Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O Trypanosoma cruzi é o agente etiológico da doença de Chagas, transmitida através de insetos vetores triatomíneos durante a alimentação no hospedeiro vertebrado. Os triatomíneos ingerem numa única alimentação cerca de 10 mM de heme ligado à hemoglobina. O heme é uma importante molécula no metabolismo dos organismos. Um mecanismo intracelular importante no controle de sua homeostase é a degradação enzimática pela Heme Oxigenase (HO) formando biliverdina (Bv), monóxido de carbono e ferro. Como esta enzima não está presente no genoma de T. cruzi, esse trabalho tem por objetivo identificar uma atividade funcional de HO neste parasito, uma vez que dados do nosso laboratório mostram a presença de biliverdina nas incubações dessas células com heme. No presente trabalho testamos o efeito do SnPPIX (inibidor da HO-1), CoPPIX (indutor da HO-1) e Bv sobre a proliferação da forma epimastigota do parasito. A adição de SnPPIX diminuiu a proliferação do parasito na tanto na ausência quanto na presença de heme. Quando a Bv foi adicionada à cultura esse efeito foi revertido; a Bv aumenta a proliferação celular na presença de heme. Por outro lado, a adição de CoPPIX não interferiu na proliferação. Posteriormente, mostramos através da técnica de immunoblotting, utilizando anticorpo monoclonal contra a HO-1, um aumento da expressão de uma proteína em resposta ao heme. Diferentemente das HO-1 já descritas que possuem massa molecular de 32 kDa, a única banda reconhecida pelo anticorpo apresenta 45 kDa. Analisamos também a expressão da HO-1 na presença de CoPPIX, SnPPIX e biliverdina, e somente o CoPPIX foi capaz de modular os níveis de expressão da HO-1. A análise estrutural através da técnica de imunocitoquímica mostrou uma maior expressão da enzima na presença de heme, e que a HO-1 de T. cruzi pode ter mais de uma localização, apresentando marcação citoplasmática e glicossomal. A fim de investigar a sequência da HO-1 de T. cruzi, o DNA genômico foi extraído para amplificação por PCR do gene da HO-1 utilizando oligonucleotídeos desenhados no genoma de T. cruzi. Os dois pares de oligonucleotídeos utilizados nao foram capazes de amplificar uma sequência equivalente a uma HO. Em seguida, utilizamos a técnica de imunoprecipitação, seguida de immunoblotting, com anticorpo anti-HO-1, com objetivo de concentrar a proteína alvo, e observamos um aumento significativo do imunocomplexo nas células tratadas com heme 300 mM, cerca de 2 vezes em relação ao controle. Dando seguimento à tentativa de identificação da HO-1 de T. cruzi, utilizamos a técnica de espectrometria de massa a partir de eletroforese unidimensional, que mostrou uma grande alteração do perfil protéico na presença de heme, mas futuros experimentos são necessários, como eletroforese 2D, para a identificação da proteína alvo / Trypanosoma cruzi, the ethiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate host. These hematophagous insects ingest blood about 6 to 12 times its original weight, reaching in a single meal about 10 mM heme bound to hemoglobin. Heme (iron protoporphyrin IX) is an important molecule in metabolism of all living organisms. One important intracellular mechanism to control heme homeostasis is its enzymatic degradation by heme oxygenase (HO). HO catalyzes the degradation of heme to biliverdin (Bv), carbon monoxide and iron. HO is absent in T. cruzi genome, thus we have been investigating the presence of a functional HO in this parasite, since our previous results showed a presence of biliverdin in heme-treated epimastigotes. In the present work, we evaluated the effect of SnPPIX, a HO-1 inhibitor, CoPPIX, a HO inducer, and Bv upon T. cruzi epimastigotes proliferation. The addition of SnPPIX decreased the parasite proliferation in the absence or in the presence of heme. When Bv was added to the culture this effect was reversed; Bv increases the parasite proliferation in the presence of heme. On the other hand, CoPPIX did not interfered on proliferation. Furthermore, we showed through immunoblotting, using an anti-HO-1 monoclonal antibody, an increase in the protein expression in heme-treated epimastigotes. Differently of described HO-1 that has a mass molecular of a 32 kDa, we showed a 45 kDa protein, the only band recognize by