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Orientador: Mário José Abdalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T22:12:54Z (GMT). No. of bitstreams: 1 Mittestainer_FrancineCappa_M.pdf: 2379142 bytes, checksum: cb6178fdb335fdabb4a29e4767a5c1b5 (MD5) Previous issue date: 2011 / Resumo: A infusão da insulina durante a inflamação aguda melhora os resultados clínicos, mas o exato mecanismo desse efeito benéfico da insulina ainda não foi bem compreendido. Estudos recentes mostraram que a insulina tem um efeito antiinflamatório de modo que o hormônio também exibe um efeito inibidor sobre a mediação da transcrição de NF-kB em células mononucleares. Assim, o objetivo do presente estudo foi investigar os efeitos agudos da administração de insulina regular sobre a modulação de proteínas da via IKK/IkB/NF-kB e das proteínas chave da via de sinalização da insulina em tecido hepático, muscular e adiposo, a expressão de NF-kB nesses tecidos através de ELISA, o efeito do tratamento com insulina em macrófagos extraídos do peritônio de ratos e a influência dos inibidores da PI3K e MAPK na via inflamatória. Ademais, fizemos um ensaio da proteína fosfatase PP2A usando a imunoprecipitação com anticorpos anti-IKKbeta. Para o experimento, ratos machos adultos Wistar compuseram aleatoriamente 4 grupos diferentes. Três deles foram submetidos à infusão de insulina na veia porta e, então foram estimulados em 1, 3 e 5 minutos, enquanto o outro grupo (0) não foi estimulado com insulina. Em nossos resultados, observamos um aumento da fosforilação de IR, IRS1 e Akt induzida pela insulina nos três tecidos estudados. Em paralelo, houve uma redução na fosforilação de IKK e IkB após o estimulo da insulina. A ativação de NF-kB p65 no núcleo das células mostrou a redução na fosforilação do IKK e IkB no fígado, músculo e tecido adiposo. Na cultura de macrófagos tratada com insulina, após a adição dos inibidores específicos para PI3K e MAPK, observamos um aumento na fosforilação de IKK e IkB. Nossos resultados também mostraram que após estímulo da insulina houve um aumento na atividade da proteina fosfatase PP2A associada ao IKK nos três tecidos estudados. Desta forma, podemos sugerir que insulina pode induzir uma interação entre PP2A e IKK, o que resultará em mais desfosforilação de IKK e, assim, uma inibição da atividade da proteína quinase, revelando um potencial mecanismo de ação efeito anti-inflamatório da insulina / Abstract: Insulin infusion during acute inflammation improves clinical the outcomes but the exact mechanism of this beneficial effect is unclear. Recent studies have suggested that insulin has an anti-inflammatory effect in such a way that this hormone exhibits an inhibitory effect on the mediation of transcription of NF- kB in mononuclear cells. Thus, the aim of this study was to investigate the acute effects of regular insulin administration in modulation of IKK/IkB/NF-kB pathway and insulin signaling pathway (IR, IRS1 and Akt) and the DNA binding of NF-kB p65 in liver, muscle and adipose tissue of rats and also analyze the effect of treatment with insulin on macrophages of rats and the influence of PI3K and MAPK inhibitors on pathway. And finally, we analyze a phosphatase assay by using an immunoprecipitation with anti-IKKbeta antibody and PP2A phosphatase assay. For the experiment, male adult Wistar rats were divided in 4 groups. Three of them were submitted to insulin injection in the portal vein and were stimulated for 1, 3 and 5 minutes and the other group (0) was not stimulated (saline). In our results, we observed an increase in insulin-induced phosphorylation of IR, IRS1 and Akt during insulin injection in the three tissues studied. In parallel, there was a reduction in the phosphorylation of IKK and IkB after insulin stimulation. The DNA binding of NF-kB p65 in the cell nucleus showed a reduction of the IKK and IkB phosphorylation after insulin injection in liver, muscle and adipose tissue. In macrophages culture of treated with insulin and when added the specific inhibitor for PI3K and MAPK we observed an increased on phosphorylation of IKK and IkB. Our results also showed that after insulin stimulus there was an increase in PP2A phosphatase activity associated with IKK in the three tissues studied. Thus, we can suggest that insulin might induce an interaction between PP2A and IKK, which will result in more IKK dephosphorylation and thus an inhibition of this protein kinase activity, showed a possible anti-inflammatory effect of insulin / Mestrado / Mestre em Ciências

付記する学位プログラム名: 京都大学卓越大学院プログラム「メディカルイノベーション大学院プログラム」 / 京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第25222号 / 薬科博第184号 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 井垣 達吏, 教授 掛谷 秀昭 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-06-09T21:26:35Z No. of bitstreams: 2 Dissertação - Kelly de Souza Fernandes - 2016: 2715711 bytes, checksum: a2c065301e1443bb424fa23c738e43d2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-06-10T13:24:18Z (GMT) No. of bitstreams: 2 Dissertação - Kelly de Souza Fernandes - 2016: 2715711 bytes, checksum: a2c065301e1443bb424fa23c738e43d2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-06-10T13:24:18Z (GMT). No. of bitstreams: 2 Dissertação - Kelly de Souza Fernandes - 2016: 2715711 bytes, checksum: a2c065301e1443bb424fa23c738e43d2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2016-02-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Miltefosine (MT) is a alkylphospholipid originally developed for treatment of breast cancer and other solid tumors. It is currently used in the treatment of leishmaniasis, an infectious parasitic disease caused by protozoa of the genus Leishmania, being the first oral drug adopted for this purpose. However, its mechanism of action remains unclear. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled lipid (5-DOXIL stearate) and a thiol-specific spin label (4-maleimido-TEMPO) in the membrane of axenic amastigotes of L.(Leishmania) amazonensis and peritoneal macrophages from Balb/c mice showed that MT causes significant increase in membrane dynamics at similar concentrations that inhibit parasite growth or are cytotoxic to macrophage. Although these alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ~80% parasites and macrophages death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. Cell viability, analyzed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide tetrazolium) reduction assay, showed that 50% inhibitory concentration (IC50) of MT depends on the cell concentration used in the assay. This dependence was analyzed using a theoretical equation involving biophysical parameters such as the partition coefficient of watermembrane and MT concentrations on the membrane and in the aqueous medium. The data showed that cells more sensitive to MT are respectively: erythrocytes, Leishmania promastigotes and Leishmania amastigotes and macrophage. The IC50 value of MT for 4 x 107 parasites/mL was 24,35 M. For the same cell concentration, a significant alteration was detected in the membrane lipid fluidity of parasites to 15 M of MT. The EPR spectra of spinlabeled membrane-bound proteins were consistent with more expanded and solvent exposed protein conformations, suggesting a detergent-like action, with a possible formation of micelle-like structures around polypeptide chains. / A Miltefosina (MT) é um alquilfosfolipídio originalmente desenvolvido para o tratamento de câncer de mama e outros tumores sólidos. Atualmente é utilizada no tratamento da leishmaniose, uma doença infecto-parasitária causada pelo protozoário do gênero Leishmania, sendo o primeiro fármaco de uso oral aprovado para tal fim. No entanto, seu mecanismo de ação ainda é pouco conhecido. A espectroscopia de Ressonância Paramagnética Eletrônica (RPE) de um marcador de spin lipídico (5-DOXIL estearato) e outro específico de grupo tiol (4-maleimido-TEMPO) na membrana de amastigotas axênicas de L. (Leishmania) amazonensis e de macrófagos extraídos de peritônio de camundongos BALB/c, mostrou que a MT causa um aumento significativo na dinâmica destas membranas para concentrações similares àquelas que inibem o crescimento do parasito ou que são citotóxicas ao macrófago. Embora essas alterações possam ser detectadas usando um marcador de spin lipídico, nossos resultados experimentais indicaram que a MT interage predominantemente com a componente proteica da membrana. Utilizando um método para incorporação rápida da MT na membrana, esses efeitos foram medidos imediatamente após o tratamento. Ensaios de citotoxicidade, estimada via contagem microscópica de células vivas e mortas, indicaram que ~80% dos parasitos e dos macrófagos estão mortos na concentração de MT no qual a espectroscopia de RPE detectou uma mudança significativa na dinâmica da membrana. A viabilidade celular, quantificada pela redução do sal MTT ((3-4,5- Dimetiltiazol-2yl)-2,5 difenil brometo de tetrazolina), mostrou que a concentração de MT que inibe 50% da proliferação celular (IC50) depende da concentração celular usada no ensaio. Esta dependência foi analisada utilizando uma equação teórica envolvendo parâmetros biofísicos tais como o coeficiente de partição membrana-água e concentrações de MT na membrana e no meio aquoso. Os dados mostraram que as células mais sensíveis à MT são respectivamente: eritrócitos, formas promastigotas e amastigotas da Leishmania e macrófagos. O valor do IC50 da MT para 4 x 107 parasitos/mL foi de 24,35 M. Para esta mesma concentração de células, foi detectada uma alteração significativa na fluidez lipídica da membrana dos parasitos com 15 M de MT. Os espectros de RPE do marcador de spin ligados a proteínas da membrana foram consistentes com as conformações mais expandidas e dinâmicas das proteínas expostas no solvente, sugerindo uma ação semelhante à de detergente, com uma possível formação de estruturas tipo micelas em volta das cadeias polipeptídicas.

Interferons are known cytokines that display antiviral, anti-proliferative and immuno-modulatory functions in the host. Interferon-gamma (Ifnγ) is the only type II family interferon that binds to the heterodimeric receptor consisting of two subunits, IfnγR1 and IfnγR2. This specific interaction between Ifnγ and its receptor triggers the activation of Janus Kinase (Jak) – Signal Transducer and Activator of Transcription (Stat) pathway. This triggers a cascade of events leading to modulation of a wide variety of genes resulting in a plethora of responses including antimicrobial activities, induction of Major Histocompatibility Complex encoded molecules etc. The impact of Ifnγ in regulating host defense is observed in patients who lack functional IFNγ or its receptor as they succumb to less virulent strains of intracellular bacteria such as Mycobacterium and Salmonella. Also, mice lacking important downstream signaling components such as Stat1 and Interferon Regulated Factor 1 (Irf1) are known to be highly susceptible to a variety of bacterial and viral infections. Consequently, studies on uncharacterized signaling and regulatory molecules downstream to Ifnγ are of great interest. The modulatory functions of Ifnγ have been attributed to its ability to regulate the expression of a vast number of genes in a Stat1 and Irf1 dependent manner. Also, gene regulation in response to Ifnγ in a target cell such as mouse hepatoma cell line, H6, can be categorized broadly into two subsets: Reactive Oxygen Species (ROS) - Reactive Nitrogen Intermediates (RNI) dependent (e.g. Nos2, Catalase, Id2 etc.) as well as ROS – RNI independent (e.g. Tap1, Lmp2 etc.). However, the effect of Ifnγ induced ROS and RNI in the regulation of the expression of genes at the level of transcriptome and how these could impact cellular and host responses are not well characterized. To investigate these questions, we standardized an in vitro Ifnγ responsive primary cell culture system using mouse adherent peritoneal macrophages (PMs). It needs to be highlighted that this study has, primarily, utilized unstimulated resident PMs. The adherent cells from the peritoneal cavity were positive for macrophage markers such as F4/80 and CD11b, but negative for granulocyte marker Gr1. Also, PMs were resistant to Ifnγ induced cell death, unlike cell lines such as the mouse fibroblast cell line L929, for the time points studied. There are three distinct parts to this study involving the system of PMs: I. To understand the contribution of Nitric Oxide (NO) in regulating the expression of novel Ifnγ responsive genes, PMs from C57BL/6 mice and mice lacking nitric oxide synthase 2 (Nos2-/-), the enzyme isoform responsible for the generation of NO in PMs, were stimulated with Ifnγ for 8 h and microarray analysis was performed. Detailed analysis led to identification of several annotated genes that were uniquely regulated in C57BL/6 and Nos2-/- PMs. Further analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database identified several differentially regulated pathways. Interestingly, a large number of metabolism related pathways, Butirosin and neomycin, Galactose, Phenylalanine and glyoxylate and dicarboxylate, were specifically up-regulated in the C57BL/6 PMs treated with Ifnγ. Similarly, other metabolism related pathways were differentially regulated by Ifnγ in PMs from C57BL/6 and Nos2-/- mice. One of the pathways that was up regulated in a Nos2 dependent manner in the C57BL/6 PMs upon Ifnγ treatment was that of circadian rhythm, which consisted of genes Per1, Bhlhb2 and Bhlhb3. All three are known circadian rhythm regulators, with Bhlhb2 and Bhlhb3 being transcriptional repressors that bind to E-box consensus sequence (CANNTG) as heterodimers, using the basic helix-loop-helix (bHLH) domains, along with other transcriptional regulators. Both Bhlhb2 and Bhlhb3 were up regulated at RNA and protein levels in a kinetic manner upon Ifnγ treatment in L929 cells. Studies with inhibitors to ROS and RNI revealed that up regulation of Bhlhb2 and Bhlhb3 was RNI, but not ROS, dependent in response to Ifnγ. Interestingly, the RNI inhibitor, NG-Methyl-L-Arginine (LNMA) rescued Ifnγ induced ROS. On the other hand, ROS inhibitors, e.g. Apocyanin and polyethylene glycol Catalase (PEG-Catalase), did not affect the nitrite amounts in the supernatant. These experiments suggested that RNI was upstream to ROS in L929 cells and both contributed to Ifnγ induced cell death. The knockdown of Bhlhb3 using specific siRNAs in the untreated L929 cells increased Bhlhb2 amounts, but not vice versa. This observation is consistent with the fact that Bhlhb3 is a known repressor of Bhlhb2. However, this repression of Bhlhb2 by Bhlhb3 was not detected upon Ifnγ treatment in L929 cells possibly because of heterodimerization of Bhlhb3 with other Ifnγ induced transcriptional modulators. Finally, knockdown of either of the proteins did not affect induced nitrite but decreased ROS amounts resulting in significant rescue of Ifnγ induced cell death of L929 cells. Thus, Bhlhb2 and Bhlhb3 are novel Ifnγ induced proteins that are NO dependent and contribute to Ifnγ induced cell death. Ifnγ and Nos2 are known to elicit antibacterial defense in the host. Interestingly, recent studies have implicated circadian rhythm to regulate bacterial infection in mice. Therefore, regulation of both Bhlhb2 and Bhlhb3 upon Ifnγ treatment and during Salmonella enterica Serovar Typhimurium (S. typhimurium) infection in the bone marrow derived macrophages (BMDMs) was performed. Both Bhlhb2 and Bhlhb3 were induced in a Nos2 dependent manner upon Ifnγ addition in BMDMs. Similar to L929 cells, Bhlhb3 repressed Bhlhb2 in the untreated BMDMs. Also, infection of BMDMs with S. typhimurium increased the protein amounts of Bhlhb2, while repressing Bhlhb3. Importantly, knockdown of Bhlhb2 resulted in higher colony forming units (CFU), whereas knockdown of Bhlhb3 reduced CFU in BMDMs 18 h post infection with S. typhimurium. Thus, Bhlhb2 induced whereas Bhlhb3 repressed antibacterial defense in BMDMs. The exact mechanism downstream to these two proteins and their inter-relationship in regulating S. typhimurium infection is of considerable interest and will be studied in future. II. Macrophages are known to produce a large number of different cytokines and chemokines upon activation. To identify novel cytokines and chemokines that may be differentially regulated in response to Ifnγ, a protein array was performed using the supernatants of C57BL/6 PMs treated with and without Ifnγ. Chemokine Ccl3 was found to be repressed by Ifnγ in the supernatant of PMs. Further analysis using Enzyme Linked Immuno-Sorbent Assay (ELISA) revealed that both Ccl3 and Ccl4, highly homologous proteins that chemo-attract almost all types of leukocytes, were repressed upon Ifnγ treatment. This response was ligand and cell specific as Lip polysaccharide (LPS) stimulation of PMs and Ifnγ stimulation of thioglycollate elicited PMs did not result in repression of Ccl3 and Ccl4. Also, studies with fludarabine, an inhibitor to Stat1, revealed that the repression of Ccl3 and Ccl4 as well as induction of Cxcl10 in response to Ifnγ was Stat1 dependent. Importantly, the use of LNMA as well as PMs from Nos2-/- mice established the role of Nos2 in the repression of Ccl3 and Ccl4, but not Cxcl10 induction, in response to Ifnγ. Furthermore, activation of p38 Mapk, but not Jnk, was downstream to Nos2 activation and contributed functionally to the repression of Ccl3 and Ccl4 in response to Ifnγ. Finally, the transcriptional repressor, Activating Transcription Factor 3 (Atf3), was induced in a Stat1-Nos2-p38 Mapk dependent manner and knockdown of Atf3 using siRNAs established the functional role of the same in the repression of Ccl3 and Ccl4 in response to Ifnγ. Further, to understand the regulation of Ccl3 and Ccl4, their modulation upon S. typhimuirum infection of BMDMs was performed. Apart from regulating the CFU in BMDMs, Ifnγ and Nos2 functionally repressed Ccl3 and Ccl4 upon S. typhimurium infection. Oral infection of mice with S. typhimurium was performed and mice lacking Ifnγ and Nos2 were found to have greater CFU in their organs as well as more leukocytes in the infected liver sections in comparison to the infected C57BL/6 mice. Importanly, absence of Ifnγ as well as Nos2 increased the amounts of Ccl3 and Ccl4 in the sera upon S. typhimurium infection in comparison to the C57BL/6 infected mice. Overall, this part of the study identified Ifnγ and Nos2 to repress chemokines Ccl3 and Ccl4 in macrophages and in mice upon S. typhimurium infection. III. While working on the above mentioned studies, it was noticed that addition of Ifnγ to PMs induced in a dose and time dependent manner aggregation of cells. Experiments with LPS, TG PECs and BMDMs established that Ifnγ induced aggregation of PMs was ligand and cell type specific. A panel of cell surface integrins and selectins were screened for regulation upon Ifnγ addition, namely Icam1, Lfa1, CD11b, P-selectin and E-selectin. Most of the cell surface integrins were repressed by Ifnγ in a kinetic manner. Interestingly, CD11b as well as E-Selectin co-localized to the sites of interactions between the PMs upon Ifnγ treatment. Studies with Reopro, a purified F(ab’)2 to glycoprotein GPIIb that is also known to functionally block CD11b, revealed the functional contribution of CD11b during Ifn induced aggregation of PMs. Further, studies with specific inhibitors identified RNI, but not ROS, to contribute to Ifnγ induced PM aggregation. Also, lack of Nos2 in PMs upon Ifnγ treatment resulted in minimal aggregation together with morphological changes, e.g. flattening of cells. Since differences in the morphology of PMs was observed in the absence of Nos2 upon Ifnγ treatment, the regulation and roles of cytoskeleton proteins, Actin and tubulin, during Ifnγ induced aggregation of PMs was studied. Upon Ifnγ stimuli, actin and tubulin get stabilized. On the other hand, the absence of Nos2 leads to depolymerization of actin, while tubulin failed to stabilize to the membrane, in response to Ifnγ. Further, addition of actin and tubulin depolymerizing agents, Cytochalasin D and Colcemid respectively, decreased Ifnγ induced aggregation of PMs. Live cell imaging studies revealed that PMs needed actin, but not tubulin or CD11b, for mobility. Upon Ifnγ treatment, PMs from C57BL/6 mice exhibited reduced mobility and aggregated with each other. The Nos2-/- PMs exhibited lower mobility compared to C57BL/6 PMs and, upon Ifnγ treatment, underwent morphological changes with time, e.g. flattening. On the other hand, Nos2 is important for endogenous mobility and maintaining the cellular morphology in response to Ifnγ. To understand the physiological relevance of our observations, oral infection of C57BL/6 and Nos2-/- mice with S. typhimurium was performed. Four days post infection, no significant differences in the number of peritoneal cells were found. Importantly, PMs from infected Nos2-/- mice had higher CFU in comparison to C57BL/6 mice. However, the amounts of cytokines such as Ifnγ, Tnfα, Il6 and Il1β in the peritoneal lavage were not significantly different between the two infected strains. Interestingly, PMs isolated from infected Nos2-/- mice displayed distinct morphology, e.g. flattening. In comparison, infected C57BL/6 PMs aggregated when cultured for 24 h in vitro. In the future, it will be interesting to address the functional roles of aggregates of macrophages during physiologically relevant responses such as combating intracellular bacterial infection. This part of the study adds a new dimension to the ability of Ifnγ in the regulation of macrophage-macrophage interactions and their roles during intracellular bacterial infections. Overall, the present study has elucidated hitherto uncharacterized roles of Nos2 and mechanisms involved in regulation of novel functional responses of PMs to Ifnγ and during S. typhimurium infection.