FEBS Course on

Human and Microbial Genomics:

DNA Arrays applied to Human Pathogens & Diseases

Athens , March 30^th - April 7^th, 2006

Hellenic Pasteur Institute
127, Vas. Sofias Avenue 11521, Athens , Greece

ABSTRACTS

Keynote Lecture I
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From Aristotle to Watson and Crick
Georges Cohen
Pasteur Institute, Paris , France

This historical overview will bring us from the Greek antiquity to the today's era of modern genetics.

•  HEREDITY : Aristotle and Hippocrates
- Nehemiah Grew and Camerarius: The sexuality of higher plants (1676-1694).
- Linnaeus (1760). Cotton Mather: Hybrids in Maize (1716)
- Kôlreuter (1761-1766): Systematic study of plant hybrids
- Charles Darwin (1809 -1882)
- Naudin (1855-1869)
- Gregor Mendel (1822-1884)
- Thomas Hunt Morgan and the Drosophila school (1866–1945)
- Sturtevant, Muller and Bridges (Classical Book, 1915)

2) ENCOUNTER OF GENETICS AND BIOCHEMISTRY
- Lucien Guenot (1903)
- Archibald Garrod (1908), Bateson, Landsteiner
-Sturtevant Caspari: Mosaicism in the eye color of Drosophila (1920-1933)
- Ephrussi and Beadle: Transplantation in Drosophila, Imaginal Disks (1935-1936)
- Beadle and Tatum: Neurospora crassa (1941)

3 ) THE CHEMICAL NATURE OF GENES
- Friedrich Miescher (1871)
- Altmann (1889)
- Wilson (1985)
- Albrecht Kossel (1853-1927)
- Ascoli and Levene (1900-1903)
- Erwin Chargaff (1950)
- Grifith and Pneumococcus (1928), Dawson (1931)
- Avery and his collaborators (1944)

4) THE LAST SERIOUS ANTI-GENETIC MOVEMENT
Trofim Denissovitch Lysenko (1898-1976) and the agricultural genetics in URSS

5 ) THE SOCIAL IMPLICATIONS OF HUMAN GENETICS .

The generally held view is that the rise of genomics and post-genomics corresponds to a fading of the reductionist vision which dominated biology over the previous decades. We will challenge this view by showing that the structural side of molecular biology has never before been as active as it is now.

The present transformations reflect the increasing necessity to link the molecular explanations with other modes of explanation, i.e. physical and Darwinian explanations. This necessity is the mere result of the success of the molecular approach, which has reached its limits. The physical studies of isolated molecules, functional genomics, evo/devo biology and the development of synthetic biology are different examples of the need to merge molecular explications with the physical and evolutionary explanations that we will describe in this lecture.

What will be the future of this active process of synthesis and combination? The rise of a new discipline, similar to the rise of molecular biology in the 1930s and 1940s? The parallel between the views that were dominant at that time, and those associated with the entry into the post-genomic era, might support this hypothesis. Another possibility is that the linking of these views will lead to huge advances in different areas of biological research, without bringing about the major synthesis that so many look for.

In a few days, the tenth anniversary of the publication of the whole genome sequence of the Eukaryotic model species, the yeast Saccharomyces cerevisiae , will be celebrated. Taking advantage of this date, I would like to analyze aspects of the role of Genetics and Genomics and their relationship for the study of this model organism.

In this aim, I shall go back to the past to describe milestones concerning this species: the pre-sequencing genetic landscape, the early beginnings of the Genomics era, the first analysis of the sequence and its unforeseen discoveries. I shall also consider the years where the series of post-genomics resources were specially rich in the case of yeast (sets of strains bearing a deletion of each of 6000 putative genes, sets of PCR products of each gene, two-hybrid systematic screening, sets of DNA hybridization microarrays…) have begun to generate a huge quantity of results.

Today, the high throughput genomic analysis has become a reality for yeast as for a large range of species but continues to provide food for the debate concerning small-scale and large-scale research: Is the goal of Genomics to serve geneticists? Can Genomics elucidate gene function? Is the promise of the functional Genomics to complete the encyclopedia of a cell? I shall present these different points of view and discuss them.

References:
- Dujon B (1996) The yeast genome project: what did we learn?, Trends Genet. (7):263-70.
- Goffeau A (2000) Four years of post-genomic life with 6,000 yeast genes, FEBS Lett, 480(1):37-41
- Hoheisel JD (2006) Microarray technology: beyond transcript profiling and genotype analysis, Nature reviews , (7): 200-210,
- Hughes TR Robinson MD, Mitsakakis N and Johnston M (2004) The promise of functional genomics: completing the encyclopedia of a cell, Current Opinion in Microbiology (7):546–554
- Johnston M& Fields S (2000) Grass-roots genomics nature genetics , (24):5-7
- Mortimer RK and Schild D (1980) Genetic map of Saccharomyces cerevisiae , Microbiol Rev. 44(4): 519–571.
- Schena M, Shalon D, Davis RW, Brown PO (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray, Science ;(270): 467-70.

Overview of a DNA microarray experiment

Jean-Yves Coppée & Béatrice Regnault
Platform DNA Arrays, Pasteur Institute, Paris
Email: jycoppee@pasteur.fr & regnault@pasteur.fr

The array technology has become a standard tool in many genomic research laboratories. DNA arrays have deeply changed the approach to biological research. Instead of working on a gene-by-gene basis, scientists can now study tens of thousands of genes at once. In this presentation, we will give a general overview of the various steps of a DNA array experiment: the fabrication of the arrays the preparation of the labeled targets, the detection and quantification of the hybridization signals, the data analysis and handling.

Two technologies will be presented: the Affymetrix arrays and the microarrays spotted onto glass slides.

DNA chip experiments generate thousands of measurements that represent variations of gene expression levels between different biological conditions. These data must be carefully analysed in order to extract the most of the biological information they carry. A gene expression data analysis involves several steps which are experiment design setting, data preprocessing and normalization, differential analysis, unsupervised and supervised clustering.

Statistical and data analysis methods are applied at each of these steps in order to make the most of this huge amount of data. Although a large number of analysis softwares are freely available and easy to use, some basic notions are necessary to understand the analysis process, choose the right method and apply it properly.

Three one-hour lectures will be proposed during the course. The first one will provide students with basic statistical terms and concepts such as mean, median, variance, normal distribution, etc. The second lecture will explain the basis of hypothesis testing and its application to differential analysis. Unsupervised clustering techniques will be considered in the third lecture.

