In this study, two symbiotic fungi of Southern Pine Beetle (SPB), Entomocorticium peryii and Entomocorticium sp.A were evaluated in terms of polyphenol oxidase (PPO) production. The effect of different inhibitors, inducers and assay parameters such as temperature and pH on enzyme activity were investigated and maximum PPO activity was observed at 30&deg / C, pH 8.0 and when tannic acid was used as an inducer. Copper-chelator salicyl hydroxamic acid (SHAM) and pcoumaric acid, both indicated as inhibitors of tyrosinase and catechol oxidase significantly reduced the activity. For biochemical characterization studies, the enzyme was concentrated by ultrafiltration. To determine type of the enzyme, activity staining after Native-PAGE was carried out. Type of polyphenol oxidase produced by E. peryii and E. sp.A was determined as catechol oxidase by activity staining. However higher activity was observed on hydroquinone (p-diphenol) rather than catechol (o-diphenol). The enzyme obeys Michealis-Menten kinetics with Km and Vmaxvalues being 10.72 mM hydroquinone and 59.44 U/ml for E. peryii and 8.55 mM hydroquinone and 73.72 U/ml for E. sp.A respectively..

A large proportion of the world’s nitrogen needs is derived from symbiotic nitrogen fixation (SNF), which contributes substantially to agricultural sustainability. The partnership between legumes and rhizobia result in the formation of specialised structures called root nodules. Within these nodules SNF is supported by the sucrose transported from the leaves to the nodules for respiration. The end products of SNF in soybean (Glycine max (L.) Merr.) root nodules, namely ureides, are transported to the upper parts of the plant to supply nitrogen. Symbiotic nitrogen fixation provides a vital advantage for the production of soybean compared with most grain crops in that soybean fixes the nitrogen required for its growth and for the production of the high-protein content in seed and oil. The process of SNF is dramatically affected by drought, salt, cold and heavy metal stresses. Since SNF is such an important yield-determining factor, a lack in understanding these complexes inevitably delays progress towards the genetic improvement of soybean genotypes and also complicates decisions with regard to the suitability of certain genotypes for the various soybean producing areas in South Africa. The largest soybean producing areas in South Africa are situated at high altitudes, with minimum daily temperatures which can be critically low and impeding the production of soybean. Soybean is chilling sensitive, with growth, development and yield being affected negatively at temperatures below 15°C. Dark chilling (low night temperature) stress has proved to be one of the most important restraints to soybean production in South Africa. Among the symptoms documented in dark chilling sensitive soybean genotypes are reduced growth rates, loss of photosynthetic capacity and pigment content, as well as premature leaf senescence and severely inhibited SNF. Existing knowledge about stress-induced nodule senescence is based on fragmented information in the literature obtained in numerous, and often diverse, legume species. The precise nature and sequence of events participating in nodule senescence has not yet been fully explained. The main objectives of this investigation were to characterise the natural senescence process in soybean nodules under optimal growth conditions and to characterise the alteration of the key processes of SNF in a chilling sensitive soybean genotype during dark chilling. Moreover, to establish whether recovery in nodule functionality following a long term dark chilling period occured, or whether nodule senescence was triggered, and if sensitive biochemical markers of premature nodule senescence could be identified. A known chilling sensitive soybean genotype, PAN809, was grown under controlled growth conditions in a glasshouse. To determine the baseline and change over time for key parameters involved in SNF, a study was conducted under optimal growing conditions over a period of 6 weeks commencing 4 weeks after sowing. The cluster of crown nodules were monitored weekly and analysis included nitrogenase activity, ureide content, respiration rate, leghemoglobin content, sucrose synthase (SS) activity and sucrose content. Further investigations focused on induced dark chilling effects on nodule function to determine the alterations in key parameters of SNF. Plants were subjected to dark chilling (6˚C) for 12 consecutive nights and kept at normal day temperatures (26˚C). The induced dark chilling was either only shoot (SC) exposure or whole plant chilling (WPC). These treatments were selected since, in some areas in South Africa cold nights result not only in shoot chilling (SC) but also in low soil temperatures causing direct chilling of both roots and shoots. To determine if premature nodule senescence was triggered, the recovery following 12 consecutive nights of chilling treatment was monitored for another 4 weeks. It was established that