Made available in DSpace on 2015-04-17T14:49:12Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1477687 bytes, checksum: 499775300ecca263fde140f8d1c98bb3 (MD5) Previous issue date: 2010-09-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The aim of this work was to evaluate the PAHs contamination in muscular tissue and bile of Mugil liza fishes from Guanabara Bay. The Guanabara Bay is an estuarine ecosystem with high anthropogenic influence which receives chronic pollution from Rio de Janeiro city. It was determined 38 compounds (16 PAHs EPA + alkylated ones) in muscular tissue samples of 41 fishes captured from August 2008 to October 2008. Statistical analyzes showed that the data concentration has a normal distribution and drawn from the same population, with mean concentration of 127 ng/g and standard deviation of 18 ng/g to the 16 PAHS EPA. The mean concentration for the 38 compounds was 274 ng/g and standard deviation of 59 ng/g. It was determinated 13 metabolites of PAHs of 30 Mugil liza fishes (n=30) captured in the inner part of the Guanabara Bay. The metabolites determined were OH-Naphthalenes, OH-Biphenyl, OH-Fluorene, OH-Phenantrenes, OH-Pyrene, OH-Chrysene, OH-Benz(a)pyrene. It was observed in this study a normal distribution of concentrations for both set of data: bile PAH metabolites and PAH muscular tissue. This indicates the existence of two data populations. The mean concentration of PAH metabolites obtained was 1,039 ng/g and standard deviation of 624 ng/g. These data can be used as back ground to the inner part of Guanabara Bay. The bioindicator in this study, Mugil liza fish, has shown to be effective to evaluate the contamination and exposition of Guanabara Bay ecosystem to PAH. / Este trabalho teve como objetivo a avaliação da contaminação por HPAs em peixes provenientes da Baía de Guanabara através da identificação e quantificação desses compostos em tecido muscular e de seus metabólitos em bile. A Baía de Guanabara é um ecossistema estuarino de forte influência antropogênica que recebe poluição crônica da região metropolitana do Rio de Janeiro. Foram analisados 41 amostras de peixes da espécie Mugil liza (Tainha) coletados na Baía de Guanabara no período de agosto a outubro de 2008. Nos tecidos musculares foram quantificados 38 compostos (16 HPAs EPA e seus respectivos alquilados). A análise estatística revelou que esse grupo de dados é proveniente de uma mesma população com distribuição normal, com valor médio de concentração de 127 ng/g e desvio-padrão populacional de 18 ng/g para o Σ 16 HPAs e concentração média populacional para o Σ 38 HPAs de 274 ng/g e desvio-padrão de 59 ng/g. Em sequência, foi realizada a avaliação do nível de exposição a que os organismos aquáticos da Baía de Guanabara estão submetidos, através da análise de 30 amostras de bile de peixes da espécie Mugil Liza provenientes de currais da área de fundo da Baía de Guanabara, onde foram quantificados 13 metabólitos individuais de HPAs. Os metabólitos determinados foram 1-OH-Naftaleno, 2-OH-Naftaleno, 2-OH-Bifenila, 3-OH-Fluoreno, 1-OH-Fenantreno, 2-OH-Fenantreno, 3-OH-Fenantreno, 4-OH-Fenantreno, 9-OH-Fenantreno, 1-OH-pireno, 1-OH-criseno, 3-OH-benzo(a)pireno e 9-OH-benzo(a)pireno. O valor médio de concentração do somatório dos metabólitos foi de 1.039 ng/g e desvio-padrão populacional de 623 ng/g. Esses dados podem ser utilizados como valores característicos para a região já que apresentaram uma distribuição normal. As concentrações determinadas encontraram-se dentro da faixa de outras regiões com influência antropogênica elevada, com aporte crônico de hidrocarbonetos. O bioindicador utilizado neste estudo, o peixe da espécie Mugil liza, mostrou-se efetivo para a avaliação da contaminação por HPAs em um ecossistema como a Baía de Guanabara.

