Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2012-2013. / Le réchauffement climatique est un problème de plus en plus préoccupant pour la communauté scientifique. La plupart des experts affirment qu'une solution temporaire à ce problème consiste à réduire les émissions de CO2. Un moyen efficace permettant de réduire les émissions de CO2 tout en continuant à exploiter les ressources mondiales de pétrole et de charbon est la capture du CO2. La dernière décennie a vue naître une technologie des plus novatrices dans le domaine de la capture du CO2 soit l'utilisation de l'anhydrase carbonique, une enzyme catalysant la réaction d'hydratation du CO2 de façon très efficace (kh » ÎOV1 ). Ce projet de maîtrise présente une étude cinétique de l'hydratation du CO2 en présence de l'anhydrase carbonique humaine de type II (l'hCA II) immobilisée, réalisée à l'aide d'un microréacteur enzymatique. Présentement, l'utilisation de cette enzyme à des fins industrielles est très limitée. Le travail présenté ici est innovateur; à notre connaissance, aucune étude similaire impliquant l'enzyme immobilisée n'est disponible dans la littérature ouverte. Cette étude cinétique contribuera ultérieurement au design d'un réacteur monolithique enzymatique destiné à la capture du CO2. La réalisation de cette étude cinétique a nécessité plusieurs étapes préliminaires : la production de l'enzyme, l'immobilisation de l'enzyme et la caractérisation de l'immobilisation. Les résultats expérimentaux ont démontré que la contribution du biocatalyseur sur la réaction globale d'hydratation du CO2 augmente avec l'augmentation du débit volumétrique et l'augmentation de la concentration initiale en molécules de tampon non protonées, ainsi qu'avec la diminution de la concentration initiale en CO2. Un modèle cinétique de l'hydratation catalytique du CO2 par l'hCA II immobilisée basé sur un mécanisme Quad Quad Iso Ping-pong aléatoire a aussi été développé grâce aux résultats expérimentaux.

Une alternative intéressante pour la capture du dioxyde de carbone réside dans l'utilisation de l'anhydrase carbonique pour produire du bicarbonate. Un des goulots d'étranglement du procédé se situe au niveau de la quantité élevée d'enzyme nécessaire. Pour résoudre ce problème, un système d'expression inductible d'anhydrase carbonique humaine de type II utilisant Pichia pastoris (P. pastoris) a été développé, ce qui permettrait éventuellement une production intégrée à moindre coût de l'enzyme. Les cinétiques de croissance et de production du nouveau système d'expression ont été caractérisées par le moyen de cultures en bioréacteur complètement automatisé. Deux constructions ont été sélectionnées et comparées dans cette étude. Une production de l'ordre du gramme d'enzyme par litre, purifiée selon deux étapes simples (clarification et concentration) a été obtenue. Le coût du milligramme de protéine obtenue est estimé à moins d'un dollar.

Le Groupe d'expert Intergouvernemental sur l'Évolution du Climat (GIEC) dans son rapport de 2005 sur le piégeage et le stockage du CO2 fait une revue exhaustive des procédés de capture démontrés et implémentés industriellement ou en développement. Un procédé novateur ayant suffisamment de potentiel pour justifier des études plus poussées échappe toutefois à cette revue et consiste en l'utilisation d'une enzyme dont la cinétique est extrêmement rapide, l'anhydrase carbonique, comme catalyseur de l'hydratation de CO2. La présente étude propose donc une revue des procédés enzymatiques de capture de CO2 et une caractérisation expérimentale de la capture de CO2 en colonne garnie à contre courant avec de l'anhydrase carbonique immobilisée. La découverte la plus importante résultant de cette étude est que l'utilisation de cette enzyme immobilisée en colonne garnie à contre courant n'est pas une bonne configuration de réacteur due à des réactions compétitives en phase homogène liquide qui limitent la quantité de CO2 accessible à l'enzyme. / In its 2005 report on CO2 capture and storage, the International Panel on Climate Change (IPCC) makes an exhaustive review of capture processes that are demonstrated, industrially implemented or still in development. However, an innovative approach that has sufficient potential to justify detailed investigation escaped the attention of the IPCC. It consists in utilizing an enzyme which kinetics is extremely fast, namely carbonic anhydrase, as a catalyst in C02 hydration. The present study deals with a review of enzymatic CO2 capture processes and an experimental characterization of C02 capture in packed counter current column using immobilized carbonic anhydrase. The principal finding of this study is that the use of immobilized carbonic anhydrase in packed counter current column is not a good reactor configuration because of concurrent homogeneous liquid phase reactions that limit the amount of CO2 accessible to the enzyme.

