Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-25T15:11:49Z No. of bitstreams: 2 Dissertação - Mariane Brom Sobreiro - 2017.pdf: 16013047 bytes, checksum: 6d41859718e06adc6294f5de038bc631 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-25T15:12:31Z (GMT) No. of bitstreams: 2 Dissertação - Mariane Brom Sobreiro - 2017.pdf: 16013047 bytes, checksum: 6d41859718e06adc6294f5de038bc631 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-25T15:12:31Z (GMT). No. of bitstreams: 2 Dissertação - Mariane Brom Sobreiro - 2017.pdf: 16013047 bytes, checksum: 6d41859718e06adc6294f5de038bc631 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Considering the rate of increase in average annual temperature and the seasonality of rainfall in several regions of the country, investigations on the mechanisms of plant’s response to low water availability become relevant. Tabebuia Alliance species - monophyletic clade of the Bignoniaceae family are commonly known as ipe – are distribuited in areas with different soil and climatic conditions. This feature makes them an interesting model to understand mechanisms tolerance’s to abiotic stresses. For each species there were two groups: control group, which had maintained irrigation; low water availability group, which irrigation was stopped and the experiment continued until the subtrate reached 40% of field capacity. The main objective of this work was to identify differentially expressed genes (DEG) in four species - two from the Brazilian savannah (T. aurea and Handroanthus ochraceus) and two from seasonally dry forests (H. impetiginosus and H. serratifolius). Then, RNA was extracted from the plants for sequencing on the Illumina platform with paired-end sequences of 100 base pairs (bp). Sequences were evaluated for quality control and mapped onto the genome of H. impetiginosus to identify DEG using the R software. The DGEs obtained by DESeq2 were subjected to functional enrichment analysis and potential changes in the level of expression of genes encoding enzymes of particular metabolic pathways. In all tools, H. serratifolius species showed the highest number of DGE (4908 noDESeq2), while H. ochraceus had the lowest number of DGE (6 in DESeq2). Functional enrichment analyzes demonstrated that the species presented, individually or collectively, typical responses of low water availability such as decrease of photosynthetic rate, increase of proline and increase of starch degradation. Although species share some responses, the complexity of organisms does not allow them to exhibit identical behaviors. / Considerando-se o aumento crescente da temperatura média anual e a sazonalidade das chuvas em diversas regiões do país, investigações acerca dos mecanismos de resposta vegetais à baixa disponibilidade hídrica tornam-se pertinentes. Espécies da Aliança Tabebuia – clado monofilético da família Bignoniaceae são comumente conhecidas como ipê – possuem uma ampla distribuição, ocupando áreas com diferentes condições edafoclimáticas. Esta característica torna-as um modelo interessante para se entender os mecanismos de tolerância a estresses abióticos. Sob este cenário, o presente trabalho teve como principal objetivo a identificação de genes diferencialmente expressos (GDE) em quatro espécies – das quais duas são de cerrado stricto sensu (T. aurea e Handroanthus ochraceus) e duas de floresta estacional (H. impetiginosus e H. serratifolius). Para cada espécie houve dois grupos experimentais: grupo controle, o qual teve a irrigação mantida; grupo de baixa disponibilidade hídrica, o qual a irrigação foi interrompida e o experimento mantido até que o subtrato atingisse 40% da capacidade de campo. Em seguida, extraiu-se o RNA das plantas para sequencimento em plataforma Illumina com sequências do tipo “paired-end” de 100 pares de base (pb). As sequências foram avaliadas para o controle de qualidade e alinhadas no genoma de H. impetiginosus para identificação de genes diferencialmente expressos (GDEs) com o uso de pacotes do R. Os GDEs obtidos pelo DESeq2 foram submetidos a análise de enriquecimento funcional e potenciais alterações no nível de expressão de genes que codificam enzimas de determinadas vias metabólicas. Em todas as ferramentas, a espécie H. serratifolius apresentou o maior número de GDEs (4908 noDESeq2), enquanto H. ochraceus apresentou o menor número de GDEs (6 no DESeq2). As análises de enriquecimento funcional demonstraram que as espécies apresentaram, individual ou coletivamente, respostas típicas de baixa disponibilidade hídrica como diminuição de taxa fotossintética, aumento de prolina e aumento da degradação de amido. Ainda que as espécies compartilhem algumas respostas, a complexidade dos organismos não permite que exibam comportamentos idênticos.