the phase of optimum nitrogenase activity under optimal growing conditions occurred during 4 to 6 weeks after sowing where after a gradual decline commenced. This decline was associated with a decline in nitrogenase protein content and an increase in ureide content. The stability of SS activity and nodule respiration showed that carbon-dependent metabolic processes were stable for a longer period than previously mentioned parameters. The negative correlation that was observed between nitrogenase activity and nodule ureide content pointed towards the possible presence of a feedback inhibition trigger on nitrogenase activity. A direct effect of dark chilling on nitrogenase activity and nodule respiration rate led to a decline in nodule ureide content that occurred without any limitations on the carbon flux of the nodules (i.e. stable sucrose synthase activity and nodule sucrose content). The effect on SC plants was much less evident but did indicate that currently unknown shoot-derived factors could be involved in the minor inhibition of SNF. It was concluded that the repressed rates of respiration might have led to increased O2 concentrations in the nodule, thereby inhibiting the nitrogenase protein and so the production of ureides. It was found that long term chilling severely disrupted nitrogenase activity and ureide synthesis in nodules. Full recovery in all treatments occurred after 2 weeks of suspension of dark chilling, however, this only occurred when control nodules already commenced senescence. This points toward reversible activation of the nitrogenase protein with no evidence in support of premature nodule senescence. An increase in intercellular air space area was induced by long term dark chilling in nodules, specifically by the direct chilling of nodules (WPC treatment). The delayed diminishment of intercellular air space area back to control levels following dark chilling may be an important factor involved in the recovery of nitrogenase activity because enlarged air spaces would have favoured gaseous diffusion, and hence deactivation of nitrogenase, in an elevated O2 environment (due to supressed nodule respiration rates). These findings revealed that dark chilling did not close the diffusion barrier, as in the case of drought and other stress factors, but instead opened it due to an increase in air space areas in all regions of the nodule. In conclusion, this study established that dark chilling did not initiate premature nodule senescence and that SNF demonstrated resilience, with full recovery possible following even an extended dark chilling period involving low soil temperatures. / Thesis(PhD (Botany))--North-West University, Potchefstroom Campus, 2013

A large proportion of the world’s nitrogen needs is derived from symbiotic nitrogen fixation (SNF), which contributes substantially to agricultural sustainability. The partnership between legumes and rhizobia result in the formation of specialised structures called root nodules. Within these nodules SNF is supported by the sucrose transported from the leaves to the nodules for respiration. The end products of SNF in soybean (Glycine max (L.) Merr.) root nodules, namely ureides, are transported to the upper parts of the plant to supply nitrogen. Symbiotic nitrogen fixation provides a vital advantage for the production of soybean compared with most grain crops in that soybean fixes the nitrogen required for its growth and for the production of the high-protein content in seed and oil. The process of SNF is dramatically affected by drought, salt, cold and heavy metal stresses. Since SNF is such an important yield-determining factor, a lack in understanding these complexes inevitably delays progress towards the genetic improvement of soybean genotypes and also complicates decisions with regard to the suitability of certain genotypes for the various soybean producing areas in South Africa. The largest soybean producing areas in South Africa are situated at high altitudes, with minimum daily temperatures which can be critically low and impeding the production of soybean. Soybean is chilling sensitive, with growth, development and yield being affected negatively at temperatures below 15°C. Dark chilling (low night temperature) stress has proved to be one of the most important restraints to soybean production in South Africa. Among the symptoms documented in dark chilling sensitive soybean genotypes are reduced growth rates, loss of photosynthetic capacity and pigment content, as well as premature leaf senescence and severely inhibited SNF. Existing knowledge about stress-induced nodule senescence is based on fragmented information in the literature obtained in numerous, and often diverse, legume species. The precise nature and sequence of events participating in nodule senescence has not yet been fully explained. The main objectives of this investigation were to characterise the natural senescence process in soybean nodules under optimal growth conditions and to characterise the alteration of the key processes of SNF in a