A conexina 32 (Cx32) é uma estrutura protéica que constitui os canais que promovem as comunicações intercelulares via junções comunicantes (GJIC), permitindo difusão de pequenas moléculas citoplasmáticas de uma célula à outra. Este trabalho objetivou os estudos destas estruturas devido a sua importância em processos hepáticos, mais especificamente, a fibrose hepática. O presente estudo foi realizado através da administração oral da droga hepatotoxica dimetilnitrosamina (DMN) em ratas Wistar duas vezes por semana em dias consecutivos no prazo de cinco semanas. A necropsia destes animais foi realizada após cinco semanas da última administração da droga e revelou um quadro de fibrose hepática, em contra partida aos resultados obtidos em um grupo controle com a mesma quantidade de animais. O material fibrótico foi submetido à análise imunohistoquímica que revelou uma presença preferencial de Cx32 dispersa no citoplasma, o que pode levar à hipótese de problemas no mecanismo de transporte citoplasmático destas estruturas, em contrapartida ao material pertencente ao grupo controle que evidenciou a presença das Cx32 na membrana plasmática formando placas juncionais. Quando submetido à análises moleculares o fígado fibrótico revelou uma diminuição da expressão gênica embora o produto protéico deste material quando comparado ao grupo controle não tenha se mostrado diminuído. / The connexin 32 (Cx32) is a proteic structure that constitute the channels that promote the cell communication by means of the gap junction (GJIC), allowing the diffusion of short cytoplasmic molecules from a cell to another. This work aimed to study these structures due to their importance in the hepatic metabolic processes. The hepatic fibrosis was triggered by the oral administration of dimethylnitrosamine (DMN) in the female rat Wistars twice a week in consecutive days during five weeks. The necropsy of these animals was carried out after the last drug administration. They presented a hepatic fibrosis state. The fibrotic material was submitted to the imunohistochemical analysis, which showed a preferencial presence of Cx32 in the cytoplasm, whereas in the control group the Cx32 was located at the membranes, in the junctional plaques. The molecular analysis showed a decrease of the genic expresson of the fibrotic material, however the proteic product wasn? t reduced in comparison with the control group as it was shown by western blot. We concluded that the fibrotic state introduced a disturbance in the intracellular distribution and genic expression of the connexin 32.

This study has two aspects. One concerns the development of a sensitive differential pulse polarographic (DPP) method and its use to conduct a preliminary study of natural waters to detect the presence of nitrosoamines, and the second, where the study was carried out in greater detail, is the investigation of the kinetics of reduction of N-nitrosodimethylamine (NDMA) in aqueous solution. An optimization study was first conducted to identify the conditions that give the greatest sensitivity. Conditions investigated were different supporting electrolytes and solvent mixtures. Supporting electrolytes used were 0.10 M solutions of acids, bases and salts, and the solvent mixtures studied were ethanol/water mixtures. The effect of various supporting electrolytes and various ethanol/water mixtures on peak current was studied in order to select the conditions that give the greatest sensitivity for the determination of nitrosoamine concentrations. The lowest nitrosoamine concentration that can be detected in a solution (limit of detection) was determined. The optimum supporting electrolytes for N-nitrosoamines and the C-nitrosoamine were respectively found to be 0.10 M H2S04 and 0.10 M NaOH. Aqueous solutions were also found to give greater sensitivity than ethanol/water mixtures. The presence of nitrosoamines in some natural water samples was then checked using differential pulse polarography (DPP).Concentrations of nitrosoamines in these water samples were determined using the optimum conditions. The analyte analytes studied were 4-nitroso-N,N-dimethylaniline (4NDMA); Nnitrosodibutylamine (NDBA),N-nitrosodi-n-propylamine (NDPA) and N-nitrosodiphenylamine (NDPhA). All four nitrosoamines were detected in some of the selected natural water samples . The kinetics of the electrochemical reduction of NDMA was studied using controlled potential coulometry, linear scan voltammetry (LSV) and cyclic voltammetry (CV). Controlled potential coulometry was first used to determine the number of electrons transferred (n) in the reduction reaction. Two moles of electrons were found to be required for the reduction of one mole of NDMA solution, at a stationary mercury drop electrode (SMDE). The major product obtained, identified by UVNIS spectroscopy, was dimethylamine (DMA). / Ph.D. (Chemistry) North-West University, Mafikeng Campus, 2005