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The difference in rainfall throughout the year is a recurring problem that the plants in the field must contend as a result of the moments of drought to which they can be submitted. In the Caatinga, this problem is even greater, since the rains are very sparse and irregular, causing drought for months. The plants of the Caatinga have several adaptations to resist such conditions. This work aimed to evaluate physiological parameters of two endemic species of the Caatinga, the |catingueira| (Poincianella pyramidalis) and |pinhão-bravo| (Jatropha mollissima), in search to discover what strategies these species adopt to survive the drought. Two collections, one in the dry season and one in the wet, were made to evaluate parameters of water relations, concentrations of organic solutes and photosynthetic pigments, chlorophyll a fluorescence and gas exchange. The |catingueira| shown to have been damaged by drought, with a significant reduction in their rates of water relations, particularly in the relative water content, which reached values below 60%, and the performance indices of chlorophyll a fluorescence. The |pinhão-bravo| was able to maintain their high tissue turgor, but the rates of fluorescence and net photosynthesis demonstrated that he was affected by drought since their photosynthetic parameters suffered very significant reductions. / A diferença de precipitação ao longo do ano é um problema recorrente que as plantas em campo precisam enfrentar, em decorrência dos momentos de déficit hídrico aos quais elas podem ser submetidas. Na Caatinga, esse problema é ainda maior, já que as chuvas são muito esparsas e irregulares, ocasionando meses a fio de estiagem. As plantas da Caatinga possuem diversas adaptações para suportar tais condições. Esse trabalho teve por objetivo avaliar parâmetros fisiológicos de duas espécies endêmicas da Caatinga, a catingueira (Poincianella pyramidalis) e o pinhão-bravo (Jatropha mollissima), em busca de descobrir que estratégias essas espécies adotam para sobreviver ao período de estiagem. Foram feitas duas coletas, uma na estação seca e outra na chuvosa, para avaliar parâmetros de relações hídricas, concentrações de solutos orgânicos e de pigmentos fotossintéticos, fluorescência de clorofila a e trocas gasosas. A catingueira demonstrou ter sido bastante afetada pela seca, com redução significativa nos seus parâmetros de relações hídricas, sobretudo no teor relativo de água, que atingiu valores abaixo de 60%, e nos índices de desempenho da fluorescência da clorofila a. O pinhão-bravo conseguiu manter a turgescência de seus tecidos alta, mas os índices da fluorescência e da fotossíntese líquida demonstraram que ele também foi afetado pelo déficit hídrico, uma vez que seus parâmetros fotossintéticos sofreram reduções bastante significativas.

Massive parallel sequencing (MPS) data analysis tasks are often computationally demanding and their execution time would take too long using standard computing machines. Thus there is a need for parallelization of this tasks and ability to execute them on a sufficiently powerful computing machines. In the first chapter we describe a newly created platform for resequencing analysis of MPS data - MOLDIMED and novel annotation tool, which is ready to deploy on HPC infrastructure. The second chapter describes MPS approaches in Diamond-Blackfan anaemia (DBA), which is predominantly underlined by mutations in genes encoding ribosomal proteins (RP); however, its etiology remains unexplained in approximately 25% of patients. We performed panel sequencing of all ribosomal genes in DBA patient without previously known molecular pathology. A novel heterozygous RPS7 mutation coding RPS7 p.V134F was found in one female patient and subsequently confirmed in two asymptomatic family members, in whom mild anemia were detected on further examination. Subsequently, we performed whole transcriptome analysis in all family members and patient with RPS7 mutation in comparison with healthy control group and with DBA patients with known mutation in RPS19. We observed dysregulation mainly in signal pathways of translation,...

