Decay kinetics of bacteria in biological wastewater treatment systems are vital to efficient design and operation of treatment plants.  Of special concern are decay characteristics of fecal indicator organisms, which can aid design of wastewater treatment processes to eliminate fecal pathogens.  This study focuses on characterizing the decay of three strains of the key fecal indicator bacterium, Escherichia coli, and comparing microbial techniques for quantifying decay rates.  Traditional metrics for monitoring decay include volatile solids and plate counts, which may not provide the full picture of specific decay dynamics. In particular, the viable-but-not-culturable growth phase is challenging to assay.  Recently, more specific molecular techniques have been developed, such as DNA and RNA extraction with qPCR and RT-qPCR, ATP assays and live/dead cell-staining.  However, these assays have not been widely accepted or bench-marked against traditional techniques.  It is expected that molecular assays will generate kinetic constants that better reflect the viability and activity of the bacteria as they decay, generating a more integrated understanding of the decay process.  Cells grown in pure culture were spiked into microreactors and subjected to a variety of time/temperature treatments in both buffered pure culture and sludge matrices.  These scenarios were intended to mimic on a small scale typical waste treatment processes, more specifically pasteurization, thermophilic digestion and land application.  Results indicate decay curves similar to traditional curves but with constants that vary with respect to the strains and the characterization methods used.  The foundation laid by this work can be utilized in further studies involving other organisms in a variety of environmental scenarios. / Master of Science

La Directive Cadre Européenne sur l'eau impose d'évaluer la qualité des cours d'eau au moyen d'indicateurs chimiques et biologiques dont les diatomées font partie. Les indices basés sur la composition taxonomique et l'abondance relative des taxa de diatomées sont robustes. Cependant, de nombreux échantillons doivent être analysés chaque année alors que l'identification de ces micro-algues en microscopie optique est difficile à cause des incertitudes taxonomiques, et nécessite temps et expertise. Ainsi, des améliorations peuvent encore être apportées pour faciliter le suivi en routine de la qualité de l'eau. Les techniques de biologie moléculaire sont des outils efficaces pour identifier les microorganismes et pourraient donc être utilisées pour améliorer l'identification des diatomées. Les objectifs de cette thèse étaient donc de compléter les connaissances sur la taxonomie des diatomées d'eau douce par des méthodes moléculaires et de progresser dans le développement d'un outil moléculaire permettant l'identification des diatomées dans des échantillons naturels, en vue de son utilisation en bioindication. L'étude de la taxonomie de plusieurs groupes de diatomées a été réalisée en combinant des approches morphologiques et des approches moléculaires. Nos travaux ont montré les capacités des séquences ADN pour discriminer les taxa de diatomées et révéler leurs relations phylogénétiques. L'utilisation de séquences ADN a montré que les critères morphologiques utilisés pour identifier les diatomées ne correspondaient pas systématiquement à leurs relations phylogénétiques. L'utilisation de différents marqueurs a permis des discriminations à différents niveaux taxonomiques. Nos résultats ont également révélé l'importance de combiner des approches complémentaires, morphologiques et moléculaires, pour améliorer notre compréhension des relations entre les différents taxa de diatomées et ainsi stabiliser leur taxonomie. Les séquences ADN permettant une discrimination des taxa de diatomées, nous avons testé un outil moléculaire de séquençage haut-débit, le pyroséquençage 454, dans le but d'identifier les taxa composant les communautés de diatomées. Nous avons ainsi assemblé des bases de séquences de référence bénéficiant d'une identification taxonomique. Nous avons également participé au développement d'outils bioinformatiques nécessaires à l'analyse des données de pyroséquençage. Enfin, nous avons pu tester ces outils pour établir des inventaires taxonomiques de diatomées dans des communautés artificielles (mélanges de souches) et dans des communautés environnementales (biofilms d'eau douce). Ces études ont prouvé le potentiel du pyroséquençage 454 pour étudier les communautés de diatomées à des niveaux taxonomiques précis. La comparaison de différents marqueurs nucléiques a révélé que le marqueur rbcL était le marqueur le plus adapté à l'identification des diatomées par pyroséquençage. En effet, en prenant en compte les bases de séquences de référence, la reproductibilité et les biais de la méthode ainsi que le pouvoir résolutif