the HO-1 antibody. HO-1 expression analysis in the presence of CoPPIX, SnPPIX and biliverdin, showed that only CoPPIX was able to modulate its expression level. Ultrastructural immunocytochemistry analysis suggests a higher expression of the enzyme in heme-treated epimastigotes, and that T. cruzi HO-1 might have a dual distribution, since the anti-HO-1 antibody labeled both cytosol and glycosomes. In order to investigate the T. cruzi HO-1 gene sequence, we isolated genomic DNA from T. cruzi for PCR amplification using primers designed as from the parasite genome. Unfortunately, the two pairs of the designed oligonucleotides tested were unable to amplify a sequence equivalent to a HO-1. In order to isolate the target protein, immunoprecipitation was performed and subsequently immunoblotted with anti-HO-1 antibody. The immunocomplex level was two-fold higher in heme-treated cells. Following the attempt to identify a HO-1 in T. cruzi, we used mass spectrometry from unidimensional electrophoresis. We showed a significant protein profile modification in heme-treated epimastigotes, but further experiments will be necessary, such as mass spectrometry from bidimensional electrophoresis, for identification of the target protein

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As doenças cardiovasculares representam a principal causa de morte nos países ocidentais. Dentre essas doenças, a aterosclerose é que mais se destaca, sendo caracterizada pelo acúmulo de células musculares lisas vasculares (CMLV). O efeito patológico das CMLV em resposta a diferentes estímulos pode acarretar em disfunções nestas células. É notável que a aterosclerose ocorra principalmente em vasos sinuosos onde ocorre um forte turbilhonamento do fluxo sanguíneo, que pode acarretar em hemólise e, consequentemente, acúmulo de heme livre. Além disso, no processo de aterogênese as moléculas de adesão, principalmente integrinas, são de crucial importância durante a resposta de CMLV. Nesse trabalho nosso objetivo inicial foi avaliar o efeito do heme livre nas funções de CMLV, bem como os mecanismos moleculares por trás desses efeitos. Em uma segunda parte, investigamos o envolvimento da integrina α1ß1 no efeito da Angiotensina II (Ang II) em CMLV. Nós observamos que o heme livre é capaz de induzir a proliferação e migração de CMLV via espécies reativas de oxigênio (ERO) provenientes da NADPHoxidase (NADPHox). Adicionalmente vimos que o heme ativa vias de sinalização redox-sensíveis relacionadas à proliferação celular, como MAPKinases e o fator de transcrição NFκB. Também observamos que há uma ligação entre a NADPHox e o sistema heme oxigenase (HO), uma vez que o heme induz a expressão de HO-1 e o pré-tratamento das CMLV com inibidores de HO levam ao aumento tanto o efeito proliferação quanto a indução de ERO promovidas pelo heme. Além disso, vimos que o efeito contra-regulatório promovido pela HO ocorre devido as metabolites do heme: biliverdina, bilirrubina e monóxido de carbono. Por último, quando bloqueamos tanto a NADPHox quanto o sistema HO o heme não teve efeito algum na proliferação de CMLV. Em um segundo estudo, observamos que o efeito da Ang II sobre a migração de CMLV foi inibido quando as células foram pré-tratadas com o ligante da integrina α1ß1, a desintegrina Obtustatina. A seguir observamos que o efeito da Ang II na ativação de FAK e na colocalização actina-ILK é dependente da integrina α1ß1, que possivelmente ativa PKCα, uma vez que vimos que a produção de ERO induzida por Ang II foi inibida pela Obtustatina. Vimos que a indução da expressão de ILK por Ang II em CMLV é dependente da integrina α1ß1 e também observamos que a Obtustatina inibibiu o desacoplamento de ILK da FAK, uma vez que a Obtustatina bloqueou a fosforilação de FAK induzida por Ang II (processo crucial para o desacoplamento da ILK). Nós também observamos que a Ang II induz, via integrina α1ß1, a fosforilação de AKT e a diminuição da expressão de p21, provavelmente via ILK. Corroborando estes dados, nós mostramos que o pré-tratamento com Obtustatina induziu um estacionamento na fase G0 e diminuição da proliferação de CMLV tratadas com Ang II. Portanto, mostramos nesse trabalho que o heme livre induz a ativação de CML via NADPHox, que é elegantemente contra-regulado pelo sistema HO. Além disso, sugerimos que a integrina α1ß1 pode ser um importante alvo molecular para o desenvolvimento de intervenções mais efetivas para a aterosclerose. / Cardiovascular diseases represent the major mortality reason in western countries. Among these diseases, atherosclerosis is the most prominent one, which is characterized by vascular smooth muscle cell (VSMC) accumulation. The pathological effect of VSMC in response to different stimuli is able to induce VSMC dysfunctions. Notably, this cardiovascular disease occurs mainly in sinuous vessels with turbulent blood flow, which may lead to hemolysis and consequent free heme accumulation. Furthermore, in atherogenesis the adhesion molecule, mainly integrins, were of crucial importance during the VSMC response. In this work our aim was to elucidate the effect of free heme in VSMC, as well the molecular mechanisms underlying this process. In a second part, we investigated the role of α1ß1 integrin in Angiotensin II (Ang II) effect on VSMC. We observed that free heme is able to induce VSMC proliferation in a Reactive Oxygen Species (ROS) derived from NADPHoxidase (NADPHox) dependent manner. Additionally, heme activates proliferation-relationed redox-sensitive signaling routes, such as MAPKinases and the transcription factor NFκB. It was also observed a critical crosstalk between NADPHox and heme oxygenase (HO) system, once heme induces HO-1 expression and VSMC pretreatment with HO inhibitors increased heme proliferative effect and ROS production. Accordingly, we observed that the counter-regulatory effect promoted by HO occurs due heme metabolites: biliverdin, bilirubin and carbon monoxide. Finally, when both NADPHox and HO system were blocked, heme had no effect on VSMC proliferation. In a second part, we observed that the chemotactic effect of Ang II on VSMC was abolished when the cells were pretreated with the α1ß1 integrin ligand, the disintegrin Obtustatin. Then, we observed that the Ang II effect on FAK activation and actin-ILK colocalization is integrin α1ß1 dependent, which possibly activates PKCα, once we observed that the ROS production induced by Ang II was inhibited by Obtustatin. We demonstrated by western blotting that ILK induction by Ang II is dependent of α1ß1 integrin and we also observed that Obtustatin inhibited the uncoupling of ILK to FAK, once Obtustatin blocked the FAK phosphorylation induced by Ang II (crucial process to ILK uncoupling). We also observed that Ang II induced, via α1ß1 integrin, AKT phosphorylation, and p21 expression reducement, probably via ILK. Corroborating these data we demonstrated that the pretreatment with Obt induced G1 phase arrest and diminishment of VSMC proliferation treated with AngII. Thus we showed that free heme induces VSMC activation via NADPHox, which is elegantly counter-regulated by HO-1. Furthermore, we suggest that α1ß1 integrin may be an important target molecule to the development of more effective therapeutic interventions in atherosclerosis.

This thesis is focused on the study of glycosphingolipids in the rat liver in different types of cholestasis and the effect of oxidative stress on changes in the composition and localization of gangliosides. First, it was necessary to optimize the immunochemical detection of glycosphingolipids. GM1 ganglioside was selected as a representative of a large glycolipid family. We found that minimum water content in the fixing solution was a key condition for fixation of histological sections. Optimized method of GM1 detection was subsequently used in in vivo experiments. We have demonstrated that estrogen-induced cholestasis characterized by high concentrations of bile acids and increased oxidative stress caused changes in the synthesis and distribution of liver gangliosides. HMOX induction is associated with a reduction in oxidative stress level and accompanied by normalization in GSL content. In experiments with obstructive cholestasis, we found that changes in the distribution and synthesis of gangliosides were not strictly specific to a particular type of cholestasis. We assume that it represents a general mechanism of hepatoprotection. We also confirmed the important role of bilirubin, product of HMOX reaction, in protection of hepatocytes against oxidative damage caused by high concentrations of...

Orientadores: Mônica Barbosa de Melo, Fernando Ferreira Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T00:19:46Z (GMT). No. of bitstreams: 1 Gil_GislenePereira_M.pdf: 2808239 bytes, checksum: 22a11f67b6d086bf9bab0100ff802ec7 (MD5) Previous issue date: 2012 / Resumo: Embora a anemia falciforme (AF) resulte da homozigosidade de uma única mutação, no codon 6 do locus da ?-globina, fenotipicamente, essa doença é muito heterogênea, de modo que diferentes pacientes podem apresentar evoluções clínicas significativamente distintas. As complicações nestes pacientes normalmente são decorrentes de acometimento vascular causado pelo acúmulo de hemácias falcizadas nos vasos sanguíneos. Um dos eventos que vem sendo associados a complicações em diversas doenças é o mecanismo de estresse oxidativo, o qual apresenta- se exacerbado em pacientes com AF. Dentre as fontes de estresse oxidativo nestes pacientes estão os eventos de vaso-oclusão e isquemia reperfusão, os quais são muito frequentes. O estresse oxidativo em níveis elevados pode danificar várias moléculas e posteriormente prejudicar o organismo. Alguns polimorfismos em enzimas envolvidas na via de estresse oxidativo foram associados com doenças vasculares como, hipertensão, doença arterial coronária, doença arterial periférica. Considerando que os pacientes com AF apresentam complicações decorrentes de acometimento vascular, esses polimorfismos podem estar contribuindo para as várias manifestações clínicas e, consequentemente, para a gravidade da doença. Este projeto se propôs a avaliar três polimorfismos em genes envolvidos no mecanismo de estresse oxidativo em pacientes com AF e testar a associação com o grau de gravidade da doença. Os polimorfismos analisados foram o C242T e -930 A/G no gene CYBA e o -413 T/A no gene HMOX-1. Os genótipos foram identificados por meio das técnicas de PCR e sequenciamento direto, e os escores de gravidade foram obtidos através de índices de severidade, em 169 pacientes. O genótipo AA do polimorfismo -930 A/G apresentou-se associado com escores baixos de gravidade, obtidos através do índice pediátrico, nas crianças com anemia falciforme. O genótipo CT do polimorfismo C242T esteve associado com crises álgicas e o genótipo TT do polimorfismo -413 T/A mostrou-se associado a níveis elevados de HbF, através de análises dos dados de todos os pacientes. De acordo com este estudo, sugerimos novos marcadores genéticos, os quais podem estar direta ou indiretamente envolvidos com a gravidade da doença em pacientes com AF na população brasileira. Estudos futuros em grandes coortes necessitam ser realizados para confirmar esses resultados / Abstract: Although sickle cell anemia (SCA) results from the homozygosity for a single mutation at codon 6 of the ?-globin locus, phenotypically this disease is very heterogeneous, so that different patients may have significantly different clinical outcomes. The complications in these patients are usually caused by vascular impairment caused by the accumulation of sickled erythrocytes in the blood vessels. One event that has been associated with complications in several diseases is oxidative stress, which has increased levels in SCA patients. Among the sources of oxidative stress in these patients are the vaso-occlusion and ischemia reperfusion events, which are very common. Oxidative stress at high levels can damage various molecules and subsequently damage the organism. Some polymorphisms of enzymes involved in the oxidative stress pathway have been associated with vascular diseases as: hypertension, coronary heart disease, peripheral arterial disease. Considering that the complications in SCA patients are due to vascular involvement, these polymorphisms may be contributing to the various clinical manifestations and consequently to the severity of the disease. This project proposes to evaluate three polymorphisms in genes involved in the mechanism of oxidative stress in SCA patients and test the association with the severity of the disease. The analyzed polymorphisms were C242T and -930 A/G in the CYBA gene and the -413 T/A in the HMOX-1 gene. The genotypes were identified through PCR and direct sequencing, and severity scores were obtained by severity indexes in 169 patients. The AA genotype of the -930 A/G polymorphism was associated with low severity scores, obtained from the pediatric index, in children with sickle cell anemia. The CT genotype of the C242T polymorphism was associated with pain crisis and the TT genotype of the -413 T/A polymorphism was associated with high levels of HbF, after analyzing the data from all patients. According to this study, new genetic markers can be suggested, which may be, directly or indirectly, involved with disease severity in patients with SCA in the Brazilian population. Future studies in larger cohorts have to be conducted to confirm these results / Mestrado / Clinica Medica / Mestre em Clinica Medica

Type 1 diabetes (T1D) is an autoimmune disease whereby the insulin-producing β-cells of the pancreas are destroyed by the immune system, possibly related to an inappropriate immune reaction to dietary antigens and/or microbes in the gut. We previously observed a deficit in gut-resident CD163+ M2 anti-inflammatory macrophages in BioBreeding diabetes-prone (BBdp) rats. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme of the CD163 pathway and through the breakdown of toxic heme releases potent antioxidants. We hypothesized that the treatment of animals with cobalt protoporphyrin (CoPP), an inducer of HO-1 expression, would inhibit development of T1D through modulation of the CD163/HO-1 pathway and increase M2 macrophages. HO-1 expression was significantly increased in the pancreas and gut. T1D incidence was inhibited in CoPP-treated rats and these animals showed an unexpected increase in cells expressing CD68 (an M1 pro-inflammatory macrophage marker) in the pancreas and gut. CoPP induced the expression of cathelicidin anti-microbial peptide (CAMP) in the jejunum, which co-localized with CD163+ (M2) macrophages. KLF4, an M2 macrophage-specific transcription factor, was significantly upregulated in the pancreas and jejunum of CoPP-treated animals and co-localized with CD68 and HO-1 in the pancreas. We conclude that HO-1 induction prevented T1D through modulation of the gut immune system and potential recruitment of a unique population of anti-inflammatory M2 macrophages in the gut and pancreas

Gangliosides are sialic acid-containing glycosphingolipids located on the cell surface of all animal cell types. They play a role as receptor molecules, share in cell-to-cell interaction and protect the cell against harmful environmental factors by increasing of rigidity of cell surface. This diploma thesis studies an influence of experimental cholestasis on hepatic ganglioside composition. Cholestasis was induced by bile duct ligation in Wistar rats. A significant increase of total lipid bound sialic acid and b-series gangliosides (GD1b, GT1b, event. GD3) was found in cholestatic liver when compared with controls. These results found in obstructive cholestasis correspond with the results Majer et al. Biomed. Chromatogr., 21, 446-450 (2007), described in 17α− ethinylestradiol induced cholestasis, but the increase of b-series gangliosides was milder in our study. As a second point, an effect of modulated heme-oxygenase 1 (HO-1) activity was investigated in cholestatis induced bile duct ligation (HO-1 activator- hemine, HO-1 inhibitor- Sn-mesoporphyrin). An increase of a total lipid sialic acid was found in Sn-mesoporphyrin treated animals, but a decrease of some a- and b- series gangliosides was observed. In group with activated HO-1 total sialic acid increased, but the composition of gangliosides...

Acute kidney injury (AKI) is a major public health concern, which contributes to serious hospital complications, chronic kidney disease (CKD) and even death. Renal ischemia- reperfusion injury (IRI) remains a leading cause of AKI.IRI combines major cell stress, significant burst of free radicals, and strong inflammatory responses leading to extensive cell injury, necrosis, and late interstitial fibrosis. Moreover, IRI- induced AKI releases pro-inflammatory cytokines (e.g. IL-1β, TNF-α, IL-6) that induce a systemic inflammatory response, resulting in pro-inflammatory cells recruitment and remote organ damage. AKI is associated with poor outcomes, particularly when extrarenal complications or distant organ injuries occur.The stress-responsive enzyme, heme oxygenase-1 (HO-1) mediates protection against renal IRI and may be preventively induced using hemin prior to renal insult. This HO-1 induction pathway called hemin preconditioning is largely known in the literature to be effective.We first confirmed that hemin-induced HO-1 improved renal outcomes after IRI (i.e. fewer renal damage, renal inflammation and oxidative stress). We then demonstrated that this protective pathway mitigated AKI-induced ALI, a major extrarenal complication after renal IRI, through modulation of systemic and lung inflammation.Afterwards, we focused on the specific contribution of myeloid HO-1 to renal IRI, which remains poorly characterized. We therefore investigated the contribution of myeloid HO-1 to renal IRI using mice with myeloid-restricted deletion of HO-1 (HO-1M-KO). We observed that myeloid HO-1 appeared to be a critical regulator of the earliest phases of IRI (i.e. higher plasma creatinine, tubular damage, and renal inflammation/oxidative stress in HO-1M-KO mice).As a link between the severity of renal injury and the risk maladaptive repair leading to CKD has been established, we thereby decided to focus on tubular repair and fibrosis deposition upon IRI. We identified that myeloid HO-1 prevented maladaptive repair and subsequent CKD through modulation of cell-cycle and autophagy regulatory proteins.We then showed that hemin-mediated protection requires specific expression of HO-1 within myeloid cells. We therefore identified CD11b+ F4/80lo macrophages as the main protective myeloid source of HO-1 upon renal IRI. Interestingly, we observed this myeloid cell sub- population in the kidney and spleen, suggesting that protective effects might be provided by both tissue-resident and infiltrating/circulating HO-1+ myeloid cells.Based on its promising cytoprotective effects when giving preemptively, we investigated the use of hemin-induced myeloid HO-1 as a strategy to mitigate established AKI. However, due to its chemical structure and oxidative properties, hemin worsened IRI-induced AKI. We thereby identified that hemin had a dual effect on renal IRI, protective or deleterious, depending on the timing of its administration.Altogether, this work suggests that myeloid HO-1 plays a critical role in the modulation of IRI- induced AKI by improving short- and long-term functional outcomes after renal IRI. We conclude that hemin-induced myeloid HO-1 pathway might be an efficient preventive strategy in many renal IRI situations with predictable AKI such as renal transplantation or partial nephrectomy. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Gangliosides are sialic acid-containing glycosphingolipids located on the cell surface of all animal cell types. They play a role as receptor molecules, share in cell-to-cell interaction and protect the cell against harmful environmental factors by increasing of rigidity of cell surface. This diploma thesis studies an influence of experimental cholestasis on hepatic ganglioside composition. Cholestasis was induced by bile duct ligation in Wistar rats. A significant increase of total lipid bound sialic acid and b-series gangliosides (GD1b, GT1b, event. GD3) was found in cholestatic liver when compared with controls. These results found in obstructive cholestasis correspond with the results Majer et al. Biomed. Chromatogr., 21, 446-450 (2007), described in 17ethinylestradiol induced cholestasis, but the increase of b- series gangliosides was milder in our study. As a second point, an effect of modulated heme-oxygenase 1 (HO-1) activity was investigated in cholestatic rats (HO-1 activator- hemine, HO- 1 inhibitor- Sn- mesoporphyrin). An increase of a total lipid sialic acid was found in Sn-mesoporphyrin treated animals but without significant changes in gangliosides composition. Lipid sialic acid and gangliosides were not changed in animals with hemine activated HO-1. Expression of mRNA of key...

This thesis is focused on the study of glycosphingolipids in the rat liver in different types of cholestasis and the effect of oxidative stress on changes in the composition and localization of gangliosides. First, it was necessary to optimize the immunochemical detection of glycosphingolipids. GM1 ganglioside was selected as a representative of a large glycolipid family. We found that minimum water content in the fixing solution was a key condition for fixation of histological sections. Optimized method of GM1 detection was subsequently used in in vivo experiments. We have demonstrated that estrogen-induced cholestasis characterized by high concentrations of bile acids and increased oxidative stress caused changes in the synthesis and distribution of liver gangliosides. HMOX induction is associated with a reduction in oxidative stress level and accompanied by normalization in GSL content. In experiments with obstructive cholestasis, we found that changes in the distribution and synthesis of gangliosides were not strictly specific to a particular type of cholestasis. We assume that it represents a general mechanism of hepatoprotection. We also confirmed the important role of bilirubin, product of HMOX reaction, in protection of hepatocytes against oxidative damage caused by high concentrations of...

1 Abstract In this thesis I have focused on the role of lipids in the pathogenesis of liver diseases, specifically on cholestasis and non-alcoholic fatty liver disease (NAFLD). The first major aim was to clarify the changes in liver ganglioside metabolism in various types of cholestasis and to elucidate the role of heme oxygenase-1 (HMOX1) and associated oxidative stress. The second objective was to determine the effects of n-3 polyunsaturated fatty acids (n-3 PUFA) administration on NAFLD development in a rodent dietary model of NAFLD and in patients with metabolic syndrome and NAFLD. Our results suggest that increased ganglioside biosynthesis and their re-distribution might represent a general protective mechanism of hepatocytes under cholestatic conditions (both estrogen-induced and obstructive aetiology). These changes are closely related to oxidative stress and might protect hepatocytes against deleterious effect of accumulated bile acids. The lack of HMOX1 activity and subsequent oxidative stress potentiate pathological changes in the liver and resulted in tissue-specific modulation of synthesis and re-distribution of gangliosides (in vivo and in vitro). Contrary to it, HMOX1 activation has an opposite effect and may represent a general hepatoprotective mechanism. We have proven that observed changes...