La cholestase est l'une des manifestations les plus courantes des lésions induitespar des médicaments. Dans 40% des cas elle n’est pas prévisible; une meilleure prédictibilité représente donc un défi majeur. Tout d'abord, nous avons démontré que les cellules hépatiques humaines HepaRG différenciées sont un modèle approprié pour étudier la cholestase induite par les médicaments en comparant la localisation et l’activité des transporteurs d'influx et d'efflux avec les hépatocytes humains primaires. Tous les transporteurs d'efflux et d’influx testés ont été correctement localisés au niveau des membranes canaliculaire (BSEP, MRP2, MDR1 et MDR3) et basolatéral (NTCP, MRP3) et sont fonctionnels. En outre, ces cellules expriment également les enzymes qui métabolisent les acides biliaires (ABs) et ont la capacité de les synthétiser et de les conjuguer avec la taurine, la glycine et le sulfate, à un taux similaire à celui des hépatocytes primaires. Des changements ont été observés sur la répartition des ABs totaux après traitements de cellules HepaRG par un médicament cholestatique, la cyclosporine A (CsA), de manière concentration- dépendante. L'inhibition de l'efflux et de l'influx de taurocholate a été observée après 15 min et 1 h respectivement. Ces premiers effets ont été associés à la dérégulation de la voie des cPKC et l'induction d’un stress du réticulum endoplasmique puis d’un stress oxydant. Nous avons également montré pour la première fois une accumulation intracellulaire d’ABs endogènes avec un médicament cholestatique in vitro. En outre, notre travail apporte des preuves que la motilité des canalicules biliaires (BC) est indispensable à la clairance des ABs. La voie ROCK et le complexe actomyosine sont fortement impliqués. Nous avons fourni la première démonstration que la voie ROCK et les dynamiques des BC sont des cibles majeures des composés cholestatiques. Nos données devraient contribuer à l'élaboration de méthodes de screening pour la prédiction précoce des effets secondaires induits par les médicaments cholestatiques. / Cholestasis is one of the most common manifestations of drug-induced liver injury (DILI). Since up to now it is unpredictable in 40% of all cases its accurate prediction represents a major challenge. First, we validated that differentiated HepaRG human liver cells are a suitable in vitro model to study drug-induced cholestasis, by comparing localization of influx and efflux transporters and their functional activity in these cells and primary human hepatocytes. All tested influx and efflux transporters were correctly localized to canalicular (BSEP, MRP2, MDR1, and MDR3) or basolateral (NTCP, MRP3) membrane domains and were functional. In addition, the HepaRG cell line also exhibits bile acids (BAs) metabolizing enzymes and has the capacity to synthesize BAs and to further amidate these BAs with taurine and glycine as well as sulfate, at a rate similar to that of primary hepatocytes. Concentration- dependent changes were observed in total BAs disposition after treatment of HepaRG cells by the cholestatic drug cyclosporine A (CsA). Inhibition of efflux and uptake of taurocholate was evidenced as early as 15 min and 1 h respectively. These early effects were associated with deregulation of cPKC pathway and induction of endoplasmic reticulum stress that preceded generation of oxidative stress. We also showed for the first time intracellular accumulation of endogenous BAs by a cholestatic drug in vitro. In addition, our work brings evidences that motility of bile canaliculi (BC) is essential for BAs clearance where ROCK pathway and actomyosin complex are highly implicated. We provided the first demonstration that ROCK pathway and BC dynamics are major targets of cholestatic compounds. Our data should help in the development of screening methods for early prediction of drug-induced cholestatic side effects.

