and identification of functional pathways.

46

P.30

Ms6 Gp1 behaves as a chaperone assisting LysA delivery to its substrate

Francisco Martins, Adriano Gigante, Maria João Catalão, José Moniz-Pereira and Madalena Pimentel

Centro de Patogénese Molecular – Unidade dos Retrovírus e Infecções Associadas, Faculty of Pharmacy, University of Lisbon, Portugal

Mycobacteriophage Ms6 is a temperate double-stranded DNA (dsDNA) phage that infects the non-pathogenic Mycobacterium smegmatis. Similarly to what happens with all other dsDNA phages studied so far, Ms6 must compromise host cell integrity in order to release its progeny at the end of the lytic cycle. Ms6 lytic operon is organized into five genes. In addition to the endolysin (lysA) and 46errei-like genes (gp4 and gp5), two accessory lysis genes are found, gp1 and gp3 (lysB), which reflects a novel mechanism of phage mediated lysis. LysB encodes an enzyme with lipolytic activity whereas gp1 encodes a chaperone-like protein. Gp1 interacts with LysA and enables its access to the peptidoglycan layer in a 46errei-independent manner. However, some aspects concerning Gp1 role in the lytic process are not completely clear. In this work we present data obtained using two different recombinant mycobacteriophages constructed by the recently developed Bacteriophage Recombineering of Electroporated DNA (BRED) technology. Infection of M. smegmatis with a gp1 defective Ms6 shows decreased plate efficiency as well as lower burst-size when compared to the wild-type phage. Cell fractionation assays upon infection of M. smegmatis with Ms6 carrying gp1 and lysA fused to tag sequences reveals that Gp1 is present in every cell compartment, while LysA seems to be restricted to the cell wall. Furthermore, Gp1 expression precedes LysA production along the infectious cycle. Overall, these results show that Gp1 has an important role in the whole process, being required for an efficient lysis of M. smegmatis. In addition, Gp1 ubiquitous distribution is in agreement with its chaperone features and with the fact that it assists LysA translocation across the cytoplasmic membrane. The study of bacteriophages opens new perspectives regarding the treatment of bacterial infections and, in this case, it may also contribute to the understanding of the secretion pathways used by mycobacteria.

47

P.31

Identification of CFTR traffic regulators as a source of novel cystic fibrosis therapeutic targets

Hugo M. Botelho1,2, Shehrazade Dahimène1,2, Inna Uliyakina1,2, Beate Neumann2, Christian Tischer2,

Rainer Pepperkok2, Margarida D. Amaral1

1University of Lisboa, Faculty of Sciences (FCUL), BioFIG – Center for Biodiversity, Functional & Integrative Genomics, Lisboa, Portugal

2Cell Biology Biophysics Unit and Advanced Light Microscopy Facility (ALMF), European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

Cystic Fibrosis (CF) is the most common genetic disease in Caucasians, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel. About 90% of CF cases are caused by F508del-CFTR, a misfolded mutant which is retained in the endoplasmic reticulum and prematurely targeted for degradation. The resulting defects in transepithelial chloride transport may severely impact several organs, most notably the airways. Manipulation of cell proteostasis in order to enhance F508del-CFTR traffic holds significant potential for CF therapeutics: even small amounts of F508del-CFTR rescue to the plasma membrane can alleviate the disease phenotype because the channel exhibits residual chloride transport activity, which can be further stimulated by the FDA-approved potentiator Ivacaftor. Therefore, genes promoting wt-CFTR and/or rescuing F508del-CFTR to the cell surface upon knock-down are candidate therapeutic targets for CF. To identify novel CFTR traffic factors we developed a mCherry-CFTR construct encompassing an extracellular Flag-tag stably expressed in A549 alveolar epithelial cells or CFBE airway epithelial cells under the control of an inducible (Tet-ON) promotor [1]. The fluorescence ratio of immunodetected Flag versus mCherry was used to quantify the fraction of CFTR in the plasma membrane of individual cells under siRNA knock-downs and thus the trafficking efficiency. Screening one third of a druggable genome siRNA library revealed 178 siRNAs enhancing and 126 siRNAs significantly inhibiting wt-CFTR traffic in A549 cells. A secondary (validation) screen confirmed hits including COPI coat components, G protein-coupled receptors, chaperones and proteins involved in sterol binding, ?-catenin 47erreira47, nuclear/chromatin structuring, cytoskeletal dynamics, cell differentiation and channel regulation in both cell lines. Preliminary results have identified several siRNAs rescuing F508del-CFTR to the plasma membrane. The relevance of our results in the context of basic cell biology and CF therapeutics will be discussed. Acknowledgements Cystic Fibrosis Foundation grant 7207534, TargetScreen2 project EU-FP6-2005-LH-7-037365 (to MDA and RP), PTDC/SAU-GMG/122299/2010 from FCT/MCTES (PIDDAC) (to MDA) and Pest-OE/BIA/UI4046/2011 centre grant (to BioFIG). CFF post-doc fellowship (to HMB). [1] Almaça, J. et al. (2011) Methods Mol Biol 742, 249-264

48

P.32

Splicing factors in CD5 Mrna alternative polyadenylation

Inês Lago-Baldaia1, Nair Lopes1, Inês Boal-Calvalho1, Rita G Domingues2, Danica Drpic2, Telmo Henriques2, Vânia Glória1, Isabel Pereira Castro1, Alexandre M Carmo2,5, Alexandra Moreira1

1Gene Regulation Group and 2Cell Activation and Gene Expression Group at IBMC–Instituto de

Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; 5ICBAS–Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal

The tight relation between the different Mrna processing mechanisms and transcription suggests that the intervenient protein factors have multiple functions in more than one of those mechanisms. The pre-Mrna 3’end processing is required for the stability, nuclear export and proper translation of the mature mRNAs, playing a major role in gene expression. Most mammalian genes have more than one polyadenylation site (PAS) that can be differentially selected. Alternative polyadenylation (APA) yields different Mrna isoforms that may produce different protein isoforms, or may have different 3’UTR lengths and consequently possess different regulatory elements, as microRNAs target sites and RNA-binding protein binding sites. APA is influenced by several factors, namely protein factors, the strength of cis sequences and chromatin features, which can vary under different cellular conditions. CD5 is a cell surface protein constitutively expressed in T-cells and in a subset of B cells, B1a cells, that works as an inhibitor effector of the TCR and BCR and is involved in autoimmune and lymphoproliferative disorders. We mapped the Mrna 3’ends of CD5 in a Jurkat cell line and identified three major Mrna isoforms differing on the 3’UTR length – Pa1, Pa2 and Pa3. Although the strongest polyadenylation (Pa) signal determines the longest Mrna isoform (Pa3), this is the least expressed and has the lower translation efficiency. We have also studied the role of splicing factors with predicted binding sites in the CD5 3’UTR in Pa signal choice and preliminary data suggests that PTB and SRSF6 (Srp55) favor the selection of the shortest Mrna isoforms (Pa1), as their knock down by siRNAs leads to an increase of the Pa1 isoform. Additionally, SRSF3 (Srp20), which has putative binding sites throughout the CD5 3’UTR and has already been reported with a function in APA, seems to have an inhibitory function in the levels of all Mrna isoforms. A possible model for regulation of CD5 APA by these factors will be discussed. This work is funded by FEDER through Programa Operacional Fatores de Competitividade – COMPETE – and by National Funds through FCT – Fundação para a Ciência e a Tecnologia – project ref: PTDC/SAU-GMG/116621/2010 and FCOMP-01-0124-FEDER-022718 (Pest-C/SAU/LA0002/2011)

49

P.33

An Hydrogen Peroxide sensor in an anaerobic bacterium: Desulfovibrio vulgaris Hildenborough PerR

Vasco Ribeiroa,Isabel Silvaa, David Sardinhaa, Alice Pereiraa, Pedro Tavaresa and Cristina Timóteoa

a REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de

Lisboa, 2829-516 Caparica, Portugal

Desulfovibrio (D.) vulgaris Hildenborough is an anaerobic bacterium which has a peroxide regulon repressor protein (PerR). This protein belongs to the super-family of the iron uptake repressor (Fur) and controls the response to oxidative stress, being a sensor of the presence of hydrogen peroxide. Due to the difficulties of expressing a soluble form of D. vulgaris PerR we used recombinant DNA technology to express a fusion of two proteins: small ubiquitin like modifier (SUMO) and PerR, in which SUMO helped to express a soluble fusion protein, suitable for purification. Currently we are testing different methods of purification of the soluble protein using high performance liquid chromatography (HPLC), so that we can characterize it biochemically with and without the SUMO tag. The functionality of a Desulfovibrio vulgaris Hildenborough PerR without a SUMO tag, solubilized from inclusion bodies, was evaluated through the development of a DNA binding activity assay. The tests showed that the protein, when incubated with Zn2+ and Fe2+, is able to bind DNA, thus adopting its native structure. In the future we intend to study the interaction of the soluble PerR with the DNA, in different conditions.

50

P.34

Regulation of Mrna metabolism by U2AF65 splicing factor – novel mechanisms for the coordination of gene expression?

Isabel Peixeiro, Samuel Casaca, Margarida Gama‐Carvalho

Gene Expression and Bioinformatics Unit, BioFIG – Center for Functional and Integrative Genomics,

Faculty of Sciences, University Of Lisbon, Portugal

In eukaryotes, gene expression is a highly controlled process that requires a coordinated regulation at several levels. Post-transcriptional processes can quickly impact cell function through the modulation of Mrna transport, stability and translation. RNA-binding proteins (RBPs) were shown to determine the fate of several Mrna targets, thus affecting multiple cellular processes. We have previously demonstrated that U2AF65 and PTB, two mammalian splicing factors that recognize pyrimidine tracts, associate with a discrete subset of cellular mRNAs. The functional classification of these interaction profiles revealed underlying Mrna populations encoding proteins involved in common cellular functions. This strongly suggests that their expression is coordinated through overlapping post-transcriptional networks defined by U2AF65 and PTB. In fact, the Mrna population associated with U2AF65 shows a significant enrichment for molecules that encode proteins involved in RNA processing and cell cycle regulation. Here we characterize the function of U2AF65 as a modulator of the Mrna metabolism by taking advantage of the lambda N-based tethering system. The artificial tethering of U2AF65 downstream of the coding sequence (CDS) resulted in downregulation of the luciferase reporter. Furthermore, we demonstrate that overexpression of U2AF65 has an inhibitory effect on the expression of a reporter gene fusing the luciferase CDS with the 3’UTR of U2AF65, without affecting the reporter Mrna levels. The 3’UTR of U2AF65 presents several predicted U2AF65 binding motifs that bind to recombinant U2AF65. In fact, we show that overexpression of exogenous U2AF65 results in decreased endogenous U2AF65 protein levels, suggesting the existence of a feedback regulatory mechanism. These observations point to U2AF65 as a negative regulator of gene Mrna metabolism, in contrast with its known role as an enhancer of splicing and 3’end processing.

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P.35

Studying the interaction of dengue virus capsid protein inhibitor pep14-23 with membranes

André F. Faustino1, Gabriela M. Guerra1, Axel Hollmann1, Miguel A.R.B. Castanho1, Fábio C.L.

Almeida2, Andrea T. Da Poian2, Nuno C. Santos1,Ivo C. Martins1.

1 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal; 2 Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Dengue virus (DENV) causes a mosquito-borne disease that affects millions, causing 20,000 deaths worldwide every year. With outbreaks occurring in tropical and increasingly in temperate regions and without effective therapies available, a better understanding of DENV life cycle is of utmost importance. DENV capsid © protein interaction with host lipid droplets (LDs) is essential for viral replication. We studied this interaction before [1,2], developing pep14-23, a patented peptide inhibitor of this interaction. In this study, we evaluated pep14-23 (and the corresponding C protein region) interaction with phospholipids. The combination of bioinformatics analysis with experimental tensiometry, circular dichroism and zeta potential data shows that pep14-23 binds anionic phospholipids, acquiring ?-helical structure. Given LDs negative charge, pep14-23 inhibition of DENV C may involve a similar interaction mechanism with LDs phospholipids (which may also occur in the corresponding intrinsically disordered region of DENV C protein). This data may help to design future pep14-23 based therapies for DENV and related flaviviruses.

