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Ein Schlüsselprozess in der Immunantwort ist die durch das proinflammatorische Zytokin Interferon-gamma (IFNg) induzierte transiente Akkumulation von neu synthetisierten defekten Proteinen, die durch Anknüpfen von Polymeren des Proteins Ubiquitin (Ub) post-translational modifiziert werden. Die Ubiquitinierung ist das Schlüsselsignal für den Abbau dieser Proteine. Die Abbauprodukte dienen unter anderem als Quelle für die Prozessierung von Antigenen. Um die frühe Immunantwort besser zu verstehen, wurden im Rahmen dieser Arbeit die Identität und Charakteristika dieser neu synthetisierten Proteine, sowie die Topologie ihrer post-translationalen Modifizierung durch Ub untersucht. Dazu wurde die massenspektrometrischen Analyse ubiquitinierter Proteine weiterentwickelt, indem die experimentellen Konditionen auf deren Analyse optimiert wurden. Insbesondere konnte gezeigt werden, dass die kombinierte Verdauung der Ub-Konjugate mittels zweier Peptidasen die Identifizierung der Proteinpeptide und der Indikatorpeptide für Ubiquitinierungsstellen entscheidend verbessert. Es wurde demonstriert, dass eine selektive Isotopenmarkierung der neu synthetisierten Proteine möglich ist. Mit Hilfe dieser Methode gelang es, Veränderungen der Ub Modifikationen bezüglich der Topologie sowie quantitative Unterschiede der ubiquitinierten Proteine aus humanen HeLa-Zellen in An- und Abwesenheit von IFNg zu identifizieren. Nach Induktion durch IFNg wurden drei polyubiquitinierte, drei mono- oder polyubiquitinierte und 111 potentiell ubiquitinierte Proteine identifiziert. Diese Proteine zeigten, dass keine generelle Ubiquitinierungspräferenz für die durch IFNg verstärkt transkribierten Gene besteht. Die Ergebnisse dieser Arbeit erweitern das Verständnis der frühen zellulären Immunantwort und tragen zum Verständnis von Krebs- und Autoimmunerkrankungen sowie chronischen Entzündungsprozessen bei. Möglicherweise bilden sie die Grundlage für die Weiterentwicklung entsprechender Therapien. / A key process within the immune response of organisms is the transient accumulation of newly synthesized defective proteins affected by the proinflammatory cytokine interferon-gamma(IFNg). These proteins are post-translationally modified by attachment of polymers of the protein ubiquitin (Ub), which represents the key signal for targeting them to degradation. The resulting peptides can serve as sources for antigen processing. In order to get an insight into the details of the cellular early immune response, the identity and characteristics of the newly synthesized proteins and the topology of their post-translational modification with Ub were investigated. An essential progress of the mass spectrometric approach was achieved by optimizing the experimental conditions with regard to the requirements of the analysis of the targeted proteins. In particular, it could be shown that a combined digestion of Ub-conjugates with two peptidases leads to an improved detection of protein peptides and indicator peptides for ubiquitination sites. It was shown that a selective isotopic labeling of the newly synthesized proteins was possible. With this method, a decisive step forward was made in the understanding of changes of the Ub modifications with respect to the topology and in clarifying the quantitative differences between Ub-conjugates from IFNg treated and untreated human HeLa cells. Three IFNg induced polyubiquitinated proteins, three mono- or polyubiquitinated as well as 111 potential Ub-substrates could be identified. These proteins did not show any general ubiquitination preference for genes whose transcription is enhanced in presence of IFNg. The results obtained in this work help to broaden and refine the general picture of the early cellular immune response. They contribute to the knowledge on molecular processes of cancer, autoimmune diseases or chronic inflammation and, potentially, can give hints for the continued development of corresponding therapies.

La thèse présentée décrit la préparation et l’étude d’auto-assemblages élaborés à partir de conjugués amphiphiles Tat-b-poly(triméthylène carbonate) (Tat-b-PTMC) doués de propriétés d’internalisation cellulaire conférées par le segment peptidique Tat. L’objectif principal de ces travaux était d’établir et de comprendre les liens entre la structure macromoléculaire, les caractéristiques colloïdales et l’activité biologique de ces systèmes. Les efforts de précision moléculaire et de caractérisation fournispour la synthèse des chimères Tat-b-PTMC a permis de corréler finement leurs structures chimiquesaux paramètres physico-chimiques des nanoparticules obtenues. Grâce à une approche expérimentale transverse combinant des études de biologie cellulaire et de biophysique, le mécanisme d’interaction in vitro de ces nanoparticules avec les cellules HeLa a pu être en partie élucidé. Enfin, un camouflage électrostatique pH-sensible a été mis au point pour tenter de moduler leur activité et d’augmenter leur sélectivité vis-à-vis de l’environnement tumoral. / This thesis work deals with preparation and study of cell-penetrating self-assemblies from amphiphilicpolymer-b-peptide Tat-b-poly(trimethylene carbonate) conjugates. Tat-b-PTMC chimeras withtunable hydrophilic fractions were synthesized, thoroughly characterized and self-assembled inaqueous buffer into size-tunable, highly monodisperse core-shell nanoparticles, presenting a full Tatcorona. Their physico-chemical profiles were assessed by complementary imaging (AFM, TEM) andscattering techniques (multiangle DLS, SANS) and correlated with their molecular architectures.Their transduction ability in vitro on HeLa cells and interaction mode with phospholipid membraneswere studied with a view to correlate their physico-chemical profiles with their biological properties.This interdisciplinary approach partially shed light on the interactions at play in the cellular uptakeprocess. With the ultimate goal of improving pharmacological characteristics, we finally endeavoredto develop an ON/OFF PEGylation strategy to harness the cell penetrating power of thosebiomacromolecular self-assembled systems.

Photo sensitizers showing visible light induced DNA cleavage activity are of current importance for medicinal applications related to photodynamic therapy (PTD) considering greater skin penetration of light near 700 nm. While organic molecules and complexes of 4d-5d metal ions are extensively studied for their DNA photo-damage properties in UV and visible light, the chemistry of 3D metal complexes showing visible light-induced DNA cleavage activity is relatively unexplored efforts have been made in this thesis work to design new ternary copper (II) complexes having a-amino acids Such copper (II) complexes with tunable coordination geometry could find potential applications in PDT. Ternary Copper (II) complexes containing L-methionine, S-methy1-L-cysteine and phenanthroline bases are prepared and characterized. They display DNA binding and visible light induced DNA cleavage activity. An enhancement of the DNA cleavage activity is observed for analogous ternary copper (II) complexes contained L-lysine with a pendant cationic amine moiety as a photo-induced DNA Cleavage activity using binary and ternary copper (II) complexes of L-arginine and phenanthroline bases. We have observed AT selective DNA binding and visible light –induced DNA cleavage activity. The crescent-shaped bis-arginine Copper (II) complex mimics the natural antiviral antibiotic netropsin. T o investigate the role of the pendant groups of the amino acids, we have explored the DNA binding and DNA cleavage activity of analogues L-glutamine and L-asparagine complexes. We have prepared ternary copper (II) complexes containing two photosensitizers, viz., L-tryptophan (L-trp) and dipyridoquininoxaline/dipyridophenazine to achieve double strand breaks forming linear DNA. Complex [Cu(L-trp)(dppz)(H2O)+ shows a stacking arrangement of the indole and dppz rings giving a separation that fits with the base pair separation of ds-DNA. Photosensitizes in these complexes approach two different complementary stands of the ds-DNA, leading to double strand breaks and formation of linear DNA. Mechanistic studies on the DNA photocleavage reactions reveal the formation of singlet oxygen(1O2)species by a type-II pathway in preference to the hydroxyl radical generation. A process leading to an efficient DNA cleavage activity on visible light irradiation. The observation of sequence selectivity and double strand DNA cleavage on red light exposure by national design of the complexes is significant considering importance of the results in the chemistry of photodynamic therapy of cancer. The results of this dissertation open up new avenues for designing and developing 3d metal-based photosensitizers with potential utility in nulcleic acid chemistry.

Les défauts de génome mitochondrial provoquent des maladies neuromusculaires, pour lequel aucun traitement efficace n'a été mis au point. La plupart des mutations mitochondriales sont hétéroplasmique, ce qui signifie que l'ADN mitochondrial (ADNmt) de type sauvage et muté coexistent dans la même cellule, et le changement de proportion entre deux types d'ADNmt pourrait rétablir les fonctions mitochondriales. Le but du projet était le développement du système pour cibler l'ARN thérapeutique dans les cellules humaines vivantes. Au cours de ma thèse j'ai synthétisé une série de nouveaux ARN anti-réplicatifs contenant modifications chimiques pour augmenter leur stabilité dans la cellule, et mis au point la nouvelle méthode de synthèse chimique des molécules d'ARN contenant cholestérol fixé par l'intermédiaire d'un pont biodégradable. Ces ARN étaient capable de pénétrer dans les cellules humains, d'être adressées dans les mitochondries et de diminuer la proportion d' ADNmt muté. / Defects in mitochondrial genome cause neuromuscular diseases, for which no efficient therapy has been developed. Since most mitochondrial mutations are heteroplasmic, wild type and mutated mitochondrial DNA (mtDNA) coexist in the same cell, and the shift in proportion between two mtDNA types could restore mitochondrial functions. The aim of the project was development of carrier-free system for targeting the therapeutic mitochondrially importable RNA into living human cells. During my PhD study, I have synthesized a set of new anti-replicative RNAs containing various chemical modifications, aiming to increase their stability in the cell, and developed a new method for the chemical synthesis of RNA molecules containing cholesterol attached through a biodegradable bridge. Cholesterol containing antireplicative RNAs were characterised by efficient cellular uptake, partial colocalisation with mitochondria and ability to decrease the proportion of mutant mtDNA.

Les anticorps monoclonaux (mAbs) sont des glycoprotéines complexes possédant de nombreuses micro-hétérogénéités qui peuvent influencer leur efficacité dans l’organisme. Il est par conséquent nécessaire de développer des méthodes analytiques robustes, sensibles et spécifiques pour les caractériser avec la plus grande précision. L’objectif de cette thèse a été de développer des méthodes analytiques permettant la caractérisation fine et à différents niveaux d’un anticorps monoclonal, le cetuximab, ainsi qu’un anticorps monoclonal conjugués à un principe actif, le brentuximab vedotin, sur des couplages direct ou indirect de l’électrophorèse capillaire et la spectrométrie de masse. Dans une première partie, une approche middle-up protéomique du cetuximab a été réalisé sur le couplage indirect CZE-UV/MALDI-MS afin de séparer et caractériser les variants de charges du fragment F/2 et F(ab)’2 ainsi que la caractérisation top-down des fragments Fc/2. Ensuite une nouvelle stratégie indirecte CZE-UV/nanoESI-MS a été développée pour permettre la caractérisation fine de ce mAbs partiellement digéré. Enfin un couplage direct par CESI-MS a été développé pour permettre l’analyse rapide et précise du cetuximab middle-up. Dans une deuxième partie, la combinaison d’analyse de mAbs d’intact, middle-up et bottom-up protéomique a été réalisée sur le couplage CZE-UV/nanoESI-MS et CESI-MS. Cela a permis la caractérisation à différent niveau du brentuximab vedotin. Cette méthodologie a permis l’analyse du DAR, l’identification de fragments conjugués, la caractérisation simultanée de la séquence complète de l’anticorps, d’un grand nombre de modifications post-traductionnelles, la caractérisation des peptides conjugués ainsi que l’identification d’ions diagnostiques du principe actif. / Monoclonal antibodies (mAbs) are highly complex glycoproteins having a lot of micro-heterogeneities which can influence their effectiveness. As a consequence, it is necessary to develop robust analytical methods, sensitive and specific to characterize them with high accuracy. The purpose of this thesis was to develop analytical methods allowing the multi-level characterization of monoclonal antibody (cetuximab), and antibody drug conjugates (brentuximab vedotin), using on-online or off-line capillary electrophoresis – mass spectrometry coupling. In the first section, a middle-up proteomic approach of cetuximab was carried out using Off-line CZE-UV/MALDI-MS coupling to separate and to characterize Fc/2 and F(ab)’2 charge variants. A top-down characterization of Fc/2 fragments was also employed. Then a new strategy off-line CZE-UV/nanoESI-MS was used to allow the characterization of this partially digest mAbs. Finally, an online coupling by CESI-MS was developed to allow the fast and accurate analysis of middle-up cetuximab. In a second part, the combination of intact, middle-up and bottom-up proteomic carried out on CZE-UV/nanoESI-MS and CESI coupling allowed the most exhaustive characterization of brentuximab vedotin. This methodology allowed the analyze of DAR, the identification of fragments drug conjugates, the simultaneous characterization of the complete structure of antibody, a significant number of post-translational modifications, all peptides drug conjugates and the identification of diagnostic ions.

Chapter 1. Supramolecular Gels and their Applications Supramolecular gels are viscoelastic materials composed of a solid like three dimensional fibrillary network that is embedded in a liquid. Supramolecular gels are derived from low molecular weight compounds (typically MW < 3000). In the 1990s, the investigations on gels were mainly focused on designing new gelator molecules. However, during the last decade, research focus shifted towards designing functional gels and their applications. As a result of extensive work in this area, gels have been found to have varied applications in the templated synthesis of inorganic nanomaterials, hybrid materials, light harvesting systems, as responsive system and sensors, and also in drug delivery, tissue engineering etc. This chapter gives an introduction to supramolecular hydrogels/organogels and relevant bile acid chemistry touching upon the gelation properties of the bile acid derivatives. Diverse applications of the supramolecular gels are also illustrated with several examples. Scheme 1. Various applications of functional supramolecular gels Chapter 2. Bile Acid derived novel Hydrogelators Part 1. Hydrogelation of Bile acid protected Amino acids and Hybrid Materials Hydrogels from low molecular weight molecules have significant importance in biomedical applications. In this chapter, we report injectable hydrogel formation from bile acid conjugates of various amino acids. Hydrogel formation was found to be dependent on multiple factors such as bile acid backbone structure, linkage between the bile acid and the amino acid, pH etc. Single crystal structures of lithocholyl phenylalanine, lithocholyl-glycine, lithocholyl-L valine and lithocholyl-L alanine were also determined. Finally, the hydrogel frameworks were utilized to produce hybrid materials with Gold and ZnO nanoparticles. Scheme 2. (a) Crystal structure of LC-LF-OH gelator molecule, (b) photograph of gel, (c) SEM and (d) AFM image of LC-LF-OH xerogel Part 2. Hydrogelation of bile acid-dipeptide conjugates and in situ synthesis of silver and gold nanoparticles in the hydrogel matrix Fabricating supramolecular hydrogels with embedded metal nanostructures are important for the design of novel hybrid nanocomposite materials for diverse applications such as bio sensing and chemo sensing platforms, catalytic and antibacterial functional materials etc. Supramolecular self-assembly of bile acid-dipeptide conjugates have led to the formation of new supramolecular hydrogels. Gelation of these molecules depends strongly on the hydrophobic character of the bile acids. Ag+ and Au3+ salts were incorporated in the hydrogels, and photo reduction and chemical reduction led to the in situ generation of Ag and Au NPs in these supramolecular hydrogels without the addition of any external stabilizing agent. The color, size and shape of silver nanoparticles formed by photo reduction depended on the amino acid residue on the side chain. Furthermore, the hydrogel-Ag nanocomposite was tested for its antimicrobial activity. Scheme 3. Bile acid based dipeptide hydrogelators and soft hybrid materials Chapter 3. Sonogels of bile salts of In(III): use in the formation of self-templated indium sulfide nanostructures In this chapter, facile hydrogel formation by Indium(III) cholate and deoxy cholate are reported. When In(III) solution was added to aqueous solutions of sodium cholate and sodium deoxy cholate and sonicated, the mixtures formed gels. The gels thus obtained were translucent/turbid and thermos irreversible. Rheological measurements showed that all of them could be classified as viscoelastic soft solids. Scanning electron microscopy and atomic force microscopy showed typical entangled three dimensional fibrous networks. The In-Ch hydrogel were further used to prepare nanostructured In2S3 in which the cholate units possibly acted as a surfactant to confine the growth of the Nano flakes. Scheme 4. In-Ch hydrogel (Photograph and SEM image of In-Ch gel) Chapter 4. Palladium-Hydrogel Nanocomposite for C-C Coupling Reactions Supported metallic nanoparticles are important composite materials owing to their enormous potential for applications in various fields. This chapter describes the in situ formation of palladium nanoparticles in a calcium-cholate (Ca-Ch) hydrogel by reduction with sodium cyan borohydride. The hydrogel matrix appeared to assist the controlled growth as well as stabilization of palladium nanoparticles. The palladium nanoparticle/Ca-Ch hydrogel hybrid was characterized by scanning and transmission electron microscopy, atomic force microscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy. Furthermore, PdNP/Ca-Ch hybrid xerogel was shown to act as an active catalyst for Suzuki reaction under aqueous aerobic conditions, up to 4 cycles. This PdNP/Ca-Ch xerogel retained its catalytic activities on storage for several months. Scheme 5. Palladium-hydrogel nanocomposite for C-C coupling reactions in water Chapter 5. Sensitization of Terbium/Europium in self-assembled cholate hydrogel: An approach towards the detection of amine vapours "Luminescent" lanthanides have intrinsic low molar absorptivity, although this problem can be addressed by complexing the lanthanide ion with suitable chelating ligands which improve the luminescence properties drastically. However the design of such systems often involves careful planning and laborious synthetic steps. It is therefore desirable to have a simpler way to sensitize lanthanides with high efficiency. It was observed in our group that trivalent lanthanides formed hydrogels on the addition of sodium cholate. This chapter describes the discovery of the several biphenyl derivatives (such as 4-biphenylcarbaxaldehyde, 4-acetylbiphenyl) for sensitization of Tb(III) and Eu(III) in lanthanide hydrogels. Sensitization of Tb(III) and Eu(III) were observed by doping was characterized by scanning and transmission electron microscopy, atomic force microscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy. Furthermore, PdNP/Ca-Ch hybrid xerogel was shown to act as an active catalyst for Suzuki reaction under aqueous aerobic conditions, up to 4 cycles. This PdNP/Ca-Ch xerogel retained its catalytic activities on storage for several months. Scheme 6. Schematic representation of the sensitization process (the arrangement of themolecules in the gel fiber is arbitrary)(For figures pl refer the abstract pdf file)

Ferrocene is an important molecule in the field of chemical biology due to its stability, unique redox property and significant lipophilicity for better cellular delivery. The medicinal importance of ferrocene is well recognized after its successful incorporation into breast cancer drug tamoxifen and antimalarial drug chloroquin. Designing ferrocene conjugated transition metal complexes is an interesting area of research in the field of photodynamic therapy, a new modality of light activated cancer treatment. The objective of the present thesis work is to develop photoactive ferrocene conjugates showing DNA photocleavage and photocytotoxic activity. We have synthesized the ferrocene conjugated imidazophenanthroline derivative which exhibits visible light induced DNA photocleavage activity and photocytotoxicity in HeLa cell line. The corresponding phenyl analogue is found to be inactive. Polypyridyl platinum(II) complexes of ferrocenyl as well as phenyl moiety are prepared and studied their interactions with calf thymus DNA. The cytotoxicity of the complexes enhance significantly upon irradiation of UV-A light of 365 nm. To enhance the photodynamic potential and to understand the role of organometallic ferrocenyl moiety, ferrocene conjugated terpyridyl copper(II) complexes having planar phenanthroline bases are prepared. The interaction of these complexes with duplex DNA and their photo-induced DNA cleavage and anticancer activity in HeLa cancer cells are studied. The complexes are able to generate ROS in the presence of visible light which causes DNA damage as well as cell death. The importance of ferrocenyl moiety is evidenced from the less activity of the corresponding phenyl analogues complex. We have prepared copper(II) complexes of ferrocenyl methyl dipicolylamine ligand to understand the role of terpyridyl moiety. These complexes lacking any conjugation between the copper(II) and the ferrocenyl moiety are found to be less active compared to the terpyridyl conjugated system. The copper(II) complexes are found to show undesirable dark cytotoxicity in the presence of cellular thiols like GSH. To overcome the dark toxicity problem and to understand the mechanistic aspects of DNA photocleavage and photocytotoxicity, a series of binary ferrocene conjugated terpyridyl complexes of Fe(II), Co(II), Cu(II) and Zn(II) are prepared and their DNA photocleavage and anticancer activity studied. The zinc(II) complex having redox-active ferrocenyl moiety and redox-inactive zinc(II) center exhibits significant PDT effect with low dark toxicity compared to its copper(II) analogue. The ferrocenyl moiety plays an important role towards showing photocytotoxic activity since its phenyl analogue is inactive in nature. Finally, the present thesis work opens up a new strategy for designing and developing new ferrocene based metal complexes as novel photosensitizers for PDT applications.

La mise au point de techniques de fractionnement des moûts de Sauvignon blanc par chromatographie de partage centrifuge et par chromatographie Flash, a permis de mettre en évidence la présence de deux « nouvelles » formes précurseurs du 3SH (le S-3-(hexan-1-al)-glutathion et l'acide S-3-glutathionyl-hydroxyhexanesulfonique). L’ensemble de ces résultats a permis de confirmer la contribution majeure des S-conjugués au potentiel en 3SH des moûts. Le S-3-(hexan-1-ol)-L-cystéine, le S-3-(hexan-1-ol)-glutathion ainsi que les deux nouvelles formes identifiées, permettent d’expliquer près de 65% du 3SH libéré dans les vins. Par ailleurs, nous avons montré que l’ensemble des formes précurseurs appartenait à la même voie d’assimilation de la levure et que le S-3-(hexan-1-ol)-L-cystéine est un intermédiaire indispensable à la biotransformation des S-conjugués au glutathion, précurseurs du 3-sulfanylhexanol. De plus, le pourcentage de biotransformation des différents S-conjugués en 3SH semble directement lié à leur position dans le flux métabolique. / The development of fractionation techniques in Sauvignon Blanc musts such as centrifugal partition chromatography and flash chromatography, permitted to identify two precursor forms of 3-sulfanylhexanol : the S-3-(hexan-1-al)-glutathione, and the S-3-glutathionyl-hydroxy hexanesulfonic acid. These results confirmed the major contribution of S-conjugates in the potential liberation of 3-sulfanylhexanol in musts. The two newly identified forms along with S-3-(hexan-1-ol)-L-cysteine and S-3-(hexan-1-ol)-glutathione, explain up to 65% of the total release of 3-sulfanylhexanol. Futhermore, we showed that all precursor forms belonged to the same assimilation pathway of yeast and the S-3-(hexan-1-ol)-L-cysteine is an essential intermediate in the metabolism of S-glutathione conjugates, which is a precursor of 3-sulfanylhexanol . Finally, the biotransformation percentage of different S-conjugates of 3-sulfanylhexanol seems directly related to their position in the metabolic flow.

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