The successful crowdsourcing model and gamification design of Stack Overflow (SO) Q&A platform have attracted many programmers to ask and answer technical questions, regardless of their level of expertise. Researchers have recently found evidence of security vulnerable code snippets being possibly copied from SO to production software. This inspired us to study how reliable is SO in providing secure coding suggestions. In this project, we automatically extracted answer posts related to Java security APIs from the entire SO site. Then based on the known misuses of these APIs, we manually labeled each extracted code snippets as secure or insecure. In total, we extracted 953 groups of code snippets in terms of their similarity detected by clone detection tools, which corresponds to 785 secure answer posts and 644 insecure answer posts. Compared with secure answers, counter-intuitively, insecure answers has higher view counts (36,508 vs. 18,713), higher score (14 vs. 5), more duplicates (3.8 vs. 3.0) on average. We also found that 34% of answers provided by the so-called trusted users who have administrative privileges are insecure. Our finding reveals that there are comparable numbers of secure and insecure answers. Users cannot rely on community feedback to differentiate secure answers from insecure answers either. Therefore, solutions need to be developed beyond the current mechanism of SO or on the utilization of SO in security-sensitive software development. / Master of Science / Stack Overflow (SO), the most popular question and answer platform for programmers today, has accumulated and continues accumulating tremendous question and answer posts since its launch a decade ago. Contributed by numerous users all over the world, these posts are a type of crowdsourced knowledge. In the past few years, they have been the main reference source for software developers. Studies have shown that code snippets in answer posts are copied into production software. This is a dangerous sign because the code snippets contributed by SO users are not guaranteed to be secure implementations of critical functions, such as transferring sensitive information on the internet. In this project, we conducted a comprehensive study on answer posts related to Java security APIs. By labeling code snippets as secure or insecure, contrasting their distributions over associated attributes such as post score and user reputation, we found that there are a significant number of insecure answers (644 insecure vs 785 secure in our study) on Stack Overflow. Our statistical analysis also revealed the infeasibility of differentiating between secure and insecure posts leveraging the current community feedback system (eg. voting) of Stack Overflow.

Les clones de visage 3D peuvent être utilisés dans de nombreux domaines tels que l'interaction homme-machine et comme prétraitement dans des applications telles que l'analyse de l'émotion. Toutefois, ces clones doivent avoir la forme du visage bien modélisée tout en conservant les spécificités des individus et ils doivent être sémantiques. Un clone est sémantique quand on connaît la position des différentes parties du visage (yeux, nez ...). Dans notre technique, nous utilisons un capteur RVB-Z pour obtenir les spécificités des individus et un modèle déformable de visage 3D pour marquer la forme du visage. Pour la reconstruction de la forme, nous inversons le processus utilisé classiquement. En effet, nous réalisons d'abord le fitting puis la fusion de données. Pour chaque trame de profondeur, nous gardons les parties appropriées de données appelées patchs de forme. Selon le positionnement de ces patchs, nous fusionnons les données du capteur ou les données du modèle déformable de visage 3D. Pour la reconstruction de la texture, nous utilisons des patchs de forme et de texture pour préserver les caractéristiques de la personne. Ils sont détectés à partir des cartes de profondeur du capteur. Les tests que nous avons effectués montrent la robustesse et la précision de notre méthode. / 3D face clones can be used in many areas such as Human-Computer Interaction and as pretreatment in applications such as emotion analysis. However, such clones should have well-modeled facial shape while keeping the specificities of individuals and they should be semantic. A clone is semantic when we know the position of the different parts of the face (eyes, nose...). In our technique, we use a RGB-D sensor to get the specificities of individuals and 3D Morphable Face Model to mark facial shape. For the reconstruction of the shape, we reverse the process classically used. Indeed, we first perform fitting and then data fusion. For each depth frame, we keep the suitable parts of data called patches. Depending on the location, we merge either sensor data or 3D Morphable Face Model data. For the reconstruction of the texture, we use shape and texture patches to preserve the person's characteristics. They are detected using the depth frames of a RGB-D sensor. The tests we perform show the robustness and the accuracy of our method.

This report discusses clones with nullary operations and the corresponding relational clones, both defined on arbitrary non-empty sets. The relationship between such clones and clones in the usual sense, i.e. without nullary operations, is investigated, and in particular the latter type of clones is located in the lattice of all clones. By means of two pairs of kernel and closure operators, a framework is developed that allows to transfer statements about usual clones to statements about clones with nullary constants. In this respect, familiar operators and constructions from clone theory, like the operators Pol and Inv, the closure operators belonging to the clone lattices, and the different variants of local closure operators on sets of relations and operations, respectively, are translated from the usual setting to the more general one and vice versa. The applicability of the presented machinery is demonstrated using the example of the theorem characterising Galois closed sets w.r.t. Pol-Inv as local closures of clones and relational clones, respectively.

C. elegans est infecté par divers agents pathogènes ; bactéries, champignons et virus. Lors d'une infection fongique, C. elegans surexprime de nombreux gènes codant pour des peptides antimicrobiens (AMP). Le principal objectif de ma thèse était de construire un réseau de régulation génique intégré représentant l'induction de ces gènes AMP pendant l'infection. Pour trouver les principales composantes du réseau de régulation, via un criblage ARNi du génome entier (Zugasti et al 2016), nous avons identifié 278 clones Nipi (pour "Absence d'induction de peptides antimicrobiens après infection") qui abrogent l’induction d’AMP. En utilisant "CloneMapper" (Thakur et al. 2014), nous avons identifié 338 gènes cibles pour ces clones. Nous avons montré que les voies de MAPK sont au cœur de l'induction des AMP. Parmi les 50 gènes arbitrairement sélectionnés et surexprimant les AMP, nous en avons validé 48 en utilisant Fluidigm. Pour attribuer des fonctions aux gènes identifiés dans ces études à haut débit, nous avons développé un outil d'enrichissement fonctionnel pour la communauté C.elegans (MS en préparation). Nous avons utilisé cet outil pour analyser les cibles du criblage ARNi sur le génome entier et sur d'autres bases de données concernant divers agents pathogènes. Nous avons fait une analyse de l'enrichissement fonctionnel des cibles ChIPseq de CEBP-1, un facteur de transcription lié à la régulation de la réponse immunitaire innée (Kim et al, Soumis). Enfin, pour mieux comprendre l'interaction entre l'hôte et le pathogène, nous avons séquencé, assemblé, annoté et analysé le génome de D. coniospora. Nous avons identifié plusieurs facteurs de virulence potentiels dans ce génome. / C. elegans is infected by diverse pathogens, including bacteria, fungi and viruses. Upon fungal infection, C. elegans up-regulates the expression of many antimicrobial peptide (AMP) genes. The main aim of my thesis was to build an integrated gene regulatory network representing the induction of these AMP genes upon infection. To find the main/backbone components of the regulatory network, through a genome-wide RNAi screen (Zugasti et al. 2016), we identified 278 Nipi (for “no induction of antimicrobial peptides after infection”) clones that abrogate AMP induction. Using “CloneMapper” (Thakur et al. 2014), we identified 338 target genes for these clones. We showed that MAPK pathways are central to the induction of AMPs. We also characterized the transcriptional changes provoked by infection using RNA-sequencing and identified more than 300 genes that are dynamically up-regulated after infection, including 13 AMPs. We validated 48 (96%) of 50 arbitrary selected up-regulated genes using Fluidigm. To assign functions to genes identified in these high-throughput studies, we developed a functional enrichment tool for C.elegans community (MS in preparation). We used this tool to analyse the genome-wide RNAi screen targets and other pathogen-related datasets. We did functional enrichment analysis of ChIPseq targets of CEBP-1, TF linked to the regulation of the innate immune response (Kim et al., submitted). Finally, to understand better the interaction between host and pathogen, we sequenced, assembled, annotated and analysed the D. coniospora genome (Lebrigand et al. 2016). We identified various potential virulence factors in the fungal genome.

Les clones de visage 3D peuvent être utilisés dans de nombreux domaines tels que l'interaction homme-machine et comme prétraitement dans des applications telles que l'analyse de l'émotion. Toutefois, ces clones doivent avoir la forme du visage bien modélisée tout en conservant les spécificités des individus et ils doivent être sémantiques. Un clone est sémantique quand on connaît la position des différentes parties du visage (yeux, nez ...). Dans notre technique, nous utilisons un capteur RVB-Z pour obtenir les spécificités des individus et un modèle déformable de visage 3D pour marquer la forme du visage. Pour la reconstruction de la forme, nous inversons le processus utilisé classiquement. En effet, nous réalisons d'abord le fitting puis la fusion de données. Pour chaque trame de profondeur, nous gardons les parties appropriées de données appelées patchs de forme. Selon le positionnement de ces patchs, nous fusionnons les données du capteur ou les données du modèle déformable de visage 3D. Pour la reconstruction de la texture, nous utilisons des patchs de forme et de texture pour préserver les caractéristiques de la personne. Ils sont détectés à partir des cartes de profondeur du capteur. Les tests que nous avons effectués montrent la robustesse et la précision de notre méthode. / 3D face clones can be used in many areas such as Human-Computer Interaction and as pretreatment in applications such as emotion analysis. However, such clones should have well-modeled facial shape while keeping the specificities of individuals and they should be semantic. A clone is semantic when we know the position of the different parts of the face (eyes, nose...). In our technique, we use a RGB-D sensor to get the specificities of individuals and 3D Morphable Face Model to mark facial shape. For the reconstruction of the shape, we reverse the process classically used. Indeed, we first perform fitting and then data fusion. For each depth frame, we keep the suitable parts of data called patches. Depending on the location, we merge either sensor data or 3D Morphable Face Model data. For the reconstruction of the texture, we use shape and texture patches to preserve the person's characteristics. They are detected using the depth frames of a RGB-D sensor. The tests we perform show the robustness and the accuracy of our method.

L'identification des visages est primordiale lors de nos interactions sociales. Ainsi, notre comportement change suite à l'identification de la personne avec laquelle nous interagissons. De plus, les travaux en psychologie et en neurosciences ont observé que le traitement cognitif face à un visage familier diffère de celui que nous avons face à un visage inconnu.D'une autre part, les dernières techniques de rendu 3D et les dernières avancées des scans 3D ont permis la création de visages virtuels photo-réalistes modélisant des personnes réelles existantes. La tendance actuelle pour modéliser des humains virtuels est de se baser sur des techniques d'acquisition de données réelles (issues de scans et de sessions de capture de mouvement). Par conséquent, les recherches et applications en humains virtuels ont connu un intérêt croissant pour ces clones virtuels (des agents ayant un aspect familier ou du moins reconnaissable). Les clones virtuels sont donc de plus en plus répandus dans des interfaces homme-machine et dans l'industrie audio-visuelle.L'étude de la perception et de l'interaction avec des clones virtuels est donc devenue nécessaire pour appréhender la conception et l'évaluation de cette technologie. En effet, très peu d'études se sont penchées sur l'évaluation de la fidélité de ces clones virtuels. L'objectif de cette thèse consiste à explorer cet axe de recherche en examinant le processus de perception de la fidélité d'un visage virtuel, clone d'une personne réelle (que l'on connait ou non).Nos travaux répondent à plusieurs questions de recherche: Quels sont les éléments qui nous permettent d'évaluer la ressemblance du clone virtuel avec son référent? Parmi les multiples possibilités de techniques de rendu, d'animation et d'acquisition de données qu'offre l'informatique graphique, quelle est la meilleure combinaison pour assurer le plus haut degré de fidélité perçue ? L'apparence visuelle n'est cependant qu'une des composantes qui interviennent dans la reconnaissance de personnes familières. Les autres composantes comprennent ainsi l'expressivité mais aussi le traitement des connaissances que nous avons sur cette personne (par exemple sa manière particulière d'évaluer une situation émotionnelle et de l'exprimer via son visage).Nos contributions apportent des éléments de réponse à ces questions à plusieurs niveaux. Nous avons défini un cadre conceptuel identifiant les principaux concepts pertinents pour l'étude de la fidélité d'un visage virtuel. Nous avons aussi étudié l'aspect visuel de la fidélité à travers l'exploration de différentes techniques de rendu. Nous avons étudié dans une autre étape l'impact de la familiarité dans le jugement de la fidélité. Finalement, nous avons proposé un modèle informatique individuel basé sur une approche cognitive des émotions qui permet de guider l'animation expressive du clone virtuel.Ces travaux de thèse ouvrent des perspectives pour la conception et l'amélioration de clones virtuels, mais aussi plus généralement des interfaces homme-machine basées sur des agents expressifs. / Face identification plays a crucial role in our daily social interactions. Indeed, our behavior changes according to the identification of the person with whom we interact. Moreover, several studies in Psychology and Neurosciences have observed that our cognitive processing of familiar faces is different from the cognitive processing of unfamiliar faces.Creating photorealistic an animated human-like face of a real person is now possible thanks to recent advances in Computer Graphics and 3D scan systems. Recent rendering techniques are challenging our ability to distinguish between computer generated faces and real human faces. Besides, the current trend to model virtual humans is to involve real data collected using scans and motion capture systems. Research and applications in virtual humans have experienced a growing interest in so-called virtual clones (agents with a familiar aspect or at least recognizable). Virtual clones are therefore increasingly used in human-machine interfaces and in the audiovisual industry. Studies about the perception and interaction with virtual clones are therefore required to better understand how we should design and evaluate this kind of technology. Indeed, very few studies have tried to evaluate virtual clones' fidelity with respect to the original human (hereafter called “the referent”). The main goal of this thesis is to explore this line of research. Our work rises several research questions: What are the features of the virtual clone that enable us to evaluate the resemblance between a virtual clone and its referent? Among several possibilities of rendering, animation and data acquisition techniques offered by Computer Graphics, what is the best combination of techniques to ensure the highest level of perceived fidelity?However, visual appearance is not the only component that is involved in recognizing familiar people. The other components include facial expressiveness but also the possible knowledge that we have about the referent (e.g. his particular way of assessing an emotional situation and expressing it through his face).Our contributions provide answers to these questions at several levels. We define a conceptual framework identifying the key concepts which are relevant for the study of the fidelity of a virtual face. We explore different rendering techniques. We describe an experimental study about the impact of familiarity in the judgment of fidelity. Finally, we propose a preliminary individual computational model based on a cognitive approach of emotions that could drive the animation of the virtual clone.This work opens avenues for the design and improvement of virtual clones, and more generally for the human-machine interfaces based on expressive virtual agents.

Le mycobiome digestif comprend l’ensemble des espèces de champignons contenu dans le tube digestif. Au cours de cette thèse, nous avons réalisé dans un premier temps une revue de la littérature sur le mycobiome digestif afin d’établir le répertoire des espèces de champignons décrites dans le tube digestif et leur implication dans les infections digestives et systémiques. Puis dans un second temps, notre travail expérimental a combiné l’approche culture et l’approche moléculaire basée sur le séquençage des clones pour explorer la diversité du mycobiome digestif. Nous avons répertorié une variété d’amorces PCR dans la littérature ciblant le gène 18S rRNA et ITS rRNA. En appliquant ces outils moléculaires et la culture à l’analyse d’une selle chez un sujet obèse nous avons détecté 16 espèces de champignons dont 11 espèces sont associées à l’alimentation avec 8 espèces de champignons observées pour la première fois dans les selles.Ensuite, la même approche appliquée à une selle collectée chez un sujet anorexique a permis de détecter 8 espèces de champignons dont 5 espèces associées au régime alimentaire du sujet et 3 espèces retrouvées pour la première fois dans les selles humaines.Dans un dernier temps en utilisant la même méthodologie, nous nous sommes intéressés à la diversité du mycobiome en fonction de l’origine géographique des sujets. Pour cela, nous avons exploré la communauté du mycobiome dans les selles provenant des quatre continents Afrique, Asie, Amérique et l’Europe. Dans ce travail, nous avons détecté 40 espèces de champignons provenant de l’environnement dont certains pathogènes opportunistes. / The human gut mycobiome, comprising of all fungal species detected in the gut. The Human Microbiome Project and the Metagenomics of the Human intestinal tract projects has led to new interest in the study of the human gut mycobiome. Recently, culture-independent approaches including PCR-based molecular clone libraries sequencing and high-throughput sequencing allowed to explore the diversity of gut mycobiota. In this thesis, firstly, we reviewed fungal species described in the human gut and their implication in systemic infections. We reported from literature fungal species described in healthy individuals compared to repertoire described in diseased individuals.Secondly, in our experimental work we used molecular and culture approaches to explore gut mycobiota diversity related to host physiology. We selected various set of PCR primers from literature targeting 18S rRNA gene and ITS rRNA gene. Combining molecular and culture tools in stool specimen from an obese individual we detected 16 fungal species, 11 were linked to food and 8 species were found for first in the human stools. Using the same approaches in an anorexic individual stool we identified 8 fungal species, five were associated to subjected diet collected and three fungal species were observed for the first time in the human stools. From these two studies, we observed that the gut mycobiome diversity is part associated to diet.Using the same methodology, we to explored gut mycobiota diversity according to different geographical locations. For this, fungal diversity was screened in stools samples from four continents Africa, America, Asia and Europe.

O presente estudo analisou a influência de clones do híbrido de Eucalyptus grandis x Eucalyptus urophylla, com densidades básicas e cargas de álcali ativo diferentes, no processo de polpação e branqueamento. As características e propriedades das polpas não-branqueadas, licor preto residual, branqueamento, morfologia e propriedades físico-mecânicas das polpas branqueadas foram analisadas para avaliar os efeitos dos fatores clone e carga de álcali ativo. As polpações foram realizadas aplicando-se cargas de álcali ativo de 12,5%, 13,5% e 14,5%. Em seguida, as polpas não-branqueadas foram submetidas a uma pré-deslignificação com oxigênio e branqueadas através da seqüência D0EOPD1. As polpas branqueadas foram refinadas em moinho PFI e analisadas quanto às resistências físico-mecânicas. Os resultados obtidos indicam que a madeira do clone G-31 apresentou menor densidade básica e teor de holocelulose, e maiores teores de extrativos totais e lignina Klason comparativamente à madeira do clone C-41. Os clones mostraram rendimentos bruto e depurado semelhantes. O clone G-31 produziu polpa com menor número kappa na menor carga alcalina e polpa com maior número kappa na maior carga alcalina revelando uma importante interação significativa clone x álcali ativo. Os fatores álcali ativo, clone e interação clone x álcali ativo tiveram influências nos parâmetros avaliados na polpação. Os licores residuais dos clones apresentaram valores semelhantes de pH, teor de sólidos solúveis, densidade e álcalis residuais. O clone G-31 consumiu mais álcali ativo residual. O fator que exerceu maior influência nestes parâmetros foi a carga de álcali ativo. O branqueamento mostrou para o clone G-31 maior eficiência na prédeslignificação com oxigênio e menor número kappa. A viscosidade intrínseca da celulose branqueada e a alvura foram mais elevadas para o clone C-41. O aumento da carga alcalina reduziu ligeiramente a alvura do clone G-31 e elevou a alvura do clone C-41. Os fatores álcali ativo, clone e interação clone x álcali ativo tiveram influências nos parâmetros avaliados no branqueamento. A fibra da polpa branqueada do clone G-31 apresentou, significativamente, maiores comprimento, espessura da parede, índice de enfeltramento, fração parede e índice de Runkel; e menores diâmetro do lume, coeficiente de flexibilidade e "coarseness". O menor valor de "coarseness" mostrou fibras mais leves na polpa do clone G-31. O fator clone exerceu a maior influência nas propriedades físicas e mecânicas da polpa branqueada. A polpa branqueada do clone G-31 apresentou, significativamente, menor número de revoluções no moinho PFI para atingir o índice de tração de 70 N.m/g, baixo grau Schopper Riegler para atingir este nível de tração e gerou folhas com valores mais elevados de volume específico e opacidade. Estas características e propriedades permitiram concluir que a polpa do clone G-31 foi a mais atrativa e indicada para a fabricação de papéis de imprimir e escrever. A polpa branqueada do clone C-41 apresentou valores mais elevados de volume específico aparente e ascensão capilar Klemm quando analisadas sem refino. Estas propriedades foram mais evidentes nas cargas alcalinas mais elevadas. A polpa do clone C-41 mostrou características mais favoráveis para a fabricação de papéis para fins sanitários. / The present study has analyzed the influences of Eucalyptus grandis x Eucalyptus urophylla hybrid clones with different specific gravity and active alkali charges in the bleaching and pulping process. The characteristics and properties of unbleached pulp, residual black liquor, bleaching, bleached fiber morphology and bleached pulp physical-mechanical properties were analyzed to evaluate the effects of the active alkali charge and clone factors. The pulping were realized applying active alkali charges of 12,5%, 13,5% and 14,5%. Then, the unbleached pulps were submitted to an oxygen delignification and bleached with the sequence D0EOPD1. The bleached pulps were refined using PFI mill and the physical-mechanical properties were analyzed. The results showed that G-31 wood clone presented lower specific gravity and holocellulose content and higher total extractives and Klason lignin contents when compared to C-41 wood clone. Both of them showed similar pulp yield total and screening. The G-31 clone produced pulp with lower kappa number in the lowest alkaline charge and pulp with higher kappa number in the highest alkaline charge showing an important significant interaction between clone and active alkali. The factors active alkali, clone and clone x active alkali interaction influenced in the evaluated parameters in the pulping. The clones residual black liquor showed similar values of pH, soluble solids content, density and residual alkalis. The G-31 clone consumed more residual active alkali. The factor that exerted the highest influence in these parameters was the active alkali charge. The bleaching showed that the G-31 clone has higher efficiency in the oxygen delignification and lower kappa number. The bleached pulp viscosity and brightness were higher for C-41 clone. The increase in the alkaline charge reduced slightly the G-31 clone brightness and elevated the C-41 clone brightness. The factors active alkali, clone and clone x active alkali interaction influenced in the bleaching parameters evaluated. The fiber of the G-31 bleached pulp showed, significantly, higher length, wall thickness, felting index, wall ratio and Runkel index, and lower lumen diameter, flexibility coefficient and coarseness. The lowest value of coarseness showed lighter fibers in the G-31 pulp. The factor clone exerted the highest influence in the physical and mechanical properties of the bleached pulp. The G-31 bleached pulp showed, significantly, lowest revolutions number in the PFI mill to reach tensile index of 70 N.m/g, low Schopper Riegler degree to reach this tensile index and generated sheets with higher values to bulk and opacity. These characteristics and properties allow to conclude that G-31 pulp was the most attractive and indicated to produce printing and writing papers. The bleached pulp of C-41 clone showed higher values of bulk and capillarity Klemm when analyzed without refining. These properties were more evident in the highest alkaline charges. The C-41 pulp showed characteristics more favorable to the production of tissue papers.

Este trabalho descreve um novo modelo murino para o estudo da infecção crônica pelo T. cruzi usando o clone Sylvio X10/4. A infecção crônica de camundongos C3H/HePAS é caracterizada por intensas lesões inflamatórias no coração que podem ser comparáveis às observadas na doença de Chagas humana. Lesões moderadas também estavam presente na musculatura estriada esquelética desses animais. No coração dos animais crônicos foram detectados parasitas viáveis, confirmando a hipótese de que o desenvolvimento da patologia cardíaca na doença de Chagas está diretamente relacionada com a persistência do T. cruzi no tecido inflamado. Em contraste, camundongos A/J cronicamente infectados desenvolvem lesões no fígado e no músculo estriado, enquanto que no coração, não foram observados lesões nem parasitas. A análise fenotípica das gerações F1 e F2 (A/J X C3H/HePAS) sugere que a predisposição genética para desenvolver lesões teciduais na infecção pelo T. cruzi é heterogênea uma vez que a patologia no coração e no fígado é segregada na geração F2. Nossos resultados corroboram a hipótese que a heterogeneidade na patologia observada em pacientes com a doença de Chagas (ausência ou presença de lesões cardíacas ou digestivas) pode ser atribuída a fatores genéticos. / This work describes a novel murine model of the chronic infection by T. cruzi using the clone Sylvio X10/4. The infection in the C3H/HePAS mouse strain is characterized by intense inflammatory lesions in the heart, which can be comparable to the observed in the human Chagas\' disease. Moderate striated muscle lesions are also present in C3H/HePAS mice. In the heart of the chronic animals viable parasites were detected, confirming the hypothesis that the development of the heart pathology in the Chagas\' disease is related to parasite persistence in the inflamed tissue. By contrast, in infected A/J mice, develop lesions in the liver and skeletal muscle, while in the heart, lesions nor parasites were not observed. The phenotypic analysis of the generations F1 and F2 (A/J X C3H/HePAS) mice suggests that the genetic predisposition to develop the inflammatory lesions caused by T. cruzi is heterogeneous because the heart and liver pathology segregate in the F2 generation. These findings raise the hypothesis that the pathology heterogeneity observed in humans with Chagas\' disease (absence and presence of cardiac or digestive chronic lesions) can be attributed to host genetic factors.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Bovine viral diarrhea virus (BVDV) is a worldwide pathogen associated with important losses to livestock production. Most of these losses come from reproductive disorders and from the ability of the virus to produce persistent infections following in utero infection of the fetus. A number of reverse genetics methodologies have been used for BVDV in order to better understand the biology of the virus, which allowed the elucidation of a number of biological features including virus replication, host-virus interaction, immune response, and the pathogenesis of fetal infection. The present study describes the construction, characterization and manipulation of an infectious clone out of a non-cytophatic Brazilian BVDV strain IBSP4-ncp. The cDNA recombinant clone was constructed by yeast homologous recombination with a low-copy vector, from three genomic fragments comprising the open reading frame (ORF). The two untranslated regions (5' and 3' UTR) were replaced by the respective UTRs of the reference strain NADL. The constructed vector was transcribed in vitro and the resulting RNA was transfected on MDBK cells to rescue infectious virus. The rescued viruses (IC-pBSC_IBSP4-ncp#2 and #3) were maintained for ten passages in tissue culture and characterized in vitro, showing replication dynamics, focus size and morphology similar to those of the parental IBSP-4. Genomic analysis revealed five point mutations in the gene coding for Npro protein, resulting in amino acid changes. These mutations probably reflect an adaptation of the virus to the heterologous UTRs. The infectious clone IC-pBSC_IBSP4-ncp#2 was further used for the construction of a recombinant virus expressing the Gaussia luciferase (Gluc) reporter gene. The reporter gene was inserted between the Npro and Core genes, being flanked by an upstream linker and a downstream sequence of the Foot and Mouth Disease virus protease (FMDV2Apro) for accurate protein processing. The recombinant vector was in vitro transcribed and the RNA was transfected on MDBK cells. Recombinant infectious viruses were rescued (IC-pBSC_IBSP4-ncpGluc#3 and #4) and characterized in vitro, showing replication dynamics, focus size and morphology similar to those of the parental IBSP-4 clone. The Gluc reporter gene was accurately expressed and processed by the recombinant virus during 15 passages in tissue culture. These studies revealed that the infectious clone constructed herein can be easily manipulated and is able to carry in its genome heterologous genes up to 555 base pairs in length in a stable fashion and without interference with its replication efficiency. Thus, the constructed clone may be very useful for genetic manipulation towards studying different aspects of the BVDV biology and its interactions with the host, and for the development of vaccine strains with attenuated phenotype and/or with antigenic markers. / O vírus da diarreia viral bovina (BVDV) é um patógeno de bovinos distribuído mundialmente, associado com importantes perdas econômicas. As maiores perdas devem-se aos problemas reprodutivos causados pela infecção, e pela capacidade do vírus de causar persistência após infecção fetal no terço inicial da gestação. Para entender melhor a biologia desse vírus, sistemas de genética reversa foram desenvolvidos e tem permitido a elucidação de vários aspectos da replicação viral, interação vírus hospedeiro, resposta imune e patogenia da infecção fetal. O presente estudo relata a construção, caracterização e manipulação de um clone infeccioso, a partir da cepa brasileira não-citopática IBSP4-ncp. O clone de DNA recombinante foi construído pela técnica de recombinação homóloga em levedura, utilizando um vetor de baixo número de cópias, construído a partir de três fragmentos genômicos, que compreendiam a fase aberta de leitura (open reading frame, ORF) do vírus. As duas regiões não traduzidas (5 e 3 UTR) foram substituídas pelas respectivas UTRs da cepa de referência NADL. O vetor construído foi transcrito in vitro e o RNA obtido foi transfectado em células MDBK para recuperação de vírus infecciosos. Os vírus recuperados (CI-pBSC_IBSP4-ncp#2 e #3) foram mantidos por 10 passagens em cultivo celular e caracterizados in vitro, apresentando dinâmica de replicação, tamanho e morfologia de focos similares ao vírus parental IBSP-4. A análise do genoma por sequenciamento revelou cinco mutações pontuais no gene Npro, com trocas de aminoácidos, provavelmente refletindo uma adaptação do vírus às UTRs heterólogas. O clone infeccioso construído CIpBSC_ IBSP4-ncp#2, foi então utilizado para a construção de um vírus recombinante expressando o gene repórter Gaussia luciferase (Gluc). O gene repórter foi inserido entre os genes Npro e Core do vírus. Para o processamento da proteína repórter, uma sequência ligante foi adicionada anteriormente ao gene, e a sequência da protease do vírus da Febre Aftosa (FMDV2Apro) foi inserida após o gene. O vetor recombinante construído foi transcrito in vitro e o RNA obtido foi transfectado em células MDBK. Vírus recombinantes infecciosos foram recuperados (CI-pBSC_IBSP4-ncpGluc#3 e #4) e caracterizados in vitro, apresentando dinâmica de replicação, tamanho e morfologia de focos similares ao vírus obtido do clone infecioso. O gene repórter Gluc foi corretamente expresso e processado pelo vírus recombinante durante 15 passagens em cultivo celular. Com os resultados obtidos nestes estudos, conclui-se que o clone infeccioso construído pode ser facilmente manipulado e é capaz de carrear em seu genoma, e expressar de forma estável, genes heterólogos com até 555 pares de base, que parecem não interferir com sua capacidade replicativa. Dessa forma, o clone obtido pode ser muito útil para manipulação genética visando estudar diferentes aspectos da biologia do BVDV e de suas interações com o hospedeiro, assim como para a produção de cepas vacinais com fenótipo atenuado e/ou com marcadores antigênicos.