Zugl.: Kiel, Universiẗat, Diss., 2009.

Dortmund, Universiẗat, Diss., 2001.

Made available in DSpace on 2014-06-11T19:24:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-10-30Bitstream added on 2014-06-13T20:12:22Z : No. of bitstreams: 1 biondo_ga_me_botfm.pdf: 10870076 bytes, checksum: c768a26cf85de27fd0f5359da74435ca (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Paracoccidioides brasiliensis, é o agente da paracoccidioidomicose, a micose profunda endêmica na América Latina. Produção de eicosanóides, como prostaglandinas e leucotrienos, durante as infecções fúngicas tem mostrado desempenhar um papel crítico na sobrevivência e/ou crescimento fúngico, bem como na modulação da resposta imune do hospedeiro. As células hospedeiras são uma fonte desses mediadores, porém outra fonte em potencial pode ser o próprio fungo. O objetivo do nosso estudo foi avaliar se cepas do P. brasiliensis com diferentes graus de virulência (Pb18, Pb265, BT79, Pb192) produzem, prostaglandina E2 (PGE2) e leucotrieno B4 (LTB4). Além disso, verificamos se P. brasiliensis pode usar fontes exógenas de ácido araquidônico (AA), bem como as vias metabólicas dependentes das enzimas cicloxigenase (COX) e lipoxigenase (5-LO), para a produção de PGE2 e LTB4, respectivamente. Finalmente, uma possível associação entre esses eicosanóides e a viabilidade do fungo também foi avaliada. Demonstramos, usando ensaios de Elisa, que todas as cepas de P. brasiliensis, independente do seu grau de virulência, produzem altos níveis de PGE2 e LTB4 após 4h cultura que foram reduzidos após 8h. No entanto, em ambos os tempos da cultura, foram detectados níveis mais elevados de eicosanóides, quando as culturas foram complementadas com uma fonte exógena de AA. Diferentemente, o tratamento com indometacina, um inibidor da COX, ou MK886, um inibidor da 5-LO, induz a uma redução nos níveis de PGE2 e LTB4, respectivamente, bem como a viabilidade do fungo. Os dados fornecem evidências de que P. brasiliensis tem a capacidade de metabolizar, tanto AA endógeno como exógeno por vias dependentes da COX e 5-LO enzimas que participam da produção de PGE2 e LTB4, respectivamente, que são indispensáveis para a sua viabilidade. / Paracoccidioides brasiliensis, is the agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. Production of eicosanoids, such as prostaglandins and leukotrienes, during fungal infections is theorized to play a critical role on fungal survival and/or growth as well as on host immune response modulation. Host cells are one source of these mediators; however another potential source may be the fungal itself. The purpose of our study was to assess whether P. brasiliensis strains with different degree of virulence (Pb18, Pb265, PbBT79, Pb192) produce both, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4). Moreover, we asked if P. brasiliensis can use exogenous sources of arachidonic acid (AA), as well as metabolic pathways dependent on cyclooxygenase ( COX) and lipooxygenase (5-LO ) enzymes, for PGE2 and LTB4 production , respectively. Finally, a possible association between these eicosanoides and fungus viability was assessed. We have demonstrated, using Elisa assays, that all P. brasiliensis strains, independently of their virulence degree, produce high PGE2 and LTB4 levels on 4h culture that were reduced after 8 h. However, in both culture times, higher eicosanoides levels were detected when culture medium was supplemented with exogenous AA. Differently, treatment with indomethacin, a COX inhibitor, or MK886, a 5-LO inhibitor, induces a reduction on PGE2 and LTB4 levels, respectively, as well as in fungus viability. The data provide evidence that P. brasiliensis has the capacity to metabolize either endogenous or exogenous AA by pathways dependent on COX and 5-LO enzymes for producing PGE2 and LTB4, respectively, that are critical for its viability.

