Thesis (Ph. D.)--University of Florida. 1981. / Description based on print version record. Typescript. Vita. Includes bibliographical references (leaves 68-71).

Les Bradyrhizobium photosynthétiques sont capables d'induire la formation de nodules fixateurs d'azote chez certaines légumineuses du genre Aeschynomene. La découverte récente que certaines de ces souches ne possèdent pas les gènes canoniques nodABC indique l'existence d'un nouveau processus symbiotique rhizobium-légumineuse indépendant des facteurs Nod. L'objectif de ce travail de thèse a consisté à avancer dans la compréhension des mécanismes mis en jeu lors de cette nouvelle interaction. Dans un premier temps, à travers différentes approches cytologiques, le processus par lequel la bactérie infecte la plante en l'absence de facteurs Nod a été décrit. Dans un deuxième temps, afin de mettre en évidence les bases moléculaires de cette interaction, une banque de 15 000 mutants Tn5 de la souche ORS278 a été criblée sur plante. Ce criblage a permit l'identification de plus d'une centaine de gènes bactériens intervenant durant le processus symbiotique. Les résultats obtenus nous ont conduits à proposer un modèle dans lequel la mise en place de la symbiose Nod-indépendante impliquerait, d'une part, la synthèse bactérienne d'une cytokinine permettant le déclenchement de l'organogenèse nodulaire, et d'autre part, d'autres signaux bactériens intervenant dans l'étape de reconnaissance avec la plante hôte. Enfin, nous avons mis en place une technique de transformation génétique d'Aeschynomene et validé cet outil à travers l'étude de l'expression hétérologue de la noduline précoce MtENOD11. Il peut à présent être envisagé de conduire des études fonctionnelles sur Aeschynomene en vue de caractériser la voie de signalisation Nod-indépendante. / The photosynthetic Bradyrhizobium are able to induce the formation of nitrogen-fixing nodules in some legumes of the Aeschynomene genus. The recent discovery that some of these strains lack the canonical nodABC genes indicates the existence of a new symbiotic rhizobium-legume process that is independent of Nod factors. The aim of this work was to improve our understanding of the mechanisms involved in this new interaction. First, through various cytological approaches, the process by which the bacterium infects the plant in the absence of Nod factors has been described. Second, in order to decipher the molecular basis of this interaction, a library of 15,000 Tn5 mutants of the ORS278 strain was screened on plant. This screening allowed the identification of about one hundred bacterial genes involved in this symbiotic process. These results led us to propose a model in which the establishment of the Nod-independent symbiosis involves, on one han d, the synthesis of a bacterial cytokinin that triggers nodule organogenesis, and on the other hand, others bacterial signals that permit the recognition with the host plant. Finally, we developed a genetic transformation procedure of Aeschynomene and we validated this tool by studying the heterologous expression of the early nodulin MtENOD11. Now, functional studies on Aeschynomene are possible to permit the characterization of the Nod-independent signaling pathway.

Les légumineuses sont capables de développer des organes symbiotiques, les nodules, qui hébergent des bactéries du sol appelées rhizobia. Au sein des nodules les rhizobia intracellulaires se différencient en bactéroïdes capables de réduire l'azote atmosphérique en ammonium au bénéfice de la plante. En contrepartie, la plante alimente la bactérie en sources de carbone. Des études récentes sur le modèle symbiotique Medicago/Sinorhizobium ont montré dans les nodules la forte présence d'une grande diversité de peptides appelés NCR qui sont similaires aux peptides antimicrobiens (AMP) impliqués dans l'immunité innée. Ces NCR sont responsables du maintien de l'homéostasie entre les cellules hôtes et la forte population bactérienne qu'elles contiennent. Bien que certains NCR sont de vrais AMP, capable de tuer des bactéries in vitro, dans les nodules ils induisent plutôt une différenciation terminale caractérisée par une élongation cellulaire, une amplification du génome, une perméabilité membranaire et une perte des capacités de division de la bactérie. Néanmoins le mode d'action des NCR reste à élucider. Au cours de ma thèse j'ai participé à la caractérisation des processus de différenciation dans le modèle Aeschynomene, une légumineuse tropicale, Bradyrhizobium.Dans un premier temps, une nouvelle classe de NCR a été identifiée chez différentes espèces d'Aeschynomene. Ces NCR sont responsables de la différenciation des Bradyrhizobium via un processus similaire à celui décrit chez Medicago. Ces résultats suggèrent une évolution convergente des processus de différenciation chez les Dalbergioïdes (Aeschynomene) et le clade des IRLC (Medicago).Ensuite, pour identifier les fonctions bactériennes requises lors de la différenciation, j'ai criblé 53 mutants Tn5 d'Aeschynomene indica fix- . Huit gènes bactériens dont la mutation inhibe ou affecte le processus de différenciation ont été identifiés. Parmi eux, je me suis focalisé sur la DD-CPase une enzyme de modification du peptidoglycane et sur 2 gènes impliqués dans l'homéostasie du phosphate.La caractérisation du gène DD-CPase1 a permis de démontrer que le remodelage du peptidoglycane est requis pour une différenciation correcte des bactéroïdes chez les plantes hôtes qui produisent des NCR, en général, et chez Aeschynomene en particulier. Ces résultats suggèrent une interaction possible entre DD-CPase1 et des NCR conduisant à l'endoréplication des bactéroïdes.Enfin, j'ai étudié les propriétés physiologiques et symbiotiques des mutants pstC et pstB. Les mutants Tn5 des gènes pstC et pstB de la souche ORS285 de Bradyrhizobium sont sévèrement affectés par la carence en phosphate en culture pure et leurs propriétés symbiotiques (différenciation, réduction de l'azote) sont fortement réduites. Des analyses fonctionnelles plus approfondies de l'opéron Pst devraient permettre une meilleure compréhension du lien entre l'homéostasie du phosphate et l'efficience symbiotique dans l'interaction Aeschynomene-Bradyrhizobium.Mes travaux ont permis d'élargir nos connaissances sur l'évolution de la symbiose en montrant que le modus operandi impliquant des peptides dérivés de l'immunité innée utilisée par certaines légumineuses pour maintenir leur population bactérienne intracellulaire sous contrôle est plus répandue et ancienne qu'on ne le pensait et a été utilisée par l'évolution à plusieurs reprises. De plus différentes cibles bactériennes pouvant participer au processus de différenciation ont également été identifiées. / The legume species are able to form symbiotic organs, the nodules, that house soil bacteria called rhizobia. Within these nodules intracellular rhizobia differentiate into bacteroids, which are able to reduce atmospheric dinitrogen to ammonium for the benefit of the plants. In counterpart, the plants provide carbon sources to the bacteria. Recent studies on symbiotic model Medicago-Sinorhizobium showed that the nodules of M. truncatula produce a massive diversity of peptides called NCRs, which are similar to antimicrobial peptides (AMPs) of innate immune systems. These NCRs are responsible in maintaining the homeostasis between the host cells in the nodules and the large bacterial population they contain. Although many NCRs are genuine AMPs, which kill microbes in vitro, in nodule cells they do not kill the bacteria but induce them into the terminally differentiated bacteroids characterized by cell elongation, genome amplification, membrane permeability and loss of cell division capacity. However, the action mode of NCRs is still an open question. During my PhD thesis I focused on the identification of plant and bacterial functions required for bacteroid differentiation in the Aeschynomene-Bradyrhizobium model.Firstly, a new class of cysteine rich peptides (NCR-like) was identified in tropical aquatic legumes of the Aeschynomene genus, which belong to the Dalbergioid clade. These peptides govern terminal bacteroid differentiation of photosynthetic Bradyrhizobium spp. This mechanism is similar to the one previously described in Medicago suggesting that the endosymbiont differentiation in Dalbergioid and ILRC legumes is convergently evolved.Secondly, in order to identify the bacterial functions involved in bacteroid differentiation, I screened 53 fix- Tn5 mutants of the ORS278 strain on Aeschynomene indica. This screening allowed identify 8 bacterial genes, which inhibit or disorder the bacteroid differentiation. Among these identified genes, I focused on DD-CPase encoding a peptidoglycan-modifying enzyme and two genes pstC and pstB belonging to Pst-system.The characterization of DD-CPase gene demonstrated that the remodeling peptidoglycan enzyme, DD-CPase1, of Bradyrhizobium is required for normal bacteroid differentiation in host legumes that produce NCRs, in general, and in Aeschynomene spp., in particular. This prompts a possibility of direct interaction of DD-CPase1 with NCRs leading to endoreduplication of the bacteroids.Finally, I have investigated the physiological and symbiotic properties of different mutants of pstC and pstB genes. The Tn5 mutants of pstC and pstB genes of Bradyrhizobium sp. strain ORS278 severely affected symbiosis on A. indica and A. evenia. Further functional studies on pst-operon will provide deeper understanding the correlation between phosphate homeostasis and nitrogen fixation efficiency in Aeschynomene-Bradyrhizobium symbiosis.This study broadens our knowledge on the evolution of symbiosis by showing that the modus operandi involving peptides derived from innate immunity used by some legumes to keep their intracellular bacterial population under control is more widespread and ancient than previously thought and has been invented by evolution several times.

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-01-20T16:48:22Z No. of bitstreams: 2 Dissertação - Lorena Lana Camelo Antunes - 2015.pdf: 8402044 bytes, checksum: 843f4a12f15a60c2c393753516d13b0d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-21T06:37:38Z (GMT) No. of bitstreams: 2 Dissertação - Lorena Lana Camelo Antunes - 2015.pdf: 8402044 bytes, checksum: 843f4a12f15a60c2c393753516d13b0d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-01-21T06:37:38Z (GMT). No. of bitstreams: 2 Dissertação - Lorena Lana Camelo Antunes - 2015.pdf: 8402044 bytes, checksum: 843f4a12f15a60c2c393753516d13b0d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Subfamilies Caesalpinioideae, Mimosoideae, and Papilionoideae are circumscribed in the Leguminosae, which is cosmopolitan, encompasses approximately 750 genera and 19.500 species, and is the third largest taxon of the Angiosperms. In Brazil, it is the richest family, with 2.756 species and 213 genera, which makes it very important ecologically. Papilionoideae, its largest subfamily, with 478 genera, 13.800 species, and 28 tribes, is monophyletic and traditionally recognized for the papilionaceous flowers, leaves usually imparipinnate or trifoliolate, and legume fruits. Among Papilionoideae tribes, Dalbergieae sensu lato is one of the largest, encompassing 49 genera and ca. 1.325 species, distributed in the clades Adesmia, Pterocarpus, and Dalbergia. The latter is the most diverse, with 706 species distributed in 17 genera, among which Aeschynomene stands out for its complex taxonomy, paraphyletic nature, and high representativeness (ca. 180 species). In Brazil, Aeschynomene is the most diverse genus of Dalbergieae sensu lato, with 49 species. Nonetheless, the taxonomic knowledge of this genus in Brazilian flora is scarce, moreover considering its species that occur in the central part of the country. Based on usual methods, the taxonomic study of Aeschynomene in the state of Goiás. This taxonomic study resulted in the registration of 25 species, one of them new to science (A. veadeirana Silva M. J. & Antunes L. L. C.), nine new occurrences, three synonymizations, and one lectotypification. This dissertation reveals the diversity of Aeschynomene in the state of Goiás and furnishes information to understand the infrageneric and evolutionary relationships in the genus. / As subfamílias Caesalpinioideae, Mimosoideae e Papilionoideae são circunscritas à Leguminosae que é cosmopolita e reúne aproximadamente 750 gêneros e 19.500 espécies, sendo o terceiro maior táxon de Angiospermas. No Brasil, é a mais rica família, com 2.756 espécies e 213 gêneros, o que a torna bastante importante ecologicamente. Papilionoideae, sua maior subfamília, com 478 gêneros, 13.800 espécies e 28 tribos, é monofilética e tradicionalmente reconhecida por suas flores papilionáceas, folhas usualmente imparipinadas ou trifolioladas e frutos do tipo legume. Entre as tribos de Papilionoideae, Dalbergieae sensu lato é uma das maiores, agregando 49 gêneros e cerca de 1.325 espécies, distribuídas nos clados Adesmia, Pterocarpus e Dalbergia, sendo o último o mais diverso com 706 espécies em 17 gêneros, entre os quais se destaca Aeschynomene por sua taxonomia complexa, natureza parafilética e elevada representatividade (ca. 180 espécies). No Brasil, Aeschynomene é o gênero mais diverso de Dalbergieae sensu lato, com 49 espécies. No entanto, o conhecimento taxonômico desse gênero na flora brasileira é escasso, sobretudo se consideradas as suas espécies ocorrentes na parte central do país. Com base em metodologia usual, foi realizado o estudo taxonômico de Aeschynomene no Estado de Goiás. Como resultado deste estudo foram registradas 25 espécies, uma das quais é nova para a ciência (A. veadeirana Silva M. J. e Antunes L. L. C.), nove novas ocorrências, propostas três sinonimizações e uma lectotipificação. Esta dissertação revela a diversidade de Aeshynomene no estado de Goiás e propicia subsídios para o entendimento das relações infragenéricas e evolutivas do gênero.

Les Légumineuses ont développé une interaction symbiotique avec des bactéries du sol, les rhizobia, qui fixent l’azote atmosphérique et le transfèrent à la plante sous forme assimilable.Cette interaction a lieu, au sein des nodosités, des organes racinaires où les bactéries intracellulaires se différencient en bactéroïdes. Chez Medicago truncatula, ces bactéroïdes correspondent à un stade de différentiation terminale corrélée à une endoréplication de leur génome, une augmentation de la taille des cellules, une modification des membranes et une faible capacité à se propager. Cette différentiation est induite par des facteurs de la plante appelés NCR (Nodule-specific Cysteine Rich). Les peptides NCRs ressemblent à des défensines, des peptides antimicrobiens ayant une activité antimicrobienne in vitro, tuant des bactéries. Ainsi, un élément clef dans la différenciation des bactéroïdes est la protéine bactérienne BacA, un transporteur membranaire qui confère une résistance contre l’activité antimicrobienne des peptides. Dans le cadre de ce travail de thèse, j’ai montré que l'expression des NCR est soumise à une régulation stricte et qu’ils sont activés dans trois vagues dans les cellules symbiotiques polyploïdes.Les mécanismes de contrôle par la plante sur les rhizobia intracellulaires demeurent à ce jourpeu connus et le seul modèle étudié, au début de ce travail de thèse, restait l'interaction entre M. truncatula et S. meliloti. Je me suis donc intéressée à la symbiose de certaines Légumineuses tropicales du genre Aeschynomene appartenant au clade des Dalbergoïdes où jemontre qu’ils utilisent une classe différente de peptides riches en cystéine (NCR-like) pour induire la différenciation des bactéroïdes. Ce mécanisme est analogue à celui décrit précédemment chez Medicago qui était jusqu'à présent supposé être limitée aux légumineuses appartenant au clade des IRLC. J’ai également montré que Bradyrhizobium, symbionte d’Aeschynomene possèdent un transporteur de type ABC homologues à BacA de Sinorhizobium nommé BclA. Ce gène permet l'importation d'une variété de peptides comprenant des peptides NCR. En l'absence de ce transporteur, les rhizobiums sont incapables de se différencier et de fixer l'azote.Cette étude a permis d'élargir nos connaissances sur l'évolution de la symbiose en montrant qu’au cours de l’évolution, deux clades de Légumineuses relativement éloignés (IRLC et Dalbergoïdes) aient convergé vers l’utilisation de peptides de l’immunité innée afin de contrôler leur symbionte bactérien et d’en tirer un bénéfice maximal au cours de l’interaction symbiotique. / The ability of legumes to acquire sufficient nitrogen from the symbiosis with Rhizobium relies on the intimate contact between the endosymbiotic, intracellular rhizobia, called bacteroids, and their host cells, the symbiotic nodule cells. A well-studied example is the symbiotic nitrogen fixing bacterium Sinorhizobium meliloti, which nodulates the legume Medicago truncatula. Nodules of M. truncatula produce an enormous diversity of peptides called NCRs which are similar to antimicrobial peptides (AMPs) of innate immune systems. These NCRs are involved in maintaining the homeostasis between the host cells in the nodules and the large bacterial population they contain. Although many NCRs are genuine AMPs which kill microbes in vitro, in nodule cells they do not kill the bacteria but induce them into the terminally differentiated bacteroid state involving cell elongation, genome amplification, membrane fragilization and loss of cell division capacity. Protection against the antimicrobial action of NCRs by the bacterial BacA protein is critical for bacteroid survival in the symbiotic cells and thus for symbiosis. As a part of my PhD thesis, I have shown that the differentiation of the symbiotic cells in M. truncatula is associated with a tremendous transcriptional reprogramming involving hundreds of genes, mainly NCR genes, which are only expressed in these cells. Although the extensive work on the model M. truncatula/S. meliloti, little is known how the plant controls its intracellular population and imposes its differentiation into a functional form, the bacteroids in other symbiotic systems.In my PhD work, I provide several independent pieces of evidence to show that tropical legumes of the Aeschynomene genus which belong to the Dalbergoid legume clade use a different class of cysteine rich peptides (NCR-like) to govern bacteroid differentiation. This mechanism is similar to the one previously described in Medicago which was up to now assumed to be restricted to the advanced IRLC legume clade, to which it belongs. I have also shown that the Bradyrhizobium symbionts of Aeschynomene legumes possess a multidrug transporter, named BclA, which mediates the import of a diversity of peptides including NCR peptides. In the absence of this transporter, the rhizobia do not differentiate and do not fix nitrogen. BclA has a transmembrane domain of the same family as the transmembrane domain of the BacA transporter of Rhizobium and Sinorhizobium species which is known to be required in these rhizobia to respond to the NCR peptides of IRLC legumes. Again this is a mechanism which is analogous to the one described in S. meliloti the symbiont of Medicago.This study broaden our knowledge on the evolution of symbiosis by showing that the modus operandi involving peptides derived from innate immunity used by some legumes to keep their intracellular bacterial population under control is more widespread and ancient than previously thought and has been invented by evolution several times.

CAPES / A família Fabaceae (Leguminosae) é constituída por 750 gêneros e mais de 18000 espécies, distribuídas em todo o mundo. No Brasil esta é representada por aproximadamente 175 gêneros e 1500 espécies. As espécies da família Fabaceae são particularmente ricas em flavonoides e compostos biossinteticamente relacionados, como rotenóides e isoflavonoides. Alcalóides, terpenóides e esteróides são exemplos de outras classes de substâncias que ocorrem em muitos exemplares da família. Dentre as espécies representantes desta família na região de Porto Rico-PR, área de proteção ambiental, está Aeschynomene fluminensis e Machaerium hirtum. Uma revisão bibliográfica destas espécies indicou a ausência de estudos fitoquímicos e biológicos, o que motivou a escolha das mesmas para investigação. Ainda foi verificado que M. hirtum é utilizada na medicina popular contra tosse, diarréia e câncer. O estudo fitoquímico para as duas espécies foi iniciado a partir da obtenção dos extratos brutos metanólicos das folhas e dos galhos, por maceração, e as obtenções de frações destes extratos a partir de partições em solventes de polaridades diferentes. As frações obtidas foram submetidas a purificações cromatográficas em colunas de gel de sílica e/ou Sephadex LH-20. Os tratamentos cromatográficos realizados para as frações de A. fluminensis levaram ao isolamento dos flavonóides canferitrina, α-raminoisorobina, quercitrina, 3-O-apiofuranosil-7-O-raminopiranosil-canferol e avicularina, da procianidina A2; da flavanona iso-hemifolina; da isoflavona daidzeína e do açúcar 3- O-metil-quiro-inositol. O estudo das frações obtidas de M. hirtum, resultou no isolamento das flavonas isovitexina e swertisina; do alcalóide 4-hidróxi-N-metilprolina; dos triterpenos friedelan-3-ona e lupen-20(29)-3β-ol e da mistura dos esteróides estigmasterol e sitosterol, a qual também foi encontrada em A. fluminensis. A elucidação das estruturas foi realizada mediante análise de dados espectroscópicos de RMN de 1H e 13C e de técnicas bidimensionais gCOSY, ii gHSQC e gHMBC) e por comparação com os dados relatados na literatura. Os extratos brutos e frações de A. fluminensis foram submetidos a ensaios de atividade anti-inflamatória no modelo edema de orelha e se mostraram todos ativos, sendo mais significativos os extratos e frações hexânicas das folhas e dos galhos. Nos ensaios de atividade da enzima de mieloperoxidase (MPO) apresentaram resultados significativos todas as frações de A. fluminensis com maiores atividades as frações hexânicas e CHCl3 das folhas e dos galhos. Os extratos brutos e todas as frações de M. hirtum apresentaram bons resultados quando submetidos a ensaios de atividade anti-inflamatória no modelo edema de orelha, para a substância 4-hidróxi-N-metil-prolina este ensaio resultou em redução moderada. Na atividade da enzima de mieloperoxidase apresentaram resultados significativos os extratos e todas as frações de M. hirtum, sendo mais ativas as frações hexânicas e AcOEt das folhas e dos galhos e CHCl3 dos galhos. A mistura swertisina e isovitexina e a substância 4-hidróxi-N-metil-prolina apresentaram redução pronunciada da enzima. O teste de pleurisia foi significativo apenas para o extrato bruto das folhas de M. hirtum. No teste da atividade antitumoral de A. fluminensis sobre células Caco-2 foi observado que a fração CHCl3 dos galhos e a canferitrina apresentaram os resultados mais promissores. / The Fabaceae family (Leguminosae) comprises 750 genera and over 18000 species, distributed worldwide. In Brazil it is represented by approximately 175 genera and 1500 species. The Fabaceae species are particularly rich in flavonoids and biosynthetically related compounds, such as isoflavonoids and rotenoids. Alkaloids, terpenoids and steroids are examples of other classes of substances that occur in many samples of the family. Aeschynomene fluminensis and Machaerium hirtum are among the representative species of this family in the region of Porto Rico-Paraná state, an environmental protection area. A literature review of these species indicated the absence of phytochemical and biological studies, which motivated their choice for investigation. It was also found that M. hirtum is used in folk medicine against cough, diarrhea and cancer. The phytochemical study for both species was initiated after obtaining the methanol crude extracts of leaves and twigs, by percolation, and the varieties of extract fractions by means of partitions in different polarities solvents. The obtained fractions were subjected to purification on chromatographic columns of silica gel and/or Sephadex LH-20. The chromatographic treatments performed for A. fluminensis fractions led to the isolation of the following flavonoids: kaempferitrin, α-rhamnoisorobin, quercitrin, 3-Oapiofuranosyl- 7-O-rhamnopyranosyl-kaempferol and avicularin, A2 procyanidin; the flavanone iso-hemipholin; the isoflavone daidzein and the 3-O-methyl-chiro-inositol sugar. The study of the obtained fractions of M. hirtum resulted in the isolation of the isovitexin and swertisin flavones; of the 4-hydroxy-N-methyl-proline alkaloid, the friedelan-3-one and lupen-20(29)-3β-ol triterpenes and the stigmasterol and sitosterol steroids mixture, which was also found in A. fluminensis. The elucidation of the structures was performed by analysis of spectroscopic data of 1H and 13C NMR and bidimensional techniques (gCOSY, gHSQC and gHMBC) and by comparison with the data reported in the literature. The crude extracts and the A. fluminensis iv fractions were tested for anti-inflammatory activity in the ear edema model and showed all active; the most significant were the extracts and hexane fractions from leaves and twigs. The tests of the myeloperoxidase (MPO) enzyme activity showed significant results for all A. fluminensis fractions with more activities for the CHCl3 and hexane fractions of leaves and twigs. The crude extracts and all M. hirtum fractions showed good results when tested for anti-inflammatory activity in the ear edema model; for the substance 4-hydroxy-N-methyl-proline this test resulted in moderate reduction. For the activity of the myeloperoxidase enzyme, the M. hirtum extracts and all fractions showed significant results, being more active the hexane and EtOAc fractions of leaves and twigs and CHCl3 of twigs. The swertisin and isovitexin mixture and the substance 4-hydroxy-N-methyl-proline showed pronounced enzyme reduction. The pleurisy test was significant only for the crude extract of M. hirtum leaves. When testing the A. fluminensis antitumor activity on Caco-2 cells, it was observed that the CHCl3 fraction of the twigs and the kaempferitrin showed the most promising results.

CAPES / A família Fabaceae (Leguminosae) é constituída por 750 gêneros e mais de 18000 espécies, distribuídas em todo o mundo. No Brasil esta é representada por aproximadamente 175 gêneros e 1500 espécies. As espécies da família Fabaceae são particularmente ricas em flavonoides e compostos biossinteticamente relacionados, como rotenóides e isoflavonoides. Alcalóides, terpenóides e esteróides são exemplos de outras classes de substâncias que ocorrem em muitos exemplares da família. Dentre as espécies representantes desta família na região de Porto Rico-PR, área de proteção ambiental, está Aeschynomene fluminensis e Machaerium hirtum. Uma revisão bibliográfica destas espécies indicou a ausência de estudos fitoquímicos e biológicos, o que motivou a escolha das mesmas para investigação. Ainda foi verificado que M. hirtum é utilizada na medicina popular contra tosse, diarréia e câncer. O estudo fitoquímico para as duas espécies foi iniciado a partir da obtenção dos extratos brutos metanólicos das folhas e dos galhos, por maceração, e as obtenções de frações destes extratos a partir de partições em solventes de polaridades diferentes. As frações obtidas foram submetidas a purificações cromatográficas em colunas de gel de sílica e/ou Sephadex LH-20. Os tratamentos cromatográficos realizados para as frações de A. fluminensis levaram ao isolamento dos flavonóides canferitrina, α-raminoisorobina, quercitrina, 3-O-apiofuranosil-7-O-raminopiranosil-canferol e avicularina, da procianidina A2; da flavanona iso-hemifolina; da isoflavona daidzeína e do açúcar 3- O-metil-quiro-inositol. O estudo das frações obtidas de M. hirtum, resultou no isolamento das flavonas isovitexina e swertisina; do alcalóide 4-hidróxi-N-metilprolina; dos triterpenos friedelan-3-ona e lupen-20(29)-3β-ol e da mistura dos esteróides estigmasterol e sitosterol, a qual também foi encontrada em A. fluminensis. A elucidação das estruturas foi realizada mediante análise de dados espectroscópicos de RMN de 1H e 13C e de técnicas bidimensionais gCOSY, ii gHSQC e gHMBC) e por comparação com os dados relatados na literatura. Os extratos brutos e frações de A. fluminensis foram submetidos a ensaios de atividade anti-inflamatória no modelo edema de orelha e se mostraram todos ativos, sendo mais significativos os extratos e frações hexânicas das folhas e dos galhos. Nos ensaios de atividade da enzima de mieloperoxidase (MPO) apresentaram resultados significativos todas as frações de A. fluminensis com maiores atividades as frações hexânicas e CHCl3 das folhas e dos galhos. Os extratos brutos e todas as frações de M. hirtum apresentaram bons resultados quando submetidos a ensaios de atividade anti-inflamatória no modelo edema de orelha, para a substância 4-hidróxi-N-metil-prolina este ensaio resultou em redução moderada. Na atividade da enzima de mieloperoxidase apresentaram resultados significativos os extratos e todas as frações de M. hirtum, sendo mais ativas as frações hexânicas e AcOEt das folhas e dos galhos e CHCl3 dos galhos. A mistura swertisina e isovitexina e a substância 4-hidróxi-N-metil-prolina apresentaram redução pronunciada da enzima. O teste de pleurisia foi significativo apenas para o extrato bruto das folhas de M. hirtum. No teste da atividade antitumoral de A. fluminensis sobre células Caco-2 foi observado que a fração CHCl3 dos galhos e a canferitrina apresentaram os resultados mais promissores. / The Fabaceae family (Leguminosae) comprises 750 genera and over 18000 species, distributed worldwide. In Brazil it is represented by approximately 175 genera and 1500 species. The Fabaceae species are particularly rich in flavonoids and biosynthetically related compounds, such as isoflavonoids and rotenoids. Alkaloids, terpenoids and steroids are examples of other classes of substances that occur in many samples of the family. Aeschynomene fluminensis and Machaerium hirtum are among the representative species of this family in the region of Porto Rico-Paraná state, an environmental protection area. A literature review of these species indicated the absence of phytochemical and biological studies, which motivated their choice for investigation. It was also found that M. hirtum is used in folk medicine against cough, diarrhea and cancer. The phytochemical study for both species was initiated after obtaining the methanol crude extracts of leaves and twigs, by percolation, and the varieties of extract fractions by means of partitions in different polarities solvents. The obtained fractions were subjected to purification on chromatographic columns of silica gel and/or Sephadex LH-20. The chromatographic treatments performed for A. fluminensis fractions led to the isolation of the following flavonoids: kaempferitrin, α-rhamnoisorobin, quercitrin, 3-Oapiofuranosyl- 7-O-rhamnopyranosyl-kaempferol and avicularin, A2 procyanidin; the flavanone iso-hemipholin; the isoflavone daidzein and the 3-O-methyl-chiro-inositol sugar. The study of the obtained fractions of M. hirtum resulted in the isolation of the isovitexin and swertisin flavones; of the 4-hydroxy-N-methyl-proline alkaloid, the friedelan-3-one and lupen-20(29)-3β-ol triterpenes and the stigmasterol and sitosterol steroids mixture, which was also found in A. fluminensis. The elucidation of the structures was performed by analysis of spectroscopic data of 1H and 13C NMR and bidimensional techniques (gCOSY, gHSQC and gHMBC) and by comparison with the data reported in the literature. The crude extracts and the A. fluminensis iv fractions were tested for anti-inflammatory activity in the ear edema model and showed all active; the most significant were the extracts and hexane fractions from leaves and twigs. The tests of the myeloperoxidase (MPO) enzyme activity showed significant results for all A. fluminensis fractions with more activities for the CHCl3 and hexane fractions of leaves and twigs. The crude extracts and all M. hirtum fractions showed good results when tested for anti-inflammatory activity in the ear edema model; for the substance 4-hydroxy-N-methyl-proline this test resulted in moderate reduction. For the activity of the myeloperoxidase enzyme, the M. hirtum extracts and all fractions showed significant results, being more active the hexane and EtOAc fractions of leaves and twigs and CHCl3 of twigs. The swertisin and isovitexin mixture and the substance 4-hydroxy-N-methyl-proline showed pronounced enzyme reduction. The pleurisy test was significant only for the crude extract of M. hirtum leaves. When testing the A. fluminensis antitumor activity on Caco-2 cells, it was observed that the CHCl3 fraction of the twigs and the kaempferitrin showed the most promising results.

Made available in DSpace on 2014-08-20T13:44:35Z (GMT). No. of bitstreams: 1 dissertacao_andre_pich_brunes.pdf: 980824 bytes, checksum: 90e2fb8b525b06494bd014358a4889ab (MD5) Previous issue date: 2012-10-15 / The objective of this study was to evaluate the allelopathic potential of aqueous extract of leaves of different rice cultivars in germination and seed vigor of weeds Digitaria, Aeschynomene and red rice and search the mycoflora of S. montevidensis in order to identify species of pathogenic fungi with the potential to act as bioherbicide and verify their specificity as to plants of irrigated rice. The first experiment was conducted in a completely randomized design with four replications in a factorial design, where the first factor tested of fresh leaves of rice cultivars (BRS Querência, Irga 424, Roxinho and Cica 07) and factor B concentrations of the extract compared (0, 25, 50, 75 and 100%). Evaluations of germination, first count of germination, velocity of germination, shoot length and root, dry mass of shoot and root. In the second experiment, the experimental design was completely randomized with five replications and the treatments arranged in a factorial design where the first factor was composed of two species of fungi (Fusarium semitectum and Fusarium oxyosporum), previously isolated from lesions found in plants collected during phase of suvery in the region of Pelotas paddy and factor B compared six levels of Spore concentration (0, 1x103, 1x104, 1x105, 1x106, 1x107 spores ml-1) applied at the stadium where the plants had a leaf arrowhead and sagittate in rice plants at growth stage R4. Extracts from leaves of rice cultivars Cica 07, Querência, Roxinho and Irga 424, in high concentrations, reduce germination and seed vigor of Digitaria and Aeschynomene. The isolates of F. oxyosporum and F. semitectum are not effective in controlling S. montevidensis when applied in a unique way and the developmental stage of seedling leaf with a sagittate, however, when applied at flowering stage of rice plants, and cause losses in yield and reduction in root length.Keywords: Oryza sativa, Aeschynomene sp., Digitaria sp., Sagittaria montevidensis. / O objetivo do trabalho foi avaliar o potencial alelopático do extrato aquoso de folhas de diferentes cultivares de arroz irrigado na germinação e no vigor de sementes de milhã, angiquinho e arroz-vermelho e pesquisar a micobiota infestante de Sagittaria montevidensis, a fim identificar espécies de fungos fitopatogênicos com potencial para atuação como bioherbicida e verificar sua especificidade quanto à cultura do arroz irrigado. O primeiro experimento foi conduzido em delineamento inteiramente casualizado, com quatro repetições, em esquema fatorial, onde o fator A testou extrato de folhas frescas das cultivares de arroz (BRS Querência, Irga 424, Roxinho e Cica 07) e o fator B comparou concentrações do extrato (0, 25, 50, 75 e 100%). Avaliaram-se a germinação, primeira contagem da germinação, índice de velocidade de germinação, comprimento da parte aérea e de raiz e massa seca da parte aérea e de raiz. No segundo experimento o delineamento experimental utilizado foi inteiramente casualizado, com cinco repetições, sendo os tratamentos arranjados em esquema fatorial onde o fator A foi composto por duas espécies de fungos (Fusarium semitectum e oxyosporum), isolados previamente das lesões encontradas nas plantas coletadas durante a fase de levantamento na região orizícola de Pelotas e o fator B comparou seis níveis de concentração de esporos (0;1x103; 1x104; 1x105; 1x106 e 1x107 esporos ml-1) aplicados no estádio em que as plantas de sagitária apresentavam uma folha sagitada e nas plantas de arroz no estádio de desenvolvimento R4. Extratos de folhas de arroz das cultivares Cica 07, Querência, Roxinho e Irga 424 em elevadas concentrações, reduzem a germinação e o vigor de sementes de milhã e angiquinho. Os isolados de F. oxyosporum e F. semitectum não são efetivos no controle de S. montevidensis quando aplicados de forma singular e no estádio fenológico de plântula com uma folha sagitada, entretanto, quando aplicados na fase de floração das plantas de arroz irrigado, causam perdas de produtvidade e redução no comprimento radicular.

A spontaneous outbreak of a neurological disease in swine caused by the ingestion of Aeschynomene indica seeds and the reproduction of the disease in the same animal species are reported. The natural outbreak occurred in a pig-raising facility in the central region of the state of Rio Grande do Sul, Brazil. On the premises there were 100 pigs (20 breeding sows and 80 young weaned pigs from several categories) that were fed a ration made by mixing 50% of corn bran, 25% of soy bean bran, 5% comercial premix with minerals and vitamins and 20% of broken rice contaminated with 40% of A. indica seeds. Although all 100 pigs apparently ingested the same ration only 45-day-old pigs were affected; morbidity, mortality and lethality rates were respectively 25%-40%, 8,5%-20%; and 25%-66%. Clinical signs appeared ≈ 24 hours after the start of the feeding of A. indica seeds contaminated ration and included variable degrees of incoordinated gait, falls, sternal recumbency with the hind limbs in a wide base stance, lateral recumbency and death. It was not possible to ascertain how many pigs recovered nor the time frame of recovery. One pig was necropsied in the premises. The intoxication was reproduced in 5 young pigs (A-E) which were fed a ration containing 10% (Pig A), 15% (Pig B) and 20% (Pig C-E) of A. indica seeds and in one adult pig (Pig F) which was fed a ration with 16.5% of A. indica seeds. Pigs A, B and F were euthanatized and Pigs C-E died of an acute disease respectively 16, 21 and 24 hours after the start of the experiment. Clinical signs were similar to those observed in pigs of the spontaneous outbreak. Necropsy findings included marked hyperemia of encephalic leptomeninges of all pigs, large amounts of A. indica seeds in the stomach and small intestine of Pigs C-E and, reddening of the intestinal wall and bloody intestinal content. A hematoma was observed in the lungs of Pig C. The histopathological findings in the brain of pigs fed rations with larger concentrations (20%) of A. indica seeds (C-E) included congestion edema and hemorrhage and swollen capillary endothelia in several nuclei and in the telencephalic cortex. In the Pig F, fed a ration with 16,5% of A. indica seeds the lesions were similar in distribution but more severe. In swine fed rations containing 10% and 15% of A. indica in the ration, histopathological changes in the brain consisted of discreet focal symmetrical areas of malacia in which closely packed gitter cells and astrocytosis, and capillaries with swollen endothelium obliterated the normal neuropil. The symmetrical malacic foci from the ingestion of A. indica seeds in swine affected cerebellar and vestibular nuclei, putamen, and the mesencephalic oculomotor and red nuclei. This indicates that the A. indica seeds ingestion is responsible for this neurological condition in swine and that the clinical outcome and pathological changes are dose-dependent. / Relata-se um surto espontâneo de intoxicação em suínos pela ingestão de sementes de Aeschynomene indica e a reprodução da doença nessa espécie animal. O surto espontâneo ocorreu numa propriedade de criação de suínos localizada na região central do Rio Grande do Sul. Nessa propriedade havia 100 suínos (20 matrizes e 80 suínos jovens de várias categorias). Os suínos eram alimentados com uma ração feita na propriedade pela mistura de 50% farelo de milho, 25% de farelo de soja, 5% de um suplemento vitamínico mineral de origem comercial e 20% quirera de arrroz contaminada por 40% de sementes de A. indica. Embora aparentemente todos os 100 suínos tenham recebido a mesma ração, apenas os suínos de 45 dias de idade foram afetados; as taxas de morbidade, mortalidade e letalidade foram respectivamente 25%-40%, 8,5%-20% e 25%-66%. Os sinais clínicos apareceram ≈ 24 horas após o início da administração da ração contendo sementes de A. indica e incluíam vários graus de incoordenação no andar, quedas, decúbito esternal com membros pélvicos posicionados afastados entre si, decúbito lateral e morte. Não foi possível determinar quantos suínos se recuperaram e quanto tempo levou a recuperação. Um suíno foi necropsiado na propriedade. A doença foi reproduzida em 5 suínos jovens (AE) alimentados com uma ração contendo 10% (Suíno A), 15% (Suíno B) e 20% (Suínos CE) de sementes de A. indica e em um suíno adulto (Suíno F) que recebeu uma ração com 16,5% das sementes de A. indica. Os sinais clínicos foram semelhantes aos observados nos suínos do surto espontâneo. Os Suínos A, B e F foram submetidos à eutanásia e os Suínos C-E morreram de uma doença aguda respectivamente 16, 21 e 24 horas após o início do experimento. Os achados de necropsia incluíam acentuada hiperemia das leptomeninges em todos os suínos, grandes quantidades de sementes de A. indica no estômago e intestino delgado dos Suínos C-E e avermelhamento transmural da parede do intestino e conteúdo intestinal sanguinolento. Um hematoma foi observado no pulmão do Suíno C. Os achados histopatológicos no encéfalo dos suínos alimentados com as maiores concentrações (20%) de sementes de A. indica (C-E) consistiram de congestão, edema, hemorragia e tumefação dos endotélios capilares em diversos núcleos e no córtex telencefálico. No Suíno F que recebeu uma ração com 16,5% de sementes, as lesões foram semelhantes em distribuição, porém mais intensas. Nos suínos alimentados com concentrações menores (10% e 15%) das sementes de A. indica, as alterações histológicas no encéfalo consistiam de áreas bem definidas de malacia focal simétrica; nessas áreas a neurópila normal era obliterada por numerosos macrófagos espumosos dispostos em aposição estreita, astrocitose e capilares com endotélios tumefeitos. Os focos de malacia focal simétrica em suínos intoxicados com sementes de A. indica afetavam os núcleos cerebelar e vestibular, a substância negra, o putâmen e os núcleos mesencefálicos, oculomotor e núcleo vermelho. Esses dados indicam que ingestão de sementes de A. indica é responsável por essa condição neurológica em suínos e que o desenlace clínico e as alterações patológicas são dependentes da dose.