du marqueur, l'utilisation du rbcL a permis la meilleure estimation de la composition en diatomées d'échantillons complexes. Des progrès devront encore être faits avant de pouvoir utiliser les outils moléculaires pour évaluer la qualité de l'eau par les diatomées. Cependant nos différentes études permettront de guider les prochaines analyses de manière à aboutir à un suivi de la qualité de l'eau basé sur des inventaires moléculaires des taxa de diatomées. / The European Water Framework Directive requires assessing the river quality using chemical and biological indicators among which are diatoms. Indices based on taxonomic composition and relative abundance of diatom taxa are robust. However, thousands of diatom samples are analyzed every year while microscopic identification is difficult due to taxonomic uncertainties, and requires time and expertise. Thus, it is still possible to improve the routine monitoring of water quality. The molecular biology techniques are accurate tools to identify microorganisms and could be used to enhance diatom identification. The objectives of this thesis were therefore to improve our knowledge on the freshwater diatom taxonomy by molecular methods and to progress in the development of a molecular tool in order to identify diatoms in natural samples, thus improving bioindication tools. The taxonomic study of several groups of diatoms was achieved by combining morphological and molecular approaches. Our results showed the capacity of DNA sequences to discriminate diatom taxa and revealed their phylogenetic relationships. The use of DNA sequences showed that the morphological criteria used to identify diatoms do not correspond systematically to their phylogenetic relationships. The use of different markers allowed discrimination at different taxonomic levels. Our results also revealed the importance of combining complementary approaches, morphological and molecular, to improve our understanding of the relationships between different diatom taxa and thus stabilize their taxonomy. As DNA sequences allowed discrimination of diatom taxa, we tested a molecular tool of high throughput sequencing, 454 pyrosequencing, in order to identify the diatom composition of communities. We assembled reference libraries of sequences linked to taxonomic identification and we contributed to the development of bioinformatic tools required to analyze data from pyrosequencing. Finally, we tested these tools to establish taxonomic inventories of diatoms in artificial communities (mixtures of strains) and environmental communities (freshwater biofilm samples). The studies showed the potential of 454 pyrosequencing to accurately analyze diatom communities. The comparison of different nucleic markers revealed that the rbcL marker was the most suitable to identify diatoms using pyrosequencing. Indeed, taking into account reference libraries, reproducibility and bias of the method, and the resolving power of marker, the use of rbcL allowed the best estimation of the diatom composition in complex samples. Improvements will be necessary to use molecular tools in order to assess water quality using diatoms. However our studies lead the way for future experiments in order to achieve a monitoring of water quality based on molecular inventories of diatom taxa.

Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-02-06T20:08:28Z No. of bitstreams: 2 Dissertação - Mariana Natalice de Siqueira - 2014..pdf: 2279133 bytes, checksum: 6e154dbf06fec9fd7fd142269ced0028 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-02-19T14:38:22Z (GMT) No. of bitstreams: 2 Dissertação - Mariana Natalice de Siqueira - 2014..pdf: 2279133 bytes, checksum: 6e154dbf06fec9fd7fd142269ced0028 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-19T14:38:22Z (GMT). No. of bitstreams: 2 Dissertação - Mariana Natalice de Siqueira - 2014..pdf: 2279133 bytes, checksum: 6e154dbf06fec9fd7fd142269ced0028 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-08-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Eugenia klotzschiana Berg is a species of Myrtaceae with restricted distribution in the Cerrado. Its fruits present nutritional potential and can be used in extractive way, as raw material for production of jams, jellies and juices. In this context, it is necessary to evaluate population-genetic aspects in order to provide information to assist in targeting strategies for management and conservation. To assess the genetic variability in a population context microsatellite markers have been used in recent years. The sequences flanking the microsatellite regions of the genome are highly conserved between related species, enabling the portability of these markers, reducing thereby the costs of developing the same for each species under study. In this context, the aim of this study was to test the potential for transferability to the genome of E. klotzschiana of genic microsatellite markers developed for Eucalyptus, as well as to characterize the genetic variability in their populations. For this purpose, DNA was extracted from leaf tissue of E. klotzschiana individuals and used to test the cross 120 pairs of amplification primers. Samples from seven locations were used to characterize the population genetic variability. The amplification products were analyzed on agarose and polyacrylamide capillary electrophoresis gel in different stages. Of total primers tested, 67 (55.83%) did not amplify, 42 (35%) had many nonspecific amplification products and 11 (9.2%) were successfully transferred. Of the 11 transferred eight showed polymorphism. For the eight polymorphic markers, the probability of identity was high (2.02 x10-3) and the power of paternity exclusion was moderated (0.74). In this population the average value of expected heterozygosity (He) was 0.28 and observed heterozygosity (Ho) was 0.44, as in subpopulations of He mean values ranged from 0.18 to 0.27 and the mean values Ho ranged from 0.36 to 0.53, respectively. No significant for the fixation index (f) were observed in the populations values indicating allele frequencies below the expected Hardy-Weinberg proportions.The extent of genetic divergence among subpopulations showed relatively high genetic differentiation, with a value of θ equal to 0.20, and peerto- peer values ranging between 0.00 and 0.34. The strategy of transferability of microsatellite markers was efficient to generate a panel of polymorphic microsatellite markers and learn a little of the genetic variability of the species. It was possible to verify that there is a low genetic variability within subpopulations for biomarkers. However, this work is a starting point for future studies to better understand the reproductive biology of the species Eugenia klotzschiana. / Eugenia klotzschiana Bergé uma espécie da família Myrtaceae com distribuição restrita no Cerrado. Seus frutos apresentam potencial alimentício e podem ser utilizados de forma extrativista,como matéria-primapara produção de doces, geleias e sucos. Neste contexto, torna-se necessário a avaliação de aspectos genético-populacionais a fim de disponibilizar informações que auxiliem no direcionamento de estratégias de manejo e conservação. Marcadores microssatélites têm sido utilizados nos últimos anospara avaliar a variabilidade genética em um contexto populacional. As sequências que flanqueiam as regiões microssatélites no genoma encontram-se altamente conservadas entre espécies relacionadas, o que possibilita a transferibilidade destes marcadores, reduzindo, desta forma, os custos de desenvolvimento dos mesmos para cada uma das espécies que se pretende estudar. Nesse contexto, o objetivo do presente estudo foi testar o potencial de transferibilidade para o genoma de Eugenia klotzschiana, de marcadores microssatélites gênicos desenvolvidos para Eucalyptus, assim como caracterizar a variabilidade genética existente em suas populações. Para tanto, o DNA foi extraído a partir de tecido foliar de indivíduos de E. klotzschianae utilizado para testar a amplificação cruzada de 120 pares de primers. Amostras oriundas de sete localidades foram utilizadas para a caracterização da variabilidade genética populacional. Os produtos de amplificação foram analisados em gel de agarose, poliacrilamida e emeletroforese capilar, em diferentes etapas. Do total deprimers testados, 67 (55,83%) não amplificaram, 42 (35%) apresentaram muitos produtos de amplificação inespecíficos e 11 (9,2%) foram considerados transferidos com sucesso. Dos 11 transferidos, oito apresentaram polimorfismo. Para os oito marcadores polimórficos, a probabilidade de identidade foi alta (2,02x10-3) e o poder de exclusão de paternidade foi moderado (0,74). Na população, o valor médio da heterozigosidade esperada (He) foi de 0,28 e a heterozigosidade observada (Ho) foi de 0,44, já nas subpopulações os valores médios de He variaram de 0,18 a 0,27 e os valores médios de Ho variaram de 0,36 a 0,53.Não foram observados valores significativos para o índice de fixação (f) nas subpopulações, indicando que as frequências alélicas seguem as proporções esperadas pelo equilíbrio de Hardy-Weinberg. A medida de divergência genéticaentre as subpopulações apresentou diferenciação genética relativamente alta,com valor de θigual a0,20, e com valores par-a-par variando entre 0,00 e 0,34. A estratégia de transferibilidade de marcadores microssatélites foi eficiente para gerar um painel de marcadores microssatélites polimórficos e conhecer um pouco da variabilidade genética da espécie. Foi possível verificar que existe uma baixa variabilidade genética dentro das subpopulações para os marcadores avaliados. Todavia, esse trabalho é um ponto de partida para futuros estudos que permitam conhecer melhor a biologia reprodutiva da espécie Eugenia klotzschiana.

L'occupation quasi constante des environnements intérieurs (en moyenne 90% du temps), expose en permanence les occupants à une large variété de microorganismes présents dans l'air de ces espaces. En raison de difficultés technologiques liées à la collecte et à l'analyse, ce domaine scientifique reste pourtant quasiment vierge et ce, malgré les retombées possibles dans le domaine de la santé. Le manque est particulièrement marqué, en ce qui concerne l'exposition des individus aux aérosols microbiens et plus globalement la gestion sanitaire de la qualité de l'air des espaces clos. Cette étude a pour objectif la caractérisation de la diversité microbienne de l'air de différents espaces clos collectifs par une approche qualitative et quantitative. L'ensemble de l'étude a porté sur des environnements sensibles d'un point de vue des occupants (Hôpital), de la densité de fréquentation (Le Musée du Louvre) ou d'un temps d'exposition prolongé (Bureau).L'originalité de cette thèse a résidé dans l'association d'une stratégie d'échantillonnage représentative des environnements étudiés et d'outils analytiques pertinents permettant d'étudier la microflore de l'air indépendamment de la cultivabilité des micro-organismes. Pour la première fois, un séquençage haut débit (pyroséquençage 454) a été appliqué à des échantillons d'air intérieur permettant d'accéder à une diversité microbienne rare comme le sont les espèces pathogènes. Les résultats montrent une diversité microbienne plus riche que celle habituellement observée par des méthodes culturales. Plusieurs micro-organismes impliqués dans des problématiques sanitaires ont été retrouvés (Borrelia spp., Burkholderia spp., Legionella spp., Neisseria spp. et Mycobacterium spp.). Les résultats mettent en évidence une stabilité à la fois spatiale et temporelle pour les bactéries retrouvées dans l'air intérieur. Cette stabilité est à la fois qualitative (structure des communautés microbiennes) et quantitative (abondance des microorganismes). La forte présence de séquences d'origine humaine permet de considérer l'Homme comme le principal élément orientant la microflore bactérienne de l'air intérieur. Des « cores species » signant l'air intérieur anthropisé ont pu être identifiées. / The constant occupation of indoor environments (average 90% of the time), constantly confront the occupants to a wide variety of microorganisms from the air of these spaces. Due to technological difficulties related to the collection and the analysis of airborne microorganisms, this field of study remains scanty, despite the potential health impact. The lack is particularly pronounced in terms of understanding of the risks of contamination of people by bioaerosols and overall health management of air quality of confined spaces.This study aims to characterize dynamics of the microbial diversity of different indoor environments. The entire study involved representative environments (hospital, office and museum).The originality of this thesis is the combination of a representative sampling strategy on environments studied and of analytical tools relevant to study the microflora of the indoor air regardless of the culturability of microorganisms.. For the first time, a high throughput sequencing (454 pyrosequencing) was applied to samples of indoor air in order to assess microbial diversity and pathogenic species..Several microorganisms implicated in health problems were found (Borrelia spp., Burkholderia spp. ,Legionella spp., Neisseria spp. and Mycobacterium spp.).The results give a different and more varied qualitative picture than that usually observed by cultural methods. The results show a stability of both spatial and temporal microflora of indoor air. This stability is both qualitative (microbial community structure) and quantitative (abundance of microorganisms). Man can be considered as the main factor driving the indoor air microflora due to the strong presence of sequences of human origin.'Cores species' signing the antropogenic indoor air were identified.

Polychlorinated biphenyls (PCBs) are toxic and persistent anthropogenic contaminants. Concern on their adverse health effects has led to their regulation in air, water and/or soil in addition to sludge. Hence, removal of PCBs in various matrices, including transformer oils (TO) is a priority. This study aims to investigate PCB-118 and Aroclor 1254 toxicity and dechlorination by varying certain critical experimental components including electron donor (sludge or fatty acids), inocula (unacclimated or acclimated culture) and the doses of PCB and TO under anaerobic conditions. Anaerobic toxicity assays (ATA) reactors, lab-scale anaerobic batch digesters and sediment microcosms were used for this purpose. Increase in PCB-118 and TO doses affected anaerobic digester performance by negatively influencing methanogenesis, while favoring dechlorination only with the increase in PCB-118 dose. Up to 22% PCB-118 removal was attained with unacclimated culture. Studies with acclimated cultures showed Grasse River (GR) sediment to be the most active when compared to Fox River and Baltimore Harbor sediments. In GR sediment microcosms, PCB-118 and Aroclor 1254 removal efficiencies decreased when TO was present (1%), while 10% TO inhibited PCB dechlorination. Waste activated sludge was shown to be an effective electron donor, similar to fatty acids. Aroclor 1254 dechlorination was dechlorinated through removal of flanked meta and para chlorines, however, dechlorination pathways appeared to differ according to the presence/absence of TO. No ortho or unflanked chlorines were removed. Molecular tools (qPCR and DHPLC) were used to confirm the presence of active PCB dechlorinators. Dechlorination of PCBs was shown to be growth-linked.

