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The adhesion G protein–coupled receptor (aGPCR) GPR126/ADGRG6 plays an important role in several physiological functions, such as myelination or peripheral nerve repair. This renders the receptor an attractive pharmacological target. GPR126 is a mechano-sensor that translates the binding of extracellular matrix (ECM) molecules to its N terminus into a metabotropic intracellular signal. To date, the structural requirements and the character of the forces needed for this ECM-mediated receptor activation are largely unknown. In this study, we provide this information by combining classic second-messenger detection with single-cell atomic force microscopy. We established a monoclonal antibody targeting the N terminus to stimulate GPR126 and compared it to the activation through its known ECM ligands, collagen IV and laminin 211. As each ligand uses a distinct mode of action, the N terminus can be regarded as an allosteric module that can fine-tune receptor activation in a context-specific manner.

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Malgré quarante ans d’épidémie causée par le VIH-1, aucun traitement curatif n’est actuellement disponible. Un obstacle majeur à l’éradication du virus est la persistance de cellules infectées ensemençant le réservoir viral. Les immunothérapies représentent une voie prometteuse afin de contrôler la charge virale tel que suggéré par de récentes études animales et essais cliniques récents. Les thérapies à base d’anticorps ont le potentiel d’éliminer les cellules infectées par la réponse cytotoxique dépendante des anticorps (ADCC). Lors de l’infection au VIH, les protéines accessoires virales Nef et Vpu jouent un rôle primordial dans l’évasion des réponses ADCC en régulant la conformation de la glycoprotéine d’enveloppe (Env) à la surface des cellules infectées. Dans son état natif, Env stabilise une conformation dite «fermée», lui conférant une résistante à la reconnaissance par les anticorps non-neutralisants (nnAbs). Ces anticorps ont le potentiel d’éliminer les cellules infectées par ADCC lorsqu’Env interagit avec son récepteur CD4 et stabilise des conformations dites «ouvertes». Nef et Vpu bloquent cette éventualité en régulant négativement le récepteur CD4 de la surface cellulaire, empêchant une interaction prématurée entre Env et CD4. Tout comme le récepteur CD4, de petites molécules mimant le CD4 (CD4mc) permettent de sensibiliser les cellules infectées à la réponse ADCC en induisant des changements structurels dans Env, exposant les épitopes reconnus par les nnAbs. Afin de mieux comprendre comment les CD4mc sensibilisent les cellules infectées à la réponse ADCC, nous avons étudié le rôle des différentes classes d’anticorps ciblant l’Env chez les personnes vivant avec le VIH (PVVIH). Nos résultats ont identifié une famille d’anticorps ciblant une région conservée de la gp41 comme jouant un rôle majeur dans la réponse ADCC des plasmas des PVVIH en présence de CD4mc. Ces résultats ont mené au développement d'un nouveau cocktail composé de trois familles de nnAbs et de CD4mc augmentant l’élimination de cellules infectées par la réponse ADCC. Lors de l’étude de la susceptibilité des cellules infectées à la réponse ADCC, nous avons découvert un autre mécanisme par lequel la protéine accessoire Vpu protège les cellules infectées de la réponse cytotoxique des cellules NK. En effet, nous avons observé que Vpu diminue l’expression de surface de CD48, le ligand du récepteur co-activateur 2B4 des cellules NK. Nous avons démontré l’importance fonctionnelle de cette régulation négative afin d’échapper à la réponse ADCC. Les résultats de cette thèse approfondissent nos connaissances sur les mécanismes gouvernant la susceptibilité des cellules infectées à la réponse ADCC, ce qui permettra de développer de nouvelles approches visant à purger les réservoirs viraux à l’aide d’immunothérapies à base d’anticorps. / Despite forty years of research, there is still no curative treatment available for HIV-1. The persistence of latently infected cells seeding the viral reservoir is a major obstacle to viral eradication. Immunotherapies represent a promising avenue for controlling the viral load as suggested by recent animal studies and clinical trials. Antibody-based therapies have the potential to eliminate infected cells through the antibody-dependent cytotoxic response (ADCC). During HIV infection, the viral accessory proteins Nef and Vpu play a critical role in protecting infected cells from ADCC by downregulating CD4 which would otherwise prematurely “open-up” the HIV-1 envelope glycoproteins (Env), rendering infected cells susceptible to ADCC mediated by non-neutralizing antibodies (nnAbs). Stemming from this observation, we use small CD4 mimetic (CD4mc) to “open” Env, rendering HIV-1-infected cells susceptible to ADCC by nnAbs. To move this strategy forward, we studied the contribution of different classes of nnAbs in people living with HIV to eliminate infected cells. We identified a family of nnAbs targeting a conserved region of gp41 that plays a major role in CD4mc-mediated ADCC. These results led to the development of a new cocktail composed of three nnAbs and CD4mc with very potent ADCC activity. While studying the susceptibility of infected cells to ADCC, we uncovered another immune evasion strategy mediated by Vpu, which downregulates the cell-surface expression of CD48, the ligand of the NK-cell co-activating receptor 2B4. We found that Vpu-mediated CD48 downregulation contributes to the resistance of HIV-1-infected cells to ADCC. Altogether, the results presented in this thesis provides a better understanding of the susceptibility of HIV-1 infected cells to ADCC. This new information would likely be helpful in the development of new approaches aimed at purging viral reservoirs using antibody-based immunotherapies.

Computer modeling of enzymes is a valuable complement to experiments. Quantum chemical studies of enzymatic reactions can provide a detailed description of the reaction mechanism and elucidate the roles of various residues in the active site. Different reaction pathways can be analyzed, and their feasibility be established based on calculated energy barriers. In the present thesis, density functional theory has been used to study the active sites and reaction mechanisms of three different enzymes, cytosine deaminase (CDA) from Escherichia coli, ω-transaminase from Chromobacterium violaceum (Cv-ωTA) and dinitrogenase reductase-activating glycohydrolase (DraG) from Rhodospirillum rubrum. The cluster approach has been employed to design models of the active sites based on available crystal structures. The geometries and energies of transition states and intermediates along various reaction pathways have been calculated, and used to construct the energy graphs of the reactions. In the study of CDA (Paper I), two different tautomers of a histidine residue were considered. The obtained reaction mechanism was found to support the main features of the previously proposed mechanism. The sequence of the events was established, and the residues needed for the proton transfer steps were elucidated. In the study of Cv-ωTA (Paper II and Paper III), two active site models were employed to study the conversion of two different substrates, a hydrophobic amine and an amino acid. Differences and similarities in the reaction mechanisms of the two substrates were established, and the role of an arginine residue in the dual substrate recognition was confirmed. In the study of DraG (Paper IV), two different substrate-binding modes and two different protonation states of an aspartate residue were considered. The coordination of the first-shell ligands and the substrate to the two manganese ions in the active site was characterized, and a possible proton donor in the first step of the proposed reaction mechanism was identified. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Platelet-activating factor acetylhydrolase (PAFAH) is elevated in atherosclerosis and may play a role in pathogenesis of this disease. Molecular mechanisms regulating the expression of this lipoprotein-associated PLA2 are indistinct. Mildy oxidized low density lipoprotein (oxLDL) and monocytes (the primary source of PAFAH) are co-localized in early atheromas. Monocytes are activated by oxidized phospholipids (oxPL) in the oxLDL particle. We hypothesized that oxPL-activated monocytes are the source of increased levels of PAFAH in atherosclerosis. We found that PAFAH expression is significantly induced by OxPAPC and in particular long-chain fractions of oxPAPC in monocytes and cytokine-differentiated DC, but not cytokine-differentiated MO. Furthermore, spontaneously differentiated MO and DC from monocytes of non-periodontitis and aggressive periodontitis subjects, oxPAPC induced PAFAH in DC alone. 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphocholine (PEIPC) is a particularly bioactive component of long-chain oxPAPC fractions that binds the prostaglandin receptor subtypes DP1 and EP2. We revealed using selective agonists and antagonists of these receptors that DP1 and EP2 are required for the induction of PAFAH expression. OxPAPC stimulates IL-6 release from monocytes and this cytokine is required for oxPAPC-induced PAFAH expression. We next tested the hypothesis that oxPAPC did not induce PAFAH in MO because a key component of the signaling machinery was lacking. Flow cytometric and immunoblot analyses demonstrated that MO express very low levels of IL-6 receptor in comparison to DC and monocytes. Based on these observations, we propose that long-chain oxPL induce PAFAH expression by binding DP1 and/or EP2 and stimulating IL-6 production. These data strongly support the hypothesis that oxLDL-activated DC are the source of high PAFAH levels in atherosclerosis. Platelet activating factor (PAF) is the inflammatory phospholipids for which PAFAH is named. PAF has been shown by other investigators to induce the expression of PAFAH. In our physiologically relevant monocytes, PAF suppresses PAFAH transcription and expression. 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine (POVPC) is a short-chain oxPL that signals through the PAF receptor. Our preliminary data suggest that like PAF, POVPC suppresses PAFAH expression in monocytes. Further investigation into the effects of the short-chain oxPL are warranted. Our data support the hypothesies that oxPL-activated DC are the source of high PAFAH levels in atherosclerosis.

Classical frontal teaching, where pupils are supposed to listen to the teacher and make notes is becoming stereotypical. Therefore I decided to focus on a topic where I can apply more teaching methods both in Geography lessons and Biology ones. The goal of this work was to create a teaching guide including necessary materials covering the topic of American natural lanscapes and afterwards to evaluate the contribution of these activities. The teaching materials were tested in two classes in The Basic School in Dolní Břežany. Both classes underwent a pretest, the first posttest (immediately after the experimental lessons), the second posttest (six weeks after the experimental lessons) and pupils also tried activities in which they had to apply their achieved knowledge. The data were evaluated using statistical analyses where the results showed a progress of gained knowledge between the pretest and the first posttest, whereas their knowledge did not significantly differ between the first and second posttest. It can be concluded that pupils broadened their knowledge, which was deeper than at the beginning and the pupils were able to retain this knowledge for a longer time. Key words: North America, South America, natural lanscapes, Biology, Geography, activating teaching methods, curriculum integration