Dada a importância da ciência no desenvolvimento humano, a adoção da alfabetização científica como meta educacional mundial e o papel fundamental da educação formal nesse sentido, muito se tem feito para promover a educação continuada dos professores de ciências. Se se discute quais habilidades e competências são importantes para a atividade didática, o bom conhecimento do conteúdo é consenso. A análise dos registros do curso a distância Bioquímica das Drogas, planejado, ministrado, avaliado e aprimorado neste trabalho e oferecido a professores de Biologia, Química e Ciências da rede pública de ensino do Estado de São Paulo, revelou que: (1) o modelo de ensino em ambientes virtuais e de aprendizagem colaborativa pode ser adequadamente aplicado na formação continuada de professores, embora com restrições relacionadas ao letramento digital; (2) a participação de pós-graduandos na equipe didática trouxe valiosa contribuição para o projeto e para sua formação didática em EaD; (3) os professores têm diversas concepções alternativas em bioquímica, sobretudo com relação à complexidade da estrutura de proteínas e às inter-relações dos metabolismos de carboidratos, lipídeos e proteínas e (4) as intervenções didáticas realizadas foram eficientes para promover a aprendizagem de concepções científicas sobre o tema, incluindo as relacionadas às concepções alternativas detectadas. / Given the importance of Science to human development, the adoption of scientific literacy as a global educational goal, and the major role of formal education, many efforts have been made to promote continuing education of science teachers. While the discussion on which didactic skills are essential to teacher education still endures, mastering the subject matter remains a consensus. This work focuses on designing, implementing, and improving the distant education course Biochemistry of Drugs, offered to science teachers of public schools in São Paulo, Brazil. The analysis of the course records revealed that: (1) the collaborative learning model in virtual environments is adequate to teachers\' continuing education, although with some restrictions concerning digital literacy issues; (2) joining graduate students to the teaching staff contributed positively to the project itself as well as to the didactic training of the latter in distance education; (3) school teachers displayed several alternative conceptions in biochemistry, remarkably on the topics of protein structure and the correlation of the metabolism of carbohydrates, lipids and proteins; (4) the instruction efficiently facilitated learning of biochemical concepts, including those related to the misconceptions detected.

Les antibiotiques sont les médicaments les plus utilisés dans la médecine moderne. Depuis leurs découvertes, ils ont drastiquement changé la façon dont les infections sont traitées. Toutefois, à travers le processus d’adaptation, les bactéries deviennent éventuellement résistantes aux antibiotiques. Malgré leur omniprésence dans la biosphère, l’émergence de souches résistantes est favorisée par le mauvais usage des antibiotiques, ce qui crée une menace importante pour la santé publique. Les antibiotiques actuelles perdent graduellement leur efficacité, et vue le faible nombre de nouvelles molécules développées, la priorité est donnée pour la découverte de nouvelles stratégies capables de combattre les pathogènes. Les nouvelles cibles thérapeutiques idéales doivent exercer une faible pression évolutive, diminuer la virulence et être unique aux microorganismes. Une façon d’atteindre cet objectif est d’interférer dans la régulation et l’homéostasie du Fer chez les bactéries. La biodisponibilité du Fer a fortement influencé l’émergence de la vie sur terre et les stratégies évolutives qu’elle a adoptée. Ce qui a mené à l’apparition d’un mécanisme central de détection du Fer assurant la régulation de cet élément de haute importance. Ce senseur est un point faible que nous pourrons exploiter dans notre combat contre les infections bactériennes. La protéine Fur, pour « Ferric Uptake Regulator », est un régulateur de transcription métal dépendant qui est impliqué dans vaste réseau de régulation contrôlant principalement l’homéostasie du Fer et l’expression de facteurs de virulence. Le travail présenté dans ce manuscrit complète les études précédentes sur des inhibiteurs de la protéine Fur en utilisant une approche combinée théorique et expérimentale grâce a des expériences de XAS, SAXS et MALLS associé a de la modélisation moléculaire. Nous décrivons pour la première fois la structure de Fur d’E. coli ainsi que la structure d’un tétramère de Fur d’un mutant de P. aeruginosa. Par ailleurs, les profils d’énergie libre des protéines Fur de différentes espèces ont été déterminé, pour des complexes tetramériques ou dans le cas de dimères liés à l’ADN, permettant une compréhension préliminaire de leur mécanistique. Les informations structurales obtenues grâce aux travaux présentés dans ce manuscrit permettront de mieux comprendre les mécanismes d’inhibition des protéines Fur ainsi que fournir de nouvelles opportunités pour le développement de molécules a visée thérapeutique. / The most commonly prescribed drugs in human medicine are antibiotics. Since their discovery, they have drastically impacted the way we treat infections. However, a bacterium eventually becomes resistant to antimicrobial treatment through the natural process of adaptative evolution. Even if resistant bacteria are omnipresent in the biosphere, their emergence rate is accelerated by the misuse of antimicrobial agents leading to the public health threat we are facing now. As currently available antimicrobial agents lose their effectiveness and very few new drugs are being developed, a breakthrough in new strategies to fight pathogens should be a priority. Ideal new therapeutic targets should exert weak evolutionary pressure, disarm or weaken the pathogen and be unique to microorganisms. One way to do so is by interfering with the iron regulation and its homeostasis within Bacteria. The bioavailability of iron strongly influenced early life and the metabolic strategies that sustained it. A central iron sensing mechanism evolved to ensure the regulation of such an important element. Sadly for bacteria this sensor became an exploitable weakness in our battle against infection. The “Ferric Uptake Regulator” is a metal dependent transcription regulator with a large regulatory network controlling iron homeostasis and bacterial virulence. This work continues previous investigations on Fur inhibitors using a combined experimental and theoretical approach by performing XAS, SAXS and MALLS experiments together with computer simulations. We describe for the first time the structures of Fur from E. coli in addition to a tetrameric Fur structure of a mutant from P. aeruginosa. Moreover, free energy profiles of Fur proteins, as tetramers or dimers bound to DNA, from different species were generated and key residues involved in the interactions determined, providing mechanistic insights into Fur complexes. The structural information gathered from this work will be used to better understand inhibition mechanisms of Fur proteins providing new opportunities to overcome drug development challenges.