A conexina 32 (Cx32) é uma estrutura protéica que constitui os canais que promovem as comunicações intercelulares via junções comunicantes (GJIC), permitindo difusão de pequenas moléculas citoplasmáticas de uma célula à outra. Este trabalho objetivou os estudos destas estruturas devido a sua importância em processos hepáticos, mais especificamente, a fibrose hepática. O presente estudo foi realizado através da administração oral da droga hepatotoxica dimetilnitrosamina (DMN) em ratas Wistar duas vezes por semana em dias consecutivos no prazo de cinco semanas. A necropsia destes animais foi realizada após cinco semanas da última administração da droga e revelou um quadro de fibrose hepática, em contra partida aos resultados obtidos em um grupo controle com a mesma quantidade de animais. O material fibrótico foi submetido à análise imunohistoquímica que revelou uma presença preferencial de Cx32 dispersa no citoplasma, o que pode levar à hipótese de problemas no mecanismo de transporte citoplasmático destas estruturas, em contrapartida ao material pertencente ao grupo controle que evidenciou a presença das Cx32 na membrana plasmática formando placas juncionais. Quando submetido à análises moleculares o fígado fibrótico revelou uma diminuição da expressão gênica embora o produto protéico deste material quando comparado ao grupo controle não tenha se mostrado diminuído. / The connexin 32 (Cx32) is a proteic structure that constitute the channels that promote the cell communication by means of the gap junction (GJIC), allowing the diffusion of short cytoplasmic molecules from a cell to another. This work aimed to study these structures due to their importance in the hepatic metabolic processes. The hepatic fibrosis was triggered by the oral administration of dimethylnitrosamine (DMN) in the female rat Wistars twice a week in consecutive days during five weeks. The necropsy of these animals was carried out after the last drug administration. They presented a hepatic fibrosis state. The fibrotic material was submitted to the imunohistochemical analysis, which showed a preferencial presence of Cx32 in the cytoplasm, whereas in the control group the Cx32 was located at the membranes, in the junctional plaques. The molecular analysis showed a decrease of the genic expresson of the fibrotic material, however the proteic product wasn? t reduced in comparison with the control group as it was shown by western blot. We concluded that the fibrotic state introduced a disturbance in the intracellular distribution and genic expression of the connexin 32.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Abstract: Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFN-γ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFN-γ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFN-γ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in H SC activation. Liver fibrosis was induced in Socs1[superscript -/-]Ifng[superscript -/-] mice with dimethylnitrosamine or carbon tetrachloride. Ifng[superscript -/-] and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1[superscript -/-]Ifng[superscript -/-] mice showed elevated serum ALT levels and increased liver fibrosis com-pared to mice Ifng[superscript -/-]. The latter group showed higher alanine aminotransferase (ALT) levels and fibrosis than C57BL/6 controls. The livers of Socs1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. Socs1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from Socs1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of Socs1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFN-γ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability. / Résumé: Le suppresseur de la signalisation des cytokines 1 (SOCS1) est un régulateur indispensable de la signalisation de l'IFN-γ et a été aussi impliqué dans la régulation de la fibrose hépatique. Cependant, on ne sait pas si les fonctions anti-fibrotiques sont médiées directement dans le foie