chilling sensitive soybean genotype during dark chilling. Moreover, to establish whether recovery in nodule functionality following a long term dark chilling period occured, or whether nodule senescence was triggered, and if sensitive biochemical markers of premature nodule senescence could be identified. A known chilling sensitive soybean genotype, PAN809, was grown under controlled growth conditions in a glasshouse. To determine the baseline and change over time for key parameters involved in SNF, a study was conducted under optimal growing conditions over a period of 6 weeks commencing 4 weeks after sowing. The cluster of crown nodules were monitored weekly and analysis included nitrogenase activity, ureide content, respiration rate, leghemoglobin content, sucrose synthase (SS) activity and sucrose content. Further investigations focused on induced dark chilling effects on nodule function to determine the alterations in key parameters of SNF. Plants were subjected to dark chilling (6˚C) for 12 consecutive nights and kept at normal day temperatures (26˚C). The induced dark chilling was either only shoot (SC) exposure or whole plant chilling (WPC). These treatments were selected since, in some areas in South Africa cold nights result not only in shoot chilling (SC) but also in low soil temperatures causing direct chilling of both roots and shoots. To determine if premature nodule senescence was triggered, the recovery following 12 consecutive nights of chilling treatment was monitored for another 4 weeks. It was established that the phase of optimum nitrogenase activity under optimal growing conditions occurred during 4 to 6 weeks after sowing where after a gradual decline commenced. This decline was associated with a decline in nitrogenase protein content and an increase in ureide content. The stability of SS activity and nodule respiration showed that carbon-dependent metabolic processes were stable for a longer period than previously mentioned parameters. The negative correlation that was observed between nitrogenase activity and nodule ureide content pointed towards the possible presence of a feedback inhibition trigger on nitrogenase activity. A direct effect of dark chilling on nitrogenase activity and nodule respiration rate led to a decline in nodule ureide content that occurred without any limitations on the carbon flux of the nodules (i.e. stable sucrose synthase activity and nodule sucrose content). The effect on SC plants was much less evident but did indicate that currently unknown shoot-derived factors could be involved in the minor inhibition of SNF. It was concluded that the repressed rates of respiration might have led to increased O2 concentrations in the nodule, thereby inhibiting the nitrogenase protein and so the production of ureides. It was found that long term chilling severely disrupted nitrogenase activity and ureide synthesis in nodules. Full recovery in all treatments occurred after 2 weeks of suspension of dark chilling, however, this only occurred when control nodules already commenced senescence. This points toward reversible activation of the nitrogenase protein with no evidence in support of premature nodule senescence. An increase in intercellular air space area was induced by long term dark chilling in nodules, specifically by the direct chilling of nodules (WPC treatment). The delayed diminishment of intercellular air space area back to control levels following dark chilling may be an important factor involved in the recovery of nitrogenase activity because enlarged air spaces would have favoured gaseous diffusion, and hence deactivation of nitrogenase, in an elevated O2 environment (due to supressed nodule respiration rates). These findings revealed that dark chilling did not close the diffusion barrier, as in the case of drought and other stress factors, but instead opened it due to an increase in air space areas in all regions of the nodule. In conclusion, this study established that dark chilling did not initiate premature nodule senescence and that SNF demonstrated resilience, with full recovery possible following even an extended dark chilling period involving low soil temperatures. / Thesis(PhD (Botany))--North-West University, Potchefstroom Campus, 2013

Na natureza, as orquídeas são totalmente dependentes de fungos micorrízicos para germinar. Estes fungos podem penetrar nas células das raízes e formar pélotons, os quais, quando digeridos pela planta, providenciam açúcares simples para o embrião. Durante a fase aclorofilada de desenvolvimento da plântula, orquídeas são obrigatoriamente dependentes dos fungos; algumas continuam assim por toda vida enquanto outras se tornam facultativamente responsivas à colonização. O objetivo deste trabalho foi identificar quantos clados de fungos podem estabelecer associação micorrízica com Coppensia doniana (sin. Oncidium donianum), uma orquídea amplamente distribuída nos arredores de Campos do Jordão e, demonstrar como as características morfológicas dos isolados, quando analisadas com ferramentas de