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Apoptosis is essential for development and the maintenance of cellular homeostasis and is frequently dysregulated in disease states. Proteins of the BCL-2 family are key modulators of this process and are thus ideal therapeutic targets. In response to diverse apoptotic stimuli, the pro-apoptotic member of BCL-2 family, BAX, redistributes from the cytosol to the mitochondria or endoplasmic reticulum and primes cells for death. The structural changes that enable this lethal protein to transition from a cytosolic form to a membrane-bound form remain poorly understood. Elucidating this process is a necessary step in the development of BAX as a novel therapeutic target for the treatment of cancer, as well as autoimmune and neurodegenerative disorders. A three-part study, utilizing computational modeling and biological assays, was used to examine how BAX, and similar proteins, transition to membranes. The first part tested the hypothesis that the C-terminal α9 helix regulates the distribution and activity of BAX by functioning as a "molecular switch" to trigger conformational changes that enable the protein to redistribute from the cytosol to mitochondrial membrane. Computational analysis, tested in biological assays, revealed a new finding: that the α9 helix can dock into a hydrophobic groove of BAX in two opposite directions – in a self-associated, forward orientation and a previously, unknown reverse orientation that enables dimerization and apoptosis. Peptides, made to mimic the α9-helix, were able to induce the mitochondrial translocation of BAX, but not when key residues in the hydrophobic groove were mutated. Such findings indicate that the α9 helix of BAX can function as a "molecular switch" to mediate occupancy of the hydrophobic groove and regulate the membrane-binding activity of BAX. This new discovery contributes to the understanding of how BAX functions during apoptosis and can lead to the design of new therapeutic approaches based on manipulating the occupancy of the hydrophobic groove. The second and third parts of the study used computational modeling to examine how the helical stability of proteins relates to their ability to functionally transition. Analysis of BAX, as a prototypical transitioning protein, revealed that it has a broad variation in the distribution of its helical interaction energy. This observation led to the hypothesis tested, that proteins which undergo 3D structural transitions during execution of their function have broad variations in the distribution of their helical interaction energies. The result of this study, after examination of a large group of all-alpha proteins, was the development of a novel, predictive computational method, based on measuring helical interactions energies, which can be used to identify new proteins that undergo structural transitioning in the execution of their function. When this method was used to examine transitioning in other members the BCL-2 family, a strong agreement with the published experimental findings resulted. Further, it was revealed that the binding of a ligand, such as a small peptide, to a protein can have significant stabilizing or destabilizing influences that impact upon the activation and function of the protein. This computational analysis thus contributes to a better understanding of the function and regulation of the BCL-2 family members and also offers the means by which peptide mimics that modulate protein activity can be designed for testing in therapeutic endeavors.

Ziel der vorliegenden Arbeit ist die Erschließung eines neuen, synthetischen Zugangs zu linearen und makrocyclischen Systemen, die als Strukturelement eine Cycloheptatrieneinheit besitzen. Hierbei kann das Cycloheptatrien aufgrund seiner zahlreichen Transformationsmöglichkeiten als ein molekularer Schalter angesehen werden. Durch photochemische Reaktionen könnte auf diese Weise der Informationsgehalt supramolekularer Systeme verändert werden. Um eine verbesserte Photoschaltbarkeit zu gewähren, werden bisarylsubstituierte Cycloheptatriene benötigt. Verbrückte Aryltropyliumsalze werden durch Umsetzung mit Anilinderivaten in verbrückte Bisarylcycloheptatriene überführt. Die Makrocyclisierung mit verbrückten Carbonsäurechloriden führt, unter den Bedingungen des Verdünnungsprinzips, zu amidischen Ringverbindungen. / The object of the following thesis is the development of a new synthetic approach to linear or makrocyclic systems, which contain cycloheptatriene as a structural element. Cyclohepta-triene could be used as a molecular switch, due to the fact that there are several possible transformations. Using photochemical reactions there could be an easy change of order and involed information in supramolecular systems. Bisarylcycloheptatrienes enables the photo-active system to switch more easy. Bridged arylcycloheptatrienylium salts react with anilines to bridged bisarylcycloheptatrienes. Makrocyclisation under high dilution conditions with bridged chlorocarbonacids leads to cyclic systems containing the needed structural element.

Molecular switch systems that activate gene expression by a small molecule are effective technologies that are widely used in applied biological research. Previously, two orthogonal ligand receptor pairs (OLRP) were developed as potential molecular switch systems by modifying nuclear receptors, ligand-activated transcription factors, to bind and activate gene expression with the synthetic ligand LG335 and not with the natural ligand 9-cis retinoic acid (9cRA). The two OLRP previously discovered were RXR variant 130 (I268A, I310A, F313A, and L436F) (also known as GR130) and the RXR variant QCIMFI (Q275C, I310M, and F313I) and (also known as GRQCIMFI). The OLRP were further developed into molecular switches to provide controlled gene expression and potentially benefit gene therapy applications by replacing the DNA binding domain (DBD) with a Gal4 DBD, a yeast transcription factor. Both molecular switches are able to bind Gal4 RE in response to LG335 and activate expression of a luciferase or GFP reporter gene in either a two- or one-component system. When characterizing the GR130 variant in the two-component system, no activation was observed with the natural ligand 9cRA, and the variant displayed a 19±5-fold activation and a 50 nM EC50 value in the presence of LG335. When the GRQCIMFI variant was evaluated in the two-component system, activation was observed in the presence of LG335 with a 10 nM EC50 value and a 6±2-fold induction, and 9cRA induced activation only at the highest concentration. The GRQCIMFI variant was also characterized with the one-component system containing the reporter gene GFP in a transient transfection as well as through retroviral transduction, displaying green fluorescence in 30% of the cells in the presence of 10 µM LG335. Several attempts were made to improve the molecular switch system. The VP16 activation domain was fused to GRQCIMFI in an effort to increase the fold induction; however, the addition of the VP16 created a constitutively active protein. Another approach to improve the molecular switch incorporated error-prone PCR to discover a new variant, Q275C, I310M, F313I, L455M (QCIMFILM), which displayed a 10-fold increase in sensitivity towards LG335 with a 5 nM EC50 value. Examination of the L455 position in the crystal structure of RXR revealed this residue is located outside of the ligand binding pocket on helix 12 (H12), but is able to significantly enhance receptor function. In fact, the single variant, L455M, was able to enhance receptor activation, compensate for a nonfunctional variant, as well as influence coactivator association. The long-term goal of this research is to develop a gene regulation system that would be used in human gene therapy trials. In the process of creating this system a deeper assessment of the nuclear receptor structure and function is made, which can be used for the enhancement and development of transcriptional regulation mechanisms.

Biochemical reactions make up most of the activity in a cell. There is inherent stochasticity in the kinetic behavior of biochemical reactions which in turn governs the fate of various cellular processes. In this work, the precision of a method for dimensionality reduction for stochastic modeling of biochemical reactions is evaluated. Further, a method of stochastic simulation of reaction kinetics is implemented in case of a specific biochemical network involved in maintenance of long-term potentiation (LTP), the basic substrate for learning and memory formation. The dimensionality reduction method diverges significantly from a full stochastic model in prediction the variance of the fluctuations. The application of the stochastic simulation method to LTP modeling was used to find qualitative dependence of stochastic fluctuations on reaction volume and model parameters. / text

Our target is the engineering of a light-gate molecular switch for the artificial ion channel, which will enable artificial ion channels to operate successfully in microfluidic systems, biomimetic sensors and various technical devices. A stable but reversible switch mechanism design is crucial, because the artificial ion channels known to date are lacking any control mechanism. Our artificial molecular switch is divided in two parts: the body part (calixarene) and a gate part based on light-responsive azo groups. The key to the controlling mechanism is the conformational change between cis and trans isomers, which is translated into movement of the gate. The gate is very robust and can either block or let the ions pass the molecular switch. Patch clamp investigations indicate successful integrations of gated artificial ion channels into lipid membranes. / Unser Ziel ist die Entwicklung eines lichtinduzierten Molekularschalters für künstliche Ionenkanäle, der als Nanobauteil für die Entwicklung von Sensoren in mikrofluiden Systemen, in biomimetischen Sensoren und in verschiedenen technischen Baugruppen eingesetzt werden soll. Für ein stabiles und zugleich reversibles System ist der Schaltmechanismus entscheidend, da die künstlichen Ionenkanäle bisher – soweit bekannt – keinen Regelmechanismus besitzen. Unser künstlicher molekularer Schalter setzt sich aus einem Rumpfteil (Calix[4]resorcinaren) und einer Schalteinheit, basierend auf lichtempfindlichen Azogruppen, zusammen. Die Schalteinheit ist sehr widerstandsfähig, kann den Ionenfluss blockieren oder die Ionen durch den Ionenkanal passieren lassen. Durch Bestrahlung wird die Kanalaktivität unterdrückt und reversibel wiederbelebt. Mittels Patch-Clamp-Untersuchungen wird das Schalten der synthetischen Ionenkanäle überprüft.

Le travail présenté dans ce mémoire est dédié à l'élaboration de complexes de ruthénium(II) photo-commutables originaux incorporant le couple photochrome Diméthyldihydropyrène (DHP) / Cyclophanediène (CPD), pour de futurs dispositifs moléculaires optoélectroniques.Dans le but de réaliser des systèmes complexes pouvant reproduire les fonctions d'un circuit logique, une stratégie intéressante repose sur l'association de molécules photo-commutables et de complexes métalliques. Cependant, cette stratégie se confronte à certains obstacles majeurs, généralement associés à la perte des propriétés de commutation des photochromes organiques. En réponse aux précédents résultats confirmant cette tendance, deux nouvelles familles de complexes terpyridiniques de ruthénium(II) à base de DHP ont été synthétisées. Sachant que la présence de fonction pyridinium améliore significativement les propriétés d'isomérisation du cœur DHP, les centres métalliques ont été connectés soit via un lien benzyle pyridinium (Ru-Lpy+tpy) soit via un lien aryle pyridinium (Ru-LZincke). Bien que fonctionnant à plus faible énergie, le complexe Ru-LZincke présente des performances amoindries, en revanche les complexes Ru-Lpy+tpy affichent une préservation notable de leurs propriétés de commutation. Suite à la découverte d'une photo-réactivité originale favorisée par la présence du centre métallique, une famille analogue à base de complexes bipyridiniques de ruthénium(II) (Ru-Lpy+bpy) a été étudiée. Même si les mécanismes ne sont pas complètement rationalisés, les complexes Ru-Lpy+bpy se sont révélés être de remarquables candidats pour la réalisation de photo-commutateurs réversibles quantitativement dans le domaine du visible. / The work of this thesis is devoted to the development of original photo-switchable ruthenium(II) complexes incorporating the photochromic Dimethyldihydropyrene (DHP) / Cyclophanediene (CPD), for future optoelectronic molecular devices.In order to realize complex systems capable of reproducing the functions of a logic circuit, an interesting strategy is based on the association of photo-switchable molecules and metal complexes. However, this strategy is confronted with some major obstacles, generally associated with the loss of the switching properties of organic photochromes. In response to previous results confirming this trend, two new families of DHP-based terpyridine ruthenium(II) complexes have been synthesized. Since the presence of pyridinium function significantly improves the isomerization properties of the DHP core, the metal centers were connected either via a pyridinium benzyl linkage (Ru-Lpy+tpy) or via an aryl pyridinium linkage (Ru-LZincke). Although operating at lower energy, Ru-LZincke complex exhibits lessened performance, whereas Ru-Lpy+tpy complexes exhibit a notable preservation of their switching properties. Following the discovery of an original photo-reactivity favored by the presence of metal center, an analogue family based on ruthenium (II) bipyridine complexes (Ru-Lpy+bpy) was studied. Even if the mechanisms are not completely rationalized, Ru-Lpy+bpy complexes have proved to be remarkable candidates for the realization of quantitatively reversible photo-switches in the visible domain.

Les bactéries utilisent un mode de communication appelé quorum sensing pour régulerl’expression des gènes en fonction de la densité de population et contrôler ainsi de façonmulticellulaire des processus tels que la sporulation, la compétence ou la virulence. Chez les bactériesà Gram-positif, le quorum sensing repose principalement sur la production, la sécrétion et la détectionde petits peptides de signalisation.Le projet porte sur l’étude du système quorum sensing: NprR/NprX chez Bacillus cereus, oùNprR est l’effecteur qui reconnait spécifiquement le peptide de signalisation NprX. NprR est uneprotéine bi-fonctionnelle. Seule, elle agit en tant qu’inhibiteur de la sporulation, en complexe avecNprX, elle perd sa fonction initiale au profit d’une activité facteur de transcription impliquée dans lavirulence. NprR appartient à une famille d’effecteurs de quorum sensing appelée RNPP (Rap, NprR,PlcR et PrgX) encore mal caractérisée au niveau structural. Mon projet de thèse a consisté en l’analysestructure-fonction du système NprR/NprX.Pour comprendre la régulation fonctionnelle de NprR par NprX, des études en solution (SECMALSet DLS) ont permis de mettre en évidence un switch moléculaire qui repose sur un changementd’oligomérisation. Ainsi NprX fait basculer NprR d’une conformation Apo dimérique à uneconformation compléxée tétramérique.L’étude structurale par cristallographie a aboutit à la résolution de la structure du complexeNprR/NprX. L’analyse de ce tétramère suggère la reconnaissance de 2 sites distincts sur l’ADN.L’étude structurale par SAXS, a quant à elle, permis de proposer une conformation dimérique de laforme Apo NprR, modèle conforté grâce à une étude par mutagénèse dirigée des résidus d’interface. Ils’agit d’un mode de dimérisation semblable à celui des protéines Rap (membres de la famille RNPP).La caractérisation par ITC de l’interaction NprR/NprX avec différentes formes du peptide,ainsi que l’analyse de la poche de fixation du complexe, ont permis de mieux comprendre la spécificitéd’interaction et de mettre en évidence deux résidus clés de l’effecteur : l’Asn275 essentielle à lafixation du peptide et l’Arg 126 essentielle à l’activation de la fonction facteur de transcription.Ces travaux ont contribué à une meilleure compréhension du système quorum sensingNprR/NprX grâce à l’élucidation du switch moléculaire contrôlé par NprX mais aussi à une meilleureconnaissance de la famille d’effecteurs RNPP. / Bacteria use a communication mode named quorum sensing to regulate gene expression depending on the population density and thus to control processes such as sporulation, competence or virulence in a multicellular manner. In Gram-positive bacteria, the quorum sensing relies mostly on the production, the secretion and the detection of small signaling peptides. The project focuses on the study of the quorum sensing system NprR/NprX in Bacillus cereus, where NprR is the effector, which recognizes specifically the signaling peptide NprX. NprR is a bi-functional protein. In the absence of peptide, it acts as a sporulation inhibitor while in complex with NprX, it acts as transcription factor implicated in virulence. NprR belongs to a family of quorum sensing effectors named RNPP (for the first identified members: Rap, NprR, PlcR and PrgX) still not well characterized at a structural level. My PhD project consisted to perform the structure/function analysis of the NprR/NprX system. To understand the functional regulation of NprR by NprX, I carried out different studies in solution (SEC-MALS and DLS). These results allowed me to highlight a molecular switch based on a changing of the oligomerisation state of the protein. NprX binding switches NprR from an Apo dimeric conformation to a tetrameric complex. The structural study by crystallography led to the resolution of the tetrameric NprR/NprX complex structure. The analysis of this tetramer suggests the recognition of 2 DNA binding sites. The structural study of the dimeric conformation of Apo NprR by SAXS, allowed me to propose a model similar to that of the Rap dimers (members of RNPP family). This model is supported by a directed mutagenesis study of interface residues. The characterization by ITC of the NprR/NprX interaction with different forms of the peptide, as well as the analysis of the binding pocket in the complex, led to a better understanding of the specificity of the interaction. Two key residues of the effector were highlighted: Asn275, essential to peptide binding and Arg126, essential to the activation of the transcription factor function. These results have contributed to a better understanding of the NprR/NprX quorum sensing system thanks to the elucidation of the molecular switch controlled by NprX but also in a better knowledge of the RNPP effectors family.

Lors de ce travail de thèse, nous avons effectué des recherches sur des chromophores organiques. Notre étude s’est portée sur la commutation des propriétés optiques linéaires et non linéaires via l’étude d’interrupteurs organiques multi-adressables. Notre étude s’est portée plus particulièrement sur la famille des dérivés de l’indolino-oxazolidine. Ce type de molécule présente l’avantage d’avoir deux voix de commutations distinctes par photochromisme et acidochromisme. Nous avons recherché le lien entre la structure des molécules et leurs propriétés optiques par une étude combinée originale théorie/expérience avec l’utilisation de calculs quantiques sophistiqués et par diffusion hyper-Rayleigh de la lumière. / In this work we have investigated the commutation of linear and non linear optical properties in organic multifunctional switches. Indolino-oxazolidine compounds and their derivatives were chosen as example of multifunctional systems, because they combine both acidochromic and photochromic properties. The relationships between the molecular structure of these compounds and their optical properties were established by means of high level the quantum chemical approach and hyper-Rayleigh Scattering of light approaches.

In the past, aminocyclohexanol rings have been successfully utilized as pH-triggered molecular switches in various trans-2-aminocyclohexanol derivatives. By changing the groups on the amine nitrogen, these models provided a wide pH range in which a switch can occur. The pH-induced switch of conformation was monitored by 1H-NMR spectroscopy. The models were also incorporated into the bilayer membrane of liposome structures and tested for their ability to disrupt their membrane upon their conformational flip induced by a decrease in pH. In this work, the amide bond has been studied as a molecular switch and various amide derivatives have been tested for their potential as lipid-like compounds that also exhibit a pH-sensitive conformational flip. The conformational analysis of these compounds was achieved by various NMR techniques and NMR acid-base titration studies were utilized to estimate the pKa of a number of the compounds described.