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H Διπλωματική Εργασία αυτή επικεντρώνεται στο θέμα της Υπερηχητικής Απεικόνισης του ανθρώπινού σώματος με την παρατήρηση της δεύτερης αρμονικής που πηγάζει από την μη-γραμμική αλληλεπίδραση των υπερήχων με τους ιστούς που φωτίζονται από ένα παλμικό σήμα με φέρουσα συχνότητα εκπομπής f0. Η επεξεργασία των σημάτων λήψης στην ζώνη γύρω από την συχνότητα 2f0 και η απεικόνισή τους με συμβατικό τρόπο (b-mode) επιτρέπει την επίτευξη καλλίτερης χωρητικής διακριτικής ικανότητας. Η μελέτη ακολουθεί δύο παράλληλες μεθόδους: α) την κυματική ανάλυση του φαινομένου σκέδασης από μια μη γραμμική σφαίρα των υπερηχητικών κυμάτων και β) την εκτέλεση πειραματικών μετρήσεων με χρήση συστήματος υπερηχογράφου που έχει "ανοικτή" αρχιτεκτονική. Τόσο τα θεωρητικά όσο και τα πειραματικά αποτελέσματα επιβεβαιώνουν την χρησιμότητα της μεθόδου σαν μια εναλλακτική ιατρική απεικονιστική τεχνική. / The present Diploma Thesis is focused on the study of using 2nd harmonic ultrasound imaging of tissues as an alternate imaging technology in biomedical engineering.The principle of using of 2nd harmonic imaging is based on the illumination of a tissue medium with a frequency of f0 and then receive and prosess the signals with the same transducer at 2fo frequency. In order to derive specific conclusions this imaging problem is treated both theoreticaly and experimentally in the framework of the present Diploma Thesis.

<p>Malaria is a widespread disease caused by parasites of the genus <i>Plasmodium</i>. Each year 500 million clinical cases are reported resulting in over one million casualties. The most lethal species, <i>P. falciparum</i>, accounts for ~90% of the fatal cases and has developed resistance to chloroquine. The resistant strains are a major problem and calls for novel drugs.</p><p>In this thesis, the process of computational inhibitor design is illustrated through the development of <i>P. falciparum</i> aspartic protease inhibitors. These proteases, called plasmepsins, are part of the hemoglobin degradation chain. The hemoglobin is degraded during the intraerythrocytic cycle and serves as the major food source. By inhibiting plasmepsins the parasites can be killed by starvation.</p><p>Novel inhibitors with very high affinity were found by using a combination of computational and synthetic chemistry. These inhibitors were selective and did not display any activity on human cathepsin D. The linear interaction energy (LIE) method was utilized in combination with molecular dynamics (MD) simulations to estimate free energies of binding. The MD simulations were also used to characterize the enzyme–inhibitor interactions and explain the binding on a molecular level.</p><p>The influence of the partial charge model on binding free energy calculations with the LIE method was assessed. Two semiempirical and six <i>ab initio</i> quantum chemical charge derivation schemes were evaluated. It was found that the fast semiempirical charge models are equally useful in free energy calculations with the LIE method as the rigorous <i>ab initio</i> charge models.</p>

Malaria is a widespread disease caused by parasites of the genus Plasmodium. Each year 500 million clinical cases are reported resulting in over one million casualties. The most lethal species, P. falciparum, accounts for ~90% of the fatal cases and has developed resistance to chloroquine. The resistant strains are a major problem and calls for novel drugs. In this thesis, the process of computational inhibitor design is illustrated through the development of P. falciparum aspartic protease inhibitors. These proteases, called plasmepsins, are part of the hemoglobin degradation chain. The hemoglobin is degraded during the intraerythrocytic cycle and serves as the major food source. By inhibiting plasmepsins the parasites can be killed by starvation. Novel inhibitors with very high affinity were found by using a combination of computational and synthetic chemistry. These inhibitors were selective and did not display any activity on human cathepsin D. The linear interaction energy (LIE) method was utilized in combination with molecular dynamics (MD) simulations to estimate free energies of binding. The MD simulations were also used to characterize the enzyme–inhibitor interactions and explain the binding on a molecular level. The influence of the partial charge model on binding free energy calculations with the LIE method was assessed. Two semiempirical and six ab initio quantum chemical charge derivation schemes were evaluated. It was found that the fast semiempirical charge models are equally useful in free energy calculations with the LIE method as the rigorous ab initio charge models.

Indications that existing parameter sets of extended Linear Interaction Energy (LIE) models are transferable between lipases from Rhizomucor Miehei and Thermomyces Lanigunosus in complex with a small set of vinyl esters are demonstrated. By calculat- ing energy terms that represents the cost of forming cavities filled by the ligand and the complex we can add them to a LIE model with en established parameter set. The levels of precision attained will be comparable to those of an optimal fit. It is also demonstrated that the Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) methods are in- applicable to the problem of calculating absolute binding energies, even when the largest source of variance has been reduced.

An alternative formulation of the governing equations of a dynamical system, called the multi-scale localized perturbation method, is introduced and derived for the purpose of solving complex geophysical flow problems. Simulation variables are decomposed into background and perturbation components, then assumptions are made about the evolution of these components within the context of an environmental flow in order to close the system. Once closed, the original governing equations become a set of one-way coupled governing equations called the "delta form" of the governing equations for short, with one equation describing the evolution of the background component and the other describing the evolution of the perturbation component. One-way interaction which arises due to non-linearity in the original differential equations appears in this second equation, allowing the background fields to influence the evolution of a perturbation. Several solution methods for this system of equations are then proposed. Advantages of the delta form include the ability to specify a complex, temporally- and spatially-varying background field separate from a perturbation introduced into the system, including those created by natural or man-made sources, which enhances visualization of the perturbation as it evolves in time and space. The delta form is also shown to be a tool which can be used to simplify simulation setup. Implementation of the delta form of the incompressible URANS equations with turbulence model and scalar transport within OpenFOAM is then documented, followed by verification cases. A stratified wake collapse case in a domain containing a background shear layer is then presented, showing how complex internal gravity wave-shear layer interactions are retained and easily observed in spite of the variable decomposition. The multi-scale localized perturbation method shows promise for geophysical flow problems, particularly multi-scale simulation involving the interaction of large-scale natural flows with small-scale flows generated by man-made structures. / Master of Science / Natural flows, such as those in our oceans and atmosphere, are seen everywhere and affect human life and structures to an amazing degree. Study of these complex flows requires special care be taken to ensure that mathematical equations correctly approximate them and that computers are programmed to correctly solve these equations. This is no different for researchers and engineers interested in studying how man-made flows, such as one generated by the wake of a plane, wind turbine, cruise ship, or sewage outflow pipe, interact with natural flows found around the world. These interactions may yield complex phenomena that may not otherwise be observed in the natural flows alone. The natural and artificial flows may also mix together, rendering it difficult to study just one of them. The multi-scale localized perturbation method is devised to aid in the simulation and study of the interactions between these natural and man-made flows. Well-known equations of fluid dynamics are modified so that the natural and man-made flows are separated and tracked independently, which gives researchers a clear view of the current state of a region of air or water all while retaining most, if not all, of the complex physics which may be of interest. Once the multi-scale localized perturbation method is derived, its mathematical equations are then translated into code for OpenFOAM, an open-source software toolkit designed to simulate fluid flows. This code is then tested by running simulations to provide a sanity check and verify that the new form of the equations of fluid dynamics have been programmed correctly, then another, more complicated simulation is run to showcase the benefits of the multi-scale localized perturbation method. This simulation shows some of the complex fluid phenomena that may be seen in nature, yet through the multi-scale localized perturbation method, it is easy to view where the man-made flows end and where the natural flows begin. The complex interactions between the natural flow and the artificial flow are retained in spite of separating the flow into two parts, and setting up the simulation is simplified by this separation. Potential uses of the multi-scale localized perturbation method include multi-scale simulations, where researchers simulate natural flow over a large area of land or ocean, then use this simulation data for a second, small-scale simulation which covers an area within the large-scale simulation. An example of this would be simulating wind currents across a continent to find a potential location for a wind turbine farm, then zooming in on that location and finding the optimal spacing for wind turbines at this location while using the large-scale simulation data to provide realistic wind conditions at many different heights above the ground. Overall, the multi-scale localized perturbation method has the potential to be a powerful tool for researchers whose interest is flows in the ocean and atmosphere, and how these natural flows interact with flows created by artificial means.

<p>Accurate calculations of free energies for molecular association and solvation are important for the understanding of biochemical processes, and are useful in many pharmaceutical applications. In this thesis, molecular dynamics (MD) simulations are used to calculate thermodynamic properties for solvation and ligand binding.</p><p>The thermodynamic integration technique is used to calculate p<i>K</i>_a values for three aspartic acid residues in two different proteins. MD simulations are carried out in explicit and Generalized-Born continuum solvent. The calculated p<i>K</i>_a values are in qualitative agreement with experiment in both cases. A combination of MD simulations and a continuum electrostatics method is applied to examine p<i>K</i>_a shifts in wild-type and mutant epoxide hydrolase. The calculated p<i>K</i>_a values support a model that can explain some of the pH dependent properties of this enzyme.</p><p> Development of the linear interaction energy (LIE) method for calculating solvation and binding free energies is presented. A new model for estimating the electrostatic term in the LIE method is derived and is shown to reproduce experimental free energies of hydration. An LIE method based on a continuum solvent representation is also developed and it is shown to reproduce binding free energies for inhibitors of a malaria enzyme. The possibility of using a combination of docking, MD and the LIE method to predict binding affinities for large datasets of ligands is also investigated. Good agreement with experiment is found for a set of non-nucleoside inhibitors of HIV-1 reverse transcriptase.</p><p>Approaches for decomposing solvation and binding free energies into enthalpic and entropic components are also examined. Methods for calculating the translational and rotational binding entropies for a ligand are presented. The possibility to calculate ion hydration free energies and entropies for alkali metal ions by using rigorous free energy techniques is also investigated and the results agree well with experimental data.</p>

Métodos computacionais são usados para gerar estruturas de complexo proteína-ligante e estimar suas afinidades. Esse trabalho investigou como as diferentes representações da flexibilidade proteica afetam as poses obtidas por ancoragem molecular e as afinidades atribuídas a essas poses. Os mutantes L99A e L99A/M102Q da lisozima T4 foram escolhidos como sistemas modelo. Um descritor para predição de afinidades baseado na aproximação de energia de interação linear (LIE) foi parametrizado especificamente para ligantes da lisozima e foi usado para estimar as afinidades. A proteína foi representada como um grupo de estruturas cristalográficas ou de estruturas de trajetória de dinâmica molecular. O campo de força OPLS-AA para modelar a proteína e os ligantes e a aproximação de Born generalizada para modelar o solvente foram empregados. O descritor de afinidades parametrizado resultou em desvios médios entre afinidades experimentais e calculadas de 1,8 kcal/mol para um conjunto de testes. O descritor teve desempenho satisfatório na separação entre poses cristalográficas e poses falso-positivo e na identificação de poses falso-positivo. Experimentos de agrupamento de complexos realizados com o objetivo de reduzir o custo computacional para estimar afinidades apresentaram resultados insatisfatórios. As melhores aproximações da teoria do ligante implícito propostas aqui para estimar afinidades consideram conjuntos de estruturas de receptor com o mesmo peso. Configurações de ligante também apresentam o mesmo peso ou são dominadas por uma única configuração. A representação da flexibilidade requer um tratamento estatístico adequado para estimativa de afinidades. Aqui, a associação entre LIE e a teoria do ligante implícito mostrou-se frutífera. / Computational methods are used to generate protein-ligand complex structures and estimate their binding affinities. This work investigated how different representations of protein flexibility affect poses obtained by molecular docking and the affinities attributed to these poses. T4 lysozyme mutants L99A and L99A/M102Q were chosen as model systems. A descriptor for prediction of affinities based on linear interaction energy (LIE) approximation was parametrized specifically to lysozyme ligands and was used to estimate affinities. The protein was represented as a group of crystal structures or as structures from a molecular dynamics trajectory. OPLS-AA force field was used to model protein and ligands and the Generalized Born approximation was used to model solvent. The parametrized affinity descriptor resulted in average deviations between experimental and calculated affinities of 1.8 kcal/mol for a test set. Descriptor performance was satisfactory in the separation between crystal poses and false-positive ones and in the identification of false-positive poses. Clustering of complexes was tried out to reduce computational cost to estimate affinities, but results were poor. The best approximations to the implicit ligand theory proposed here in order to estimate affinities consider groups of receptor structures with the same weight. Ligand configurations also have the same weight or are dominated by only one configuration. The representation of protein flexibility requires an adequate statistical treatment when used to estimate affinities. Here, the linking between LIE and the implicit ligand theory proved itself useful.

Ce travail porte sur l'étude de la cohérence entre champs triplets générés par interaction non linéaire d'ordre trois. Un protocole indirect, basé sur l'étude du champ issu de la somme de fréquences des champs triplets dans un cristal non linéaire a été envisagé. Une modélisation théorique en formalisme quantique de l'évolution des champs, de leur génération à leur recombinaison, a été développée, donnant lieu à une recherche exhaustive des signatures de corrélations susceptibles d'émerger de notre protocole. Les expériences menées, à savoir la génération non linéaire bi-stimulée et la recombinaison des champs ainsi générés, sont en accord avec notre modèle théorique et permettent de mettre en évidence le masquage des corrélations entre les champs dans ce régime fortement injecté, ces corrélations étant contenues dans les fluctuations quantiques, alors négligeables, des opérateurs champs. Le calcul théorique prédit une signature de corrélations fortes entre les photons triplets dans la cas d'un génération par fluorescence paramétrique, et plus faible dans le cas d'une génération mono-stimulée. / This work deals about the study of coherence between triple photon beams generated by a third order non linear interaction. A protocol, based on the study of the field arising from sum-frequency of the triplet fields in a non linear crystal, was proposed. A theoretical model, in the quantum formalism, was developed, leading to an exhaustive research of the potential signature of the correlations. All the possible schemes were studied : triple fields generation from parametric fluorescence or from stimulated interaction, and recombination of two or three of the triple fields. The experiments we led, i.e. the bi-stimulated non linear generation and the recombination of the so-born fields, agree with our theoretical model and put into light the correlations hiding in this highly stimulated regime, since these correlations lie in the quantum fluctuations of the fields operators. The theoretical calculations predicts a strong correlations signature when the fields are generated from parametric fluorescence, and a weaker one in the case of a generation mono-stimulated.

Accurate calculations of free energies for molecular association and solvation are important for the understanding of biochemical processes, and are useful in many pharmaceutical applications. In this thesis, molecular dynamics (MD) simulations are used to calculate thermodynamic properties for solvation and ligand binding. The thermodynamic integration technique is used to calculate pKa values for three aspartic acid residues in two different proteins. MD simulations are carried out in explicit and Generalized-Born continuum solvent. The calculated pKa values are in qualitative agreement with experiment in both cases. A combination of MD simulations and a continuum electrostatics method is applied to examine pKa shifts in wild-type and mutant epoxide hydrolase. The calculated pKa values support a model that can explain some of the pH dependent properties of this enzyme. Development of the linear interaction energy (LIE) method for calculating solvation and binding free energies is presented. A new model for estimating the electrostatic term in the LIE method is derived and is shown to reproduce experimental free energies of hydration. An LIE method based on a continuum solvent representation is also developed and it is shown to reproduce binding free energies for inhibitors of a malaria enzyme. The possibility of using a combination of docking, MD and the LIE method to predict binding affinities for large datasets of ligands is also investigated. Good agreement with experiment is found for a set of non-nucleoside inhibitors of HIV-1 reverse transcriptase. Approaches for decomposing solvation and binding free energies into enthalpic and entropic components are also examined. Methods for calculating the translational and rotational binding entropies for a ligand are presented. The possibility to calculate ion hydration free energies and entropies for alkali metal ions by using rigorous free energy techniques is also investigated and the results agree well with experimental data.