A Araucaria angustifolia é uma conífera nativa do Brasil. Em função da sua intensa exploração florestal, a espécie ocupa apenas 2% de sua vegetação natural. Neste sistema, a aplicação de técnicas biotecnológicas, como a embriogênese somática, podem ser integradas a programas de melhoramento genético e conservação. A similaridade entre a embriogênese somática e zigótica, tem sido utilizada para o estabelecimento de estudos visando o aperfeiçoamento do cultivo in vitro dos embriões somáticos, bem como para um maior conhecimento dos aspectos moleculares e fisiológicos que regulam a embriogênese. O metabolismo de poliaminas (PAs), mais especificamente putrescina, espermidina e espermina, tem se mostrado como fundamental para a compreensão e evolução da embriogênese zigótica e somática. Entretanto, a biossíntese das PAs e seu envolvimento nos vários processos biológicos que regulam a embriogênese, são pouco conhecidas em coníferas. Inserido nessa perspectiva, o presente trabalho teve como objetivo o estudo do metabolismo de PAs durante três estádios de desenvolvimento da semente (contendo as fases da embriogênese inicial até a tardia) e na proliferação de linhagens embriogênicas com diferentes potenciais embriogênicos de A. angustifolia. Foram investigados: a) os perfis de PAs (livres e conjugadas) e aminoácidos; b) determinação da via preferencial da biossíntese de putrescina, através da atividade enzimática da arginina descarboxilase (ADC) e ornitina descarboxilase (ODC); c) identificação e caracterização do padrão de expressão dos genes envolvidos no metabolismo de PAs; e d) a identificação das relações entre os perfis de PAs e aminoácidos presentes nas sementes das matrizes, e sua potencial influência nas fases de indução, proliferação e maturação dos embriões somáticos. Durante a embriogênese zigótica, a expressão dos genes AaADC (arginina descarboxilase) e AaSAMDC (S-adenosilmetionina descarboxilase) aumentaram no estádio cotiledonar, juntamente com o aumento de PAs. A biossíntese da putrescina é realizada preferencialmente via ADC, enquanto que a citrulina foi o principal aminoácido presente nas sementes. Em relação ao metabolismo de PAs nas culturas embriogênicas, os dados obtidos demonstraram que a arginina e ornitina parecem ter diferentes funções em cada linhagem testada. Na linhagem com alto potencial embriogênico, a arginina parece estar associada com a ativação dos genes relacionados ao catabolismo de PAs (AaPAO2, AaCuAO e AaALDH), enquanto que esse efeito não foi observado na linhagem bloqueada. A ODC tem uma maior atividade na linhagem responsiva, enquanto que na linhagem bloqueada, as atividades da ADC e ODC são similares. Dependendo da matriz foram observados diferentes perfis de PAs e aminoácidos, sendo estes perfis relacionados com as taxas de indução, proliferação e desenvolvimento dos embriões somáticos. Putrescina total, ornitina e asparagina foram os metabólitos diferencialmente identificados entre as matrizes, os quais podem ser propostos como marcadores bioquímicos para a seleção de matrizes com alto potencial para a embriogênese somática. Os resultados obtidos fornecem informações relevantes e inéditas sobre o metabolismo de PAs e aminoácidos na embriogênese zigótica e somática de A. angustifolia, bem como fornece novos subsídios para o aprimoramento das condições artificiais utilizadas para o desenvolvimento dos embriões somáticos / The Araucaria angustifolia is a native conifer species of Brazil. Due to its intense exploitation, the species cover only 2% of its original forest area. In this system, biotechnological tools, like somatic embryogenesis, may be integrated into breeding and conservation programs. The similarity between zygotic and somatic embryogenesis have been used to establishment of studies in order to optimization of somatic embryos in vitro culture, as well as for a better understanding of physiologic and molecular aspects that modulates the embryogenesis. The metabolism of polyamines (PAs), specifically putrescine, spermidine and spermine, has been demonstrated as fundamental for the comprehension and evolution of zygotic and somatic embryogenesis. However, the biosynthetic pathways of PAs and their involvement in various biological process that regulate the embryogenesis are little known in conifers. Inserted in this perspective, the aim of the current work was to study the metabolism of PAs during three seeds development stages (containing the early till late embryogenesis phases) and in proliferation of cell lines with different embryogenic potential of A. angustifolia. Were investigated: a) PAs (free and conjugated) and amino acids profiles; b) determination of preferential pathway for putrescine biosynthesis, through enzymatic activity of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC); c) identification and