par SOCS1 ou par la modulation de l'IFN-γ, qui est connu pour son effet atténuateur de la fibrose hépatique. En outre, il est possible que SOCS1 contrôle la fibrose hépatique par la régulation des cellules stellaires hépatiques (CSH), un acteur clé dans la réponse fibrogénique. Alors que les voies d'activation des CSH ont été bien caractérisées, les mécanismes de régulation ne sont pas encore clairs. Les buts de cette étude étaient de dissocier la régulation de la réponse fibrogénique hépatique médiée par SOCS1 et celle dépendante de IFN-γ et d'élucider les fonctions régulatrices de SOCS1 dans l'activation des CSH. La fibrose hépatique a été induite chez des souris Socs1[indice supérieur -/-]Ifng[indice supérieur -/-] par la diméthylnitrosamine ou le tétrachlorure de carbone. Les souris Ifng[indice supérieur -/-] et C57BL6 ont servi comme contrôles. Après les traitements fibrogéniques, les souris Socs1[indice supérieur -/-]Ifng[indice supérieur -/-] ont montré des niveaux sériques élevés d'alanine aminotransférase (ALT) ainsi que l'augmentation de la fibrose du foie par rapport à des souris Ifng[indice supérieur -/-]. Le dernier groupe a montré des niveaux plus élevés d'ALT et de fibrose par rapport aux souris C57BL6 contrôles. Les foies des souris déficientes en Socs1 ont montré une fibrose septale, qui a été associée à une augmentation de l'accumulation des myofibroblastes et à un dépôt abondant du collagène. Les foies déficients en SOCS1 ont montré une expression accrue de gènes codant pour l'actine musculaire lisse, le collagène et les enzymes impliquées dans le remodelage de la matrice extracellulaire, à savoir les métalloprotéinases de la matrice et l'inhibiteur tissulaire des métalloprotéinases. Les CSH primaires de souris déficientes en Socs1 ont montré une prolifération accrue en réponse à des facteurs de croissance tels que le HGF, EGF et le PDGF. Aussi, les foies fibrotiques de souris déficientes en Socs1 ont montré une expression élevée du gène PDGFB. Pris ensemble, ces données indiquent que SOCS1 contrôle la fibrose hépatique indépendamment de l'IFN-γ et qu'une partie de cette régulation peut se produire en régulant la prolifération des HSC et en limitant la disponibilité des facteurs de croissance.

N-Nitrosodimethylamine (commonly known as NDMA) is a probable human carcinogen that has been recognized as an emerging drinking water contaminant in recent years. Previous studies have shown that certain N-containing organic compounds may form NDMA in reaction with chlorine or monochloramine and the NDMA yield is affected by the structure of the organic-N compounds, water conditions and treatment parameters. Many amine-based water treatment polymers contain organic-N functional groups and thus have been suspected as potential NDMA precursors in water treatment systems. The purpose of this research was to systematically assess the potential NDMA formation from different structural types of water treatment polymers in reactions with various oxidants and probe the possible factors that influence the NDMA formation. Robust analytical methods for detection of NDMA and the well-known NDMA precursor dimethylamine (DMA) in the reaction samples were established. The cationic polyacrylamide (cationic PAMS), aminomethylated polyacrylamide (Mannich), poly-diallyldimethylammonium chloride (polyDADMAC) and polyamine polymers were evaluated in reactions with nitrite, free chlorine, monochloramine or chlorine dioxide in aqueous solutions at circumneutral pH and room temperature conditions. This study employed high dosages of polymer and oxidant and long reaction time in order to assess the maximum potential to form NDMA. A range of operational parameters that may affect the above reactions were also evaluated.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-03T19:11:49Z No. of bitstreams: 1 Rodrigo Andrade Lesoes vasculares intra-hepaticas....pdf: 3334038 bytes, checksum: 67ec7a5a26604cc2e6ee412582500748 (MD5) / Made available in DSpace on 2012-08-03T19:11:49Z (GMT). No. of bitstreams: 1 Rodrigo Andrade Lesoes vasculares intra-hepaticas....pdf: 3334038 bytes, checksum: 67ec7a5a26604cc2e6ee412582500748 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Lesões vasculares são importantes na patogênese de muitas das doenças agudas e crônicas do fígado e estão relacionadas às principais doenças hepáticas no nosso meio, como esquistossomose, hepatites e cirrose. Estudos recentes