Cette thèse décrit la Chimie Covalente Dynamique (CCD) des échanges imine/imine, Knoevenagel/imine et Knoevenagel/Knoevenagel. La L-proline est un excellent organocatalyseur pour la formation de Bibliothèques Covalentes Dynamiques (BCDs). Cependant, l’interconversion entre des dérivées Knoevenagel de l’acide diméthylbarbiturique et des imines se déroule rapidement sans catalyseur. Une nouvelle classe de CCD basée sur des échanges par substitutions nucléophiles (SN2/SN2’) entre des sels d’ammonium quaternaires et des amines tertiaires est développée, impliquant la catalyse par l’iodure. Les réactions d’échange entre des sels de pyridinium et un dérivé de pyridine génèrent des liquides ioniques dynamiques. Enfin, la sélection cinétique et thermodynamique de la formation d’imines dans la CCD est réalisée en solution aqueuse e ten solvant organique. / This thesis describes the dynamic covalent chemistry (DCC) of imine/imine, Knoevenagel/imine, and Knoevenagel/Knoevenagel exchange. L-proline is shown to be an excellent organocatalyst to accelerate the formation of dynamic covalent libraries (DCLs). The interconversion between Knoevenagel derivatives of dimethylbarbituric acid and imines is found to occur rapidly in the absence of catalyst. A new class of DCC based on nucleophilic substitution (SN2/SN2’) component exchange between quaternary ammonium salts and tertiary amines is developed, by the use of iodide as a catalyst. The exchange reactions between pyridinium salts and a pyridine derivative generate dynamic ionic liquids. Finally, kinetic and thermodynamic selection of imine formation in a DCC is perfomed in aqueous solution and organic solvent.

L’élucidation des interactions non-covalentes des complexes biologiques revêt d’une importance majeure dans la compréhension du fonctionnement cellulaire. L’objectif de ce travail de thèse est d’approfondir les développements de la spectrométrie de masse (MS) pour l’étude de ces complexes, que ce soit par MALDI-MS (la désorption-ionisation laser assistée par matrice) ou par ESI-MS (l’ionisation électrospray). Ce travail s’est articulé autour de trois axes : i) étude de la stœchiométrie et de la topologie du complexe SAGA HAT (Spt-Ada-Gcn5 Acétyltransferase, module Histone Acétyl Transferase) par pontage chimique couplé à la MS ; ii) suivi de la dimérisation des complexes formés par RAR-RXR (récepteur de l’acide rétinoïque - récepteur X des rétinoïdes) avec différents ADNs ; iii) mesure de la constante de dissociation des complexes RXR-ligand. Les méthodologies développées ont permis de repousser le potentiel de la MS et d’obtenir des informations structurales des complexes biologiques. / Elucidation of non-covalent interactions of biological complexes takes on great importance for the understanding of cellular function. The purpose of this thesis is a further development of mass spectrometry (MS) for the study of these complexes, either by MALDI-MS (matrix-assisted laser desorption-ionization) or by ESI-MS (electrospray ionization). This work was focused on three main lines: i) study of the stoichiometry and the topology of SAGA HAT (Spt-Ada-Gcn5 Acetyltransferase, Histone Acetyl Transferase module) complex by chemical cross-linking coupled to MS; ii) monitoring the dimerization of the complexes formed by RAR-RXR (retinoic acid receptor - retinoid X receptor) with different DNAs; iii) measuring the dissociation constant of RXR-ligand complexes. The developed methodologies made it possible to expand the potential of MS and get insight into structure of biological complexes.

Les récepteurs moléculaires sont des architectures maintenues par des liaisons covalentes et capables de fixer sélectivement des substances (ioniques et/ou moléculaires) au moyen d’interactions intermoléculaires diverses, aboutissant ainsi à la formation d’un assemblage d’au moins de deux espèces nommé complexe moléculaire. Au cours de ce travail, des stratégies de synthèse de dérivés de calix[4]arène et de son analogue thiacalix[4]arène en conformation 1,3-alternée ont été mises au point. Ces composés ont été fonctionnalisés par des groupements pyridine, catéchol, imidazole, pyrazole et pour la première fois oxamate. Les composés obtenus ont été caractérisés à la fois en solution par RMN et à l’état cristallin. Les propriétés complexantes vis-à-vis des métaux de transition en solution sont présentées. Pour certains dérivés, le pouvoir extractant envers les métaux de transition a été étudié et discuté. Enfin, la formation de réseaux de coordination à l’état cristallin par auto-assemblage de dérivés de calixarène ou de thiacalixarène et le cation argent est présentée et commentée. / Molecular receptors are preorganised architectures held by covalent bonds and capable of binding selectively ionic and / or molecular substrates via various intermolecular interactions, leading to the formation of molecular complexes composed of at least two species.In this work, the synthesis of a library of calix[4]arene and its analogue thiacalix[4]arene in 1,3-alternate conformation based ligands and tectons has been investigated. These two types of platforms have been equipped with pyridine, catechol, imidazole, pyrazole and, for the first time, oxamate units as coordinating sites. All compounds prepared were characterized in solution and in some cases in the crystalline state. Their binding propensity in solution towards transition metals has been determined and discussed. For some derivatives, their metal extracting properties have been investigated and presented. Finally, the formation of extended periodic architectures of the coordination network type in the crystalline state by self-assembly of calixarene based tectons and silver cation was achieved and presented.

Ce travail s’inscrit dans le domaine des cages moléculaires, édifices dont la géométrie définit unecavité tridimensionnelle capable de contenir d’autres entités.L’objectif de cette thèse est la synthèse de cages moléculaires covalentes possédant despropriétés d’encapsulation et une taille modulables. Les cages synthétisées avec succèsincorporent des porphyrines de zinc ou bases libres, ainsi que huit ligands triazoles périphériques.Des molécules ont pu être encapsulées par coordination aux porphyrines ou par interactions π, cequi démontre la capacité de la cage à s’adapter à l’invité grâce à sa flexibilité. La coordinationd’ions argent(I) permet de passer d’une conformation aplatie à une conformation ouverte. Lecaractère commutable de la cage a été démontré par décoordination de ces ions. Enfin, lacoordination d’ions argent(I) aux ligands de la cage augmente la stabilité de deux ligandsditopiques, le DABCO et la pipérazine, au sein de la cavité. Elle permet aussi l’accès de la cavité àdes molécules non encapsulées en l’absence d’argent(I). Ces résultats démontrent le contrôleallostérique de l’encapsulation de molécules par un stimulus chimique. / This work belongs to the field of molecular cages, hollow structures enclosing a three-dimensionalcavity able to encapsulate other molecules.The goal of this thesis include the synthesis of molecular covalent cages with a controllablecavity size able to perform switchable guest encapsulation. A DABCO-templated click reactionafforded three flexible covalent cages, endowed with two zinc(II) or free-base porphyrins and eightperipheral triazole ligands. Various guest molecules could be encapsulated by coordination toporphyrins or π-π interactions, in an induced-fit mechanism, probing the ability of the cage to adaptto the guest thanks to its flexibility. Coordination of silver(I) ions to the peripheral triazoles allow toswitch from a flattened to an open and locked conformation. Removal of the ions proved thecommutability of the cage. Finally, coordination of silver(I) ions to triazole ligands increase thestability of two ditopic ligands, DABCO and piperazine, inside the cavity. It also allowed the accessto the cavity of molecules that were not otherwise encapsulated. These results shows the allostericcontrol of guest encapsulation by a chemical stimulus.

This thesis titled, “Nature of Local Interactions at cisPro-Aro Peptide Sequences in Proteins: Evidences for van der Waals type Interactions. Design and Synthesis of Novel Covalent Surrogates for the Peptide Hydrogen Bond”, describes two important studies. The first is to gain a thorough understanding of the nature of interactions that govern cisPro stability at Pro-Aro sequences, which described in the first four chapters. The final chapter describes the synthesis of novel 4-carbon covalent surrogates for the peptide H-bonding interaction. Chapter 1: Local Interactions Governing cisPro Stability: Refining the Model Peptides Chapter 1 Section A: Understanding the role of inter-side chain CH•••Aro interaction in cis-trans isomerization at Pro-Aro and Aro-Pro Sequences. This chapter is divided into two sections. In the first section an exhaustive overview of earlier investigations into the nature of local interactions at Xaa-cisPro-Aro and Aro-cisPro-Xaa peptide sequences, by various groups, are discussed. Most studies have found evidence for the close assemblage between side chains of residues flanking cisPro motifs, when at least one of them is an aromatic group. An electronic C-H•••π nature has been proposed for these assemblies and they are proposed to influence the cisPro stability. We highlight those features in these studies that indicate that these interactions are not always electronically tunable, are insensitive to presence of strong chaotropes in the solvent and occur at protein sequences lacking Pro or cisPro; all of which contradict the electronic C-H•••π model for these inter-side chain assemblages and their perceived influence on cisPro stability. Chapter 1 Section B: Investigation of the Nature of H Xaa•••Aro interaction at Xaa-Pro-Pro-Phe-sequences In Section B, we design and synthesize Pro-Aro containing short peptide models to investigate the nature of local C-H•••Aro interactions in them. We synthesize a series of homologous Pro-Pro-Aro containing peptides (modeled based on earlier studies) and investigate the relative populations of its four Xaa-Pro rotamers using extensive 1D and 2D NMR techniques including TOCSY, HSQC and ROESY. We find several drawbacks that make this a relatively deficient model. Firstly, their relative populations of the rotamers (the most important data for current investigation) cannot be determined with high fidelity as they are dependent on the solvent polarity, solute concentration and chemical shift degeneracy of crucial NMR signals for the rotamers. Importantly, the populations of a few rotamers are influenced by strong 13-membered ring backbone H-bonds. Notably, some of the cisPro rotamers do not even contain the inter-side chain assembly, whose nature is under investigation. Design of novel models – unconstrained by H-bonds We design the Acyl-Pro-Pro-Aro-OMe peptides that lack the possibility of forming the 13-membered ring H-bonded structures. Thorough 1D and 2D NMR analyses of these models reveal that strong Type VI β-turn type 10-membered ring H-bonds are formed in the rotamers of these models – hence precluding their applications for current study. Interestingly, the relative rotamer populations are strongly influenced by solvent polarity and are entirely different from those of the corresponding C-terminal amide models. We further discover that the Pro-Pro-Aro motif is not essential to express the inter-side chain interactions – Ala-Pro-Aro are sufficient. Formation of the 10-membered H-bonding interactions, however, are not precluded. Chapter 2: Design and Synthesis of Acyl-Pro-Phe-OMe: Novel models to investigate the role of HαXaa•••Aro interactions on Xaa-cisPro-Aro stability. Chapter 2 Section A: Design, Synthesis and Conformational Analysis of Ibu-Pro-Phe-OMe Chapter 2 is divided into two sections. In Section A, we replace the amino acid at the N-terminal of the putative Pro residue with simple isosteric isobutyryl group, the resulting minimalist dipeptide model shows the exclusive influence of desired inter-side chain interactions in the cisPro rotamer. Solvent polarity and temperature coefficient studies reveal that absence of any intramolecular H-bonding or Oπ* interactions in it. 1D and 2D NMR analyses clearly indicate the close proximity between the side chains of Ibu and Phe exclusively in the cisPro rotamer. The Kc/t value decreases upon mutation of Phe to Ala. All these features favor the Ibu-Pro-Phe-OMe as an ideal minimalistic model for investigating the nature of Ibu•••Ph assemblages in the cisPro rotamer. Chapter 2 Section B: Investigation of CH•••Aro /Alp•••Alp interactions in Ibu-cisPro-Xaa-OMe In Section B, the 1D and 2D NMR analyses of the complete set of the aliphatic and aromatic analogues Ibu-Pro-Xaa-OMe were investigated. DMSO-d6 was found to be the best solvent for mimicking both the folded and the unfolded local environments of these short peptide sequences. The HαIbu•••Aro assemblage is observed in Aro analogues, but cannot be electronically tuned. The aliphatic analogues also surprisingly contain the HαIbu•••Alp interactions! The Kc/t values (cisPro %) increase in the aliphatic analogues too, where the aliphatic side chain is long. Increase in cisPro stability is not due to ring current effects or intramolecular H-bonds or Oπ* interactions. It seems to be due to van der Waals type interactions between the involved side chains, either of which need not be aromatic in nature. Chapter 3: Nature of Inter-Side Chain Interactions at Acyl-cisPro-Aro Sequences: Evidence for van der Waals Interactions Chapter 3 Section A: Investigation of nature of inter-side chain interactions in R-CO-cisPro-Phe-OMe Chapter 3 has two sections. Section A describes the systematic design and synthesis of Acyl-Pro-Phe-OMe homologues where first the steric bulk and hence the surface area of the aliphatic side chain of the acyl group is varied. Interaction of the phenyl ring of Phe seems to occur with the Cα-Cβ σ-bond of the acyl group. Branching at either Cα or Cβ seems to destabilize the cisPro rotamer. Aliphatic•••Aromatic interactions overwhelm the cisPro rotamer population to be greater than that of transPro. In the analogues where the acidity of the acyl Cα-H bond is increased, the Kc/t does not increase correspondingly. The Δδ(trans-cis) ppm shifts of HαAcyl protons are dependent exclusively on its acidity rather than on the Kc/t values. In carbamyl-Pro, which entirely lack the HαAcyl proton, the Kc/t values are significantly high and improve as the aliphatic surface on the alkoxy group increases. Introduction of benzyloxy carbamyl group at Pro renders almost the same Kc/t values as that of ethyloxy carbamate. All these data contradict the C-H•••π interaction model and strongly support a van der Waals type interaction between the Acyl (preceding cisPro) group’s Xα-Yβ σ-bond and the Aro or Alp side chains (succeeding cisPro). Chapter 3 Section B: Evidence for the Van der Waals nature of Inter Side Chain (Acyl•••S.C.Aro/Alp) interactions- Determination of Interactions energies In Section B, a thorough investigation of both aliphatic•••aliphatic and aliphatic•••aromatic interactions on the background of homologous Acyl-Pro-Aro/Alp-OMe peptide models is undertaken. These models uniquely allow the delineation of contribution of the van der Waals interactions and the ring current effects to the cis/trans isomerization in these peptides. We see that the energy of the van der Waals component of these aliphatic•••aliphatic and aliphatic…aromatic interactions increase linearly with increase in Kc/t, in both DMSO-d6 and D2O. On other hand, energy from the ring current effects largely remains invariant. The Acyl•••Aro/Alp interactions are not hydrophobic and are facilitated by conformational effects. Chapter 4: Crystallographic evidence for van der Waals interaction-mediated stabilization of cisPro conformers Chapter 4 Section A: Systematic crystallization and crystal structure analyses of homologous Xaa-cisPro-Alp and Xaa-cisPro-Aro rotamers: Evidence for van der Waals interactions Chapter 4 has two sections, both of which present crystallographic evidence for the van der Waals nature of the Xaa•••Aro interactions at Xaa-cisPro-Aro sequences. Section A describes the unique crystal structures of five of the Acyl-Pro-Alp-OMe analogues that have been synthesized in the current study. All of them remarkably crystallize with two features: 1) the Acyl-Pro peptide bond adopts the cisPro rotamer in all; and 2) the aliphatic side chains of the acyl group and the Alp side chain are involved in van der Waals type interactions. The cisPro rotamers of even the bulkiest motifs, namely Ibu-Pro-Val-OMe, Piv-Pro-Ile-OMe and Piv-Pro-Leu-OMe crystallize, stabilized by van der Waals packing between aliphatic groups of the acyl and the Leu/Ile/Val side chains. Where the side chains are not long enough to make sub-van der Waals contacts with each other, their acyl C′-Cα σ-bond rotations are restricted due to Oσ* interactions involving the charge on the acyl carbonyl O. Where this occurs, the short space between the acyl and Alp side chains are filled in by aliphatic groups from neighbouring molecules at sub van der Waals distances. The Pro, Alp and χ1(Alp) dihedral angles are restricted to narrow range of values, irrespective of the length of Alp side chain, indicating that this backbone conformation is a conformational minimum when i+3i backbone H-bond is removed, with Pro at the i+1st position. This is further substantiated in Piv-Pro-Gly-OMe, which crystallizes in trans-Pro form, but still adopts similar backbone dihedral angles in spite of lacking any Alp side chain for interactions with the acyl group. Three of the Acyl-Pro-Aro-OMe models also crystallize in cisPro rotamer forms – both exhibit van der Waals type contacts between the Acyl group and backbone of Phe, rather than the aromatic ring of Phe. The phenyl ring of Phe may or may not form intramolecular Ph•••Pro inter-side chain contacts – which is not a pre-requisite for cisPro stabilization. No C-H••• interactions are observed anywhere in these peptides – van der Waals type contacts alone predominate in all cases. There are no abnormal distortions in bond angles or lengths even in the most sterically hindered cases, signifying that the conformations of these cisPro rotamers involving aliphatic•••aliphatic type contacts are natural minima. Chapter 4 Section B: Mining the PDB for Statistical Evidence of van der Waals interactions Section B of chapter 4 describes the data mining and statistical analyses of Xaa-cisPro-Phe, Xaa-cisPro-Val and Xaa-cisProLeu sequences in the PDB. The PEARL program was used to mine the PDB data. The overall frequency of 5.3% for appearance of cisPro among all Xaa-Pro peptide bonds, improves when Xaa is Phe or Tyr. However, several anomalies highlight the need for refining the analyses set to only those sequences where the side chains of Xaa and Aro/Alp face each other. In this refined set, clearly, inter side chain Xaa•••Alp/Aro contacts take precedence over even Aro•••Pro interactions at Aro-cisPro sequences (where Xaa is Aro). The Phe and the Leu side chains induce similar conformational effects on the preceding Xaa-Pro backbone. So does Val. Strong aliphatic•••aliphatic inter side chain contacts at van der Waals distances are observed to flank cisPro in several proteins. Substitution at the Cα of Xaa governs the proximity of the approaching side chain of Alp / Aro residue. The Cα-H of Xaa steers away from the Aro side chain at Xaa-Pro-Phe sequences, as the Aro group gets closer to it – implying the absence of ordered C-H••• contacts between them. There is consistent parallel alignment between Cα-Cβ -bond of Xaa and the C -C bond of the approaching side chain of Alp or Aro group – clearly highlighting the presence of van der Waals type interactions between them. All these evidences clearly point towards the van der Waals nature of local interactions at cisPro-Aro/Alp peptide sequences. Chapter 5: A novel 4-carbon covalent surrogate model for peptide H-Bond Chapter 5 describes the design and synthesis of novel 4-carbon covalent surrogates for the peptide H-bond (HBS). These surrogates would allow the unique constraining of two peptide strands in their extended conformations. The covalent HBS contain four orthogonal functional groups for independent extension at all of the four ends – similar to an endogenous inter-strand peptide H-bond. The synthesis of the surrogate is achieved by directly using natural chiral amino acid derivatives, beginning from amino alcohols obtained from reduction of desired amino acids. Suitably N-protected alcohols undergo oxidation to aldehyde followed by Grignard addition of allyl magnesium bromide, TBDMS protection of the homoallylic alcohol and reductive ozonolysis of the olefin to get a primary alcohol which is subject to Fukuyama-Mitsunobu reaction with desire protected peptide. The residue preferences that produce strongest inter-strand H-bonds were explored. The designed 4-carbon covalent HBS was incorporated using this methodology in a Gramicidin-S analogue, its first structural mimic containing only a single turn motif. This HBS model will have wide applications for constraining peptides in a number of secondary structures.

Ce travail réalisé dans le cadre de l’initiative africaine de l’IUCr porte sur l’étude structurale par diffraction des rayons X de quelques architectures élaborées par association, via des interactions faibles, des anions {[M(C2O4)3]3-,M = Cr, Fe} et des cations complexes à base de la 2-picolylamine (amp) métaux de transition (Co2+, Cu2+ et Mn2+). L’architecture à base de l’ion Co2+ est bidimensionnelle et présente des feuillets ondulés constitués de chaines bimétalliques de chiralité différente où les deux ions complexes ([Cr(C2O4)3]3- et [Co(amp)3]3+ ) sont connectés par des liaisons hydrogène. Ces feuillets hébergent des molécules d’eau qui forment des clusters dodécamèriques aux caractéristiques nouvelles. Le composé déshydraté se réhydrate rapidement dans l’air ambiant et les deux états possèdent des couleurs différentes. Plusieurs cycles de déshydratation-réhydratation n’altèrent pas la qualité cristalline du composé. L’architecture à base des ions Cu2+ possède également des feuillets mais présente une ondulation plus forte que celle de l’architecture au cobalt. Ces couches sont constituées de chaines formées de cations dimériques [Cu2(amp)4Cl]3+ et d’anions {[M(C2O4)3]3-,M = Cr, Fe}. Les deux composés sont iso-structuraux et leur architecture présente des canaux monodimensionnels qui contiennent des molécules d’eau qui forment des clusters hexamèriques. Le composé subit des transitions de phase entre la basse température (100K) et la température de déshydratation (341K) avec une perte de la symétrie. Le composé se réhydrate plus difficilement que celui à base de l’ion cobalt(III). L’ion Mn2+ ne donne pas l’architecture escomptée mais un polymère de coordination nouveau / This work, realized under the IUCr initiative, framework involves the structural study via X-ray diffraction, of some heteromolecular architectures formed by the association through non-covalent bonds, between the tris (oxalato) chromate (III) and tris (oxalato) ferrate (III) anions {[M(C2O4)3]3-, M = Cr, Fe} and the cationic complex of the 2-picolylamine (amp) and transition metal (Co2 +, Cu2 + and Mn2 +). Co2 + ion builds two-dimensional corrugated layers made of bimetallic chiral chains where the two different complex ions ([Cr(C2O4)3]3- and [Co(amp)3]3 +) are connected by hydrogen bonds. These layers, connected by weak hydrogen interactions, host between them, water molecules which build dodecameric clusters having new characteristics. The dehydrated compound has different structure and color and is able to quickly reabsorb water molecules from surrounding to regenerate the initial compound despite that it has no pores. Several cycles of this process do not seriously affect the crystalline quality of this compound. The compound obtained with the Cu2 + ion also has a two-dimensional framework. Their layers are formed between the dimeric cation [Cu2 (amp) 4Cl]3 + and the anion {[M(C2O4)3]3-,M = Cr, Fe}. Both compounds are iso-structural; their frameworks are formed via π - - - π interactions and build 1D channels which contain water molecules forming hexameric clusters. The compound undergoes a phase transition between 100 K and the dehydration temperature (341K). During this dehydration, a loss of symmetry of the compound is recorded and rehydration process is more difficult than for cobalt(III)-framework. The use of Mn2+ ions does not give the expected architecture but a new coordination polymer

Cerca de 20 % dos casos familiares de esclerose lateral amiotrófica (ELAf) são causados por mutações na enzima Cu,Zn-superóxido dismutase (SOD1). Inicialmente se supôs que as enzimas mutantes teriam a atividade SOD comprometida, entretanto isto não foi comprovado. Atualmente, considera-se que as enzimas mutantes adquiram propriedades tóxicas. Quais seriam estas propriedades e como levariam à degeneração do neurônio motor são questões ainda não respondidas. Neste trabalho, comparamos neuroblastomas humanos transfectados com SOD1 G93A associada à ELAf (SH-SY5YG93A), e SOD1 selvagem (SH-SY5YWT) com células parentais (SH-SY5Y) em relação ao crescimento, viabilidade, produção basal de oxidantes, atividades SOD e peroxidásica e modificações estruturais da SOD. As células transfectadas apresentaram aumento na taxa de crescimento e na produção basal de oxidantes. As células SH-SY5YWT e SH-SY5YG93A mantiveram a expressão de SOD1 e atividade consistente com o aumento esperado de duas vezes, em estágios iniciais de cultura. A atividade peroxidásica do homogenato da célula SH-SY5YG93A foi maior. Após quatro semanas, a linhagem SH-SY5YG93A manteve a expressão de SOD1, mas as atividades dismutásica e peroxidásica diminuíram. A expressão de SOD1 aumentou a proporção de formas alteradas de SOD1, como enzima reduzida, multímeros formados por ponte dissulfeto e formas insolúveis em detergente, particularmente na linhagem SH-SY5YG93A. Entre estas formas insolúveis, identificamos um dímero covalente de SOD. Estas formas alteradas provavelmente são responsáveis pela ativação do proteassomo e estresse do retículo endoplasmático, verificados nas células transfectadas. Concluindo, a superexpressão da SOD1 foi suficiente para elevar as formas imaturas e oligomerizadas de SOD1 e a oxidação basal, e a mutação G93A ressaltou estes processos. / Some familial ALS (fALS) are caused by mutations in the Cu,Zn-superoxide dismutase enzyme (SOD1). It was thought that the mutated enzymes would have impaired SOD activity, but this has not been corroborated so far. Presently, it is more accepted that the mutated enzymes acquire a new toxic function. What this new toxic function is and how it relates to the degeneration of motor neurons remains debatable. Here, we compared human neuroblastoma cells transfected with fALS mutant G93A (SH-SY5YG93A) or wild-type SOD1 (SH-SY5YWT) with parent cells (SH-SY5Y) in regard to growth, viability, basal oxidant production, SOD and peroxidase activities, and SOD forms. Transfected cells presented increased growth rate and basal oxidant production. SH-SY5YWT and SH-SY5YG93A cells in early culture stage showed SOD expression and activity consistent with the expected two-fold increase; SH-SY5YWT homogenates showed increased peroxidase activity. After four weeks, SH-SY5YG93A maintained SOD1 expression levels but peroxidase and dismutase activities were lower. SOD1 expression increased the levels of altered SOD1 forms such as the reduced enzyme, disulfide multimers and detergent-insoluble forms, particularly in SH-SY5YG93A cells. Among the insoluble forms a covalent SOD dimer was identified. These altered SOD forms are probably responsible for proteasome activation and endoplasmatic reticulum stress response verified in transfected cells. In conclusion, SOD1 over-expression was sufficient to increase intracellular immature and oligomerized SOD1 forms and basal oxidation and the G93A mutation enhanced these processes.

Für medizinische Anwendungen sind Stammzellen aufgrund ihrer Eigenschaften (Selbsterneuerung, hohe Proliferationsrate und Differenzierungsmöglichkeit in verschiedene Zelltypen) beispielsweise in den Bereichen des regenerativen Gewebeersatzes und der Zelltherapie sehr interessant. In vivo umgibt die Stammzellen eine definierte Mikroumgebung, die sie unterstützt sich zu teilen, ihren undifferenzierten Status aufrecht zu erhalten und Tochterzellen für das Wachstum, die routinemäßige Erneuerung oder den Ersatz von Gewebe zu produzieren. Diese Mikroumgebungen werden als Stammzellnischen bezeichnet. Für die Kultivierung von Stammzellen in vitro muss die in vivo-Situation möglichst getreu nachgestaltet werden. Ziel der Forschung ist die Schaffung einer künstlichen Umgebung, die sowohl die funktionellen Eigenschaften einer Nische besitzt als auch frei von Risiken xenogener Pathogene oder Gewebeunverträglichkeiten für die Anwendung am humanen Organismus eingesetzt werden kann. Einen Ansatz dafür bietet beispielsweise die Kopplung von Faktoren, die für den Erhalt der Stammzelleigenschaften notwendig sind, an synthetische Oberflächen. Ausgehend vom Bedarf an Kultur- oder Modellsystemen für die Expansion von (embryonalen) Stammzellen sollte in dieser Arbeit analysiert werden, ob alternierende Maleinsäureanhydrid (MA)-Copolymere ein geeignetes Trägersystem für die biofunktionelle kovalente Immobilisierung spezifischer Zytokine sind und dadurch unter anderem als künstliche Stammzellnische Anwendung finden können. MA-Copolymere eignen sich aufgrund ihrer spontanen Reaktion mit Aminogruppen für die Immobilisierung von Proteinen. Das Zytokin LIF (Leukemia inhibitory factor) existiert in vivo auch in immobilisierter Form und ist in embryonalen Mausstammzellen (mESC) allein in der Lage, das Stammzellpotential dieser Zellen zu erhalten. Aus diesem Grund ist LIF für die Analyse der Aufgabenstellung geeignet. Nach der Charakterisierung LIF-modifizierter Oberflächen wurde die biologische Verfügbarkeit des kovalent immobilisierten Zytokins mit Hilfe von LIF-sensitiven Fibroblasten und mESC der Linie R1 überprüft. Anschließend wurde im Mausmodell in vivo der Erhalt der Pluripotenz der mESC durch immobilisiertes LIF analysiert. Dafür standen die Oberflächen Poly(ethylen-alt-maleinsäureanhydrid) (PEMA) und Poly(octadecen-alt-maleinsäureanhydrid) (POMA) jeweils ohne und mit Polyethylenglykol (PEG7)-Modifizierung zur Verfügung, an die LIF kovalent gekoppelt wurde. Zusätzlich wurde LIF physisorptiv an einer Kollagen-Fibronektin-Matrix über hydrolysiertem POMA immobilisiert. Mit Hilfe von radioaktiv markiertem LIF konnte gezeigt werden, dass die Gesamtbeladungsmenge mit Zytokin von den Eigenschaften der MA-modifizierten Träger abhing. Auf PEMA konnten mit steigenden Immobilisierungskonzentrationen höhere Belegungsdichten an der Oberfläche erreicht werden, die im analysierten Bereich eine lineare Abhängigkeit zeigten. Aufgrund der starken Quellung in wässrigen Lösungen war eine Einlagerung von LIF-Molekülen in die Polymerschicht möglich und führte bei hohen Immobilisierungskonzentrationen auch nach 3 Tagen Inkubation mit proteinhaltigem Medium noch zur Verdrängung nicht kovalent gebundener Zytokinmoleküle aus PEMA-Oberflächen. Obwohl ein Teil des LIF in die Polymerschicht eindrang, war der Großteil der Moleküle für einen spezifischen Antikörper zugänglich. Hydrophobe Oberflächen mit POMA konnten bei hohen Immobilisierungskonzentrationen weniger LIF binden und zeigten Sättigungsverhalten der Oberflächen bei einer Belegungsdichte von 178 ng/cm^2 LIF. Eine Freisetzung von LIF nach mehr als 3 Tagen konnte nicht beobachtet werden. Gleichzeitig war hier aufgrund der hydrophoben Polymerseitenketten die Antikörperzugänglichkeit deutlich reduziert. Wegen des geringen Quellungsverhaltens von POMA in wässrigen Lösungen konnte eine Einlagerung des immobilisierten Zytokins in die Polymerschicht aber ausgeschlossen werden. Die kovalente LIF-Immobilisierung über PEG7-Spacer führte im Vergleich zu den nicht PEG-modifizierten Oberflächen PEMA und POMA zu jeweils geringeren Belegungsdichten, ohne dabei den Charakter der Abhängigkeit von der Immobilisierungskonzentration zu verändern (linear für PEMA+PEG7, Sättigung für POMA+PEG7). Die schlechte Antikörperzugänglichkeit von immobilisiertem LIF auf POMA konnte durch die Einführung des PEG7-Spacers deutlich verbessert werden und erreichte einen Wert ähnlich dem der hydrophilen PEMA-Oberflächen. Kovalent immobilisiertes LIF zeigte auf den vier MA-Oberflächen homogene und definiert einstellbare Belegungsdichten auf den einzelnen Proben. Die physisorptive Immobilisierung von LIF an extrazelluläre Matrixkomponenten auf hydrolysiertem POMA führte zu inhomogenen und bereits bei geringen Immobilisierungskonzentrationen instabilen Belegungsdichten. Die Einstellung definierter Belegungsdichten und die homogene Verfügbarkeit des Zytokins sind für die spätere Anwendung bei der Kultivierung wichtig, da so allen Zellen die gleiche definierte Zytokindosis unabhängig von der Oberflächencharakteristik präsentiert wird und Populationsunterschiede vermieden werden. LIF-sensitive Mausfibroblasten der Linie NIH3T3 reagierten auf immobilisiertes LIF mit der Aktivierung des Signalwegproteins STAT3. Durch den direkten Vergleich von STAT3-Aktivierungsprofilen nach Stimulation mit gelöstem oder immobilisiertem LIF konnte gezeigt werden, dass durch beide Präsentationsformen innerhalb der ersten 15 Minuten nach Stimulationsbeginn eine starke Aktivierung von STAT3 erfolgt, die anschließend wieder abklingt. Die Profile beider Präsentationsformen unterschieden sich in ihren Intensitäten nur bei der starken STAT3-Aktivierung. Dabei ergaben sich bei gelöstem LIF aufgrund der größeren Kontaktfläche mit Zytokin (gesamte Zelloberfläche) etwas stärkere Aktivierungen. Durch die sehr ähnlichen Aktivierungsprofile konnte nachgewiesen werden, dass das Zytokin LIF für Zellen zugänglich an MA-Copolymere mit und ohne Spacer-Modifizierung immobilisiert werden kann. Dabei lag ein Teil der Moleküle in einer Konformation und Orientierung gebunden vor, die die Funktionalität des Zytokins erhalten konnten. Zwischen den Oberflächen mit kovalenter LIF-Immobilisierung konnten keine wesentlichen Unterschiede in der STAT3-Aktivierung festgestellt werden. LIF war an all diesen Oberflächen für die LIF-sensitiven NIH3T3 Mausfibroblasten biologisch verfügbar. LIF-abhängige embryonale Mausstammzellen (mESC) reagierten nach 72 Stunden LIF-Stimulation mit der Aktivierung von STAT3. Bei Belegungsdichten ab 8 ng/cm^2 kovalent immobilisiertem LIF auf POMA mit und ohne PEG7-Spacer konnten ähnliche Aktivierungen wie durch die Stimulation mit gelöstem LIF festgestellt werden. Dies bestätigte die biofunktionelle LIF-Immobilisierung. Zwischen den POMA-Oberflächen mit und ohne PEG7 war dabei kein deutlicher Unterschied erkennbar. Eine reduzierte Zugänglichkeit des Antikörpers auf POMA beeinflusste demnach die biologische Verfügbarkeit des Zytokins für die mESC nicht. Der Erhalt des Stammzellpotentials durch kovalent an POMA gebundenes LIF konnte in vitro durch die Präsenz von Oct4 im Zellkern der mESC nachgewiesen werden. Durch die instabile Immobilisierung bei physisorptiver Assoziation des Zytokins an Matrixkomponenten über hydrolysiertem POMA reduzierte sich der Erhalt des Stammzellpotentials auf diesen Oberflächen stark. Kovalent immobilisiertes LIF dagegen konnte auch während der Kultur über mehrere Passagen hinweg die Pluripotenz der murinen ESC erhalten. Nach der Fusion mit Blastozysten beteiligten sich diese kultivierten Zellen in vivo erfolgreich an der Bildung von Chimären. Dabei konnten keine Unterschiede der Chimärenhäufigkeit zwischen der Kultivierung der mESC mit gelöstem oder kovalent an POMA immobilisiertem LIF festgestellt werden. Kovalent an MA-Copolymere immobilisiertes LIF ist demnach in der Lage, gelöstes LIF vollständig zu ersetzen und über mehrere Passagen hinweg allein das Stammzellpotential von mESC zu erhalten. Die Experimente zeigten, dass sich MA-Copolymere für die funktionelle kovalente Immobilisierung von Signalmolekülen eignen. Dabei konnten keine starken Unterschiede bei der Reaktion der Zellen auf die Oberflächen PEMA oder POMA festgestellt werden. Auch die Einführung eines zusätzlichen Spacers war für die Signaltransduktion nach Stimulation mit kovalent immobilisiertem LIF nicht notwendig. Für künftige Arbeiten zur kovalenten Immobilisierung von LIF an MA-Copolymeren ist deshalb aus Stabilitäts- und Effizienzgründen die Oberfläche POMA zu bevorzugen. Diese Favorisierung kann jedoch aufgrund der unterschiedlichen Tertiärstruktur anderer Proteine und ihrer verschiedenen Steifigkeiten sowie bei der Verwendung anderer Zelltypen nicht automatisch für ein anderes Modellsystem übernommen werden. Die Verwendung hydrophiler Oberflächen oder die Kopplung über Spacer sollte demnach in Abhängigkeit vom zu immobilisierenden Protein und den auszusiedelnden Zellen geprüft werden. Die vorgestellte Kopplungsmethode umgeht die Modifikation des Proteins sowie Behandlungen zur Vernetzung des Zytokins. Die Immobilisierungsreaktion ist bei Raumtemperatur und Umgebungsdruck sowie unter sterilen Bedingungen durchführbar. Immobilisierte Zytokine werden homogen kovalent an der Oberfläche gebunden und sind dort für die Zellen zugänglich. Außerdem ermöglicht die Einstellung definierter Belegungsdichten die gezielte Applikation von Zytokindosen. MA-Copolymere sind somit nicht nur für die Kultivierung von Stammzellen unter Erhaltung des Stammzellstatus einsetzbar, sondern eignen sich auch für Differenzierungsstudien. Teilergebnisse dieser Arbeit wurden publiziert unter K. Alberti, R.E. Davey, K. Onishi, S. George, K. Salchert, F.P. Seib, M. Bornhäuser, T. Pompe, A. Nagy, C.Werner, and P.W. Zandstra. Functional immobilization of signaling proteins enables control of stem cell fate. Nat Methods, 5(7):645–650, Jul 2008 und T. Pompe, K. Salchert, K. Alberti, P.W. Zandstra, and C. Werner. Immobilization of growth factors on solid supports for the modulation of stem cell fate. Nat Protocols, 5(6):1042–1050, Jun 2010. / In vitro cultivation of (embryonic) stem cells requires a defined environment. Together different properties as cytokine supplement, extracellular matrix composition or topographic design can mimic this stem cell niche in an artificial system. For mouse embryonic stem cells the cytokine leukemia inhibitory factor (LIF) is able to keep those cells in undifferentiated state and to enhance self renewal without the supplement of other factors. In vivo LIF exists in both diffusible and extracellular matrix immobilized form. This work investigates whether LIF can be immobilized covalently to alternating maleic anhydride (MA)-copolymers in a functional manner. When bioavailable, covalently immobilized LIF should be able to interact with specific cytokine receptor subunits and provide information to keep murine embryonic stem cells in a pluripotent state. In aqueous solution with neutral pH (such as phosphate buffered saline, PBS) and ambient temperature and pressure MA-copolymers react spontaneously with aminogroups and therefore represent a useful support for covalent protein immobilization. Depending on the choice of co-monomer, properties of copolymer vary: ethylene results in hydrophilic poly-(ethylene-alt-maleic anhydride) (PEMA), octadecene in more hydrophobic poly-(octadecene-alt-maleic anhydride) (POMA). LIF can be covalently immobilized onto the MA-copolymers as shown by radiolabeling experiments. The amount of cytokine coupled to PEMA increased linear whereas on POMA saturation could be observed for higher concentrations. A subsequent coupling of a polyethylene glycol spacer (PEG7) further modified the properties and led to more hydrophilic surfaces. The amount of LIF per area decreased in comparison to MA-copolymers without the spacer but the graph characteristics remained unaltered (linear for PEMA+PEG7, saturation for POMA+PEG7). During the first three days in buffer solution supplemented with bovine serum albumin, unbound LIF was displaced and the amount of immobilized cytokine remained stable. This Stability after preincubation allowed to immobilize required amounts of LIF per area. Although hydrophilic surfaces with PEMA showed swelling behavior resulting in increased layer thickness after incubation in PBS, accessibility to LIF for an antibody was not impaired. The amounts per area detected by radiolabeling method and using the antibody were similar and indicated that LIF was not covered by copolymers. For cell culture addition of diffusible as well as immobilized growth factors or cytokines requires dosage control. Frequently it is necessary to provide homogeneous distribution of the factor of interest. In the present study analysis by fluorescence microscopy confirmed homogeneity for surfaces with covalently immobilized LIF (iLIF) but not for LIF physisorbed to extracellular matrix components collagen type I and fibronectin. LIF transduces signals via the JAK/STAT pathway. Preliminary experiments with LIF-sensitive fibroblasts showed similar activation of STAT3 after stimulation with immobilized or diffusible LIF. The results of STAT3 activation revealed an activation profile with high intensities within the first 15 minutes for both immobilized and diffusible LIF followed by decrease. STAT3 activation profiles were similar on different surfaces and independent of LIF presentation mode. These results revealed that fibroblasts could recognize covalently immobilized LIF onto MA-copolymers and were able to activate STAT3. In the absence of LIF mESC start to differentiate within 24 to 36 hours and loose their pluripotency. To confirm the functional immobilization of LIF mouse embryonic stem cells (mESC) were cultivated on iLIF-modified POMA or POMA+PEG7 surfaces for 72 hours and stained for activated STAT3. Results showed a dose-dependent activation increasing with the iLIF amount per area. Higher amounts (8 and 75 ng/cm^2) of iLIF activated STAT3 similar to 10 ng/ml diffusible LIF. Introduction of PEG7 spacer did not further increased STAT3 activation. Both, the amount of ESC marker Oct4 and the percentage of Oct4-positive cells increased with higher amounts of iLIF and showed similar results as obtained with 10 ng/ml diffusible LIF. Murine ESC cultivated on LIF physisorbed to matrix components expressed similar amounts of transcription factor Oct4 compared to unstimulated cells. STAT3 activation and Oct4 expression in the absence of diffusible cytokine indicated a functional covalent immobilization of LIF. To confirm the pluripotency, mESC were stimulated for 6 to 8 subcultures only with iLIF, cell aggregates were fused with mouse embryos and implanted in pseudopregnant surrogate mothers. Three weeks after birth the contribution of mESC aggregates to chimera was evaluated. ESC stimulated with iLIF only contributed to chimera formation with around the same frequency as mESC cultivated with 10 ng/ml diffusible LIF. Thus, iLIF maintained pluripotency of mESC during in vitro expansion and could replace diffusible LIF. As shown by the experiments, MA-copolymers provide a support to covalently immobilize cell signaling molecules in a functional manner. This method of coupling does not need any protein modification or cross-linking treatment after protein incubation. Reaction can be carried out under sterile conditions at ambient temperature and pressure. The immobilized ligand is distributed equally on the supporting copolymer and the adjustment of required ligand amounts is possible. These properties characterize MA-copolymers as a suitable support to immobilize cell signaling molecules not only for keeping the stem cell fate but also for differentiation studies. Parts of this work were published: K. Alberti, R.E. Davey, K. Onishi, S. George, K. Salchert, F.P. Seib, M. Bornhäuser, T. Pompe, A. Nagy, C.Werner, and P.W. Zandstra. Functional immobilization of signaling proteins enables control of stem cell fate. Nat Methods, 5(7):645–650, Jul 2008. T. Pompe, K. Salchert, K. Alberti, P.W. Zandstra, and C. Werner. Immobilization of growth factors on solid supports for the modulation of stem cell fate. Nat Protocols, 5(6):1042–1050, Jun 2010.

The connection of molecular monomers through non-covalent interaction (e.g. hydrogen bonding, π-π interaction, coordination bonding) enables the formation of supramolecular polymers. The major advantage of these bonds is their reversibility and consequently their ability to self-assembly or to disconnect depending on given conditions. This thesis examines the self-assembly through quadruple hydrogen bonding, which is strong and the resulting structures stable. Ureidopyrimidinon A and aromatic amines were always used as the starting compound for the preparation of monofunctional and bifunctional ureidopyrimidinones.

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