<p>U eksperimantalnom postupku disertacije mi&scaron;evi NMRI soja su tretirani infuzom biljke Aristolochia clematitis. Sasu&scaron;eni listovi, grane i plodovi biljke potopljeni su u ključalu vodu i ostavljeni 3-5 sati da stoje, a potom su profiltrirani kroz filter papir. Pravljen je rastvor biljke/vode od 10g/ 1000ml (1%), 20g/ 1000ml (2%) i 40g/ 1000ml (4%). Različite koncentracije infuza su date mi&scaron;evima da piju u neograničenoj količini u periodu od 7 nedelja. Tako su formirane tri ispitne grupe, prva koja je primala 1% infuz, druga 2% infuz i treća 4% infuz i kontrolna grupa koja je dobijala samo vodu da pije. U svakoj grupi je bilo 20 životinja. Tako je razvijen animalni model hronične toksičnosti. Na kraju eksperimenta je urađena patohistolo&scaron;ka analiza bubrega, makroskopski pregled organa i merenje diureze tokom trajanja eksperimenta. Urađena je kompletna analiza urina koja podrazumeva utvrđivanje: boje, izgleda, pH, specifične težine, proteina i sedimenta urina. Analize urina ponavljane su na svakih 7 dana u toku 7 nedelja istraživanja. Na kraju eksperimenta urađena je analiza biohemijskih parametara (glukoza, urea, kreatinin, mokraćna kiselina, ukupni bilirubin, direktni bilirubin, ukupni tj. totalni proteini, natrijum i kalijum) i analiza kompletne krvne slike. Utvrđeno je da je Aristolochia clematitis izrazito nefrotoksična biljka. Utvrđene su patohistolo&scaron;ke promene tubula i intersitcijuma NMRI mi&scaron;a, koje su bile najveće u ispitnoj grupi koja je primala najaču dozu. Ustanovljene&nbsp; patohistolo&scaron;ke promene su slične opisanim patohistolo&scaron;kim promenama tubulointersticijuma bolesnika obolelih od Balkanske endemske nefropatije. Nije ustanovljeno postojanja karcinoma gornjeg urotrakta. Makroskopskim pregledom prilikom obdukcije eksperimentalnih životinja nisu ustanovljene značajnije promene bubrega. Do&scaron;lo je prvo do izrazitog porasta diureze u prvoj, odnosno drugoj nedelji praćenja, kod druge i treće eksperimentalne grupe, da bi nakon 7 nedelja istaživanja diureza u svim ispitnim grupama bila manja od kontrolne grupe. Postoji porast ureje na kraju istraživanja, koji je dvostruko veći u trećoj eksperimentalnoj grupi u odnosu na kontrolnu. Postoji izrazit pad mokraćne kiseline na kraju istraživanja kod eksperimentalne grupe 3. Postoji izrazit pad granulocita u leukocitarnoj formuli u svim ispitnim grupama, a najveći je u trećoj ispitnoj grupi. Kako je do&scaron;lo do pada relativnih vrednosti granulocita, tako je do&scaron;lo do porasta relativnih vrednosti limfocita u prvoj i drugoj ispitnoj grupi. U trećoj ispitnoj grupi je pad granulocita praćen izrazito velikim povećanjem relativnog broja bazofilnih granulocita. Postoji značajan pad specifične težine urina na kraju istraživanja u drugoj i trećoj eksperimentalnoj grupi. Proteinurija je bila čest nalaz svim eksperimentalnim grupama, dok je bila odsutna ili samo u tragu u kontrolnoj grupi. Na kraju eksperimenta je utvrđen znatni porast broja kristala fosfata u eksperimentalnim grupama. Cilindri su se pojavljivali samo u nalazu urina u trećoj ispitnoj grupi. Najveći broj promena urina je utvrđen u trećoj eksperimentlanoj grupi.</p> / <p>In the experimental procedure of dissertation, NMRI strain mice were treated with infusion of plants Aristolochia clematitis. Dried leaves, branches and fruit plants are submerged in boiling water and left to stand for 3-5 hours, and then filtered through filter paper. It was made a solution of the plant / water of 10g / 1000ml (1%), 20g / 1000ml (2%) and 40g / 1000ml (4%). Different concentrations of infusions were given to mice to drink an unlimited amount for a period of 7 weeks. So we formed the three test groups, the first who received 1% infusion, the second received 2% infusion and third received 4% infusion and a control group that received only water to drink. In each group there were 20 animals. Thus, developed