<p dir="ltr">Proteins and their interactions with each other, with nucleic acids, and with other molecules are foundational to all known forms of life. The three-dimensional structures of these interactions are an essential component of a comprehensive understanding of how they function. Molecular-biological hypothesis formulation and rational drug design are both often predicated on a particular structure model of the molecule or complex of interest. While experimental methods capable of determining atomic-detail structures of molecules and complexes exist, such as the popular X-ray crystallography and cryo-electron microscopy, these methods require both laborious sample preparation and expensive instruments with limited throughput. Computational methods of predicting complex structures are therefore desirable if they can enable cheap, high-throughput virtual screening of the space of biological hypotheses. Many common biomolecular contexts have largely been blind spots for predictive modeling of complex structures. In this direction, docking methods are proposed to address extreme conformational change, nonuniform environments, and distance-geometric priors. Flex-LZerD deforms a flexible protein using a novel fitting procedure based on iterated normal mode decomposition and was shown to construct accurate complex models even when an initial input subunit structure exhibits extreme conformational differences from its bound state. Mem-LZerD efficiently constrains the docking search space by augmenting the geometric hashing data structure at the core of the LZerD algorithm and enabled membrane protein complexes to be efficiently and accurately modeled. Finally, atomic distance-based approaches developed during modeling competitions and collaborations with wet lab biologists were shown to effectively integrate domain knowledge into complex modeling pipelines.</p>

No description available.

Broadband Dielectric Spectroscopy (BDS) in combination with a nanostructured electrode arrangement – which circumvents the conventional need to evaporate metal electrodes onto soft matter – is used to study the molecular dynamics of several glass forming materials confined in nanometric (> 5 nm) layers. Other complementary experimental tools employed in this work include spectroscopic vis-Ellipsometry (SE), AC-chip calorimetry (ACC), X-ray reflectrometry (XRR), Differential Scanning Calorimetry (DSC) and Atomic Force Microscopy (AFM). The latter is used to characterize the topography of the samples and to determine their thicknesses. Under the conditions of annealing samples (Tg + 50K) in high oil-free vacuum (10E-6 mbars) for at least 12 h and carrying out measurements in inert (dry nitrogen or argon) atmosphere, it is found for all studied thin layers that the structural relaxation, and hence the dynamic glass transition – in its mean relaxation times – remains within a margin ±3 K from the respective bulk behaviour. It is revealed, inter alia, that the one-dimensional confinement of thin films introduces restrictions on other (slower) molecular relaxation processes which manifest, depending on the specific system under investigation, as (i) an interruption of the end-to-end (normal mode) fluctuation of the chains, or (ii) a slowing down of the delta-relaxation when the system is cooled towards glass-formation. Furthermore, (iii) evidence is provided to show that the dimensionality of confinement plays a significant role in determining the resulting dynamics. A molecular understanding of these findings is given, and the discussion presented with respect to the on-going international debate about dynamics in confinement.

In general, this dissertation is concerned with modeling of mechanical behavior of protein molecules. In particular, we focus on coarse-grained models, which bridge the gap in time and length scale between the atomistic simulation and biological processes. The dissertation presents three independent studies involving such models. The first study is concerned with a rigorous coarse-graining method for dynamics of linear systems. In this method, as usual, the conformational space of the original atomistic system is divided into master and slave degrees of freedom. Under the assumption that the characteristic timescales of the masters are slower than those of the slaves, the method results in Langevin-type equations of motion governed by an effective potential of mean force. In addition, coarse-graining introduces hydrodynamic-like coupling among the masters as well as non-trivial inertial effects. Application of our method to the long-timescale part of the relaxation spectra of proteins shows that such dynamic coupling is essential for reproducing their relaxation rates and modes. The second study is concerned with calibration of elastic network models based on the so-called B-factors, obtained from x-ray crystallographic measurements. We show that a proper calibration procedure must account for rigid-body motion and constraints imposed by the crystalline environment on the protein. These fundamental aspects of protein dynamics in crystals are often ignored in currently used elastic network models, leading to potentially erroneous network parameters. We develop an elastic network model that properly takes rigid-body motion and crystalline constraints into account. This model reveals that B-factors are dominated by rigid-body motion rather than deformation, and therefore B-factors are poorly suited for identifying elastic properties of protein molecules. Furthermore, it turns out that B-factors for a benchmark set of three hundred and thirty protein molecules can be well approximated by assuming that the protein molecules are rigid. The third study is concerned with the polymer mediated interaction between two planar surfaces. In particular, we consider the case where a thin polymer layer bridges two parallel plates. We consider two models of monodisperse and polydisperse for the polymer layer and obtain an analytical expression for the force-distance relationship of the two plates. / text

A abordagem de modos normais de baixa frequência na descrição das flutuações conformacionais dos estados nativos das proteínas globulares tem ajudado na caracterização das suas funções biológicas. Vários métodos teóricos e experimentais têm sido empregados para a determinação destas flutuações internas. Estes movimentos podem ser caracterizados pelo fator Debye-Waller (fator-B), correspondente à mobilidade local do resíduo em nível atômico. A análise de modos normais utilizando os modelos de rede elástica (ENM) demonstra ser uma técnica robusta. Fatores-B experimentais são reproduzidos teoricamente por meio desta técnica em tempos computacionais relativamente curtos, mostrando-se competitiva com as técnicas mais sofisticadas. O modelo de rede elástica é uma abordagem t ipo coarse-grain na qual a proteína no seu estado enovelado é representada por uma rede elástica tridimensional de carbonos conectados por molas. As molas representam as interações ligantes e não ligantes entre os carbonos . Neste trabalho, inicialmente, estudamos os modelos de rede elástica já conhecidos na literatura. Em seguida, realizamos um estudo comparativo entre eles. Neste estudo, comprovamos que os modelos pfGNM e pfANM apresentam melhor correlação com os fatores-B experimentais que os os modelos GNM e ANM tradicionais. Desenvolvemos também uma nova abordagem, a qual intitulamos número de contatos ponderados anisotrópica (AWCN). Mostramos que a abordagem AWCN apresenta um desempenho significativamente melhor que o modelo de rede elástica anisotrópica tradicional. Por fim, realizamos um estudo de caráter investigativo do comportamento do peso das interações entre resíduos. Este estudo re velou que, para os modelos WCN e AWCN, a correlação exibe o seu valor máximo para interações ponderadas $1/R^p$, entre resíduos $i$ e $j$j, para valores de $p$ em torno de 2. Nos modelos pfGNM e pfANM a correlação é maximizada para dois valores de $p$, o primeiro em torno de 2 e o segundo em torno de 4,75, indicando que a ponderação pelo recíproco do quadrado da distância, usualmente empregada na literatura, pode não ser adequada para obter a melhor correlação. / Low frequency normal mode approach to describe conformational fluctuations of globular proteins has helped to characterize their biological functions. Various theoretical and experimental methods have been employed to det ermine the magnitudes of those internal motions. Those motions can be characterized by the Debye-Waller factor (B-factor), co rresponding to the local mobility of the residue at the atomic level. Normal mode analysis using elastic network models (ENM) has demonstrated to be a robust technique. Experimental B-factors has been reproduced theoretically by means of this techniq ue in a short computational time and it has been shown to be competitive with more sophisticated techniques. The ENM is a coarse-grained approach in which the protein is represented by a three-dimensional elastic network of alpha-carbon atoms connect ed by springs. Springs represent bonded and non-bonded interactions between the alpha-carbon atoms. In this work, we study th e elastic network models known in the literature. Next, we perform a comparative study between them. We show that the pfGNM a nd pfANM models present better correlation with experimental B-factors than the traditional GNM and ANM models. We also devel op a new approach, which we entitled anisotropic weighted contact number (AWCN). We show that it presents results significantly better than the traditional anisotropic elastic network model. Finally, we perform a study of investigative character of the behavior for the weight of the interactions between residues. This study revealed that, for the WCN and AWCN models, the correlation exhibits its maximum value for weighted interactions $1/R^p$, between residues $i$ and $j$, for values of $p$ around 2. In the pfGNM and pfANM models the correlation is max imized for two values of $p$, the first one around 2 and the second one around 4.75. This indicates that the weighting by the reciprocal of the square of the distance, usually employed in the literature, may not be appropriate to obtain the best correlation.

Sistemas alostéricos são característicos de proteínas com um ou mais estados de equilíbrio. Nesse sentido, uma enzima passa por modificações de sua atividade quando um substrato cooperativo se liga a um estado ou outro (1). Estes estados são reconhecidos por possuírem uma conformação mais estável e coexistirem num ensemble. Este trabalho sustenta que tais proteínas oscilem naturalmente entre esses estados. Experimentos de difração de raios-X e RMN, que proporcionam parâmetros de deslocamento anisotrópicos e tempos de relaxação de spin nuclear, já demonstram a coexistência de ambos estados em solução e descrevem o movimento como uma mudança de equilíbrio populacional dos confórmeros (2). Também é possível desenvolver métodos numéricos, como o cálculo de modos normais e a simulação de dinâmica molecular, para associar a geometria proteica a um movimento sobre determinado potencial de campos de força. O sistema adotado para o desenvolvimento desses estudos é a enzima alostérica Glucosamina-6-fosfato Desaminase. Características que defendem seu uso são sua reversibilidade catalítica, rápido equilíbrio cinético e muito baixa afinidade do estado T por ligantes. Sua estrutura também já foi resolvida por experimentos de cristalografia, identificando ambos estados alostéricos. E a caracterização das mudanças estruturais entre os estados T e R está bem estabelecida, identificando diferentes subunidades a distintos graus de rotação e prevendo uma oscilação de baixa frequência entre eles (3). Resultados obtidos neste projeto constituem: (a) uma dinâmica de 100ns partindo do estado T de toda a proteína (hexamérica) solvatada explicitamente, formando um ensemble NVT de 92000 átomos através do programa NAMD, usando o campo de forças CHARMM; (b) análise de componentes principais aproveitando esta dinâmica e usando algoritmos do programa Gromacs; (c) e análise de modos normais, em que os cálculos de minimização de energia foram feitos pelo programa Gromacs sob o campo de forças ENCADV, no vácuo. Análises desses resultados envolvem cálculos de RMSDs e flutuações, trajetórias calculadas para os autovetores oriundos de NMA ou de PCA, fatores de Debye-Waller e a confirmação visual (e gráficos de distância entre resíduos) de aproximação a um estado ou outro. Como a prévia caracterização da movimentação alostérica, identificava duas regiões para cada monômero como representativas de corpos rígidos, também é desenvolvida uma análise por tensores de inércia. Espera-se que, ao longo do tempo, essas subunidades se comportem como corpos quase rígidos e os movimentos destas regiões rígidas correspondam a uma maior representatividade da transição alostérica. Assim, a caracterização dos tensores seria capaz de filtrar movimentos de mais alta frequência que constituem ruído em relação a movimentos funcionais da proteína. - Algoritmos para cálculos matriciais dos tensores foram escritos em Fortran e em TCl. / Allosteric systems are characteristic of proteins with one or more equilibrium states. Such an enzyme experiences a modification of its activity when a cooperative substrate binds to a state or another, thus, establishing a change in population equilibrium (1). These states are recognized by having a more stable conformation, and they coexist in an ensemble. X-ray diffraction and NMR experiments already demonstrated this dynamic equilibrium, and simulation methods, as molecular dynamics and normal mode analysis, generally provide a more complete proof (2).The allosteric enzyme Glucosamine-6-Phosphate Deaminase appeared to be a good model to better understand the equilibrium dynamics as essential to the protein function, given its reversibility of the catalysis and rapid-equilibrium kinetic mechanism. It also has the structure elucidated for both its conformers (3). A computational approach would now give better perspective on how the conformational changes occur. A set of results of this latter kind were obtained: (a) a 100ns dynamic starting at the hexameric T conformer, explicitly solvated, building a NVT ensemble using NAMD program and CHARMM force field; (b) a principal components analysis making use of the calculated dynamic and of the Gromacs algorithms; (c) and normal mode analysis of the T conformer structure (pdb code 1fsf) minimized with Gromacs program using ENCADV vacuum force field. Not only the conventional analyses for these results (fluctuations and projections) were taken, but also an inertia tensor analysis was developed. As the allosteric conformational change, for this protein, was described by the displacement of only two rigid body subunits³, its description by inertia tensors should act as a filter for the high frequency and functionally uninteresting motions, which normally constitute only noise.

The objectives of the thesis work were to study theinteraction between embedded piezoceramic transducers andcomposite structures as well as determine techniques tosimplify the Lamb waves analysis. Firstly, this studyconsidered the design of the embedded piezoceramic transducers.Secondly, the effect of the embedded transducer on thecomposite strength as well as the influence of the mechanicallyloaded composite on the characteristics of the embeddedtransducer were investigated. Finally, to simplify the analysisof such complex Lamb wave responses, two techniques weredeveloped. They were based on the wavelet technique and amodelling technique, respectively. The design of the embedded piezoceramic transducers wasimproved by reducing the stress concentrations in the compositeas well as in all components constituting the piezoceramictransducer, that is, the piezoceramic element, interconnectorand conductive adhesive. The numerical analysis showed that thethickness of the interconnector had no significant influence onthe stress state of the piezoceramic transducer. It was alsofound that a compliant conductive adhesive reduced the stressconcentration located at the edge of the piezoceramic element.The structural integrity of composites embedded with theimproved piezoceramic transducer was investigated. Theexperiments, performed in tensile and compressive staticloading, indicated that the strength of the composite was notsignificantly reduced by the embedded piezoceramic transducer.Further investigations were conducted to evaluate theperformance of the improved piezoceramic transducer used as aLamb wave generator embedded in composites subjected tomechanical loading. The tests were conducted in tensile andcompressive static loading as well as fatigue loading. Thestudy showed a large working range of the embedded piezoceramictransducer. A post processing technique based on the waveletswas further assessed in the detection of damage and in thedamage size evaluation. A new wavelet basis was developedspecially for processing the Lamb wave response. This method,focused on the wavelet coefficients from the decomposition Lambwave response, showed promising results in evaluating thedamage size. The wavelets offered a sensitive tool to detectsmall damage, compared to other detection methods, improvingthe damage detection capabilities. The other technique wasdevoted to the simplification of the generated Lamb waves bythe use of multi-element transducers. The transducers weredesigned using both a normal-mode expansion and a FE-method.This technique allowed reducing the effect of a Lamb wave modetowards another. This technique was successfully implemented ina damage detection system in composites. <b>Keywords:</b>Embedded piezoceramic, transducer, composite,structural integrity, health monitoring, damage detection, Lambwaves, wavelets, normal-mode expansion, FE-method

<p>The objectives of the thesis work were to study theinteraction between embedded piezoceramic transducers andcomposite structures as well as determine techniques tosimplify the Lamb waves analysis. Firstly, this studyconsidered the design of the embedded piezoceramic transducers.Secondly, the effect of the embedded transducer on thecomposite strength as well as the influence of the mechanicallyloaded composite on the characteristics of the embeddedtransducer were investigated. Finally, to simplify the analysisof such complex Lamb wave responses, two techniques weredeveloped. They were based on the wavelet technique and amodelling technique, respectively.</p><p>The design of the embedded piezoceramic transducers wasimproved by reducing the stress concentrations in the compositeas well as in all components constituting the piezoceramictransducer, that is, the piezoceramic element, interconnectorand conductive adhesive. The numerical analysis showed that thethickness of the interconnector had no significant influence onthe stress state of the piezoceramic transducer. It was alsofound that a compliant conductive adhesive reduced the stressconcentration located at the edge of the piezoceramic element.The structural integrity of composites embedded with theimproved piezoceramic transducer was investigated. Theexperiments, performed in tensile and compressive staticloading, indicated that the strength of the composite was notsignificantly reduced by the embedded piezoceramic transducer.Further investigations were conducted to evaluate theperformance of the improved piezoceramic transducer used as aLamb wave generator embedded in composites subjected tomechanical loading. The tests were conducted in tensile andcompressive static loading as well as fatigue loading. Thestudy showed a large working range of the embedded piezoceramictransducer. A post processing technique based on the waveletswas further assessed in the detection of damage and in thedamage size evaluation. A new wavelet basis was developedspecially for processing the Lamb wave response. This method,focused on the wavelet coefficients from the decomposition Lambwave response, showed promising results in evaluating thedamage size. The wavelets offered a sensitive tool to detectsmall damage, compared to other detection methods, improvingthe damage detection capabilities. The other technique wasdevoted to the simplification of the generated Lamb waves bythe use of multi-element transducers. The transducers weredesigned using both a normal-mode expansion and a FE-method.This technique allowed reducing the effect of a Lamb wave modetowards another. This technique was successfully implemented ina damage detection system in composites.</p><p><b>Keywords:</b>Embedded piezoceramic, transducer, composite,structural integrity, health monitoring, damage detection, Lambwaves, wavelets, normal-mode expansion, FE-method</p>

Sistemas alostéricos são característicos de proteínas com um ou mais estados de equilíbrio. Nesse sentido, uma enzima passa por modificações de sua atividade quando um substrato cooperativo se liga a um estado ou outro (1). Estes estados são reconhecidos por possuírem uma conformação mais estável e coexistirem num ensemble. Este trabalho sustenta que tais proteínas oscilem naturalmente entre esses estados. Experimentos de difração de raios-X e RMN, que proporcionam parâmetros de deslocamento anisotrópicos e tempos de relaxação de spin nuclear, já demonstram a coexistência de ambos estados em solução e descrevem o movimento como uma mudança de equilíbrio populacional dos confórmeros (2). Também é possível desenvolver métodos numéricos, como o cálculo de modos normais e a simulação de dinâmica molecular, para associar a geometria proteica a um movimento sobre determinado potencial de campos de força. O sistema adotado para o desenvolvimento desses estudos é a enzima alostérica Glucosamina-6-fosfato Desaminase. Características que defendem seu uso são sua reversibilidade catalítica, rápido equilíbrio cinético e muito baixa afinidade do estado T por ligantes. Sua estrutura também já foi resolvida por experimentos de cristalografia, identificando ambos estados alostéricos. E a caracterização das mudanças estruturais entre os estados T e R está bem estabelecida, identificando diferentes subunidades a distintos graus de rotação e prevendo uma oscilação de baixa frequência entre eles (3). Resultados obtidos neste projeto constituem: (a) uma dinâmica de 100ns partindo do estado T de toda a proteína (hexamérica) solvatada explicitamente, formando um ensemble NVT de 92000 átomos através do programa NAMD, usando o campo de forças CHARMM; (b) análise de componentes principais aproveitando esta dinâmica e usando algoritmos do programa Gromacs; (c) e análise de modos normais, em que os cálculos de minimização de energia foram feitos pelo programa Gromacs sob o campo de forças ENCADV, no vácuo. Análises desses resultados envolvem cálculos de RMSDs e flutuações, trajetórias calculadas para os autovetores oriundos de NMA ou de PCA, fatores de Debye-Waller e a confirmação visual (e gráficos de distância entre resíduos) de aproximação a um estado ou outro. Como a prévia caracterização da movimentação alostérica, identificava duas regiões para cada monômero como representativas de corpos rígidos, também é desenvolvida uma análise por tensores de inércia. Espera-se que, ao longo do tempo, essas subunidades se comportem como corpos quase rígidos e os movimentos destas regiões rígidas correspondam a uma maior representatividade da transição alostérica. Assim, a caracterização dos tensores seria capaz de filtrar movimentos de mais alta frequência que constituem ruído em relação a movimentos funcionais da proteína. - Algoritmos para cálculos matriciais dos tensores foram escritos em Fortran e em TCl. / Allosteric systems are characteristic of proteins with one or more equilibrium states. Such an enzyme experiences a modification of its activity when a cooperative substrate binds to a state or another, thus, establishing a change in population equilibrium (1). These states are recognized by having a more stable conformation, and they coexist in an ensemble. X-ray diffraction and NMR experiments already demonstrated this dynamic equilibrium, and simulation methods, as molecular dynamics and normal mode analysis, generally provide a more complete proof (2).The allosteric enzyme Glucosamine-6-Phosphate Deaminase appeared to be a good model to better understand the equilibrium dynamics as essential to the protein function, given its reversibility of the catalysis and rapid-equilibrium kinetic mechanism. It also has the structure elucidated for both its conformers (3). A computational approach would now give better perspective on how the conformational changes occur. A set of results of this latter kind were obtained: (a) a 100ns dynamic starting at the hexameric T conformer, explicitly solvated, building a NVT ensemble using NAMD program and CHARMM force field; (b) a principal components analysis making use of the calculated dynamic and of the Gromacs algorithms; (c) and normal mode analysis of the T conformer structure (pdb code 1fsf) minimized with Gromacs program using ENCADV vacuum force field. Not only the conventional analyses for these results (fluctuations and projections) were taken, but also an inertia tensor analysis was developed. As the allosteric conformational change, for this protein, was described by the displacement of only two rigid body subunits³, its description by inertia tensors should act as a filter for the high frequency and functionally uninteresting motions, which normally constitute only noise.

Submitted by Pedro Silva Filho (pfsilva@ipen.br) on 2018-01-31T16:47:23Z No. of bitstreams: 0 / Made available in DSpace on 2018-01-31T16:47:23Z (GMT). No. of bitstreams: 0 / Asma é uma doença de elevada prevalência e morbidade, constituindo-se um importante problema de saúde pública em todo o mundo. Muitos diagnósticos médicos por imagem dependem da utilização de raio X; entretanto, no caso da asma, o diagnóstico é clínico, particularmente na criança. Os pacientes são frequentemente submetidos a procedimentos radiológicos, preferencialmente o raio X de tórax. A escassez de dados na literatura referentes aos riscos da exposição à radiação ionizante em pacientes com asma e os possíveis benefícios relacionados à oportunidade de coleta de informações justificaram a investigação no programa municipal infantil de controle da asma de Ipatinga, Minas Gerais (Programa Respirar). Foi realizado estudo retrospectivo referente ao ano de 2014, com caso controle, do qual participaram 363 pacientes do Programa Respirar e número semelhante de controle, sem diferença sociodemográfica significativa entre os grupos. Encontramos que uma criança do grupo respirar tem 1,59 vez mais chance de realizar um raio X, e para o raio X de tórax essa chance aumenta para 6,56 vezes. A maioria dos raios X de tórax foi solicitada nas visitas aos serviços de urgência, mas 90% dos raios X realizados e laudados no grupo respirar e 84,2% do grupo controle revelaram resultado normal ou com alterações típicas, possibilitando o questionamento a respeito da indicação do exame. / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP