Les propriétés mécaniques des polymères et des copolymères à blocs ont été étudiées par simulation de type dynamique moléculaire (modèle billes-ressorts). Les échantillons polymères ont été générés par la méthode de « radical like polymerisation ». Ces échantillons ont été soumis à des essais de traction uniaxiaux et triaxiaux dans le but d’étudier leurs réponses mécaniques. Dans la première partie de ce travail on a comparé deux méthodes de traction : « méthode de traction homogène» et la traction « pilotée par les bords » de l’échantillon. Les résultats montrent que les deux méthodes sont équivalentes à faible vitesse de traction. Le changement de distance entre enchevêtrement dans un polymère modèle sous traction est analysé, les résultats montrent que le désenchevêtrèrent des chaines est plus prononcé lorsque la déformation de l’échantillon est uniaxiale du fait de la relaxation latérale de l’échantillon. La nucléation des cavités dans les polymères amorphes soumis à une déformation triaxial a été également étudiée. On a trouvé que les cavités se forment dans des zones qui sont caractérisées par un faible module d’incompressibilité élastique. Ces zones sont identifiables dès le début de la déformation à une température très basse (T~0K). La seconde partie de ce travail se concentre sur la simulation de la réponse mécanique des copolymères à blocs. L’influence de l’architecture moléculaire sur le comportement mécanique de l’échantillon a été analysée. Les résultats montrent que le comportement mécanique des échantillons est piloté par le taux des chaines liantes qui assurent la transmission des contraintes entre les phases. Le flambement des lamelles dans les copolymères à blocs a été également étudié, l’influence de la taille de l’échantillon et de la vitesse de déformation sur la réponse mécanique de l’échantillon a été explorée. Les résultats montrent un changement de mode du flambement selon la vitesse de déformation imposée. Un nouveau modèle qui prend en compte le facteur cinétique du flambement est proposé pour décrire la compétition entre les modes. / We use molecular dynamics simulation of a coarse grained model to investigate the mechanical properties of homogenous polymers and lamellar block copolymers. Polymer samples have been generated using “radical like polymerisation” method. These samples were submitted to uniaxial and triaxial tensile tests in order to study their mechanical responses. First we compare two tensile test methods: the “homogenous deformation method” and the “boundary driven deformation method”. We find that the two methods lead to similar results at low strain rate. The change of the entanglement network in polymer sample undergoing a tensile deformation was investigated. We have found that the sample exhibits an increase of its entanglement length in uniaxial deformation test compared to triaxial deformation one. Our finding was interpreted by the pronounced chain disentanglement observed in the uniaxial deformation test due to the lateral relaxation of the sample. The cavity nucleation in amorphous polymers has been also studied. We have found that the cavities nucleate preferentially in zones that exhibit a low elastic bulk modulus. These zones can be identified from the initial undeformed state of the sample at low temperature (T~0K). The second part of the work focused in the simulation of the mechanical response of block copolymers. The influence of chain architecture on the mechanical properties was investigated: our finding reveals an important role of the bridging molecules (cilia chains and knotted loop chains) on the stress transmission between phases at high strain. The initiation of plasticity in copolymer samples was also studied. The role of the buckling has been found to be determinant in the mechanical response of the sample The dependence of the buckling instability with the sample size and the deformation rate was investigated. We have found that the fundamental (first) mode of buckling develops at relatively low strain rate whereas at high strain rate the buckling of the sample occurs with the second or higher mode of buckling. A new model that takes into account the buckling kinetic was developed to describe this competition between the buckling modes.

As cisteíno-proteases da família da papaína desempenham funções essenciais em processos biológicos, entre eles o desenvolvimento e crescimento do organismo, vias de sinalização celular e apoptose, invasão de parasitas em células hospedeiras. Assim, trata-se de uma classe de proteínas de grande interesse para as indústrias farmacêuticas, sendo utilizada como alvo para o tratamento de doenças como o câncer e metástases, osteoporose. Disfunções relacionadas ao sistema imune, doenças parasitárias como malária, leishmaniose, doença do sono e doença de Chagas. Esta última é uma enfermidade considerada negligenciada pelas grandes indústrias farmacêuticas, sem nenhum tratamento eficaz e seguro disponível, que gera um problema econômico de mais de sete bilhões de dólares anuais devido à perda de mão de obra e gastos com tratamento para amenizar os efeitos da doença. A cisteíno protease cruzaína de Trypanosoma cruzi, causador da Doença de Chagas, desponta como um alvo validado na busca de novos fármacos contra essa enfermidade. Essa enzima apresenta um par de aspartatos que interagem entre si, para os quais foi predito um pKa de 7, sendo possível a forma desprotonada desse par em condições biológicas. Neste caso, pode levar à exposição de uma nova cavidade por meio do movimento da alça entre os resíduos 57-62, segundo as simulações de dinâmica molecular desse trabalho, que se trata de uma possível candidata a ponto de seletividade de inibidores de cisteínoproteases de parasitos em relação às suas ortólogas em Homo sapiens que não possuem o par de aspartatos. Em pH ácido, foi mostrado por meio de análise de componentes principais de simulações de dinâmica molecular que as cisteíno protease apresentam uma restrição gradual na amostragem conformacional do sítio ativo quando complexadas com as formas não covalente e covalente de inibidores derivados de dipeptidil nitrilas. Isso sugere que esse sistema segue o modelo de seleção conformacional para flexibilidade de proteína. Notou-se também que o perfil de restrição de ligantes que inibem na faixa de nmol.L-1 difere daqueles a µmol.L-1 , o que possibilitou a construção de uma árvore de decisão para identificar os complexos que apresentam afinidade a nmol.L-1 . / The papain-like cysteine proteases are essentials for biological process, performing important roles on the parasite development, growth and also in the parasite invasion process on the host cell, in cellular signaling pathways and apoptosis, among others. Thus, the pharmaceutical industry widely uses this class of protein as target for the development of new drugs, against cancer and metastasis, osteoporosis and immune system disorders, resulting in many approved drugs. Additionally, these enzymes are validated target against parasitic diseases as leishmaniose, malaria and African and American trypanosomiasis. The last one, also known as Chagas\' disease, is neglected disease for which, further a century form this discovery, there is no effective and safe chemotherapy and is responsible for an economic loss of around seven billion dollars in the world per year due to the health care and lost productivity from infected people. Faced with this situation, the Cruzain, a cysteine protease from the Trypanosoma cruzi, the causative agent of Chagas\' disease, is emerging as interesting and validated target to the search for new drugs against this sickness. This enzyme has a pair of interactiong Asp for which was predicted a pKa of 7, by computational methods. By this way, this pair under neutral to alkaline pH adopts the deprotonated form, which exposed a new cavity through a movement of loop of residues 57-62, as we showed here by molecular dynamics simulations. This cavity emerges as a possible selectivity point of the cruzain inhibitors, once Homo sapiens enzynes does not present the aspartic acid - aspartate pair. In condition of acidic pH, principal component analysis of molecular dynamics simulations showed a gradual reduction of the conformational space covered by the active site of cruzain, cathepsin K and cathepsin L in it free form and complexed with dipeptidyl nitrilelike molecules in it noncovalent e covalent forms. This suggests these systems follows the conformational selection model of protein flexibility. Furthermore, we observed the ligands that inhibits the protein at nmol.L-1 induces the protein flexibility in a similar way, while the µmol.L-1 ones leads to another pattern. That made possible the construction of a decision tree which is able to identify nmol.L-1 from µmol.L-1 complexes.

As alquilfosfocolinas (APC) são uma classe de fármacos derivados de fosfolipídios endógenos que apresentam potencial antitumoral. Diferentemente de outros fármacos antitumorais que agem no DNA da célula, as APCs têm como primeiro local de ação a membrana plasmática e proteínas de sinalização, como a PKC. O objetivo desse trabalho é elucidar, através de metodologias computacionais, os possíveis mecanismos de ação das APCs que provocam hemólise, inibição da PKC e interação com membranas celulares. Inicialmente, a toxicidade de um conjunto de 34 APCs foi estudada pelos métodos quimiométricos de Análise de Agrupamentos Hierárquicos (HCA) e Componentes Principais (PCA). As moléculas foram simuladas com dinâmica molecular (DM) e propriedades físico-químicas e estruturais foram calculadas para os confôrmeros de menor energia. Após aplicação de HCA e PCA, as APCs foram divididas em 3 grupos, de acordo com suas características estruturais. Os resultados sugerem que a presença de grupos catiônicos volumosos, ou anéis como adamantila e ciclohexila, aumentam a hemólise de compostos de cadeia alquílica longa. Anéis macrocíclicos como ciclopentadecila parecem ser importantes para o potencial hemolítico de compostos com cadeia alquílica curta. Com relação a compostos sem anéis e de cadeia linear, grupos catiônicos menos volumosos parecem favorecer a hemólise. Na próxima etapa do estudo, 7 derivados de APC, com diferentes grupos catiônicos, foram selecionados e ancorados no domínio C2 da PKCα. O intuito foi mapear resíduos de aminoácidos importantes para a interação dos ligantes com a enzima, e comparar com o modo de ligação do ativador endógeno fosfatidilserina (PS). Mais uma vez, HCA e PCA foram aplicados para extrair informação relevante do mapeamento. Os resultados mostraram que as cadeias laterais de Pro188, Asn189, Arg216, Trp247, Asp249 e Thr250 não permitem a aproximação adequada do ligante, o que impede que a porção fosforila se coordene com um dos átomos de cálcio. A porção catiônica da PS, em contrapartida, consegue estabelecer ligação-hidrogênio com Asn189 de forma a posicionar os oxigênios da fosforila para interagir, ao mesmo tempo, com o átomo de cálcio. Com menos pontos de coordenação, a afinidade de ligação do cálcio pela PKCα diminui e a ativação da enzima fica comprometida, interrompendo toda a cascata de sinalização que depende dela. A parte final desse trabalho se dedicou ao estudo da interação da miltefosina com diferentes bicamadas lipídicas sob o ponto de vista termodinâmico. Oito bicamadas de diferentes fosfolipídios foram simuladas por DM e a interação energética da miltefosina foi calculada por Umbrella Sampling. Os resultados mostraram que a miltefosina apresenta maior partição em bicamadas contendo colesterol, sendo a miscibilidade nesses sistemas cerca de 76 vezes maior que os valores encontrados para bicamadas sem colesterol. Além disso, verificou-se que a internalização da miltefosina é mais fácil em regiões contendo lipídeos poli-insaturados, provavelmente devido ao empacotamento mais frouxo da bicamada. Os dados sugerem que a miltefosina age principalmente em rafts lipídicos e que células contendo mais lipídicos poli-insaturados podem incorporar maior quantidade do fármaco. / Alquilfosfocolines (APCs) comprise a class of drugs with antitumor activity derived from endogenous phospholipids. Differently from other drugs whose primary site of action is the DNA, APCs act firstly in the plasma membrane and signaling proteins, such as PKC. The main objective of this work is to elucidate, via computational approaches, the possible mechanisms of actions that cause hemolysis, PKC inhibition and interaction with cellular membranes. Initially, a set of 34 APCs was studied by means of Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA). The molecules were simulated by means of molecular dynamics simulations (MD) and molecular and structural properties were calculated for the lowest-energy conformer. After HCA and PCA methodologies, the set was divided into 3 groups according to their structural features. The findings suggest that the presence of bulky cationic moieties, or the adamantyl and cyclohexil rings, increase the hemolytic potential of compounds with long alkyl chains. Macrocyclic rings, such as cyclopentadecyl, seem to be important to elevate the hemolysis of compounds with short alkyl chains. Regarding linear carbon chain derivatives with no ring substitution, less bulky cationic head groups seem to favor hemolysis. In the next step of this work, 7 APC derivatives were selected and docked in the C2 domain of PKCα. The aim now was to map the residues relevant for ligands interaction compared to the binding mode of the endogenous activator, phosphatidylserine (PS). HCA and PCA were again applied in order to extract relevant information from the mapping. The results showed that the lateral chains of Pro188, Asn189, Arg216, Trp247, Asp249 and Thr250 do not allow the proper approximation of the ligands, impeding the phosphoryl moiety from coordinating with one of the calcium atoms. On the other hand, the cationic moiety of PS forms hydrogen-bonding with Asn189 in order to position the oxygens to interact, at the same time, with a calcium atom. With less coordination sites, calcium binding affinity diminishes and the enzyme activation is compromised, interrupting the signaling cascade. The final part of this work was dedicated to the study of miltefosine interaction with different lipid bilayers from the thermodynamics standpoint. Eight bilayers were simulated with MD and the energetic interaction was calculated via Umbrella Sampling simulations. The findings showed that miltefosine has higher partition in bilayers containing cholesterol, with miscibility of about 76 times higher than the values referring to bilayers without cholesterol. Moreover, it was observed that the internalization of miltefosine is facilitated in regions containing polyunsaturated lipids, probably due to the looser packing. The data suggest that miltefosine acts primarily in lipid rafts, and that cells containing more polyunsaturated lipids in their membranes can incorporate higher quantities of this drug.

As cisteíno-proteases da família da papaína desempenham funções essenciais em processos biológicos, entre eles o desenvolvimento e crescimento do organismo, vias de sinalização celular e apoptose, invasão de parasitas em células hospedeiras. Assim, trata-se de uma classe de proteínas de grande interesse para as indústrias farmacêuticas, sendo utilizada como alvo para o tratamento de doenças como o câncer e metástases, osteoporose. Disfunções relacionadas ao sistema imune, doenças parasitárias como malária, leishmaniose, doença do sono e doença de Chagas. Esta última é uma enfermidade considerada negligenciada pelas grandes indústrias farmacêuticas, sem nenhum tratamento eficaz e seguro disponível, que gera um problema econômico de mais de sete bilhões de dólares anuais devido à perda de mão de obra e gastos com tratamento para amenizar os efeitos da doença. A cisteíno protease cruzaína de Trypanosoma cruzi, causador da Doença de Chagas, desponta como um alvo validado na busca de novos fármacos contra essa enfermidade. Essa enzima apresenta um par de aspartatos que interagem entre si, para os quais foi predito um pKa de 7, sendo possível a forma desprotonada desse par em condições biológicas. Neste caso, pode levar à exposição de uma nova cavidade por meio do movimento da alça entre os resíduos 57-62, segundo as simulações de dinâmica molecular desse trabalho, que se trata de uma possível candidata a ponto de seletividade de inibidores de cisteínoproteases de parasitos em relação às suas ortólogas em Homo sapiens que não possuem o par de aspartatos. Em pH ácido, foi mostrado por meio de análise de componentes principais de simulações de dinâmica molecular que as cisteíno protease apresentam uma restrição gradual na amostragem conformacional do sítio ativo quando complexadas com as formas não covalente e covalente de inibidores derivados de dipeptidil nitrilas. Isso sugere que esse sistema segue o modelo de seleção conformacional para flexibilidade de proteína. Notou-se também que o perfil de restrição de ligantes que inibem na faixa de nmol.L-1 difere daqueles a µmol.L-1 , o que possibilitou a construção de uma árvore de decisão para identificar os complexos que apresentam afinidade a nmol.L-1 . / The papain-like cysteine proteases are essentials for biological process, performing important roles on the parasite development, growth and also in the parasite invasion process on the host cell, in cellular signaling pathways and apoptosis, among others. Thus, the pharmaceutical industry widely uses this class of protein as target for the development of new drugs, against cancer and metastasis, osteoporosis and immune system disorders, resulting in many approved drugs. Additionally, these enzymes are validated target against parasitic diseases as leishmaniose, malaria and African and American trypanosomiasis. The last one, also known as Chagas\' disease, is neglected disease for which, further a century form this discovery, there is no effective and safe chemotherapy and is responsible for an economic loss of around seven billion dollars in the world per year due to the health care and lost productivity from infected people. Faced with this situation, the Cruzain, a cysteine protease from the Trypanosoma cruzi, the causative agent of Chagas\' disease, is emerging as interesting and validated target to the search for new drugs against this sickness. This enzyme has a pair of interactiong Asp for which was predicted a pKa of 7, by computational methods. By this way, this pair under neutral to alkaline pH adopts the deprotonated form, which exposed a new cavity through a movement of loop of residues 57-62, as we showed here by molecular dynamics simulations. This cavity emerges as a possible selectivity point of the cruzain inhibitors, once Homo sapiens enzynes does not present the aspartic acid - aspartate pair. In condition of acidic pH, principal component analysis of molecular dynamics simulations showed a gradual reduction of the conformational space covered by the active site of cruzain, cathepsin K and cathepsin L in it free form and complexed with dipeptidyl nitrilelike molecules in it noncovalent e covalent forms. This suggests these systems follows the conformational selection model of protein flexibility. Furthermore, we observed the ligands that inhibits the protein at nmol.L-1 induces the protein flexibility in a similar way, while the µmol.L-1 ones leads to another pattern. That made possible the construction of a decision tree which is able to identify nmol.L-1 from µmol.L-1 complexes.

As alquilfosfocolinas (APC) são uma classe de fármacos derivados de fosfolipídios endógenos que apresentam potencial antitumoral. Diferentemente de outros fármacos antitumorais que agem no DNA da célula, as APCs têm como primeiro local de ação a membrana plasmática e proteínas de sinalização, como a PKC. O objetivo desse trabalho é elucidar, através de metodologias computacionais, os possíveis mecanismos de ação das APCs que provocam hemólise, inibição da PKC e interação com membranas celulares. Inicialmente, a toxicidade de um conjunto de 34 APCs foi estudada pelos métodos quimiométricos de Análise de Agrupamentos Hierárquicos (HCA) e Componentes Principais (PCA). As moléculas foram simuladas com dinâmica molecular (DM) e propriedades físico-químicas e estruturais foram calculadas para os confôrmeros de menor energia. Após aplicação de HCA e PCA, as APCs foram divididas em 3 grupos, de acordo com suas características estruturais. Os resultados sugerem que a presença de grupos catiônicos volumosos, ou anéis como adamantila e ciclohexila, aumentam a hemólise de compostos de cadeia alquílica longa. Anéis macrocíclicos como ciclopentadecila parecem ser importantes para o potencial hemolítico de compostos com cadeia alquílica curta. Com relação a compostos sem anéis e de cadeia linear, grupos catiônicos menos volumosos parecem favorecer a hemólise. Na próxima etapa do estudo, 7 derivados de APC, com diferentes grupos catiônicos, foram selecionados e ancorados no domínio C2 da PKCα. O intuito foi mapear resíduos de aminoácidos importantes para a interação dos ligantes com a enzima, e comparar com o modo de ligação do ativador endógeno fosfatidilserina (PS). Mais uma vez, HCA e PCA foram aplicados para extrair informação relevante do mapeamento. Os resultados mostraram que as cadeias laterais de Pro188, Asn189, Arg216, Trp247, Asp249 e Thr250 não permitem a aproximação adequada do ligante, o que impede que a porção fosforila se coordene com um dos átomos de cálcio. A porção catiônica da PS, em contrapartida, consegue estabelecer ligação-hidrogênio com Asn189 de forma a posicionar os oxigênios da fosforila para interagir, ao mesmo tempo, com o átomo de cálcio. Com menos pontos de coordenação, a afinidade de ligação do cálcio pela PKCα diminui e a ativação da enzima fica comprometida, interrompendo toda a cascata de sinalização que depende dela. A parte final desse trabalho se dedicou ao estudo da interação da miltefosina com diferentes bicamadas lipídicas sob o ponto de vista termodinâmico. Oito bicamadas de diferentes fosfolipídios foram simuladas por DM e a interação energética da miltefosina foi calculada por Umbrella Sampling. Os resultados mostraram que a miltefosina apresenta maior partição em bicamadas contendo colesterol, sendo a miscibilidade nesses sistemas cerca de 76 vezes maior que os valores encontrados para bicamadas sem colesterol. Além disso, verificou-se que a internalização da miltefosina é mais fácil em regiões contendo lipídeos poli-insaturados, provavelmente devido ao empacotamento mais frouxo da bicamada. Os dados sugerem que a miltefosina age principalmente em rafts lipídicos e que células contendo mais lipídicos poli-insaturados podem incorporar maior quantidade do fármaco. / Alquilfosfocolines (APCs) comprise a class of drugs with antitumor activity derived from endogenous phospholipids. Differently from other drugs whose primary site of action is the DNA, APCs act firstly in the plasma membrane and signaling proteins, such as PKC. The main objective of this work is to elucidate, via computational approaches, the possible mechanisms of actions that cause hemolysis, PKC inhibition and interaction with cellular membranes. Initially, a set of 34 APCs was studied by means of Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA). The molecules were simulated by means of molecular dynamics simulations (MD) and molecular and structural properties were calculated for the lowest-energy conformer. After HCA and PCA methodologies, the set was divided into 3 groups according to their structural features. The findings suggest that the presence of bulky cationic moieties, or the adamantyl and cyclohexil rings, increase the hemolytic potential of compounds with long alkyl chains. Macrocyclic rings, such as cyclopentadecyl, seem to be important to elevate the hemolysis of compounds with short alkyl chains. Regarding linear carbon chain derivatives with no ring substitution, less bulky cationic head groups seem to favor hemolysis. In the next step of this work, 7 APC derivatives were selected and docked in the C2 domain of PKCα. The aim now was to map the residues relevant for ligands interaction compared to the binding mode of the endogenous activator, phosphatidylserine (PS). HCA and PCA were again applied in order to extract relevant information from the mapping. The results showed that the lateral chains of Pro188, Asn189, Arg216, Trp247, Asp249 and Thr250 do not allow the proper approximation of the ligands, impeding the phosphoryl moiety from coordinating with one of the calcium atoms. On the other hand, the cationic moiety of PS forms hydrogen-bonding with Asn189 in order to position the oxygens to interact, at the same time, with a calcium atom. With less coordination sites, calcium binding affinity diminishes and the enzyme activation is compromised, interrupting the signaling cascade. The final part of this work was dedicated to the study of miltefosine interaction with different lipid bilayers from the thermodynamics standpoint. Eight bilayers were simulated with MD and the energetic interaction was calculated via Umbrella Sampling simulations. The findings showed that miltefosine has higher partition in bilayers containing cholesterol, with miscibility of about 76 times higher than the values referring to bilayers without cholesterol. Moreover, it was observed that the internalization of miltefosine is facilitated in regions containing polyunsaturated lipids, probably due to the looser packing. The data suggest that miltefosine acts primarily in lipid rafts, and that cells containing more polyunsaturated lipids in their membranes can incorporate higher quantities of this drug.

Proteins and DNA are essential to life as we know it and understanding their function is understanding their structure and dynamics. The importance of the latter is being appreciated more in recent years and has led to the development of novel interdisciplinary techniques and approaches to studying protein function. Three techniques to study protein structure and dynamics have been used and combined in different ways in the context of this thesis and have led to a better understanding of the three systems described herein. X-ray crystallography is the oldest and still arguably most popular technique to study macromolecular structures. Nuclear magnetic resonance (NMR) spectroscopy is a not much younger technique that is a powerful tool not only to probe molecular structure but also dynamics. The last technique described herein are molecular dynamics (MD) simulations, which are only just growing out of their infancy. MD simulations are computer simulations of macromolecules based on structures solved by X-ray crystallography or NMR spectroscopy, that can give mechanistic insight into dynamic processes of macromolecules whose amplitudes can be estimated by the former two techniques. MD simulations of the model protein GB3 (B3 immunoglobulin-binding domain of streptococcal protein G) were conducted to identify origins of discrepancies between order parameters derived from different sets of MD simulations and NMR relaxation experiments.The results highlight the importance of time scales as well as sampling when comparing MD simulations to NMR experiments. Discrepancies are seen for unstructured regions like loops and termini and often correspond to nanosecond time scale transitions between conformational substates that are either over- or undersampled in simulation. Sampling biases can be somewhat remedied by running longer (microsecond time scale) simulations. However, some discrepancies persist over even very long trajectories. We show that these discrepancies can be due to the choice of the starting structure and more specifically even differences in protonation procedures. A test for convergence on the nanosecond time scale is shown to be able to correct for many of the observed discrepancies. Next, MD simulations were used to predict in vitro thermostability of members of the bacterial Ribonuclease HI (RNase H) family of endonucleases. Thermodynamic stability is a central requirement for protein function and a goal of protein engineering is improvement of stability, particularly for applications in biotechnology. The temperature dependence of the generalized order parameter, S, for four RNase H homologs, from psychrotrophic, mesophilic and thermophilic organisms, is highly correlated with experimentally determined melting temperatures and with calculated free energies of folding at the midpoint temperature of the simulations. This study provides an approach for in silico mutational screens to improve thermostability of biologically and industrially relevant enzymes. Lastly, we used a combination of X-ray crystallography, NMR spectroscopy and MD simulations to study specificity of the interaction between Drosophila Hox proteins and their DNA target sites. Hox proteins are transcription factors specifying segment identity during embryogenesis of bilaterian animals. The DNA binding homeodomains have been shown to confer specificity to the different Hox paralogs, while being very similar in sequence and structure. Our results underline earlier findings about the importance of the N-terminal arm and linker region of Hox homeodomains, the cofactor Exd, as well as DNA shape, for specificity. A comparison of predicted DNA shapes based on sequence alone with the shapes observed for different DNA target sequences in four crystal structures when in complex with the Drosophila Hox protein AbdB and the cofactor Exd, shows that a combined ”induced fit”/”conformational selection” mechanism is the most likely mechanism by which Hox homeodomains recognize DNA shape and achieve specificity. The minor groove widths for all sequences is close to identical for all ternary complexes found in the different crystal structures, whereas predicted shapes vary between the different DNA sequences. The sequences that have shown higher affinity to AbdB in vitro have a predicted DNA shape that matches the observed DNA shape in the ternary complexes more closely than the sequences that show low in vitro affinity to AbdB. This strongly suggests that the AbdB-Exd complex selects DNA sequences with a higher propensity to adopt the final shape in their unbound form, leading to higher affinity. An additional AbdB monomer binding site with a strongly preformed binding competent shape is observed for one of the oligomers in the reverse complement strand of one of the canonical (weak) Hox-Exd complex binding site. The shape preference seems strong enough for AbdB monomer binding to compete with AbdB-Exd dimer binding to that same oligomer, suggested by the presence of both binding modes in the same crystal. The monomer binding site is essentially able to compete with the dimer binding site, even though binding with the cofactor is not possible, because its shape is very close to the ideal shape. A comparison of different crystal structures solved herein and in the literature as well as a set of molecular dynamics simulations was performed and led to insights about the importance of residues in the Hox N-terminal arm for the preference of certain Hox paralogs to certain DNA shapes. Taken together all these insights contribute to our understanding of Hox specificity in particular as well as protein-DNA interactions in general.

Yan, Changqing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 92-97). / Abstracts in English and Chinese. / ABSTRACT (ENGLISH) --- p.iii / ABSTRACT (CHINESE) --- p.iv / ACKNOWLEDGMENTS --- p.v / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.ix / LIST OF TABLES --- p.x / Chapter CHAPTER ONE: --- BACKGROUND INFORMATION --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Deoxyribonucleic Acid --- p.2 / Chapter 1.3 --- DNA Studies --- p.9 / Chapter 1.4 --- Cisplatin Studies --- p.11 / Chapter 1.5 --- Scope of the Thesis --- p.13 / Chapter CHAPTER TWO: --- METHODOLOY AND COMPUTATION --- p.16 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Molecular Dynamics Simulation --- p.16 / Chapter 2.3 --- Quantum Mechanics Calculation --- p.23 / Chapter 2.4 --- Verification of Methodology --- p.25 / Chapter 2.4.1 --- Backbone Torsion Angles --- p.25 / Chapter 2.4.2 --- N7-N7 Distance --- p.30 / Chapter 2.4.3 --- Location of HOMO --- p.33 / Chapter 2.5 --- Summary --- p.35 / Chapter CHAPTER THREE: --- UNDERSTANDING OF THE CISPLATIN-DNA CROSSLINKS --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- MO Analysis --- p.37 / Chapter 3.3 --- Potential Binding Products with the Ligand --- p.37 / Chapter 3.3.1 --- "1,2-d(GpG) Intrastrand Crosslink" --- p.43 / Chapter 3.3.2 --- "l,2-d(ApG) Intrastrand Crosslink" --- p.43 / Chapter 3.3.3 --- "l,3-d(GpXpG) Intrastrand Crosslink" --- p.44 / Chapter 3.3.4 --- d(GpC)d(GpC) Interstrand Crosslink --- p.44 / Chapter 3.3.5 --- d(GpXpC)d(GpXpC) Interstrand Crosslink --- p.44 / Chapter 3.3.6 --- Summary --- p.45 / Chapter 3.4 --- Potential Binding Products Analysis --- p.47 / Chapter 3.4.1 --- Site Identification Convention --- p.47 / Chapter 3.4.2 --- Potential Binding Products Analysis --- p.48 / Chapter 3.4.3 --- Applications --- p.53 / Chapter 3.5 --- Cisplatin-DNA Crosslink Products Analysis --- p.56 / Chapter 3.5.1 --- "1,2-d(GpG) and l,2-d(ApG) Intrastrand Crosslinks" --- p.61 / Chapter 3.5.2 --- "l,3-d(GpXpG) Intrastrand and d(GpXpC)d(GpXpC) Interstrand Crosslinks" --- p.62 / Chapter 3.5.3 --- d(GpC)d(GpC) Interstrand Crosslinks --- p.63 / Chapter 3.5.4 --- Platination at Terminal Positions --- p.65 / Chapter 3.6 --- Summary --- p.65 / Chapter CAHPTER FOUR: --- CONCLUDING REMARKS --- p.67 / APPENDIX I: BACKBONE TORSION ANGLES AND SUGAR RING CONFORMATIONS OF THE OPTIMIZED GEOMETRIES --- p.69 / APPENDIX II: BACKBONE TORSION ANGLES OF THE EXPERIMENTAL SEQUENCES FROM NUCLEIC ACID DATABASE (NDB) --- p.77 / REFERENCES --- p.92

The conventional Verlet table neighbor list algorithm is improved to reduce the number of unnecessary inter-atomic distance calculations in molecular simulations involving large amount of atoms. Both of the serial and parallelized performance of molecular dynamics simulation are evaluated using the new algorithm and compared with those using the conventional Verlet table and cell-linked list algorithm. Results show that the new algorithm significantly improved the performance of molecular dynamics simulation compared with conventional neighbor list maintaining and utilizing algorithms in serial programs as well as parallelized programs. / Singapore-MIT Alliance (SMA)

Microcantilever sensor has gained much popularity because of its high sensitivity and selectivity. It consists of a micro-sized cantilever that is usually coated on one side with chemical/biological probe agents to generate strong attraction to target molecules. The interactions between the probe and target molecules induce surface stress that bends the microcantilever. This current work applied the molecular dynamics simulation to study the microcantilever system. Lennard-Jones potentials were used to model the target-target and target-probe interactions and bond bending potentials to model the solid cantilever beam. In addition, this work studied the effect of probe locations on the microcantilever deflection. The simulation results suggest that both target-target and target-probe interactions as well as the probe locations affect the arrangement of the bonds; in term of the bonding number, the area containing the bonded molecules, and the distances between them. All these factors influence the microcantilever deflection.

Carbohydrates are abundant in nature and have functions ranging from energy storage to acting as structural components. Analysis of carbohydrate structures is important and can be used for, for instance, clinical diagnosis of diseases as well as in bacterial studies. The complexity of glycans makes it difficult to determine their structures. NMR spectroscopy is an advanced method that can be used to examine carbohydrates at the atomic level, but full assignments of the signals require much work. Reliable automation of this process would be of great help. Herein studies of Escherichia coli O-antigen polysaccharides are presented, both a structure determination by NMR and also research on glycosyltransferases which assemble the polysaccharides. The computer program CASPER has been improved to assist in carbohydrate studies and in the long run make it possible to automatically determine structures based only on NMR data. Detailed computer studies of glycans can shed light on their interactions with proteins and help find inhibitors to prevent unwanted binding. The WaaG glycosyltransferase is important for the formation of E. coli lipopolysaccharides. Molecular docking analyses of structures confirmed to bind this enzyme have provided information on how inhibitors could be composed. Noroviruses cause gastroenteritis, such as the winter vomiting disease, after binding human histo-blood group antigens. In one of the projects, fragment-based docking, followed by molecular dynamics simulations and binding free energy calculations, was used to find competitive binders to the P domain of the capsid of the norovirus VA387. These novel structures have high affinity and are a very good starting point for developing drugs against noroviruses. The protein targets in these two projects are carbohydrate binding, but the techniques are general and can be applied to other research projects. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Submitted. Paper 5: Manuscript. Paper 6. Manuscript.