Introdução: A alteração dos níveis normais de marcadores séricos e urinários ocorre na presença de dano renal associado à uropatia obstrutiva. Valores séricos e e urinários de TGF beta1 e CA 19-9 ainda não foram avaliados em modelo experimental de uropatia obstrutiva. Material e Métodos: Ratos foram divididos em sete grupos: referência, sham operation, nefrectomia unilateral, ligadura completa de ureter unilateral, obstrução parcial de ureter unilateral, obstrução parcial de ambos ureteres, nefrectomia unilateral associada à obstrução parcial do ureter contralateral. Morfometria renal e ureteral, concentrações séricas e urinárias de TGF beta1 e CA 19-9 e expressão tecidual renal de CA 19-9 foram analisadas. A correlação destes marcadores com os grupos submetidos a obstrução completa, obstrução parcial ou sem obstrução foi realizada. Resultados: Achados anatomopatológicos correlacionaram-se positivamente à intensidade da obstrução ureteral e negativamente aos níveis urinários de CA 19-9. Subexpressão acentuada do CA 19-9 foi observada em unidades renais com obstrução completa. Não foram encontradas diferenças estatisticamente significativas para os marcadores TGF beta1 urinário, TGF beta1 sérico e para o CA 19-9 sérico Conclusões: O CA 19-9 urinário correlacionou-se negativamente com o grau de obstrução ureteral. A análise imuno-histoquímica demonstrou a expressão do CA 19-9 no citoplasma das células epiteliais tubulares, sugerindo produção renal do marcador. O TGF beta1 sérico e urinário não apresentaram modificações de acordo com o grau de severidade e tempo de obstrução, o que pode estar relacionado a remodelamento renal menos intenso em resposta à uropatia obstrutiva nestes ratos / Introduction: Abnormal levels of serum and urinary markers occur in the presence of renal damage associated to obstructive uropathy. Urinary and serum TGFbeta1 and CA 19- 9 have not yet been evaluated in an experimental model of obstructive uropathy. Material and Methods: Rats were divided into seven groups: reference, sham operation, unilateral nephrectomy, complete unilateral ureteral obstruction, partial unilateral ureteral obstruction, partial bilateral ureteral obstruction, and unilateral nephrectomy with contralateral partial ureteral obstruction. Kidney and ureter morphometry, TGFbeta1 and CA 19-9 serum and urinary concentrations and CA 19-9 renal tissue expression were analysed. Correlation of these markers to complete, partial obstruction or unobstructed groups was performed. Results: Pathological findings correlated positively with the degree of ureteral obstruction, but negatively with urinary CA 19-9 levels. Marked underexpression of CA 19-9 was observed in kidneys with complete ureteral obstruction. No statistically significant differences were found for urinary and serum TGFbeta1 and also for serum CA 19-9. Conclusions: Urinary CA 19-9 correlated negatively with ureteral obstruction grade. Immunohistochemistry depicted CA 19-9 expression on epithelial tubular cells cytoplasm, suggesting renal origin. Serum and urinary TGFbeta1 did not show alterations in response to severity and length of urinary obstruction, which might be associated with less intense renal remodeling

Pacientes submetidos por longos períodos à diálise peritoneal (DP) podem evoluir com fibrose e redução da capacidade de ultrafiltração da membrana peritoneal (MP). Essas alterações da MP são desencadeadas pela exposição prolongada às soluções de diálise peritoneal, peritonites de repetição e irritantes químicos que induzem inflamação, neoangiogênese e fibrose da MP. A ativação da via Transforming Growth Factor (TGF-beta)/Smad é um fundamental mecanismo mediador da fibrogênese peritoneal. Sendo assim, drogas que inibam a via TGF-beta/Smad são de especial interesse no tratamento da FP. O ácido valpróico (VPA) é um inibidor das histona desacetilases (iHDAC), enzimas que regulam a conformação da cromatina e a expressão gênica, com atividade anti-inflamatória e antifibrótica. O presente estudo tem como objetivo principal avaliar o efeito do VPA em um modelo experimental de fibrose peritoneal em ratos. Vinte e quatro ratos Wistar machos (peso inicial de 280 - 320g) foram dividos em 3 grupos experimentais: CONTROLE (n=8), animais normais que receberam injeções de salina intraperitoneal (IP); FP (n=8), animais que recereberam injeções IP de gluconato de clorexidina (GC) diariamente por 15 dias para indução de fibrose peritoneal; FP+VPA (n=8), animais com FP e tratados com VPA. O ácido valpróico (300mg/kg) foi administrado por gavage diariamente por 15 dias, simultaneamente à indução de fibrose peritoneal. Ao fim dos experimentos, amostras do tecido peritoneal foram coletadas para realização de histologia, imunho-histoquímica (IH), imunofluorescência (IF) e biologia molecular. A análise da MP dos animais do grupo FP revelou um espessamento significativo da camada submesotelial devido ao acúmulo de matriz extracelular e infiltrado inflamatório. O tratamento com VPA foi capaz de prevenir significativamente o espessamento da MP, mantendo a espessura do peritôneo do grupo FP+VPA similar a do grupo CONTROLE. Com relação à função peritoneal, a administração de VPA evitou a queda da ultrafiltração e aumento do transporte peritoneal de glicose induzidos pelo GC. Além disso, o VPA impediu o aumento da expressão de miofibroblastos e de fatores associados à fibrose (TGF-beta, FSP-1 e fibronectina) induzidos pelo GC. Interessantemente, o VPA reduziu de maneira significativa a expressão da Smad3, mediador intracelular crítico da sinalização TGF-beta/Smad, em relação ao grupo FP. Por outro lado, os animais tratados com VPA apresentaram um aumento da expressão peritoneal de fatores antifibróticos como a BMP-7 e Smad7, proteínas que contrarregulam as ações do TGF-beta. Além de atenuar a fibrose peritoneal, o VPA apresentou efeitos anti-inflamatório e antiangiogênico, demonstrado pela menor expressão de citocinas pró-inflamatórias, fatores quimiotáticos para macrófagos (MCP-1) e VEGF no grupo FP+VPA quando comparado ao grupo FP. Em resumo, o VPA foi capaz de bloquear o espessamento por fibrose da MP e preservar a sua função, além de proteger o peritônio contra a neoangiogênese e inflamação. Além disso, o VPA induziu um aumento da expressão de fatores antifibróticos na MP. Os resultados apresentados neste trabalho chamam a atenção para mecanismos envolvidos nas modificações da MP induzidas pela DP ainda pouco explorados e que podem constiuir potenciais alvos na prevenção do desenvolvimento da fibrose peritoneal associada à DP / Long term peritoneal dialysis (PF) can induce peritoneal fibrosis and loss of ultrafiltration capacity of peritoneal membrane (PM). These peritoneal changes are due to prolonged exposure to peritoneal dialysis solutions, chemical irritants and acute peritonitis episodes that induce inflammation, neoangiogenesis and PM fibrosis. The Transforming Growth Factor (TGF-?) is the main mediator involved in the development of peritoneal fibrosis. Thus, drugs that inhibit the TGF-?/Smad pathway or inflammation are of particular interest in the treatment of PF. Valproic acid (VPA) is an histone deacetylase (HDAC) inhibitor. HDACs are enzymes that regulate chromatin conformation and gene expression. Recent studies have described HDACi as promising drugs in the treatment of inflammatory and fibrotic diseases. The main aim of this study was to evaluate the effect of VPA in an experimental model of peritoneal fibrosis in rats. Twenty four Wistar rats (initial weight of 280-320g) were divided into three experimental groups: CONTROL (n = 8), normal animals that received only saline ip; FP (n = 8), peritoneal fibrosis was induced by daily Gluconate Clorhexedine (GC) intraperitoneal (IP) injections for 15 days; FP+VPA (n = 8), animals with peritoneal fibrosis and treated with VPA. Daily valproic acid (300mg/kg) doses were administered by gavage simultaneously with the induction of peritoneal fibrosis in the FP+VPA group. At the end of experiments, the animals were submitted to euthanasia and samples of peritoneal tissue were collected for histology, immunocytochemistry, immunofluorescence, and molecular biology. Also, a functional peritoneal test was performed. The FP group showed a significant thickening of PM due to the accumulation of extracellular matrix and inflammatory cellular infiltration. VPA treatment was able to significantly prevent PM thickening, maintaining the peritoneal thickness of the VPA group similar to that of the CONTROL group. The VPA administration also preserved peritoneal function in the FP+VPA group, avoiding the reduction of ultrafiltration and increasing of peritoneal glucose transport induced by GC. According to the histological changes mentioned above, the VPA hampered the upregulation of the pro-fibrotic genes (TGF-beta, FSP-1, and fibronectin) and increase in the myofibroblasts expression induced by GC injections. Interestingly, the peritoneal expression of phosphorylated Smad3 detected by immunohistochemistry and Smad3 mRNA was significantly higher in the FP group. However, this effect was attenuated by VPA treatment. On the other hand, VPA was able to induce an increase in the expression of the antifibrotic factors, such as BMP-7 and Smad7, in the peritoneal membrane. Besides its antifibrotic activity, VPA also showed anti-inflammatory and anti-angiogenic effects. Animals of the FP+VPA group showed a significant reduction of the PM expression of pro-inflmmatory cytokines, macrophage chemoattractants and, VEGF expression when compared with FP group. In conclusion, we have shown that VPA inhibits the progression of peritoneal fibrosis in a CG-induced peritoneal fibrosis model in rats. VPA inhibited different and important mechanisms involved in peritoneal membrane modifications induced by PD, as activation of TGF-beta/Smad pathway, inflammation, and angiogenesis. Notably, VPA induced the expression of antifibrotic factors. Our results are very interesting and shed lights on a new perspective for the treatment of peritoneal fibrosis. However, this is an exploratory study and future studies are needed before to translate this experimental finding into clinical application

Em virtude dos pobres resultados obtidos no tratamento do Câncer de Pulmão, seja em estágios iniciais ou na doença avançada localmente, há a necessidade de se desenvolver marcadores moleculares e imunohistoquímicos que possam prever o comportamento tumoral. Ácido Hialurônico (HA) é um componente da matriz extracelular, responsável pela hidratação e manutenção do equilíbrio osmótico tecidual. Concentrações de HA estão elevadas em vários tipos de cânceres, incluindo pulmão. Hialuronidases (HAases), são uma família de enzimas relacionadas com a propagação de infecções bacterianas, toxinas de venenos e progressão tumoral. A quebra do HA em pequenos fragmentos (3-25 dissacarídeos) promovidos pela ação das HAases tipo Hyal1, Hyal2 e Hyal3, está relacionada à promoção do câncer através da indução da angiogênese e estímulo a proliferação através de ativação da via tirosina quinase. Algumas isoformas de HAases, descritas como produto de splicing alternativo, possuem atividade enzimática diversificada. A heterogeneidade de expressão das HAases foi identificada em alguns tipos de câncer e pode ser correlacionada com o comportamento diferenciado dos tumores. Em uma primeira instância, o perfil de expressão das HYAL foi avaliado em tecidos pulmonares tumorais e normais de 69 tumores ressecados de pacientes com adenocarcinomas (ADC) e carcinomas de células escamosas (CCE) oriundos do Hospital das Clinicas e Hospital do Câncer AC. Camargo. A expressão da HYAL1- selvagem (wt) e variantes 1 a 5, HYAL2-wt, HYAL3-wt e variantes 1 a 3 foi identificada por PCR e seqüenciamento direto. Diferentes proporções de HYAL3-wt e variantes foram expressas em tecidos pulmonares tumorais e controles. HYAL1-wt esteve associada com prognóstico desfavorável e HYAL3-v1 com prognóstico favorável. Diante dos resultados obtidos dos tumores de pacientes do Hospital das Clínicas e Hospital AC. Camargo, prosseguimos a investigação para estudar a imunoexpressão das