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Les associations symbiotiques constituent un moteur majeur de la diversification écologique et évolutive des organismes métazoaires. Chez les insectes, elles ont conduit, au cours de l’évolution, à l’émergence d’un nouveau type cellulaire spécialisé dans l’hébergement des bactéries symbiotiques, les bactériocytes. Ces cellules demeurent une énigme fascinante de la symbiose, les processus déterminant leur développement, leur morphogenèse et leur dégénérescence restant encore méconnus. Dans cette étude, nous avons utilisé la symbiose entre le puceron Acyrthosiphon pisum et son endosymbiote obligatoire, Buchnera aphidicola, comme système modèle. En combinant des approches inédites de cytométrie en flux et d’imagerie cellulaire, nous avons démontré une régulation fine et coordonnée des dynamiques de croissance et de dégénérescence des bactériocytes et des symbiotes bactériens, en accord avec les besoins physiologiques de l’insecte. De l’embryon à l’âge adulte, les cellules symbiotiques croissent de manière exponentielle, répondant aux besoins nutritionnels de l’hôte qui nécessite pour son développement de grandes quantités d’acides aminés essentiels produits par le métabolisme bactériocytaire. Avec la sénescence du puceron, les bactériocytes diminuent en nombre, en taille et subissent une dégénérescence progressive. Ce processus dégénératif ne montre pas les signes classiques de l’apoptose. Il résulte d’une hypervacuolisation cytoplasmique, dérivée du réticulum endoplasmique, déclenchant une cascade de réponses cellulaires dont l’activation des voies autophagique et lysosomale. Ce phénomène de mort cellulaire non-apoptotique en deux étapes, rappelant la paraptosis, n’a jamais été décrit chez les insectes et sa découverte ouvre la voie à l’étude des régulations agissant sur l’homéostasie bactériocytaire. Dans le dernier volet de cette thèse, nous avons procédé à l’étude fonctionnelle du gène PAH, fortement exprimé dans les bactériocytes et potentiellement impliqué dans la régulation de leur homéostasie. Les résultats obtenus n’ont pas révélé de phénotype bactériocytaire, après ARN interférence, mais ont permis de démontrer un rôle essentiel de ce gène dans la morphogenèse des insectes. / Symbiotic associations constitute a driving force in the ecological and evolutionary diversification of metazoan organisms. Over the evolution, they have led to the emergence, in insects, of a novel eukaryotic cell type, the bacteriocytes, specialized in harboring symbiotic bacteria. These cells constitute a fascinating enigma in cell biology, as the processes underpinning their development, morphogenesis and degeneration remain still unsolved. In my PhD thesis, we have used the nutritional symbiosis between the aphid, Acyrthosiphon pisum, and its obligate endosymbiont, Buchnera aphidicola, as a model system. We have first developed a novel approach for counting symbiotic bacteria, based on flow cytometry, and showed that the endosymbiont population increases exponentially throughout aphid nymphal development, with a growing rate that has never been characterized by indirect molecular techniques. Using histology and imaging techniques, we have shown that bacteriocytes also increase significantly in number and size during nymphal development. Once adulthood is reached, the dynamics of symbiont and host cells is reversed: the number of endosymbionts decreases progressively and bacteriocytes start to degenerate. These results show a coordination of the cellular dynamics between bacteriocytes and primary symbionts, and reveal a fine-tuning of aphid symbiotic cells to the nutritional demand imposed by the host physiology throughout development. Interestingly, the degenerative process that bacteriocytes undergo with aging exhibits morphological features distinct from the evolutionary conserved apoptotic cell deaths. It originates from an extensive ER-derived hypervacuolation, triggering a cascade of cellular stress responses including the activation of autophagy and lysosomal pathways. This stepwise non-apoptotic cell death, sharing several features with paraptosis, has hitherto never been characterized in insects and its discovery opens the way to the identification of the molecular mechanisms acting on bacteriocyte homeostasis. In the last part of this PhD project, we have proceeded to the characterization of the PAH gene functions in aphid physiology, using an RNA interference (RNAi) approach. Our results show that, even though this gene is highly expressed in bacteriocytes, it is not involved in the regulation of their homeostasis. Nevertheless, we have demonstrated a new role for this metabolic gene in insect embryonic development and morphogenesis.

The diploma thesis deals with the development of the market for protein crops. The literature research deals one hand with the distribution and morphology of legumes, and on second hand with the structure and chemical composition of seeds of selected species (pea, soybean and lupine). The literature research also covers the production of protein concentrates and their commercialization. The practical part is mainly engaged in the analysis of market developments of selected species of legumes in the Czech Republic, especially during the years 2000 - 2015, in comparison with the market development of the individual European states (Germany, Austria, Poland, Slovakia and France). The outcome of this diploma thesis is the ordination of statistical data available in clear graphical and tabular outputs, it does not miss time series evaluation of development of individual crops in both the Czech Republic and other selected countries.

