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It has been observed that confusion exists in literature concerning the meaning and use of the term efficacy. Confusion is worsened by the use of the term as a general term describing agonist activity. The meaning of the terms intrinsic activity, efficacy and intrinsic efficacy as used in theoretical models of drug action was investigated. The classical occupation model, the two-state model, the ternary complex model (including conformational change and ideas surrounding G-proteins) and the operational model were studied in order to understand the historical and current usage of these terms. Although efficacy estimates are often reported as a molecular property, it was shown that agonist activity is tissue dependent and cannot be fully portrayed by an efficacy estimate. It was found that efficacy has a different definition in each model. This is not always recognized in literature. It was suggested that the term efficacy should only be used in the context of a specific model / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2007.

Organic-inorganic nanostructured composites are nowadays integrated in the field of material science and technology. They are used as advanced materials directly or as precursors to novel composites with potential applications in optics, mechanics, energy, catalysis and medicine. Many properties of these complex materials depend on conformational rearrangements in their inherently dynamic organic parts. The focus of this thesis is on the study of the molecular mobility in ordered nanostructured composites and lyotropic mesophases and also on the development of relevant solid-state NMR methodologies. In this work, a number of new experimental approaches were proposed for dipolar NMR spectroscopy for characterizing molecular dynamics with atomic-level resolution in complex solids and liquids. A new acquisition scheme for two-dimensional dipolar spectroscopy has been developed in order to expand the spectral window in the indirect dimension while using limited radio-frequency power. Selective decoupling of spin-1 nuclei for sign-sensitive determination of the heteronuclear dipolar coupling has been described. A new dipolar recoupling technique for rotating samples has been developed to achieve high dipolar resolution in a wide range of dipolar coupling strength. The experimental techniques developed herein are capable of delivering detailed model-independent information on molecular motional parameters that can be directly compared in different composites and their bulk analogs. Solid-state NMR has been applied to study the local molecular dynamics of surfactant molecules in nanostructured organic-inorganic composites of different morphologies. On the basis of the experimental profiles of local order parameters, physical motional models for the confined surfactant molecules were put forward. In layered materials, a number of motional modes of surfactant molecules were observed depending on sample composition. These modes ranged from essentially immobilized rigid states to highly flexible and anisotropically tumbling states. In ordered hexagonal silica, highly dynamic conformationally disordered chains with restricted motion of the segments close to the head group have been found. The results presented in this thesis provide a step towards the comprehensive characterization of the molecular states and understanding the great variability of the molecular assemblies in advanced nanostructured organic−inorganic composite materials. / <p>QC 20150225</p>

While great strides have been made in treating many classes of human disease, the late-onset neurodegenerative diseases continue to elude modern medicine. These diseases, which include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), the transmissible spongiform encephalopathies (TSEs), and amyotrophic lateral sclerosis (ALS), involve accumulation of insoluble aggregates of one or more causative proteins, leading to progressive loss of central nervous system neurons, progressively worsening neurological symptoms, and eventual patient death. All of these diseases are currently incurable and fatal. In the case of ALS, progressive death of upper and lower motor neurons leads to full-body paralysis, respiratory difficulty, and patient death. Of the subset of ALS cases showing familial inheritance, approximately 20% are caused by mutations in the SOD1 gene, encoding the Cu, Zn superoxide dismutase (SOD1). These mutations do not have the common property of impairing SOD1's normal function as a free radical scavenger. Instead, they are thought to increase the protein's likelihood of misfolding and aggregating via a poorly-understood aggregation cascade. It is believed that species populated along the misfolding and aggregation pathway may prove to be good targets for therapies designed to block accumulation of downstream toxic species, or to prevent aberrant protein-protein interactions responsible for neurotoxicity. In this thesis, several new techniques are developed to enable detailed elucidation of the SOD1 unfolding and misfolding pathways. Time-resolved measurements collected during SOD1 unfolding or misfolding of release of bound Cu and Zn, of changes in intrinsic fluorescence, of exposure of hydrophobic surface area, and of alterations in the chemical environment of histidine residues, are presented. A new mathematical analysis technique named the Analytical Laplace Inversion Algorithm is developed for rapid extraction of mechanistic information from these time-resolved signals. These tools are applied to the construction of the most detailed models to date of the unfolding and misfolding mechanisms of WT and ALS-causing mutant SOD1. The models presented identify several well-populated unfolding and misfolding intermediates that could serve as good targets for therapies designed to address the fundamental molecular mechanisms underlying SOD1-associated ALS, and to treat what is currently a devastating and incurable disease.

Cette thèse a pour but de développer le pendant in silico des étapes clés du Fragment-Based Drug Design (FBDD), et ce dans le cadre plus général du développement de l'outil S4MPLE. Le FBDD génère des ligands drug-like à partir de petites molécules (fragments). Après une étape de validation de S4MPLE et de sa fonction d’énergie, un recentrage autour du FBDD est réalisé, à travers le docking puis l'optimisation virtuelle de fragments par growing ou linking (G/L). Cette stratégie reposesur 1) la création d’une chimiothèque focalisée en connectant un ou deux fragment(s) avec des linkers pré-générés, et 2) l’échantillonnage avec S4MPLE des composés chimères dans le site avec des contraintes. Des simulations de G/L plus ou moins ambitieuses (site flexible, ajout de H2O libres) permettent de valider cette approche avec des études rétrospectives basées sur des données expérimentales. La dernière phase de la thèse a consisté à appliquer ce protocole in silico à un projet de l’entreprise. / This work aims to develop in silico methods targeting the key stages of Fragment-Based Drug Design (FBDD), participating to the development of the molecular modeling tool S4MPLE. Briefly, FBDD generates ıdrug-likeı ligands from small organic molecules called fragments. After a validation step of S4MPLE and its energy function, the work focused on FBDD: molecular docking of fragments and their subsequent virtual optimization. The latter mimics standard evolution strategies in FBDD(growing and linking). This in silico approach involves among other two key stages 1) building of a focused library by plugging in pre-generated linkers into reference fragments using rules and 2) sampling of these new compounds under atomic and binding site constraints. Validation simulations, relying on known experimental data, included ıclassicalı growing / linking and more challenging ones (site flexibility, free waters). Finally, this strategy is applied to one project of the company.

Made available in DSpace on 2015-07-22T20:49:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-29. Added 1 bitstream(s) on 2015-08-11T03:26:18Z : No. of bitstreams: 1 Publico-13280.pdf: 1828173 bytes, checksum: df8fb9928c37e920c5f9a2281ba9c092 (MD5) / O presente trabalho trata do estudo conformacional de S-nitrosotióis com potencial atividade biológica, 2–acetamido-3-metil-3-nitrosossulfanil-N-arilbutanamidas, e de seus tióis precursores, 2–acetamido-3-mercapto-3-metil-N-arilbutanamidas. As conformações de menor energia dos S-nitrosotióis e tióis em estudo são estabilizadas por ligações de hidrogênio intramoleculares que promovem uma maior estabilidade dos confôrmeros. A análise geométrica do grupo R-SNO mostra que esses compostos preferem a conformação trans. O cálculo das interações orbitalares pelo método NBO (Natural Bond Orbital) para as 2–acetamido-3-mercapto-3-metil-N-arilbutanamidas mostrou que as mesmas são estabilizadas pelas seguintes interações: no (N2) → &#61552;&#61482; (C3-O4) e no(N10) → (C11-O12). Os resultados de NBO para os S-nitrosotíois mostraram que a interação hiperconjugativa é bastante efetiva nas conformações estáveis desses compostos, enfraquecendo a ligação que resulta no aumento do comprimento da ligação S-N em S-Nitrosotióis. A forte delocalização , induz caráter parcial a ligação S-N. A fraca ligação S-N indica uma forte delocalização do par de elétrons do O(NO) devido a interação, que é responsável pelo alongamento da ligação S-N, aumentando e a potencial capacidade do óxido nítrico ser liberado. / We carried out a conformational study on the S-nitrosothiols (R-SNO), 2-acetamido-3-methyl-3-(nitrososulfanyl)-N-arylbutanamides and their thiol precursors 2-acetamido-3-mercapto-3-methyl-N-arylbutanamides. The lowest energy conformation for both compounds is stabilized by intramolecular hydrogen bonds. Trans conformation was determined as the predominant conformation after geometrical analysis of R-SNO. Orbital interactions for 2-acetamido-3-mercapto-3-methyl-N-arylbutanamides were calculated using Natural Bond Orbital (NBO) methodology. Calculations indicated that orbital interactions for these compounds are stabilized by the following interactions: no (N2) → &#61552;&#61482; (C3-O4) and no(N10) → (C11-O12). NBO results showed that the hyperconjugative interaction is very effective, weakening the σ bond and resulting in increasing length of the S-N bond in R-SNO. The strong delocalization induces partial character to the S-N bond. The bond S-N indicates a strong delocalization of the electron pair of O(NO) due to interaction. This interaction is responsible for the elongation of the S-N bond which increases the ability of the compound to release nitric oxide (NO). Based on the enhanced capacity to release NO by these compounds, our findings suggest that both compounds may display biological activity. / TEDE / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2015-07-22T20:50:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-01-27. Added 1 bitstream(s) on 2015-08-11T03:25:32Z : No. of bitstreams: 1 Publico-00399a.pdf: 1787027 bytes, checksum: 6cb684251c1894e3685b611ace5c9056 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:32Z : No. of bitstreams: 2 Publico-00399a.pdf: 1787027 bytes, checksum: 6cb684251c1894e3685b611ace5c9056 (MD5) Publico-00399b.pdf: 1285455 bytes, checksum: 52050426f68cebcd4b7e00081ce49a38 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:32Z : No. of bitstreams: 3 Publico-00399a.pdf: 1787027 bytes, checksum: 6cb684251c1894e3685b611ace5c9056 (MD5) Publico-00399b.pdf: 1285455 bytes, checksum: 52050426f68cebcd4b7e00081ce49a38 (MD5) Publico-00399c.pdf: 2029206 bytes, checksum: 480bbfb2deea834d9cc92a52def4805e (MD5) / A gomesina (Gm) é um potente peptídeo antimicrobiano catiônico. Este peptídeo e seu análogo linear [Ser2,6,11,15]-Gm, com menor atividade lítica, foram sintetizados pela metodologia da fase sólida, empregando-se a estratégia t-Boc. Neste estudo, investigamos a interação da Gm e da [Ser2,6,11,15]-Gm com vesículas unilamelares gigantes (GUVs), compostas por membranas lipídicas de POPC ou POPC/POPG, através de microscopias óticas de contraste de fase e fluorescência. As análises se dividiram em duas diferentes partes. Na primeira, observou-se o efeito da injeção de uma solução peptídica, com micropipeta, na vizinhança das GUVs. Como resultado da interação peptídeo/lipídio, as GUVs foram desestabilizadas e romperam-se rapidamente, sem observação de formação de poros estáveis durante todo o experimento. Nos estudos controles, em ausência de peptídeo, as GUVs não romperam de forma espontânea. Peptídeos marcados com a sonda fluorescente rodamina (Rh) foram injetados e mostram que a Gm primeiramente acumula-se na superfície da vesícula, na qual, então, pequenas partículas de alto-contraste são formadas e ocorre, eventualmente, a destruição das GUVs. Este fato leva-nos a especular que a Gm rompe as membranas via modo carpete. Na segunda parte, uma solução contendo GUVs foi misturada a crescentes concentrações peptídicas para quantificação da ação lítica destes peptídeos em vesículas de diferentes composições lipídicas. O efeito de atividade lítica observado foi maior que 90% em baixas concentrações tanto de Gm quanto de [Ser2,6,11,15]-Gm em GUVs compostas de POPC, lipídio eletricamente neutro, e de uma mistura de POPG, negativamente carregados, a POPC em diferentes proporções. Estudos conformacionais foram feitos por duas técnicas espectroscópicas distintas: dicroísmo circular (CD) e fluorescência. A Gm e seu análogo linear aparentemente interagiram com as cargas negativas de SDS nas concentrações acima e abaixo da CMC. Os espectros de CD da [Ser2,6,11,15]-Gm em água apresentaram uma banda fortemente negativa em 198 nm, indicando tratar-se de uma estrutura desordenada, como esperado para peptídeos livres em solução, e não de uma dobra beta como apresentada pela Gm. A partir dos resultados obtidos, foi possível concluir que ambos os peptídeos analisados interagem com vesículas compostas por fosfolipídios e induzem o vazamento de seu conteúdo interno dependentemente da carga de membrana destas, o que corrobora estudos prévios que sugerem que as interações eletrostáticas com a bicamada lipídica dos micro-organismos representam um importante papel no mecanismo de ação da Gm. / TEDE / BV UNIFESP: Teses e dissertações

Les structures cristallographiques de macromolécules comme les protéines, obtenues par la biologie structurale sont des modèles statiques. Or, c'est la flexibilité et la dynamique de ces macromolécules qui sont généralement responsables de leurs fonctions. La simulation permet d'explorer cette flexibilité lors de différents phénomènes qui ont lieu dans ces systèmes : une réaction chimique, des interactions avec une petite molécule… Simuler de tels phénomènes est un défi car la dynamique moléculaire classique ne permet pas de les observer. Des algorithmes permettent d'accélérer l'échantillonnage des dynamiques pour lever cette limitation et de calculer les barrières d'activation pour de tels phénomènes. Simultanément, le choix du niveau de calcul est crucial car il faut concilier la taille importante des systèmes, la nature des interactions et les phénomènes électroniques impliqués. Dans ce travail, différentes méthodes, dont principalement la métadynamique soit au niveau classique ou quantique, ou encore en combinant les deux niveaux quantique/classique, seront utilisées pour modéliser quatre processus complexes : des changements de conformations d'une protéine, des interactions entre métalloprotéine et inhibiteur, des réactions en solution et dans une enzyme. / Crystallographic structures of macromolecules, such as proteins, obtained by structural biology are static models. However, flexibility and dynamics of macromolecules are generally responsible for their functions. Modeling allows us to explore this flexibility in different phenomena that take place in these systems: a chemical reaction, interaction with a small molecule... Modeling such phenomena is a challenge because they cannot be observed by classical molecular dynamics. Algorithms can accelerate sampling of dynamics to simulate these events and calculate their activation barriers. Simultaneously, the choice of the level of calculation is crucial because it must merge with the size of the systems, the nature of interactions and the electronic phenomena involved.In this thesis, some methods, mainly metadynamics at classical level, quantum or the hybrid quantum/classical level, will be used to model four complex processes: conformational changes of proteins, metalloprotein/inhibitor interactions, reactivity in solution and enzymatic reactivity.

Protein misfolding is considered to be the major pathogenic mechanism in homocystinuria due to cystathionine beta-synthase (CBS) deficiency. The aim of this work was to study molecular mechanisms underlying protein misfolding of CBS mutants. Firstly, we studied spatial arrangement of normal human CBS protein. Using data from differential covalent labeling of solvent-exposed aminoacid residues, we identified interdomain contact area between the catalytic core and the regulatory domain in human CBS, and we subsequently generated the structural model of the full-length CBS. In the next step, we studied evolutionary divergence of CBS protein structures. We performed phylogenetic analysis that revealed unique spatial arrangement of CBS enzyme in nematodes; the domain architecture of CBS in Caenorhabditis elegans was studied experimentally in more detail. Finally, we determined conformational properties of a representative set of human CBS mutants that exhibited in various extent affected formation of tetramers and decreased catalytic activity. Using thermolysin-based proteolytic techniques for analysis of nine mutants expressed in E.coli, we found that an unfolded structure is a common intermediate occurring in CBS misfolding. The importance of protein unfolding for pathogenesis of CBS deficiency was...

A radioterapia externa pós-operatória em mulheres diagnosticadas com câncer de mama em estágio inicial é tido como um procedimento padrão, no entanto durante o planejamento para irradiação do volume alvo as possíveis incertezas dosimétricas introduzidas dado o movimento respiratório intrínseco da paciente são desconsideradas. Este estudo avalia não apenas a influência da respiração na distribuição tridimensional da dose, mas como essa distribuição se modifica dado a técnica radioterápica empregada para o tratamento. Três técnicas de planejamento foram analisadas: a radioterapia conformacional tridimensional (3D-RT) com filtros, a radioterapia com intensidade modulada (IMRT) usando planejamento direto e o IMRT inverso. A fim de simular o movimento de contração e expansão da caixa torácica, utilizou-se uma plataforma com amplitudes de oscilação pré-determinadas, sendo a frequência de oscilação provida por uma fonte de tensão variável. Para simular a mama usou-se objetos simuladores semiesféricos preenchidos com gel dosimétrico (MAGIC-f). Os planejamentos para cada técnica foram realizados sobre a mesma tomografia computadorizada (CT) do objeto simulador preenchido com água no modo estático. Foram produzidos três lotes de dosímetro gel para o projeto, cada lote foi irradiado com uma técnica radioterápica diferente, sendo que cada lote inclui cinco objetos simuladores e um conjunto de nove tubos de calibração preenchidos com gel MAGIC-f. O primeiro dos objetos simuladores é utilizado como referência, o segundo é irradiado no modo estático, os demais são irradiados em diferentes amplitudes, respectivamente: 0,34 cm, 0,88 cm e 1,22 cm. A informação volumétrica de dose foi obtida utilizando imagens por ressonância magnética nuclear (IRMN), para cada lote foram adquiridos IRMN com sequência multi spin echo e os mapas de relaxometria, que são associados à dose, foram extraídos em um software desenvolvido e aprimorado pelo nosso grupo de pesquisa. A comparação quantitativa dos mapas de relaxometria dos objetos simuladores em movimento em relação ao modo estático foi realizado pelo índice gamma tridimensional (3% / 3mm / 15% Threshold). Para o 3D-RT a porcentagem de pontos aprovados do objeto estático em relação ao oscilante na amplitude de 0,34 cm foi de 96,44%, para amplitude de 0,88 cm foi de 93,23% e para amplitude de 1,22 cm foi de 91,65%. Para o IMRT direto a porcentagem de pontos aprovados do objeto estático em relação ao oscilante na amplitude de 0,34 cm foi de 98,42%, para amplitude de 0,88 cm foi de 95,66% e para amplitude de 1,22 cm foi de 94,31%. Para o IMRT inverso a porcentagem de pontos aprovados do objeto estático em relação ao oscilante na amplitude de 0,34 cm foi de 94,49%, para amplitude de 0,88 cm foi de 93,51% e para amplitude de 1,22 cm foi de 86,62%. A partir dos resultados, infere-se que a movimentação respiratória de baixa amplitude, para tratamentos de câncer de mama, não é um fator preocupante para a rotina clínica, porém o aumento da amplitude da oscilação aumenta a inomogeneidade de dose e pode afetar os parâmetros dosimétricos da cobertura do volume alvo em relação ao planejamento do tratamento. Observou-se em conjunto que a distribuição de dose se modifica claramente com a técnica em uso e no caso do IMRT inverso para amplitude de oscilação de 1,22 cm a aprovação no índice gamma foi menor que 90% / External postoperative radiotherapy in women diagnosed with early stage breast cancer is considered as a standard procedure, however during planning for target volume irradiation as possible dosimetric uncertainties reabsorption of the patient\'s intrinsic respiratory movement are disregarded. This study evaluates not only the influence of respiration on the three-dimensional distribution of the dose but how this distribution is modified due to the radiotherapy technique used for treatment. Three planning techniques were analyzed: three-dimensional conformational radiotherapy (3D-RT) with filters, intensity-modulated radiotherapy (IMRT) using direct planning and inverse IMRT. In order to simulate the movement of contraction and expansion of the chest wall, a platform with predetermined oscillation amplitudes was used, the oscillation frequency was provided by a variable voltage source. To simulate the breast, semi-spherical simulator objects filled with dosimetric gel (MAGIC-f) were used. The plannnings for each technique were performed on the computerized tomography (CT) of the simulator object filled with water in static mode. Three batches of gel dosimeters were prepared for the project, each batch was irradiated with a different radiothermic technique and comprised five simulator objects and a set of nine calibration tubes filled with MAGIC-f gel. The first simulator objects is used as reference, the second is irradiated in the static mode, the others are irradiated using different amplitudes, respectively: 0,34 cm, 0,88 cm and 1,22 cm. Volumetric dose information was obtained using Nuclear Magnetic Resonance Imaging, each batch was scanned with a multi spin echo sequence and the dose-related relaxometry maps were extracted in a software developed and improved by our Group of research. The quantitative comparison of the relaxometry maps of the moving simulator objects with respect to the static mode was performed by the three-dimensional gamma index (3% / 3mm / 15% threshold). For the 3D-RT, the percentage of approved points of the static object with respect to the oscillator in the amplitude of 0.34 cm was 96.44%, for amplitude of 0.88 cm was 93.23% and for amplitude of 1.22 cm was 91.65%. For the direct IMRT the percentage of approved points of the static object in relation to the oscillator in the amplitude of 0.34 cm was 98.42%, for amplitude of 0.88 cm was 95.66% and for amplitude of 1.22 cm was 94.31%. For the inverse IMRT, the percentage of approved points of the static object in relation to the oscillator in the amplitude of 0.34 cm was 94.49%, for amplitude of 0.88 cm was 93.51% and for amplitude of 1.22 cm was 86.62%. From the results, it is inferred that a low-amplitude respiratory movement, for breast cancer treatments, is not a worrying factor for clinical routine, however, increasing the amplitude of the oscillation increases the inomogeneity of the dose and this affects the dosimetry parameters of the target volume coverage. It was observed that the dose distribution changes with the technique in use and in the case of the inverse IMRT for amplitude of oscillation of 1.22 cm, less than 90% of points were approved in the gamma index evaluation