<p>Upon activation mast cells release numerous proinflammatory mediators. With this feature, mast cells play an important role in host defense against pathogens, and are involved in tissue remodeling and wound healing. However, in cases of excessive inflammation the effects of mast cells are detrimental. This is observed in allergy, asthma, rheumatoid arthritis, atherosclerosis, certain types of heart failure, and in several other chronic destructive inflammations. Mast cell numbers are typically increased at inflammatory sites. There they act both directly, as effector cells, and in a regulatory manner, secreting agents that recruit and activate other immune cells.</p><p>The studies presented here investigated mechanisms regulating mast cell numbers at sites of inflammation, focusing on cell migration and regulation of survival/apoptosis. We report that SCF-induced mast cell migration requires p38 MAP kinase activity. Moreover, we found that SCF-mediated mast cell survival is regulated through downregulation of the proapoptotic Bcl-2 family member Bim, as well as through phoshorylation of Bim. SCF seems to control Bim protein levels via FOXO transcription factors, and to induce phosphorylation of Bim via the Mek/Erk and the PI3-kinase/Akt signaling pathways. Furthermore, mast cell death triggered by deprivation of SCF and/or IL-3 involves the Bim protein, as demonstrated using <i>bim</i>-/- mast cells. Additional studies revealed that IgE-receptor activation, which occurs in allergy, promotes both prosurvival and proapoptotic signaling events. This includes upregulation of Bim and the prosurvival Bcl-X_L and A1, as well as phosphorylation of Akt, FOXO factors, GSK-3β, IκB-α, Bad, and Bim. The simultaneous stimulation of prosurvival and proapoptotic signaling events could be a way to fine-tune the fate of mast cells after IgE-receptor activation and degranulation.</p><p>The new insights about mechanisms involved in mast cell migration and regulation of survival/apoptosis might prove useful for future efforts to design new drugs to be used for mast cell-associated diseases.</p>

Mast cells are long-lived effector cells of importance for both acute and chronic inflammations. Mast cells can be activated in many different ways, leading to the release of inflammatory mediators. In contrast to most other inflammatory cells, activated mast cells have the capacity to recover, regranulate and thereby be activated again. In this thesis I have investigated the mechanisms involved in regulating activation-induced mast cell survival. We have found that cross-linking of FcεRI-bound IgE with an antigen (IgER-CL) induces a survival program in mast cells. Upon IgER-CL, mouse and human mast cells upregulate the pro-survival Bcl-2 family gene A1/Bfl-1. A1-/- mast cells degranulate upon FcεRI activation but they cannot recover most likely due to the lack of A1. Sensitized and provoked A1-/- mice exhibit lower amounts of mast cells compared to littermate controls. In contrast to mast cells, no Bfl-1 expression or survival promotion can be detected in basophils after IgER-CL. Another mast cell secretagogue, an adenosine receptor agonist, neither promoted upregulation of A1 nor survival. Although it is well established that mast cell survival is dependent on stem cell factor (SCF), it has not been described how this process is regulated. We have found that SCF promotes survival through Akt-mediated inhibition of the forkhead transcription factor FOXO3a and its transcriptional target Bim, a BH3-only pro-apoptotic protein. SCF-treatment prevents upregulation of Bim protein expression and leads to an upregulation of Bim phosphorylation through PI3-kinase and MEK-dependent pathways. Overexpression of FOXO3a causes an upregulation of Bim and induces mast cell apoptosis, even in the presence of SCF. Taken together, the work in this thesis demonstrates that A1/Bfl-1 and Bim play key roles in mast cell survival. These findings might be of importance in understanding the mechanisms of mast cell longevity and hence for possible new therapeutics used for mast cell-associated inflammations.

Upon activation mast cells release numerous proinflammatory mediators. With this feature, mast cells play an important role in host defense against pathogens, and are involved in tissue remodeling and wound healing. However, in cases of excessive inflammation the effects of mast cells are detrimental. This is observed in allergy, asthma, rheumatoid arthritis, atherosclerosis, certain types of heart failure, and in several other chronic destructive inflammations. Mast cell numbers are typically increased at inflammatory sites. There they act both directly, as effector cells, and in a regulatory manner, secreting agents that recruit and activate other immune cells. The studies presented here investigated mechanisms regulating mast cell numbers at sites of inflammation, focusing on cell migration and regulation of survival/apoptosis. We report that SCF-induced mast cell migration requires p38 MAP kinase activity. Moreover, we found that SCF-mediated mast cell survival is regulated through downregulation of the proapoptotic Bcl-2 family member Bim, as well as through phoshorylation of Bim. SCF seems to control Bim protein levels via FOXO transcription factors, and to induce phosphorylation of Bim via the Mek/Erk and the PI3-kinase/Akt signaling pathways. Furthermore, mast cell death triggered by deprivation of SCF and/or IL-3 involves the Bim protein, as demonstrated using bim-/- mast cells. Additional studies revealed that IgE-receptor activation, which occurs in allergy, promotes both prosurvival and proapoptotic signaling events. This includes upregulation of Bim and the prosurvival Bcl-XL and A1, as well as phosphorylation of Akt, FOXO factors, GSK-3β, IκB-α, Bad, and Bim. The simultaneous stimulation of prosurvival and proapoptotic signaling events could be a way to fine-tune the fate of mast cells after IgE-receptor activation and degranulation. The new insights about mechanisms involved in mast cell migration and regulation of survival/apoptosis might prove useful for future efforts to design new drugs to be used for mast cell-associated diseases.

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2017-11-09T15:57:04Z No. of bitstreams: 1 Dissertação_ODONTO_ Ernesto Santos Sousa Neto.pdf: 4765427 bytes, checksum: ae3f93ce46ff32b7ce466869740090de (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-11-17T14:44:34Z (GMT) No. of bitstreams: 1 Dissertação_ODONTO_ Ernesto Santos Sousa Neto.pdf: 4765427 bytes, checksum: ae3f93ce46ff32b7ce466869740090de (MD5) / Made available in DSpace on 2017-11-17T14:44:34Z (GMT). No. of bitstreams: 1 Dissertação_ODONTO_ Ernesto Santos Sousa Neto.pdf: 4765427 bytes, checksum: ae3f93ce46ff32b7ce466869740090de (MD5) / Fapesb / O presente trabalho teve como objetivo estudar, por meio da imunohistoquímica, a participação de componentes celulares estromais a exemplo dos mastócitos (mast cell triptase), miofibroblastos (alfa-SMA) e macrófagos (CD163) e componentes vasculares (CD34 e D2-40) em uma série de tumores odontogênicos queratocísticos (TOQ), na tentativa de fornecer subsídios para compreender a interação entre esses componentes e o comportamento biológico distinto desta lesão. Para fins comparativos, cistos radiculares (CRs) e folículos pericoronários (FPs) também foram incluídos. A amostra foi composta por 30 TOQs, 15 CRs e 07 FPs. Para a avaliação dos mastócitos, miofibroblastos e macrófagos, foram quantificadas as células imunorreativas aos marcadores mast cell triptase, alfa-SMA e CD163, respectivamente, em 10 campos (400x). Os índices angiogênico e linfangiogênico foram avaliados por meio da densidade microvascular (MVD) e densidade microvascular linfática (MVDL) dos microvasos marcados, respectivamente, pelos marcadores CD34 e D2- 40. Foi utilizado o teste ANOVA com pós-teste de correção de Tukey, para análise estatística entre os marcadores segundo o tipo de lesão. Para a análise da correlação entre os marcadores dentro da mesma lesão, utilizamos a correlação de Pearson. Para a associação, no TOQ, entre os marcadores e a presença de inflamação, utilizamos o Teste T de Student. A análise da expressão do marcador mast cell triptase revelou existir diferenças significativas (p<0,05) entre a densidade de células mastocitárias presentes nos CRs (22,7) em relação aos FPs (1,23) e TOQs (7,39), e entre a densidade de mastócitos presentes no TOQs e FPs. Houve também diferença significativa (P<0,05) entre a densidade de miofibroblastos em TOQs (29,25) em relação aos CRs (4,66) e os FPs (1,50). A diferença da densidade de miofibroblastos entre os CRs e FPs não foi significativa (p>0,05). A respeito dos macrófagos, não houve diferenças significativas (p= 0,084) entre as densidades de macrófagos presentes nos TOQs (2,31), CRs (1,42) e FPs (0,14). Na avaliação do índice angiogênico, não houve diferenças significativas da MVD entre as três lesões estudadas. No entanto, para o índice linfangiogênico, houve diferença significativa (p<0,05) entre a MVDL presentes nos TOQs (11,64) em relação aos CRs (4,19) e aos FPs (0,167). A diferença entre a MVDL dos CRs em relação aos FPs não foi estatisticamente significante (p>0,05). Não foi encontrada associação significativa (p>0,05) entre os marcadores estudados com a presença de inflamação no TOQ. No presente trabalho, encontramos uma correlação positiva e moderada entre os marcadores mast cell triptase e o CD34 nos TOQ (p = 0,025). Já nos CRs encontramos uma correlação inversa e moderada entre os marcadores SMA x CD34 (p = 0,017). Nos FPs também encontramos uma correlação inversa entre os mesmos marcadores anteriores, porém forte (p=0,049). Embora os componentes celulares estromais e microvasculares aqui representados por mastócitos, miofibroblastos, macrófagos CD163 positivos e vasos CD34 e D240, respectivamente, sejam importantes para manutenção do arcabouço estrutural do TOQ e CR, existiu uma interação significante entre mast cell triptase e CD34 no TOQ e entre CD34 e o alfa –SMA no CR.

