Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-06T17:04:41Z No. of bitstreams: 2 Dissertação - Renata Vieira Bueno - 2013.pdf: 10302942 bytes, checksum: c2dc51ba669f5ba3c0e30c60904d357d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-09T15:54:05Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Renata Vieira Bueno - 2013.pdf: 10302942 bytes, checksum: c2dc51ba669f5ba3c0e30c60904d357d (MD5) / Made available in DSpace on 2014-10-09T15:54:05Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Renata Vieira Bueno - 2013.pdf: 10302942 bytes, checksum: c2dc51ba669f5ba3c0e30c60904d357d (MD5) Previous issue date: 2013-01-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Tuberculosis (TB) is a chronic infectious and contagious disease with high epidemiological rates. The rise of multi- and extensively drug-resistant strains as well as the side effects and the long term treatment become urgent the development of novel therapy options. The enzyme thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt) is essential to DNA synthesis and cell replication. Moreover, this enzyme has unique structural characteristics among TMPKs family, emerging as a potential target to rational design of novel anti-TB agents. The present work had as objective the application of Computer Aided Drug-Design (CADD) strategies, using a set of 109 thymidine analogues inhibitors of TMPKmt selected from the literature, aiming to elucidate the structural features relevant to the biological activity of this set of compounds and generate models able to predict the activity of untested compounds. Methodologies of 2D-QSAR (HQSAR), 3-D-QSAR (CoMFA and CoMSIA), QM/MM docking and bioisosteric fragment replacement were performed for proposing new TMPKmt inhibitors. The final models of HQSAR, CoMFA and CoMSIA exhibit good internal and external consistency, presenting good correlation ability and prediction of biological activity. The HQSAR contribution maps and the contour maps of CoMFA and CoMSIA provided important information about structural features related to affinity, such as the favorable presence of hydrophobic and less bulky substituents on thymine ring and more bulky, electronegative, hydrophilic and hydrogen acceptors on sulfone of naphtosultam ring. Gathering the information provided, it was planned nine new compounds as potential TMPKmt inhibitors, which showed optimized affinity and physicochemical properties. / A tuberculose (TB) é uma doença infecto-contagiosa crônica com altas taxas epidemiológicas. O surgimento de cepas multi- e extensivamente resistentes aos fármacos utilizados bem como os efeitos adversos e a longa duração do tratamento tornam urgente o desenvolvimento de novas opções terapêuticas. A enzima timidina monofosfato quinase de Mycobacterium tuberculosis (TMPKmt) é essencial para a síntese de DNA e replicação celular. Além disso, esta enzima possui características estruturais únicas na família de TMPKs, sendo um alvo potencial para o planejamento racional de novos agentes anti-TB. O presente trabalho objetivou a aplicação de estratégias de planejamento de fármacos auxiliado por computador (CADD), utilizando um conjunto de 109 análogos de timidina inibidores de TMPKmt selecionados da literatura, buscando-se elucidar os requisitos estruturais relevantes à atividade biológica dessa classe de compostos e gerar modelos capazes de prever a atividade de compostos ainda não testados. Utilizou-se metodologias de QSAR-2D (HQSAR), QSAR-3D (CoMFA e CoMSIA), docking QM/MM e substituição bioisostérica de fragmentos para a proposição de novos inibidores da TMPKmt. Os modelos finais de HQSAR, CoMFA e CoMSIA possuem elevada consistência interna e externa, apresentando bom poder de correlação e predição da atividade biológica. Os mapas de contribuição de HQSAR e os mapas de contorno de CoMFA e CoMSIA forneceram informações importantes sobre características estruturais relacionadas à afinidade, tais como a presença de substituintes hidrofóbicos e pouco volumosos no anel timina favorecerem a atividade, assim como a presença de grupos volumosos e com alta densidade eletrônica, hidrofílicos e aceptores de ligação de hidrogênio no grupo sulfona do anel naftosutâmico. Utilizando-se as informações obtidas, planejou-se nove novos compostos como possíveis inibidores de TMPKmt, que apresentaram afinidade e propriedades físico-químicas otimizadas. Estudos de docking evidenciaram que os dois hits mais potentes apresentam interações no sítio ativo da TMPKmt capazes de desestabilizar o processo catalítico entre a enzima e o substrato natural, indicando que os compostos propostos são potenciais inibidores da TMPKmt.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-11-08T17:21:55Z No. of bitstreams: 1 2016 - Carolina Gurski Olanda.pdf: 2816027 bytes, checksum: 2bb43929b846b2fad139c3570efabf00 (MD5) / Made available in DSpace on 2017-11-08T17:21:55Z (GMT). No. of bitstreams: 1 2016 - Carolina Gurski Olanda.pdf: 2816027 bytes, checksum: 2bb43929b846b2fad139c3570efabf00 (MD5) Previous issue date: 2016-09-30 / CAPES / Dengue is an infectious disease caused by an arbovirus which is transmitted by the mosquito Aedes aegypti. In Brazil, due to the rainy season in the summer, it is common the occurrence of epidemics. In recent years, the number of cases in the country is growing at an alarming rate, increasing not only the number of records, but also the number of deaths. Until this date, there is no antiviral treatment that is effective, only measures for the sweetening of symptoms. The viral enzyme RNA-dependent RNA polymerase (NS5) plays a role of great importance for viral replication. This project aims to rational planning of C-nucleosides 1,2,3 triazole derivatives of ribavirin (1 - [(2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) dioxolan-2- yl] -1H-1,2,4-triazole-3-carboxamide), a potent inhibitor of the replication of the flavivirus, proposed inhibitors. To achieve this, was used molecular docking methods and semi-empirical calculations for the study of interactions between the derivatives proposed by our group and the two enzymes. The realization of this study will serve as basis for the synthesis of most promising products, which in the future may lead to the creation of a prototype of an antiviral drug that contributes to the treatment of all forms of dengue, as well as in preventing it. / A dengue ? uma doen?a infecciosa causada por um arbov?rus, que ? transmitido pelo mosquito Aedes aegypti. No Brasil, devido aos per?odos chuvosos no ver?o, ? comum a ocorr?ncia de epidemias. Nos ?ltimos anos, o n?mero de casos da doen?a no pa?s vem crescendo de forma alarmante, aumentando n?o s? a quantidade de registros, mas tamb?m o n?mero de ?bitos. At? o momento, n?o h? tratamento antiviral que seja efetivo, apenas medidas para a ameniza??o dos sintomas. A enzima viral RNA polimerase dependente de RNA (NS5) exerce papel de grande import?ncia para a replica??o viral. Este projeto tem como objetivo o planejamento racional de C-nucleos?deos 1,2,3 triaz?licos derivados da ribavirina (1-[(2R,3R,4S,5R)-3,4-dihidroxi- 5-(hidroximetil)oxolan-2-il]-1H-1,2,4-triazol-3-carboxamida), um potente inibidor da replica??o dos flaviv?rus, inibidores propostos das enzimas NS5. Para se alcan?ar este objetivo, ser?o usados os m?todos de docking molecular e de c?lculos semi-emp?ricos para o estudo das intera??es entre os derivados propostos por nosso grupo e as duas enzimas. A realiza??o desse estudo servir? como base para a s?ntese dos derivados mais promissores, que futuramente poder?o levar ? cria??o de um prot?tipo de um f?rmaco antiviral, que contribua para o tratamento de todas as formas da dengue, assim como na preven??o da mesma.

O mercado da indústria petrolífera tem aumentado consideravelmente seus investimentos em projetos, dada a importância desse setor para o desenvolvimento de um país. O elevado número de projetos faz com que seus objetivos sejam cada vez mais difíceis de serem alcançados, seja pela competição de recursos, pela alta complexidade ou mesmo pela interferência entre eles. A área de logística, um dos elos da cadeia produtiva desse mercado especialmente a de transporte marítimo, possui projetos caracterizados pela manutenção preventiva dos navios existentes, denominada de docagem, e pela construção de novos navios. Verifica-se, nos projetos de docagem, uma diferença entre os objetivos planejados e os alcançados, indicando a necessidade de melhorias e, por se tratar de projetos, podem-se utilizar os conceitos e ferramentas de gestão de projetos e de gestão de riscos para essa finalidade. Este trabalho apresenta um estudo dos eventos de risco do projeto de docagem de navios petroleiros, indicando os de maior relevância, suas causas e conseqüências, e a metodologia, ferramentas e técnicas utilizadas. / The oil industry has substantially increased its investments on projects given the importance of this sector to the development of a country. The large quantity of projects has turning their objectives more and more hard to be achieved either by the competition for resources or by their high complexity or even by interference among the projects themselves. At one of the links of the production chain of this industry, the logistic area, more specifically the maritime transportation, the projects are related to the preventive maintenance of the existing ships, which is called docking, and to the building of new ships. A difference is noticed on the docking projects regarding the planned objectives and those achieved which indicates the necessity of improvements. As dockings are considered projects, the concepts and tools of project and risk management can be used to fulfill such a necessity. This paper presents a study of the risk events of the docking projects for oil tankers identifying the most important ones, shows their causes and consequences and the used methodology, tools and techniques.

Desde a identificação do vírus da imunodeficiência humana (HIV, do inglês Human Immunodeficiency Virus) como agente causador da Síndrome da Imunodeficiência Adquirida (AIDS ? do inglês Acquired Immunodeficiency Syndrome), a busca para tratamentos seguros e eficazes contra o HIV transformou-se no principal foco para a descoberta de uma nova droga em todo o mundo. A AIDS aparece como um dos principais problemas de saúde pública para as próximas décadas, onde será o maior determinante de mortalidade na faixa etária entre 20 e 50 anos em praticamente todos os países do mundo. Tendo como objetivo relacionar a atividade de compostos biflavonóides anti-HIV-1 com algumas de suas propriedades moleculares, serão utilizados métodos de Mecânica Molecular e Química Quântica. O método de cálculo semi-empírico AM1 foi empregado para calcular um conjunto de propriedades moleculares dos 14 compostos biflavonóides com atividade anti-HIV-1. A seguir utilizar-se-á métodos estatísticos com a finalidade de separar os 14 compostos em duas classes, ativos e não ativos, de forma que se relacione qual as propriedades, dentre as calculadas, são responsáveis pela atividade dos compostos biflavonóides estudados. As técnicas estatísticas utilizadas foram a Análise de Componentes Principais (PCA: Principal Components Analysis), Análise Hierárquica de Agrupamentos (HCA: Hierarquical Clusters Analysis) e Análise de Discriminates por Passos (SDA: Stepwise Discriminant Analysis). Os estudos com PCA, HCA, e SDA mostraram que as variáveis HOMO (Highest Occupied Molecular Orbital - Orbital Molecular Ocupado de Maior Energia), LUMO (Lowest Unoccupied Molecular Orbital ? Orbital Molecular Desocupado de Menor Energia), e Área superficial são responsáveis pela separação dos compostos com alta e baixa atividade anti-HIV-1. O comportamento destas três propriedades pode ser útil na tentativa de se obter outros compostos biflavonóides com elevada atividade inibidora anti-HIV-1. / A set of 25 quinone compounds with anti-trypanocidal activity was studied by using the Density Functional Theory (DFT) method in order to calculate electronic atomic and molecular properties to be correlated with the biological activity. The chemometric methods Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA), Stepwise Discriminant Analysis (SDA), Kth nearest neighbor (KNN) and Soft Independent Modeling of Class Analogy (SIMCA) were used to obtain possible relationships between the calculated descriptors and the biological activity studied and predict the anti-trypanocidal activity of new quinone compounds from a test set. Four descriptors were responsible for the separation between the active and inactive compounds: T5 (torsion angle), HOMO-1 (energy of the first molecular orbital below HOMO), QTS1 (sum of absolute values of the atomic charges) and VOLS2 (volume of the substituent at region B). These descriptors give information on the kind of interaction that occurs between the compounds and the biological receptor. The prediction study was done with a set of three new quinone compounds by using the PCA, HCA, SDA, KNN and SIMCA. Beside the five chemometric methods, the neural network method was used by employing the backpropagation algorithm. The four variables (T5, QTS1, VOLS2 and HOMO-1) were employed to validate the models constructed previously. The architecture of networks consisting of four neurons at input layers, ten neurons at intermediary layers and two neurons at output layers was adopted to observe the root mean square error between the true and desired output over the entire training set. The percentage of correct classification was 87.5%, and only one compound was predicted wrong in the test set, which indicates that the model is robust and could be able to make predictions. The docking studies were carried out with two different programs in the approach of ligands: the Autodock and FlexX. The docking results on trypanothione reductase enzyme showed that all studied compounds stay at second hydrophobic pocket in the outer region of the active site called the Z-site. The residues that could be specifically involved in the binding of ligands are Lys62, Thr66, Thr397, Thr463, Leu399, Ser464, Glu466 and Glu467, where the residues Thr66, Thr463 and Leu399 are conserved in different trypanothiones and could be used for the development of selective inhibitors against to the parasite enzyme.

The emergence and proliferation of Gram-negative bacteria expressing β-lactamases is a significant threat to human health. β-Lactamases are enzymes that degrade the β-lactam antibiotics (e.g., penicillins, cephalosporins, monobactams, and carbapenems) that we use to treat a diverse range of bacterial infections. Specifically, β-lactamases catalyze a hydrolysis reaction where the β-lactam ring common to all β-lactam antibiotics and responsible for their antibacterial activity, is opened, leaving an inactive drug. There are two groups of β-lactamases: serine enzymes that use an active site serine residue for β-lactam hydrolysis and metalloenzymes that use either one or two zinc ions for catalysis. Serine enzymes are divided into three classes (A, C, and D), while there is only one class of metalloenzymes, class B. Clavulanic acid, sulbactam, and tazobactam are β-lactam-based BLIs that demonstrate activity against class A and C β-lactamases; however, they have no activity against the class A KPC and MBLs, NDM and VIM. Avibactam and vaborbactam are novel BLIs approved in the last two years that have activity against serine carbapenemases (e.g., KPC), but do not inhibit MBLs. The overall goals of this project were to use X-ray crystallography to study the catalytic mechanism of serine β-lactamases with β-lactam antibiotics and to understand the mechanisms behind the broad-spectrum inhibition of class A β-lactamases by avibactam and vaborbactam. This project also set out to find novel inhibitors using molecular docking and FBDD that would simultaneously inactivate serine β-lactamases and MBLs commonly expressed in Gram-negative pathogenic bacteria. The first project involved examining the structural basis for the class A KPC-2 β-lactamase broad-spectrum of activity that includes cephalosporins and carbapenems. Three crystal structures were solved of KPC-2: (1) an apo-structure at 1.15 Å; (2) a complex structure with the hydrolyzed cephalosporin, cefotaxime at 1.45 Å; and (3) a complex structure with the hydrolyzed penem, faropenem at 1.40 Å. These complex structures show how alternative conformations of Ser70 and Lys73 play a role in the product release step. The large and shallow active site of KPC-2 can accommodate a wide variety of β-lactams, including the bulky oxyimino side chain of cefotaxime and also permits the rotation of faropenem’s 6-alpha-1R-hydroxyethyl group to promote carbapenem hydrolysis. Lastly, the complex structures highlight that the catalytic versatility of KPC-2 may expose a potential opportunity for drug discovery. The second project focused on understanding the stability of the BLI, avibactam, against hydrolysis by serine β-lactamases. A 0.83 Å crystal structure of CTX-M-14 bound by avibactam revealed that binding of the inhibitor impedes a critical proton transfer between Glu166 and Lys73. This results in a neutral Glu166 and neutral Lys73. A neutral Glu166 is unable to serve as a general base to activate the catalytic water for the hydrolysis reaction. Overall, this structure suggests that avibactam can influence the protonation state of catalytic residues. The third project centered on vaborbactam, a cyclic boronic acid inhibitor of class A and C β-lactamases, including the serine class A carbapenemase KPC-2. To characterize vaborbactam inhibition, binding kinetic experiments, MIC assays, and mutagenesis studies were performed. A crystal structure of the inhibitor bound to KPC-2 was solved to 1.25 Å. These data revealed that vaborbactam achieves nanomolar potency against KPC-2 due to its covalent and extensive non-covalent interactions with conserved active site residues. Also, a slow off-rate and long drug-target residence time of vaborbactam with KPC-2 strongly correlates with in vitro and in vivo activity. The final project focused on discovering dual action inhibitors targeting serine carbapenemases and MBLs. Performing molecular docking against KPC-2 led to the identification of a compound with a phosphonate-based scaffold. Testing this compound using a nitrocefin assay confirmed that it had micromolar potency against KPC-2. SAR studies were performed on this scaffold, which led to a nanomolar inhibitor against KPC-2. Crystal structures of the inhibitors complexed with KPC-2 revealed interactions with active site residues such as Trp105, Ser130, Thr235, and Thr237, which are all important in ligand binding and catalysis. Interestingly, the phosphonate inhibitors that displayed activity against KPC-2, also displayed activity against the MBLs NDM-1 and VIM-2. Crystal structures of the inhibitors complexed with NDM-1 and VIM-2 showed that the phosphonate group displaces a catalytic hydroxide ion located between the two zinc ions in the active site. Additionally, the compounds form extensive hydrophobic interactions that contribute to their activity against NDM-1 and VIM-2. MIC assays were performed on select inhibitors against clinical isolates of Gram-negative bacteria expressing KPC-2, NDM-1, and VIM-2. One phosphonate inhibitor was able to reduce the MIC of the carbapenem, imipenem 64-fold against a K. pneumoniae strain producing KPC-2. The same phosphonate inhibitor also reduced the MIC of imipenem 4-fold against an E. coli strain producing NDM-1. Unfortunately, no cell-based activity was observed for any of the phosphonate inhibitors when tested against a P. aeruginosa strain producing VIM-2. Ultimately, this project demonstrated the feasibility of developing cross-class BLIs using molecular docking, FBDD, and SAR studies.

Die Alzheimer'sche Erkrankung wird derzeit durch die Gabe von Acetylcholinesterase- Inhibitoren (AChEI) symptomatisch behandelt. Durch die AChE-Hemmung steht mehr Acetylcholin (ACh) für die Neurotransmission zur Verfügung. Bei Progression der Erkran-kung nimmt der Anteil an AChE drastisch ab, so dass die Enzymisoform Butyrylcholin- esterase (BChE) die Hydrolyse des Neurotransmitters ACh übernimmt. In späten Phasen der Alzheimer'schen Erkrankung ist daher der Einsatz selektiver BChE-Hemmer erfolgsver- sprechend. Inhibitoren können verschiedene Bindestellen in der Cholinesterase-Bindetasche adressie-ren und dadurch in dieser stabilisiert werden. Zu den Bindestellen zählen die katalytisch aktive Stelle (CAS) am Ende eines 20 Å langen Bindetaschentunnels, die Oxyanion-Vertie-fung, die Cholinbindestelle, sowie die periphere anionische Bindestelle (PAS), welche am Bindetascheneingang lokalisiert ist. In der vorliegenden Arbeit wurden durch in silico Dockingstudien gezielt Protein-Ligand- Interaktionen untersucht, um Strukturmerkmale hochaffiner Inhibitoren von Cholinesterasen zu identifizieren. Damit soll die zukünftige Entwicklung von Cholinesteraseinhibitoren hinsichtlich der Affinität zum Enzym verbessert werden. Ferner dienten synthetische Untersuchungen eines Naturstoffes dazu, Chinazoliniumverbindungen als Leitstruktur für die Inhibition der Cholinesterasen zu etablieren. Für hochaffine tri- und tetrazyklische aminsubstituierte AChE-selektive Chinazolin- und Chinazolinoninhibitoren sollte die bevorzugte Orientierung der Liganden in der Bindetasche ermittelt werden. Hierfür ist die Lokalisation des Aminsubstituenten in der CAS (invertierter Bindemodus) oder die dortige Bindung des Chinazolin-/Chinazolinongerüstes (klassischer Bindemodus) denkbar. Anhand eines präferierten einheitlichen Bindemodus sollten die Struktur-Aktivitäts-Beziehungen erklärt werden. Dockingstudien zeigten die klare Präferenz für den invertierten Bindemodus, bei dem der Aminsubstituent in der Nähe der CAS platziert wird. Ein strukturelles Merkmal für hochaffine Inhibitoren ist ein unter Assaybedingungen protoniertes Amin, welches eine Kation-π-Wechselwirkung zu dem Indolringsystem des Tryptophans der Cholinbindestelle eingehen kann. Für das Ligandengrundgerüst wurde lediglich für tetrazyklische Verbindungen eine π-π-Interaktion mit der peripheren Bindestelle (PAS) am Bindetascheneingang identifiziert. Der Datensatz umfasste auch chirale Chinazolinon- und Chinazolinderivate mit hydrierter C=N-Doppelbindung, die eine schwächere Affinität zu AChE zeigten. Diese ist vermutlich auf das nicht-planare Ligandengrundgerüst zurückzuführen, da vor allem für tetrazyklische chi-rale Verbindungen die Stabilisierung des Ligandengrundgerüstes durch π-π-Interaktionen am Bindetascheneingang aufgrund der Sterik entweder gar nicht, oder nur für ein Enantio-mer möglich ist. Aufgrund der nanomolaren Affinität der achiralen Chinazolin- und Chinazolinonverbindungen wurden weitere gerichtete Wechselwirkungen in der Bindetasche erwartet. Derartige Wechselwirkungen konnten in Form von Wasserstoffbrücken durch die Verwendung von sieben ausgewählten strukturellen Wassermolekülen im Docking identifiziert werden. Durch diese Wassermoleküle werden Wasserstoffbrücken vom Ligandengrundgerüst zum Protein vermittelt. Diese Wechselwirkungen scheinen essentiell für die Stabilisierung hoch-affiner Chinazolin- und Chinazolinoninhibitoren in der AChE-Bindetasche zu sein. Zwei photochrome Bis-Tacrin-Konstitutionsisomere (Ring-geöffnete und Ring-geschlossene Form) inhibieren die AChE und zeigen einen unterschiedlichen Effekt in der Hemmung der Amyloid-β Fibrillenbildung. Die Fibrillenbildung wird durch eine unbesetzte periphere Bindestelle (PAS) am Eingang der AChE-Bindetasche katalysiert, weshalb eine unterschiedliche Interaktion der Liganden mit ebendieser Bindestelle vermutet wird. Dockingstudien lieferten für beide Konstitutionsisomere einen ähnlichen Bindemodus, der vor dem Hintergrund der ähnlichen IC50-Werte von 4.3 und 1.8 nM für die Ring-geöffnete und Ring-geschlossene Form plausibel erscheint. Durch die Auswahl einer geeigneten Röntgenstruktur wurden Dockinglösungen erhalten, bei denen ein Tacrinsubstituent in der PAS bindet und dort π-π-Interaktionen mit einem Tryptophan und einem Tyrosin eingeht. Eine solche Lage des PAS-bindenden Tacrinsubstituenten ist energetisch bevorzugt und drückt sich durch bessere Scores gegenüber Dockinglösungen, bei denen dieser auf der Protein-oberfläche lokalisiert ist, aus. Der andere Tacrinsubstituent bindet in der CAS wie dies von bereits kristallisierten Tacrinderivaten bekannt ist. Mittels molekulardynamischer Simulati-onen wurde die Stabilität der Protein-Dockinglösungs-Komplexe beider Konstitutionsiso-mere verglichen. Dabei wurde die bessere Stabilisierung des CAS-bindenden Tacrinsubsti-tuenten für die Ring-geöffnete Form des Liganden ermittelt. Ferner zeigt sich für die Ring-geöffnete Inhibitorform während der Simulation der Einstrom von sechs Wassermolekülen in einen Hohlraum der PAS. Dies hat zur Folge, dass der PAS-bindende Tacrinsubstituent während der Hälfte der Simulationszeit durch Wasserstoffbrücken in der PAS stabilisiert wird. Ein Wasserstoffbrückennetzwerk diesen Ausmaßes kann für die Ring-geschlossene Inhibitorform nicht ermittelt werden. Die bessere Hemmung der Amyloid-β Fibrillenbildung der Ring-geöffneten Inhibitorform wird daher auf die bessere Stabilisierung des Liganden durch Wasserstoffbrücken in der AChE-Bindetasche zurückgeführt. Für carbamatsubstituierte Tetrahydrochinazolinverbindungen sollten die bevorzugten Interaktionen in der BChE-Bindetasche ermittelt werden. Die Carbamatverbindungen sind pseudo-irreversible Inhibitoren und zeigen eine zeitabhängige Hemmung mit diversen Interaktionszuständen zwischen Protein und Ligand. Darüber hinaus stellen Dockingstudien in der BChE bislang eine Herausforderung dar, da es derzeit nur zwei Röntgenstrukturen dieses Enzyms mit reversiblen Liganden gibt, weshalb kaum Studien zur Identifikation einer geeigneten Bewertungsfunktion durchgeführt werden können. Im Docking wurde sich für die Analyse des reversiblen Anlagerungskomplexes entschieden, da das Docking des tetraedrischen Übergangszustandes energetisch entartete Dockinglösungen lieferte. Eine weitere Herausforderung stellte die Größe der BChE-Bindetasche dar, die auch im reversiblen Docking entartete Dockinglösungen lieferte. Aufgrund einer ähnlichen Übertragungsrate aller getesteten Inhibitoren wurde eine konservierte Lage des Carbamates in der Bindetasche angenommen. Deshalb wurde eine repräsentative Dockinglösung einer Referenzverbindung als Ausgangspose für einen Modelling-Ansatz gewählt, die hinsichtlich der Interaktionen in der Bindetasche ausgewählt wurde. Diese Interaktionen sind: 1) Eine Wasserstoffbrückendistanz zwischen der Carbamat-Carbonylgruppe und der Oxyanion-Vertiefung sowie 2) eine Distanz, die den nucleophilen Angriff des Serins auf den Carbamatkohlenstoff erlaubt. Im Modelling-Ansatz wurde die repräsentative Bindepose dazu verwendet die entsprechenden Inhibitoren in der Bindetasche aufzubauen. Die bevorzugte Position der N-Methylgruppe wurde für beide Enantiomere über die berechneten Spannungsenergien der Bindeposen abgeschätzt. Für die S-Enantiomere ergab sich die präferierte Bindung mit quasi-„axialer“ Methlygruppe und für die R-Enantiomere mit quasi-„äquatorialer“ Stellung dieser. Die Carbamatstrukturen liegen somit mit der Heptylkette in der Acyltasche und die Ligandengrundgerüste werden in einer Seitentasche der BChE-Bindetasche platziert, in der hydrophobe Wechselwirkungen dominieren. Zusätzlich zu den hochaffinen Chinazolinonverbindungen sollten artverwandte Chinazolini-umverbindungen als Leitstruktur für Cholinesteraseinhibitoren untersucht werden. Zunächst erfolgten Studien zur chemischen Reaktivität und Stabilität des Naturstoffes Dehydroevodiamin (DHED) sowie seines Benz-Derivates (Benz-DHED). Insbesondere Benz-DHED war unter den bisher verwendeten und in der Literatur beschriebenen Synthesemethoden instabil. Die Untersuchungen erforderten daher zunächst die Einführung einer geeigneten Syntheseroute, in diesem Fall die Oxidation mit KMnO4, einhergehend mit der Verbesserung der Ausbeute und ohne Nebenproduktbildung. Für die zukünftige Synthese von Derivaten wurde die Verwendung einer geeigneten Lewis-Säure-labilen Schutzgruppe herausgearbeitet. Die untersuchten Chinazoliniumverbindungen zeigen die Eigenschaft, dass sie in Abhängigkeit der Reaktionsbedingungen in zwei Formen (Ring-geöffnet und Ring-geschlossen = Chi-nazoliniumsalz) isoliert werden können. Mittels UV/Vis-Untersuchungen wurde das Gleich-gewicht dieser Spezies aufgeklärt und in wässrigen alkalischen Lösungen die Anreicherung einer dritten, bislang nicht in diesem Zusammenhang beschriebenen, Spezies beobachtet. Als biologisch aktive Spezies konnte die Chinazoliniumform identifiziert werden. In Dockingstudien der Chinazoliniumform von Benz-DHED, nach dem für Carbamatverbindungen entwickelten Modelling-Ansatz, konnte auch hierfür die Stabilisierung der Docking- lösung über eine Wasserstoffbrücke in der BChE-Bindetasche zu einem strukturellen Wassermolekül identifiziert werden. Dies verdeutlicht erneut, dass die Berücksichtigung von Wassermolekülen in Dockingstudien dazu dienen kann zusätzliche Protein-Ligand-Interaktionen festzustellen. Auf Grundlage der Forschung zu Chinazoliniumverbindungen kann die zukünftige Inhibitorentwicklung von Strukturen basierend auf dieser Substanzklasse erfolgen. Die durchgeführten synthetischen und theoretischen Studien liefern wichtige Beiträge zum Verständnis der Wechselwirkungen zwischen Inhibitoren und Cholinesterasen, die in der zukünftigen Inhibitorentwicklung Anwendung finden können. / Alzheimer's disease is currently treated symptomatically by the administration of acetyl-cholinesterase inhibitors (AChEI). By AChE inhibition more acetylcholine (ACh) is available for neurotransmission. During disease progression the amount of AChE drastically decreases, so that the enzyme butyrylcholinesterase (BChE) takes over hydrolysis of this neurotransmitter. In late stages of Alzheimer's disease, the use of selective BChE in-hibitors may therefore be advantageous. Inhibitors can adress different binding sites in the cholinesterase binding pocket. The bind-ing sites includes the catalytic active site (CAS), which is located at the end of a 20 Å long binding gorge, the oxyanion hole, the choline binding site and the peripheral anionic site (PAS), located at the entrance of the binding gorge. In the present study, specific protein-ligand interactions were investigated by means of in silico docking studies to identify structural features of high-affinity inhibitors of cholinester-ases. Thereby the aim is to improve the affinitiy of newly developed cholinesterase- inhibitors. Furthermore, synthetic studies of a natural product served to establish a new lead compound for cholinesterase inhibitors. For high affinity tri- and tetracyclic amine substituted AChE-selective quinazoline and quinazolinone inhibitors, the aim was to determine the preferred ligand orientation in the binding site. To this end, the localization of the amine substituents in the CAS (inverted binding mode) or the binding of the quinazoline-/quinazolinone moiety there (classical binding mode) is conceivable. The aim was to explain structure-activity-relationships by the identified preferred and consistent binding mode. Docking studies showed a clear preference for the inverted binding mode in which the amine substituent is placed in the vicinity of the CAS. A structural characteristic of high affinity inhibitors is a protonated amine which can form a cation-π-interaction to the tryptophan indole ring system of the choline binding site. Furthermore, a π-π-interaction with the peripheral binding site (PAS) has been identified for the ligand backbone of tetra-cyclic inhibitors. The data set also included chiral quinazolinone- and quinazoline derivatives with hydrogenated C=N-double bond, which showed weaker affinity to AchE compared to non chiral compounds. The weaker affinity is probably due to the non-planar ligand back-bone. Due to steric reasons, especially for tetracyclic chiral compounds, stabilisation of the ligand backbone through π-π interactions is either not possible at all, or possible only for one enantiomeric form. Due to the nanomolar affinity of non chiral quinazoline and quinazolinone compounds, other directional interactions between protein and ligand were expected. Several inter- actions were identified in form of hydrogen bonds through the use of conserved water molecules in docking. These water molecules mediate hydrogen bonds from the ligand backbone to the protein. These interactions seem to be essential for the stabilization of high affinity quinazoline and quinazolinone inhibitors in the AChE binding site. Two photochromic bis-tacrine constitutional isomers (ring-open and ring-closed form) inhibit AChE and show a different effect with regard to the inhibition of amyloid-β fibril formation. Fibril formation is catalyzed by an unoccupied peripheral site (PAS) at the entrance of the AChE-binding pocket, which is why a different interaction of the ligands is expected with this binding site. Docking studies provided a similar binding mode for both constitutional isomers, which appears plausible for the ring-open and ring-closed form in the light of similar IC50-values of 4.3 and 1.8 nM. By selecting a suitable crystal structure, docking solutions were obtained in which one tacrine substituent is placed in the PAS and undergoes π-π-interactions with a tryptophan and a tyrosine. This placement of the PAS-binding tacrine-substituent is energetically favored which is expressed through better scores, compared to docking solutions where the tacrine-substituent is placed on the protein surface. The other tacrine substituent binds in the CAS in a manner which is known from other already crystalized tacrines. Molecular dynamic simulations were subsequently used for stability comparison of the protein-docking solution-complex of both isomers. Here, the better stabilization of the CAS-binding tacrine-substituent for the ring-open form was determined. The complex with the ring-open form shows the influx of six water molecules in a cavity of the PAS at the beginning of the simulation. As a consequence, the PAS-binding tacrine substituent is stabilized by hydrogen bonds to these water molecules during half of the simulation time. A hydrogen bond network of this magnitude was not observed for the ring-closed form of the inhibitor. The better inhibition of amyloid-β fibril formation by the ring-open form may be due to better stabilization of this ligand through hydrogen bonds in the AChE-binding site, which is not observed for the ring-closed form. Preferred interactions could also be identified for carbamate-based quinazoline inhibitors in the BChE-binding site. These carbamate compounds are pseudo-irreversible inhibitors and show a time-dependent inhibition with several interaction possibilities between protein and ligand. Docking studies in the BChE are challenging, as there currently exist only two X-ray structures of this enzyme with reversible ligands. Therefore, it is difficult to identify a suitable scoring function for the data set under investigation. Docking of the tetrahedral transition state delivered energetically degenerate docking solutions wherefore the aim was to investigate the reversible attachment complex in more detail. Thereby the challenge was the size of the BChE-binding pocket, which also supplied degenerate docking solutions in the reversible docking. Due to a similar carbamoylation rate of all the inhibitors tested, a conserved position of the carbamate moiety in the binding pocket was presumed. Therefore, a representative docking solution of a reference compound, which was selected in the binding pocket in terms of interactions, was chosen for a modeling approach. These interactions are 1) A hydrogen bond distance between the carbamate carbonyl and the oxyanion hole and 2) a distance between the carbamate carbon and the serine which allows for a nucleophilic attack and thus the transfer of the carbamate moiety onto the enzyme. For the modeling approach, the representative binding pose was used to construct the appropriate inhibitor structures. The preferred position of the N-methyl group was calculated for both enantiomers via the tension energy of the corresponding binding poses. For S-enantiomers the preferred posi-tion of the methyl group is quasi-"axial" and for R-enantiomers quasi-"equatorial". The conserved binding mode thus is characterized by the heptyl chain being placed in the acyl pocket and the ligand scaffold in a side pocket of the BChE-binding site where hydrophobic interactions dominate. In addition to high affinity quinazoline and quinazolinone inhibitors, it was intended to make quinazolinium compounds accessible as lead compounds for future cholinesterase inhibitors. Synthetic studies initially focused on chemical reactivity and stability of the natural product dehydroevodiamine (DHED) and its benz-derivative (benz-DHED). In particular, benz-DHED was unstable under the synthesis conditions applied so far and described in the literature. The investigations therefore required introduction of a suitable synthetic route, in this case oxidation with KMnO4, accompanied by improvement of yield and no by-product formation. For future syntheses of derivatives, the use of a Lewis-acid-labile protecting group is suggested. Depending on the reaction conditions, the quinazolinium compounds can be isolated in two forms (ring-opened and ring-closed = quinazolinium-salt). By means of UV/Vis-studies, the equilibrium of these two forms was elucidated. In aqueous alkaline solutions the enrichment of a third, hitherto in this context undescribed species, was observed. These studies helped in identification of the quinazolinium-form as biologically active species. Docking studies of this form, using the modeling-approach which was evolved for carbamates, showed a possible stabilization of this compound via a hydrogen bond mediated by a struc-tural water molecule in the BChE-binding site. The role of water here again shows that consideration of water molecules in docking studies might be able to describe preffered binding modes by means of additional protein-ligand-interactions better, compared to docking studies without explicit water molecule consideration. Based on the basic research carried out for quinazolinium compounds, future inhibitor development based on this substance class can be performed. Finally, the herein conducted synthetic and theoretical studies provide important contributions to the understanding of the interaction between inhibitors and cholinesterases, which can be used for future inhibitor development.

This report describes a docking system to allow autonomous battery charging of a mobile robot, and a Web interface that allows long-term unaided use of a sophisticated mobile robot by untrained Web users around the world. The docking system and Web interface are applied to the biologically inspired RatSLAM system as a foundation for testing both its long-term stability and its practicality for real-world applications. While there are existing battery charging and Web interface systems for mobile robots, the developed solution combines the two, resulting in a self-sufficient robot that can recharge its own batteries and stay accessible from the Web. Existing mobile robots on the Internet require manual charging by a human operator, leading to significant periods when the robot is offline. Furthermore, since the robot may be operational for extended periods without powering down, it may perform learning operations that require significantly longer operation than a single battery-recharge cycle would allow. The implemented Web interface makes use of the RatSLAM navigation system. RatSLAM provides the onboard intelligence for the robot to navigate to the user-supplied goal locations (such as “go to location X”), despite long paths or obstacles in the environment. The majority of the existing robot interfaces on the Internet provide direct control of the robot (such as “drive forward”) and therefore the users suffer greatly from the inherent delays of the Internet due to the time lag between performing an action and seeing the feedback. Instead, the robot in this study uses an onboard intelligent navigation system to generate all low-level commands. Due to the minimal input required to give high-level commands to the robot, the system is robust to the long and highly unpredictable delays of Internet communication. Traditional methods of autonomous battery charging for mobile robots have had limited reliability, often due to the mechanical docking system requiring a highly precise connection. Therefore, the mechanical design of the implemented battery charging system improves reliability by allowing for a significantly larger navigation error. In addition, the robot uses a standard vision sensor for both the long-range and short-range stages of navigation to the battery charger, compared to the many systems that require an omnidirectional camera and a high-resolution Laser range finder for this process. The result of this study is a public web interface at "http://ratslam.itee.uq.edu.au/robot.html" (currently offline), where any Web user in the world can watch and control the live mobile robot that is using RatSLAM for navigation, as it drives in its laboratory environment without human assistance.

Les flavonoïdes sont des composés poly-hydroxylés d'origine végétale, connus pour leurs vertus pour la santé. Afin d'obtenir des dérivés plus stables et solubles dans des formulations hydrophobes tout en conservant les activités biologiques des molécules d'origine, une solution consiste à acyler ces composés de manière régiosélective. Ceci peut être accompli en utilisant des lipases comme catalyseurs, en milieu organique. Grand nombre d'études expérimentales sur ces bioprocédés sont disponibles, mais aucune d'entre elles n'apporte d'explication, au niveau moléculaire, de la sélectivité de ces réactions d'acylation. Le but de cette étude est d'appliquer différents outils de simulation moléculaire pour mieux comprendre, au niveau moléculaire, les propriétés de sélectivité de l'acétylation de trois flavonoïdes (quercétine et ses dérivés glycosylés isoquercitrine et rutine), en utilisant les lipases CALB et PCL. D'abord, des simulations de docking ont été appliquées, afin d'obtenir les positions et les orientations les plus probables des flavonoïdes dans la cavité des lipases préalablement acétylées. Ensuite, des simulations de dynamique moléculaire ont été exécutées sur les complexes obtenus par docking, afin d'étudier stabilité structurale des complexes sur une période de temps et notamment la stabilité des interactions enzyme-substrats. Enfin, des simulations basées sur une approche de chimie quantique (DFT) ont été appliquées pour évaluer la réactivité chimique des flavonoïdes dockées dans les complexes. Les premières tendances observées aux cours des simulations ont présenté une bonne corrélation avec les résultats expérimentaux d'acétylation. Globalement, les résultats obtenus ont montré que la sélectivité de ces réactions dépend de l'orientation des substrats (flavonoïde et acétate) dans la cavité catalytique de la lipase, des interactions intermoléculaires stabilisant ces substrats et de la réactivité chimique intrinsèque des groupements OH des flavonoïdes se situant à proximité des résidus catalytiques.

Le docking moléculaire est une tâche complexe, difficile à appréhender pour une personne seule. C'est pourquoi, nous nous proposons d'étudier la distribution cognitive des charges de travail à travers la collaboration. Une plate-forme distribuée de déformation moléculaire interactive a été mise en place afin d'étudier les avantages mais aussi les limites et les contraintes du travail collaboratif étroitement couplé. Cette première étude, basée sur trois expérimentations, a permis de valider l'intérêt d'une approche collaborative pour des tâches complexes à fort couplage. Cependant, elle a mis en évidence des conflits de coordination ainsi que des problématiques liées à la dynamique d'un groupe. Suite à cette première étude, nous avons proposés une nouvelle configuration de travail associée à des métaphores de communication haptiques afin d'améliorer la communication et les interactions entre les différents collaborateurs. Une dernière expérimentation avec des biologistes a permis de montrer l'utilité de la communication haptique pour le travail collaboratif sur des tâches complexes à fort couplage.

Les introns de groupe II forment une classe d'ARN connus avant tout pour leur activité ribozymique, qui leur permet de catalyser leur propre réaction d'épissage. Sous certaines conditions, ces introns peuvent s'exciser des ARN précurseurs dont ils font partie et assurer la ligation des exons qui les bordent sans l'aide d'aucune protéine. Les introns de groupe II sont généralement excisés sous forme d'un lariat, semblable à celui formé par les introns des prémessagers nucléaires, dont l'épissage est assurée par le spliceosome. De telles similarités dans le mécanisme d'épissage suggèrent que les introns de groupe II et les introns des prémessagers nucléaires pourraient avoir un ancêtre évolutif commun.Malgré leurs séquences très diverses, les introns de groupe II peuvent être définis par une structure secondaire commune, hautement conservée. Celle-ci est formée de six domaines (domaine I à domaine VI ; D1-D6), émergeant d'une roue centrale. L'épissage des introns de groupe II comprend deux étapes, et autant de réactions de transestérification, qui produisent les exons liés et l'intron excisé sous forme lariat. Il est généralement admis que la structure du ribozyme subit des changements conformationnels entre les deux étapes de l'épissage et que le domaine VI est un acteur clé dans ce phénomène. Cependant, malgré l'identification d'un certain nombre d'interactions tertiaires entre domaines, ni la RMN, ni les études faisant appel à des modifications chimiques ne sont parvenues à déterminer l'environnement immédiat, au niveau du site actif du ribozyme, de l'adénosine qui sert de point de branchement de la structure en lariat, ainsi que des nucléotides qui entourent cette adénosine au sein du domaine VI. A l'aide d'analyses phylogénétiques et d'une modélisation moléculaire tridimensionnelle, nous avons identifié plusieurs sections du ribozyme susceptibles de constituer le site de fixation du domaine VI au cours de l'étape de branchement. Des mutations ont été introduites dans ces sites de fixation potentiels et la cinétique de réaction des ARN mutants résultants a été déterminée. Afin de démontrer formellement l'interaction du domaine VI avec le site récepteur le plus probable, une molécule de ribozyme dont la réaction de branchement est assurée par l'addition d'oligonucléotides ADN ou ARN qui positionnent correctement le domaine VI vis-à-vis de son partenaire a été construite. En combinant l'information apportée par différentes expériences de ce type, nous avons pu générer un modèle à résolution atomique du complexe formé par le domaine VI, son site de branchement et le reste de l'intron au moment où l'épissage est initié.