La symbiose ectomycorhizienne se définie comme une association mutualiste entre les racines des plantes ligneuses et le mycélium de champignons du sol. Elle est majoritaire dans les écosystèmes forestiers de l'hémisphère nord et permet l'amélioration de la nutrition hydrominérale des plantes ligneuses, notamment lorsque la disponibilité en ressources se fait rare. Des données transcriptomiques et génomiques du champignon ectomycorhizien Hebeloma cylindrosporum ont permis d'identifier différents gènes codant pour des protéines capables de transporter des nutriments. L'implication éventuelle de ces systèmes de transport dans la nutrition potassique et phosphatée ectomycorhize-dépendante de la plante hôte Pinus pinaster reste à évaluer. Dans cette étude, deux gènes candidats codant pour des systèmes de transport de potassium (K+), HcTrk1 et HcSKC, et deux autres pour des transporteurs de phosphate inorganique (Pi), HcPT1.1 et HcPT2, ont été analysés. Des approches permettant la localisation dans l'ectomycorhize des transcrits (hybridation in situ) et des protéines (fusion traductionnelle) de ces candidats ont été générés. Ces différents outils ont permis de montrer que le transporteur HcTrk1 et le canal HcSKC étaient respectivement localisés dans l'ectomycorhize au niveau des sites de prélèvement et de relargage du K+. D'autre part, des lignées transgéniques d'H. cylindrosporum sur- et/ou sous-exprimant ces gènes ont été produites afin de voir si la nutrition végétale en K+ et en Pi était impactée. Ainsi, l'utilisation en mycorhization de lignées surexprimant HcTrk1 et sous-exprimant HcSKC ont montré une diminution de la nutrition potassique et de l'homéostasie du phosphore de la plante hôte. Les mêmes types d'approches ont été utilisés avec les transporteurs de Pi HcPT1.1 et HcPT2. En utilisant des lignées transgéniques, il a ainsi pu être montré que la surexpression de HcPT1.1 en condition carencée décrite précédemment était liée à l'activité de son promoteur. Quant au transporteur HcPT2, des analyses préliminaires de localisation suggèrent qu'il pourrait être impliqué dans le prélèvement de Pi du sol et dans son relargage au niveau du réseau de Hartig. Des études complémentaires restent cependant à mener. Enfin, cinq autres systèmes de transport de K+ et trois de phosphate ont été récemment identifiés à partir du génome d'H. cylindrosporum, ouvrant la voie à la dissection fine des mécanismes moléculaires régissant la nutrition potassique et phosphatée ectomycorhize dépendante de P. pinaster. / Ectomycorrhizal symbiosis is defined as a mutual association between the roots of woody plants and the mycelium of soil fungi. This symbiosis is widespread in northern forests and plays a major role in nutrient and water uptake of woody plants, especially when resources become scarce. Transcriptomic and genomic data of the ectomycorrhizal fungus Hebeloma cylindrosporum allowed the identification of several genes coding for nutrient transport proteins. Their putative involvement in ectomycorrhiza-dependent potassium and phosphate nutrition of the host plant Pinus pinaster needs to be assessed. In this study, two candidate genes coding for potassium (K+) transport systems, HcTrk1 and HcSKC, and two other genes coding for inorganic phosphate (Pi) transporters, HcPT1.1 and HcPT2, were analyzed. Molecular approaches allowing the localization of transcripts (in situ hybridization) and proteins (translational fusion) of these candidates in ectomycorrhiza were obtained. These tools allowed us to show that the HcTrk1 transporter and the HcSKC channel were localized in K+ uptake and release sites of the ectomycorrhiza, respectively. In order to know whether these proteins play a role in plant K+ and Pi nutrition, H. cylindrosporum transgenic lines with up- and/or down-regulated expression of candidate genes were produced. In mycorrhizal assays, the use of fungal strains with up- or down- regulated expression of HcTrk1 and HcSKC, respectively, affects the K+ nutrition and phosphorus homeostasis of the host plant. The same approaches were used for HcPT1.1 and HcPT2 Pi transporters. Therefore, using transgenic strategies, we demonstrated that the previously shown up-regulation of HcPT1.1 expression under Pi shortage is related to its promoter activity. Concerning HcPT2, preliminary localization analysis suggested that this transporter might be involved in Pi uptake from soil and in release in the Hartig net. However, complementary studies are needed. Five and three novel K+ and Pi transport systems, respectively, were identified from the recent genome accession of H. cylindrosporum, opening the way to a fine dissection of molecular mechanisms controlling the ectomycorrhiza-dependent K+ and phosphate nutrition of Pinus pinaster.

L'arthrose (OA) est une maladie articulaire chronique qui résulte de changements complexes dans le phénotype des chondrocytes. La présence de microcristaux contenant du phosphate dans les zones de cartilage lésées suggère que le métabolisme phosphocalcique contribue en partie aux modifications du phénotype chondrocytaire au cours de la maladie. De nombreuses études ont montré que des concentrations élevées en Phosphate Inorganique extracellulaire (ePi) ou en PyroPhosphate Inorganique (ePPi) ont respectivement un effet activateur ou répressif sur la minéralisation du cartilage articulaire. Comme le Fibroblast Growth Factor 23 (FGF23) régule les concentrations de Pi, FGF23 semble être un candidat aux modifications phénotypiques observées dans l'OA. De plus, il a récemment été mis en évidence que l’ePPi prévient la dédifférenciation in vitro des chondrocytes articulaires chez le rat, un effet provoqué par la production de PPi par la protéine Ank. Cela suggère que l’ePPi pourrait être un candidat pour prévenir les modifications du phénotype chondrocytaire. Premièrement, nous avons montré que l’expression de FGF23 est plus importante dans du cartilage lésé que dans du cartilage sain. Sous stimulation croissante de FGF23, les chondrocytes humains OA présentent une expression soutenue des marqueurs d’hypertrophie tels que COL10A1, VEGF et MMP13. Nous avons également démontré que l’expression de MMP13 est fortement dépendante de FGFR1 mais indépendante de Klotho et qu’elle est fortement régulée par la voie MEK/ERK et dans une moindre mesure par la voie PI3K/AKT. Deuxièmement, nous avons montré que FGF23 est produit de façon plus importante au cours de la différenciation des ATDC5 et qu’une stimulation par FGF23 augmente la minéralisation et l’expression des marqueurs d’hypertrophie, et ce, d’autant plus fortement en présence d’une stimulation par du Pi dans ces cellules. Dans la seconde partie, nous avons montré que des chondrocytes humains OA stimulés par du PPi présentent une expression diminuée des composants collagéniques de la matrice et une expression augmentée des MMPs, de la fibronectine et des intégrines. Une stimulation par le PPi active de façon importante la voie p38 et dans une moindre mesure la voie ERK pour réguler l’expression de ses gènes cibles et notamment MMP13 d’une manière Ank indépendante. Enfin, nous avons démontré qu’une stimulation par FGF23 entraine une augmentation de l’expression de Pit-1, ENPP1 et ANK ainsi que la production de PPi par les chondrocytes humains OA. Les résultats obtenus dans cette étude démontrent que le FGF23 permet localement une différenciation des chondrocytes OA vers un phénotype hypertrophique et peut potentiellement être considéré comme un facteur aggravant de l’OA. Contrairement aux données préliminaires chez le rat, le PPi permet un remodelage matriciel des chondrocytes humains OA et pourrait potentiellement contribuer aux effets pro-hypertrophiques du FGF23 / Osteoarthritis (OA) is the most common form of chronic joint disease, characterized by cartilage degeneration that results from complex changes in the chondrocyte phenotype. The presence of phosphate-containing microcrystals in the injured cartilage areas suggests the contribution of the phosphocalcic metabolism in the phenotype switch of chondrocytes during the disease. Numerous studies have shown that elevated concentrations of extracellular inorganic phosphate (ePi) or inorganic pyrophosphate (ePPi) have, respectively, activating or repressive mineralizing effects on articular cartilage. As Fibroblast Growth Factor 23 (FGF23) plays a major role in regulating concentrations of Pi, FGF23 is an attractive candidate to participate in the phenotype switch of the articular chondrocyte observed in OA. Moreover, we recently demonstrated that ePPi also prevents the in vitro dedifferentiation of articular chondrocyte in rats, an effect mostly triggered by Ank-induced release of PPi. This suggests that PPi may be an attractive candidate to prevent the phenotype switch of the articular chondrocyte. Firstly, we showed that FGF23 expression was higher in OA samples than in healthy one. When stimulated with increasing concentrations of FGF23, human OA chondrocytes displayed a sustained expression of markers of hypertrophy such as COL10A1, VEGF and MMP13. We demonstrated further, that MMP13 expression was mainly dependent on FGFR1 and independent of Klotho and was strongly regulated by the MEK/ERK cascade and to a lesser extent by the PI-3K/AKT pathway. Secondly, we showed that FGF23 and FGFRs were produced more importantly during ATDC5 differentiation and that FGF23 stimulation increased hypertrophic markers expression and mineralization in a synergic manner with Pi. In the second part, we showed that human OA chondrocytes stimulated with PPi displayed a decreased expression of collagen components of the matrix and sustained expression of MMPs, fibronectin and integrins. We demonstrated further that PPi stimulation mostly activates p38 pathway and to a lesser extent ERK pathway to regulate the expression of its target genes in an Ank-independent manner. Finally, we demonstrated that FGF23 stimulation increased Pit-1, ENPP1 and Ank expressions and PPi production by human OA chondrocyte. Altogether, the results obtained in this study demonstrate that FGF23 locally promotes differentiation of OA chondrocytes towards a hypertrophic phenotype and may therefore be considered as an aggravating factor for OA. In contrast to previous data obtained in rats, we demonstrated that PPi promotes matrix-remodeling of human OA chondrocytes and might contribute to FGF23 pro-hypertrophic effect

The synergistic effect of nitric oxide toward dephosphorylation reactions involving phosphate esters was the subject of investigation in this research. Sodium nitroprusside under UV irradiations at 254nm, 365nm and white light was utilized as nitric oxide donor in solutions. The effects of cobalt trimethylenediamine and nitroprusside towards dephosphorylation of nitrophenylphosphate and pyrophosphate which were modeled as organophosphate ester substrates were also investigated. The activated substrate models showed more rate enhancement than the unactivated models in all cases. The direct interaction of nitric oxide with the phosphorus centre is presumed to be the reason for enhanced hydrolysis. This study demonstrates the possible role of nitric oxide in decontamination reactions of poorly biodegradable phosphate esters in the biosphere. / Chemistry / M. Sc. (Chemistry)

Made available in DSpace on 2015-03-26T13:40:05Z (GMT). No. of bitstreams: 1 texto completo.pdf: 466360 bytes, checksum: e0d21936cd393cfb2e58f6a6e09e5149 (MD5) Previous issue date: 2007-03-16 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objective of this work was to evaluate the impact of the herbicides ametryn and trifloxysulfuron-sodium, singly or combined, and 2,4-D, on the soil microrganisms and the mineral nutrition of the sugar-cane plants. In the first trial, it was evaluated the activity of inorganic phosphate-solubilizing microorganisms and the numbers of fungi and bacteria in the rhizosphere of sugar cane and, in the second, the respiratory rate, microbial biomass, metabolic quotient, and total C-CO2 evolved from the soil. In the third trial, the growth and the macro and micronutrients concentrations were evaluated in the foliar tissues of the sugar-cane plants after the herbicide application. In the three trials, foliar tissue samples and rhizospheric and non-rhizospheric soil samples were collected and immediately analyzed at 15, 30, 45, and 60 days after spraying (DAS). At 15 DAS, all herbicides tested led to reductions in the numbers of fungi in the soil. 2,4-D caused a reduction in the densities of bacterial populations in the rhizosphere at all times of evaluation, evidencing a higher sensitivity of bacteria to this compound. The soil microbial biomass was sensitive to ametryn applied singly or combined, especially up to 30 DAS. These treatments resulted in the highest values of metabolic quotient at 45 and 60 DAS. The foliar concentrations of N, P and Mg were not affected for the herbicide use. At 60 DAS, trifloxysulfuron-sodium lead to increase of 22.10% in the biomass accumulation of shoots sugar cane plants. Trifloxysulfuron-sodium and 2,4-D led to higher inorganic phosphate solubilization at 15, 30, and 45 DAS, and 15 and 30 DAS, respectively, without affecting the microbial biomass. This work evidences that herbicide application in the shoots of sugar cane affects the foliar concentrations of macro and micronutrients and the soil microorganisms in the rhizosphere. / Objetivou-se neste trabalho avaliar a microbiota do solo e a nutrição mineral da cana-de-açúcar após aplicação dos herbicidas 2,4-D, ametryn, trifloxysulfuronsodium e ametryn+trifloxysulfuron-sodium. No primeiro ensaio, avaliou-se a atividade e a densidade populacional dos microrganismos solubilizadores de fosfato inorgânico (MSFI) e totais no solo rizosférico da cana-de-açúcar e, no segundo, foram avaliadas a taxa respiratória, a biomassa microbiana e o quociente metabólico do mesmo solo. No terceiro ensaio, avaliaram-se o crescimento e as concentrações de macro e micronutrientes nos tecidos foliares das plantas de cana-de-açúcar após a aplicação dos herbicidas. Nos três ensaios foram realizadas avaliações aos 15, 30, 45 e 60 dias após a aplicação (DAA) dos herbicidas. Todos os herbicidas provocaram redução na densidade populacional fúngica do solo somente no período inicial de avaliação. O 2,4-D foi mais tóxico às bactérias do solo até aos 60 DAA. Analisando o quociente metabólico, ressaltaram-se condições estressantes para a microbiota do solo quando este foi tratado com ametryn isolado e em mistura com trifloxysulfuronsodium. A atividade dos MSFI foi favorecida pela aplicação do 2,4-D até os 30 DAA e o trifloxysulfuron-sodium estimulou essa atividade por todo período de avaliação. As concentrações de N, P e Mg nos tecidos foliares não foram afetados pela utilização dos herbicidas. Em plantas tratadas com trifloxysulfuron-sodium observou-se acréscimo de 22,10% no acúmulo de massa seca da parte aérea das plantas aos 60 DAA. Este trabalho evidenciou que a aplicação dos herbicidas na parte aérea de plantas de cana-de-açúcar afetou a concentração dos nutrientes foliares, a densidade populacional de microrganismos, atividade de solubilização de fosfato, a taxa respiratória, a biomassa e o quociente metabólico na rizosfera.