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Les travaux réalisés au cours de cette Thèse avaient pour but de caractériser au niveau fonctionnel la protéine FAM110B, identifiée au laboratoire il y a plusieurs années par une méthode de sélection d’éléments génétiques suppresseurs destinée à rechercher de nouveaux gènes impliqués dans la sensibilité à un inhibiteur de Topoisomérase II, la 9-hydroxyéllipticine. Localisée au niveau cytoplasmique et très conservée chez les mammifères, FAM110B est essentielle à la survie comme le montre le blocage en phase S des cellules dans lesquelles son expression est transitoirement diminuée. Sa répression conduit d’ailleurs à l’inhibition de plusieurs voies impliquées dans la prolifération cellulaire comme les voies Wnt, Notch ou TGF-. Les résultats que nous avons obtenus suggèrent que ce rôle dans la prolifération peut être régulé par l’interaction de FAM110B avec la β-caténine. Cette interaction régule le niveau d’expression de la β-caténine et/ou sa localisation, ce qui a pour conséquence directe de moduler l’expression de ses gènes cibles impliqués dans la prolifération cellulaire. Nous avons également démontré que FAM110B intervient dans les processus de migration cellulaire en régulant directement ou indirectement l’expression de la E-cadhérine par la modulation sélective de l’expression d’un de ses répresseurs, Slug. L’augmentation de l’expression de la E-cadhérine dans des cellules sousexprimant FAM110B est accompagnée d’une diminution de l’expression de la N-cadhérine, un phénomène qui est fréquemment observé lors de la reverse EMT, passage d’un stade mésenchymateux à un stade épithélial au cours duquel, des cellules à caractère invasif et métastatique acquièrent des propriétés adhésives associées à une perte de leur propriétés de migration et d’invasion. Enfin, nous avons pu démontrer que FAM110B est également impliquée dans la sensibilité cellulaire à divers agents anticancéreux. Sa répression induit une sensibilisation à la camptothécine et au cisplatine alors qu’elle confère une résistance aux poisons de tubuline (taxol et vincritine) et aux inhibiteurs de Topoisomérases II par diminution du nombre de complexes de clivage ADN-enzyme associée à une réduction du niveau de Topo2 dont on ne connait pas encore l’origine. L’ensemble de nos résultats confirment l’importance de FAM110B dans la migration et la prolifération cellulaire ainsi que dans la réponse aux stress induits par diverses classes d’agents anticancéreux. De ce fait, FAM110B peut être considérée comme une nouvelle cible potentielle en cancérologie et son inhibition être utilisée pour potentialiser l’action de thérapeutiques existantes tels que les dérivés de la camptothécine ou les dérivés du platine qui sont largement utilisés en clinique. / FAM110B is a new protein that was identified several years ago in our laboratory by a functional screen, the selection of genetic suppressor elements (GSEs), which goal was to identify new genes involved in the cellular sensitivity to the topoisomerase II inhibitor 9-hydroxyellipticine. FAM110B is localized in the cytoplasm and is extremely conserved across mammals. We found that FAM110B is essential for cell survival, as its transient repression induces a blockage in the S phase of the cell cycle. Its repression also induces the inhibition of various pathways involved in the regulation of cell proliferation, such as Wnt, Notch or TGF-. Our results suggest that its role in cell proliferation relies on FAM110B interaction with β-catenin. This interaction regulates β-catenin expression and its subcellular localization, which directly impacts on the expression of its target genes involved in cell proliferation. We have also demonstrated that FAM110B is involved in cell migration by regulating the expression of E-cadherin via the specific modulation of one of its repressors, Slug. Increase in E-cadherin expression in cells with downregulated FAM110B is accompanied by a decrease in N-cadherin expression, a phenomenon which is reminiscent of a reverse EMT i.e. a mesenchymal to epithelial transition which is characterized by a loss of invasiveness and metastatic potential. Finally, we also showed that FAM110B is involved in the regulation of the cellular sensitivity to various anticancer agents. Transient repression of FAM110B sensitizes cells to camptothecin and cisplatin, whereas it confers a resistant phenotype to tubuline poisons (taxol and vincristine) and Top2 inhibitors. This latter effect is accompanied by a reduction in DNA-Topo2 cleavage complexes due to a reduction in Topo2 levels by a mechanism which is not fully elucidated. Together, our results confirm the importance of FAM110B in essential processes such as migration, cell proliferation, and cell response to various stresses induced by chemotherapeutic agents. Therefore, FAM110B can be considered as a new potential target for cancer treatment and its inhibition can also be used to potentiate existing treatments such as camptothecin derivatives and platinum compounds that are widely used in the clinic.