Les nanoémulsions (NEs) huile/eau peuvent être utilisées en tant que systèmes de délivrance des médicaments pour l’encapsulation des substances actives hydrophobes afin d’améliorer leur stabilité et leur biodisponibilité. Néanmoins, leur stabilisation nécessite l’utilisation de concentrations plus importantes de tensioactifs par rapport aux émulsions conventionnelles en raison de l’augmentation de la surface spécifique. La plupart des tensioactifs synthétiques couramment utilisés dans la formulation des émulsions sont potentiellement irritants, voire toxiques. Cela entrave l'application thérapeutique des NEs en particulier pour les traitements à long terme. L'objectif de cette thèse est alors de formuler des NEs pharmaceutiques huile/eau stabilisées par un biopolymère, la beta-lactoglobuline (beta-lg), à la place des tensioactifs synthétiques.Les NEs ont été préparées par homogénéisation à haute pression (HHP). La composition de la formulation et les conditions du procédé ont été optimisées afin d’obtenir des gouttelettes nanométriques dans des NEs stables. Les résultats ont montré que les NEs les plus stables, avec une taille de gouttelettes < 200 nm, ont été obtenues quand 5 m/m% de l’huile ayant la viscosité la plus faible ont été utilisés en tant que phase huileuse, 95 m/m% de la solution de beta-lg à une concentration de 1 m/m% ont été utilisés en tant que phase aqueuse et 4 cycles d’HPH de 100 MPa ont été appliqués. Cette formulation a été stable contre les phénomènes de croissance de gouttelettes pendant au moins 30 jours grâce à un film interfacial quasiment purement élastique. La gomme xanthane, un polysaccaride naturel, a été ajoutée à la formulation optimale à une concentration de 0,5 m/m% en tant qu’agent épaississant. Cela a permis d’obtenir une texture crémeuse avec un comportement rhéofluidifiant. Dans cette dernière formulation, la vitesse de migration des gouttelettes a été considérablement réduite et la stabilité des NEs a été améliorée.Les effets du procédé d’HPH sur les différents niveaux de structure de la protéine ont été évalués à l’aide de méthodes spectroscopiques, chromatographiques et électrophorétiques. L’influence de ce traitement sur ses propriétés interfaciales et émulsionnantes a également été étudiée. L’efficacité émulsionnante optimale a été obtenue quand les conditions d’HPH n’ont pas altéré la structure de la beta-lg, ni ses propriétés interfaciales. Néanmoins, un traitement d’HHP excessif (300 MPa/5 cycles) a induit des modifications structurelles, principalement une transformation des feuillets beta en structures désordonnées, une large perte dans le cœur hydrophobe, et une agrégation importante par des liaisons disulfure intermoléculaires. La beta-lg modifiée par l’HHP a montré une hydrophobie de surface plus importante conduisant à une vitesse d’adsorption à l’interface huile/eau plus élevée et une formation plus précoce d’un film interfacial. La dénaturation de la protéine par ce traitement à haute pression, qui a été effectuée avant le processus d’émulsification, n'a pas modifié de façon significative l'efficacité émulsionnante. La réduction de l’efficacité a été probablement plutôt induite par la dénaturation simultanée avec l’émulsification sous conditions d’écoulement très turbulent.L’intérêt de la formulation développée en tant que véhicule pour un modèle de substance active hydrophobe a été étudié avec l’isotrétinoïne (IT), usuellement utilisé pour le traitement de l’acné sévère. La formulation développée a permis d’encapsuler 0,033 m/m% d’IT sans aucune modification de la stabilité du système. Environ 10 % de l’IT ajoutée ont été solubilisés dans la phase aqueuse en association avec la protéine libre en excès. L’IT encapsulée dans les gouttelettes huileuses a été plus stable contre la photo-isomérisation que celle associée à la protéine libre. La formulation développée apparait prometteuse en tant que système de délivrance de l’IT pour une application cutanée. / Oil-in-water nanoemulsions can be used as drug delivery systems for the encapsulation of hydrophobic active substances in order to increase their solubility and their bioavailability. However, due to their higher specific area, their stabilization requires higher surfactant concentrations compared to conventional emulsions. Most of the synthetic surfactants commonly used in emulsion formulation are potentially irritant and even toxic, which hinders the therapeutic application of nanoemulsions especially during long-term treatment. The objective of this thesis is thus to formulate pharmaceutical oil/water nanoemulsions stabilized by a biopolymer, beta-lactoglobulin (beta-lg), instead of synthetic surfactants. Nanoemulsions were prepared by high pressure homogenization (HPH). The formulation composition and the process conditions were optimized in order to obtain nanometric droplets within stable nanoemulsions. The results showed that the most stable nanoemulsions, with droplet size inférieure à 200 nm, were obtained when 5 w/w% of the oil with the lowest viscosity value was used as the oily phase, 95 w/w% of beta-lg solution at a concentration of 1 w/w% was used as the aqueous phase, and 100 MPa of homogenization pressure was applied for 4 cycles. This formulation was stable against droplet growth phenomena during 30 days at least, thanks to a quasi purely elastic interfacial film. Xanthan gum, a natural polysaccharide, was added to the optimal formulation as a texturizing agent at a concentration of 0.5 w/w%. This allowed obtaining a cream texture with a shear thinning behavior. In this formulation, the migration rate of droplets was considerably reduced and the nanoemulsions stability was enhanced.The effects of the homogenization process on the different levels of the protein structure were assessed by spectroscopic, chromatographic and electrophoretic methods. The influence of this treatment on its interfacial and emulsifying properties was also investigated. The optimal emulsifying efficiency was obtained when the homogenization conditions did alter neither the structure of beta-lg nor its interfacial properties. However, an excessive HPH treatment (300 MPa/5 cycles) introduced structural modifications, mainly from beta-sheets into random coils, wide loss in lipocalin core, and protein aggregation by intermolecular disulfide bridges. HPH modified beta-lg displayed higher surface hydrophobicity inducing a higher adsorption rate at the O/W interface and an earlier formation of an elastic interfacial film. Structural and interfacial properties modifications by HPH denaturation appeared qualitatively similar to that of the heat denaturation with, however, differences in extent. Protein denaturation by a high pressure treatment that was performed before the emulsification process did not alter significantly its emulsifying efficiency. The reduction in the efficiency was rather induced by the simultaneous denaturation with the emulsification under high turbulent flow.The efficiency of the developed formulation as a vehicle for a model hydrophobic active substance was studied using isotretinoin, usually used for the treatment of severe acne. The developed formulation was able to encapsulate 0.033 w/w of isotretinoin without any modification on the system stability. About 10 % of the added isotretinoin was solubilized in the aqueous phase associated with the free protein in excess. Isotretinoin encapsulated in the oily droplets was more stable against photo isomerization than the one associated to the excess protein in the aqueous phase. The developed formulation seems promising as a drug delivery system of isotretinoin for a dermal application.