Parmi les espèces de levures du clade CTG, certaines sont responsables de candidoses tandis que d’autres présentent des potentiels en biotechnologie. Depuis quelques années, nous assistons à une intensification des recherches sur ces levures. Cependant, leur code génétique particulier a ralenti la mise au point d’outils génétiques pour la plupart d’entre elles. Cette thèse vise à développer des outils moléculaires standardisés pour un grand nombre d’espèces de levures du clade CTG. Nous avons d’abord conçu des vecteurs d’expression adaptés à l’espèce M. guilliermondii. Par la suite, nous avons caractérisé le gène de résistance à l’acide mycophénolique IMH3.2 afin de l’utiliser comme marqueur de sélection lors de la transgénèse d’espèces du clade CTG. Enfin, nous avons mis au point une série de vecteurs permettant la manipulation génétique de ces espèces. Ce travail a conduit à la conception d’une large gamme d’outils utilisable dans un grand nombre de ces levures, pré-requis essentiel aux futurs recherches en mycologie médicale et au développement de stratégies de biologie synthétique. / The fungal CTG clade includes well-known yeasts of clinical importance and/or biotechnological potential. Thus, albeit being intensively studied over the last 30 years, their uncommon genetic code precludes the use of the widely available markers and reporter systems for genetic approaches in these microorganisms. We provide here a toolbox to genetically manipulate a wide range of CTG clade species. Firstly, we developed a new series of versatile controllable expression vectors for M. guilliermondii. After, we characterized MPA-resistant gene IMH3.2 et used it as a drug resistance marker in several yeast species. Finaly, we provide a molecular toolbox suitable to genetically manipulate a broad range of prominent species from the CTG clade. This versatile toolkit represents a new starting point for successful developments of research in medical mycology in the CTG clade but also will expedite synthetic biology strategies in these microorganisms for biotechnological applications.

Os fitoplasmas são organismos procariotos sem parede celular, que habitam as células do floema das plantas e são transmitidos, principalmente, por cigarrinhas sugadoras de floema. Este patógeno é responsável por causar doenças em diversas espécies vegetais, provocando a expressão de diversos sintomas, entre os quais podem ser destacados a redução do tamanho foliar, amarelecimento das folhas, superbrotamento de ramos e enfezamento da planta hospedeira. Entre os anos de 2013 e 2014, plantas de três espécies vegetais com sintomas característicos de infecção causada por fitoplasmas foram observadas no estado de São Paulo. O DNA total foi extraído a partir de plantas de couve-flor com sintomas de nanismo, malformação da inflorescência e necrose dos vasos do floema; de plantas da ornamental conhecida por árvore-da-felicidade com sintomas amarelecimento e redução do limbo foliar; e de plantas de Alho-poró com sintomas de enfezamento. Por meio do teste de PCR conduzido em sua maioria com os primers P1A/16S-SR foi possível detectar fitoplasmas nas três espécies vegetais testadas. Os fragmentos genômicos correspondentes ao 16S rRNA dos fitoplasmas foram sequenciados e identificados. Nas plantas de couve-flor e da árvore-da-felicidade foram identificados fitoplasmas afilados ao grupo 16SrVII-B, através da análise virtual de RFLP, cálculo do coeficientes de similaridade (F) e análise filogenética. Para ambas as espécies, este é o primeiro relato da ocorrência de representantes deste grupo, nas condições brasileiras. Nas plantas de Alho-poró foram identificados fitoplasmas afiliados ao subgrupo 16SrIII-J e ao grupo 16SrXIII, com base na análise dos genes 16S rRNA, SecY e proteína ribossomal, conduzidas através de análise virtual de RFLP, valores de coeficientes de similaridade e análise filogenética. Para o fitoplasma membro do grupo 16SrXIII foram identificadas quatro estirpes, uma delas afiliada ao subgrupo 16SrXIII-E e as demais como prováveis representantes de novos subgrupos. O presente estudo se constitui em uma contribuição ao conhecimento de novos patossistemas envolvendo fitoplasmas, bem como à caracterização molecular de fitoplasmas baseadas em diferentes genes marcadores, atualmente usados para a classificação de representantes deste grupo emergente de agentes causais de doenças de plantas. / The phytoplasmas are prokaryotic organisms without cell walls, which inhabit the phloem cells of plants and are transmitted mainly by leafhoppers sucking the phloem. This pathogen is responsible for causing diseases in various plant species, leading to expression of many symptoms, among which can be highlighted the reduction of leaf size, leaf yellowing, shoots proliferation and stunting of the plant host. Between the years 2013 and 2014, three plant species with characteristic symptoms of infection caused by phytoplasma were observed in São Paulo. Total DNA was extracted from cauliflower plants with symptoms of stunting, malformation of the inflorescence and necrosis of the phloem; ornamental plants known for Ming Aralia with symptoms yellowing and little leaves; and leek plants with symptoms of stunting. Through the PCR test conducted mostly with the primers P1A/16S-SR was possible to detect phytoplasmas in the three species of plants. The genomic fragments corresponding of 16S rRNA gene of the phytoplasma were sequenced and identified. In plants of cauliflower and Ming Aralia were identified phytoplasmas belonging to 16SrVII-B group through virtual RFLP analysis, calculation of similarity coefficients (F) and phylogenetic analysis. For both species, this is the first report of the occurrence of representatives of this group, in Brazilian conditions. In leek plants were identified phytoplasmas affiliated with 16SrIII-J subgroup, and the 16SrXIII group, based on the analysis of the 16S rRNA, SecY and ribosomal protein genes, conducted through virtual analysis of RFLP, similarity coefficient values and phylogenetic analysis. For the phytoplasma member of group 16SrXIII were identified four strains, one affiliated with 16SrXIII-E subgroup and the others as probable representatives of new subgroups. This study constitutes a contribution to knowledge of new pathosystems involving phytoplasmas and the molecular characterization of phytoplasma based on different marker genes, currently used for the classification of representatives of this emerging group of plant pathogens.

Os fitoplasmas são organismos procariotos sem parede celular, que habitam as células do floema das plantas e são transmitidos, principalmente, por cigarrinhas sugadoras de floema. Este patógeno é responsável por causar doenças em diversas espécies vegetais, provocando a expressão de diversos sintomas, entre os quais podem ser destacados a redução do tamanho foliar, amarelecimento das folhas, superbrotamento de ramos e enfezamento da planta hospedeira. Entre os anos de 2013 e 2014, plantas de três espécies vegetais com sintomas característicos de infecção causada por fitoplasmas foram observadas no estado de São Paulo. O DNA total foi extraído a partir de plantas de couve-flor com sintomas de nanismo, malformação da inflorescência e necrose dos vasos do floema; de plantas da ornamental conhecida por árvore-da-felicidade com sintomas amarelecimento e redução do limbo foliar; e de plantas de Alho-poró com sintomas de enfezamento. Por meio do teste de PCR conduzido em sua maioria com os primers P1A/16S-SR foi possível detectar fitoplasmas nas três espécies vegetais testadas. Os fragmentos genômicos correspondentes ao 16S rRNA dos fitoplasmas foram sequenciados e identificados. Nas plantas de couve-flor e da árvore-da-felicidade foram identificados fitoplasmas afilados ao grupo 16SrVII-B, através da análise virtual de RFLP, cálculo do coeficientes de similaridade (F) e análise filogenética. Para ambas as espécies, este é o primeiro relato da ocorrência de representantes deste grupo, nas condições brasileiras. Nas plantas de Alho-poró foram identificados fitoplasmas afiliados ao subgrupo 16SrIII-J e ao grupo 16SrXIII, com base na análise dos genes 16S rRNA, SecY e proteína ribossomal, conduzidas através de análise virtual de RFLP, valores de coeficientes de similaridade e análise filogenética. Para o fitoplasma membro do grupo 16SrXIII foram identificadas quatro estirpes, uma delas afiliada ao subgrupo 16SrXIII-E e as demais como prováveis representantes de novos subgrupos. O presente estudo se constitui em uma contribuição ao conhecimento de novos patossistemas envolvendo fitoplasmas, bem como à caracterização molecular de fitoplasmas baseadas em diferentes genes marcadores, atualmente usados para a classificação de