Made available in DSpace on 2015-04-14T14:51:23Z (GMT). No. of bitstreams: 1 447380.pdf: 675913 bytes, checksum: bfb87e6d734478e3c7bb6275c3132761 (MD5) Previous issue date: 2013-02-26 / Liver fibrosis is the wound healing response to repeated injury of the liver. It is characterized by disruption of the liver architecture associated with increased expression of extracellular matrix components. Hepatic stellate cells (HSC) play a key role in liver fibrogenesis. In normal liver, HSC are quiescent and its main function is to store vitamin A. During liver injury, these cells undergo activation, become myofibroblasts and acquire fibrogenic properties. Activation of PPARγ (peroxisome proliferator-activated receptor gamma) and inhibition of fibrogenic molecules are potential strategies to block HSC activation and differentiation. Aware that the process of hepatic fibrosis involves inflammatory mediators, various anti-inflammatory substances have been studied in an attempt to revert fibrosis. The purpose of this study was to investigate the in vitro effects of fructose-1,6-bisphospahte (FBP) on HSC phenotype. The results demonstrated that FBP induced quiescent phenotype in HSC via PPARγ activation. Significant decrease in type I collagen mRNA expression was observed in the first 24h of treatment. These events preceded the reduction of TGF-β1 (transforming growth factor-beta) and total collagen secretion. Thus, FBP promoted downregulation of HSC activation by its antifibrotic and anti-inflammatory actions. These findings demonstrate that FBP may have potential as a novel therapeutic agent for the treatment of liver fibrosis. / A fibrose hep?tica ? a resposta cicatricial do f?gado a les?es continuadas, caracterizada pelo rompimento da arquitetura hep?tica associada ao aumento da express?o dos componentes da matriz extracelular. As c?lulas estreladas hep?ticas (HSC) desempenham um papel fundamental no processo de fibrog?nese. No f?gado normal, as HSC encontram-se em sua forma quiescente de dep?sito de vitamina A. Durante a les?o hep?tica, essas c?lulas passam por uma ativa??o fenot?pica, tornam-se miofibroblastos e adquirem propriedades fibrog?nicas. O processo de fibrose hep?tica envolve v?rios mediadores inflamat?rios e, portanto, subst?ncias anti-inflamat?rias tem sido empregadas na tentativa de reverter a fibrose e bloquear a ativa??o e diferencia??o das HSC. A ativa??o de PPARγ (receptor ativado por proliferador de peroxissomo Gama) e a inibi??o de mol?culas fibrog?nicas s?o poss?veis estrat?gias para estes fins. O objetivo deste estudo foi investigar os efeitos in vitro da frutose-1,6-bisfosfato (FBP) sobre o fen?tipo das HSC. Os resultados demonstraram que a FBP ? capaz de induzir o fen?tipo quiescente das HSC via ativa??o de PPARγ. Foi observado nas primeiras 24h de tratamento uma diminui??o significativa da express?o de mRNA de col?geno tipo I. Posteriormente, houve uma redu??o do col?geno total e de TGF-β1 (fator de transforma??o do crescimento beta). Assim, a FBP diminui o estado de ativa??o das HSC por suas a??es antifibr?ticas e anti-inflamat?rias. Estas descobertas demonstram que a FBP pode ser um potencial novo agente terap?utico para o tratamento de fibrose hep?tica.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-10-09T19:45:36Z No. of bitstreams: 1 475655 - Texto Completo.pdf: 1136903 bytes, checksum: 1a2ad879662fb2a0203c2418ab7dc36f (MD5) / Made available in DSpace on 2015-10-09T19:45:36Z (GMT). No. of bitstreams: 1 475655 - Texto Completo.pdf: 1136903 bytes, checksum: 1a2ad879662fb2a0203c2418ab7dc36f (MD5) Previous issue date: 2015-05-15 / Liver fibrosis is a complex disease that is caused by inappropriate tissue repair due to the deposition of connective tissue. When a chronic lesion affects the liver, regenerative response fails and hepatocytes are replaced with abundant extracellular matrix (ECM). The imbalance between production and degradation of ECM will result in the accumulation of proteins that change normal liver architecture, and thus its functionality. The main source of ECM is the activated hepatic stellate cell (HSC). In order, to clarify possible therapeutic approaches to the disease, the this work aimed to evaluate the possible antifibrotic action of Pluchea sagitallis (Lam.) Cabrera on an activated HSC immortalized lineage (GRX). Our results demonstrated that the P. sagittalis aqueous extract at 0.039 and 0.078 mg/mL concentrations was able to reduce cell growth and proliferation. Regarding to oxidative stress evaluation, there was no statistically significant difference between the treated group and the control. Staining with OilRed-O (ORO) showed a statistically significant increase in intracellular lipid content after 5 days of treatment, exerting in vitro effect on the GRX phenotypic change of activated towards the quiescent state. These results were confirmed by colorimetric quantification of lipid content. Regarding the TGF-?1 and collagen production, there were no statistically significant differences observed between the groups. In conclusion, the P. sagittalis aqueous extract reduces the growth and proliferation of GRX cells and induces the reversal of activated towards a quiescent phenotype. There was no decrease in cell proliferation either by necrosis or by apoptosis via activation of the senescence. Thus, our data suggest that the extract showed an antifibrotic effect, possibly by activating phenotype reversal. / A fibrose hep?tica apresenta uma patog?nese complexa causada por reparo tecidual inadequado devido ? deposi??o de tecido conectivo. Quando um dano cr?nico acomete o f?gado, a resposta regenerativa falha e os hepat?citos s?o substitu?dos por matriz extracelular (ECM) excedente. Assim, o desequil?brio entre a degrada??o e a produ??o de ECM acarretar? no ac?mulo dessas prote?nas que alteram a arquitetura normal do f?gado e, consequentemente, sua funcionalidade. A principal fonte de ECM ? a c?lula estrelada hep?tica (HSC) ativada. Sendo assim, na tentativa de elucidar poss?veis abordagens terap?uticas para a doen?a, o objetivo desse trabalho foi avaliar a poss?vel a??o antifibr?tica do extrato aquoso de Pluchea sagitallis (Lam.) Cabrera sobre uma linhagem imortalizada de HSC ativadas (GRX). Nossos resultados demonstraram que as concentra??es de 0,039 e 0,078 mg/mL do extrato aquoso de P. sagitallis foram capazes de diminuir o crescimento e a prolifera??o celular. Quanto ? avalia??o do estresse oxidativo, n?o foi observada diferen?a estatisticamente significativa entre o grupo tratado e controle. A colora??o com oil red (ORO) mostrou aumento significativo do conte?do lip?dico intracelular ap?s 5 dias de tratamento, indicando efeito in vitro sobre a mudan?a fenot?pica em linhagem GRX, do estado ativado para o estado quiescente. Esses resultados foram confirmados pela quantifica??o colorim?trica de lip?dios. Em rela??o ? produ??o de TGF-?1 e col?geno total, n?o foram observadas diferen?as estatisticamente significativas entre os grupos. Concluindo, o extrato aquoso da P. sagittalis diminuiu o crescimento e a prolifera??o das c?lulas GRX e induziu a revers?o do fen?tipo ativado para quiescente. A diminui??o na prolifera??o celular n?o ocorreu nem por necrose nem por ativa??o da apoptose e senesc?ncia. Sendo assim, nossos resultados sugerem que o extrato apresenta um efeito antifibr?tico, possivelmente pela via que ativa a revers?o do fen?tipo.

Made available in DSpace on 2015-04-14T14:51:35Z (GMT). No. of bitstreams: 1 461390.PDF: 2830978 bytes, checksum: c3b746f15a8c1946dc08a31f60e01821 (MD5) Previous issue date: 2014-03-10 / Hereditary hemochromatosis (HH) is a genetic disease where iron balance is deregulated and this metal accumulates in the liver, causing toxic effects and fibrosis. Fibrosis is an exacerbated wound-healing response with extracellular matrix (ECM) deposition. Hepatic stellate cells (HSC), when activated, are the main responsible for ECM production. Fructose-1,6-bisphosphate (FBP) is a sugar and possess innumerous beneficial effects, like enhance cell antioxidant potential, protects liver from damage and reverts the phenotype of activated HSC. Because of this, we aimed to test the effects of FBP in immortalized HSC line (GRX) exposed to free iron (Fe) tempting to simulate what occurs in patients with HH.Fe (1mg/L) treatment for 8 days increased cell growth, whereas Fe + FBP (1mg/L + 0.6mM) decreased cell proliferation to levels below of control. LDH activity, apoptosis rate and cell cycle were not altered in any group. Oil Red-O (ORO) staining showed a decrease in lipid content when GRX cells were Fe-treated (1mg/L) for 8 days. In Fe + FBP (1mg/L + 0.6mM), GRX cells showed increased lipid content and characteristics of quiescent HSC. PPAR-γ expression was diminished on Fe group and same as control on Fe + FBP group. On the contrary, Fe treatment rose Col-1 expression and Fe + FBP reversed it to control levels. TGF-β1 was unaltered in Fe group. However, on Fe + FBP group, TGF-β1 levelswas far bellow of control and Fe-treated group, showing an antifibrotic activity. FBP didn t present antioxidant activity by DPPH assay. Ferrozine assay showed a decreased absorbance after 120 min in all FBP-tested doses, demonstrating that FBP is an iron chelator. These data demonstrate that FBP reverse the phenotype of GRX cells even when in Fepresence and that this could be caused by regulation of PPAR-γ and COL-1. In conclusion, FBP diminished the growth rate and reversed the phenotype of GRX cell, showing a possible antifibrotic effect. / Hemocromatose heredit?ria (HH) ? uma doen?a gen?tica onde o balan?o do ferro est? desregulado e esse metal se acumula no f?gado, causando efeitos t?xicos e, principalmente, fibrose. Fibrose ? uma resposta de cicatriza??o exacerbada com dep?sito de matriz extracelular (ECM). C?lulas estreladas hep?ticas (HSC) quando ativadas s?o as maiores respons?veis pela produ??o de ECM. Frutose-1,6-bisfosfato (FBP) ? um a??car e possui in?meros efeitos ben?ficos, como melhorar o potencial antioxidante da c?lula, proteger o f?gado de les?o e reverter o fen?tipo de HSC ativadas. Por causa disso, nosso objetivo foi testar os efeitos da FBP em uma linhagem imortalizada de HSC (GRX) expostas a ferro livre (Fe), na tentativa de simular o que ocorre em pacientes com HH.O tratamento com Fe (1mg/L) por 8 dias aumentou a prolifera??o celular enquanto o tratamento com Fe + FBP (1mg/L + 0.6mM) a diminuiu para n?veis menores que os do controle. A atividade da LDH, taxa de apoptose e ciclo celular n?o foi alterada em nenhum grupo. A colora??o com OilRed-O (ORO) mostrou uma diminui??o na quantidade de lip?dio intracelular quando as c?lulas foram tratadas com Fe por 8 dias. No grupo Fe + FBP, houve um aumento do conte?do lip?dico e as c?lulas apresentaram caracter?sticas morfol?gicas de c?lulas quiescentes. A express?o de PPAR-γ foi diminu?da no grupo Fe e igual ao controle no grupo Fe + FBP. Ao contr?rio, o Fe aumentou os n?veis de express?o de Col?geno tipo I (Col-1) e o tratamento concomitante com FBP reverteu esse efeito, ficando igual ao controle. A produ??o de TGF-β1 se manteve inalterada no grupo Fe e foi menor que o controle no tratamento com Fe + FBP, mostrando uma atividade antifibr?tica da FBP. O teste de DPPH mostrou que a FBP n?o possui atividade antioxidante em nenhuma dose testada. O teste de Ferrozine mostrou uma diminui??o da absorb?ncia depois de 120 minutos de incuba??o de FBP + Fe em todas as doses testadas, mostrando que a FBP ? um quelante de ferro.Esses dados demonstram que FBP reverte o fen?tipo das c?lulas GRX mesmo quando em presen?a do Fe e que isso pode ser causado pela regula??o da express?o do PPAR-γ e COL-1.Em conclus?o, a FBP diminuiu o crescimentoe reverteu o fen?tipo de c?lulas GRX, mostrando um poss?vel efeito antifibr?tico.

Made available in DSpace on 2015-04-14T13:34:54Z (GMT). No. of bitstreams: 1 422850.pdf: 2953890 bytes, checksum: a9a608f4e09d0de94b994f35e62d2827 (MD5) Previous issue date: 2010-03-22 / Introdu??o: Evid?ncias crescentes t?m demonstrado que o transplante de c?lulas-tronco pode tornar-se uma terapia eficaz em hepatopatias. A inje??o sist?mica de c?lulas-tronco tem demonstrado que um percentual significativo dessas c?lulas fica retido no pulm?o e no ba?o. Assim, a pun??o percut?nea da veia porta orientada por ultrassonografia com Doppler colorido, em ratos, para administra??o de c?lulas-tronco apresenta-se como uma alternativa. O objetivo deste estudo foi avaliar a exequibilidade da t?cnica de pun??o percut?nea da veia porta e a efic?cia do transplante intraportal de c?lulas mononucleares de medula ?ssea em ratos com cirrose induzida. Metodologia: Para a execu??o do experimento, foram selecionados 24 ratos f?meas da ra?a Wistar com cirrose induzida atrav?s da ligadura do ducto biliar. Quatorze dias ap?s a ligadura, os animais foram preparados com tricotomia abdominal, seda??o e orienta??o do procedimento por ecografia abdominal com Doppler colorido para facilitar a localiza??o da veia porta. A fra??o mononuclear obtida da medula ?ssea de ratos machos doadores foi infundida utilizando uma agulha de gauge 30, com pun??o direta pela parede abdominal por via transhep?tica at? a veia porta, junto do hilo do f?gado. No 28? dia do experimento, os ratos foram sacrificados e avaliou-se o repovoamento hep?tico de c?lulas-tronco atrav?s da an?lise imunohistoqu?mica das c?lulas portadoras do cromossoma Y e a quantidade de c?lulas atrav?s da citometria de fluxo. Resultados: Os resultados obtidos atrav?s da infus?o de c?lulas-tronco diretamente na veia porta, orientada por ultrassonografia com Doppler colorido, evidenciaram que, dos 24 ratos estudados, sete animais (29,2%) morreram nas primeiras 24 horas. Houve um significativo repovoamento hep?tico com c?lulas da medula ?ssea do doador em 77,8% dos ratos estudados e uma efic?cia de 31,5%. Conclus?o: A t?cnica de pun??o percut?nea da veia porta mostrou-se de f?cil execu??o e com visualiza??o do material no interior do sistema portal durante o procedimento, embora aproximadamente 1/3 dos animais tenham morrido no per?odo relacionado ao procedimento. A infus?o da fra??o mononuclear da medula ?ssea diretamente na veia porta de ratos cirr?ticos permitiu a nida??o, fus?o e/ou fagocitose das c?lulas do animal doador no f?gado do receptor, sugerindo potencial terap?utico no tratamento da cirrose humana.

Made available in DSpace on 2015-04-14T13:35:51Z (GMT). No. of bitstreams: 1 453321.pdf: 2208938 bytes, checksum: 36179d9e8ff3571a84f104fd9b7bd8ef (MD5) Previous issue date: 2013-12-18 / Fibrosis is a chronic liver disease that is a major cause of human mortality and is characterized by the accumulation of extracellular matrix in response to chronic liver injury. Important causes of chronic liver injury are: viral hepatitis, metabolic diseases, autoimmune diseases and exposure to chemicals, such as alcohol or drugs. The GRX cells are a representative line of hepatic stellate cells (HSC), which is associated with development of fibrosis, in the last stage is the cirrhosis. In healthy liver, these cells exhibit a phenotype or quiescent lipocyte characterized by its hability to store lipid droplets. Gallic acid is involved in several biological processes such as cell growth inhibition and apoptosis also has a variety of pharmacological actions, including antioxidant activity, anti-inflammatory, antimicrobial and antitumor. The aim of this study was to investigate the in vitro effects of gallic acid on the phenotype of HSC. The results showed that gallic acid is able to reduce cell proliferation, induce quiescent phenotype in HSCs by increasing lipid droplets, probably by activating peroxisome proliferator-activated receptor gama, decrease of transforming growth factor 1 signaling and decreased expression of collagen type I. These results demonstrate that the gallic acid may be a novel therapeutic agent for treating hepatic fibrosis. / A fibrose ? uma doen?a cr?nica do f?gado que representa uma das maiores causas de mortalidade humana e ? caracterizada pelo ac?mulo de matriz extracelular em resposta ? les?o hep?tica cr?nica. Importantes causas de les?es hep?ticas cr?nicas s?o: hepatites virais, doen?as metab?licas, doen?as autoimunes e exposi??o a subst?ncias qu?micas, como ?lcool ou drogas. As c?lulas GRX s?o uma linhagem representativa das c?lulas estreladas hep?ticas (HSC), que est? associada ao desenvolvimento da fibrose que, em ?ltimo est?gio, ? a cirrose. No f?gado saud?vel, estas c?lulas apresentam um fen?tipo quiescente ou lipoc?tico, caracterizado pela sua capacidade de armazenar got?culas lip?dicas. O ?cido g?lico est? envolvido em v?rios processos biol?gicos, tais como a inibi??o do crescimento celular e apoptose, al?m de possuir uma variedade de a??es farmacol?gicas, incluindo as atividades antioxidantes, anti-inflamat?rias, antimicrobiana e antitumoral. O objetivo deste estudo foi investigar os efeitos in vitro do ?cido g?lico sobre o fen?tipo das HSC. Os resultados obtidos mostraram que o ?cido g?lico ? capaz reduzir a prolifera??o celular, induzir o fen?tipo quiescente nas HSCs pelo aumento de got?culas lip?dicas, provavelmente pela ativa??o do receptor ativado por proliferador de peroxissomo gama, bloqueio da sinaliza??o de fator de transforma??o do crescimento beta 1 e diminui??o da express?o do col?geno tipo I. Estes resultados demonstram que o ?cido g?lico pode ser um novo agente terap?utico para o tratamento de fibrose hep?tica.

In this dissertation, I investigate the impact of the extended religious and political elements in the ancient Near East of the Late Bronze period that influenced the reigns of Hattušili III, his consort, Queen Puduhepa, circa 1267 to 1237 BCE and their son Tudhaliya IV circa 1237 to 1228 BCE. As rulers of the Hittites, they were not the greatest and most influential royals, like the great Suppiluliuma I circa 1322 to 1344 BCE, but their ability to adopt an eclectic approach similar to that of their great predecessors regarding religion, politics, international diplomacy and signing treaties made this royal triad a force to be reckoned with in the ancient Near East. Therefore, central to this investigation will be the impact of Hattušili III’s usurpation of the throne and Puduhepa’s role in the Hurrianisation of the state cult and pantheon. Also included is a brief investigation into the continuation of the reorganisation and restructuring of the Hittite state cult and local cult inventories by Tudhaliya IV and his mother Puduhepa after the death of Hattušilli III. By researching this royal triad, their deities, their Hurro-Hittite culture and the textual evidence of their rule, it becomes possible to assemble some of the elements that impacted on their rule. I have used available transliterated translated texts and pictures to support and illustrate the investigation of this complex final period in the history of the Hittite Empire. / Biblical and Ancient Studies / MA (Ancient Near Eastern Studies)