No description available.

<p>Interactions between nucleic acid substrates and the proteins and enzymes that bind and catalyze them are ubiquitous and essential for reading, writing, replicating, repairing, and regulating the genomic code by the proteomic machinery. In this dissertation, computational molecular engineering furthered the elucidation of spatial-temporal interactions of natural nucleic acid binding proteins and enzymes and the creation of synthetic counterparts with structure-function interactions at predictive proficiency. We examined spatial-temporal interactions to study how natural proteins can process signals and substrates. The signals, propagated by spatial interactions between genes and proteins, can encode and decode information in the temporal domain. Natural proteins evolved through facilitating signaling, limiting crosstalk, and overcoming noise locally and globally. Findings indicate that fidelity and speed of frequency signal transmission in cellular noise was coordinated by a critical frequency, beyond which interactions may degrade or fail. The substrates, bound to their corresponding proteins, present structural information that is precisely recognized and acted upon in the spatial domain. Natural proteins evolved by coordinating substrate features with their own. Findings highlight the importance of accurate structural modeling. We explored structure-function interactions to study how synthetic proteins can complex with substrates. These complexes, composed of nucleic acid containing substrates and amino acid containing enzymes, can recognize and catalyze information in the spatial and temporal domains. Natural proteins evolved by balancing stability, solubility, substrate affinity, specificity, and catalytic activity. Accurate computational modeling of mutants with desirable properties for nucleic acids while maintaining such balances extended molecular redesign approaches. Findings demonstrate that binding and catalyzing proteins redesigned by single-conformation and multiple-conformation approaches maintained this balance to function, often as well as or better than those found in nature. We enabled access to computational molecular engineering of these interactions through open-source practices. We examined the applications and issues of engineering nucleic acid binding proteins and enzymes for nanotechnology, therapeutics, and in the ethical, legal, and social dimensions. Findings suggest that these access and applications can make engineering biology more widely adopted, easier, more effective, and safer.</p> / Dissertation

La capacité des lectines à reconnaître spécifiquement des glycoconjugués à la surface de cellules en font des outils de diagnostic biomédical pour les pathologies associées à des changements de glycosylation (inflammation, du cancer ...). De par leur interaction avec les glycosphingolipides, ces protéines peuvent aussi être utilisées pour étudier le trafic membranaire. Toutefois, un nombre réduit de lectines sont actuellement disponibles, limitant leur utilisation dans les biotechnologies et la recherche. Le but de ma thèse est d'une part de concevoir des néo-lectines de valence et topologie contrôlées pour comprendre l'effet de la multivalence sur le mécanisme d'endocytose, et d'autre part de concevoir des lectines de spécificité modulable afin de les utiliser dans la reconnaissance spécifique des cellules tumorales.RSL est une lectine à fucose de la bactérie Ralstonia solanacearum qui a une structure en β-propeller formée par l'association de trois monomères présentant deux sites de liaison très similaires. Cette protéine trimérique et hexavalente a été choisie comme structure de base pour la conception de néolectines. Des RSLs trivalentes ont été produites par mutation d'un acide aminé essentiel pour la stabilisation du fucose. Leur caractérisation a démontré qu'ils avaient perdu la capacité d'invaginer la membrane plasmique. Une protéine de même structure que RSL mais monomérique a été ingénierée, puis une librairie de plus de 13 mutants de valence présentant différentes topologies a été créée. L'analyse de tous les mutants a permis de démontrer que la formation de tubules dans les membranes dépend plus de la distance entre les sites que du nombre de sites.Nous avons ensuite mis au point un protocole de bio-informatique afin de prédire l'orientation et la conformation d'oligosaccharides fucosylés dans les sites de fixation de plusieurs lectines à fucose. Les affinités relatives ont pu être calculées avec une bonne corrélation avec les valeurs expérimentales. La modélisation et la structure cristallographique des complexes entre RSL et les oligosaccharides Lewis X et Sialyl Lewis X indiquent un changement conformationnel du glycanne très inhabituel lors de l'interaction, donnant ainsi des pistes pour la conception de mutants de plus haute spécificité. / The ability of lectins to specifically recognize glycoconjugates on cell surface makes them excellent biomedical diagnostics tools for diseases associated with glycosylation changes (e.g inflammation, cancer, etc.). Furthermore, because of their interaction with glycosphingolipids, lectins may also be used to study membrane trafficking. However, only small number of lectins are currently available, limiting their use in biotechnology and research. The aim of my thesis was first to develop neolectins with controlled valency and topology to understand the effect of multivalency on the endocytosis mechanism, and second to design lectins with tuned specificity for the recognition of tumor cells.RSL is a fucose binding lectin from the bacterium Ralstonia solanacearum which has a β-propeller structure that is formed by the association of three monomers each having two very similar binding sites. This trimeric and hexavalent protein was chosen as the scaffold structure for the design of neolectins. Trivalent RSLs were created by mutating an amino acid with essential role in fucose binding. Characterization showed that these mutants lost the ability to invaginate the plasma membrane. In addition, monomeric RSL was engineered and a library of more than 13 mutants, with different topologies and valencies, was created. Analysis of these mutants showed that the formation of tubules in the membrane depends mostly on the distance between the sites rather than on the number of sites.Then we developed a bioinformatic protocol to predict the orientation and conformation of fucosylated oligosaccharides in the binding sites of several fucose binding lectins. The relative affinities could be calculated with a good correlation to experimental values. Both the model and the crystal structures of RSL complexed with sialyl Lewis X and Lewis X oligosaccharides indicate a very unusual conformational change of the glycan during the interaction. These studies pave the way for the design of mutants with higher specificity.

Les matériaux hybrides organiques-inorganiques forment une classe de systèmes dans lesquels plusieurs types de molécules organiques peuvent être insérées au sein d'une structure dite d’accueil, souvent de nature inorganique. C'est dans ce contexte que s'inscrit notre étude théorique de ces matériaux, utilisant les méthodes de la théorie de la fonctionnelle de la densité (DFT), jointes aux techniques de dynamique moléculaire (MD) dans l'approche dite de dynamique moléculaire ab initio. Nous avons développé un protocole permettant d'étudier l'évolution des propriétés de matériaux hybrides spécifiques sous l'effet de stimuli extérieurs comme la pression ou la température. Nous effectuons une étude théorique d’un matériau de référence dans la chimie des matériaux hybrides lamellaires: l'hydroxyde acétate de cuivre Cu2 (OH)3 (CH3 COO). Nous avons obtenu une transition d’un état antiferromagnétique à un état ferromagnétique sous l’effet d’une pression proche de 2 GPa, en bon accord avec la valeur expérimentale (1,2 GPa). Ensuite, nous appliquons ces méthodes à l’étude de nouveaux matériaux hybrides lamellaires simples contenant des molécules de fluorènes mono- et di- phosphoniques. / Ab-initio molecular dynamics (AIMD) refers to a set of state-of-the-art computational methods combining molecular dynamics with density functional theory. It is the basis of what could be called a «Virtual laboratory approach». In this work, we use the Car-Parrinello Molecular Dynamics (CPMD) scheme for investigating the properties of Copper Hydroxide Acetate system, a typical organic-inorganic hybrid material. We determine the corresponding atomic structure as well as several of its chemical and magnetic properties. Recent experimental achievements provide accurate XRD measurements enabling the study of magneto-structural properties of Cu2 (OH)3 (CH3 COO). compound. The pressure-induced magnetic transition, observed experimentally (at 1,2 GPa) in this material has been successfully reproduced (close to 2 GPa), thereby, highlighting the role of structural optimizations in theoretical treatments of such materials. Our aim is to elucidate the complex interplay between structural properties, interfacial inte r facial chemistry and magnetic behaviors of various nanoscale structured materials both at the local (atomic) and bulk (crystal) levels. We focus on copper hydroxide-based hybrid materials spanning through different organic components (such as fluorene mono- or di-phosphonic molecules) considered prototypical and very promising in the field of hybrid multifunctional materials.