representantes deste grupo emergente de agentes causais de doenças de plantas. / The phytoplasmas are prokaryotic organisms without cell walls, which inhabit the phloem cells of plants and are transmitted mainly by leafhoppers sucking the phloem. This pathogen is responsible for causing diseases in various plant species, leading to expression of many symptoms, among which can be highlighted the reduction of leaf size, leaf yellowing, shoots proliferation and stunting of the plant host. Between the years 2013 and 2014, three plant species with characteristic symptoms of infection caused by phytoplasma were observed in São Paulo. Total DNA was extracted from cauliflower plants with symptoms of stunting, malformation of the inflorescence and necrosis of the phloem; ornamental plants known for Ming Aralia with symptoms yellowing and little leaves; and leek plants with symptoms of stunting. Through the PCR test conducted mostly with the primers P1A/16S-SR was possible to detect phytoplasmas in the three species of plants. The genomic fragments corresponding of 16S rRNA gene of the phytoplasma were sequenced and identified. In plants of cauliflower and Ming Aralia were identified phytoplasmas belonging to 16SrVII-B group through virtual RFLP analysis, calculation of similarity coefficients (F) and phylogenetic analysis. For both species, this is the first report of the occurrence of representatives of this group, in Brazilian conditions. In leek plants were identified phytoplasmas affiliated with 16SrIII-J subgroup, and the 16SrXIII group, based on the analysis of the 16S rRNA, SecY and ribosomal protein genes, conducted through virtual analysis of RFLP, similarity coefficient values and phylogenetic analysis. For the phytoplasma member of group 16SrXIII were identified four strains, one affiliated with 16SrXIII-E subgroup and the others as probable representatives of new subgroups. This study constitutes a contribution to knowledge of new pathosystems involving phytoplasmas and the molecular characterization of phytoplasma based on different marker genes, currently used for the classification of representatives of this emerging group of plant pathogens.

Les ARN double brin (ARNdb) sont les molécules clés initiatrices du RNA silencing, à partir desquelles les différentes classes de petits ARN (sRNAs), conférant la séquence spécificité de ce mécanisme, vont être produit. Chez la plante modèle Arabidopsis thaliana, le clivage des divers ARNdb en sRNAs est opéré par quatre enzymes de type RNase III, nommées DCL1 à DCL4, dont l’activité peut être assistée par des protéines fixant l’ARNdb (DRBs). Au cours de cette thèse, j’ai pu caractériser la fonction d’une nouvelle DRB, DRB7.2. Les résultats obtenus m’ont permis de démontrer que cette protéine régule la production d’une classe particulière de sRNAs endogènes, les endoIR-siRNAs, en séquestrant spécifiquement leurs précurseurs ARNdb, inhibant ainsi leur clivage par les différents DCLs. En parallèle, j’ai également développé des outils moléculaires afin d’étudier le mode d’action du DCL le plus polyvalent chez les plantes, DCL4. La caractérisation détaillée de ces outils a permis de révéler le rôle clé de déterminant structuraux distinct (protéiques ou nucléiques) impliqués dans la spécificité de reconnaissance et de clivage des divers substrats ARNdb par cette enzyme. / RNA silencing is initiated by double-stranded RNA (dsRNA) molecules that will be processed into various classes of small RNAs (sRNAs), which confer the sequence-specificity of this mechanism. In the model plant Arabidopsis thaliana, dsRNA processing is mediated by four distinct RNaseIII-like enzymes, named DCL1 to DCL4, which can be assisted by dsRNA-binding proteins (DRBs). During my PhD, I was able to characterize in details the function of a new DRB protein, DRB7.2. Our results revealed that this protein regulates the accumulation of a specific class of endogenous sRNAs, the endoIR-siRNAs, by selectively sequestering their dsRNA precursors and inhibiting their cleavage by the DCLs. In parallel, I also developed molecular tools to study the mode of action of the most versatile DCL in plants, DCL4. Detailed characterization of these tools revealed key roles of distinct structural determinants (at the protein or RNA level), implicated in the specificity and cleavage efficiency of the various dsRNA susbtrates by DCL4.