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réalisé en cotutelle avec la Faculté des Sciences de Tunis, Université Tunis El Manar. / Le pois chiche (Cicer arietinum L.) a l’avantage de pouvoir assimiler l'azote atmosphérique grâce à son association symbiotique avec des bactéries du genre Mesorhizobium. Malgré cet effet bénéfique sur les systèmes culturaux, le pois chiche réduit parfois la productivité du blé qui la suit. Cet effet négatif du pois chiche pourrait provenir d’une réaction allélopathique à ses exsudats racinaires ou résidus, ou de changements inopportuns dans la communauté microbienne du sol induits par la plante. L'amélioration des interactions symbiotiques du pois chiche pourrait améliorer la performance économique et environnementale des systèmes culturaux basés sur le blé. L’objectif à long terme de ce travail est d'améliorer l’influence du pois chiches sur son environnement biologique et sur la productivité du système cultural. À court terme, nous voulons 1) vérifier l'effet des champignons endophytes sur la performance de cultivars de pois chiche de type desi et kabuli, particulièrement en conditions de stress hydrique, ainsi que sur celle d’une culture subséquente de blé dur, 2) identifier des cultivars de pois chiche capables d’améliorer la qualité biologique de sols cultivés, 3) vérifier que des composés biologiquement actifs sont présents dans les racines des différents cultivars de pois chiches et 4) définir la nature de l’activité (stimulation ou inhibition) des ces composés sur les champignons endomycorhiziens à arbuscules (CMA), qui sont des microorganismes bénéfiques du sol reconnus. L’inoculation du pois chiche avec des champignons endophytes indigènes en serre a augmenté la tolérance à la sécheresse du cultivar de type kabuli à feuille simple CDC Xena et amélioré la nutrition azotée et phosphatée d’un cultivar de type desi, cv. CDC Nika, cultivé en conditions de stress hydrique. La germination des graines de blé dur fut meilleure lorsque celles-ci étaient semées dans les débris de pois chiche inoculé de type kabuli. Le sol dans lequel le génotype de pois chiche à feuille simple CDC Xena fut cultivé mais duquel tout le matériel végétal de pois chiche fut retiré a fortement inhibé la germination des semences de blé dur, ce qui suggère un effet des exsudats racinaires sur la communauté microbienne du sol associée à cette variété de pois chiche. En champ, les cultivars de pois chiche ont influencé différemment la composition des communautés de champignons de la rhizosphère. Les espèces de champignons pathogènes étaient infréquentes et les espèces saprotrophiques et de CMA étaient fréquentes dans la zone des racines du cultivar de type desi CDC Anna. L’effet des composés contenus dans les fractions séparées par HPLC et solubles en solution de méthanol à 25% et 50% de l’extrait racinaire de ce cultivar sur la germination de spores de CMA a été testé in vitro. Les deux espèces de CMA utilisées ont répondu différemment à l’exposition aux composés testés, révélant un mécanisme impliqué dans l’association préférentielle entre les plantes hôtes et les CMA qui leurs sont associés. Nous concluons que le génotype de pois chiche influence la composition de la communauté microbienne qui lui est associée et que cette influence est reliée au moins en partie aux molécules bioactives produites par les racines de la plante. D’autre part, la productivité du pois chiche et de la culture subséquente pourrait être favorisée par la manipulation de leurs champignons endophytes par inoculation. / Chickpea (Cicer arietinum L.) has the ability to bring free N into cropping systems, but is only a fair rotation crop, leading to lower yield in following wheat crops, as compared to medic, vetch or lentil. The negative effects of a chickpea plant on the following wheat crops could come from chickpea root exudates, their residues or their influence on the soil microbial community. The identification of chickpea cultivars best able to promote soil biological quality and the growth of a subsequent crop in rotation will help farmers in selecting better crop rotations and, thus, will improve crop management in soil zone growing chickpea. The global objective