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Mast cell tumors (MCT) are neoplastic tumors of mast cells of unknown etiopathogenesis, representing up to 21% of all canine cutaneous cancers. This tumor varies widely in biological presentations and behaviors, ranging from benign to extremely aggressive, leading to metastasis and death. The cytological identification of MCT is a highly recommended method for initial detection of the tumor, it is inexpensive, can be performed with conscious animal and allows to suspect a tumor of high degree according to the cellular features. In addition to the histological grading, which is the gold standard for establishing the prognosis of MCT, additional markers are recommended, among them the AgNOR count, an indicator of cell proliferation. Additionally, some studies focus on the relationship between MCT and vitamin D, since mast cell tumors express vitamin D receptors (VDR). Thus, the aim of this research is to characterize the occurrence of canine MCT diagnosed by cytological examination in the region of Santa Maria/RS, to describe a protocol for determining AgNOR index in cytological samples of MCT and to investigate the relationship between vitamin D levels and AgNOR index in dogs with this tumor, since the AgNOR index is already consolidated as a prognostic marker for mastocytoma and vitamin D has been reported as a target for MCT management. Regarding to the occurrence profile of this tumor, we found results similar to those described in the literature, with some peculiarities referring to breeds and clinicopathological findings, allowing to suspected of MCT and planning more appropriate therapeutic interventions for each case. Referring to the AgNOR index, a protocol adapted from techniques already described was applied to cytological samples. Also has been observed that vitamin D and AgNOR index in dogs with MCT are negatively and moderatelycorrelated (P=0,011), suggesting that vitamin D levels can be measured in dogs with this tumor. In addition, follow-up studies of dogs with MCT and monitoring their outcoming are necessary, so that the vitamin D can be proposed as a prognostic marker and therapeutic agent for this neoplasm. / Mastocitomas são proliferações neoplásicas de mastócitos, de etiopatogênese desconhecida, que representam até 21% de todos os tumores cutâneos caninos. Este tumor possui uma grande variedade de apresentações e comportamentos biológicos, os quais vão desde benignos a extremamente agressivos levando à metástase e morte. A identificação citológica do mastocitoma um método bastante recomendado para detecção inicial do tumor, é de baixo custo, pode ser realizada com o animal consciente e permite suspeitar de um tumor de alto grau conforme as características celulares. Além da graduação histológica, que é o padrão-ouro para estabelecer o prognóstico do mastocitoma, marcadores adicionais são recomendados, como a contagem de AgNOR, um indicador de proliferação celular. Adicionalmente, alguns estudos enfocam a relação entre mastocitoma e vitamina D, uma vez que mastocitomas expressam receptores de vitamina D (VDR). Assim, a finalidade desta pesquisa é caracterizar a ocorrência de mastocitoma canino diagnosticado por exame citológico na região de Santa Maria/RS, descrever um protocolo para determinação de índice AgNOR em amostras citológicas de mastocitoma e investigar a relação entre níveis de vitamina D e índice AgNOR em cães com este tumor, visto que o índice AgNOR já é consolidado como marcador prognóstico para o mastocitoma e a vitamina D vem sendo apontada como alvo de intestigação neste tumor. Em relação ao perfil de ocorrência do mastocitoma canino, neste trabalho encontraram-se resultados semelhantes aos descritos na literatura, com algumas particularidades referentes a raças e achados clínico-patológicos, permitindo o direcionamento para suspeita de mastocitoma e planejamento de intervenções terapêuticas mais adequadas para cada caso. Referente ao índice AgNOR, foi especificado um protocolo adaptado, a partir de técnicas já descritas, aplicável a amostras citológicas. Também pode-se observar que a vitamina D e o índice AgNOR em cães com mastocitoma estão correlacionados de forma negativa e moderada (P=0,011), sugerindo-se que os níveis de vitamina D sejam avaliados nos cães com este tumor. Em adição, estudos de acompanhamento de cães com mastocitoma e monitoramento de seu tempo de sobrevida são necessários para que a dosagem de vitamina D possa ser proposta como marcador prognóstico e agente terapêutico para esta neoplasia

Submitted by HELENA RIBEIRO SOUZA null (helena.riber@hotmail.com) on 2016-08-11T15:45:49Z No. of bitstreams: 1 Dissertação Helena R Souza.pdf: 28021261 bytes, checksum: cfa8bae6ea9a632c8ff6462cc740385a (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-12T19:10:30Z (GMT) No. of bitstreams: 1 souza_hr_me_sjrp.pdf: 28021261 bytes, checksum: cfa8bae6ea9a632c8ff6462cc740385a (MD5) / Made available in DSpace on 2016-08-12T19:10:30Z (GMT). No. of bitstreams: 1 souza_hr_me_sjrp.pdf: 28021261 bytes, checksum: cfa8bae6ea9a632c8ff6462cc740385a (MD5) Previous issue date: 2016-07-29 / O processo de reparo de lesões térmicas pode ser dividido nas fases de inflamação, proliferação e remodelação. No seu processo de desgranulação, os mastócitos (MCs) liberam fatores quimiotáticos, citocinas e proteases, como triptase e quimase, para o meio extracelular, contribuindo na degradação da matriz lesada e síntese de uma nova. Além disso, estudos indicam que os MCs armazenam a proteína anti-inflamatória anexina A1 (AnxA1), envolvida na inflamação, apoptose, crescimento e diferenciação celular. Contudo, não se conhecem relatos da expressão e função da AnxA1 no reparo de queimaduras. Diante disso, os objetivos desse estudo foram quantificar e avaliar a ativação, acúmulo de histamina e heterogeneidade para triptase e quimase dos MCs, bem como, verificar a expressão da AnxA1, quantificar macrófagos, e dosar as citocinas TNF-α, IL-1β, IL-6, IL-10 e a quimiocina MCP-1 em modelo de queimadura de segundo grau em ratos Wistar. Para o desenvolvimento da lesão os animais foram anestesiados e submetidos à aplicação de um bloco metálico aquecido no dorso. Os animais foram divididos em grupos controles e tratados com pomada de sulfadiazina de prata a 1% (SDP 1%). As análises foram realizadas em 3, 7, 14 e 21 dias após injúria, e também em fragmentos de pele normal. Avaliações macroscópicas e histopatológicas da cicatrização confirmaram as características de queimadura de segundo grau e a melhor evolução da cicatrização nos animais tratados. A quantificação dos MCs mostrou grande quantidade de células intactas na pele normal e redução significante dessas células nas fases de inflamação (dia 3) e de proliferação (dia 7). Nas fases de proliferação de matriz (dia 14) e remodelação (dia 21) foi verificada maior quantidade de MCs nos animais controles e essas células foram observadas, principalmente, desgranuladas no dia 14 e intactas no dia 21. Ainda, no grupo tratado aos 7 dias e em ambos os grupos aos 14 dias, foi encontrada diferença entre MCs com grande quantidade de histamina e MCs totais. A análise da heterogeneidade dos MCs revelou maior número de MCs positvos para triptase (MCT) do que para quimase (MCQ) na pele normal e redução de MCTs e MCQs nas primeiras fases da cicatrização. Contudo, na fase de remodelação, muitos MCQs foram observados no grupo controle. A expressão da AnxA1 foi fraca na pele normal com aumento nas fases de inflamação e de proliferação. Aos 21 dias após lesão, a expressão da AnxA1 foi maior nos animais tratados com SDP 1%, no estroma e também em regiões epiteliais próximas à neogênese dos anexos cutâneos. As análises das citocinas mostraram aumento das pró-inflamatórias TNF-α, IL-1β e IL-6, e da citocina anti-inflamatória IL-10, nas fases iniciais do reparo, especialmente nos dias 3 e 7. O mesmo foi observado com relação a quimiocina MCP-1 e a quantificação de macrófagos. Nossos resultados evidenciaram diferenças no número de MCs e na imunomarcação da AnxA1 entre os grupos estudados, moduladas pelo tratamento com a SDP 1% e indicam que essas células e proteínas podem ser alvos terapêuticos no processo de regeneração de queimaduras. / The repair process of thermal injury can be divided into phases of inflammation, proliferation and remodeling. The mast cells (MCs) in their degranulation process, release chemotactic factors, cytokines and proteases, as tryptase and chymase, to the extracellular environment contributing to the degradation of damaged matrix and synthesis of a new one. Moreover, studies indicate that MCs store the antiinflammatory protein annexin A1 (AnxA1) which involved in inflammation, apoptosis, growth and cell differentiation. However, there are no known reports of the expression and function of AnxA1 in burns repair. Thus, the objectives of this study were to quantify, assess the activation, histamine accumulation and heterogeneity for tryptase and chymase of MCs, as well as, check the expression of AnxA1, associated with macrophages quantification and the levels of TNF-α, IL-1β, IL-6, IL-10 and MCP-1 in a second degree burn model in rats. For the development of the lesions, animals were anesthetized for applying a water-heated metal block in the dorso. The animals were divided into control and treated or not with the cream of silver sulfadiazine 1% (SDP 1%). The analyzes were performed on 3, 7, 14 and 21 days after injury, and also in normal skin fragments. Macroscopic and histopathological evaluations of healing confirmed the burning characteristics of second degree and the better progress of wound repair in treated animals. Quantification of MCs showed large amount of intact cells in normal skin and a significant reduction of these cells in the stages of inflammation (day 3) and proliferation (day 7). In the phases of matrix proliferation (day 14) and remodeling (day 21) it was observed increased amount of MCs, only in the control animals, and these cells were mainly degranulated on day 14, but intact on day 21. Also, in the treated group on day 7 and in both groups on day 14, it was found difference between MCs with large amounts of histamine and total MCs. The heterogeneity analysis revealed more MCs positves for tryptase (MCT) than for chymase (MCC) on normal skin and reduced MCTs and MCQs in the early stages of healing. However, in the remodeling phase, many MCCs were observed in the control group. The expression of AnxA1 was low in normal skin and increased in the stages of inflammation and proliferation. On 21 days after injury, the expression of AnxA1 was higher in animals treated with SDP 1% in the stroma and epithelial cells especially in regions close to neogenesis of skin attachments. Analysis of the inflammatory mediators showed an increase pro-inflammatory TNF-α, IL-1β and IL-6 and anti-inflammatory IL-10 cytokines in the early stages of the repair, especially on days 3 and 7. The same was observed for MCP-1 chemokine and quantification of macrophages. Our results showed differences in the number of MCs and AnxA1 expression between groups, that were modulated by treatment with the SDP 1%, indicating that these cells and protein can be used as therapeutic targets in burns regeneration process.

Os mastócitos participam do sistema imunológico liberando mediadores farmacologicamente ativos. A principal via de ativação dos mastócitos é através do receptor de alta afinidade para a imunoglobulina E (FcRI). A ativação dos mastócitos via FcRI culmina com a liberação de mediadores. A enzima PLD atua sobre fosfolipídios hidrolisando a fosfatidilcolina em ácido fosfatídico e colina. A PLD é ativada após o estímulo via FcRI e possui um papel importante na transdução do sinal em mastócitos. Existem duas isoformas da enzima PLD, a PLD1 e a PLD2 que são expressas, diferentemente, de acordo com o tipo celular. Ambas as isoformas podem estar expressas numa mesma célula, apenas uma ou nenhuma. Neste estudo foram utilizadas células RBL-2H3 transfectadas para a super expressão PLD2 nas formas catalítica ativa (CA) e inativa (CI). O papel da PLD2 foi examinado nestas células com o objetivo de elucidar sua atuação no processo de secreção incluindo o aparelho de Golgi e os grânulos secretores. As células CA e CI possuem maior atividavidade de -hexosaminidase total, porém quando estimuladas mostram uma deficiência na liberação desta enzima, quando comparadas com as células selvagens. A PLD2 nas células CA, CI, VET e RBL-2H3 está localizada no citosol, sendo abundante na região justanuclear, principalmente nas células CI, sugerindo uma associação com o aparelho de Golgi. A dupla marcação com o mAb AA4, que imunomarca gangliosídeos derivados do GD1b da membrana plasmática e com anti-PLD2, mostrou que esta enzima não se localiza na membrana plasmática. A dupla marcação com anti-PLD2 e anti-GM130 mostrou que as áreas de maior concentração da PLD2 se co-localizam com o aparelho de Golgi, especialmente nas células CI. A marcação com anti-GM130 e os experimentos com microscopia eletrônica de transmissão mostraram que o aparelho de Golgi está organizado nas células CA e desorganizado nas células CI, onde se encontra disperso no citoplasma. Ainda, as células CI expressam menos GM130 em comparação com as demais linhagens celulares. Quando a produção de PA pela PLD está inibida pelo 1-Butanol, as células CA apresentam as mesmas características fenotípicas das células CI. A incubação das CI com PA resulta na reestruturação do aparelho de Golgi. A manutenção estrutural do aparelho de Golgi, também está relacionada com os microtúbulos. Nas células CI o centro organizador de microtúbulos é dificilmente identificado. Os microtúbulos nas células CI são desordenados em comparação com as demais linhagens celulares. Estes resultados mostram que a produção de PA pela PLD2 é importante na organização de microtúbulos e na manutenção da estrutura do aparelho de Golgi. As alterações celulares relacionadas com os microtúbulos e o aparelho de Golgi afetam o processo secretor nestas células e, provavelmente, em outros tipos de células secretoras. Estes achados poderão levar a novas estratégias terapêuticas para controlar a liberação de mediadores durante processos alérgicos e inflamatórios. / Mast cells are components of the immune system that liberate a wide variety of pharmacologically active mediators. The principle method of activating mast cells is through the high affinity receptor for IgE (FcRI). This activation then culminates with the release of mediators. Phospholipase D (PLD) acts on phospholipids, hydrolyzing phosphatidylcholine to phosphatidic acid (PA) and choline. PLD is activated following stimulation via FcRI and plays an important role in signal transduction in mast cells. PLD has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type where none, one or both may be expressed. RBL-2H3 cells, a mast cell line, transfected to super express catalytically active (CA) and inactive (CI) forms of PLD2 were used in the present study. The role of PLD2 was examined in these cells in order to clarify the action of PLD2 in the secretory process. Although the CA and CI cells posses a greater total -hexosaminidase activity, when stimulated these cells release less -hexosaminidase than cells transfected with empty vector or wild type RBL-2H3 cells. In all cell lines, PLD2 was dispersed throughout the cytoplasm with a concentration in the juxtanuclear region suggesting an association of PLD2 with the Golgi apparatus. Double labeling with anti-PLD2 and mAb AA4, which recognizes gangliosides derived from GD1b on the plasma membrane, showed that PLD2 was not associated with the plasma membrane. When the cells were double labeled with anti-PLD2 and anti-GM130, which labels the cis-Golgi saccules, PLD2 does colocalize with the Golgi apparatus, especially in CI cells. Labeling with anti-GM130 alone as well as experiments employing transmission electron microscopy revealed that the Golgi apparatus is well organized in the CA cells, but is disorganized and dispersed in the cytoplasm in the CI cells. By Western Blotting, the CI cells also expressed less GM130 than the other cell lines. When the production of PA by PLD2 was inhibited by 1-Butanol, the Golgi apparatus of the CA cells presented the