Avec la mondialisation et l'industrialisation de l'alimentation, la fraude et les cas de contamination des aliments peuvent avoir un impact international et entraîner des conséquences de grande envergure à la fois sur l'économie et la santé des consommateurs (Cubero-Leon et al., 2014). La fraude et l'authentification sont donc devenues des sujets émergents dans le secteur alimentaire. D'autant que les fraudes sont de plus en plus sophistiquées pour contourner au mieux les contrôles et donc de plus en plus difficiles à détecter par des analyses classiques. Les aliments issus de l'agriculture biologique (AB ou bio) font d'ailleurs partie des aliments qui risquent le plus de faire l'objet de fraude. Mais la traçabilité des aliments est principalement garantie par des moyens administratifs (règlement UE 178/2002). C'est pourquoi il est nécessaire de recourir à des techniques analytiques avancées pour détecter les produits non-conformes et pour garantir la traçabilité et l'authenticité des aliments, notamment ceux issus de l'AB. Notre étude est basée sur l'hypothèse que les traitements, associés à différents types d'agriculture, ont un impact mesurable sur la microflore des aliments. L'objectif principal était de pouvoir utiliser l'environnement microbien des aliments pour les discriminer en fonction de leur mode de production. La PCR-DGGE, un outil moléculaire d'écologie microbienne, pourrait servir à discriminer les modes de production d'aliments par analyse des profils génétiques des ADNr bactériens et fongiques. L'analyse des profils génétiques microbiens de nectarines, pêches, bananes et de pommes a montré qu'il était possible de différencier les fruits en fonction de leur mode de production. La robustesse de notre méthodologie a été démontrée en comparant les résultats obtenus sur deux années de récolte successives. Létude des variation intra-parcellaires ont également permis de démontrer que les fruits bio pouvaient être différenciés des conventionnels indépendamment de leur position dans la parcelle (centre vs bord) ou encore sur l'arbre. Les différences observées au niveau de la structure des communautés microbiennes étaient donc suffisamment importantes pour conclure que les traitements appliqués ont un impact significatif sur ces communautés. De plus, l'identification des espèces microbiennes obtenues après PCR-DGGE et NGS a révélé que certains groupes microbiens (fongiques et bactériens) pourraient être spécifiques aux aliments bio. Cependant, l'effet terroir est un critère important à prendre en compte dans la mise en place d'un outil d'authentification des aliments bio. Une application sur le terrain serait donc difficile à prévoir si elle est parcelle-spécifique. Cette étude s'inscrit à la base de la mise au point d'un outil analytique qui pourrait permettre de répondre aux besoins des professionnels de l'industrie alimentaire en termes d'authenticité et de sûreté alimentaire, en particulier pour aider les organismes certificateurs à contrôler et authentifier les aliments bio. Cette étude a également permis d'enrichir les connaissances actuelles sur l'écosystème microbien des fruits en fonction des pratiques agricoles. / Globalization of trades and industrialization of food have increased the occurrence of food fraud. Cases of food contamination now have a global impact and lead to far-reaching consequences both on the economy and the health of consumers (Cubero-Leon et al., 2014). Thus, fraud and authentication became important topics in the food sector. Especially as food frauds are becoming more sophisticated to bypass controls and are therefore more difficult to detect by classical approaches. Organic foods are part of foods that are the most likely to be subject of fraud. But traceability of foods is mainly performed by administrative means (UE Regulation 178/2002). That is why it is necessary to resort advanced analytical techniques to detect non-compliant products and to ensure traceability and authentication of foods, including those from organic agriculture. Our study is based on the hypothesis that treatments associated to various farming types have a measurable impact on food microflora. That is why, the main objective of this study was to use the microbial environment of foods to discriminate them according to their production mode. PCR-DGGE, a molecular tool of microbial ecology, could be used as to discriminate food production modes using bacterial and fungal rDNA profiles. The analysis of microbial genetic profiles of nectarines, peaches, bananas and apples showed that it was possible to differentiate fruits according to their farming types. It was possible to verify the robustness of our methodology by comparing results obtained on two successive harvest years. We estimated also the intra-plot variations and observed that organic apples could be discriminated from conventional ones independently upon their position in the field (centre or border) or even on the tree. The observed differences in microflora between organic and conventional apples were significant enough to conclude that the applied treatments have a significant impact on this microflore. Furthermore, the analysis of DNA sequences obtained from PCR-DGGE and NGS allowed some microbial groups (fungal and bacterial) to be identified as specific to organic foods. However, the “terroir effect” is an important criterion to take into account for the implementation of an authentication tool for organic products. So, an application in the field would be difficult to predict if it is plot-specific. This study constitutes the basis for the development of an analytical tool that could meet the needs of food industry professionals in terms of authenticity and food safety, especially to assist certifying bodies to control and authenticate organic food products. This study enabled also to enrich the existing knowledge on the microbial ecosystem of fruits from different agricultural practices.