Les dérivés arène-carboxyliques sont des matériaux intéressants dans le but d’obtenir une cellule solaire organique efficace à base de cristaux liquides : Ils présentent à la fois des propriétés électroniques modulables en fonction de la structure chimique, une tendance aux larges plages de températures de présence d’une mésophase colonnaire et souvent un alignement homéotrope au contact de surfaces polaires telles que l’ITO ou le verre. Nous montrons avec cette thèse que des couples donneur-accepteur colonnaires à température ambiante peuvent être obtenus, avec des propriétés suffisamment complémentaires pour prévoir des dissociations d’excitons en charges séparées à leur jonction dans des cellules solaires. / Carboxylic derivatives of polycyclic aromatics are interesting materials in order to obtain efficient organic solar cells based on liquid crystals: They have adjustable electronic properties depending on the chemical structure, a tendency to form a columnar mesophase over wide ranges of temperatures and often a homeotropic alignment in contact with polar surfaces such as ITO or glass. We show with this thesis that donor-acceptor pairs of room-temperature columnar liquid crystals with adjusted properties can be obtained in view of efficient exciton dissociation into separate charges at their junction in solar cells.

<p dir="ltr">Plant immunity to pathogens involves a network of genetic, molecular, and cellular processes that culminate in activation of responses that restrict pathogen ingress and reduce diseases symptoms. Mechanisms of pathogen recognition, signaling, and activation of immune responses are well understood especially for (hemi) biotrophic pathogens. By contrast, there is paucity of knowledge on immune signaling for responses to broad host necrotrophic fungi such as <i>Botrytis cinerea (Botrytis</i><i>)</i>. Plant resistance to such pathogens is genetically complex with no complete resistance observed in any host species. However, genetic variation for quantitative resistance has been documented although the molecular mechanisms are poorly understood. In the current study, we focused on functional dissection of components of tomato immune signaling underlying quantitative resistance to <i>Botrytis</i>. Tomato BIK1-family receptor-like cytoplasmic kinases (RLCKs) were studied using gene edited mutants, and subsequent molecular, biochemical, and genomic characterizations of the mutants and the corresponding proteins. In addition, <i>Botrytis</i> induced transcriptome of Arabidopsis and tomato were compared to explain observed differences in their resistance to the fungus. The results from these studies are described in four chapters. Chapter 1 provides a review of RLCKs and their function in plant responses to biotic and abiotic stresses. Chapter 2 presents the genetic, molecular, and biochemical characterization of tomato RLCK, TPK1B RELATED PROTEIN KINASE (TPK09) in fungal resistance and responses to light stress. Chapter 3 highlights observations on the functions of TPK1B RELATED PROTEIN KINASE7 (TPK07) in fungal and bacterial resistance. Lastly, Chapter 4 covers comparative transcriptome analysis of Arabidopsis and tomato responses to <i>Botrytis</i>.</p><p dir="ltr">Specialized cell-surface receptors mediate the perception of environmental changes. A subset of plant cell surface receptors recognizes pathogen-associated molecular patterns (PAMPs), which are immunogenic pathogen or host-derived molecules, peptides (phytocytokines), lipids, and carbohydrates. PAMPs are perceived by surface receptors designated as pattern recognition receptors (PRRs) that are categorized as receptor like kinases (RLKs) or receptor like proteins (RLPs). Recognition of PAMPs through PRR is an evolutionarily conserved pathway that aids plants in specific recognition of pathogens. The signaling events initiated by PRRs are connected to PRR-associated RLCKs, which amplify the signal and activate other regulatory proteins. PRR-RLCK activation is linked to immune messengers ROS, Ca⁺, and MAPKs based on extensive research in Arabidopsis which also established the paradigm for RLCKs functions in integrating signals from various PRRs. However, insufficient attention was given to RLCK functions in crop plant responses to biotic and abiotic stressors and, thus, limited data exists on RLCKs from crops of agronomic and horticultural significance. To address this gap, we conducted genetic, genomic, and molecular studies on the biological functions of tomato TPK1B RELATED KINASES TPK09 and TPK07.