Since experiment design and data normalization involve methods that may be chip technology dependent, these topics will be discussed during the workshops and practical courses. Supervised clustering is far beyond the scope of this course and will not be considered.

Fever and chills in applying micro array technology to the study of Plasmodium

Peter H. David
Parasite Molecular Immunology Unit, URA CNRS 2581, Pasteur Institute, Paris
Email: pdavid@pasteur.fr

The scourge of malaria is worsening, with billions of exposed individuals and millions of deaths occurring yearly. Towards the establishment of novel approaches to parasite control, significant advances have been made in research on the biology of Plasmodium falciparum, the human parasite responsible the most severe form of disease. In the past, Plasmodium may often have been looked upon as a mere assortment of “promising vaccine candidates”; today, a major research challenge is to confer biological significance to the massive amount of information provided by the sequence of the P.falciparum genome, published in 2002.

P. falciparum goes through a series of different developmental stages during its complex life cycle which takes place in two hosts: an Anopheles mosquito and Homo sapiens. In the latter, the parasite first develops in the liver; its progeny–a novel developmental stage- then invades red blood cells in the circulation, where it undergoes successive 48h cycles of multiplication.

DNA microarrays can provide important information on the mechanisms of genetic regulation which allows the parasite to exploit successive biological niches offered by its hosts. Whole genome transcriptional analysis of the parasite during different stages of its development cycle has been performed with different types of oligonucleotide-based microarrays. A striking observation is that the majority of P. falciparum genes are regulated throughout the parasite cycle. For example, during the 48h parasite development in the red cell, the relative abundance of individual mRNAs varies in a continuous mode, with a single maximum and a single minimum. Such a highly streamlined gene expression pattern introduces experimental challenges, since well controlled time-course experiments with tightly synchronized parasites are required to address questions such as the consequences of gene inactivation on transcription, or gene regulation responses during parasite adaptation to environmental factors. Genes with correlated temporal expression patterns are thought to be associated with similar functions, but in the case of P.falciparum, gene clustering approaches have mainly led to hypothesis which remains to be verified experimentally. Although microarray data reflect the existence of significant transcriptional regulation, P.falciparum transcription factors have been difficult to identify leading to speculation on the role played by post-transcriptional regulation mechanisms. The latter could underlie observations such as discrepancies between the parasite transcriptome and proteome, or the prevalence of anti-sense RNA.

In addition to studies on gene expression, P.falciparum DNA microarrays have been applied to comparative genomic analysis. Early work performed with laboratory strains adapted to long term culture showed the feasibility of such an approach which has recently been used for the study of field isolates. Comparative genome hybridization is now an efficient tool to analyze P.falciparum polymorphism on a genome-wide scale.

Complex inheritance is defined as the variability in phenotype expression that is attributed both to the inheritance of combinations of alleles at multiple loci and to environmental exposures. In the case of complex diseases a polygenic component is interacting with environmental factors to cause phenotypic variation, translated by a genetic disorder. Some common diseases belong to this category : breast cancer, diabetes and other autoimmune diseases, as well as heart disease, high blood pressure, Alzheimer disease, arthritis, and obesity.

Multifactorial inheritance is also associated with heritable traits such as fingerprint patterns, height, eye color, skin color, but also in some cases with susceptibility/resistance to infectious agents.

The central problem of complex inheritance has been to map genes responsible for disease susceptibility. To this end the advances made in the past 10 years endowed significantly with the identification of disease loci and in some cases with the isolation of the contributing genes. The often minor effects of complex disease genes to the phenotype of a given syndrome have hampered studies towards gene isolation.

The vast wealth of data produced by genome sequencing projects represents a new challenge in the efforts to describe genome function and to attempt to identify genes involved in complex diseases. Indeed functional genomics, a field of molecular biology, makes use of the large amount of data and by using high-throughput techniques like microarrays, proteomics, metabolomics and mutation analysis offers the potential to describe the function and interaction of genes implicated in the pathogenesis of complex diseases.

We have applied functional genomics to the study of the immune events taking place at the early stages of autoimmune condition, prior to the appearance of clinical signs, in type 1 diabetes (T1D). We first established a sub phenotype allowing the early selection of NOD (Non obese Diabetes) mice, which will develop T1D later on and applied functional genomics.

We established that E-IAA (Early Insulin AutoAntibodies) between 3 to 5 weeks of age correlate with early T1D and define the first autoimmune window for disease pathogenesis (Melanitou et al , 2004, J. Immunol; 173, 6603). We have used this marker to select animals E-IAA positive or negative and perform differential gene expression analysis in the PaLN (pancreatic lymph nodes).

Between the 12,986 genes included in the microchip (Affymetrix, MG_U74Av2), 125 showed significant differential gene expression patterns between the two samples, with 90 genes up-regulated and 35 down regulated in the E-IAA positive PaLN. Several interesting aspects of the obtained data will be discussed.

This data set corresponding to the careful selection of individuals after sub phenotypic attribution as described, might represent the “ breadcrumb trail ” for deciphering the early mechanisms responsible for autoimmune disturbance.

Complete genomes sequencing as a first step provide a large amount of data and above all, point out the importance of the regulation since, for example, only 35 thousands genes must resolve entirely the complexity of the mankind. With the systematic sequencing of cDNA libraries and the advent of microarray analysis, it has become possible to describe, at the tissue level, the expression pattern of thousands of genes already known or yet to be characterized. As an example, multitissue experiments using microarrays provided a broad source of information and constituted a first step in the description of gene expression profiles. Actually, these approach largely used to define expression profiles of various tumor allows to better classified them. Large scale analysis is undoubltedly a powerful tool to address fundamental questions, generate new hypothesis and open new way of our understanding of the cell or tissue physiology, and ultimately lead to new resources for therapy.

There are different levels of gene regulation, the starting point being the transcription, then post-transcriptional events occur before that the translation start with its own different check points and feedback to the genome. Large scale expression profiling used in divers physiopathologic situation indicate the set of genes of which expression are disturb or not in these conditions and above all lead to the classification of normal or sick tissue providing information of the tissue, organs, cells or disease molecular aetiology. We will talk about divers aspects and concepts of these approaches, and particularly the intrinsic difficulties and also its limit leaning on some examples drawn from our experiences (1, 2, 3, 4).

