As cores sempre exerceram fascínio sobre a humanidade e, por toda a história, os compostos coloridos sempre foram considerados ferramentas atrativas nas atividades comerciais. Os corantes sintéticos são amplamente utilizados na indústria têxtil, nas impressões de papel e fotografia, nas indústrias farmacêuticas, alimentícias e de cosméticos. Estes compostos são considerados importantes contaminantes ambientais, representando sérios riscos à flora, fauna e ao ser humano. Apesar da grande quantidade de corantes disponíveis, os estudos sobre a toxicidade desses compostos são escassos e pouco se sabe a respeito dos efeitos genotóxicos destas substâncias. Dentro deste contexto, o presente trabalho avaliou o potencial genotóxico dos corantes Sudan III, Vat Green 3, Reactive Orange 16 e Reactive Black 5, utilizando o Ensaio de Micronúcleos em células HepG2. Os corantes Sudan III e Reactive Orange 16 não induziram aumento, estatisticamente significativo, no número total de micronúcleos em relação aos controles, indicando assim que estes corantes não são capazes de induzir mutações cromossômicas no tipo celular e condições testadas. Entretanto, os corantes Vat Green 3 e Reactive Black 5 induziram mutagenicidade, concentrações de 10,0 e 25,0 ?g/mL, e 0,1; 0,25; 0,5 e 1,0 ?g/mL, respectivamente, demonstrada por um efeito concentraçãodependente, no qual há um aumento de MNs até a concentração de 25,0 ?g/mL para o Vat Green 3 e 0,5 ?g/mL para o Reactive Black 5 com p<0,05. Não foram observadas diferenças significativas entre os IDNs calculados para cada tratamento e controle dos corantes testados, indicando que esses corantes não interferem na proliferação celular das HepG2. Dessa forma, conclui-se que dos quatro compostos analisados, os corantes têxteis Vat Green 3 e Reactive Black 5 são capazes de induzir mutações cromossômicas em células HepG2 e, o potencial mutagênico do Reactive Black 5 é maior que o do Vat Green 3 no sistema celular avaliado, uma vez que foi capaz de induzir mutações, em concentrações menores. Os resultados obtidos neste trabalho permitem concluir que cada um desses importantes contaminantes ambientais deve ser avaliado individualmente a fim de proteger o meio ambiente, garantindo assim a proteção da saúde humana. / The colors have always caused fascination in mankind. Throughout history, colored compounds have always been considered attractive tools in industrial activities. The synthetic dyes are widely used in textile industry, paper and photography printing, in pharmaceutical, food, and cosmetic industries. These compounds are considered important environmental contaminants, and they can cause serious risks to wildlife and humans. Despite the large number of dyes available, studies on the toxicity of these compounds are scarce and little is known about the genotoxic effects of these substances. This study evaluated the genotoxic potential of the dyes Sudan III, Vat Green 3, Reactive Orange 16 and Reactive Black 5 using the micronucleus assay in HepG2 cells. The dyes Sudan III and, Reactive Orange 16, do not induce an increase statistically significant, in the total number of micronuclei when compared to controls. This result shows that these dyes are not able to induce chromosomal mutations in the cell type under the conditions tested. However, the dyes Vat Green 3 and Reactive Black 5 induced mutagenicity, following a dose-response effect, in which there is an increase of micronuclei until the concentration of 25.0 ?g/mL for Vat Green 3 and 0.5 ?g/mL for Reactive Black 5, with p <0.05. There were no significant differences between the NDI calculated for each treatment and control of the dyes studied, indicating that these dyes do not interfere in HepG2 cell proliferation. Thus, the textile dyes Vat Green 3 and Reactive Black 5 are able to induce chromosomal mutations in HepG2 cells, and the dye Reactive Black 5 is more mutagenic than the dye Vat Green 3, since it induced mutations in cellular system tested at lower concentrations. The results of this study indicate that each one of these important environmental contaminants should be assessed individually in order to protect the environment, thus ensuring the protection of human health.

Selenoproteine spielen eine wichtige Rolle in der antioxidativen Abwehr und bei Immunreaktionen. Der Selen(Se)metabolismus wird von Hepatozyten gesteuert, die das Se-Transportprotein Selenoprotein P (SEPP) synthetisieren und sezernieren. SEPP nimmt bei kritischen Erkrankungen, z. B. Sepsis ab und führt zu niedrigen Se-Spiegeln. Sepsis triggert die übermäßige Produktion von proinflammatorischen Zytokinen. Aminoglykosid-Antibiotika (AG), die oft bei schwerer Sepsis eingesetzt werden, induzieren Fehlinterpretationen der mRNA inklusive des Stoppcodons UGA welches für die Selenoprotein-Biosynthese notwendig ist. Es wurden daher die molekularen Wechselwirkungen zwischen den Zytokinen IL-6, IL-1b und TNFa, AG und dem Se-Status mit der Biosynthese in Leberzelllinien untersucht. IL-6 führte zu einer starken Reduktion der SEPP-mRNA und einer dosisabhängigen Reduktion von SEPP. Parallel dazu reduzierte IL-6 das Transkriptlevel, die Proteinexpression und die Enzymaktivität der Typ-I-Dejodase (DIO1). Auf die Expression der antioxidativ-wirkenden Glutathionperoxidasen (GPX) wirkte IL-6 isozymspezifisch; während die Transkriptkonzentrationen von GPX2 anstiegen und die von GPX4 abnahmen, blieb GPX1 unbeeinflusst. Die IL-6-abhängigen Effekte bestätigten sich auch in Reportergenassays von SEPP-, DIO1-, GPX2- und GPX4-Promotorkonstrukten. Um die Wirkungen von AG auf die Selenoprotein-Translation besser zu verstehen, wurden die SECIS-Elemente von GPX1-, GPX4- und SEPP-Transkripten in ein Reportersystem kloniert und auf eine Regulation durch AG und Se analysiert. Die Ergebnisse zeigen, dass der korrekte Se-Einbau vom Se-Status, von der AG-Konzentration und dem spezifischen SECIS-Element abhängig ist. Auf transkriptionaler und translationaler Ebene führten AG zu stark erhöhten SEPP-Spiegeln, während die Expression und Enzymaktivität von GPX und DIO1 nur in geringerem Ausmaß beeinflusst wurden. Eine Analyse der Se-Beladung zeigte, dass der Se-Gehalt von SEPP stark durch AG reduziert und vom Se-Status abhängig war. / Selenoproteins play important roles in antioxidant defence and immunoregulation. Selenium (Se) metabolism is controlled by hepatocytes synthesizing and secreting the Se-transporter selenoprotein P (SEPP) declining in critical illness, e.g., sepsis. Sepsis triggers excessive production of pro-inflammatory cytokines. Aminoglycoside (AG) antibiotics applied in sepsis in induce mRNA misinterpretation including the stop codon UGA required during selenoproteins biosynthesis. The molecular interplay between the cytokines IL-6, IL-1b and TNFa, AG and Se-status on selenoprotein expression was investigated in hepatic-derived cell lines. IL-6 strongly reduced the level of SEPP mRNA and secreted SEPP in a dose-dependent manner. Likewise, expression of selenoenzyme type 1 deiodinase (DIO1) declined at the transcript, protein and enzyme activity level. The effects of IL-6 on the expression of antioxidative-acting glutathione peroxidases (GPX) were isozyme-specific; while transcript level of GPX2 increased and those of GPX4 decreased, GPX1 remained unaffected. IL-6-dependent effects were reflected in reporter gene experiments of selenoprotein promoter constructs. Characterising the effects of AG on selenoprotein translation, the SECIS-elements of GPX1, GPX4 and SEPP transcripts were cloned into a reporter system and analysed for their response to AG and Se. The results indicate that the correct co-translational Se-insertion depends on the Se-status, AG concentration and the specific SECIS-element. At both transcriptional and translational levels, SEPP levels were strongly increased in response to AG, whereas the expression and enzyme activity of GPX and DIO1 were affected to a lower degree. Analysis Se-status indicate that the Se-content of SEPP was strongly reduced by AG and depends on Se-status.

