Ureases são enzimas que possuem atividades biológicas independentes de suas funções enzimáticas, como indução de exocitose e atividade inseticida. Rhodnius prolixus é um dos modelos de inseto sensíveis a essa toxicidade. Quando testada em tecidos isolados de R. prolixus, a urease de Canavalia ensiformis (JBU) causa diferentes efeitos deletérios modulados por eicosanóides. A via de biossíntese dos eicosanóides envolve fosfolipases A2 (PLA2), que são enzimas responsáveis pela liberação de ácido araquidônico (AA). Uma vez liberado, AA pode ser oxigenado na via da ciclooxigenase, a qual produz prostaglandinas (PGs). Em insetos, a resposta imune compreende as reações humoral e celular e é centralmente modulada por eicosanóides, principalmente PGs. Em busca de uma ligação entre o efeito tóxico de JBU e reações imunológicas em insetos, estudamos os efeitos desta toxina em hemócitos de R. prolixus. Descobrimos que JBU ativa a agregação de hemócitos, tanto após injeção na hemolinfa quanto em células in vitro. Em ensaios in vitro, observamos que os inibidores da síntese de eicosanóides, dexametasona e indometacina, inibem a agregação induzida por JBU, sugerindo que produtos da ciclooxigenase modulam a reação. Após hemócitos cultivados serem tratados com JBU, sítios imunorreativos anti-JBU, danos no citoesqueleto e agregação de núcleos foram observados nas células. Também identificamos e clonamos os transcritos de dois genes de PLA2s de R. prolixus, Rhopr-PLA2III e Rhopr-PLA2XII. Analisamos os perfis de expressão de ambos os genes, e observamos que os transcritos são amplamente distribuídos em vários tecidos, porém em diferentes níveis. Utilizando a estratégia de RNAi, silenciamos a expressão de Rhopr-PLA2XII em ninfas de R. prolixus em uma média de 70% entre variados tecidos. Observamos que a toxicidade de JBU foi diminuída em mais de 50% em ninfas com a expressão de Rhopr-PLA2XII diminuída, indicando que o gene está ligado ao efeito tóxico de JBU nesse inseto. No presente estudo demonstramos pela primeira vez a capacidade de uma urease de induzir a agregação de hemócitos. Da mesma forma, os genes de PLA2 aqui apresentados foram os primeiros a serem identificados e a terem seus transcritos clonados em um vetor da doença de Chagas. Nossos resultados estão de acordo com estudos anteriores que demonstraram que eicosanóides modulam a toxicidade de JBU, contribuindo para a compreensão dos mecanismos entomotóxicos de ação dessas proteínas multifuncionais. / Ureases are multifunctional enzymes that display biological activities independent of their enzymatic function, including induction of exocytosis and insecticidal effects. Rhodnius prolixus, a major vector of Chagas’ disease, is one of the known susceptible models of this toxicity. When tested in isolated tissues of R. prolixus, jack bean urease (JBU) causes different deleterious effects modulated by eicosanoids. The eicosanoid biosynthetic pathway involves phospholipase A2 (PLA2) enzymes that are responsible for releasing arachidonic acid (AA), along with other products. Once released, AA may enter different enzymatic oxygenation pathways, among which is the cyclooxygenase pathway, yielding products such as prostaglandins (PGs). In insects, the immune response comprises cellular and humoral reactions, and is centrally modulated by eicosanoids, mainly PGs. Searching for a link between JBU toxic effect and immune reactions in insects we have studied the effects of this toxin on R. prolixus hemocytes. We have found that JBU triggers aggregation of the hemocytes either after injection in the hemocoel as well as in isolated cells. In vitro assays in presence of the eicosanoid synthesis inhibitors dexamethasone and indomethacin counteracted JBU's effect, indicating eicosanoids, more specifically cyclooxygenase products, are likely to modulate the reaction. Cultured cells were treated with JBU and immune reactive sites were found in the cells along with damage to the cytoskeleton. The highest concentration of JBU used in the cultured cells also led to nuclei aggregation of adherent hemocytes. We have also identified two genes and cloned the transcripts with complete coding sequences of two PLA2s from R. prolixus, Rhopr- PLA2III and Rhopr-PLA2XII. We have analyzed their expression profiles and found the transcripts to be distributed in various tissues, but at different levels. We have knocked down 70% of Rhopr-PLA2XII expression in 5th instar R. prolixus, and JBU's toxicity was decreased by more than 50%, indicating that Rhopr-PLA2XII gene is linked to JBU's toxic effect in this insect. This was the first time effects of an urease on insect hemocytes were demonstrated. The PLA2 genes here presented were also the first to be identified and to have their transcripts cloned in a Chagas’ disease vector. Our findings support previous data demonstrating that eicosanoids modulate JBU's toxicity, and contribute to the understanding of the mechanisms of entomotoxic action of this multifunctional protein.