Les électrodes modifiées avec cyclodextrines ayant des grandes performances analytiques représentent une alternative intéressante pour le développement de (bio)capteurs électrochimiques dans un domaine attractif de la recherche pour différentes applications, et notamment les analyses pharmaceutiques et biomédicales. Le but de cette étude a été de développer de nouvelles électrodes modifiées avec β-cyclodextrine pour des applications biomédicales et pharmaceutiques. L'influence de la β-cyclodextrine a été étudiée en solution et à la surface d’électrodes.L'influence de la β-cyclodextrine en solution aqueuse sur le comportement électrochimique de certaines substances pharmaceutiques (l’acide ascorbique, l'acide urique, la caféine, la théophylline, l'aminophylline et l'acétaminophène) a été étudiée en utilisant des méthodes électrochimiques et spectrales, mettant en évidence la formation de complexes d'inclusion.Diverses techniques ont été utilisées pour la modification des électrodes avec β-cyclodextrine: l'incorporation de la β-cyclodextrine dans la pâte de carbone et le piégeage dans des films polymères de polyéthylèneimine. Ces capteurs ont permis la détermination simultanée de l'acide ascorbique et de l'acide urique. Les électrodes modifiées ont été aussi appliquées pour le dosage de l'acide ascorbique dans deux produits pharmaceutiques et pour l’évaluation quantitative de l'acide ascorbique et de l'acide urique dans l’urine humaine avec de bonnes performances.Deux types de biocapteurs basés sur une nouvelle nanostructure de graphène ont été élaborés avec de l'oxyde réduit de graphène, de β-cyclodextrine et de tyrosinase en utilisant la méthode couche par couche et l’électropolymérisation. Les nouveaux nanocomposites ont été caractérisés par des techniques spectrales, microscopiques et électrochimiques. Les biocapteurs optimisés ont été appliquées avec succès pour la détermination du catéchol et de la dopamine dans des produits pharmaceutiques et des échantillons biologiques avec une bonne récupération.La solubilisation dans l'eau de certains nouveaux fluorophores (quatre nouvelles tétrazines) en utilisant la -cyclodextrine et de nanoparticules d'or modifiées avec β-cyclodextrine a été signalée. Les assemblages supramoléculaires redox ont été caractérisés dans l'eau par analyses électrochimiques et de fluorescence. L'immobilisation de tétrazines sur différents types d'électrodes modifiées par polypyrrole-cyclodextrine a été également réalisée et examinée par techniques électrochimiques, spectroscopiques et microscopiques.Une autre contribution originale est la combinaison de la lithographie avec de nanosphères utilisant des billes de latex avec différents diamètres (900 et 100 nm), avec l’électropolymérisation du monomère pyrrole-Ru(II). Des micro et nanostructures très organisées ont été réalisées en présentant de meilleures6propriétés pour le film photosensible de poly [Ru(II)-pyrrole]. Par ailleurs, le film de poly [Ru(II)-pyrrole] a été modifié avec d'autres types de dérivés de pyrrole qui présentent de propriétés de complexation utiles pour l’immobilisation des biomolécules à la surface de l'électrode. / The cyclodextrin modified electrodes with enhanced analytical performances represent an attractive promise for the future development of electrochemical (bio)sensors and remain a very active field of research for a wide range of applications in many areas, including pharmaceutical and biomedical analysis. The aim of this study was to develop novel cyclodextrin modified electrodes for pharmaceutical and biomedical applications. The β-cyclodextrin influence was investigated both in solution and immobilized at the electrode surface.The influence of β-cyclodextrin on the electrochemical behavior of some pharmaceuticals (ascorbic acid, uric acid, caffeine, theophylline, aminophylline, and acetaminophen) in aqueous solutions was studied by using electrochemical and spectral methods, which highlighted the inclusion complexes formation.Various techniques were used for the electrode modification with β-cyclodextrin, such as: the incorporation in carbon paste and the entrapment in polymeric films (polyethylenimine). These sensors allowed the simultaneous determination of ascorbic and uric acids. The modified electrodes were also applied for the dosage of ascorbic acid in two pharmaceutical products and for the ascorbic and uric acids quantification in human urine with good performances.Two types of biosensors based on a new nanostructured graphene framework were developed with reduced graphene oxide, β-cyclodextrin and tyrosinase by using either layer by layer method or electropolymerization. These new nanocomposites were characterized by spectral, microscopic and electrochemical techniques. The optimized biosensors were successfully applied for catechol and dopamine determination in pharmaceutical products, serum and urine samples with good recoveries.The solubilization in water of some new fluorophores (four new synthesized tetrazines) by using -cyclodextrin and gold nanoparticles modified with β-cyclodextrin was reported. The redox supramolecular assemblies were characterized in water by electrochemical and fluorescence measurements. The immobilization of tetrazines onto various types of electrodes modified with polypyrrole-cyclodextrin was also achieved and examined by electrochemical, microscopic and spectroscopic techniques.Another original contribution is the combination of nanosphere lithography by using latex beads with different diameters (900 and 100 nm), with the electropolymerization of a Ru(II)-pyrrole monomer. The achievement of highly organized micro and nanostructures showed enhanced features for the photosensitive electrogenerated poly-[RuII-pyrrole] films. Furthermore, poly-[RuII-pyrrole] film was modified with other types of pyrrole derivatives presenting complexation properties in order to immobilize biomolecules at the electrode surface.

Les invadosomes sont des structures d’invasion plastiques et dynamiques qui interagissent avec leur microenvironnement. Ils possèdent différentes fonctions telles que l’adhésion, la mécanotransduction ou encore la dégradation de la matrice extracellulaire (MEC). Mon travail de thèse s’est concentré sur i) l’étude globale de la composition des invadosomes par spectrométrie de masse et ii) sur l’impact d’éléments du microenvironnement dans la formation de ces structures d’invasion.i) Les invadosomes sont des complexes multi-protéiques dont tous les partenaires ne sont pas encore totalement identifiés. Au laboratoire, une nouvelle approche combinant la microdissection laser suivie d’une analyse par spectrométrie de masse, a été développée. Cette technique a été appliquée à l’étude des invadosomes rosettes. Nous avons ainsi mis en évidence une nouvelle fonction associée aux invadosomes, en les définissants comme des sites actifs de traduction protéique. Les invadosomes cependant, sont des structures plastiques dont la formation et la morphologie sont modulées par différents éléments de l’environnement. Nous souhaitons à présent déterminer les partenaires communs et spécifiques entre les différentes organisations des invadosomes afin d’identifier les molécules impliquées dans cette plasticité.ii) La formation des invadosomes peut être induite par différents éléments du microenvironnement comme des facteurs de croissance ou encore la composition et la rigidité de la MEC. Le TGF-β est un facteur de croissance impliqué dans la formation des invadosomes, dans la promotion de la rigidité de la MEC et dans la fibrose hépatique pouvant mener au développement du carcinome hépatocellulaire. Nous avons alors étudié l’impact du TGF-β dans la formation des invadosomes linéaires en contexte de collagène de type I. Nous montrons que le TGF-β module la machinerie moléculaire associée aux invadosomes linéaires en induisant l’expression de DDR1 et MT1-MMP, ainsi que des éléments impliqués dans leur formation tels que le collagène I. Ces modulations sont dépendantes de la voie de signalisation canonique du TGF-β passant par Smad4 et favorisent la formation et l’activité des invadosomes linéaires. De plus, le TGF-β induit une surexpression de la LOXL2 qui est une enzyme de réticulation du collagène, augmentant la rigidité de la matrice ce qui favorise la formation des invadosomes.Les résultats obtenus durant ma thèse auront permis de mieux définir les éléments impliqués dans la composition et la formation des invadosomes. / Invadosomes are plastic and dynamic invasive structures interacting with the microenvironement. Those structures are involved in several functions as adhesion, mecanotransduction and degradation of the extracellular matrix (ECM). My PhD work focuses on i) the study of the invadosomes composition by mass spectrometry and ii) on the impact of microenvironmental elements on the formation of those invasive structures.i) Invadosomes are multi-protein complexes in which all partners are not yet fully identified. In the laboratory, a new approach combining laser micordissection followed by mass spectrometry analysis was developed. This technique has been applied to the study of invadosome rosettes. We have demontrasted a new function associated with indosomes, defining them as active sites of protein translation. Invadosomes, however, are plastic structures whose formation and morphology are modulated by different elements of the environment. We now wish to determine the common and specific partners between the different invadosomes organizations in order to identify the molecules involved in their plasticity.The invadosomes formation can be induced by different elements of the microenvironment such as growth factors or the composition and rigidity of the ECM. TGF-β is a growth factor involved in the invadosomes formation, in the ECM rigidity and in liver fibrosis that can lead to the development of hepatocellular carcinoma. We have studied the impact of TGF-β in the formation of linear invadosomes in the context of type I collagen. We show that TGF-β modulates the molecular machinery associated with linear invadosomes by inducing the expression of DDR1 and MT1-MMP, as well as elements involved in their formation, such as collagen I. These modulations are dependent on the TGF-β canonical signaling pathway through Smad4 and promote the formation and activity of linear invadosomes. In addition, TGF-β induces an overexpression of LOXL2, which is a collagen cross-linking enzyme, increasing the matrix stiffness and promotes the formation of these structures.Taken together, these results enabled us to better define the elements involved in the composition and formation of invadosomes.

Title: Raman optical activity and conformational flexibility of peptides in solution Author: Jana Hrudíková Department: Institute of Physics of Charles University Supervisor: Doc. RNDr. Vladimír Baumruk, DrSc. Supervisor's e-mail address: baumruk@karlov.mff.cuni.cz Abstract: Molecular flexibility can significantly modify Raman and ROA spectral intensities, band positions and the ROA signs. Taking into account dynamic aspects of behavior of studied molecules in solution via conformational averaging therefore seems to be crucial for spectral interpretation. The first of studied models, histidine, plays an important role in metallo-enzymatic reactions and peptide folding, due to its imidazole ring. ROA spectra of His at different pH, His complexed with Cu2+ and dipeptides His- Gly and Gly-His were recorded on the spectrometer built at the Institute of Physics of the Charles University as a first step of the subsequent study. The second studied system, a cyclic hexapeptide c-(Phe-D-Pro-Gly-Arg-Gly-Asp), serves as a convenient model for β- hairpin and anti-parallel β-sheet. It was previously studied by means of VCD and IR. From molecular dynamics simulations 10 peptide geometries were selected for spectral modeling. The Raman and ROA spectra were calculated ab initio. For a model fragment Phe-D-Pro, which...

Fibroblast growth factor 21 (FGF21) is a unique peptide hormone involved in the energy homeosta- sis, as well as in the regulation of glucose and lipid metabolism. Numerous animal studies suggest that FGF21 may be used as a potential treatment for obesity and type 2 diabetes mellitus. It was found out, that FGF21 counteracts the development of obesity presumably by increasing energy expenditure through activation of thermogenesis in brown and white adipose tissue. FGF21 apparently also inhibits lipolysis. However, the specific mechanism of action of FGF21 is not clear. In our experiments we studied the antiobesogenic effects of FGF21 on mice model of diet-induced obesity at thermoneutrality. It is assumed that this model approach (in contrast to housing mice at standard laboratory temperature) mimics closely the metabolic status of humans. During the 4- to 8-day FGF21 treatment we observed a gradual reduction of lipid content in the brown and white adipose tissue and liver, especially in combination with β3-adrenergic stimulation. We have confirmed that FGF21 inhib- its lipolysis and also stimulates browning in certain adipose tissue depots. Furthermore, we have found that the effect of FGF21 on fatty acid secretion by adipose tissue is not mediated by changes in the fatty acid re-esterification...

β-N-acetylhexosaminidases (EC 3.2.1.52, GH 20) are retaining exo-glycosidases that in vivo cleavage both β-N-acetylglucosamine (GlcNAc) or β-N-acetylgalactosamine (GalNAc) residues fom glycostructures. Under suitable reaction conditions, these enzymes are able to synthesize the glycosidic bond in good yields. Substitution of selected amino acid(s) in the emzyme active site by site-directed mutagenesis may change the enzyme's substrate specificity or suppress the hydrolytic activity of the enzyme in favor of synthesis. The present thesis deals with three mutant β-N-acetylhexosaminidases from Talaromyces flavus, in which the amino acid residues responsible for binding to C-4 hydroxyl of the substrate (Arg218, Glu546) were exchanged for amino acids proposed on the basis of molecular modeling. The effect of introduced single point mutations on substrate specificity of prepared enzymes was studied. Mutant β-N-acetylhexosaminidases were heterologously expressed in Pichia pastoris and characterized. Furthermore, transglycosylation reactions with these enzymes were performed. The prepared carbohydrate products were characterized by NMR.

Pyrrolo [2,1-c] [1,4] benzodiazepine (PBD) is a class of natural products obtained from various actinomycetes which have both anti-tumor and antibiotic activities and can bind to specific sequences of DNA. PBD-dilactam was initially produced using isatoic anhydride and (L)-proline which was then converted to the PBD-thiolactam using Lawesson's reagent. Reaction of thiolactam with hydrazine in ethanol afforded PBD-11-hydrazinyl. Condensation of 11-hydrazinyl PBD with aldehydes possessing various substitutions was performed to obtain (S,E)-11-[2-(arylmethylene) hydrazono] pyrrolo [2,1-c] [1,4] benzodiazepine derivatives. 1HNMR, 13C-NMR, DEPT, IR, GC-MS and X-ray crystallography were used for the characterization. Inhibition activity of the products were carried out using TEM-1, AmpC and P99 β-lactamases. A minimal inhibition growth of 25% was observed for one of the selected PBDs on cancer cell line. A promising result was observed on preliminary cannabinoid binding activity test on one of the compounds.

Endogenous cardiac protection against acute ischemia/reperfusion injury can by increased by cardiac adaptation to various forms of chronic hypoxia. Chronic hypoxia induces a large variety of adaptive changes in the myocardium that could be considered as protective, but the exact mechanism of increased ischemic tolerance is unknown. Different studies suggest that catecholamine release and their effect on -adrenergic signaling after adaptation to chronic hypoxia contributes to cardioprotection. In this study we focused on characterization of -adrenergic receptors ( -ARs) in the myocardium of rats after adaptation to three different hypoxic conditions: 1. intermittent normobaric hypoxia - INH/R (23 h hypoxia, 1 h reoxygenation), 2. intermittent normobaric hypoxia - INH (8 h hypoxia, 16 h normoxia), 3. continuous normobaric hypoxia - CNH (24 h hypoxia). We compared how each hypoxic model affects the total number of -adrenergic receptors and proportion of individual subtypes ( 1-and 2-ARs) in the left and right ventricles compared control normoxic rats. The INH model had apparently no effect on -ARs in either ventricles. On the other hand, adaptation to INH/R and CNH was accompanied by a significant decrease (by about 25%) in the total number of -adrenergic receptors in the right ventricles. Our present...

The first project in this dissertation describes the total synthesis of yaku’amide A analogues. Natural product yaku’amide A possesses potent anticancer activity and exhibits a novel mode of action. However, due to its complex asymmetrical isoleucine dehydroamino acids, the synthesis of this polypeptide poses a unique challenge. Despite the efficient synthesis developed in our lab, the total synthesis of this natural product remains lengthy. In order to simplify the overall synthesis, symmetrical dehydroamino acids were incorporated to replace the dehydroisoleucine residues yielding two analogues of yaku’amide A that closely resembles the conformation of the natural product. Biological testing of the simplified analogues disclosed similar potency to that of yaku’amide A. The second part of this dissertation focuses on the influence that dehydroamino acids have on secondary structures. Peptides have an important role in medicine despite their limitations due to poor bioavailability and stability. Therapeutic use of peptides can be enhanced by designing new strategies to improve the proteolytic stability of these compounds. Attempts to increase peptide stability using trisubstituted and tetrasubstituted dehydroamino acids (ΔAAs) have been reported. Similarly, modified ΔAAs should also help tune the electronic and steric properties of peptides, while improving proteolytic stability. However, studies of peptides containing modified ΔAAs and are scarce. This project describes the synthesis and studies of incipient 310 helical tetrapeptides containing dehydroamino acids. Bulky and cyclic ΔAAs were demonstrated to alter the conformation of these tetrapeptides and impart greater stability against proteolysis and thiol additions. We believe these results can be a powerful tool to design peptide drug candidates with high proteolytic resistance and stability. tetrasubstituted dehydroamino acids (ΔAAs) have been reported. Similarly, modified ΔAAs should also help tune the electronic and steric properties of peptides, while improving proteolytic stability. However, studies of peptides containing modified ΔAAs and are scarce. This project describes the synthesis and studies of incipient 310 helical tetrapeptides containing dehydroamino acids. Bulky and cyclic ΔAAs were demonstrated to alter the conformation of these tetrapeptides and impart greater stability against proteolysis and thiol additions. We believe these results can be a powerful tool to design peptide drug candidates with high proteolytic resistance and stability.