One of these examples is the ability to classify biological samples according to their particular transcription patterns, this approach called "expression profile" considers the resemblance or non resemblance of biological samples rather than that of the genes themselves. This approach provides new classifications of tumors based on transcriptome, in relation with prognosis. They show that the systematic use of array testing holds great promise to improve the prediction of prognosis and chemo sensitivity of breast cancer (in our case) and to provide new therapeutic targets. Our results indicate that gene expression profiling can predict clinical outcome and lead to a more precise classification of breast tumors (5, 6). Application of expression profiling to other research fields will open new way of understanding complex biological systems (7, 8, 9).

Another application is the gene discovery which is one of the first applications. Here, we compare the expression profile between two or several states in order to select genes with unknown function involved in one process (10, 11, and 12). We applied this approach to the thymus a primary lymphoid organ involved in differentiation of T lymphocytes. Its stroma creates several distinct microenvironments which control defined steps in T cell development. We ground on this study on a differential screening between different knock-out mouse models, and this allow to isolate some candidates potentially involved in these differentiation processes.

The third approach is to use the profiling as a tool to phenotype an organ. We applied this concept to the thymus to virtually micro dissect this complex organ (13). One of the goals is, on one hand to use this profile to phenotype thymus from genetically engineered mice, and on the other hand to understand the complex mechanisms of T cell development. This major site for T-lymphocyte maturation is anatomically divided into three regions, the sub capsular region, the cortex and the medulla. Each of these compartments forms a specialized stromal microenvironment that is crucial to control T-cell development. To get an integrated view of these processes, both whole thymi from genetically engineered mice together with purified thymocytes were analyzed. Using mice exhibiting various transcriptional perturbations and developmental blockades, we performed a transcriptional micro dissection of the organ. Multiple signatures covering both cortical and medullar stroma as well as various thymocyte maturation intermediates were clearly defined. Beyond the definition of histological and functional signatures (proliferation, rearrangement), we provide the first evidence that such an approach may also highlight the complex cross-talk events that occur between maturing T cells and stroma. Our data constitute a useful integrated resource describing the main gene networks set up during thymocyte development and a first step toward a more systematic transcriptional analysis of genetically modified mice.

References

1 - Nguyen C, Rocha D, Granjeaud S, Baldit M, Bernard K, Naquet P and Jordan BR. Differential gene expression in the murine thymus assayed by quantitative hybridization of arrayed cDNA clones. Genomics, 1995, 29: 207-216,
2 - Bernard K, Auphan N, Granjeaud S, Victorero G, Schmitt-Verhulst AM, Jordan BR and Nguyen C. Multiplex messenger assay: Simultaneous, quantitative measurement of expression of many genes in the context of T cell activation. N. A. R.,1996, 24 (8):1435-1442,
3 - Loriod B, Victorero G and Nguyen C. (Book). cDNA mico-(and macro) arrays with radioactive detection. Methods in Molecular Biology, Ed B Jordan, Spring –Verlag, Tiergartenstr.17, D-69121 Heidelberg .
4 - Bertucci F, Bernard K, Loriod B, Yi-Chung-Chang, Granjeaud S, Birnbaum.D, Nguyen C, Peck K and Jordan BR. Sensitivity issues in DNA arrays-based expression measurements: Advantages of nylon microarrays for small samples. Human Molecular Genetics, 1999, vol. 8, N°9 : 1715-1722
5 - Bertucci F, Houlgatte R, Benziane A, Granjeaud S, Adelaide J, Tagett R, Loriod B, Jacquemier J, Viens P, Jordan B, Birnbaum D, Nguyen C. Gene expression profiling of primary breast carcinomas using arrays of candidate genes. Hum Mol Genet. 2000 Dec 12;9(20):2981-91.
6 - Bertucci F, Nasser V, Granjeaud S, Eisinger F, Adelaide J, Loriod B, Tagett R, Giaconia A, Benziane A, Devilard E, Jacquemier J, Viens P, Nguyen C, Birnbaum D, Houlgatte R. Gene expression profiling in primary breast cancer and survival of patients treated with adjuvant chemotherapy. Human Hum Mol Genet. 2002 Apr 15;11(8):863-72.
7 - Magrangeas F., Nasser V., Avet-loiseau H., Loriod B, Decaux O., Granjeaud S., Bertucci F., Birnbaum D., Nguyen C., Bataille R, Houlgatte R., and S. Minvielle. Gene expression profiling of multiple myeloma according to immunoglobuli types and light chain subtypes reveals molecular portraits in relation to the pathogenesis of the disease. Blood. 2003 Mar 6
8 - L. Shaffer, Andreas Rosenwald, Elaine M. Hurt, Jena M. Giltnane, Lloyd T. Lam, Oxana K. Pickeral,. and Louis M. Staud. Signatures of the Immune Response. Immunity, Vol. 15, 375–385, September, 2001.
9 - Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, Boldrick JC, Sabet H, Tran T, Yu X, Powell JI, Yang L, Marti GE, Moore T, Hudson J, Lu L, Lewis DB, Tibshirani R, Sherlock G, Chan WC, Greiner TC, Weisenburger DD, Armitage JO, Warnke R, Levy R., Wilson W., Grever MR., Byrd JC, Botstein D., Brown PO, Staudt LM. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. 2000 Nature 403:503-511
10 - Carrier A, Nguyen C, Victorero G, Granjeaud S, Rocha D, Bernard K, Miazec A, Ferrier p, Malissen M, Malissen B and Jordan BR. Differential gene expression in CD3- epsilon and Rag- 1 deficient thymuses : Definition of a set of genes potentially involved in thymocyte maturation.. Immunogenetics, 1999, 50:255-270
11 - Wurbel M A, Philippe J M, Nguyen C.,Victorero G., Freeman T., Wooding P., Miazek A., Mattei M.G., Malissen M., Jordan B.R., Malissen B., Carrier A. And P. Naquet. The chemokine TECK is expressed by thymic and intestinal epithelial cells and attracts double and single psitive thymocytes expressing the Teck receptor CCR9.. EJI, 2000 Jan;30(1):262-271
12 - Ramialison, M., Mohr, E., Nal, B., Saboul, T., Carrier, A., Tagett, R., Granjeaud, S., Nguyen, C., Gautheret, D., Jordan, B. R. and Ferrier, P. Expression profiling in mouse fetal thymus reveals clusters of coordinately expressed genes that mark individual stages of T cell ontogeny. Immunogenetics, (2002) 54:469–478
13 - Ross et al Nature genetics 2000, 24, 227-35.

Dissecting the response of hematopoietic cells to ionizing radiation exposure with cDNA microarrays

Diana Tronik-Le Roux
Laboratoire de Génomique et Radiobiologie de l'Hématopoïèse. Service de Génomique Fonctionnelle.
Commissariat à l'Energie Atomique. 2 rue Gaston Crémieux. 91057 Evry. France.
E-mail: diana.le-roux@cea.fr

Multicellular organisms encounter daily variations in their environment that can adversely affect their physiological functions. To maintain their integrity, individuals respond to these stress situations by triggering a complex integrated network of protein signaling that geared towards cytoprotective measures. In this study by using high-throughput technology, we have initiated the elucidation of the complex transcriptional processes that are triggered by hematopoietic cells in response to DNA damage resulting from IR exposure. Hematopoiesis is an example of a developmental process which entails homing of stem cells in different anatomic locations, differentiation, proliferation, and maturation events. This system is finely regulated to respond rapidly and specifically to modifications of the number of circulating cells or to environmental modifications. One of the best known responses is the rapid eradication of hematopoietic cells after IR exposure; however, the molecular mechanisms that control the susceptibility of these cells to radiation exposure are not completely elucidated. To identify the molecular programs underlying this process, we have applied a genome wide analysis strategy to monitor transcriptional changes occurring in hematopoietic cells present in the bone marrow and spleen. The results highlight transcriptional modulations identified as early as 1 hour after IR exposure. Many of the identified genes were never described before as playing a role in the IR response. Clustering the modulated genes using a QT clustering method led to select 2 specific clusters of up-regulated genes in the spleen. Cluster 1 includes transcripts that are rapidly but transiently up-regulated such as those coding for DNA repair enzymes. Cluster 2 includes genes for which the high level of transcripts persists at least for 3 hours. This cluster includes many known p-53 target genes such as p21 and proapoptotic activities such as Bax and Mdm2. Promoter analysis revealed that the majority of genes included in this cluster possess p-53 responsive elements. This suggests that these genes might be co-regulated and might constitute novel targets of the tumor suppressor gene. This hypothesis was assessed by chromatin immunoprecipitation.

The results of the transcriptional analysis were then compared to those elicited by purified bone marrow hematopoietic cells and non hematopoietic tissues. The data show a highly tissue specific transcriptional activity mainly elicited by the modulation of several transcription factors and a variety of yet uncharacterized stress signals. Assessing important interactions between gene products and pathways revealed a clear differential IR response displayed by both tissues.

Altogether this approach has the potential for unraveling molecular networks and finding novel and specific molecules involved in the early response of cells to radiation exposure and might facilitate the derivation of clinically useful targets for patient benefit.

Microarray data standards and ArrayExpress, an international repository for Microarray Data at the EBI

Helen Parkinson
European Bioinformatics Institute, EMBL-EBI Wellcome Trust Genome Campus, Hinxton CB10 1SD , UK.
Email: parkinson@ebi.ac.uk

The lecture will cover the current state of microarray data standards, formats and ontologies and the history of these and the MGED society (Brazma, Hingamp et al. 2001) . The data content standard MIAME will be explained, the format MAGE-ML and tools that generate it will be introduced and the MGED ontology (Whetzel 2006) and it's successor FuGO - the functional genomics ontology will be demonstrated in the context of submission and query tools.

There will be an introduction to EBI services, the ArrayExpress (Parkinson, Sarkans et al. 2005) database, linked resources such as Uniprot, Ensembl and Expression Profiler (Kapushesky, Kemmeren et al. 2004) (Kasprzyk, Keefe et al. 2004) .

Links to web based tutorial resources on data retrieval and data submissions will be provided for participants.

References

Brazma, A., P. Hingamp, et al. (2001). "Minimum information about a microarray experiment (MIAME)-toward standards for microarray data." Nat Genet 29 (4): 365-71.

Kapushesky, M., P. Kemmeren, et al. (2004). "Expression Profiler: next generation--an online platform for analysis of microarray data." Nucleic Acids Res 32 (Web Server issue): W465-70.

Kasprzyk, A., D. Keefe, et al. (2004). "EnsMart: a generic system for fast and flexible access to biological data." Genome Res 14 (1): 160-9.

Parkinson, H., U. Sarkans, et al. (2005). "ArrayExpress--a public repository for microarray gene expression data at the EBI." Nucleic Acids Res 33 Database Issue : D553-5.

Patricia L. Whetzel , H. P., Helen C. Causton , Liju Fan , Jennifer Fostel , Gilberto Fragoso , Laurence Game , Mervi Heiskanen , Norman Morrison , Philippe Rocca-Serra , Susanna-Assunta Sansone, Chris Taylor , Joseph White , and Christian J. Stoeckert, Jr. (2006). "The MGED Ontology; a resource for semantics-based description of microarray experiments." Bioinformatics .

Stoeckert, C. J. a. P., H (2003). "The MGED ontology: a framework for describing functional genomics experiments." Comparative and Functional Genomics 4 : 127-132.

Development of bioinformatic tools are of crucial necessity to store and analyse the data which are now routinely produced by post genomic technologies. We have developed a set of tools to handle microarray analyses and compare transcriptome data produced by our public platform (http://transcriptome.ens.fr). For instance the Laboratory Information Management System (LIMS) database is constructed as modules allowing separating storage of raw microarray data from all steps involved in their analysis. This structure offers to the user a very flexible and powerful tool that can be adapted to his specific needs. We also developed Doelan, an automated tool to check the quality of produced DNA microarrays [1]. The reports generated by Doelan will help microarray platforms in their quality approach such as ISO 9001 certification. In addition to microarray quality, normalisation of the results is a critical step of microarray data analysis. Various normalisation methods exist but the influence of the method chosen on the data is hard to assess. We developed Goulphar, an R script, to offer biologists with an easy to use interface to access and monitor data normalisation.

The group also focuses on creating tools to help the community make the most of microarray data. Yeast Microarray Global Viewer (yMGV) has been designed to provide biologists with meaningful information from yeast genome-wide expression data [2]. Thus far around 1300 transcriptome states are available in yMGV and a second yeast ( S. pombe ) has been added [3]. Another tool called MiCoViTo [4] has also been developed as a methodology to compare the gene networks in various microarray experiments under a range of conditions. To achieve this, MiCoViTo use a graph theory based approach.

From the very beginning the aim of this platform was to be actively involved in the propagation of the know-how and in the distribution of the products issued from its activity. A massive effort has been made to put online tutorials for common software and to create programs to assist the user in each step of the analyses. We hope that this integration of microarray data will help the scientific community working on expression experiment.

References

[1] Jourdren L. and S. Le Crom (2005). Doelan: a solution for quality control monitoring of microarray production. Bioinformatic, . 21 (22) : 4194-495.

[2] S. Le Crom, F. Devaux, C. Jacq and P. Marc (2002). yMGV: helping biologists for yeast microarray data mining. Nucleic Acid Research, 30(1) p76-79.

[3] G. Lelandais, S. Le Crom, F. Devaux, S. Vialette, GM. Church, C. Jacq and P. Marc (2004). yMGV: a cross-species expression data mining tool. Nucleic Acids Research , 32 Database issue : D323-325.

[4] Lelandais G., Marc P., Vincens P., Jacq C. and S. Vialette (2004). MiCoViTo: a tool for gene-centric comparison and visualization of yeast transcriptome states. BMC Bioinformatics , 5 (1) : 20.

Microarray in infectious diseases: from basic research to vaccine design
Renata Grifantini
Chiron-Vaccines , Via Fiorentina 1 , 53100 Siena, Italy
Email: Renata_Grifantini@chiron.com    

DNA and protein microarrays are powerful technologies to study the biology of bacterial pathogens and to support the identification of antigen candidates for vaccine development. In the presentation will be provided some hints on the utility of DNA microarray to understand the bacterial infectious mechanisms through the analysis of whole bacterial transcriptome under in vitro conditions that simulate host environment. Examples will be provided on how the analysis of bacterial transcription profile facilitates vaccine design. A second topic of the presentation will focus on the use of protein array in antigen discovery. Examples will be provided on the application of this technology for antigen identification by screening of human sera from infected patients.    

References

Role of FNR and FNR-regulated, Sugar Fermentation Genes in Neisseria meningitidis infection. Erika Bartolini, Elisabetta Frigimelica, Serena Giovinazzi, Giuliano Galli, Yazdani Shai, Caroline Genco, Jo Anne Welsch, Dan M. Granoff, Guido Grandi and Renata Grifantini. Mol Microbiol, 2006, in press  

Characterization of a novel Neisseria meningitidis Fur and iron-regulated operon required for protection from oxidative stress: utility of DNA microarray in the assignment of the biological role of hypothetical genes. Grifantini R, Frigimelica E, Delany I, Bartolini E, Giovinazzi S, Balloni S, Agarwal S, Galli G, Genco C, Grandi G. Mol Microbiol., 2004, 54:962-79.  

Identification of iron-activated and -repressed Fur-dependent genes by transcriptome analysis of Neisseria meningitidis group B. Grifantini R, Sebastian S, Frigimelica E, Draghi M, Bartolini E, Muzzi A, Rappuoli R, Grandi G, Genco CA. Proc Natl Acad Sci U S A., 2003, 100:9542-7  

Gene expression profile in Neisseria meningitidis and Neisseria lactamica upon host-cell contact: from basic research to vaccine development. Grifantini R, Bartolini E, Muzzi A, Draghi M, Frigimelica E, Berger J, Randazzo F, Grandi G. Ann N Y Acad Sci., 2002, 975:202-16.  

Previously unrecognized vaccine candidates against group B meningococcus identified by DNA microarrays. Grifantini R, Bartolini E, Muzzi A, Draghi M, Frigimelica E, Berger J, Ratti G, Petracca R, Galli G, Agnusdei M, Giuliani MM, Santini L, Brunelli B, Tettelin H, Rappuoli R, Randazzo F, Grandi G. Nat Biotechnol., 2002, 20:91 -21.

The massive and rapid increase in the amount of human genome-scale DNA sequencing and the parallel development of methods to exploit these data, drive the biomedical research today in a significant transition. Technologies that generate information about the genome are being used to explore changes in gene expression and related proteins following exposure to chemicals, xenobiotics, drugs etc. Toxicogenomics and toxicoproteomics refereed as the application of the functional genomic technologies and the genome-wide data in toxicology, enabling the study of adverse effects of toxic substrates in relation to structure and activity of the genome. The principal evident underlying on this fact is that a specific pattern of altered gene expression is related with each substrate properties, especially toxicological properties and it will be revealed using holistic approaches analyzing samples from exposure organisms, tissues or cells. In “omics” studies, developing techniques such as microarrays, two dimensional gel electrophoresis (2-DE) and mass spectrometry in conjunction with advanced analysis software and the availability of databases offer a powerful set of tools to investigate the response to specific stimuli. For toxicogenomic studies, microarrays seem to be the most popular technique. Nevertheless when such a technology is used, the toxic effect complexity of a singe substrate or a mixture of substrates has to be taken into account plus the fact that these effects are time, dose and target (organism, tissue, cell) related. In relation to an a priori hypothesis, gene expression profiling applied to toxicology has the potential to reveal the molecular pathways, cellular processes and tissue disturbances that mediate the adverse responses to a toxicant. For the complete study of a toxic effect by microarrays, a three or four dye cDNA labeling procedure was developed permitting the simultaneous analysis of three or four samples at the same time on the same slide. Toxicoproteomics seek to identify critical proteins and pathways in biological systems that are affected by and respond to adverse exposures using global protein analysis technologies. 2-DE accompanied by mass spectrometry remains the most important technology in the study of proteins and supports toxicology in understanding the biological effects of different kinds of exposures in living systems. In contrast to microarrays, proteomic approaches have the advantage of studying toxicity signatures in DNA and RNA free samples such as serum, plasma and other biofluids. Nowadays, the extensive use of toxicogenomics and toxicoproteomics provide a better understanding of the mechanisms of toxicity and facilitate the prediction of toxicity of chemicals, drugs or unknown compounds. Furthermore, these approaches provide specific opportunities for the identification of more precise exposure indicative and mechanism-related molecular biomarkers and overall improvement of the risk assessment process at various stages such as the development of novel predictive models identifying human health hazards.

References

1. Heijne WH, Kienhuis AS , van Ommen B, Stierum RH, Groten JP. Systems Toxicology: application of toxicogenomics, transcriptomics, proteomics, and metabolomics in toxicology. Expert Rev. Proteomics 2(5): 767-780, 2005.

2. Lee KM, Kim JH, Kang D. Design issues in toxicogenomics using DNA microarray experiment. Toxicol Appl Pharmacol 207 (2S): 200-208, 2005.

3. Ekins S, Nikolsky Y, Nikolskays T. Techniques: application of systems biology to absorption, distribution, metabolism, excretion and toxicity. Trends Pharmacol Sci 24(4): 202-209, 2005

4. Tsangaris GT, Botsonis A, Politis I, Tzortzatou-Stathopoulou F. Evaluation of cadmium-induced transcriptome alterations by three color cDNA labelling microarray analysis on a T-cell line. Toxicology 178: 135-160, 2002.

5. Fountoulakis M Application of proteomics technologies in the investigation of the brain.. Mass Spectrom Rev 23(4):231-258, 2004.

6. Witzmann FA, Bai F, Hong SM, Liu S, Pedrick N, Ringham H, Tan J. Gels and more gels: probing toxicity. Curr Opin Mol Ther 6(6): 608-615, 2004

Plenary Lecture I
______________________________________________________________________________

The impact of post genomics on our understanding of parasitism: where do we stand?

Geneviève Milon
Unité d'Immunophysiologie et Parasitisme Intracellulaire ,
Institut Pasteur, 25-28 rue du Dr Roux, 75015, Paris, France
Email: gmilon@pasteur.fr

The very initial event leading to the establishment of parasitism as a life style of the organisms named “parasites” has been the encounter among these organisms and the hosts on which strictly relies their perpetuation. Over the week we shall have been sharing the present understanding of the life traits displayed by Plasmodium spp within the two hosts on which relies their perpetuation namely the blood – feeding Anopheles spp and the Anopheles' blood source ie laboratory rodents, humans .Of note Anopheles spp relies on blood derived nutrients/signals for achieving a complete gonotrophic cycle.

Regardless of the exact dating of the beginning of hematophagy – a process coupled to morphological modification of mouthparts of the insect lineages- , we are now aware that their salivary glands contain cocktails of pharmacologic reagents that affect blood clotting, platelet aggregation, vascular bed features, the immune system – the inflammatory /counterinflammatry processes included - the angiogenesis of the blood sources some being members of the Homo lineage(s). With the development of transcriptome analysis, the salivary compositional diversity of several hematophagous arthropods is being revealed at a fast pace; however, the majority of these proteins have no known function ( Ribeiro, J. M., and Francischetti, I. M. 2003 ,Schneider and James 2006 ).

In this lecture, will be shared those determinants that facilitated establishment of the Leishmania and its development within each of the two major organisms on which relies their perpetuation : (1)the blood-feeding insects –sand flies - that act as Leishmania hosts and vectors-(2) the Leishmania mammalian hosts that also act as blood -sources of the blood-feeding sand flies. This short lecture is also designed to present research questions by identifying the important subjects that still lack relevant data.

Posters

Poster H2
Banica Leontina (Email: rcg@cantacuzino.ro )
“ Molecular dysfunctions in peripheral T lymphocytes freshly isolated from patients with systemic Lupus Erythematosus “
Leontina Mirela Banica , Alina Nicoleta Besliu, Gina Pistol, Maria Stefanescu, Cristiana Matache, “Cantacuzino” National Institute of Research and Development for Microbiology and Immunology, Advanced Studies Center, Cellular Receptor Group 103 Splaiul Independentei, 70100 Bucharest, Romania

Poster H3
Bulavait Aiste (Email: aisteb@ibt.lt )
“The Expression of West Nile Virus Derived Proteins in Yeast S. cerevisiae”
Aiste Bulavait and Kstutis Sasnauskas
Institute of Biotechnology , V.Graiciuno 8-206, LT-02241, Vilnius , Lithuania

Poster H4
Karray-Chouayekh Sondes (Email: sondeskarray@yahoo.fr )
“Molecular caracterisation of brca1 gene in hereditary breast cancer“
Sondes Karray-Chouayekh 1 , Raja Mokdad-Gargouri 1 , Jamel Daoud 2 , Mounir Frikha and Ali Gargouri 1 1-LGME-CBS, 2- service of radiotherapy CHU Habib Bourguiba Sfax, 3- cabinet of cancerology Sfax, Tunisia

Poster H5
Krjutskov Kaarel (Email: karlakrj@ut.ee )
“Universal Primer APEX: flexible and medium-scale genotyping method“
Kaarel Krjutskov and Andres Metspalu
Dep. of Biotechnology, Riia 23 – room 205, TARTU 51010 , Estonia

Poster H6
Martsenyuk Olga (Email: o.p.martsenyuk@imbg.org.ua )
“ Association of glutathione S-transferase activity with polymorphic methylenetetrahydrofolate reductase and glutathione S-transferase genes in human placenta “
Martsenyuk Olga Petrivna, Slonchak A. M., Teplyuk N.M., Sazonova L.Y., Obolenskaya M.Yu.
Institut of molecular biology and genetics NAS of Ukraine, Kyiv National Taras Shevchenko University , Kyiv 03142

Poster H7
Panayiotou Andrie (Email: andriepa@cing.ac.cy )
“ Risk factors for the development of early unstable atherosclerotic plaque -The Cyprus study “
A. Panayiotou (1), N.Georgiou (2), M. Griffin (2), D. Bond (2), T. Tyllis (2) , S. E Humphries (3), A.N. Nicolaides(1,2).
1. University of Cyprus , Nicosia , Cyprus . 2.Vascular Screening and Diagnostic Center , Nicosia , Cyprus . 3.Cardiovascular Genetics, UCL, London , UK . Postal address: 1 Chiou street , Agioi Omologites, 1086, Nicosia , Cyprus .

Poster H8
Pogorelko Gennady (Email: gpogorelko@yandex.ru )
“Transgenic plants as the way to discover and validate novel genes and their functions“
Pogorelko GV , Fursova OV, Ogarkova OA, Tarasov VA
N.I. Vavilov Institute of General Genetics Russian Academy of Sciences, Gubkin Str., 3, Moscow 119991 GSP-1, Russia

Poster H9
Rolo Anabela (Email: anpiro@ci.uc.pt )
“Hyperglycemia decreases mitochondrial function: The regulatory role of mitochondrial biogenesis“
Carlos M. Palmeira1, Anabela P. Rolo1 , Jessica Berthiaume2, J. A. Bjork2 and Kendall B. Wallace2.
1Center for Neurosciences and Cell Biology, Department of Zoology, university of Coimbra , 3004-517 Coimbra , Portugal. 2Department of Biochemistry & Molecular Biology, University of Minnesota School of Medicine, Duluth, MN, USA.

Poster H10
Shostak Kateryna (Email: Kateryna.Shostak@gmail.com )
“Gene expression changes associated with human glioma development “
K. Shostak, V . Dmitrenko, T. Bukreieva, O. Simyrenko, O. Boyko, Y. Zozulya, V. Kavsan
Institute of Molecular Biology and Genetics, 150 Zabolotnogo str., Kiev , 03143, Ukraine

Poster H11

Sikora Marta (Email: martas@ibch.poznan.pl )
“ Metabolism of homocysteine thiolactone and N-homocysteinylation of proteins “
Marta Sikora , Jaroslaw Zimny, Andrzej Guranowski, Tomasz Twardowski and Hieronim Jakubowski.
Institute of Bioorganic Chemistry, Polish Academy of Sciences Noskowskiego 12/14, 61-704 Pozna Poland

Poster H12
Sivitskaya Larysa (Email: silarissa@yandex.ru )
“ Human mitochondrial DNA “
Sivitskaya L.N ., Kushnerevich E.I.,Zhidkov I.V.,Danilenko N.G.,Davydenko O.G.
Akademicheskaya st.27, 220072 Minsk Belarus

Poster H13
Skindersoe Mette ( msk@biocentrum.dtu.dk )
“ Prokaryotic-eukaryotic interaction: Pseudomonas aeruginosa and the immune system “
Skindersoe Mette Elena
Centre for Biomedical Microbiology, Building 301, Technical University of Denmark , DK-2800 Kgs. Lyngby, Denmark

Poster H14
Swiercz Aleksandra (Email: aswiercz@cs.put.poznan.pl )
“ DNA Sequencing by Hybridization via Genetic Search “
Jacek Blazewicz, Ceyda Oguz, Aleksandra Swiercz and Jan Weglarz
Institute of Computer Science, Poznan University of Technology Ul. Piotrowo 2 60-965 Poznan , Poland

Poster H15
Szabo Kornelia (Email: szabok@mail.derma.szote.u-szeged.hu )
“ Monitoring the gene expression changes of human keratinocytes in response Propionibacterium acnes “
Kornelia Szabo1 , Istvan Nagy1, Zoltan Hegedus2, Laszlo Hackler Jr2, Laszlo Puskas2, Lajos Kemeny1
1. Department of Dematology and Allergology, University of Szeged , 2. Biological Research Center of the Hungarian Academy of Sciences

Poster H16
Tokovenko Bogdan (Email: boga@bigmir.net )
“ Arrays analysis in multi-cell systems biology studies “
Institute of Molecular Biology and Genetics, Zabolotnogo 150 Ukraine , 03143 Kyiv

Poster H17
Tyczewska Agata Anna (Email: agatat@ibch.poznan.pl )
“ Searching of short RNAs that influence activities of chosen ribonucleases “
Agata Tyczewska , Tomasz Twardowski, Marek Figlerowicz
Institute of Bioorganic Chemistry, Polish Academy of Sciences
Noskowskiego St. 12/14, 61-704 Poznan Poland

Poster H18
Voskarides Constantinos (Email: kvoskar@cing.ac.cy )
"Genetic and clinical investigation of familial hematuria. Many patients develop progressive chronic renal failure from focal segmental glomerular sclerosis"
Constantinos Voskarides 1 , Loukas Damianou 2 , Vassos Neocleous 3 , Ioanna Zouvani 4 , Stalo Christodoulidou 1 , Valsamakis Hadjiconstantinou 1 , Kyriakos Kyriakou 3 , Kyriacos Ioannou 5 , Charalampos Patsias 6 , Efstathios Alexopoulos 7 , Alkis Pierides 5 , C. Constantinou Delta 1,3
1 Department of Biological Sciences, University of Cyprus, 2 Department of Nephrology, Evangelismos Hospital, Athens, Greece, 3 The Cyprus Institute of Neurology and Genetics, Nicosia, 4 Department of Histopathology, Nicosia General Hospital, 5 Department of Nephrology, Nicosia General Hospital, 6 Department of Nephrology, Larnaca General Hospital, 7 Nephrology Clinic, Medicine Faculty, Aristotle University of Thessalloniki,Greece.

Group M

Poster M1
Bauchart Philippe (Email: p.bauchart@mail.uni-wuerzburg.de )
“ Analysis of the zoonotic risk of extraintestinal pathogenic Escherichia coli “
Philippe Bauchart, Ulrich Dobrindt, and Jörg Hacker
Institut für Molekulare Infektionsbiologie, Röntgenring 11, 97070 Würzburg ,, Germany

Poster M2
Berglund Eva (Email: eva.berglund@ebc.uu.se )
“ Genome variation of Bartonella grahamii strains “
Eva Berglund, Olga Vinnere Pettersson, Martin Holmberg and Siv Andersson
Department of Molecular Evolution, Evolutionary Biology Centre, Uppsala University ,
752 36 Uppsala , Sweden ; Department of Medical Sciences, Section for Infectious Diseases, Uppsala University Hospital , 751 85 Uppsala , Sweden

Poster M3
Blomberg Christel (Email: christel.blomberg@smi.ki.se )
“ Comparative genomics of clinical isolates of Streptococcus pneumoniae using microarray technology “
Christel Blomberg 1,2, Karin Sjöström1,2, Jessica Vallhagen1,2, Jenny Fernebro1,2, Monica Andersson1, Eva Morfeldt1, Staffan Normark,1,2 Birgitta Henriques Normark1,2.
1Swedish institute for infectious disease control, Solna, 2. Microbiology and Tumor biology Centre, Karolinska Institutet , Sweden . Smittskyddsinstitutet (SMI) /Swedish Institute for Infectious Disease Control Avd.fBakteriologi(Bakt)/Bacteriology, rum 359, Nobelsväg 18 S-171 82 Solna

Poster M4
Brehony Carina , (Email: carina.brehony@zoo.ox.ac.uk )
“ Genetic characterisation of European meningococcal disease isolates “
Carina Brehony ,
Peter Medawar Building , Department of Zoology University of Oxford , OX1 3PS , UK

Poster M5
Dziurdza Beniamin (Email: bdziurdza@cs.put.poznan.pl )
“ The parallel genetic algorithm for designing DNA randomizations in a combinatorial protein experiment “
J. Blazewicz*,**, B.Dziurdza*,**, W.T. Markiewicz**, C. Oguz***
* - Institute of Computing Science, Poznan University of Technology Piotrowo 2, 60-965 Poznan, Poland, ** - Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland *** - Department of Industrial Engineering, Koc University, Rumeli Feneri Yolu, 34450 Sariyer, Istanbul, Turkey

Poster M6
Eyangoh Sara (Eamil: eyangoh@pasteur-yaunde.org )
“ Molecular characterisation of Mycobacterium ulcerans strains isolated in Cameroon “
Centre Pasteur du Cameroun, B.P. 1274 Yaoundé, Cameroon

Poster M7
Falduto Maria (Email: mariafalduto@hotmail.com )
“Detection of Novel Immunoprotective Cryptococcal Antigens by lambda Phage Display“
M. Falduto 2 , G. Tuscano1, G. Garufi1, K. Genovese2, G. Di Salvo2, A. Ruggeri2,G. Teti2 and F.Felici1 1. Department of Microbiology, Genetics and Molecular Sciences, University of Messina 2. Department of Pathology and Experimental Microbiology, University of Messina

Poster M8
Guerfali Fatma (Email: mariafalduto@hotmail.com )
“ Say it loudly to be seen: How Leishmania parasite interacts with macrophage. Transcriptome analysis using SAGE “
Guerfali F (1,3), Laouini D(1,3), Guizani L(1,3)*, Ottones F(2,3)*, Ben-Aissa K(1,3), Marti J(2,3) et Dellagi K(1,3).
* These authors contributed equally to this work.
(1) Laboratoire d'Immunologie, de Vaccinologie et de Génétique Moléculaire, Institut Pasteur de Tunis, Tunisie.
(2) Groupe d'Etudes des Transcriptomes, Institut de Génétique Humaine, UPR CNRS 1142, Montpellier, France.
(3) Laboratoire International Associé, Bioingénieurie Moléculaire, CNRS.

Poster M9
Gvozdevskiy Nikolay (zasulich80@yahoo.com)
“ Characteristics of the indel genome polymorphism of the Russian Mycobacterium tuberculosis population “
NA Gvozdevskiy1 , TL Azhikina1, IG Shemyakin2, ED Sverdlov1
1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russ.Acad.Sci., Moscow, Russia, 117997; 2 State Research Center for Applied Microbiology, Obolensk, Moscow reg., Russia, 142279

Poster M10
Ibarz-Pavon Ana-Belen ( anabelen.ibarz-pavon@medawar.ox.ac.uk )
“ Effect of the meningococcal conjugate serogroup C-polysaccharide vaccine on the carriage of Neisseria meningitidis “
Ibarz-Pavon AB ; Urwin R and Maiden, MCJ
University of Oxford , OX1 3PS , UK

Poster M11
Loh Edmund (Email: edmund.loh@molbiol.umu.se )
“ Role of small regulatory RNAs and Riboswitches in Listeria monocytogenes “
Edmund Loh 1, Pascale Cossart 2 and Jörgen Johansson 1.
1Department of Molecular Biology, Umeå University , 90187 Umeå , Sweden .
2Interactions Bactéries-Cellules, Institut Pasteur, 75015, Paris, France INSERM U604

Poster M12
Oloomi Mana (Email: Manaoloomi@yahoo.com )
“ Study on Molecular and Immunological aspects of bacterial toxins and its subunits as therapeutic agents Mana Oloomi and Saeid Bouzari
Molecular Biology Unit, Pasteur Institute of Iran , Pasteur Ave , Tehran-Iran 13164

Poster M13
Saadaoui Imène (Email: imen_saadaoui@yahoo.fr )
“ Isolation, characterization of a new hyper toxic strain of B. thuringiensis and investigation of the mutation and domain which are responsible “
Imen Saadaoui, Soluad Rouis and Samir Jaoua
Centre of Biotechnology of Sfax Laboratory of Biopesticides BP"K" 3038 Sfax , Tunisia

Poster M14
Schauer Kristine (Email: schauer@pasteur.fr )
“ Global identification of direct NikR target genes in Helicobacter pylori “
Kristine Schauer , Jean-Michel Thiberge, Hilde de Reuse, and Agnes Labigne.
Unité de Pathogénie Bactérienne des Muqueuses,
Institut Pasteur, 28, rue du Docteur Roux , 75724 PARIS Cedex 15, France

Poster M15
Skouri-Gargouri Houda (Email: houda.skouri@cbs.rnrt.tn )
“ Purification and characterization of a novel antifungal peptide from Aspergillus sp “
Houda Skouri-Gargouri & Ali Gargouri
Centre de Biotechnologie de Sfax BP"K" 3038_SFAX, Tunisia

Poster M16
Slavova-Azmanova Neli (Email: nellystz@yahoo.com )
“ Biochemical characteristics of bacterial UMP kinases - potential target molecules for structure based drug design “
Neli Slavova-Azmanova , Cecile Evrin, Liliane Assairi, Hristo Najdenski and Anne-Marie Gilles
Department of Pathogenic bacteria, The Stephan Angeloff Institute of Microbiology
Bulgarian Academy of Sciences, Acad. G. Bonchev Str, Bl 26, Sofia 1113, Bulgaria

Poster M17
Tsitoura Panagiota ( panagiota@pasteur.gr )
“ Development of viral systems for heterologous expression and self-assembly of HCV core protein".
P. Tsitoura 1 , U. Georgopoulou 1 , S. Pêtres 2 , A. Epstein 3 , E. Tsitoura 3 and P. Mavromara 1
1 Molecular Virology Laboratory, Hellenic Pasteur Institute, Greece
2 . Plate forme H5 - Institut Pasteur, France
3 UMR 5534 CNRS-Université Claude Bernard Lyon 1, France
Postal address: Hellenic Pasteur Institute, Vas.Sofias 127
Athens , 11521 Greece