O Histoplasma capsulatum (H.capsulatum) é um fungo dimórfico patogênico e responsável por graves lesões pulmonares, as quais se caracterizam pelo acúmulo de leucócitos ao redor do fungo, resultando na formação de granulomas. A infecção ocorre principalmente pela inalação de conídios ou pequenos fragmentos de micélio que alcançam os alvéolos, onde se transformam em leveduras, que é a forma patogênica do fungo. Na resposta imune do hospedeiro, as integrinas participam nos mecanismos fagocíticos, essenciais na resposta à histoplasmose. As 2integrinas contêm uma cadeia 2, também conhecida como CD18, comum a várias moléculas de adesão, e uma cadeia variável. Até o momento foram identificadas quatro cadeias distintas: L, a qual forma o dímero L2, também conhecido como LFA-1 (do inglês leukocyte function antigen-1) ou CD11aCD18; m, formando m2, chamado Mac-1 (do inglês macrophage differentiation antigen 1) ou CR3 (do inglês complement receptor 3) ou CD11bCD18; x, formando x2, CD11cCD18, gp150, 95 ou CR4 (do inglês complement receptor 4) e a cadeia d, formando d2, CD11dCD18. Neste trabalho, investigamos o papel da molécula CD18 em macrófagos alveolares (MAs) e macrófagos peritoneais (MPs) nas funções efetoras contra H. capsulatum e a relação do leucotrieno B4 (LTB4) nestas respostas. Inicialmente confirmamos que MAs e MPs provenientes dos animais CD18low, expressam baixa porcentagem de CD11bCD18 (CR3). Demonstramos que, como esperado, MAs e MPs de ambos os grupos fagocitam mais leveduras opsonizadas com complemento do que não opsonizadas. Surpreendentemente, MAs de animais CD18low fagocitam 136% mais leveduras opsonizadas do que MAs de C57BL/6. Também, MPs destes animais fagocitam aproximadamente 240% mais leveduras quando infectados com H. capsulatum e opsonizados, quando comparados aos MPs de C57BL/6. A adição de LTB4 aumenta a atividade fagocítica em 520% por MAs de animais C57BL/6 e 200% por MAs de CD18low, enquanto que a adição de LTB4 aumentou a fagocitose dos MPs de animais C57BL/6 em 600% vezes quando comparado aos MPs de CD18low. Este fenômeno foi inibido pela pré-incubação destas células com antagonista específico do receptor BLT1 apenas em animais C57BL/6. A adição de LTB4 na cultura de MPs reduziu a porcentagem de morte das leveduras apenas nos animais C57BL/6. Os animais CD18low produzem espontaneamente mais LTB4 e apresentaram um grande aumento na produção de óxido nítrico quando comparados aos animais C57BL/6. Pacientes acometidos pela Doença Granulomatosa Crônica (DGC) possuem deficiência congênita da molécula CD18. Células fagocíticas isoladas do sangue periférico de pacientes com DGC foram incubadas com leveduras opsonizadas e assim como macrófagos de animais deficientes de CD18, fagocitam mais leveduras opsonizadas (900%) ou não (300%), quando comparado com células de indivíduos sadios. Sugerimos que a molécula CD18 tem importante participação nos mecanismos efetores da imunidade inata, por mecanismo dependente de mediadores lipídicos, como o LTB4, no controle dos mecanismos de defesa contra H. capsulatum. / Histoplasma capsulatum (H. capsulatum) is a pathogenic dimorphic fungus and its infection is characterized by accumulation of leukocytes and granuloma formation. Infection occurs mainly by fungal inhalation that reaches the alveoli, which became yeast (the pathogenic form). in the immune response of host, integrins participate in phagocytic mechanisms, fundamental in the response against histoplasmosis.,8^2-integrin has a 02 chain known as CD18, usual to many adhesion molecules, and a variable a chain. Until the moment, it was identified four variable a chains: aL, that constitutes the dimer aL,82, also known as LFA-1 (leukocyte function antigen) or CD11aCD18; am forming the a^m,B^2 or Mac-1 (macrophage differentiation antigen 1) and CR3 (complement receptor 3) and CD11bCD18; ax, constituting the dimer Able CD11cCD18, gp150, 95 or CR4 (complement receptor 4) and ad chain, that constitutes a^d,8^2, CD11dCD18. In the present study, we sought to investigate the effect of CD18 in alveolar (AMs) and peritoneal macrophages (PMs) effecter functions against H. capsulatum and the relation of LTB^4 in those responses. We confirm that AMs and PMs of CD18\'°^W mice have low expression of ,32-integrin compared to wild type mice (WT). We demonstrate that, as expected, AMs and PMs from WT and CD18\'°^W, phagocytosed more complement (C)-opsonized yeasts than the unopsonized yeasts. Surprisingly, AMs from CD18\'°^Wmice phagocytosed 136% more (C)-opsonized yeasts than AMs obtained from WT. Also, PMs of CD18^b°^W mice phagocytosed 240% more (C)-opsonized yeasts than PMs of WT. The addition of LTB^4, increases the phagocytic activity by AMs of WT mice in 520% and by AMs from CD18\'°^W mice in 200%, while the addition of LTB^4 only increased the phagocytosis of C-opsonized H. capsulatum by PMs of C57BL/6 mice in 600%, when compaired with PMs from CD18\'°^W mice. This phenomenon was inhibited by pretreatment of these cells with an especific BLT1 receptor antagonist only in PMs from C57BU6 mice. The addition of LTB^4 in the culture of MPs reduced the percentage of death of yeasts in animals C57BL/6. CD18\'°^W mice, spontaneously produce more LTB^4 and showed a large increase in the production of nitric oxide when compared to C57BU6. Patients affected by Chronic Granulomatous Disease (DGC) have congenital deficiency of the CD18 molecule. Phagocytic cells isolated from peripheral blood of patients with DGC were incubated with C-opsonized yeasts and as well as macrophages from CD18\'°^W, phagocytosed more C-opsonized yeasts (900%) or not (300%) when compared with cells from healthy individuals.Therefore, we suggest that the CD18 molecule has important participation in the effector mechanisms of innate immunity, a mechanism dependent on lipid mediators such as LTB^4, to control these mechanisms in defense against H. capsulatum.

Leucotrienos (LTs) são mediadores lipídicos derivados do ácido araquidônico. Existem evidências que receptores da imunidade inata interagem com receptores para LTs amplificando funções efetoras de macrófagos. Investigamos se LTs modulam a fagocitose e a atividade microbicida via receptores Manose, Dectina -1 e PTX3 em macrófagos alveolares (AMs) e os mecanismos moleculares envolvidos. Nossos resultados mostram que: 1) AMs sintetizam LTs quando fagocitam C. albicans, Zy e Zy-PTX3; 2) LTs potencializam a fagocitose de C. albicans e Zy, mas não de Zy-PTX3. Este efeito dos LTs depende: do reconhecimento via receptor manose (LTB4) e Dectina-1 (LTD4); da integridade de lipid rafts; da ação em mecanismos de polimerização de actina; do aumento dos níveis de F-actina por inativação da Cofilina-1; da ativação das LIMKs, que regulam Cofilina-1; da ativação de PKC<font face=\"Symbol\">d e PI3K3) LTs aumentam a capacidade de AMs em matar C. albicans por ativação de NADPH oxidase. Em conjunto, mostramos que LTs potencializam programas de sinalização específicos para determinados PRRs. / Leukotrienes (LTs) are lipid mediators derived from arachidonic acid. There is evidence that innate immunity receptors and leukotrienes receptors interact and amplify macrophage effector functions. We investigated if LTs receptors modulate phagocytosis and microbicidal activity mediated by Mannose receptor, Dectin-1 and PTX3 in alveolar macrophages (AMs) and the molecular mechanisms involved. Our results showed that: 1) AMs produce LTs when stimulated with C.albicans, Zy, Zy-PTX3; 2) LTs enhance phagocytosis of C.albicans and Zymosan, but not Zy-PTX3. This is dependent on: recognition via mannose receptor (LTB4) and Dectin-1 (LTD4); integrity of lipid rafts; its action on actin polimerization mechanisms; enhancement of F-actin levels by induction of Cofilin-1 inactivation; activation of LIMKs that regulate Cofilin-1; activation of PKC<font face=\"Symbol\">d and PI3K3) LTs enhance killing of C.albicans by activation of NADPH oxidase. Taken together, our results showed that LTs specifically influence signaling programs of keys PRRs.

