Les rétrovirus endogènes (ERV) sont constitutifs du génome des eucaryotes et représentent environ 400000 loci dans le génome humain divisés en différentes familles. Ces HERV (Human ERV) sont pour la majorité silencieux en contexte physiologique excepté dans le placenta mais présentent une activité transcriptionnelle en contexte pathologique comme par exemple dans les cancers. Il est difficile de comprendre de façon systématique les mécanismes de régulation/dérégulation des HERV et leur implication en contexte physiopathologique car il n’existe à ce jour aucun critère permettant de distinguer qu’elles sont les longues terminaisons répétées (LTR) transcriptionnellement actives dans l’ensemble de ces éléments de régulation. Nous avons développé deux générations de puces à ADN haute densité afin d’appréhender quelles étaient les LTR réactivées dans les cancers et de comprendre les mécanismes sous-jacents à la transcription des HERV. Avec la première version de la puce HERV, nous avons notamment identifié six loci de la famille HERV-W différentiellement exprimés dans le cancer testiculaire dont le locus ERVWE1 qui code pour la syncytine-1 impliquée dans la morphogénèse placentaire. L’analyse de l’ADN des tumeurs et des tissus sains adjacents démontre que l’hypométhylation des régions U3 promotrices est un pré-requis à l’activation des HERV. La deuxième version de la puce HERV a été utilisée pour une recherche de biomarqueurs pronostiques dans le cancer du poumon non à petites cellules. Ceci a permis de mettre en évidence des réactivations de HERV dans certains échantillons cancéreux et illustre la difficulté d’une telle approche au regard des disparités inter-individus / Endogenous Retroviruses (ERVs) are inherited part of the Eukaryotic genomes, and represent about 400,000 loci in the Human genome divided in distinct families. The majority of HERVs (Human ERV) are mainly silent in most physiological contexts excepted in placenta, whereas a significant expression is observed in pathological contexts such as cancers. It is difficult to understand HERV (de)regulation mechanisms and their implication in physio-pathological contexts, as there is no criteria defining transcriptional active promoters HERV long terminal repeats (LTRs) among all these regulatory élements. We developed two versions of highdensity DNA microarray to specifically detect LTR reactivated in cancers and try to understand transcription mechanism of HERV. With the first version of HERV-microarray, we identified six HERV-W loci over-expressed in testicular cancer, including the domesticated ERVWE1 locus which produces an envelope protein dubbed Syncytin-1 associated with placenta development. The analysis of DNA from tumoral versus normal tissue reveals that hypomethylation of U3 promoters in tumors is a prerequisite of HERV activation. The second version of HERV-microarray was used to identify prognosis biomarkers in non small cell lung cancer. This study identified HERV reactivation in some samples and highlighted difficulties of such approach due to inter-individuals disparities

<p>A novel technology for protein detection, proximity ligation, has been developed along with improved methods for <i>in situ</i> synthesis of DNA microarrays. Proximity ligation enables a specific and quantitative transformation of proteins present in a sample into nucleic acid sequences. As pairs of so-called proximity probes bind the individual target protein molecules at distinct sites, these reagents are brought in close proximity. The probes consist of a protein specific binding part coupled to an oligonucleotide with either a free 3’- or 5’-end capable of hybridizing to a common connector oligonucleotide. When the probes are in proximity, promoted by target binding, then the DNA strands can be joined by enzymatic ligation. The nucleic acid sequence that is formed can then be amplified and quantitatively detected in a real-time monitored polymerase chain reaction. This convenient assay is simple to perform and allows highly sensitive protein detection. Parallel analysis of multiple proteins by DNA microarray technology is anticipated for proximity ligation and enabled by the information carrying ability of nucleic acids to define the individual proteins. Assays detecting cytokines using SELEX aptamers or antibodies, monoclonal and polyclonal, are presented in the thesis.</p><p>Microarrays synthesized <i>in situ</i> using photolithographic methods generate impure products due to damaged molecules and interrupted synthesis. Through a molecular inversion mechanism presented here, these impurities may be removed. At the end of synthesis, full-length oligonucleotides receive a functional group that can then be made to react with the solid support forming an arched structure. The 3’-ends of the oligonucleotides are then cleaved, removing the impurities from the support and allowing the liberated 3’-hydroxyl to prime polymerase extension reactions from the inverted oligonucleotides. The effect of having pure oligonucleotides probes compared to ones contaminated with shorter variants was investigated in allele specific hybridization reactions. Pure probes were shown to have greater ability to discriminate between matched and singly mismatched targets at optimal hybridization temperatures.</p>

A novel technology for protein detection, proximity ligation, has been developed along with improved methods for in situ synthesis of DNA microarrays. Proximity ligation enables a specific and quantitative transformation of proteins present in a sample into nucleic acid sequences. As pairs of so-called proximity probes bind the individual target protein molecules at distinct sites, these reagents are brought in close proximity. The probes consist of a protein specific binding part coupled to an oligonucleotide with either a free 3’- or 5’-end capable of hybridizing to a common connector oligonucleotide. When the probes are in proximity, promoted by target binding, then the DNA strands can be joined by enzymatic ligation. The nucleic acid sequence that is formed can then be amplified and quantitatively detected in a real-time monitored polymerase chain reaction. This convenient assay is simple to perform and allows highly sensitive protein detection. Parallel analysis of multiple proteins by DNA microarray technology is anticipated for proximity ligation and enabled by the information carrying ability of nucleic acids to define the individual proteins. Assays detecting cytokines using SELEX aptamers or antibodies, monoclonal and polyclonal, are presented in the thesis. Microarrays synthesized in situ using photolithographic methods generate impure products due to damaged molecules and interrupted synthesis. Through a molecular inversion mechanism presented here, these impurities may be removed. At the end of synthesis, full-length oligonucleotides receive a functional group that can then be made to react with the solid support forming an arched structure. The 3’-ends of the oligonucleotides are then cleaved, removing the impurities from the support and allowing the liberated 3’-hydroxyl to prime polymerase extension reactions from the inverted oligonucleotides. The effect of having pure oligonucleotides probes compared to ones contaminated with shorter variants was investigated in allele specific hybridization reactions. Pure probes were shown to have greater ability to discriminate between matched and singly mismatched targets at optimal hybridization temperatures.

Motivation: As the human genome project progresses and some microbial and eukaryotic genomes are recognized, numerous biotechnological processes have attracted increasing number of biologists, bioengineers and computer scientists recently. Biotechnological processes profoundly involve production and analysis of highthroughput experimental data. Numerous sequence libraries of DNA and protein structures of a large number of micro-organisms and a variety of other databases related to biology and chemistry are available. For example, microarray technology, a novel biotechnology, promises to monitor the whole genome at once, so that researchers can study the whole genome on the global level and have a better picture of the expressions among millions of genes simultaneously. Today, it is widely used in many fields- disease diagnosis, gene classification, gene regulatory network, and drug discovery. For example, designing organism specific microarray and analysis of experimental data require combining heterogeneous computational tools that usually differ in the data format; such as, GeneMark for ORF extraction, Promide for DNA probe selection, Chip for probe placement on microarray chip, BLAST to compare sequences, MEGA for phylogenetic analysis, and ClustalX for multiple alignments. Solution: Surprisingly enough, despite huge research efforts invested in DNA array applications, very few works are devoted to computer-aided optimization of DNA array design and manufacturing. Current design practices are dominated by ad-hoc heuristics incorporated in proprietary tools with unknown suboptimality. This will soon become a bottleneck for the new generation of high-density arrays, such as the ones currently being designed at Perlegen [109]. The goal of the already accomplished research was to develop highly scalable tools, with predictable runtime and quality, for cost-effective, computer-aided design and manufacturing of DNA probe arrays. We illustrate the utility of our approach by taking a concrete example of combining the design tools of microarray technology for Harpes B virus DNA data.

