Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-08-18T11:18:15Z No. of bitstreams: 1 texto completo.pdf: 483031 bytes, checksum: fdee08ea45b1d6006db79159f59bcb16 (MD5) / Made available in DSpace on 2016-08-18T11:18:15Z (GMT). No. of bitstreams: 1 texto completo.pdf: 483031 bytes, checksum: fdee08ea45b1d6006db79159f59bcb16 (MD5) Previous issue date: 2015-07-13 / Existe um crescente interesse na utilização de microalgas para a produção de biocombustíveis, alimentos e outros produtos de valor comercial. As microalgas possuem grande capacidade de fixar CO2 atmosférico e acumular carbono, sobretudo na forma de amido e lipídeos. Entretanto, ainda pouco é conhecida a regulação da biossíntese de amido e lipídeos ao longo do curso diário da fotossíntese e as relações das variações desses metabólitos com o crescimento e a produção final de biomassa. Assim, faz-se necessário uma maior compreensão das vias metabólicas e sua regulação para compreender a fisiologia e os mecanismos envolvidos na biossíntese de amido e lipídeos. Dessa forma o presente trabalho objetivou estudar a biossíntese e degradação do amido, de açúcares e lipídeos ao longo do dia. Foram selecionadas duas espécies de microalgas verdes com diferentes taxas de crescimento e contrastantes quanto a produção de amido e lipídeos totais. Com base nestes critérios foram utilizadas Chlamydomonas reinhardtii CC125, que possui um elevado acúmulo de amido e baixo teor lipídico, e Monoraphidium irregulare BR023, que possui menor conteúdo de amido e maior conteúdo de lipídeo. A cepa de M. irregulare apresentou ao final do cultivo 2,5x106 células/mL, sendo inferior ao verificado para o cultivo de C. reinhardtii (3,5x106 células/mL). Para proteínas foram encontrados valores médios inferiores em M. irregulare aos observadoss em C. reinhardtii. Comportamento similar entre as cepas foi observado para os teores de aminoácidos totais. Verificou-se menores taxas de síntese e degradação de amido para M. irregulare em ambas as fases quando comparado com as respectivas taxas para C. reinhardtii. Foram observadas maiores intensidades de fluorescência para lipídeos em M. irregulare, o que indica que esta espécie apresenta maior teor de lipídeo durante o cultivo em relação a C. reinhardtii. Conclui-se que: (i) os teores de proteínas e aminoácidos estão relacionados diretamente com a taxa de crescimento celular; (ii) lipídeo apresenta variação constante ao longo do dia; (iii) amido apresenta comportamento em ritmo circadiano; (iv) a taxa de degradação mais rápida do amido acompanha o aumento do crescimento, como em C. reinhardtii. / There is a growing interest in using microalgae as a resource for biofuel production, food and other value products. Microalgae have great ability to fix atmospheric CO2 and accumulate carbon, mostly in the form of starch and lipids. However, it is still little known regulation of starch biosynthesis and lipid along the daily course of photosynthesis and the relationship of variations of these metabolites to the growth and final biomass production. Thus, a greater understanding of metabolic pathways it is necessary and its regulation to understand the physiology and the mechanisms involved in the biosynthesis of starch and lipids. Thus the present study investigated the biosynthesis and degradation of starch, sugars and lipids throughout the day. Two species of green microalgae with different growth rates and contrasting as the production of starch and total lipids selected. Based on these criteria were used Chlamydomonas reinhardtii CC125, which has a high accumulation of starch and low- fat, and Monoraphidium irregulare BR023, which has a lower starch content and higher content of lipid. The strain of M. irregulare showed at the end of cultivation 2.5x106 cells/ml, being lower than that for the cultivation of C. reinhardtii (3,5x106 cells/ml). For proteins found average in M. irregulare average values lower to observed for the C. reinhardtii. Similar behavior was observed among strains for total amino acid content. It was found lower rates of starch synthesis and degradation M. irregulare in both phases compared to the respective rates for C. reinhardtii. There was higher fluorescence intensities for lipids in M. irregulare, indicating that this species has a higher lipid content during cultivation. It concludes that: (i) the levels of proteins and amino acids are directly related to the rate of cell growth; (ii) lipid presents constant change throughout the day; (iii) starch shows behavior circadian rhythm; (iv) the faster degradation rate of the starch accompanies increased growth, as in C. reinhardtii.

Since 2010 IS has transformed itself from a terroristgroup to an insurgency organisation that held territory from Mosul in Iraq to Aleppo in Syria. Through this conquest of territory it has proven it’s capabilities in launching conventional style offenses but also it use of irregular tactics and the use of different terrorist acts using suicide bombers and vehicle-borne improvised explosions. This study examines IS warfare between 2010 and 2015 and examens if David Galulas theory ’Insurgency doctrin’ and Frank Hoffmans theory ’Hybrid warfare’ can describe IS warfare through this period. This analysis shows that IS partially can be described through the insurgency doctrin theory as an insurgency following the steps that it descibes. It also shows that while IS follows the steps it also continued to use blind terrorism and selektive terrorism through the whole campaigns. Further more the analysis also shows that IS can be described through Frank Hoffmans ’Hybrid warfare’ theory. IS used conventional means such as military weapons and equipment along with artillery, tanks, anti-tank weapons in both conventional and irregular ways. It’s extensive use of IED, VBIED IS used technology in an ortodox way by rebuildning vehicles and drones into mobile IED’s but also vehicles into light fighting vehicles by mounting heavy machineguns on pickups. This study also concludes that IS can not be described as an hybrid actor becuse it strives at building a regulare army. The conclusion of this study is that IS warfare can be described as an insurgent uprising with hybrid elements. Since 2010 IS has transformed itself from a terroristgroup to an insurgency organisation that held territory from Mosul in Iraq to Aleppo in Syria. Through this conquest of territory it has proven it’s capabilities in launching conventional style offenses but also it use of irregular tactics and the use of different terrorist acts using suicide bombers and vehicle-borne improvised explosions. This study examines IS warfare between 2010 and 2015 and examens if David Galulas theory ’Insurgency doctrin’ and Frank Hoffmans theory ’Hybrid warfare’ can describe IS warfare through this period. This analysis shows that IS partially can be described through the insurgency doctrin theory as an insurgency following the steps that it descibes. It also shows that while IS follows the steps it also continued to use blind terrorism and selektive terrorism through the whole campaigns. Further more the analysis also shows that IS can be described through Frank Hoffmans ’Hybrid warfare’ theory. IS used conventional means such as military weapons and equipment along with artillery, tanks, anti-tank weapons in both conventional and irregular ways. It’s extensive use of IED, VBIED IS used technology in an ortodox way by rebuildning vehicles and drones into mobile IED’s but also vehicles into light fighting vehicles by mounting heavy machineguns on pickups. This study also concludes that IS can not be described as an hybrid actor becuse it strives at building a regulare army The conclusion of this study is that IS warfare can be described as an insurgent uprising with hybrid elements

Les champignons mycorhizien à arbuscules (CMA) sont des organismes pouvant établir des symbioses avec 80% des plantes terrestres. Les avantages d'une telle symbiose sont de plus en plus caractérisés et exploités en agriculture. Par contre, jusqu'à maintenant, il n'existe aucun outil permettant à la fois l'identification et la quantification de ces champignons dans le sol de façon fiable et rapide. Un tel outil permettrait, entre autres, de mieux comprendre les dynamiques des populations des endomycorhizes dans le sol. Pour les producteurs d'inoculum mycorhiziens, cela permettrait également d'établir un suivi de leurs produits en champs et d'avoir un contrôle de qualité de plus sur leurs inoculants. C'est ce que nous avons tenté de développer au sein du laboratoire du Dr. Hijri. Depuis environ une trentaine d'années, des outils d'identification et/ou de quantification ont été développés en utilisant les profiles d'acides gras, les isozymes, les anticorps et finalement l'ADN nucléaire. À ce jour, ces méthodes d’identification et de quantification sont soit coûteuses, soit imprécises. Qui plus est, aucune méthode ne permet à la fois la quantification et l’identification de souches particulières de CMA. L’ADN mitochondrial ne présente pas le même polymorphisme de séquence que celui qui rend l’ADN nucléaire impropre à la quantification. C'est pourquoi nous avons analysé les séquences d’ADN mitochondrial et sélectionné les régions caractéristiques de deux espèces de champignons mycorhiziens arbusculaires (CMA). C’est à partir de ces régions que nous avons développé des marqueurs moléculaires sous forme de sondes et d’amorces TaqMan permettant de quantifier le nombre de mitochondries de chacune de ces espèces dans un échantillon d’ADN. Nous avons ensuite tenté de déterminer une unité de quantification des CMA, soit un nombre de mitochondries par spore. C’est alors que nous avons réalisé que la méthode de préparation des échantillons de spores ainsi que la méthode d’extraction d’ADN avaient des effets significatifs sur l’unité de quantification de base. Nous avons donc optimisé ces protocoles, avant d’en e tester l’application sur des échantillons de sol et de racines ayant été inoculés avec chacune des deux espèces cibles. À ce stade, cet outil est toujours semi-quantificatif, mais il permet 9 l’identification précise de deux espèces de CMA compétentes dans des milieux saturés en phosphore inorganique. Ces résultats , en plus d’être prometteurs, ont permis d’augmenter les connaissances méthodologiques reliées à la quantification des CMA dans le sol, et suggèrent qu’à cause de leurs morphologies différentes, l’élaboration d’un protocole de quantification standardisé pour toutes les espèces de CMA demeure un objectif complexe, qui demande de nouvelles études in vivo. / Arbuscular Mycorrhizal Fungi (AMF) are able to form symbiosis with approximately 80% of plant species, including most important corps. This symbiosis is known as Arbuscular Mycorrhiza which has been largely used in agriculture to promote plant growth by enhancing minerals uptake and protecting plants against biotic and abiotic stresses. Despite the ecological and economical importance of this symbiosis, specific markers for spore quantification of commercial AMF strains by quantitative real-time PCR (qPCR) are extremely limited and none has been rigorously validated for quality control of manufactured products such as biofertilizers. Most of the existing AMF markers developed either for AMF identification or for AMF quantification purposes are based on either morphological characters, on Fatty Acids Methyl Esters (FAME) profiles, on specific isozymes or antibodies, or on nuclear DNA. It has been shown that mitochondrial (mt) genomes are conserved among AMF species. This allowed us to use mt genomes in order to develop efficient isolate-specfic markers. Using the alignments of 11 complete AMF mt genomes, a qPCR assay using a hydrolysis probes designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of the model R. irregularis isolate DAOM197198 and another probe was designed in nad1-cox2 intergene specific to Glomus cerebriforme. The specificity tests performed, using standard PCR and qPCR, clearly showed that the primers specifically amplified the isolate DAOM-197198 or Glomus cerebriforme DAOM220722. Quantification assays were successfully undertaken on unknown samples in liquid suspensions, commercial solid formulations and soil samples to show the accuracy and reproducibility of the assays. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate Glomus cerebriforme DAOM220722. The latter could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.

