O paradigma de sistemas de moléculas auto-replicantes é o modelo de quase-espécies, no qual as moléculas são representadas por seqüências binárias de tamanho L e o mecanismo de replicação é suposto imperfeito. Em particular, cada seqüência é gerada corretamente com probabilidade Q = qL, onde q é a probabilidade de cópia exata por dígito. Um dos resultados mais intrigantes no modelo para o relevo de replicação de pico único, no qual há apenas um tipo de molécula com vantagem seletiva a em relação aos outros tipos, é a observação de um limiar de erro a partir do qual toda informação biológica relevante é perdida. A transição de limiar de erro verificada para Qc = l /a pode ser visualizada como uma transição de fase do tipo ordem-desordem. Verificamos que a largura dessa transição decresce com L de acordo com L-1. Concluímos também que as grandezas físicas de interesse são bem descritas por meio de funções de escala. Elaboramos ainda uma versão estocástica (isto é, tamanho de população N finito) para o modelo de quase-espécies, no qual a dinâmica é descrita por uma cadeia de Markov. Mostramos que o tempo característico &#964 para o desaparecimento de seqüências mestras na população obedece uma relação de escala bem definida. A transição em nosso modelo é constatada através da divergência de &#964 em Qc no limite de N &#8594 &#8734 ,sendo que a largura da transição decresce de acordo com N -1/2. Em nossa abordagem não utilizamos nenhuma definição arbitrária para o limiar de erro para população finita. Como solução para o problema da crise de informação associada ao limiar de erro estudamos o modelo de hiperciclos. Neste modelo, as macromoléculas se replicam com o auxílio de outros membros do hiperciclo por meio do mecanismo de catálise. Estudamos analiticamente a propagação de erro no hiperciclo e obtemos os diagramas de fases no espaço de parâmetros para vários tamanhos de hiperciclo n. Esses diagramas descrevem as regiões de estabilidade das diversas soluções de estado estacionário do sistema. Constatamos que para hiperciclos com n &#8804 4 existe um limiar de erro menor que aquele verificado no modelo de quase-espécies. Desde que o suporte catalítico realizado por uma molécula no hiperciclo pode ser considerado de fato um comportamento altruísta, modelos para evolução do altruísmo como, a teoria de seleção de grupos, têm sido utilizados no contexto de evolução pré-biótica. Aqui investigamos a evolução da produção de enzimas e os efeitos de sinergia utilizando esses conceitos. / The quasi-species model is the paradigm of systems composed of self-replicating molecules, which are represented by sequences of fixed length L. The replication machinery is assumed to be imperfect. Particularly, each sequence is copied exactly with probability Q=qL, where q denotes the probability of exact copy per digit. One of the most intriguing results of the model for the single-peak replication landscape, which considers the existence of a master sequence that has a selective advantage a in comparison to the other types, is the occurrence of an error threshold phenomenon beyond the biological information is completely lost. The error threshold transition can be viewed as an order-disorder phase-transition. We investigated the sharpness of the threshold and found that its characteristics persist across a range of Q of order L-1 about Qc. Other physical quantities of interest are also well described by universal functions. We formulate a stochastic version (i.e., finite population size N) for the quasispecies model, in which a Markov chain defines the dynamics. We show that the characteristic time r for the disappearance of master sequences in the population obeys a well defined scaling relation. The transition in this model is signalized by the divergente of t at Qc in the limit N &#8594 &#8734, and the sharpness of the transition decreases like N -1/2. In our approach we do not use any arbitrary definition of the error threshold for finite population. As a solution for the information crisis associated to the error threshold, here we considered the hypercycle model, where the macromolecules self-replicate with the catalytic support of the other members of the hypercycle. We study analytically the error propagation in the hypercycle and obtain the phases diagrams in the space of parameters for several hypercycle sizes n. These diagrams describe the stability regions of the steady state solutions of the system. We find that for hypercicle size n &#8804 4 the error threshold is smaller than that for the quasispecies model. Since the catalytic support developed by a molecule is in fact an altruistic behavior, some models for the evolution of altruism, for instance, the selection group theory, have been investigated in the context of pre-biotic evolution. Specifically, here we analyze the evolution of enzyme production and the effects of synergism.

