English Abstract Shortly after the identification of HIV as a causative agent of AIDS, an aspartic protease was identified in the viral genetic information. The very same time protease has become one of the dominant therapeutical targets in AIDS therapy. The introduction of protease inhibitors into the antiretroviral therapy has led to a significant improvement in the quality and length of life of HIV patients. However, the virus is still able to effectively prevent the impact of an inhibitor via generating inhibitor-resistant mutated protease variants. Thus, there is a constant need for novel types of inhibitors that would be capable of effectively blocking these resistant variants and simultaneously not supporting the development of novel resistant viral strains. One way to identify such inhibitors could be searching for compounds interacting with the enzyme at different sites than the active cavity, via the mechanisms of noncompetitive or uncompetitive inhibition. The group of compounds called metallacarboranes - inorganic compounds consisting of carbon, boron, hydrogen and metall ion - were shown to exhibit such an activity against HIV-1 protease. However, for further optimization of these inhibitors, detailed biophysical investigation of the enzyme-inhibitor complex is needed. This work focuses on the...

Die Arbeit beschreibt die Auffindung und Charakterisierung einer FDH aus Rhodococcus jostii. Das Enzym konnte erfolgreich als Cofaktor-Regenerierungssystem eingesetzt werden. Darüber hinaus sind verschiedene Mutagenese-Experimente beschrieben, welche eine Optimierung des Enzyms für die industrielle Anwendung ermöglichten.:1. Einleitung 1 1.1. Allgemeine Einführung 1 1.1.1. Eigenschaften von Enzymen 1 1.1.2. Veränderungen von Enzymeigenschaften durch Mutagenese 3 1.1.3. Einsatz von Enzymen 4 1.2. Cofaktor-Regenerierung 5 1.2.1. Cofaktoren 5 1.2.2. Prinzip und Bedeutung der Cofaktor-Regenerierung 7 1.2.3. Enzymatische Cofaktor-Regenerierung 8 1.3. Formiatdehydrogenasen 11 1.3.1. Metallabhängige Formiatdehydrogenasen 12 1.3.2. NAD-abhängige Formiatdehydrogenasen 15 1.4. Anwendung von NAD-abhängigen FDH 27 1.5. Motivation und Zielsetzung 30 2. Material und Methoden 32 2.1. Materialien und Geräte 32 2.1.1. Geräte 32 2.1.2. Spezialchemikalien 33 2.1.3. Enzyme 33 2.1.4. Kitsysteme 33 2.1.5. Chromatographiesäulen 34 2.1.6. Weitere Verbrauchsmaterialien 34 2.2. Nukleinsäuren und Bakterienstämme 34 2.2.1. Bakterienstämme 34 2.2.2. Spenderorganismen 34 2.2.3. Plasmide 35 2.2.4. Oligonukleotide 35 2.3. Kultivierung von Mikroorganismen 36 2.3.1. Medien 36 2.3.2. Kultivierung von E. coli-Stämmen 37 2.3.3. Kultivierung von E. coli-Stämmen in Mikrotestplatten 37 2.3.4. Rekombinante Genexpression im 250 mL-Maßstab 37 2.3.5. Rekombinante Genexpression in Mikrotestplatten 38 2.3.6. Stammhaltung 38 2.4. Molekularbiologische Methoden 38 2.4.1. Isolation genomischer DNA aus Bakterien 38 2.4.2. Plasmidisolation 38 2.4.3. Agarose-Gelelektrophorese 38 2.4.4. Polymerase-Kettenreaktion (PCR) 39 2.4.5. Gibson-Assembly 41 2.4.6. Restriktionsansätze 42 2.4.7. Isolation und Reinigung von DNA-Fragmenten aus dem Agarosegel 42 2.4.8. Transformationstechniken 43 2.4.9. Mutagenesetechniken 44 2.4.10. Sequenzierung von DNA-Fragmenten 44 2.4.11. Herstellung kompetenter E. coli-Zellen 45 2.5. Biochemische Methoden 46 2.5.1. Einstellung der pH-Werte von Puffersubstanzen 46 2.5.2. Denaturierende SDS-Polyacrylamid-Gelelektrophorese 46 2.5.3. Proteinquantifizierung 47 2.5.4. Zellaufschluss 48 2.5.5. Proteinreinigung 49 2.5.6. Größenausschlusschromatographie (SEC) 50 2.6. Enzymatische Testverfahren 51 2.6.1. Berechnung der Enzymaktivität 51 2.6.2. Bestimmung der Enzymaktivität im 1 mL-Maßstab 51 2.6.3. Bestimmung der Enzymaktivität in Mikrotestplatten 52 2.6.4. Bestimmung der Enzymstabilität bei unterschiedlichen Temperaturen 52 2.6.5. Bestimmung der Enzymstabilität in Anwesenheit von org. Lösungsmitteln 53 2.6.6. Enzymstabilitätseffekte von Schwermetallen 53 2.6.7. Cofaktor-Regenerierungssysteme 53 2.6.8. Enzymatische Reduktion von Kohlenstoffdioxid 55 2.7. Analysemethoden 55 2.7.1. Hochleistungsflüssigkeitschromatographie (HPLC) 55 2.7.2. Gaschromatographie (GC) 56 2.8. Bioinformatische Methoden 57 2.8.1. In silico-Arbeiten mit DNA-Sequenzen 57 2.8.2. In silico-Arbeiten mit Proteinen 57 2.8.3. In silico-Analyse von Proteineigenschaften 57 2.8.4. Erstellung von Homologiemodellen 58 3. Ergebnisse und Diskussion 59 3.1. Identifizierung hochaktiver NAD-abhängiger Formiatdehydrogenasen 59 3.1.1. Bioinformatische Auswahl von FDH-Sequenzen 59 3.1.2. Vorauswahl potentiell hochaktiver FDH-Varianten 64 3.1.3. Bereitstellung potentiell hochaktiver FDH-Varianten und Bestimmung der kinetischen Parameter 71 3.1.4. Fazit 75 3.2. Proteinbiochemische Charakterisierung der RjFDH 77 3.2.1. Allgemeine Charakteristika 77 3.2.2. Substratspektrum und Inhibition der RJFDH 83 3.2.3. Beeinflussung der Enzymaktivität der RjFDH durch Metallionen 84 3.2.4. Cofaktor-Nutzung der RjFDH 86 3.2.5. Reduktion von CO2 91 3.2.6. Temperaturabhängigkeit und Thermostabilität der RjFDH 97 3.2.7. Einfluss des pH-Werts auf die RjFDH 101 3.2.8. Einfluss von Lösungsmitteln auf die Enzymaktivität 104 3.2.9. Fazit 108 3.3. Leistung der RjFDH als Cofaktor-Regenerierungssystem 111 3.3.1. Cofaktor-Regenerierung in Kombination mit der Alkoholdehydrogenase A 111 3.3.1. Cofaktor-Regenerierung in Kombination mit der Leucin-Dehydrogenase aus Lysinibacillus sphaericus 113 3.3.2. Fazit 115 3.4. Veränderung der Enzymeigenschaften der RjFDH 116 3.4.1. Einfluss von Einzelmutationen auf die thermostabilität der RjFDH 116 3.4.2. Einfluss von Cystein-Resten auf die Enzymstabilität 127 3.4.3. Einfluss des C-terminalen Bereichs der RjFDH auf die Enzymaktivität 141 3.4.4. Veränderung der Cofaktorspezifität der RjFDH 150 Anhang 167 Literaturverzeichnis 174 Eidesstattliche Erklärung 191

In der vorliegenden Arbeit wurde die bisher kaum in der Literatur beschriebene Stoffklasse der Prostaglandin-Glycerolester (PG-G) in Hinblick ihrer biochemischen Wirkmechanismen genauer charakterisiert. Einige Studien beschreiben PG-G als relevante Mediatoren im Rahmen sowohl inflammatorischer als auch nozizeptiver Prozesse. Dieser Sachverhalt macht diese Stoffklasse zum interessanten Gegenstand aktueller pharmakologischer Forschung, dessen Grundlage die Identifizierung und Charakterisierung entsprechender Rezeptoren darstellt. Im Rahmen unserer Arbeit, identifizierten wir als ersten Schritt das Target des PGE2-G. Weder Prostaglandinrezeptoren noch bekannte orphane GPCR konnten durch die Stimulation mit PGE2-G aktiviert werden. Jedoch konnten in bestimmen Zelllinien wie RAW264.7 und H1819 Effekte durch PGE2-G erzielt werden, während andere Zelllinien keine Reaktionen zeigten. Dies machten wir uns zu Nutzen und sequenzierten das Transkriptom dieser Zelllinien, um so durch subtraktive Analysen Rezeptoren einzugrenzen, welche voraussichtlich als Target von PGE2-G fungieren. Als vielversprechender Kandidat exprimierten wir den UDP-Rezeptor P2Y6 heterolog in HEK293-Zellen und wiesen mittels Zellkultur-basierter Assays den Agonismus von PGE2-G am P2Y6 nach. Weitere Untersuchungen bestätigten diese hochspezifische Bindung zwischen beiden Interaktionspartnern. Auffällig war jedoch die ausordentlich hohe Potenz von PGE2-G, welche schon bei picomolaren Stoffkonzentrationen eine Aktivierung von P2Y6 initiiert. Insbesondere in Hinblick auf den deutlich höheren EC50-Wert von UDP stellt sich die Frage, inwiefern dies die Signaltransduktionsmechanismen und die damit einhergehenden physiologischen Effekte moduliert. Es stellte sich weiterhin die Frage, wie der P2Y6 die Bindung zweier chemisch unterschiedlichen Substanzen realisieren kann. Ziel dieser Arbeit war es nun, die Struktur von P2Y6 auf molekularbiologischer Ebene zu untersuchen, um ein Verständnis über die Art und Weise der Bindung der unterschiedlichen Agonisten zu erhalten.:Entzündungsreaktionen ................................................................................................................. 6 Prostaglandine ............................................................................................................................... 7 Prostaglandin‐Glycerolester und die Identifizierung ihrer Rezeptoren ......................................... 9 Prostaglandin‐Glycerolester ........................................................................................................9 G‐Protein‐gekoppelte Rezeptoren (GPCR) ............................................................................... 11 Orphane GPCR .......................................................................................................................... 12 Screening potentieller Targets von Prostaglandin‐E2‐Glycerolester ........................................ 13 Der Pyrimidinrezeptor P2Y6 .......................................................................................................... 14 Nucleotidrezeptoren ................................................................................................................ 14 Pharmakologische Bedeutung von Nucleotidrezeptoren ........................................................ 15 P2Y6 .......................................................................................................................................... 15 Fragestellung ................................................................................................................................ 18 Publikationen ................................................................................................................................... 20 Zusammenfassung der Ergebnisse und Diskussion .......................................................................... 50 Prostaglandin‐E2‐Glycerolester ist ein endogener Agonist am P2Y6 .......................................... 