We hypothesized that with exercise training and the subsequent attenuation of hyperglycemia, β-cell adaptation to worsening insulin resistance would be maintained. Also, because classical stress-activated systems and oxidative stress are involved in hepatic insulin resistance we examined if exercise would be associated with improvements in hepatic markers of oxidative stress and inflammation. Exercise maintained fasted hyperinsulinemia and preserved normoglycemia in male Zucker diabetic fatty (ZDF) rats. β-cell function calculations indicate prolonged β-cell adaptation in exercised animals. Such improved β-cell function was associated with increased β-cell mass. Hypertrophy and replication contributed to expansion of β-cell mass; exercised animals had increased β-cell size and bromodeoxyuridine (BrdU) incorporation rates versus controls. Furthermore, we observed augmented β-cell-specific immunohistochemical staining of GLUT2 and Akt/PKB in exercised versus sedentary controls. We also observed large cytoplasmic ubiquitinated structures which form in response to oxidative stress in pancreatic tissue samples from hyperglycemic ZDF rats. In the exercised groups such aggregate numbers were reduced to numbers compared to those seen in younger non-diabetic basal ZDF animals and age-matched lean Zucker rats. With respect to the liver we investigated whether exercise alters kinases such as c-Jun NH2-terminal kinase (JNK) and IKKβ (as evidenced by IκBα levels) and related insulin receptor substrate-1 (IRS-1) serine phosphorylation which are associated with hepatic insulin resistance in obesity. On average, exercised animals ran 5250m/day which improved insulin sensitivity based on the homeostasis model assessment for insulin resistance (HOMA-IR) calculations, and maintained fed and fasted glucoregulation and glucose tolerance. Ten weeks of running decreased whole-body markers of inflammation and oxidative stress in the blood and in the liver. Exercise lowered circulating interleukin-6 (IL-6), haptoglobin, malondialdehyde (MDA) levels, and protein oxidation in the liver. Exercise reduced phosphorylated JNK (pJNK) indicating decreased JNK activity; in accordance serine phosphorylated IRS-1 was reduced in exercised rats. In conclusion, improvements in glucoregulation were associated with increased β-cell compensation at least in part due to a reduction in oxidative stress. Furthermore, we show exercise attenuates development of hyperglycemia in ZDF rats in association with decreases in plasma and hepatic markers of inflammation, oxidative stress, JNK activation, and serine phosphorylation of IRS-1.

No description available.

Thesis (doctoral)--University of Lund, 1981. / Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:23:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-07Bitstream added on 2014-06-13T18:48:32Z : No. of bitstreams: 1 duarte_all_me_jabo.pdf: 292637 bytes, checksum: 6ba54a997864af0075b8c619c900785e (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A técnica de biópsia hepática guiada por videolaparoscopia em caprinos foi realizada em 12 animais machos, castrados, hígidos, sem raça definida e com idades de seis a 12 meses. Os animais foram submetidos à anestesia intravenosa e a laparoscopia consistiu na introdução da agulha de Veress no flanco direito para provocar pneumoperitôneo com dióxido de carbono e permitir a inserção da cânula e trocarte para introdução do endoscópio na cavidade abdominal acoplado a uma microcâmera e ao monitor de vídeo por um cabo de fibra ótica. Após a visualização do fígado, foi introduzida a agulha de biópsia hepática tru-cut no décimo primeiro espaço intercostal, a aproximadamente 12cm ventral à coluna vertebral, para punção e remoção de fragmento do lobo hepático direito. Em seguida, o pneumoperitônio foi desfeito e as incisões cutâneas foram suturadas. Os fragmentos hepáticos colhidos foram fixados em solução de Bouin por 24 horas e processados de forma rotineira para inclusão em parafina. Foram feitas avaliações clínicas e exames hemáticos no pré-jejum e às 24, 48 e 72 horas após a intervenção cirúrgica. Houve leucocitose com neutrofilia e aumento do fibrinogênio plasmático nas primeiras 24 horas pós-operatórias, indicando a ocorrência de processo inflamatório intra-abdominal discreto e fugaz, não mais observado na 488 hora pós-cirúrgica. A biópsia hepática com agulha tru-cut guiada por videolaparoscopia foi considerada segura e eficaz para uso em caprinos, permitindo a obtenção de fragmentos hepáticos suficientes para exame histopatológico. / The technique of videolaparoscopic guided hepatic biopsy was performed in 12 castrated male goats, healthy, without defined breed and aging from six to 12 months. Each animal received total intravenous anesthesia and was submitted to the laparoscopic procedure that consisted in the insertion of a Veress needle in the right flank in order to cause a pneumoperitoneum with carbon dioxide. This procedure was followed by cannulation and then by introduction of a rigid endoscope coupled with a microcamera. Afier visualization of the liver, a tru-cut hepatic biopsy needle was applied in the eleventh intercostal space about 12cm ventral to the spinal column for punch and removal of a fragment from the right hepatic lobe. Soon afier the drainage of the pneumoperitoneum, the small skin incisions were sutured. The biopsy fragments were fixed in Bouin's solution for 24 hours and then submitted to paraffin inclusion histologic routine. Clinical evaluation and hematologic exams were made during the pre-fasting period and again at 24, 48 and 72 hours afier the surgery. There was observed leucocytosis with increased neuthrophils and plasmatic fibrinogen levels at 24 hours afier the procedure, indicating the presence of a discrete intraabdominal inflammatory process that was no more observed at 48 hours. The videolaparoscopic guided hepatic biopsy with a tru-cut needle was considered efficacious and secure to be used in goats, allowing the obtention of hepatic tissue fragments suitable for histopathological examination.

Orientador: Germano Francisco Biondi / Banca: Noeme Sousa Rocha / Banca: Roberto de Oliveira Roça / Banca: Milton Hisashi Yamamura / Banca: Ademir Benedito da Luz Pereira / Resumo: O objetivo deste estudo foi determinar a freqüência de ocorrência de lesões hepáticas provocadas por larvas de helmintos em eqüídeos abatidos em matadouro-frigorífico no Paraná, diagnosticado durante o exame post mortem e estudo macro e microscópico das lesões hepáticas. Foram pesquisados nos meses de abril de 2003 a dezembro de 2004, 38.363 animais, procedentes de diversos estados da federação. A freqüência de ocorrência de granulomas no fígado foi revelada em 14.443 (37,64%), com aderências e manchas 6.645 (17,32%) e sem lesões macroscópicas 17.275 (45.03%). Macroscopicamente, foram verificados fígados com nódulos calcificados, presença de manchas esbranquiçadas, aderências e pontas soltas em forma de "linhas" sobre a cápsula hepática. Não foram encontradas larvas nos fígados. Histologicamente, os fragmentos hepáticos revelaram na microscopia óptica, células inflamatórias com o predomínio de eosinófilos ao redor dos granulomas, com moderada quantidade de macrófagos e presença de fibroblastos. / Abstract: The aim of this study was investigate the hepatic lesions caused by migratory larvae of the helminths. The diagnostic of the lesions were done at post mortem during the slaughter of the horses in Paraná State. The survey was developed during the april 2003 to april 2004. The horses came from diferent regions or states of Brazil. The frequency of occurrence reveled 14.443 (37,64%) hepatic lesions with parasitic granuloma, 6.645 (17,32%) with spot on the hepatic capsule and 17.275 (45.03%) without lesions. Macroscopic analysis reveled presence of calcifications nodules, adhesions, fibrous tags over widely distributed areas on the hepatic capsule. The most evidence observed on the tissue were widespread nodules many of which were coalesced. No larvae were found in the liver. Fragments hepatic were processed for histologic and reveled extensively infiltrated with eosinophils and fibrosis in tissue sections. / Doutor

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.

A capacidade extraordinária de crescimento do tecido hepático pode ser estimulada por dois processos, a hepatectomia parcial ou pelo fornecimento de substâncias hepatotróficas. Neste trabalho, foi adotada a segunda opção para tratar fígados de rata com fibrose, verificando-se o efeito dos fatores hepatotróficos sobre o quadro histopatológico, a proporção volumétrica do colágeno e a condição funcional do fígado de ratas Wistar com fibrose induzida por dimetilnitrosamina. Após 5 semanas de tratamento. Os animais foram submetidos à biópsia hepática e divididos em 2 grupos: o Grupo C recebeu 40 ml/kg de solução fisiológica por via intraperitoneal a cada 12h, durante 10 dias consecutivos e o Grupo F recebeu fatores hepatotróficos na mesma dosagem e freqüência que as do Grupo C. O sangue foi colhido momentos antes do sacrifício e em seguida amostras dos fígados foram levadas à rotina de fixação e inclusão para microscopia de luz. O exame histopatológico mostrou a presença de lesões características de fibrose hepática, tais como: nódulos regenerativos, proliferação de ductos biliares, infiltrado inflamatório com presença de células ovais e megalocitose de hepatócitos, sendo que os animais do Grupo F apresentaram redução do quadro acima descrito. O peso dos animais do grupo F aumentou 13%, o peso do fígado aumentou 12,2% e o peso do fígado em relação ao peso da carcaça aumentou 16%. A densidade volumétrica do colágeno no lóbulo hepático reduziu-se em 43% no grupo F, enquanto que no Grupo C houve aumento de 6%. O colágeno perivascular na veia centrolobular aumentou 42,7% no grupo C e reduziu-se 43,4% no grupo F. No espaço porta o colágeno perivascular aumentou 6,7% no grupo C e reduziu 67,3% no grupo F. A concentração sérica de albumina no Grupo C foi de 3,8g/l e no Grupo F de 4,7g/l. A proteína total foi 6,1g/l no Grupo C e no Grupo F de 7g/l. A aspartato aminotransferase AST foi 148,3 U/l no Grupo C e 94,9 U/lno Grupo F, a alanina aminotransferase ALT foi 86,9 U/l no Grupo C e 44,1 U/l no Grupo F. Concluiu-se assim, que os fatores hepatotróficos injetados intraperitonealmente, reduziram o quadro histopatológico e a densidade volumétrica do colágeno, promovendo melhor resposta da função hepática no Grupo F quando comparado com o Grupo C. / The extraordinary capacity of growth of hepatic tissue can be stimulated by two processes, by the partial hepatectomy or by hepatotrophic substance supply. In this work, it was adopted the second option to treat rats with induced-fibrosis, verifying the effect of the hepatotrophic factors on the histopathological evaluation, the ratio volumetric of the collagen and the functional condition of the liver of Wistar rats with induced-fibrosis by dimetilnitrosamine. After 5 weeks of treatment, the animals had been submitted to hepatic biopsies and divided in 2 groups: Group C received 40 ml/kg of physiological solution by intraperitoneal application each 12h, during 10 consecutive days and Group F received hepatotrophic factors in same dosage and frequency of Group C. Blood collection was proceeded moments before the sacrifice and samples of the liver had been submitted to the light microscopy routine of fixation and inclusion. The histopathologic evaluation showed the presence of characteristic injuries of hepatic fibrosis, such as: regenerative nodules, biliar ducts proliferation, inflammatory infiltrated with presence of oval cells and hepatocyte megalocytes, and the animals of Group F presented reduction of the above description. The weight of the animals of group F increased 13%, the weight of the liver increased 12.2% and the weight of the liver in relation to the weight of the carcass increased 16%. The volumetric collagen density in hepatic lobe decreased 43% in group F, while in Group C it icreased 6%. The central lobular vein perivascular collagen increased in 42.7% in group C and decreased 43.4% in group F. In the portal space, the perivascular collagen increased 6.7% in group C and reduced 67.3% in group F. The serum concentration of albumen in Group C was 3,8g/l and in Group F 4,7g/l. The total protein was 6,1g/l in Group C and in Group F 7g/l. The aspartate aminotransferase AST was 148,3 U/l in 94,9 Group C and U/l in Group F, alanine aminotransferase ALT was 86,9 U/l in 44.1 Group C and U/l in Group F. We concluded the intraperitoneal hepatotrophic factors injection had reduced the histopathological signs and the collagen volumetric density, promoted better recovery of the hepatic function in Group F when compared to Group C.

During chronic and severe acute liver injury, regeneration is thought to occur through hepatic progenitor cells (HPCs). Understanding the regulation of HPCs may offer therapeutic opportunities to enhance liver regeneration. HPCs are associated with an increase in laminins in the extracellular matrix. Laminins are heterotrimeric proteins, composed of an alpha, beta and gamma chain. There are 5 alpha chains with different distributions and functions, but the relative contributions of these in HPC-mediated liver regeneration are not known. My aims were to describe the laminin alpha chains associated with the HPC response and to define the functional effects of specific laminin chains on HPCs. I examined the laminin alpha chains in two mouse models of HPC activation: a transgenic model using conditional deletion of Mdm2 in hepatocytes, and a dietary model using 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). The laminin alpha 5 (Lama5) chain is significantly upregulated in both models and forms a basement membrane which surrounds the progenitor cells. I have also demonstrated Lama5 expression in the ductular reaction seen in human liver disease. Using primary mouse cell cultures, I have shown that Lama5 is produced predominantly by the HPCs themselves, rather than by stellate cells. The HPCs express the cell surface receptor alpha-6 beta-1 integrin, a binding partner of Lama5. I then studied the functional effects of matrix on cell behaviour in vitro using recombinant laminins and a line of spontaneously immortalised mouse HPCs. Compared to other laminin chains, Lama5 selectively promotes HPC adhesion and spreading. These effects are partially blocked by antibodies against beta-1 integrin. Lama5 also significantly enhances HPC migration, resulting in an increase in cell migration. Furthermore, only Lama5 enhances HPC survival in serum-free medium, with an increase in cell viability. Culturing HPCs on HPCs maintained in culture on plastic synthesise Lama5 chain. Knock-down of endogenous Lama5 production using siRNA results in reduced proliferation and increased hepatocytic differentiation, with increased albumin production. I then studied the effects in vivo using transgenic Cre-lox mouse strains that allow conditional knock-out of either laminin alpha 5 or beta-1 integrin in HPCs. The effects of gene deletion were examined in healthy mice and two dietary models of HPC activation: the DDC diet and a choline-deficient, ethionine-supplemented (CDE) diet. Although these experiments were limited by a low number of experimental animals and low recombination rates, there was a suggestion of impaired HPC expansion associated with loss of laminin alpha 5. There was also a significant increase in hepatocellular injury and fibrosis in response to the DDC diet seen with loss of laminin alpha 5 expression. Laminin alpha 5-containing matrix is deposited around HPCs during liver regeneration and supports progenitor cell attachment, migration and maintenance of an undifferentiated phenotype. This work identifies a novel target for enhancing liver regeneration.

Orientador: Rejane Maria Tommasini Grotto / Resumo: A progressão da fibrose hepática somada à infecção pelo vírus da hepatite C (VHC) tem sido associada à resposta imunológica permanente. O estudo no repertório de anticorpos Anti-VHC na progressão da fibrose hepática foi explorado através de ferramentas de sequenciamento em larga escala (NGS) possibilitando uma análise de repertórios altamente variáveis como as porções variáveis VH (cadeia pesada) e Vk (cadeia leve) das imunoglobulinas, e a determinação de famílias e subfamílias dos genes V-D-J associadas à resposta humoral encontrada nas diferentes fases da doença proporcionam uma ferramenta importante no entendimento da resposta imune frente à infecção viral pelo VHC. As porções VH e Vk das imunoglobulinas foram obtidas a partir da amplificação de RNA de sangue de paciente VHC positivos e com diferentes graus de fibrose, e sequenciadas na plataforma Illumina® Miseq, fornecendo uma grande variabilidade de sequências que foram pré-processadas por ferramentas de bioinformática e analisadas em dois bancos de anticorpos diferentes: IgBlast (NCBI) e IMGT® quanto às famílias e subfamílias mais expressas. A expressão restrita de algumas famílias e subfamílias: IGHV1, IGHV3, IGHV4 e subfamílias já descritas em vários estudos associados ao VHC corrobora com nossos achados de que existe uma tendência do uso de algumas subfamílias como: IGHV1-2, IGHV1-8, IGVH1-69, IGHV3-11, IGHV3-21, IGHV3-23, IGVH3-30, IGHV4-4, IGHV4-34 IGHV4-39 na cadeia pesada; assim como IGkV3-15 e IGkV3-20 na cadei... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The progression of hepatic fibrosis in the hepatitis C virus (HCV) infection has been linked with permanent immune response. The study in the repertoire of Anti-HCV antibodies in the progression of hepatic fibrosis was analysed for large-scale sequencing (NGS) tools enabling a highly variable analysis such as the VH (heavy chain) and Vk (light chain) portions of immunoglobulins and the determination of families and subfamilies of the V-D-J genes in the humoral response found in the different phases of the disease, a great tool in the understanding of the immune response to HCV viral infection. The VH and V portions of the immunoglobulins were obtained from the amplification of HCV positive HCV patient blood with different degrees of fibrosis and sequenced on the Illumina® Miseq platform, providing a large sequence variability that was preprocessed by tools of bioinformatics and analyzed in two different antibody banks: IgBlast (NCBI) and IMGT® for the most expressed families and subfamilies. The restricted expression of some families: IGHV1, IGHV3, IGHV4 and subfamilies already described in several HCV-related studies confirm our findings that there is a tendency to use some subfamilies, such as: IGHV1-2, IGHV1-8, IGVH1- 69, IGHV3-11, IGHV3-21, IGHV3-23, IGVH3-30, IGHV4-4, IGHV4-34 IGHV4-39 in the heavy chain; as well as IGkV3-15 and IGkV3-20 in the light chain, but the subfamilies: IGHV1-8, IGHV3-11, IGHV4-39, IGkV1-5, IGkV1-12, IGkV1-39 were also among the most expressed, i... (Complete abstract click electronic access below) / Mestre

Many individuals presumed to have non-alcoholic fatty liver disease (NAFLD) consume moderate amounts of alcohol; however, little is known regarding patterns of alcohol use and how drinking behaviors may influence liver fat. We conducted a cross-sectional study of 2,475 participants of the Framingham Heart Study who underwent computed tomography (CT) to define liver fat. We performed multivariable-adjusted logistic regression models for the association between different alcohol drinking patterns, including the average alcoholic drinks/week, frequency of alcohol use, usual quantity of alcohol consumed, maximum drinks consumed in 24 hours, and binge drinking behavior, and CT-defined hepatic steatosis. We excluded heavy alcohol users defined as women who drink > 14 drinks/week and men who drink > 21 drinks/week. We also performed an analysis specific to beverage type (beer, wine, or liquor/spirit drinks).The prevalence of hepatic steatosis in our study sample (mean age ± standard deviation (SD) 49.8±10.2, 50.3% women) was 17.5%. Among individuals with presumed NAFLD, binge drinking occurred in 25.4% of individuals. In adjusted models, the odds of hepatic steatosis increased by 20% for each SD increase in the number of alcoholic drinks consumed per week (OR 1.20; 95% confidence interval (CI) 1.08, 1.36). Frequency of alcohol use (drinking days/week) was also associated with hepatic steatosis (OR 1.09; 95% CI 1.03, 1.15). The odds of hepatic steatosis increased by 15% for each SD increase in the maximum drinks per week (OR 1.15; 95% CI 1.02, 1.30). In the beverage specific analysis, alcohol use patterns were associated with hepatic steatosis among beer drinkers, but no significant associations were observed among wine drinkers. Conclusions: Even after excluding heavy alcohol users from our sample, alcohol use contributed to liver fat, which suggests alcohol-related liver fat may be present among individuals presumed to have NAFLD. Additional prospective studies are needed to validate our findings and to determine if more comprehensive alcohol use screening tools should be used in practice or clinical trial settings. / 2020-06-03T00:00:00Z