Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2018-01-08T16:01:25Z No. of bitstreams: 1 viniciusdelimafreitas_tese.pdf: 8494219 bytes, checksum: 35b61ce25b380b2ebf2433cf44ff18e5 (MD5) / Made available in DSpace on 2018-01-08T16:01:25Z (GMT). No. of bitstreams: 1 viniciusdelimafreitas_tese.pdf: 8494219 bytes, checksum: 35b61ce25b380b2ebf2433cf44ff18e5 (MD5) Previous issue date: 2017-05-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Introduction: The prevalence of hepatic steatosis and non-alcoholic fatty liver disease (NAFLD) in the Brazilian population is not known, and there are few studies about this disease in the country. Lifestyle modification, including physical activity and exercise are first line recommendation for the tratment of patients with NAFLD. Aim: To evaluate the efficacy of the Supervised physical activity and exercise program on-site and distance-supervised with duration of 12-month for patients with non-alcoholic steatohepatitis (NASH). Methods: This is a prospective, longitudinal, open cohort study including consecutive patients who had a histological diagnosis of NASH in the last 12 months and who were followed up at the outpatient NAFLD clinic. Exclusion criteria were: patients with concomitant liver diseases who could lead to steatosis; history or active significant alcohol intake such as equal or greater than 210g / week for men and 140g / week for women; drugs known to be related to NAFLD; untreated hypo or hyperthyroidism; pevious bariatric surgery; obesity equal or greater than grade III; binge eating or other uncontrolled psychiatric disorder. The patients were studied withing a pre-stated protocol study including clinical and laboratory evaluation, as well as the Baecke questionnaire and the six minute walk test (6MWT), before and after participation in the physical conditioning program. Descriptive statistics, Student's tes and the Mann-Whitney test, were performed for parametric and non-parametric variables as apropriated. The significance level adopted was p-value >0.05. Results: From the 15 included patients, three of them did not complete the multidisciplinary program during the 12-month study period. Thus, the total sample analyzed was 11 patients, that is, 73.33% of included patients. The 5% goal for body weight loss was not reached, however low density lipoprotein (LDL) presented significant reduction at the end of the study (p = 0.0130). The distance-supervised program was chosen by all patients and walking was the main physical activity (66.67%), followed by soccer. The 6-min walk distance (6MWD) was sgnificantly higher at trhee and six month when compared with basal distance at the entry of the study. Conclusion: The distancesupervised physical activity and exercise program had high adherence and was effective in improving the functional capacity for patitients with NASH. On the other hand, there was partial improvement for biochemical and antropometric variables. Aditionally, this is a distance-supervised life-style modification program with low cost and high potential cost-benefit for patients with DHGNA and NASH attended on the National Health System. / Introdução: A prevalência da Esteatose Hepática (EH) e da Doença Hepática Gordurosa Não Alcoólica (DHGNA) na população brasileira não é conhecida, e são poucos os estudos sobre esta doença no país. A mudança no estilo de vida representa a principal recomendação para o tratamento da DHGNA, assim, a atividade física e o exercício físico são ferramentas eficientes no combate à dislipidemia e acúmulo de gordura no fígado. Objetivo: Avaliar os efeitos do programa de condicionamento físico supervisionado in loco e supervisionado à distância com duração de 12 meses em pacientes com Esteatohepatite não alcoólica (NASH). Casuística e Método: Trata-se de um estudo de coorte aberto prospectivo, longitudinal, no qual foram estudados, pacientes em acompanhamento nos ambulatórios de DHGNA do Hospital de Base de São José do Rio Preto, que tiveram o diagnóstico histológico de NASH nos últimos 12 meses. Os critérios de exclusão apresentados foram: pacientes com outras doenças hepáticas concomitantes que possam cursar com esteatose; história prévia de ingestão alcoólica igual ou superior a 210g/semana para homens e 140g/semana para mulheres; medicação conhecidamente relacionada com a etiologia de DHGNA; hipotireoidismo ou hipertireoidismo não tratado; pós-operatório de cirurgia bariátrica; obesidade maior ou igual ao grau III; compulsão alimentar ou outro distúrbio psiquiátrico não controlado. Os pacientes foram analisados em protocolo de avaliação clínica e laboratorial, como o questionário de Baecke e o Teste de Caminhada de 6 minutos (TC6), antes e após a participação no programa de condicionamento físico em estudo. A estatística descritiva foi composta pelas variáveis paramétricas e não paramétricas (média, desvio padrão). As comparações entre os valores basais e, após a intervenção do programa de condicionamento físico foram efetuadas pelo teste t de Student (dados pareados) e teste não paramétrico de Mann-Whitney, com nível de significância (valores de p) inferior a 0,05. Resultados: Dos 15 pacientes incluídos no estudo, três pacientes não concluíram o programa multidisciplinar no período de 12 meses. Assim, a amostra total analisada foi de 11 pacientes, isto é, 73,33% dos incluídos no estudo. O programa supervisionado a distância foi escolhido por todos os pacientes avaliados tendo a caminhada como atividade física mais praticada (66,67%), seguido do futebol. A meta de perda de 5% do peso corporal não foi atingida ao final do estudo, e a lipoproteína plasmática de baixa densidade (LDL) apresentou redução significante (Tempo 4, p=0,0130) durante o estudo. A Distância Percorrida no Teste (DTC6) foi maior nos Tempos 1 e 2 quando comparado ao Tempo 0, com diferença significante (p < 0,05). Conclusão: O programa de condicionamento físico supervisionado à distância teve alta adesão e foi eficaz para a melhora da capacidade funcional de pacientes com NASH. A melhora foi parcial para os parâmetros bioquímicos e antropométricos. Adicionalmente, este programa de condicionamento físico, monitorado à distância, tem baixo custo e é de facil implantação no Sistema Único de Saúde, com alto potencial de custo-benefício para pacientes com DHGNA e NASH, que poderão ser maiores a longo prazo.

Non-alcoholic fatty liver disease (NAFLD) results from an unequal uptake/storage and export/oxidation of lipids within the liver and is often a secondary disease to type II diabetes (22). NAFLD causes this imbalance of lipids by altering glucose and lipid metabolism, which corresponds to a decrease in mitochondrial function leading to failure of the liver. One established treatment for type II diabetes and NAFLD is the drug metformin, which has similar properties to the newly discovered CTRP 3 protein which is part of a group of bioactive molecules secreted by adipose tissue, collectively termed adipokines (2-4). Both have similar effects on hepatic glucose and lipid metabolism and both specifically suppress hepatic gluconeogenesis (11, 17, 27, 29). The revolutionary Seahorse extracellular flux analyzer was used to measure the metabolism of H4IIE hepatocytes without use of radiolabeling (1). By detecting the Oxygen Consumption Rate (OCR) of hepatocytes the level of metabolic function within mitochondria can be measured. Once an effective protocol was established using this new technology, hepatocytes treated with metformin had a significantly lower OCR compared to control treated hepatocytes treated. However, H4IIE hepatocytes treated with metformin and palmitate had a significant increase in OCR and eventually equilibrated with the lower OCR of hepatocytes solely treated with metformin. With similar effect, hepatocytes treated with CTRP3 and palmitate caused a drastic increase in OCR while hepatocytes treated with only CTRP3 had a decrease in OCR. This suggests that CTRP3 increases fatty acid oxidation which decreases lipid concentrations within hepatocytes which could mean future protection of liver against NAFLD. In conclusion, our Seahorse XF analyzer models compare metformin and CTRP3’s similarities and suggest the possible liver protective functions of CTRP3. Our results will aid in future research of CTRP3 to further determine its possible uses as a treatment for liver-associated diseases.

Alcoholic fatty liver disease (AFLD) is a significant public health concern. Excessive alcohol (ethanol) consumption causes liver cell damage and death, which results in eventual failure of the liver and death. AFLD is the number one cause of liver-related mortality in the United States. Our lab works with the novel protein C1q TNF Related Protein 3 (CTRP3), which inhibits non-alcoholic fatty liver disease, however the effects on AFLD are unknown. Therefore, the purpose of this experiment is to determine if CTRP3 prevents ethanol-induced liver cell death. The H4IIE rat hepatoma cell line was chosen for experimentation as a cell culture model of liver tissue. To determine a suitable alcohol level H4IIE cells were treated with 50, 100, and 200 mmol of ethanol for 18-24 hours. Trypan Blue was used to identify the dead/damaged cells, as only dead/damaged cells will be stained blue with this protocol. We observed that 100 mmol of ethanol consistently induced ~10% mortality rate in these cells. Next, we tested the ability of CTRP3 to reduce ethanol-induced mortality. We added purified CTRP3 protein to the cell media along with the 100 mmol ethanol treatment. The addition of CTRP3 reduced the amount of alcohol-induced cell death/damage in the H4IIE cell line by approximately 60%. Our next goal was to determine how CTRP3 reduces ethanol-induced death. The Akt signaling pathway is a well-known inhibitor of cell death. Therefore, to determine if CTRP3 attenuated ethanol-induced cell damage/death through activation of the Akt signaling pathway, another set of cells was treated with 100 mmol of ethanol and CTRP3 (with or without insulin). Western blots were used to compare the amount of active Akt (phosphorylated) in the CTRP3-treated and non-treated cells. A Western blot utilizes an electric current to separate denatured protein samples on a SDS-page gel, separating the proteins based on size. The smaller the protein the faster it migrates across the gel. The proteins are then transferred to a membrane for analysis, through exposure to commercial antigens and chemiluminescence imaging. There was no change in the amount of total or active Akt between the samples treated with or without CTRP3. We conclude that CTRP3 protects liver cells from ethanol-induced damage/death, but not through activation of the Akt pathway.

Alcohol abuse is the third leading cause of preventable death in the United States. Excessive intake of alcohol can result to alcoholic fatty liver disease, the number one cause of live related mortalities in the US. The outlining purpose for this project is to determine the alcohol-induced changes in the liver cell protein signaling. For this project, we treated H4IIE rat hepatoma cells (with 100 and 200 mM ethanol overnight). H4IIE cells were chosen because they are a commonly used liver cell culture line that maintains characteristics of intact liver cells. After treatment we collected and prepared the cells for protein signaling analysis, using standard western blotting procedure. A western blot detects relative quantity of proteins in a sample. Briefly, protein samples are separated by size through electrophoresis, smaller proteins move faster through the gel so that the larger proteins are toward the top and smaller towards the bottom. The proteins are then transferred to a nitrocellulose membrane and protein concentration is detected by chemiluminescence. We chose to examine the effects of ethanol on the activation of the key regulator of metabolic signaling, Protein Kinase B/Akt (Akt). Based on our results, ethanol has no effect on the total amount of Akt in the H4IIE liver cells. However, ethanol significantly attenuates insulin-induced activation of Akt in a dose-dependent manner, as seen by a reduction in the amount of phosphorylated Akt. Therefore, we conclude that treatments that increase Akt activation may be a viable option for the treatment of alcoholic fatty liver disease.

Over the past five decades, per capita caloric intake has increased by approximately 28% in the United States. A hallmark of the current standard American diet is an excess of energy sources from saturated fat and refined carbohydrates. High energy diets such as the “Western” diet cause numerous pathologies, including non-alcoholic fatty liver disease (NAFLD), high blood pressure, dyslipidemia, and peripheral insulin resistance. High energy diets also negatively impact the hippocampus, a brain area important for learning and memory. It is not surprising, then, that high energy diets impair hippocampal-dependent memory. The experiments in this dissertation investigate possible diet-induced consequences that may contribute to the impairing effects of high energy diets on hippocampal-dependent memory. Our initial experiments found that diet-induced NAFLD impairs hippocampal-dependent memory, but these cognitive deficits were not due to decreases in insulin-like growth factor-1 (IGF-1) or hippocampal insulin signaling. Next, we found that a high energy diet increased the ability of epinephrine to increase blood glucose concentrations, indicating a novel way in which high energy diets impair liver function. The final set of experiments found that high energy diets do not necessarily impair memory but instead may prevent the memory-enhancing effects of acute stress. Taken together, these results indicate that high energy diets interact with acute stress to negatively impact hippocampal-dependent memory, and that hippocampal insulin resistance and IGF-1are not likely involved.

Bakgrund: Levercirros är ett sjukdomstillstånd som har hög mortalitet om inte en levertransplantation genomförs. Väntelistan till transplantation är lång samtidigt som organbehovet ökar. Syfte: Syftet med studien är att beskriva patientens upplevelser och livskvalitet i väntan på en levertransplantation. Metod: En litteraturöversikt valdes där 9 vetenskapliga artiklar granskades och analyserades enligt Fribergs modell (2006). Resultat: Beskedet om att bli uppsatt på väntelista präglades av både positiva och negativa känslor; glädje, lättnad, oro samt tveksamhet. Vid väntan uppstod fysiska och psykiska begränsningar. Olika kategorier framkom som var tydliga i de granskade artiklarna; att bli uppsatt på väntelista, att uppleva begränsad frihet, att ha behov av information och supportsamt att uppleva fysiska och psykiska begränsningar. Slutsats: Genomgående sänkt livskvalitet visas hos leversjuka människor som väntar på en levertransplantation vilket stämmer överens med tidigare forskning. Genom vårdpersonalens djupare förståelse för patienten i dennes väntan och dess fysiska och psykiska påverkan kan livskvaliteten samt upplevelsen av väntan förbättras. / Background: Liver cirrhosis is a condition that has a high mortality unless a livertransplantation is carried out. The waiting list for transplantation is long while the need for organs increases. Purpose: The purpose of this study is to describe patient’s experiences and quality of life while awaiting a liver transplant. Method: A literature review was chosen where 9 scientific articles were reviewed andanalyzed according to Friberg's model (2006). Results: The diagnosis and the registration on the waiting list were marked by both positive and negative emotions; joy, relief, worry and doubt. During the waiting period physical and mental limitations were present. Different categories emerged which were evident in the reviewed articles, to be put on waiting list, to experience limited freedom, to have needs for information and support, to experience physical and mental limitations. Conclusion: Consistently lower quality of life having a liver disease and for people waiting for a liver transplant which agree with earlier studies. By health professionals gaining a deeper understanding of the patient's situation while waiting and its physical and psychological impact, the quality of life and the experience of waiting can be improved.

Nonalcoholic fatty liver disease (NAFLD) has become a worldwide, chronic liver disease of increasing clinical significance. It is closely associated with the rising epidemics of obesity and insulin resistance. Up to 17% of the United States population may progress from the disease stage characterized as simple, benign steatosis to the more severe, inflammatory stage of nonalcoholic steatohepatitis (NASH). This progression occurs through 2nd 'hits' of increased oxidative stress and inflammation to a liver that has been sensitized by lipotoxic stress. NASH is also characterized by increased collagen deposition resulting in fibrosis and architectural rearrangement of the liver. Progressive NAFLD is currently recognized as an important contributor to the development of cryptogenic cirrhosis and subsequent liver-related mortalities (estimated at 30-40% in these patients).The pathological progression of NAFLD, as described by the 'two hit' hypothesis, characterizes the different stages of liver injury. However, the mechanism(s) responsible for the progression to NASH are unknown. Profiling global gene expression and metabolite patterns in human liver samples representing the full spectrum of progressive human NAFLD may reveal potential mechanisms of progressive disease. Human liver samples representing each stage of NAFLD progression were analyzed by methodologies such as high-throughput microarrays, high resolution mass spectrometry, and protein immunoblot techniques. Bioinformatics tools and gene expression/regulation database software were utilized in several studies to characterize the altered hepatic profiles of these patients. Hepatic transcriptomic profiles of ADME (absorption, distribution, metabolism and elimination) and ER (endoplasmic reticulum) stress response genes exhibited initiated hepatoprotective responses in patients with NASH. The endogenous pathways of BA (bile acid) synthesis and BCAA (branched chain amino acid) metabolism also showed evidence of coordinately regulated alterations in response to disease-induced stress in NASH. The transcriptional regulation of the investigated pathways was confirmed by transcription factor binding sites enrichment analysis. The collective response to hepatic stress in human NAFLD, demonstrates a coordinated, hepatoprotective intent that may be utilized for future therapeutics in the battle against progressive liver disease.

Inter-individual variability in drug response is a significant clinical concern and may lead to the development of adverse drug reactions, which are currently a top-ten cause of death in the United States. Recently, the manifestation of disease, which may alter normal physiological function, has gained increased attention for its role as a contributing factor in the development of inter-individual responses to drugs. One such disease, known as nonalcoholic fatty liver disease (NAFLD), is the most common chronic liver disease in Western society and represents a spectrum of clinical morbidities that range from the usually benign simple fatty liver to the more advanced nonalcoholic steatohepatitis (NASH). Prior investigations have identified liver-specific alterations in xenobiotic transporter and metabolizing enzymes in NASH, which lead to the functional disruption of drug disposition. To identify a useful model(s) that is representative of hepatic transporter expression profiles in humans with NASH, gene and protein expression profiles of liver membrane transporters were assayed across several commonly used experimental rodent models of the disease. NASH models that were representative of the human condition developed global, adaptive changes in transporter regulation in the liver, which was not present in models that failed to recapitulate human profiles. Specifically, decreased expression of hepatic uptake transporters was coupled with an induction of efflux transporters, which may serve as a hepatoprotective response by limiting hepatic exposure to potentially harmful substances during times of tissue stress. To link a possible molecular mechanism for these hepatic adaptations in NASH, the role of the oxidative stress-activated transcription factor, Nrf2, was investigated. A functional Nrf2 regulatory element was identified within the eighth intron of the human ABCC3 transporter gene, implicating Nrf2 activation in NASH as a contributor to the coordinated induction of hepatic efflux transporters in the disease. To further clarify the effects of NASH on renal membrane transporter regulation, a thorough analysis of gene and protein expression was conducted with the validated rodent models used previously. Following the manifestation of disease, a global induction of renal efflux was observed, suggesting a compensatory, coordinated response of membrane transporters in the kidney upon disease induction. The functional consequences of liver and kidney xenobiotic transporter dysregulation was shown to disrupt the disposition of the environmental toxicant, arsenic. Specifically, NASH results in increased excretion of arsenic into urine as well as altered hepatic and renal exposure. These findings are associated with hepatic and renal transporter dysregulation and demonstrate for the first time that NASH alters the disposition of environmental toxicants. In summary, these studies contribute novel findings that identify liver and kidney-specific adaptations in disease that may contribute to global alterations in xenobiotic disposition thereby increasing the likelihood of developing adverse drug reactions in patients with NASH.

Aufgabe dieser Arbeit war es, den Serumspiegel von Pentraxin-3-Werten im Blutplasma bei Lebererkrankungen zu bestimmen und mit anderen diagnostischen Parametern zu vergleichen. Vorausgegangene Studien hatten bereits darauf hingewiesen, dass PTX3-Werte als früh verfügbare und aussagekräftige Marker bei Entzündungskrankheiten in verschiedenen Organen hilfreich sein können. Es wurden zwei Patientenkollektive miteinander verglichen. Von 25 Patienten ohne Lebertransplantation und von 39 Patienten nach Lebertransplantation wurden die PTX3-Werte mit Hilfe des „Quantikine® Human Pentraxin 3/TSG-14 Immunoassay“ gemessen. Die Werte der Patienten nach Lebertransplantation wurden noch unterteilt in die Zeiträume drei oder mehr Monate sowie fünf oder mehr Monate nach der Transplantation. Die statistische Prüfung erfolgte mit Hilfe des t-Tests. Das Patientenkollektiv mit Lebererkrankungen wies hochsignifikante Steigerungen des PTX3-Wertes gegenüber dem Normwert auf. Das jeweilige Patientenalter sowie das Geschlecht der untersuchten Personen besaßen nur einen geringfügigen Einfluss auf die PTX3-Konzentration. Bei Patienten mit Lebertransplantation hatten sich die PTX3-Werte nach drei oder mehr Monaten weitgehend normalisiert, weitere Messungen nach fünf oder mehr Monaten bestätigten diese Tendenz, die geringfügige Differenz zum Normwert war nicht mehr statistisch signifikant. Die PTX3-Messwerte der an Lebererkrankungen leidenden Patienten wurden zudem mit anderen klinisch-diagnostischen Parametern verglichen. Im Vergleich mit CRP, ALT, AST, AP und GGT zeigte die statistische Analyse signifikante und hochsignifikante Unterschiede mit stärkeren Erhöhungen der PTX3-Werte. Daraus lässt sich ableiten, dass PTX3 eine zuverlässige Rolle bei der klinischen Diagnose von Lebererkrankungen einnehmen kann.

The biological activity of α-linolenic acid (ALA) is poorly understood and primarily associated with its conversion to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). This study used the Δ6 desaturase knockout (D6KO) mouse, which lacks Δ6 desaturase and therefore cannot convert ALA, to evaluate the independent effects of ALA on preventing non-alcoholic fatty liver disease (NAFLD). First, the capacity of very long chain fatty acids to rescue the D6KO lipid profile was established. Next, to evaluate the independent effects of ALA, D6KO or wild-type mice were fed diets containing lard, canola, flaxseed, or fish oil. Following treatment, liver phospholipid fatty acid composition was evaluated and livers were scored for steatosis and inflammation. Glucose tolerance was also evaluated. D6KO mice fed ALA-rich diets had lower liver lipid accumulation, lower hepatic inflammation (8 weeks) and improved glucose tolerance (20 weeks) relative to lard-fed D6KO mice. Overall, this thesis supports an independent biological role for ALA. / D.W.L. Ma is funded by the Canola Council of Canada, Natural Sciences and Engineering Research Council of Canada and the Canada Foundation for Innovation Leaders Opportunity Fund with matching from the Ontario Research Fund; Jessica Monteiro is funded Ontario Graduate Scholarship.