P. Carvalho et al., 2012, J Virol, 86:2096 2. Martins et al., 2012, Biochem J, 444:405

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P.36

AUG-Proximal CFTR nonsense mutations are NMD resistant and induce translation reinitiation

João P. Amorim1, Margarida D. Amaral1 and Anabela S. Ramalho1

1University of Lisboa, Faculty of Sciences, BioFIG – Centre for Biodiversity and Functional and

Integrative Genomics, Lisboa, Portugal

Cystic Fibrosis (CF), the most common autosomic disease in Caucasians, is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. CFTR gene variants introducing premature termination codons (PTC) usually lead to the loss of the respective transcripts via the quality-control mechanism termed Nonsense-Mediated Mrna Decay (NMD). However, not every PTC-containing Mrna is susceptible to NMD. Indeed, analysis of ?-globin Mrna has shown that mutations in the first exon were NMD-insensitive. Recent studies proposed two mechanisms for NMD escape: i) by promoting translation reinitiation downstream of the PTC or ii) by interaction of the poly(A) binding protein, cytoplasmic 1 (PABPC1) with the ribosome during premature termination, inhibiting degradation mediated by Up-frameshift suppressor 1 (UPF1). Previously we showed (Ramalho et al, 2009) that deletion of CFTR start codon generated an N-terminal truncated protein with residual function [1]. Here, we aimed to assess if naturally occurring AUG-proximal nonsense mutations in CFTR are NMD-resistant and, if so, what is the underlying mechanism. We generated stable HEK293 cell lines expressing CFTR mini-genes comprising the full CFTR Cdna sequence containing nonsense mutations and containing the adjacent introns. We show by RT-PCR that Q2X, S4X or Q39X-containing CFTR transcripts are able to escape NMD, yielding stable mRNAs. Furthermore, by Western Blot we detected N-truncated CFTR proteins. However G542X (ex11) transcripts induced NMD since no Mrna was detected. Taken together, our data support that CFTR containing AUG-proximal PTCs are indeed NMD-insensitive and this is possibly due to their capability to reinitiate translation.

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P.37

Deciphering the genetic architecture of haemolysis in sickle cell anaemia

Andreia Coelho1, Alexandra Dias2, Anabela Morais3, Emanuel Ferreira1, Isabel Picanço1, Baltazar Nunes4, Paula Faustino1, João Lavinha1

1Departamento de Genética Humana, Instituto Nacional de Saúde Ricardo Jorge, Lisboa, (INSA);2Departamento de Pediatria, Hospital Prof Doutor Fernando Fonseca, Amadora;

3Departamento de Pediatria, Hospital de Santa Maria, Lisboa; 4Departamento de Epidemiologia, INSA; Portugal.

“Sickle-cell anaemia (SCA) is a clinically heterogeneous autosomal recessive monogenic chronic anaemia characterized by recurrent episodes of severe vaso-occlusion, haemolysis and infection. Several genetic and environmental modifiers have been suggested to modulate the onset and course of SCA. As part of a wider research on the development and validation of vaso-occlusion early predictors in SCA, we have studied the association between haemolysis biomarkers (LDH, total 53erreira53 and reticulocyte count) and the inheritance of genetic variants of ten candidate genes in a series of 99 paediatric SS patients (median current age of 9.9 years) followed-up in two general hospitals in Greater Lisboa area (median follow-up/patient of 5.0 years). Although in a large number of tests a seemingly significant (i.e., p<0.05) association was observed, only the following ones were confirmed upon correction for the false discovery rate: a) An elevated LDH was associated to haplotype 7 within VCAM1 gene. b) A lower total 53erreira53 was associated to the 3.7kb deletion at HBA gene, rs2070744_T allele and haplotypes 3 and 4 at NOS3 gene and haplotype 9 within VCAM1 gene and rs3783598_G and rs3917024_T alleles at VCAM1 gene promoter. c) A diminished reticulocyte count was associated to the 3.7kb deletion at HBA gene, whereas an elevated count was associated to rs1984112_G allele at CD36 gene. Furthermore, at the phenotypic level all three haemolysis biomarkers were positively associated to left ventricle dilation, a common chronic complication of SCA. On the whole, our findings suggest a complex genetic architecture for the haemolytic endophenotype in SCA involving multiple pathways, namely control of vascular cell adhesion, NO synthesis and erythrocyte volume and haemoglobinisation. Further mechanistic studies are needed to explore these avenues leading to a better understanding of the inter- and intra-individual clinical variability of SCA. Acknowledgement: Work partially funded by FCT grants PIC/IC/83084/2007 and CIGMH.”

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P.38

Semantic Similarity in the Biomedical Domain

João D. Ferreira & Francisco M. Couto joao.ferreira@lasige.di.fc.ul.pt (corresponding author), fcouto@di.fc.ul.pt

Departamento de Informática, Faculdade de Ciências da Universidade de Lisboa

One of the most important aspects in molecular biology is the ability to determine whether two molecules are related to each other. For instance, in genetics, similarity between genetic products is often associated with one or more functions being shared among them, in chemistry, similarity in molecular structure correlates to a similar biological role. It is possible to compare these entities directly from their structure, by comparing aminoacid sequences or the graph representation of molecules, however, these methods do not always reflect biological similarity: for example, L-serine and D-serine have very different biological roles. Moreover, in some cases, such as when a comparison of biological roles is needed, there is not an easy way to extract a similarity measure: how to generate a mathematical representation of a function? Ontologies can fill this gap. Ontologies represent knowledge by means of simple statements expressing a relation between concepts: for example, “<vasodilation> is part of <blood circulation>”, “<lithium sulfate> has role <antidepressant>”. Thus, ontologies allow computers to automatically explore the meaning behind concepts. One of the technologies enabled by the use of ontologies is indeed the calculation of similarity between the concepts they represent, a technology also known as semantic similarity. Since “<arm> is a <limb>” and “<leg> is a <limb>”, they are more similar than, e.g. an <arm> and <torso>. We can therefore create measures to compare the entities represented by these concepts. The usefulness of this technology, however, transcends the simple comparison of ontological concepts. With the help of Gene Ontology annotations (e.g. “<CFTR> has function <ion transporter>”), proteins themselves gain a machine-readable semantics that can be used to compare proteins not only by their sequence (using classical methods such as BLAST) but by their functions as well.

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P.39

Nucleic acid delivery vectors derived from viral proteins: Cell Penetrating Peptides vs. Supercharged Proteins

João Miguel Freire1, Ana Salomé Veiga1, Inês Rego de Figueiredo1, Beatriz G. de la Torre2, Nuno C.

Santos1, David Andreu2, Andrea T. Da Poian3 and Miguel A.R.B. Castanho1

1 – Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal 2 – Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona

Biomedical Research Park, Spain 3 – Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil

“Successful gene therapy depends critically on developing efficient vectors to deliver genetic material into cells. Cell-penetrating peptides (CPP) are being optimized as delivery systems for nucleic acids, and are alternatives to the use of viral vectors. Recently, supercharged proteins were identified as a class of proteins that efficiently deliver functional macromolecules into mammalian cells in in vivo models 1, 2. In this work we show that selected viral proteins, particularly capsid proteins such as those of flaviviruses with a high net charge/mass ratio (> +1.07/kDa), are not only a source of naturally occurring supercharged proteins but good templates to design novel CPP. Two novel CPP, pepR and pepM, were designed and synthesized based on two conserved domains of the supercharged dengue virus (DENV) capsid © protein. Interactions between DENV C protein-derived peptides and nucleic acid, and lipid membrane interactions were studied through lipid vesicle fusion, aggregation and leakage assays. Nucleic acid delivery (ssDNA primer, siRNA and GFP plasmid) was analyzed by confocal microscopy, as well as by flow cytometry at 4°C and 37°C in BHK21, HepG2 cells and astrocytes. The translocation kinetics of both peptides was monitored by a novel real-time flow cytometry methodology, which showed that DENV C protein and both CPP (pepR and pepM) use distinct cellular internalization routes. Altogether, the results showed that viral capsid-derived peptides serve as good templates for novel CPP-based nucleic acid delivery strategies exploring different routes for cell entry. They also show a natural occurrence of supercharged proteins among structural viral proteins, which may be explored as potent nucleic acid delivery tools. This ubiquity found on flaviviruses capsid proteins raises the hypothesis that these supercharged proteins may have biological roles that arise from their intrinsic ability to penetrate cells. Additionally, new quantitative methodologies are presented to study the kinetics and the mechanisms of delivery by drug delivery formulations. 1.Cronican, J. J. et al. Chem. Biol. 18, 833–838 (2011). 2.Thompson, D. B., Villaseñor, R., Dorr, B. M., Zerial, M. & Liu, D. R. Chem. Biol. 19, 831–843 (2012).”

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P.40

Molecular regulation of wood formation in Eucalyptus

Jorge Paiva

Instituto de Investigação Cientítica Tropical Understanding the molecular mechanisms of the formation of wood and other forest products and plant-environment interactions can contribute for high productive and healthy forest plantations. In this context my research has been conducted aiming to study the molecular mechanisms of regulation of wood formation and other forest products, and the adaptation of planted forests to adverse environmental conditions, in the frame of global climate change, taking as main model the Eucalypts species. These species represents the major planted species in the world with increased interest on pulp and paper production to the emergent areas of bio-fuels and bio-materials. Moreover, the recent availability of the E. grandis genome sequence (www.phytozome.net) as boost the interest of using these species as a forest tree model. Research have been supported by several projects funded by Fundação para a Ciência e a Tecnologia (Portugal), allowed us to expand the knowledge of the Eucalyptus genome by focusing on regions potentially involved in the determination of wood properties, namely pulp yield and lignin content or at genome wide approach to study the dynamics of wood forming transcriptome regulation or on the identification of candidate genes implied on the biosynthesis and molecular regulation of cell walls, representing an important step to provide new insights into the nature of the molecular machinery involved in wood formation and most importantly in the identification of key players determining the variability of wood characteristics and its end-uses, and in particular for the development of genomic resources for Eucalyptus community. Acknowledgments: This work has been partially supported by Fundação para a Ciência e Tecnologia (Portugal): PTDC/AGR-GPL/098179/2008, “microEGO”; PTDC/AGR-GPL/66564/2006, “GenEglobwq”, P-KBBE TREEFORJOULES (P-KBBE/AGR_GPL/0001/2010), and Pest-OE/EQB/ LA0004/2011], and the INTEREG IVB SudoE project Interbio. The author acknowledges the research contract Ciencia 2008 program (FCT)/POPH (QREN) and the valuable contribution of Ana Teresa Freitas (INESC-ID, Portugal), Pedro Fevereiro (IBET, Portugal), José Carlos Rodrigues (IICT, Portugal), and Jacqueline Grima-Pettenati (LRSV, France).

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P.41

CRiSPy evolution of venom

Kartik Sunagar, Warren E. Johnson, Stephen J. O’Brien, Vitor Vasconcelos and Agostinho Antunes

P. CIMAR/CIIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Rua dos Bragas, 177,

4050-123 Porto, Portugal 2. Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre,

4169-007, Porto, Portugal 3. Laboratory of Genomic Diversity, National Cancer Institute, Frederick, MD 21702-1201, USA.