The differentiation, in an infant with cholestasis, between extrahepatic biliary atresia (EHBA) and the neonatal hepatitis syndrome (NHS) is important in that laparotomy is always indicated in EHBA but is undesirable in NHS. This differentiation is particularly difficult in those infants with complete cholestasis. Hepatobiliary scintigraphy is a commonly used investigation in infants with obstructive jaundice. The scintigraphic demonstration of excretion into the gut excludes extrahepatic obstruction, however, absence of excretion may be due to EHBA, severe cholestasis with patent extrahepatic bile ducts or poor uptake of the agent, and is therefore not diagnostic. This study has examined the quantitative measurement of the hepatic uptake of p-butyl IDA and Sn colloid, and an estimation of liver shape, in a group of patients with complete cholestasis in whom conventional scan interpretation, based on excretion into the-gut, would not be useful. The scans were recorded as dynamic studies and the resultant time-activity curves were subjected to curve fitting to calculate a rate constant for uptake of radiopharmaceutical. Liver shape was determined from the anterior static image of the colloid scan. The results show a significant difference between the EHBA and the NHS patients in the rate of uptake of p-butyl IDA, in the ratio of the rate of uptake of p-butyl IDA/the rate of uptake of colloid and in the measurements used to express liver shape. Using this method of scan interpretation, a diagnostic accuracy of 85% was achieved in this study of patients who clinically, and on scan, had no evidence of bile flow. Hepatic scintigraphy is therefore a useful investigation in the diagnostic work-up of infants presenting with obstructive jaundice even when bile flow is completely absent.

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Kyeong-Ok Chang / Caliciviruses are important pathogens of humans and animals. Noroviruses are major causes of foodborne gastroenteritis cases, but their research is hindered due to the inability to grow human noroviruses in cell culture. Detailed studies on entry events of caliciviruses are lacking and may be crucial for development of cell culture models. We examined the entry events of caliciviruses using porcine enteric calicivirus (PEC), feline calicivirus (FCV) and murine norovirus-1 (MNV-1). PEC replication in LLC-PK cells requires bile acid in the medium, but the mechanism is not well understood. Our studies showed that bile acids are required in the early stage of virus replication, and while internalization of PEC is not dependent of them, they are required for endosomal escape and successful replication. Further examination on virus entry, we demonstrated that endosomal acidification and cathepsin L activity are essential in the replication of PEC, FCV and MNV-1. The results showed that inhibition of endosomal acidification or cathepsin L activity led to retention of viruses in the endosomes. Also we demonstrated that recombinant cathepsin L cleaved structural protein of PEC, FCV or MNV-1, which suggests that the enzyme may facilitate uncoating viruses in endosomes. In addition to bile acids, we found that a cold shock treatment during virus entry supported PEC replication by facilitating the endosomal escape. While PEC alone did not induce ceramide formation, bile acids or cold shock treatment induce ceramide formation on endosomes through activation acid sphingomyelinase (ASM), and this event was crucial for virus replication because inhibition of ASM blocked ceramide formation and significantly reduced PEC replication. Incubation of FCV or MNV-1 with cells led to ceramide formation during virus entry, and inhibition of ASM also significantly reduced their replication. Inhibition of ASM led to endosomal retention of PEC, FCV or MNV-1 during virus entry, which may be the reason for the reduction of viral replication. These studies revealed the important and common events during calicivirus entry for successful replication, including virus endosomal escape, cathepsin L activity and ASM/ceramide formation. This detailed information may provide clues for understanding the replication of fastidious caliciviruses and for potential therapeutic targets.

No description available.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

The optical design for a dual modality endoscope based on piezo scanning fiber technology is presented including a novel technique to combine forward-viewing navigation and side viewing OCT. Potential applications include navigating body lumens such as the fallopian tube, biliary ducts and cardiovascular system. A custom cover plate provides a rotationally symmetric double reflection of the OCT beam to deviate and focus the OCT beam out the side of the endoscope for cross-sectional imaging of the tubal lumen. Considerations in the choice of the scanning fiber are explored and a new technique to increase the divergence angle of the scanning fiber to improve system performance is presented. Resolution and the necessary scanning density requirements to achieve Nyquist sampling of the full image are considered. The novel optical design lays the groundwork for a new approach integrating side-viewing OCT into multimodality endoscopes for small lumen imaging.

[pt] O petróleo é uma mistura complexa de diversos compostos, sendo os hidrocarbonetos seus principais constituintes, chegando a atingir 98% da composição total. Entre os hidrocarbonetos, os hidrocarbonetos policíclicos aromáticos (HPA) destacam-se por serem, em geral, resistentes à biodegradação e persistentes no ambiente, sendo potencialmente carcinogênicos ao homem e aos organismos. A presença destes compostos nos ecossistemas, de origem natural ou proveniente de fontes antropogênicas, e seus efeitos podem ser monitorados utilizando organismos do próprio ambiente. Nos ecossistemas aquáticos, a determinação de metabólitos de HPA na bílis de peixes que habitam o ambiente pode ser usada como indicadora de exposição ambiental recente. Assim, o desenvolvimento e a validação de métodos usualmente empregados na determinação de metabólitos de HPA em bílis de peixes têm contribuição e aplicação na elaboração de diagnósticos ambientais sobre a contaminação por petróleo e derivados nos sistemas aquáticos. Este trabalho otimizou, validou e comparou dois métodos para a determinação de 1- hidroxipireno em bílis de peixes. Um método usando a cromatografia a liquido de alta eficiência com detector de fluorescência (HPLC-F) e o outro usando a espectroscopia de fluorescência convencional. Os métodos foram validados com base nos parâmetros linearidade, sensibilidade, limite de detecção, limite de quantificação, repetitividade, reprodutibilidade e incluindo os cálculos das incertezas associadas. Nos dois casos, as fontes de contribuição mais significativas para a incerteza dos métodos foram as soluções-padrão. A incerteza padrão combinada associada ao método utilizando a cromatografia a líquido foi menor que a associada ao método utilizando fluorescência molecular. Entretanto, o método em fluorescência, apesar de ser semi-quantitativo e de apresentar incerteza maior, tem tempo de análise e custos de operação e manutenção menores, sendo indicado para análises preliminares. / [en] Petroleum is a complex mixture of several compounds. Among its principal components there are hydrocarbons which account to up to 98% of the total composition. Among the hydrocarbons, there are polycyclic aromatic hydrocarbons (PAH), which are more resistant to biodegradation, quite persistent in the environment and potentially carcinogenic to humans and marine organisms. The presence of these compounds on ecosystems, from natural origin or from anthropogenic sources, and their efluorescênciaects, can be monitored using living organisms of the environment. In aquatic ecosystems, the determination of PAH metabolites, in fish bile from the environment can be used as an indicator of recent environmental exposure. Thus, the development and validation of methods usually employed in the determination of PAH metabolites in the bile of fish have assistance in the preparation and implementation of diagnoses on the environmental contamination by petroleum and derivatives in aquatic systems. In this work two methods for the determination of 1- hydroxypyrene in fish bile were improved, validated and compared. A method using the high liquid chromatography with fluorescence detector (HPLC-F) and the other using fixed fluorescence spectroscopy. The methods were validated based on parameters linearity, sensitivity, limit of detection, limit of quantification, repeatability, reproducibility. In addition, calculations of the uncertainties associated to the 1-hydroxypyrene measurement were made. In both cases, the sources of most significant contribution to the methods uncertainty were the standard solutions. The combined standard uncertainty associated with the method using the liquid chromatography was lower than that associated with the method using molecular fluorescence. The fluorimetric method can be used as a semi-quantitative tool and presented greater uncertainty. However, this method has lower analysis time and costs of operation and maintenance, being quite suitable for preliminary analysis.