estatística multivariada, podem ser úteis para a taxonomia destes fungos. Dez plantas foram amostradas em um sítio com vegetação típica de campos de altitude, junto ao Parque Estadual de Campos do Jordão. Fungos foram isolados pela transferência asséptica de cortes de raízes contendo pélotons para meios de cultura BDA modificados. Três clados de fungos foram formados, tanto analisando as características qualitativas das culturas quanto as quantitativas. Os clados foram identificados como dois morfotipos do gênero Ceratorhiza (fase anamórfica de Ceratobasidium) e uma Rhizoctonia-uninucleada. O sequenciamento da região ITS produziu resultados idênticos a estes, mostrando os mesmos três clados. Todas as sequências tiveram alta correlação com sequências de Ceratobasidium depositadas no Genbank, o que sugere uma alta afinidade de Coppensia doniana com este gênero. Também ficou demonstrado que os dados morfológicos, quando associados à estatística multivariada são uma ferramenta útil na taxonomia polifásica de Rhizoctonia spp. As sequências dos isolados de Coppensia doniana também foram comparadas com as de isolados de outras orquídeas, dentro da subtribo Oncidiinae, incluindo: Ionopsis utricularioides e Psygmorchis pussila, coletadas na região do Valle del Cauca Colômbia e isolados de 10 Ionopsis utricularioides, Oncidium altissimum e Tolumnia variegata, estudados por Otero (2002, 2004, 2007), em diferentes regiões de Porto Rico, Costa Rica, Cuba e Panamá. Esta última análise veio a comprovar a preferência de orquídeas da subtribo Oncidiinae por fungos do gênero Ceratobasidium, apesar de que os clados obtidos no Brasil e na Colômbia foram distantes filogeneticamente dos clados previamente estudados na América Central. Representantes dos três clados obtidos de C. doniana em Campos do Jordão foram também testados quanto à capacidade de induzir germinação em suas sementes. Todos isolados testados tiveram sucesso na germinação das sementes, levando as plântulas a estádios avançados de desenvolvimento após 30 dias, o que indica um alto potencial para utilização biotecnológica destes isolados para a germinação das sementes destas orquídeas, tanto para a floricultura comercial quanto para programas de reintrodução de espécies de orquídeas ameaçadas de extinção. / In nature, orchids are fully dependent on mycorrhizal fungi for germination. These fungi can penetrate root cells and form pelotons, whose digestion provides simple sugars for the embryo. During the achlorophyllous seedling stage, orchids are obligatory dependent on the fungi, and some species remain so through life, while others become facultatively responsive to fungal infection. The aim of this study was to identify how many fungal clades can establish mycorrhizal associations with Coppensia doniana, a widespread orchid from Campos do JordãoBrazil, and to demonstrate how their morphological features, analyzed with multivariate statistics, can be useful for classification. Ten plants were sampled in an Araucaria forest near Campos do Jordão. Fungi were isolated by transferring surface disinfected root segments containing pelotons to PDA culture medium. Three main fungal clades were formed by qualitative and quantitative morphological data. They were identified as two morphotypes of Ceratorhiza (anamorphic stage of Ceratobasidium) and one uninucleated Rhizoctonia. The ITS sequencing corroborates this identification, since the same three clades were found. All sequences were highly correlated to Ceratobasidium ITS data deposited at the Genebank, suggesting a high affinity between this species of Oncidiinae and Ceratobasidium. It also could be shown that morphological data associated with multivariate statistics can be a useful tool in fungal multi-level taxonomy. C. doniana sequences were also compared to sequences obtained from isolates of other orchids, belonging to the sub-tribe Oncidiinae, including: Ionopsis utricuarioides and Psygmorchis pussila, collected in Valle del Cauca Colombia and isolated from I. utricularioides, Oncidium altissimum and Tolumnia variegata, studied by Otero (2002, 2004, 2007) in different regions of Puerto Rico, Costa Rica and other Caribbean islands. This last analysis confirmed the preference of this Oncidiinae sub-tribe for Ceratobasidium, although isolates obtained in Brazil or Colombia belong to different clades from those previously studied in Puerto Rico, Costa Rica, Panama and Cuba. 12 Fungi representing the three clades obtained from C. doniana in Campos do Jordão were also tested for their ability to induce germination of C. doniana seeds, with a positive response for all of them, being able to bring the seedlings to advanced development stages in 30 days. These results suggest a high biotechnological potential of these isolates, to be used in orchid symbiotic germination for commercial flower production or for the reintroduction of endangered Brazilian orchid species.