Hyal 1 e 3 e HAS 1, 2 e 3 nos CCE e ADC. Observamos que a intensidade de expressão de Hyal 3 foi maior pelas células tumorais quando comparada aos controles, porém esta diferença foi marginalmente significante. Já o resultado da análise da freqüência de imunoexpressão das Hyal 1 e 3, e HAS1, 2 e 3 demonstrou expressão na maioria dos espécimes tumorais e controles. A associação entre as variáveis foi testada e evidenciou imunoexpressão concomitante de HYAL e HAS nos tumores. O modelo matemático de sobrevida , controlado para sexo, idade e estadiamento mostrou risco de morte associado com adenocarcinoma sólido e imunoreatividade para HAS2 e HAS3. Para validar os resultados obtidos, sobretudo com a imunoexpressão das Hyal e HAS nos CCE e ADC, estudamos a população de pacientes do Hospital Universitário de Coimbra. Documentamos pela primeira vez uma via pela qual a hiperexpressão de HAS3 e Hyal 3 respectivamente por células epiteliais neoplásicas e mesenquimais, podem favorecer a invasão nos ADC e CCE. Surpreendentemente, demonstramos que a imunoexpressão de HAS1 e 3 pelas células epiteliais neoplásicas confere mais agressividade aos ADC acinares e papilares, mas uma expressão negativa de HAS1 pelas células mesenquimais confere um papel protetor a MEC auxiliando-a a evitar a invasão pelas células tumorais em ambos os tipos subtipos histológicos. A interação entre a expressão das hialuronidades e sintases do àcido hialurônico foi avaliada em relação à expressão de proteínas da transição epitélio-mesênquimal nos tumores de pacientes do Hospital Universitário de Coimbra. Hyal, HAS, E-caderina e TGF- modularam uma via invasiva tumorinduzida nos ADC e CCE de pulmão, e estiveram associados a um espectro diferente de agressividade, uma vez que houve uma relação inversa entre a expressão de biomarcadores epiteliais e mesenquimais. Enquanto a hiperexpressão de HAS1 e HAS3 provê uma agressividade aos CCE e ADC, uma hiperexpressão de TGF- e E-caderina, confere um efeito protetor à MEC ao evitar a invasão por células tumorais em ambos os tipos histológicos. Comparamos os níveis de imunoexpressão das Hyal1 e 3 e HAS 1, 2 e 3 nos tumores ressecados no Hospital das Clínicas e Hospital AC. Camargo com os níveis obtidos em tumores do Hospital Universitário de Coimbra. Verificamos que a imunoexpressão das HAS 1, 2, 3 e Hyal1 foi significativamente maior nos tumores de pacientes do Hospital Universitário de Coimbra, enquanto que a imunoexpressão de Hyal 3 foi significativamente maior nos tumores de pacientes brasileiros. Por todas essas razões, nossos resultados sugerem que estratégias direcionadas à modulação dos níveis de HYAL1-wt e HYAL3-v1, da hiper imuno expressão de HAS3 e Hyal 3 respectivamente por células epiteliais neoplásicas e mesenquimais, da alta síntese de HAS3 e Hyal 1, ou a resposta local baixa de TGF- e E-caderina, poderão ter grande impacto no câncer de pulmão / Given the poor results obtained in the treatment of Lung Cancer, in early stages or locally advanced disease, there is a need to develop molecular markers and immunohistochemical studies that can predict tumor behavior. Hyaluronic Acid (HA) is a component of extracellular matrix is responsible for hydration and maintenance of tissue osmotic equilibrium. Concentrations of HA are elevated in several types of cancers, including lung. Hyaluronidases (HAases) are a family of enzymes involved in the spread of bacterial toxins, poisons and tumor progression. The breakdown of HA into small fragments (3-25 disaccharides) promoted by the action of type HAases Hyal1, Hyal 2 and Hyal 3 is related to the promotion of cancer by inducing angiogenesis and stimulate proliferation through activation of the tyrosine kinase. Some isoforms HAases, described as the product of alternative splicing, have diverse enzymatic activity. The heterogeneity of expression of HAases was identified in some cancers and can be correlated with the different behavior of tumors. In a first instance, the expression profile of Hyal spliced forms was evaluated in tumor and normal lung tissue of 69 tumors resected from patients with adenocarcinomas(ADC) and squamous cell carcinomas (SqCC) from the Hospital das Clínicas and Hospital AC. Camargo. Gene expression of HYAL1 wild-type (wt) and variants 1 to 5 HYAL2-wt, and HYAL3-wt and variants 1 to 3 was identified by PCR and direct sequencing. Different proportions of HYAL3-wt and variants were expressed in tumor and normal lung tissue. HYAL1-wt was associated with unfavorable prognosis and HYAL3-v1 with favorable prognosis. Given the genetic abnormalities found in tumors of patients from Hospital das Clinicas and Hospital AC. Camargo, we continued our research to study the expression of Hyal 1.3 and HAS 1, 2, 3 in squamous cell carcinomas and adenocarcinomas. We observed that the intensity of expression of Hyal 3 was higher in tumor cells compared to controls, but this difference was marginally significant. Since the result of frequency analysis of immunoreactivity of Hyal 1 and 3, and HAS1, 2 e 3 showed expression in the majority of tumor samples and controls. The association between variables was tested and showed concomitant immunoexpression of the HAS and HYAL in tumors. The mathematical model of survival, adjusted for sex, age and staging showed risk of death associated with adenocarcinoma and solid and HAS3 HAS2 immunoreactivity.To validate the results, especially with the immunostaining of Hyal and HAS in squamous cell carcinomas and adenocarcinomas of the lung, the patient population studied at the University Hospital of Coimbra. Documented for the first time a route by which the overexpression of HAS3 and Hyal 3 respectively by neoplastic epithelial and mesenchymal cells may favor the invasion in ADC and SqCC, respectively. Surprisingly, we demonstrated that hyper HAS1 and 3 immunoreactivity by neoplastic epithelial cells confers more aggressiveness to the ADC acinar and papillary, but a negative expression of HAS1 by mesenchymal cells confers a protective role ECM-helping to prevent the invasion by tumor cells in