Orientador: Valéria Marçal Felix de Lima / Resumo: A leishmaniose canina (CanL) é causada pelo parasito intracelular Leishmania infantum. Devido ao alto parasitismo na pele o cão é considerado o principal reservatório urbano da L. Infantum. A prostaglandina-E2 (PGE2) possui propriedades reguladoras potentes do sistema imunológico e pode se ligar aos receptores EP1, EP2 e EP4 que geram ativação celular ou EP3 que gera inibição de resposta celular. Na CanL o papel regulador da PGE2 ainda não foi estudado, por isso, os parâmetros foram avaliados em cultura de leucócitos esplênicos de cães com CanL. Avaliando o nível de PGE2, seus receptores, e seu efeito modulador sobre a PGE2 na atividade da arginase, NO2, citocinas IL-10, IL-17 e TNF-α e carga. Para isso utilizamos seus agonistas, antagonista e inibidor. Nossos resultados mostraram que a expressão do receptor EP2 diminuiu nos leucócitos esplênicos dos cães com CanL quando comparado aos cães saudáveis. Observamos que o NO2 diminuiu quando tratados com os agonistas dos receptores de PGE2 (EP1/EP2/EP3), antagonista dos receptores de PGE2 (AH-6809) e inibidor de COX-2 (NS-398). A concentração das citocinas TNF-α e a IL-17 diminuíram quando tratadas com agonista do receptor de PGE2 (EP2), e quando estimuladas com a PGE2. A carga parasitária diminuiu na cultura de leucócitos esplênicos de cães com CanL estimulados com PGE2. Concluímos que a infecção por Leishmania modula os receptores de PGE2 em cães, e que a ligação da PGE2 aos receptores pode ativar a capacidade microbicida das cé... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

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Kam Yiu-wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 200-215). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iii / Publications --- p.iv / Abbreviations --- p.v / Contents --- p.vii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- G protein-coupled receptors --- p.1 / Chapter 1.1.1 --- Introduction --- p.1 / Chapter 1.1.2 --- G protein-coupled receptors --- p.2 / Chapter 1.1.3 --- Heterotrimeric G-proteins --- p.4 / Chapter 1.1.4 --- Second messenger systems --- p.5 / Chapter 1.1.5 --- Mechanism of GPCR activation --- p.6 / Chapter 1.1.6 --- GPCR cross talk --- p.8 / Chapter 1.2 --- Receptor theory --- p.10 / Chapter 1.2.1 --- Introduction --- p.10 / Chapter 1.2.2 --- Two-state model --- p.11 / Chapter 1.2.3 --- Three-state model --- p.12 / Chapter 1.2.4 --- Extended ternary complex model --- p.12 / Chapter 1.2.5 --- Multiple receptor state --- p.13 / Chapter 1.2.6 --- Constitutively active mutant receptors --- p.14 / Chapter 1.3 --- Agonist trafficking --- p.15 / Chapter 1.3.1 --- Introduction --- p.15 / Chapter 1.3.2 --- Effect of agonist efficacy on receptor coupling --- p.17 / Chapter 1.3.3 --- Effect of receptor expression level on receptor coupling --- p.17 / Chapter 1.3.4 --- Receptor promiscuity --- p.18 / Chapter 1.3.5 --- Agonist-induced conformational changes --- p.19 / Chapter 1.3.5.1 --- Conformational induction --- p.19 / Chapter 1.3.5.2 --- Conformational selection --- p.19 / Chapter 1.3.6 --- Receptor/G-protein system --- p.20 / Chapter 1.3.7 --- Implication of agonist trafficking --- p.21 / Chapter 1.4 --- Receptor switching --- p.22 / Chapter 1.4.1 --- Introduction --- p.22 / Chapter 1.4.2 --- Receptor switching --- p.23 / Chapter Chapter 2 --- Prostacyclin receptors --- p.34 / Chapter 2.1 --- General properties of prostacyclin --- p.34 / Chapter 2.1.1 --- Synthesis of prostacyclin --- p.34 / Chapter 2.1.2 --- Prostacyclin and its mimetics --- p.34 / Chapter 2.1.3 --- Characterization of IP-receptors --- p.36 / Chapter 2.1.3.1 --- Classification of