4. Theodosius Dobzhansky Center for Genome Bioinformatics, St. Petersburg University, St. Petersburg, Russia

Cysteine-rich Secretory proteins (CRISPs) are glycoproteins found exclusively in vertebrates and have broad diversified functions. They are theorized to play important functions in mammalian reproduction and reptilian venom, where they disrupt homeostasis of the prey through several mechanisms, including among others, blockage of cyclic nucleotide-gated and voltage-gated ion channels and inhibition of smooth muscle contraction. We evaluated the molecular evolution of CRISPs in toxicoferan reptiles at both nucleotide and protein levels relative to their non-venomous mammalian homologues. We show that the evolution of CRISP gene in these reptiles is significantly influenced by positive selection, and in snakes (?=3.84) more than in lizards (?=2.33), while mammalian CRISPs were under strong negative selection (CRISP1 ?=0.55, CRISP2 ?=0.40 and CRISP3 ?=0.68). The use of ancestral sequence reconstruction, mapping of mutations on the three-dimensional structure and detailed evaluation of selection pressures, suggests that the toxicoferan CRISPs underwent accelerated evolution aided by strong positive selection and directional mutagenesis, while their mammalian homologues are constrained by negative selection. Nucleotide and amino acid-level selection analyses identified 41 positively selected sites in snakes and 14 sites in lizards. Most of these sites are located on the molecular surface (nearly 76% in snakes and 79% in lizards) while the backbone of the protein retains a highly conserved structural scaffold. Nearly 46% of the positively selected sites occur in the cysteine-rich domain of the protein. This directional mutagenesis, where the hotspots of mutations are found on the molecular surface and functional domains of the protein, acts as a diversifying mechanism for the exquisite biological targeting of CRISPs in toxicoferan reptiles. Finally, our analyses suggest that the evolution of toxicoferan-CRISP venoms might have been influenced by the specific predatory mechanism employed by the organism. CRISPs in Elapidae, which mostly employ neurotoxins have experienced less positive selection pressure (?=2.86) compared with the ‘non-venomous’ colubrids (?=4.10) that rely on grip and constriction to capture the prey, and the Viperidae, a lineage that mostly employs haemotoxins (?=4.19). Relatively lower omega estimates in Anguimorph lizards (?=2.33) than snakes (?=3.84) suggests that lizards probably depend more on pace and powerful jaws for predation than venom.

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P.42

Towards understanding the role of PTEN genes in P. patens

Laura Saavedra1, Rita Catarino1, Tobias Heinz2, Ingo Heilmann2, Magdalena Bezanilla3 and Rui Malhó1

1Universidade de Lisboa, Faculdade de Ciencias de Lisboa, BioFIG, 1749-016, Lisboa, Portugal. 2 Universität Halle, Institut für Biochemie, 06120 Halle (Saale), Germany.

3 Department of Biology, University of Massachusetts, Massachusetts, USA.

Presenting author email address:llborelli@fc.ul.pt

Phosphoinositides (PPIs) are minor membrane lipids which play important roles in many signal transducing pathways in eukaryotic cells such as cell growth and proliferation, cytoskeleton organization, vesicle trafficking, regulation of ion channels and nuclear 58eneraliz pathways. The immediate precursor of all PPIs is phosphatidylinositol and phosphorylation of the lipid head group by the action of phosphoinositide kinases results in the generation of seven PPIs species. We are focusing on phosphatase and tensin homolog (PTEN) genes, which in animal cells hydrolyzes PtdIns(3,4,5)P3, a potent second messenger that elicit cellular behaviours that favour oncogenesis. Loss of PTEN creates a state in which the PI3K pathway is constitutively active stimulating cell division, increases cell size and inhibits apoptosis. Interestingly, PtdIns(3,4,5)P3 is the only known PPI so far not detected in any plant system, and the enzymes that synthesizes PtdIns(3,4,5)P3 in animal cells do not exist in plants. P. patens has four PTEN genes ubiquitously expressed during the whole moss life cycle. Using a knock-in approach, we found that at subcellular level, PTENs are found in the cytosol and in the nucleus, and they are mainly expressed in tip growing cells such as caulonema and rhizoids, but as well as in buds. Knock-outs for these genes were obtained, and our preliminary results show that triple pten KO caulonemal cells display a higher rate of cell division and show an early switch from the juvenile protonemal stage to the development of adult gametophores, supporting the role of PpPTENs as a suppressor of cell growth.

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P.43

Disclosing the role of DNA methylation in salt tolerance/response mechanisms in rice

Liliana Ferreira1, João Maroco2, Mª Margarida Oliveira1, Ana Paula Santos1

1Genomics of Plant Stress Laboratory, Instituto de Tecnologia Química e Biológica, Quinta do Marquês, Av. Da República, 2780-157 Oeiras, Portugal

2UIPES, ISPA-Instituto Universitário, Rua Jardim do Tabaco, nº34 1149-041 Lisboa , Portugal

E-mail: lferreira@itqb.unl.pt Epigenetics refers to changes in gene expression without modification on the underlying DNA sequence. These modifications have the potential of affecting plant physiology to respond to rapid and unexpected environmental conditions but thus far the mechanisms underlying that interplay have been poorly addressed. Our previous cytologic analysis of rice interphase nuclei using Fluorescence In Situ Hybridization (FISH) allowed detecting major reorganization of highly condensed heterochromatic domains upon imposition of salt stress. Now, the big questions are how global DNA methylation changes in response to salt stress and how those specifically affect the methylation profile of salt stress responsive genes. To unveil how DNA methylation impacts salt stress response, we have used rice varieties that cope differently with salt stress: IR29 and Nipponbare that are salt-sensitive and Pokkali and FL478, which are salt tolerant. Expression profile of genes codifying for DNA methyltransferases and DNA demethylases revealed a decrease of the former and an increase of the later in response to salt stress. ELISA assays with antibody specific for 5-methylcytosine and MeDIP-seq shown that DNA methylation levels are strongly reduced in leaves upon salt stress in particular in salt tolerant rice varieties. In contrast, roots had considerably lower levels of DNA methylation and were not much affected by salt stress suggesting a more primed state to respond to stress conditions. Distinct patterns of methylation in tolerant versus sensitive rice varieties were also observed by immunolocalization analysis in interphase nuclei. Taken together, the gathered results so far point out to an involvement of DNA methylation in rice response/adaptation to salt stress.

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P.44

Effect of the intron 22 inversion mutation of the f8 gene in hemophilia A on the structure and function of the FVIII protein.

Linda MEDDAH

Université des sciences et de la technologie d’Oran Mohamed BOUDIAF

Hemophilia A (HA) is a hereditary X-linked disorder of blood coagulation caused by a deficiency of factor VIII that the micro-inversion of intron 22 represents the most frequent mutation in hemophilia A with severe form (40-50% of cases). First , we have initiated to the molecular biology techniques (DNA extraction, DNA assay and PCR Long Range) to explore the micro-inversion of intron 22 of F8 gene in 13 patients with severe HA from Western Algeria. In the second time, we provide an “in silico” study protocol using free software based on different analytical methods as Multalin, SIFT, PolyPhen and structural (Swiss-Pdb Viewer). This protocol was used to detect causal mutations in two patients with severe HA from western Algeria and predict their deleterious effects on the FVIII protein. We have identified two causal mutations: an intronic mutation on the splice donor site which is probably responsible for a skipping of exon 14 and 60enerali mutation responsible for a conformational change of the FVIII protein. Both mutations are predicted to be responsible for the severe form of the disease in two patients.

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P.45

The impact of rpiA overexpression on plasmid biopharmaceutical production by Escherichia coli GALG20, a pgi-gene knockout strain.

Luís A. M. Carreira1,2, Geisa A. L. Gonçalves1,2, Duarte M. F. Prazeres1,2 and Gabriel A. Monteiro1,2.

1 Department of Bioengineering, Instituto Superior Técnico (IST), Lisbon, Portugal 2 Center for

Biological and Chemical Engineering, IBB-Institute for Biotechnology and Bioengineering, IST, Lisbon, Portugal

The interest in plasmid DNA (Pdna) as a biopharmaceutical increased due to its attractive potential application in gene therapy and DNA vaccines. Currently, most of the Pdna production processes use strains of Escherichia coli (E. coli) that are known to be effective producers of recombinant proteins. However, these strains may not be the most favorable when plasmid DNA is the final product. Recently, new discoveries in the field of strain engineering for Pdna production demonstrate the importance of strain genetic background in the creation of new E. coli strains for Pdna production. The E. coli host strain GALG20 was specifically designed for Pdna production by knocking out the endA, recA and pgi genes in the wild-type strain MG1655. As a consequence of the pgi-mutation, the GALG20 strain preferentially uses the pentose phosphate (PP) pathway as the main via for glucose consumption. This innovative mutation leads to an increase of plasmid production and to a reduction of acetate produced during fermentation [1]. Nevertheless, it is not known whether other enzymes in the PP pathway are limiting extra synthesis of Pdna by GALG20. In this work, the impact of overexpression of the rpiA gene in the novel strain GALG20 will be analyzed. The rpiA gene codes for ribose-5-phosphate isomerase A, and is responsible for nucleotide synthesis in the PP pathway. Previous work demonstrated that overexpression of rpiA increased Pdna yields, using a plasmid expression system in E. coli BL21 [2]. Thus, the replacement of rpiA promoter, directly in the genome of GALG20 strain, could potentially enhance nucleotide synthesis and consequently increase Pdna production. KEYWORDS: Plasmid biopharmaceuticals, DNA vaccine, Escherichia coli, Metabolic engineering, Strain engineering. References [1] Goncalves, G. A., Prazeres, D. M.F., Monteiro, G. A., Prather, K. L., De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production. Appl Microbiol Biotechnol 2013, 97, 611-620. [2] Wang, Z., Xiang, L., Shao, J., Wegrzyn, A., Wegrzyn, G., Effects of the presence of ColEI plasmid DNA in Escherichia coli on the host cell metabolism. Microb Cell Fact 2006, 5.

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P.46

Dynamics of Akt activation during mouse embryo development: distinct subcellular patterns distinguish proliferating versus differentiating cells.

Luís Marques and Sólveig Thorsteinsdóttir

Centro de Biologia Ambiental / Departamento de Biologia Animal, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal

Instituto Gulbenkian de Ciência, Oeiras, Portugal.

(Corresponding authors at: Centro de Biologia Ambiental/Departamento de Biologia Animal, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisbon, Portugal. Tel.: +351 217500212;

fax.; +351 217500028.)

KEYWORDS: Akt; mouse embryo; proliferation; differentiation; confocal imaging; 3D reconstruction.

Akt is a highly conserved serine-threonine protein kinase which has been implicated in a wide variety of cellular functions, from the regulation of growth and metabolism, to activation of pro-survival pathways and cell proliferation, and promotion of differentiation in specific cell types. However, very little is known about the spatial and temporal pattern of Akt activity within cells and whether this pattern changes as cells enter and proceed in their differentiation programs. To address this issue we profiled Akt activation in E8.5 – E13.5 mouse embryos and in C2C12 cells. We used a commercial antibody against Akt, phosphorylated on one of its activating residues, Thr-308, and performed high resolution confocal imaging of the immunofluorescence in labeled embryos. We observe strong Akt activity during mitosis in the dermomyotome, the neuroepithelium and some mesenchymal cells. This burst of activity fills the whole cell except for heterochromatin-positive areas in the nucleus. A surge in activity during mitosis is also observed in subconfluent C2C12 cells. Later on in the differentiation programs of skeletal muscle and neural cells, derivatives of the dermomyotome and neuroepithelium, respectively, we find robust, sustained Akt activity in the cytoplasm, but not in the nucleus. Concomitantly with skeletal muscle differentiation, Akt activity becomes concentrated in the sarcomeric Z-disks whereas developing neurons maintain a uniform cytoplasmic pattern of activated Akt. Our findings reveal unprecedented cellular and subcellular details of Akt activity during mouse embryo development, which is spatially and temporally consistent with proposed functions for Akt in mitosis and myogenic and neural differentiation and/or survival. Our results thus demonstrate a subcellular change in the pattern of Akt activation when skeletal muscle and neural progenitor cells cease dividing and progress in their differentiation programs.