both types histological subtypes.The interaction between the expression of hialuronidades and hyaluronic acid synthases was evaluated for protein expression of epithelial-mesenchymal transition in tumor patients at the University Hospital of Coimbra. Hyaluronidase, hyaluronan synthase, Ecadherin, and TGF- modulated via an invasive tumor-induced in the ADC and SqCC lung, and were associated with a different spectrum of aggressiveness, since there was an inverse relationship between the expression of epithelial biomarkers and mesenchymal cells. While overexpression of HAS1 and HAS3 provides an aggressiveness to SqCC and ADC, an overexpression of TGF- and E-cadherin confers a protective effect by preventing the ECM invasion by tumor cells in both histological types. We compared the levels of immunostaining Hyal 1, 3 and HAS1, 2 and 3 in tumors resected at the Hospital AC. Camargo, and the levels obtained in tumors of the Hospital Universitário de Coimbra. We found that the immunostaining of HAS 1, 2, 3 and Hyal1 was significantly higher in tumors from patients of Coimbra, while Hyal 3 immunoreactivity was significantly. higher in tumors of patients in Brazil. For all these reasons, our results suggest that strategies directed at modulating the levels of HYAL1-wt and HYAL3-v1, the immunohistochemical expression of HAS3 and Hyal 3 respectively by neoplastic epithelial and mesenchymal cells, the synthesis of HAS3 and Hyal1 or the local response of low TGF- and E-cadherin, may have great impact on lung cancer

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-07-01T13:51:16Z No. of bitstreams: 1 karinehelenadesouzalopes.pdf: 1140714 bytes, checksum: b276a86b4727bee8ef060477fc027e6b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-13T16:01:04Z (GMT) No. of bitstreams: 1 karinehelenadesouzalopes.pdf: 1140714 bytes, checksum: b276a86b4727bee8ef060477fc027e6b (MD5) / Made available in DSpace on 2016-07-13T16:01:04Z (GMT). No. of bitstreams: 1 karinehelenadesouzalopes.pdf: 1140714 bytes, checksum: b276a86b4727bee8ef060477fc027e6b (MD5) Previous issue date: 2012-08-27 / O laser de baixa intensidade e o ultrassom terapêutico têm se mostrado opções para modulação da cicatrização, porém, os mecanismos de ação destas técnicas não são bem esclarecidos. Para avaliar os efeitos da laserterapia e do ultrassom terapêutico em feridas cutâneas cirúrgicas em ratos Wistar (n=24), foram utilizados quatro grupos: I (controle), II (LLLT), III (ultrassom) e IV (laserterapia e ultrassom). No décimo dia, as lesões foram fotografadas e medidas e, após removidas excisionalmente no momento da eutanásia, foram processadas para avaliação histopatológica para avaliação da densidade e organização das fibras colágenas; avaliação histomorfométrica para quantificação da angiogênese e infiltrado inflamatório; e imunoistoquímica, para expressão de TGFβ1. As amostras dos grupos tratados exibiram aspecto macroscópico mais maduro em relação ao grupo não tratado, sem diferença significativa no fechamento das lesões; microscopicamente, os resultados sugeriram que a laserterapia exerceu melhor efeito imunomodulador quando utilizado isoladamente e que o ultrassom terapêutico mostrou maior potencial angiogênico. A avaliação imunoistoquímica revelou que a maioria das células inflamatórias na área cicatricial não expressava TGFβ1. Ainda, embora a laserterapia e a aplicação do ultrassom atuem diretamente na redução do infiltrado, as terapias concomitantes não potencializam o efeito observado quando aplicadas isoladamente; apesar do tempo de fechamento das feridas não ter sido influenciado pelas terapias isoladas ou associadas, todos os tratamentos favoreceram a organização da matriz extracelular colagenosa. A LLLT isoladamente e a combinação de ambas as técnicas possibilitaram a reepitelização das feridas submetidas a estas modalidades terapêuticas. A maioria das células que migraram ou entraram em proliferação na área cicatricial não expressavam o TGFβ1, sugerindo que o controle do infiltrado inflamatório exercido pela LLLT e pelo UST não é modulado por esta citocina. / Low level laser therapy (LLLT) and therapeutic ultrasound have been demonstrated to be options for healing modulation, but the mechanisms of action involved in these processes are not clear. The effects of laser therapy and therapeutic ultrasound on surgical skin wounds in Wistar rats (n=24) were evaluated using four groups: I (control), II (LLLT), III (ultrasound) and IV (laser therapy and ultrasound). On the tenth day, the wounds were photographed and measured, and after excision at the moment of euthanasia, they were processed for histopathological evaluation to assess the density and organization of collagen fibers. In addition, histomorphometric evaluations were conducted to quantify angiogenesis and inflammatory infiltrates, and immunohistochemistry was performed to assess TGF1 expression. The samples from the treated group had a more mature macroscopic appearance compared to the untreated group, with no significant difference in wound closure. Microscopically, the results suggested that the laser therapy had a better immunomodulatory effect when used alone and that the therapeutic ultrasound showed a higher angiogenic potential. The immunohistochemical evaluation revealed that most of the inflammatory cells in the scar area did not express TGF1. Still, although the application of laser therapy and ultrasound act directly in the reduction of infiltration, the concomitant therapies do not potentiate the effect observed when applied in isolation, despite the time of wound closure was not influenced by therapy alone or associated, all treatments favored collagenous extracellular matrix organization. The LLLT alone and the combination of both techniques allowed the re-epithelialization of wounds under these treatment modalities. The majority of cells that migrated in the proliferation or entered in the cicatricial area is not expressed TGFβ1, suggesting that the control exercised by the inflammatory infiltrate and the LLLT UST is not modulated by this cytokine.

Orientador: Ricardo Della Coletta / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T21:07:52Z (GMT). No. of bitstreams: 1 Bufalino_Andreia_D.pdf: 2552060 bytes, checksum: 9156e1c08764322bea090809a2b166d5 (MD5) Previous issue date: 2013 / Resumo: Ativina A é um membro da família dos fatores de crescimento transformante-? e sua expressão têm sido associados ao desenvolvimento tumoral. Contudo, sua expressão, função e mecanismos de regulação, por exemplo, via folistatina, no carcinoma espinocelular (CEC) oral é parcialmente conhecida. Diante disto, nosso estudo teve como objetivo avaliar a participação de ativina A na tumorigênese dos CECs orais. Para alcançar este objetivo, a expressão imuno-histoquímica de ativina A foi analisada em 115 amostras de CEC oral e sua expressão foi correlacionada com características clínico-patológicas e de sobrevida. In vitro a influência de ativina A sobre os principais eventos biológicos relacionados à tumorigênese oral foi verificada por 3 abordagens: 1) exposição da linhagem celular HaCAT a ativina A recombinante nas concentrações de 0, 1, 10 e 100 ng/ml, 2) tratamento das linhagens tumorais LN2 com folistatina recombinante nas concentrações de 0, 1, 10 e 100 ng/ml e 3) silenciamento estável da expressão de ativina A na linhagem LN2 com RNA de interferência (shINHBA). A expressão aumentada de ativina A em CECs orais foi significantemente correlacionada com a presença de metástases regionais (estádio N, p=0,034), tumores classificados como pobremente diferenciados (p=0,013) e demonstrou ser preditiva de um menor período de sobrevida livre de doença em 5 anos (HR: 1,74; 95% CI: 1,39-2,97; p=0,016). Os resultados dos estudos in vitro revelaram que ativina A apresenta um efeito pleotrópico no controle dos principais eventos associados à tumorigênese oral. A exposição das células HaCAT a ativina A resultou em um significante bloqueio da morte celular por apoptose e necrose, promoveu alteração do padrão de expressão dos marcadores da transição epitélio mesenquimal (TEM), aumentou a adesão celular aos componentes da matriz extracelular (MEC) e induziu a invasão e migração celular. Por outro lado, o tratamento das células LN2 com folistatina foi capaz de induzir significantemente a apoptose e a morte celular por necrose, reduzir a proliferação celular, alterar o padrão de expressão dos marcadores da TEM, além de reduzir a adesão celular aos componentes da MEC e os potenciais invasivo e migratório. O bloqueio de ativina A com a transdução estável de shINHBA na linhagem tumoral LN2 promoveu significantemente a apoptose e a morte por necrose, alterou a expressão dos marcadores da TEM de maneira similar aos efeitos da folistatina e reduziu a proliferação, invasão, migração e motilidade celular, que foi avaliada por formação de filopódios e lamelipódios. Interessantemente, o bloqueio com shINHBA significantemente facilitou a adesão das células LN2 aos componentes da MEC, diferente do que foi observado no tratamento com folistatina. Em conclusão, os resultados deste estudo sugerem que a ativina A regula eventos biológicos essenciais para a tumorigênese oral e é um fator prognóstico independente de sobrevida livre de doença em pacientes com CECs orais / Abstract: Activin A is a member of the transforming growth factor-? family and its deregulated expression has been described in different cancers. However, its expression, function and regulatory mechanisms, particularly via follistatin, in oral squamous cell carcinoma (OSCC) are partially known. The aim of the present study was to evaluate the role of activin A in the promotion of oral tumorigenesis. To achieve this goal, immunohistochemical expression of activin A was analyzed in 115 samples of OSCCs and its expression was correlated with clinicopathological features and outcome. In vitro, the influence of activin A on oral tumorigenesis was determined by 3 different approaches: 1) exposition of HaCaT cells to recombinant activin A in different concentrations (0, 1, 10 and 100 ng/ml), 2) treatment of LN2 tumor cells with recombinant follistatin in different concentrations (0, 1, 10 and 100 ng/ml) and 3) stable knockdown of activin A expression in the LN2 tumor cells by using interferencing RNA (shINHBA). Increased activin an expression in OSCCs was significantly correlated with the presence of regional metastases (stage N, p=0.034), poorly differentiated tumors (p=0.013), and shown to be predictive of a shortened disease-free survival (HR: 1.74, 95% CI: 1.39-2.97, p=0.016). In vitro studies showed a pleotropic effect of activin an on control of key events associated with oral tumorigenesis. Activin A resulted in a reduction of cell death by apoptosis and necrosis, promoted changes in the expression of epithelial-mesenchymal transition (EMT) markers, increased cell adhesion to extracellular matrix (ECM) components and induced cell invasion and migration in HaCAT cells. On the other hand, the inhibition of activin a using follistatin induced apoptosis and cell death by necrosis, reduced cell proliferation, changed the expression of EMT markers, reduced cell adhesion to ECM components and reduced the cell invasion and migration in LN2 cells. The activin a knockdown with shINHBA stable transduction in the tumor cell line LN2 significantly promoted death by apoptosis and necrosis changed the expression of EMT markers, and decreased cell proliferation, invasion, migration and motility evaluated by lamellipodia and filopodia formation. Interestingly, knockdown with shINHBA significantly promoted the adhesion of cells to ECM components in LN2 cells, different to the results observed in the treatment with follistatin. In conclusion, our results suggest that activin A regulate biological events essential for oral tumorigenesis, and is an independent prognostic factor for disease-free survival in patients with OSCCs / Doutorado / Patologia / Doutora em Estomatopatologia