Orientador: Maria Marluce dos Santos Vilela / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T19:21:41Z (GMT). No. of bitstreams: 1 Tani_SergioMassayuki_M.pdf: 3275240 bytes, checksum: cc4115cf1f3cfbc43979ab57c10fcc10 (MD5) Previous issue date: 2005 / Resumo: O sistema complemento apresenta imaturidade funcional em crianças mais jovens, com o sistema imune inato mais ativado em recém-nascidos e lactentes jovens. Infecções crônicas e oportunistas concomitantes, como na Síndrome da Imunodeficiência Adquirida (SIDA), causam disfunção na atividade fagocitária de células mononucleares. A atividade fagocitária de monócitos in vitro , por índice fagocitário (porcentagem de células com fagocitose efetuada em uma lâmina) e capacidade fagocitária (número de partículas fagocitadas em 100 células), foi estudada em 58 crianças soro-reversoras, separadas em faixas etárias, para zymosan não incubado, zymosan incubado com soro de doadores normais e do próprio paciente e com hemácias de carneiro incubadas com anticorpos de coelho antieritrócitos de carneiro, respectivamente para os receptores CR1, CR3 e Fc. Foram avaliados parâmetros hematológicos (hemograma completo), sistema T (TCD4+ e TCD8+), sistema B (níveis séricos de imunoglobulinas) e profilaxias com zidovudina e com sulfametoxazol e trimetoprima. Um grupo de crianças infectadas pelo vírus da imunodeficiência humana do tipo I (HIV-1) foi utilizado como referência para comparações. A metodologia estatística constou de: análise descritiva com tabelas de freqüências, testes não paramétricos de Mann-Whitney e de Kruskal-Wallis, teste de correlação não linear com o coeficiente de Spearman. O nível de significância adotado foi de p<0,05. O estudo foi aprovado pelo Comitê de Ética em Pesquisa da Faculdade de Ciências Médicas da Unicamp. Na comparação do índice fagocitário das crianças soro-reversoras para zymosan incubado com soro normal e do próprio paciente, pelas vias CR1 (CD35) e CR3 (CD11bCD18), foram observados valores menores (p=0,004 e p=0,001, no grupo total e p=0,027 e p=0,021, no grupo de crianças soro-reversoras maiores de 24 meses, respectivamente) em relação ao grupo de referência. Associações negativas: capacidade fagocitária de monócitos com zymosan não incubado, pela via das lectinas, com idade das crianças soro-reversoras (p=0,014); função fagocitária de monócitos para zymosan incubado com soro normal e do paciente, via receptores CR1 (CD35) e CR3 (CD11b CD18), pela ativação da via alternativa com zidovudina (Rs=-0,509; Rs=-0,344; Rs =-0,342; Rs=-0,328). Associações positivas: capacidade fagocitária de monócitos vias CR1 (CD35) e CR3 (CD11b CD18) e idade (p=0,026) e para níveis séricos de IgA (Rs=0,277). Valores de hemoglobina, leucócitos totais e TCD4+ foram menores (p<0,003, p<0,006 e p<0.004, respectivamente), e valores de TCD8+, IgA, IgG e IgM foram maiores (p<0,001) no grupo de referência. Nas crianças soro-reversoras, 55,2% receberam profilaxia com zidovudina e 79,3%, com sulfametoxazol e trimetoprima. O amadurecimento do sistema imune ocorreu com o aumento da idade e a atividade fagocitária de monócitos foi mais estimulada em crianças infectadas pelo HIV-1, na comparação com crianças soro-reversoras / Abstract: The complement system presents functional immaturity in young children and the innate immune system is more activated in newborns and infants. Chronics and opportunists infections, as well as Acquired Immunodeficiency Syndrome (AIDS), cause phagocytic activity¿s disfunction on the mononuclear cells. Monocyte function was evaluated in 58 exposed seroreverter children with an assay blood monocyte phagocytosis for zymosan and sheep red blood cells, mediated by the CR1, CR3 and Fc receptors, respectively. The zymosan assay was conducted with non-incubated zymosan and incubated zymosan with patient serum or serum from a normal blood donor pool. The phagocytic index (percentage of cells having phagocytosis on a slide) and the phagocytic capacity (number of phagocytes particles in a 100 cells counts) were determined. Complete blood count, lymphocyte subsets determination (TCD4+ and TCD8+), immunoglobulin levels (IgA, IgG and IgM) and prophylaxis with zidovudine and sulfametoxazol and trimetoprim were studied. The results were compared with the reference group (HIV-1 infected children). Seroreverter phagocytic index for incubated zymosan by CR1 (CD35) and CR3 (CD11bCD18) showed inferior results when compared to the reference group. Negative associations: phagocytic capacity with not-incubated zymosan, by lectin pathway activation with seroreverter children¿s age; and incubated zymosan on CR1 and CR3 receptors by activation of alternative pathway from complement system with zidovudine. Positive associations: phagocytic capacity by CR1 and CR3 receptors with seroreverter children¿s age and IgA serum levels. Seroreverter children presents higher values of hemoglobin, total leukocytes and TCD4+, and inferior values of TCD8+, IgA, IgG and IgM, when compared to the reference group. The immune system maturation occurred with age increased and the monocytes function was more stimulated in HIV-1 infected children / Mestrado / Pediatria / Mestre em Saude da Criança e do Adolescente