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P.47

Tauroursodeoxycholic acid prevents MPTP-induced dopaminergic cell death in a mouse model of Parkinson’s disease

M Castro-Caldas1,2*, A Neves Carvalho 1,3*, E Rodrigues1,4, C J Henderson5, C R Wolf 5, C M P

Rodrigues1,4, M J Gama1,4§

1 Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto 1649-003 Lisbon, Portugal

2 Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa 2829-516 Caparica, Portugal

3 Centre of Ophthalmology, Institute of Biomedical Research in Light and Image – IBILI, Faculty of Medicine, University of Coimbra, Azinhaga de Santa Comba, Celas 3000-548 Coimbra, Portugal

4 Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal

5 Division of Cancer Research, Medical Research Institute, Level 9, Jacqui Wood Cancer Centre, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, United Kingdom

Mitochondrial dysfunction and oxidative stress are implicated in the neurodegenerative process in Parkinson’s disease (PD). Moreover, c-Jun N-terminal kinase (JNK) plays an important role in dopaminergic neuronal death in substantia nigra pars compacta. Tauroursodeoxycholic acid (TUDCA) acts as a mitochondrial stabilizer and anti-apoptotic agent in several models of neurodegenerative diseases. Here, we investigated the role of TUDCA in preventing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurodegeneration in a mouse model of PD. We evaluated whether TUDCA modulates MPTP-induced degeneration of dopaminergic neurons in the nigro-striatal axis, and if that can be explained by regulation of JNK phosphorylation, reactive oxygen species (ROS) production, glutathione S-transferase (GST) catalytic activation and Akt signaling, using C57BL/6 glutathione S-transferase pi (GSTP) null mice. TUDCA efficiently protected against MPTP-induced dopaminergic degeneration. We have previously demonstrated that exacerbated JNK activation in GSTP null mice resulted in increased susceptibility to MPTP neurotoxicity. Interestingly, pre-treatment with TUDCA prevented MPTP-induced JNK phosphorylation in mouse midbrain and striatum. Moreover, the anti-oxidative role of TUDCA was demonstrated by impairment of ROS production in the presence of MPTP or MPP+, both in vivo and in vitro. Finally, results herein suggest that the survival pathway activated by TUDCA involves Akt signaling, including downstream Bad phosphorylation and NF-?B activation. We conclude that TUDCA is neuroprotective in an in vivo model of PD, acting mainly by modulation of cellular redox thresholds. These results open new perspectives for the pharmacological use of TUDCA, as a modulator of neurodegeneration in PD. This work was supported by Fundação para a Ciência e a Tecnologia (FCT) and FEDER through grants PPCDT/SAU-FCF/58171/2004 and Pest-OE/SAU/UI4013/2011, and PhD fellowship SFRH/BD/39897/2007.

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P.48

Integrating survey and molecular approaches to better understand the origin of endemic flora in the Cape Verde Islands

Romeiras M.M.1,2,*, Monteiro F.2, Fernandes C.3, Duarte M.C.1

1IICT-Tropical Research Institute, Trav. Conde da Ribeira, 9, 1300-142 Lisbon, Portugal 2BioFIG,

University of Lisbon, Faculty of Sciences, Campo Grande, 1749-016 Lisbon, Portugal 3Direção Geral do Ambiente, Ministério do Ambiente de Cabo Verde

The well-known “Biodiversity Hotspots” have been defined based on endemic taxa, massive habitat loss and vulnerability to extinction. The great number of endemic plants (ca. 900 species) has made the Macaronesian Islands (Azores, Madeira, Selvages, Canaries and Cape Verde) an outstanding area for studies of evolution and speciation. From a molecular systematic perspective, oceanic archipelago floras such as those of the Canary Islands and Hawaii have attracted considerable interest during the last two decades, with molecular data providing valuable new insights into the relationships of insular floras. However, little is known about the evolution of the endemic flora within the Cape Verde and in order to fully understand the origin plant diversity in these islands two projects were recently funded: FCT (PTDC/BIA-BIC/4113/2012) and Mohamed Bin Zayed Species Conservation Fund (MBZ nº 12255026). This poster will present the state of the art insights in several different fields and approaches from biogeography, phylogeny, and evolution of plant diversity in Cape Verde.

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P.49

Neuronal Protection in Parkinson’s Disease: is there a role for Gst pi?

Andreia Neves Carvalho1,2, Margarida Castro-Caldas1,4, Carla Marques2, Elsa Rodrigues1,3,Colin J. Henderson5, C. Roland Wolf 5, Paulo Pereira2, Maria João Gama1,3

1 Research Institute for Medicines and Pharmaceutical Sciences – Imed.UL, Faculty of Pharmacy,

University of Lisbon, Av. Prof. Gama Pinto 1649-003 Lisbon, Portugal. 2 Centre of Ophtalmology and Vision Science, Institute of Biomedical Research in Light and

Image – IBILI, Faculty of Medicine, University of Coimbra, Azinhaga de Sta. Comba, Celas 3000-548 Coimbra, Portugal.

3 Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.

4 Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa 2829-516 Caparica, Portugal

5 Division of Cancer Research, Medical Research Institute, Level 9, Jacqui Wood Cancer Centre, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, United Kingdom.

Parkinson’s disease (PD) is a movement disorder resulting from severe dopamine (DA) deficiency arising from dopaminergic neuronal degeneration. Mitochondrial dysfunction, oxidative stress and failure of proteolytic pathways are involved in PD pathogenesis. Glutathione S-Transferase pi (GSTP), whose expression is regulated by the nuclear factor erythroid 2 related factor 2 (Nrf2), is a phase II drug metabolizing enzyme that catalyze the conjugation of reduced glutathione to electrophilic groups on substrate molecules. The main objective of our past and ongoing work is to characterize the putative neuroprotective role of GSTP against MPTP-induced oxidative stress. So far, we demonstrated that GSTP is an endogenous regulator of c Jun N terminal kinase (JNK) activity. In mice brain, GSTP is able to bind JNK, through direct protein protein interaction, inhibiting its activation, and consequently preventing its downstream effects on triggering cell death pathways. We have further characterized GSTP neuroprotective function by demonstrating that, GSTP potentiates Kelch ECH associating protein 1 (Keap1) S-glutathionylation following MPTP administration. As Keap1 is the major endogenous regulator of Nrf2, Keap1 S glutathionylation results in the subsequent Nrf2 pathway activation, and increased expression of GSTP. As the UPS is also a critical player in the regulation of the Nrf2 elicited antioxidant response, we investigated the modifications in UPS function, upon MPTP induced oxidative stress and proteasome inhibition, in wild-type and GSTP null mice brain. We concluded that different components of the UPS have different susceptibilities to oxidative stress and, importantly GSTP null mice display increased susceptibility to UPS damage and inactivation upon MPTP induced oxidative stress. In conclusion, our results provided new insights into the molecular mechanisms involved in DA neuronal loss and identified novel mechanisms involved in GSTP elicited neuronal protection. This work was supported by grants PPCDT/SAU-FCF/58171/2004, PTDC/SAU-MMO/57216/2004 and Pest-OE/SAU/UI4013/2011 and PhD fellowship SFRH/BD/39897/2007 from Fundação para a Ciência e a Tecnologia

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P.50

Yeast Two-Hybrid System: TCTEX1D4 interactome, a protein is known by the company it keeps

Maria João Freitas, Luís Korrodi-Gregório, Margarida Fardilha

Signal Transduction Laboratory, Centre for Cell Biology, Biology Department and and Department,

University of Aveiro, Aveiro, Portugal

For the past few years, molecular biology has promoted several distinct areas such as medicine and energy production. The identification of protein-protein interactions is essential to unveil protein functions, understand signaling pathways and ultimately identify possible therapeutic targets. In 1989, Song and Fields developed a revolutionary molecular biology technique, the Yeast Two-Hybrid (YTH) system. This method was one of the first that allowed the detection of protein-protein interactions in vivo, using Saccharomyces cerevisiae as a model system. Until today, the YTH suffered several modifications that allowed high throughput screen protein-protein interactions in a cell or tissue based systems. Combination of YTH with a biochemical protein-protein interaction approach is essential to validate the interaction. The contribution of the YTH in molecular biology is reflected in the countless interactomes unraveled by this technique. Helicobacter pylori, Saccharomyces cerevisae and a partial Human interactome are a few examples. One of the appeals of building an interactome by YTH is the possibility of unveiling possible protein functions by characterizing it´s interactome. A testis YTH of TCTEX1D4, a dynein light chain, was performed with the goal of identifying novel functions of this protein. TCTEX1D4/TCTEX1D2 interaction was further validated by blot overlay and immunocytochemistry studies. A possible function in acrosome reaction was identified.

67

P.51

Exploring a link between mycobacteria structure and virulence

C. Silva1, J. Perdigao1, E. Alverca2, A.P. Matos3, P.A. Carvalho4,I. Portugal1, L. Jordao5*

1 CPM, URIA, Faculdade de Farmácia da Universidade de Lisboa 2 Instituto Nacional de Saúde Dr Ricardo Jorge (INSA), DAS

3 Serviço de Anatomia Patológica, Centro Hospitalar de Lisboa Central, Hospital Curry Cabral 4ICEMS, Departamento de Bioengenharia, Instituto Superior Técnico, Universidade Técnica de

Lisboa, 5 INSA, DDI

*corresponding author maria.jordao@insa.min-saude.pt

Tuberculosis (TB) is a major health problem. The emergence of multidrug resistant (MDR) Mycobacterium tuberculosis (Mtb) isolates confounds treatment strategies. In Portugal, cases of MDR-TB are reported annually with increased incidence noted in Lisbon. The majority of these MDR-TB cases are due to closely-related mycobacteria known collectively as Lisboa family and Q1 cluster. The genetic determinants linked to drug resistance have been exhaustively studied resulting in the identification of family and cluster specific mutations. Nevertheless little is known about other factors involved in drug resistance development. Here we focused on the study of morphological and structural features of Mtb isolates, collected during 2008-2009 in Lisbon, in order to complement the genetic analysis. For this propose scanning and transmission electron microscopy techniques were used. Particular attention was given to Lisboa family and Q1 cluster isolates since together they account for the majority of reported MDR-TB cases. This analysis allowed the identification of structural differences, such as cell envelope thickness, between Mtb clinical isolates, which are correlated with antibiotic resistance. The infection of human monocyte derived macrophages allowed us to document the relative selective advantage of Lisboa family isolates over other circulating Mtb isolates.

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P.52

Measuring transcript abundance of candidates from selected gene families: a first glimpse on the molecular basis of postharvest biocontrol in pears

Maria Manuel Cristóvão, Luis F. Goulao and Claudia Sánchez

Agri4Safe/BioTrop – IICT, Tapada da Ajuda 1349-017 Lisboa, Portugal INIAV, Av. República, Quinta do Marquês 2784-505 Oeiras, Portugal

GIRM –IPL, Santuário da Nossa Senhora dos Remédios 2520-641 Peniche, Portugal Understanding the relationship between the traits affecting a given physiological response can be tackled by tracing the changes in the expression of responsible genes. During ‘Rocha’ pear postharvest storage, an Aureobasidium pullulans isolate proved to be an effective bio-control agent against blue mold decay, but the mechanisms underlying its antagonistic activity to induce host defence responses remain to be elucidated. After controlled Penicillium infection, with and without antagonistic bio-control protection, a set of candidate genes with roles on selected metabolic pathways (pathogen response, anti-oxidative stress, ethylene biosynthesis, cell wall loosening/reinforcing and phenylpropanoid pathway routing) was investigated on its transcript accumulation in a time-course assay, by quantitative real-time PCR (qrt-RT-PCR) using the geometric average of the transcription levels of four reference genes for data normalization. A significant modification of the patterns of gene expression from all treated samples when compared to control conditions was observed, suggesting an antagonist effect by activation of resistance mechanisms in the host tissues. The results provided indications about the most responsive metabolic pathways and are useful to assist the selection of the appropriate time-points to elect for further –OMICs studies. Acknowledgments: Project funding by FCT trough project PTDC/AGR-ALI/118477/2010: SafeFruit – Aureobasidium pullulans, a biofungicide for the control of postharvest decay in pears: insights into its action mechanisms, from MCTES, Portugal.

