Orientador: Cristiano Gallina Moreira / Resumo: As infecções do trato urinário são frequentes no mundo todo, sendo a Escherichia coli Uropatogênica (UPEC) o patógeno responsável pela maior parte dos casos de cistite e pielonefrite aguda. A patogenicidade das UPECs está relacionada a expressão de diversos fatores de virulência, sendo a regulação da expressão desses fatores mediada por moléculas sinalizadoras químicas que permitem a comunicação célula-célula inter e intra-reinos, o que facilita o processo de colonização e estabelecimento da patogênese. Um dos sistemas responsáveis por essas cascatas de sinalização é composto por uma proteína sensora de membrana (QseC) e outra reguladora de resposta citoplasmática (QseB), constituindo o sistema de dois componentes QseBC, capaz de reconhecer sinais produzidos pelo hospedeiro e por outras bactérias, levando a regulação da expressão de genes de virulência do patógeno. Estudos realizados pelo nosso grupo, evidenciaram atenuação da virulência de patógenos Gram-negativos na ausência do gene qseC, levando ao desenvolvimento de moléculas que atuassem inibindo essa via como o LED209. O objetivo do presente trabalho foi caracterizar cepas multi-droga resistentes (MDR) de UPECs obtidas a partir de isolados clínicos, além de investigar, in vitro e in vivo, a participação da via QseBC na patogênese e na virulência de cepas de UPECs e isolados clínicos MDR, com o uso do composto LED209 na atenuação da virulência frente a esses patógenos. Também constituiu o objetivo, o uso do ácido 3,4-di-... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Urinary tract infections are found commonly worldwide, whereas Uropathogenic Escherichia coli (UPEC) is the most prevalent pathogen, responsible for utmost cases of cystitis and acute pyelonephritis. The pathogenicity of UPECs is related to the expression of several virulence factors, and the regulation of the expression of these factors is mediated by chemical signaling molecules. The communication allow inter-intra-kingdom and cell-to-cell facilitates the process of colonization and establishment of pathogenesis. QseBC two-component system is capable of recognizing signals produced by the host leading to regulation of pathogen virulence gene expression. This system consists of membrane sensing protein (QseC) and a cytoplasmic response regulator (QseB) that mediate the entire cascade of virulence genes. Studies conducted by our group showed attenuation of the virulence of Gram-negative pathogens in the absence of the qseC gene, leading to the development of molecules that act by inhibiting this pathway such as LED209. The aim of the present study was to characterize UPEC multidrug-resistant strains (MDR) from clinical isolates. Investigate, in vitro and in vivo, the involvement of the QseBC pathway in the pathogenesis and virulence of UPEC strains and clinical isolates. The use of LED209 and the 3,4-dihydroximandelic acid (DHMA), norepinephrine intermediate metabolite, as chemoattractant, were employed to attenuate of virulence against these pathogens. The results showed a h... (Complete abstract click electronic access below) / Mestre

Submitted by PATRICK DA SILVA null (patrick_dasilva@ymail.com) on 2016-09-01T14:44:37Z No. of bitstreams: 1 Dissertação Patrick da Silva.pdf: 3098856 bytes, checksum: 3fb93fb0061cbc5d3f1d4f4066945e07 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-09-01T18:20:53Z (GMT) No. of bitstreams: 1 silva_p_me_arafcf.pdf: 3098856 bytes, checksum: 3fb93fb0061cbc5d3f1d4f4066945e07 (MD5) / Made available in DSpace on 2016-09-01T18:20:53Z (GMT). No. of bitstreams: 1 silva_p_me_arafcf.pdf: 3098856 bytes, checksum: 3fb93fb0061cbc5d3f1d4f4066945e07 (MD5) Previous issue date: 2016-07-19 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Sinalização química em bactérias patogênicas é um mecanismo empregado para interagir com o hospedeiro e sua respectiva microbiota. Através desta interação ocorre a regulação dos mecanismos de virulência. Estudando o mecanismo de sinalização química do sistema de 2-componentes QseBC em Salmonella enterica serovar Typhimurium foi aberta uma nova perspectiva para desvendar os mecanismos de patogenicidade. Dentre estes, uma nova proteína VisP (Virulence and stress-related Periplasmic protein) foi reportada. Seu papel inicial na interação com a enzima LpxO em S. Typhimurium foi anteriormente demonstrada. O antígeno-O da camada de LPS fornece proteção contra as defesas do hospedeiro e, particularmente, o comprimento de sua cadeia exerce