Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted. Errata sheet inserted.

Thesis--Uppsala.

Thesis--University of Helsinki. / Includes bibliographies.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted. Errata sheet inserted.

Liver injury stimulates hepatocyte proliferation, regenerating the liver through self-replication. In cases where there is severe, repetitive, parenchymal damage, as seen in human chronic liver disease, hepatocyte mediated regeneration becomes impaired. In this setting it is currently unclear whether endogenous biliary epithelial cells can repopulate the hepatocyte compartment. This thesis therefore aimed to address this point by lineage tracing the main two liver epithelia populations on a background of impaired hepatocyte regeneration. To impair regeneration, an Itgb1 transgene was specifically deleted, conditionally, from the hepatocyte epithelium. Long-term loss of β1-Integrin alone or with additional injury caused an epithelial ductular reaction of biliary origin. Alongside β1-Integrin ablation, the hepatocyte epithelium was also labelled with a heritable ROSA26LSLtdTomato reporter. Impaired hepatocyte regeneration mediated by β1- integrin ablation resulted in 25% of hepatocytes becoming tdTomato negative (non-hepatocyte derived). To verify that the non-hepatocyte mediated regeneration was originating from the biliary epithelium, anti-Itgb1 RNAi was administrated to K19CreERT LSLtdTomato mice. Resulting in tdTomato positive hepatocytes that had differentiated from the labelled tdTomato positive biliary epithelial cells. In summary, this thesis demonstrates that hepatocyte β1-Integrin ablation combined with toxic damage causes marked ductular reactions and results in a substantial regeneration of functional hepatocytes from the biliary epithelium.

Orientador: Cecilia Amelia Fazzio Escanhoela / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T22:27:25Z (GMT). No. of bitstreams: 1 Silva_GustavoHenriqueda_D.pdf: 4225256 bytes, checksum: 6254dc5eb8ce2f1f9c9ac0ffbce99d41 (MD5) Previous issue date: 2009 / Resumo: A esteatose hepática não relacionada ao alcoolismo pode ocorrer isoladamente ou fazer parte da doença hepática gordurosa não alcoólica (DGHNA). Esta abrange um amplo espectro de alterações morfológicas, variando desde a esteatose até um estágio mais grave, acompanhado por fibrose, podendo chegar à cirrose. O objetivo do nosso estudo foi avaliar e caracterizar a esteatose de causa não definida na infância, por meio de análise morfológica e morfométrica do tecido hepático. Dezoito biópsias provenientes de 16 pacientes com idade variando de 3 meses a 12 anos e 9 meses, com vaga dor abdominal e/ou mínima hepatomegalia associada a aumento discreto e persistente das enzimas hepáticas, foram analisadas através de microscopia de luz e eletrônica de transmissão. Nestes detectou-se esteatose macro e microvesicular "pura", ou seja, não acompanhada de fibrose ou de quaisquer outras alterações histológicas. Na microscopia de luz foi realizada a determinação semi-quantitativa da intensidade da esteatose total (macro e microvesicular), com classificação de 1 a 4 e a estimativa da porcentagem de hepatócitos afetados pela esteatose microvesicular em relação aos hepatócitos afetados pela macrovesicular; na microscopia eletrônica calculou-se a densidade mitocondrial por hepatócito e a área mitocondrial média utilizado-se em ambos os casos o programa TPS Dig versão 1.30. Dez pacientes com 1 a 14 anos de idade e com diagnóstico de normalidade em biópsia hepática foram utilizados como grupo controle. Os resultados obtidos mostraram predomínio de esteatose microvesicular entre os pacientes estudados (61%), com elevação significativa da área mitocondrial, sendo 1,52 ± 0,08 µm2 a área média para o grupo controle, 1,49 ± 0,07 µm2 para o grupo de esteatose macrovesicular e 2,92 ± 0,36 µm2 para o grupo de esteatose microvesicular. Não foram observadas alterações significativas na densidade mitocondrial, seja relacionada à esteatose microvesicular predominante ou à macrovesicular (72 ± 2 mitocôndrias/hepatócitos para o grupo controle, 70 ± 11 mitocôndrias/hepatócitos para o grupo de esteatose macrovesicular e 66 ± 8 mitocôndrias/hepatócitos para o grupo de esteatose microvesicular). Tampouco se observou correlação entre as variações dos valores das transaminases com ambos os tipos de esteatose, uma vez que para o grupo de esteatose microvesicular os valores médios foram AST = 62.9 ± 35.0, ALT = 125.9 ± 129.0 e GGT = 210.5 ± 278.3 e para o grupo de esteatose macrovesicular AST = 82.1 ± 97.5, ALT = 57.4 ± 41.4 e GGT = 185.8 ± 141.7. No grupo de estudo, a esteatose "pura", com pouca ou nenhuma sintomatologia clínica, associada ou não a alterações de transaminases, não relacionada à obesidade ou doença metabólica conhecida, mostrou-se predominantemente microvesicular e relacionada a aumento do volume mitocondrial. Nas hepatopatias mitocondriais primárias (HMP), principalmente por defeitos na cadeia respiratória, este tipo de alteração é comum. Devido a estas alterações mitocondriais, esta esteatose não deve ser denominada "pura" podendo, inclusive, corresponder à porção mais distal no longo espectro da DGHNA. Destacamos que, apesar da pouca manifestação clínica e laboratorial destas crianças, é fundamental o acompanhamento sistemático destas, havendo necessidade de maior atenção aos casos de esteatose na infância com predomínio da forma microvesicular. / Abstract: Hepatic steatosis unrelated to alcoholism may occur separately or be part of fatty liver disease (NAFLD). It encompasses a wide spectrum of morphologic alterations, ranging from steatosis to a more severe stage accompanied by fibrosis and cirrhosis. The aim of our study was to assess and characterize childhood steatosis of not defined cause carrying out morphologic and morphometric analysis of liver tissue. Eighteen biopsies from 16 patients with age varying from 3 months to 10 years, with vague abdominal pain and/or minimal hepatomegaly associated with slight and persistent increase in hepatic enzymes had been analyzed through light microscopy and transmission electron microscopy. On those patients, "pure" steatosis was detected, meaning it was not followed of fibrose or any other significant histological alterations. In light microscopy total steatosis was semi-quantified with estimative of macro- and microvesicular steatosis percentage and determination of predominant steatosis, classified from 1 to 4 and estimate the percentage of hepatocytes affected by steatosis microvesicular in comparison to hepatocytes affected by macrovesicular; in electron microscopy it was determined the degree of mitochondrial density in the hepatocytes and mean mitochondrial surface area using in both cases the TPS Dig version 1.30. Ten patients between 1 and 14 years old who had a normal diagnosis on liver biopsy were used as a control group. The results had shown microvesicular predominance of steatosis in the studied patients (61%) with significant increase of mitochondrial surface area values: 1,52 ± 0,08 µm2 in the control group, 1,49 ± 0,07 µm2 in the macrovesicular steatosis group and 2,92 ± 0,36 µm2 in the microvesicular steatosis group. Significant alterations in mitochondrial density were not observed, neither related to predominant micro nor macrovesicular steatosis (72 ± 2 mitochondrias/hepatocytes in the control group, 70 ± 11 mitochondrias/hepatocytes in the macrovesicular steatosis group and 66 ± 8 mitochondrias/hepatocytes in the microvesicular steatosis group). A correlation between variations in transaminases levels and both types of steatosis was also not observed, since for the microvesicular steatosis group the average values was AST = 62.9 ± 35.0, ALT = 125.9 ± 129.0 and GGT = 210.5 ± 278.3 and for the macrovesicular steatosis AST = 82.1 ± 97.5, ALT = 57.4 ± 41.4 e GGT = 185.8 ± 141.7. In our study group, "pure" steatosis, with little or no clinical symptoms, associate or not to alterations of transaminases and that did not correlate with obesity or known metabolic diseases is predominantly microvesicular and was related to increased mitochondrial volume. In the primary mitochondrial hepatopathies (PMH), especially for respiratory chain defects, this alteration is common. Due to these probable mitochondrial alterations, this steatosis doesn't have to be called "pure", being able, also, to correspond to the portion distal in the long specter of the NAFLD. It is important to emphasize that although the small number of clinical and laboratorial manifestations of these children, it is essential their systematic accompaniment. There is necessity of more attention to the cases with steatosis in pediatric group where microvesicular form predominance occurs. / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Abstract Cytochrome P450 2A6 (CYP2A6) is involved in the 7-hydroxylation of coumarin, C-oxidation of nicotine, and the metabolism of tobacco specific nitrosamines. Initially in 1995 Fernandez-Salguero et al. reported a genotyping method for three alleles: CYP2A6*1 (wild-type), CYP2A6*2 (variant 1), and CYP2A6*3 (variant 2). Later studies presented in this thesis indicated that the original genotyping method produces erroneous results for the CYP2A6*3 allele due to unspecific PCR conditions and previously unknown CYP2A6*1B allele. Furthermore, the CYP2A6*2 allele genotyping caused erroneous genotypes (CYP2A6*2/*2 was misclassified as CYP2A6*1/*2). In this work, new PCR based genotyping methods were developed for CYP2A6*2 and for several new alleles (CYP2A6*1B, CYP2A6*4A/*4D and CYP2A6*5). In population-based studies, the deletion alleles (pooled as CYP2A6*4) turned out to be more prevalent among Asians (15.1%) than Caucasians (0.5%). The frequencies of the other inactive alleles varied within 0–3% in both populations. Asians totally lacked the CYP2A6*2 allele, whereas Caucasians lacked the CYP2A6*5 allele. The frequencies of two wild-type alleles, CYP2A6*1A and CYP2A6*1B alleles were 66.5% and 30.0% in Caucasians, and 43.2% and 40.6% in Asians, respectively. Correlation studies between the phenotype, as tested by the administration of coumarin, and the genotype demonstrated that individuals with the CYP2A6*2/*2 genotype were totally defective, while CYP2A6*1/*2 subjects exhibited intermediate and CYP2A6*1/*1 subjects full capablility of producing 7-hydroxycoumarin. Upon phenotyping with nicotine, individuals with the CYP2A6*1/*2 or CYP2A6*1/*4 genotype were shown to have a lower enzyme activity (one fourth of the normal activity), compared to those with the CYP2A6*1/*1 genotype. Defective CYP2A6 activity has been hypothesised to reduce the risk of environmentally (especially tobacco smoke) induced diseases either by decreasing production of genotoxic metabolites or by preventing addiction to tobacco smoking. However, in our case-control studies on Spanish patients with liver cirrhosis (n = 83) and liver cancer (n = 90) and their controls (n = 237) no significant association between the CYP2A6 genotypes and disease proneness was found. The odds ratio (OR) for developing liver cancer was was 1.4 (95% confidence interval [CI] 0.5–3.7) for genotypes containing at least one CYP2A6*2 allele. For liver cancer the respective OR was 1.3 (95% CI 0.4–4.5). Similarly, no statistically association between CYP2A6 alleles and the risk of lung cancer was observed in our Finnish study population cinsisting of 177 cases and 1089 controls; the OR for combined CYP2A6 variant allele containing genotypes (CYP2A6*1/*2 and CYP2A6*1/*4) was 1.19 (95% CI 0.56–2.45). Our studies therefore do not indicate any major modifying role for the CYP2A6 genotypes in individual susceptibility to environmentally induced diseases.

Obesity in adults and children is associated with increased cardiovascular mortality and morbidity. This is forecast to increase markedly in the next decade as childhood obesity is a burgeoning epidemic. Excess weight is clearly associated with insulin resistance, increased circulating triglycerides, and hypertension, all of these are related to progressive heart and liver disease. Ectopic fat deposition within organs is reported to cause lipotoxicity, which may lead to dysfunction and disease, but there have been few human studies to confirm this. This doctoral thesis set out to study the early pathophysiology of obesity in adults and children using in vivo magnetic resonance (MR) imaging and spectroscopy to assess the composition and function of the heart and liver in lean and obese individuals. The central tenet of this project was to establish and validate a clinically viable method for measuring the fat content of viscera safely and accurately, and to determine a normal range for the triglyceride content of the heart and liver. The initial study demonstrated that the heart remodels in response to weight loss, with over 20% reduction in LV mass, confirming that excess weight is genuinely a modifiable risk factor. Then, using spectroscopy, it was established that the healthy myocardium has a median triglyceride content of 0.37% (IQR 0.24% - 0.47%), which increases linearly in overweight and obese adults. Obesity, in the absence of any confounders, was also associated with a 10% reduction in cardiac contractile function. In comparison, healthy liver median lipid content was 0.67% (IQR 0.44% – 0.88%), which increased in obese adults to 2.9% (IQR 1.6% - 7.6%). There was a graded association between ectopic fat deposition in the liver and dyslipidaemia in adults, characterised by increased circulating triglycerides and reduced high-density lipoprotein. This dyslipidaemia may impair reverse cholesterol transport, and thus could be expected to exacerbate weight gain. Among obese and overweight subjects, there were some with severe steatosis and evidence of coexistent hepatic inflammation and fibrosis. To verify the accuracy of these spectroscopic measures for ectopic fat, a blinded, prospective comparison of non-invasive assessment of unselected liver disease in liver biopsy patients was completed. Liver disease presents with one or more of steatosis, fibrosis and haemosiderosis, all of which are associated with adverse cardiovascular outcomes. Fifty patients were recruited, and interobserver variability among pathologists was measured for histological reference standards for fat, fibrosis and iron deposition. MR measures of each of these metrics predicted the fibrosis, steatosis and haemosiderosis scores accurately. This enabled precise tissue characterisation of all forms of liver disease, including steatohepatitis, with one non-invasive test, to allow the diagnosis and monitoring of hepatic conditions. Lastly, all these new biomarkers of early cardiac and liver disease associated with excess weight were applied to obese and lean children, to understand whether ectopic fat played a substantial role in early life. Obese children had increased ectopic fat in their hearts and livers, as well as impaired strain, evidence of dyslipidaemia, and in some cases evidence of active steatohepatitis, comparable to adults with severe disease. The thesis therefore demonstrates that in vivo magnetic resonance techniques can be used for accurate measurement of visceral lipid content. Furthermore, there is evidence of significant ectopic fat deposition in both adults and children, with evidence of organ dysfunction, which raises the possibility that cardiovascular magnetic resonance may be of value to risk stratify obese individuals based on organ involvement. Finally, the developed methods may have broader applicability and offer a promising new method for the non-invasive diagnosis of chronic liver disease in other clinical settings.

Obesity is increasing worldwide. The addition of excess calorie intake and unhealthy human behavior leads to also other diseases such as metabolic syndromes and liver disease such as non-alcoholic fatty liver disease. Furthermore, the accumulation of fat triggers specific mechanisms that, if prolonged, can cause tissue damage. For instance, the innate immune system becomes agitated in patients with NAFLD or obesity due to excessive fat accumulation. This leads to inflammation in specific tissues and infiltration of other immune cells. One of the main immune cells are neutrophils, which secrete a protease enzyme called protease neutrophil elastase. Interestingly, there have been studies conducted that have shown that when neutrophil elastase is knocked out in mouse models that mimic NAFLD, there seems to be a protective effect occurring in the body and lessen tissue scarring. A possible explanation, and the aim of this thesis, is to explore if autophagy is regulated and thus plays a role in protecting liver from inflammation and fibrosis. Western blotting approach was used to test this hypothesis. The protein samples that are used are extracted from neutrophil elastase knockout mice that have been fed a high-fat high-fructose diet and compare them to samples from wild-type control mice that have been fed a normal chow diet, high-fat diet, and high fat high fructose diet. The results indicated that potential upregulation of the autophagy pathway in the liver of neutrophil elastase knockout mice and more studies would need before accurately and reliably acknowledging the alternation of the autophagy pathway in the liver from mouse model of NAFLD and when neutrophil elastase is knocked out. / 2023-12-10T00:00:00Z

No description available.