In this work, we were interested in studying the effect of two main metabolic factors, hypoxia and extracellular ATP metabolism, on the effector function of T helper subsets. The major oxygen sensor is HIF-1α which is continuously degraded in the presence of oxygen but is stabilized in hypoxia, leading to transcription of genes involved in cellular adaptation to low oxygen level. Our data show that the proportion of IFN-& / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les nucléotides, et tout particulièrement l’ATP, sont capables de moduler la fonction de l’un des principaux acteurs de la réponse immunitaire à savoir les cellules dendritiques (DC). Les DC expriment à leur surface de nombreux récepteurs P2X et P2Y dont le P2Y11, couplé à la voie de l’AMP cyclique (AMPc) et impliqué dans l’immunomodulation. L’ATP est capable, par son action sur le P2Y11, d’induire une semi-maturation des DC caractérisée entre autre par une diminution de la sécrétion d’IL-12 et une augmentation de la sécrétion d’IL-10. De plus, des données antérieures obtenues au laboratoire montrent que l’ATP confère également des propriétés immunosuppressives aux DC en stimulant l’expression de la thrombospondine-1 (TSP-1) et de l’indoléamine-2,3-dioxygénase (IDO).<p><p>Dans ce contexte, notre projet visait à établir un profil d’expression génique en réponse à l’ATP dans des DC humaines issues de monocytes (MoDC) afin d’avoir une vue globale de l’action de l’ATP sur les DC. Dans un premier temps, nous avons donc réalisé une étude cinétique comparant les profils d’expression génique en réponse à l’ATPγS, un agoniste plus stable que l’ATP, et à la prostaglandine E2 (PGE2), un activateur de l’AMPc induisant une semi-maturation des DC, par la technique de microarray. L’analyse de ces profils a mis en évidence une action précoce et large de l’ATPγS sur les MoDC. Un grand nombre de régulations obtenues ont confirmé les effets déjà connus de l’ATP sur les DC. Par ailleurs, nous avons confirmé par différentes techniques plusieurs nouvelles cibles de l’ATP impliquées dans l’inflammation et la réponse immunitaire (ex. CSF-1, NRP-1, VEGF).<p><p>En analysant plus en détail ces profils, nous avons observés la régulation de plusieurs gènes dont VEGF, AREG, EREG et HB-EGF, intervenant dans les processus d’angiogenèse et de tumorigenèse. Parmi ceux-ci, le gène AREG codant pour l’amphiréguline, un ligand de l’EGF récepteur (EGFR) était le gène le plus régulé dans notre profil microarray. Pour la première fois, nous avons démontré que les DC traitées à l’ATPγS en présence de LPS constituaient une importante source d’amphiréguline capable de stimuler la croissance de cellules musculaires lisses et de cellules tumorales LLC (Lewis Lung Carcinoma) in vitro. <p>Parallèlement, nous avons étudié l’implication des cellules dendritiques traitées à l’ATPγS sur la croissance tumorale in vivo. Pour ce faire, nous avons coinjecté, à des souris C57 black/6, des cellules tumorales LLC avec des surnageants issus de BMDC traitées au LPS ou au LPS+ATPγS. De cette façon, nous avons mis en évidence que des surnageants issus de BMDC traitées au LPS+ATPγS induisaient une augmentation significative de la masse tumorale par rapport aux surnageants issus de BMDC traités au LPS seul. Au moyen d’un anticorps bloquant anti-amphiréguline, nous avons démontré que cette augmentation de la masse tumorale était due à l’importante sécrétion d’amphiréguline par les BMDC traitées au LPS+ATPγS. De plus, nous avons observé une augmentation du nombre de vaisseaux positifs pour l’α-SMA, un des marqueurs des cellules musculaires lisses, dans les tumeurs issues de la coinjection de cellules LLC avec des surnageants de BMDC traitées au LPS+ATPγS. Pour finir, nous avons montré que les DC au sein des tumeurs LLC expriment non seulement le récepteur EGFR mais également l’amphiréguline. Ces différents résultats observés mettent en avant l’importance de l’ATP extracellulaire dans la croissance tumorale par son action sur les DC. <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Les biocapteurs sont des moyens d’analyse en plein essor à la fois rapides, sélectifs et peu coûteux applicables à des domaines extrêmement variés (environnement, santé, agroalimentaire,…). Dans ce type d’outil, un élément sensible de nature biologique (anticorps, enzyme, microorganisme, ADN…) doté d’un pouvoir de reconnaissance pour un analyte ou un groupe d’analytes est associé à un transducteur pouvant être de type électrochimique, optique ou thermique.Dans ce travail, nous nous sommes intéressés au développement de trois biocapteurs pour la détection de substances biologiquement actives. Le premier permet la détermination simultanée de l’adénosine triphosphate (ATP) et du glucose par ampérométrie, le deuxième celle de la créatine kinase, et le troisième est un biocapteur conductimétique pour la quantification de l’ATP. Dans les deux premiers biocapteurs, deux enzymes (l’hexokinase et la glucose oxydase) sont immobilisées à la surface de microélectrodes constituées d’un disque de platine. Le troisième biocapteur est basé sur l’immobilisation de l’hexokinase sur des microélectrodes interdigitées en or. L’immobilisation est réalisée dans tous les cas par co-réticulation des enzymes en présence d’albumine de sérum bovin à l’aide de glutaraldehyde. Les caractéristiques analytiques des biocapteurs ont été déterminées et différentes procédures ont été développées pour l’analyse d’échantillons réels. Les biocapteurs ont pu être appliqués avec succès à la quantification de l’ATP, du glucose et de la créatine kinase dans des préparations pharmaceutiques et du sérum sanguin / Biosensors are rapid, selective and inexpensive devices that combine a biological recognition element, the so-called bioreceptor (e.g. enzymes, antibodies, DNA or microorganisms) to a physical transducer (e.g. electrochemical, optical, thermal or piezoelectrical). They can be used to detect one specific analyte or one family of analytes for a wide range of applications (e.g. environment, food, health). In this work, the detection of biologically active substances was targeted. A biosensor system for simultaneous determination of adenosine triphosphate (ATP) and glucose, a biosensor for creatine kinase analysis, and a novel conductometric biosensor for ATP determination were developed. In the first two biosensors, two enzymes (hexokinase and glucose oxidase) were immobilized at the surface of platinum disc microelectrodes for amperometric detection. The third biosensor was based on hexokinase immobilized onto gold interdigitated microelectrodes for conductometric detection. In all cases, the enzymes were co-immobilized with bovine serum albumin by cross-linking using glutaraldehyde. Analytical characteristics of the biosensors were determined and different procedures were developed for real samples analysis. The biosensors could be successfully applied to the determination of ATP, glucose, and creatine kinase in pharmaceutical samples and blood serum

L’ATP est l’agoniste naturel du récepteur P2Y11. Ce nucléotide ne peut cependant pas être utilisé comme agent thérapeutique car, in vivo, il s’hydrolyse rapidement en ADP ou en AMP qui ne possèdent qu’une faible activité pour le récepteur. D’où l’intérêt de disposer d’analogues de synthèse moins sensibles à l’hydrolyse et possédant une affinité égale ou supérieure à celle de l’ATP.<p><p>Le premier objectif que nous nous sommes fixés au cours de notre thèse de doctorat fut de mettre au point un schéma de synthèse permettant d’obtenir des analogues de l’adénosine-5’-triphosphate [1] portant un motif thioalkyle ou thioaryle en position 2 de la base ainsi qu’un groupement dichlorométhylène entre les phosphores b et g & / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

<p>This work studies the effect of dopamine (DA) and adenosine triphosphate (ATP) on lipid membranes using a number of complementary experimental methods. These methods include Dynamic Light Scattering to measure electrostatic surface potentials, solid-state Nuclear Magnetic Resonance to measure the degree of lipid acyl chain order, Electron Paramagnetic Resonance to measure changes in membrane viscosity, and X-ray diffuse scattering to measure structural and material parameters of lipid bilayers. It is shown that both DA and ATP have a measurable affinity to the lipid-water interface even in the absence of specialized biological receptors. These results are important for understanding the function of DA and ATP in cellular processes.</p>

Biological processes are so complicated that to understand the mechanisms underlying the functioning of biomolecules it is inevitable to study them from various perspectives and with a wide range of tools. Understanding the function at the molecular level obviously requires the knowledge of the three dimensional structure of the biomolecules. Experimentally this can be obtained by techniques such as X‐ray crystallography and NMR studies. Computational biology has also played an important role in elucidating the structure function relationship in biomolecules. Computationally one can obtain the temporal as well as ensemble behavior of biomolecules at atomic level under conditions that are experimentally not accessible. Molecular dynamics(MD) study is a