an animal model of chronic toxicity. At the end of the experiment was performed histopathological analysis of kidneys, macroscopic examination of organs and measuring urine output during the experiment. We performed a complete analysis of urine, which is the determination of: color, appearance, pH, specific gravity, protein and urine sediment. Urinalysis were repeated every 7 days during the 7 weeks of the study. At the end of the experiment were analyzed for biochemical parameters (glucose, urea, creatinine, uric acid, total bilirubin, direct bilirubin, total proteins, sodium and potassium) and analysis of the complete blood count. It has been found that Aristolochia clematitis is extremely nephrotoxic plant. Identified histopathological changes of tubules and interstitium of NMRI mouse, which were the biggest in the test group receiving biggest dose. Established histopathological changes are similar to those described by pathological changes of tubulointerstitial injury of patients with Balkan endemic nephropathy. Not established the existence of cancer of the upper urinary tract. Macroscopic examination at autopsy of experimental animals, did not determine significant changes in the kidneys. There is first an enormous increase in diuresis in the first and second week of follow-up, in the second and third experimental groups retrospectively, that after 7 weeks of research, diuresis in all test groups was lower than the control group. There is an increase of urea at the end of the research, which is twice higher in the third experimental group compared to the control. There is a marked decrease in uric acid at the end of the research in the experimental group 3. There is a marked decrease in granulocytes in the leukocyte formula in all test groups, and the highest in the third test group. As the decline in the relative values of granulocytes, so there has been a rise in the relative values of lymphocite in the first and second test group. In the third test group, granulocyte drop was accompanied by a extremely large increase in the relative number of basophils. There is a significant drop in specific gravity of urine at the end of the research in the second and third experimental group. Proteinuria is a common finding to all experimental groups, while it was absent or only in traces in the control group. At the end of the experiment was determined to increase significantly the number of phosphate crystals in the experimental groups. The cylinders have appeared only in the urine in the third test group. The greatest number of changes in the urine is determined in the third experimental group.</p>

Muitas espécies do gênero Aristolochia (Familia Aristolochiaceae) têm sido usadas na medicina tradicional e folclórica como medicamentos e tônicos, as quais demonstravam atividades farmacológicas de interesse clínica e medica como anti-hemorrágica, anti-parasita, antibacteriano, antifúngico, analgésico, antitumoral entre outras. Visando a obtenção de mais informações sobre essas plantas e na procura por substâncias com efeitos antiofídicos, neste trabalho avaliou-se à ação de extratos aquoso, metanólico e de acetato de etila de folhas e caule contra as ações tóxicas da peçonha de Bothrops asper, ambos procedentes do Panamá e contra o efeito miotóxico da peçonha de Bothrops jararacussu e das miotoxinas BthTX-I (isolada de B. jararacussu) e Mtx-II (isolada de B. asper). O extrato das folhas em acetato de etila apresentou a melhor inibição da atividade fosfolipásica da peçonha de B. asper, demonstrando inibição de 45%, 35% e 33% nas proporções de 1:5, 1:10 e 1:30 (m/m), respectivamente. Enquanto que, o extrato de caule em acetato de etila demonstrou maior eficácia na neutralização da atividade coagulante, e, além disso, inibiu 96%, 92% e 87% do edema, da miotoxicidade e hemorragia induzidas pela peçonha de B. asper, respectivamente. Os percentuais