characterization of gene expression profile of genes related to metabolism of PAs; and d) identification of the relationship between PAs and amino acids profiles in seeds of mother plants, and their potential influence in initiation, proliferation and maturation phases of somatic embryos. During the zygotic embryogenesis, AaADC (arginine decarboxylase) and AaSAMDC (S-adenosylmethionine decarboxylase) genes were up-regulated at cotyledonary stage along with the increasing of PAs. The biosynthesis of putrescine is performed preferentially by ADC pathway, while citrulline was the main amino acid recorded during the seed development. Regarding the metabolism of PAs in embryogenic cultures, the data demonstrated that arginine and ornithine seem to have different functions in each cell line tested. In cell line with high embryogenic potential, arginine seems to be associated to activation of genes related to PAs catabolism (AaPAO2, AaCuAO e AaALDH), while in blocked cell line this effect was not observed. ODC has a higher enzymatic activity in responsive cell line, while in blocked cell line, both ADC and ODC activities are similar. Depending of mother plant, were observed different PAs and amino acids profiles, being these profiles related with the rate of initiation, proliferation and maturation of somatic embryos. Total putrescine, ornithine and asparagine were the differentially metabolites identified between the mother plants, which can be proposed as biochemical marker to select mother plant with high potential to somatic embryogenesis. The results obtained provide relevant and inedited information about the metabolism of PAs and amino acids in zygotic and somatic embryogenesis of A. angustifolia, as well as provide news subsidies for optimization of in vitro conditions for somatic embryos development

Due to the immense utilization in food and industry, there is enormous commercial and scientific interest to manipulate canola (Brassica napus L.) seed oil. Seed oil accretions are influenced by genes involved in embryo and seed development. FUSCA3 (FUS3) and LEAFY COTYLEDON1 (LEC1) are well-known transcription factors involved during seed and embryo development. The main objective of this project was to evaluate the role of these genes during seed storage deposition and microspore-derived embryogenesis in B. napus. For this purpose, six BnLEC1 transgenic lines and three BnFUS3 TILLING mutant lines were generated. The over expression of BnLEC1 significantly increased the seed oil content, while the down regulation of BnLEC1 or mutation of BnFUS3 reduced the level of seed oil. Experimental alterations of BnLEC1 and BnFUS3 triggered transcriptional modifications in enzymes taking part in sucrose transport and metabolism, glycolysis, and fatty acid (FA) biosynthesis. These changes are suggestive of a greater carbon pool to FA biosynthesis in tissues over-expressing BnLEC1, and a reduced carbon flux available for the synthesis of FA in BnLEC1 down regulators and BnFUS3 tilling mutants. While the elevated oil content induced by BnLEC1 was not accompanied by alterations in FA composition, oil nutritional value, or glucosinolate (GLS) levels, suppression of BnLEC1 reduced seed oil accumulation and raised levels of GLS, possibly through the transcriptional regulation of BnST5a (Sulphotransferase5a), the last GLS biosynthetic enzyme. BnFUS3 tilling mutant seeds had increased levels of linoleic acid, possibly due to the reduced expression of ω-3 FA DESATURASE (FAD3). The effects of altered expression of BnLEC1 and BnFUS3 were also assessed during microspore-derived embryogenesis. Substantial structural abnormalities, accompanied by changes in transcript levels of several embryo marker genes were observed in embryos in which the expression of BnLEC1 or BnFUS3 was altered. The changes in oil level and FA profiles observed in the transformed microspore-derived embryos followed a similar trend to that described in seeds. Collectively, these observations suggest that manipulation of BnLEC1 and BnFUS3 can be employed as a tool to enhance seed oil production and quality in B. napus. / February 2017

Le cancer de la prostate (CaP) est le cancer le plus fréquent chez l'homme et représente la deuxième cause de mortalité par cancer. Cette pathologie est sensible aux androgènes des stades localisés aux stades métastatiques. Après le traitement des formes avancés de ce cIl est admit aujourd'hui que les androgènes seuls ne sont pas suffisants pour déclencher le cancer de la prostate. En effet, le rôle des œstrogènes dans la carcinogenèse prostatique est suggéré par plusieurs études. L'activation de la voie de signalisation Hedgehog (Hh) joue un rôle important dans le développement de plusieurs cancers, y compris le CaP. Une meilleure compréhension des mécanismes qui régulent l'activation de cette voie dans le CaP est nécessaire afin de définir de nouvelles stratégies thérapeutiques plus efficaces.Dans ce travail, nous mettons en évidence l'interaction entre la voie Hh et les hormones stéroïdiennes dans les cellules prostatiques. Nous avons observé : i) une activation de la voie Hh par l'œstrogène (sulfate d'œstrone (SE1)), atténuée par l'anti-œstrogène (ICI) et par l'inhibiteur de la voie Hh (KAAD-cyclopamine), ii) une régulation négative de la voie Hh par l'androgène (dihydrotestostérone (DHT)) et l'œstrogène (17β-œstradiol (E2)). Nous avons démontré que l'inhibition de la voie Hh induite par DHT et E2 est dépendante des récepteurs des androgènes (RA). Cependant, l'effet de SE1 sur la voie Hh pourrait être dépendante des récepteurs des œstrogènes (ER). Enfin, nous avons observé une inhibition de l'activité des RA par KAAD-cyclopamine. Les dérivés de cyclopamine pourraient donc représenter une nouvelle classe d'agents thérapeutiques ciblant le RA dans le cancer de la prostate. Une meilleure caractérisation des cibles potentielles de ces molécules semble être intéressante. / Prostate cancer (PCa) is the most frequent male malignancy and the second most common cause of cancer-related death in men. This cancer is androgen sensitive in its development and progression to metastatic disease. Despite this, increasing evidence suggest that androgens alone are not able to induce PCa and estrogen signaling has a key role in prostate cancer progression. Hedgehog (Hh) pathway activation is important in the growth and development of various carcinomas including PCa. A better understanding of Hh pathway regulating mechanisms in PCa is important in order to identify new therapeutic strategies for this pathology. In this study we investigate the interaction between Hh pathway and steroid hormones in prostate cells. We showed: i) Hh pathway activation by the estrogen (estrone sulfate E1S), attenuated by the anti-estrogen (ICI) and by the Hh pathway inhibitor (KAAD-cyclopamine) ii) Hh pathway negative regulation by the androgen (dihydrotestostérone (DHT)) and the estrogen (17β-estradiol (E2)). Moreover, we showed that Hh pathway inhibition is androgen receptor (AR) dependent. However, E1S effect on this pathway might be estrogen receptor (ER) dependent. Finally, our results suggest that targeting AR signaling by cyclopamine derivatives could be promising therapeutic alternative in prostate cancer, which needs a further investigation.

Chez les métazoaires, la transcription est inactive durant les étapes précoces du développement embryonnaire. Chez Drosophila melanogaster, des études récentes de l'activation du génome zygotique (AGZ) ont mis en évidence l'implication de quelques acteurs moléculaires (Zelda, STAT92E), mais les mécanismes régulateurs généraux restent à découvrir. En appliquant des méthodes bioinformatiques à l'analyse de données à haut débit de différentes sources, j'ai recherché de nouveaux éléments cis-régulateurs impliqués dans l'AGZ. Tout d'abord, par l'analyse de données transcriptomiques, j'ai sélectionné un groupe de gènes activés pendant l'AGZ. L'analyse de leurs régions non codantes a mis en évidence neuf motifs, dont trois correspondent à des motifs connus (Zelda, Trl et facteur TTK). La recherche systématique de ces motifs m'a permis de prédire des modules cis-régulateurs (CRMs) potentiels pour lesquels j'ai défini un environnement chromatinien spécifique en analysant des profils d'occupation de (co-) facteurs de transcription pertinents et d'histones modifiées (ChIP-seq) ainsi que des profils d'ouverture de la chromatine. L'ensemble de ces résultats m'a permis de définir un modèle de régulation de l'AGZ et de sélectionner des régions candidates pour une validation expérimentale. / In metazoa, transcription is silent during early embryogenesis. In Drosophila melanogaster, recent studies of the zygotic genome activation (ZGA) highlighted the implication of few molecular actors (Zelda, STAT92E), but general regulatory mechanisms remains to be understood. Applying bioinformatics analyses on different type of high-throughput data, I searched for new cis-regulatory elements involved in ZGA.First, through analysis of transcriptome data, I selected a group of genes activated during ZGA. Analysis of their non-coding sequences highlighted nine motifs, of which three correspond to known binding motifs and factors (Zelda, Trl and TTK). Systematic research of these motifs led to the prediction of putative cis-regulatory modules (CRMs) for which I defined a specific chromatin environment analyzing occupancy profiles of relevant transcription (co-) factors and modified histones, as well as profiles of chromatin opening. Altogether, these results allowed me to define a regulatory model of the ZGA and select candidate regions for further experimental validation.