50 Die Bindungstasche des P2Y6 ....................................................................................................... 50 Evolutionäre Konservierung der Agonisten‐Promiskuität........................................................ 50 Simulation der Ligandenbindung im Homologiemodell........................................................... 51 Experimentelle Prüfung der Interaktionspartner ......................................................................... 52 Interaktionspartner beider Agonisten: R103, Y107, R287 ....................................................... 53 Interaktionpartner mit UDP: Y262 ........................................................................................... 53 Interaktionspartner mit PGE2‐G: Y75, N109, S291, N293 ........................................................ 53 Korrelation der experimentellen Daten mit einem Homologiemodell ........................................ 53 Ausblick ........................................................................................................................................ 54 Literatur ............................................................................................................................................ 56 Anhang ............................................................................................................................................. 62 Darstellung des eigenen Beitrags ..................................................................................................... 85 Erklärung über die eigenständige Abfassung der Arbeit ................................................................. 87 Lebenslauf ........................................................................................................................................ 88 Publikationen und Vorträge ............................................................................................................. 90 Danksagung ...................................................................................................................................... 91

In der vorliegenden Arbeit wurden die molekularen Grundlagen für die Agonistspezifität des G-Protein-gekoppelten Rezeptors GPR34 untersucht. Mittels verschiedener funktioneller Versuche konnte an ausgewählten Orthologen des Rezeptors gezeigt werden, dass, im Gegensatz zu publizierten Daten, Lysophosphatidylserin (Lyso-PS) nicht der natürliche Agonist des GPR34 ist. Lediglich an einigen cyprinoiden Subtypen des GPR34 hat Lyso-PS surrogat-agonistische Effekte. Anhand eines detaillierten evolutionären Vergleichs von Orthologen konnten Bereiche des Rezeptors ermittelt werden, welche an der Ligandenbindung, und damit an der Agonistspezifität des GPR34 beteiligt sind. Durch Übertragung dieser Bereiche vom Karpfen-GPR34-Subtyp 2a auf den humanen GPR34 konnte dieser zu einem Lyso-PS-sensitiven Rezeptor modelliert werden. Weiterhin wurde Aminoethyl-Carbamoyl-ATP (EDA-ATP) als inverser Agonist an cyprinoiden Orthologen des GPR34 identifiziert. Die Erweiterung des möglichen Ligandenspektrums von Lipiden zu Nukleotidderivaten gibt Hinweise auf die Promiskuität der Bindungsstelle des GPR34. Die Ergebnisse zeigen, dass Lyso-PS nur eine zufällige Aktivität an einigen Orthologen des GPR34 hat. Mit Identifizierung eines Nichtlipides als invers-agonistischen Liganden ist die Suche nach dem natürlichen Liganden des GPR34 noch nicht abgeschlossen und sollte auf weitere chemische Entitäten ausgeweitet werden. / Lyso-PS (lyso-phosphatidylserine) has been shown to activate the G(i/o)-protein-coupled receptor GPR34. Since in vitro and in vivo studies provided controversial results in assigning lyso-PS as the endogenous agonist for GPR34, we investigated the evolutionary conservation of agonist specificity in more detail. Except for some fish GPR34 subtypes, lyso-PS has no or very weak agonistic activity at most vertebrate GPR34 orthologues investigated. Using chimaeras we identified single positions in the second extracellular loop and the transmembrane helix 5 of carp subtype 2a that, if transferred to the human orthologue, enabled lyso-PS to activate the human GPR34. Significant improvement of agonist efficacy by changing only a few positions strongly argues against the hypothesis that nature optimized GPR34 as the receptor for lyso-PS. Phylogenetic analysis revealed several positions in some fish GPR34 orthologues which are under positive selection. These structural changes may indicate functional specification of these orthologues which can explain the species- and subtype-specific pharmacology of lyso-PS. Furthermore, we identified aminoethyl-carbamoyl ATP as an antagonist of carp GPR34, indicating ligand promiscuity with non-lipid compounds. The results of the present study suggest that lyso-PS has only a random agonistic activity at some GPR34 orthologues and the search for the endogenous agonist should consider additional chemical entities.