O paradigma de sistemas de moléculas auto-replicantes é o modelo de quase-espécies, no qual as moléculas são representadas por seqüências binárias de tamanho L e o mecanismo de replicação é suposto imperfeito. Em particular, cada seqüência é gerada corretamente com probabilidade Q = qL, onde q é a probabilidade de cópia exata por dígito. Um dos resultados mais intrigantes no modelo para o relevo de replicação de pico único, no qual há apenas um tipo de molécula com vantagem seletiva a em relação aos outros tipos, é a observação de um limiar de erro a partir do qual toda informação biológica relevante é perdida. A transição de limiar de erro verificada para Qc = l /a pode ser visualizada como uma transição de fase do tipo ordem-desordem. Verificamos que a largura dessa transição decresce com L de acordo com L-1. Concluímos também que as grandezas físicas de interesse são bem descritas por meio de funções de escala. Elaboramos ainda uma versão estocástica (isto é, tamanho de população N finito) para o modelo de quase-espécies, no qual a dinâmica é descrita por uma cadeia de Markov. Mostramos que o tempo característico &#964 para o desaparecimento de seqüências mestras na população obedece uma relação de escala bem definida. A transição em nosso modelo é constatada através da divergência de &#964 em Qc no limite de N &#8594 &#8734 ,sendo que a largura da transição decresce de acordo com N -1/2. Em nossa abordagem não utilizamos nenhuma definição arbitrária para o limiar de erro para população finita. Como solução para o problema da crise de informação associada ao limiar de erro estudamos o modelo de hiperciclos. Neste modelo, as macromoléculas se replicam com o auxílio de outros membros do hiperciclo por meio do mecanismo de catálise. Estudamos analiticamente a propagação de erro no hiperciclo e obtemos os diagramas de fases no espaço de parâmetros para vários tamanhos de hiperciclo n. Esses diagramas descrevem as regiões de estabilidade das diversas soluções de estado estacionário do sistema. Constatamos que para hiperciclos com n &#8804 4 existe um limiar de erro menor que aquele verificado no modelo de quase-espécies. Desde que o suporte catalítico realizado por uma molécula no hiperciclo pode ser considerado de fato um comportamento altruísta, modelos para evolução do altruísmo como, a teoria de seleção de grupos, têm sido utilizados no contexto de evolução pré-biótica. Aqui investigamos a evolução da produção de enzimas e os efeitos de sinergia utilizando esses conceitos. / The quasi-species model is the paradigm of systems composed of self-replicating molecules, which are represented by sequences of fixed length L. The replication machinery is assumed to be imperfect. Particularly, each sequence is copied exactly with probability Q=qL, where q denotes the probability of exact copy per digit. One of the most intriguing results of the model for the single-peak replication landscape, which considers the existence of a master sequence that has a selective advantage a in comparison to the other types, is the occurrence of an error threshold phenomenon beyond the biological information is completely lost. The error threshold transition can be viewed as an order-disorder phase-transition. We investigated the sharpness of the threshold and found that its characteristics persist across a range of Q of order L-1 about Qc. Other physical quantities of interest are also well described by universal functions. We formulate a stochastic version (i.e., finite population size N) for the quasispecies model, in which a Markov chain defines the dynamics. We show that the characteristic time r for the disappearance of master sequences in the population obeys a well defined scaling relation. The transition in this model is signalized by the divergente of t at Qc in the limit N &#8594 &#8734, and the sharpness of the transition decreases like N -1/2. In our approach we do not use any arbitrary definition of the error threshold for finite population. As a solution for the information crisis associated to the error threshold, here we considered the hypercycle model, where the macromolecules self-replicate with the catalytic support of the other members of the hypercycle. We study analytically the error propagation in the hypercycle and obtain the phases diagrams in the space of parameters for several hypercycle sizes n. These diagrams describe the stability regions of the steady state solutions of the system. We find that for hypercicle size n &#8804 4 the error threshold is smaller than that for the quasispecies model. Since the catalytic support developed by a molecule is in fact an altruistic behavior, some models for the evolution of altruism, for instance, the selection group theory, have been investigated in the context of pre-biotic evolution. Specifically, here we analyze the evolution of enzyme production and the effects of synergism.

