Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2016-08-08T16:42:04Z No. of bitstreams: 1 brunaluanamarcial.pdf: 8389291 bytes, checksum: 93256796394730c4d8f7a200fe2be582 (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: tentar corrigir a fórmula no resumo on 2016-08-09T11:35:20Z (GMT) / Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2016-08-09T11:47:58Z No. of bitstreams: 1 brunaluanamarcial.pdf: 8389291 bytes, checksum: 93256796394730c4d8f7a200fe2be582 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-08-09T11:51:59Z (GMT) No. of bitstreams: 1 brunaluanamarcial.pdf: 8389291 bytes, checksum: 93256796394730c4d8f7a200fe2be582 (MD5) / Made available in DSpace on 2016-08-09T11:52:00Z (GMT). No. of bitstreams: 1 brunaluanamarcial.pdf: 8389291 bytes, checksum: 93256796394730c4d8f7a200fe2be582 (MD5) Previous issue date: 2013-07-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As Metaloproteinases de Matriz (MMPs) desempenham um papel crucial nos processos fisiológicos e condições patológicas tais como progressão tumoral e metástases. Nos últimos anos, tem sido proposto uma série de inibidores de MMPs (IMMPs), incluindo as Tetraciclinas Quimicamente Modificadas (CMTs), as quais têm apresentado resultados promissores em ensaios com modelos pré-clínicos de câncer. No entanto, até o presente não havia nenhum estudo da estrutura-atividade desses compostos. Neste sentido, na presente tese foram combinadas as metodologias teóricas da Teoria do Funcional da Densidade (DFT), Docking molecular e simulação de Dinâmica Molecular (DM) para elucidar o mecanismo de interação das CMTs com o sítio ativo das MMPs. Primeiramente a estrutura e estabilidade dos modelos moleculares miméticos do sítio ativo da MMP-2 representados por: [Zn(LHn)(H2O)2]2+x (n = 0, 1, 2 e x = -2, -1, 0) e [Zn(L)(His)3], onde L representa CMT1, -3, -4, -7 e -8, foram calculados usando DFT. O efeito do solvente e do pH foram também avaliados. Os resultados sugerem que a coordenação com o zinco ocorra no sítio VI(O11O12), o qual é igualmente favorável para a CMT-3 (log β = 15,9) e CMT-8 (log β = 21,3). Esta análise inicial evidenciou que a atividade das CMTs como inibidoras de MMP estaria diretamente relacionada à sua estrutura. Na segunda fase do trabalho foi investigada a interação das CMTs diretamente com o sítio ativo da MMP-2 (1QIB) combinando os métodos de docking molecular, simulações de DM e cálculos de energia livre. Um novo conjunto de parâmetros para os átomos de Zn(II) estrutural e catalítico da enzima foi calculado, validado e depositado dentro do campo de forças AMBER. As CMTs foram otimizadas no nível B3LYP/6-31G(d) e “docadas” dentro do sítio ativo da MMP-2. As melhores soluções foram minimizadas e submetidas a 12ns de simulação de DM. A energia livre de ligação foi calculada usando as metodologias de integração termodinâmica e MMPBSA. Os principais resultados mostram que as CMTs coordenam-se ao Zn(II) através do sítio VI(O11-O12), como proposto experimentalmente. Com exceção do análogo CMT-3 que está envolvido em contatos hidrofóbicos e interações de van der Waals com aminoácidos do bolso S1’da enzima e ainda interage com o Zn(II) pelo O11. A análise da energia livre de ligação revela uma maior afinidade entre o análogo CMT-3 com o sítio ativo da MMP-2, seguido pela CMT-7 e CMT-8, sendo que a diferença de energia varia apenas de 3 kcal mol1. De acordo com os resultados, é possível inferir que a ocupação do canal S1’ seria um fator fundamental para a resposta biológica da CMT-3, enquanto que as demais CMTs, por impedimento estérico, não entram no túnel hidrofóbico da enzima. Os resultados apresentados nesta tese tratam do primeiro estudo em nível molecular da interação de CMTs com metaloproteínas, como contribuição para a compreensão das observações experimentais, sobretudo dos testes biológicos, e ainda uma referência direta para o desenho de novos análogos das CMTs com maior potencial biológico. / Matrix Metalloproteinases (MMPs) play a critical role in physiological processes and pathological conditions such as tumor progression and metastasis. In recent years, a number of MMP inhibitors (MMPIs) have been proposed, including the chemically modified tetracyclines (CMTs), which have been evaluated in preclinical cancer models showing promising results. However, until present there were no studies on the structure-activity relationship for these compounds. In this sense, the present thesis focuses on the direct interaction of CMTs with the active site of the MMPs. Density Functional Theory (DFT), molecular docking and molecular dynamics (MD) simulations were accomplished for seven CMT derivatives. Firstly the structure and stability of mimetic molecular models of active site of MMP-2 represented by: [Zn(LHn)(H2O)2]2+x (n = 0, 1, 2 and x = -2, -1, 0) and [Zn(L)(His)3], where L=CMT-1, CMT-3, CMT-4, CMT-7 and CMT-8, were calculated at DFT level. The solvent and pH effects were also evaluated. The results suggested that the coordination at the O11-O12 site (site VI) was quite favorable to CMT-3 (log β = 15,9) and CMT-8 (log β = 21,3). This initial analysis showed that the activity of CMTs as inhibitors of MMP is directly related to its structure. In the second stage of the study, we investigated the interaction of CMTs directly with the active site of MMP-2 (1QIB). New sets of parameters were proposed for structural and catalytic zinc atoms in order to study MMPs and their complexes by means of the AMBER force field. The CMTs ligands were optimized at the B3LYP/6-31G(d) and docked onto the active site of MMP-2. The best solutions were minimized and subjected to 12ns MD simulation. The binding free energy was calculated using the methods of thermodynamic integration and MM-PBSA. The main results showed that the CMTs bind to the catalytic zinc of the MMP-2 enzyme through the O11-O12 moiety (site VI) as proposed experimentally. The exception was the CMT-3 analogue which is involved in hydrophobic contacts and van der Waals interactions with amino acids of the S1’ pocket in the MMP-2 enzyme and also interacts with the Zn (II) by O11. The binding energy calculated in solution predicts the CMT-3 complexes as the most favorable, followed by the CMT-7 and CMT-8 analogues, whose energy difference is within 3 kcal mol-1. These results suggest that the occupancy of S1’ pocket is an important molecular feature for biological response of CMT-3, while the other CMTs do not enter the hydrophobic tunnel of the enzyme due to the steric hindrance. The results presented in this thesis are the first step towards understanding the mechanism of CMTs as MMP inhibitors at a molecular level and could be a significant contribution to the understanding of experimental observations, especially the biological tests and the CMTs action mechanism as MMP inhibitors, allowing the design of new analogues with enhanced biological potency.

L'interférométrie optique en astronomie est une technique permettant de sonder les objets observés à une résolution spatiale impossible à atteindre avec un télescope unique. Ce domaine d'instrumentation entre dans une nouvelle phase avec l'arrivée de nouveaux instruments combinant de très grands télescopes sur des bases allant jusqu'à 200m. AMBER (Astronomical Multi BEam Recombiner) est l'instrument recombineur à trois télescopes du VLTI (Very Large Telescope Interferometer) qui apporte à la fois de la résolution spectrale et des capacités d'imagerie sur cette infrastructure à la pointe de la recherche astrophysique. Je présente dans cette thèse mon apport au traitement du signal de l'instrument AMBER afin d'obtenir les observables nécessaires à l'exploitation scientifique des données. J'y présente l'approche dans le plan image qui a été utilisée et validée sur le ciel avec cet instrument, insi que les observables différentielles chromatiques que j'ai développées spécifiquement. Les observables différentielles chromatiques consistent à comparer la mesure de franges enregistrées simultanément entre deux longueurs d'ondes proches. Cela permet de reconstruire une fraction très substancielle de l'information de phase de l'objet, perdue par les estimateurs classiques jusque là utilisés en interférométrie. Je développe aussi le potentiel de ces observables ainsi que leur application pratique sur l'étude de divers objets astrophysiques dont les étoiles à enveloppe et les objets binaires.

Bacteriophage Lambda (λ) has played a historical role as an essential model contributing to our current understanding of molecular genetics. Lambda’s major capsid protein “gpD” occurs on each capsid at 405 to 420 copies per phage in homotrimeric form and functions to stabilize the head and likely to compact the genomic DNA. The interesting conformation of this protein allows for its exploitation through the genetic fusion of peptides or proteins to either the amino or carboxy terminal end of gpD, while retaining phage assembly functionality and viability. The lytic nature of λ and the conformation of gpD in capsid assembly makes this display system superior to other display options. Despite previous reports of λ as a phage display candidate, decorative control of the phage remains an elusive concept. The primary goal of this study was to design and construct a highly controllable head decoration system governed by two genetic conditional regulation systems; plasmid-mediated temperature sensitive repressor expression and bacterial conditional amber mutation suppression. The historical λ Dam15 conditional allele results in a truncated gpD fragment when translated in nonsuppressor, wild-type E. coli cells, resulting in unassembled, nonviable progeny. I sequenced the Dam15 allele, identifying an amber (UAG) translational stop at the 68th codon. Employing this mutant in combination with a newly created isogenic cellular background utilizing the amber suppressors SupD (Serine), SupE (Glutamine), SupF (Tyrosine) and Sup— (wild type), we sought to control the level of incorporation of undecorated gpD products. As a second dimension, I constructed two separate temperature-inducile plasmids whereby expression of either D or D::eGFP was governed by the λ strong λ CI[Ts]857 temperature-sensitive repressor and expressed from the λ PL strong promoter. Our aim was to measure the decoration of the λ capsid by a D::gfp fusion under varying conditions regulated by both temperature and presence of suppression. This was achieved utilizing this controllable system, enabling the measurement of a variable number of fusions per phage based on diverse genetic and physical environments without significantly compromising phage viability. Surprisingly, both SupE and SupF showed similar levels of Dam15 suppression, even though sequencing data indicated that only SupE could restore the native gpD sequence at amino acid 68 (Q). In contrast, SupD (S), conferred very weak levels of suppression, but imparted an environment for very high decoration of gpD::eGFP per capsid, even at lower (repressed) temperatures. The presence of albeit few wild-type gpD molecules allowed for an even greater display than that of the perceived “100%” decoration scenario provided by the nonsuppressor strain. It appears that the lack of wild-type gpD does not allow for the space required to display the maximum number of fusions and in turn creates an environment that affects both phage assembly and therefore phage viability. Finally, the use of Western blotting, confirmed the presence of gpD::eGFP fusion decoration by employing a polyclonal anti-eGFP antibody. The significance of this work relates to the unique structure of λ’s capsid and its ability to exploit gpD in the design of controlled expression, which is guiding future research examining the fusion of different therapeutic peptides and proteins. Furthermore this approach has important implications specifically for the design of novel vaccines and delivery vehicles for targeted gene therapy in which steric hindrance and avidity are important concerns. The execution of this project employed basic bacterial genetics, phage biology and molecular biology techniques in the construction of bacterial strains and plasmids and the characterization of the phage display system.

No description available.

Bacteriophage Lambda (λ) has played a historical role as an essential model contributing to our current understanding of molecular genetics. Lambda’s major capsid protein “gpD” occurs on each capsid at 405 to 420 copies per phage in homotrimeric form and functions to stabilize the head and likely to compact the genomic DNA. The interesting conformation of this protein allows for its exploitation through the genetic fusion of peptides or proteins to either the amino or carboxy terminal end of gpD, while retaining phage assembly functionality and viability. The lytic nature of λ and the conformation of gpD in capsid assembly makes this display system superior to other display options. Despite previous reports of λ as a phage display candidate, decorative control of the phage remains an elusive concept. The primary goal of this study was to design and construct a highly controllable head decoration system governed by two genetic conditional regulation systems; plasmid-mediated temperature sensitive repressor expression and bacterial conditional amber mutation suppression. The historical λ Dam15 conditional allele results in a truncated gpD fragment when translated in nonsuppressor, wild-type E. coli cells, resulting in unassembled, nonviable progeny. I sequenced the Dam15 allele, identifying an amber (UAG) translational stop at the 68th codon. Employing this mutant in combination with a newly created isogenic cellular background utilizing the amber suppressors SupD (Serine), SupE (Glutamine), SupF (Tyrosine) and Sup— (wild type), we sought to control the level of incorporation of undecorated gpD products. As a second dimension, I constructed two separate temperature-inducile plasmids whereby expression of either D or D::eGFP was governed by the λ strong λ CI[Ts]857 temperature-sensitive repressor and expressed from the λ PL strong promoter. Our aim was to measure the decoration of the λ capsid by a D::gfp fusion under varying conditions regulated by both temperature and presence of suppression. This was achieved utilizing this controllable system, enabling the measurement of a variable number of fusions per phage based on diverse genetic and physical environments without significantly compromising phage viability. Surprisingly, both SupE and SupF showed similar levels of Dam15 suppression, even though sequencing data indicated that only SupE could restore the native gpD sequence at amino acid 68 (Q). In contrast, SupD (S), conferred very weak levels of suppression, but imparted an environment for very high decoration of gpD::eGFP per capsid, even at lower (repressed) temperatures. The presence of albeit few wild-type gpD molecules allowed for an even greater display than that of the perceived “100%” decoration scenario provided by the nonsuppressor strain. It appears that the lack of wild-type gpD does not allow for the space required to display the maximum number of fusions and in turn creates an environment that affects both phage assembly and therefore phage viability. Finally, the use of Western blotting, confirmed the presence of gpD::eGFP fusion decoration by employing a polyclonal anti-eGFP antibody. The significance of this work relates to the unique structure of λ’s capsid and its ability to exploit gpD in the design of controlled expression, which is guiding future research examining the fusion of different therapeutic peptides and proteins. Furthermore this approach has important implications specifically for the design of novel vaccines and delivery vehicles for targeted gene therapy in which steric hindrance and avidity are important concerns. The execution of this project employed basic bacterial genetics, phage biology and molecular biology techniques in the construction of bacterial strains and plasmids and the characterization of the phage display system.

The diploma thesis presents an overview of the extinct representatives of Blattidae, the still living cockroach family. Six already described species, belonging to three genera, have been confirmed. All of them lived during Cenozoic. Eight new morphotypes from Myanmar amber were recognized. Cenomanian Burmite with its exceptional state of preservation, allows a detailed study of the morphology of the inclusions. Six morphototypes belong to the family Blattidae. One morphotype belongs to the derived family Olidae studied herein. One female morphotype belongs to the still living family Ectobiidae, based on the presence of an attached ootheca. The work specifies the previous molecular analysis (J2-K1) and determines the divergence of Blattidae during Early Cretaceous. Any evidence and interpolations before 127Ma is not substantiated. Phylogenetic analysis confirmed Blattidae being parafyletic in respect to the family Olidae. The work emphasizes the need for changes in systematics of the Mesoblattinidae, the stem of the Blattidae. Amber is confirmed as the product of coniferous trees of the Araucariaceae family. Key words: Blattidae, taxonomy, phylogeny, Cenomanian, Myanmar amber

The tudlov amber has been the only known fossil resin from the Czech Republic, in which the biological inclusions are preserved, and at the same time the only Czech fossil resin occurring not in the area of the Bohemian Massif, but in outer arch of the Outer Western Carpathians. So far, only single findings of fossil Hymenoptera and Diptera have been published. In contrast to mostly Cretaceous ambers of the Bohemian Massif, the age of tudlov amber is usually estimated from the late Paleocene to mid Eocene. This diploma thesis provides an overview of the fossil organisms found in the tudlov amber in the past two decades and it is also the most comprehensive text dedicated to the palaeodiversity of embedded organisms from this remarkable fossil resin. The presence of various microscopic fungi is proven, as well as the presence of angiosperm trichomes, mites and insects (prevailed by Hymenoptera and Diptera). The discovery of a hymenopteran family Stigmaphronidae (Apocrita) is the first Cenozoic evidence of this family, formerly considered as extinct by the end of Cretaceous. Finding of an isolated gymnosperm twig with wood anatomical characters close to genus Glyptostrobus allows to outline some conclusions about the paleoecosystem. The Eocene age of the resin is confirmed by the combination of...

The aim of the study was to analyse where different organic molecules situated themselves in relation to the water surface of a water microdroplet and use the resulting data to compare three different forcefields in the simulation package GROMACS. The forcefields used were: General AMBER forcefield (GAFF), Optimized potentials for liquid simulations - all atoms (OPLS-AA), and CHARMM general force field (CGenFF). A library of 146 molecules were simulated using molecular dynamics. Out of the 146 molecules only 65 resulted in useful data for the comparison of the forcefields. The molecules were placed in the centre of a water microdroplet and their movements were simulated for a duration of 1 ns. The trajectories and positions of the molecules were stored and from each simulation a density profile was generated, showing where the molecules situated themselves. The distance from the peak of the density profile to the water surface was calculated and compared between the different forcefields. To analyse the data further some of the molecules were divided into subsets based on their functional groups to see if any trends were visible. Although inconclusive, the data suggested that different forcefields were more or less agreeable depending on the functional group of the molecules, for example OPLS-AA differed from CGenFF and GAFF in the case of alcohols.

The diploma thesis presents an overview of the extinct representatives of Blattidae, the still living cockroach family. Six already described species, belonging to three genera, have been confirmed. All of them lived during Cenozoic. Eight new morphotypes from Myanmar amber were recognized. Cenomanian Burmite with its exceptional state of preservation, allows a detailed study of the morphology of the inclusions. Six morphototypes belong to the family Blattidae. One morphotype belongs to the derived family Olidae studied herein. One female morphotype belongs to the still living family Ectobiidae, based on the presence of an attached ootheca. The work specifies the previous molecular analysis (J2-K1) and determines the divergence of Blattidae during Early Cretaceous. Any evidence and interpolations before 127Ma is not substantiated. Phylogenetic analysis confirmed Blattidae being parafyletic in respect to the family Olidae. The work emphasizes the need for changes in systematics of the Mesoblattinidae, the stem of the Blattidae. Amber is confirmed as the product of coniferous trees of the Araucariaceae family. Key words: Blattidae, taxonomy, phylogeny, Cenomanian, Myanmar amber

This study covers how Amber Heard and Johnny Depp were portrayed during their defamation trial in Virginia 2022 by the British newspaper The Sun. The research questions include both how Amber Heard and Johnny Depp were portrayed during the timeframe as well as how they were perceived as a couple. The research questions also cover if there were any differences in how The Sun were framing them during the time period. The method that was used was a thematic analysis of all the articles that The Sun published in the time period that the trial took place, the 11th of April 2022 to the 1st of June 2022. To better help our understanding of the subject we used framing theory and celebrity studies as the theoretical framework. The framing theory also includes a subcategory which is the theory about the ideal victim. Since both Johnny Depp and Amber Heard accused each other of abuse, it was relevant to include if one of them were portrayed as a victim. In the thematic analysis we created three categories based on the research questions. Different themes were found in the articles and were placed as subcategories to the three main categories. The first category was about how the relationship between Johnny Depp and Amber Heard was portrayed. Three themes were found: The relationship as a circus, Difference in fame and The trial. The second category was about how Johnny Depp was portrayed. Three themes were identified: Depp as the perpetrator, Superstar Johnny Depp and Innocent or a bully? The third category depicts how Amber Heard was portrayed. Four themes were identified: Heard accused of lying, Heard as the perpetrator, Heard ridiculed by the public and Heard innocent? The results of the study conclude that both Depp and Heard have been portrayed as perpetrators and victims all though Depp was given more attention in the articles than Heard. Another conclusion is that some of the articles describe the case as Depp suing Heard for lying, in contrast to the articles where the case is described as Depp suing Heard for libel.