Le remodelage de la chromatine et la modification des histones jouent des rôles très importants dans l’établissement et la reprogrammation de l’état de l’expression génique. Il reste largement inconnu concernant les mécanismes de la régulation de ces processus chromatiniens dans le contrôle de l’expression génique impliquée dans le développement de la plante et son adaptation à l’environnement. Mon sujet de thèse se focalise sur l’analyse fonctionnelle d’un facteur de remodelage de la chromatine de type Chromodomain/Hélicase/DNA-binding 1 (CHD1) d’Arabidopsis, appelé CHR5 et une histone démethylase qui est spécifiquement impliquée dans la démethylation de l’histone H3 lysine 4 (H3K4), appelée JMJ15. Dans la première partie de cette étude, nous avons montré que le gène CHR5 est activé au cours de l’embryogénèse et que son expression se maintient élevé dans les tissues/organes en développement. L’analyse de mutants révèle que la perte de fonction de ce gène fait réprimer l’expression de gènes régulateurs de la maturation de l’embryon tels que LEC1, ABI3 et FUS3 pendant le développement des graines, et fait baisser l’accumulation des protéines de réserve. L’analyse de double mutants a permis de démontrer une fonction antagoniste entre CHR5 et PKL, une protéine du groupe « CHD3 », dans l’activité du promoteur de gènes régulateurs du développement de l’embryon et l’accumulation de réserve de graine. Nous avons montré que la protéine CHR5 s’associe directement avec les promoteurs d’ABI3 et FUS3 et que la mutation du gène CHR5 conduit à l’augmentation de présence de nucléosome dans la région du départ de transcription. Ces résultats suggèrent que CHR5 est impliquée dans le positionnement de nucléosome pour stimuler l’expression de gènes de la maturation de l’embryon, ce qui est contrebalancé par l’action de PKL au cours du développement de l’embryon. La deuxième partie de cette étude a permis de montrer que l’expression du gène de l’histone démethylase JMJ15 manifeste une forte spécificité tissulaire. L’analyse de mutants du gène a permis de l’identification de 2 allèles de gain de fonction (avec surexpression du gène), et un allèle de perte de fonction. La surexpression du gène réduit la croissance d’hypocotyle et de tige de la plante avec accumulation de lignine dans la tige, mais le perte de fonction du gène ne produise pas de phénotype apparent. Par ailleurs, la surexpression du gène renforce la tolérance de la plante au stress salin, alors la perte de fonction du gène rend la plante plus sensible. L’analyse du transcriptome a révélé beaucoup plus de gènes réprimés qu’activés par la surexpression du gène JMJ15. Ces gènes réprimés sont préférentiellement marqué la H3K4me2 ou H3K4me3, parmi lesquels beaucoup codent de facteurs de transcription. Ces données suggèrent que l’induction de JMJ15 pourrait réguler le programme de l’expression génique qui coordonne la restriction de la croissance de la plante et la tolérance au stress. Ces travaux de thèse a permis ‘identifier quelques nouveaux éléments dans la compréhension de la fonction de régulateurs chromatiniens dans l’expression génique de la plante. / Chromatin remodeling and histone modification play important roles in the establishment and dynamic regulation of gene expression states. However, little is known regarding to the regulatory mechanism of chromatin modification and remodeling that control gene expression involved in plant development and responses to environmental cues. My thesis work concerns functional analysis of an Arabidopsis Chromodomain/Helicase/DNA-binding 1 (CHD1) type chromatin remodeling gene known as CHR5 and a histone demethylase gene that specifically removes methyl groups from methylated histone H3 lysine 4 (H3K4me), called JMJ15 in regulating chromatin structure or in resetting chromatin modifications that control the expression of plant developmental and stress responsive genes. In the first part of the study we found that CHR5 expression is activated during embryogenesis and remained to be expressed in developing organs/tissues. Analysis of mutants revealed that loss-of-function of the genes led to decreased expression of key embryo maturation genes LEC1, ABI3 and FUS3 in developing seeds and reduced seed storage protein accumulation. Analysis of double mutants revealed an antagonistic function between CHR5 and PKL, a CHD3 gene, in embryo gene promoter activity and seed storage protein accumulation. CHR5 was directly associated with the promoters of ABI3 and FUS3 and chr5 mutations led to increased nucleosome occupancy near the transcriptional start site. The results suggest that CHR5 is involved in nucleosome occupancy to regulate embryo identity genes expression, which is counterbalanced by PKL during embryo development. The second part of this study showed that expression of JMJ15 was restricted to a few tissues during vegetative growth. The jmj15 gain-of-function mutations reduced the length of seedling hypocotyls and inflorescence stems with higher accumulation of lignin in the stem, while the loss-of-function mutants did not show any visible phenotype. The gain-of-function mutants enhanced salt tolerance, whereas the loss-of-function mutants were more sensitive to salt. Transcriptomic analysis revealed a much higher number of genes down-regulated in JMJ15 over-expression plants, which are highly enriched for H3K4me3 and H3K4me2. Among the down-regulated genes, many encode transcription regulators of stress responsive genes. The data suggest that increased JMJ15 levels may regulate the gene expression program that may coordinate plant growth restrains and enhances stress tolerance. Taken together, my thesis work brought a few new elements to the current understanding of chromatin regulators function in plant gene expression.