diferenciados na neutralização das ações tóxicas da peçonha de Bothrops asper, revelam diferentes perfis do potencial antiofídico de Aristolochia sprucei. Um dos componentes bioativos foi isolado do extrato de caule desta planta por CLAE, e a caracterização química, por ressonância magnética nuclear, demonstrou ser o ácido aristolóquio que inibiu a atividade miotóxica das peçonhas de B. jararacussu e de B. asper em 80% e 85% e assim como a atividade miotóxica da BthTX-I e Mtx-II em 64% e 60%, respectivamente. A atividade hemolítica indireta da peçonha de B. asper foi inibida em 43% pelo o ácido aristolóquio. A análise dos espectros de dicroísmo circular e os estudos de interação por modelagem molecular sugerem que o ácido aristolóquio forma um complexo com a Mtx-II de B. asper inibindo sua atividade. A ligação do ácido aristoloquio com as miotoxinas (MjTX-1, BthTX-II) modificou a forma e a intensidade dos espectros de dicroísmo circular da miotoxina e induziu alterações na porcentagem dos diversos domínios que constituem a estrutura secundária desta miotoxina. Os resultados obtidos confirmam que os extratos de A. sprucei possuem propriedades antiofídicas e sugerem a necessidade de aprofundar estudos que permitam utilizar com segurança os extratos e o principio ativo isolado como suplementos dos antisoros para aumentar a eficácia na neutralização dos efeitos tóxicos locais da peçonha das serpentes. / A lot of species of genus Aristolochia (Familia Aristolochiacheae) have been used in traditional medicine and folk, such as medicaments and tonics, which show pharmacological activities of clinic and medical interest, like antihemorragic, antiparasitic, antibacterial, antifungic, analgesic, antitumoral between others. Expecting to get more information about these plants and in the search by substances with antiophidic effects, in this work was evaluated the action of aqueous, metanolic and ethyl acetate extracts from leaves and stems of Aristolochia sprucei against the toxic action of Bothrops asper venom, both native from Panamá and against the myotoxic effect of Bothrops jararacussu venom and BthTX1 (isolated from B. jararacussu) and Mtx-II (isolated from Bothorps asper). The leaves extracts in ethyl acetate showed the best inhibition registered of PLA2 activity of venom de B. asper showing inhibition of 45 %, 35 % and 33 %, in proportion (m/m) of 1:5, 1:10 and 1:30 respectively. As regards to stem extract in ethyl acetate, it showed high efficacy in neutralization of coagulant activity, besides It inhibited 96 %, 92 % and 87 % of edema, myotoxicity and hemorrhage induced by B. asper venom, respectively. One of bioactives components was isolated from stem extract of this plant by CLAE and the chemical characterization by nuclear magnetic resonance, this showed that the compound is the aristolochic acid. This compound inhibited the myotoxic activity of B. jararacussu and B. asper venom in 80 % and 85 %, so like myotoxic activity of BthTx-I and MTx-II in 64 % and 60 % respectively. The indirect hemolytic activity of B. asper venom was inhibited in 43 % by the aristolochic acid. The analyze of spectrum of circular dichroism and the studies of interaction by molecular modelagem suggest that the aristolochic acid forms a complex 1:1 with the miotoxin inhibiting their activity. The joint of aristolochic acid with the miotoxins (MjTX-1, BthTx-II) changes the way and the intensity of spectra from dichroism circular of miotoxin and It induced alteration in percentage of several domains that constitute a secondary structure from this toxin. The results obtained confirm that the extracts of A. sprucei have antiophidic properties and it suggest the necessity of deepen studies that allow to use with safety the extracts and the isolated active principle, like antiserum supplements to increase the efficacy in the neutralization of local toxics effects of snakes venoms.