same phenotypic characteristics as that of the CI cells. Conversely, incubation of the CI cells with PA resulted in the reorganization of the Golgi apparatus. The structural maintenance of the Golgi apparatus is also related to microtubules. In the CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized in the cytoplasm as compared to the other cell lines. These results show that the production of PA by PLD2 is important in the arrangement of the microtubules and in maintaining the structure of the Golgi apparatus. Alterations in the distribution of the microtubules and the structure of the Golgi apparatus in the CI cells affect the secretory process in these cells, and such alterations may affect the secretory process in other cell types as well. The findings presented here may lead to new therapeutic strategies to control the production and release of mediators during allergic and inflammatory processes.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2016-12-19T17:52:15Z No. of bitstreams: 2 Tese - Maurício Barcelos Costa - 2014.pdf: 740997 bytes, checksum: fec1f12612ff6a41fbbbee9640600a35 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Rejected by Luciana Ferreira (lucgeral@gmail.com), reason: Júlio, falta o arquivo da tese. on 2016-12-26T12:31:54Z (GMT) / Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-01-02T15:41:57Z No. of bitstreams: 2 Tese - Maurício Barcelos Costa - 2014.pdf: 10731375 bytes, checksum: d59433128feb9bc7facacb81cb311a98 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-03T09:40:55Z (GMT) No. of bitstreams: 2 Tese - Maurício Barcelos Costa - 2014.pdf: 10731375 bytes, checksum: d59433128feb9bc7facacb81cb311a98 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-03T09:40:55Z (GMT). No. of bitstreams: 2 Tese - Maurício Barcelos Costa - 2014.pdf: 10731375 bytes, checksum: d59433128feb9bc7facacb81cb311a98 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-02-26 / Leprosy is a complex, chronic, infectious dermato-neurological disease that affects the skin and peripheral nerves especially during acute immune-inflammatory episodes known as type 1/T1R and type 2/T2R reactions. There is no experimental model for leprosy and leprosy skin lesions have been extensively used to unravel its multifaceted immunopathological mechanisms.This study investigated in situ expression of two distinct cell populations with important immunoregulatory roles: T regulatory (Treg) cells and mast cells (MC) in diverse skin diseases with emphasis on leprosy T1R and T2R. For the Treg cell study, 154 skin biopsies from 114 participants belonging to 3 groups were investigated: 1. Leprosy (n=74), 56 T1R (28-paired biopsies reactionfree/reactional from the same patient, 28 single reactional biopsy), 18 T2R (12 pairedreaction-free/reactional lesions, 6 single reactional biopsy); 2. Dermatoses: (n=29) noninfectious and cutaneous infectious diseases; 3. Normal controls: skin fragment of mammoplasty from healthy females that had cosmetic surgery. Double immunohistochemical detection of Treg cells was performed with automated platform for CD25 and Foxp3 staining. Quantifications of double immunostained Treg cells was performed (values expressed by mm2 ) blinded to the participants’ clinical status. For the mast cell study 80 skin biopsies from 3 groups were investigated: 40 newly diagnosed untreated leprosy patients (18 reaction-free, 11 T1R, 11 T2R), 29 patients with other dermatoses (the same as for Treg study) and 11 normal skins. Toluidine blue stained intact and degranulated MC counts/mm2 ; streptavidin-biotin-peroxidase immunostaining was used to detect tryptase/try+ and chymase/chy+ MCs and their density (median optical density) was evaluated. Results: Treg study: Not one CD25+ Foxp3+ Treg cell was seen in any of the 11 normal skin sections while variable numbers were detected in skin diseases (p<0.0001); the number of double stained cells was higher in infectious compared to non-infectious diseases (p=0.008). Treg cell numbers were comparable between leprosy and other infectious dermatoses (p=0.157) Treg cell counts in reactional lesions were higher than in reaction-free leprosy lesions (p<0.002). Paired biopsies of T1R or T2R reactional/reaction-free lesions showed xxvii increased numbers of Treg during T1R compared to reaction-free lesions from the same patient (p< 0.001). Treg cell median in T1R developed during MDT was slightly higher compared to T1R developed at diagnosis in naïve patients (p=0.047). There was a trend in increasing Treg cell numbers from the tuberculoid to borderline-lepromatous form, which showed the highest median value of Tregs, however this difference was not significant (p>0.8). Mast cell study: Infectious and non-infectious skin lesions showed higher numbers of degranulated than intact MC both for leprosy and other dermatoses, compared to normal skin. The numbers of degranulated MC were higher than intact MC regardless of the leprosy form (from tuberculoid/TT to lepromatous/LL), regardless of the occurrence of leprosy reactions (reactional and reaction-free) and regardless of the type of reaction (T1R/T2R). Try+ MC numbers and density were higher than chy+ MC in leprosy, in reaction-free and reactional lesions, particularly in T2R, but not in other dermatoses. Conclusions: Higher Treg numbers seen in T1R suggest Treg role in suppressing the exacerbated cell-mediated phenomenon that causes T1R. Differential expression/ of try+ and chy+ MC subsets was seen in leprosy compared to other skin diseases and to normal skin. However, neither leprosy form nor leprosy reaction was associated with MC changes in lesions suggesting that the Mycobacterium leprae infectious process per se dictates MC expression in leprosy skin lesions. / A hanseníase é uma complexa doença dermato-neurológica, crônica, de causa infecciosa que afeta a pele e os nervos periféricos, especialmente durante os episódios imunoinflamatórios agudos conhecidos com reações tipo 1/RT1 e tipo 2/RT2. Não existe modelo experimental para hanseníase e as lesões de pele têm sido extensivamente usadas para desvendar os multifacetados mecanismos imunopatológicos associados com a doença. Este estudo investigou a expressão in situ de duas distintas populações celulares que apresentam importante papel imunorregulatório: células Treg (Treg) e mastócitos (MC) em pele normal e diversas doenças cutâneas com ênfase nas reações hansênicas RT1 e RT2. Para o estudo de Tregs foram utilizadas 154 biópsias cutâneas de 114 participantes de três grupos: 1. Hanseníase (n=74), 56 RT1 (28-biópsias pareadas do mesmo paciente sem reação/durante reação, 28 biópsias únicas de RT1), 18 RT2 (12 biópsias pareadas sem reação/durante reação, 6 biópsias únicas de RT2); 2. Dermatoses: (n=29) doenças cutâneas não infecciosas e infecciosas. Controles Normais: fragmentos de peles obtidos de mamoplastias eletivas em mulheres saudáveis. Imunomarcação dupla CD25+ Foxp3+ de células Treg foi realizada em plataforma automatizada. Quantificação das células Treg duplo positivas foram feitas sem conhecimento da condição clínica do paciente (valores expressos em mm2 ). Para o estudo dos MC 80 biópsias de 3 grupos foram investigadas: 40 pacientes com hanseníase recém diagnosticados não tratados (18 sem reação, 11 RT1, 11 RT2), 29 pacientes com outras dermatoses e 11 biópsias de pele normal. Quantificação de MC intactos e desgranulados corados por azul de toluidina/mm2 ; imunomarcação streptavidina-biotina-peroxidase foi empregada para detectar MC triptase/try+ e chimase/chy+ e a densidade ótica (mediana da densidade ótica) foi avaliada. Resultados: Estudo das Tregs: Nenhuma célula Treg CD25+ Foxp3+ foi identificada em nenhuma das 11 amostras de pele normal, enquanto um número variável de Tregs foi identificado nas diversas doenças cutâneas (p<0.0001); o número de células Treg duplo positivas foi maior nas doenças infecciosas comparado com as não-infecciosas (p=0.008). Medianas de Treg entre hanseníase e outras doenças infecciosas foram semelhantes (p=0.157). Quantificação de Tregs em lesões reacionais foram maiores do que as sem reação (p<0.02). Nas biópsias pareadas de lesões de xxv RT1/sem reação ou RT2/sem reação, números maiores de Treg foram vistos durante a RT1 quando comparados com a não reacional do mesmo paciente (p< 0.001). Mediana de Treg em RT1 desenvolvidas durante MDT foi ligeiramente superior comparada a RT1 desenvolvida em pacientes sem de tratamento (p=0.047). Observou-se uma tendência de aumento no número das Tregs do polo tuberculoide em direção ao lepromatoso, mais especificamente até a forma borderline-lepromatosa que apresentou a maior mediana da quantificação de Treg, entretanto esta diferença não foi estatisticamente significativa (p>0.8). Estudo dos Mastócitos: lesões cutâneas de origem infecciosa e não infecciosa mostraram números aumentados de mastócitos desgranulados do que intactos tanto na hanseníase como nas outras dermatoses quando comparados com pele normal. Os números de mastócitos (MC) desgranulados foram maiores do que intactos, independente da forma de hanseníase (do polo tuberculoide/TT ao lepromatoso/LL), independente da ocorrência de reações hansênicas (lesão reacional/sem reação) e independente do tipo de reação (RT1/RT2). Número e densidade de mastócito triptase positivo (MC try+ ) estão aumentados em relação aos quimase positivo (MC chy+ ) na hanseníase, em pacientes com e sem reação, particularmente na RT2, mas não nas outras dermatoses. Conclusões: aumento nas Treg detectados durante RT1 sugerem papel supressor dessas células em eventos associados à resposta imune celular exacerbada, responsáveis pela RT1. Expressão diferencial das subpopulações de MC try+ e chy+ foi observada na hanseníase em relação a outras doenças cutâneas e pele normal. Entretanto, nem a forma da hanseníase, nem a ocorrência de reação hansênica, estava associada a mudanças nas subpopulações de MC nas lesões sugerindo que o processo infeccioso pelo Mycobacterium leprae per se direciona a expressão de MC nas lesões cutâneas da hanseníase.

Les mastocytes sont des cellules immunitaires présentes dans tous les tissus de l'organisme. Ils ont été depuis longtemps associés aux réponses allergiques, mais ces cellules sont aussi des acteurs majeurs de la réponse inflammatoire. La dégranulation des mastocytes, ou exocytose des granules sécrétoires, est un mécanisme d'action important de ces cellules. Au laboratoire, nous avons mis au point une méthode innovante qui nous permet de suivre en temps réel la dynamique de cette dégranulation. Cette méthode repose sur l'utilisation de l'avidine couplé à un fluorochrome qui se lie spécifiquement à la matrice des granules. Nous avons recherché les modalités de la dégranulation lorsque les mastocytes sont activés par un ligand cellulaire comme des cellules cibles recouvertes d'anticorps. Nous avons pu mettre en évidence, de façon surprenante, que les mastocytes dégranulent de manière polarisée contre une cellule cible opsonisée avec des anticorps de type IgE ou IgG en mettant en place un nouveau mécanisme que nous avons nommé ADDS (Antibody Dependent Degranulatory Synapse). L'ADDS est caractérisée par une signalisation du récepteur RFc et une dépolymérisation du cytosquelette cortical d'actine locales. De plus, cette synapse peut aussi avoir lieu lorsque le mastocyte est au contact d'un parasite opsonisé comme Toxoplasma Gondii, et induit la mort du parasite et la libération de cytokines et chimiokines pro-inflammatoires. Nous avons ensuite analysé la dynamique de la dégranulation à l'échelle " single cell " et sa régulation par des facteurs inflammatoires. Grâce à une approche de " cell barcoding " et de cytométrie en flux en temps réel, nous avons pu mettre en évidence que la dégranulation induite par l'agrégation des RFc?I était contrôlée par deux mécanismes : un premier qui règle le nombre de mastocytes qui dégranulent et un second qui régule l'intensité de la dégranulation. L'interleukine 33 peut finement potentialiser ces deux mécanismes en augmentant le pourcentage de mastocytes qui dégranulent et l'intensité de la dégranulation. L'IL-33 induit ainsi l'émergence de cellules hautement inflammatoires. Dans un second axe de recherche, nous avons étudié quel pouvait être l'impact des prostaglandines dans le dialogue entre mastocytes et lymphocytes T CD4+ Helper (TH). Nos résultats indiquent un rôle insoupçonné de la prostaglandine D2 et la prostaglandine E2 comme des acteurs importants pour la production d'interleukine 17 par les LTH. En conclusion, mon travail de thèse nous a permis de révéler l'existence de la synapse dégranulatoire des mastocytes, de nouveaux mécanismes contrôlant la dégranulation et d'identifier les mastocytes comme une source importante de prostaglandines impliquées dans la polarisation des LTH. / Mast cells are tissue-resident immune cells particularly enriched in regions exposed to the external environment. They have been associated, for a long time, with allergic disorders but these cells are also major effectors of inflammatory response. The degranulation process, or granule exocytosis, is one of the main effector functions of mast cells and it has been implicated in various biological processes. In our laboratory, we have developed a new method to monitor live mast cell degranulatory response. This approach is based on fluorescent avidin that binds selectively mast cell granule matrix. Thanks to this method, we have investigated the degranulatory response following mast cell activation by cell bound antigens. We have shown that mast cells can undergo polarized degranulation toward IgE- or IgG-opsonized cells in a new mechanism we named ADDS (Antibody Dependent Degranulatory Synapse). The ADDS is characterized by a local signaling of FcR receptors and cortical actin depolymerisation. Moreover, this synapse takes place when mast cells are triggered by opsonized parasite Toxoplasma Gondii. It leads to the death of the parasite and the release of pro-inflammatory cytokines and chemokines. We next analyzed the mast cell degranulatory response dynamics at the single cell level and its regulation by alarmin IL-33. Using cutting-edge "cell barcoding" approach and live flow cytometry, we showed that Fc?RI mediated degranulatory response is controlled by two mechanisms: a first one that sets the frequency of degranulated mast cells and a second one that regulates the magnitude of the degranulation. The IL-33 fine tunes these two mechanisms by augmenting both the frequency of degranulated mast cells and the extent of individual mast cell degranulation. Our results indicate that interleukin 33 induces the emergence of high inflammatory mast cells. In a second axis of research, we analysed the influence of prostaglandins during the cooperation between mast cells and CD4+ T helper cells. Our results indicate that prostaglandin D2 and E2 produced by mast cells are important inducers of interleukin 17 by CD4+ T helper cells. Taken together, my thesis work revealed that mast cells can form degranulatory synapses, new mechanisms that control the degranulatory response and identified mast cells as a source of pro-IL-17 prostaglandins during their cooperation with CD4+ T helper cells.

Signal Transducers and Activators of Transcription (STATs) are latent transcription factors that mediate several cellular responses. This protein family consists of seven members, STAT1 – 6 including two closely related molecules, STAT5a and STAT5b, that show 96% amino acid sequence homology and are critical for lymphoid, myeloid and erythroid cell development and function. Activated STAT proteins dimerize and translocate to the nucleus, where they bind to high-affinity DNA motifs to modulate gene expression. We recently identified STAT5b as the critical regulator of IgE-mediated cytokine production in mast cells. STAT5b knockout (KO) cells show decreased sensitivity to IgE-mediated passive systemic anaphylaxis accompanied with decreased production of IL-6 and IL-13 compared to wild type counterparts. Interestingly, STAT5b KO mice demonstrated elevated levels of serum IgE but a normal response to histamine-mediated passive systemic anaphylaxis. The current work demonstrates that STAT5b regulates mast cell function both in vivo and in vitro. Additionally, activated STAT proteins can also form tetramers through an N-terminal domain-mediated oligomerization process when bound to low-affinity tandem motifs. Dr. Warren Leonard’s laboratory generated STAT5a-STAT5b double knock-in (DKI) mice in which STAT5 proteins are phosphorylated and can form dimers but not tetramers. We have now found that bone marrow-derived mast cells from STAT5 DKI mice are defective in IgE-induced cytokine and chemokine production and exhibit defective stem cell factor (SCF)-induced migration and survival responses in vitro. Similarly, IgE-mediated passive systemic anaphylaxis is decreased in STAT5 DKI mice. These data indicate that Stat5 tetramers are critical for some aspects of mast cell function in allergic and inflammatory disease.