Orientador: Cláudio Chrysostomo Werneck / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T04:57:03Z (GMT). No. of bitstreams: 1 Nery-Diez_AnaClaudiaCoelho_D.pdf: 21192177 bytes, checksum: 572bd54be1e7496c1fd325e94812ecc1 (MD5) Previous issue date: 2013 / Resumo: As mutações no gene da fibrilina-1, presente na fibra elástica, estão relacionadas à síndrome de Marfan, doença genética autossômica dominante. Acredita-se que a maioria dos seus sintomas esteja relacionada a uma hiper-ativação do fator TGF-?. Quando camundongos modelo para síndrome são tratados com losartan apresentam uma melhora significativa nos sinais clínicos. Assim, neste estudo, investigamos o papel de fibrilina-1 na análise de trombose arterial em modelos de camundongos para a síndrome de Marfan (Fbn1mg?/+). Foram analisados a formação de trombos, os níveis de plaquetas, APTT, PT e TT, agregação e adesão plaquetária, parâmetros hemodinâmicos, níveis de TGF-?, atividade das MMPs, bem como a morfologia das plaquetas, das fibras elásticas e dos trombos. Foi observado que os animais Fbn1mg?/+ necessitam de cerca de 120±21,07 minutos para formarem o trombo, enquanto os animais selvagens precisam de 58±7,16 minutos. Mas, quando os animais Fbn1mg?/+ foram tratados com anti-hipertensivos losartan e captopril, ocorreu uma recuperação no tempo de formação do trombo, com redução de 57,5% e 67,5% no tempo, respectivamente. Além disso, constatou-se que os animais Fbn1mg?/+ apresentam um aumento na atividade das MMPs e TGF-? ativos, quando tratados com os anti-hipertensivos, houve uma diminuição apenas na atividade das MMPs. Ademais, não foi verificada diferença significativa entre todos os outros parâmetros analisados. Este estudo sugere que, de alguma forma, as drogas interferem na remodelação da matriz elástica, devido a uma diminuição da atividade das metaloproteinases de matriz e, consequentemente, leva á recuperação na formação do trombo / Abstract: Recent works show that an increased activation of the TGF-? is associated with most of the symptoms of the Marfan syndrome. Studies using mouse models of Marfan treated with losartan have been shown to prevent the degradation of the elastic matrix. Other investigators suggest that the metalloproteinases and the noncanonical ERK signaling are involved in the breakdown of the elastic fiber, which results in the aneurysm. In this study we investigated the role of fibrillin-1 in the arterial thrombosis model using mouse models of Marfan. We analyzed thrombus formation, platelet levels, APTT, PT and TT time, hemodynamic parameter, TGF-? levels, MMP activity as well as platelets, elastic fibers morphology and thrombus. This study demonstrated that Fbn1mg?/+ mice take about 120±21,07 minutes for the thrombus to be formed when compared with wild type, 58±7,16. When these Fbn1mg?/+ mice were treated with antihypertensive losartan and captopril the time taken for the thrombus formation was reduced in 57,5% and 67,5%, respectively. The activity of metalloproteinases and activity TGF-? was increased in the Fbn1mg?/+, however no significant difference between the hemodynamic parameters, levels of totals TGF-?, morphological platelets, elastic fiber and thrombus were observed. Finally, the results suggested that fibrillin-1 interferes with this process and antihypertensive affect the physiology of Fbn1mg?/+ mice. This study suggested that somehow drugs interfere with elastic matrix remodeling due to a decrease in the activity of matrix metalloproteinases which results in the recovery time of thrombus formation / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Orientador: Maria Julia Marques / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T07:19:08Z (GMT). No. of bitstreams: 1 Moreira_DrielendeOliveira_M.pdf: 2075338 bytes, checksum: 00bb5917e4f176b73c58453340cb3a5e (MD5) Previous issue date: 2012 / Resumo: A Distrofia muscular de Duchenne (DMD) é uma doença caracterizada pela fraqueza muscular progressiva que leva à insuficiência respiratória e cardíaca, resultando em morte por volta dos 30 anos de idade. No camundongo mdx, modelo experimental da DMD, os músculos diafragma e cardíaco são severamente afetados apresentando fibrose semelhante à observada na patologia humana. O objetivo deste trabalho foi investigar os efeitos do tratamento a longo prazo com suramina, uma droga anti-fibrótica, nos músculos diafragma e cardíaco de camundongos mdx idosos. Camundongos mdx (n=20; 8 meses de idade) receberam injeções intraperitoneais de suramina (60 mg/kg), durante 3 meses. Controles mdx (n=20; 8 meses) e C57BL/10 (n=18; 8 meses) foram injetados com solução salina. Os camundongos da linhagem C57BL/10 expressam distrofina e são utilizados como controle da linhagem mdx. A suramina diminuiu os níveis de CK e atenuou a perda da força muscular. No músculo diafragma, a suramina reduziu a área de fibrose e a mionecrose. No músculo cardíaco, houve redução da fibrose, da inflamação e melhora significativa de parâmetros funcionais cardíacos (amplitude das ondas P, Q, R e S do eletrocardiograma). Sugere-se que a suramina possa ser potencialmente útil nas distrofinopatias, atenuando a miopatia nos músculos mais afetados, o coração e o diafragma, nos estágios tardios da doença / Abstract: Duchenne muscular dystrophy (DMD) is a disease characterized by progressive muscle weakness leading to respiratory and cardiac failure, resulting in death around 30 years of age. In the mdx mice model of DMD, diaphragm and cardiac muscles are severely affected in the later stages of the disease, showing intense fibrosis similar to that observed in human pathology. The aim of the present study was to investigate the effects of long-term treatment with suramin, an anti-fibrotic agent, in the diaphragm and cardiac muscles of the mdx mice. Mdx mice (n=20; 8 months of age) received intraperitoneal injections of suramin (60 mg/kg) for 3 months. Mdx controls (n=20; 8 months) and C57BL/10 (n=18; 8 months old) were injected with saline. C57BL/10 mice express dystrophin and are the control strain for the mdx mice. Suramin decreased CK levels and reduced the loss of muscle strength. Suramin reduced fibrosis and myonecrosis in diaphragm. In the cardiac muscle, suramin decreased fibrosis, inflammation and improved cardiac functional parameters (P, Q, R and S waves of the electrocardiogram). It is suggested that suramin may be a potential therapy for distrophinopaties, attenuating the dystrophic phenotype of the most affected cardiac and diaphragm muscles of the mdx mice, during later stages of the disease / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

The heat shock response (HSR) is a robust cellular reaction to mitigate protein damage from heat and other challenges to the proteome. This protective molecular program in humans is controlled by heat shock transcription factor 1 (HSF1). Activation of HSF1 leads to the induction of an array of cytoprotective genes, many of which code for chaperones. These chaperones, known as heat shock proteins (HSPs), are responsible for maintaining the functional integrity of the proteome. HSPs achieve this by promoting proper folding and assembly of nascent proteins, refolding denatured proteins, and processing for degradation proteins and aggregates which cannot be returned to a functional conformation. The powerful ability of the heat shock response to promote cell survival makes its master regulator, HSF1, an important point of research. To garner a better understanding of HSF1, we reviewed the role of the highly dynamic HSF1 protein structure and investigated how HSF1 affects cancer cell behavior and drug response. Cancers can be characterized in part by abhorrent replication, self-sufficient growth signaling, invasion, and evasion of apoptosis. HSF1 has been found to promote proliferation, invasion, and drug resistance in several types of cancer; including lung and ovarian cancer. Ovarian cancer has elevated levels of HSF1, but the role of HSF1 in ovarian cancer behavior had not been previously examined. Researching the role of HSF1 in ovarian cancer is merited, because treatment outcomes are poor due to the high frequency of late stage detection and drug resistance. We hypothesized that HSF1 is important in the malignant growth and drug resistance of ovarian cancer. We have created ovarian cancer cell lines with inducible knockdown of HSF1 to investigate how HSF1 contributes to the behavior of ovarian cancer. This allowed us to examine the behavior of cells in the absence HSF1. Both 2D and 3D spheroid tissue culture models were used to study how HSF1 contributes to the growth and invasion of ovarian cancer cells after treatment with the transforming growth factor β (TGFβ) cytokine. Additionally, we studied how HSF1 reduction modulates the response to multiple therapeutic drugs. Our research shows that HSF1 induces epithelial-mesenchymal transition (EMT) in a 3D growth model. Our work also demonstrates that reduction of HSF1 sensitizes ovarian cancer cells to multiple drugs.

La fibrose pulmonaire peut être induite par différentes agressions comme certaines chimiothérapies et notamment la bléomycine. Elle est souvent idiopathique (FPI) sans étiologie retrouvée. Cette maladie, qui n’a pas de traitement efficace et un pronostic sombre, débute dans les régions sous-pleurales. Elle est caractérisée par la présence de myofibroblastes responsables de la synthèse de la matrice extracellulaire qui va s’accumuler de façon disproportionnée. Ce dépôt excessif de collagène conduira à une perte des fonctions mécaniques du poumon et une limitation des échanges gazeux. L’origine des myofibroblastes est encore discutée mais une hypothèse récente propose que les cellules épithéliales puissent, sous l’influence de Transforming Growth Factor (TGF)-β1, une cytokine majeure du processus fibrosant, se transformer en myofibroblastes par un procédé nommé transition épithélio-mésenchymateuse (EMT). Dans ce travail, nous nous sommes intéressés dans un premier temps au rôle de la plèvre dans l’initiation de la FPI en développant un nouveau modèle de fibrose pleurale, puis nous avons étudié l’implication de la petite protéine de choc thermique HSP27 dans l’EMT. Enfin, nous nous sommes intéressés à la toxicité d’un dérivé de la bléomycine, la déglyco-bléomycine. Nous avons montré dans le premier projet que la bléomycine, quelque soit son mode d’administration, pouvait, si elle était associée à la présence de nanoparticules de carbone dans l’espace pleural (particules présentes dans la fumée de cigarette et dans la pollution), induire une fibrose pleurale progressive. Cette fibrose pleurale ne se limitait pas à une accumulation de collagène au niveau de la plèvre mais se propageait dans les régions sous-pleurales. Cette invasion de la fibrose s’expliquait par la capacité des cellules mésothéliales à subir une EMT. Dans une deuxième étude nous avons montré qu’HSP27 était fortement surexprimée au niveau de la plèvre et dans le parenchyme pulmonaire lors de la fibrose pulmonaire idiopathique et également dans nos différents modèles animaux de fibrose pulmonaire et pleurale. Nous avons montré in vitro qu’HSP27 était aussi fortement surexprimée au cours de l’EMT et que sa surexpression seule suffisait à induire une EMT. Au contraire, l’inhibition de son expression in vitro bloquait l’induction d’une l’EMT par le TGF-β1 et in vivo empêchait le développement de la fibrose pleurale et la migration des cellules mésothéliales dans le parenchyme pulmonaire. Enfin dans la troisième partie de notre travail, nous avons démontré chez le rongeur que la déglyco-bléomycine, dérivé de la bléomycine, avait le même effet anti-tumoral que cet anticancéreux mais sans sa toxicité pulmonaire fibrosante. Notre travail met en lumière le rôle probable de la plèvre dans l’initiation de la fibrose pulmonaire idiopathique. Nous espérons que nos travaux sur HSP27 et sur la déglyco-bléomycine vont pouvoir rapidement déboucher sur des applications thérapeutiques chez l’homme. / Pulmonary fibrosis could be induced by a number of injuries like chemotherapy (i.e. bleomycin) or could be idiopathic (IPF). This disease has currently no treatment. IPF classically starts in sub-pleural areas and is characterized by the presence of myofibroblasts producing the extracellular matrix that will accumulated into the parenchyma resulting in impaired lung functions and respiratory failure. The origin of the myofibroblasts is still debated but one recent hypothesis suggests that epithelial cells could become myofibroblasts through the epithelial-to-mesenchymal transition (EMT). This process is initiated by Transforming Growth Factor (TGF)-β1 one of the most, if not the most important cytokine involved in fibrotic process. In this work we focused on the role of the pleura in the onset of IPF and developed a new pleural fibrosis model in mice. We studied the implication of the heat shock protein 27 (HSP27) in pulmonary fibrotic processes. We then focused on the pulmonary toxicity of the deglyco-bleomycin a product derived from the anticancerous bleomycin. We demonstrated in the first part of our work that bleomycin was able, in combination with the presence of carbon nanoparticules in the pleural space (found in cigarette’s smoke and pollution), to induce a progressive pleural fibrosis. This pleural fibrosis was associated with a progression of the disease within the subpleural area as observed in IPF patients. This invasion within the parenchyma was explained by the fact that mesothelial cells undergo an EMT. In the second part of our work, we showed that HSP27 was overexpressed in the parenchyma and the pleura of IPF patients and also in all our pleural and pulmonary fibrosis models in rodents. Furthermore we established in vitro that HSP27 was also overexpressed during EMT and that its overexpression was sufficient to induce EMT. Additionally HSP27 inhibition blocks TGF-β1 induced EMT in vitro and blocks pleural fibrosis development and mesothelial cells migration within the parenchyma in vivo. In the last project we demonstrated that deglyco-bleomycin had the same anti-tumoral effect than bleomycin in vivo and was devoided from its pulmonary toxicity. This work highlights the potential role of the pleura in initiating IPF and may open fields for the development of new therapeutics by preventing pulmonary fibrosis initiation but also progression.

The Smad proteins transduce the signal of transforming growth factor-β (TGF-β) and related factors from the cell surface to the nucleus. Following C-terminal phosphorylation by a corresponding receptor kinase, the R-Smad proteins form heteromeric complexes with Smad4. These complexes translocate into the nucleus, bind specific transcriptional activators and DNA, ultimately modulating gene expression. Though studied through a variety of means, the stoichiometry of the R-Smad/Smad4 complex is unclear. We investigated the stoichiometry of the phosphorylation-induced R-Smad/Smad4 complex by using acidic amino acid substitutions to simulate phosphorylation. Size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry analysis revealed that the R-Smad/Smad4 complex is a heterotrimer consisting of two R-Smad subunits and one Smad4 subunit. In addition, a specific mechanism for phosphorylation-induced R-Smad/Smad4 complex formation was studied. Although it had been previously established that part of the mechanism through which phosphorylation induces Smad oligomerization is through relieving MH1-domain mediated autoinhibition of the MH2 (oligomerization) domain, it is also evident that phosphorylation serves to energetically drive Smad complex formation. Through mutational and size exclusion chromatography analysis, we established that phosphorylation induces oligomerization of the Smads by creating an electrostatic interaction between the phosphorylated C-terminal tail of one R-Smad subunit in a Smad trimer with a basic surface on an adjacent R-Smad or Smad4 subunit. The basic surface is defined largely by the L3 loop, a region that had previously been implicated in R-Smad interaction with the receptor kinase. Furthermore, the Smad MH2 domain shares a similar protein fold with the phosphoserine and phosphothreonine-binding FHA domains from proteins like Rad53 and Chk2. Taken together, these results suggest that the Smad MH2 domain may be a distinct phospho serine-binding domain, which utilizes a common basic surface to bind the receptor kinase and other Smads, and takes advantage of phosphorylation-induced allosteric changes dissociate from the receptor kinase and oligomerize with other Smads. Finally, the structural basis for the preferential formation of the R-Smad/Smad4 heterotrimeric complex over the R-Smad homotrimeric complex was explored through X-ray crystallography and isothermal titration calorimetry. Crystal structures of the Smad2/Smad4 and Smad3/Smad4 complexes revealed that specific residue differences in Smad4 compared to R-Smads resulted in highly favorable electrostatic interactions that explain the preference for the interaction with Smad4.