Le rat (Rattus spp.) est une source de nombreux pathogènes zoonotiques responsables de morbidité et de mortalité dans le monde. Ces espèces sont particulièrement problématiques en santé publique car leur mode de vie synanthrope favorise la proximité rat-Homme et la transmission potentielle de pathogènes. Selon l'approche « une seule santé », la surveillance sanitaire des rats et d'autres espèces animales sensibles devrait contribuer à améliorer la santé de ces dernières et de l'Homme. Notre objectif était de développer la surveillance des zoonoses associées aux rats chez une espèce source (R. norvegicus) et chez des espèces cibles (bovins, chiens et porcs) en tant que sentinelles de l'exposition de l'Homme. L'intérêt de méthodes de détection dont la micro-puce à ADN développée dans le cadre du projet européen « WildTech » et l'investigation de la distribution du risque étaient les thèmes majeurs de ces travaux. Ils ont été documentés à partir de 181 rats capturés dans le Rhône entre 2010 et 2013 et, de données diagnostiques de leptospiroses animales enregistrées au Laboratoire des Leptospires – Lyon entre 2008 et 2012. Les méthodes de détection directes et indirectes utilisées à des fins de surveillance ont montré leur intérêt par la mise en évidence de quatre pathogènes potentiellement zoonotiques chez les rats (Hantavirus Séoul, virus de l'hépatite E, Leptospira spp. et Toxoplasma gondii). Malgré la spatialisation hétérogène des statuts infectieux, Leptospira spp. et l'hantavirus Séoul étaient les dangers prédominants avec respectivement, 26%, CI95%=20%-33% et 14%, CI95%=8%-20% de rats infectés par ces agents. Leur distribution spatiale a été caractérisée par des indices socio-économiques et, dans le cas des infections par les leptospires, une étude approfondie des souches circulantes a montré que leur persistance relevait de facteurs locaux, intrinsèques à la colonie. L'étude des leptospiroses animales (chiens et bovins) suggère leur exposition accrue au sérogroupe Australis, leur distribution spatiale hétérogène et une croissance significative de l'incidence annuelle canine. Ces trois observations également rapportées chez l'Homme soulignent l'intérêt de la surveillance de ces espèces en tant que sentinelles. Les informations obtenues par l'ensemble des méthodes appliquées contribuent à une meilleure compréhension de l'épidémiologie des zoonoses associées aux rats et de la leptospirose en particulier, afin d'orienter la mise en œuvre de leur surveillance et les décisions de santé publique à venir. / Rats (Rattus spp.) are a source of a number of zoonotic pathogens responsible for morbidity and mortality worldwide. These species are particularly problematic with regards to rat associated health risks because rats are living in close contact with people leading to potentially rat disease transmission. Based on the "One Health" approach, surveillance of zoonotic pathogens in rats and other susceptible hosts should help to improve animal and human health. Our aim was to develop the surveillance of rat-associated zoonoses in a source species (Rattus norvegicus) and, in some target populations (cattle, dogs and pigs) as sentinels of human exposure. The screening methods including DNA microarray developed for the purpose of the "WildTech" project and the spatial distribution of the risk were the major themes in this work. They have been documented based on 181 rats captured in the administrative unit “département du Rhône” between 2010 and 2013 and, diagnostic data of leptospirosis in cattle, dogs and pigs, recorded at "Laboratoire des Leptospires – Lyon" between 2008 and 2012. The application of various screening methods (direct and indirect) for the purpose of surveillance were relevant and detected four potentially zoonotic pathogens circulating in rats, (hantavirus Seoul, hepatitis E virus, Leptospira spp. and Toxoplasma gondii). Although the location of infected rats varied among a short geographic distance, Leptospira spp. and hantavirus Seoul were the predominant hazard with respectively 26%, IC95% = 20% -33% and 14%, IC95% = 8% -20% of infected rats. Their spatial distribution could be characterized with socio-economic indices and, regarding Leptospira-infected rats, a further study shown that the maintenance of strains was related to local and intrinsic factors. The study of leptospirosis in dogs and cattle revealed their increased exposure to the serogroup Australis, their heterogeneous spatial distribution and the significant increase of annual incidence in dogs. The same trends were observed in humans which underlines the relevance of surveillance of animal leptospirosis as sentinels of human exposure. All together, the information obtained contributes to a better understanding of the epidemiology of rat-associated zoonoses to support implementation of surveillance and public health decisions in the future.

L'objectif de cette thèse est d'explorer l'inflammation et l'infection au niveau du transcriptome, à l'aide de la technologie des puces à ADN. Pour cela, nous avons dans un premier temps travaillé sur des données publiques. Nous avons construit une base de données de signatures transcriptionnelles annotées, et développé un logiciel modulaire d'analyse. Ce logiciel permet d'explorer aisément les données publiques en effectuant des recherches par nom de gène ou par mots-clés. Nous avons ensuite exploré la modulation temporelle de l'expression des gènes du parenchyme pulmonaire dans un modèle murin d'inflammation aiguë par injection d'acide oléique. Dans un second modèle murin d'infection par Coxiella burnetii, nous avons analysé le rôle du sexe dans la modulation de la réponse transcriptionnelle hépatique, et identifié des voies métaboliques impliquées dans le contrôle de l'infection. Dans un troisième modèle in-vitro d'infection par différentes souches du virus de la grippe, nous avons identifié une signature transcriptionnelle commune de réponse à l'infection. Par une approche bio-informatique originale, cette signature a conduit à l'identification de nouveaux anti-viraux à large spectre, dont l'efficacité a été démontrée in-vitro sur les souches utilisées pour l'analyse, et sur la souche H1N1, responsable de la dernière pandémie grippale. Enfin, nous avons analysé les modulations du transcriptome lors de pneumonies associées à la ventilation mécanique compliquant l'évolution de sujets traumatisés graves admis en réanimation. / The goal of this PhD is to explore inflammation and infection at the transcriptome level, using DNA microarrays. In order to do so, we first analyzed public data. We built a database with annotated transcriptional signatures and developed a modular analysis software to query this database. This software allows to easily explore public data with requests based on gene names or annotation keywords. We then explored the temporal modulation of lung gene expression following oleic acid injection in a murine model. In a second murine model of infection with Coxiella burnetii, we analyzed the influence of sex-related modulation in the hepatic transcriptional response after infection and identified several pathways implicated in the control of infection. In a third model of in-vitro infection with various Influenza virus strains, we identified a shared transcriptional signature in response to cell infection. Using an original in-silico methodology, this signature allowed us to identify new broad-spectrum antivirals. Efficacy of these molecules was demonstrated in-vitro against the strains used to define the signature, and also against the new pandemic H1N1 SOIV strain. Finally, we analyzed the transcriptional modulation occurring in whole blood samples from trauma patients hospitalized in intensive care unit, and whose evolution was complicated with ventilator-associated pneumonia.

A análise da expressão gênica através de dados gerados em experimentos de microarrays de DNA vem possibilitando uma melhor compreensão da dinâmica e dos mecanismos envolvidos nos processos celulares ao nível molecular. O aprimoramento desta análise é crucial para o avanço do conhecimento sobre as bases moleculares das neoplasias e para a identificação de marcadores moleculares para uso em diagnóstico, desenho de novos medicamentos em terapias anti-tumorais. Este trabalho tem como objetivos o desenvolvimento de modelos de análise desses dados, propondo uma nova forma de normalização de dados provenientes de microarrays e dois modelos para a construção de redes regulatórias de expressão gênica, sendo uma baseada na conectividade dinâmica entre diversos genes ao longo do ciclo celular e a outra que resolve o problema da dimensionalidade, em que o número de experimentos de microarrays é menor que o número de genes. Apresenta-se, ainda, um pacote de ferramentas com uma interface gráfica de fácil uso contendo diversas técnicas de análise de dados já conhecidas como também as abordagens propostas neste trabalho. / The analyses of DNA microarrays gene expression data are allowing a better comprehension of the dynamics and mechanisms involved in cellular processes at the molecular level. In the cancer field, the improvement of gene expression interpretation is crucial to better understand the molecular basis of the neoplasias and to identify molecular markers to be used in diagnosis and in the design of new anti-tumoral drugs. The main goals of this work were to develop a new method to normalize DNA microarray data and two models to construct gene expression regulatory networks. One method analyses the dynamic connectivity between genes through the cell cycle and the other solves the dimensionality problem in regulatory networks, meaning that the number of experiments is lower than the number of genes. We also developed a toolbox with a user-friendly interface, displaying several established statistical methods implemented to analyze gene expression data as well as the new approaches presented in this work.

Devido à grande diversidade de microrganismos desconhecidos no meio ambiente, 99% deles não podem ser cultivados nos meios de cultura tradicionais dos laboratórios. Para isso, projetos metagenômicos são propostos para estudar comunidades microbianas presentes no meio ambiente, a partir de técnicas moleculares, em especial o seqüenciamento. Dessa forma, para os próximos anos é esperado um acúmulo de seqüências produzidas por esses projetos. As seqüências produzidas pelos projetos genomas e metagenomas apresentam vários desafios para o tratamento, armazenamento e análise, como exemplo: a busca de clones contendo genes de interesse. Este trabalho apresenta uma abordagem algébrica que define e gerencia de forma dinâmica as regras para a seleção de clones em bibliotecas genômicas e metagenômicas, que se baseiam em álgebra de processos. Além disso, uma interface web foi desenvolvida para permitir que os pesquisadores criem e executem facilmente suas próprias regras de seleção de clones em bancos de dados de seqüências genômicas e metagenômicas. Este software foi testado em bibliotecas genômicas e metagenômicas e foi capaz de selecionar clones contendo genes de interesse. / Due to the wide diversity of unknown organisms in the environment, 99% of them cannot be grown in traditional culture medium in laboratories. Therefore, metagenomics projects are proposed to study microbial communities present in the environment, from molecular techniques, especially the sequencing. Thereby, for the coming years it is expected an accumulation of sequences produced by these projects. Thus, the sequences produced by genomics and metagenomics projects present several challenges for the treatment, storing and analysis such as: the search for clones containing genes of interest. This work presents an algebraic approach that defines it dynamically and manages the rules of the selection of clones in genomic and metagenomic libraries, which are based on process algebra. Furthermore, a web interface was developed to allow researchers to easily create and execute their own rules to select clones in genomic and metagenomic sequence database. This software was tested in genomics and metagenomics libraries and it was able to select clones containing genes of interest.

This work concerns with data analysis of DNA microarrays by using cluster analysis. It explains biological terms - gene expression and DNA microarray. Next, it contains mathematical and informatical description of clustering methods and describes a way to apply these methods to microarrays data. Next, the work contains implementation's detail of clustering methods k-means, DBSCAN and introduces an original clustering algorithm Strom++. Then, description of implementation and application manual follow. Finally, accomplished results are evaluated.

Le corps humain est un ensemble de microflores où cohabitent bactéries, archées, virus et eucaryotes. Ces écosystèmes complexes sont appelés microbiotes. Parmi ceux-ci figure le microbiote intestinal qui compte 1011 à 1014 bactéries/g de selle. Les modifications de la flore intestinale peuvent être à l'origine de pathologies comme les diarrhées infectieuses. Il s'agit d'un véritable problème de santé publique puisqu'environ 2.16 millions de décès sont liés à cette pathologie chaque année. Les virus intestinaux jouent un rôle prépondérant mais les infections bactériennes restent également importantes. Le diagnostic de ces infections bactériennes reste compliqué puisque le microbiote intestinal comporte 75% d'espèces non cultivables. De plus, on ne dispose pas réellement d'une liste exhaustive des bactéries pouvant être responsables de diarrhées infectieuses. Nous avons donc choisi d'étudier le microbiote intestinal dans des selles normales et pathologiques, par une approche polyphasique alliant une étape préliminaire de concentration des selles diarrhéiques par la lyophilisation, à des techniques de culture et des méthodes de biologie moléculaire. Pour cela nous avons mis au point une nouvelle technologie pour la détection des entéropathogènes par hybridation sur puce à ADN permettant la détection des bactéries et des virus ADN entéropathogènes, en présence d'un témoin archae. Notre outil permet le diagnostic multiplexe des diarrhées infectieuses puisque nous avons correctement identifié un adénovirus et la bactérie Campylobacter jejuni présents dans une même selle. / The human body is a collection of microflora where cohabit bacteria, archaea, viruses and eukaryotes. These complex ecosystems are called microbiota. Among these is the intestinal microbiota that counts 1011 to 1014 bacteria/g of stool. Changes in the intestinal flora can cause of pathologies such as infectious diarrhea. This is a real public health problem since about 2.16 million deaths are related to this disease each year. Enteric viruses play a preponderant role but bacterial infections are also important. The diagnosis of bacterial infections is complicated because the intestinal microbiota includes 75% non-cultivable species. In addition, there is not really a list of bacteria could be responsible for infectious diarrhea. We therefore decided to study the intestinal microbiota in normal stool and also pathological stools by a polyphasic approach combining a preliminary step of diarrheal stools concentration by lyophilization, with cultivation techniques and molecular biology methods. We developed a new technology for the detection of enteropathogens by hybridization on DNA microarray for the detection of bacteria and enteric viruses (DNA) in the presence of a control archaea. Our tool allows multiplexed diagnostic of infectious diarrhea since we correctly identified an adenovirus and Campylobacter jejuni present in a same sample. This is the first DNA microarray for multiplex detection of bacteria and viruses (DNA) enteropathogens. An improvement of our protocol for nucleic acid extraction is proposed to allow the detection of RNA viruses such as rotavirus and calicivirus which are currently dominant.