</p><p dir="ltr">To determine the function of TPK09 and TPK07, mutant alleles of these two RLCKs were generated through CRISPR-Cas9 gene editing. Loss of function mutants of <i>tpk09</i> exhibited increased susceptibility to <i>Botrytis</i> but showed no altered responses to the bacterial pathogen <i>P. syringae</i>. Plants carrying mutant alleles displayed reduced immune gene expression and impaired accumulation of reactive oxygen species in response to chitin and flg22. In addition to <i>Botrytis</i> and several plant hormones, the expression of <i>TPK09</i> gene is induced by light but suppressed by darkness. The exposure of wildtype tomato plants to light-emitting diodes (LEDs) reduced hypocotyl length but <i>tpk09</i> mutants were insensitive. Furthermore, <i>tpk09</i> mutants also exhibited increased accumulation of H₂O₂ and extensive necrosis, suggesting a disturbance in cellular homeostasis in response to changes in light spectra, ultimately leading to enhanced susceptibility to <i>Botrytis</i>. Although the global impact of TPK09 on <i>Botrytis</i> induced transcriptome was limited, the expression of the tomato negative regulator of cell death SlBI-like1 gene was significantly reduced in the mutant, particularly in response to the combined effect of <i>Botrytis</i> and LED light. The data suggest TPK09 regulates SlBI-like1 gene expression, but the mechanism is unclear. Further, the impairment of the light stress response in <i>tpk09</i> mutants was substantiated by a reduction in chlorophyll content and damage to the photosynthetic machinery, along with a clear reduction in the expression of genes related to light harvesting and photosynthesis. Regulatory network analysis using RNA-seq data identified TPK09 regulated genes related to stress and oxidative damage. This was further supported from proteomic studies of tomato TPK09-HA transgenic plants. Immunoprecipitation coupled with mass spectrometry revealed that proteins associated with photosynthesis and photosystem subunits were enriched in TPK09 overexpressing plant. Our data establishes that TPK09 is required for fungal resistance and light stress by maintaining functional photosynthetic machinery and ROS homeostasis.</p><p dir="ltr">We also studied the function of tomato TPK07 which is another member of the RLCK-VII family. Through analysis of <i>tpk07</i> mutants, we show that TPK07 it required for resistance to both <i>Botrytis</i> and <i>P. syringae</i> expressing the cysteine protease type III effector AvrPphB which cleaved TPK07 protein. However, further studies are needed to understand the biological function of this cleavage. The expression of TPK07 was induced by <i>Botrytis</i> and the bacterial PAMPs flg22, flg28, and csp22. TPK07 belongs to the RLCK VII-5 subfamily and clusters in the same clade with Arabidopsis PBL34 (AT5G15080), PBL35 (AT3G01300), and PBL36 (AT3G28690) that functions in immunity to <i>P. syringae</i>. In addition, <i>tpk07</i> mutants showed reduced accumulation of ROS in response to chitin and lipopolysaccharide. Moreover, TPK07 is a plasma membrane-localized kinase with a unique N-terminal sequence, offering a research avenue for future structural analysis to understand its contribution in pathogen responses.</p><p dir="ltr">In the last chapter, we conducted comparative analyses of <i>Botrytis</i> induced transcriptome of Arabidopsis and tomato to explain the observed differences between the two plant species in their resistance to <i>Botrytis</i>. Over the years, we observed tomato is more susceptible to <i>Botrytis</i> than Arabidopsis under the same disease assay conditions, but the mechanism is not known. We sought to gain insight into the immune responses of the two species and identify targets for future functional analyses that can explain the differential pathogen responses. Many separate studies<i> </i>have demonstrated<i> </i>that <i>Botrytis</i> causes extensive transcriptional reprogramming in Arabidopsis and tomato. Our results from the comparative studies of in-house generated data identified differentially expressed genes (DEGs), Gene Ontology terms, and metabolic pathways that are specific or shared between the two species. Interestingly, some genes show distinct expression pattern in tomato and Arabidopsis consistent with previous observation that some genes in the two species show contrasting disease resistance functions. Genes with distinct expression patterns warrant further studies to better understand differences in host immune responses between the two species. In addition, transcription factors (TFs) and regulatory hub genes that could be important for further studies were identified.</p><p dir="ltr">In sum, our data establishes the functions of two tomato RLCKs in fungal resistance, interactions between fungal resistance and plant responses to light, and the conserved and contrasting gene expression profiles of tomato and Arabidopsis genes, laying the foundation for future studies.</p>

La fluorescence excitée à deux photons est actuellement largement utilisée pour l’imagerie de tissus biologiques, mais la faible sensibilité des fluorophores utilisés en microscopie confocale (excitation à un photon) à une excitation à deux photons (ADP) rend nécessaire la conception et la synthèse de nouveaux fluorophores spécifiques pour la microscopie de fluorescence par excitation bi-photonique (MFEB). Mon travail de thèse a ainsi porté sur l’ingénierie moléculaire (conception, synthèse et caractérisations) de nouveaux fluorophores pour la MFEB. Nous nous sommes particulièrement intéressés à des systèmes unidimensionnels (1D) de petite taille comportant des systèmes π étendus autour d’un cœur rigide (dicétopyrrolopyrrole ou DPP) et entourés de différents systèmes électro-actifs. Nous avons modifié par la suite les fluorophores précédents de manière à pouvoir les conjuguer à des molécules d’intérêt biologique, comme des protéines. Ces fluorophores bio-conjugables ont été greffés sur un peptide du virus HIV étudié au laboratoire : TAT (Trans-Activator of Transcription). L’imagerie par microscopie biphotonique a été effectuée avec succès sur des cellules HeLa. Nous nous sommes ensuite tourné vers la mise au point de nouvelles sondes multimodales pour associer la MEBP à une autre modalité d’imagerie : la résonance magnétique nucléaire et la microscopie électronique (imagerie corrélative). Pour ce faire nous avons développé des colorants fluorescents par excitation bi-photonique comportant une entité paramagnétique ou dense aux électrons (nanoparticules de magnétite, ion gadolinium III ou atomes lourds comme le platine et l’or). / Two-photon induced fluorescence is nowadays widely used for the imaging of biological tissues. The classical fluorophores used in confocal microscopy exhibit low sensitivity to two-photon excitation for the two-photon excitation microscopy (TPEM), led the researchers towards the development of new fluorophores, specifically engineered for TPEM. This manuscript describes our work on conception, synthesis and characterizations of new one-dimensional fluorophores based on dicétopyrrolopyrrole (DPP) central core, surrounded by various electro-active systems through π conjugated systems. We also modified such fluorophores to be able to conjugate them to molecules of biological interest, such as proteins. These bio-conjugable fluorophores were grafted on a peptide of HIV virus studied in our laboratory: TAT (Trans-Activator of transcription). The imaging by TPEM was successfully performed on HeLa cells. In addition we developed new multimodal probes for the correlative light electronic microscopy and for the correlative imaging fluorescence microscopy/ Magnetic resonance imaging (MRI). Theses multimodal probes associate a fluorescent moiety based on the DPP core associated to a paramagnetic or electron dense entity (magnetite nanoparticles, gadolinium III or heavy atoms such as platinum or gold).

L’utilisation de chromophores absorbant à deux photons (ADP) pour des applications en photothérapie dynamique (PDT) et en imagerie de fluorescence présente de nombreux avantages. Les propriétés non-linéaires de ces chromophores permettent notamment d’améliorer la longueur de pénétration dans les organismes vivants ainsi que la résolution. Pour des applications in-vivo la biocompatibilité de ces chromophores lipophiles doit aussi se poser. Une étude d’ingénierie pour le développement de chromophores pour la PDT-ADP en utilisant des atomes de brome comme groupe générateur d’oxygène singulet est décrite. Différents paramètres dont le nombre et la position des atomes de brome sur la chaîne carbonée, la longueur de conjugaison, la géométrie des chromophores ont été étudiés. Cette étude permet de mettre en évidence l’importance de la position des substituants bromes et de la symétrie sur le rendement de croisement inter-système.Les observations spectroscopiques faites lors de l’étude d’ingénierie ont permis de développer des chromophores pour la microscopie de fluorescence à deux photons. La biocompatibilité est apportée grâce à un polymère d’(hydroxyethyl)acrylate. Ce polymère permet de créer une coquille hydrosolubilisante covalente. Ces chromophores ont été utilisés pour faire de l’imagerie de vascularisation cérébrale de haute résolution. Une observation particulière sur un chromophore, marquage des cellules endothéliales des parois des vaisseaux sanguins intravitaux ainsi que les applications en résultant sont présentées. Des stratégies visant l’amélioration de la sélectivité des systèmes polymères/chromophores pour des applications intravitales, comme le traitement des tumeurs cancéreuses sont décrites. Une stratégie de modification des fonctions hydroxy des chaînes polymères par des groupements imidazoliums est présentée. L’étude de complexation des polymères avec l’ADN et les études spectroscopiques in-cellulo ont été réalisées. / The use of two-photon absorbing (TPA) chromophore for applications in photodynamic therapy (PDT) and fluorescence imaging provides many advantages. The non-linear properties make it possible to increase both observation depth in animals and 3D resolution. Nevertheless, for in-vivo applications, improving bio-compatibility of these inherently lipophilic chromophore is a challenge. The development of new chromophores for PDT-TPA using a molecular engineering approach using bromide substituents as singlet oxygen generators is described. Parameters like position and number of bromide, the conjugated length and chromophore symmetry are studied. The study shows the importance of bromide atom position and of the symmetry on the inter system crossing efficiency. During the engineering study, spectroscopic observation and rationalization permit to envision the design of new chromophores for two photon laser scanning fluorescent microscopy. Bio-compatibility of these chromophores is provided by (hydroxyethyl)acrylate polymer, which provides a covalent water-soluble shell. These chromophores are used to make high resolution image of cerebral vascularization. One of these chromophores shows intravital specific interaction with endothelial cells in blood vessels. Some applications of the chromophore are described. Strategies to increase the intravital selectivity of polymer/chromophores units towards cancer cells and tumor are presented. A modification of hydroxyl function by imidazolium group is described. This new chromophore is evaluated towards its complexation properties with DNA and in cellulo spectroscopic studies.

<p dir="ltr">Bacterial Wilt, caused by <i>Ralstonia solanacearum</i>, is among the most devastating plant diseases in the world. This pathogen causes significant loss in crops such as tobacco, potato, and tomato. <i>R. solanacearum</i> root infection and xylem colonization determine disease outcome. To date, little is known about the defense mechanism mediated by roots to prevent <i>R. solanacearum</i> vascular colonization during the initial infection stages. Plant early responses are important since they may impact disease outcomes<i>.</i><i> </i>Here, we report the formation of root hairs and primary root growth inhibition in tomato seedlings as <i>Ralstonia</i>-induced phenotypes that depend on tomato genotype and <i>Ralstonia</i> species. The <i>Ralstonia</i>-induced root phenotypes are independent of a functional type III secretion system and exopolysaccharide production (EPS). We also found that <i>R. solanacearum</i><i> </i>K60 infection increased auxin levels throughout the root meristem in wilt-susceptible tomato roots. Our data suggest proper auxin signaling and transport are important for susceptibility to <i>R. solanacearum</i> K60. Blocking auxin transport pharmacologically or genetically led to fewer wilting symptoms, suggesting that auxin is important during early infection stages and disease outcomes in tomato. We previously found that a tomato mutant defective in auxin transport and signaling, known as <i>diageotropica</i> (<i>dgt</i>), has enhanced resistance to <i>R. solanacearum</i> K60. We characterized the resistant response in the <i>dgt</i> mutant, and we found that the resistant response in the <i>dgt</i> mutant may be due to increased lignin content preventing pathogen vasculature colonization. <i>DGT</i> encodes a cyclophilin protein that regulates auxin transport and signaling. Mutations in the cyclophilin DGT promote resistance to <i>R. solanacearum</i> K60. DGT has been reported to regulate auxin transport and signaling. However, the molecular mechanism regarding how DGT mediates these processes is still unknown. We used Yeast Two-Hybrid to identify candidate protein interactors, and we found that SlbZIP1/SlbZIP29, Sl14-3-3, and SlMYB110 may interact with DGT to regulate both development and defense responses. Understanding the role of DGT, auxin, and lignin in defense responses to <i>R. solanacearum</i> K60 in tomato is necessary for Solanaceae crop improvement.</p>

No description available.