Orientador: Maria Aparecida Marin Morales / Coorientador: Paula Suares Rocha / Banca: Thaís Cristina Casimiro Fernandes / Banca: Grasiela Dias de Campos Severi Aguiar / Resumo: Os ecossistemas aquáticos são os ambientes mais comprometidos pela poluição derivada de emissões de substâncias tóxicas presentes em efluentes industriais e domésticos e em resíduos agrícolas. Essa contaminação pode atingir tanto a água quanto os sedimentos dos ambientes aquáticos e induzir diversos efeitos adversos aos organismos vivos expostos. A bacia do rio Corumbataí, localizada no interior do estado de São Paulo (Brasil), apresenta grande importância econômica e ambiental para estado. As águas deste rio sofrem diversos impactos ambientais caracterizados pelo descarte de efluentes urbanos e industriais das cidades localizadas próximas à sua margem, bem como da ação de atividades agrícolas, principalmente da cultura de cana-de-açúcar. Sendo assim, este rio recebe uma quantidade considerável de substâncias potencialmente tóxicas, que vêm comprometendo a qualidade de suas águas e de seus sedimentos. O presente estudo teve como objetivo avaliar a citotoxicidade, genotoxicidade e mutagenicidade de amostras de águas e de sedimentos do rio Corumbataí - SP, em pontos situados entre as cidades de Rio Claro e Piracicaba. Foram realizadas duas coletas, uma na estação chuvosa e quente e outra na estação seca e fria, em quatro pontos distintos do rio: P1) à montante da cidade de Rio Claro; P2) à jusante da cidade de Rio Claro; P3) à jusante de Santa Gertrudes e da confluência do rio Corumbataí com o Ribeirão Claro; P4) à montante da cidade de Piracicaba. As avaliações foram realizadas por meio de bioensaios desenvolvidos com os organismos testes Allium cepa e com cultura de hepatócitos de truta arco-íris da linhagem RTL-W1. Para os bioensaios realizados com A. cepa, foram realizados testes de índice mitótico (IM), de aberrações cromossômicas (AC) e de indução de micronúcleos (MN), em células meristemáticas e testes de indução de MN em células F1. Para os bioensaios realizados com cultura de... / Abstract: Aquatic ecosystems are the most affected by pollution from toxic waste emissions of industrial, domestic and agricultural effluents. This contamination may occur in both water and sediments of aquatic environments and induce many adverse effects to the living organisms. The Corumbataí river basin, located within the state of São Paulo (Brazil), has great economic and environmental importance to the state. The waters of this river suffer from various environmental impacts characterized by the disposal of municipal and industrial effluents from cities located close to its border. Thus, this river receives a considerable amount of potentially toxic substances. Due to its importance for public water supply in the region this study aimed to evaluated the cytotoxic, genotoxic and mutagenic effects of water and sediments samples from this water resource, by means of bioassays. For this study, two sampling campaigns were performed, one in the warm and rainy season and the other in the cold and dry season. Samples were collected in Corumbataí River, in four different sites: P1) upstream the municipality of Rio Claro, P2) downstream the municipality of Rio Claro; P3) downstream the confluence of Corumbataí River and Ribeirão Claro; P4) upstream the municipality of Piracicaba. The evaluations were performed through bioassay with the Allium cepa test organisms and culture of hepatocytes from rainbow trout RTL - W1. According to obtained results in this work, it was possible to observed that water and sediments Corumbataí have genotoxic and mutagenic characteristics. The most significant results were recorded for the points P2 and P3, located near the cities of Rio Claro and Santa Gertrudes. Our results also indicated that samples collected at P3 were more affected, probably due to influence of discharges of industrial and domestic effluents from the city of St. Gertrudes , which is considered the largest ceramic hub of America / Mestre

Lipoxygenasen sind nichthämeisenhaltige Dioxygenasen, die die Bildung von Hydroperoxiden aus molekularem Sauerstoff und mehrfach ungesättigten Fettsäuren katalysieren. Im Rahmen dieser Arbeit wurden verschiedene Lipoxygenase-Ligand-Wechselwirkungen untersucht: a) Durch gezielte Substratveränderungen und ortsgerichtete Mutagenese konnte gezeigt werden, dass Fettsäuren normalerweise mit dem Methylende in der Bindungstasche tierischer Lipoxygenasen gebunden werden. Unterschiedliche Positionsspezifitäten basieren demzufolge auf dem Volumen der Substratbindungstasche (Volumenhypothese). Darüber hinaus konnte bei der 15-Lipoxygenase eine inverse Substratbindung (Carboxylgruppe im aktiven Zentrum) durch Modifikation beider Enden der Fettsäure erzwungen werden, wodurch ausschließlich 5-Lipoxygenierung katalysiert wurde. b) Untersuchungen mit dem Hemmstoff Ebselen ergaben unterschiedliche Hemmmechanismen für verschiedene Enzymzustände. Die Hemmung der Lipoxygenase im Grundzustand (Fe[II]) erfolgt durch kovalente Bindung und Veränderung der Eisenligandensphäre irreversibel nach einem nicht-kompetitiven Mechanismus. Dagegen wird die aktive Lipoxygenase (Fe[III]) nur noch kompetitiv durch Ebselen gehemmt. c) Die Membranbindung der tierischen 15-Lipoxygenase erfolgt über hydrophobe Wechselwirkungen, vermittelt durch oberflächenexponierte, hydrophobe Aminosäuren aus beiden Enzymdomänen. Die Expression einer enzymatisch aktiven Trunkationsmutante, der die N-terminale Domäne fehlt, zeigte, dass diese nicht essentiell ist für die Membranbindung. / Lipoxygenases are nonheme iron-containig dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxy derivatives. Here, the interaction of lipoxygenases with various ligands was investigated: a) From substrate modifications and site directed mutagenesis it was concluded that fatty acids are bound with their methyl end in the substrate binding pocket of mammalian lipoxygenases. The positional specificity is therefore related to different volumes of this binding pocket (space-based hypothesis). For the 15-lipoxygenase an inverse substrate binding (carboxy terminus in the pocket) could be forced by simultaneous modification of both ends of the fatty acid. This lead to an exclusive 5-lipoxygenation by the 15-lipoxygenase. b) The mechanism of lipoxygenase inhibition by ebselen depends on the enzyme's state. The groundstate lipoxygenase (containing Fe[II]) is irreversibly inhibited in a non-competetive manner due to covalent modification and alteration of the iron ligand sphere. The active enzyme (containing Fe[III]) on the other hand is only competetively inhibited. c) The membrane binding of the mammalian 15-lipoxygenase is based on hydrophobic interactions mediated by solvent exposed, hydrophobic amino acid residues of both enzyme domains. The expression of an enzymatically active truncation mutant, which lacks the entire N-terminal domain, showed that this domain is not essential for membrane binding.

Escherichia coli patogênicas extraintestinais (ExPEC) causam colibacilose aviária, infecções do trato urinário e meningite neonatal em humanos. A cepa ExPEC MT78 é virulenta in vitro e in vivo, e possui a habilidade de invadir células eucarióticas. Para melhor entender o fenótipo invasivo dessa cepa, foi criada uma biblioteca de mutantes aleatórios pela técnica de mutagênese marcada com assinatura, e os mutantes foram selecionados negativamente em ensaio de invasão a fibroblastos aviários. Mutantes atenuados apresentaram mutação em genes do operon da fímbria do tipo 1 e nos genes de metabolismo de açúcares treA e bglB. Foram feitos mutantes específicos para o gene que codifica a enzima trealase periplasmática (TreA) e para o operon de metabolismo de β-glicosídeos (bgl). O mutante MT78ΔtreA apresentou, frente à cepa selvagem, diminuição na capacidade de adesão e invasão a fibroblastos aviários, na expressão da fímbria do tipo 1 e na capacidade de colonizar a bexiga de camundongos em modelo de infecção urinária. O mutante MT78Δbgl também apresentou, frente à cepa selvagem, diminuição na capacidade de adesão e invasão a fibroblastos aviários e na capacidade de colonizar a bexiga de camundongos em modelo de infecção urinária, mas não mostrou alteração na expressão da fímbria do tipo 1, medida por aglutinação de leveduras. Os resultados deste trabalho mostraram que a trealase periplasmática afeta a expressão da fímbria do tipo 1 e a virulência in vivo da cepa ExPEC MT78, enquanto o operon do metabolismo de β-glicosídeos afeta a virulência in vivo da cepa ExPEC MT78 por um mecanismo ainda não-elucidado. / Extraintestinal pathogenic E. coli (ExPEC) cause colibacillosis in birds, urinary tract infections and neonatal meningitis in humans. The ExPEC strain MT78 is virulent in vitro and in vivo, and is able to invade eukaryotic cells. In order to better understand the invasive phenotype of this strain, a library of random mutants was made using the signature-tagged mutagenesis approach. The mutants were negatively selected in invasion assays of avian fibroblasts. Attenuated mutants presented mutation in the type 1 fimbria operon and in the genes of sugar metabolism treA and bglB. Specific mutants were created for the periplasmic trehalase (TreA) gene and for the β-glycosides metabolism operon (bgl). The MT78ΔtreA mutant displayed, in comparison with the wild type strain, a reduction on the capacity of adhesion and invasion to avian fibroblastos, on type 1 fimbriae expression and on the capacity to colonize the bladder in a murine model of urinary tract infection. The MT78Δbgl mutant, compared to the wild type strain, also displayed a reduction on the capacity of adhesion and invasion to avian fibroblastos and on the capacity to colonize the bladder in a murine model of urinary tract infection, but did not show any difference on type 1 fimbriae expression as detected by yeast agglutination. Taken together, our results show that the periplasmic trehalase affects type 1 expression and in vivo virulence of the ExPEC strain MT78, and the operon for β-glycosides metabolism affects in vivo virulence by an as yet unidentified mechanism.

Submitted by Irvana Coutinho (irvana@ufpa.br) on 2012-12-12T13:51:59Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AvaliacaoVitroPossIveis.pdf: 1249677 bytes, checksum: 36d6e43acaaa9c2bacd886ccc1729b0f (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2012-12-20T13:18:24Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AvaliacaoVitroPossIveis.pdf: 1249677 bytes, checksum: 36d6e43acaaa9c2bacd886ccc1729b0f (MD5) / Made available in DSpace on 2012-12-20T13:18:24Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AvaliacaoVitroPossIveis.pdf: 1249677 bytes, checksum: 36d6e43acaaa9c2bacd886ccc1729b0f (MD5) Previous issue date: 2012 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / A artemisinina é uma substância extraída da planta chinesa Artemisia annua L., sendo bastante utilizada na medicina natural como um terapêutico em várias patologias. Já o artemer é uma substância sintetizada a partir da artemisinina. Estas drogas se enquadram em um grupo especial de moléculas denominadas de lactonas sesquiterpênicas sendo amplamente administradas na terapêutica da malária. Embora sejam considerados eficientes anti-maláricos, muito pouco se sabe sobre os efeitos genotóxicos e citotóxicos destes fármacos. Portanto, no presente trabalho, avaliamos os efeitos citotóxicos, genotóxicos e mutagênicos da artemisinina e do artemeter em cultura de linfócitos humanos por meio do ensaio cometa, do teste do micronúcleo e do ensaio de citotoxicidade para detecção de necrose e apoptose por marcação fluorescente diferencial com laranja de acridina/brometo de etídio (LA/BE), respectivamente. Nossos resultados demonstraram um aumento significativo (p<0,05) no índice de dano do DNA avaliado pelo ensaio do cometa, bem como na frequência de micronúcleos em ambas as substâncias testadas. Foi observado também, que apenas a artemisinina induziu um aumento estatisticamente significativo (p<0.05) no número de células necróticas nos linfócitos em 48 h de tratamento. Desta forma, demonstrou-se em nosso trabalho, que estas duas drogas exercem efeitos citotóxicos, genotóxicos e mutagênicos em culturas de linfócitos humanos, nas condições avaliadas. Nossos dados apontam a necessidade de cautela no uso de tais medicamentos, uma vez que efeitos genotóxicos/mutagênicos podem aumentar o risco de carcinogênese. / Artemisinin is a substance extracted from the Chinese plant Artemisia annua L., and widely used in natural medicine for a treatment of various diseases. Artemether is a substance synthesized from artemisinin. These drugs belong to a special group of molecules called sesquiterpene lactones widely administered in the treatment of malaria. Although considered effective anti-malarial drugs, very little is known about the genotoxic and cytotoxic effects of these drugs. Therefore, in the present study, we evaluated the genotoxic, mutagenic and cytotoxic effects of artemisinin and artemether in cultured human lymphocytes using the comet assay, the micronucleus test and a cytotoxicity assay for detection of necrosis and apoptosis by fluorescent differential acridine orange/ethidium bromide (LA/BE), respectively. Our results showed a significant increase (p<0.05) in the rate of DNA damage measured by comet assay and in the micronucleus frequency after treatment with both drugs. It was also observed that only artemisinin induced a statistically significant increase (p<0.05) in the number of lymphocytes with death by necrosis 48 h after treatment. Thus, it was shown in our work that these two drugs exert mutagenic, genotoxic and cytotoxic effects in cultured human lymphocytes under the conditions evaluated. Our data indicate the need for caution in the use of such drugs, since genotoxic/mutagenic effects may increase the risk of carcinogenesis.

Estudos ambientais têm demonstrado que substâncias geradas por processos antropogênicos podem causar efeitos prejudiciais interferindo no equilíbrio natural do ecossistema. Manguezais exercem funções essenciais nos ciclos biológicos e constituem Área de Proteção Permanente no Brasil. Infelizmente, eles estão sendo degradados acima do seu limite de suporte, levando a uma redução das áreas remanescentes no mundo. Este trabalho apresenta os resultados de mutagenicidade e genotoxicidade observados em quatro amostragens (PI, V, O e PII) entre 2009 e 2010, relacionados com metais e hidrocarbonetos policíclicos aromáticos (HPA) em sedimento de mangue para a caracterização dos valores de referência. Os testes de genotoxicidade foram feitos a partir de hemócitos do caranguejo Goniopsis cruentata, coletados em um ecossistema potencialmente não poluído do Brasil, localizado no sul do Rio de Janeiro (Parati/RJ), chamado de "Saco do Mamanguá". Coleta, armazenamento e manipulação dos sedimentos e material biológico de cinco pontos de amostragem (M1- M5) foram processados de acordo com normas norte-americanas reconhecidas. A identificação das substâncias químicas foi realizada com extratos de sedimentos e utilizada no bioensaio Salmonella/microssoma (Kado). A avaliação de potenciais danos genotóxicos estabelecidos foi realizada através do Teste de Micronúcleo, que apresentou valores significativos na amostra V. Resultados negativos foram observados para as cepas de Salmonella typhimurium TA97, TA98, TA100 e TA102, tanto na ausência quanto na presença de fração de metabolização exógena de mamíferos (S9 mix 4%) em todas as análises. A quantificação por cromatografia gasosa com detecção por espectrometria de massas dos 16 HPA prioritários em termos de conservação ambiental apresentou valores baixos nas duas primeiras amostragens (PI e V) e nulos nas coletas seguintes (O e PII), nos mesmos pontos. De acordo com os valores utilizados nos Estados Unidos e Canadá como referência, os detectados por nós não são considerados como toxicantes ambientais positivos, com exceção do Benzo(a)pireno, que em M1V apresentou valores um pouco acima do limite a partir do qual já podem ser observados pequenos efeitos na biota. A análise dos metais (Cd, Cr, Cu, Fe, Mn, Ni, Pb e Zn) por Espectrometria de Absorção Atômica inicialmente realizada com a água intersticial foi melhor interpretada a partir da matriz sedimento. Este estudo contribuirá com a implementação de indicadores para valores de referência em mangue. / Environmental studies have shown that substances generated by anthropogenic processes can cause adverse effects by interfering with the ecosystem natural balance. Mangroves perform essential functions in biological cycles and are Permanent Protection Area in Brazil. Unfortunately, they are being degraded above its support limit, leading to a reduction of the remaining areas in the world. This paper presents the results of mutagenicity and genotoxicity observed in four samples (PI, V, O, and PII) between years 2009 and 2010 linked to metals and polycyclic aromatic hydrocarbons (PAHs) in mangrove sediment in order to characterize reference values. The genotoxicity tests were made from the hemocytes Goniopsis cruentata collected in a potential unpolluted ecosystem of Brazil, located in the southern Rio de Janeiro (Parati/RJ), called "Saco do Mamanguá. Collection, storage, manipulation of sediment and biological material from five sampling sites (M1-M5) were processed according to U.S. standards recognized. The identification of the chemicals was performed with extracts of sediments and bioassay used in the Salmonella / microsome (Kado). The assessment of potential genotoxic damage were done down through the Micronucleus Test, and showed significant values in the sample V. Negative results were observed for the Salmonella typhimurium strains TA97, TA98, TA100 and TA102, both in the absence or presence of exogenous fraction of mammalian metabolism (S9 mix 4%) for all analysis. Quantification by Gas chromatography-mass spectrometry of the 16 priority PAHs in terms of environmental conservation, presented low values in the first two samples (V and PI) and null value in the following collections (and the IIP), in the same points. According to United States and Canada references, we detected chemicals not regarded as a positive environmental toxicants, with the exception of benzo(a)pyrene, which showed values in M1V just above the threshold at which already small effects can be observed in the biota. The analysis of metals (Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn) by Atomic Absorption Spectrometry initially performed with the pore water was best interpreted from the sediment matrix. This study will contribute to the implementation of indicators to benchmarks in the swamp.

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-12-17Bitstream added on 2014-06-13T18:41:12Z : No. of bitstreams: 1 ambrosio_jb_dr_rcla.pdf: 849914 bytes, checksum: 8eebea30948cfc32353c99fa5c95f4e0 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O Brasil é o maior produtor de cana-de-açúcar no mundo. Dentre os Estados brasileiros, São Paulo representa 60% de todo o açúcar produzido e 70% das exportações. Pela alta produção de cana-de-açúcar no Estado de São Paulo e o consequente consumo elevado de agrotóxicos, este trabalho teve como objetivo investigar os efeitos citotóxicos, genotóxicos e mutagênicos de baixas concentrações de dois agrotóxicos utilizados nesta cultura, o herbicida sulfentrazona e o inseticida imidaclopride, tanto em testes individuais quanto associados entre si. As avaliações foram feitas pelos testes de aberrações cromossômicas (AC) e de micronúcleos (MN) em Allium cepa; teste do MN e ensaio do cometa em células HepG2; e teste de Ames com Salmonella typhimurium. Para melhor avaliar o modo de ação dos agrotóxicos, a técnica de hibridização in situ fluorescente (FISH) com sonda pan-centromérica foi associada ao teste do MN realizado com células HepG2. Os testes com A. cepa indicaram genotoxicidade para todas as concentrações de imidaclopride, após um período de recuperação de 48 horas. A maior indução de pontes e aderências cromossômicas sugeriu uma ação clastogênica para o inseticida. Esta hipótese foi confirmada nos testes com HepG2, pela técnica de FISH, uma vez que a maioria dos MN (76,6%) não apresentou sinal fluorescente, indicando presença de fragmentos cromossômicos. Para o herbicida sulfentrazona, foram observadas AC para todas as concentrações, considerando os diferentes tempos de tratamento. A indução de MN foi observada para a menor concentração, para todos os tempos de tratamento, sugerindo que os danos induzidos por essa concentração persistem ao longo do ciclo celular. A maior concentração testada do herbicida causou uma diminuição no índice mitótico... / Brazil is the largest producer of sugarcane in the world. Among the Brazilian states, São Paulo represents 60% of all sugar produced and 70% of exports. Due to the high production of sugarcane in São Paulo state and the great pesticide consumption, this work investigated the cytotoxic, genotoxic and mutagenic effects of low concentrations of two pesticides utilized in sugarcane culture: the herbicide sulfentrazone and the insecticide imidacloprid, both individually and combined. The evaluations were made by chromosome aberrations (CA) and micronucleus (MN) tests in Allium cepa, MN and comet assay in HepG2 cells and Ames test with Salmonella typhimurium. Associated to the MN test in HepG2 cells, the fluorescent in situ hybridization (FISH) technique with pan-centromeric probe was applied in order to better evaluate the mode of action of the agrochemicals. The tests with A. cepa indicated genotoxicity for all concentrations of imidacloprid, after a recovery period of 48 hours. The higher induction of bridges and chromosome stickiness suggests a clastogenic action for the insecticide. This hypothesis was confirmed in the tests with HepG2, by FISH, since most of the MN (76.6%) did not present fluorescent signal, indicating presence of chromosomal fragments. For the herbicide sulfentrazone, CA were observed in all concentrations, in the different treatment times. The induction of MN was observed for the lowest concentration for all the treatment times, suggesting that the damages induced by this concentration persist throughout the cell cycles. The highest concentration tested of the herbicide caused a decrease in the mitotic index of the cells after 72 hours recovery period. This effect may be associated with the high amount of induced chromosome stickiness. The mixture of pesticides induced cytotoxicity, genotoxicity... (Complete abstract click electronic access below)