Ce travail de thèse est une contribution à l’étude phytochimique par Chromatographie de Partage Centrifuge (CPC), de trois plantes utilisées en médecine traditionnelle au Soudan : Aristolochia bracteolata (plante entière), Ziziphus spina-christi (feuilles) et Hydnora abyssinica (rhizomes). Ce travail a permis de mettre au point trois méthodologies de purification par CPC, applicables au fractionnement des acides aristolochiques, des flavonoïdes ou des proanthocyanidols (PAC). Dans ce contexte, la première partie de ce manuscrit est consacrée aux notions générales portant sur la CPC. La deuxième partie porte sur l’étude d’Aristolochia bracteolata. Cette plante est utilisée en médecine traditionnelle, malgré la présence d'acides aristolochiques qui confèrent une néphrotoxicité élevée. Ce travail a permis de mettre au point une méthode innovante pour l’isolement et la purification, avec un très haut niveau de pureté, des acides aristolochiques I, II et IIIa à partir d’un extrait brut, en une étape par CPC en mode d’échange d’ions forts (SIX-CPC). L’acide aristolochique IIIa n’avait jamais été décrit dans cette plante auparavant. Ces résultats ont fait l’objet d’une publication en 2015 dans Separation and Purification Technology. Dans la troisième partie de cette thèse, la CPC a été appliquée à l’isolement de flavonosides présents dans Z. spina-christi. Nous appuyant sur l’expérience du laboratoire dans l’extraction par CPC des flavonosides du Ginkgo biloba, nous proposons une méthodologie de purification utilisant les systèmes de solvant biphasiques EtOAc/n-BuOH/MeOH/H2O et EtOAc/n-BuOH/H2O à différents ratios en fonction de la polarité des flavonosides. Dans la dernière partie, l’étude phytochimique de Hydnora abyssinica a mis en évidence la présence de PACs, polymères de hauts poids moléculaires de flavanols. La méthodologie de fractionnement CPC, précédée d’un pré-fractionnement sur résine LH-20, a permis l’isolement pour la première fois dans cette plante de la katsumadine et du rhodioloside. / This work was a contribution to the phytochemical study of three Sudanese medicinal plants: Aristolochia bracteolata (Whole plant), Ziziphus spina-christi (Leaves) and Hydnora abyssinica (Rhizomes). The specificity of this research program was to emphasize the application of Centrifugal Partition Chromatography (CPC) for the fractionation of these plants. Three specific CPC methodologies were developed for the purification of either aristolochic acids, flavonoids or proanthocyanidins (PACs). In this context, the first part of this manuscript was devoted to the presentation of the CPC methodology. The second part focused on the fractionation of crude extract of Aristolochia bracteolata. This plant is used in traditional medicine, in spite of the presence of aristolochic acids that confer a high nephrotoxicity. In this work was developed an innovating procedure for the isolation and purification in high purity of aristolochic acids I, II and IIIa, in one step from crude extract, using Strong Ions eXchange CPC (SIX-CPC). These results were published in 2015 in Separation and Purification Technology. In the third part, the flavonosides present in Z. spina-christi were isolated using CPC, either in normal or reverse elution mode, using two phases solvent systems EtOAc/n-BuOH/MeOH/H2O or EtOAc/n-BuOH/H2O with different ratios. In the last part, the phytochemical study of Hydnora abyssinica led to the fractionation of PACs, polymers of high molecular weight of flavanols. The CPC fractionation methodology, preceded by LH-20 resin pre-fractionation, allowed the isolation of katsumadine and rhodioloside.

Muitas espécies do gênero Aristolochia (Familia Aristolochiaceae) têm sido usadas na medicina tradicional e folclórica como medicamentos e tônicos, as quais demonstravam atividades farmacológicas de interesse clínica e medica como anti-hemorrágica, anti-parasita, antibacteriano, antifúngico, analgésico, antitumoral entre outras. Visando a obtenção de mais informações sobre essas plantas e na procura por substâncias com efeitos antiofídicos, neste trabalho avaliou-se à ação de extratos aquoso, metanólico e de acetato de etila de folhas e caule contra as ações tóxicas da peçonha de Bothrops asper, ambos procedentes do Panamá e contra o efeito miotóxico da peçonha de Bothrops jararacussu e das miotoxinas BthTX-I (isolada de B. jararacussu) e Mtx-II (isolada de B. asper). O extrato das folhas em acetato de etila apresentou a melhor inibição da atividade fosfolipásica da peçonha de B. asper, demonstrando inibição de 45%, 35% e 33% nas proporções de 1:5, 1:10 e 1:30 (m/m), respectivamente. Enquanto que, o extrato de caule em acetato de etila demonstrou maior eficácia na neutralização da atividade coagulante, e, além disso, inibiu 96%, 92% e 87% do edema, da miotoxicidade e hemorragia induzidas pela peçonha de B. asper, respectivamente. Os percentuais diferenciados na neutralização das ações tóxicas da peçonha de Bothrops asper, revelam diferentes perfis do potencial antiofídico de Aristolochia sprucei. Um dos componentes bioativos foi isolado do extrato de caule desta planta por CLAE, e a caracterização química, por ressonância magnética nuclear, demonstrou ser o ácido aristolóquio que inibiu a atividade miotóxica das peçonhas de B. jararacussu e de B. asper em 80% e 85% e assim como a atividade miotóxica da BthTX-I e Mtx-II em 64% e 60%, respectivamente. A atividade hemolítica indireta da peçonha de B. asper foi inibida em 43% pelo o ácido aristolóquio. A análise dos espectros de dicroísmo circular e os estudos de interação por modelagem molecular sugerem que o ácido aristolóquio forma um complexo com a Mtx-II de B. asper inibindo sua atividade. A ligação do ácido aristoloquio com as miotoxinas (MjTX-1, BthTX-II) modificou a forma e a intensidade dos espectros de dicroísmo circular da miotoxina e induziu alterações na porcentagem dos diversos domínios que constituem a estrutura secundária desta miotoxina. Os resultados obtidos confirmam que os extratos de A. sprucei possuem propriedades antiofídicas e sugerem a necessidade de aprofundar estudos que permitam utilizar com segurança os extratos e o principio ativo isolado como suplementos dos antisoros para aumentar a eficácia na neutralização dos efeitos tóxicos locais da peçonha das serpentes. / A lot of species of genus Aristolochia (Familia Aristolochiacheae) have been used in traditional medicine and folk, such as medicaments and tonics, which show pharmacological activities of clinic and medical interest, like antihemorragic, antiparasitic, antibacterial, antifungic, analgesic, antitumoral between others. Expecting to get more information about these plants and in the search by substances with antiophidic effects, in this work was evaluated the action of aqueous, metanolic and ethyl acetate extracts from leaves and stems of Aristolochia sprucei against the toxic action of Bothrops asper venom, both native from Panamá and against the myotoxic effect of Bothrops jararacussu venom and BthTX1 (isolated from B. jararacussu) and Mtx-II (isolated from Bothorps asper). The leaves extracts in ethyl acetate showed the best inhibition registered of PLA2 activity of venom de B. asper showing inhibition of 45 %, 35 % and 33 %, in proportion (m/m) of 1:5, 1:10 and 1:30 respectively. As regards to stem extract in ethyl acetate, it showed high efficacy in neutralization of coagulant activity, besides It inhibited 96 %, 92 % and 87 % of edema, myotoxicity and hemorrhage induced by B. asper venom, respectively. One of bioactives components was isolated from stem extract of this plant by CLAE and the chemical characterization by nuclear magnetic resonance, this showed that the compound is the aristolochic acid. This compound inhibited the myotoxic activity of B. jararacussu and B. asper venom in 80 % and 85 %, so like myotoxic activity of BthTx-I and MTx-II in 64 % and 60 % respectively. The indirect hemolytic activity of B. asper venom was inhibited in 43 % by the aristolochic acid. The analyze of spectrum of circular dichroism and the studies of interaction by molecular modelagem suggest that the aristolochic acid forms a complex 1:1 with the miotoxin inhibiting their activity. The joint of aristolochic acid with the miotoxins (MjTX-1, BthTx-II) changes the way and the intensity of spectra from dichroism circular of miotoxin and It induced alteration in percentage of several domains that constitute a secondary structure from this toxin. The results obtained confirm that the extracts of A. sprucei have antiophidic properties and it suggest the necessity of deepen studies that allow to use with safety the extracts and the isolated active principle, like antiserum supplements to increase the efficacy in the neutralization of local toxics effects of snakes venoms.

Present doctoral thesis contributed to understanding of mechanistic principles of two enzymes participating in the process of carcinogenesis; DNA polymerase  (pol ) and cytochromes P450 (CYP). Pol  is part of the DNA base-excision repair mechanism (BER). The primary role of pol  in, the BER mechanism, is inserting a new nucleotide into a DNA strand according to Watson-Crick base pairing rules. Pol  plays an important role in the process of carcinogenesis, approximately 30 % of human tumors express pol  mutants. The ability of pol  to discriminate between "right" and "wrong" nucleotide during the insertion process is called fidelity. We employed computational methods to elucidate molecular basis of the fidelity of pol . First, the relative free energy calculation method LRA was employed to compare differences in free energies between the "right" and "wrong" nucleotide during its insertion into DNA. The results indicated a better stabilization of transition-state of the nucleophilic substitution catalyzed by pol  in the case of the "right" versus "wrong" nucleotide. This difference resulted in an 80-fold contribution to its fidelity. Further, computational methods FEP and LIE were used to examine how mutations effect fidelity of pol . Results were than correlated with experimental data...

La néphropathie aux plantes chinoises (CHN) est une maladie rénale grave qui a été décrite pour la première fois en 1993 chez des patientes ayant suivi un régime amaigrissant à base d’extraits de plantes chinoises (Aristolochia fangchi) contenant des acides aristolochiques (AA). Cette néphropathie se caractérise par une atrophie tubulaire et une fibrose interstitielle aboutissant à l’urémie terminale et se complique fréquemment de cancers des voies urinaires. Au moment d’initier ce travail, il subsistait toujours un large débat quant au rôle étiologique réel des acides aristolochiques dans la genèse de cette maladie. En effet, les gélules à visée amaigrissante contenaient d’autres substances potentiellement néphrotoxiques. Mais surtout, il n’existait aucune preuve expérimentale que les AA pouvaient induire une fibrose rénale interstitielle.<p>Dans la première partie de ce travail, nous démontrons que l’injection par voie sous-cutanée d’AA à la dose de 10 mg/Kg/jour à des rats Wistar mâles en déplétion sodée entraîne l’apparition au 35ème jour d’une atrophie tubulaire, d’une fibrose interstitielle et d’une insuffisance rénale, reproduisant ainsi les anomalies caractéristiques de la CHN. Nous avons ensuite montré que la dexfenfluramine, substance anorexigène à action de type sérotoninergique prise concomitamment par les patientes atteintes de CHN, ne potentialise pas la toxicité rénale des AA. Enfin, la stimulation du système rénine angiotensine (SRA) par la déplétion sodée ou l’inhibition de celui-ci par un traitement pharmacologique ne modifie pas la fibrose interstitielle ni l’insuffisance rénale induite par les AA.<p>En conclusion, nous avons réussi à développer un modèle in vivo de fibrose rénale interstitielle induite par les AA. Dès lors nous avons apporté la preuve expérimentale de l’implication des AA dans le développement de la CHN. Ce modèle a permis de démontrer que les autres éléments potentiellement néphrotoxiques contenues dans la cure d’amaigrissement (dexfenfluramine, diurétique, laxatif) n’influençaient pas l’évolution de la fibrose interstitielle, ce qui confirme que la prise isolée d’AA suffit à expliquer le développement de la CHN. Cette confirmation à d’importantes implications en santé publique dans la mesure où des plantes contenant des acides aristolochiques font toujours partie des phytothérapies traditionnelles. De plus, il est apparu que, dans ce modèle, les mécanismes de la fibrose rénale interstitielle pouvaient être largement indépendants du SRA. Enfin, de par sa durée limitée et sa grande reproductibilité, ce modèle constitue un outil expérimental d’avenir pour l’étude des mécanismes physiopathologiques de la fibrose rénale interstitielle en général.<p> / Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished