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First published online: 2012-03-10 / 名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成24年4月27日 森谷隆氏の博士論文として提出された

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-10-03T12:12:48Z No. of bitstreams: 1 THIAGO JOSÉ FIGUEIRA RAMOS.pdf: 1733228 bytes, checksum: c1606fa7a4e1569d3cc660ae62c32331 (MD5) / Made available in DSpace on 2013-10-03T12:12:48Z (GMT). No. of bitstreams: 1 THIAGO JOSÉ FIGUEIRA RAMOS.pdf: 1733228 bytes, checksum: c1606fa7a4e1569d3cc660ae62c32331 (MD5) Previous issue date: 2012-10-10 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A silicose é uma doença ocupacional causada pela inalação de sílica em sua forma livre e cristalina. A inflamação e fibrose pulmonar constituem as principais características da patologia, que envolve uma variedade de mediadores inflamatórios, como o TNF-α, um mediador de ações amplas e que exerce seus efeitos em um grande número de tipos celulares. LASSBio-468 é um análogo da talidomida já descrito como capaz de modular a produção de TNF-α e inibir o choque endotóxico e artrite reumatóide em modelos animais. Neste estudo, nós investigamos o potencial efeito do LASSBio-468 em modelo de silicose experimental em camundongos. Animais Swiss-webster machos (18 – 20g) foram instilados intranasalmente com uma suspensão de sílica (10 mg/50 μL) e veículo (salina). O tratamento consistiu na administração por via oral de LASSBio-468 (12,5 – 100 mg/kg) e talidomida (50 mg/Kg) durante 7 dias consecutivos, do vigésimo primeiro ao vigésimo oitavo dia após a instilação de sílica. Foram analisadas a função pulmonar (resistência e elastância) e hiperreatividade das vias aéreas à aerolização de metacolina (3 – 27 mg/mL), através de pletismógrafo invasivo de corpo inteiro (Finepoint, Buxco System), além de análises morfológicas e morfométricas do tecido pulmonar. A produção de colágeno tecidual foi acessada pelo método de sircol, enquanto que a quantificação de citocinas/quimiocinas foi realizada pelo ensaio de ELISA. A técnica de imunohistoquímica permitiu a identificação da população de miofibroblastos no pulmão. Através de uma digestão mecânica e enzimática do tecido pulmonar, obtivemos uma cultura primária de miofibroblastos que, assim como macrófagos alveolares AMJ2C11 e células epiteliais A549, foram pré-tratados com diferentes concentrações de LASSBio-468 e talidomida, estimulados com IL-13 ou sílica e avaliados quanto a proliferação celular, viabilidade e produção de mediadores inflamatórios in vitro. Demonstramos que animais silicóticos apresentaram níveis basais elevados de resistência pulmonar e elastância, bem como hiperreatividade frente à aerolização de metacolina. Resposta inflamatória tecidual, extensiva deposição de colágeno, formação de granuloma e produção de quimiocinas (KC and MCP-1) e citocinas (TNF- αand TGF-β) também foram detectadas em pulmões de animais silicóticos, assim como o aumento do número de miofibroblastos no tecido. O tratamento com LASSBio-468 reduziu o comprometimento da função pulmonar e hiperreatividade, formação de granulomas, expressão de miofibroblastos e deposição de colágeno no tecido pulmonar de animais doentes. Miofibroblastos oriundos de animais silicóticos apresentaram basal de proliferação superior, sendo responsivos também ao estímulo mitogênico da IL-13, que foi atenuado frente ao pré-tratamento com LASSBio-468. A estimulação de macrófagos alveolares e células epiteliais com sílica promoveu a liberação de TNF-α e IL-8, respectivamente, sendo o LASSBio-468 capaz de inibir de forma significativa esta produção. Em conjunto, nossos resultados mostraram que o tratamento com LASSBio-468 foi capaz de reduzir de forma curativa o comprometimento da função pulmonar e resposta granulomatosa, através de ações sobre células específicas, indicando que o composto em questão parece constituir uma promissora ferramenta para o tratamento de doenças pulmonares fibróticas crônicas como a silicose. / Silicosis, one of the oldest occupational diseases in the world, is a consequence of long-term exposure to inhaled dust containing silica in its free and crystalline form. Lung interstitial inflammation and fibrosis are the main features of the disease, involving a wide range of chemical mediators such as TNF-α. This is a pleiotropic molecule which exerts its effects on many cell types. LASSBio-468 is a thalidomide analogue which modulates TNF-α production and inhibits endotoxic shock and arthritis in animal models. In this study we investigated the effect of LASSBio-468 on experimental silicosis in mice. Anesthetized male Swiss-Webster mice (18-20g) received intranasal (i.n.) instillation of silica (10 mg/50 μL) and vehicle (saline). Treatment consisted of oral administration of the LASSBio-468 (12,5 - 100 mg/kg) and thalidomide (50 mg/Kg) during 7 consecutive days, from day 21 to 28 post-silica. The analyses included lung function (resistance and elastance) and airways hyperreactivity to aerosolized metacholine (3 -27 mg/mL) measured by whole body invasive plestimography (Finepoint, Buxco System). Morphological and morphometrical analyses included classical dyes such as Hematoxylin-Eosin and Picrus-Sirius. Collagen content and cytokine/chemokine generation were quantified by Sircol technique and ELISA, respectively. Immunohistochemistry was employed to identify the lung myofibroblastic population. Primary murine myofibroblasts cells were obtained after a mechanical and enzymatic digestion of the lung from saline and silicotic mice. Myofibroblasts, AMJ2C11 alveolar macrophages and A549 epithelial cells were pre-treated with LASSBio-468 and Thalidomide, stimulated with IL-13 or silica and evaluated thought proliferation, viability and production of inflammatory mediators in vitro. We showed that silicotic mice exhibited increased basal levels of lung resistance and elastance as well as airways hyperreactivity to methacholineaerosolization. Tissue inflammatory response, extensive collagen deposition, granuloma formation and chemokine (KC and MCP-1)/cytokine (TNF-α and TGF-β) generation were also detected in the silicotic lungs, as well as an increasing of myofibroblasts expression in the lung. Administration of LASSBio- 468 into silicotic mice reduced the decline lung function and airways hyperreactivity, myofibroblasts numbers, tissue collagen deposition and granulomatous area. The generation of cytokines and chemokines was also suppressed by the drug. The primary lung myofibroblasts obtained from silicotic mice showed a superior basal proliferation and IL-13, a pro-fibrotic cytokine, stimulated the (3H) incorporation in vitro, which was diminished by the treatment with LASSBio-468. Stimulation of alveolar macrophages and epithelial cells by silica in vitro increased the release of TNF-α and IL-8, respectively, and LASSBio-468 was able to reduce these inflammatory mediators production. The compound modulated key cells functions without decline in cell death. Altogether our findings show that the treatment of silicotic mice with LASSBio-468 reduced curatively the decline in lung function and granulomatous response, throught actions on myofibroblasts, macrophages and epithelial cells, indicating that this compound constitutes a promising tool for the treatment of chronic fibrotic pulmonary diseases such as silicosis.

Periodontitis potentially contributes to many systemic diseases. Specific gram-negative bacteria such as Porphyromonas gingivalis (P.g) and Treponema denticola (T.d) contribute to the initiation and progression of periodontal disease via factors such as NF-κB, TNF-α, IL-1β, and CXCL1. Down-regulation of these factors by natural compounds or short hairpin RNAs (shRNAs) reduces periodontal bacteria-induced inflammation. Our preliminary data indicated that P.gingivalis / lipopolysaccharide (LPS) stimulates chemokine CXCL1 production in macrophages. CXCL1 stimulates LPS-induced TNF-α expression, resulting in inflammation. We hypothesize that inhibiting the expression of CXCL1 will reduce LPS-induced TNF-α production. We recently demonstrated that a CXCL1 shRNA inhibits LPS-stimulated CXCL1 production in macrophages and leads to reduced expression of LPS-stimulated pro-inflammatory cytokines TNF-α and IL-1β. This indicates that CXCL1 shRNAs have potential as inhibitors of LPS-induced inflammation. Further studies are needed to confirm these as well as to identify the signaling pathway.

Die vorliegende Untersuchung hatte zur Zielsetzung, bei Kühen die Unterschiede zwischen hypokalzämischen und normokalzämischen Festliegern zu analysieren. Es sollte geklärt werden, welche klinischen und labordiagnostischen Veränderungen, außer der Kalzium- (Ca) Konzentration, dem normokalzämische Festliegen zugrunde liegen. Des Weiteren wurden die TNF-α-, Haptoglobin- (Hp-) und TEAC-Konzentrationen in Beziehung zum Festliegen, den Mineralstoffkonzentrationen sowie hinsichtlich diagnostischer Information geprüft. Außerdem wurden die Festlieger mit Nachgeburtsverhaltung (Ret. sec.) und die Kühe mit Exitus letalis labordiagnostisch genauer analysiert.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-09-19T12:33:33Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação de Mestrado com Ficha Catalográfica - Leonilda (6).pdf: 1607291 bytes, checksum: 83e1d52bc2be1f64f2a2a27dc1f58889 (MD5) / Made available in DSpace on 2016-09-19T12:33:33Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação de Mestrado com Ficha Catalográfica - Leonilda (6).pdf: 1607291 bytes, checksum: 83e1d52bc2be1f64f2a2a27dc1f58889 (MD5) Previous issue date: 2016-02-18 / A infecção pelo Vírus da Imunodeficiência Humana 1 (HIV-1) tem como característica clássica a depleção de linfócitos T (LT) CD4+, que quando não tratada culmina na Síndrome da Imunodeficiência Adquirida (AIDS). Com o surgimento terapia antirretroviral (TARV), na década de 80, ocorreu uma verdadeira revolução. A TARV, apesar de não eliminar o vírus, consegue manter a carga viral em níveis indetectáveis, melhorando a qualidade de vida dos portadores. No entanto, entre 15-30% dos indivíduos em uso regular de TARV e com carga viral indetectável não consegue recuperar os níveis de LT CD4+ (Recuperação Imunológica). Estudos apontam que a apoptose pode estar envolvida na diminuição dos LT CD4+ e que variações genéticas como polimorfismos de base única (SNPs) em moléculas envolvidas nas vias da apoptose podem levar a diferentes respostas imunológicas do indivíduo à infecção. Neste sentido, o presente estudo se propôs a investigar o papel de SNPs (rs1800692, rs767455, rs2270926, rs8904, rs1800629) em genes codificadores de proteínas que ativam a via extrínseca da apoptose através do TNF-α/TNFR1, e sua relação com a recuperação imunológica de pacientes com uso regular de TARV. Foram estudados 113 pacientes HIV positivos, atendidos e tratados no Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), em uso de TARV por um ano e com carga viral indetectável, os quais foram divididos em dois grupos: sucesso imunológico (controle) e falha imunológica (caso). Não foram observadas associações entre os polimorfismos dos genes estudados com o sucesso ou falha imunológica apresentada pelos indivíduos fazendo uso regular da TARV. Também não foi observada nenhuma associação entre a falha imunológica e as variáveis clínicas: peso, etnia, idade, gênero e esquema terapêutico. Apesar da ausência de associações dos SNPs estudados com a falha imunológica, alguns fatores limitantes do estudo devem ser considerados (pequeno número amostral, não inclusão de outras moléculas da via estudada), se fazendo necessário novos estudos de réplica em outras populações. / Infection by Human Immunodeficiency Virus 1 (HIV-1) has as a classic feature the depletion of T lymphocytes (TL) CD4 + that, when untreated, culminates in Acquired Immunodeficiency Syndrome (AIDS). With the advent of HAART (Highly Active Antiretroviral Therapy), in the 80s, there was a real revolution. The ART, although it doesn't eliminate the virus, it can keep the viral load in undetectable levels, improving the quality in patient's' lifes. However, 15-30% of individuals regularly using HAART and with undetectable viral load, cannot recover CD4 + LT levels (Immune Recovery). Studies have shown that apoptosis may be involved in CD4 + depletion and genetic variations, such as single nucleotide polymorphisms (SNPs), in molecules involved in apoptosis pathways can lead to different immune responses from the individuals to the infection. Therefore, the present study aims to investigate the role of SNPs (rs1800692, rs767455, rs2270926, rs8904, rs1800629) in genes encoding proteins that activate the extrinsic pathway of apoptosis, by TNF-α / TNFR1, and its relationship with immune recovery of patients in regular use of HAART. We studied 113 HIV-positive patients assisted and treated by the Institute of Integrative Medicine Professor Fernando Figueira (IMIP), who were under HAART for at least one year and with undetectable viral load, which we divided into two groups: immunological success (control) and immunological failure (case). There were no associations between the polymorphisms of the studied genes with the immune failure presented by individuals making regular use of HAART. Also, it has been observed no associations between the immunologic failure with the clinical variables: weight, ethnicity, age, gender and treatment regimen. Despite the absence of associations of SNPs studied with immunological failure, some limitations of the study should be considered (small sample size, no inclusion of other molecules of the studied pathway), thus new replica studies in other populations are necessary.

The involvement of the pro-inflammatory cytokine, tumour necrosis factor alpha (TNF-α) and the sympathetic nervous system in the development of delayed-onset muscle soreness has not been established. I assessed the effect of etanercept, a TNF- α inhibitor, and atenolol, a β1-receptor antagonist, on DOMS induced in the quadriceps muscle. Thirteen male subjects reported to the exercise laboratory on three separate occasions, 6-15 weeks apart. In a randomised, double-blind cross-over format, I administered etanercept (25mg), atenolol (25mg) or placebo, one hour before the exercise. Subjects then completed four sets of 15 repetitions at 80% of their one repetition maximum (1RM) on a 45° inclined leg press machine. Muscle strength changes were detected by remeasuring the subject’s 1RM 24h, 48h and 72h after the exercise. Sensitivity to pressure of the quadriceps muscle was measured using a pressure algometer before and 24h, 48h and 72h after exercise. The subject’s perception of the pain was measured with the visual analogue scale and McGill Pain Questionnaire. Muscle tumour necrosis factor-alpha concentration was measured before exercise and then 2h and 24h after exercise in four subjects. Muscle strength was impaired 24h and 48h after exercise regardless of agent administered (P < 0.001). At 72h after exercise, muscle strength was significantly improved (P < 0.01) in subjects receiving etanercept and atenolol compared to those receiving placebo. The subject’s were significantly more sensitive to pressure applied to the quadriceps 24h, 48h and 72h after exercise compared to before exercise, regardless of agent administered (P < 0.001). The VAS was elevated significantly at all three time intervals, with no difference after etanercept or atenolol administration compared to that of placebo. There was no significant difference in the muscle TNF-α concentration between any of the time intervals or between subjects receiving placebo and etanercept (P=0.065). The administration of atenolol and etanercept, at the regimen used, had no effect on the soreness associated with DOMS.

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2015-10-22T15:14:10Z No. of bitstreams: 3 license_text: 21328 bytes, checksum: 683d9883b2ad62ac3b8bafc566b2e600 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2015LucianaKnabbendeOliveiraBeckerDelving.pdf: 1574790 bytes, checksum: b751dea1371239a23970a2767f3789ba (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2015-11-18T11:37:45Z (GMT) No. of bitstreams: 3 license_text: 21328 bytes, checksum: 683d9883b2ad62ac3b8bafc566b2e600 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2015LucianaKnabbendeOliveiraBeckerDelving.pdf: 1574790 bytes, checksum: b751dea1371239a23970a2767f3789ba (MD5) / Made available in DSpace on 2015-11-18T11:37:45Z (GMT). No. of bitstreams: 3 license_text: 21328 bytes, checksum: 683d9883b2ad62ac3b8bafc566b2e600 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2015LucianaKnabbendeOliveiraBeckerDelving.pdf: 1574790 bytes, checksum: b751dea1371239a23970a2767f3789ba (MD5) / Na medicina tradicional, as plantas tem sido utilizadas para o tratamento de diversas doenças ao longo dos séculos e são consideradas uma das maiores fontes para o desenvolvimento de novas drogas. Com o objetivo de contribuir com o conhecimento cientifico no estudo e desenvolvimento de novas drogas procurou-se com este trabalho, identificaros principais constituintes fitoquímicosda Calyptranthes grandifolia, avaliar seus potenciais citotóxicos e antioxidantes e também identificar e avaliar o efeito terapêutico, através de vias específicas e já conhecidas por seu envolvimento no processo inflamatório, como o TNF- α. Para todos os experimentos, foram utilizados extratos etanólico e hexânico das folhas da C.grandifolia, coletada no entorno do município de Lajeado.Foi realizada a análise fitoquímica de esteroides/terpenoides, taninos, flavonoides, cumarinas, quinonas, alcaloides e saponinas, utilizando metodologias descritas na literatura. O potencial citotóxico in vitro foi avaliado pelo método de Alamar Blue, utilizando células não metabolizadoras CHO-K1 e a atividade antioxidante foi realizada pelo método2,2-difenil-1-picrilhidrazila (DPPH). A análise da liberação do TNF-α utilizou células RAW 264.7, tratadas com os extratos vegetais e posterior estimulação com lipopolissacarídeo (LPS). A quantificação de liberação do TNF-α foi feita pelo método de ELISA. Os resultados dos ensaios indicaram a presença de esteroides/terpenoides, taninos e flavonoides no extrato etanólico. O potencial citotóxico, não apresentou citotoxicidade considerável no extrato etanólico e manteve a viabilidade celular aumentada na maioria das concentrações, com uma diminuição não significativa da viabilidade em 200 μg/mL, de forma semelhante para o extrato hexânico a redução da viabilidade celular pode ser observada somente na concentração de 200 μg/mL. A atividade antioxidante esteve presente apenas no extrato etanólico de maneira dose-dependente, com IC50 de 21.3±1.7μg/mL.O extrato etanólico também apresentou melhores resultados em relação à inibição do TNF-α, tendo sido significativa na concentração de 200 μg/mL, frente ao controle positivo (LPS). O extrato hexânico não apresentou inibição da liberação do TNF-α na concentração de 200 μg/mL de forma significativa quando comparada ao LPS. Os resultados deste estudo mostram indícios de atividades anti-inflamatórias nos extratos testados, havendo um melhor desempenho do extrato etanólico. A presença de polifenóis e esteroides/terpenoides encontradas neste extrato podem ser a resposta para uma melhor avaliação das atividades antioxidantes e anti-inflamatórias. Estudos futuros podem indicar um potencial anti-inflamatório promissor para esta planta, lembrando ainda que processos inflamatórios fazem parte do processo de carcinogênese, havendo portanto uma porta de entrada para maiores pesquisas avaliando o potencial anticâncer da C. grandifolia. / In traditional medicine, plants have been used in the treatment of several diseases throughout centuries and are considered as one of the largest sources for the development of new drugs. However, the bioactive components and action mechanisms are not entirely known. Aiming to contribute with scientific knowledge towards the study and development of new drugs, this work sought to identify their main phytochemical components, evaluate their cytotoxic and antioxidant potentials, as well as identifying and evaluating the therapeutic effect of Calyptranthesgrandifolia, through specific and known paths for their involvement in the inflammatory and carcinogenic process, such as TNF- α. Ethanolic and hexanic extracts from C. grandifolialeaves, gathered within the Lajeado municipality, were used in all experiments. The phytochemical analysis of steroids/terpenoids, tannins, flavonoids, coumarins, quinones, alkaloids and saponins were characterized using methodologies described in literature. The in vitro cytotoxic potential was evaluated through the Alamar Blue method using non drug-metabolizing CHOK1 cells and the antioxidant activity was performed through the DPPH method. The analysis of TNF-α release used RAW 264.7 cells, treated with vegetal extracts and posterior stimulation with LPS. The quantification of TNF-α release was quantified using ELISA method. The experiment results indicated the presence of steroids/terpenoids, tannins and flavonoids in the ethanolic extract. The cytotoxic potential did not present considerable cytotoxicity in the ethanolic extract and maintained increased cellular viability in most concentrations, only presenting a slight reduction in viability at 200 μg/mL. Similarly, cellular viability reduction could only be observed in the hexanic extract at the 200 μg/mL concentration. The antioxidant activity was only present in the ethanolic extract at a dose-dependant manner, with an IC50 value of 21.3±1.7μg/mL. The ethanolic extract also presented better results concerning the inhibition of TNF-α, given that it was significant at the 200 μg/mL concentration in comparison with the positive control (LPS). The hexanic extract also presented higher TNF-α inhibition at the 200 μg/mL concentration, however, there was no significant difference in comparison with LPS. The results of this study demonstrate that there are evidences of antiinflammatory activity in the tested extracts, with better performance in the ethanolic extract. The presence of polyphenols and steroids/terpenoids found in this extract may be the answer for its better evaluation concerning antioxidant and anti-inflammatory activities. Future studies may indicate a promising anti-inflammatory potential for this plant, also reminding that inflammatory processes are part of the carcinogenesis process, therefore, this is an entry path for larger researches evaluating the anticarcinogenic potential in C. grandifolia extracts.

Submitted by SARAH MARIA BARNEZE COSTA null (sarahbarneze@hotmail.com) on 2017-04-27T05:04:44Z No. of bitstreams: 1 Dissertação Final para o Repositório da Unesp - Aluna Sarah Barneze.pdf: 2655925 bytes, checksum: 922eb75a13d7d9ff08b061e4361675e1 (MD5) / Rejected by Luiz Galeffi (luizgaleffi@gmail.com), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: Incluir o número do processo de financiamento nos agradecimentos da dissertação/tese. Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-05-03T14:13:29Z (GMT) / Submitted by SARAH MARIA BARNEZE COSTA null (sarahbarneze@hotmail.com) on 2017-05-03T16:11:16Z No. of bitstreams: 1 Dissertação Final para o Repositório da Unesp - Aluna Sarah Barneze.pdf: 2656758 bytes, checksum: 41757de66dd6c78c3165310b774a5fcb (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-05-03T16:16:11Z (GMT) No. of bitstreams: 1 costa_smb_me_bot.pdf: 2656758 bytes, checksum: 41757de66dd6c78c3165310b774a5fcb (MD5) / Made available in DSpace on 2017-05-03T16:16:11Z (GMT). No. of bitstreams: 1 costa_smb_me_bot.pdf: 2656758 bytes, checksum: 41757de66dd6c78c3165310b774a5fcb (MD5) Previous issue date: 2017-02-16 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A regulação da remodelação óssea ocorre por meio de fatores locais e sistêmicos. Entre os fatores locais estão as citocinas: Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL), presente nos osteoblastos, e Receptor Activator of Nuclear Factor Kappa B (RANK), presente nos osteoclastos, além de outras citocinas como o Tumor Necrosis Factor Alpha (TNF-α) que podem agir no processo de deposição e/ou reabsorção óssea in vivo. Entre os fatores sistêmicos que participam da remodelação óssea estão os hormônios estrógeno (E2) e triiodotironina (T3). O objetivo do trabalho foi verificar a ação do E2, infrafisiológico (simulando a menopausa) e T3, suprafisiológico (simulando hipertireoidismo) em osteoblastos humanos derivados de células troncos mesenquimais (CTMs) na síntese proteica de RANKL e TNF-α. Os osteoblastos foram incubados por 72 horas na presença de E2 em dose fisiológica (E2F/10-8M) e infrafisiológica (E2I/10-9M), e T3 em dose fisiológica (T3F/10-9M) e suprafisiológica (T3S/10-8M), e quantificada a matriz mineralizada óssea e a síntese proteica de RANKL e TNF-α por Western Blot. A análise estatística dos dados foi realizada pelo teste de variância ANOVA complementada pelo teste de Tukey, sendo o nível de significância considerado p<0,05. O tratamento de E2 em dose E2F (1,96± 0,48; p<0,05) e E2I (3,18±0,31; p<0,001) elevou a síntese proteica de RANKL comparado ao controle (C) (1,00±0,32), e de TNF-α em dose de E2F (3,61±0,45; p<0,05) e E2I (2,45±0,07; p<0,05) também aumentou em relação ao C (1,13±0,19). Assim como o E2, o T3 elevou os níveis proteicos de RANKL em T3F (1,18±0,10; p<0,05) e T3S (1,14±0,004; p<0,05) comparado ao controle (1,00±0,02), porém, TNF-α diminui em dose de T3S (0,82±0,09; p<0,05) relacionado ao T3F (1,00±0,09) e C (0,99±0,04). Com isso, E2I diminuiu a matriz mineralizada óssea e aumentou a síntese de ambas as proteínas estudadas, o que nos leva a especulação de que a reabsorção óssea observada na menopausa seja via RANKL e TNF-α. Por outro lado, na mimetização de hipertireoidismo com o T3S, observamos diminuição da matriz mineralizada óssea, supressão da síntese proteica de TNF-α e aumento de RANKL, o que nos leva a inferir que o mecanismo de reabsorção óssea no hipertireoidismo seja via RANKL. / The regulation of bone remodeling is intermediated by local and systemic factors. The cytokines are among the local factors: Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL), present in osteoblasts, and Receptor Activator of Nuclear Factor Kappa B (RANK), present in osteoclasts, in addition to other cytokines such as Tumor Necrosis Factor Alpha (TNF-α) that can act in the process of bone deposition and/or resorption in vivo. Hormones are among the systemic factors involved in bone remodeling, estrogen (E2) and triiodothyronine (T3). The aim of the study was to verify the action of E2, infraphysiological (simulating menopause) and T3, supraphysiological (simulating hyperthyroidism) in human osteoblasts derived from mesenchymal stem cells (CTMs) in the protein synthesis of RANKL and TNF-α. Osteoblasts were incubated during 72 hours in the presence of E2 at physiological dose (E2F/10 -8 M) and infraphysiological (E2I/10 -9 M), and T3 at physiological (T3F/10 -9 M) and supraphysiological (T3S/10-8 M) and quantified the bone mineralized matrix and the protein synthesis of RANKL and TNF-α by Western Blot. Statistical analysis of the data was performed by the ANOVA variance test complemented by the Tukey test, and the significance level was considered p<0.05. The treatment of E2F in E2F dose (1.96 ± 0.48, p<0.05) and E2I (3.18 ± 0.31; p <0.001) increased RANKL protein synthesis compared to control (C) (1.00 ± 0.32), and TNF-α in the dose of E2F (3.61 ± 0.45; p<0.05) and E2I (2.45 ± 0.07; p <0.05) also increased in relation to C (1.13 ± 0.19). As with E2, T3 increased RANKL protein levels in T3F (1.18 ± 0.10, p <0.05) and T3S (1.14 ± 0.004; p <0.05) compared to control (1 , 00 ± 0.02), but TNF-α decreased in dose T3S (0.82 ± 0.09; p <0.05) related to T3F (1.00 ± 0.09) and C (0, 99 ± 0.04). Thus, E2I decreased the bone mineralized matrix and increased the synthesis of both proteins studied which leads to speculation that bone resorption observed at menopause by RANKL and TNF-α pathway. On the other hand, in the mimicry of hyperthyroidism with T3S, we observed a reduction of the bone mineralized matrix, suppression of TNF-α protein synthesis and increase of RANKL, which leads us to infer that the mechanism of action for bone resorption in hyperthyroidism is by RANKL. / FAPESP: 2014/15529-0

The overall objective of this research was to investigate the status of tumor necrosis factor-α (TNF-α), and molecules associated with its signaling, in the pathological state of hepatic steatosis. The effect of NSAID piroxicam, a cancer preventive agent also known to affect TNF-α signaling on hepatic steatosis, was also investigated. The biological state of the tissue was assessed by examining the expression of TNF-α signaling molecule in whole tissue, as well as in hepatic lipid raft. Lipid rafts are dynamic assemblies of cholesterol and sphingolipids, microdomains that form in the exoplasmic leaflet of the biological membranes shown to play a role in compartmentalization, modulation and integration of the cell signaling. In the present research, Zucker obese rats were used as a model of human obesity and insulin resistant state. These rats exhibit hepatic steatosis in adulthood similar to those noted in obese individuals. Female Zucker obese and lean rats (5 weeks old) were fed a semisynthetic diet with or without piroxicam (150 ppm). Zucker lean counterparts served as control. After 8 weeks of feeding, rats were euthanized and liver from each animal was collected. Liver tissue from each animal was processed for histology and biochemical analysis which included lipids and proteins (COX-1 and 2, TNF-α, TNF-RI and RII, IKK-β, IκB-α and NF-κB). Liver histology and the level of total lipids confirmed that Zucker obese rats had hepatic steatosis, which was further augmented by piroxicam treatment. Whole tissue protein expression, using western blot, showed that the steatotic liver differed from non-steatotic livers by having lower levels of TNF-RII. TNF-RII showed a trend which was inversely proportional to the pathological state of the tissue. The obese-piroxicam liver had the lowest level of TNF-RII and lean livers had the highest (p<0.05). The total NF-κB level was higher in the obese and obese-piroxicam groups compared to the lean or lean-piroxicam groups (p<0.05). Piroxicam treatment lowered the level of NF-κB in obese and lean livers. IκB-α was higher in obese livers than in lean livers. The nuclear level of NF-κB by western blot analysis showed the same pattern as noted in the whole tissue homogenate. However, the difference in the level between obese and lean was marked. The obese nuclei contained two to three fold higher levels of NF-κB protein than the lean liver nuclei. IκB-α level was significantly higher in the obese liver tissues and nuclei than their lean counterparts. While transcriptionally active NF-κB was higher (p<0.05) in the obese livers than in the lean livers, the difference between obese and lean groups was not as significant as that noted for the level of NF-κB assessed by western blot. This suggests that the proportion of active NF-κB present in the nuclear fraction is much higher in the lean than in the obese nuclei. Lipid raft was extracted and identified successfully from obese and lean livers. The total caveolin and flotillin levels were significantly higher in the liver lipid rafts of the obese-piroxicam than that of the other groups. This is the group that also exhibited higher steatosis. Piroxicam treatment significantly decreased the level of caveolin in the lean liver and significantly increased the level of flotillin in the obese liver. While COX-1 was not detectable, however, the level of COX-2 and TNF-RII in lipid raft was opposite to the level noted in the whole tissue homogenate. TNFRII was highest in the obese-piroxicam lipid raft and lowest in the lean-piroxicam lipid raft. TNF-RII, COX-2, IκB-α and NF-κB proteins were the molecules profoundly affected by the pathological state of the tissue and piroxicam treatment. This research is the first to report the presence of IκB-α in the nuclear compartment with a higher level in the nuclei and whole tissue in the obese liver than in the lean liver. This research demonstrates that TNF-α to NF-κB axis is altered in steatotic liver, and analysis of lipid rafts in steatotic and non-steatotic liver demonstrates that lipid rafts play a distinct role in modifying the biological availability of key proteins in the pathological state of liver steatosis.