Ureases são enzimas que possuem atividades biológicas independentes de suas funções enzimáticas, como indução de exocitose e atividade inseticida. Rhodnius prolixus é um dos modelos de inseto sensíveis a essa toxicidade. Quando testada em tecidos isolados de R. prolixus, a urease de Canavalia ensiformis (JBU) causa diferentes efeitos deletérios modulados por eicosanóides. A via de biossíntese dos eicosanóides envolve fosfolipases A2 (PLA2), que são enzimas responsáveis pela liberação de ácido araquidônico (AA). Uma vez liberado, AA pode ser oxigenado na via da ciclooxigenase, a qual produz prostaglandinas (PGs). Em insetos, a resposta imune compreende as reações humoral e celular e é centralmente modulada por eicosanóides, principalmente PGs. Em busca de uma ligação entre o efeito tóxico de JBU e reações imunológicas em insetos, estudamos os efeitos desta toxina em hemócitos de R. prolixus. Descobrimos que JBU ativa a agregação de hemócitos, tanto após injeção na hemolinfa quanto em células in vitro. Em ensaios in vitro, observamos que os inibidores da síntese de eicosanóides, dexametasona e indometacina, inibem a agregação induzida por JBU, sugerindo que produtos da ciclooxigenase modulam a reação. Após hemócitos cultivados serem tratados com JBU, sítios imunorreativos anti-JBU, danos no citoesqueleto e agregação de núcleos foram observados nas células. Também identificamos e clonamos os transcritos de dois genes de PLA2s de R. prolixus, Rhopr-PLA2III e Rhopr-PLA2XII. Analisamos os perfis de expressão de ambos os genes, e observamos que os transcritos são amplamente distribuídos em vários tecidos, porém em diferentes níveis. Utilizando a estratégia de RNAi, silenciamos a expressão de Rhopr-PLA2XII em ninfas de R. prolixus em uma média de 70% entre variados tecidos. Observamos que a toxicidade de JBU foi diminuída em mais de 50% em ninfas com a expressão de Rhopr-PLA2XII diminuída, indicando que o gene está ligado ao efeito tóxico de JBU nesse inseto. No presente estudo demonstramos pela primeira vez a capacidade de uma urease de induzir a agregação de hemócitos. Da mesma forma, os genes de PLA2 aqui apresentados foram os primeiros a serem identificados e a terem seus transcritos clonados em um vetor da doença de Chagas. Nossos resultados estão de acordo com estudos anteriores que demonstraram que eicosanóides modulam a toxicidade de JBU, contribuindo para a compreensão dos mecanismos entomotóxicos de ação dessas proteínas multifuncionais. / Ureases are multifunctional enzymes that display biological activities independent of their enzymatic function, including induction of exocytosis and insecticidal effects. Rhodnius prolixus, a major vector of Chagas’ disease, is one of the known susceptible models of this toxicity. When tested in isolated tissues of R. prolixus, jack bean urease (JBU) causes different deleterious effects modulated by eicosanoids. The eicosanoid biosynthetic pathway involves phospholipase A2 (PLA2) enzymes that are responsible for releasing arachidonic acid (AA), along with other products. Once released, AA may enter different enzymatic oxygenation pathways, among which is the cyclooxygenase pathway, yielding products such as prostaglandins (PGs). In insects, the immune response comprises cellular and humoral reactions, and is centrally modulated by eicosanoids, mainly PGs. Searching for a link between JBU toxic effect and immune reactions in insects we have studied the effects of this toxin on R. prolixus hemocytes. We have found that JBU triggers aggregation of the hemocytes either after injection in the hemocoel as well as in isolated cells. In vitro assays in presence of the eicosanoid synthesis inhibitors dexamethasone and indomethacin counteracted JBU's effect, indicating eicosanoids, more specifically cyclooxygenase products, are likely to modulate the reaction. Cultured cells were treated with JBU and immune reactive sites were found in the cells along with damage to the cytoskeleton. The highest concentration of JBU used in the cultured cells also led to nuclei aggregation of adherent hemocytes. We have also identified two genes and cloned the transcripts with complete coding sequences of two PLA2s from R. prolixus, Rhopr- PLA2III and Rhopr-PLA2XII. We have analyzed their expression profiles and found the transcripts to be distributed in various tissues, but at different levels. We have knocked down 70% of Rhopr-PLA2XII expression in 5th instar R. prolixus, and JBU's toxicity was decreased by more than 50%, indicating that Rhopr-PLA2XII gene is linked to JBU's toxic effect in this insect. This was the first time effects of an urease on insect hemocytes were demonstrated. The PLA2 genes here presented were also the first to be identified and to have their transcripts cloned in a Chagas’ disease vector. Our findings support previous data demonstrating that eicosanoids modulate JBU's toxicity, and contribute to the understanding of the mechanisms of entomotoxic action of this multifunctional protein.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-09-13T19:35:23Z No. of bitstreams: 1 alice_h_r_silva_ioc_bp_0043_2010.pdf: 4947781 bytes, checksum: 760e0eb04ceddb221c9b8ee3a04c2bd4 (MD5) / Made available in DSpace on 2012-09-13T19:35:23Z (GMT). No. of bitstreams: 1 alice_h_r_silva_ioc_bp_0043_2010.pdf: 4947781 bytes, checksum: 760e0eb04ceddb221c9b8ee3a04c2bd4 (MD5) / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O município de Oriximiná (PA), situado à margem esquerda do rio Trombetas na Amazônia Oriental, é um dos mais importantes da Calha Norte, pois com a extração de bauxita em Porto Trombetas, torna-se o principal pólo de extração de minério do mundo. Apresenta uma posição geográfica estratégica, por fazer fronteira com os países da Guiana e Suriname, e limites com o estado de Roraima. O Estado do Pará, onde está inserido politicamente, tem apresentado o maior número de casos