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In the current work it was developed a technology for the production of probiotic microparticles where three formulations containing Lactobacillus acidophilus La-14 were prepared by external ionic gelation, using sodium alginate as the primary coating material, also adding to the capsule resistant starch (Hi-maize), and chitosan. The aim of this study was to evaluate the microcapsules in wet and dry form, analyzing the resistance of microorganisms to the drying process by freeze-drying, storage at room temperature (25° C), cooling (7° C), and freezing (-18° C) for 135 days for the wet microcapsules and 60 days for lyophilized microcapsules, and "in vitro" tolerance when inoculated in solutions of pHs simulating the human gastrointestinal tract, besides the morphology of the microcapsules by optical and electronical microscopy of scanning, as well as the average diameter. After the drying by freeze-drying there was significant logarithmic reduction for all treatments, indicating that for a better viability it is necessary the addition of a cryoprotectant agent. Regarding the viability assessed by the storage time for the wet microcapsules, the room temperature kept for 135 days the viability of the microcapsules, and the addition of prebiotic and chitosan in the capsule and improved significantly the viability. For freezing temperatures and cooling also showed better results for the treatments that contained the composition the addition of prebiotic and chitosan. Analyzing the lyophilized microcapsules, the temperature was more harmful to the viability of the microorganisms, and the temperature of refrigeration and freezing was viable for 60 days for the treatments with addition of prebiotic and chitosan. Regarding to the tests in vitro simulating the gastrointestinal conditions, both wet and lyophilized microcapsules were resistant to acid pH increasing their viability as increasing pH, whereas to the wet microcapsules the number of viable cells for all treatments was 106 log UFC/g, being within the required standards so that benefits occur exercised by the probiotics. In relation to the diameter, the wet microparticles had diameters less than 70.37 μm for both treatments, while lyophilized exhibited larger diameters in function of hydration and swelling. The microparticles developed in this study may be a viable alternative for obtaining a probiotic food product be incorporated into half, to allow a higher survival of the bacteria. / No presente trabalho foi desenvolvida uma tecnologia para a produção de micropartículas probióticas onde três formulações contendo Lactobacillus acidophilus La-14 foram elaboradas por gelificação iônica externa, utilizando alginato de sódio como principal material de revestimento, adicionando-se também à cápsula amido resistente (Hi-maize) e quitosana. O objetivo deste estudo foi avaliar as microcápsulas na forma úmida e liofilizada, analisando a resistência dos microrganismos ao processo de secagem por liofilização, de estocagem a temperatura ambiente (25° C), de refrigeração (7° C), e de congelamento (-18° C) por 135 dias para as microcápsulas úmidas e 60 dias para as microcápsulas liofilizadas, e a tolerância in vitro quando inoculados em soluções de pHs simulando o trato gastrointestinal humano, além da morfologia das microcápsulas por microscopia ótica e eletrônica de varredura, bem como o diâmetro médio. Após a secagem por liofilização houve redução logarítmica significativa para todos os tratamentos, indicando que para uma melhor viabilidade é necessário à adição de um agente crioprotetor na formulação das microcápsulas. Em relação à viabilidade avaliada pelo tempo de estocagem para as microcápsulas úmidas, a temperatura ambiente manteve durante 135 dias a viabilidade das microcápsulas, sendo que a adição de prebiótico e quitosana na cápsula melhorou positivamente a viabilidade. Para as temperaturas de congelamento e refrigeração também houve melhores resultados para os tratamentos que continham na composição a adição de prebiótico e quitosana. Analisando as microcápsulas liofilizadas, a temperatura ambiente foi a mais nociva para a viabilidade dos microrganismos, e as temperaturas de refrigeração e congelamento foram viáveis por 60 dias para os tratamentos com adição de prebiótico e quitosana. Em relação aos testes in vitro simulando as condições gastrointestinais, tanto as microcápsulas úmidas como as liofilizadas foram resistentes ao pH ácido aumentando sua viabilidade conforme aumento do pH, sendo que para as microcápsulas úmidas o número de células viáveis para todos os tratamentos foi 106 log UFC/g, estando dentro dos padrões exigidos para que ocorram os benefícios exercidos pelos probióticos. Em relação ao diâmetro, as micropartículas úmidas apresentaram diâmetros inferiores a 70,37 μm para ambos os tratamentos, enquanto as liofilizadas apresentaram diâmetros maiores em função da hidratação e intumescimento. As micropartículas desenvolvidas neste estudo podem ser um meio alternativo e viável para a obtenção de um produto probiótico a ser incorporado em alimentos, de modo a permitir uma maior sobrevivência das bactérias.

La symbiose entre les légumineuses et les Rhizobiacées est la source d’azote fixé la plus importante pour le bon fonctionnement des écosystèmes naturels et agricoles. Très étudiée chez des légumineuses modèles, certains aspects de cette interaction restent peu connus ; c’est le cas des mécanismes génétiques et fonctionnels qui contrôlent la spécificité hôte-symbiote. Il n’y a que peu d’études globales consacrées à ce phénomène, et les gènes symbiotiques sont très peu connus chez les espèces non-modèles. Dans ce contexte, nous avons étudié un cas de changement de spécificité symbiotique remarquable chez des espèces phylogénétiquement proches du genre Lupinus (Fabacées). Tout d’abord, la reconstruction et l’analyse de génomes chloroplastiques complets a permis de camper le cadre évolutif de la symbiose en générant de nouveaux marqueurs d’intérêt pour clarifier la phylogénie et l’évolution des lupins. A partir d’une expérimentation d’inoculation croisée impliquant trois espèces de lupins méditerranéens et deux souches compatibles et incompatibles de Bradyrhizobium, une approche RNA-Seq a permis de produire les premiers nodulomes de lupin et d’identifier les gènes symbiotiques. L’analyse des gènes différentiellement exprimés a montré que la spécificité symbiotique affecte non seulement la voie de signalisation et de régulation de la symbiose, mais également une diversité de voies métaboliques associées. Enfin, l’étude de la dynamique évolutive et fonctionnelle de quelques gènes a mis en évidence l’impact et l’importance des phénomènes de duplication aux différents niveaux de la cascade génétique symbiotique. / Legumes-Rhizobia symbiosis is the most important fixing nitrogen source for the good functioning of both natural and agricultural ecosystems. Although, it is extensively studied in model legumes, some aspects of this interaction remain unclear, such as the genetic and functional mechanisms controlling the host-symbiont specificity. Large scale studies of this process are scarce and symbiotic genes are not well described in non-model species. In this context, the effect of symbiotic specificity was investigated in phylogenetically close relative species belonging to the Lupinus genus (Fabaceae). First, the reconstruction and analysis of complete chloroplast genomes allowed us to generate new and useful markers for clarifying the Lupinus phylogeny in order to lighten the evolutionary context of the symbiosis. Following a cross-inoculation experiment of three Mediterranean lupine species with two compatible or incompatible Bradyrhizobium strains, a RNA-Seq approach allowed the reconstruction of the first lupine nodulomes and the identification of lupine symbiotic genes. The analysis of differentially expressed genes revealed that the symbiotic specificity affects not only the signalling and regulatory symbiotic pathways, but also diverse associated metabolic pathways. Finally, evaluating the evolutionary and functional dynamics of genes highlighted the importance of gene and genome duplication events at different steps of the symbiotic genetic pathway.

En cas de carence azotée, les légumineuses sont capables de mettre en place une symbiose avec des bactéries du sol fixatrices d’azote appelées rhizobia. Cette symbiose a lieu dans un organe appelé nodosité où les bactéries sont endocytées et appelées bactéroïdes. Certains clades de légumineuses imposent un processus de différenciation à leurs bactéroïdes qui agrandissent considérablement et deviennent polyploïdes, menant à des morphotypes bactériens allongés ou sphériques. Au cours de cette thèse, j’ai étudié la différenciation des bactéroïdes de Bradyrhizobium spp. en association avec Aeschynomene spp.. Les bactéroïdes de ces plantes présentent des degrés de différenciation distincts qui dépendent de l’espèce hôte. Mes données suggèrent que les bactéroïdes les plus différenciés sont aussi les plus efficaces. J’ai cherché à savoir quels facteurs procaryotes pourraient être impliqués dans les adaptations des bactéroïdes au processus de différenciation et à leurs divers hôtes, le tout en lien avec cette différence d’efficacité symbiotique au travers d’approches globales sans a priori de type -omiques. Les conditions considérées sont des bactéroïdes de différents morphotypes et des cultures libres de référence. Les fonctions activées en conditions symbiotiques ont été identifiées, ainsi que les gènes spécifiques d’un hôte donné. Des analyses fonctionnelles des gènes d’intérêt ont également été menées. Les mutants bactériens n’ont toutefois pas présenté de phénotype symbiotique drastique, montrant ainsi l’existence de réseaux de gènes complexes menant à la résilience des génomes de rhizobia. / In case of nitrogen starvation, legume plants establish a symbiotic interaction with nitrogen-fixing soil bacteria called rhizobia. This interaction takes place in nodules where the symbionts are internalized and become bacteroids. Some legume clades also impose a differentiation process onto the bacteroids which become enlarged and polyploid, leading to elongated or spherical morphotypes. During my PhD work, I have studied bacteroid differentiation of Bradyrhizobium species in association with Aeschynomene spp.. These bacteroids display distinct differentiation levels depending on the plant host, and my analyses suggest that the most differentiated ones are also the most efficient. I investigated the bacterial factors potentially involved in the adaptations to differentiation and host-specificity, and related to the higher efficiency of the most differentiated bacteroids using global-omics approaches without a priori. The analyzed conditions were bacteroids of distinct morphotypes and free-living reference cultures. Activated functions under symbiotic conditions were identified, as well as host-specific ones. Functional analyses were performed on genes of interest. However, the bacterial mutants did not display drastic symbiotic phenotypes, showing the existence of complex gene networks leading to high resilience of rhizobial genomes.

<p>C₄-dicarboxylic acids appear to be metabolized via the TCA cycle in N₂-fixing bacteria (bacteroids) within legume nodules. In <em>Sinorhizobium meliloti</em> bacteroids from alfalfa, NAD⁺-malic enzyme (DME) is required for symbiotic N₂-fixation and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont <em>Rhizobium leguminosarum</em> pyruvate synthesis can occur via either the DME pathway or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK), pyruvate kinase (PYK), and pyruvate dehydrogenase. Here we report that <em>dme</em> mutants of <em>Sin</em>or<em>hizobium sp</em>. NGR234 formed root nodules on a broad range of plants and that the level of N₂-fixation varied from 90% to 20% of wild type depending on the host plants inoculated. NGR234 bacteroids had significant PCK activity and while single <em>pckA</em> and single <em>dme</em> mutants fixed N₂ on <em>Macroptilium atropurpureum</em> and <em>Leucaena leucocephala</em> (albeit at a reduced rate), a <em>pckA</em> <em>dme</em> double mutant had no N₂-fixing activity (Fix^-). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix^- phenotype of <em>S. meliloti dme </em>mutants may be specific to the alfalfa-<em>S. meliloti </em>symbiosis. We therefore examined the ME-like genes <em>azc3656 </em>and <em>azc0119 </em>from <em>Azorhizobium caulinodans</em>, as <em>azc3656 </em>mutants were previously shown to form Fix^- nodules on the tropical legume <em>Sesbania rostrata</em>. We found that purified AZC3656 protein is an NAD (P)⁺-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N₂ fixation in <em>A. caulinodans </em>and <em>S. meliloti</em>, in other rhizobia this activity can be bypassed via another pathway(s).</p> <p>In <em>S. meliloti</em> both malic enzymes DME and TME share similar apparent <em>K_m</em>s for substrate and cofactors, but differ in their responses to TCA cycle intermediates, with DME activity inhibited by acetyl-CoA and induced by succinate and fumarate. Previous results in our laboratory indicated that DME is essential for symbiotic N₂ fixation, while TME fails to functionally replace DME. One possible reason for it is that a high ratio of NADPH/NADP⁺in<em> S. meliloti </em>bacteroids prevents TME from functioning in nodules. We sought to lower the<em> </em>NADPH/NADP⁺ratio by overexpressing a soluble pyridine nucleotide transhydrogenase (STH). However, metabolite measurements indicated that overproducing STH failed to lower the ratio of NADPH/NADP⁺ in<em> S. meliloti</em>.</p> <p>Previous studies assumed that DME and TME might play different roles in central carbon metabolism. To gain insight of their physiological functions, genome-wide microarray analysis was conducted in <em>S. meliloti</em> single<em> dme and</em> <em>tme</em> mutants grown on glucose or succinate. The most striking changes of gene expression were observed in <em>S. meliloti</em> <em>dme</em> mutants grown on succinate. The functions of upregulated genes suggested that DME might play an important role in regulating TCA cycle intermediates, important for the maintenance of metabolic flux through TCA cycle during C₄-dicarboxylate oxidation. However, changes of gene expression found in <em>tme </em>mutants were not significant enough to predict the physiological functions of TME protein in central carbon metabolism.</p> / Doctor of Philosophy (PhD)

The purpose of the research was to determine how educators could be motivated to implement internet technology in education. The literature study highlighted the need for e-learning while suggesting that educator support would not be achieved easily. The empirical study, however, revealed that at the international school examined, educators accept internet learning and demonstrate a willingness to introduce it into their pedagogy, were a blended approach to be adopted. Technological and psychological barriers had already been breached, as e-learning was taking place in a non-threatening environment. Educators were being empowered to experiment with e-learning in their subject areas. The study revealed that, having already embraced e-learning methodology educators would continue to do so if they could clearly perceive the benefits to be achieved. The study also showed that given a technologically nurturing environment, it would not be difficult to motivate educators to introduce internet technology into their pedagogy. / Educational Studies / M.Ed. (Adult Education)

Nas últimas décadas, a incidência de infecções hospitalares causadas por bactérias Gramnegativas multidrogarresistentes (MDR) vem crescendo de maneira vertiginosa em todo o mundo, de modo que a Organização Mundial de Saúde (OMS) recentemente reconheceu essas infecções como uma preocupação mundial devido ao seu impacto negativo sobre as taxas de mortalidade intra-hospitalar e dos custos da assistência à saúde, afetando tanto os países desenvolvidos quanto os em desenvolvimento. Atualmente considera-se que a higienização das mãos, o uso racional de antimicrobianos e o isolamento de contato são as principais medidas disponíveis para contenção desse avanço. Porém, elas são apenas parcialmente efetivas e de implementação trabalhosa e onerosa. Assim, considera-se necessário o desenvolvimento de formas mais simples e eficientes paralidar com esse problema. No presente estudo, nos propusemos a avaliar o impacto da administração de um produto simbiótico a pacientes colonizados e/ou infectados por bactérias Gram-negativas MDR sobre as taxas de descolonização desses patógenos no trato digestivo. Trata-se de um ensaio clínico randomizado, duplamente cego, controlado com placebo, envolvendo 101 pacientes hospitalizados com colonização prévia por bactérias Gram-negativas MDR, demonstrada por meio de cultura seletiva de swab retal, cuja intervenção consistiu na administração oral ou enteral diária de 1010 unidades de Lactobacillus bulgaricus e 1010 unidades de Lactobacillus rhamnosus associados a fruto-oligossacarídeos durante (FOS) 7 dias. O desfecho primário do estudo foi a descolonização completa do trato digestivo posterior à intervenção, que, na análise do tipo \"intenção de tratar modificada\" foi de 16,7% (8/48) no grupo experimental e 20,7% (11/53) no grupo controle (p=0,600). Na análise \"per protocol\", a descolonização completa do trato observada foi de 18,9% (7/37) no grupo experimental e 23,3% (7/30) no grupo controle (p=0,659). Em uma análise multivariada por meio de modelo de regressão logística o uso do simbiótico não influenciou significativamente o risco de descolonização completa do trato digestivo (OR= 0,80, IC 95%= 0,28-2,27, p= 0,678). A ocorrência de eventos adversos de natureza leve a moderada foi semelhante entre os grupos: 7,55% no grupo que utilizou placebo e 6,25% no grupo sob intervenção (p= 1,000). Nenhum evento adverso grave potencialmente relacionado às medicações de estudo foi observado. Nas condições estudadas, os dados obtidos pelo estudo nos levam à conclusão de que o simbiótico estudado demonstrou-se inefetivo na descolonização do trato digestivo de pacientes previamente colonizados por bactérias Gram-negativas MDR. / In recent decades the incidence of multidrug resistant (MDR) Gram-negative nosocomial infections has been dramatically raising in the whole world. The World Health Organization (WHO) recently recognized nosocomial infections due to MDR pathogens as a global concern due to its negative impact on patients, health-care workers and health-care institutions, affecting developed countries as well as developing ones. They negatively impact in-hospital mortality and health-care related costs. Hand hygiene promotion, antibiotic stewardship and contact precautions are the main available measures to control such MDR Gram-negative organisms in hospitals. However, they are only partially effective as well as difficult to be implemented and expensive. Therefore, simpler and more effective actions are thought to be helpful and urgent. In the present study, we analyzed the impact of the administration of a symbiotic product on patients harboring Gram-negative multidrug-resistant bacteria upon the subsequent rates of decolonization of these pathogens from the gastro-intestinal tract.This is a double-blinded and placebo controlled randomized clinical trial evaluating the oral/enteral daily administration of 1010 units of Lactobacillus bulgaricusplus 1010 units of Lactobacillus rhamnosus associated with fructo-oligosacharide (FOS), or placebo, for 7 days, to 101 patients previously colonized by MDR Gram-negative bacteria, identified through selective culture of rectal swab. The primary study outcome was the rate of complete decolonization of the MDR microorganism from the gastro-intestinal tract following the intervention. In the \"modified intention to treat\" analysis, decolonization rates observed were 16.7% (8/48) in the experimental group and 20.7% (11/53) in the placebo group (p=0,600). In the \"per protocol\" analysis, decolonization rates were 18.9% (7/37) in the experimental group and 23.3% (7/30) in the placebo group (p=0,659). In a logistic regression model, symbiotic use did not produce any impact on the chance of decolonization (OR=0.80, CI95%=0.28-2.27, p=0.678). Mild to moderate adverse events occured similarly in both the placebo (7.55%) and the experimental group (6.25%), (p=1,000). No severe adverse event potentially related to the medications was detected during the study period. In the present study conditions, the results obtained lead to the conclusion that the studied symbiotic proved to be ineffective to decolonize patients harboring multidrug resistant Gram-negative bacilli.