As células derivadas de fígado embrionário tanto de animais quanto de humanos tem sido cada vez mais estudas devido ao seu potencial antiinflamatório, imunomodulador e regenerativo, demonstrado as mesmas bipotencial de diferenciação em hepatócitos e colangiocitos. Na presente pesquisa utilizou-se células derivadas de fígados embrionários de ratos com 14,5 dias de gestação. As células apresentaram marcadores de células progenitoras hepáticas, bem como marcadores de células hepáticas e biliares diferenciadas confirmando, seu bipotencial. A terapia celular utilizando as células supracitadas, reduziu significativamente a progressão da fibrose hepática, diminuindo a inflamação e ainda estimulando a regeneração hepática de ratos submetidos à cirrose por ligadura do ducto biliar. As análises realizadas mediante avaliação quantitativa pela técnica de morfometria, demonstraram redução da deposição de fibras de colágeno, bem como menor proliferação de ductos biliares nos animais tratados. Os resultados foram ainda complementados por analise semiquantitativa, a qual avaliou a intensidade da necroinflamação dos tecidos hepáticos analisados, apontando menor escore de inflamação dos animais tratados. As células poderão ter efeito benéfico para o tratamento de doenças hepáticas crônicas, que estimulam a formação de fibrose. A cirrose é o estágio final comum à doenças hepáticas crônicas por causadas por fatores de diversas etiologias. Esta ocupa a decima quarta causa mundial de mortalidade em humanos, sendo que o único tratamento definitivo atualmente é transplante do órgão. Entretanto o número de transplantes está longe de suprir a demanda atual, visto que há um déficit de doadores do órgão. Terapias que possam oferecer uma alternativa de tratamento confiável, segura e acessível são bastante oportunas. Nossos resultados sugerem que as células utilizadas neste trabalho podem modular a fibrogênese, e consequentemente retardar o estabelecimento da cirrose em doenças hepáticas crônicas. / Studies on human and animal embryonic liver stem cells have been growing due to its anti-inflammatory, immunomodulatory and regenerative potential. These cells show also a bipotential do differentiate into hepatocytes and cholangiocytes. In the present study, it was used rodent embryonic liver with 14.5 of gestation. The cells presented hepatic progenitor, as well adult hepatic and biliary cells markers, confirming their bipotential. Previous studies with these cells in therapy decreased hepatic fibrosis progression in rat models submitted to cirrhosis by biliary duct ligation. Quantitative analysis was performed by morphometry showed decreased collagen fibers deposition and lower proliferation of biliary ducts in treated animals. Results were complemented with semiquantitative analysis with evaluation of necroinflammation of the analyzed hepatic tissues, in which a decreased inflammation score was observed. Cirrhosis is a common final stage for chronic hepatic diseases caused by different factors in several etiologies. It occupies the 14th world cause of mortality in human. However, the number of liver transplants is insufficient for current demand, caused by deficit in organs donors. Therapies that could offer an alternative for a reliable, safe and accessible treatment is opportune. Our results suggest that cells used in this study can modulate fibrogenesis and consequently delay the establishment of cirrhosis in chronic liver diseases.

Research into the gut microbiome has revealed the widespread influence that microbial species have on their host. Host genetics and environmental factors influence the abundance and diversity of the bacterial species living within the gastrointestinal tract. When the normal composition of the gut microbiota is altered, a dysbiotic state incurs. Inflammatory bowel disease (IBD) is a chronic/relapsing inflammatory disorder of the intestinal mucosa, which is characterized by a state of dysbiosis. Despite the large amount of information studying the role dysbiosis has in the pathogenesis of IBD, it is not clear how the altered microbial composition of the gut in IBD patients leads to susceptibility to enteric pathogens such as Clostridium difficile. This study aims to highlight the features of the gastrointestinal tract that are modified as a result of dysbiosis in the IBD population, and how these features facilitate colonization by C. difficile and symptom development. Review of the available literature demonstrated that the depletion of Clostridial cluster XIVa in IBD-associated dysbiosis alters bile acid metabolism and butyrate fermentation in the colon, ultimately promoting germination of C. difficile spores and weakening the gut's immune response against toxin-mediated inflammation. From continued research into the gut microbiota, more will be understood of how these microbial organisms influence human health and disease.