Pentameric ligand-gated ion channels (pLGICs) are membrane-embedded receptors found extensively in pre- and post-synaptic membranes throughout the nervous system where they play an important role in neurotransmission. The function of the prototypic pLGIC, the nicotinic acetylcholine receptor (nAChR) is highly sensitive to changes in its lipid environment, while other pLGICs display varying lipid sensitivities. This thesis presents a multidisciplinary investigation into the features of the transmembrane domain (TMD) that determine the unique functional and physical traits of different pLGICs. Using two prokaryotic homologues of the nAChR, ELIC and GLIC, as models, I focus on the outermost, lipid-exposed α-helix, M4, which, despite being distant from the primary allosteric pathway coupling agonist binding to channel gating, exercises significant control over channel function. Here, I present evidence that M4 acts as a lipid sensor, detecting changes in the surrounding lipids and transmitting these changes to the channel pore via contacts with the adjacent TMD α-helices, M1 and M3, and/or with structures in the extracellular domain. Using ELIC and GLIC chimeras, I first show that the TMD is the main driver of pLGIC thermal stability. I then demonstrate that the M4 α-helices in each channel play different roles in channel maturation and function, which suggests a divergent evolutionary path. Following this, I show that the M4 C-terminus is essential to both maturation and function in GLIC, while in ELIC its role is less defined, again showcasing possible evolutionary differences. Building on these findings, I examined the role of aromatic residues at the M4 – M1/M3 interface, and found that they predictably determine the interactions between M4 and M1/M3. Notably, the addition of aromatic residues to enhance M4-M1/M3 interactions in ELIC promotes channel function, while the elimination of aromatic residues at the M4-M1/M3 interface in GLIC is detrimental to channel function. Furthermore, I show that these same aromatics alter the strength of pLGIC lipid sensing and the sensitivity to certain disease-causing mutations, both indicating that aromatic residues are key players in channel function, stability and modulation. Finally, I and my collaborators identified and characterized a novel desensitization-linked lipid binding site in ELIC. Extensive mutagenesis studies coupled with biophysical measurements allowed us to develop a model describing how lipid binding influences the rates of ELIC desensitization to shape the agonist-induced response.

Le virus respiratoire syncytial humain (hRSV) est le principal agent pathogène responsable des bronchiolites. Le complexe ARN polymérase (RdRp), du hRSV, nécessaire à la réplication de son génome, est composé a minima de la sous-unité catalytique (L), de son principal cofacteur qu’est la phosphoprotéine (P) et de la nucléoprotéine (N) qui assure l’encapsidation du génome viral. Le cœur de mon projet doctoral a été l’étude dynamique et structurale de domaines des protéines N et P du hRSV ainsi que leurs interactions avec certaines protéines cellulaires principalement par résonance magnétique nucléaire.Dans un premier temps j’ai étudié une potentielle interaction entre 2 domaines appartenant à la protéine N et à la protéine cellulaire Tax1BP1 impliquée notamment dans la régulation de l’autophagie. Ensuite, j’ai entrepris une étude structurale et dynamique de hRSV-P isolée notamment dans le but de déterminer des contacts transitoires au sein de la protéine et d’obtenir la structure tridimensionnelle du domaine d’oligomérisation de P. Enfin, j'ai participé à la caractérisation de l’interaction entre la protéine hRSV-P et le cofacteur de transcription du hRSV hRSV-M2-1, puis entre hRSV P et la phosphatase cellulaire PP1α, afin d’en cartographier les régions de contacts. / Human respiratory syncytial virus (hRSV) is the main pathogen responsible for bronchiolitis. The RNA polymerase complex (RdRp) of hRSV, necessary for the replication of its genome, is composed at least of the catalytic subunit (L), its main cofactor phosphoprotein (P) and nucleoprotein (N), which encapsidates the viral genome. At the heart of my doctoral project was the dynamic and structural study of domains of the proteins N and P of the hRSV as well as of their interactions with several cellular proteins, mainly by nuclear magnetic resonance.Firstly, I studied a potential interaction between 2 domains belonging to the N protein and to the Tax1BP1 cellular protein involved notably in regulation of autophagy. Secondly, I undertook a structural and dynamic study of isolated hRSV-P in order to determine transient contacts within the protein and to obtain the three-dimensional structure of the P oligomerization domain. Last, I participated in the characterization of the interaction between the hRSV-P protein and the hRSV transcription cofactor hRSV-M2-1, and between hRSV P and the cellular phosphatase PP1α to map the contact regions.

Proteins are involved in almost all functions in a living cell, and functions of proteins are realized by their tertiary structures. Protein three-dimensional structures can be solved by multiple experimental methods, but computational approaches serve as an important complement to experimental methods for comparing and analyzing protein structures. Protein structure comparison allows the transfer of knowledge about known proteins to a novel protein and plays an important role in function prediction. Obtaining a global perspective of the variety and distribution of protein structures also lays a foundation for our understanding of the building principle of protein structures. This dissertation introduces our computational method to compare protein 3D structures and presents a novel mapping of protein shapes that represents the variety and the similarities of 3D shapes of proteins and their assemblies. The methods developed in this work can be applied to obtain new biological insights into protein atomic structures and electron density maps.

The work concerns to structural characterization of two important proteins of 2'-5' oligoadenylate pathway participating in an immune response of organism to a viral infection. Studied proteins were ankyrin domain of mouse RNase L, the C-terminal part of human phosphodiesterase 12 and the complete human phosphodiesterase 12. The proteins were characterized by Raman spectroscopy, infrared spectroscopy, electronic circular dichroism, dynamic light scattering and in addition by two non-spectroscopic methods- differential calorimetry and electrophoresis. For each protein the secondary structures, thermal stability, weight of oligomers and generally a basic characterization by above mentioned methods were provided.

To what extent conformational preference of short peptide sequences within proteins determine their three-dimensional structure? Large-scale quantum chemical calculations coupled with modern solvation methods represent unique set of tools to elucidate key determinants of the biomolecular structure ab initio. Full conformational sampling was performed on model systems representing short peptide fragments. The computed data reveal some of the underlying physico-chemical principles determining the spatial structure of proteins, and provide very important data for finding and tuning the optimal algorithm that may provide a full coverage of (ideally all) low-energy conformers. Keywords: Conformational space, peptide fragments, protein structure, solvation methods, Ramachandran plot, DFT-D3 methods

Drug repositioning applies existing drugs to new disease indications. A prerequisite for drug repurposing is drug promiscuity - a drug's ability to bind to several targets, possibly leading to side effects on the other hand. One reason for drug promiscuity is binding site similarity between (otherwise unrelated) proteins. In this thesis, a new algorithm for remote binding site similarity assessment and its application to the whole of the Protein Data Bank (PDB) is presented, forming the base for off-target identification and drug repositioning. The present thesis contributes to a long-standing debate on the reasons for drug promiscuity, being one of the pioneer studies investigating these from a protein structural point of view. Except for a small influence of flexibility, the analysis of all promiscuous drugs in the PDB revealed that drug properties are of minor importance. However, a strong correlation between promiscuity and binding site similarity of protein targets is found (r = 0.81), suggesting binding site similarity as the main reason for drug promiscuity. For 71 % of the promiscuous drugs at least one pair of their targets' binding sites is similar and for 18 % all are similar. In order to overcome issues in detection of remotely similar binding sites, a score for binding site similarity is developed: LigandRMSD measures the similarity of the aligned ligands and uncovers remote local similarities in proteins. It can be applied to arbitrary binding site alignments and also works on distinct ligands on a structural proteome scale. To answer the question on which other targets might be hit when targeting a particular protein, an all-to-all binding site alignment of 32,202 protein structures is analyzed. Of the hundreds of million possible protein pairs, 0.27 % were found to have similar binding sites. Extrapolating to the human proteome, for one human protein are 54 proteins with a similar binding site expected on average. Clearly, this is in contrast to the one drug-one target paradigm in drug development. Based on these data, disadvantageous off-targets can be uncovered and drug-repositioning candidates inferred. The enormous potential is demonstrated with the example of Viagra, proposing it for repositioning to Alzheimer's disease and prostate cancer. The findings in this thesis question the established single-target dogma in drug discovery. Drugs are triggered to modulate multiple targets simultaneously by the widespread binding site similarity. With the presented pipeline, drug targets can be reliably predicted: Starting from a target protein, additional targets are predicted based on binding site similarity and prioritized according to the resulting ligand structural overlap. Identifying drug targets helps to understand severe side effects and opens the door for drug repositioning.

This study is part of a project which aims to understand evolution of genetic code together with structural and functional analysis of prebiotic proteins. The repertoire of amino acids in the first proteins was probably developing in time and it influenced the development of structure and function of today's proteins. First amino acid alphabet was apparently only half of the size of present alphabet, which contains twenty amino acids. These ten amino acids were probably prebiotically available from endogenous and exogenous sources. This work includes cell-free expression and purification of two randomized protein libraries (containing approximately 1011 variants) with various amino acid composition and following comparison of their propensity to form secondary (using circular dichroism) and tertiary (using proteolytical analysis of sequences) structures. First library contains only ten probably prebiotically available amino acids; second library contains all twenty amino acids in today's genetic code. This project could help us understand benefits of genetic code expansion in terms of developing structure in protein sequences. The whole research could theoretically contribute a few basic questions not only in the fields of protein evolution but also in areas of synthetic biology or protein...