um papel essencial. A montagem do antígeno-O possui o sistema Wzz, o qual determina o comprimento final de sua cadeia polissacarídica, e também apresenta uma distribuição tri-modal. Forma cadeias curtas (S-OAg), longas (L-OAg) e muito longas (VL-OAg) de antígeno-O. As proteínas WzzST e WzzfepE regulam, respectivamente, a síntese das cadeias L-OAg e VL-OAg. Neste estudo, os genes wzzST, wzzfepE, visP e lpxO foram mutados, via mutagênese λ Red, obtendo simples e duplos mutantes. Dados preliminares evidenciaram que há um aumento nas cadeias VL-OAg e L-OAg no mutante ΔvisP, e reciprocamente há diminuição de L-OAg em ΔlpxO. Foram feitos ensaios para avaliar a motilidade, invasão em células epiteliais e sobrevivência intracelular em macrófagos com as amostras obtidas pelos ensaios de mutagênese. Foi evidenciado uma redução de 1,2 e 1,0 ordem de magnitude nos processos de invasão e sobrevivência intracelular, respectivamente, no mutante ΔvisP em relação à amostra selvagem, conforme já descrito, e também uma redução de 70,61% na motilidade comparada com à amostra selvagem. Observou-se que a deleção dos genes wzzfepE e lpxO no mutante ΔvisP faz com que a bactéria retorne ao fenótipo da amostra selvagem nos processos de motilidade, invasão em células epiteliais e sobrevivência intracelular em macrófagos. Este efeito de complementação fenotípica não ocorre no mutante duplo ΔwzzST, apresentando os mesmos níveis do mutante simples ΔvisP, apesar deste possuir um aumento na expressão de genes envolvidos na motilidade e invasão celular comparado com o mutante ΔvisP. Aparentemente, há a atenuação destes processos patogênicos em bactérias com um alto nível de VL-OAg, levando-nos a hipotetizar que este tipo de cadeia de antígeno-O possui relevância na patogênese de S. Typhimurium. WzzfepE e LpxO apresentaram uma evidente participação nos processos de motilidade, invasão celular e sobrevivência intracelular via VisP, e possivelmente o antígeno-O de comprimento muito longo (VL-Oag) apresenta um papel de inibição nestes processos, além disso, evidenciou-se que VisP desempenha uma função relevante na montagem das cadeias de antígeno-O e na patogênese de S. Typhimurium. / Chemical signaling is a mechanism employed by several bacterial species to interact within surrounding microbiota and their host. Upon this interaction the pathogenic bacteria regulate their virulence traits. The two-component system QseBC was described on chemical signaling in Salmonella enterica serovar Typhimurium, and a novel branch of pathogenic cascade regulation was revealed. Among these mechanisms a novel protein was described, VisP (Virulence and stress related Periplasmic protein). VisP interacts with LpxO enzyme on the periplasm. The O-antigen of the LPS layer provides protection against host defenses, and particularly its chain’s length plays an essential role. The O-antigen assembly has the Wzz system, which determines the O-antigen final chain length, and also presents a tri-modal distribution. It forms short (S-OAg), long (L-OAg) and very-long (VL-OAg) O-antigen chains. The WzzST and WzzfepE proteins respectively regulate the L-OAg and VL-OAg synthesis. In this study, wzzST, wzzfepE, visP and lpxO genes were mutated via λ Red mutagenesis, obtaining single and double-mutants. Our preliminary data have shown that VisP increases VL-OAg and L-OAg, conversely LpxO diminishes L-OAg chain length. The motility, epithelial cell invasion and macrophage intracellular survival and replication were assessed with the mutants obtained. The ΔvisP presented 1,2 and 1,0 order of magnitude reduction in cell invasion and intracellular macrophage replication, respectively, comparing with WT S. Typhimurium, as described, and 70,61% less motility rate than WT. Mutating wzzfepE or lpxO genes in ΔvisP recovers the motility, epithelial cell invasion and macrophage intracellular survival and replication WT phenotypes. Nonetheless, this effect does not occur with wzzST gene deletion in ΔvisP, as this double-mutant presents the same phenotypes of ΔvisP single-mutant, although this deletion raises expression rates of motility and cell invasion related genes significantly. Apparently, these pathogenic processes are attenuated within samples with VL-Oag high levels, taking us to hypothesize that there is a correlation between this O-antigen chain length and S. Typhimurium pathogenesis. WzzfepE and LpxO presented an important role on motility, epithelial cells invasion and macrophage intracellular survival through VisP, and probably the VL-OAg inhibits these processes. Furthermore, VisP plays a relevant function on O-antigen chain assembly and pathogenesis of S. Typhimurium. / CNPq: 134434/2014-5

Les kelps sont des espèces clés dans les écosystèmes marins tempérés, elles vivent en milieu intertidal le long des côtes où elles sont souvent exposées à des herbivores. Comme la plupart des organismes sessiles, les kelps ont développé des stratégies de défense contre l’herbivorie. Chez la laminaire Laminaria digitata, l’élicitation par les oligoguluronates induit des régulations transcriptomiques et l’activation de voies métaboliques, menant à la production et la libération de métabolites dans l’environnement, tels que des aldéhydes. De plus, cette espèce d’algue brune a montré des réponses systémiques suite à une élicitation. Des composés solubles dans l’eau de mer environnant les forêts de laminaires peuvent aussi modifier leur physiologie en réponse à l’élicitation. Une telle signalisation chimique à distance lors des réponses de défense rappelle les phénomènes de protection systémique et de potentialisation chez les plantes. Dans les forêts de laminaires, celle-ci pourrait jouer un rôle écologique majeur dans la structuration des algues marines et des communautés d’herbivores. Dans le contexte des interactions kelp/herbivores, nous avons étudié les réponses de défense et les processus de signalisation chez deux espèces emblématiques de laminaires originaires des côtes atlantiques et du Pacifique Sud, impliquées dans des interactions spécialisées avec leurs herbivores, L. digitata en interaction avec Patella pellucida (Bretagne, France) et Lessonia spicata avec Scurria scurra (Chili). Des approches combinées de métabolomique et de bio-essais ont été réalisées pour élucider la nature de potentiels signaux chimiques et leurs rôles biologiques et écologiques dans la défense des algues face aux herbivores. Les résultats ont montré que, en réponse à un broutage, les kelps présentent des modifications métaboliques telles que l’induction de voies de biosynthèse des acides gras, des oxylipines et d’acides aminés, ainsi que des modifications transcriptomiques. De plus, nous avons constaté que les laminaires co-incubées avec des algues broutées présentaient des changements de leur endo-métabolome en comparaison à des algues broutées, et semblaient moins consommées lors d’un broutage ultérieur, suggérant une perception et une intégration des composés algaux induits par l’herbivorie par les algues voisines. Des études de terrain et en laboratoire ayant précédemment mis en évidence une libération massive d’aldéhydes dans l’air et l’eau de mer après des stress biotiques et abiotiques, nous avons exploré les rôles biologiques d’une signalisation de type aldéhyde dans les interactions kelp/herbivores. Nous avons montré que les aldéhydes pouvaient modifier le métabolisme d’une algue par l’activation du métabolisme des acides gras, et que l’application de 4-HHE sur L. digitata pouvait réduire significativement une consommation algale ultérieure par les herbivores. Ainsi, ces résultats participent à une meilleure compréhension des réponses de défense suite à un broutage chez les laminaires, et confortent l’existence d’une signalisation à distance dans les interactions avec les herbivores, qui impliquerait des composés de défense émis par les algues, dont les aldéhydes. / Kelps are founding species of temperate marine ecosystems, they evolve in the intertidal coastal areas where they are often challenged by herbivores. As most of sessile organisms, kelps develop defensive strategies against herbivory. In the kelp Laminaria digitata, elicitation by oligoguluronates induced regulations of transcription and metabolic pathways, leading to production and release of metabolites, such as aldehydes, in the environment. Moreover, this kelp species feature systemic responses upon defense elicitation and waterborne compounds surrounding a kelp bed have shown to modify its defense physiology. Such distant chemical signaling during defense responses are reminiscent of systemy and priming mechanisms in plants. In marine kelp beds, it should have a major ecological role in structuring marine algal and herbivore communities. In the context of kelp/herbivore interactions, we investigated the defense responses and signaling processes in two emblematic kelp species from north Atlantic and South Pacific coasts, involved in specialized interactions with their herbivores, L. digitata in interaction with Patella pellucida (Brittany, France) and Lessonia spicata with Scurria scurra (Chile). Combined approaches of metabolomics and bio-assays were carried out to decipher the nature of the putative chemical cues, and their biological and ecological roles in algal defense against herbivores. Results showed that, in response to grazing, kelps featured metabolic modifications such as induction of fatty acids, oxylipins and amino acids biosynthetic pathways, as well as transcriptomic modifications. Moreover, we observed that kelps co-incubated with grazed algae exhibited endo-metabolome changes compared to grazed one, and seemed to be less consumed during a subsequent grazing, suggesting the perception and integration of herbivory-induced algal compounds in neighboring algae. As field and lab studies previously highlighted a strong release of aldehydes into the air and the seawater following biotic and abiotic stresses, we further explored the biological roles of aldehyde-based signaling in kelp/herbivore interactions. We found that aldehydes could modify algal metabolism, including the activation of fatty acid metabolism, and that the application of 4-HHE on L. digitata could significantly reduce later algal consumption by herbivores. Thus, these results contribute to a better understanding of defense responses following grazing in kelps, and revealed the occurrence of distance signaling during interaction with herbivores, which could involve algal emitted compounds, such as aldehydes.

Orientador: Elza Guimarães / Resumo: Nessa tese estudamos duas espécies melitófilas de Bignoniaceae, Jacaranda caroba e J. decurrens, que coocorrem em formações savânicas de cerrado e que se mostraram modelos interessantes para explorarmos aspectos ecológicos e evolutivos relacionados à sua reprodução sexual, tendo em vista que apresentam uma complexa rede de interações com mutualistas e antagonistas. A sobreposição geográfica dessas duas espécies nos permitiu avaliar padrões de coexistência relacionados à dinâmica de florescimento, bem como seu anúncio floral relacionado à atração de visitantes florais mutualistas e antagonistas. Além disso, essa interação com mutualistas e antagonistas possibilitou averiguar o impacto da atuação de antagonistas que roubam néctar sobre a polinização de ambas as espécies e como os danos realizados por abelhas roubadoras de néctar interferem na sinalização visual e química aos polinizadores. Também avaliamos a exploração de néctar por diferentes grupos funcionais de visitantes florais de J. caroba e J. decurrens. Por fim, considerando que o sucesso reprodutivo está diretamente relacionado com a eficiência da polinização investigamos o sistema reprodutivo dessas duas espécies e o efeito da qualidade do pólen transferido entre flores associado ao comportamento dos polinizadores durante o forrageamento, buscando compreender os fatores intrínsecos relacionados à seleção mecanismos de autoincompatibilidade nas populações naturais amostradas. / Abstract: In this dissertation we studied two mellithiphilous Bignoniaceae species, Jacaranda caroba and J. decurrens, that co-occur in savanic cerrado formations. This is an interesting model to explore ecological and evolutionary aspects related to their sexual reproduction taking into account that they present a complex network of interactions with mutualists and antagonists. These species‟ geographical overlap allowed us to evaluate patterns of coexistence related to the flowering dynamics, as well as its floral display related to the attraction of mutualists and antagonists‟ floral visitors. Besides, their interactions with mutualists and antagonists allowed us to evaluate the impact of nectar-robbing antagonists on both species and how damage by nectar-robber bees interferes with visual and chemical signaling to pollinators. We also evaluated the nectar exploitation by different functional groups of J. caroba and J. decurrens floral visitors. Finally, considering that these species‟ reproductive success and the effect of pollinator behavior during foraging on the quality of the pollen that is transferred among flowers. Considering that species‟ reproductive success is directly related to the pollination efficiency, we investigated the reproductive system of these two species and the effect of the quality of the pollen transferred between flowers associated to the pollinators‟ behavior during the foraging, trying to understand the intrinsic factors related to mechanisms of self-in... (Complete abstract click electronic access below) / Doutor