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P.53

NGSOnto – keeping track of the NGS pipeline process using an ontological approach and its application on molecular epidemiology

Mickael Silva, Catia Vaz, Alexandre Francisco, Mário Ramirez, Francisco M Couto, João André Carriço

Nowadays, microbial typing methods are fundamental to clinical microbiology, being tools for outbreak detection and tracking, and for providing valuable information in the study of the evolution of virulence factors such as antimicrobial resistance mechanisms. With the advances in sequencing technologies, namely in the Whole Genome Sequencing techniques, it has become imperative to connect the information present in the former sequence based microbial typing methods and the new high sequence throughput data.Another critical issue is the lack of interoperability and querying facilities through web services, on the currently available microbial typing databases, which are spread out through several institutes worldwide. Considering these issues and the previous development of a microbial typing identification ontology (TypOn), a Next Generation Sequencing (NGS) ontology was developed. This ontology was built in order to describe a NGS pipeline process, being interoperable with the TypOn ontology as well as other ontologies. The Web Ontology Language (OWL) was used to build the ontology, following a Basic Formal Ontology 2.0 structure and using RDF format data to save information. The data mapped on the ontology may be later queried using a SPARQL endpoint made available or through the RESTFul webservice that is under development. The NGSOnto, aims at capturing the workflow of all the processes involved , in order to ensure the reproducibility of the entire process, focusing particularly in the data analysis steps, downstream of the raw sequence data. In this way it will allow the validation of the use of NGS as bona fide source of data for microbial typing, by providing the basis to determine if the two results are comparable.

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P.54

Proteomic analysis of Quercus suber – Pisolithus tinctorius ectomycorrhizal symbiosis

Mónica Sebastiana1, Joana Martins2, Andreia Figueiredo1, Filipa Monteiro1, Catarina Franco 2, Ana Coelho2

1 Plant Systems Biology Lab, FCUL, Center for Biodiversity, Functional and Integrative Genomics,

Portugal 2 Mass Spectrometry Laboratory, ITQB, Portugal

Ectomycorrhizal (ECM) symbiosis – the interaction of roots and soil fungi- is essential for the life and health of trees in temperate and boreal forests, where it plays a major role in nutrient cycling and in functioning of the forest ecosystem. The fungus delivers soil minerals, particularly phosphorous and nitrogen, to the host roots. The plant, in return, transfers photo assimilates to the fungus. Besides increasing plant growth, this symbiosis brings also other benefits to the plant, like a more efficient uptake of water, higher resistance to pathogens, or tolerance to contaminated soils. In contrast to transcriptional approaches, there are few reports on ECM symbiosis studies using proteomics. Our current research aims to characterize plant and fungal proteins differentially expressed during the establishment of ECM by examining qualitative and quantitative differences in protein composition between the individual partners and fully developed ECM. The ECM fungus Pisolithus tinctorius was established in pure culture and used for the production of fungal inoculum for mycorrhization of Cork Oak seedlings in the nursery. A proteome analysis based on DIGE technology is being conducted in order to characterize proteins that are induced and repressed when comparing ECM roots with non mycorrhizal roots and axenically grown fungal mycelium.

71

P.55 Effect of peanut extraction protocols on yield of crude protein, quantities of allergens Ara

h 1 and 2, and detection in subsequent experiments NicoleE. Walczyk,†,§ Euan Tovey,# Pen C.Smith,§ and Thomas H.Roberts*,†,⊗

†Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia §School of Biological Sciences, University of Sydney, Sydney, Australia

#Woolcock Institute for Medical Research, Sydney, Australia ⊗Department of Plant and Food Sciences, University of Sydney, Sydney, Australia

Globally, there is an increasing prevalence of allergy to peanuts. Extraction protocols for preparing extracts vary widely. No empirical study has determined the impact of buffers and experimental conditions on the quantity of peanut allergens and their detection in subsequent experiments, necessary for a 71eneralized71ion. We tested the influence of the extraction buffer and other common experimental conditions on the extraction efficiency of crude protein and Ara h 1 and 2 as well as the outcome of subsequent methods of analysis, including Western blots. After extracting peanut proteins using a wide range of extraction buffers with different Ph values, de-fatting reagents, extraction times and temperatures, and centrifugation acceleration (g), the crude protein was quantified and Ara h 1 and 2 content determined using ELISA kits. Qualitative differences in protein abundances were further tested with 1D- and 2D-gel electrophoresis and Western blotting using peanut allergic serum. The defatting reagent, buffer composition and their Ph values substantially influenced the extraction efficiency of crude protein and Ara h 1 and 2. Buffers conferring denaturating and/or reducing conditions dramatically influenced the detection of Ara h 1 and 2 in ELISAs. The extraction buffer had a significant impact on the recognition of protein spots IgE from human serum in Western blotting. Different centrifugation times and temperatures did not influence the crude protein and Ara h 1 and 2 extraction efficiency, although Ara h 1 extraction was susceptible to extraction time. No protein was extracted from seed coats using standard methods. This study contributes towards 71eneralized71ion of the measurement of peanut allergens by comparing extraction protocols empirically and demonstrating the critical importance of specific extraction variables.

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P.56

Reserpine Inhibits the CFTR Chloride Channel in Caco-2 Monolayers

Nikhil T Awatade 1,2, Margarida Ramos1, Margarida D Amaral1 and John P Winpenny2

1 University of Lisboa – Faculty of Sciences, BioFiG Centre, Portugal;2 Biological Sciences and Norwich Medical School, Biomedical Research Centre, University of East Anglia, Norwich Research Park,

Norwich, Norfolk, UK . Secretory 72enerali continues to be an enormous health problem worldwide. Pharmacological block of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regarded as a potential therapeutic target for secretory 72enerali. The main objective of this study was to determine the action of the plant derived compound, reserpine, on the CFTR chloride channel. The effect of reserpine on the CFTR chloride current was investigated using the short circuit current (Isc) technique on monolayers of Caco-2 cells grown on permeable supports. Monolayers had a transepithelial resistance (RT) of 467 ± 40 ?/cm2 and an initial Isc of 4.5 ± 1.2 ?A/cm2 (n=22). Initial experiments on intact Caco-2 monolayers suggested that a 15 min, apical preincubation with 50?M reserpine resulted in an inhibition of the 10 ?M forskolin-stimulated Isc (n=3). All further experiments were carried out in nystatin-permeabilised Caco-2 monolayers. Basolateral addition of 10?M Forskolin resulted in an increase in Isc of 68 ± 12.6 ?A/cm2 (n=3) which was completely inhibited by apical addition of 50?M GlyH101. Forskolin-stimulated (10?M) Isc was inhibited by apical addition of reserpine (10-50?M) in a concentration dependent manner (n=1). Further investigation of this result demonstrated that the forskolin-stimulated Isc was inhibited by 71% (n=3) after a 15min, apical preincubation with reserpine (50 ?M). A 15min, basolateral preincubation with reserpine (50 ?M) also resulted in a 43% (n=3) inhibition of the forskolin-stimulated Isc. Taken together, the data suggest that reserpine potentially targets the CFTR chloride channel and may be useful for further development as an antidiarrhoeal agent.

73

P.57

Role of 2CysPrx and PrxIIB in H2O2 antioxidant system under heat stress

Patrícia Vidigal1, Ana Montserrat Martin-Hernandez2, Cèlia Guiu-Aragonés2, Sara Amâncio1, Luísa Carvalho1

1 DRAT/CBAA, Instituto Superior de Agronomia, Universidade Técnica de Lisboa. Tapada da Ajuda. 1349-017 Lisboa, Portugal

2 Investigación y Tecnología Agroalimentarias (IRTA) – Centre for Agrigenomics Research (CRAG), Campus UAB – Edificio CRAG, Bellaterra – Cerdanyola del Vallès, 08193 Barcelona, Spain

(Patrícia Vidigal: pvidigal@isa.utl.pt) The effect of post-transcriptional suppression of 2Cys Peroxiredoxin (2CysPrx) and Type-II Peroxiredoxin B (PrxIIB) in the antioxidant system under control (22ºC) and heat stress (48ºC) was analyzed. To study 2CysPrx function, several mutagenic approaches were made, but none involving PrxIIB. However, these approaches resorting to antisense and double mutants were not able to prevent accumulation of 2CysPrx protein at wild-type levels due to translational and post-translational regulation. Virus-induced-gene-silencing (VIGS) that is based on post-transcriptional gene silencing (PTGS) is a technique that allows studying gene function without plant transformation. Here we show that using VIGS vector based on tobacco rattle virus (TRV), 2CysPrx and PrxIIB post-transcriptional regulation mechanisms were successfully affected in Nicotiana benthamiana. The impairment of 2CysPrx post-transcriptional regulation mechanism in control conditions caused a severe oxidative stress, reflected by an increase in hydrogen peroxide (H2O2) and oxidized redox state of glutathione and ascorbate. Also in control conditions, silencing of PrxIIB led to a mild increase of H2O2, to a reduced ascorbate pool, and an oxidized glutathione pool. Abscisic acid (ABA)-biosynthesis and ABA-insensitive genes revealed that the regulation of both 2CysPrx and PrxIIB are ABA-independent. Relative expression of antioxidant system genes under control and heat stress conditions in both TRV:2CysPrx and TRV:PrxIIB plants showed that 2CysPrx regulation appears to be mediated by an ascorbate–glutathione H2O2 redox-state dependent mechanism and that PrxIIB regulation might be related with peroxisome-H2O2-metabolism dependent on ascorbate.

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P.58

The role of fibronectin matrix in regulating segmentation in the vertebrate embryo

Patrícia Gomes Almeida1, Ana Lina Rodrigues1, Isabel Palmeirim2 and Sólveig Thorsteinsdóttir1,3

1Centro de Biologia Ambiental/Departamento de Biologia Animal, Faculdade de Ciências, Universidade de Lisboa, Portugal; 2 Centro de Biomedicina Molecular e Estrutural, Universidade do

Algarve, Faro, Portugal; 3 Instituto Gulbenkian de Ciência, Oeiras, Portugal. Somitogenesis is one of the most dynamic and complex processes of vertebrate embryogenesis. The periodic segmentation of the presomitic mesoderm (PSM) leads to the formation of somites, precursors of the axial skeleton and musculature. Temporal control of somite formation is defined by the cyclic expression of molecular clock genes, with each cycle defining the space and time at which a new pair of somites forms. One of the players regulating vertebrate development is the extracellular matrix (ECM). More than providing mechanical support, the ECM also regulates cell physiology, morphology, and differentiation, by controlling gene expression in cooperation with other signaling pathways. One of the most abundant ECM molecules during early development is fibronectin. Notably, mouse, zebrafish and Xenopus embryos with deficiencies in fibronectin matrices fail to form somites. Our group has demonstrated that an intact fibronectin matrix surrounding the PSM is crucial for somitogenesis in the chick embryo (Rifes et al., 2007). Isolated PSMs retaining a source of fibronectin during a 6 hour culture period are able to form epithelial somites in the absence of other structures. Conversely, culturing PSMs in the absence of a fibronectin matrix abrogates somitogenesis and also perturbs the molecular segmentation of the PSM. Thus fibronectin may be an active player in both somite patterning and morphogenesis. We are presently addressing how the fibronectin matrix regulates the molecular and morphological segmentation of the PSM. This will increase our understanding of how a fibronectin ECM drives morphogenesis during development. This work was financed by FCT project PTDC/SAU-OBD/103771/2008.

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P.59

Cortical microtubule and ß-tubulin organization in developing maize (Zea mays L.) endosperm transfer cells and starchy endosperm cells

Sara Rocha1, Artur da Câmara Machado1, Paula Sampaio2, Roberto Salema2,3, Paulo Monjardino1,*

1Instituto de Biotecnologia e Bioengenharia – Centro de Biotecnologia dos Açores, Universidade dos Açores, Rua Capitão João de Ávila, 9700-042 Angra do Heroísmo,

Portugal. 2Instituto de Biologia Molecular e Celular, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.

3Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre S/N, 4150-180 Porto, Portugal.

*Corresponding author: e-mail 75ener@uac.pt, telephone +351295402200 and Fax +351295402201.

The most basal endosperm transfer cells (MBETCs) in maize have both flange and reticulate ingrowths that are located on different cell walls, but the mechanisms underlying the differentiation of these ingrowths are not well understood. We investigated the organization of cortical microtubules and ß-tubulin in these cells during endosperm development using confocal laser scanning microscopy, using the starchy endosperm cells (SCs), which lack such ingrowths, as a reference. The microtubules associated with flange ingrowths formed long and predominantly longitudinal bundles, whereas those associated with reticulate ingrowths formed short and curvilinear bundles that appeared to surround the ingrowths. The ß-tubulin complexes were primarily located adjacent to the microtubule bundles in both the flange and reticulate ingrowths. In the SCs, the initially randomly-distributed microtubules formed bundles in a net-like array during differentiation but later separated into individual microtubules which were organized in tight parallel arrays in the mature cells. The ß-tubulin complexes were also randomly distributed in the immature cells, later forming discrete bundles and then spreading through the cell mirroring the behavior of the microtubules. These data allowed us to propose new models for the contribution of microtubules and ß-tubulin to reticulate and flange ingrowth formation in MBETCs and to cell wall formation in maize SCs during endosperm development.

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P.60

Bioinformatics awareness through training provision in Portugal

Pedro L. Fernandes, Instituto Gulbenkian de Ciência, Oeiras, PT Cath Brooksbank, European Bioinformatics Institute, Hinxton, UK

Access to affordable training on computational methodologies in Biology in the last 20 years has had a very positive impact in Portugal. This is due to a fortunate combination of circumstances, investments and decision-making. Consequently the density of bioinformatics-aware researchers in Portugal is relatively high. Contributing to this, the Instituto Gulbenkian de Ciência (IGC) has provided bioinformatics services since 1991, and launched a regular training programme in 1, the GTPB (http://gtpb.igc.gulbenkian.pt) [1]. Key to the success of this activity were the various forms of support provided by EMBL via its Hinxton Outstation, the European Bioinformatics Institute it would be unfair to single it out and 76ene the contributions of tens of individuals that helped to 76enerali and actually deliver the training. However, in the context of the present celebration, we need to 76eneraliz EMBL’s support in helping to reach critical condition to make things happen, namely by allowing instructors to accept invitation to teach, by enrolling in large projects like BioSapiens that held a summer course for beginners, like Brenda and SLING that supported the creation of BTN [2] and financed roadshows and workshops, by attaching to EMBnet for more than 20 years as a node, by helping to create ELIXIR for European infrastructure, by supporting GOBLET, etc. Training is essential to catalyse adoption of best practices in research [3] and is all the more essential in bioinformatics, where the rapid growth in size and complexity of resources makes it challenging for researchers to keep up [4]. Altogether, IGC’s training activities have touched thousands of people. Here we present a summary of its history and impact. References: [1] Fernandes PL, “”The GTPB training programme in Portugal””, Brief Bioinform (2010) 11(6): 626-634, first published online October 21, 2010 doi:10.13/bib/bbq063. [2] Schneider MV, et al, Bioinformatics Training Network (BTN): a community resource for bioinformatics trainers.Brief Bioinform. 2012 May,13(3):383-9. Doi: 10.1093/bib/bbr064. Epub 2011 Nov 22. Review. [3] Via A, et al, “”Best practices in bioinformatics training for life scientists”” Brief Bioinform. 2013 Jun 25. [Epub ahead of print] doi:10.1093/bib/bbt043 [4]Fernandes P, et al “”Training Experimental Biologists in Bioinformatics”” Advances in Bioinformatics (open access), Volume 2012, Article ID 672749, doi:10.1155/2012/672749”

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P.61

Centromere-independent accumulation of enerali at ectopic heterochromatin sites induces chromosome stretching during anaphase

Oliveira, R.*1,2, Kotadia, S.*3, Mirkovic, M.2, Bowlin, K.3, Eichinger, C.1, Nasmyth, K.1, and Sullivan, W.

1–Department of Biochemistry, University of Oxford, South Parks Road, Oxford Ox13QU, United

Kingdom 2– Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780--?156 Oeiras, Portugal

3 – Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064.

Although chromosomes contain small segments of heterochromatic regions along chromosomearms, large stretches of heterochromatin containing highly repetitive sequences and spanning several megabases are almost exclusively found around the centromeric region. The reason remains elusive and whether or not the presence of highly dense heterochromatic sites distal to the centromere can have deleterious effects to the cells has not been extensively investigated. Here we show that engineered chromosomes from Drosophila melanogaster containing large portions of heterochromatin embedded in euchromatic regions display significant stretching during anaphase. Such stretching is caused by ectopic cohesion at these sites that is mediated by the cohesion complex, a ring--?like protein complex that entraps sister DNA molecules inside its proteinaceous ring. We demonstrate that cohesion is preferentially loaded at heterochromatin regions early in S--? Phase and is maintained at these regions until anaphase, independent of centromere proximity. This accumulation occurs independent of S--?phase progression and results from a preferential loading of cohesion rather than selective protection. These results illustrate a novel mechanism by which chromosomes containing long stretches of heterochromatin distant from the centromere may compromise the fidelity of chromosome segregation.”

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P.62

Intra-strain phenotypic and genomic variability of the commercial Saccharomyces cerevisiae strains Zymaflore VL1 recovered from vineyard environments

Ricardo Franco-Duarte1, Laura Carreto2, Inês Mendes1, Brigitte Cambon3, Sylvie Dequin3, Manuel A.S.

Santos2, Margarida Casal1, Dorit Schuller1

1 CBMA (Centre of Molecular and Environmental Biology) / Department of Biology / University of Minho, Portugal,

2 RNA Biology Laboratory, CESAM, Biology Department, Aveiro University, Campus Universitário de Santiago, Aveiro

3 UMR Sciences pour l’Oenologie, Microbiologie, INRA, Montpellier, France. ricardofilipeduarte@bio.uminho.pt

The use of commercial Saccharomyces cerevisiae wine strains as fermentation starters has been extensively 78eneralized over the past two decades. These strains are used by wineries every harvest in large quantities. We showed that such strains are disseminated from the winery and their permanence in nature induced genetic changes, not found among a control group of isolates that derived from clonal expansion of the commercial “mother” strain. The objective of the present study was to evaluate by comparative genome hybridization on array (Acgh) the genome variations among four isolates of the commercial strain S. cerevisiae Zymaflore VL1, re-isolated from vineyards surrounding the wineries where this strain was applied, in comparison to the commercial “mother” strain. Data analysis showed genetic differences among the recovered isolates in comparison with the “mother” strain: amplification of 14 genes related with mitosis (SHE1), meiosis (HFM1), lysine biosynthesis (LYS14), galactose (GAL1) and asparagine catabolism (ASP3-2), and amplification of eight Ty elements. A phenotypic screening was performed considering 28 physiological tests. Seven phenotypic traits distinguished the recovered strains from the “mother” strain which was unable to grow at 18ºC, but evidenced some growth in the presence of CuSO4(5Mm) and SDS 0.01%(v/v). Variable growth patterns were found for NaCl(1.5M), KHSO3(300mg/L) and wine supplemented with glucose (0.5% and 1%w/v). Sequencing and genome comparison of the five isolates is currently underway. We hypothesize that the transition from nutrient-rich musts to nutritionally scarce natural environments induces adaptive responses and microevolutionary changes promoted by Ty elements. These changes (and possibly others as well) may contribute to intra-strain phenotypic variability. This work was funded by the fellowship SFRH/BD/48591/2008. Financial support was also obtained from FEDER funds through the program COMPETE and by national funds through FCT by the project FCOMP-01-0124-008775.

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P.63

Glycine uptake in brain nerve terminals is decreased by BDNF through a reduction in GlyT2 membrane insertion

Rita I Aroeira, Ana M Sebastião and Cláudia A Valente

Institute of Pharmacology and Neurosciences, Faculty of Medicine and Neurosciences Unit, Institute

of Molecular Medicine, University of Lisbon, Portugal. Glycine transporter 2 (GlyT2) is localized in the nerve terminals of glycinergic neurons, promoting glycine uptake into the neurons and ensuring the refilling of glycinergic vesicles. Brain-derived neurotrophic factor (BDNF) activates its high affinity TrkB receptors, which occur in two isoforms, full length (TrkB-FL) and truncated (TrkB-T1/T2). After BDNF binding to TrkB receptor, several intracellular cascades are triggered, specifically PLC, Akt and MAPK signaling pathways. To determine if BDNF modulates glycine uptake mediated by GlyT2 in synaptosomes obtained from rat hippocampus, [3H]glycine uptake assays were performed. We showed that BDNF decreased glycine uptake mediated by GlyT2. Upon synaptosomes incubation with BDNF, GlyT2 displayed a lower Vmax value and a similar Km constant when compared to BDNF absence. Our results also demonstrated that both TrkB receptor isoforms, TrkB-FL and TrkB-T, are present in synaptosomes. However, we revealed that the BDNF effect is TrkB-FL dependent. Firstly, the tyrosine kinase inhibitor, k252a, was able to restore glycine uptake. Furthermore, the BDNF action was lost when specific inhibitors of TrkB signaling pathways, namely U73122, LY294002 and U0126 (inhibitors of PLC, Akt and MAPK pathways, respectively) were used. Moreover, synaptosomes pre-incubation with monensin, a transporter recycling inhibitor, prevented the BDNF action upon glycine uptake, suggesting that BDNF reduces GlyT insertion in the plasma membrane. In conclusion, our results showed that BDNF effect upon glycine uptake in brain synaptosomes requires the activation of the TrkB-FL receptor and associated signaling pathways and occurs by inhibiting GlyT2 membrane incorporation. Supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal. Rita Aroeira has a PhD fellowship (SFRH/BD/62831/2009) from FCT. BDNF was a gift from Regeneron.

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P.64

Towards understanding the role of PTEN genes in P. patens

Laura Saavedra1, Rita Catarino1, Tobias Heinz2, Ingo Heilmann2, Magdalena Bezanilla3 and Rui Malhó1

1Universidade de Lisboa, Faculdade de Ciencias de Lisboa, BioFIG, 1749-016, Lisboa, Portugal. 2 Universität Halle, Institut für Biochemie, 06120 Halle (Saale), Germany. 3 Department of Biology,

University of Massachusetts, Massachusetts, USA. Presenting author email address: llborelli@fc.ul.pt

Phosphoinositides (PPIs) are minor membrane lipids which play important roles in many signal transducing pathways in eukaryotic cells such as cell growth and proliferation, cytoskeleton organization, vesicle trafficking, regulation of ion channels and nuclear 80ignaling pathways. The immediate precursor of all PPIs is phosphatidylinositol and phosphorylation of the lipid head group by the action of phosphoinositide kinases results in the generation of seven PPIs species. We are focusing on phosphatase and tensin homolog (PTEN) genes, which in animal cells hydrolyzes PtdIns(3,4,5)P3, a potent second messenger that elicit cellular behaviours that favour oncogenesis. Loss of PTEN creates a state in which the PI3K pathway is constitutively active stimulating cell division, increases cell size and inhibits apoptosis. Interestingly, PtdIns(3,4,5)P3 is the only known PPI so far not detected in any plant system, and the enzymes that synthesizes PtdIns(3,4,5)P3 in animal cells do not exist in plants. P. patens has four PTEN genes ubiquitously expressed during the whole moss life cycle. Using a knock-in approach, we found that at subcellular level, PTENs are found in the cytosol and in the nucleus, and they are mainly expressed in tip growing cells such as caulonema and rhizoids, but as well as in buds. Knock-outs for these genes were obtained, and our preliminary results show that triple pten KO caulonemal cells display a higher rate of cell division and show an early switch from the juvenile protonemal stage to the development of adult gametophores, supporting the role of PpPTENs as a suppressor of cell growth.

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p.65

Quaking A – a post-transcriptional regulator of early patterning in zebrafish

Fernandes, S.F.1,2,3, Fior, R.1,2, Gama-Carvalho, M.3,4 and Saúde, L.1,2

1Instituto de Medicina Molecular e Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina da Universidade de Lisboa;

2Instituto Gulbenkian de Ciência; 3Gene Expression and Bioinformatics Unit, Center for Biodiversity, Functional & Integrative Genomics, Faculdade de Ciências, Universidade de Lisboa, Portugal;

4Faculdade de Ciências, Universidade de Lisboa, Portugal Embryonic development is a highly controlled process, both spatially and temporally, and in many developmental processes this control relies on mechanisms of post-transcriptional regulation, namely the modulation of Mrna stability and translation efficiency. This study is focused on the post-transcriptional regulator QuakingA (QkA). This RNA binding protein belongs to the STAR protein family, which has been implicated in many developmental processes including mesoderm formation and muscle fiber development. In addition, during the paraxial mesoderm segmentation process (somitogenesis), qkA is expressed in the paraxial mesoderm and somites. However the contribution of QkA to somitogenesis has not been explored. We set out to assess the effects of QkA loss-of-function on somitogenesis, using the zebrafish as a model system. We preformed a preliminary characterization of the QkA loss-of-function phenotype, using a qkA morpholino, which revealed not only alterations in somite size but also in the left-right positioning of the internal organs and in the number of otoliths (endolymphatic infillings of the inner ear). A candidate target of QkA – Cadherin11 (cdh11) – was identified as a potential mediator of one or more of the QkA loss-of-function phenotypes. Cadherin11 is expressed in the left-right patterning – associated organ the Kupfer’s vesicle and has been linked to kinocilia and otolith formation. Preliminary western blot assays also reveal that Cdh11s’ protein levels are increased when QkA is depleted. We are currently using a morpholino against cdh11, together with the qkA morpholino, to determine if any of the quaking loss-of-function phenotypes can be rescued by restoring the Cdh11 levels.

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Potential involvement of Smurf2 in altered regulation of the TGF-beta signaling pathway in Cystic Fibrosis disease

Sara Canato, Margarida Telhada, Carlos Farinha, Margarida Amaral, Luka Clarke

University of Lisboa – Faculty of Sciences, BioFIG – Center for Biodiversity, Functional & Integrative

Genomics, Lisboa, Portugal

Cystic Fibrosis (CF) is an autosomic recessive lethal disorder resulting from a defect in the CFTR gene, which encodes an epithelial chloride channel. The F508del mutation is the most common cause of CF and leads to CFTR retention in the endoplasmic reticulum, after which it is prematurely degraded thus precluding the mutant from reaching the cell surface. SMURF2 is an E3-ubiquitin ligase involved in the negative regulation of the TGF-beta 82ignaling pathway, by promoting degradation of TGF-beta mediators such as SMADs and TGF-beta receptors. TGF-beta in CF is associated with neutrophilic inflammation and diminished lung function and it has been reported that protein levels of SMAD3, a TGF-beta mediator, are reduced. Given the anti-inflammatory properties of TGF-beta and the hyper-inflammatory phenotype observed in CF we investigated the negative feedback by E3-ubiquitin ligases in the context of TGF-beta signaling (SMURF1, SMURF2 and NEDD4L) in polarized CF Bronchial Epithelial (CFBE) cell models. Firstly, using real-time quantitative PCR (Qpcr), we demonstrate that the F508del mutation is not sufficient to induce significant differential expression of those 3 E3-ubiquitin ligases. However, when stimulated with TGF-beta CFBE cells expressing F508del-CFTR showed a 2-fold increase in both SMURF2 Mrna and protein levels. Interestingly, we also detected increased expression of SMAD7, a SMURF2 partner also participating in the inhibition of TGF-beta 82ignaling. Taking these data together, we thus hypothesize that increased levels of SMURF2 are responsible for diminished SMAD3 expression, thus providing a partial explanation for the hyper-inflammation observed in the CF airways. Supported by Pest-OE/BIA/UI4046/2011 grant (BioFIG).

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A derivative of the natural compound kakuol affects DNA relaxation of topoisomerase IB

Silvia Castelli1*, Sara Vieira1*, Ilda D’Annessa1, Prafulla Katkar1, Loana Musso2, Sabrina Dallavalle2 and Alessandro Desideri1

* the authors equally contributed to the work

1University of Rome Tor Vergata, Rome, Italy

2Università di Milano, Milan, Italy

Topoisomerases IB are anticancer and antimicrobial targets whose inhibition by several natural and synthetic compounds has been documented over the last three decades. Here we show that kakuol, a natural compound isolated from the rhizomes of Asarum sieboldii, and a derivative analogue are able to inhibit the DNA relaxation mediated by the human enzyme. The analogue is the most efficient one and the inhibitory effect is enhanced upon pre-incubation with the enzyme. Analysis of the different steps of the catalytic cycle indicates that the inhibition occurs at the cleavage level and does not prevent DNA binding. Molecular docking shows that the compound preferentially binds near the active site at the bottom of the catalytic residue Tyr723, providing an atomistic explanation for its inhibitory activity.

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Cross talks between amyloid-forming proteins in neurodegeneration

Sofia B. Carvalho1, Sónia S. Leal1, Isabel Cardoso2, Hugo M. Botelho1, Günter Fritz3 and Cláudio M. Gomes1

1Instituto Tecnologia Quimica e Biológica, UNL, Oeiras, Portugal

2 Instituto Biologia Celular e Molecular, Porto, Portugal 3 Department of Neuropathology, University of Freiburg, Germany

E-mail: gomes@itqb.unl.pt URL: www.itqb.unl.pt/pbfs

Protein misfolding and conformational changes are a cornerstone of neurodegenerative diseases involving formation and deposition of toxic protein oligomers. Although mutations favor protein aggregation, physiological factors such as labile metal ions within the cellular environment are play also a prominent role in aggregation processes (1). This communication summarizes our recent progresses on the analysis of amyloid formation by S100 proteins, which are upregulated in neurodegenerative proteinopathies such a Alzheimer’s, PD and ALS. These proteins form amyloid-like structures (2) characterized by the typical Thioflavin T fluorescence and FT-IR fingerprints. We have found that calcium and zinc, two major players in the chemical biology of the glutamatergic synapse influence the fibrillation pathway and aggregate morphology. Also, we have recently elicited that S100A6, which is overexpressed in AD and ALS, is itself amyloidogenic and seeds SOD1 aggregation, shortening its nucleation process (3). 1. Leal et al (2012) Coordination Chemistry Reviews 256(19-20):2253-2270. 2. Fritz et al (2010) FEBS journal 277(22):4578-4590 and Carvalho et al (2013) submitted 3. Botelho et al. (2012) 287(50):42233-42”

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Microbial Stress to Metals and ROS

Cláudia Nóbrega1, Iris L. Ziober1, Célia Silveira1, Bart Devreese2, Manolis Matzapetakis3, Sofia R.

Pauleta1,*

1 REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade

Nova de Lisboa, 2829-516 Caparica, Portugal. 2 Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, 9000 Ghent, Belgium. 3Instituto de Tecnologia Química e

Biológica, Universidade Nova de Lisboa, Av. Da República, 2780-157 Oeiras, Portugal.

*Email: srp@fct.unl.pt

The study of bacterial copper homeostasis, especially copper tolerance mechanisms, has gain interest due to the widespread use of copper compounds as bactericide in agriculture or as disinfectants in the food industry. This general use will undoubtedly lead to the selection of copper-resistance strains of bacteria and the dissemination of plasmid-borne copper resistance genes among different bacterial pathogens with important implications in disease control efforts. Moreover, mechanisms of copper tolerance have recently been linked to the molecular mechanism of pathogenesis. During infection, bacteria are exposed to different stresses, as reactive oxygen species and fluctuations in metal ions concentration. Under the scope of copper resistance, we have been investigating the molecular mechanism of copper resistance in a bacterial system, Marinobacter hydrocarbonoclasticus. Host immune response to pathogenic microorganisms, frequently employes oxidative stress in the form of reactive oxygen species (ROS). Some of the most common ROS found in biological systems involve the superoxide anion, hydrogen peroxide (H2O2) and hydroxyl radical. Pathogenic bacteria from the species Neisseria gonorrhoeae and Neisseria 85ignaling85es, that cause gonorrhea and meningitis, respectively, have developed numerous defense mechanisms to cope with oxidative stress of the constant exposure to ROS. These mechanisms are essential for cell survival taking since ROS can cause damage to DNA, proteins and cell membranes. One of these mechanisms is based on the family of bacterial cytochrome c peroxidases. These periplasmatic enzymes catalyze the conversion of H2O2 to water using haem co-factors [3] and c-type cytochromes or small type copper proteins of the respiratory chain as the electron donor.

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A ClpB chaperone knockout mutant of Mesorhizobium 86ignal shows a delay in the root nodulation of chickpea plants

Clarisse Brígido1, Marta Robledo2, Esther Menéndez2, Pedro F. Mateos2, Solange Oliveira1

1Laboratório de Microbiologia do Solo, ICAAM (Instituto de Ciências Agrárias e Ambientais

Mediterrânicas), Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal 2Departamento de Microbiología y Genética, Centro Hispano Luso de Investigaciones Agrarias,

Universidad de Salamanca, Salamanca, 37185, España

During the establishment of a successful symbiosis with legumes, rhizobia have to deal with several environmental stress conditions outside and within the root nodule. Several molecular chaperones are known to be involved in bacteria stress response. To investigate the role of chaperone ClpB in rhizobia stress tolerance as well as in the rhizobia-plant symbiosis process, the clpB gene from a chickpea microsymbiont, strain Mesorhizobium 86ignal LMS-1, was identified and a knockout mutant was obtained. The ClpB knockout mutant was tested to several abiotic stresses showing that it was unable to grow after a heat shock and it was more sensitive to acid shock than the wild-type strain. A plant growth assay performed to evaluate the symbiotic performance of the clpB mutant showed a higher proportion of ineffective root nodules obtained with the mutant than with the wild-type strain. Nodulation kinetics analysis showed a 6-8 day delay in nodule appearance in plants inoculated with ?clpB mutant. Analysis of histological sections of nodules formed by the clpB mutant showed that most of the nodules were immature, with a low amount of bacteroids. No differences in the root infection abilities of GFP-tagged clpB mutant and wild-type strains were detected by confocal spectral microscopy. To our knowledge, this is the first study that evidences the involvement of the chaperone ClpB from rhizobia in the symbiotic nodulation process. Acknowledgments This work was supported by FCT-Fundação para a Ciência e a Tecnologia (PTDC/BIO/80932/2006), co-financed by EU-FEDER (FCOMP-01-0124-FEDER-007091). C. Brígido acknowledges a FCT fellowship (SFRH/BD/30680/2006).

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Chestnut transcriptome after Phytophthora cinnamomi challenge

Susana Serrazina1*, Carmen Santos2, Helena Machado2, Cátia Pesquita3, Rita Costa2, Maria Salomé Pais2 and Mónica Sebastiana

1: Plant Molecular Biology and Biotechnology Lab, Center for Biodiversity, Functional and Integrative Genomics (BioFIG), Edifício C2, Campus da Faculdade de Ciências da Universidade de Lisboa, Campo

Grande 1749-016 Lisboa- Portugal. 2: INIAV I.P., Molecular Biology Laboratory, Av da República, 2780-159 Oeiras –Portugal. 3: Informatics Department, Faculdade de Ciências da Universidade de

Lisboa, Portugal *e-mail: smserrazina@fc.ul.pt

The European chestnut, an important forest species for the economy of Portugal, covers a total area of 2.53 million hectares, including 0.53 million hectares devoted to fruit production. Castanea sativa is declining since the beginning of the XX century due to P. cinnamomi and P. cambivora, with considerable negative effects on fruit production and ecosystems. To help elucidate the defense mechanisms in chestnut to the ink disease, we applied 454 pyrosequencing to four Cdna libraries, including 3 clones of the sensitive species C. sativa and 6 clones of the resistant species C. crenata, inoculated and mock-inoculated with P. cinnamomi. This produced 771030 reads, with 15683 contigs generated for C. sativa and 16828 contigs for C. crenata. The annotation revealed that the resistant species upregulates more genes related to defense response after inoculation than C. sativa. Also those genes are expressed at a higher level in inoculated tissues when compared to the control. GO annotation points to the transcriptomes’ representativity in response to oxidative stress, 87ignaling and cell wall organization, antioxidant activity and transcription factor activation after pathogen presence. C. crenata up-regulates transcripts coding for proteins that act on cell wall and membrane. GO annotation also indicates that the libraries are suitable for the purpose. These results bring us close to identifying which genes and pathways are involved in the response of the resistant species to P. cinnamomi and the response differences between Japanese and European chestnut. Based on the outcomes of this project we can generate molecular markers for genetic diversity data within the Fagaceae and assist tree improvement programs to fungal pathogens. Acknowledgments: This work was supported by FCT – PTDC/AGR-CFL/101707/2008.

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Analysis of the effect of the two in cis CFTR splice variants c.2562T>G and c.2657+5G>A

Susana Igreja1, Teresa Casals2, Margarida D. Amaral1, Anabela S. Ramalho1

1University of Lisboa, Faculty of Sciences, BioFIG – Centre for Biodiversity, Functional and Integrative Genomics, Campo Grande-C8 1749-016 Lisboa. Portugal

2Human Molecular Genetics Group, Bellvitge Biomedical Research Institute (IDIBELL). 08908 Barcelona. Spain

Cystic fibrosis (CF) is the most common, lethal autosomal recessive disease, with an incidence of 1 case per 2,500 births. Clinically, CF is dominated by the severe impairment of the respiratory tract, the main cause of morbidity and mortality, with airway obstruction by thick mucus and chronic infections, eventually leading to progressive loss of lung function. Other CF symptoms include pancreatic insufficiency, elevated sweat electrolytes and male infertility. Despite impressive advances related to the molecular basis of CF, life expectancy and quality of life are still limited for CF patients (~37 years). CF is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding a chloride channel, expressed at the apical membrane of epithelial cells and regulated by Camp-dependent protein kinase A. Approximately 1,900 CFTR mutations have been described and most are presumed to be pathogenic. A significant fraction of CF-causing mutations affects pre-Mrna splicing. The potential pathogenic nature of many non-obvious splicing variants has been neglected. Here, we aimed to provide insights on a new CFTR complex allele carrying the coding SNP c.2562T>G (exon 15) and the splicing mutation c.2657+5G>A (intron 16) in cis, thus testing if c.2562T>G has an effect on CFTR splicing. We investigated for the occurrence of abnormal splicing using an adequate in vitro minigene model mimicking the in vivo situation. The splicing products were analised both at the RNA and protein levels. Our results, show that the minigene carrying the c.2562T>G SNP alone showed a similar 88ehavior, at the RNA and protein level, as that of a WT-CFTR minigene. A minigene carrying the c.2657+5G>A mutation alone, produced three transcripts: a WT transcript, a transcript lacking exon 16 and a transcript lacking both exons 15 and 16. Exon 16 skipping is the main aberrant transcript resulting from this mutation. The double mutant (c.2562T>G and c.2657+5G>A) produced the same transcripts as the single mutant c.2657+5G>A. Overall, these in vitro data show that the c.2562T>G SNP does not seem to impact on splicing and/or CFTR function/expression when occurring in cis with the c.2657+5G>A splicing mutation. Work supported by ISCiii FIS/FEDER PI080041, Fundación Sira Carrasco, Spain and, partially by Pest-OE/BIA/UI4046/2011 BioFig centre grant (POCTI/FCT/PIDDAC, PT) and Ciência2008 fellowship (FCT, PT) to ASR.

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Protein misfolding at the mitochondria: lessons from fatty acid oxidation diseases

Bárbara J. Henriques, Tânia G. Lucas, Cláudio M. Gomes

Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa Av. República 127, 2780-756 Oeiras, Portugal. Tel: +351 214469332.

E-mail: gomes@itqb.unl.pt URL: www.itqb.unl.pt/pbfs Loss-of-function metabolic diseases such as mitochondrial fatty acid oxidation defects are paradigmatic examples of protein folding disorders. In these cases, missense mutations are often the prevalent genetic lesions causing defectively folded proteins. How these folding defects translate into distinct clinical phenotypes and the how small molecules can effectively rescue protein function have been central to our research. Using ETF and ETF:QO proteins as models to study these process in MADD (OMIM 231680), in the last years we experimentally established how ETF/ETF:QO mutations affect protein folding and assembly (1), impair catalytic activity generating ROS (2), disrupt protein: protein interactions and influence the recognition by molecular chaperones (3). Another significant finding has been the establishment of a molecular rationale for the therapeutic action of riboflavin (Vitamin B2) as a pharmacological chaperone (4), together with other mitochondrial metabolites (5). We also set up Drosophila as a tool to investigate MADD-like severe phenotypes, establishing the proof of concept for its use as disease model (6). (1) Henriques et al (2009) J Biol Chem 284, 4222-4229. (2) Rodrigues and Gomes (2012) Free radical Biol Med 53, 12-19 (3) Henriques et al (2010) BBA-MBD 1802, 1070-1077. (4) Henriques et al (2010) Current Med Chem 17, 3842-3854. (5) Lucas et al (2011) BBA 1812, 1658-1663. (6) Alves et al (2012) BBA 1822, 1284-1292.”

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Magnetic labeling and detection of cfDNA for cancer diagnosis

Tomás Dias

INESC-MN & BERG-IBB

The measurement of cell-free DNA (cfDNA) in blood circulation has been targeted as a promising non-invasive methodology in the field of cancer diagnostics and therapy follow-up. Acting as a liquid biopsy, the measurement of cfDNA levels, mutations on genes and alterations on methylation patterns, can be assessed as potential biomarkers avoiding the need of unnecessary invasive biopsies for the follow-up of patients. Therefore, the combined identification of decisive biomarkers is critical to ensure a reliable outcome of a given disease. In this work, a novel microarray-like strategy is proposed to evaluate the cfDNA integrity in plasma samples from cancer donor patients. Two particular cfDNA fragments of different sizes (115-bp and 247-bp) and of significant importance in cancer diagnosis were targeted for PCR amplification after cfDNA extraction from plasma samples. During the reaction, a biotin and a 5’ phosphate group were incorporated per newly synthesized fragments allowing to generate single-stranded targets after a digestion step with lambda exonuclease. By means of a streptavidin-biotin interaction, the single stranded targets were labeled with streptavidin-coated superparamagnetic nanoparticles of 250 nm and hybridized with complementary probes immobilized on top of magnetoresistive sensors. With this approach, the magnetically labeled DNA targets were detected due to the fringe field originated from the magnetic labels. Concentrations in the picomolar range were detected making this approach suitable for the detection of low copy number targets.

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Alternative splicing of tumour-related Rac1b is regulated by upstream signalling pathways

Vânia Gonçalves and Peter Jordan

Departamento de Genética, Instituto Nacional de Saúde 'Dr. Ricardo Jorge', Lisboa

The small GTPase Rac1 regulates signalling pathways controlling actin-dependent cell motility as well as gene transcription. An alternative splicing variant Rac1b is overexpressed in a subset of colorectal tumours and cooperates with mutant B-Raf to sustain tumour cell viability. The alternative splicing mechanism regulating Rac1b expression involves two antagonistic splicing factors, ASF/SF2 and SRp20. Using a Rac1 minigene approach and siRNA-mediated depletion, we identified ASF/SF2 as an enhancer of endogenous Rac1b splicing whereas SRp20 acts as a silencer. Inhibition of the PI3-kinase pathway increased protein levels of ASF/SF2 and promoted Rac1b generation. By contrast, depletion of endogenous protein kinase SRPK1 led to decreased Rac1b expression. Together, these data indicate that altered upstream signaling pathways in colorectal cancer cells will target splicing factors that regulate alternative splicing of the small GTPase Rac1."

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An Hydrogen Peroxide sensor in an anaerobic bacterium: Desulfovibrio vulgaris Hildenborough PerR

Vasco Ribeiroa, Isabel Silvaa, David Sardinhaa, Alice Pereiraa, Pedro Tavaresa and Cristina Timóteoa

a REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de

Lisboa, 2829-516 Caparica, Portugal

Desulfovibrio (D.) vulgaris Hildenborough is an anaerobic bacterium which has a peroxide regulon repressor protein (PerR). This protein belongs to the super-family of the iron uptake repressor (Fur) and controls the response to oxidative stress, being a sensor of the presence of hydrogen peroxide. Due to the difficulties of expressing a soluble form of D. vulgaris PerR we used recombinant DNA technology to express a fusion of two proteins: small ubiquitin like modifier (SUMO) and PerR, in which SUMO helped to express a soluble fusion protein, suitable for purification. Currently we are testing different methods of purification of the soluble protein using high performance liquid chromatography (HPLC), so that we can characterize it biochemically with and without the SUMO tag. The functionality of a Desulfovibrio vulgaris Hildenborough PerR without a SUMO tag, solubilized from inclusion bodies, was evaluated through the development of a DNA binding activity assay. The tests showed that the protein, when incubated with Zn2+ and Fe2+, is able to bind DNA, thus adopting its native structure. In the future we intend to study the interaction of the soluble PerR with the DNA, in different conditions.

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Using an mRNA-based approach to detect rare CFTR mutations

Verónica Felicio1, Margarida Amaral1 and Anabela Ramalho1

1University of Lisboa, Faculty of Sciences, BioFIG - Centre for Biodiversity, Functional and Integrative Genomics, Lisboa, Portugal

Cystic fibrosis (CF) is the most common lethal autosomal recessive disease among the caucasian population, with an incidence of 1 in 2500-6000 newborns. This disease is caused by mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene which contains 27 exons and codes for the CFTR protein. Due to the large extension of CFTR gene (~190kb) and the large number of gene variants reported (>1900) there's a need to establish quick methods for mutation analysis. Usually this is achieved by limiting analysis to the most common mutations. However, this often leads to difficulties in CF genotype identification, especially in populations with high prevalence on non-p.F508del mutations. We have designed a mRNA-based protocol using just 9 RT-PCR reactions, hereby the complete CFTR coding region is analysed. Moreover, one of the reactions (exons 11-13) is based on ARMS for p.F508del mutation, which allows easy detection of this mutation. We have applied this protocol to genotype 12 CF patients with absence or just residual CFTR-mediated Cl- secretion in rectal biopsies1 and only 1 CFTR mutation identified by routine methods. Using this strategy we detected mRNA alterations in 75% of the patients and identified both mutations, confirmed by genomic DNA sequencing. For 2 siblings we detected a cryptic exon between exons 10-11 resulting from a mutation far deep into IVS 10 (c.1584+18672A>G), unlikely to be detected by CFTR exon sequencing. The other mutations detected were: p.G576A, c.1717-1G>A, c.1812-1G>A, c.3272-26A>G, c.3120+1G>A (2P) (legacy nomenclature). We conclude that this is a rapid, robust and inexpensive method to detect rare mutations that can be easily used after a first screen. Furthermore it already demonstrates the functional consequences of mutations in case they occur at the RNA level. Supported by PEst-OE/BIA/UI4046/2011 BioFig centre grant (POCTI/FCT/PIDDAC, Portugal), Gilead MED-2012-131 and Ciência2008 fellowship (FCT, Portugal) to ASR. 1. Sousa et al (2012) PLoS One 7:e47708.