of this research is to improve the fitness of chickpea crops to their biological environment and to improve the ability of the plant to enhance soil biological quality. The specific objectives were (1) to verify that the productivity of chickpea and subsequent crops could be promoted through the inoculation by some indigenous endophytic fungi particularly under drought stress conditions (2) to verify the existence of variation in the rhizospheric associations of field-grown chickpea, as it is a necessary condition for the selection of genotypes with improved compatibility with beneficial microorganisms. (3) to identify the biologically active compounds present in the root extracts of chickpea cultivars with contrasting phenotypes, and assess their effect on beneficial and pathogenic soil microorganisms. The greenhouse experiments show that inoculation with indigenous endophytes increased drought tolerance of the unifoliate Kabuli chickpea CDC Xena and the N and P nutrition of the drought stressed Desi chickpea CDC Nika. Inoculation of both Kabuli chickpea varieties with indigenous endophytes improved wheat seeds germination in tissues amended soil. Residue-free soil previously growing the unifoliate Kabuli chickpea CDC Xena strongly inhibited durum seed germination suggesting an effect of root exudates on the soil microbial community, with this Kabuli chickpea variety. In a field experiment, the fungal diversity in cultivated Prairie dryland appeared to host a large array of fungal groups known to reduced plant nutrient, water and biotic stresses, and chickpea genotypes influenced differently the composition and biomass of the soil microbial community. The Desi chickpea CDC Anna was associated with high diversity of arbuscular mycorrhizal fungi (AMF) and culturable fungi, favored the proliferation of soil bacteria and fungal genus hosting biocontrol agents, and developed high AM root colonization level, as compared to the three Kabuli genotypes examined. The HPLC fractions of the roots of chickpea cultivar CDC Anna were recovered and the effects of these fractions on AM fungal spore germination were assayed in multi-well plates. Root extract fractions affect in a different ways the percentage of spores’ germination of Glomus etunicatum and Gigaspora Rosea. We concluded that the genotype of chickpea plants influences the composition of the associated microbial community, and this influence may be related to molecular signals produced by the plants. Furthermore, the productivity of chickpea and subsequent crops could be promoted through the inoculation with indigenous endophytic fungi.

Der Klimawandel bedroht Ökosysteme auf der ganzen Welt. Besonders der Anstieg in Länge, Intensität und Häufigkeit von Dürren kann bedeutenden Einfluss auf den globalen Kohlenstoffkreislauf haben. Die Frage, ob Pflanzen und Mikroorganismen anfällig gegenüber ökologischem Stress wie Dürren sind, wurde bereits in vielen Studien für verschiedene Ökosysteme und mit verschiedenen Ansätzen untersucht, aber Analysen von Dürreauswirkungen, die ober- und unterirdische Interaktionen von Pflanzen und Mikroorganismen mit einbeziehen, sind eher selten. Deshalb wird in der vorliegenden Studie die Frage erörtert, wie Trockenheit und/oder Hitze die Interaktionen von Pflanzen und Mikroorganismen in Bezug auf ihre Kohlenstoff-Verbindung beeinflussen. Dies dient zur Bestimmung der Stärke der Pflanze-Mikroorganismen-Kohlenstoff-Verbindung, wenn das Ökosystem an seine Grenzen gebracht wird. Der Fokus liegt deshalb auf durch Trockenstress und Hitze hervorgerufenen Veränderungen in der ober-unterirdischen Kohlenstoff-Dynamik in zwei vom Klimawandel bedrohten Ökosystemen. Es wurde untersucht, wie extreme Klimaereignisse, deren Häufigkeit in Zukunft weiter ansteigen soll, die Kohlenstoff-Verbindung zwischen Pflanzen und Mikroorganismen beeinflusst und wie mikrobielle Gemeinschaften unter diesen Umständen reagieren, um die Resistenz und Reaktionsmechanismen von Ökosystemen im zukünftigen Klimawandel besser vorhersagen zu können. In Kapitel 4 wurde ein Buchenwaldunterholz-Ökosystem untersucht. Buchenwaldmonolithen wurden einem extremen Klimaereignis (Trockenheit und/oder Hitze) ausgesetzt. Die Stärke der Pflanze-Mikroorganismen-Kohlenstoff-Verbindung und Veränderungen in der mikrobiellen Gemeinschaftsstruktur und -aktivität wurden mithilfe von stabilen 13C Isotopenmethoden und Ansätzen auf molekularer Basis, wie 16S rRNA- und Phospholipid-Analysen, bestimmt. In Kapitel 5 wurde ein kleines aquatisches Ökosystems untersucht. Zwei emerse aquatische Makrophyten, Phragmites australis und Typha latifolia, wurden in einem Mesokosmos-Experiment mit Sediment aus einem Soll einer einmonatigen Dürre ausgesetzt. Mithilfe einer 13CO2 Pulsmarkierung, sowie PLFA- und nicht-strukturbildenden Kohlenhydrat-Analysen wurde Kohlenstoff von den Blättern in die Wurzeln bis ins Sediment verfolgt, wo er teilweise in mikrobielle Phospholipide eingebaut wird. Diese Studie hat gezeigt, dass die zwei untersuchten Ökosysteme Trockenstress und Hitze relativ gut widerstehen können, zumindest kurzfristig, und dass das Kohlenstoff-Kontinuum, beziehungsweise die Verbindung zwischen ober- und unterirdischen Gemeinschaften, auch unter starkem Stress intakt bleibt. Zusammenfassend scheint es, dass Ökosysteme stark von einem funktionierenden Pflanze-Boden/Sediment-Mikroorganismen Kohlenstoff-Kontinuum abhängen und versuchen, es auch unter starkem Stress zu erhalten, was möglicherweise dazu beiträgt, dem Anstieg von extremen Dürreperioden aufgrund des Klimawandels besser zu widerstehen. / Climate change is threatening ecosystems around the world. Especially the increase in duration, intensity, and frequency of droughts can have a considerable impact on the global carbon cycle. The question whether plants and microbes are susceptible to environmental stress like drought has been assessed in many studies for different ecosystem types and by using numerous approaches, but research on drought effects that includes above- and belowground interactions is rather scarce. Therefore, the present study assesses the question of how drought and/or heat influence the interactions of plants and microbes, especially the carbon coupling, in order to determine the strength of plant-microbe carbon linkages when an ecosystem is pushed to its limits. The focus of this study thus lies on changes in aboveground-belowground carbon dynamics and the subsequent effects on the soil microbial community under drought and/or heat stress in two climate-threatened ecosystems. It was evaluated how extreme climate events, that are predicted to be more frequent in the near future, affect the carbon coupling between plants and microorganisms and how microbial communities respond under these circumstances, in order to be able to better predict ecosystem resistance and response mechanisms under future climate change. In chapter 4 a beech forest understory ecosystem was investigated. An extreme climate event (drought and/or heat) was imposed on beech forest monoliths and the strength of the plant-microbe carbon linkages and changes in the microbial community structure and activity were determined by using stable 13C isotope techniques and molecular-based approaches like 16S rRNA and microbial phospholipid-derived fatty acid (PLFA) analysis. In chapter 5 a small aquatic ecosystems was investigated. Two emergent aquatic macrophytes, Phragmites australis and Typha latifolia, were grown on kettle hole sediment and then exposed to a month-long summer drought in a mesocosm experiment. By conducting a 13CO2 pulse labeling as well as PLFA and non-structural carbohydrate analyses, the fate of carbon was traced from the plant leaves to the roots and into the sediment, where some of the recently assimilated carbon is incorporated into microbial PLFAs. Overall, this study showed that the two investigated ecosystems can endure environmental stress like heat and drought relatively well, at least in the short-term, and that the carbon continuum, or the linkage between above- and belowground communities, remained intact even under severe stress. In conclusion, it seems that ecosystems strongly depend on and try to maintain a functional plant-soil/sediment microorganism carbon continuum under drought, which might help to withstand the increase in extreme drought events under future climate change.

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