Le développement cranio-facial est un processus complexe qui implique interactions tissulaires et différenciations cellulaires. La façon dont ces processus sont coordonnés lors de l'embryogenèse reste évasive. Perturber ce développement coordonné provoque un large éventail de malformations. Afin de trouver de nouveaux gènes impliqués dans le développement de la tête, un criblage phénotypique a été réalisé par mutagenèse. L'une des lignées de souris obtenues montre des malformations au niveau des arches pharyngées (AP), qui sont les précurseurs de la tête. Ces mutants meurent à mi gestation, due à des problèmes vasculaires. La mutation ponctuelle générée a été localisé dans le gène Mortalin. Mon travail de thèse vise à comprendre comment Mortalin contrôle le développement embryonnaire. Mortalin est exprimée de manière ubiquitaire, puis son expression augmente au niveau des AP, dans les tissus musculaires et nerveux. Pour déterminer les mécanismes moléculaires affectées chez ce mutant, un profil d'expression génique a révélé l'induction de gènes impliqués dans la réponse au stress du réticulum endoplasmique (RE), appelée UPR, dont le rôle est de rétablir l'homéostasie du RE. Mortalin est impliqué dans le contrôle de l'UPR en interagissant avec BiP, un régulateur direct de cette voie. L'activation soutenue de l'UPR entraîne l'apoptose, ce que nous observons chez nos mutants. De plus, l'analyse du cycle cellulaire indique que la phase S est plus longue chez le mutant, suggérant que Mortalin régule le cycle cellulaire. Ainsi, l'ensemble des données suggère que Mortalin est nécessaire pour la survie des cellules au cours du développement. / The development of the head is a complex process that involves tissue interaction and cellular differentiation. Precisely how these processes are coordinated during embryogenesis remains elusive. Disruption of this coordinated development causes a wide range of malformations. In order to find new genes involved in the development of the head, a phenotype-driven ENU screen was performed. One of the mouse lines generated exhibits small pharyngeal arches (PAs), which are the main precursors of the head. Mutant embryos die around mid-gestation, most likely as a result of defective vasculature. We mapped the ENU-mediated point mutation within Mortalin. My thesis work aims to understand how Mortalin controls embryonic development. Mortalin is ubiquitously expressed before mid-gestation. Then its expression increases in the PAs and cranial ganglia. In older embryos, mortalin is expressed in muscle and nervous tissue. To determine which molecular mechanisms are affected in the mutant, gene expression profil revealed the induction of genes involved in the response to endoplasmic reticulum (ER) stress, called UPR. The role of the UPR is to restore homeostasis in the ER. I found that Mortalin regulates the UPR by interacting with BiP, a direct regulator of this pathway. Sustained activation of UPR leads to apoptosis, which is observed in our mutant. Cell cycle has been analyzed to investigate the cause of the reduced embryonic size in our mutant. The length of the S phase was found longer in the mutant, indicating that Mortalin also regulates cell cycle. All together, these data suggest that Mortalin is required for cell survival during development, in part by controlling the UPR.

Araucaria angustifolia é uma espécie de conífera native do Brasil, com importância econômica, social e ecológica. Devido a sua intensa exploração ao longo dos anos, atualmente a espécie cobre apenas 2% do sua área florestal original. Neste sistema, a embriogênese somática pode ser integrada em programas de melhoramento e conservação. Além disso, a similaridade entre a embriogênese zigótica e somática tem sido usada para desenvolver estudos baseados em biologia de sistemas, a fim de otimizar o desenvolvimento do embrião somático in vitro, bem como para gerar uma melhor compreensão dos eventos moleculares, bioquímicos e fisiológicos que modulam a formação do embrião. O metabolismo dos carboidratos é uma rota central que desempenha um papel importante durante o crescimento e desenvolvimento das plantas. Além de seu papel essencial como substrato no metabolismo de carbono e energia, os açúcares também desempenham papéis importantes como moléculas sinalizadoras. Para A. angustifolia, os bancos de dados de transcriptoma e proteoma identificaram o metabolismo de carboidratos como uma via importante na modulação do processo embriogênico. Assim, o objetivo principal deste trabalho foi estudar o metabolismo de carboidratos durante três estádios da embriogênese zigótica (globular, cotiledonar e maduro) e nas fases de proliferação e maturação de linhagens celulares embriogênicas com potencial embriogênico contrastante (responsiva e bloqueada). Para tanto, foram analisados os perfis de carboidratos não estruturais e monossacarídeos de parede celular, bem como a identificação e caracterização dos principais genes e proteínas envolvidos nas respostas mediadas por carboidratos, na homeostase de comunicação célula-a-célula e na modulação do metabolismo de sacarose, amido, rafinose e parede celular. Adicionalmente, um banco de dados de metaboloma foi gerado e integrado ao transcriptoma e proteoma de A. angustifolia através de redes de co-expressão, em uma abordagem de biologia de sistema. As respostas mediadas por carboidratos que ocorrem durante a embriogênese somática de A. angustifolia se assemelham às que ocorrem nos estádios iniciais da embriogênese zigótica. Além disso, o acúmulo de sacarose e amido durante o desenvolvimento embrionário foi modulado pelas respostas de detecção de açúcar e sinalização, destacando este processo como uma característica importante que direciona a responsividade das linhas celulares embriogênicas. Associado a isso, a seletividade mediada pela comunicação de plasmodesmas e transporte vesicular na linhagem celular responsiva, apareceu como importante controle de diferenciação e desenvolvimento de tipos celulares. Embora o mecanismo completo da embriogênese somática de A. angustifolia não tenha sido completamente elucidado, nossas análises sobre as alterações metabólicas (metaboloma) durante a embriogênese zigótica e somática indicam que as redes regulatórias envolvidas no crescimento e desenvolvimento estão altamente interconectadas aos níveis de metabólito, proteína e transcrito, mostrando altas correlações entre alvos envolvidos no metabolismo de carboidratos. Os resultados obtidos fornecem informações relevantes e inéditas sobre o metabolismo dos carboidratos na embriogênese zigótica e somática de A. angustifolia, bem como fornecem subsídios para a otimização das condições in vitro para o desenvolvimento de embriões somáticos. / Araucaria angustifolia is a native conifer species of Brazil, that with economic, social and ecological importance. Due to its intense exploitation, the species cover only 2% of its original forest area. In this system, somatic embryogenesis may be integrated into breeding and conservation programs. Beside this, the similarity between zygotic and somatic embryogenesis have been used to develop studies based on system biology, in order to optimize the in vitro somatic embryo development, as well as to generate a better understanding of molecular, biochemical and physiological events that modulate the embryogenesis. Carbohydrates metabolism is a central route that plays an important role during plant growth and development. In addition to its essential role as a substrate in carbon and energy metabolism, sugars also play important roles as signal molecules. For A. angustifolia, transcriptome and proteome databases identified carbohydrates metabolism as an important pathway in the modulation of embryogenic process. Thus, the main objective of this work was to study the carbohydrates metabolism during three zygotic embryogenesis stages (globular, cotyledonal and mature) and in proliferation and maturation phases of embryogenic cell lines with contrasting embryogenic potential (responsive and blocked). To achieve this purpose, the profiles of non-structural carbohydrates and cell wall monosaccharides were generated, as well as the identification and characterization of the main genes and proteins involved in carbohydrate-mediated responses, cell-to-cell communication homeostasis and modulation of sucrose, starch, raffinose and cell wall metabolism. Additionally, a metabolome database was generated and integrated with A. angustifolia transcriptome and proteome through co-expression networks in a system biology approach. The carbohydrate-mediated responses that occur during A. angustifolia somatic embryogenesis resembled those occurring in the early stages of zygotic embryogenesis, where the main responses that affect the targeting of the tissue differentiation of the seed occur. Beside this, sucrose and starch accumulation during embryo development were modulated by sugar sensing and signaling responses, highlighting this process as an important trait that directs the responsiveness of embryogenic cell lines. Associated to this, the selectivity mediated by plasmodesmata communication and vesicular transport in the responsive cell line, appeared as an important control of cell types differentiation and development. Even though the complete mechanism of A. angustifolia somatic embryogenesis has not been completely elucidated, our analyses about the metabolic changes (metabolome) during zygotic and somatic embryogenesis indicate that the regulatory networks involved in growth and development are highly inter-connected at the metabolite, protein and transcript levels, showing high correlations between targets involved in carbohydrate metabolism. The results obtained provide relevant and inedited information about the carbohydrates metabolism in A. angustifolia zygotic and somatic embryogenesis, as well as provide news subsidies for optimization of in vitro conditions for somatic embryos development.