Les interactions protéine - protéine sont au centre de nombreux processus biologiques, et représentent des cibles thérapeutiques pertinentes pour le traitement de certaines maladies. La conception de molécules antagonistes visant à inhiber ces interactions requiert la reconnaissance spécifique d’une des surfaces protéiques impliquées. Les foldamères de type oligoamides de quinoline constituent de bons candidats. Leur production et leur fonctionnalisation sont relativement aisées. Ils adoptent des structures hélicoïdales semblables à celles rencontrées dans les protéines. Grâce à différentes techniques biophysiques (CD, SPR, diffraction des rayons X), nous montrons que ces molécules sont aptes à reconnaître une surface protéique. Deux protéines cibles ont été choisies : l’interleukine 4 et l’anhydrase carbonique humaine II.La stratégie retenue dans ce travail (ancrage du foldamère à la protéine via un bras espaceur) nous a permis d’obtenir des informations structurales sur les interactions foldamère – protéine avant toute optimisation de ces interactions. La première structure 3D d’un complexe foldamère de quinoline complexée à une protéine est décrite. / Protein-protein interactions are a central issue in biological processes and represent relevant therapeutic targets for the treatment of diseases. The design of antagonistic molecules directed towards the disruption of these interactions requires the specific recognition of protein surfaces. Quinoline oligoamide foldamers present all the properties to reach that point. They are easily synthesized and fold into helices (similar to α helices) which can be decorated. Thanks to biophysical studies (CD, SPR, RX diffraction), we demonstrate that these molecules are able to recognize protein surfaces. Two proteins have been studied: the human interleukin 4 and the human carbonic anhydrase II.The applied strategy (keeping the foldamer close to the protein surface via a linker) allowed us to gather structural information about foldamer protein interactions before any strong binding is established. The first crystal structure between a protein and an aromatic amide foldamer is reported.

A novel concept for protein recognition has been developed. The recognition unit is a hybrid molecule obtained by conjugation of a small organic molecule to a synthetic polypeptide selected from a 16-membered set of 42 amino acid residue sequences. The sequences are unordered and have no prior relation to the target proteins. The concept is based on the hypothesis that a small set of sequences capable of hydrophobic interactions, hydrogen bonding and electrostatic interactions can yield a binder for any selected protein, provided that the small molecule shows medium affinity or better and is reasonably selective. The concept has been illustrated by the design, synthesis and evaluation of binders for three different proteins, the C-reactive protein, CRP, human Carbonic anhydrase II, HCAII, and Acetylcholine esterase, AChE. Highly efficient binders for CRP have been developed by conjugation of a derivative of the natural ligand, phosphocholine, to the side chain of one of the amino acids in each polypeptide. The binders in the set show a wide range of affinities for CRP and the tightest binder, 4-C10L17-PC6, binds almost irreversibly. Selected binders have been evaluated in human serum, where they capture CRP with high selectivity.High-affinity binders have been developed for HCAII, and the selectivity evaluated by extraction of the protein from blood. The binder 4-C37L34-B, a polypeptide conjugated to a spacered benzenesulphonamide residue, was able to extract Carbonic anhydrases specifically and to discriminate between the two isoforms of human Carbonic anhydrase. The conjugation of an acridine derivative to a polypeptide via a 14 atom spacer has been shown to yield a binder with high affinity and selectivity for AChE. The selectivity was demonstrated by extraction of AChE from Cerebrospinal fluid. This thesis focuses on the development of a fast and reliable procedure for the construction, selection and evaluation of protein binders, with the ambition to develop a technology that is applicable to the development of binders for all proteins.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13429号 / 論医博第2233号 / 新制||医||1053(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 妹尾 浩, 教授 川口 義弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

[pt] No contexto da redução da concentração de CO2 na atmosfera e utilizar o mesmo na síntese de produtos de interesse, buscou-se sintetizar COFs com propriedades biomiméticas da enzima anidrase carbônica capaz de converter CO2. O chamado Tppa-NO2-COF foi planejado a partir dos blocos de construção triformilfluoroglucinol e 2-nitro 1,4-fenilenodiamina (comercialmente disponível). O triformilfluoroglucinol foi previamente sintetizado pela reação de Duff, entretanto outras metodologias alternativas foram testadas de forma a melhorar as condições reacionais e o custo atrelado ao processo. Em decorrência da não reprodutibilidade das metodologias sintéticas do Tppa-NO2-COF reportadas na literatura, foi necessário um processo de otimização (variando tipo e quantidade de solvente, concentração do ácido, condição reacional, entre outros). A influência do grupamento nitro no Tppa-NO2 foi igualmente avaliada, através da síntese do material análogo sem o nitro, o chamado Tppa-1-COF, o qual foi obtido com área de 434 m2/g, condizente com o dado reportado na literatura. Outra metodologia abordada para a síntese desses materiais foi a sonificação (sonochemistry) que demonstrou ser uma excelente alternativa para a síntese dos materiais de maneira eficaz e rápida. Entretanto, o material foi obtido com cristalinidade e área específica baixas e muito variáveis (de 40 a 628 m2/g), o que, por definição não pode ser chamado de COF, sendo então tratado como Covalent Organic Network (CON). Apesar da adversidade, seguiu-se com as modificações pós-sintéticas no Tppa-NO2-CON, realizando a redução do grupamento nitro e consequente reação do grupo amina para formar linkers que permitissem a atividade biomimética à enzima via ligação peptídica ou via triazol, capazes de se coordenar ao íon zinco. Otimizações ainda se fazem necessárias para a obtenção do COF, bem como estudos mais aprofundados nas modificações pós sintéticas e de adsorção de CO2, aplicação na conversão de CO2 a bicarbonato e outros produtos de interesse para a síntese orgânica. / [en] In the context of reducing the concentration of CO2 in the atmosphere and using it in the synthesis of products of interest, we sought to synthesize COFs with biomimetic properties of the carbonic anhydrase enzyme capable of converting CO2. The so-called Tppa-NO2-COF was designed from the building blocks triformylfluoroglucinol and 2-nitro 1,4-phenylenediamine (commercially available). Triformylfluoroglucinol was previously synthesized by the Duff reaction, however other alternative methodologies were tested in order to improve the reaction conditions and the cost linked to the process. Due to the non-reproducibility of synthetic methodologies of Tppa-NO2-COF reported in the literature, an optimization process (varying type and amount of solvent, acid concentration, reaction condition, among others) was necessary. The influence of the nitro group on Tppa-NO2 was also evaluated, through the synthesis of an analogous material without nitro, the so-called Tppa-1-COF, which was published with an area of 434 m2/g, consistent with what is reported in the literature. Another approach to synthetic materials for sonification (sonochemistry) includes being an excellent alternative for an efficient and rapid synthesis of materials. However, the material was found with low and highly variable crystallinity and specific area (from 40 to 628 m2/g), which, by definition, cannot be called COF, being treated as Covalent Organic Network (CON). Despite the adversity, post-synthetic modifications in the Tppa-NO2-CON followed, performing the reduction of the nitro group and consequent reaction of the amine group to form ligands that would allow a biomimetic activity to the enzyme via peptide bond or via triazole, from coordinate with the zinc ion. Optimizations are still necessary to obtain the COF, as well as in-depth studies on post-synthetic modifications and CO2 adsorption, application in the conversion of CO2 to bicarbonate and other products of interest to organic synthetic.

Fluorescent labeling is a method used for visualization of various types of biomolecules including proteins and protein complexes. However, the effect of protein labeling on protein structure and functions has not been investigated so far. The goal of the diploma thesis was to examine an influence of NHS-fluorescein binding on structure and function of human carbonic anhydrase I (hCA-I). The particular aims of this work were to prepare recombinant 15N-hCA-I which was used for NMR structure analysis of carbonic anhydrase upon fluorescent labeling. Furthermore, enzyme activity was measured in order to find out a correlation between the concentration of NHS- fluorescein and protein function. In addition, the reaction mixtures were systematically analyzed by ESI FT-ICR mass spectrometry. The analysis revealed experimental conditions for fluorescent labeling of human carbonic anhydrase I with minimal effect on protein structure and function. The results of this study show that the calculation of molar excess of NHS-fluorescein cannot rely on a simple procedure provided by manufacturer. However, due to decrease of enzyme activity upon fluorescent labeling, it is better to take into count the influence of NHS-fluorescein concentration on the relative enzymatic activity. Moreover, the calculation of molar...

Introdução: As estatinas são drogas amplamente utilizadas na prevenção primária e secundária de doenças cardiovasculares, por reduzirem o nível de colesterol. Porém alguns pacientes podem apresentar elevação da creatinofosfoquinase (CPK) e sintomas musculares relacionados ao seu uso. Além da CPK, outros marcadores de dano muscular podem apresentar alterações. Este estudo analisou a concentração dos marcadores bioquímicos, CKMB e anidrase carbônica III (CAIII) e sua relação com a presença de miosite. Métodos: Foram selecionados pacientes em tratamento com estatinas e com elevação da CPK. Foram realizadas as determinações de CKMB e CAIII e analisadas as variáveis clínicas e laboratoriais destes pacientes. Resultados: Cerca de 10% dos pacientes em tratamento com estatina apresentaram elevações de CPK acima 1x o limite superior de normalidade (LSN). Desses, 50,4% apresentaram sintomas musculares, definido como miosite. O uso de sinvastatina [OR=2,24 (IC95%:1,47-3,42)], o índice de massa corpórea > 28 Kg/m2 [OR=1,06 (IC95%: 1,01-1,10)] e a CKMB > 1xLSN [OR=1,59 (IC95%: 1,02-2,49)] apresentaram-se como preditores independentes para a ocorrência de miosite. A CKMB aumentada foi observada em 36,2% dos pacientes (7,17±4,4 ng/mL). Os pacientes com e sem miosite apresentaram valores semelhantes de CAIII (211,3±93,4pg/mL vs 204,0±84,6pg/mL; p=0,549). Pacientes diabéticos apresentaram elevações significantes de CKMB em relação aos não diabéticos (4,8±4,6ng/mL vs 3,5±2,4ng/mL; p=0,0006) e não apresentaram diferenças quanto à presença de miosite. Conclusão: A CKMB apresentou alteração em parte dos pacientes tratados com estatinas e foi um preditor independente para a presença de miosite. A CAIII não foi considerada um bom marcador de dano muscular na população deste estudo / Introduction: Statins are drugs widely used in primary and secondary prevention of cardiovascular diseases, due to the decreasing effect on cholesterol level. However, some patients may present elevated levels of creatine phosphokinase (CK) and muscle symptoms related to statin use. In addition to CK, other markers of muscle damage may present changes. This study analyzed the concentration of biochemical markers, CKMB and carbonic anhydrase III (CAIII) and related them to the presence of myositis. Methods: Patients on statin therapy and CK elevation were selected. CKMB and (CAIII) assays were performed and the clinical and laboratory variables of these patients were analyzed. Results: About 10% of the patients receiving statin therapy (6692) presented CK elevations above 1x upper reference limit (URL). Muscular symptoms, defined as myositis, were presented in 50.4% of these patients. Use of simvastatin [OR=2,24 (IC95%:1,47-3,42)], a body mass index > 28 kg / m2 [OR = 1.06 (95% CI: 1.01-1, 10)] and a concentration of CKMB > 1x URL [OR = 1.59 (95% CI: 1.02-2.49)] presented as independent predictors for the occurrence of myositis. Increased CKMB was observed in 36.2% of patients (7.17 ± 4.4 ng / mL). Patients with and without myositis had similar CAIII values (211.3 ± 93.4pg / mL vs 204.0 ± 84.6pg / mL, p = 0.549). Diabetic patients showed significant elevations of CKMB compared to non-diabetic patients (4.8 ± 4.6 ng / mL vs. 3.5 ± 2.4 ng / mL, p = 0.0006) and did not present differences regarding the presence of myositis. Conclusion: CKMB level changed in part of the patients treated with statins and this enzyme was an independent predictor for the presence of myositis. CAIII was not considered a good marker of muscle damage in the studied population

This thesis describes the target selection, cloning, expression, purification, crystallization, structure and biochemical characterization of five essential Mycobacterium tuberculosis (Mtb) proteins. The search for drugs against the causal agent of tuberculosis is urgently needed and the targeting of essential genes is necessary to fulfill this goal. The crystal structures of carbonic anhydrases (CA) Rv1284 and Rv3588c have been determined to 2.0 and 1.7 Å resolution, respectively. Rv3588c, in contrast to Rv1284, is an active β-CA that shows two different active site conformations and pH-dependent oligomerization states. Rv1295 is an active threonine synthase with an unusually high pH optimum; the structure has been solved to 2.5 Å resolution, based on which a modification to the reaction mechanism published previously is proposed. Mtb has a thick and impermeable cell envelope that constitutes an efficient barrier against drugs. One of the essential components of the envelope is mycolic acid (MA). The inhibition of enzymes participating in its synthesis would be lethal for Mtb. Rv0636, a formerly unknown-function protein has β-hydroxyacyl-ACP dehydrase activity which is essential for MA synthesis. Co-expression with partners notably improves its solubility. Around 55% of Mtb proteins have unknown function. Rv3778c is one of them and its three-dimensional structure has been determined to 1.8 Å resolution. Studies aimed at the elucidation of its biochemical function are shown. A pathway not yet reported in Mtb is also suggested.