Mit der vorliegenden Habilitationsschrift habe ich den Versuch unternommen, die beiden Themenkomplexe meiner bisherigen wissenschaftlichen Tätigkeit als Beispiele für die Rolle von Modellen in der klinischen Forschung zu verwenden. Den Ansto§ dazu gaben Diskrepanzen, die mir in der Auseinandersetzung mit eigenen Ergebnissen und Beobachtungen im Umfeld dieser Themenkomplexe aufgefallen sind: der Rolle kontaminierender Tumorzellen in der Hochdosistherapie maligner Tumoren einerseits und dem Enzym Dipeptidylpeptidase IV (DPP IV) andererseits. Die beobachteten Diskrepanzen sind Ausdruck konkurrierender pathophysiologischer oder therapeutischer Modelle, und die Präferenz eines bestimmten Modells scheint nicht rein rational erklärbar. Welche Faktoren tragen jedoch zur Entscheidung für oder gegen ein bestimmtes Modell bei? Ich möchte den Umgang mit wissenschaftlichen Modellen anhand der genannten Themenkomplexe aus meiner Sicht erörtern. Anschlie§end soll ein Entwurf skizziert werden, in dem die der Entscheidung für oder gegen ein therapeutisches Modell zugrundeliegende Motivationslage besser verständlich wird und die Intentionalität klinischer Forschung auf den Patienten hin berücksichtigt. / In the thesis presented here, I have taken the challenge to use the topics of my scientific work to discuss the role that models appear to exert in clinical science. This decision arose from discrepancies that became evident in the comparative assessment of my own studies in relation with the surrounding scientific context: the role of tumor cells contaminating peripheral blood or progenitor cell harvests as part of a high-dose chemotherapy regimen on the one hand, and the enzyme dipeptidyl peptidase IV (DPP IV) on the other. The observed discrepancies appear to result from competing pathophysiological or therapeutic models, and the preference or rejection of one model apparently cannot be explained solely by rational factors. I will discuss the application of models in the context of the topics which my scientific work has been focusing on, and I will develop a draft proposal which will render the individual motivational status underlying the decision in favor of or against a distinct model easier to understand, with attention to the intentionality of clinical research towards the patient.

Der Austausch von genetischem Material über horizontalen Gentransfer, stellt einen wichtigen Mechanismus in der bakteriellen Evolution dar. Legionella pneumophila Stämme codieren für verschiedene Typ IV Sekretionssysteme (T4SS) und integrative konjugative Elemente, die zur genomischen Variabilität der intrazellulären Erreger beitragen. L. pneumophila Corby codiert auf der genomischen Insel Trb-1 für ein funktionelles Konjugations- und T4ASS. Trb-1 ist innerhalb des tRNAPro Gens integriert und kann in einer chromosomalen oder zirkulären episomalen Form existieren. Zusätzlich zu den trb/tra Genen sind auf der Insel eine Integrase (int-1) und die Gene lvrRABC der Legionella vir Region (lvr) lokalisiert. Durch die Deletion von int-1 konnte gezeigt werden, dass die Exzision von Trb-1 unter Beteiligung der Integrase erfolgt. Zudem wurde in dieser Arbeit zum ersten Mal demonstriert, dass die lvr-Region, vor allem der putative Phagen-Repressor LvrR an der Regulation der Exzision von Trb-1 beteiligt ist. Die Konjugation von Trb-1 in L. oakridgensis, hatte keinen Effekt auf die in vivo Fitness der Transkonjuganten in humanen Makrophagen. Die genomischen Inseln LpcGI-1 und LpcGI-2 codieren für ein neues putatives GI-T4SS. Für LpcGI-2 konnte erstmals gezeigt werden, dass das T4SS funktionell ist und die Konjugation der genomischen Insel in einen anderen L. pneumophila Stamm vermitteln kann. LpcGI-2 kann anschließend ortsspezifisch in das Genom der Transkonjuganten integriert werden. LpcGI-1 und LpcGI-2 werden vom tRNAThr bzw. tRNAMet Gen flankiert und können in verschiedenen chromosomalen und zirkulären, episomalen Formen existieren. Die Exzision von LpcGI-2 erfolgt ähnlich zu Trb-1, in Abhängigkeit einer ortsspezifischen Integrase. Im Genom von Lp Corby wurden zwei weitere genomische Inseln (LpcGI-Asn und LpcGI-Phe) identifiziert. In silico Analysen zeigten zudem, dass genomische Inseln mit einer Ähnlichkeit zu Trb-1, LpcGI-2 bzw. LpcGI-1 im Genus Legionella verbreitet sind. / Exchange of genetic information by horizontal gene transfer is an important mechanism for the evolution of bacterial genomes. Legionella pneumophila strains encode different type IV secretion systems and integrative conjugative elements contribute to the variability of the intracellular pathogen. The genomic island Trb-1 of L. pneumophila Corby encodes a functional conjugation and T4ASS. Trb-1 is integrated within the tRNAPro gene and can exist in a chromosomal or an episomal circular form. In addition to the trb/tra genes, a site-specific integrase (int-1) and a Legionella vir region (lvrRABC) are also localized on the genomic island. By deleting the int-1 gene, it could be demonstrated that the excision and of Trb-1 is integrase dependent. Furthermore, in this work it was shown for the first time that the lvr region and especially the putative phage repressor LvrR, is involved in the regulation of Trb-1 excision. Conjugation of Trb-1 in L. oakridgensis does not influence the in vivo fitness of the transconjugants in human macrophages. The genomic islands LpcGI-1 and LpcGI-2 encode a new putative T4SS. For the first time it could be demonstrated, that the T4SS localized on LpcGI-2 is functional. Although LpcGI-2 could be mobilized and transferred via conjugation to another L. pneumophila strain, followed by the site-specific integration into the genome of the transconjugants. LpcGI-1 and LpcGI-2 are flanked by the tRNAThr or tRNAMet gene respectively. Both islands can exist in different chromosomal and episomal forms. The excision of LpcGI-2 occurs similar to Trb-1 in an integrase dependent manner. Two additional genomic islands (LpcGI-Asn and LpcGI-Phe) could be identified in the genome of Lp Corby. Moreover, data of the in silico analysis demonstrated, that genomic islands similar to Trb-1, LpcGI-2 and LpcGI-1 are distributed within the genus Legionella.

In den letzten Jahren gibt es großes Interesse am SiGeSn Materialsystem aufgrund seines Potentials für die Verwendung in der Optoelektronik, Elektronik und Photovoltaik. Während jedoch die binären Verbindungshalbleiter Si(x)Ge(1-x) und Ge(1-y)Sn(y) schon intensiv untersucht wurden, sind die Materialeigenschaften des ternären Verbindungshalbleiters Ge(1-x-y)Si(x)Sn(y) und Nanostrukturen basierend auf diesem Verbindungshalbleiter noch weitgehend unbekannt. In dieser Arbeit werden drei theoretische/theoretisch-experimentelle Studien zur Untersuchung des SiGeSn Materialsystems vorgestellt. In einer Studie wird die Abhängigkeit der Größe der direkten Bandlücke von der Zusammensetzung des Ge(1-x-y)Si(x)Sn(y) Verbindungshalbleiters untersucht. Basierend auf Messungen der Rutherford Rückstreuung, Röntgenbeugung und Photolumineszenz (PL) von Ge(1-x-y)Si(x)Sn(y) Proben mit an Ge angepassten Gitterkonstanten wird die Abhängigkeit von Größe der direkten Bandlücke und der Materialkomposition mit einer quadratischen Gleichung beschrieben. Weiterhin wird die Bandanordnung der elementaren Halbleiter Si, Ge und Sn an Grenzflächen untersucht. Anhand von Kohn-Sham basierter Density Functional Theory (DFT) in Kombination mit Local Density Approximation (LDA) berechneten Bandstrukturen von Grenzflächen zwischen Elementarhalbleitern wird der Versatz im Valenzband zwischen Si, Ge und Sn untersucht. Es wird gezeigt, dass aufgrund zu kleiner Bandlücken resultierend aus dem Kohn-Sham-Ansatz in Verbindung mit der LDA ein unphysikalischer „Broken Gap“ Versatz zwischen Ge und Sn Bändern entsteht. In einer dritten Studie werden die PL-Spektren von Ge Quantentöpfen mit Si Barrieren untersucht. Um die Abhängigkeit der PL-Spektren von Anregungsintensität und Temperatur zu verstehen, wird ein selbstkonsistentes Effektives-Massen-Model entwickelt. Mit diesem Model ist es möglich den Einfluss von Temperatur und Bandauffüllung auf das PL-Spektrum zu untersuchen. / The SiGeSn semiconductor material system has recently attracted great interest due to its prospective potential for use in optoelectronics, electronics, and photovoltaics. While the binary alloy Si(x)Ge(1-x) and Ge(1-y)Sn(y) have already been well studied, the properties of bulk and heterostructures involving the Ge(1-x-y)Si(x)Sn(y) ternary alloy are largely unknown. In this thesis, we present the results of three theoretical/experimental-theoretical investigations concerning the SiGeSn material system. First, we investigate the compositional dependence of the direct band-gap of Ge(1-x-y)Si(x)Sn(y) alloys. Based on Rutherford backscattering, x-ray diffraction, and photoluminescence (PL) measurement of Ge(1-x-y)Si(x)Sn(y) alloys lattice-matched to Ge, we describe the compositional dependence of the band gap using a quadratic equation. We predict Ge(1-x-y)Si(x)Sn(y) alloys lattice-matched to Ge to be direct-band-gap semiconductors for Sn concentrations larger than 12%. Secondly, we investigate the band alignment between the elemental semiconductors Si, Ge, and Sn. Performing bulk and interface calculations using density functional theory (DFT) in combination with the local density approximation (LDA), we attempt to calculate the valence band offset between the elemental semiconductors. We find that the Kohn-Sham based DFT-LDA calculations are flawed by the underestimation of the band-gaps of the elemental semiconductors, which leads to a false broken gap band alignment between Ge and Sn. Third, we study the PL of ultrathin Ge multiple quantum well (multiple-QW) structures grown on Si. To understand the excitation density and temperature related shifts of the PL spectra of the sample, we develop a self-consistent multivalley effective mass model. Using second-order perturbation theory, we calculate the indirect phonon-assisted radiative spontaneous recombination rate together with the no-phonon peak energy and compare our results to the experimental results.

Made available in DSpace on 2015-03-04T18:51:16Z (GMT). No. of bitstreams: 1 Anexos-Final.pdf: 7146890 bytes, checksum: bb4f151b856a0b67e03a2261753fccfe (MD5) Previous issue date: 2008-10-31 / Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior / The type IV secretion system can be classified as a large family of macromolecule transporters divided in three recognized sub-families involved in different bacterial functions. The major sub-family of T4SS is the conjugation system, which allows transfer of genetic material as a nucleoprotein via cell contact among bacteria. Analogously to bacterial conjugation, the T4SS can transfer genetic material from bacteria to eukaryotic cells; such is the case of T-DNA transfer of Agrobacterium tumefaciens to host plant cells. The system of effector proteins transport constitutes the second sub-family, being indispensable for infection processes of several mammalian and plants pathogens. The third sub-family corresponds to the DNA uptake/release system involved in genetic transformation competence, independently of cell contact, as it was described to the systems VirB/D4 from Campylobacter jejuni and ComB form Helicobacter pylori. Several essential features of T4SS are well known, but the knowledge in support of an uncomplicated classification or proper protein annotation of system subunits remains confusing, which in same cases can avoid making inferences about evolution of the system in bacterial species. The purpose of this work was to organize, classify and integrate the knowledge about T4SS through building a database devoted to this bacterial secretion system. The T4SS database was created using the SGBD MySQL and Perl programming language and with a web interface (HTML/CGI) that gives access to the database. Currently, this database hold genomic data from 43 bacteria and 10 plasmids acquired from the GenBank NCBI, these organisms comprise groups from Actionobacteria to Gram-negative Proteobacteria including symbiotic and pathogenic bacteria. By applying Bidirectional Best-Hits method was possible to get a core set of 75 clusters with 974 proteins involved in the T4SS. Also, during this procedure BlastP, Muscle e ClustalW algorithms were applied. The database was manually annotated supported by cross references built-in the T4SS annotation pages, such as the UniProtKB/Swiss-Prot, COG, InterPro and TCDB as well as by the methods for signal peptide and transmembrane regions prediction. All T4SS protein records scattered into 75 ortholog clusters were organized into five different classes of type IV secretion system proteins: (i) Type IVA Mpf/T4CP; (ii) Type IVA Dtr; (iii) F-type plasmid; (iv) IncP-1-type plasmid; (v) Type IVB Icm/Dot. All 974 proteins were annotated into 68 well-known families, which can be involved in conjugation, effector translocator, DNA uptake/release or even can be bifunctional proteins. Also, by using the Maximum Likelihood method were built 70 unrooted phylogenetic trees that represents just 70 clusters instead of 75, this is due to five clusters had only two protein sequences, five unrooted phylogenetic trees were built for each group of first hierarchical classification, one unrooted phylogenetic trees including proteins from archetype systems of all groups, one unrooted phylogenetic trees from 16S sequence of each organism and one rooted tree including a sequence from a Gram-positive bacteria as an external group. The phylogenetic analyses show that some proteins of T4SS are more divergent than others, which indicate that for a particular function few sequence mutations were needed, but other proteins required many sequence mutations to get another functions. Thus, these results proved that proteins belong to the same cluster show different functions: conjugation, DNA uptake/release or effector translocator. Consequently, it was possible verify that similar functions were grouped together within phylogenetic tree, which allowed to annotate a probable function of some uncharacterized proteins, that is possibly due to the sequence similarity may reveal a similar evolution to get the same function. Thus, the phylogenetic trees allowed confirming the protein annotation as well as inferring whether uncharacterized proteins would encompass a known function. The T4SS database will be an open access, given to the users searching and submission sequence tools, which will permit to get insights about classification and phylogeny of T4SS sequence of interest. T4SS Database is accessible at the URL http://www.t4ss.lncc.br. / O T4SS pode ser classificado como uma família de transportadores de macromoléculas envolvidos em diferentes funções bacterianas. A maior subfamília do T4SS é a do sistema de conjugação, o qual permite a transferência de material genético entre bactérias. Analogamente à conjugação, o sistema pode transferir material genético entre bactérias e eucariotos, tal como a transferência de T-DNA de Agrobacterium tumefaciens. O sistema de transporte de proteínas efetoras constitui uma segunda subfamília do T4SS, sendo indispensável nos processos de infecção de vários patógenos de mamíferos e plantas. A última subfamília corresponde ao sistema DNA-uptake/release" que funciona independente de contato com uma célula alvo, representado pelos sistemas VirB/D4 de Campylobacter jejuni e ComB de Helicobacter pylori. Muitas características básicas do T4SS são bem conhecidas, entretanto o conhecimento para a classificação simples e intuitiva ou a anotação apropriada das proteínas ainda não está claro, impedindo em alguns casos estabelecer correlações evolutivas deste sistema em bactérias. O objetivo deste trabalho foi o de organizar, classificar e integrar o conhecimento do T4SS através da construção de um banco de dados especializado para este sistema secretório bacteriano. O banco de dados T4SS foi criado utilizando o SGBD MySQL e a linguagem de programação Perl e com uma interface web (HTML/CGI) que fornece acesso ao banco. Este banco consta atualmente com 43 genomas bacterianos e 10 plasmídeos obtidos do GenBank NCBI, estes organismos vão desde Actinobactérias até Proteobactérias Gram-negativas, incluindo simbiontes e patogênicos. Foi utilizada a metodologia do Bidirectional Best-Hits", com a qual foi possível obter um conjunto mínimo de 75 clusters" com 974 proteínas envolvidas no T4SS. Também, durante este procedimento foram utilizados os algoritmos BlastP, Muscle e ClustalW. O banco foi anotado manualmente utilizando referências cruzadas incluídas nas páginas de anotação do T4SS, tais como UniProtKB/Swiss-Prot, COG, InterPro e TCDB e métodos para predição de regiões de peptídeos sinal e transmembrana. As análises do banco T4SS permitiram criar uma classificação hierárquica e funcional para as proteínas do T4SS, consistindo em cinco grupos: (i) Type IVA Mpf/T4CP; (ii) Type IVA Dtr; (iii) F-type plasmid; (iv) IncP-1-type plasmid; (v) Type IVB Icm/Dot). As 974 proteínas foram anotadas em 68 famílias conhecidas, as quais podem estar envolvidas em conjugação, transferência de T-DNA, transferência de proteínas efetoras, DNA-uptake/release" ou bem serem proteínas bifuncionais. Também, através do método de máxima verossimilhança foram geradas 70 árvores filogenéticas não enraizadas (NR) representando apenas 70 clusters, já que cinco clusters apresentaram apenas duas seqüências de proteínas, cinco árvores filogenéticas NR foram criadas para cada grupo da primeira categoria hierárquica, uma árvore NR com representantes de todos os grupos, uma árvore NR gerada a partir das seqüências 16S de cada organismo e uma árvore de um cluster incluindo uma seqüência de bactéria Gram-positiva como grupo externo. As análises filogenéticas mostram que determinadas proteínas do sistema são mais divergentes que outras, indicando que para uma determinada função poucas mutações de seqüências foram necessárias, já outras proteínas precisaram de maiores mutações para adquirir outras funções. Por isso, verifica-se que proteínas de um mesmo cluster apresentam diferentes funções: conjugação, DNA-uptake/release", traslocadores de proteínas efetoras. Conseqüentemente, foi possível verificar que funções semelhantes se agruparam juntas nas árvores filogenéticas, permitindo anotar uma função provável das proteínas ainda não caracterizadas ( unknown"), isto possivelmente devido a que em virtude de sua semelhança de seqüências, possivelmente evoluíram para realizar a mesma função. Assim, as arvores possuíram a finalidade de confirmar a anotação e contribuíram permitindo inferir se os unknown" ou probable" podem ser de uma determinada classificação funcional. O banco T4SS será de uso público, oferecendo ao usuário ferramentas de buscas e submissão de seqüências, as quais permitirão inferir respostas sobre a classificação e filogenia da seqüência T4SS de interesse. O banco de dados T4SS pode ser acessado na URL: http://www.t4ss.lncc.br.

Xanthomonas axonopodis pv. citri (Xac) é o causador do cancro de plantas cítricas. Entre os potenciais fatores de virulência codificados por Xac, está o Sistema de Secreção do Tipo IV (T4SS), um grande complexo multiprotéico que atravessa o periplasma e as membranas interna e externa de bactérias Gram-negativas. O T4SS está envolvido com secreção de proteínas e/ou DNA para o meio extracelular ou diretamente no interior da célula do hospedeiro. Este Sistema requer tipicamente 12 proteínas para realizar suas funções: VirB1-VirB11 e VirD4. O T4SS codificado pelo cromossomo de Xac está aparentemente incompleto, devido a não codificar nenhuma proteína com similaridade de seqüência a VirB7. Os objetivos deste trabalho são estudar a estrutura, função e interações das proteínas do T4SS de Xanthomonas. Foram clonados 23 genes que codificam proteínas ou domínios relacionados ao T4SS, e os polipeptídeos foram produzidos de forma recombinante em E. coli. Treze deles foram purificados e submetidos a estudos estruturais, espectroscópicos e de interações proteína-proteína. A estrutura em solução de Xac262224-139 foi resolvida, apresentando uma região N-terminal desenovelada de aproximadamente 30 resíduos e um domínio globular. Este polipeptídeo oligomeriza em troca química rápida na escala de tempo de RMN e o seu N-terminal desenovelado reconhece o domínio C-terminal de VirB9 (VirB9154-255) em troca lenta. Análise de RMN demonstrou que VirB9154-255 possui uma estrutura flexível em solução, sofrendo uma marcante mudança conformacional na presença de Xac262224-139. Ambas proteínas se tornam rígidas após a interação. Xac2622 é o equivalente a VirB7 em Xanthomonas, baseado na localização do seu gene no lócus do T4SS, localização subcelular predita do polipeptídeo codificado e sua interação com VirB9. Porém, diferente de outras proteínas da família VirB7, Xac2622 possui um domínio globular adicional, com topologia e estrutura similares a domínios presentes apenas em proteínas associadas à membrana externa de bactérias Gram-negativas. Nocaute do gene xac2622, contudo, não afetou a virulência de Xac na infecção de plantas de laranja pêra. O domínio enovelado de Xac2622 foi cristalizado, e os cristais obtidos difrataram até uma resolução de 1,0 Å, pertencendo ao grupo espacial C2221. O modelo preliminar possui Rfactor de 0,121 e Rfree de 0,147. Foram obtidos cristais de outras 3 proteínas relacionadas ao T4SS de Xac, porém somente um deles difratou em alta resolução (2,0 Å, pertencendo ao grupo espacial C2). O potencial sinal de secreção pelo T4SS de Xanthomonas é um domínio C-terminal conservado de aproximadamente 115 resíduos, encontrado nos substratos putativos do T4SS. Caracterizamos um destes domínios, presente na proteína Xac2609, e ele é intrinsicamente desestruturado. Essa observação pode ter implicações funcionais, visto que os substratos são desenovelados antes de sua passagem pelo canal de secreção do T4SS / Xanthomonas axonopodis pv. citri (Xac) is a gram-negative bacterial phytopathogen that infects citrus. One possible virulence determinant is a chromosomally encoded Type IV Secretion System (T4SS), a multiprotein complex that spans the bacterial periplasm and both inner and outer membranes. The T4SS is used by some bacteria to secrete proteins and/or DNA to the extracellular milieu or the host interior. The model T4SS from Agrobacterium tumefaciens is made up of twelve structural proteins: VirB1-VirB11 and VirD4. The Xanthomonas T4SS is apparently incomplete because of the lack of a polypeptide with sequence similarity to VirB7. The aim of this project is the study of structure-function relationships in the Xanthomonas T4SS. Twenty-three T4SS protein-coding genes, including full-length proteins or domains, were cloned and the proteins were produced in different E. coli strains. Thirteen polypeptides were purified and some of them were submitted to structural, spectroscopic and protein-protein interaction studies. We used NMR to solve the solution structure of Xac262224-139 which consists of an unfolded N-terminal segment of ~30 residues followed by a globular domain. Xac262224-139 oligomerizes in fast exchange at the NMR time scale and interacts via its unfolded N-terminus with the VirB9 C-terminus (VirB9154-255) in slow exchange. NMR analysis showed that VirB9154-255 has a flexible structure in solution. However, this polypeptide undergoes a significant conformational modification in the presence of Xac2622,24-139 and both proteins become rigid upon interaction. Xac2622 is the Xanthomonas VirB7, based on the chromosomal localization of its gene, predicted subcellular localization and protein interaction analysis. But surprisingly, unlike other VirB7 proteins, Xac2622 has an extra C-terminal folded domain whose topology and structure are strikingly similar to that of periplasmic domains found in outer membrane proteins of many bacterial Secretion Systems. Knockout of the xac2622 gene, however, does not affect the Xac virulence in orange leaf infection assays. The Xac2622 folded domain was also crystallized, and these crystals diffracted up to 1.0 Å resolution and belong to the space group C2221. The preliminary refined model has Rfactor of 0.121 and Rfree of 0.147. Crystals of three other T4SS proteins have been obtained, but only one of them diffracted to high resolution (2.0 Å; space group C2). Xac2610 is a hypothetical protein whose gene is located in the T4SS locus, and its interactions were studied with VirB9, VirB11 and Xac2609, a putative T4SS substrate. The potential T4SS secretion signal is a conserved, approximately 115 residues, C-terminal domain found in the putative substrates of the Xanthomonas T4SS. This sequence mediates interactions with VirD4. We have characterized this domain from one substrate and it is mainly unfolded. This observation may have functional implications, as the substrates are unfolded before their secretion through the T4SS channel

Made available in DSpace on 2017-07-24T19:38:12Z (GMT). No. of bitstreams: 1 Gustavo Marciniuk.pdf: 9151594 bytes, checksum: 94a7264c77b0567f7224ef931267fdab (MD5) Previous issue date: 2014-03-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This work refers to the development of a simple and innovative method for the synthesis of ternary composites of different proportions based on amorphous carbon, polyaniline and manganese (IV) oxide for their future use in electrochemical supercapacitors. The process presented here consists, essentially, of 3 parts: (i) at first, it has been performed the oxidative treatment of amorphous carbon and the separated synthesis of pure MnO2 and polyaniline, when all the materials involved were structurally characterized by XRD, FTIR, RAMAN, DLS and SEM, and electrochemically characterized by cyclic voltammetry measurements, in order to identify its structural, morphological and electrochemical properties and then compared with those presented by the future ternary composites; (ii) synthesis of a binary C/MnO2 composite by the anchoring of the MnO2 particles on the oxidized sites of the amorphous carbon, which represents the first stage on the formation of the ternary composites. Its purpose was to identify possible structural and electrochemical changes caused by MnO2 surface layer in the comparison of the binary composite with its individual components. Therefore, this composite was characterized by the same methods mentioned above; (iii) synthesis and characterization of ternary composites on different proportions, based on the initial formation of binary C/MnO2 followed by in situ polymerization of aniline monomers on their surface structure. The purpose of the synthesis in different proportions is to obtain a response profile when small changes in the quantity of the constituent materials are introduced in the ternary composites. The characterization of the ternary composites were carried out by XRD, FTIR, RAMAN, DLS and SEM, which allow to identify possible structural and morphological changes showed by composites compared to the individual components and, by cyclic voltammetry measurements, when it was possible to notice the electrochemical behavior of these composites and define their specific capacitance values in parallel to their processes of charge storage and ions diffusion. / Neste trabalho, buscou-se desenvolver uma metodologia simples e inovadora para a síntese de compósitos ternários de diversas proporções baseados em carbono amorfo, polianilina e óxido de manganês (IV) visando sua futura utilização em supercapacitores eletroquímicos. O trabalho consiste basicamente de 3 partes; (i) inicialmente realizou-se o tratamento oxidativo do carbono amorfo e a síntese individual dos compostos MnO2 e PAni, onde todos os materiais foram caracterizados estruturalmente por DRX, FTIR, RAMAN, DLS e MEV e eletroquimicamente por medidas de voltametria cíclica, afim identificar suas propriedades estruturais, morfológicas e eletroquímicas e compará-las com as apresentadas pelos futuros compósitos ternários; (ii) síntese de um compósito binário C/MnO2 pelo ancoramento das partículas de MnO2 sobre sítios oxidados do carbono amorfo, a qual configura a 1ª etapa da formação dos compósitos ternários. Sua finalidade foi de identificar as possíveis mudanças estruturais e eletroquímicas ocasionadas pela presença da camada superficial de MnO2 quando comparado o compósito binário aos seus componentes individuais. Para tanto, este compósito foi caracterizado pelos mesmos métodos apresentados acima; (iii) síntese e caracterização de compósitos ternários de diferentes proporções baseados na inicial formação do binário C/MnO2 seguido da polimerização in situ dos monômeros de anilina sobre sua estrutura superficial. A abordagem da síntese em diferentes proporções torna possível a obtenção de um perfil de resposta quando são introduzidas nos compósitos ternários pequenas modificações na quantidade dos materiais constituintes. A caracterização dos compósitos ternários foi realizada pelas técnicas de DRX, FTIR, RAMAN, DLS e MEV, as quais permitem identificar possíveis mudanças estruturais e morfológicas apresentadas pelos compósitos frente aos componentes individuais, e, por medidas de voltametria cíclica, onde foi possível observar o comportamento eletroquímico destes compósitos e definir seus valores de capacitância específica juntamente com seus processos de armazenamento de carga e difusão de íons.

La voie de réparation NHEJ (Non-Homologous End-Joining) est une voie majeure de réparation des cassures double-brin chez l’homme. Les protéines de cette voie interagissent et forment des complexes dynamiques dont les mécanismes moléculaires sont encore largement méconnus. Nous avons dans un premier temps mis au point des protocoles de production à l’échelle de plusieurs milligrammes des protéines cœur de la voie NHEJ en cellules d’insecte à l’aide du système MultiBac. Nous avons ainsi purifié les complexes Ku70/Ku80 et Ligase4/XRCC4 et les protéines Cernunnos et Artemis à homogénéité. Des essais de cristallisation, des études par SAXS et des analyses par microscopie électronique ont été réalisés sur différents complexes formés par ces protéines cœur du NHEJ. Nous avons également caractérisé par chromatographie d’exclusion de taille et calorimétrie, les interactions effectuées entre les protéines de la voie NHEJ. L’ensemble de ces travaux a permis d’établir des bases biochimiques solides en vue des études structurales et fonctionnelles de la voie NHEJ chez l’homme. / Human DNA repair pathway NHEJ (Non-Homologous End-Joining) is a major pathway of double-strand breaks repair. The proteins involved in this pathway interact and form dynamic complexes whose molecular mechanisms are largely unknown. Firstly, we established protocols to be able to purify milligrams of those NHEJ pathway core proteins using MultiBac insect cells system. We then purified Ku70/Ku80 and Ligase4/XRCC4 complexes, Artemis and Cernunnos to homogeneity. Crystallogenesis assays, SAXS experiments and Transmission Electronic Microscopy experiments have been performed on several complexes formed by these core NHEJ proteins. We also characterized the interactions between these proteins by Size Exclusion Chromatography and Isothermal Calorimetry. These experiments have led to biochemical results sufficient to establish a solid basis to initiate the structural and functional study of the Human NHEJ Pathway.

A autoxidação de [Ni(cyclam)]2+ (cyclam = 1,4,8,11-tetraazociclotetradecano) induzida por S(IV) foi estudada acompanhando-se espectrofotometricamente, em função do tempo, a formação de [Ni(cyclam)]3+ em soluções saturadas com oxigênio contendo: [Ni(cyclam)]2+ 6,0 x10-3 mol L-1, [Ni(cyclam)]3+ (inicial) 8,0x10-6 mol L-1, cyclam 6,0x10-3 mol L-1, S(IV) (1,0 - 5,0)x10-4 mol L-1 e HClO4 1,0 mol L-1 a T = 25,0 °C e força iônica 1,0 mol L-1. A reação de autoxidação exibe um comportamento autocatalítico no qual o período de indução depende da concentração inicial de Ni(III). O estudo cinético da redução de Ni(III) por S(IV), sob condições anaeróbicas, e a autoxidação de Ni(II) mostraram que a etapa determinante da velocidade da reação é a redução de Ni(III) por S(IV) para produzir o radical SO3•-, que reage com o oxigênio dissolvido produzindo SO5•- o qual rapidamente reoxida o Ni(II). Os resultados mostram claramente um ciclo redox que depende do balanço entre a concentração de S(IV) e oxigênio. Quando a reação se processa na presença de Co(III) (10-6 a 10-4 mol L-1) e de Mn(II) (10-3 a 10-2 mol L-1) constatou-se a ocorrência de efeito sinérgico positivo na formação de [Ni(cyclam)]3+. / The autoxidation of [Ni(cyclam)]2+, cyclam = 1,4,8,11-tetraazacyclotetradecane, accelerated by sulfur dioxide was studied by spectrophotometrically monitoring the formation of [Ni(cyclam)3+ under the conditions: [Ni(cyclam)]2+ = 6.0 x 10-3 mol L-1; initial [Ni(cyclam)]3+ = 8.0 x 10-6 mol L-1; [cyclam] = 6.0 x 10-3 mol L-1 ; [S(IV)] = (1.0 - 5.0) x10-4 mol L-1 and 1.0 mol L-1 perchloric acid in oxygen saturated solutions at 25.0 °C and ionic strength 1.0 mol L-1. The autoxidation reaction exhibits an autocatalytic behavior in which the induction period depends on the initial Ni(III) concentration. The kinetic study of the reduction of Ni(III) by S(IV), under anaerobic conditions, and autoxidation of Ni(II) showed as the rate-determining step the reduction of Ni(III) by S(IV) to produce SO3•- radical, which reacts with dissolved oxygen to produce SO5•- and rapidly oxidizes Ni(II). The results clearly show a redox cycling process which depends on the balance of S(IV) and oxygen concentration. The sinergystic effect of the Co(III) and Mn(II) was studied. Both exhibit a positive synergistic effect in the sulfite-induced aLrtoxidation of Ni(II).

Neste trabalho experimental, objetivou-se avaliar comparativamente a alteração dimensional de três diferentes materiais utilizados na confecção de troqueis: gesso tipo IV Fuji Rock (GC), resina epóxica industrial Sikadur 32 (Sika) e resina epóxica Tri-Epoxy Die Material (Tri-Dynamics). Foram confeccionados dois modelos-padrão em aço inoxidável. Um desses modelos foi confeccionado de modo a simular um preparo para coroa total em um pré-molar. O outro modelo foi confeccionado 2,0mm maior em todas as dimensões em relação ao primeiro de modo a proporcionar um alívio uniforme para a base leve do material de moldagem. Trinta troquéis foram obtidos a partir de moldes em silicona de adição (Aquasil Dentsply) utilizada com a técnica de dois passos. Os troqueis foram divididos em grupos de dez para cada material. As alterações foram mensuradas através das medições da altura e dos diâmetros da base e do topo dos troqueis obtidos, com o auxílio de um Projetor de Perfis Deltronic DV-114 com leitura em software. As medidas obtidas foram dispostas em tabelas e analisadas estatisticamente. Após avaliação pode-se concluir que houve diferença estatisticamente significante entre todos os grupos testados. Os troquéis de gesso tipo IV Fuji Rock (GC) contraíram nas medições da altura e do diâmetro do topo, e expandiram nas medições do diâmetro da base, apresentando diferença estatisticamente significativa em todas as comparações com o grupo controle (metal). Os troquéis de resina epóxica Tri-Epoxy (Tri-Dynamics) contraíram nas medições da altura e do diâmetro do topo, e expandiram nas medições diâmetro da base, apresentando diferença estatisticamente significante somente no diâmetro do topo nas comparações com o grupo controle (metal). Os troquéis de resina epóxica Sikadur 32 (Sika) contraíram nas medições da altura e do diâmetro do topo, e expandiram nas medições do diâmetro das base, apresentando diferença estatisticamente significante em todas as comparações com o grupo controle (metal). / The aim of this study was to comparatively evaluate the dimensional changes of three different materials used in the production of dies: gypsum type IV - Fuji Rock (CG), industrial epoxy resin - Sikadur 32 (Sika) and epoxy resin - Tri-Epoxy Die Material (Tri-Dynamics). Two standard models of stainless steel have been made. One of these models was prepared to simulate a full crown preparation on a premolar. The other model was prepared 2.0 mm larger in all dimensions compared to the first one so as to provide an uniform relief for impression material mild base. Thirty dies have been made from molds in vinyl polysiloxane (Aquasil - Dentsply) using the two steps technique. Dies were divided into groups of ten for each material. Changes were evaluated by measuring the height and diameter of bottom and top of the dies with the aid of a Deltronic DV-114 Profiles Projector with reading software. The resultant measurements were arranged into tables and statistically analyzed. After evaluation, it was concluded that there was a statistically significant difference among all tested groups. Gypsum Fuji Rock type IV (GC) dies shrank in tops height and diameter measurements, and expanded the bottom diameter measurements, showing statistically significant differences when compared to control group (metal). Epoxy resin Epoxy-Tri (Tri-Dynamics) dies shrank in tops height and diameter and expanded in bottom diameter measurements, showing statistically significant difference only in tops diameter when compared to control group (metal). Epoxy resin Sikadur 32 (Sika) dies shrank in tops height and diameter measurements and expanded in bottoms diameter measurements, showing a statistically significant difference when comparing to control group (metal).

<p>U okviru ove disertacije izvr&scaron;eno je preparatvno izolovanje etarskog ulja li&scaron;ća ginka (<em>Ginkgo biloba</em> L.) destilacijom pomoću vodene pare, u cilju određivanja pojedinih fizičko-hemijskih parametara. Oficinalnim postupkom određen je sadržaj etarskog ulja u li&scaron;ću ginka i iznosi 0,0083%. Li&scaron;će ginka je ekstrahovano klasičnim rastvaračima tj. sme&scaron;om alkohol-voda, pri čemu je koncentracija alkohola iznosila 40% (m/m). Primenom navedenog rastvarača, izvr&scaron;ena je vi&scaron;estupna protivstrujna ekstrakcija (u pet stupnjeva) pri čemu je dobijen tečni ekstrakt (<em>Extracta fluida</em>) sa relativno visokim sadržajem ekstraktivnih materija (17,06%). Tečni ekstrakt je direktno kori&scaron;ćen za dobijanje suvog ekstrakta li&scaron;ća ginka (<em>Extracta sicca</em>)primenomvsu&scaron;nie sa raspr&scaron;ivanjem (spray dryer). Kvalitativna i kvantitativna karakterzacija izvr&scaron;ena je primenom postupka tečne hromatografije na tankom sloju (HPTLC) i određen sadržaj ukupnih flavonoida sračunatih na rutin, a i na katehin, a određen je i sadržaj ukupnih fenola sračunatih na hlorogensku kiselinu. Glavni deo doktorske disertacije predstavlja ekstrakcija sistema li&scaron;će ginka-ugljenik (IV)-oksid pod pritiskom, pri čemu je ispitivan uticaj stepena usitnjenosti droge na prinos ekstrakcije, i kori&scaron;ćen koeficijent brze i spore ekstrakcije kao mera kinetičkog pona&scaron;anja ekstrakcije. Dobijeni rezultati ispitivanja su pokazali značajan uticaj stepena usitnjenosti droge na brzinu ekstrakcije, pogotovo, kod ekstrakcije natkritičnim ugljenik (IV) oksidom. Radi izbora optimalnog protoka ekstragensa ispitivani su sledeći protoci 0,095, 0,194 i 0,277 kg/h . Na osnovu prinosa ekstrakcije usvojeno je da je protok ekstragensa od 0,194 kg/h optimalan. Prinos ekstrakcije je ispitivan kori&scaron;ćenjem dva postupka ekstrakcije, i to: ekstrakcija tečnim ugljenik (IV) -oksidom (temperatura ispod kritične temperature Tc= 31,1^oC, a pritisak ne&scaron;to ispod ili iznad kritičnog pritiska p_c=73,8 bar), i ekstrakcija natkritičnim ugljenik (IV)- oksidom (pritisak i temperatura iznad kritičnih vrednosti pritiska i temperature). U natkritičnoj oblasti promenom pritiska se značajno menjaju svojstva ekstragensa, povećava se sposobnost rastvaranja, dielektrična konstanta i dr. Ispitivan je uticaj temperature na prinos ekstrakcije (izotermni proces), pri čemu se dobijaju rezultati koji se ne mogu objasniti jednostavno, analizirajući samo uticaj gustine rastvarača na moć rastvaranja, već se za obja&scaron;njenje dobijenih rezultata uključuje i uticaj napona pare ekstrahovane komponente, tako da rastvorljivost komponente može da se poveća, smanji ili ostane ista sa povećanjem temperature na konstantnom pritisku, u zavisnosti koji uticaj je dominantniji. Kod izotermnih postupaka prinos ekstrakcije raste sa povećanjem pritiska ekstrakcije, &scaron;to je u saglasnosti sa teoretskim principima. S druge strane, ekstrakti dobijeni pri vi&scaron;im pritiscima imaju manji sadržaj etarskog ulja &scaron;to se obja&scaron;njava činjenicom da veći pritisci imaju veću moć rastvaranja glavnih komponenata kao i komponenata kao &scaron;to su smole, voskovi i masna ulja. Kvalitativnom i kvantitativnom analizom selektovanih ekstrakata dobijenih tečnim (130 bar, 20^oC, 3 h) i natkritičnim (100 bar, 40^o C, 4 h) ugljenik (IV)-oksidom, kao i etarska ulja izolovana iz ovih ekstrakata nađeno je da ispitivani uzorci sadrže nalkane, račvaste alkane i jedinjenja sa kiseonikom, među kojima posebno mesto zauzimaju fenoli sa zasićenim i nezasićenim alkil ostacima.<br />Izvr&scaron;ena je uporedna anliza etarskog ulja dobijenog destilacijom li&scaron;ća ginka pomoću vodene pare i etarskih ulja dobijenih iz selektovanih ekstrakata. Interesantan je podatak koji se odnosi na sadržaj etarskog ulja u drogi određen direktnom destilacijom droge pomoću vodene pare i izdvajanjem etarskih ulja iz ekstrakata takođe destilacijom pomoću vodene pare. U ovom drugom slučaju dobija se, preračunavanjem, sadržaj etarskog ulja u drogi vi&scaron;estruko veći (4-10 puta) od sadržaja, određenog oficinalnim postupkom, u drogi. Ova pojava se obja&scaron;njava uvođenjem pojma &bdquo;vezano&ldquo; etarsko ulje i &bdquo;slobodno&ldquo; etarsko ulje. Ekstrakcijom ugljenik (IV)-oksidom pod pritiskom tzv. vezano etarsko ulje se oslobađa voskova i masnog ulja &scaron;to se odražava na njegovu povećanu količinu u CO₂-ekstraktima. Na kraju u ovoj disertaciji izvr&scaron;eno je modelovanje ekstrakcionog sistema li&scaron;će <em>Ginkgo biloba</em> &ndash; ugljenik (IV)-oksid pod pritiskom kori&scaron;ćenjem model jednačine Naika i saradnika, modifikovane model jednačine Reverchon i Sesti-Osseo kao i model, če&scaron;će kori&scaron;ćen, koji je predložila Sovov&aacute;. Kori&scaron;ćeni modeli mogu relativno uspe&scaron;no da se koriste za opisivanje ekstrakcionog sistema li&scaron;će <em>Ginkgo biloba</em> &ndash; ugljenik (IV)-oksid pod pritiskom. Radi iznalaženja najpovoljnijih uslova ekstrakcije primenjen je metod odzivne povr&scaron;ine variranjem parametra ekstrakcije (pritisak, temperatura i vreme ekstrakcije). Na osnovu eksperimentalnih rezultata dobijen je polinom drugog reda za izračunavanje optimalnog prinosa ekstrakcije i određeni su najpovoljniji uslovi ekstrakcije kao i međusobni uticaji pojedinih parametra.</p> / <p>Within this thesis preparative isolation of essential oil from leaves of ginkgo (<em>Ginkgo biloba</em> L.) by steam distillation was carried out, in order to determine some physico-chemical parameters. The essential oil content in the leaves of ginkgo wass determined by an officinal procedure and its value is 0.0083%. Ginkgo leaves were extracted with conventional solvents, i.e. alcohol-water mixture, where the alcohol concentration was 40% (w/w). The forementioned solvent was used to carry out a multistage counter-current extraction (five stages) by which the liquid extract (<em>Extracta fluida</em>) with a relatively high content of extracts (17.06%) was obtained. The liquid extract was directly used to obtain a dry extract of ginkgo leaves (<em>Extracta sicca</em>) by spay drying. The qualitative and quantitative characterisation was based on the proceedings of liquid thin layer chromatography (HPTLC), the content of total flavonoids expressed as rutin, and as catechin, and the total phenol content expressed as chlorogenic acid was determined. The main part of the doctoral thesis is the extraction system of ginkgo leaves-carbon (IV) oxide under pressure, in which the effect of the drug particle size on the extraction yield was studied, and as a measure of the kinetic behavior of extraction the coefficient of fast and slow extraction was used. The obtained results showed a significant effect of the drug particle size on the speed of extraction, particularly, the extraction with supercritical carbon (IV) oxide. For the purpose of selecting the optimal flow of the solvent several flowrates were investigated 0.095, 0.194 and 0.277 kg/h. Based on the yield of extraction the solvent flowrate of 0.194 kg/h was assumed as optimal. The extraction yield was investigated using two extraction procedures: extraction with liquid carbon (IV) oxide (temperature below the critical temperature Tc = 31.1 ⁰C and pressure slightly below or above the critical pressure p_c = 73.8 bar), and extraction with supercritical carbon (IV) oxide (pressure and temperature above the critical values of pressure and temperature). In the supercritical area the change in pressure significantly the influences the properties of the solvent, increases the ability of dissolving, dielectric constant, etc. The effect of temperature on extraction yield was examined (isothermal process), where the obtained results can not be explained simply by analyzing only the effect of solvent density on the power of dissolution, but to explain these results the effect of vapor pressure of the extracted components must be included, so that the solubility of the component may increase, decrease or remain the same with increasing temperature at constant pressure, depending on which influence is dominant. In isothermal processes the extraction yield increases with increasing pressure of extraction, which is consistent with theoretical principles. On the other hand, extracts obtained at high pressures have a lower content of essential oils which can be explained by the fact that higher pressures have a greater power of dissolution of the main components as well as components such as resins, waxes and fatty oils. The qualitative and quantitative analysis of the selected extracts obtained with liquid (130 bar, 20 ⁰C, 3 h) and supercritical (100 bar, 40 ⁰C, 4 h) carbon (IV) oxide, and essential oils isolated from these extracts showed that the studied samples contain n-alkanes, branched alkanes and compounds with oxygen, including phenols with saturated and unsaturated alkyl residues. A comparative analysis of the essential oil obtained by distillation of ginkgo leaves by steam and essential oil obtained from selected extracts was carried out. An interesting fact concerning the essential oil content in the drug determined by direct distillation of drugs by steam and separating the essential oils from extracts also produced by steam distillation. In the other case, by calculation, the obtained essential oil content in the drug is several times higher (4-10 times) of content, determined by an fficinal procedure, in the drug. This phenomenon is explained by introducing the notion of &quot;linked&quot; essential oil and &quot;free&quot; essential oil. The extraction with carbon (IV) oxide under pressure the so called linked essential oil is released from waxes and fatty oils which is reflected in its increased amount in CO₂-extracts. At the end of this dissertation the modeling of the extraction system leaves of <em>Ginkgo biloba</em> - carbon (IV) oxide under pressure was carried out using the model equation of Naik and associates, the modified model equation of Reverchon and Sesti Osseo and also like a frequently used model proposed Sovov&aacute;. The mentioned models can be relatively used to describe the extraction system leaves of <em>Ginkgo biloba</em> - carbon (IV) oxide under pressure. In order to find the most favorable conditions of extraction, the response surface methodology varying extraction parameters was used (pressure, temperature and extraction time). Based on the experimental results a second order polynom for the calculation of the optimum yield of extraction was obtained and the extraction conditions and the mutual influence of some parameters were determined.</p>