La germination des spores est une étape essentielle dans le cycle de vie de la majorité des champignons filamenteux. Les champignons mycorhiziens à arbuscules (CMA) forment un certain nombre de propagules infectieuses différentes qui augmentent leur potentiel à coloniser les racines. Parmi elles se trouvent les spores extraracinaires et intraracinaires. La paroi cellulaire des spores joue un rôle majeur dans la survie de ces propagules en étant une barrière physique et osmotique. Puisque une cellule peut faire des ajustements considérables dans la composition et la structure de sa paroi, en réponse aux conditions environnementales, il est possible que les parois des spores intraracinaires et extraracinaires montrent des propriétés mécaniques et osmotiques différentes affectant leur germination et leur survie. Pourtant, contrairement à la connaissance de la génétique moléculaire et de la formation de la paroi cellulaire des CMA, peu d’information est disponible au sujet de ces propriétés mécaniques. Les informations sur la germination des CMA dans des conditions hypertoniques sont aussi rares, et les modèles expérimentaux ne séparent généralement pas les effets directs de la forte pression osmotique externe sur la germination des champignons et les effets attribuables aux plantes. Cette étude avait pour but de répondre à deux importantes séries de questions concernant le comportement des spores mycorhiziennes. Nous avons d'abord déterminé la relation entre la composition de la paroi cellulaire, la structure et les propriétés mécaniques du champignon modèle Glomus irregulare (isolat DAOM 197198). La micro-indentation a été utilisée pour mesurer quantitativement les propriétés mécaniques de la paroi cellulaire. La composition (contenu de chitine et de glomaline) de la paroi cellulaire a été quantifiée par immunofluorescence tandis que la microscopie optique a été utilisée pour mesurer l'épaisseur de la paroi cellulaire. La densité locale en glomaline et l’épaisseur de la paroi étaient significativement plus élevées pour les parois des spores extraracinaires alors que la densité locale en chitine et la rigidité n’ont pas montré de variations entre les spores extraracinaires et intraracinaires. La grande variabilité dans les paramètres étudiés nous a empêchés de cibler un facteur principal responsable de la force totale de la paroi lors de la compression. La diminution des concentrations de chitine et de glomaline a été corrélée à l'évolution de la paroi du champignon au cours de son cycle de vie. On a aussi observé une composition différentielle des couches de la paroi: les polymères de chitine et de glomaline furent localisés principalement dans les couches externes et internes de la paroi, respectivement. Dans la deuxième partie de notre travail, nous avons exploré les effets directs d'engrais, par rapport à leur activité de l'eau (aw), sur la germination des spores et la pression de turgescence cellulaire. Les spores ont été soumises à trois engrais avec des valeurs de aw différentes et la germination ainsi que la cytorrhyse (effondrement de la paroi cellulaire) des spores ont été évaluées après différents temps d'incubation. Les valeurs de aw des engrais ont été utilisées comme indicateurs de leurs pressions osmotiques. L'exposition des spores de Glomus irregulare au choc osmotique causé par les engrais dont les valeurs de aw se situent entre 0,982 et 0,882 a provoqué des changements graduels au niveau de leur cytorrhyse et de leur germination. Avec l'augmentation de la pression de turgescence externe, la cytorrhyse a augmenté, tandis que le taux de germination a diminué. Ces effets ont été plus prononcés à des concentrations élevées en éléments nutritifs. La présente étude, bien qu’elle constitue une étape importante dans la compréhension des propriétés mécaniques et osmotiques des spores de CMA, confirme également que ces propriétés dépendent probablement de plusieurs facteurs, dont certains qui ne sont pas encore identifiés. / Spore germination is an essential developmental stage in the life cycle of many filamentous fungi. Arbuscular mycorrhizal fungi (AMF) form a number of different infectious propagules that increase their potential to colonize roots. Among them are extraradical and intraradical spores. The spore cell wall plays a major role in the survival of these propagules by being a physical and osmotic barrier. Because a cell can make considerable adjustments to the composition and structure of its wall in response to environmental conditions, it is possible that intraradical and extraradical spore walls show different mechanical and osmotic properties affecting their survival and germination. However, in contrast to the knowledge on the genetics and molecular composition of AMF cell wall, little is known about its mechanical properties. Information on the germination of AMF under hypertonic