La régulation fine des mécanismes d’élimination des ARN messagers (ARNm) au sein des cellules contribue au contrôle de l’expression génétique ainsi qu’à l’adaptation rapide des niveaux de transcrits en réponse à divers événements cellulaires ou stimuli externes. Elle intervient ainsi dans différents aspects de la physiologie cellulaire : différentiation, prolifération, homéostasie, inflammation ou encore défense anti-parasitaire. Les ARNm eucaryotes matures sont protégés d’une dégradation incontrôlée par une coiffe et une queue poly(A), à chacune de leurs extrémités. Le premier événement amorçant la dégradation des ARNm est le raccourcissement de la queue poly(A) par le complexe CCR4/Not par un processus appelé déadénylation. Ensuite, la coiffe 5’ est éliminée pendant l’étape de « decapping » qui est considérée comme une étape cruciale, irréversible et extrêmement contrôlée, nécessaire à la dégradation rapide du corps du messager par Xrn1. L’étape de “decapping” est effectuée via le recrutement d’un complexe protéique formé de l’enzyme Dcp2 et de son co-activateur essentiel Dcp1. Cependant, ce complexe n’est que peu actif et nécessite de nombreux co-facteurs pour être pleinement efficace. Ces facteurs comprennent l’anneau LSm1-7 qui reconnaît l’extrémité 3’ des ARNm déadénylés et interagit avec Pat1, une protéine plateforme qui recrute l’hélicase Dhh1 et les protéines activatrices du decapping Edc1-2-3. Tous ces facteurs sont organisés au sein d’un réseau d’interaction complexe et dynamique qui, dans certaines conditions, colocalise dans les P-bodies, des foyers cytoplasmiques impliqués dans la dégradation des ARNm et dans la répression de la traduction.Même si de nombreuses études ont révélé l’importance des interactions protéine/protéine dans le processus de decapping, peu d’informations sont disponibles sur les mécanismes moléculaires du recrutement et d’activation de Dcp2 par ses différents co-facteurs. De même, en raison de l’absence de structure de Dcp2 en complexe avec un ARNm coiffé, les détails moléculaires de la reconnaissance et du clivage de la coiffe sont inconnus. Mon projet de thèse a pour but de répondre à ces questions par l’étude fonctionnelle et structurale des acteurs du decapping, en utilisant les protéines de la levure Saccharomyces cerevisiae comme système modèle, puisque la plupart des acteurs du decapping sont conservés au sein des eucaryotes. Dans ce but, j’ai exprimé par génie génétique et isolé la majorité des facteurs impliqués dans le “decapping” et reconstitué plusieurs sous complexes comprenant Dcp2 et ses différents cofacteurs. / MRNA decay is a highly regulated process allowing cells to rapidly adapt their abundance of transcripts to environmental conditions. Eukaryotic mRNAs are protected from uncontrolled decay by a cap structure (m7GpppX) and a poly(A) tail at their 5’ and 3’ ends, respectively. The first event initiating the 5’ to 3’ degradation pathway is the shortening of the poly(A) tail by the CCR4/Not complex through a process known as deadenylation. Then the 5’ cap is degraded during the decapping step, which is considered as a crucial and irreversible step before rapid degradation of RNAs. Decapping is accomplished by the recruitment of a protein complex formed by the Dcp2 catalytic subunit and its activator Dcp1. However, this complex has a low intrinsic decapping activity and requires several accessory factors to be fully efficient. These include the Lsm1-Lsm7 complex that binds to the 3’ end of deadenylated mRNAs and promotes decapping. This complex binds to Pat1, a scaffolding protein recruiting other accessory proteins such as Dhh1 and Edc1-3 proteins (Enhancer of Decapping), which favor decapping. After efficient removal of the cap, Xrn1 (the major cytoplasmic 5’-3’ exonuclease) is recruited and degrades the resulting uncapped RNAs. Interestingly, all these proteins are part of dynamic and multifunctional protein assemblies that, under conditions, localize into cytoplasmic foci known as P-bodies.Although many studies have revealed the importance of these protein/protein interactions, little is known concerning the mechanisms of recruitment and activation of the decapping enzyme by its numerous co-factors. Moreover, in the absence of Dcp2 in complex with a capped RNA, molecular details of cap recognition and cleavage are lacking. My thesis project aims at answering these open questions with the structural and functional studies of the decapping machinery, using yeast Saccharomyces cerevisiae as a model organism, as most of decapping actors are well conserved among eukaryotes. For this purpose, I expressed and purified the majority of the decapping factors and reconstituted several sub-complexes including Dcp2 and its cofactors.

Indiana University-Purdue University Indianapolis (IUPUI) / Mononuclear molybdopterin enzymes are a large class of enzymes that are present in all phyla of life. All pterin containing enzymes posses a molybdopterin cofactor made up of a molybdenum metal center coordinated directly by a dithiolene ligand, which is appended to a pyranopterin cofactor. The majority of these enzymes catalyze oxygen atom transfer reactions that are concomitant with a transfer of two-electrons. We are hypothesizing that by altering the oxidation states of the dithiolene, the reactivity of the cofactor can be tuned for different substrates. This investigation focuses on the synthesis and characterization of oxo-MoIV(dithiolene) complexes that possess a fully reduced dithiolene ligand (dithiolene) and a fully oxidized dithiolene ligand (dithione). These complexes are designed to represent the asymmetry of the dithiolene ligand that is observed in the crystal structures of the DMSO reductase family. Asymmetric oxo-MoIV(dithiolene) complexes exhibit a unique structural property, a large fold angle along the S•••S vector of the dithione ligand. These complexes also show a positive solvatochromic effect in a range of polar to nonpolar solvents. The rich electrochemical properties of these redox active complexes and other characterization details such as IR, and NMR studies will be presented. Effects on the reactivity of these complexes using biologically relevant substrates will be discussed. The oxygen atom transfer reactivity has been probed by mass spectrometry and NMR spectroscopy. The presented complexes aide in highlighting the effect redox state of the dithiolene ligand has in modulating reactivity

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The goal of this research was to develop a series of mixed-metal supramolecular complexes through systematic component variation to better understand the role of structural modification on basic chemical and photochemical properties including photocatalysis of H₂O to H₂. Varying bidentate polypyridyl terminal ligands (TL), non-chromophoric halides (X), or number of Ru(II) light absorbers (LA) tunes the electrochemical, spectroscopic, photophysical, and photochemical properties within the supramolecular architecture. Ru(II),Rh(III),Ru(II) trimetallics of the design [{(TL)₂Ru(dpp)}₂RhX₂](PF₆)₅ (TL = phen = 1,10-phenanthroline or Ph₂phen = 4,7-diphenyl-1,10-phenanthroline; dpp = 2,3-bis(2-pyridyl)pyrazine; X = Cl⁻ or Br⁻) covalently couple two Ru(II) LAs to a central Rh(III) electron collector (EC) through dpp polyazine bridging ligands (BL). Ru(II),Rh(III) bimetallics of the design [(TL)₂Ru(dpp)RhCl₂(TL′)](PF₆)₃ (TL = Ph₂phen or bpy = 2,2′-bipyridine; TL′ = Ph₂phen or tBu2bpy = 4,4′-Di-tert-butyl-2,2′-bipyridine) couple only one Ru(II) LA to a Rh(III) metal center through the dpp BL. The Ru(II),Rh(III),Ru(II) trimetallic and Ru(II),Rh(III) bimetallic complexes are synthesized using a building block approach, permitting facile modification of the supramolecular architecture throughout molecular assembly. Electrochemical analysis of both architectures displays a Ru-based HOMO tuned by TL identity (RuII/III = +1.62 V and +1.58 V vs. Ag/AgCl for TL = phen and Ph₂phen, respectively) and a Rh-based LUMO tuned by X identity (RhIII/II/I = -0.35 V and -0.32 V vs. Ag/AgCl for X = Cl⁻ and Br⁻, respectively). Modification of TL′ at Rh(III) within the bimetallics provided varying LUMO identity. The trimetallics and bimetallics are efficient light absorbers throughout the UV and visible with π⟶ π* intraligand (IL) transitions in the UV and Ru(dπ)⟶ligand(π*) metal-to-ligand charge transfer (MLCT) transitions in the visible. While X identity does not vary the light absorbing properties within Ru(II),Rh(III),Ru(II) trimetallics, TL identity and the number of Ru(II) LAs strongly impacts spectral coverage and the extinction coefficient. Photoexcitation of the Ru(dπ)⟶dpp(π*) ¹MLCT results in near unity population of the weakly emissive, short-lived Ru(dπ)⟶dpp(π*) ³MLCT excited state, which is efficiently quenched by intramolecular electron transfer to populate a non-emissive Ru(dπ)⟶Rh(dσ*) metal-to-metal charge transfer (³MMCT) excited state. Photolysis of the complexes in the presence of the sacrificial electron donor N,N-dimethylaniline (DMA) results in multi-electron collection at Rh, thereby converting Rh(III) to Rh(II) to Rh(I) accompanied by halide loss at each step. This establishes the Ru(II),Rh(III),Ru(II) and Ru(II),Rh(III) complexes as photochemical molecule devices (PMD) for photoinitiated electron collection (PEC). The ability of these systems to undergo multiple redox cycles, absorb light efficiently, populate photoreactive excited states, and collect electrons at a reactive Rh metal center fulfills the requirements for H₂O reduction photocatalysts. Photolysis of trimetallic or bimetallic complexes at 470 nm in the presence of DMA and H₂O substrate yields photocatalytic H2 production. Within [{(TL)₂Ru(dpp)}₂RhX₂]⁵⁺ trimetallics (TL = phen or Ph₂phen; X = Cl⁻ or Br⁻), varying the TL from phen to Ph₂phen and X from Cl⁻ to Br⁻ yielded the most active and robust photocatalyst with [{(Ph₂phen)₂Ru(dpp)}₂RhBr₂]⁵⁺ producing 44 ± 6 mL H₂, 610 ± 90 mol H₂/mol Rh catalyst, and 7.3% maximum quantum efficiency (max. ΦH₂) in a DMF solvent system after 20 h photolysis. The proposed mechanism of PEC suggests bimetallic systems might be prepared that are active photocatalysts. Ru(II),Rh(III) bimetallics are synthetically more challenging and the energetic proximity of dpp(π*) and Rh(dσ*) orbitals make electronic tuning with steric protection of the photogenerated Rh(I) difficult. Within [(TL)₂Ru(dpp)RhCl₂(TL′)]³⁺ bimetallics (TL = Ph₂phen or bpy; TL′ = Ph₂phen or tBu₂bpy), a careful balance of steric and electronic effects was required to produce active photocatalysts. The bimetallic [(Ph₂phen)₂Ru(dpp)RhCl₂(Ph₂phen)]³⁺ produces 1.1 ± 0.07 mL H₂, 81 ± 5 TON, and 0.88% max. ΦH₂ in a DMF solvent system after 20 h photolysis. This establishes the [(Ph₂phen)₂Ru(dpp)RhCl₂(Ph₂phen)]³⁺ complex as the first Ru(II),Rh(III) bimetallic to function as a homogeneous single-component H₂O reduction photocatalyst. This dissertation reports the detailed analysis of the electrochemical, spectroscopic, photophysical, and photocatalytic properties of [{(TL)₂Ru(dpp)}₂RhX₂]⁵⁺ trimetallic (TL = phen or Ph₂phen; X = Cl or Br) and [(TL)₂Ru(dpp)RhCl₂(TL′)]³⁺ bimetallic (TL = Ph₂phen or bpy; TL′ = Ph₂phen or tBu₂bpy) supramolecular complexes. The design of the molecular architecture and the intrinsic properties of each component contribute to the overall function and efficiency of these systems. The careful design, meticulous synthesis and purification, detailed characterizations, and methodical experimentation have led to an in-depth understanding of the properties and factors needed for more efficient photocatalytic reduction of H₂O to H₂. / Ph. D.

Les systèmes complexes se caractérisent par l’émergence de phénomènes macroscopiques qui ne s’expliquent pas uniquement par les propriétés de leurs composantes de base. La synchronisation est l’un de ces phénomènes par lequel les interactions entre des oscillateurs engendrent des mouvements collectifs coordonnés. Une représentation sous forme de graphe permet de modéliser précisément les interactions, alors que les oscillations se décrivent par des dynamiques non linéaires. Étant donné le lien subtil entre le graphe et la dynamique des oscillateurs, il est difficile de prédire l’émergence de la synchronisation. L’objectif de ce mémoire est de développer de nouvelles méthodes pour simplifier les systèmes complexes d’oscillateurs afin de révéler les mécanismes engendrant la synchronisation. À cette fin, nous introduisons des notions de la théorie des graphes et des systèmes dynamiques pour définir la synchronisation sur des bases mathématiques. Nous présentons ensuite des approches existantes sophistiquées pour réduire la dimension de dynamiques d’oscillateurs. Ces techniques sont toutefois limitées lorsque les dynamiques évoluent sur des graphes plus complexes. Nous développons alors une technique originale—spécialement adaptée pour les graphes aux propriétés plus hétérogènes—pour réduire la dimension de dynamiques non linéaires. En plus de généraliser des approches récentes, notre méthode dévoile plusieurs défis théoriques reliés à la simplification d’un système complexe. Entre autres, la réduction de la dimension du système se bute à une trichotomie : il faut favoriser la conservation des paramètres dynamiques, des propriétés locales du graphe ou des propriétés globales du graphe. Finalement, notre méthode permet de caractériser mathématiquement et numériquement l’émergence d’états exotiques de synchronisation. / Complex systems are characterized by the emergence of macroscopic phenomena that cannot be explained by the properties of their basic constituents. Synchronization is one of these phenomena in which the interactions between oscillators generate coordinate collective behaviors. Graphs allow a precise representation of the interactions, while nonlinear dynamics describe the oscillations. Because of the subtle relationship between graphs and dynamics of oscillators, it is challenging to predict the emergence of synchronization. The goal of this master’s thesis is to develop new methods for simplifying complex systems of oscillators in order to reveal the mechanism causing synchronization. To this end, we introduce notions of graph theory and dynamical systems to define synchronization on sound mathematical basis. We then present existing sophisticated approaches for reducing the dimension of oscillator dynamics. Yet, the efficiency of these techniques is limited for dynamics on complex graphs. We thus develop an original method—specially adapted for graphs with heterogeneous properties—for reducing the dimensions of nonlinear dynamics. Our method generalizes previous approaches and uncovers multiple challenges related to the simplification of complex systems. In particular, the dimension reduction of a system comes up against a trichotomy: one must choose to conserve either the dynamical parameters, the local properties of the graph, or the global properties of the graph. We finally use our method to characterize mathematically and numerically the emergence of exotic synchronization states.