IP-receptors --- p.36 / Chapter 2.1.3.2 --- Distribution of IP-receptors in the peripheral system --- p.36 / Chapter 2.1.3.3 --- Distribution of IP-receptors in the central nervous system --- p.38 / Chapter 2.1.3.4 --- Structure of IP-receptors --- p.39 / Chapter 2.1.4 --- Anti-thrombotic role of prostacyclin --- p.40 / Chapter 2.1.5 --- Cytoprotective role of prostacyclin --- p.41 / Chapter 2.1.6 --- Role of prostacyclin in adipogenesis --- p.42 / Chapter 2.2 --- Coupling capacity of IP-receptors --- p.43 / Chapter 2.2.1 --- Introduction --- p.43 / Chapter 2.2.2 --- Interaction with Gs --- p.44 / Chapter 2.2.3 --- Interaction with Gi --- p.45 / Chapter 2.2.4 --- Interaction with Gq --- p.45 / Chapter 2.2.5 --- Interaction with PPARs --- p.43 / Chapter 2.2.6 --- IP-receptor isoprenylation --- p.49 / Chapter 2.3 --- Regulation of IP-receptors --- p.50 / Chapter 2.3.1 --- Mechanism of signal termination --- p.50 / Chapter 2.3.2 --- Desensitization of IP-receptors --- p.51 / Chapter 2.3.3 --- Internalization of IP-receptors --- p.53 / Chapter Chapter 3 --- Materials and solutions --- p.59 / Chapter 3.1 --- Materials --- p.59 / Chapter 3.2 --- "Culture media, buffers and solutions" --- p.53 / Chapter 3.2.1 --- Culture media --- p.63 / Chapter 3.2.2 --- Buffers --- p.64 / Chapter 3.2.3 --- Solutions --- p.65 / Chapter Chapter 4 --- Methods --- p.67 / Chapter 4.1 --- Cell culture --- p.67 / Chapter 4.2 --- Mammalian cell transfection --- p.68 / Chapter 4.2.1 --- Preparation of plasmid DNA --- p.68 / Chapter 4.2.2 --- Transient transfection of mammalian cells --- p.71 / Chapter 4.2.3 --- β-galactosidase assay --- p.73 / Chapter 4.2.3.1 --- Introduction --- p.73 / Chapter 4.2.3.2 --- Preparation of cell lysate --- p.73 / Chapter 4.2.3.3 --- Micro β-galactosidase assay --- p.73 / Chapter 4.3 --- Measurement of adenylate cyclase activity --- p.74 / Chapter 4.3.1 --- [3H]-adenine prelabelling method --- p.74 / Chapter 4.3.1.1 --- Preparation of columns --- p.74 / Chapter 4.3.1.2 --- Incubation of cells --- p.75 / Chapter 4.3.1.3 --- Measurement of [3H]-cyclic AMP production --- p.75 / Chapter 4.3.1.4 --- Data analysis --- p.76 / Chapter 4.3.2 --- cAMP [125I]-scintillation proximity assay (SPA) --- p.77 / Chapter 4.3.2.1 --- Introduction --- p.77 / Chapter 4.3.2.2 --- Cell lysis method --- p.77 / Chapter 4.3.2.3 --- cAMP [I25I]-scintillation proximity assay --- p.78 / Chapter 4.4 --- Measurement of phospholipase C activity --- p.78 / Chapter 4.4.1 --- Introduction --- p.78 / Chapter 4.4.1.1 --- Preparation of columns --- p.78 / Chapter 4.4.1.2 --- [3H]-inositol phosphate assay --- p.79 / Chapter 4.4.1.3 --- Measurement of [3H]-inositol phosphate production --- p.79 / Chapter 4.4.1.4 --- Data analysis --- p.80 / Chapter 4.4.2 --- "D-myo-Inositol 1,4,5-trisphosphate (IP3) [3H] assay system" --- p.81 / Chapter 4.4.2.1 --- Introduction --- p.81 / Chapter 4.4.2.2 --- Sample preparation --- p.81 / Chapter 4.4.2.3 --- "D-myo-Inositol 1,4,5-trisphosphate (IP3) [3H] assay system" --- p.82 / Chapter 4.5 --- Measurement of changes in intracellular Ca2+ concentration --- p.82 / Chapter 4.5.1 --- Introduction --- p.82 / Chapter 4.5.2 --- Cell preparation --- p.83 / Chapter 4.5.3 --- Measurement of Fura-2 fluorescence --- p.83 / Chapter 4.6 --- Radioligand binding --- p.84 / Chapter 4.6.1 --- Introduction --- p.84 / Chapter 4.6.2 --- [3H]-iloprost ligand binding --- p.85 / Chapter 4.6.3 --- Data analysis --- p.86 / Chapter 4.7 --- Cytotoxicity test using trypan blue exclusion test --- p.86 / Chapter Chapter 5 --- Multiple coupling capacity of prostacyclin receptors in CHO cells --- p.88 / Chapter 5.1 --- Introduction --- p.88 / Chapter 5.2 --- Method --- p.88 / Chapter 5.3 --- Results and Discussion --- p.89 / Chapter 5.3.1 --- IP agonist log concentration-response curves for [3H]-cAMP and [3H]-inositol phosphate production in mIP-CHO cells --- p.89 / Chapter 5.3.2 --- Effect of varying Gαs cDNA concentration on cicaprost-stimulated [3H]-cAMP and [3H]-inositol phosphate production in mlP-CHO cells --- p.90 / Chapter 5.3.3 --- Effect of varying Gαq cDNA concentration on cicaprost-stimulated [3H]-cAMP and [3H]-inositol phosphate production in mlP-CHO cells --- p.92 / Chapter 5.4 --- Conclusion --- p.95 / Chapter Chapter 6 --- Multiple coupling capacity of prostacyclin receptors in neuroblastoma cells --- p.113 / Chapter 6.1 --- Introduction --- p.113 / Chapter 6.2 --- Method --- p.114 / Chapter 6.3 --- Results and Discussion --- p.114 / Chapter 6.3.1 --- Characterization of prostanoid receptors in SK-N-SH cells --- p.114 / Chapter 6.3.2 --- Property of IP-receptor signaling in SK-N-SH cells --- p.116 / Chapter 6.3.3 --- Effect of Gαq subunits on PLC activation in SK-N-SH cells --- p.117 / Chapter 6.3.4 --- Coupling capacity of IP-receptorin rat/mouse neuroblastoma (NG108-15) cells --- p.119 / Chapter 6.4 --- Conclusion --- p.123 / Chapter Chapter 7 --- Agonist trafficking --- p.133 / Chapter 7.1 --- Introduction --- p.133 / Chapter 7.2 --- Method --- p.134 / Chapter 7.3 --- Results and Discussion --- p.134 / Chapter 7.3.1 --- Simultaneous measurement of AC and PLC activation in metabolically-labelled mIP-CHO cells --- p.134 / Chapter 7.3.1.1 --- Effect of IBMX on PLC activation --- p.135 / Chapter 7.3.1.2 --- Effect of Li+ ion on AC activation --- p.135 / Chapter 7.3.1.3 --- Separation of [3H]-adenine and [3H]-inositol using column chromatography method --- p.136 / Chapter 7.3.2 --- Measurement of AC and PLC activation using different assay systems --- p.137 / Chapter 7.3.2.1 --- cAMP 125I-Scintillation Proximity Assay System --- p.137 / Chapter 7.3.2.2 --- "D-myo-Inositol 1,4,5-trisphosphate (IP3) [3H]-assay system" --- p.138 / Chapter 7.4 --- Conclusion --- p.139 / Chapter Chapter 8 --- Regulation of prostacyclin receptor coupling --- p.147 / Chapter 8.1 --- Introduction --- p.147 / Chapter 8.2 --- Methods --- p.149 / Chapter 8.3 --- Results and Discussion --- p.150 / Chapter 8.3.1 --- Role of protein kinases on IP-receptor activity --- p.150 / Chapter 8.3.2 --- Effect of SQ22536 on IP-receptor activity --- p.155 / Chapter 8.3.3 --- Role of PKA phosphorylation site in mIP-receptor activity --- p.156 / Chapter 8.4 --- Conclusion --- p.157 / Chapter Chapter 9 --- Prostacyclin receptor isoprenylation --- p.171 / Chapter 9.1 --- Introduction --- p.171 / Chapter 9.2 --- Method --- p.172 / Chapter 9.3 --- Results and Discussion --- p.173 / Chapter 9.4 --- Conclusion --- p.175 / Chapter Chapter 10 --- IP/DP chimeric receptors --- p.178 / Chapter 10.1 --- Introduction --- p.178 / Chapter 10.2 --- Method --- p.179 / Chapter 10.3 --- Results and Discussion --- p.180 / Chapter 10.3.1 --- Property of IP/DP chimeric receptors --- p.180 / Chapter 10.3.2 --- "ID1 chimeric receptor mutant receptors (M4, M5, M6)" --- p.182 / Chapter 10.3.3 --- "Mutant mIP-receptors (Ml, M2, M3)" --- p.183 / Chapter 10.3.4 --- Comparison between M5 and ID1 receptors --- p.184 / Chapter 10.4 --- Conclusion --- p.184 / Chapter Chapter 11 --- General Discussion and Conclusions --- p.193 / Chapter 11.1 --- Introduction --- p.193 / Chapter 11.2 --- Multiple coupling capacity of prostacyclin receptors --- p.193 / Chapter 11.3 --- Factors influencing prostacyclin receptor coupling --- p.196 / Chapter 11.4 --- Prostacyclin receptor cross talk and regulation --- p.198 / References --- p.200

No description available.

Orientador: Angela Maria Victoriano de Campos Soares / Banca: Eduardo Bagagli / Banca: Adriana Pardini Vicentini Moreira / Resumo: Paracoccidioides brasiliensis, é o agente da paracoccidioidomicose, a micose profunda endêmica na América Latina. Produção de eicosanóides, como prostaglandinas e leucotrienos, durante as infecções fúngicas tem mostrado desempenhar um papel crítico na sobrevivência e/ou crescimento fúngico, bem como na modulação da resposta imune do hospedeiro. As células hospedeiras são uma fonte desses mediadores, porém outra fonte em potencial pode ser o próprio fungo. O objetivo do nosso estudo foi avaliar se cepas do P. brasiliensis com diferentes graus de virulência (Pb18, Pb265, BT79, Pb192) produzem, prostaglandina E2 (PGE2) e leucotrieno B4 (LTB4). Além disso, verificamos se P. brasiliensis pode usar fontes exógenas de ácido araquidônico (AA), bem como as vias metabólicas dependentes das enzimas cicloxigenase (COX) e lipoxigenase (5-LO), para a produção de PGE2 e LTB4, respectivamente. Finalmente, uma possível associação entre esses eicosanóides e a viabilidade do fungo também foi avaliada. Demonstramos, usando ensaios de Elisa, que todas as cepas de P. brasiliensis, independente do seu grau de virulência, produzem altos níveis de PGE2 e LTB4 após 4h cultura que foram reduzidos após 8h. No entanto, em ambos os tempos da cultura, foram detectados níveis mais elevados de eicosanóides, quando as culturas foram complementadas com uma fonte exógena de AA. Diferentemente, o tratamento com indometacina, um inibidor da COX, ou MK886, um inibidor da 5-LO, induz a uma redução nos níveis de PGE2 e LTB4, respectivamente, bem como a viabilidade do fungo. Os dados fornecem evidências de que P. brasiliensis tem a capacidade de metabolizar, tanto AA endógeno como exógeno por vias dependentes da COX e 5-LO enzimas que participam da produção de PGE2 e LTB4, respectivamente, que são indispensáveis para a sua viabilidade. / Abstract: Paracoccidioides brasiliensis, is the agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. Production of eicosanoids, such as prostaglandins and leukotrienes, during fungal infections is theorized to play a critical role on fungal survival and/or growth as well as on host immune response modulation. Host cells are one source of these mediators; however another potential source may be the fungal itself. The purpose of our study was to assess whether P. brasiliensis strains with different degree of virulence (Pb18, Pb265, PbBT79, Pb192) produce both, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4). Moreover, we asked if P. brasiliensis can use exogenous sources of arachidonic acid (AA), as well as metabolic pathways dependent on cyclooxygenase ( COX) and lipooxygenase (5-LO ) enzymes, for PGE2 and LTB4 production , respectively. Finally, a possible association between these eicosanoides and fungus viability was assessed. We have demonstrated, using Elisa assays, that all P. brasiliensis strains, independently of their virulence degree, produce high PGE2 and LTB4 levels on 4h culture that were reduced after 8 h. However, in both culture times, higher eicosanoides levels were detected when culture medium was supplemented with exogenous AA. Differently, treatment with indomethacin, a COX inhibitor, or MK886, a 5-LO inhibitor, induces a reduction on PGE2 and LTB4 levels, respectively, as well as in fungus viability. The data provide evidence that P. brasiliensis has the capacity to metabolize either endogenous or exogenous AA by pathways dependent on COX and 5-LO enzymes for producing PGE2 and LTB4, respectively, that are critical for its viability. / Mestre

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21403号 / 生博第404号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 HEJNA,James, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM