A ampla diversidade metabólica das cianobactérias é associada não somente a sua importância nos ciclos biogeoquímicos, mas também a sua distribuição global. Tal característica também é responsável pela capacidade destes organismos em produzir uma ampla variedade de substâncias de estruturas incomuns e atividades de interesse para o homem. Microcystis é um gênero cianobacteriano reconhecido como produtor de mais de duas centenas de produtos naturais, incluindo cianotoxinas. Microcystis aeruginosa é uma espécie frequentemente encontrada em florações, portanto causando preocupações sobre sua influência ecológica, especialmente em corpos d\'água doce utilizados para consumo humano. Desta forma, o objetivo deste trabalho foi o levantamento da diversidade e quantidade de metabólitos secundários que podem ser produzidos pela espécie, através de análises genômicas, além de variáveis que podem potencialmente interferir nas análises computacionais, procurando-se por padrões na espécie, e comparando-se 18 linhagens de todos os continentes. Foi encontrado o total de 235 agrupamentos relacionados ao metabolismo secundário, categorizados em 12 classes segundo as estruturas de seus produtos, nas 18 linhagens, evidenciando a riqueza de agrupamentos relacionados ao metabolismo secundário encontrados nesta espécie. Destes agrupamentos, os mais abundantes pertencem às categorias dos Terpenos, Híbridos, Bacteriocinas e NRPS. Entre as NRPS, nenhuma foi comum a todas as linhagens. Ainda, a quantidade de agrupamentos variou entre 6 e 21, e a quantidade de categorias de produtos variou entre 4 e 10, mostrando uma distribuição heterogênea de agrupamentos e tipos de metabólitos preditos. Esta distribuição heterogênea foi detalhada para melhor compreensão deste padrão encontrado na espécie. Dos agrupamentos de NRPS, os três mais frequentes foram selecionados para uma análise pormenorizada de sua estrutura e sequência: aeruginosina (15 linhagens), microcistina (11 linhagens), e micropeptina (15 linhagens). O agrupamento de micropeptina encontrado nas linhagens SPC777, TAIHU98 e PCC 9806 se mostrou amplamente dissimilar com relação à referência utilizada, potencialmente indicando um erro de identificação causado pela plataforma antiSMASH utilizada para a localização dos agrupamentos. Análises de colinearidade genômica mostram uma baixíssima sintenia entre os genomas das linhagens em análise, sugerindo frequentes eventos de reorganização genômica. Ainda, análises de pangenoma mostram um cenário em que mais genomas desta espécie são necessários para a estimativa da quantidade total de genes diferentes que a espécie pode possuir, o que é interessante para futuros estudos de procura de metabólitos secundários. Análises do genoma cerne apontam para uma estimativa segura de 1.944 genes comuns a todos os genomas desta espécie, o que corresponde entre 35% e 50% dos genes em cada linhagem. Análises estatísticas apontam para diferentes graus de interferência não linear da quantidade de sequências contíguas na observação de diferentes padrões de outras características genômicas, sugerindo precaução nas expectativas com relação ao metabolismo secundário em caso de linhagens em que a montagem gênica ultrapasse o limite superior aproximado de 100 sequências contíguas. / The wide metabolic diversity of cyanobacteria is associated not only with their importance in biogeochemical cycles, but also with their global distribution. Such a feature is also responsible for the ability of these organisms to produce a wide variety of substances with unusual structures and activities of interest to man. Microcystis is a cyanobacterial genus recognized as a producer of more than two hundred natural products, including cyanotoxins. Microcystis aeruginosa is a species frequently found in cyanobacterial blooms, thus causing concerns about its ecological influence, especially in freshwater bodies used for human consumption. In this way, the objective of this work was the survey of the diversity and quantity of secondary metabolites that can be produced by the species, through genomic analyzes, besides variables that can potentially interfere in the computational analyzes, searching for patterns in the species, and comparing 18 strains from all the continents. A total of 235 clusters, categorized in 12 classes according to the structure of their products, were found in the 18 strains, evidencing the richness of clusters related to the secondary metabolism found in this species. Of these clusters, the most abundant belong to the categories of Terpenes, Hybrids, Bacteriocins and NRPS. Among NRPS, none were common to all strains. Also, the number of groups ranged from 6 to 21, and the number of product categories ranged from 4 to 10, showing a heterogeneous distribution of predicted groupings and types of metabolites. Such a heterogeneous distribution was detailed for a better understanding of this pattern found in the species. Of the NRPS clusters, the three most frequent were selected for a detailed analysis of their structure and sequence: aeruginosin (15 strains), microcystin (11 strains), and micropeptin (15 strains). The micropeptide cluster found in the SPC777, TAIHU98 and PCC 9806 strains was widely dissimilar to the reference, só potentially indicating an identification error caused by the antiSMASH platform used to locate the clusters. Genomic collinearity analyzes showed a very low synteny among the genomes of the strains under analysis, suggesting frequent events of genomic reorganization. Also, pangenome analyzes show a scenario in which more genomes of this species are needed for the estimation of the total amount of different genes the species may possess, which is interesting for future studies conserning secondary metabolites. Coregenome analyzes point to a reliable estimate of 1,944 genes common to all genomes of this species, which corresponds to 30% up to 50% of the genes in each strain. Statistical analyzes point to different degrees of non-linear interference of the number of contiguous sequences on the observation of different patterns of other genomic characteristics, suggesting necessary caution about expectations regarding the secondary metabolism in case of strains in which the gene assembly exceeds the approximate upper limit of 100 contiguous sequences.

No description available.

Les bactéries du genre Streptomyces produisent de nombreux métabolites secondaires, dont certains possèdent des propriétés intéressantes en agriculture et en pharmaceutique. Avec le développement de la génomique, de nombreux outils bioinformatiques de recherche de groupes de gènes du métabolisme secondaire ont été développés au cours de la dernière décennie pour explorer les génomes. Ces outils sont basés sur la recherche de similarité de séquences et de ce fait, les clusters atypiques, constitués de gènes non caractérisés, ne peuvent être détectés par ces approches. L'isolement de tels clusters nécessite donc la mise en œuvre de nouvelles stratégies. La comparaison d’espèces d'Actinomycetes proches a révélé que les îlots génomiques, régions présentes dans un seul génome, sont très souvent enrichis en gènes du métabolisme secondaire. Nous avons participé (en collaboration avec les équipes d’Olivier Lespinet et de Pierre Leblond et Bertrand Aigle) au développement d’un outil, Break Viewer, permettant de localiser les îlots génomiques en comparant des génomes proches de Streptomyces. Cet outil a permis l'identification d'un îlot non détecté par les approches classiques, îlot dont l'étude a montré qu'il contenait un groupe de gènes du métabolisme secondaire. L’étude de ce groupe de gènes a montré qu'il dirige la synthèse de trois composés, le produit majoritaire étant le sphydrofurane. Une analyse fonctionnelle du cluster sphydrofurane a permis de déterminer les gènes impliqués dans la biosynthèse et la régulation de la biosynthèse du sphydrofurane et de proposer un modèle préliminaire pour la biosynthèse de ce métabolite. / Streptomyces are soil-dwelling bacteria that produce numerous secondary metabolites, some of which have interesting properties in agriculture and pharmaceuticals. With the development of genomics, many bioinformatics tools to search genomes for secondary metabolism gene clusters have been developed over the last decade. These tools are based on sequence similarity searches and therefore atypical clusters, consisting of uncharacterized genes, cannot be detected by these approaches. The isolation of such atypical clusters therefore requires the implementation of new strategies.Comparing closely related Actinomycetes species revealed that genomic islands (regions that are present in one genome only), are often enriched in secondary metabolite genes. We participated (in collaboration with the team of Olivier Lespinet and the team of Pierre Leblond and Bertrand Aigle) to the development of a new tool, Break Viewer, to locate genomic islands by comparing the genomes of closely related Streptomyces. This tool allowed the identification of an island, undetected by conventional approaches, island whose study showed that it contained a secondary metabolism gene cluster. The study of this cluster has shown that it directs the synthesis of three compounds, the major product being sphydrofuran. A functional analysis of the sphydrofuran gene cluster allowed us to identify the genes involved in the biosynthesis and regulation of sphydrofuran and to propose a preliminary model for the biosynthesis of this metabolite.

Ma thèse comporte deux volets: d’une part, le développement de ligands ciblant les protéines antiapoptotiques et d’autre part, l’étude par RMN des protéines CGC impliquées dans la biosynthèse de la congocidine, métabolite secondaire chez Streptomyces.La famille de protéines Bcl-2 est impliquée dans un des processus clé de la mort cellulaire programmée, appelée l’apoptose mitochondriale. Elle se divise en membres anti-apoptotiques (Bcl-2, Bcl-xL, Mcl-1) et pro-apoptotiques (Bak, Bax et les «BH3»).Ces molécules vont réguler l’apoptose en maintenant ou non l’intégrité de la membrane mitochondriale. En réponse à un signal de stress, les «BH3» neutralisent les antiapoptotiques et activent les pro-morts, leur permettant de former des pores sur la membrane mitochondriale. Ce phénomène aboutit au relargage du cytochrome c dans le cytosol et à l’activation de la cascade des caspases dont la finalité est la destruction de la celluleLa pertinence de l’étude des Bcl-2 s’observe de manière croissante depuis les années 1990. En effet, une surexpression des membres pro-survie de cette famille (Bcl-2, Bcl-xL, Mcl-1, BFL1 etc...) a été observée dans de nombreux cancers. Suite à ce constat, cibler ces molécules est devenue une piste prometteuse en cancérologie par le développement d’inhibiteurs des protéines pro-survie, l’objectif étant de restaurer l’apoptose dans les cellules tumorales.Dans cette perspective, différentes stratégies thérapeutiques ont été imaginées:(i) la thérapie génique avec l’Oblimersen, un ADN antisens développé par Genta, qui inhibe l’expression de la Bcl-2. Néanmoins, les résultats précliniques sont décevants.(ii) l’utilisation de peptides (ou peptidomimétiques) imitant les sentinelles «BH3» comme antagonistes de l’interaction Bcl-xL/Bak. Il faut souligner le concept des «stappled peptides», permettant la stabilisation des hélices par cyclisation des chaînes latérales. Si certaines de ces molécules synthétisées semblent très actives, aucune n’est encore en étude clinique.(iii) le développement de petites molécules non peptidiques, issues d’un criblage systématique in vitro ou in silico et se caractérisant par une grande variété structurale. Parmi ces molécules, certaines sont synthétiques comme l’ABT-737 et l’ABT-263 élaborés par les laboratoires Abbott, grâce à la méthode d’assemblage des fragments (fragment-based drug design) aidée par des études SAR by NMR (structure activity relationship). D’autres sont issues de produits naturels comme le (R)-Gossypol, le TW-37, la sanguinarine ou l’Obatoclax. En 2008, 11 composés étaient en phase préclinique ou clinique et les résultats pour certains d’entres eux semblaient plus prometteurs que pour l’Oblimersen.Ces stratégies ont permis de mettre au point un certain nombre de composés ciblant les protéines anti-apoptototiques. Si certains de ces composés sont actuellement en phase de tests cliniques, les plus prometteurs (ABT) ont démontré une efficacité uniquement vers certains des protéines anti-apoptotiques laissant place à un phénomène d’échappement des cellules cancéreuses.Un criblage réalisé à l’ICSN par l’équipe de F. Guéritte (Pôle Substances Naturelles Plantes) a permis d’identifier une nouvelle classe de molécules ayant une affinité de l’ordre du μM pour Bcl-xL. Parmi ces composés, deux présentent une attractivité d’un point de vue structural qui rend faisable leur synthèse chimique:(i) la meiogynine A, un sesquiterpène dimère de structure originale isolé des écorces de Meiogyne cylindrocarpa, une plante de Malaisie.(ii) le drimane, un sesquiterpène isolé en grandes quantités du genre zygogynum, une espècede Nouvelle-Calédonie.Ainsi, des collaborations ont été établies au sein de l’ICSN, réunissant diverses expertises (chimie, biologie, physicochimie et modélisation) afin de dégager les synergies souhaitables.Dans la perspective de la conception rationnelle d’analogues aux propriétés améliorées ciblant l’ensemble des membres anti-apoptotiques de la famille Bcl-2, ma contribution est de choisir les cibles biologiques (Bcl-xL et Mcl-1), de les obtenir pures et marquées en isotopes stables afin de réaliser par RMN et modélisation moléculaire une étude structurale des complexes protéines/ligands et de définir un modèle d’interaction.Le deuxième volet de ma thèse, à dominante fondamentale, a pour objectif de caractériser par RMN les médiateurs enzymatiques d’une voie de biosynthèse d’un métabolite secondaire, la congocidine issue des bactéries Streptomyces Ambofaciens.La congocidine présente des propriétés antivirales et anticancéreuses de par sa capacité à se fixer à l’ADN. Cependant, du fait de sa forte toxicité, cette molécule ne peut pas être utilisée directement à des fins thérapeutiques.L’analyse des groupes de gènes impliqués dans la biosynthèse de la congocidine a mis en évidence 24 gènes. Seuls certains intermédiaires réactionnels ont été identifiés. Cependant, le rôle précis des produits de ces gènes n’est pas encore bien défini.Ainsi, en collaboration avec l’équipe de JL Pernodet à l’Université d’Orsay, nous nous sommes intéressés à deux enzymes en particulier intervenant dans la synthèse de la congocidine, les protéines CGC-10 et CGC-19.L’objectif de cette collaboration est d’utiliser la spectroscopie RMN couplée à la modélisation moléculaire sous contraintes expérimentales afin de déterminer la structure de ces deux protéines. Nous souhaitons apporter des informations sur l’éventuelle présence de motifs structuraux au sein de ces protéines afin de mieux comprendre leur fonction et de définir à quel moment de la voie de biosynthèse elles interviennent.Concernant la protéine CGC-10, nous avons conçu un plasmide optimisé que nous avons fait synthétiser. Le gène obtenu a été cloné dans un vecteur d’expression choisi par nos collaborateurs (pQE30).Concernant la protéine CGC-19, nous allons en décrire les étapes d’expression et de purification qui nous ont permis d’enregistrer l’ensemble des expériences 3D-triple résonnance nécessaires à la détermination de la structure de la protéine. De plus, il a été mis en évidence la présence d’une modification post-traductionnelle de type phosphopanthéténylation au sein de cette protéine. Nous avons produit l’enzyme responsable de cette modification, la sfp, afin de pouvoir effectuer la réaction et suivre l’effet de la modification sur les spectres RMN de la protéine et donc sur la structure.Ce projet, qui s’inscrit dans une perspective de recherche à plus long terme, a pour objectif de caractériser précisément le mécanisme de production de la congocidine. A travers cette démarche, il s’agit de combiner la biologie moléculaire (modification en amont les gènes) à la chimie afin d’obtenir des molécules différentes aux propriétés améliorées et non toxiques. / My PhD thesis contains two parts: development of ligands against anti-apoptotic proteins and structural study of CGC proteins involved in the biosynthesis of congocidine, a Streptomyces Ambofaciens secondary metabolite.The first project concerns the NMR study of the interactions between the anti-apoptotic proteins and two potential ligand candidates, the Meiogynine and the Drimane. These two terpenoïds, identified from ICSN’s chemical library screening against the Bcl-xL protein, have shown a significant inhibiting activity, thus opening promising perspectives for the treatment of cancer cells overexpressing anti-apoptotic proteins. In fact, as these compounds are considerably smaller than the binding site, our objective is to introduce modifications (such as elongation of their structure, functionalization with hydrophilic groups etc.) that may improve their binding properties as well as their delivery and bioavailability.Following to the successful recombinant expression and purification, necessary to obtain labelled targets (15N/13C), our preliminary NMR studies suggested a rather universal action of our candidates, capable to bind not only to Bcl-xL but also to the other major anti-apoptotic protein, Mcl-1. Titration experiments revealed significant perturbations of the HSQC protein NMR spectra with the progressive disappearance of several protein HN and ligand signals, confirming dissociation constants at the µM region for both targets. However, the intermediate chemical exchange NMR regime observed, associated with the weak ligand solubility, poses severe difficulties for the structural elucidation of the complexes by classical NMR methods.In this work alternative approaches for the localisation of the ligands in the hydrophobic cleft of both target proteins will be presented.Oligopyrroles are secondary metabolites synthesized by Streptomyces bacteria. This family of natural products, composed by one or more pyrrole-2-carboxamide groups is characterized by a variety of biological activities such as antiviral, antitumor and antibiotic functions.One of the best-known metabolites is the congocidine, extensively studied due to its capacity to bind into the minor groove of the DNA double helix, with strong sequence specificity. However, because of its strong toxicity, this molecule cannot be directly used for therapeutic purposes.The analysis of the groups of genes involved in congocidine biosynthesis brought to light 24 genes, but their precise role is not yet well defined. We were particularly interested in two enzymes: the proteins called CGC-10 and CGC-19. For the recombinant expression of the first one, we designed an optimized insert which was cloned in an expression vector pQE30.Concerning CGC-19, the stages of expression and purification, which allowed us to obtain doubly-labeled protein, as well as the 3D NMR experiments for spectral assignment and structure elucidation, will be discussed.Furthermore, we were interested in the holo- state of this protein obtained through a post-traductional modification (phosphopanthéténylation). To this, we produced the enzyme responsible for this modification, Sfp, carry out the reaction in vitro and follow the effect of the modification at the NMR spectra.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-01-26T10:49:56Z No. of bitstreams: 1 2015 - Rafaela Scalise Xavier de Freitas.pdf: 1344462 bytes, checksum: 1e3669fc0b4da746d7a41a31525f3492 (MD5) / Made available in DSpace on 2017-01-26T10:49:56Z (GMT). No. of bitstreams: 1 2015 - Rafaela Scalise Xavier de Freitas.pdf: 1344462 bytes, checksum: 1e3669fc0b4da746d7a41a31525f3492 (MD5) Previous issue date: 2015-06-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / This survey was divided into two chapters, the first one producing and characterizing Urochloa humidicola methanol extract in order to present secondary metabolites classes and bromathological and chemical composition. These metabolites have several functions within the plant and are associated to defense system, protecting the environment where they have been living. These compounds have been used on animal feed in the reason presenting antimicrobial properties that could be employed for inducing ruminal fermentation. Phytochemical screening tests and chemical composition of U. humidicola methanol extract and in natura plant were carried out on this present survey. The following secondary compounds classes: saponins, tannins, flavonoids, non-protein amino acids, cardiotive glycosides, steroids, tripernoids, catechins and saccharides were indentified U. humidicola methanol extract in regarding to in natura plant showed 10,20% and 5,17% crude protein concentrations, 35% and 1,51% lipids and 9,59% aand 39,92% non-fibrous carbohydrates, respectively. These results might be explained by percolation with methanol extraction methods extracting only soluble constituents transporting silted protein, lipids and ash to the extract. The second chapter of this survey aimed evaluating U. humidicola extract addition effect containing saponin associated to U. brizantha assessing gases production, (methane and carbon dioxide), ruminal kinetics, dry matter degradation and short chain fatty acids production (SCFA: acetate, propionate and butyrate), as well. Plant extracts have been an alternative inducing ruminal fermentation by secondary metabolites in the reason they are from natural sources and with no residue hazards in products like meat and milk. Ruminal fermentation induction could reduce methane production, as well as, increase acetate: propionate ratio and improve food degradation. Four U. humidicola methanol extract concentrations (0, 75, 150 and 250 g/L) on U. brizantha degradability by in vitro gases production were tested. At 150 g/L extract concentration gas production from fibrous carbohydrates was 118,21 mL. However, the highest fiber concentration rate occurred at 150 g/L. Increasing extract concentrations (75, 150 and 250 g/L) soluble fraction values were: 10,27; 7,46 and 14,07%, respectively. Effective ruminal degradability at 75, 150 and 250 g/L concentrations for passage rates for an animal in maintenance were 38,53%, 27,71% and 20,30%, respectively. Extract concentrations increase exerted a linear effect (P<0.05) on ruminal pH values being more evident at high extract concentration (250 g/L) as 5,73 and 5,43 at 12 and 24 hs, respectively. CO2 averages in regarding to incubated and degraded dry matter did not differ with extract concentrations increase at 12 hs. Methane averages in regarding to incubated and degraded dry matter were no significative by regression analysis. Treatment at 250 g/L concentration presented the lowest value for methane at 12 hs. At 75 g/L concentration, total SCFA (acetic, propionic and butyric acid) increase at 12 and 24 hs was reported. U. humidicola methanol extract different concentration addition improved U. brizantha fermentation kinects parameters at 150 g/L and 250 g/L concentrations. However, negative effect on U. brizantha dry matter degradation and ruminal pH values according to extract concentrations increase was reported. Strong correlation between pH values and dry matter degradation (p=0,61, P<0,05) was presented. Carbon dioxide concentration increased, as well as, methane production decreased U. humidicola crude methanol extract presented potential for use as ruminal fermentation promoter. New studies about U. humidicola extract employing animals for justifying its efficiency as food additive should, furthermore, be developed. / Este trabalho foi dividido em dois cap?tulos, o primeiro teve por objetivo produzir e caracterizar o extrato metan?lico de Urochloa humidicola, com o intuito de conhecer as classes de metab?litos secund?rios presentes e a composi??o qu?mico-bromatol?gica. Estes metab?litos possuem diversas fun??es dentro dos vegetais e est?o associados ao sistema de defesa, os protegendo no ambiente que vivem. Estes compostos est?o sendo utilizados na alimenta??o animal por apresentarem propriedades antimicrobianas que podem ser empregadas para induzir a fermenta??o ruminal. Para este estudo foram realizados os testes de prospec??o fitoqu?mica e as an?lises de composi??o bromatol?gica do extrato metan?lico de U. humidicola e da U. humidicola in natura. Foram identificadas as seguintes classes de compostos secund?rios: saponinas, taninos, flavonoides, amino?cidos n?o proteicos, glicos?deos cardioativos, ester?ides e tripern?ides, catequinas e sacar?deos. O extrato metan?lico de U. humidicola em rela??o ? planta in natura, apresentaram concentra??es de prote?na bruta de 10,20% e 5,17%, e mat?ria mineral de 16,14% e 8,14%, extrato et?reo de 35% e 1,51%, carboidrato n?o fibroso, 9,59% e 39,92%, respectivamente. Esse resultado pode ser explicado pelo m?todo de extra??o que foi por percola??o com metanol, extraindo somente os constituintes sol?veis carreando somente prote?na, lip?deos e cinzas para o extrato. No segundo cap?tulo deste trabalho teve como objetivo avaliar o efeito da adi??o de extrato de U. humidicola contendo saponina, associada ? Urochloa brizantha, avaliando a produ??o de gases (metano e de di?xido de carbono), a cin?tica ruminal, a degrada??o da mat?ria seca e produ??o de ?cidos graxos de cadeia curta (AGCC; acetato, propionato e butirato). Os extratos vegetais de plantas s?o uma alternativa para induzir da fermenta??o ruminal por possu?rem metab?litos secund?rios, por serem de fontes naturais e sem riscos de res?duos nos produtos como carne e leite. A indu??o da fermenta??o ruminal pode reduzir a produ??o de metano, aumentar a rela??o de acetato: propionato e melhorar a degrada??o do alimento. Foram testados quatro concentra??es de extrato metan?lico de U. humidicola (0, 75, 150 e 250 g/L) sobre a degradabilidade da U. brizantha pela produ??o de gases in vitro. Na concentra??o de 150 g/L do extrato, a produ??o de g?s proveniente dos carboidratos fibrosos, foi de 118,21 mL. No entanto, a maior taxa de degrada??o dos carboidratos fibrosos ocorreu na concentra??o 150 g/L. Com o aumento das concentra??es de extrato (75, 150 e 250 g/L) os valores da fra??o sol?vel foram de 10,27; 7,46 e 14,07% respectivamente. A degradabilidade ruminal efetiva nas concentra??es de (75, 150 e 250 g/L) para as taxas de passagem para um animal em manten?a foram de 38,53%, 27,71% e 20,30%, respectivamente. O aumento das concentra??es de extrato exerceu um efeito linear (P<0,05) sobre os valores de pH ruminal, sendo mais evidente na alta concentra??o de extrato (250 g/L) que foi de 5,73 e 5,43 nos tempos de 12 e 24 horas, respectivamente. As m?dias de CO2 com rela??o ? mat?ria seca incubada e degradada n?o diferiram entre si com o aumento das concentra??es de extrato nos tempos de 12 horas. As m?dias de metano com base na mat?ria seca incubada e degradada n?o apresentaram signific?ncia para an?lise de regress?o. O tratamento com a concentra??o de 250 g/L de extrato apresentou menor valor para metano no tempo de 12 horas. A concentra??o de extrato (75 g/L) proporcionou um aumento do total de AGCC, ?cido ac?tico, ?cido propi?nico e ?cido but?rico tanto no tempo de 12 e 24 horas. A adi??o das diferentes concentra??es de extrato metan?lico de U. humidicola melhorou os par?metros da cin?tica da fermenta??o da U. brizantha nas concentra??es de 150 e 250 g/L. Mas causou um efeito negativo sobre a degrada??o da mat?ria seca da U. brizantha e no pH ruminal com o aumento das concentra??es de extrato. Existe uma forte correla??o entre os valores de pH e degrada??o da mat?ria seca (?=0,61, P<0,05). Aumentou as concentra??es de g?s carb?nico e reduziu a produ??o de metano. O extrato metan?lico bruto de U. humidicola tem potencial para uso como indutor da fermenta??o ruminal. ? necess?rio ? purifica??o e o isolamento da saponina do extrato para comprovar o efeito ben?fico sobre a fermenta??o ruminal. S?o imprescind?veis novos estudos com o extrato de U. humidicola, utilizando animais para se comprovar a efici?ncia na utiliza??o como aditivo alimentar

Made available in DSpace on 2017-07-10T17:37:02Z (GMT). No. of bitstreams: 1 Monica_Anghinoni_Muller.pdf: 756682 bytes, checksum: 61f5a661d3ee29d68326be6cb32f6bc7 (MD5) Previous issue date: 2015-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The nematode Pratylenchus brachyurus known as nematode lesions, affects the soybean crop caused significant damage, this means that there is a need to develop alternatives that supply the control of pathogens by aggregating in productivity. Then the objective is to verify the influence of homeopathic Mercurius solubilis in different potencies in soybean plants and on control of the nematode. For this, three experiments were carried out in climatized greenhouse, testing the potencies of 6, 12, 24, 50, 100, 200 and 400CH (centesimal Hahnemannian) of Mercurius solubilis, ethanol 30% and healthy plants (untreated and not inoculated) were used as control treatment. The treatments were applied weekly from the V3 growth stage of soybeans. Three days after the first treatment, inoculation of nematodes was done. After 50 and 70 days after inoculation of the first and second experiment respectively, were made assessments of the aerial part height, stem diameter, number of pods per plant, dry weight of aerial part, dry weight of leaf + petiole + stem, dry mass of total pods, dry weight per pod, fresh weight of root, and were count juvenile, adults and eggs in the soil and in roots, and determined the reproduction factor (RF). In the third experiment, were quantified enzymes involved in secondary metabolism of plants, peroxidase (POX), phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO). The sample of roots were taken at intervals of 0, 3, 7 and 14 days after treatment (DAT) and in the 3rd DAT, inoculation was made. In laboratory was conducted a experiment to evaluate in vitro motility and mortality, a distilled water solution containing 100 ml-1 juveniles and adults were placed in plastic container and add 7 mL of in vivo treatments tested at a dilution of 0.1%. The experiments were conducted in a randomized block design. The potencies 24CH, 50CH, 200CH and 100CH reduce the number of adults and juveniles in soil, as well as the reproduction factor, furthermore 100CH is able to interfere in the productive aspects, increasing 107.5% in the number of pods when compared to the control ethanol 30%, as well as dynamization 6CH and 12CH. To POX the enzymatic activity was higher for dynamizations 6CH, 100CH and 400CH, 3, 7 and 14 DAT respectively. The PAL activity presented increases of 79.93%, 80.72% and 84.10% in dynamizations 6CH, 12CH and 24CH respectively compared to control treatment, 3 DAT. 14 days after the first treatment, 400CH dynamization showed an increase in the enzymatic activity of 53.41% and 32.21% when compared to the control ethanol 30% and absolute control respectively. The dynamization 24CH when compared to absolute control showed an increase of 41.10% in the enzymatic activity. So Mercurius solubilis may be a potential alternative for the control of the nematode / O nematoide Pratylenchus brachyurus conhecido como nematoide das lesões, afeta a cultura da soja causado danos expressivos, isso faz com que haja a necessidade de desenvolver alternativas que supram o controle dos patógenos, agregando em produtividade. Objetivou-se então, verificar a influência do medicamento homeopático Mercurius solubilis em diferentes dinamizações nas plantas de soja e no controle de P. brachyurus. Para tanto, foram conduzidos três experimentos em casa de vegetação climatizada, testando-se as dinamizações de 6, 12, 24, 50, 100, 200 e 400CH (centesimal hahnemanniana) de Mercurius solubilis, Etanol 30% e plantas sadias (não tratada e não inoculada) foram utilizadas como tratamento testemunha. Os tratamentos foram aplicados semanalmente a partir do estádio fenológico V3 da soja. Três dias após o primeiro tratamento, foi feita a inoculação dos nematoides. Decorridos 50 e 70 dias após a inoculação do primeiro e segundo experimento respectivamente, foram realizadas as avaliações de altura de parte aérea, diâmetro do coleto, número de vagens por planta, massa seca de parte aérea, massa seca de folha+pecíolo+caule, massa seca total de vagens, massa seca por vagem, massa fresca de raiz, e contagem de juvenis, adultos e ovos presentes no solo e na raiz, e determinado o fator de reprodução (FR). No terceiro experimento, foram quantificadas enzimas envolvidas no metabolismo secundário das plantas, peroxidase (POX), fenilalanina amônia-liase (FAL) e a polifenoloxidase (PFO). As coletas das amostras de raízes foram realizadas no intervalo de 0, 3, 7 e 14 dias após o tratamento (DAT) sendo que no 3º DAT foi feita a inoculação. Em laboratório foi realizado experimento in vitro para avaliação de motilidade e mortalidade, uma solução de água destilada contendo 100 juvenis e adultos mL-1 foi depositada em recipiente plástico, e adicionados 7 mL dos tratamentos testados in vivo na diluição de 0,1%. Os experimentos foram conduzidos em delineamento em blocos casualizados. As dinamizações 24CH, 50CH, 100CH e 200CH reduzem o número de juvenis e adultos presentes no solo, assim como o fator de reprodução, além disso, a dinamização 100CH é capaz de interferir em aspectos produtivos pelo aumento de 107,5% no número de vagens quando comparada à testemunha etanol 30%, assim como a dinamização 6CH e 12CH. Para POX a atividade enzimática foi superior para as dinamizações 6CH, 100CH e 400CH, em 3, 7 e 14 DAT respectivamente. A atividade de FAL apresentou incrementos de 79,93%, 80,72% e 84,10% nas dinamizações 6CH, 12CH e 24CH respectivamente em relação à testemunha absoluta, 3 DAT. 14 dias após o primeiro tratamento, a dinamização 400CH mostrou um aumento na atividade enzimática de 53,41% e 32,21% quando comparada à testemunha etanol 30% e testemunha absoluta respectivamente. A dinamização 24CH quando comparada a testemunha absoluta mostrou um acréscimo de 41,10% na atividade enzimática. Assim Mercurius solubilis pode ser uma alternativa potencial para o controle de P. brachyurus

O estudo fitoquímico de Peperomia oreophila revelou a presençca de duas lignanas furofurânicas (7R, 8R, 7R, 8R)-3,4,5-trimetóxi-3,4-metilenodioxi-5-metóxi- 8.8,7.O.9,7.O.9-lignana (1), (7R, 8R, 7R, 8R)-3,4,5-trimetóxi-3,4,5-trimetóxi- 8.8,7.O.9,7.O.9-lignana (2); as duas amidas (2E)-N-isobutil-3-(5-metóxi-7,8- benzodioxol-1-il)acrilamida (3), (2E)-N-isobutil-3-(3,4,5-trimetóxifenil)acrilamida (4), três derivados de acido cinâmico (2E)-3-(3,4,5-trimetóxifenil)acrilato de metila (5), (2Z)-3- (3,4,5-trimetóxifenil)acrilato de metila (6), (2E)-3-(5-metóxi-7,8-benzodioxol-1-il)acrilato de metila (7); os dois policetídeos fenólicos [(2E)-3,7-dimetilocta-2,6-dien-1-il]-5- metil-2-(3-metilbut-2-en-1-il)benzeno-1,3-diol (8) (inédita) e [(2E)-3,7-dimetilocta- 2,6-dien-1-il]-2,2,7-trimetil-2H-cromen-5-ol (9); de P. arifolia: o policetídeo fenólico [(2E)-3,7-dimetilocta-2,6-dien-1-il]-5-metil-2-(3-metilbut-2-en-1-il) benzeno-1,3-diol, isolada também de P. oreophila (10) (inédita); de P. urocarpa: o policetídeo fenólico 5- metil-2-[(2E,6E)-3,7,11-trimetildodeca-2,6,10-trien-1-il] benzeno-1,3-diol (11) e o ácido 2,4-dihidróxi-6-metil-3-[(2E,6E)-3,7,11-trimetildodeca-2,6,10-trien-1-il] benzóico, (12); de P. nitida: o fenilpropanoide apiol (1-alil-3,6-dimetóxi-10,11- benzodioxol) (13), os cromenos 7-hidróxi-2,2,5-trimetil-2H-cromeno-carboxilato de metila (14) e o 7-metóxi-2,2,5-dimetil-2H-cromeno-6-carboxilato de metila (15). O policetídeo 2-hidróxi-4,6-dimetóxiacetofenona, principal metabólito das folhas de P. glabella, teve sua biossíntese investigada utilizando-se como precursores o acetil-CoA e o malonil-CoA. Foram realizados estudos de otimização da atividade de policetídeo sintase (PKS) em função do pH, tempo de reação, temperatura e saturação de substratos, além de estudos da variação circadiana. Estudos de genes de PKS resultaram em amplificações cujo seqüenciamento poderá determinar a identidade dessas regiões e homologia entre as seqüências dessas Peperomias e a região KS do gene AviM de Streptomyces viridochromogenes que expressa o ácido orselínico / The phytochemical investigation carried out on Peperomia oreophila revealed the accumulation of two furofuran lignans (7R,8R,7R,8R)-3,4,5-trimethoxy-3,4- methylenedioxy-8.8, 7.O.9, 9.O.7-lignan (1), (7, 8R, 7R, 8)-3,4,5-trimethoxy-3,4,5- trimethoxy-8.8-7.O.9, 9.O.7-lignan (2); two amides (2´E)-N-isobutyl-3´-(5-methoxy-7,8- benzodioxol-1-yl) acrylamide (3), (2E)-N-isobutyl-3-(3,4,5-trimethoxyphenyl)acrylamide (4), three derivate cinâmic acid methyl (2E)-3-(3,4,5-trimethoxyphenyl)acrylate (5), methyl (2Z)-3-(3,4,5-trimethoxyphenyl)acrylate (6), methyl (2E)-3-(5-methoxy-7,8- benzodioxol-1-yl)acrylate (7); two phenolic polyketides [(2E)-3,7-dimethylocta-2,6- dien-1-yl]-5-methyl-2-(3-methylbut-2-en-1-yl)benzene-1,3-diol (8) (novel), [(2´E)-3´,7´- dimethylocta-2´,6´-dien-1-yl]-2,2,7-trimethyl-2H-chromen-5-ol (9); P. arifolia, two phenolic polyketide [(2E)-3,7-dimethylocta-2,6-dien-1-yl]-5-methyl-2-(3-methylbut-2- en-1-yl)benzene-1,3-diol, also isolated from P. oreophila (10) (novel); P. urocarpa, the two phenolic polyketides 5-methyl-2-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1- yl]benzene-1,3-diol (11) and 2,4-dihydroxy-6-methyl-3-[(2E,6E)-3,7,11-trimethyldodeca- 2,6,10-trien-1-yl]benzoic acid (12); P. nitida, the phenylpropanoid apiole 1-allyl-3,6- dimethoxy-10,11-benzodioxole (13); the chromenes methyl 7-hydroxy-2,2,5-trimethyl- 2H-chromene-6-carboxylate (14) and methyl 7-methoxy-2,2,5-trimethyl-2H-chromene-6- carboxylate (15). The polyketide 2-hydroxy-4,6-dimethoxyacetophenone, the major compound in P. glabella leaves, had its biosynthetic origin investigated using acetyl-CoA and malonyl-CoA as precursors. The enzymatic activity of polyketide synthase was optimized to pH, incubation time, temperatures and substrate saturation, in addition to the analysis of circadian variation activity. The amplifications of putative PKS genes was based on primers from AviM gene of Streptomyces viridochromogenes that express for orsellinic acid. The sequencing will enable the identification of such regions and also to study the homology to fungi PKS

Transcription factors (TFs) are global regulators of gene expression playing essential roles in almost all biological processes, and are therefore of great scientific and biotechnological interest. This project focused on functional characterisation of three DNA-binding-with-one-zinc-finger (DOF) TFs from the genetic model plant Arabidopsis thaliana, namely OBP1, OBP2 and AtDOF4;2. These genes were selected due to severe growth phenotypes conferred upon their constitutive over-expression. To identify biological processes regulated by OBP1, OBP2 and AtDOF4;2 in detail molecular and physiological characterization of transgenic plants with modified levels of OBP1, OBP2 and AtDOF4;2 expression (constitutive and inducible over-expression, RNAi) was performed using both targeted and profiling technologies. Additionally expression patterns of studied TFs and their target genes were analyzed using promoter-GUS lines and publicly available microarray data. Finally selected target genes were confirmed by chromatin immuno-precipitation and electrophoretic-mobility shift assays. This combinatorial approach revealed distinct biological functions of OBP1, OBP2 and AtDOF4;2. Specifically OBP2 controls indole glucosinolate / auxin homeostasis by directly regulating the enzyme at the branch of these pathways; CYP83B1 (Skirycz et al., 2006). Glucosinolates are secondary compounds important for defence against herbivores and pathogens in the plants order Caparales (e.g. Arabidopsis, canola and broccoli) whilst auxin is an essential plant hormone. Hence OBP2 is important for both response to biotic stress and plant growth. Similarly to OBP2 also AtDOF4;2 is involved in the regulation of plant secondary metabolism and affects production of various phenylpropanoid compounds in a tissue and environmental specific manner. It was found that under certain stress conditions AtDOF4;2 negatively regulates flavonoid biosynthetic genes whilst in certain tissues it activates hydroxycinnamic acid production. It was hypothesized that this dual function is most likely related to specific interactions with other proteins; perhaps other TFs (Skirycz et al., 2007). Finally OBP1 regulates both cell proliferation and cell expansion. It was shown that OBP1 controls cell cycle activity by directly targeting the expression of core cell cycle genes (CYCD3;3 and KRP7), other TFs and components of the replication machinery. Evidence for OBP1 mediated activation of cell cycle during embryogenesis and germination will be presented. Additionally and independently on its effects on cell proliferation OBP1 negatively affects cell expansion via reduced expression of cell wall loosening enzymes. Summing up this work provides an important input into our knowledge on DOF TFs function. Future work will concentrate on establishing exact regulatory networks of OBP1, OBP2 and AtDOF4;2 and their possible biotechnological applications. / Biologische Prozesse, wie beispielsweise das Wachstum von Organen und ganzen Organismen oder die Reaktion von Lebewesen auf ungünstige Umweltbedingungen, unterliegen zahlreichen Regulationsmechanismen. Besonders wichtige Regulatoren sind die sogenannten Transkriptionsfaktoren. Dabei handelt es sich um Proteine, die die Aktivität von Erbeinheiten, den Genen, beeinflussen. In Pflanzen gibt es etwa 2000 solcher Regulatoren. Da sie wichtige Kontrollelemente darstellen, sind sie von großem wissenschaftlichen und biotechnologischen Interesse. Im Rahmen der Doktorarbeit sollte die Funktion von drei Transkriptionsfaktoren, genannt OBP1, OBP2 und AtDOF4;2, untersucht werden. Sie wurden bei der Suche nach neuen Wachstumsregulatoren identifiziert. Als Untersuchungsobjekt diente die in der Öffentlichkeit kaum bekannte Pflanze Ackerschmalwand, lateinisch als Arabidopsis thaliana bezeichnet. Um die Funktion der Regulatoren zu entschlüsseln, wurden an der Modellpflanze genetische Veränderungen durchgeführt und die Pflanzen dann mit molekularbiologischen und physiologischen Methoden analysiert. Es zeigte sich, dass OBP1 an der Regulation der Zellteilung beteiligt ist. Alle Lebewesen sind aus Zellen aufgebaut. Gelingt es, die Zellteilung gezielt zu steuern, kann damit beispielsweise die Produktion von pflanzlicher Biomasse verbessert werden. Das OBP1-Protein übt auch einen Einfluss auf die Zellstreckung aus und beeinflusst auch auf diesem Wege das pflanzliche Wachstum. Die beiden anderen Proteine steuern Prozesse, die im Zusammenhang mit der Bildung von Pflanzeninhaltsstoffen stehen. OBP2 ist Teil eines zellulären Netzwerkes, dass die Synthese von sogenannten Glucosinolaten steuert. Glucosinolate kommen unter anderem in Broccoli und Kohl vor. Sie fungieren als Abwehrstoffe gegen Fraßinsekten. Einigen Glucosinolaten wird auch gesundheitsfördernde Wirkung zugesprochen. Das Protein AtDOF4;2 ist Komponente eines anderen Netzwerkes, dass die Bildung von Phenylpropanoiden steuert. Diese Substanzen haben strukturelle Funktion und spielen darüber hinaus eine Rolle bei der pflanzlichen Toleranz gegenüber tiefen Temperaturen. Mit der Doktorarbeit konnte das Wissen über die Transkriptionsfaktoren erheblich erweitert und die Grundlage für interessante zukünftige Arbeiten gelegt werden. Von großer Bedeutung wird es dabei sein, die Netzwerke, in die die Transkriptionsfaktoren eingebunden sind, noch besser zu verstehen. Dann wird es möglich sein, auch Teilnetzwerke gezielt zu beeinflussen, was für biotechnologische Anwendungen, beispielsweise bei der Präzisionszüchtung von nachwachsenden Rohstoffen, von zentraler Bedeutung ist.

Viele Studien beschreiben Entwicklung und Sekundärmetabolismus des filamentösen Pilzes Aspergillus nidulans, was zu einem besseren Verständnis der Regulation des Sekundärmetabolismus von Pilzen auf molekularer Ebene beisteuert. Dennoch muss ein umfassendes Bild dieser Regulation noch gezeigt werden. Aus diesem Grund geben wir in dieser Arbeit einen ausführlichen Überblick von Transkriptom und Metabolom, welcher die Antworten der lichtabhängigen Entwicklung dieses bodenbürtigen Pilzes aufzeigt. Licht begünstigt die Entwicklung asexueller Sporen und hemmt die Entstehung sexueller Fruchtkörper (Kleistothetien), die bevorzugt im Dunkeln gebildet werden. Insgesamt werden 2.014 Gene, was etwa 20 % dessen Genom entspricht, während unterschiedlicher Entwicklungsphasen in Licht und Dunkelheit differenziell exprimiert und beeinflusst. Licht kontrolliert die Entwicklung, indem es die Genexpression während 24-48 Stunden der Entwicklung erheblich induziert. Die gezielte Repression von Lichtsensor-Komplexen in der frühen sexuellen Entwicklung könnte die Genexpression und schließlich die Differenzierung des Pilzes verzögern. Die erhöhte Anzahl verzögerter Gene während der sexuellen Differenzierung zeigt eine zeitliche Übereinstimmung mit der sekundär induzierten und verzögerten Bildung von Konidien bei sexueller Entwicklung. Interessanterweise zeigen die Transkriptome vom vegetativen Wachstum und der frühen sexuellen Entwicklung ähnliche Expressionsmuster auf, was sich in den äußerst ähnlichen Phänotypen beider Phasen widerspiegelt. Ein charakteristisches Merkmal während der späten Phase asexueller Sporenbildung im Licht stellt die Expression von Genen für die Antwort auf Stress dar. Dadurch könnten Resistenzen gegenüber verschiedensten abiotischen Stressbedingungen, einschließlich UV-Bestrahlung and daraus entstehende reaktive Sauerstoffspezies, in den luftverbreiteten Konidien gebildet werden. Die Entwicklung der Pilze ist von psi (precocious sexual inducer) Faktoren, welche Prostaglandin verwandte Oxylipin-Hormone sind, abhängig. PsiC1(5,8-DiHOE) erscheint spezifisch während der frühen sexuellen Entwicklung in Dunkelheit. Im Laufe der sexuellen Entwicklung aktiviert A. nidulans viele Gene für den Zellwandabbau, einschließlich Gene für den Abbau von Pflanzen- und Bakterienzellwand sowie für die Hydrolyse von Polysacchariden. Dadurch könnten Energie und die Grundbausteine für die erfolgreiche Fertigstellung sexueller Fruchtkörper während Nährstoffmangelbedingungen mobilisiert wird. Während der späten sexuellen Entwicklung sind Sekundärmetaboliten vorhanden, welche den Schutz der Fruchtkörper und Askosporen z. B. gegen Fressfeinde gewährleisten und daher den Askosporen beim Keimen in Anwesenheit einer geringeren Anzahl von Mitstreitern unterstützt. Das Emericellamide C Metabolit wird ausgeschieden bevor die Reifung der Kleistothecien und die sexuelle Entwicklung abgeschlossen sind. Viele herunter regulierte Gene für die Synthese von Aminosäuren und die geringe Anreicherung zellulärer Aminosäuren in der späten sexuellen Entwicklung spiegeln einen Ruhezustand wider, bei welchem die Translation aufgrund des Mangels an Aminosäuren gestoppt wurde. Der Pilz initiiert den programmierten Zelltod in der späten sexuellen Entwicklung durch die Expression von Apoptose-Genen, welche mit einem Alterungsprozess einhergeht. Unsere Ergebnisse zeigen, dass während der lichtabhängigen Entwicklung des Pilzes ein beachtlicher Teil des Genoms (20 %) durch das Lichtsignal beeinflusst wird. Dieses führt zu verschiedenen Antworten einschließlich der Produktion von Sekundärmetaboliten und anderen Anpassungen, welche es zusammengenommen dem Pilz ermöglichen sich anzupassen und in den vorherrschenden Umweltbedingungen zu überleben.

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