agudos de doença de Chagas nas últimas duas décadas, cuja forma de transmissão tem sido atribuída ao consumo de frutos de palmeiras nativas. O objetivo deste trabalho foi estudar a eco-biologia dos triatomíneos desta região, até então não estudada, contribuindo com os serviços para as ações de vigilância e controle dos triatomíneos. As investigações foram realizadas em áreas silvestres e rurais utilizando o método de busca passiva, com o uso das armadilhas de Noireau e a busca ativa. O material coletado foi identificado em nível de espécie utilizando a chave dicotômica de Lent & Wygodzinsky e pela técnica da PCR Multiplex. Os triatomíneos capturados foram examinados para averiguar a presença de tripanosomatídeos e o isolado, cartacterizado por tipagem molecular. A identificação das fontes alimentares foi feita pelo método de ELISA. Dos 442 espécimes coletados, 289 foram identificados como Rhodnius robustus e 153 como Rhodnius pictipes. A taxa de infecção para tripanosomatídeos foi de 17,4% e o isolado classificado como Z3. Devido à falta de conteúdo estomacal dos insetos, trabalhou-se com pool para avaliação da fonte alimentar e, sendo assim, as 54 amostras utilizadas para o teste de ELISA foram constituídas de 118 insetos. Dos 11 anti-soros testados, apenas quatro positivaram para ave, hemolinfa, cavalo e roedor. Verificou-se uma variação morfológica nos espécimes que foram confirmados como R. robustus por PCR Multiplex. R. robustus apresentou uma associação com palmeiras do tipo inajá, que formam aglomerados, tornando-se abundantes em áreas rurais, ao contrário de R. pictipes encontrados em outras espécies de palmeiras no ambiente silvestre. A comprovação de espécies de triatomíneos na região estudada e a presença de T. cruzi em um dos espécimes coletados nas proximidades da Sede do Município evidenciou a importância da vigilância entomológica e epidemiológica na área estudada. / The municipality of Oriximiná located on the left bank of the river Trombetas, in the eastern Amazon, is one of the most important cities of Northern Rail, as with bauxite mining at Porto Trombetas, becomes the main hub of the mining world. Presents a strategic geographical position, by making the border with the countries of Guyana and Suriname, and limits to the state of Roraima. The state of Pará, where it operates politically, has submitted the largest number of acute cases of Chagas disease in the past two decades, whose mode of transmission has been attributed to consumption of fruits of native palms. This work aims to study the eco- biology of the insects of this region, hitherto not studied, contributing to the vigilance and control of triatomines. The investigations were conducted in wilderness areas and rural areas using the method of passive search, with the use of Noireau traps and active search. The material collected was identified to the species level using the dichotomous key of Lent & Wygodzinsky and Multiplex PCR, was also used to investigate the presence of tripanosomatids and the identification of the feeding habits using the ELISA method. Of the 442 specimens collected, 289 were identified as Rhodnius robustus and 153 as Rhodnius pictipes. The infection rate for trypanosomatids was 17.4% and one strain was identified as Z3. Due to the lack of gut contents of the insects, we worked with a pool for the evaluation of food source and, thus, the 54 samples used for ELISA, were composed of 118 insects. Of the 11 antisera tested, only four were positive for bird, hemolymph, horse and rodent. There was a morphological variation in specimens that were confirmed as R. robustus by Multiplex PCR. The association of R. robustus with palm type inajá, which form clusters, becoming abundant in rural environments, was confirmed as opposed to R. pictipes which was found in other palm species in the wild. Evidence of species of insects in the region studied and the presence of T. cruzi in one of the specimens, near the headquarters of the city, highlighted the importance of epidemiological and entomological surveillance in the region.

Les perturbations anthropiques en Amazonie liées au déboisement de la forêt tropicale conduisent à une mosaïque de paysages constituée de végétations secondaires (forêts secondaires, palmeraies, jachères) et de pâturages. Ces modifications favorisent la prolifération de grands palmiers héliophiles invasifs de la famille Attalea spp., palmiers qui constituent l'écotope principal des espèces de Rhodnius, punaises hématophages vectrices de Trypanosoma cruzi, agent étiologique de la Maladie de Chagas en Amérique Latine. Cette étude a porté sur différentes unités de paysage de trois communautés rurales du bas Tapajós (Amazonie brésilienne) ayant une époque d'installation différente sur le territoire (25-75 ans). Six unités de paysage ont été définies sur le terrain et appliquée via une classification supervisée à une image SPOT 5, afin d'obtenir une cartographie du risque environnemental associé à la présence de palmiers dans la région. Sur les cent trente trois palmiers disséqués appartenant aux trois espèces Attalea maripa, A. phalerata et A.speciosa, 73 (54.88%) étaient infestés par R. robustus (742 insectes récoltés). Des diminutions significatives de densité de triatomes ont été observées chez A. maripa, dans la communauté la plus récemment établie (Araipá) et dans les unités de paysage les plus anthropisées. L'infection des insectes par T. cruzi et T. rangeli a été examinée à l'aide de méthodes moléculaires (mini exon SL_IR and sno-RNA-C11). Respectivement 123 (16.57%) et 69 (9.3%) insectes dans 31 (23.3%) et 17 (13.82%) palmiers ont été identifiés positifs à T cruzi et à T.rangeli. Aucune infection n'a été trouvée dans les insectes collectés à Araipá et aucune différence significative n'a été mise en évidence entre les différentes unités de paysage. Les souches de Trypanosoma cruzi identifiées à l'aide de 4 marqueurs moléculaires (mini exon SL-IR, GPI, HSP60 et D7-24α-rRNA) appartiennent à la lignée TcId et 10 (8.13%) individus présentent une infection mixte TcI-TcII. Vingt espèces d'hôtes réparties en trois classes (mammifères, oiseaux, sauropsidés) ont été identifiées comme sources alimentaires à partir du repas sanguin contenu dans le tube digestif des insectes, à l'aide d'amorces cytochrome b, spécifiques de vertébrés. Quatre-vingts et un pourcent des repas détectés ont été effectués sur des mammifères, hôtes potentiels de T.cruzi dont Tamandua tetradactyla, source alimentaire principale. Cet hôte a été clairement identifié comme réservoir de T.rangeli ainsi que suggéré pour T.cruzi. L'analyse phylogénique réalisée à l'aide de séquences de cytochrome b démontre que les individus de Rhodnius identifiés dans la région du Tapajos appartiennent au clade II, ce qui correspond à une extension de l'aire précédemment décrite pour ce clade. L'utilisation du marqueur mitochondrial cytochrome b a permis de mettre en évidence une structuration phylogénétique (haplogroupes) non retrouvée à l'aide des marqueurs microsatellites. Ce résultat montre que l'histoire des gènes (génome mitochondrial) ne retrace pas l'histoire des individus (microsatellites, 10 locus). L'analyse de génétique des populations conduite à l'aide des deux types de marqueurs n'a pas révélé de structuration génétique au sein de la zone d'étude entre les communautés ou les unités de paysage. Cette étude met en évidence des flux géniques importants peu sensibles à la fragmentation du milieu, la dynamique d'invasion des palmiers assurant aux insectes une connectivité fonctionnelle entre les différentes unités de paysages et les communautés. La prédiction du risque environnemental lié à la situation du Tapajós va vers une augmentation du risque de transmission de la Maladie de Chagas dans cette région, du fait de l'abondance des palmiers, de leur forte connectivité, et de la présence de vecteurs et d'hôtes infectés circulant entre les différentes communautés et unités du paysage / Anthropic disturbances from deforestation of Amazon tropical forest leads to a mosaic of landscapes composed of secondary vegetation (secondary forest, palm groves, fallows) and pasture. These changes result in the proliferation of invasive heliophilous palm trees of the family Attalea spp., the principal ecotope of Rhodnius species, bloodsucking bug vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease in Latin America. The present study focuses on different land cover classes of three rural communities of the lower Tapajós (Brazilian Amazon) with different settlement times (25-75 years). Six different land-cover classes were identified on the field and applied through supervised classification on a SPOT 5 image of the study area in order to cartography environmental risk associated to palm tree presence in the area. Three hundred and thirty palms trees of three species Attalea maripa, A. phalerata and A.speciosa were dissected of which 73 (54.88%) were infested with R. robustus (742 insects collected). The distribution of palm species varied in each community, A.maripa was the only species found in the most recently settled community (Araipá). Significant decreases in bug density were observed in A.maripa, in the community most recently established (Araipá) and in the two most anthropogenic landcover classes.Infection of insects by T. cruzi and T. rangeli was examined using molecular methods (mini exon SL_IR and sno-RNA-C11). Respectively, 123 (16.57%) and 69 (9.3%) insects in 31 (23.3%) and 17 (13.32%) palms were identified as positive for T. cruzi and T. rangeli. A lack of infection was detected in Araipá but no differences were observed between the different land cover classes. The strains of Trypanosoma cruzi were identified using four distinct molecular markers (mini-exon SL-IR, GPI, HSP60 and D7-24α-rRNA) as belonging to the lineage TCI specifically TcId and 10 (8.13%) individuals showed a mixed infection TCI-TCII. Twenty host species divided into three classes were identified (mammals, birds, reptiles) were identified as food source from blood meal from the bug gut with cytochrome b primers, specific for vertebrates (25.74% of meals). Eighty-one percent of meals were conducted on mammals, potential hosts of T. cruzi, specially on Tamandua tetradactyla,identified as the main food source. This host was clearly identified as a reservoir for T.rangeli and also suggested for T. cruzi.Phylogenetic analysis performed using cytochrome b sequences, identified Rhodnius individuals in the Tapajos region within clade II, which represent an extension of the range previously described for this clade. The use of the cytochrome b marker also revealed a phylogenetic structure (haplogroups) not found using microsatellite markers. This result showed that the history of the genes (mitochondrial genome) does not match the history of individuals (microsatellites, 10 loci). Population genetics analysis conducted using both markers did not reveal genetic structure within the study area between the communities or the landcover classes. The study revealed significant gene flow, which was not restricted by the fragmentation of the environment. The invasive dynamics of Attalea palm trees provide a functional connectivity for insects to move between the different landcover classes and communities. Due to the abundance of palm trees and their high connectivity, the presence of vectors and infected hosts moving between the different communities and landcover classes, the environmental risk constituted by Attalea palm tree presence of Chagas disease in Tapajós region will presumably continue to increase

En Guyane comme dans l'ensemble de la région amazonienne, le risque de maladie de Chagas pour l'homme a longtemps été sous estimé du fait de l'absence de vecteur domestique et de l'existence d'un réservoir animal exclusivement sauvage. Le travail présenté répond aux besoins de compléter et d'actualiser les connaissances épidémiologiques disponibles sur la maladie de Chagas dans ce département , en précisant les modalités de circulation de Trypanosoma cruzi et les facteurs impliqués dans le cycle à l'origine des cas humains. Les données cumilées entre 2001 et 2023 représentent 971 spécimens de triatomes. Nous avons choisi de différencier les triatomes provenant des zones non habitées (forêts primaire ou secondaire) de ceux provenant de zones habitées, collectées dans les maisons à la faveur d'intrusion. Une espère, Panstrongylus geniculatus, s'est distinguée par son abondance (61,1%) et son omniprésence dans les différents types de milieux explorés. Au total, neuf espèces ont été retrouvées. En zone non habitée, avec une prédominance de P. geniculatus, P. lignarius, Rhodnius pictipes et R. robustus. L'infection des triatomes par T. cruzi a été étudiée, avec une mise en évidence d'un taux d'infection plus élevé chez les triatomes collectés à l'intérieur des habitations (53.9%) que chez ceux provenant des zones non habitées (46%). L'étude sur les mammifères sauvages a montré des taux d'infection élevés pour T. cruzi chez deux espèces : didelphis marsupialis (62.5%) et Philander opossum (35%). Cette étude a été complétée par l'analyse de la variabilité génétique de T. cruzi identifié par des techniques de génotypage réalisées sur du matériel obtenu à partir de triatomes, de mammifères sauvages et domestiques et des hommes. En zone non habitée trois groupes, TcI, TcII-TcV-TcVI et TcIII-TcIV, ont été caractérisés, avec une prédominance de TcI. Parmi les acteurs du cycle parasite, les chiens et P. geniculatus hébergent le groupe TcIII-TcIV à la différence du genre Rhodnius et des marsupiaux qui hébergent le groupe TcI. Nos résultats ont permis d'émettre un certain nombre d'hypothèses sur la circulation des génotypes de T. cruzi en Guyane / In French Guiana, as in the whole of Amazon region, the risk of Chagas disease to humans has long been underestimated due to the presence of non-domiciled triatomines and wild animal reservoir. Overall, this thesis aims at reactualizing and improving epidemiological knowledge about Chagas disease in French Guiana, by specific modalities of circulation of Trypanosoma cruzi and the factorsinvolved in the transmission cycle of the origin of human cases. Accumulated data, between 2001 and 2013, represent 971 triatomine specimens collected. We chose to differentiate triatomine collected in uninhabited areas (primary or secondary forests) and those frominhabited areas, collected in homes by intrusion. One species, Panstrongylus geniculatus, was distinguished by its abundance (61.1%) and its presence in different types of habitats investigated. A total of nine species were found in uninhabited area, with the predominance of four species: P. geniculatus, P. lignarius, Rhodnius pictipes and Eratyrus mucronatus. In inhabited area, five species of triatomine were collected, with the predominance of P. geniculatus, R. pictipes and R. robustus. The triatomine infection rate with T. cruzi has been studied, with a highlighting of higher rate of infection in triatomine collected inside houses (53.9%) than those from uninhabited area (46%). The study on wild mammals showed high T. cruzi infection rates for two species: Didelphis marsupialis (62.5%) and Philander opossum (35%).This study was completed by the analysis of the genetic variability of T. cruzi identified by genotyping techniques performed on material obtained from triatomine, wild and domestic mammals and humans. In uninhabited area, three groups$ TcI, TcII-TcV-TcVI and TcIII-TcIV have been characterized, with a predominance of TcIII-TcIV group. In inhabited area, only two groups were characterized (TcI andTcIII-TcIV), with a predominance of TcI. Among the actor’s transmission cycle of parasite, dogs and P. geniculatus were characterized by TcIII-TcIV group. Contrary to Rhodnius genus and marsupials were characterized by the TcI group. Our results have allowed issuing a number of assumptions about the circulation of T. cruzi genotypes in French Guiana

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho utilizou-se a técnica fluorescência de raios X usando radiação síncrotron (SR-TXRF) para estudar, quantitativamente, o transporte de cloro, potássio e cálcio na hemolinfa, urina e túbulos de Malpighi (TM) em ninfas de quinto estágio do Rhodnius prolixus (R. prolixus), considerando a excreção destes elementos em diferentes dias após o repasto sanguíneo. R. prolixus é um dos principais vetores do Trypanosoma cruzi, agente causador da doença de Chagas. R. prolixus fornece um sistema modelo particularmente útil porque seus TMs tanto secretam quanto reabsorvem íons a taxas elevadas. Os TMs filtram a hemolinfa e secretam um líquido que é muitas vezes comparado com a urina primária em vertebrados. Os resultados obtidos mostram que a concentração de potássio na urina é substancialmente maior do que na hemolinfa. A concentração de cloro na hemolinfa é menor do que na urina, mas a diferença não é tão marcada como no caso do potássio. No caso do Rhodnius é razoável interpretar a elevada concentração de potássio na urina como adaptativo para o problema de excreção imediato do inseto. A concentração de cálcio nos TMs é substancialmente maior em comparação com os valores encontrados na hemolinfa e urina. Este resultado mostra que o cálcio é retido no corpo do R. prolixus e pouco eliminado. Os resultados obtidos estão coerentes com a literatura. Avaliou-se também o efeito no transporte de Cl, K e Ca após um repasto de sangue de coelho contaminado com HgCl2 de modo a avaliar o efeito da presença deste metal tóxico no balanço iônico nos fluidos de excreção urina e hemolinfa e também pelo principal órgão de transporte, os túbulos de Malpighi. As excreções de Cl e K pela urina são afetadas pela ingestão. Este resultado é esperado levando-se em consideração a ingestão de excesso de Cl através do HgCl2. O transporte de Cl, K e Ca na hemolinfa do Rhodnius prolixus não é afetada pela ingestão de HgCl2. Nos túbulos de Malpighi, as altas concentrações de Ca obtidas foram comparáveis àquelas encontradas nos insetos controle. Pode-se concluir que SR-TXRF é um método muito promissor para análises diretas, rápidas e confiáveis para a quantificação simultânea de elementos envolvidos na regulação do transporte e em todo o sistema excretor de insetos. Além disso, o estudo do transporte e a excreção de elementos no inseto Rhodnius prolixus abrem oportunidade para a maior compreensão de efeitos da poluição em espécies de invertebrados. / In this work, we investigated changes in the concentrations of Cl, K and Ca, in 5th instar using total reflection X-ray fluorescence Rhodnius prolixus with synchrotron radiation (SR-TXRF). The elements were quantified using urine, hemolymph and Malpighian tubules samples collected on different days after a blood meal. Rhodnius prolixus is one of the most important vectors of the Trypanosoma cruzi, causative agent of Chagas? disease. R. prolixus provides a particularly useful model system because its MTs both secrete and reabsorb ions at high rates. The TMs filter hemolymph and secrete a liquid that is often compared with the primary urine in vertebrates. The experimental results showed that the concentration of potassium in the urine is substantially greater than in the hemolymph. The concentration of chlorine in the hemolymph is generally less than in the urine, but the difference is not so marked as in the case of potassium. In the case of Rhodnius, it is reasonable to interpret the high concentration of potassium in the urine as adaptive to the animals? immediate excretory problem. The concentration of calcium in the TMs is substantially greater than in both the hemolymph and the urine. This result shows that that calcium is retained in the body and not eliminated. These results are in accordance with the literature. We also investigated whether dietary mercury contamination may influence the transport of Cl, K and Ca by the hemolymph, urine and Malpighian tubules of R. prolixus fed on blood containing HgCl2. The results suggested that dietary Hg contamination may influence the Cl and K contents during excretion of the urine. It was expected considering the large amounts of chlorine ingested by Rhodnius prolixus in its meals of blood containing HgCl2. Statistical analysis showed no significant variation in all elements contents for hemolymph samples. The main conclusion which can be drawn from the results is that in all the insects studied calcium is deposited in Malpighian tubules. These observations point out that the analytical approach of the SR-TXRF method can be efficiently used to measure elements involved in the transport regulation into insect Malpighian tubules and also provides useful data concerning the biological effects of pollution on invertebrate species.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho utilizou-se a técnica fluorescência de raios X usando radiação síncrotron (SR-TXRF) para estudar, quantitativamente, o transporte de cloro, potássio e cálcio na hemolinfa, urina e túbulos de Malpighi (TM) em ninfas de quinto estágio do Rhodnius prolixus (R. prolixus), considerando a excreção destes elementos em diferentes dias após o repasto sanguíneo. R. prolixus é um dos principais vetores do Trypanosoma cruzi, agente causador da doença de Chagas. R. prolixus fornece um sistema modelo particularmente útil porque seus TMs tanto secretam quanto reabsorvem íons a taxas elevadas. Os TMs filtram a hemolinfa e secretam um líquido que é muitas vezes comparado com a urina primária em vertebrados. Os resultados obtidos mostram que a concentração de potássio na urina é substancialmente maior do que na hemolinfa. A concentração de cloro na hemolinfa é menor do que na urina, mas a diferença não é tão marcada como no caso do potássio. No caso do Rhodnius é razoável interpretar a elevada concentração de potássio na urina como adaptativo para o problema de excreção imediato do inseto. A concentração de cálcio nos TMs é substancialmente maior em comparação com os valores encontrados na hemolinfa e urina. Este resultado mostra que o cálcio é retido no corpo do R. prolixus e pouco eliminado. Os resultados obtidos estão coerentes com a literatura. Avaliou-se também o efeito no transporte de Cl, K e Ca após um repasto de sangue de coelho contaminado com HgCl2 de modo a avaliar o efeito da presença deste metal tóxico no balanço iônico nos fluidos de excreção urina e hemolinfa e também pelo principal órgão de transporte, os túbulos de Malpighi. As excreções de Cl e K pela urina são afetadas pela ingestão. Este resultado é esperado levando-se em consideração a ingestão de excesso de Cl através do HgCl2. O transporte de Cl, K e Ca na hemolinfa do Rhodnius prolixus não é afetada pela ingestão de HgCl2. Nos túbulos de Malpighi, as altas concentrações de Ca obtidas foram comparáveis àquelas encontradas nos insetos controle. Pode-se concluir que SR-TXRF é um método muito promissor para análises diretas, rápidas e confiáveis para a quantificação simultânea de elementos envolvidos na regulação do transporte e em todo o sistema excretor de insetos. Além disso, o estudo do transporte e a excreção de elementos no inseto Rhodnius prolixus abrem oportunidade para a maior compreensão de efeitos da poluição em espécies de invertebrados. / In this work, we investigated changes in the concentrations of Cl, K and Ca, in 5th instar using total reflection X-ray fluorescence Rhodnius prolixus with synchrotron radiation (SR-TXRF). The elements were quantified using urine, hemolymph and Malpighian tubules samples collected on different days after a blood meal. Rhodnius prolixus is one of the most important vectors of the Trypanosoma cruzi, causative agent of Chagas? disease. R. prolixus provides a particularly useful model system because its MTs both secrete and reabsorb ions at high rates. The TMs filter hemolymph and secrete a liquid that is often compared with the primary urine in vertebrates. The experimental results showed that the concentration of potassium in the urine is substantially greater than in the hemolymph. The concentration of chlorine in the hemolymph is generally less than in the urine, but the difference is not so marked as in the case of potassium. In the case of Rhodnius, it is reasonable to interpret the high concentration of potassium in the urine as adaptive to the animals? immediate excretory problem. The concentration of calcium in the TMs is substantially greater than in both the hemolymph and the urine. This result shows that that calcium is retained in the body and not eliminated. These results are in accordance with the literature. We also investigated whether dietary mercury contamination may influence the transport of Cl, K and Ca by the hemolymph, urine and Malpighian tubules of R. prolixus fed on blood containing HgCl2. The results suggested that dietary Hg contamination may influence the Cl and K contents during excretion of the urine. It was expected considering the large amounts of chlorine ingested by Rhodnius prolixus in its meals of blood containing HgCl2. Statistical analysis showed no significant variation in all elements contents for hemolymph samples. The main conclusion which can be drawn from the results is that in all the insects studied calcium is deposited in Malpighian tubules. These observations point out that the analytical approach of the SR-TXRF method can be efficiently used to measure elements involved in the transport regulation into insect Malpighian tubules and also provides useful data concerning the biological effects of pollution on invertebrate species.

Submitted by PPG Medicina e Ci?ncias da Sa?de (medicina-pg@pucrs.br) on 2018-09-17T18:15:32Z No. of bitstreams: 1 KARINE_KAPPAUN.pdf: 4206975 bytes, checksum: b450fa28646271e7dfac9389de975825 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-09-18T12:20:56Z (GMT) No. of bitstreams: 1 KARINE_KAPPAUN.pdf: 4206975 bytes, checksum: b450fa28646271e7dfac9389de975825 (MD5) / Made available in DSpace on 2018-09-18T12:54:15Z (GMT). No. of bitstreams: 1 KARINE_KAPPAUN.pdf: 4206975 bytes, checksum: b450fa28646271e7dfac9389de975825 (MD5) Previous issue date: 2018-07-06 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Ureases (urea amidohydrolases, EC 3.5.1.5) are nickel-dependent enzymes, widely spread in bacteria, fungi and plants, which catalyze the hydrolysis of urea to ammonia and carbon dioxide. Jaburetox is a peptide derived from a urease isoform of the Canavalia ensiformis plant. Structurally, jaburetox is an intrinsically disordered peptide, demonstrated by bioinformatics tools, and experimentally, by circular dichroism (CD) and nuclear magnetic resonance (NMR). Several biological properties of jaburetox have already been described, such as insecticidal activity, ability to interact with lipid bilayers, fungitoxic effect, among others. Soyuretox is a peptide colinear to jaburetox derived from the soybean ubiquitous urease. One of the objectives of this thesis was to carry out structural studies of the soyuretox peptide by CD and NMR, which revealed its intrinsically disordered nature and that its secondary structure content is modified in the presence of SDS micelles. Biological properties of soyuretox were evaluated. The peptide has entomotoxic activity, inducing aggregation of hemocytes from Rhodnius prolixus, the vector of Chagas disease, both in vivo and in vitro. The fungitoxic effect of soyuretox on yeast Candida albicans involves the production of superoxide anions, and the peptide was immunolocalized on the surface of the yeast. Finally, the behavioral and morphological effects of soyuretox on zebrafish larvae (Danio rerio) were evaluated, aiming to deepen the understanding of the mechanism of toxic action and the toxicological profile of this molecule, which presents biotechnological potential as a biopesticide. / Ureases (ureia amido-hidrolases; EC 3.5.1.5) s?o enzimas n?quel dependentes, amplamente distribu?das em bact?rias, fungos e plantas, que catalisam a hidr?lise da ureia ? am?nia e di?xido de carbono. O jaburetox ? um pept?deo derivado de uma isoforma de urease da planta Canavalia ensiformis. Estruturalmente, o jaburetox ? um pept?deo intrinsecamente desordenado, demonstrado por ferramentas in silico de bioinform?tica, e experimentalmente, por dicro?smo circular (CD) e resson?ncia magn?tica nuclear (RMN). Diversas propriedades biol?gicas do jaburetox j? foram descritas, tais como atividade inseticida, capacidade de interagir com bicamadas lip?dicas, efeito fungit?xico, dentre outras. O soyuretox ? um pept?deo colinear ao jaburetox, por?m derivado da urease ub?qua de soja. Um dos objetivos dessa tese foi realizar estudos estruturais do pept?deo soyuretox por CD e RMN, que revelaram sua natureza intrinsecamente desordenada e aumento no conte?do de estrutura secund?ria na presen?a de micelas de SDS. Propriedades biol?gicas do soyuretox foram avaliadas. O pept?deo tem atividade entomot?xica, induzindo agrega??o de hem?citos de Rhodnius prolixus, o vetor da doen?a de Chagas, tanto in vivo como in vitro. O efeito fungit?xico do soyuretox na levedura Candida albicans envolve a produ??o de ?nions super?xido, sendo o pept?deo imunolocalizado ligado na superf?cie da levedura. Por fim, foram avaliados os efeitos comportamentais e morfol?gicos do soyuretox em larvas de zebrafish (Danio rerio), visando aprofundar o entendimento do mecanismo de a??o t?xica e o perfil toxicol?gico dessa mol?cula, que apresenta potencial biotecnol?gico como biopesticida.

In insects, ecdysis is an important feature of growth and development and is tightly controlled by a variety of neuropeptides. In holometabolous insects, crustacean cardioactive peptide (CCAP) is one of many factors that regulate ecdysis behaviours; however, not much is known about the control of ecdysis in hemimetabolous insects. In this thesis, the CCAP-signaling pathway is shown to be essential for successful ecdysis in the hemimetabolous insect, Rhodnius prolixus. The cDNA sequence of the CCAP gene has been cloned from the R. prolixus central nervous system (CNS) and the functional role of CCAP as a neuromodulator/neurotransmitter demonstrated. Specifically, the expression of RhoprCCAP in CNS neurons producing extensive CCAP-like immunoreactive processes within the neuropile indicates that CCAP plays central roles in coordination of other neurons. RhoprCCAP also acts as a neurohomone/neuromodulator released peripherally to coordinate many tissues. Thus, CCAP-like immunoreactive processes are found in neurohemal sites and also on peripheral tissues. The RhoprCCAP receptor (RhoprCCAPR) has been cloned and shown to be a G-protein coupled receptor (GPCR). RhoprCCAPR expression is observed in the CNS and certain peripheral tissues of R. prolixus. Also, CCAP stimulates hindgut contractions and increases the heartbeat rate in a dose-dependent manner. The involvement of CCAP in R. prolixus ecdysis has been investigated. Up-regulation of the RhoprCCAP transcript in the CNS and the RhoprCCAP receptor (RhoprCCAPR) transcript in the CNS and specific peripheral tissues was observed immediately prior to ecdysis. Also, decreasing staining intensity of CCAP-like immunoreactivity in neurons immediately following ecdysis indicates the release of CCAP during ecdysis. The critical importance of the CCAP-signalling pathway was further demonstrated by knockdown of the RhoprCCAP and RhoprCCAPR transcripts utilizing double stranded RNA interference. Insects with these transcripts knocked down have high mortality (up to 84%), typically at the expected time of ecdysis, or have ecdysis extremely delayed. Taken together, this thesis demonstrates that RhoprCCAP plays a crucial role in regulating ecdysis behaviours in R. prolixus, and clearly shows the conserved nature of the CCAP-signaling pathway in ecdysis for both holometabolous and hemimetabolous insects.

In insects, ecdysis is an important feature of growth and development and is tightly controlled by a variety of neuropeptides. In holometabolous insects, crustacean cardioactive peptide (CCAP) is one of many factors that regulate ecdysis behaviours; however, not much is known about the control of ecdysis in hemimetabolous insects. In this thesis, the CCAP-signaling pathway is shown to be essential for successful ecdysis in the hemimetabolous insect, Rhodnius prolixus. The cDNA sequence of the CCAP gene has been cloned from the R. prolixus central nervous system (CNS) and the functional role of CCAP as a neuromodulator/neurotransmitter demonstrated. Specifically, the expression of RhoprCCAP in CNS neurons producing extensive CCAP-like immunoreactive processes within the neuropile indicates that CCAP plays central roles in coordination of other neurons. RhoprCCAP also acts as a neurohomone/neuromodulator released peripherally to coordinate many tissues. Thus, CCAP-like immunoreactive processes are found in neurohemal sites and also on peripheral tissues. The RhoprCCAP receptor (RhoprCCAPR) has been cloned and shown to be a G-protein coupled receptor (GPCR). RhoprCCAPR expression is observed in the CNS and certain peripheral tissues of R. prolixus. Also, CCAP stimulates hindgut contractions and increases the heartbeat rate in a dose-dependent manner. The involvement of CCAP in R. prolixus ecdysis has been investigated. Up-regulation of the RhoprCCAP transcript in the CNS and the RhoprCCAP receptor (RhoprCCAPR) transcript in the CNS and specific peripheral tissues was observed immediately prior to ecdysis. Also, decreasing staining intensity of CCAP-like immunoreactivity in neurons immediately following ecdysis indicates the release of CCAP during ecdysis. The critical importance of the CCAP-signalling pathway was further demonstrated by knockdown of the RhoprCCAP and RhoprCCAPR transcripts utilizing double stranded RNA interference. Insects with these transcripts knocked down have high mortality (up to 84%), typically at the expected time of ecdysis, or have ecdysis extremely delayed. Taken together, this thesis demonstrates that RhoprCCAP plays a crucial role in regulating ecdysis behaviours in R. prolixus, and clearly shows the conserved nature of the CCAP-signaling pathway in ecdysis for both holometabolous and hemimetabolous insects.