Made available in DSpace on 2014-08-20T14:33:03Z (GMT). No. of bitstreams: 1 dissertacao_liana_viviam_ferreira.pdf: 1066029 bytes, checksum: b8436d77d50bb76efeddb4cb96df3326 (MD5) Previous issue date: 2013-03-04 / The cultivation of edible pod peas in greenhouse and hydroponic system can be a viable alternative for obtaining high pods yield in winter and early spring crop-seasons. In addition, this crop system optimizes productive resources and causes reduced environmental impact. Currently, little information is available about edible pods pea crop, especially in greenhouse and hydroponic cultivation conditions. The adoption of this system presupposes to adequate plant density, as well as to produce knowledge and information regarding growth (dry matter production and partitioning) and yield of different genotypes. In this sense, two experiments were conducted with edible pods pea crop grown in hydroponic and greenhouse in winter/spring crop-season at the Campus of the Universidade Federal de Pelotas , in Capão do Leão, RS. The first experiment aimed to evaluate the effect of plant density on dry matter production and partitioning and yield components of edible pods pea crop 'Luana Gigante®'. Five planti densities (3.9, 4.7; 5.9, 7.8 and 11.8 plants m-2) were evaluated from May to November 2011. The second experiment aimed to characterize the growth and production dynamics of two edible pea pods genotypes ('Luana Gigante®' and 'MK10®') in a bifactorial model from May to November 2012. Genotypes composed the plots and evaluation dates composed subplots (0, 15, 30, 45, 60, 75, 95, 115 and 135 days after setting/DAS). In both experiments, biomass was quantified by dry weight of different above-ground plant organs and yield by pods fresh weight. The results obtained in the first experiment indicated that increasing of plant density in the range from 3.9 to 11.8 plants m-2 reduced linearly the growth of all organs and the individual plants pods yield. However, it increased linearly the absolute crop dry matter production and the pods yield per square meter. It did not affect the dry matter partitioning among different plant organs. The vegetative shoot plant organs were the major sinks for photoassimilates, comprising 61.5% of total plant dry matter, while the pods represented 31.7%. Among the yield components, only the number of harvested pods per plant was reduced and there were not negative effects on average pods fresh weight and the percentage of marketable pods. Therefore, we can recommend the plant density of 11.8 plants m-2 for pea crop 'Luana Gigante'. In the second experiment, it was observed that 'Luana Gigante' and 'MK10' presented a sigmoidal type plant growth curve as a function of time. MK10 presented higher vegetative shoot plant parts and crop growth than 'Luana Gigante'. 'MK10' also presented higher pods growth at 95 DAS, but both genotypes presented similar pods growth and yield at the end of the crop cycle. Pods comprised 36% and 43% of the total above-ground dry matter production, respectively, for 'MK10' and 'Luana Gigante' at the end of the crop cycle. Thus 'Luana Gigante' presents greater ability to assimilate distribution to pods growth than 'MK10'. The vegetative shoot organs are the strongest sinks for assimilates of the plant and stems are more beneficed in relation to the partition of dry matter than leaves. / A produção de ervilha torta em ambiente protegido e sistema hidropônico pode ser uma alternativa viável para a obtenção de alto rendimento de vagens no período de inverno e início de primavera, além de proporcionar melhor otimização dos recursos produtivos e reduzido impacto ambiental. Atualmente, existem poucas informações disponíveis sobre esta cultura, principalmente em condições de ambiente protegido e cultivo hidropônico. A adoção deste sistema pressupõe a adequação da densidade de plantio, assim como, produzir conhecimentos e informações referentes ao crescimento (produção e partição de massa seca) e comportamento produtivo de genótipos. Neste sentido, dois experimentos foram realizados com a cultura da ervilha torta em sistema hidropônico e ambiente protegido em ciclo de inverno/primavera no Campus da Universidade Federal de Pelotas, no município de Capão do Leão, RS. O primeiro experimento, realizado de maio a novembro de 2011, objetivou avaliar o efeito da densidade de plantio sobre a produção e a partição da massa seca e os componentes do rendimento de ervilha torta Luana Gigante® , em experimento unifatorial com cinco densidades de plantio (3,9; 4,7; 5,9; 7,8 e 11,8 plantas m-2). O segundo experimento objetivou caracterizar a dinâmica do crescimento e da produção de dois genótipos de ervilha torta ( Luana Gigante e MK10 ) em esquema bifatorial no período de maio a novembro de 2012. As parcelas foram constituídas pelos genótipos e as subparcelas pelas épocas de avaliação das plantas (aos 0, 15, 30, 45, 60, 75, 95, 115 e 135 dias após o transplante). Para ambos os experimentos, a biomassa foi quantificada através da massa seca dos diferentes órgãos aéreos da planta e o rendimento através da massa fresca de vagens. Em relação aos resultados obtidos no primeiro experimento, observou-se que o aumento da densidade de plantio no intervalo entre 3,9 a 11,8 plantas m-2 reduz o crescimento de todos os órgãos e a produtividade individual das plantas de forma linear. Porém, aumenta de forma linear a produção absoluta da massa seca da cultura bem como a produtividade por unidade de área e não afeta a partição proporcional de massa seca entre os diferentes órgãos da planta. Os órgãos vegetativos aéreos são os principais drenos de fotoassimilados, representando 61,5% da massa seca total das plantas, enquanto as vagens representam 31,7%. Entre os componentes do rendimento, somente o número de vagens colhidas por planta é reduzido, não havendo efeitos negativos sobre a massa fresca média das vagens e a porcentagem de vagens comerciais colhidas. Portanto, recomenda-se a densidade de 11,8 plantas m-2 para a ervilha torta Luana Gigante . No segundo experimento, observou-se que Luana Gigante e MK10 expressam crescimento da planta do tipo sigmoidal em relação ao acúmulo de MS ao longo do ciclo de cultivo. MK10 apresenta maior crescimento dos órgãos vegetativos aéreos e da cultura do que Luana Gigante . MK10 também apresenta maior crescimento de vagens aos 95 DAT, porém os genótipos se assemelham em relação ao crescimento e à produção de vagens ao final do ciclo de cultivo. As vagens representam 36% e 43% da massa seca aérea da planta, respectivamente de MK10 e Luana Gigante , ao final do ciclo de cultivo. Assim, Luana Gigante apresenta maior capacidade de destinar assimilados para o crescimento de vagens do que MK10 . O conjunto dos órgãos vegetativos aéreos são os principais drenos de assimilados da planta, sendo os caules priorizados na partição de MS em relação às folhas.

L’association mycorhizienne à arbuscules (AM) est le résultat d’une interaction compatible entre les génomes des deux partenaires symbiotiques. Dans ce contexte, le but de mes recherches a été de mieux caractériser le rôle des gènes de pois liés aux stades tardifs de la symbiose, PsSym36, PsSym33 and PsSym40, dans le fonctionnement de la symbiose MA (i) en étudiant l’effet des mutations de ces trois gènes sur l’expression des gènes de la plante et du champignon, et (ii) en créant les conditions pour positionner deux de ces gènes, PsSym36 and PsSym40, sur la carte génétique afin d’envisager leur clonage futur. L’expression d’un groupe de dix gènes fongiques et de huit gènes de plante, déjà décrits pour être activés durant le développement de la mycorhize, a été comparée dans les racines de pois inoculées avec G. intraradices chez les plantes de génotypes sauvages, ou les mutants Pssym36, Pssym33 et Pssym40. L’expression de la plupart des gènes fongiques a été inhibée dans les racines du mutant Pssym36 où la formation des arbuscules est avortée, tandis que l’expression de plusieurs d’entre eux a été activée lorsqu’il existe un développement plus rapide du champignon dans les racines du mutant Pssym40. Des microdisséquats obtenus à partir de racines mycorhizées du mutant PsSym40 confirment l’expression préférentielle de trois gènes de G. intraradices (SOD, DESAT et PEPISOM) dans les cellules contenant les arbuscules. L’inactivation du gène PsSym36 provoque également une inhibition des gènes de plante alors que la mutation des gènes PsSym33 and PsSym40 affecte l’expression des gènes de plante plutôt de façon temporelle. Les résultats indiquent ainsi une implication des gènes SYM de pois dans la modulation des interactions moléculaires entre la plante et le champignon impliquées au niveau de la signalisation, des échanges nutritifs ou de la régulation des réponses au stress durant la formation et/ou le fonctionnement de la symbiose AM. Les conditions pour la localisation des gènes PsSym36 and PsSym40 sur la carte génétique du pois ont été développées pour leur clonage basé sur la cartographie. En utilisant les marqueurs moléculaires obtenus, il a été possible de conclure que la localisation du gène PsSym40 réside vraisemblablement à l’extérieur des groupes de liaison I, II, III ou V de la carte génétique du pois. / The arbuscular mycorrhizal (AM) association results from a successful interaction between the genomes of the two symbiotic partners. In this context, the aim of my research was to better characterize the role of the late stage symbiosis-related pea genes PsSym36, PsSym33 and PsSym40 in the functional AM (i) by investigating the effect of mutations in the three genes on fungal and plant gene responses and (ii) by creating conditions for the localization of two of the genes, PsSym36 and PsSym40, on the pea genetic map for future map-based cloning. The expression of a subset of ten fungal and eight plant genes,previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated roots of wild type and Pssym36, Pssym33 and Pssym40 mutant pea plants. Most of the fungal genes were down-regulated in roots of the Pssym36 mutant where arbuscule formation is defective, and several were upregulated with more rapid fungal development in roots of the Pssym40 mutant. Microdissection of mycorrhizal PsSym40 roots corroborated preferential expression of the three G. intraradices genes SOD, DESAT and PEPISOM in arbuscule-containing cells. Inactivation of PsSym36 also resulted in down regulation of plant genes whilst mutation of the PsSym33 and PsSym40 genes affected plant gene responses in a more time-dependent way. Results thus indicate an implication of the investigated pea SYM genes in the modulation of plant and fungal molecular interactions linked to signaling, nutrient exchange or stress response regulation during AM symbiosis formation and functioning. Conditions for localization of the PsSym36 and PsSym40 genes on the pea genetic map were developed for their future map-based cloning. Based on the molecular markers obtained, it was possible to conclude that localization of the PsSym40 gene most likely resides outside the linkage groups I, II, III or V of the genetic map of pea. / Формирование арбускулярной микоризы (АМ) является результатом успешного взаимодействия между геномами двух симбиотических партнёров. Целью моего исследования являлось изучение роли поздних симбиотических генов гороха PsSym36, PsSym33 и PsSym40 в формировании функционального АМ симбиоза. Для этого было проведено исследование эффекта мутаций в генах PsSym36, PsSym33 и PsSym40 на экспрессию грибных и растительных генов, предположительно (по литературным данным) вовлечённых в процессы формирования АМ, а так же проведена работа по локализации генов PsSym36 и PsSym40 на генетической карте гороха для последующего более точного картирования и позиционного клонирования данных генов. Экспрессия десяти грибных и восьми растительных генов была определена в корнях растений дикого типа и PsSym36, PsSym33 и PsSym40 мутантов, инокулированных G. intraradices. В корнях PsSym36 мутанта, имеющего дефект развития арбускул, большая часть грибных генов была супрессирована, в то время как в корнях PsSym40 мутанта, для которого характерна более быстрая по сравнению с диким типом микоризация, был отмечен более высокий уровень экспрессии грибных генов. Использование метода микродиссекций позволило выделить клетки, содержащие арбускулы, из микоризованных корней мутанта PsSym40 и подтвердить, что гены G. intraradices SOD, DESAT и PEPISOM преимущественно экспрессируются в клетках, содержащих арбускулы. Мутация в гене PsSym36 также привела к подавлению экспрессии большинства вовлечённых в анализ растительных генов, тогда как мутации в генах PsSym33 и PsSym40 оказали влияние на ксперессию растительных генов в меньшей степени. Полученные результаты свидетельствуют о роли исследуемых SYM генов гороха в контролировании растительно-грибных молекулярных взаимодействий, связанных с сигналингом, обменом питательными веществами и стрессовыми реакциями в процессе формирования и функционирования АМ симбиоза. Проведённое генетическое картирование не привело к локализации генов PsSym36 и PsSym40 на генетической карте гороха. Однако разработка и использование молекулярных маркеров для картирования позволили исключить локализацию гена PsSym40 в I, II, III и V группах сцепления с высокой долей вероятности.