O tubarão-azul, Prionace glauca (Linnaeus, 1758) é uma espécie cosmopolita, de alto valor comercial, principalmente capturado por embarcações que operam em alto mar e vendido em mercados e feiras-livres. Poucos dados biológicos estão disponíveis sobre esta espécie, principalmente quanto à sua sanidade e, o conhecimento do desenvolvimento de seus órgãos linfoides pode trazer importantes informações neste contexto. Desta forma, o objetivo do trabalho é descrever o desenvolvimento embrionário dos órgãos linfoides em embriões de tubarão-azul: timo, órgão epigonal, baço e órgão de Leydig, quanto à estrutura e arquitetura macroscópica, microscópica e ultraestrutural, pelas técnicas de microscopia de luz e eletrônica de transmissão. Foram coletados cinco espécimes de cada fase representativa do desenvolvimento embrionário: I, II, III e IV e do animal adulto. O timo foi visível macroscopicamente nas fases III e IV e microscopicamente da fase I a IV. O órgão de Leydig está presente nas fases II, III e IV. O baço e o órgão epigonal estão presentes em todas as fases embrionárias e no adulto. O timo apresentou principalmente populações de timócitos em diversos estágios de maturação e melanomacrófagos, o baço apresentou melanomacrófagos linfócitos em diversos estágios de maturação, neutrófilos, trombócitos e grande quantidade de eritrócitos. O órgão epigonal apresentou um grande número de células imaturas, principalemente de linfócitos e células polimorfonucleares. A função do órgão de Leydig é perdida quando adulta, sendo substituída pelo órgão epigonal. Os resultados desse trabalho permitem sugerir que esses órgãos apresentam uma função hematopoiética desde o inicio da embriogênese até a fase adulta. / The blue shark (Prionace glauca) is a cosmopolitan species of high commercial value, easily caught by vessels operating on the high seas and sold in markets and street fairs. Few biological data are available on this species, mainly from their sanity. Studies on development of these lymphoid organs can provide important information in this regard. Thus, the aim of this study is to describe the gross, microscopic and ultraestrutural morphology of the embryonic development of lymphoid organs: thymus, epigonal organ, spleen and the Leydig organ by light microscopy and transmission electron techniques. Five specimens were collected from each representative stage of embryonic development: II, III and IV besides adult specimens. Thymus was visible macroscopically at phases III and IV and microscopically from phase I to IV. Leydig organ is presente in phases II, III and IV. Spleen and epigonal organ are present in all embrionic phases and adult. Thymus presented mainly thymocytes populations in several maturation stages and melanomacrophages, spleen presentes melanomacrophages and lymphocytes, neutrophyls, trombocytes and huge amount of erytrocytes. Epigonal organ presented many immature cells, mainly lymphocytes and polimorphonuclear cells. Leidigs organ function is lost in adulthood being replaced by the epigonal organ. The results of this work allow to suggest that these organs present a hematopoietic function since the early development until the adult phase.

A família Bromeliaceae apresenta importância ecológica e econômica, desta forma, o desenvolvimento de protocolos para a micropropagação de espécies dessa família, faz-se necessário, a fim de suprir sua demanda comercial e mesmo ecológica. A escolha do meio de cultura e do explante utilizado durante a micropropagação são fundamentais para um protocolo eficaz. Nesse contexto, o objetivo deste estudo foi avaliar as diferenças quantitativas e qualitativas no desenvolvimento de explantes de Neoregelia sp em meios de cultura e monitorar as vias morfogênicas dos propágulos obtidos em explantes foliares. Para tanto, brotos de microcepas e explantes foliares procedentes de um micro jardim clonal, foram transferidos para os meios de cultura de multiplicação MS, ½ MS e WPM, todos suplementados com 0,050 mg.L-1 ANA e 0,50 mg.L-1 de BAP, onde foram mantidos por 120 dias e submetidos a diversas análises morfofisiológicas. Paralelamente, explantes foliares foram mantidos em meio de cultura MS de multiplicação para o monitoramento das vias morfogênicas durante os processos regenerativos. Para os experimentos com brotos de microcepas verificou-se que o meio de cultura MS proporcionou a melhor taxa de multiplicação, maior crescimento dos brotos, obtendo os valores mais elevados de peso de matéria fresca e seca, além disso, apresentaram maior acúmulo de nitrogênio total e proteico. No entanto, os meios de cultura ½ MS e WPM promoveram uma taxa de multiplicação semelhante a do MS, mas com brotos menores e menos vigorosos, porém, mais homogêneos, com isso, na dependência do objetivo do cultivo in vitro, não deve ser desconsiderada a possibilidade de utilização dos meios de cultura ½ MS e WPM. Os explantes foliares não se desenvolveram bem no meio de cultura WPM, não havendo diferença entre os meios MS e ½ MS, visto que ambos apresentaram resultados satisfatórios. As análises histológicas e histoquímicas identificaram células parenquimáticas, que atuam como células-tronco, manifestando capacidade morfogênica para toti ou pluripotência, dando origem respectivamente a embriões somáticos e gemas adventícias, em resposta aos estímulos in vitro. / The Bromeliaceae family has an ecological and economic importance, therefore, the protocols development for micropropagation of species of this family becomes necessary in order to meet its business and even its ecological demand. The choice of culture medium and the explant used during micropropagation are essential for an effective protocol. Thus, the aim of this study was to evaluate the quantitative and qualitative differences in the explants development of Neoregelia sp in the culture media and monitor the morphogenetic pathways of obtained propagules from leaf explants. Consequently, shoots and leaf explants coming from microcloning garden were transferred to the MS, ½ MS and WPM multiplication culture media, all supplemented with 0.050 mg.L-1 NAA and 0.50 mg.L-1 BAP, where they were held for 120 days and submitted to morphological and physiological analysis. Therefore, leaf explants were kept on MS-medium multiplication for monitoring morphogenetic pathways during the regenerative processes. Furthermore, MS medium showed the best multiplication rate for the sprouts of the microstumps, increased growth of shoots, obtaining the highest values of fresh and dry matter weight, and also showed higher accumulation of total nitrogen and protein. However, the ½ MS and WPM culture media promoted a similar multiplication rate to the MS medium, with the development of the smaller and less vigorous shoots, but with greater homogeneity. This way, depending on the purpose of in vitro culture, their use in the micropropagation for this species should not be disregarded. The leaf explants are not well developed in WPM medium, and don\'t had significant difference between the MS and ½ MS culture medium, as both showed satisfactory results. The histological and histochemical analysis identified the presence of the parenchymatic cells, which act as stem cells, expressing morphogenic ability for toti or pluripotency, leading respectively to somatic embryogenesis or adventitious organogenesis in response to in vitro stimuli.

Araucaria angustifolia é a única conífera nativa com importância econômica no Brasil. A espécie está incluída na lista oficial de espécies de plantas ameaçadas de extinção. A embriogênese somática é considerada como uma das ferramentas biotecnológicas mais promissoras na área de biotecnologia de plantas. A identificação de marcadores, que possam ser utilizados, para a seleção de genótipos aptos ao desenvolvimento do embrião somático é altamente desejável, uma vez que o desenvolvimento apropriado do embrião somático é extremamente dependente do genótipo. O presente trabalho teve como objetivo estudar e identificar marcadores bioquímicos e moleculares associados à competência para embriogênese somática em A. angustifolia. Culturas embriogênicas de genótipos com diferentes potenciais embriogênicos apresentaram diferenças nos parâmetros bioquímicos e moleculares avaliados (Poliaminas, expressão dos genes AaSERK1 e AaPP2C, proteoma). O presente trabalho propõe a utilização destes parâmetros como possíveis marcadores do potencial embriogênico no sistema A. angustifolia. / The Araucaria angustifolia is the only native conifer species with economical importance in Brazil. Recently, the species was included in the official list of endangered plant species. Somatic embryogenesis, i.e., in vitro asexual production of embryos is considered one of the most promising biotechnological tools in the area of plant biotechnology. Since the development of somatic embryos is dependent of the genotype, the identification of markers that could be used in the selection of genotypes which can develop somatic embryos is highly desirable. The main objective of the present work is to study and identify biochemical and molecular markers associated with competence for somatic embryogenesis in A. angustifolia. The evaluation of biochemical and molecular aspects (Polyamines, AaSERK1 and AaPP2C gene expression and proteome) indicated differences between embryogenic cultures of genotypes with different embryogenic potential. This work proposes the utilization of these parameters as possible markers of embryogenic potential in the system A. angustifolia.