têm enfocado a importância da fibrose obliterativa de veias centrais, porém seu completo entendimento ainda não foi estabelecido e muitos de seus aspectos fisiopatológicos permanecem obscuros. Neste contexto, a eficácia e reprodutibilidade dos modelos animais são fundamentais e cruciais para o estudo destas alterações, permitindo avanço científico na área. Para o presente estudo analisamos as alterações histopatológicas nos fígados de 28 ratos tratados por quatro semanas com injeções intraperitoneais de dimetilnitrosamina (DMN). Nas duas primeiras semanas de tratamento com DMN os animais apresentavam alterações necro-hemorrágicas na zona 3 acinar, além da presença de espessamento fibroso subendotelial em ramos da veia central e discreta fibrose portal. Nas duas últimas semanas de tratamento pudemos observar a associação de fibrose hepática e ativação de células estreladas, com a oclusão parcial ou total dos ramos de médio calibre da veia central. Nossos achados nos permitem inferir que as alterações vasculares observadas em ramos da veia central são fundamentais para o agravamento da fibrose no órgão. Propomos ainda que o modelo experimetal de agressão hepática tóxica pela DMN se mostra adequado para o estudo destas lesões vasculares. / Vascular lesions are important features in the pathogenesis of acute and chronic liver diseases and are related to the major diseases of the organ, such as schistosomiasis, hepatitis and chirrosis. Recent studies have focused on the importance of obliterative fibrosis in central veins, but its complete understanding has not yet been established and many of its pathophysiological aspects remain unclear. In this context, efficiency and reliability of animal models are essential and crucial to the study of these changes, allowing scientific advances in the area. For this study we analyzed the histopathological changes in the livers of 28 rats treated with intraperitoneal injections of dimethylnitrosamine (DMN), for four weeks. In the first two weeks of treatment animals showed necro-hemorrhagic changes in acinar zone 3 and the presence of subendothelial fibrous thickening in the central vein branches, beyond mild portal fibrosis. In the last two weeks of treatment we observed hepatic fibrosis and stellate cell activation, associated with partial or complete occlusion of medium-caliber branches of central vein. Our findings allow us to infer that the vascular changes observed in the central vein branches are critical to the worsening of liver fibrosis. We also propose that the experiential model of toxic liver injury by DMN is adequate for the study of these vascular lesions

Este trabalho teve como objetivo desenvolver um protocolo para a diferenciação in vitro de células-tronco mesenquimais (CTM) em hepatócitos e a padronização de um modelo animal de fibrose hepática induzida por dimetilnitrosamina (DMN) para ensaios pré-clínicos de transplante de CTM. CTM isoladas de fontes variadas apresentaram morfologia fibroblastóide e aderência ao plástico e o padrão de marcadores de superfície celular esperado na análise por citometria de fluxo. A capacidade de diferenciação osteogênica e adipogênica dessas células foi comprovada pelas colorações de vermelho de alizarina, oil red e azul de toluidina, respectivamente, confirmando, que as células isoladas para este estudo se comportaram como CTM conforme proposto pela Sociedade Internacional de Pesquisa em Células-tronco. A diferenciação hepática foi avaliada quanto à morfologia e capacidade das células diferenciadas de estocar glicogênio confirmada por PAS (ácido periódico-Schiff), de sintetizar albumina confirmada por imunofluorescência, além da capacidade de expressar genes hepato-específicos verificada por ensaios de PCR em tempo real. Com base na literatura para diferenciação hepática, diferentes protocolos de um, dois e três passos foram testados. CTM humanas mostraram capacidade de produzir e estocar glicogênio e de sintetizar albumina, apenas quando diferenciadas com protocolos de três etapas, porém sem uma expressão aumentada dos genes hepato-específicos albumina, α-fetoproteína e c-Met. Uma etapa de diferenciação endodérmica, previamente aplicada à diferenciação hepática, aumentou a capacidade de produzir e estocar glicogênio das CTM diferenciadas. Para a padronização do modelo de fibrose hepática induzida por DMN, foram realizados experimentos de dose-resposta e foi verificado o efeito da