Orientador: Liana Maria Cardoso Verinaud / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T22:32:56Z (GMT). No. of bitstreams: 1 Brito_VaniaNieto_D.pdf: 17655224 bytes, checksum: 75de87a92111468f9002287b57e1c7fe (MD5) Previous issue date: 2005 / Resumo: O timo e o local de maturacao dos linfocitos T que coordenam a resposta imune apos o reconhecimento de antigenos; apesar disso, torna-se alvo em diversas infeccoes. O P. brasiliensis, causador da paracoccidioidomicose, e capaz de invadir o timo e induzir atrofia deste orgao em animais experimentais. Neste estudo, foi analisada a influencia da virulencia fungica (cepas Pb18 e Pb265), da linhagem murina (A/J e B10.A) e de citocinas pro-inflamatorias (TNF e IFN-g) nesse fenomeno. As leveduras da cepa virulenta Pb18 persistiram no timo por um periodo maior e induziram alteracoes histopatologicas mais intensas, embora o isolado Pb265, de baixa virulencia, tambem tenha causado atrofia. O padrao de atrofia, caracterizado por perda de peso do orgao, aumento na taxa de apoptose e perda de delimitacao corticomedular foi semelhante nas duas linhagens, contudo os camundongos B10.A apresentaram tambem alteracoes quantitativas nas subpopulacoes de timocitos e linfocitos T esplenicos. A infeccao fungica também provocou a depressao da resposta proliferativa frente a ConA de timocitos e esplenocitos de camundongos B10.A por um periodo mais prolongado do que de animais da linhagem A/J. Em ambas as linhagens, a infeccao provocou aumento na porcentagem de linfocitos T esplenicos de fenotipo CD4+CD8+. Houve reducao no numero de celulas timicas positivas para TNF e IFN-g apos infeccao com o P. brasiliensis. Utilizando-se camundongos deficientes para o receptor p55 de TNF ou para a producao de IFN-g, verificou-se que o TNF medeia o aumento na apoptose de timocitos pos-infeccao enquanto a perda de peso e da delimitação corticomedular sao desencadeadas pelo IFN-g. Nossos resultados indicam que a atrofia timica observada na paracoccidioidomicose experimental envolve a interacao de fatores relacionados ao fungo e ao hospedeiro, que podem influenciar o curso da doenca / Abstract: Thymus is the site of T cells maturation that orchestrates specific immune response following antigen recognition. In spite of this, it can be a target during many infectious diseases. Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, is able to invade the thymus inducing atrophy in experimental models. In this study, it was analyzed the influence of fungal virulence (Pb18 and Pb265 isolates), murine strain (A/J e B10.A) and proinflammatory cytokines (TNF e IFN-g) in this phenomenon. Yeasts from virulent strain Pb18 persisted for a larger period in the thymus and induced stronger histopathological alterations, although poorly virulent Pb265 also has induced thymic atrophy. The pattern of atrophy that embodied organ weight decreases, increases in the apoptotic levels and the loss of corticomedullary delimitation was similar in both strains. However, B10. A strain also presented quantitative changes in the thymic and splenic T cell subpopulations. This strain showed a decreased proliferative response of thymocytes and splenocytes to Concanavalin A for a larger period than A/J strain, as well. Both strains had augmentation in the percentage of splenic CD4+CD8+ T cells. There was reduction in the amount of cells positive to TNF and IFN-g in the thymus after infection with P. brasiliensis. By using mice deficient to production of IFN-g or to p55 receptor of TNF it was seen that the TNF mediates the increase of apoptosis that takes place in the thymus after the infection, whereas organ weight decreases and loss of corticomedullary delimitation are performed by IFN-g. Our results suggest that thymic atrophy observed during experimental paracoccidioidomycosis involves interaction of fungal and host related factors and, most probably, influences the course of disease / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular

A doença de Crohn é uma inflamação crônica do trato gastrointestinal. O tratamento convencional é muitas vezes desapontador. Apesar da variedade de drogas disponíveis para o tratamento da doença inflamatória intestinal, tais como: salicilatos e seus derivados, corticosteróides, antibióticos e imunossupressores, nenhuma destas mostrou ser totalmente eficaz ou definitiva para o tratamento da doença e seus surtos de exacerbação. Pesquisas têm sido desenvolvidas com o objetivo de apresentar drogas mais efetivas. Dentre estas, destacam-se as drogas biológicas. O infliximabe é um anticorpo monoclonal quimérico anti-fator de necrose tumoral alfa e está indicado na doença de Crohn refratária e fistulizante. O objetivo deste estudo visa avaliar prospectivamente os resultados e efeitos colaterais precoces e tardios do uso do anti-TNF alfa no tratamento de 60 doentes com doença de Crohn, no período de julho de 1999 a dezembro de 2005. Os doentes foram tratados com anti-TNF alfa (infliximabe), na dose de 5mg/kg de peso, aplicado por via endovenosa em intervalos de dois meses. A avaliação foi realizada por protocolo clínico que classificava os quesitos: estado geral, sintomas intestinais e doença perianal em melhor, inalterado e pior, e pelo índice de atividade da doença de Cronh. Os doentes tratados com anti-TNF alfa apresentaram mediana de duração da doença de sete anos, variando de um a 28 anos entre a data do início dos sintomas e a data de início da pesquisa. 34 doentes (56,7%) já haviam sido submetidos a uma ou mais operações abdominais e 38 (63,3%) a operações orificiais. O software utilizado para a realização dos cálculos foi o SPSS® 9.0 for Windows, sendo estatisticamente significantes os testes com p<0,05. Foram aplicadas 225 doses de anti-TNF alfa, em média, 3,7 doses por paciente num período de aproximadamente cinco anos, variando de uma a 14 doses. No tratamento inicial 76% dos pacientes responderam a droga. As principais indicações para o emprego do anti-TNF alfa foram a presença de doença perianal em 36 casos (60%) e a intratabilidade clínica em 24 casos (40%). Observou-se que após a primeira dose da medicação, os doentes com mais de dez anos de doença e submetidos à operação abdominal tiveram resultado satisfatório semelhantemente aqueles doentes com menos de cinco anos de doença e não operados com p<0,05. O índice de atividade da doença de Crohn foi em média de 189,7 antes do início do tratamento e na primeira aplicação diminuiu em média para 135,4, e progressivamente ao longo das aplicações (115, 102, 109 e 88,4 até a quinta dose), sendo o resultado estatisticamente significativo. Houve efeito colateral em 40 aplicações (17,8%), sendo os efeitos principais: eritema cutâneo, dispnéia e dor abdominal. O tratamento com anti-fator de necrose tumoral alfa, obedecidas as indicações precisas, associou-se a baixo índice de efeitos colaterais graves tendo apresentado bons resultados na resolução da doença de Crohn perianal, na melhora da sintomatologia intestinal e no estado geral dos pacientes / Crohn´s disease is a chronic inflammatory disorder of the gastrointestinal tract. Conventional treatment is many times disappointing. Besides the great number of available medications to treat inflammatory bowel diseases, such as salicilates and derivatives, corticosteroids, antibiotics and immunosuppressive agents, none of them proved to be totally efficient or the ultimate treatment for inflammatory diseases and their exacerbation. Researches have been carried out to find more effective therapeutic drugs. Among these therapeutics, biologic treatments have been in evidence. Infliximab is a chimeric IgG1 monoclonal antibody against tumor necrosis factor-alpha, and is indicated for refractory luminal and fistulizing Crohns disease. The aim of this study is to prospectively evaluate the outcome, early and late adverse events, in 60 patients diagnosed with Crohn´s disease and treated with infliximab between July 1999 and December 2005. All patients were treated with anti-TNF-alpha (infliximab), 5mg/kg/dose, intravenously, each two months. Patients were clinically evaluated using a protocol that classified the evolution of the health status, intestinal symptoms and perianal disease, as better, worse or unchanged, during the treatment. Crohn´s disease activity index was also evaluated. Patients treated with anti-TNF-alpha presented a median disease duration of seven (range 1-28) years, between the beginning of the disease symptoms and the beginning of the research protocol. Thirty-four patients (56.7%) have been submitted to one or more abdominal surgeries before, and 38 (63.3%) to anal-rectum surgeries. All statistics tests were performed with computer software Statistical Package for the Social Sciences (SPSS® 9.0) for WindowsTM, and p values of less than 0.05 were considered statistically significant. Totals of 225 anti-TNF-alpha doses were administered. The mean doses administered per patient, in a period of approximately five years, were 3.7 (range 1-14) doses. After the initial treatment, 76% of the patients achieved a response. The most frequent indications for anti-TNF-alpha was perianal disease, occurring in 36 patients (60%), and clinical failure to the conventional treatment, happening in 24 patients (40%). After the first dose of anti-TNF-alpha, patients with more than 10 years of treatment and previously submitted to abdominal surgery presented a satisfactory outcome, similar to those with less than 5 years of disease and not submitted to surgery, p<0.05. Crohn´s disease activity index showed a mean index of 189.7 before treatment, that decreased to 135.4 after the first dose, and progressively decreased with the subsequent doses (means: 115, 102, 109 and 88.4, until the fifth dose, p<0.05). Adverse events were reported in 40 administrations (17,8%) from the total. The most prevalent adverse events were: rash, dyspnoea and abdominal pain. The treatment with anti-TNF-alpha, following precise indications, was associated with a low incidence of severe adverse events and presented good outcomes in the resolution of perianal Crohn´s disease, improving intestinal symptomatology and patients´ health status

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-04-04T15:36:20Z No. of bitstreams: 2 Dissertação - Hélio Galdino Júnior - 2008.pdf: 1866044 bytes, checksum: 37a41759b9e3790adfe67161890f0dd4 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-04-04T15:38:24Z (GMT) No. of bitstreams: 2 Dissertação - Hélio Galdino Júnior - 2008.pdf: 1866044 bytes, checksum: 37a41759b9e3790adfe67161890f0dd4 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-04-04T15:38:24Z (GMT). No. of bitstreams: 2 Dissertação - Hélio Galdino Júnior - 2008.pdf: 1866044 bytes, checksum: 37a41759b9e3790adfe67161890f0dd4 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2008-02-29 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Microorganisms express conserved molecules which ones activate the innate immune system. These molecules are known as pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) and bacterial lipoproteins. The PAMPs can be recognized by Toll-like receptors (TLR). The innate immunity activation through TLR pathway induces pro-inflammatory cytokines and lipid mediators, as tumor necrosis factor (TNF) and platelet-activating factor (PAF), respectively. Several reports showed the interaction between TLR4 and PAF receptor (PAFR) signaling to the TNF production; however, the interaction between PAF and other TLR was poorly investigated. The aim of this study was to evaluate the PAF regulatory activity on TLR2-induced TNF production. Thus, Mycoplasma fermentans PG 18 lipoproteins (LAMPf), TLR2/TLR6 agonists and Pam3Cys, a synthetic lipopeptide agonist of TLR2/TLR1 were added to human whole blood cultures and TNF was evaluated by enzyme-linked immunosorbent assay. To evaluate the effects of endogenous PAF on TNF production, a PAF receptor antagonist, WEB2170 was used, and to evaluate the effect of exogenous PAF, PAF was added to the cultures. The blood cultures were also activated with Gram-positive or negative heat-killed bacteria (Staphylococcus aureus or Escherichia coli). The TLR2 expression on polymorphonuclear (PMN) and monocytes were evaluated by flow cytometry, analyzing total cellularity for PMN and CD14+ cells for monocytes. LAMPf, Pam3Cys or LPS induced TNF and the treatment with WEB2170 increased TNF production after TLR2 activation, but not after TLR4 activation. Priming of the blood cultures with PAF up regulated TLR2- induced TNF production. Addition of PAF did not alter TNF release induced by LPS. E. coli induced higher levels of TNF than S. aureus and the treatment with WEB2170 lead to a significant reduction of S. aureus-induced TNF release. However, addition of PAF did not significantly alter bacteria-induced TNF production. With E. coli neither treatment with WEB2170 nor with PAF modulated TNF release. Results indicate that PAF can increase or decrease TNF production induced by TLR2 depending on the time when PAF is combined with TLR2. The increase of the TNF production after extended priming with PAF it was not caused by an increase in TLR2 expression. Thus, it is suggested that interaction between PAFR and TLR2 signaling determines the levels of TNF release. TLR2/PAF/TNF signaling pathway can be relevant in innate immune responses against Gram positive bacteria as well as in inflammatory diseases. / Os microrganismos expressam moléculas conservadas que ativam as células do sistema imune inato. Estas são conhecidas como padrões moleculares associados aos patógenos (PAMPs), tais como o lipopolissacarídeo (LPS) e as lipoproteínas bacterianas. Estes PAMPs são reconhecidos por receptores da família dos Toll-like receptors (TLR). A ativação das células da imunidade natural via TLR induz a produção de citocinas e mediadores lipídicos pro-inflamatórios dentre eles, o fator de necrose tumoral (TNF) e o fator ativador de plaquetas (PAF), respectivamente. A modulação da produção de TNF induzida por agonista de TLR4 (o LPS), pelo PAF, é conhecida, no entanto, a interação entre PAF e outros TLR foi pouco investigada. O presente trabalho teve o objetivo de avaliar a atividade moduladora do PAF na produção de TNF induzida por agonistas de TLR2. Para isto, foram utilizados as lipoproteínas de Mycoplasma fermentans PG 18 (LAMPf), agonistas de TLR2/TLR6 e o Pam3Cys, um lipopeptídeo sintético agonista de TLR2/TLR1. Culturas de sangue total periférico humano foram ativadas com os agonistas de TLR2 ou TLR4 e o TNF foi avaliado nos sobrenadantes, por meio do ELISA. Para avaliar os efeitos do PAF endógeno na produção do TNF, foi utilizado o antagonista do receptor do PAF (PAFR), o WEB2170, e para avaliar o efeito do PAF exógeno, o PAF foi adicionado às hemoculturas. As hemoculturas também foram ativadas com bactérias inteiras (S. aureus ou E. coli) inativadas pelo calor. A expressão de TLR2 em polimorfonucleares (PMN) e monócitos foi avaliada por citometria de fluxo, analisando a celularidade total para os PMN e as células CD14+, para os monócitos. As LAMPf, Pam3Cys ou LPS induziram TNF nas hemoculturas. O tratamento com WEB2170 aumentou a produção de TNF nas hemoculturas estimuladas com agonistas de TLR2, mas não de TLR4. A pré-estimulação das hemoculturas com o PAF aumentou a produção de TNF induzida pelos agonistas de TLR2. A adição de PAF não causou alterações significantes na produção de TNF induzida pelo LPS. Nos ensaios com as bactérias inteiras, E. coli induziu maiores concentrações de TNF do que S. aureus. O tratamento com WEB2170 das hemoculturas estimuladas com S. aureus reduziu a produção de TNF, no entanto, a adição de PAF não alterou significantemente a produção de TNF nas hemoculturas estimuladas com as bactérias. Para E. coli, nem o tratamento com WEB2170, nem com o PAF alterou significantemente a produção de TNF. Os resultados indicam que o PAF pode aumentar ou diminuir a produção de TNF induzida por agonista de TLR2, dependendo do momento em que ele ativa o PAFR em relação à ativação do TLR2. O aumento da produção de TNF após prolongada pré-ativação das hemoculturas com o PAF não foi devido a um aumento na expressão de TLR2. Assim, é sugerido que a interação entre as vias bioquímicas de sinalização do PAFR e do TLR2 determina o nível de produção de TNF. A via de sinalização TLR2/PAF/TNF pode ser importante na imunidade inata contra infecções causadas por bactérias Gram positivas tão bem quanto em doenças inflamatórias.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-09-13T17:35:41Z No. of bitstreams: 2 Dissertação - Mariana Paiva Lopes - 2014 .pdf: 2192084 bytes, checksum: e551c174d821b3b398247680a94c40cd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-19T13:59:44Z (GMT) No. of bitstreams: 2 Dissertação - Mariana Paiva Lopes - 2014 .pdf: 2192084 bytes, checksum: e551c174d821b3b398247680a94c40cd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-19T13:59:44Z (GMT). No. of bitstreams: 2 Dissertação - Mariana Paiva Lopes - 2014 .pdf: 2192084 bytes, checksum: e551c174d821b3b398247680a94c40cd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-03-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The gene for tumor necrosis factor (TNF) is located in the human MHC central region known as class III (Major Histocompatibility Complex). This gene encodes an important pro- inflammatory cytokine produced primarily by macrophages, and it has been associated with several diseases, such as autoimmune and degenerative ones. Studies indicate that TNF polymorphisms may influence their level of expression and activity and therefore could influence susceptibility to tumors. Whereas the bladder urothelium can constantly be the target of inflammatory processes and as the main forms of treatment of bladder tumors are immunotherapy, the genetic profile of an individual can influence susceptibility to this type of injury. In this study, we analyzed the structure and variability of the regulatory region of the TNF gene in Brazilian samples and the results were compared with data obtained by 1000Genomes project. A study of association between polymorphisms of the promoter region of TNF and bladder carcinoma patients in the state of São Paulo in Ribeirão Preto, in our analysis was conducted there was no relationship of the data found with bladder carcinoma. In all evaluated populations we found 15 variation points, found in 11 Brazilian and 15 variation points found in the 1000Genomes data, these variation points are arranged in 20 different haplotypes. These haplotypes with comparisons involving primate sequences indicated that one allele arose before the main speciation and human dispersion. Two strains were defined by haplotype relationships and are probably very old in human evolutionary history, since all populations evaluated showed haplotypes belonging to each of these strains. The frequency of haplotype H01 is the highest among all populations evaluated. However, the haplotype H12, although at reduced frequency compared to H01 is probably the oldest haplotype in part by the second line being shared with other primates. / O gene do Fator de Necrose Tumoral (TNF) humano está localizado no MHC (Complexo Principal de Histocompatibilidade) central, região conhecida como classe III. Este gene codifica uma citocina pró-inflamatória importante, produzida principalmente por macrófagos, que tem sido relacionada com diversas doenças, como as autoimunes e as degenerativas. Estudos indicam que polimorfismos do TNF podem influenciar seu nível de expressão e atividade e, portanto, poderiam influenciar a susceptibilidade a tumores. Considerando que o urotélio vesical pode ser constantemente alvo de processos inflamatórios e que as principais formas de tratamento de tumores vesicais são imunoterápicos, o perfil genético de um indivíduo pode influenciar a susceptibilidade a esse tipo de lesão. Neste estudo analisamos a estrutura e a variabilidade da região regulatória do gene TNF em amostras brasileiras e os resultados foram comparados com dados obtidos pelo projeto 1000Genomes. Foi realizado um estudo de associação entre polimorfismos da região promotora do TNF e o carcinoma vesical em pacientes do estado de São Paulo da cidade de Ribeirão Preto, nas nossas análises não houve relação dos dados encontrados com o carcinoma vesical. Considerando todas as populações avaliadas foram encontrados 15 pontos de variação, 11 encontrados nas amostras brasileiras e 15 encontrados nos dados do projeto 1000Genomes, esses pontos de variação estão arranjados em 20 haplótipos diferentes. Esses haplótipos em conjunto com as comparações envolvendo sequências de primatas indicam que um alelo principal surgiu antes da especiação e dispersão humana. Duas linhagens foram definidas pelas relações haplotípicas e são, provavelmente, muito antigas na história evolutiva humana, já que todas as populações avaliadas apresentaram haplótipos pertencentes a cada uma destas linhagens. A frequência do haplótipo H01 é a mais alta entre todas as populações avaliadas. No entanto, o haplótipo H12, embora com frequência reduzida quando comparado com o H01, provavelmente é o haplótipo mais antigo em parte por esta segunda linhagem ser compartilhada com outros primatas.

A mucosite oral (MO) constitui uma condição dolorosa que se desenvolve entre 47% e 100% dos pacientes submetidos a transplante de células-tronco hematopoiéticas (TCTH), impactando enormemente em sua qualidade de vida. Investigar fatores preditivos para MO por meio de exames não invasivos faz-se necessário, visando melhorar a qualidade de vida dos pacientes. O objetivo do estudo foi investigar a relação dos regimes de condicionamento e dos níveis salivares de Interleucina 6 (IL- 6) e Fator de Necrose Tumoral alfa (TNF-?) com a MO, bem como investigar o impacto destes na qualidade de vida. Foram selecionados 82 pacientes submetidos a TCTH, que foram avaliados em 4 momentos diferentes: no início do condicionamento para o TCTH (M1), no dia da infusão das células (M2), após 12/20 dias do início do condicionamento para transplante autólogo e alogênico, respectivamente (M3), e após 30 dias ou na alta hospitalar para ambos (M4). Nestes momentos, foi avaliado clinicamente o grau de MO segundo critérios da Organização Mundial da Saúde (OMS), coletada saliva total e aplicados 3 questionários de avaliação de qualidade de vida em relação à MO e à saúde bucal: PROMS, OHIP-14 e OMQoL. As informações clínicas e laboratoriais foram correlacionadas através do STATA 13.0 com 5% de nível de significância. Verificou-se que a maior incidência e intensidade de MO, os piores índices de qualidade de vida e os maiores níveis de IL- 6 e TNF-? foram registrados no M3, porém não houve correlação entre as citocinas e graus de MO. Houve associação entre altos níveis salivares de IL-6 e maiores pontuações no PROMS. O regime de condicionamento mieloablativo (ML) foi relacionado à MO intensa (graus 3 e 4) e à maiores pontuações nos 3 questionários de qualidade de vida, e os escores dos questionários foram maiores conforme maior foi intensidade da MO (p<0,05). / Oral mucositis (OM) is a painful condition that develops between 47% and 100% of patients undergoing hematopoietic stem cell transplantation (HSCT), impacting greatly on their quality of life. Investigation of predictive factors for OM through noninvasive exams is necessary, in order to improve the quality of life of patients. The aim of the study was to investigate the relationship of conditioning regimens and salivary levels of Interleukin 6 (IL-6) and Tumor Necrosis Factor alpha (TNF-?) with OM, and to investigate their impact on quality of life. We selected 82 patients undergoing HSCT, which were assessed at four different moments: at the start of conditioning for HSCT (M1), on the cell infusion day (M2), after 12/20 days of the start of conditioning for autologous and allogeneic transplantation, respectively (M3), and after 30 days or at hospital discharge for both (M4). In these moments, it was clinically evaluated the degree of OM according to criteria of the World Health Organization (WHO), collected whole saliva and applied 3 questionnaires of assessment of quality of life related to OM and oral health: PROMS, OHIP-14 and OMQoL. Clinical and laboratorial data were correlated using STATA 13.0 in a 5% significance level. It was found that the highest incidence and intensity of OM, the worst indices of quality of life and higher IL-6 and TNF-? levels were found in M3, but there was no correlation between cytokines and levels of OM. There was an association between high levels of salivary IL-6 and higher scores in PROMS. The myeloablative conditioning regimen (ML) was related to intense OM (grades 3 and 4) and to highest scores in the 3 questionnaires of quality of life, and the scores of questionnaires were higher as higher was the intensity of OM (p<0,05).

Orientador: Mary Luci de Souza Queiroz / Tese ( doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T11:14:50Z (GMT). No. of bitstreams: 1 Ramos_AlineLisie_D.pdf: 13996403 bytes, checksum: 20b3e94e609f9ce6fd4214680378bb52 (MD5) Previous issue date: 2008 / Resumo: A Chlorella vulgaris (CV) é uma alga microscópica bastante rica em nutrientes. Além de alto valor nutritivo, a Chlorella tem demonstrado possuir importantes atividades terapêuticas. Vários trabalhos do nosso laboratório e outros demonstram os efeitos protetores desta alga contra infecções virais e bacterianas, tumores, entre outros. No presente trabalho avaliamos os efeitos do tratamento profilático/terapêutico com a dose de 50mg/Kg/dia de CV sobre a resposta imunohematopoética de animais normais e portadores de tumor ascítico de Ehrlich (TAE). O estudo dos mecanismos envolvidos na proteção produzida pela alga frente às alterações induzidas pelo TAE no sistema imuno-hematopoético, demonstrou aumentos significativos na atividade de células NK, na produção de citocinas com padrão Th1 (IL-2 e INF-g) e na liberação de TNF-a por células esplênicas, paralelamente a uma diminuição na produção de IL-10 (padrão Th2). Além disso, o tratamento com CV protegeu o hospedeiro da mielossupressão provocada pelo desenvolvimento tumoral, seja agindo diretamente na formação e manutenção do estroma medular, estimulando a atividade das células estromais, ou ainda estimulando a produção de citocinas reguladoras da hematopoese como a IL-6 e a IL-1a. Constatamos ainda que o tratamento com CV aumenta a atividade estimuladora de colônias (CSA) no soro de animais portadores de tumor e a capacidade proliferativa de células esplênicas. Outros resultados que merecem destaque são aqueles relacionados à ativação macrofágica. Nossos resultados demonstram que, em animais normais, o tratamento com CV promove aumento significativo nos níveis de TNF-a nos sobrenadantes das culturas de macrófagos peritoneais e esplênicos sem alterar a produção de IFN-g e IL-10. Além disso, o restabelecimento da produção normal de TNF-a, IFN-g e IL-10 por macrófagos peritoneais de animais portadores de TAE e reversão parcial dos efeitos produzidos pela presença do tumor na atividade dos macrófagos esplênicos, também foram observados. Outro efeito do CV sobre macrófagos peritoneais de animais portadores de TAE foi o estímulo da produção de H2O2 e NO2 -. Analisando nossos resultados podemos dizer que a CV além de possuir alto valor nutritivo, capacidade antioxidante e atividade anti-viral e antibacteriana, apresenta importante atividade imunomoduladora frente ao desenvolvimento tumoral, já que na presença do tumor o hospedeiro que vem recebendo Chlorella é capaz de reagir mais prontamente e com maior vigor na indução de mecanismos essenciais de defesa imunológica. Neste sentido, sugerimos que a alga Chlorella vulgaris pode ser um candidato a agente preventivo e complementar no tratamento do câncer. / Abstract: Chlorella vulgaris (CV) is a microscopic alga rich in nutrients. Besides the high nutritive value, this alga has shown important therapeutic properties. Many studies from our laboratory and others have shown the protective effects of CV against viral and bacterial infections, tumors, among others. In this work we evaluated the effects of the prophylactic/therapeutic treatment with a dose of 50 mg/Kg/day in normal and Ehrlich ascites tumor (EAT)-bearing mice. The study of the mechanisms involved in the protection produced by the CV against the EAT, induced alterations in the immune-hematopoietic system; demonstrating significant improvements in the NK cells activity, Th1 (IL-2 and INF-g) and TNF-a cytokines production by mononuclear spleen cells, concomitantly with the decrease of IL-10 (Th2) production. Moreover, treatment with CV protected the host of myelosuppression caused by tumor development, it is acting directly in the formation and maintenance of bone marrow stroma, by stimulating the activity of stromal cells, or stimulating the production of important hematopoiese regulatory cytokines like IL-6 and IL-1a. We are also certain that the treatment with CV increases the colony-stimulating activity (CSA) in the serum of tumor bearing mice and the proliferative capacity of spleen cells. Other results that deserve attention are those related to macrophage activation. Our results show that in normal mice, treatment with CV, promotes significant increase in the TNF-a levels in upernatants of the cultures of peritoneal and spleen macrophages without altering the production of IFN-g and IL-10. Moreover, restoration of the normal production of TNF-a, IFN-g and IL-10 by peritoneal macrophages from animal carriers of EAT and partial reversal of the effects produced by the presence of tumor in the splenic macrophages activity were also observed. Another effect of CV on peritoneal macrophages of tumor bearing mice was the stimulation in the production of NO2 - and H2O2. Looking at our results we can say that the alga Chorella vulgaris in addition to its high nutritional value, antioxidant capacity and antiviral and antibacterial activities, it also presents important immunomodulatory activity against tumor development, since that in the presence of the tumor, the host that has received Chlorella is able to react more promptly and with greater force in the induction of essential mechanisms of immune defense. Accordingly, we suggest that the alga Chlorella vulgaris can be a candidate for the preventive and complementary treatment of cancer by strengthening the immune system. / Doutorado / Farmacologia

Il est maintenant admis que l’activité anti-tumorale du TNF implique une destruction de la vascularisation de la tumeur par induction d’apoptose des cellules endothéliales. Si le traitement au TNF en thérapie humaine permet des rémissions complètes, il n’est applicable qu’aux membres isolés de la circulation systémique car les doses requises de TNF sont létales pour le patient. Mettre en évidence les mécanismes qui contrôlent l’apoptose de la cellule endothéliale devrait permettre de développer de nouvelles stratégies thérapeutiques favorisant l’effet thérapeutique du TNF tout en évitant le choc septique qu’il engendre.<p><p>Nous avons abordé cette question en utilisant le modèle de la cellule endothéliale d’aorte bovine (BAEC) stimulée au TNF afin d’étudier les mécanismes qui contrôlent le processus apoptotique et ce au cours des phases précoces de celui-ci.<p><p>D’une part, nous avons identifié par une approche pharmacologique au moins trois voies de contrôle négatif d’induction de mort cellulaire par le TNF dans les cellules endothéliales :la première faisant intervenir une des protéines kinases C, une seconde la PI3-kinase et une troisième la protéine kinase p38. Cette dernière semble spécifiquement activée par le TNF et pourrait donc constituer une nouvelle cible pharmacologique visant à sensibiliser les cellules endothéliales de la vascularisation de la tumeur à l’action apoptotique du TNF. <p>D’autre part nous avons mis en évidence et identifié par une approche protéomique au moins deux protéines montrant une rapide diminution de phosphorylation lorsque les cellules endothéliales sont stimulées par le TNF en présence de cycloheximide. Ces deux événements semblent en aval de l’activation d’une protéase de la famille des caspases. La première protéine est la sous-unité régulatrice de type II alpha (RIIα) de la protéine kinase A. Cependant, la relation entre cette déphosphorylation et le processus d’apoptose n’a pas pu être mise en évidence, une stimulation de l’activité PKA n’affectant pas l’induction d’apoptose des BAEC. La seconde est la protéine HDGF, connue pour être sur-exprimée dans certains cancers, mais dont la régulation par phosphorylation ainsi que son implication éventuelle lors du processus apoptotique n’avaient encore jamais été envisagées.<p><p>Enfin, nous avons voulu mettre en évidence le rôle potentiel de la protéine hnRNP K qui subit une modification post-traductionnelle précoce lors de l’apoptose des BAEC. Notre étude, menée après surexpression de la protéine étiquetée, suggère une modification autre qu’une dégradation. Cependant, il ne nous a pas été permis de lui attribuer un rôle puisque la surexpression de cette protéine n’affecte pas l’apparition de différents marqueurs associés à l’apoptose. / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Abstract Apoptosis plays a crucial part in human ovarian function from fetal development to the end of reproductive potential. Failures in the regulation of ovarian apoptosis are associated with many pathological conditions such as premature ovarian insufficiency, infertility and cancer. The purpose of the present study was to analyze the factors regulating cell survival in human fetal and adult ovaries. The fetus is exposed to maternal- and placental-derived estrogens and insufficient estrogen action has destructive effects on rodent ovarian development. We detected estrogen receptors and estrogen-converting enzymes in human fetal ovaries after primordial follicle formation, indicating that estrogens participate in human fetal ovarian development, especially after folliculogenesis. The WNT4 gene is crucial for female sexual differentiation, follicle formation and oocyte survival. We detected WNT4 in follicular cells of fetal and adult human ovaries. In addition, Wnt4- knockout mice demonstrated a dramatic loss of oocytes before birth. However, no changes were detected in protein expression patterns of common apoptosis-related proteins. The results support the possible role of WNT4 in human ovarian function and strengthen previous knowledge on the antiapoptotic role of Wnt4. Apoptosis signaling is mediated by extracellular- and mitochondria-associated- pathways, ending in caspase cascade activation and fragmentation of cellular structures. In the present study we analyzed the expression of several apoptosis-related factors and detected TRAIL, TNF, Bcl-XL, Bok and caspase-3 in human ovaries. In addition, TRAIL was found to be a potent and rapid inducer of human granulosa tumor cell (KGN) apoptosis. Lentiviral downregulation of Bok or Bcl-XL protein expression in KGN cells also resulted in significant changes in cell vulnerability to apoptosis. The results show for the first time the spatiotemporal expression patterns of TRAIL, TNF, Bcl-XL, Bok and caspase-3 in human ovaries and suggest an important functional role of TRAIL, Bok and Bcl-XL in regulation of human ovarian apoptosis. The present study offers novel information on the expression and function of cell survival factors in human ovaries. These new findings open possibilities for future clinical research in attempts to understand and treat ovarian diseases caused by imbalanced regulatory pathways of apoptosis.

Adipose tissue (AT) expansion and remodeling that maintains healthy function relies on stromal preadipocytes capable of differentiating into new adipocytes (adipogenesis). During chronic positive energy balance, a relative deficit in adipogenesis, from either a decrease in preadipocyte number or their capacity to differentiate, leads to excessive adipocyte hypertrophy and AT dysfunction. AT contains macrophages whose number and activation state is dynamically regulated with changes in AT mass. This study aims to investigate the effect of macrophage-secreted factors on preadipocyte survival. To assess the effect of macrophage-secreted factors on preadipocytes, murine 3T3-L1 preadipocytes or human primary preadipocytes were incubated with macrophage-conditioned medium (MacCM), prepared from either murine (J774A.1, RAW264.7, bone marrow-derived) or human (THP-1, monocyte-derived) macrophage models, respectively. MacCM inhibited preadipocyte apoptosis and activated pro-survival signaling in both preadipocyte models. Inhibition of PDGFR, Akt, or ERK1/2 reduced the pro-survival effect of MacCM in 3T3-L1 preadipocytes. Inhibition of reactive oxygen species (ROS) generation, or enhancement of ROS clearance, reduced MacCM-dependent 3T3-L1 preadipocyte survival. Whereas anti-inflammatory activated macrophages retained the ability to prevent preadipocyte apoptosis, pro-inflammatory activated macrophages did not. TNF-α immunoneutralization restored the survival activity of pro-inflammatory MacCM on 3T3-L1 preadipocytes. These studies reveal a novel pro-survival effect of MacCM on preadipocytes, and identify signaling molecules (PDGF, Akt, ERK1/2, and ROS) that underlie this action. Macrophage activation was found to regulate the pro-survival activity of MacCM. These in vitro cell culture studies are consistent with a model in which the extent of preadipocyte apoptosis in vivo may determine preadipocyte number and the ability of AT to expand while maintaining healthy function during chronic positive energy balance.

La dystrophie musculaire de Duchenne (DMD) est une maladie liée à l'X caractérisée par la détérioration progressive des muscles en raison de l'absence de la protéine dystrophine. Les muscles atteints chez l'homme ou dans des modèles animaux (souris mdx) présentent une fibrose, accumulation excessive de protéines de la matrice extracellulaire. Parmi les facteurs induisant la fibrose se trouvent le Facteur de Croissance de Transformation de type β (TGF-β) et le Facteur de Croissance du Tissu Conjonctif (CTGF). Ce dernier est une cible de la voie de signalisation médiée par TGF-β/SMAD et est responsable des effets profibrotiques de TGF-β. La régulation de l'expression de CTGF médiée par TGF-β dans les cellules musculaires est peu connue. Nous décrivons ici un nouvel élément de liaison SMAD situé dans la région 5’UTR du gène de CTGF, important pour l'expression de CTGF médiée par le TGF-β dans des myoblastes. De plus, nos résultats suggèrent que d'autres sites de liaison du facteur de transcription présents dans le 5’UTR du gène de CTGF sont importants pour cette expression.Par ailleurs, le Facteur de Nécrose Tumorale (TNF) est une cytokine inflammatoire présente dans les muscles atteints de DMD et est responsable de la nécrose du muscle et de l'infiltration de cellules inflammatoires. Nous montrons que l’expression du récepteur soluble TNFRI par électrotransfert (ET) dans le muscle tibialis anterior de la souris atténue l'inflammation, les dommages et la fibrose dans le muscle squelettique des souris mdx, et provoque une augmentation de la force musculaire. Par conséquent, nous proposons l'ET comme thérapie efficace anti-TNF pour le traitement de dystrophies musculaires. / The Duchenne Muscular Dystrophy (DMD) is an X-linked disease characterized by progressive damage in the muscle due to the absence of the dystrophin protein. Fibrosis, the excessive accumulation of extracellular matrix (ECM) proteins, is also present in the muscle of DMD patients and several animal models (such as the mdx mice). Among the factors that induce fibrosis are Transforming Growth Factor type β (TGF-β) and Connective Tissue Growth Factor (CTGF), the latter being a target of the TGF-β/SMAD signaling pathway and is the responsible for the profibrotic effects of TGF-β and are augmented in fibrosis tissues. Little is known about the regulation of the expression of CTGF mediated by TGF-β in muscle cells. In here, we described a novel SMAD Binding Element (SBE) located in the 5’ UTR region of the CTGF gene important for the TGF-β mediated expression of CTGF in myoblasts. In addition, our results suggest that additional transcription factor binding sites present in the 5’ UTR of the CTGF gene are important for this expression. On the other hand, the Tumor Necrosis Factor (TNF) is an inflammatory cytokine that is present in DMD muscles and is responsible for muscle necrosis and inflammatory cell infiltration. In this study, we show that the increased expression of the soluble TNF Receptor I by electrotransfer (ET) in the tibialis anterior muscle attenuates inflammation, damage and fibrosis in the skeletal muscle of the mdx mice. In addition, we found increased muscle strength in the mdx mice. Therefore, we propose that ET could be used as an efficient anti-TNF therapy for treating muscle dystrophies.