Sur le plan moléculaire, le transport de petites molécules à travers les bicouches lipidiques est un processus fondamental. L'objectif de ce travail consiste a étudier les différents mécanismes de transfert de molécules hydrophiles à travers la bicouche liposomale. La calcéine a été choisie comme molécule hydrophile modèle pour simuler la vectorisation de principes actifs. Dans une première étape, la formulation des liposomes a été optimisé en considérant ses propriétés physico-chimiques (taille, efficacité d'encapsulation, fluidité et etc.) par différentes méthodes (DSC, TEM, SAXS et etc). Les résultats montrent que la taille moyenne, le potentiel zêta, Tc, l'efficacité d'encapsulation et la fluidité, sont influencés par la composition lipidique des liposomes. Les interactions entre les molécules hydrophiles encapsulées et le vecteur ont été étudiés par spectroscopie Raman, balance de Langmuir et analyse thermique différentielle. Les résultats obtenus montrent que la calcéine est capable d'interagir avec le groupement polaire de la phosphatidylcholine, en s'intercalant entre les chaînes acyles et modifiant de ce fait l'organisation de la membrane. La perméabilité des membranes à la calcéine a d'abord été évaluée sur la base d'une cinétique du premier ordre par spectrofluorimètrie. L'effet de la composition en lipides sur la fluidité membranaire a été étudié en fonction des conditions environnementales. Un modèle simulant les conditions de la digestion a été élaboré pour estimer la vitesse de libération du calcéine à travers la bicouche liposomale, son coefficient de partage, en utilisant l'AFM et la méthode STED. Les résultats obtenus ont confirmé que la calcéine diffuse lentement à travers la membrane liposomale sans pour autant déstructurer le liposome / From a molecular point of view, transport of small molecules across lipid bilayers is a fundamental and functional process. The release of efficacious dose of bioactive-entrapped in liposome depends on different parameters such as liposome permeability, bioactive structural properties and strength of liposome / bioactive interaction. The aim of this study was investigation the possible mechanisms of hydrophilic molecules transfer through liposomal bilayer. Calcein was chosen as model of hydrophilic drugs. In the first step, we optimized liposome formulation by considering its physicochemical properties (size, encapsulation efficiency, fluidity and etc.) by different methods such as DSC, TEM, SAXS, DLS, NMR and Spectroufluremtere. The reported results show that mean size, zeta potential, Tc, entrapment efficiency and fluidity were influenced by liposome lipid composition. Then, we tried to investigate hydrophilic bioactive agents? interaction with liposome by Raman Spectroscopy, Langmuir Balance and Differential Scanning Calorimetry. The obtained results indicated that calcein is being able to interact with the choline polar-head group of the lipids but probability it could intercalate into the acyl chains and disturb the chain order. Finally, the permeability of calcein across some liposome membranes was first evaluated on the basis of the first-order kinetics by spectrofluorometer. Second, the composition/fluidity effect of liposome as well as the incubation temperature/pH effect was investigated. Furthermore, a model simulating the conditions of digestion was developed to estimate the partition coefficient and to determine the mechanism transfer through liposomal bilayer by using AFM and STED methods. The results confirmed that calcein permeates slowly through liposomal membrane by diffusion without liposome disruption

No description available.

Résumé : Les méthodes de détection de similarités de sites de liaison servent entre autres à la prédiction de fonction et à la prédiction de cibles croisées. Ces méthodes peuvent aider à prévenir les effets secondaires, suggérer le repositionnement de médicament existants, identifier des cibles polypharmacologiques et des remplacements bio-isostériques. La plupart des méthodes utilisent des représentations basées sur les atomes, même si les champs d’interaction moléculaire (MIFs) représentent plus directement ce qui cherche à être identifié. Nous avons développé une méthode bio-informatique, IsoMif, qui détecte les similarités de MIF entre différents sites de liaisons et qui ne nécessite aucun alignement de séquence ou de structure. Sa performance a été comparée à d’autres méthodes avec des bancs d’essais, ce qui n’a jamais été fait pour une méthode basée sur les MIFs. IsoMif performe mieux en moyenne et est plus robuste. Nous avons noté des limites intrinsèques à la méthodologie et d’autres qui proviennent de la nature. L’impact de choix de conception sur la performance est discuté. Nous avons développé une interface en ligne qui permet la détection de similarités entre une protéine et différents ensembles de MIFs précalculés ou à des MIFs choisis par l’utilisateur. Des sessions PyMOL peuvent être téléchargées afin de visualiser les similarités identifiées pour différentes interactions intermoléculaires. Nous avons appliqué IsoMif pour identifier des cibles croisées potentielles de drogues lors d’une analyse à large échelle (5,6 millions de comparaisons). Des simulations d’arrimage moléculaire ont également été effectuées pour les prédictions significatives. L’objectif est de générer des hypothèses de repositionnement et de mécanismes d’effets secondaires observés. Plusieurs exemples sont présentés à cet égard. / Abstract : Methods that detect binding site similarities between proteins serve for the prediction of function and the identification of potential off-targets. These methods can help prevent side-effects, suggest drug repurposing and polypharmacological strategies and suggest bioisosteric replacements. Most methods use atom-based representations despite the fact that molecular interaction fields (MIFs) represents more closely the nature of what is meant to be identified. We developped a computational algorithm, IsoMif, that detects MIF similarities between binding sites. We benchmark IsoMif to other methods which has not been previously done for a MIF-based method. IsoMif performed best in average and more consistently accross datasets. We highlight limitations intrinsic to the methodology or to nature. The impact of design choices on performance is discussed. We built a freely available web interface that allows the detection of similarities between a protein and pre-calculated MIFs or user defined MIFs. PyMOL sessions can be downloaded to visualize similarities for the different intermolecular interactions. IsoMif was applied for a large-scale analysis (5,6 millions of comparisons) to predict offtargets of drugs. Docking simulations of the drugs in the binding site of their top hits were performed. The primary objective is to generate hypotheses that can be further investigated and validated regarding drug repurposing opportunities and side-effect mechanisms.

Les virus sont des parasites intracellulaires obligatoires recrutant des cofacteurs cellulaires afin de détourner les différents processus biologiques leur permettant notamment de répliquer leur génome et de former d'autres particules virales. Si des cofacteurs cellulaires de la réplication du virus Semliki Forest ont été récemment identifiés, très peu d'études ont permis de révéler des partenaires de la réplication du proche Alphavirus Chikungunya (CHIKV). Nous avons découvert, au cours de cette étude, un recrutement d'Hélicases à domaine DExD/H au niveau de sites de réplication du CHIKV. Parmi elles, DHX9 ou RNA Helicase A (RHA), grâce à ses propriétés de liaison et de modulation de structures des ARNs ou de complexes de Ribonucléoprotéines, est impliquée dans diverses fonctions depuis la transcription, la traduction, la réplication de génomes et jusqu'à la production de particules infectieuses de nombreux virus. Dans le cas du virus Chikungunya, nous avons caractérisé une fonction provirale dans la traduction de protéines non-structurales et une fonction antivirale dans la réplication du génome. Cette double fonction opposée est manipulée par le CHIKV afin d'assurer une production de protéines non-structurales composant le complexe de réplication tout en maintenant sa réplication. Ces travaux révèlent un nouveau mécanisme de régulation de la traduction d'ARN génomique de CHIKV et apportent des éléments de compréhension dans la dynamique de passage du phénomène de traduction à l'étape de réplication du génome CHIKV. / Viruses are obligate intracellular parasites recruiting cellular cofactors to divert different biological processes enabling them to replicate their genome and to form other viral particles. If cellular cofactors of Semliki Forest virus replication have recently been identified, very few studies have revealed the replication partners of the very close Alphavirus Chikungunya (CHIKV). During this study, We have discovered recruitments of several DExD/H Box Helicases at the CHIKV replication sites. Among them, DHX9 or RNA Helicase A (RHA) through its RNA binding properties and in modulating RNA secondary structures or Ribonucleoproteins complexes, is involved in various functions from transcription, translation, replication of genomes and up to production of infectious particles of many viruses. In the case of Chikungunya virus, we have characterized a proviral function in the translation of non-structural proteins and an antiviral function in the genome replication. These opposite functions are manipulated by CHIKV to ensure production nonstructural proteins, components of the CHIKV replication complex while maintaining its replication. These works reveal a new translation regulation mechanism of CHIKV genomic RNA and bring some knowledge on the passage from the translation stage to the replication step of CHIKV genome.

No / The surface energetic parameters of carbamazepine and paracetamol have been studied using inverse gas chromatography modified to produce dry and ambient conditions within the column. The values of the dispersive component of the surface free energy (¿DS) do not change significantly at the increased relative humidity. In contrast the specific component of the free energy of adsorption (-¿GSPA) as measured by polar probes, can either remain constant or decrease by up to 10%, depending on the material and the probe. This indicates that an increase in the relative humidity causes a decrease in the surface energetics of the powder surface. It is proposed that where the water molecules are adsorbing to the same sites as the polar probes, the interaction of these probes with the surface is decreased. To identify these sites, the preferential interaction of each probe, including water, with the drug molecule has been modelled.

Na atualidade estudos direcionados para a avaliação do risco químico e do impacto ambiental produzidos por compostos químicos de uso intensivo, como agrotóxicos, petroquímicos e fármacos, vêm ganhando importância por elucidarem as implicações ao meio ambiente e à saúde pública decorrentes deste uso pela sociedade moderna de forma a estabelecer parâmetros restritivos para suas aplicações, evitando-se danos posteriores. Este Estudo compreendeu as observações de sorção e de interação do antibiótico de uso animal e agrícola oxitetraciclina (OTC) com solos brasileiros e ácidos húmicos (AH) extraídos de solos, tendo-se como objetivos gerais comprovar a sorção aos solos e aos AH e verificar a influência da matéria orgânica (MO) e do pH sobre a sorção, definindo quais os mecanismos moleculares de interação envolvidos, cabendo destacar que as tetraciclinas são uma das principais classes de antibióticos utilizadas na pecuária brasileira, tanto para tratamento de enfermidades quanto como Antibióticos Promotores de Crescimento (APC), além de serem freqüentemente aplicados na agricultura para controle de fungos e bactérias. Utilizou-se a Cromatografia Líquida de Alta Eficiência (CLAE) para a coleta de dados quantitativos que permitiram a construção de isotermas de sorção solo-OTC em pH 4,8 e AH-OTC em três valores de pH (3,1, 4,8 e 8,3) para a comprovação da sorção e da influência do conteúdo de MO no primeiro caso, e na sorção aos AH, para o segundo caso. Os solos foram caracterizados conforme procedimento padrão da EMBRAPA, enquanto que os AH foram caracterizados segundo procedimentos recomendados pela literatura por meio das técnicas instrumentais de Análise Elementar, Absorção na Região do Infravermelho Médio, Ressonância Magnética Nuclear e Ressonância Paramagnética Eletrônica. Foram utilizadas as técnicas espectroscópicas de Absorção na Região do Infravermelho (Próximo e Médio) e Absorção e Fluorescência na Região do UV-Visível, para a avaliação dos possíveis mecanismos de interação envolvidos, como ligação de hidrogênio, interação hidrofóbica e transferência de elétrons. A isoterma de Freundlich apresentou-se como um modelo matemático adequado para a verificação da sorção solos-OTC e AH-OTC, a partir da aplicação de um método cromatográfico adequado, comprovando a sorção para os dois casos, bem como o efeito do conteúdo de MO dos solos e do pH do meio sobre a capacidade e a intensidade da sorção, a qual foi quantificada como de capacidade de sorção inferior em comparação a dados obtidos em clima temperado e de alta capacidade de sorção quando comparada à legislação ambiental brasileira. Foi observado que a OTC interagiu com os AH em uma larga faixa de valores de pH, indo de 3,1 a 8,3. Contudo, o meio no qual foi possível observar de uma forma mais acentuada a interação entre AH e OTC foi o meio básico (pH 8,2), sendo notado a existência de mecanismos de interação associados e dependentes do pH do meio, como: ligação de hidrogênio, interação hidrofóbica e transferência de prótons. / At present further studies on the risk assessment of chemical and environmental impact produced by chemical-intensive, such as pesticides, petrochemicals and pharmaceuticals, have gained importance for elucidating the implications for the environment and public health arising from this use by modern society to establish restrictive parameters for their applications, avoiding further damage. This Study comprised observations of sorption and interaction of antibiotic for animal and agriculture use oxytetracycline (OTC) with brazilian soils and humic acids (HA) extracted from soil, having as objectives to prove the general sorption to soils and the AH and the influence of organic matter (OM) and pH on the sorption, which define the molecular mechanisms of interaction involved, highlighting that tetracyclines are a major classes of antibiotics used in the brazilian cattle industry, both for treatment of diseases such as antibiotic growth promoters (AGP), and frequently also applied in agriculture to control fungi and bacteria. The High Performance Liquid Chromatography (HPLC) was used to collect quantitative data that allowed the construction of sorption isotherms soils-OTC in pH 4.8 and AH-OTC in three pH values (3.1, 4, 8 and 8.3) for proof of sorption and the influence of OM content in the first case, and sorption to AH, for the second case. The soils were characterized as standard procedure of EMBRAPA, while AH were characterized according to procedures recommended by the literature by means of instrumental techniques for Elemental Analysis, Absorption in the Region of the Middle Infrared, Nuclear Magnetic Resonance and Electron Paramagnetic Resonance. The Spectroscopic Absorption in the Infrared Region (Middle) and Fluorescence and Absorption in the Region and the UV-Visible were used for the evaluation of interaction mechanisms involved, such as hydrogen bonding, hydrophobic interactions and electron transfer. The Freundlich isotherm was presented as a mathematical model suitable for the verification of soils-OTC sorption and AH-OTC sorption from the application of a chromatographic method that is suitable, proving the sorption for the two cases as well as the effect of OM content of soils and the pH on the capacity and sorption intensity, which was quantified as the lower sorption capacity in comparison with data from temperate and high sorption capacity when compared to Brazilian environmental legislation. It was observed that the OTC interacted with the HA in a wide range of pH values, ranging from 3.1 to 8.3. However, the way in which it was possible to observe a more pronounced interaction between HA and OTC was the basic medium (pH 8.2), and noted the existence of mechanisms of interaction associated and dependent on pH, such as: link hydrogen, hydrophobic interaction and proton transfer.

Molecular interaction among proteins drives the cellular processes through the formation of complexes that perform the requisite biochemical function. While some of the complexes are obligate (i.e., they fold together while complexation) others are non-obligate, and are formed through macromolecular recognition. Macromolecular recognition in proteins is highly specific, yet it can be both permanent and non permanent in nature. Hallmarks of permanent recognition complexes include large surface of interaction, stabilization by hydrophobic interaction and other noncovalent forces. Several amino acids which contribute critically to the free energy of binding at these interfaces are called as “hot spot” residues. The non permanent recognition complexes, on the other hand, usually show small interface of interaction, with limited stabilization from non covalent forces. For both the permanent and non permanent complexes, the specificity of molecular interaction is governed by the geometric compatibility of the interaction surface, and the noncovalent forces that anchor them. A great deal of studies has already been performed in understanding the basis of protein macromolecular recognition.1; 2 Based on these studies efforts have been made to develop protein-protein docking algorithms that can predict the geometric orientation of the interacting molecules from their individual unbound states. Despite advances in docking methodologies, several significant difficulties remain.1 Therefore, in this thesis, we start with literature review to understand the individual merits and demerits of the existing approaches (Chapter 1),3 and then, we attempt to address some of the problems by developing methods to infer protein quaternary structure from the crystalline state, and improve structural and chemical understanding of protein-protein interactions through biological complex prediction. The understanding of the interaction geometry is the first step in a protein-protein interaction study. Yet, no consistent method exists to assess the geometric compatibility of the interacting interface because of its highly rugged nature. This suggested that new sensitive measures and methods are needed to tackle the problem. We, therefore, developed two new and conceptually different measures using the Delaunay tessellation and interface slice selection to compute the surface complementarity and atom packing at the protein-protein interface (Chapter 2).4 We called these Normalized Surface Complementarity (NSc) and Normalized Interface Packing (NIP). We rigorously benchmarked the measures on the non redundant protein complexes available in the Protein Data Bank (PDB) and found that they efficiently segregate the biological protein-protein contacts from the non biological ones, especially those derived from X-ray crystallography. Sensitive surface packing/complementarity recognition algorithms are usually computationally expensive and thus limited in application to high-throughput screening. Therefore, special emphasis was given to make our measure compute-efficient as well. Our final evaluation showed that NSc, and NIP have very strong correlation among themselves, and with the interface area normalized values available from the Surface Complementarity program (CCP4 Suite: <http://smb.slac.stanford.edu/facilities/software/ccp4/html/sc.html>); but at a fraction of the computing cost. After building the geometry based surface complementarity and packing assessment methods to assess the rugged protein surface, we advanced our goal to determine the stabilities of the geometrically compatible interfaces formed. For doing so, we needed to survey the quaternary structure of proteins with various affinities. The emphasis on affinity arose due to its strong relationship with the permanent and non permanent life-time of the complex. We, therefore, set up data mining studies on two databases named PQS (Protein Quaternary structure database: http://pqs.ebi.ac.uk) and PISA (Protein Interfaces, Surfaces and Assemblies: www.ebi.ac.uk/pdbe/prot_int/pistart.html) that offered downloads on quaternary structure data on protein complexes derived from X-ray crystallographic methods. To our surprise, we found that above mentioned databases provided the valid quaternary structure mostly for moderate to strong affinity complexes. The limitation could be ascertained by browsing annotations from another curated database of protein quaternary structure (PiQSi:5 supfam.mrc-lmb.cam.ac.uk/elevy/piqsi/piqsi_home.cgi) and literature surveys. This necessitated that we at first develop a more robust method to infer quaternary structures of all affinity available from the PDB. We, therefore, developed a new scheme focused on covering all affinity category complexes, especially the weak/very weak ones, and heteromeric quaternary structures (Chapter 3).6 Our scheme combined the naïve Bayes classifier and point-group symmetry under a Boolean framework to detect all categories of protein quaternary structures in crystal lattice. We tested it on a standard benchmark consisting of 112 recognition heteromeric complexes, and obtained a correct recall in 95% cases, which are significantly better than 53% achieved by the PISA,7 a state-of-art quaternary structure detection method hosted at the European Bioinformatics Institute, Hinxton, UK. A few cases that failed correct detection through our scheme, offered interesting insights into the intriguing nature of protein contacts in the lattice. The findings have implications for accurate inference of quaternary states of proteins, especially weak affinity complexes, where biological protein contacts tend to be sacrificed for the energetically optimal ones that favor the formation/stabilization of the crystal lattice. We expect our method to be used widely by all researchers interested in protein quaternary structure and interaction. Having developed a method that allows us to sample all categories of quaternary structures in PDB, we set our goal in addressing the next problem that of accurately determining stabilities of the geometrically compatible protein surfaces involved in interaction. Reformulating the question in terms of protein-protein docking, we sought to ask how we could reliably infer the stabilities of any arbitrary interface that is formed when two protein molecules are brought sterically closer. In a real protein docking exercise this question is asked innumerable times during energy-based screening of thousands of decoys geometrically sampled (through rotation+translation) from the unbound subunits. The current docking methods face problems in two counts: (i), the number of interfaces from decoys to evaluate energies is rather large (64320 for a 9º rotation and translation for a dimeric complex), and (ii) the energy based screening is not quite efficient such that the decoys with native-like quaternary structure are rarely selected at high ranks. We addressed both the problems with interesting results. Intricate decoy filtering approaches have been developed, which are either applied during the search stage or the sampling stage, or both. For filtering, usually statistical information, such as 3D conservation information of the interfacial residues, or similar facts is used; more expensive approaches screen for orientation, shape complementarity and electrostatics. We developed an interface area based decoy filter for the sampling stage, exploiting an assumption that native-like decoys must have the largest, or close to the largest, interface (Chapter 4).8 Implementation of this assumption and standard benchmarking showed that in 91% of the cases, we could recover native-like decoys of bound and unbound binary docking-targets of both strong and weak affinity. This allowed us to propose that “native-like decoys must have the largest, or close to the largest, interface” can be used as a rule to exclude non native decoys efficiently during docking sampling. This rule can dramatically clip the needle-in-a-haystack problem faced in a docking study by reducing >95% of the decoy set available from sampling search. We incorporated the rule as a central part of our protein docking strategy. While addressing the question of energy based screening to rank the native-like decoys at high rank during docking, we came across a large volume of work already published. The mainstay of most of the energy based screenings that avoid statistical potential, involve some form of the Coulomb’s potential, Lennard Jones potential and solvation energy. Different flavors of the energy functions are used with diverse preferences and weights for individual terms. Interestingly, in all cases the energy functions were of the unnormalized form. Individual energy terms were simply added to arrive at a final score that was to be used for ranking. Proteins being large molecules, offer limited scope of applying semi-empirical or quantum mechanical methods for large scale evaluation of energy. We, therefore, developed a de novo empirical scoring function in the normalized form. As already stated, we found NSc and NIP to be highly discriminatory for segregating biological and non biological interface. We, therefore, incorporated them as parameters for our scoring function. Our data mining study revealed that there is a reasonable correlation of -0.73 between normalized solvation energy and normalized nonbonding energy (Coulombs + van der Waals) at the interface. Using the information, we extended our scoring function by combining the geometric measures and the normalized interaction energies. Tests on 30 unbound binary protein-protein complexes showed that in 16 cases we could identify at least one decoy in top three ranks with ≤10 Å backbone root-mean-square-deviation (RMSD) from true binding geometry. The scoring results were compared with other state-of-art methods, which returned inferior results. The salient feature of our scoring function was exclusion of any experiment guided restraints, evolutionary information, statistical propensities or modified interaction energy equations, commonly used by others. Tests on 118 less difficult bound binary protein-protein complexes with ≤35% sequence redundancy at the interface gave first rank in 77% cases, where the native like decoy was chosen among 1 in 10,000 and had ≤5 Å backbone RMSD from true geometry. The details about the scoring function, results and comparison with the other methods are extensively discussed in Chapter 5.9 The method has been implemented and made available for public use as a web server - PROBE (http://pallab.serc.iisc.ernet.in/probe). The development and use of PROBE has been elaborated in Chapter 7.10 On course of this work, we generated huge amounts of data, which is useful information that could be used by others, especially “protein dockers”. We, therefore, developed dockYard (http://pallab.serc.iisc.ernet.in/dockYard) - a repository for protein-protein docking decoys (Chapter 6).11 dockYard offers four categories of docking decoys derived from: Bound (native dimer co-crystallized), Unbound (individual subunits as well as the target are crystallized), Variants (match the previous two categories in at least one subunit with 100% sequence identity), and Interlogs (match the previous categories in at least one subunit with ≥90% or ≥50% sequence identity). There is facility for full or selective download based on search parameters. The portal also serves as a repository to modelers who may want to share their decoy sets with the community. In conclusion, although we made several contributions in development of algorithms for improved protein-protein docking and quaternary structure inference, a lot of challenges remain (Chapter 8). The principal challenge arises by considering proteins as flexible bodies, whose conformational states may change on quaternary structure formation. In addition, solvent plays a major role in the free energy of binding, but its exact contribution is not straightforward to estimate. Undoubtedly, the cost of computation is one of the limiting factors apart from good energy functions to evaluate the docking decoys. Therefore, the next generation of algorithms must focus on improved docking studies that realistically incorporate flexibility and solvent environment in all their evaluations.

Con el fin de relacionar la estructura y la actividad de series de compuestos, es importante usar descriptores moleculares relevantes. Los descriptores GRIND y VolSurf pertenecen a una nueva familia de descriptores llamado libre de alineamiento. Es decir, que no necesitan alinear los compuestos con el fin de comparar sus campos de interacciones molecular. En este estudio se ha aplicado esos descriptores para la selección de reactivos químicos a partir de una amplia base de datos. La selección se ha echo mediante un protocolo que permite maximizar la diversidad de la muestra y así obtener unos compuestos muy informativos. También se ha desarrollado nuevos descriptores de forma que están basado en los cambios de curvatura de la superficie molecular. Los resultados obtenidos indican que los nuevos descriptores de forma se integran muy bien en los descriptores GRIND originales y que permiten identificar los efectos de forma tanto favorable como desfavorable. Además, se ha desarrollado nuevos descriptores libre de alineamiento llamado 'anchor-GRIND' que usan un átomo de cada molécula como punto de referencia para la comparación de los campos de interacciones molecular. Los descriptores 'anchor-GRIND' permiten una descripción mas precisa y mas sencilla que los descriptores GRIND lo que los hace mas relevante para el análisis de ciertas familias de compuestos. / In order to correlate the differences of structure with the differences of activity of series of compounds, it is important to use relevant molecular descriptors. The GRIND and VolSurf descriptors belong to the so-called alignment-free descriptors family. In other words, they do not require to align the compounds in order to compare its molecular interaction fields. In this study, we applied these descriptors to the selection of chemical reagent from a database of compounds. The selection has been done following a protocol which allows to maximize the diversity of the sample and so to obtain some compounds highly informative. In addition we developed new shape descriptors which are based on the changes of curvature of the molecular surface. The results obtained show that the new shape descriptors are well integrated in the original GRIND descriptors. Furthermore, we designed new alignment-free descriptors called 'anchor-GRIND' which use one atom of each molecule as a reference point for the comparison of the molecular interaction fields. The 'anchor-GRIND' descriptors allow a more precise and more simple description than the GRIND descriptors, which makes them more relevant for the analysis of some families of compounds.

Una de les àrees més prometedores en recerca biomèdica i farmacèutica és el disseny molecular computacional, que intenta establir relacions entre propietats físico-químiques i activitat biològica. L'èxit d'aquestes tècniques depen críticament de la qualitat de la descripció molecular. En aquest sentit, metodologies basades en potencials d'interacció molecular (MIP) són eines útils per la comparació de compostos que presenten comportaments biològics semblants. Aquest projecte desenvolupa eines per comparar molècules basades en la caracterització de llurs MIPs. El programa de similaritat molecular MIPsim ha estat desenvolupat i aplicat a diferents problemes biològics. Aquesta tesi consisteix en quatre estudis científics que mostren l'ús del MIPSim en aliniament molecular, catalisi enzimàtica, en acoratge de molècules dins el lligand i en estudis 3D-QSAR. / One of the most promising areas in biomedical and pharmaceutical research is computer assisted molecular design, which tries to stablish relationships between physicochemical properties and biological activity. The success of these techniques depends critically on the quality of the molecular description. In this sense, methodologies based on molecular interaction potentials (MIP) are useful tools for the comparison of compounds displaying related biological behaviours. This project aims to develop tools to compare 'molecules based on the characterization 'of their MIPs. To this end, the molecular similarity program MIPSim has been further developed and applied to different biological problems. This thesis consists on four scientific studies showing the use of MIPSim for molecular alignment, enzymatic catalysis, ligand-protein docking and 3D-QSAR analyses.

Na atualidade estudos direcionados para a avaliação do risco químico e do impacto ambiental produzidos por compostos químicos de uso intensivo, como agrotóxicos, petroquímicos e fármacos, vêm ganhando importância por elucidarem as implicações ao meio ambiente e à saúde pública decorrentes deste uso pela sociedade moderna de forma a estabelecer parâmetros restritivos para suas aplicações, evitando-se danos posteriores. Este Estudo compreendeu as observações de sorção e de interação do antibiótico de uso animal e agrícola oxitetraciclina (OTC) com solos brasileiros e ácidos húmicos (AH) extraídos de solos, tendo-se como objetivos gerais comprovar a sorção aos solos e aos AH e verificar a influência da matéria orgânica (MO) e do pH sobre a sorção, definindo quais os mecanismos moleculares de interação envolvidos, cabendo destacar que as tetraciclinas são uma das principais classes de antibióticos utilizadas na pecuária brasileira, tanto para tratamento de enfermidades quanto como Antibióticos Promotores de Crescimento (APC), além de serem freqüentemente aplicados na agricultura para controle de fungos e bactérias. Utilizou-se a Cromatografia Líquida de Alta Eficiência (CLAE) para a coleta de dados quantitativos que permitiram a construção de isotermas de sorção solo-OTC em pH 4,8 e AH-OTC em três valores de pH (3,1, 4,8 e 8,3) para a comprovação da sorção e da influência do conteúdo de MO no primeiro caso, e na sorção aos AH, para o segundo caso. Os solos foram caracterizados conforme procedimento padrão da EMBRAPA, enquanto que os AH foram caracterizados segundo procedimentos recomendados pela literatura por meio das técnicas instrumentais de Análise Elementar, Absorção na Região do Infravermelho Médio, Ressonância Magnética Nuclear e Ressonância Paramagnética Eletrônica. Foram utilizadas as técnicas espectroscópicas de Absorção na Região do Infravermelho (Próximo e Médio) e Absorção e Fluorescência na Região do UV-Visível, para a avaliação dos possíveis mecanismos de interação envolvidos, como ligação de hidrogênio, interação hidrofóbica e transferência de elétrons. A isoterma de Freundlich apresentou-se como um modelo matemático adequado para a verificação da sorção solos-OTC e AH-OTC, a partir da aplicação de um método cromatográfico adequado, comprovando a sorção para os dois casos, bem como o efeito do conteúdo de MO dos solos e do pH do meio sobre a capacidade e a intensidade da sorção, a qual foi quantificada como de capacidade de sorção inferior em comparação a dados obtidos em clima temperado e de alta capacidade de sorção quando comparada à legislação ambiental brasileira. Foi observado que a OTC interagiu com os AH em uma larga faixa de valores de pH, indo de 3,1 a 8,3. Contudo, o meio no qual foi possível observar de uma forma mais acentuada a interação entre AH e OTC foi o meio básico (pH 8,2), sendo notado a existência de mecanismos de interação associados e dependentes do pH do meio, como: ligação de hidrogênio, interação hidrofóbica e transferência de prótons. / At present further studies on the risk assessment of chemical and environmental impact produced by chemical-intensive, such as pesticides, petrochemicals and pharmaceuticals, have gained importance for elucidating the implications for the environment and public health arising from this use by modern society to establish restrictive parameters for their applications, avoiding further damage. This Study comprised observations of sorption and interaction of antibiotic for animal and agriculture use oxytetracycline (OTC) with brazilian soils and humic acids (HA) extracted from soil, having as objectives to prove the general sorption to soils and the AH and the influence of organic matter (OM) and pH on the sorption, which define the molecular mechanisms of interaction involved, highlighting that tetracyclines are a major classes of antibiotics used in the brazilian cattle industry, both for treatment of diseases such as antibiotic growth promoters (AGP), and frequently also applied in agriculture to control fungi and bacteria. The High Performance Liquid Chromatography (HPLC) was used to collect quantitative data that allowed the construction of sorption isotherms soils-OTC in pH 4.8 and AH-OTC in three pH values (3.1, 4, 8 and 8.3) for proof of sorption and the influence of OM content in the first case, and sorption to AH, for the second case. The soils were characterized as standard procedure of EMBRAPA, while AH were characterized according to procedures recommended by the literature by means of instrumental techniques for Elemental Analysis, Absorption in the Region of the Middle Infrared, Nuclear Magnetic Resonance and Electron Paramagnetic Resonance. The Spectroscopic Absorption in the Infrared Region (Middle) and Fluorescence and Absorption in the Region and the UV-Visible were used for the evaluation of interaction mechanisms involved, such as hydrogen bonding, hydrophobic interactions and electron transfer. The Freundlich isotherm was presented as a mathematical model suitable for the verification of soils-OTC sorption and AH-OTC sorption from the application of a chromatographic method that is suitable, proving the sorption for the two cases as well as the effect of OM content of soils and the pH on the capacity and sorption intensity, which was quantified as the lower sorption capacity in comparison with data from temperate and high sorption capacity when compared to Brazilian environmental legislation. It was observed that the OTC interacted with the HA in a wide range of pH values, ranging from 3.1 to 8.3. However, the way in which it was possible to observe a more pronounced interaction between HA and OTC was the basic medium (pH 8.2), and noted the existence of mechanisms of interaction associated and dependent on pH, such as: link hydrogen, hydrophobic interaction and proton transfer.