Submitted by Neusa Fagundes (neusa.fagundes@unioeste.br) on 2018-03-12T14:24:25Z No. of bitstreams: 2 Renan_Melo2014.pdf: 1848176 bytes, checksum: 102deb99625acf4c43610c22cb0234f3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-03-12T14:24:25Z (GMT). No. of bitstreams: 2 Renan_Melo2014.pdf: 1848176 bytes, checksum: 102deb99625acf4c43610c22cb0234f3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-12-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Ganoderma australe is a Basidiomycete species responsible for the delignification process of the organic matter known to cause "white rot" in certain species of trees. Cytochemical studies have shown that the cell wall of fungi polysaccharides has (13)- β-glucans, (16)-β-glucans type and other complex polysaccharides. Currently, it has been highlighted that the β-glucans are polysaccharides that produce higher effects as modulators of biological functions. In previous studies, the fungus G. australe has been studied regarding the composition of the cell wall polysaccharide. SK5 polysaccharide fraction was obtained after freeze-thawing of the aqueous extraction with KOH 5%. Monosaccharide composition of SK5 fraction revealed by Gas chromatography–mass spectrometry (GC-MS) showed glucose is predominant (81.3%), with presence of other sugars (mannose, arabinose, xylose, rhamnose, fucose and galactose) at concentration of less than about 3% each. The nuclear magnetic resonance of this fraction proved to be a β-glucan with glycosidic links (1→3)-β type and probably replaced in 4-O. In this study, the biological effect of SK5 polysaccharide fraction extracted from the fungus G. australe has been evaluated in vitro cell culture of peritoneal macrophages isolated from Swiss mice. The biological effect was assessed for toxicity and cell activation, nitric oxide, cytokines (TNF-α, IL-6), superoxide anion (production and scavenging capacity) and the ability to stimulate phagocytic activity. Cell viability was affected, with approximately 29% reduction with 1.0 mg / mL and 35.5% reduction with 2.5 μg/ml SK5. There was no change in nitric oxide production, TNF-α or superoxide anion concentrations between 0.1 μg/mL and 1.0 μg/mL. The scavenging ability of superoxide anion was also not detected. There was an increase in IL-6 by about 111% with 1.0 μg/mL. In regarding to phagocyte activity was increased in all concentrations examined obtaining 52.3% with 0.25 μg/mL of polysaccharide. The results indicate that (13)-β- glucan isolated from G.australe can be classified as biological response modifier (BRM). / Ganoderma australe é uma das espécies de basidiomicetos responsáveis pelo processo de deslignificação da matéria orgânica, conhecido por causar a “podridão branca” em algumas espécies de árvores. Estudos citoquímicos demostraram que a parede celular dos fungos possui polissacarídeos do tipo β (13)-glucanas, β (16)-glucanas e outros polissacarídeos complexos. Atualmente, tem-se destacado que as β-glucanas são os polissacarídeos que produzem efeitos mais intensos como moduladores das funções biológicas. Em estudos anteriores desenvolvidos em nosso laboratório, o fungo G. australe foi estudado quanto à composição polissacarídica da parede celular. A fração polissacarídica SK5 foi obtida após gelo-degelo da extração aquosa com KOH 5%. A composição monossacarídica da fração SK5 revelada por Cromatografia Gasosa acoplada a Espectrometria de Massa (GC-MS) mostrou que glicose é predominante (81,3%), com a presença de outros açúcares (manose, arabinose, xilose, ramnose, fucose e galactose) em concentração inferior a cerca de 3% cada um. A ressonância magnética nuclear desta fração comprovou ser uma β-glucana, com ligações glicosídicas do tipo β- 1→3 e provavelmente substituídas em 4-O. Neste trabalho, o efeito biológico da fração polissacarídica SK5 extraída do fungo G. australe foi avaliado in vitro em cultura de células de macrófagos peritoneais isoladas de camundongos Swiss. Foi analisado o efeito biológico quanto à toxicidade e ativação celular, a produção de óxido nítrico, citocinas (TNF-α, IL-6), ânion superóxido (produção e capacidade sequestradora) e a capacidade de estimular a atividade fagocítica. A viabilidade celular foi significativamente afetada, com cerca de 29% de redução com 1,0 μg/mL e 35,5% de redução com 2,5 μg/mL de SK5. Não houve alteração na produção de óxido nítrico, TNF-α ou de ânion superóxido nas concentrações entre 0,1 μg/mL e 1,0 μg/mL. A capacidade sequestradora de ânion superóxido também não foi constatada. Houve aumento na produção de IL-6 em cerca de 111% com 1,0 μg/mL. Em relação à atividade fagocítica houve aumento em todas as concentrações analisadas, chegando a 52,3% com 0,25 μg/mL do polissacarídeo. Os resultados obtidos indicam que a β (13)-glucana isolada de Ganoderma australe pode ser classificada como modificadora de resposta biológica (MRB).

Crotalfina (CRF) é um peptídeo produzido do veneno da serpente Crotalus durissus terrificus com efeito antinociceptivo tipo opióide detectado apenas na presença de inflamação. Estudos sobre mecanismos de analgesia relatam a ação de opióides em células imunes e macrófagos são apontados como alvos para os efeitos destes. Este estudo caracterizou a ação da CRF sobre funções de macrófagos e avaliou a importância de seus receptores opióides nestas ações. Os resultados indicam que CRF inibe fagocitose, liberação de H2O2 e produção de NO&bull; e TNF-a, modula IL-6 e estimula IL-1b por macrófagos residentes e inflamatórios. Ensaios de expressão e ativação mostram que macrófagos residentes expressam receptores opióides k seguido de m e d e macrófagos inflamatórios expressam receptores m seguido de k e d. A CRF interferiu, de maneira distinta, com a expressão destes receptores, dependendo do estado de ativação destas células. Antagonistas destes receptores bloquearam a ação da CRF, mostrando que este peptídeo possui ação imunomodulatória mediada por receptores opióides. / Crotalphine (CRF) is a peptide isolated from the venom of Crotalus durissus terrificus with type opioid antinociceptive effect only detected in the presence of inflammation. Studies on mechanisms of analgesia reported the action of opioids in immune cells and macrophages are considered as targets for these effects. This study characterized the action of CRF on functions of macrophages and assessed the importance of opioid receptors in these actions. The results indicate that CRF inhibits phagocytosis, release of H2O2 and production of NO&bull; and TNF-a, modulates IL-6 and stimulates IL-1b production by resident and inflammatory macrophages. Expression and activation assays show that resident macrophages express k receptors followed by m and d. Inflammatory macrophages express m receptors followed by k and d. CRF interfered differently with the expression of these receptors, depending on the activation state of these cells. Antagonists of these receptors blocked the action of CRF, showing that this peptide has immunomodulatory action mediated by opioid receptors.

A tolerância materno-fetal envolve, além do sistema imune, muitas peculiaridades quanto ao tipo de placenta e placentação. A placenta em bovinos é considerada não invasiva, e como conseqüência de uma implantação superficial, sem invasão endometrial, se estabelece uma barreira placentária complexa que se interpõe entre a circulação materna e fetal. Aliado a isso, a placenta um órgão que se encontra em constante diferenciação e proliferação celular além da alta atividade metabólica, fontes constantes de estresse e desafio para o sistema imune. Atuando nesse contexto, destacamos as proteínas de choque térmico (heat shock proteins HSP), proteínas que são expressadas em condições fisiológicas mas, em situações de estresse, sua expressão aumenta. Na placenta bovina já se constatou a expressão das HSP 60 e 70; contudo notou-se uma maior peculiaridade na expressão da HSP 60, que é maior no primeiro trimestre da gestação. Observando-se o perfil da presença e expressão dessa proteína na placenta bovina e, tendo em vista a característica desse órgão em apresentar populações leucocitárias funcionalmente distintas das do sangue periférico, levantou-se a hipótese da influência desta proteína sobre as células placentárias, particularmente sobre os leucócitos no processo de tolerância materno fetal. Desta forma, esse trabalho analisou a participação da HSP 60 em mecanismos imunes junto à alguns leucócitos placentários por meio de ensaio de proliferação pela técnica de CFSE, ensaio de fagocitose e ensaios bioquímicos como peroxidação lipídica; ciclo celular e potencial de membrana mitocondrial de células placentárias em cultivo. Nossos resultados mostraram que a HSP 60 não influencia a proliferação de linfócitos e outras células placentárias nas diversas condições testadas. Em contrapartida a HSP 60 aumenta a fagocitose e apoptose no terceiro terço gestacional, bem como a produção de radicais oxidados poliinsaturados e o potencial de membrana mitocondrial. Tendo em vista esses resultados, podemos observar que a HSP 60 nas células da placenta bovina possivelmente esteja modulando a cinética da ativação de mecanismos de sinalização de morte celular programada entre as células da placenta e o sistema imunológico. Esta hipótese assegura que, no concepto a termo, as variações hormonais e o sistema imune possam estar regulando o processo para o nascimento do feto. / The maternal-fetal tolerance involves, besides the immune system, many peculiarities regarding the type of placenta and placentation. The bovine placenta is considered not invasive and, as consequence of a superficial implantation without endometrial invasion, it establishes itself as complex barrier that interposes between the maternal and fetal circulation. In addition to that, placenta is an organ that undergoes continuous differentiation and cellular proliferation besides the high metabolic activity that represents a constant source of stress and challenge for the immune system. As an important component in this context, we highlight the heat shock proteins - HSP, that are usually expressed in physiological conditions but, in situations of stress, their expression increases. In the bovine placenta the expression of HSP 60 and 70 has already been verified; however HSP 60 showed a peculiar expression behavior, with higher staining in the first trimester of pregnancy. Considering the presence and expression profiles of HSP 60 in the bovine placenta allied to the particularity of presenting functional distinct leucocytes populations, a hypothesis was raised regarding the influence of this protein on the placental cells, particularly on the leucocytes in the process of fetal-maternal tolerance. Therefore, this work analyzed the role of HSP 60 in immune mechanisms related to some placental leucocytes by several approaches like proliferation assay by CFSE technique, phagocytosis assay and biochemical assays as lipoperoxidation; cell cycle phases and mitochondrial membrane potential of placental cells in culture. Our results showed that HSP 60 does not influence the proliferation of lymphocytes or any other placental cells in various conditions tested. On the other hand, HSP 60 increases phagocytosis and apoptosis in the third trimester, as well as the production of polyunsaturated oxide radicals and the mitochondrial membrane potential. In view of these results, we may infer that HSP 60 may be modulating the kinetics of activation mechanisms for signaling programmed cellular death between placental and immune cells. This hypothesis assures that, on the termed conceptus, the hormonal variation and the immune tolerance may be responsible for regulating the parturition process.

Dictyostelium discoideum is a soil dwelling amoeba which has been widely used as a model organism to study cellular processes such as signal transduction, chemotaxis, endocytosis and exocytosis. The process of phagocytosis in Dicytostelium is largely comparable to that of neutrophils and macrophages in the mammalian system. Neutrophils and macrophages are cells of the innate immune system and they engulf infectious bacteria through phagocytosis. Dictyostelium cells uptake yeast and bacteria for their nutrition through phagocytosis, which is an actin dependent mechanism and is a target of multiple signaling inputs. Recent studies have uncovered different proteins involved in the signaling of particle and further studies are required to decipher the intricate mechanism leading to the F-actin rearrangement. Two of the proteins have previously known to be involved in the pathways regulating the F- actin rearrangement name PP2A phosphatase and LKB1 kinase The main objective of this project was to determine how these proteins are affecting the two actin driven particle uptake processes, phagocytosis and fluid uptake. We showed that ablation of PsrA gene which codes the regulatory subunit of PP2A resulted in a defective phagocytosis, whereas the fluid uptake was normal. We also showed for the first time that there was an increase in the phosphorylation of some of the PKB substrate proteins in wild type cell. Cells lacking PsrA gene displayed an aberrant phosphorylation of PKB substrate protein when compared to the wild type cells further confirming the involvement of PKB substrate in phagocytosis. Further, we looked at the effects of LKB1 kinase on phagocytosis by using a LKB1 knockdown construct introduced into wild type cells. The knock down of LKB1 resulted in a higher rate of phagocytosis while introduction of a LKB1 over expressing construct severally decreased the rate of phagocytosis indicating an inhibitory effect of LKB1. Furthermore there was an increase in the PKB substrate protein but a different pattern compared to the psrA- cells. We also carried out adhesion assays on LKB1 knockdown cells and the results showed a higher substrate adhesion as compared to the wild type cells, while psrA- cells had no adhesion defect.

As poliaminas são essenciais para o controle dos mecanismos celulares nos seres vivos. A Migalina é uma acilpoliamina isolada de A. gomesiana com atividade microbicida contra E. coli, contudo, seu efeito imunomodulador é desconhecido. Sabendo que os mediadores imunes produzidos por macrófagos são críticos para a regulação da resposta imune, investigamos o efeito in vitro da Migalina sobre estas células. Demonstramos que a Migalina não induziu citotoxicidade, mas ativou iNOS, potencializando a síntese de nitrito. Aumentou a produção de TNF-<font face=\"Symbol\">a, mas não de IL-12p40. IL-1<font face=\"Symbol\">b não foi detectada. Além disto, Inibiu a produção de TNF-<font face=\"Symbol\">a e IL-12p40 induzida por LPS e quando associada à Polimixina B não alterou a síntese de TNF-<font face=\"Symbol\">a. Macrófagos deficientes de MyD88 não produziram TNF-<font face=\"Symbol\">a em resposta a Migalina, sugerindo que a sinalização mediada por TLR via MyD88 é requerida para a produção desta citocina. Comprovamos que a Migalina modula a atividade de macrófagos através de mediadores da resposta imune inata e pode ser explorada como estratégia no controle de infecções. / Polyamines are present in most living organisms. Mygalin is an acylpolyamine isolated from A. gomesiana that showed microbicide activity against E. coli, however, its immunomodulator effect has not been explored. Immune mediators produced by macrophages are essential for the regulation of immune responses. Our results showed that murine macrophages stimulated in vitro with Mygalin didnt induce cytotoxicity, but activated iNOS, inducing nitrite synthesis. The production of TNF-<font face=\"Symbol\">a was enhanced, but IL-12p40 was not. The presence of IL-1<font face=\"Symbol\">b was not detected. In higher concentrations, Mygalin inhibited significantly the production of IL-12p40 and TNF-<font face=\"Symbol\">a induced by LPS, and its association with Polymixine B did not altered the production profile of TNF-<font face=\"Symbol\">a. Macrophages deficient in MyD88 molecule activated with Mygalin did not induce the synthesis of TNF-<font face=\"Symbol\">a. Our results showed that Mygalin is capable of modulating the macrophage activity by inducing mediators of innate immune response and can be explored as a strategy in the studies to control pathogen infections.

Les neutrophiles participent à la défense de l'hôte en phagocytant les agents pathogènes et en les détruisant via notamment la production de formes réactives de l'oxygène (FRO). Les FRO sont produites par un complexe multi- protéique :la NADPH oxydase (NOX2). Celle-ci peut s’assembler à la membrane du phagosome lors de la phagocytose mais aussi à la membrane plasmique lors de la stimulation des neutrophiles par des agents bactériens ou des médiateurs de l’inflammation. La NADPH oxydase est une arme à double tranchant; une activation excessive ou inappropriée de la NADPH oxydase génère un stress oxydant, facteur aggravant des nombreuses pathologies. Cette enzyme doit donc être finement régulée. La NADPH oxydase est activée lorsque les sous-unités cytosoliques de NOX2 (p67phox, p47phox, p40phox) et la petite GTPase Rac s’assemblent avec les sous-unités membranaires (p22phox et gp91phox) à la membrane phagosomale ou plasmique. P67phox régule le flux d'électrons qui transite via gp91phox du NADPH à O2.-. Des travaux récents indiquent que les phospholipides anioniques contribueraient à la régulation de la NADPH oxydase. De plus, Les protéines organisatrices p40phox et p47phox possèdent des domaines de liaison à ces phosphoinositides : p40phox peut se lier au phosphatidylinositol 3-phosphate (PI(3)P) et p47phox au phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Nous avons donc voulu comprendre le rôle des ces phospholipides dans la régulation de la NADPH oxydase. Dans un premier temps nous nous sommes intéressés au rôle du PI(3)P, présent au phagosome après la fermeture de celui-ci, dans l’activation de la NADPH oxydase. Nos données indiquent que p40phox fonctionne comme un adaptateur, PI(3)P dépendant, permettant de maintenir p67phox dans le complexe de la NADPH oxydase. Le PI(3)P agit comme un « timer » pour l'activation de la NADPH oxydase au phagosome. Nous avons ensuite voulu examiner le rôle du PI(3,4)P2 dans la régulation de la NADPH oxydase à la membrane plasmique. Ce lipide est formé à la membrane plasmique par phosphorylation du PI(4)P par la PI3K de classe I lors de l’activation des neutrophiles. Nous avons montré que, l'activité PI3K de classe I est nécessaire pour maintenir l’activation, intégrine-dépendante, de la NADPH oxydase à la membrane plasmique. / The NADPH oxidase of the professional phagocyte is essential for the immune system. The phagocyte NADPH oxidase, NOX2, catalyze the reduction of molecular oxygen to superoxide. Superoxide is transformed rapidly into other reactive oxygen species (ROS) which play a critical role in the killing of pathogens in host defense. Indeed neutrophils, the first cells that arrive at the site of infections, engulf pathogens in a process called phagocytosis. The production of reactive oxygen species is then triggered by the NADPH oxidase in the phagosome. The importance of ROS production is demonstrated by the recurrent bacterial and fungal infections that face patients who lack functional NADPH oxidase as in the rare genetic disorder known as the chronic granulomatous disease (CGD). Upon stimulation by bacterial peptide or in some pathological conditions, NADPH oxidase can also be activated at the phagocyte plasma membrane producing ROS in the extracellular medium. So, an excessive or inappropriate NADPH oxidase activation generates oxidative stress involve in chronic inflammation, cardiovascular disease and neurodegenerative disease. The NADPH oxidase activity should be tightly regulated. The activity of the enzyme is the result of the assembly of cytosolic subunits (p47phox, p67phox, p40phox and Rac2) with membranous subunits (gp91phox and p22phox). P67phox regulates the electron flow through gp91phox from NADPH to oxygen leading to the formation of superoxide. Recent data indicate that the anionic phospholipids are important for the NADPH oxidase regulation. Moreover, p40phox and p47phox bear a PX domain that binds respectively phosphatidylinositol3-phosphate (PI3P) and phosphatidylinositol (3,4)-bisphosphate(PI(3,4)P2). Our objective was to decipher the importance of these phosphoinositides on the NADPH oxidase activity. We first examined the role of PI3P, which is present on the cytosolic leaflet of phagosome after its sealing, in NADPH oxidase activation. Our data indicate that p40phox works as a late adaptor controlled by PI3P to maintain p67phox in the NADPH oxidase complex. Thus, PI3P acts as a timer for NADPH oxidase assembly. We then examined the role of PI(3,4)P2 in the activation of the NADPH oxidase assembled at the plasma membrane. PI(3,4)P2 and PI(3,4,5)P3 are formed at the plasma membrane, upon neutrophil activation, by phosphorylation by Class I PI3K of respectively PI4P and PI(4,5)P2. We found that class I PI3K activity is required to maintain the integrin-dependent activation of NADPH oxidase at the plasma membrane.

C. glabrata est une levure commensale de l’Homme à l’origine d’infections opportunistes chez les patients immunodéprimés. Les neutrophiles sont les premières cellules recrutées sur le site de l’infection. La production de formes réactives de l’oxygène par ces cellules, initiée dans le phagosome par la NADPH oxydase 2 est un évènement majeur de la maturation du phagosome et fait l’objet de l’étude réalisée. L’objectif de ce travail était de développer des outils expérimentaux permettant l’évaluation du stress oxydant subi par C. glabrata lors de son internalisation par les phagocytes. La levure non pathogène S. cerevisiae, phylogénétiquement plus proche de C. glabrata, a été choisie comme organisme contrôle permettant une étude comparative entre les deux levures. C. glabrata est efficacement internalisée en absence d’opsonisation, par la lignée PLB-985-neutrophile contrairement à S. cerevisiae. L’utilisation de sondes organiques pour la mesure des FRO dans le phagosome est limitée puisque que le marquage des levures par ces sondes n’est pas spécifiquement localisé. Le développement de biosenseurs de FRO à partir de protéines fluorescentes, dont la localisation est maitrisée grâce à un système de marquage, est présenté. Les études réalisées suggèrent cependant que les protéines fluorescentes subissent des modifications dans le phagosome indépendantes de la production de FRO. Des tests de viabilité effectués sur les levures après phagocytose montrent que l’élimination des levures dépend fortement de facteurs indépendants de la production de FRO. Plusieurs méthodes indiquent néanmoins que les phagosomes avec C. glabrata contiennent moins de FRO que les phagosomes avec S. cerevisiae. C. glabrata semble éliminer les FRO plus efficacement que S. cerevisiae. / C. glabrata is a human commensal yeast responsible for opportunistic infections in immunocompromised patients. Neutrophils are the first cells to be recruited to the infection site. Production of reactive oxygen species (ROS) in the phagosome by the phagocyte NADPH oxidase 2 is a major event of the phagosome maturation and is the subject of the study. The aim of this work was to develop experimental tools allowing the evaluation of the oxidative stress endured by C. glabrata during its internalization by phagocytes. The non-pathogenic yeast S. cerevisiae, phylogenetically close to C. glabrata, was chosen as a control organism allowing a comparative study between the two yeasts. Unlike S. cerevisiae, non-opsonized C. glabrata is efficiently internalized by the PLB-985 cell line. The utilization of organic dyes for the detection of ROS in the phagosome lacks precision since the staining of the yeasts by those dyes is not specifically localized. The development of ROS biosensors based on fluorescent proteins, whose localization can be controlled due to a new staining procedure, is presented here. However the results suggest that fluorescent proteins undergo modifications in the phagosome independently from the ROS production. Viability tests performed on the yeasts after phagocytosis showed that yeast removal depends mainly on factors independent from ROS production. Several methods indicate nevertheless that the phagosomes of C. glabrata contain less ROS than the phagosome of S. cerevisiae. C. glabrata appears to suppress ROS more efficiently than S. cerevisiae.