conditions is scarce, and experimental designs and methodologies have generally not allowed the direct effects of high external osmotic pressure on fungal germination to be separated from plant-mediated effects. This study had the goal to address two important sets of questions regarding the behavior of mycorrhizal spores. We first determined the relationship between cell wall composition, structure and mechanical properties of the model fungus Glomus irregulare. Micro-indentation was used to quantitatively measure the cell wall mechanical properties. Cell wall composition (chitin and glomalin content) was studied by immunofluorescence whereas optical microscopy was used to measure the cell wall thickness. Glomalin local density and wall thickness were both significantly higher for extraradical spore walls while chitin local density and rigidity were unaffected by origin of spores. High variability in results prevented us from identifying a primary factor responsible for overall wall strength during compression. Decreases of chitin and glomalin concentrations were correlated to the development of the fungal wall throughout its life-cycle. There was also differential association within the wall layers: The chitin and glomalin polymers were localized mostly in the outer and inner walls, respectively. In the second part of our work, we explored the direct effects of fertilizers, in relation to their water activity (aw), on spore germination and cellular turgor pressure. Spores were exposed to three fertilizers with different aw and spore germination and cytorrhysis of spores were assessed after different times of incubation. Water activities of the fertilizers were used as indicators of their osmotic pressures. Osmotic shock exposure of the Glomus irregulare spores to fertilizers at aw values between 0.982 and 0.882 caused gradual changes in cytorrhysis and germination. With the increase of external turgor pressure, cytorrhysis increased while the rate of germination decreased. These effects were most pronounced at high nutrient concentrations. The present investigation, while likely representing a significant step forward in understanding the mechanical and osmotic properties of AMF spores, also confirms that they might depend on many, as yet unidentified factors. Future research should examine differences in the physiology to discern reasons for such differences in spore properties.

La germination des spores est une étape essentielle dans le cycle de vie de la majorité des champignons filamenteux. Les champignons mycorhiziens à arbuscules (CMA) forment un certain nombre de propagules infectieuses différentes qui augmentent leur potentiel à coloniser les racines. Parmi elles se trouvent les spores extraracinaires et intraracinaires. La paroi cellulaire des spores joue un rôle majeur dans la survie de ces propagules en étant une barrière physique et osmotique. Puisque une cellule peut faire des ajustements considérables dans la composition et la structure de sa paroi, en réponse aux conditions environnementales, il est possible que les parois des spores intraracinaires et extraracinaires montrent des propriétés mécaniques et osmotiques différentes affectant leur germination et leur survie. Pourtant, contrairement à la connaissance de la génétique moléculaire et de la formation de la paroi cellulaire des CMA, peu d’information est disponible au sujet de ces propriétés mécaniques. Les informations sur la germination des CMA dans des conditions hypertoniques sont aussi rares, et les modèles expérimentaux ne séparent généralement pas les effets directs de la forte pression osmotique externe sur la germination des champignons et les effets attribuables aux plantes. Cette étude avait pour but de répondre à deux importantes séries de questions concernant le comportement des spores mycorhiziennes. Nous avons d'abord déterminé la relation entre la composition de la paroi cellulaire, la structure et les propriétés mécaniques du champignon modèle Glomus irregulare (isolat DAOM 197198). La micro-indentation a été utilisée pour mesurer quantitativement les propriétés mécaniques de la paroi cellulaire. La composition (contenu de chitine et de glomaline) de la paroi cellulaire a été quantifiée par immunofluorescence tandis que la microscopie optique a été utilisée pour mesurer l'épaisseur de la paroi cellulaire. La densité locale en glomaline et l’épaisseur de la paroi étaient significativement plus élevées pour les parois des spores extraracinaires alors que la densité locale en chitine et la rigidité n’ont pas montré de variations entre les spores extraracinaires et intraracinaires. La grande variabilité dans les paramètres étudiés nous a empêchés de cibler un facteur principal responsable de la force totale de la paroi lors de la compression. La diminution des concentrations de chitine et de glomaline a été corrélée à l'évolution de la paroi du champignon au cours de son cycle de vie. On a aussi observé une composition différentielle des couches de la paroi: les polymères de chitine et de glomaline furent localisés principalement dans les couches externes et internes de la paroi, respectivement. Dans la deuxième partie de notre travail, nous avons exploré les effets directs d'engrais, par rapport à leur activité de l'eau (aw), sur la germination des spores et la pression de turgescence cellulaire. Les spores ont été soumises à trois engrais avec des valeurs de aw différentes et la germination ainsi que la cytorrhyse (effondrement de la paroi cellulaire) des spores ont été évaluées après différents temps d'incubation. Les valeurs de aw des engrais ont été utilisées comme indicateurs de leurs pressions osmotiques. L'exposition des spores de Glomus irregulare au choc osmotique causé par les engrais dont les valeurs de aw se situent entre 0,982 et 0,882 a provoqué des changements graduels au niveau de leur cytorrhyse et de leur germination. Avec l'augmentation de la pression de turgescence externe, la cytorrhyse a augmenté, tandis que le taux de germination a diminué. Ces effets ont été plus prononcés à des concentrations élevées en éléments nutritifs. La présente étude, bien qu’elle constitue une étape importante dans la compréhension des propriétés mécaniques et osmotiques des spores de CMA, confirme également que ces propriétés dépendent probablement de plusieurs facteurs, dont certains qui ne sont pas encore identifiés. / Spore germination is an essential developmental stage in the life cycle of many filamentous fungi. Arbuscular mycorrhizal fungi (AMF) form a number of different infectious propagules that increase their potential to colonize roots. Among them are extraradical and intraradical spores. The spore cell wall plays a major role in the survival of these propagules by being a physical and osmotic barrier. Because a cell can make considerable adjustments to the composition and structure of its wall in response to environmental conditions, it is possible that intraradical and extraradical spore walls show different mechanical and osmotic properties affecting their survival and germination. However, in contrast to the knowledge on the genetics and molecular composition of AMF cell wall, little is known about its mechanical properties. Information on the germination of AMF under hypertonic conditions is scarce, and experimental designs and methodologies have generally not allowed the direct effects of high external osmotic pressure on fungal germination to be separated from plant-mediated effects. This study had the goal to address two important sets of questions regarding the behavior of mycorrhizal spores. We first determined the relationship between cell wall composition, structure and mechanical properties of the model fungus Glomus irregulare. Micro-indentation was used to quantitatively measure the cell wall mechanical properties. Cell wall composition (chitin and glomalin content) was studied by immunofluorescence whereas optical microscopy was used to measure the cell wall thickness. Glomalin local density and wall thickness were both significantly higher for extraradical spore walls while chitin local density and rigidity were unaffected by origin of spores. High variability in results prevented us from identifying a primary factor responsible for overall wall strength during compression. Decreases of chitin and glomalin concentrations were correlated to the development of the fungal wall throughout its life-cycle. There was also differential association within the wall layers: The chitin and glomalin polymers were localized mostly in the outer and inner walls, respectively. In the second part of our work, we explored the direct effects of fertilizers, in relation to their water activity (aw), on spore germination and cellular turgor pressure. Spores were exposed to three fertilizers with different aw and spore germination and cytorrhysis of spores were assessed after different times of incubation. Water activities of the fertilizers were used as indicators of their osmotic pressures. Osmotic shock exposure of the Glomus irregulare spores to fertilizers at aw values between 0.982 and 0.882 caused gradual changes in cytorrhysis and germination. With the increase of external turgor pressure, cytorrhysis increased while the rate of germination decreased. These effects were most pronounced at high nutrient concentrations. The present investigation, while likely representing a significant step forward in understanding the mechanical and osmotic properties of AMF spores, also confirms that they might depend on many, as yet unidentified factors. Future research should examine differences in the physiology to discern reasons for such differences in spore properties.

No description available.

No description available.