hepatectomia em modelos mistos DMN/hepatectomia. A injúria hepática e o efeito do transplante de CTM foram avaliados por análise macroscópica dos fígados, histologia das biópsias de fígados corados com HE e tricromo de Masson e parâmetros bioquímicos séricos. Alterações macroscópicas, histológicas e nos níveis séricos de fosfatase alcalina indicam a indução da fibrose hepática nos ratos Wistar tratados com DMN na dose de 10 µg/g de peso animal por três dias consecutivos durante quatro semanas, mas não observamos nenhum efeito induzido pela hepatectomia. Porém, este modelo com DMN se mostra semelhante a estágios iniciais de uma fibrose hepática. O transplante de 1 x 107 CTM de veia de cordão umbilical humano (VCUH) no modelo de injúria hepática induzida por DMN não resultou em melhora da fibrose, diminuição dos níveis séricos de fosfatase alcalina e nem em ganho de peso dos animais quando comparados aos animais tratados com PBSA após a injúria hepática (grupo placebo). Em conjunto, esses resultados sugerem que CTM humanas se diferenciam após tratamentos mais complexos, onde os indutores hepatogênicos são sequencialmente adicionados ao meio de modo a mimetizar a sinalização durante o desenvolvimento embrionário. O transplante de CTM de VCUH parece não ter efeito positivo em um modelo pré-clínico de injúria hepática similar a estágios iniciais de fibrose. Financiado por CNPq (573578/2008-7) e FAPESP (2007/54260-2). / This study aimed to develop an in vitro differentiation protocol of mesenchymal (MSC) stem cells to hepatocytes and to standardize an animal model for hepatic fibrosis induced by dimethylnitrosamine (DMN) for preclinical transplant assays of MSC. MSC isolated from various sources presented fibroblastoid morphology, plastic adherence, and the expected pattern of cell surface markers by flow cytometry analysis. The capacity of osteogenic, adipogenic and chondrogenic differentiation of these cells was confirmed by alizarin red, oil red and toluidine blue staining, respectively, confirming that the cells isolated for this study behave as MSC, as proposed by the International Society for Stem Cell Research. Hepatogenic differentiation was evaluated by analysis of cell morphology, capacity to store glycogen confirmed by PAS (periodic acid-Schiff), albumin synthesis confirmed by immunofluorescence, as well as hepatic-specific gene expression verified by real time PCR assays. Based on the published literature on hepatic differentiation, several protocols of one, two, and three steps were tested. Human MSC differentiated solely when treated in a three step-protocol, showing the ability to produce and store glycogen and synthesize albumin; however the expression of hepatic-specific genes such as albumin, α-fetoprotein and c-Met was not increased. An endoderm differentiation stage, added to the hepatic differentiation protocol, increased the capacity to produce and store glycogen of differentiated MSC. In order to standardize the model of liver fibrosis induced by DMN, dose-response experiments were performed and the effect of hepatectomy in mixed models DMN/hepatectomy was observed. Severity of liver injury and the effect of cell transplantation were evaluated by macroscopic analysis of the livers, histology of liver biopsies stained with HE and Masson\'s trichrome, and evaluation of serum biochemical parameters. The macroscopic and histological observations, and altered alkaline phosphatase serum levels indicated the success in inducing liver fibrosis in DMN-treated rats at a dose of 10 µg/g of animal weight for three consecutive days, during four weeks, without any additional effect upon hepatectomy. Transplanting 1 x 107 umbilical cord MSC in the model of liver injury induced by DMN did not result in improvement of the fibrosis, decrease of alkaline phosphatase serum levels, or in weight gain of the treated animals compared to animals treated with PBSA after liver injury (placebo group). Together, these results suggest that human MSC are capable of differentiating to hepatocyte-like cells after more complex protocols, where hepatogenic inducers are sequentially added to the medium in order to mimic signaling that occurs during fetal development. Transplantation of undifferentiated umbilical cord MSC did not have any positive effect in a preclinical liver injury model characterized by an early stage of fibrosis. Supported by CNPq (573578/2008-7) and FAPESP (2007/54260-2).

Este trabalho teve como objetivo desenvolver um protocolo para a diferenciação in vitro de células-tronco mesenquimais (CTM) em hepatócitos e a padronização de um modelo animal de fibrose hepática induzida por dimetilnitrosamina (DMN) para ensaios pré-clínicos de transplante de CTM. CTM isoladas de fontes variadas apresentaram morfologia fibroblastóide e aderência ao plástico e o padrão de marcadores de superfície celular esperado na análise por citometria de fluxo. A capacidade de diferenciação osteogênica e adipogênica dessas células foi comprovada pelas colorações de vermelho de alizarina, oil red e azul de toluidina, respectivamente, confirmando, que as células isoladas para este estudo se comportaram como CTM conforme proposto pela Sociedade Internacional de Pesquisa em Células-tronco. A diferenciação hepática foi avaliada quanto à morfologia e capacidade das células diferenciadas de estocar glicogênio confirmada por PAS (ácido periódico-Schiff), de sintetizar albumina confirmada por imunofluorescência, além da capacidade de expressar genes hepato-específicos verificada por ensaios de PCR em tempo real. Com base na literatura para diferenciação hepática, diferentes protocolos de um, dois e três passos foram testados. CTM humanas mostraram capacidade de produzir e estocar glicogênio e de sintetizar albumina, apenas quando diferenciadas com protocolos de três etapas, porém sem uma expressão aumentada dos genes hepato-específicos albumina, α-fetoproteína e c-Met. Uma etapa de diferenciação endodérmica, previamente aplicada à diferenciação hepática, aumentou a capacidade de produzir e estocar glicogênio das CTM diferenciadas. Para a padronização do modelo de fibrose hepática induzida por DMN, foram realizados experimentos de dose-resposta e foi verificado o efeito da hepatectomia em modelos mistos DMN/hepatectomia. A injúria hepática e o efeito do transplante de CTM foram avaliados por análise macroscópica dos fígados, histologia das biópsias de fígados corados com HE e tricromo de Masson e parâmetros bioquímicos séricos. Alterações macroscópicas, histológicas e nos níveis séricos de fosfatase alcalina indicam a indução da fibrose hepática nos ratos Wistar tratados com DMN na dose de 10 µg/g de peso animal por três dias consecutivos durante quatro semanas, mas não observamos nenhum efeito induzido pela hepatectomia. Porém, este modelo com DMN se mostra semelhante a estágios iniciais de uma fibrose hepática. O transplante de 1 x 107 CTM de veia de cordão umbilical humano (VCUH) no modelo de injúria hepática induzida por DMN não resultou em melhora da fibrose, diminuição dos níveis séricos de fosfatase alcalina e nem em ganho de peso dos animais quando comparados aos animais tratados com PBSA após a injúria hepática (grupo placebo). Em conjunto, esses resultados sugerem que CTM humanas se diferenciam após tratamentos mais complexos, onde os indutores hepatogênicos são sequencialmente adicionados ao meio de modo a mimetizar a sinalização durante o desenvolvimento embrionário. O transplante de CTM de VCUH parece não ter efeito positivo em um modelo pré-clínico de injúria hepática similar a estágios iniciais de fibrose. Financiado por CNPq (573578/2008-7) e FAPESP (2007/54260-2). / This study aimed to develop an in vitro differentiation protocol of mesenchymal (MSC) stem cells to hepatocytes and to standardize an animal model for hepatic fibrosis induced by dimethylnitrosamine (DMN) for preclinical transplant assays of MSC. MSC isolated from various sources presented fibroblastoid morphology, plastic adherence, and the expected pattern of cell surface markers by flow cytometry analysis. The capacity of osteogenic, adipogenic and chondrogenic differentiation of these cells was confirmed by alizarin red, oil red and toluidine blue staining, respectively, confirming that the cells isolated for this study behave as MSC, as proposed by the International Society for Stem Cell Research. Hepatogenic differentiation was evaluated by analysis of cell morphology, capacity to store glycogen confirmed by PAS (periodic acid-Schiff), albumin synthesis confirmed by immunofluorescence, as well as hepatic-specific gene expression verified by real time PCR assays. Based on the published literature on hepatic differentiation, several protocols of one, two, and three steps were tested. Human MSC differentiated solely when treated in a three step-protocol, showing the ability to produce and store glycogen and synthesize albumin; however the expression of hepatic-specific genes such as albumin, α-fetoprotein and c-Met was not increased. An endoderm differentiation stage, added to the hepatic differentiation protocol, increased the capacity to produce and store glycogen of differentiated MSC. In order to standardize the model of liver fibrosis induced by DMN, dose-response experiments were performed and the effect of hepatectomy in mixed models DMN/hepatectomy was observed. Severity of liver injury and the effect of cell transplantation were evaluated by macroscopic analysis of the livers, histology of liver biopsies stained with HE and Masson\'s trichrome, and evaluation of serum biochemical parameters. The macroscopic and histological observations, and altered alkaline phosphatase serum levels indicated the success in inducing liver fibrosis in DMN-treated rats at a dose of 10 µg/g of animal weight for three consecutive days, during four weeks, without any additional effect upon hepatectomy. Transplanting 1 x 107 umbilical cord MSC in the model of liver injury induced by DMN did not result in improvement of the fibrosis, decrease of alkaline phosphatase serum levels, or in weight gain of the treated animals compared to animals treated with PBSA after liver injury (placebo group). Together, these results suggest that human MSC are capable of differentiating to hepatocyte-like cells after more complex protocols, where hepatogenic inducers are sequentially added to the medium in order to mimic signaling that occurs during fetal development. Transplantation of undifferentiated umbilical cord MSC did not have any positive effect in a preclinical liver injury model characterized by an early stage of fibrosis. Supported by CNPq (573578/2008-7) and FAPESP (2007/54260-2).

In recent years, a compound N-nitrosodimethylamine (NDMA), a probable human carcinogen, has been identified as an emerging disinfection by-product (DBP) since its formation and detection were linked to chlorine-based disinfection processes in several water utilities in the U.S. and Canada. Numerous organic nitrogen compounds present in water may impact the formation of NDMA during disinfection. Amine-based water treatment polymers used as coagulants and flocculants have been suggested as potential NDMA precursors due to the presence of amine functional groups in their structures, as well as the possible presence of dimethylamine (DMA) residues in polymer products. To minimize the potential risk of NDMA associated with water treatment polymers, the mechanisms of how the polymers behave as NDMA precursors and their contribution to the overall NDMA formation under actual water treatment conditions need to be elucidated. This research involved a systematic investigation to determine whether amine-based water treatment polymers contribute to NDMA formation under drinking water and wastewater treatment conditions, to probe the involved reaction mechanisms, and to develop strategies to minimize the polymers NDMA formation potential. The investigation included five research tasks: (1) General screening of NDMA formation potential of commonly used amine-based water treatment polymers, (2) NDMA formation from amine-based water treatment polymers under relevant water treatment conditions, (3) Probing the mechanisms of NDMA formation from polyamine and PolyDADMAC, (4) Effect of water treatment processes on NDMA formation from amine-based water treatment polymers, and (5) Developing strategies to reduce polymers NDMA formation potential. Direct chloramination or chlorination of high doses of polymers in deionized water at longer than typical contact time was used in the general screening of the NDMA formation potential of water treatment polymers and in the studies to identify reaction mechanisms. On the other hand, realistic dosages of chloramines and polymers and contact time were used in simulating representative water treatment conditions to evaluate the contribution of polymers to the overall NDMA formation in real systems. On the basis of the study results, strategies were developed to reduce the NDMA formation potential of amine-based water treatment polymers, which include modification of polymer structures and treatment parameters.