Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-06-28T11:40:26Z No. of bitstreams: 1 renansalgadoteixeira.pdf: 5350763 bytes, checksum: 59c72fea8916127b1547f326adb2158a (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-28T12:25:26Z (GMT) No. of bitstreams: 1 renansalgadoteixeira.pdf: 5350763 bytes, checksum: 59c72fea8916127b1547f326adb2158a (MD5) / Made available in DSpace on 2016-06-28T12:25:26Z (GMT). No. of bitstreams: 1 renansalgadoteixeira.pdf: 5350763 bytes, checksum: 59c72fea8916127b1547f326adb2158a (MD5) Previous issue date: 2013-07-29 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Arctigenina é uma lignana isolada da Arctium lappa L., planta esta, pertencente a família Asteraceae que é amplamente utilizada na Medicina Tradicional Chinesa. Publicações citam inúmeras atividades farmacológicas do extrato bruto dessa planta e também da substância arctigenina, como a atividade anti-inflamatória, hepatoprotetora, hipoglicemiante, antitumoral, antimicrobiana, mas, estudos detalhados da atividade hepatoprotetora ainda não foram amplamente desenvolvidos. Portanto, o objetivo deste estudo foi o isolamento, a obtenção e identificação da arctigenina a partir dos frutos da Arctium lappa L., para avaliar a atividade citotóxica in vitro frente à cultura de células de carcinoma hepático (HepG- 2) e tambén avaliar os efeitos dessa substância isolada em lesão hepática por Ligação do Ducto Biliar (BDL). O extrato clorofórmico bruto da Arctium lappa L. foi obtido por soxhlet. Para isolar as substâncias, foi realizada coluna a vácuo e observou grande quantidade de arctiina, uma precursora da arctigenina. Para obter a arctigenina, foi necessário realizar uma hidrólise para retirada da molécula de glicose presente na arctiina. A mesma foi identificada por RMN de 1H e 13C obtendo excelente grau de pureza e adicionalmente também foi realizado um perfil cromatográfico por CLAE. Nos experimentos in vitro, a arctigenina diminuiu a viabilidade celular no teste por MTT e azul de Tripan sobre células HepG-2 e não causou citotoxicidade sobre células NIH/3T3. O teste de adesão por fornecimento de colágeno tipo I sobre HepG-2 também foi realizado para observar os mecanismos que envolvem a atividade antitumoral da arctigenina. Nos experimentos in vivo foram realizadas cirurgias de BDL em ratos Wistar, com n= 6 e grupos divididos em: sham controle, sham arctigenina, BDL controle, BDL arctigenina e BDL silimarina. O tratamento dos animais foi feitos por 48 horas e após esse período, eutanasiados e o sangue e fígado foram coletados para realizar avaliações bioquímicas séricas (AST, ALT, FA e TNF-α) e também avaliação tissular (MPO). A metodologia de extração, isolamento e obtenção da arctigenina desenvolvida foi eficaz, diminuiu gastos com solventes e com o tempo para se obter as susbstâncias. Os parâmetros de confiança analisados por CLAE demonstraram que o método analítico desenvolvido foi específico e reprodutível, o que contribui para estudos em controle de qualidade e ensaios bioanalíticos. A arctigenina demonstrou atividade citotóxica e antiadesão, sendo a atividade dose dependente para células HepG-2, com IC 50 de 90,8 μM. Nenhuma atividade sobre células NIH/3T3, o que demonstrou ação específica sobre a células HepG-2. Resultados do estudo in vivo de 48 horas não apresentaram resultados significantes quando comprados aos grupos controle e controle positivo, entretanto, levantamentos bibliográficos mostram que a arctigenina é uma substância promissora e estudos de hepatoproteção por períodos maiores complementariam a elucidação de sua atividade. / Arctigenin is a lignan isolated from Arctium lappa L., this plant, belonging to family Asteraceae is widely used in traditional chinese medicine. Publications suggest numerous pharmacological activities of crude extracts of this plant and arctigenin as anti-inflammatory activity, hepatoprotective, hypoglycemic, anticancer and antimicrobial, however detailed studies of hepatoprotective activity have not yet been widely developed. The aim of this study was the isolation and identification of arctigenin obtained from the fruits of Arctium lappa L., to evaluate the cytotoxic in vitro of arctigenin in cell culture of liver carcinoma (HepG-2) and assess the effects of this isolated compound on liver injury by ligation bile duct (BDL). The crude chloroform extract of Arctium lappa L. was obtained by soxhlet. To isolate the compounds was held vacuum column and observed high concentration of arctiin, a precursor of arctigenin. To obtain arctigenin, it was necessary to carry out a hydrolysis to remove the glucose molecule present in arctiin. After, the molecule was identified by 1H NMR and 13C, attaining excellent purity, and additionally, was carried out his chromatographic profile by HPLC.. In vitro experiments show that the arctigenin decreased cell viability by MTT assay and the Trypan blue on HepG-2 cells and did not cause cytotoxicity on NIH/3T3 cells. Adhesion testing by providing collagen type I on HepG-2 was also carried out to observe the mechanisms involving the antitumor activity of arctigenin. In vivo experiments were performed by surgery in BDL rats, with n = 6 and divided into groups: sham control, sham arctigenin, control BDL, arctigenin BDL and silymarin BDL. The treatment of animals was made for 48 hours and after this period, euthanized and blood and liver were collected for serum biochemical assessments (AST, ALT, FA and TNF-α) and also tissue rating (MPO). The methodology of extraction, isolation and obtaining arctigenin developed was effective, decreased spending solvents and the time to get compounds. The trust parameters analyzed by HPLC showed that the analytical method was specific and reproducible, which contributes to quality control studies and bioanalytical assays. The arctigenin demonstrated cytotoxic activity and anti-adhesion, and dependent dose activity in HepG-2 cells, with IC 50 of 90.8 μM. No activity on NIH/3T3 cells, demonstrating the specific action on HepG-2 cells. Results of BDL done in 48 hours showed no significant results when comparing with control groups and positive control, however, literature reports show that the arctigenin is a promising compound and hepatoprotection studies for longer periods would complement the elucidation of its activity.

I den här uppsatsen analyseras den rättsliga grunden som möjliggör för polisen att behandla personuppgifter i syfte att upprätthålla allmän ordning och säkerhet. Till att börja med konstateras att begreppet personuppgifter kan avse i princip alla uppgifter som går att hänföra till en person. När sådana uppgifter samlas in kan de utgöra ett intrång i rätten till personlig integritet, antingen till följd av uppgifternas karaktär som känsliga, eller att uppgifterna tillsammans visar på hur den enskilda lever sitt liv. Den personliga integriteten skyddas både i RF och Europakonventionen. Eftersom en förutsättning för ett demokratiskt samtal är att den enskilda har rätt att pröva sina åsikter i sin ensamhet eller tillsammans med andra utan statens inblandning får personuppgiftsbehandling även betydelse för demokratiska rättigheter så som mötes- och demonstrationsfriheten.  Polisens personuppgiftsbehandling regleras framför allt i BDL och PBDL. För att behandling ska få ske krävs att det finns en rättslig grund för behandlingen, till exempel upprätthållande av allmän ordning, samt att ändamålet med behandlingen specificeras. Behandlingen måste även vara proportionerlig till det intrång i rättigheterna som den innebär vilket framkommer genom bestämmelsen om behandlingens nödvändighet.  Gällande rekvisitet upprätthållande av allmän ordning och säkerhet framkommer att polisens uppgift att upprätthålla allmän ordning skiljer sig från uppgiften att upprätthålla allmän säkerhet, även om begreppen till viss del är svårdefinierade. Det konstateras även att all brottsbekämpande verksamhet kan anses falla inom upprätthållande av allmän ordning och säkerhet medan åtgärder av rent hjälpande natur faller utanför begreppet.  Implementeringen av dataskyddsdirektivet kritiseras utifrån att allmän ordning och säkerhet inte nämns som begrepp utan att det snarare verkar som att direktivet endast avser omfatta personuppgiftsbehandlingen för upprätthållande av allmän säkerhet. Vidare är det svårt att läsa sig till när personuppgiftsbehandlingen i syfte att upprätthålla allmän ordning och säkerhet är tänkt att användas av polisen. Dessa svårigheter kritiseras utifrån legalitetsprincipen som ställer krav på lagens kvalitet och förutsebarhet. Till följd av att bestämmelsens påverkan på grundläggande rättigheter inte vägs mot de fördelar som bestämmelsen eventuellt kan innebära för polisen kritiseras lagstiftningen även utifrån proportionalitetsprincipen.

L’encéphalopathie hépatique (EH) est une complication neuropsychiatrique de la maladie de foie telle que la cirrhose, caractérisée par des dysfonctions cognitives et motrices. Le seul traitement curatif est la transplantation hépatique (TH). Historiquement, l’EH est considérée comme un désordre métabolique réversible et il est attendu qu’il soit résolu suivant la TH. Cependant, il a été démontré que des complications neurologiques persistent chez 47% des patients transplantés. La TH est une opération chirurgicale complexe accompagnée de stress péri-opératoire telle que la perte sanguine et l’hypotension. L’hypothèse de ce projet d’étude est que l’EH minimale (EHm) rend le cerveau plus susceptible à une perte neuronale suite à une insulte hypotensive. Nous avons donc caractérisé un modèle d’hypotension chez des rats cirrhotiques avec ligation de la voie biliaire (BDL) dans lequel une hypovolémie de l’artère fémorale a été faite. Avec ce modèle, nous avons étudié l’impact de différentes pressions sanguines de 120 minutes sur le compte neuronal. Nos résultats démontrent que les BDL hypotendus à une pression artérielle moyenne de 60 mmHg et 30 mmHg ont une diminution du compte neuronal et que les neurones mourraient par apoptose (observée par la présence de caspase-3 clivée). Nous avons également déterminé que le flot sanguin cérébral était altéré chez les rats cirrhotiques BDL. Le second objectif était d’évaluer si le traitement de l’EHm par l’ornithine phénylacétate (OP) permettait d’éviter la perte neuronale chez les BDL hypotendus. Nos résultats ont démontrés que l’OP permettait de partiellement rétablir les fonctions cognitives chez les rats BDL. De plus, les rats BDL traités avec l’OP peuvent éviter la mort neuronale. Cependant, le processus apoptotique est toujours enclenché. Ce résultat suggère la possibilité de mort cellulaire retardée par l’OP. Ces résultats suggèrent que les patients cirrhotiques avec EHm sont plus susceptibles à une mort neuronale induite par hypotension. La combinaison de l’EHm et l’hypotension permet d’expliquer les complications neurologiques rencontrées chez certains patients. Le diagnostic et le traitement de ce syndrome doit donc être fait chez les patients cirrhotiques pour éviter ces complications post-TH. / Hepatic encephalopathy (HE) is a major neuropsychiatric complication caused by chronic liver disease such as cirrhosis and is characterized by cognitive and motor dysfunction. The only curative treatment to date remains liver transplantation (LT). Historically, HE has always been considered to be a reversible metabolic disorder and has therefore been expected to completely resolve following LT. However, persisting neurological complications remain a common problem affecting as many as 47% of LT recipients. LT is a major surgical procedure accompanied by intraoperative stress and confounding factors, including blood loss and hypotension. We hypothesize, in the setting of minimal HE (MHE), the compromised brain becomes susceptible to hypotensive insults, resulting in cell injury and death. To investigate this hypothesis, six-week bile-duct ligated (BDL) rats with MHE and respective controls (SHAM) were used. Blood is withdrawn from the femoral artery (inducing hypovolemia) until a mean arterial pressure of 30, 60 and 90 mmHg (hypotension) and maintained for 120 minutes. Our results demonstrated that BDL with following hypotension of 30 and 60 mmHg have a lower neuronal cell count compared to SHAM-operated animals. Furthermore, we provide evidence neuronal cell death is occurring due to apoptosis (observed by presence of cleaved caspase-3). In addition, cerebral blood flow is reduced in BDL rats compared to SHAM-operated controls. Second objective was to assess the therapeutic potential of the ammonia-lowering agent ornithine phenylacetate (OP) in preventing hypotension-induced neuronal loss in BDL rats. OP-treated BDL rats, in addition to lowering blood ammonia, also ameliorated cognitive function. However, cleaved caspase-3 levels were still elevated in the brains of OP-treated BDL rats therefore suggesting OP delays the onset of neuronal death in BDL rats. Overall, these findings strongly suggest that cirrhotic patients with MHE are more susceptible to hypotension-induced neuronal cell loss. Moreover, these results suggest a patient with HE (even MHE), with a “frail brain”, will fare worse during LT transplantation and consequently result in poor neurological outcome. Combination of MHE and hypotension may account for the persisting neurological complications observed in a number of cirrhotic patients following LT. Therefore, MHE, i) should not be ignored and therefore diagnosed and ii) merits to be treated in order to reduce the risk of neurological complications occurring post-LT

L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique dû à une dysfonction hépatique où l'ammoniaque est un facteur central. Il a déjà été rapporté que l’intoxication aiguë d'ammoniaque induise le stress oxydatif/nitrosatif. La présente étude cible à évaluer le rôle du stress oxydatif/nitrosatif dans 2 modèles de l’EH chronique : (1) l’anastomose portocave (PCA) et (2) la ligation de la voie biliaire (BDL). Ces 2 modèles sont caractérisés par une hyperammoniémie et une augmentation d’ammoniaque centrale, cependant l’œdème cérébral est trouvé seulement chez les rats BDL. Des marqueurs du stress oxydatif/nitrosatif ont été évaluées dans le plasma et cortex frontal. Un stress nitrosatif central a été observé chez les rats PCA; tandis qu’un stress oxydatif/nitrosatif systémique a été démontré seulement chez les rats BDL. Ces résultats suggèrent (1) que l’hyperammoniémie chronique n’induise pas le stress oxydatif/nitrosatif systémique et (2) qu’un synergisme existe entre l’ammoniaque et le stress oxydatif/nitrosatif, en association avec l’œdème cérébral. / Hepatic encephalopathy (HE) is a neuropsychiatric complication due to liver failure where ammonia is believed to be central in the pathogenesis. Acute ammonia intoxication has demonstrated to induce oxidative/nitrosative stress in both in vivo and in vitro models. The present study was aimed to assess the role of oxidative/nitrosative stress in 2 models of chronic liver failure/HE; 1. portacaval anastomosis (PCA) and 2. bile duct ligation (BDL). Both models are characterised with hyperammonemia and increased brain ammonia however cerebral edema is only found in BDL rats. Oxidative/nitrosative stress markers were evaluated in plasma and frontal cortex of both animal models. Central nitrosative stress was observed in PCA rats, but systemic oxidative/ntrosative stress was demonstrated only in BDL rats. The results of our study suggest i) chronic hyperammonemia does not induce oxidative stress and ii) a synergistic effect between ammonia and systemic oxidative/nitrosative stress is associated with cerebral edema.

L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique dû à une dysfonction hépatique où l'ammoniaque est un facteur central. Il a déjà été rapporté que l’intoxication aiguë d'ammoniaque induise le stress oxydatif/nitrosatif. La présente étude cible à évaluer le rôle du stress oxydatif/nitrosatif dans 2 modèles de l’EH chronique : (1) l’anastomose portocave (PCA) et (2) la ligation de la voie biliaire (BDL). Ces 2 modèles sont caractérisés par une hyperammoniémie et une augmentation d’ammoniaque centrale, cependant l’œdème cérébral est trouvé seulement chez les rats BDL. Des marqueurs du stress oxydatif/nitrosatif ont été évaluées dans le plasma et cortex frontal. Un stress nitrosatif central a été observé chez les rats PCA; tandis qu’un stress oxydatif/nitrosatif systémique a été démontré seulement chez les rats BDL. Ces résultats suggèrent (1) que l’hyperammoniémie chronique n’induise pas le stress oxydatif/nitrosatif systémique et (2) qu’un synergisme existe entre l’ammoniaque et le stress oxydatif/nitrosatif, en association avec l’œdème cérébral. / Hepatic encephalopathy (HE) is a neuropsychiatric complication due to liver failure where ammonia is believed to be central in the pathogenesis. Acute ammonia intoxication has demonstrated to induce oxidative/nitrosative stress in both in vivo and in vitro models. The present study was aimed to assess the role of oxidative/nitrosative stress in 2 models of chronic liver failure/HE; 1. portacaval anastomosis (PCA) and 2. bile duct ligation (BDL). Both models are characterised with hyperammonemia and increased brain ammonia however cerebral edema is only found in BDL rats. Oxidative/nitrosative stress markers were evaluated in plasma and frontal cortex of both animal models. Central nitrosative stress was observed in PCA rats, but systemic oxidative/ntrosative stress was demonstrated only in BDL rats. The results of our study suggest i) chronic hyperammonemia does not induce oxidative stress and ii) a synergistic effect between ammonia and systemic oxidative/nitrosative stress is associated with cerebral edema.