Les anticorps catalytiques sont étudiés pour comprendre leur rôle en conditions physiopathologiques. Ils semblent aussi représenter des outils révolutionnaires pour des études à l'interface entre la chimie, la biochimie, la biologie et immunologie. Par conséquent, la connaissance des relations de structure- fonction représente un grand intérêt. Nous avons exploré deux systèmes d'expression pour la production d'un anticorps catalytique modèle présentant une activité bêta-lactamase. Le fragment scFv recombinant a été produit dans le système d'expression procaryote. Les scFv sont souvent décrits comme des protéines difficiles à produire. Une méthode efficace a été développée pour produire de grandes quantités de scFv solubles et correctement repliés. L'anticorps catalytique entier a aussi été produit en exploitant le système d'expression eucaryote. Des cellules de mammifères ont été utilisées car elles peuvent conserver le repliement original des protéines, leur assemblage et les modifications post-traductionnelles. La structure secondaire du scFv catalytique a été analysée par dichroïsme circulaire pour s'assurer que la renaturation du scFv est en accord avec le repliement des scFv natifs. La fonctionnalité du scFv catalytique et de l'anticorps catalytique entier a été validée par deux approches : (1) le développement d'un test immuno-enzymatique (ELISA) et la résonance plasmonique de surface (RPS) et (2) le développement d'un test catalytique sensible utilisant un substrat fluorogénique. Ce travail amène à considérer de potentielles applications biotechnologiques et thérapeutiques des anticorps catalytiques.

This diploma thesis deals with the design of ox-bow lake revitalization measures, located southwest to the city Strakonice. Due to its charakteristics, ox-bow lake represents high-value biodiversity area. Recently, due to a disturbation in the aquatic regime, this area is threatened in sediment issue. Practical part of the diploma thesis is focused on the sediment and water level research and monitoring of plant and animal species. Obtained data result in particular revitalization measures.

Les récepteurs de chimiokines sont des régulateurs essentiels de la migration cellulaire dans le cadre de la surveillance immunitaire, et le développement. Les récepteurs CCR5 et CXCR4 sont de plus spécifiquement impliqués dans les métastases cancéreuses et l'infection par le VIH. Nous avons développé un système permettant de sur-exprimer ces deux RCPGs. Afin de s'affranchir des problèmes de toxicité inhérents à l'expression des protéines membranaires en bactérie notre approche de production consiste à adresser les protéines vers les corps d'inclusion d'E. coli grâce à une fusion protéique N-terminale permettant de hauts niveaux d'expression. Après purification en conditions dénaturantes, les protéines sont alors repliées en présence de surfactants originaux, les amphipoles. La validation de cette nouvelle approche pour les récepteurs des chimiokines représente un des objectifs principaux de ce travail. Afin de tester la fonctionnalité des protéines repliées, une série d'outils a été développée : des versions modifiées des chimiokines ont été produites (RANTES pour CCR5 et SDF 1a pour CXCR4). La fonctionnalité des chimiokines a été évaluée au niveau moléculaire et cellulaire. L'interaction entre le récepteur replié en amphipole et son ligand a été testé par résonance de plasmons de surface (SPR). Différents types de surfaces fonctionalisées avec le récepteur de chimiokine replié en amphipole ont été explorés au cours de ce travail. A la fin de ce projet, la production des chimiokines et de leur récepteur a été mise au point. L'accès à ces outils ouvre la voie à de futures études moléculaires telles que la compréhension de la dimérisation du récepteur ou la détermination de la stoechiométrie du complexe. / Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation and development. The GPCRs (G protein-coupled receptors) CCR5 and CXCR4 are specifically implicated in cancer metastasis and HIV-1 infection. An expression system to over-express these two GPCRs was developed. To overcome the toxicity problem of membrane protein expression in bacterial system, the production approach consists in targeting the proteins towards E. coli inclusion bodies thanks to a N-terminal fusion allowing a high yield expression. After purification under denaturing conditions, these GPCRs were then folded using original polymeric surfactants: the amphipols. The validation of this new approach for the chemokine receptor production is one of the goals of this work. In order to assess the functionality of the folded proteins, series of tools have been developed: engineered chemokine ligands (RANTES for CCR5 and SDF1a for CXCR4) were produced. The functionality of chemokines was evaluated at cellular and molecular levels. Interaction between the receptor folded in amphipols and its ligand was evaluated using Surface Plasmon Resonance (SPR) technique. Several types of surfaces, functionalized with the chemokine receptor/amphipol complex have been explored in this work. At the end of this project the productions of chemokines and their receptors has been set up. These established tools open the way to future studies, at the molecular level, in order to, for instance, investigate receptor dimerization and complex stoichiometry.