Le cancer du foie est le 6ème cancer le plus fréquent et le 2ème plus meurtrier dans le monde. Au Cambodge, en raison du contexte historique et économique, les données précises concernant cette pathologie manquent. A l'aide d'outils épidémiologiques, nous avons décrit les caractéristiques de 553 patients atteints de cancer du foie à l'hôpital Calmette à Phnom Penh, et ainsi mis en évidence l'importance de l'infection par les virus des hépatites B et C chez les sujets étudiés. Puis, nous avons documenté les connaissances de 42 de ces patients vis-à-vis de leur maladie. Nous avons détaillé leurs itinéraires thérapeutiques, mis en évidences des pratiques à risques (forte utilisation d'injections thérapeutiques et de techniques de dermabrasion), et le recours fréquent à des médecines dites traditionnelles. Nous avons ensuite tenté de comprendre les stratégies de prise en charge des patients souffrant de maladies hépatiques par les médecins traditionnels, et mis en évidence la variété des remèdes utilisés et l'importance de la perception khmère des propriétés des plantes. Enfin, à l'aide d'un modèle in vitro de culture de cellules cancéreuses hépatiques couplé à des outils d'analyse métabolomique, nous avons évalué 10 espèces médicinales, sélectionnées sur des critères bibliographiques et de terrain, et tenté d'identifier les composés potentiellement responsables de l'activité antiproliférative observée. / Liver cancer is the 6th most common and 2nd most lethal cancer in the world. In Cambodia, due to the historical and economic context, there is a lack of accurate data on this pathology. Using epidemiological tools, we described the characteristics of 553 patients with liver cancer at the Calmette Hospital in Phnom Penh, and thus highlighted the importance of infection with hepatitis B and C viruses in the subjects studied. Then we documented the knowledge of 42 of these patients about their disease. We have detailed their therapeutic itineraries, highlighted risky practices (high use of therapeutic injections and dermabrasion techniques) and the use of traditional medicines. We then attempted to understand strategies for the management of patients with liver diseases by traditional healers, and highlighted the variety of remedies used and the importance of Khmer perception of plant properties. Finally, using an in vitro model of liver cancer cell culture coupled with metabolic analysis tools, we evaluated 10 medicinal species, selected on the basis of bibliographic and field criteria, and attempted to identify the compounds potentially responsible for the antiproliferative activity observed.

As cores sempre exerceram fascínio sobre a humanidade e, por toda a história, os compostos coloridos sempre foram considerados ferramentas atrativas nas atividades comerciais. Os corantes sintéticos são amplamente utilizados na indústria têxtil, nas impressões de papel e fotografia, nas indústrias farmacêuticas, alimentícias e de cosméticos. Estes compostos são considerados importantes contaminantes ambientais, representando sérios riscos à flora, fauna e ao ser humano. Apesar da grande quantidade de corantes disponíveis, os estudos sobre a toxicidade desses compostos são escassos e pouco se sabe a respeito dos efeitos genotóxicos destas substâncias. Dentro deste contexto, o presente trabalho avaliou o potencial genotóxico dos corantes Sudan III, Vat Green 3, Reactive Orange 16 e Reactive Black 5, utilizando o Ensaio de Micronúcleos em células HepG2. Os corantes Sudan III e Reactive Orange 16 não induziram aumento, estatisticamente significativo, no número total de micronúcleos em relação aos controles, indicando assim que estes corantes não são capazes de induzir mutações cromossômicas no tipo celular e condições testadas. Entretanto, os corantes Vat Green 3 e Reactive Black 5 induziram mutagenicidade, concentrações de 10,0 e 25,0 ?g/mL, e 0,1; 0,25; 0,5 e 1,0 ?g/mL, respectivamente, demonstrada por um efeito concentraçãodependente, no qual há um aumento de MNs até a concentração de 25,0 ?g/mL para o Vat Green 3 e 0,5 ?g/mL para o Reactive Black 5 com p<0,05. Não foram observadas diferenças significativas entre os IDNs calculados para cada tratamento e controle dos corantes testados, indicando que esses corantes não interferem na proliferação celular das HepG2. Dessa forma, conclui-se que dos quatro compostos analisados, os corantes têxteis Vat Green 3 e Reactive Black 5 são capazes de induzir mutações cromossômicas em células HepG2 e, o potencial mutagênico do Reactive Black 5 é maior que o do Vat Green 3 no sistema celular avaliado, uma vez que foi capaz de induzir mutações, em concentrações menores. Os resultados obtidos neste trabalho permitem concluir que cada um desses importantes contaminantes ambientais deve ser avaliado individualmente a fim de proteger o meio ambiente, garantindo assim a proteção da saúde humana. / The colors have always caused fascination in mankind. Throughout history, colored compounds have always been considered attractive tools in industrial activities. The synthetic dyes are widely used in textile industry, paper and photography printing, in pharmaceutical, food, and cosmetic industries. These compounds are considered important environmental contaminants, and they can cause serious risks to wildlife and humans. Despite the large number of dyes available, studies on the toxicity of these compounds are scarce and little is known about the genotoxic effects of these substances. This study evaluated the genotoxic potential of the dyes Sudan III, Vat Green 3, Reactive Orange 16 and Reactive Black 5 using the micronucleus assay in HepG2 cells. The dyes Sudan III and, Reactive Orange 16, do not induce an increase statistically significant, in the total number of micronuclei when compared to controls. This result shows that these dyes are not able to induce chromosomal mutations in the cell type under the conditions tested. However, the dyes Vat Green 3 and Reactive Black 5 induced mutagenicity, following a dose-response effect, in which there is an increase of micronuclei until the concentration of 25.0 ?g/mL for Vat Green 3 and 0.5 ?g/mL for Reactive Black 5, with p <0.05. There were no significant differences between the NDI calculated for each treatment and control of the dyes studied, indicating that these dyes do not interfere in HepG2 cell proliferation. Thus, the textile dyes Vat Green 3 and Reactive Black 5 are able to induce chromosomal mutations in HepG2 cells, and the dye Reactive Black 5 is more mutagenic than the dye Vat Green 3, since it induced mutations in cellular system tested at lower concentrations. The results of this study indicate that each one of these important environmental contaminants should be assessed individually in order to protect the environment, thus ensuring the protection of human health.

The key to the complexity of higher organisms lies not in the number of protein coding genes they carry, but rather in the intrinsic complexity of the gene regulatory networks. The major effectors of transcriptional regulation are proteins called transcription factors, and in this thesis four papers describing genome-wide studies of seven such factors are presented, together with studies on components of the chromatin and transcriptome. In Paper I, we optimized a large-scale in vivo method, ChIP-chip, to study protein – DNA interactions using microarrays. The metabolic-disease related transcription factors USF1, HNF4a and FOXA2 were studied in 1 % of the genome, and a surprising number of binding sites were found, mostly far from annotated genes. In Paper II, a novel sequencing based method, ChIP-seq, was applied to FOXA2, HNF4a and GABPa, allowing a true genome-wide view of binding sites. A large overlap between the datasets were seen, and molecular interactions were verified in vivo. Using a ChIP-seq specific motif discovery method, we identified both the expected motifs and several for co-localized transcription factors. In Paper III, we identified and studied a novel transcription factor, ZBED6, using the ChIP-seq method. Here, we went from one known binding site to several hundred sites throughout the mouse genome. Finally, in Paper IV, we studied the chromatin landscape by deep sequencing of nucleosomal DNA, and further used RNA-sequencing to quantify expression levels, and extended the knowledge about the binding profiles for the transcription factors NFY and TCF7L2.

Les liposomes d'ATP incluant des ligands hépatiques pourraient contribuer à améliorer le statut énergétique du greffon hépatique. Une première phase de développement a mis en évidence la nécessité de stabiliser la forme (i) et de valider un modèle cellulaire à statut énergétique altéré (ii) afin de pouvoir tester différentes formulations liposomales d'intérêt. Afin de résoudre la première problématique, différentes stratégies ont été mises en place lors de la lyophilisation de liposomes blancs ou chargés en ATP. Le saccharose et le tréhalose ont conduit à une stabilisation de la forme avant et après lyophilisation. Néanmoins, quel que soit le procédé, la lyophilisation s'est accompagnée d'une fuite en ATP. L'ajout d'un agent inhibant la cartinine palmitoyl transferase, l'Etomoxir, s'est révélé un modèle intéressant pour moduler le niveau énergétique des HepG2 en fonction de la température et de la concentration utilisée. En conclusion, ce travail contribue à mieux comprendre les facteurs critiques liés à la lyophilisation des liposomes (i) et décrit des modèles cellulaires hépatiques à statut énergétique altéré qui pourraient être mis à profit pour tester différents principes actifs ou formes galéniques innovantes.

We can meet pathological hypoxia in the cases of hearth attack, ischemic stroke, but also during tumor invasion, thanks to insufficient angiogenesis. The activation of HIF- 1 factor during hypoxic conditions is crucial for the cell survival. This factor modulates energetic metabolism in favor of fast progressing glycolysis (with the contribution of glutaminolysis) which provides to cell enough ATP and "building blocks", while suppressing Krebs cycle and respiration because of shortage of oxygen. The thesis studies energetic metabolism of HepG2 cells (derived from liver carcinoma) which are cultivated in the media with various energetic substrates, i. e. glucose or galactose (always together with glutamine and pyruvate) under the hypoxic conditions (5% O2). HepG2 cells use particularly oxidative metabolism for ATP and "building blocks" production under the normoxic conditions while hypoxic environment causes metabolic shift in glycemic condition. Interestingly, cells cultured in galactose (glutamine) didn't switch the energy metabolism from oxidative to aerobic glycolysis such as cells cultivated in glucose, although HIF-1 factor was stabilized. We found that enhanced activity and integrity of mitochondria, enhanced maximal capacity and reserve capacity of respiration chain correlates with...

No description available.

Non-alcoholic fatty liver disease (NAFLD), characterized by lipid accumulation in the liver, is the most common cause of liver diseases in Western countries. NAFLD is a major risk factor for developing hepatocellular carcinoma (HCC); however, in vitro evaluation of hepatic cancerogenesis fails due to a lack of liver models displaying a proliferation of hepatocytes. Originally designed to overcome primary human hepatocyte (PHH) shortages, upcyte hepatocytes were engineered to obtain continuous proliferation and, therefore, could be a suitable tool for HCC research. We generated upcyte hepatocytes, termed HepaFH3 cells, and compared their metabolic characteristics to HepG2 hepatoma cells and PHHs isolated from resected livers. For displaying NAFLD-related HCCs, we induced steatosis in all liver models. Lipid accumulation, lipotoxicity and energy metabolism were characterized using biochemical assays and Western blot analysis. We showed that proliferating HepaFH3 cells resemble HepG2, both showing a higher glucose uptake rate, lactate levels and metabolic rate compared to PHHs. Confluent HepaFH3 cells displayed some similarities to PHHs, including higher levels of the transaminases AST and ALT compared to proliferating HepaFH3 cells. We recommend proliferating HepaFH3 cells as a pre-malignant cellular model for HCC research, while confluent HepaFH3 cells could serve as PHH surrogates for energy metabolism studies.

La compréhension des interactions intra- et inter-moléculaires à l’échelle atomique représente un enjeu scientifique important. A l’heure actuelle, les techniques de RMN ont déjà prouvé leur efficacité pour l’analyse de ces interactions in vitro, dans les solutions tampons. Toutefois, il a également été montré que la plupart des biomolécules ont une structure et une dynamique différentes in vivo, à l’intérieur des cellules, de celle in vitro. Il est donc crucial d’analyser les biomolécules dans leur milieu naturel, la cellule. Récemment, les progrès dans le domaine de la RMN dans les cellules ont permis de mieux comprendre la dynamique et les interactions des biomolécules présentes dans le milieu cellulaire complexe. Cependant, la biomolécule étudiée étant présente en faibles concentrations, elle possède un faible signal sur le spectre RMN, qu’il est difficile de suivre. De plus, du fait de la forte viscosité du milieu cellulaire, la relaxation rapide de l’aimantation transverse se traduit par un élargissement des raies spectrales. L’utilisation des états de spin à longs temps de vie et de la Polarisation Dynamique Nucléaire suivie par la dissolution de l’échantillon (dissolution-DNP) pourraient permettre de pallier aux problèmes d’élargissement de raies et de sensibilité. L’objectif de ce travail de thèse a été d’explorer les bénéfices des ces avancées récentes de la RMN pour l’étude des petites molécules, peptides et protéines à l’intérieur des cellules. Pour la protéine c-Src, qui appartient à la classe des protéines intrinsèquement désordonnées (IDP), la dynamique de l’ensemble des conformations de l’extrémité N-terminale a été suivie utilisant des états de spin à longs temps de vie LLS. Le signal du noyau de carbone-13 de la molécule de pyruvate a été augmenté utilisant la Polarisation Dynamique Nucléaire (DNP) afin de mieux l’observer dans le milieu cellulaire. Un peptide représentatif pour la partie active d’une autre protéine, IκBα, a été introduit dans des cellules HepG2 par l’électroporation. Les observations faites lors des ces expériences sont discutées dans la perspective de faciliter les études RMN des biomolécules à l’intérieur des cellules. / Most NMR studies are carried out in vitro, but the structure and dynamics of some biomolecules inside cells differ from those in vitro. It thus becomes interesting to analyze biomolecules such as proteins in their natural environment: the cell. Recent progress of in cell NMR allowed to better understand the behaviour of proteins: their dynamics and their interactions with other biomolecules in the cell. But the low concentration of proteins leads to low signal intensity. Moreover, the viscosity of the environment induces faster transverse relaxation, resulting in line broadening for proteins signals. The use of the Long-Lived States and Coherences (LLS and LLC, respectively) as well as dissolution Dynamic Nuclear Polarization (dissolution-DNP) can improve NMR observations in cells. LLS were used to understand and characterize the structure of the N-terminal domain of c-Src, which is intrinsically disordered. To follow the phosphorylation of proteins, a first preliminary study of a 21-aa peptides derived from IKBα electroporated into HepG2 cell lines was carried out.