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Field pea is an important annual crop due to its contribution to soil fertility and other rotational benefits. However, weeds and ascochyta blight limit pea yield, particularly in organic systems. Leafed and semi-leafless pea types differ in lodging resistance, and may affect weeds and disease through differences in canopy light penetration and air flow. Mixtures of the two leaf types may improve weed and disease suppression and yield compared with monocultures of the same cultivars. To test this hypothesis, replicated field experiments were conducted under organic and conventional management in Saskatoon and Vonda, SK, in 2011 and 2012. Mixtures of a leafed and semi-leafless cultivar, CDC Sonata and CDC Dakota, were sown in ratios of 0:100, 25:75, 50:50, 75:25, and 100:0 leafed to semi-leafless pea, at target seeding rates of 88 and 132 plants m-2. Conventionally managed plots were inoculated with ascochyta blight-infested pea straw and received overhead irrigation to encourage disease. Mixtures of 50% or more semi-leafless pea adopted the greater lodging resistance and weed suppression of the semi-leafless cultivar. Mixtures comprised of 25% leafed and 75% semi-leafless pea increased both seed and biomass yield compared with either cultivar grown alone. Yield enhancement was attributed to the leafed cultivar, whose seed yield was 76% higher in mixture than expected based on monoculture yield. Ascochyta blight epidemics were of moderate severity, and leafed and semi-leafless monocultures reached 36 and 43% necrosis in 2011, and 33 and 38% necrosis in 2012, respectively. The disease reaction of mixtures fell between the two component cultivars. At disease onset in 2012, lower light interception and shorter moisture durations coincided with the lower ascochyta blight severity of leafed monocultures. In 2011 and the later phase of the 2012 epidemic, disease severity was negatively associated with vine length, and positively associated with number of nodes and tissue senescence. Despite the advantages of leafed and semi-leafless pea mixtures, the limited selection of leafed cultivars impedes adoption of this technique by growers. For pea breeders, developing mixtures of pea lines isogenic for leaf type may increase yield compared with single cultivars.

Des semis précoces permettent d'allonger les cycles de culture du pois protéagineux et d'éviter les stress hydriques et thermiques tardifs, ce qui contribue à la maîtrise des variations de rendement. Les objectifs de ce travail sont 1) d'étudier le comportement de différents génotypes de pois au cours de la germination en réponse à la température ; 2) de déterminer l'impact des vitesses de dessiccation à température élevée sur les caractéristiques des graines, leur germination et leur conservation ; 3) d'explorer la reprise du métabolisme énergétique et la mobilisation des sucres solubles au cours de la germination et de déterminer l'impact d'une vitesse de dessiccation rapide après la phase de remplissage et des basses températures pendant la germination. Les pois de printemps Baccara et Térèse et le pois d'hiver Champagne présentent une température de base identique et très basse de -1,1°C. Le génotype Champagne a une vitesse de germination plus rapide que les deux autres génotypes, des basses températures jusqu'à l'optimum. Les vitesses de dessiccation élevées ont peu d'impact sur la germination mais affectent fortement l'aptitude à la conservation des graines, mesurée après détérioration contrôlée. Cela est associé à une augmentation de la conductivité traduisant des dégâts cellulaires, en particulier au niveau des membranes. La vitesse élevée de germination de Champagne s'explique en partie par une grande vitesse d'imbibition, permettant une respiration plus forte. Une corrélation entre la consommation en oxygène et la production d'ATP a été trouvée. Champagne présente également une capacité de la voie alternative, une utilisation d'ATP et de sucres solubles plus élevées que les génotypes de printemps, Baccara et Térèse. L'utilisation des sucres solubles des axes embryonnaires, après mobilisation des RFO, est bien corrélée avec la vitesse de germination. De fortes vitesses de dessiccation entrainent une augmentation de la respiration qui serait à relier à la mise en place de mécanismes de réparation. Les basses températures retardent le processus de germination et de reprise du métabolisme par leur action sur les systèmes enzymatiques, mais lorsque l'échelle de temps est exprimée en temps thermique, le retard disparaît. Cependant, la respiration du génotype Champagne est moins affectée par les basses températures. Les résultats obtenus ont permis de proposer un schéma global résumant les étapes clés permettant une vitesse de germination rapide et un modèlesur le rôle de l'adénylate kinase et de la phosphorylation oxydative mitochondriale dans la régulation du métabolisme en lien avec l'anhydrobiose.

This diploma work was assessed effects of different seed treatment (untreated flour from whole seed, flour from the uterus after soaking seeds, flour from the seeds sprouted cotyledon) by three species of legumes (Pisum sativum conv. Sativum L., Vicia faba L., Lupinus angustifolius L.) yield and composition of protein isolate obtained by isoelectric precipitation. It was found that the seeds of change most affects yield precipitated N (mg) and 45%. When determining the value of the yield of protein was affected by significant interactions (treatment and the type of legume seeds). The largest share was, however, precipitate in untreated and germinated lupine seeds, in which values are around 57%. Furthermore, the thesis was to reverse the precipitation of proteins, using which we obtained protein concentrates from different species of legumes. The yields of protein concentrates reach values in the range of 60-80%. Spectra of soluble proteins is clearly visible high concentration of isolated proteins. Variation of sprouted seeds and soaked that occurred during treatment of seeds for the collapse of proteins with higher molecular weight. Furthermore, these grafts are transferred protein extraction at pH 9.0 in the later produced protein isolates. After acid precipitation is observed on a spectrum that is re-soluble protein isolates.