Study of receptor-ligand pair NKR-P1F and Clrg Mouse NKR-P1F:Clr-g receptor:ligand pair is important component of the receptor "zipper" that occurs at the contact between natural killer cell and its target cell, and represents a recently discovered example of lectin-lectin interactions important for recognition among immune cell subsets. In order to study structure of these proteins and interactions between them, we have prepared pET-30a(+) bacterial expression vectors coding parts of extracellular domains of the two receptors. After induction of protein production with IPTG, the proteins precipitated into inclusion bodies, from which they could be refolded in vitro. Refolded proteins were purified using combination of ion exchange and size exclusion chromatography. NKR-P1F construct yielded only small amounts of soluble protein using standard refolding protocols. Furthermore we have experienced difficulties with reproducibility of the refolding results. In the case of Clrg the standard protocols for protein refolding were not sufficient. In order for the Clrg to fold properly, the odd cysteine which does not fit into the pattern usual for this family of receptors was substituted for serine and resulting C148S construct was shown to be more useful. Further, using (benzyldimethylammonio)propanesulfonate in...

Les anticorps catalytiques sont étudiés pour comprendre leur rôle en conditions physiopathologiques. Ils semblent aussi représenter des outils révolutionnaires pour des études à l'interface entre la chimie, la biochimie, la biologie et immunologie. Par conséquent, la connaissance des relations de structure- fonction représente un grand intérêt. Nous avons exploré deux systèmes d'expression pour la production d'un anticorps catalytique modèle présentant une activité bêta-lactamase. Le fragment scFv recombinant a été produit dans le système d'expression procaryote. Les scFv sont souvent décrits comme des protéines difficiles à produire. Une méthode efficace a été développée pour produire de grandes quantités de scFv solubles et correctement repliés. L'anticorps catalytique entier a aussi été produit en exploitant le système d'expression eucaryote. Des cellules de mammifères ont été utilisées car elles peuvent conserver le repliement original des protéines, leur assemblage et les modifications post-traductionnelles. La structure secondaire du scFv catalytique a été analysée par dichroïsme circulaire pour s’assurer que la renaturation du scFv est en accord avec le repliement des scFv natifs. La fonctionnalité du scFv catalytique et de l'anticorps catalytique entier a été validée par deux approches : (1) le développement d’un test immuno-enzymatique (ELISA) et la résonance plasmonique de surface (RPS) et (2) le développement d'un test catalytique sensible utilisant un substrat fluorogénique. Ce travail amène à considérer de potentielles applications biotechnologiques et thérapeutiques des anticorps catalytiques. / Catalytic antibodies are investigated in order to understand their role under physio-pathological situations. But they also appear to be revolutionary tools to perform studies at the interface between chemistry, biochemistry, biology and immunology. Consequently, the knowledge of structure–function relationships is of great interest. We explored two expression systems for the production of a model catalytic antibody displaying a beta-lactamase activity. The recombinant scFv fragment was produced in the prokaryotic expression system. scFv fragments are often described as proteins being laborious to produce. An efficient method was developed to produce large quantities of refolded soluble catalytic scFv. Whole catalytic antibody was also produced by exploiting eukaryotic expression system. Mammalian cells were used because they are able to retain the original protein folding, assembly and post-translational modifications. The secondary structure of the catalytic scFv has been analyzed by circular dichroism to ensure that the refolded scFv is consistent with a native scFv fold. The functionality of the catalytic scFv and whole catalytic antibody has been validated by two approaches: (1) development of enzyme-linked immunosorbant assay (ELISA) and surface plasmon resonance (SPR) approaches for testing that the binding characteristics of an inhibitory peptide have been retained, and (2) proof of the subtle catalytic properties conservation through the development of a new sensitive catalytic assay using a fluorogenic substrate. This will lead to consider potential biotechnological and therapeutic applications of catalytic antibodies.

As principais matérias primas necessárias para a preparação de testes diagnósticos são as proteínas dos patógenos que necessariamente apresentem as estruturas nativas. O objetivo do presente estudo foi a obtenção das proteínas não estruturais 1 (NS1) dos vírus da dengue (DENV) e da zika (ZIKV) a partir dos corpúsculos de inclusão (CI) produzidos em bactérias Escherichia coli. Mostramos que a combinação de alta pressão hidrostática (APH) e pH alcalino é eficiente para a solubilização de NS1-CI. A incubação em 2,4 kbar das suspensões de NS1-CI em pH alcalino mostrou-se eficiente para a solubilização da NS1. A presença de Arg promove a dissociação de oligômeros. A aplicação de 2,4 kbar às suspensões de NS1-CI em pH de 10,5 (DENV) e de 11,5 (ZIKV) na presença de Arg e um par redox, seguida de diálise em tampão em pH 8,5, foram as condições escolhidas para o reenovelamento de NS1. Obtivemos ambas NS1 com rendimentos entre 75% e 90% em relação às quantidades totais das proteínas presente nos correspondentes CI de NS1. As NS1 reenoveladas apresentaram reatividade comparável às proteínas obtidas utilizando um protocolo convencional estabelecido, com rendimentos mais de 25 vezes superiores. Foi obtido um processo altamente eficiente para o reenovelamento de NS1 apresentando características biológicas preservadas em relação a reatividade com anticorpos específicos de antígeno, incluindo soro de paciente infectado com zikv e que, portanto, podem ser usados como antígeno para o desenvolvimento de vacinas ou testes de diagnóstico. Além disso, este estudo descreve a criação de um processo inovador, que é a utilização concomitante de APH e pH alcalino, para solubilização e posterior reenovelamento de NS1-CI que podem ser utilizados para outras proteínas relevantes. / The main products for the preparation of diagnostic tests are as proteins of the pathogens that necessarily present as the native structures. The objective of the present study was to obtain non-structural proteins 1 (NS1) from dengue virus (DENV) and zika virus (ZIKV) from the inclusion bodies (IBs) produced in Escherichia coli bacteria. We show that it is a combination of high hydrostatic pressure (HHP) and alkaline pH is efficient for a solubilization of NS1-IB. A 2.4 kbar incubation of NS1-IB suspensions at alkaline pH proved to be efficient for NS1 solubilization. The presence of Arg promotes the dissociation of oligomers. The application of 2.4 kbar to the suspensions of NS1-IB at pH 10.5 (DENV) and 11.5 (ZIKV) in the presence of Arg and a redox pair, dialysis in pH 8.5 buffer were as conditions chosen for the refolding of NS1. We obtained both NS1 at yields between 75% and 90% relative to the total amounts of the proteins present in the corresponding NS1 IB. Refolded NS1 showed similar to proteins obtained using an established standard protocol, with yields more than 25 times higher. A highly efficient process for the refolding of NS1 was obtained with preserved biological features regarding reactivity with antigen-specific antibodies, including sera of zikv-infected patients and that can be used as antigen for the development of vaccines or diagnostic tests. In addition, this study describes the creation of an innovative process, which is a concomitant use of HHP and alkaline pH, for solubilization and subsequent refolding of NS1-IB that can be used for other relevant proteins.

This diploma thesis deals with returning of modified troughs of small watercourses to the natural state - revitalisation. The general idea of the river beds and water environment revitalisation will be explained in the theoretical part. It will also deal with its history, development and practical application. The practical part will contain an own project creation. It is a project of the adjusted small watercourses bed revitalisation in the level of study. A section of a fine river was used for this thesis. It is called Žďárský Stream and it belongs to the cadastral area Žďár near the Kaplice. Individual parts of the river and its basin will be mapped in this work. It will also consider the hydrological and geographical conditions and it will propose a technical solving for a local revitalisation of the adjusted river bed. It will ínclude natural factors support, the ecological-morphological improvement, the migrational permeability and other links to the river environment.

As principais matérias primas necessárias para a preparação de testes diagnósticos são as proteínas dos patógenos que necessariamente apresentem as estruturas nativas. O objetivo do presente estudo foi a obtenção das proteínas não estruturais 1 (NS1) dos vírus da dengue (DENV) e da zika (ZIKV) a partir dos corpúsculos de inclusão (CI) produzidos em bactérias Escherichia coli. Mostramos que a combinação de alta pressão hidrostática (APH) e pH alcalino é eficiente para a solubilização de NS1-CI. A incubação em 2,4 kbar das suspensões de NS1-CI em pH alcalino mostrou-se eficiente para a solubilização da NS1. A presença de Arg promove a dissociação de oligômeros. A aplicação de 2,4 kbar às suspensões de NS1-CI em pH de 10,5 (DENV) e de 11,5 (ZIKV) na presença de Arg e um par redox, seguida de diálise em tampão em pH 8,5, foram as condições escolhidas para o reenovelamento de NS1. Obtivemos ambas NS1 com rendimentos entre 75% e 90% em relação às quantidades totais das proteínas presente nos correspondentes CI de NS1. As NS1 reenoveladas apresentaram reatividade comparável às proteínas obtidas utilizando um protocolo convencional estabelecido, com rendimentos mais de 25 vezes superiores. Foi obtido um processo altamente eficiente para o reenovelamento de NS1 apresentando características biológicas preservadas em relação a reatividade com anticorpos específicos de antígeno, incluindo soro de paciente infectado com zikv e que, portanto, podem ser usados como antígeno para o desenvolvimento de vacinas ou testes de diagnóstico. Além disso, este estudo descreve a criação de um processo inovador, que é a utilização concomitante de APH e pH alcalino, para solubilização e posterior reenovelamento de NS1-CI que podem ser utilizados para outras proteínas relevantes. / The main products for the preparation of diagnostic tests are as proteins of the pathogens that necessarily present as the native structures. The objective of the present study was to obtain non-structural proteins 1 (NS1) from dengue virus (DENV) and zika virus (ZIKV) from the inclusion bodies (IBs) produced in Escherichia coli bacteria. We show that it is a combination of high hydrostatic pressure (HHP) and alkaline pH is efficient for a solubilization of NS1-IB. A 2.4 kbar incubation of NS1-IB suspensions at alkaline pH proved to be efficient for NS1 solubilization. The presence of Arg promotes the dissociation of oligomers. The application of 2.4 kbar to the suspensions of NS1-IB at pH 10.5 (DENV) and 11.5 (ZIKV) in the presence of Arg and a redox pair, dialysis in pH 8.5 buffer were as conditions chosen for the refolding of NS1. We obtained both NS1 at yields between 75% and 90% relative to the total amounts of the proteins present in the corresponding NS1 IB. Refolded NS1 showed similar to proteins obtained using an established standard protocol, with yields more than 25 times higher. A highly efficient process for the refolding of NS1 was obtained with preserved biological features regarding reactivity with antigen-specific antibodies, including sera of zikv-infected patients and that can be used as antigen for the development of vaccines or diagnostic tests. In addition, this study describes the creation of an innovative process, which is a concomitant use of HHP and alkaline pH, for solubilization and subsequent refolding of NS1-IB that can be used for other relevant proteins.

Le pronostic de certains cancers s’est considérablement amélioré avec l’arrivée sur le marché des anticorps thérapeutiques. Devant l’essor de ces nouveaux médicaments associé à l’identification de nouveaux biomarqueurs, de nouvelles perspectives émergent pour l’imagerie moléculaire in vivo. En effet, disposer de nouveaux traceurs moléculaires spécifiques de ces biomarqueurs permettra de caractériser l’hétérogénéité des cellules cancéreuses, de suivre l’expression de ces marqueurs au cours de l’évolution de la tumeur, mais également de suivre l’efficacité d’un traitement sur la régression tumorale du patient. Pour répondre à cette évolution de diagnostic moléculaire in vivo, il convient de développer de nouvelles sondes moléculaires. L'objectif de ma thèse répond à ce nouveau besoin avec l'ingénierie et le marquage d'un format d'anticorps recombinant adapté à l'imagerie in vivo : le diabody 12G4 dirigé contre le récepteur de l’hormone antimüllérienne (AMH), marqueur de certains cancers de la sphère génitale. / Prognosis of cancers dramatically improved with the development on the market of therapeutic antibodies. With the increase of these new biodrugs, associated with the identification of new biomarkers, new opportunities emerge for the in vivo molecular imaging.. Indeed, to use new molecular tracers specific of tumoral biomarkers will allow to study and characterize the cancer heterogeneity, to monitor the expression of these markers during the tumor evolution, but also to check the treatment effectiveness on patients’ tumoral regression. To answer this evolution of in vivo molecular diagnosis, it is favorable to develop new molecular probes. The aim of this thesis answers this new need with the engineering and labeling of a new recombinant antibody format suitable to in vivo imaging : the 12G4 diabody directed against the type II human receptor for the anti-Müllerian hormone, a biomarker of some cancers of the genital area.

Protein Nkr-p1b is a surface receptor of cytotoxic NK cells, that mediates inhibitory signal toward the body's own cells. In this study, the ligand binding domain of the mouse protein receptor Nkr-p1b (mNkr-p1b LBD) was prepared by recombinant expression in E. coli cells. Isolated protein was subsequently used for NMR structural analysis. Prediction of protein secondary structures ratio was carried out using three different methods (CD, PSIPRED and TALOS). Results correlate well with the structure of CTLD domain, that plays a key role in ligand binding and thus to function of Nkr-p1b receptor. We managed to prepare this protein in a form suitable for NMR experiments. Based on the data obtained by NMR spectra analysis, a preliminary model of the mNkr-p1b LBD protein structure was created. However, for more precise learning of the 3D structure accurate positions of individual atoms need to be determined by other NMR spectra evaluation in the next phase. Explaining the structure of the ligand binding domain of mNkr-p1b protein could help to better understand the complex mechanism of activation of NK cell cytotoxic activity, thereby contributing to its controlled use as a therapeutic against some viral and tumor diseases.