Glutathione transferases (GSTs) are good candidates for investigations of enzyme evolution, due to their broad substrate specificities and structural homology. The primary role of GSTs is to act as phase II detoxifying enzymes protecting the cell from toxic compounds of both endo- and exogenous origins. The detoxification is conducted via conjugation with glutathione (GSH), which facilitates their removal from the body. The work presented in this thesis has supported a theory for enzyme evolution when the multiple pathway to novel functions can been seen to involve a “generalist” state from which “specialist” states with a new activities can evolve. The generalist has broader specificity and lower activity than the specialist. The term quasi-species is used for a group or cluster of enzyme variants with similar functional properties, and this entity has been suggested as the fittest group for further evolution. This is based on studies of the evolution of new GST variants in two generation. Three diverging clusters or quasi-species, with diverging substrate selectivity, were identified from a GST M1/M2 library, by using directed evolution (family DNA shuffling), multiple substrate screening and multivariate statistics as tools. One of the clusters was M1-like and the other was M2-like, both functionally and structurally. The third quasi-species diverged orthogonally from the parent-like distributions. Its functional character can be referred to as a “generalist” as it had lower activities with most of the substrates assayed except for epoxy-3-(4-nitrophenoxy)-propane (EPNP) and p-nitrophenyl acetate (pNPA). Another round of family DNA shuffling was made with selected variants from the “generalist” quasi-species. From the second generation three quasi-species emerged with diverging functions and sequences. The major cluster contained enzyme variants that represented a direct propagation of the generalists. Diverging from the generalists was a cluster with high specificity with isothiocyanates (ITCs). Increased ITC specificity and decreased epoxide specificity was observed among the novel variants (specialists). The change in functional properties was attributed to a Tyr116His substitution in the active site. These results demonstrate the usefulness of multivariate analysis in the quest for novel enzyme quasi-species in a multi-substrate space, and how minimal changes in the active site can generate distinctive functional properties. An application of our method could be identification of enzyme quasi-species that have lost their sensitivity with alternative inhibitors.

Les traitements actuels contre le virus de l’hépatite B (VHB) combinent un ou plusieurs analogues de nucléos(t)ides qui inhibent directement la réplication virale en bloquant l’étape de transcription inverse. Ces traitements très efficaces sont pourtant confrontés à l’émergence de virus résistants à ces traitements. Ces résistances sont la conséquence de l’émergence et la sélection de mutants parfois complexes présentant des mutations à la fois dans le gène de la polymérase (pol) et de l’enveloppe virale. Les objectifs principaux de ce doctorat ont été d’étudier la sensibilité des variants résistants du VHB vis-à-vis d’analogues de nucléos(t)ides et de nouveaux composés nonnucléos(t)idiques agissant contre la nucléocapside, mais également de comparer les performances virales de différents mutants afin de comprendre le processus de sélection des mutants qui s’opère chez le patient sous pression thérapeutique. Ces études ont caractérisé la sensibilité de certaines mutations de résistance aux analogues de nucléos(t)ides, de souligner l’importance des modifications de l’enveloppe dues aux mutations de résistance dans le processus d’émergence et de sélection des variants dans la quasi-espèce virale et d’identifier de nouvelles molécules antivirales efficaces permettant, en combinaison avec les analogues de nucléos(t)ide, de diminuer fortement les phénomènes de résistance du VHB. Mieux comprendre les phénomènes de résistance, les procédés d’émergence, de sélection et de transmission des mutants du VHB pour élaborer les meilleures stratégies cliniques de combinaisons thérapeutiques peut réduire considérablement le nombre de personnes touchées par ce virus / Current therapies against the hepatitis B virus (HBV) combine one or more nucleoside analogues that directly inhibit viral replication by blocking reverse transcription step. These treatments are very effective, however, faced with the emergence of viruses resistant to these treatments. These resistances are the result of the emergence and selection of mutants with mutations can be complex in both the polymerase gene (pol) and the viral envelope. The main objectives of this PhD was to study the sensitivity of resistant HBV variants vis-à-vis similar nucleos(t)ides and new compounds non-nucleos(t)idic acting against the nucleocapsid, but also compare the performance of different viral mutants to understand the process of selection of mutants that occurs in patients under therapeutic pressure. These studies have characterized the sensitivity of some resistance mutations to nucleoside analogues, to highlight the importance of the envelope changes due to resistance mutations in the process of emergence and selection of variants in the quasispecies virus and to identify new effective antiviral drugs may allow, in combination with nucleoside analogues, to greatly reduce the phenomenon of HBV resistance. Better understanding the phenomenon of resistance, the processes of emergence, selection and transmission of HBV mutants to develop the best clinical strategies of combination therapy can significantly reduce the number of people affected by this virus

Le virus de l'influenza aviaire hautement pathogène (IAHP) de sous-type H5N1 a causé de nombreuses pertes humaines, animales et économiques à travers le monde, notamment en Asie du Sud-Est. Son potentiel pandémique est une source d'inquiétude majeure en santé publique. Au Cambodge, l'infection est enzootique, et a causé la mort de 16 personnes depuis sa première détection en 2004 dans le pays, dont 8 pour la seule année 2011. Bien que l'hypothèse de la transmission directe hôte-hôte (animal-animal ou animal-homme) soit privilégiée, de récentes études semblent clairement incriminer certains éléments constitutifs de l'environnement dans le cycle de transmission du virus. Cependant, peu de données sont actuellement disponibles sur le sujet. Le travail de cette thèse a consisté en grande partie à apporter quelques réponses aux nombreuses questions soulevées. Des méthodes de détection du virus H5N1 dans l'environnement ont été mises au point, validées, et utilisées pour la détection de virus dans des prélèvements environnementaux collectés sur des sites d'épizooties au Cambodge. Le rôle de passereaux, capturés pour la réalisation de certains rituels bouddhistes en Asie, dans la dissémination du virus aux populations aviaires et humaines, a également été étudié. En parallèle, des données importantes du mode d'évolution du virus H5N1 au sein d'hôtes aviaires, jusqu'alors inexistantes, ont été apportées par l'étude des quasi-espèces du virus. L'ensemble des résultats rassemblés dans cette thèse souligne l'importance du rôle de l'environnement dans la dissémination et la transmission du virus IAHP H5N1. / The Highly Pathogenic Avian Influenza (HPAI) virus, subtype H5N1, has caused important human, animal and economical and losses in all countries affected, especially in Southeast Asia. Its pandemic potential is a major public health concern. In Cambodia, the infection is enzootic, and has caused 16 human fatalities since its first detection in the country in 2004, out of which 8 occurred in 2011. Although the hypothesis of direct host-to-host (animal-to-animal or animal-to-human) transmission is commonly accepted, recent studies clearly identified some environmental components as sources for avian and/or human contamination with H5N1 virus. Nonetheless, only few data are currently available on this topic. The work presented in this thesis aimed at better describing the role of the environment in the transmission cycle of the H5N1 virus. H5N1 virus detection methods in the environment were designed, validated and used for the detection of virus in environmental samples collected during epizootic outbreaks in Cambodia. The role of the Merit Release Birds, used during some common Buddhist rituals in Asia, in the dissemination of the virus to avian and human populations was also studied. In parallel, important and novel data regarding the evolution of the H5N1 virus within avian hosts were provided by quasi-species studies. The findings described in this thesis emphasize the relevance of the role of the environment in the dissemination and transmission of the HPAI H5N1 virus.

Le but de cette thèse fut d’étudier l’influence de l’environnement sur l’évolution des génomes de baculovirus. Nous avons d’abord caractérisé génétiquement la population naturelle d’AcMNPV par séquençage haut-débit et établi par des bioessais la sensibilité de 4 espèces hôtes au virus. Ensuite, une évolution expérimentale de 10 cycles fut mise en place sur les 4 espèces hôtes, à partir d’une population naturelle d’AcMNPV. Elle nous a permis de caractériser phénotypiquement et génotypiquement les lignées de 10ème génération. Cette expérience nous a montré des trade-off de virulence pour chaque lignée : pour augmenter leur virulence pour l’hôte sur lequel elles ont évolué, les lignées ont perdu en potentiel adaptatif généraliste. De plus, la diversité intra-populationnelle a diminué pour toutes les lignées en fonction de la sensibilité des hôtes. Enfin, en corrélant tous ces résultats nous avons mis en évidence des positions spécifiques du génome, impliquées dans l’adaptation à l’hôte. / The purpose of this thesis was to study the influence of the environment on the evolution of baculovirus genomes. We first genetically characterised the AcMNPV natural population by high-throughput sequencing and established the susceptibility of 4 hosts to the virus by bioassays. Then, the AcMNPV natural population was subjected to experimental evolution on the 4 host species for 10 cycles. The 10th generation of the evolved viral lines were then phenotypically and genotypically characterised. This experiment showed a virulence trade-off for each line: to increase their virulence to the host on which they evolved, the lines have lost generalist adaptive potential. Furthermore, intra-population diversity decreased for all the lines regardless of host susceptibility. Lastly, by correlating all these results we found specific genome positions involved in host adaptation.