technique that can be used to obtain information of the dynamic behavior of the biomolecules. Dynamics of large systems like proteins can be investigated by classical force fields. However, the changes at the level of covalent bond involve the reorganization of electron density distribution which can be addressed only at Quantum mechanical level. In the present thesis, some of the biological systems have been characterized both at the classical and quantum mechanical level. The systems investigated by MD simulations and the insights brought from these studies are presented in Chapters 3 and 4. The unusual bonds such as pyrophosphate linkage in ATP and short strong hydrogen bonds in proteins, investigated through high level quantum chemical methods, are presented in Chapters 5, 6 and 7. Part of this thesis is aimed to address some important issues related to the dynamics of Tryptophanyl tRNA synthetase (TrpRS) which belongs to classic of aminoacyl‐tRNA synthetases (aaRS). aaRSs are extremely important class of enzymes involved in the translation of genetic code. These enzymes catalyze the aminoacylation of tRNAs to relate the cognate amino acids to the anticodon trinucleotide sequences. aaRSs are modular enzymes with distinct domains on which extensive kinetic and mutational experiments as well as structural analyses have been carried out, highlighting the role of inter‐domain communication (Alexander and Schimmel, 2001). The overall architecture of tRNA synthetases consists of primarily two domains. The active site domain is responsible for the activation of an amino acid with ATP in synthesizing an enzyme‐bound aminoacyl‐adenylate, and transfer of the aminoacyl‐adenylate intermediate to the 3’end of tRNA. The second domain is responsible for selection and binding of the cognate tRNA. aaRSs are allosteric proteins in which the binding of tRNA at the anticodon domain influences the activity at the catalytic region. These two binding sites are separated by a large distance. One of the aims of this thesis is to characterize such long distance communication (allosteric communication) at atomic level in Tryptophanyl tRNA synthetase. This is achieved by generating ensembles of conformations by MD simulations and analyzing the trajectories by novel graph theoretic approach. Graph and network based approaches are well established in the field of protein structure analysis for analyzing protein structure, stability and function (Kannan and Vishveshwara, 1999; Brinda and Vishveshwara, 2005). The parameters such as clusters, hubs and shortest paths provide valuable information on the structure and dynamics of the proteins. In this thesis, network parameters are used for the analysis of molecular dynamics MD) simulation data, to represent the global dynamic behavior of protein in a more elegant way. MD simulations are performed on some available (and modeled) structures of TrpRS bound to a variety of ligands, and the protein structure networks( PSN) of non‐covalent interactions are characterized in dynamical equilibrium. The ligand induced conformational changes are investigated through structure networks. These networks are used to understand the mode of communication between the anticodon domain and the active site. The interface dynamics is crucial for the function of TrpRS (since it is a functional dimer) and it is investigated through interface clusters. The matter embodied in the thesis is presented as 9 chapters. Chapter 1 lays the suitable background and foundation for the study, surveying relevant literature from different fields .Chapter 2 describes in detail the various materials, methods and techniques employed in the different analyses and studies presented in this thesis. A brief description of well‐known methods of molecular dynamics simulations, essential dynamics calculations, cross correlation maps, conformational clustering etc.is presented. The methods for constructing protein structure graphs and networks, developed in our lab, are described in detail. The use of network parameters for the analysis of MD simulation data to address the problem of communication between the two distal sites is also presented. Some descriptions of the ab initio quantum mechanical methods, which are used to investigate the unusual bonds in biomolecules, are also presented in this chapter. Chapter 3 is devoted in discussing the results from several normal as well as high temperature MD simulations of ligand‐free and ligand bound Bacillus stearothermophilus Tryptophanyl‐tRNA synthetase (bsTrpRS). The essential modes of the protein in the presence of different ligands are captured by essential dynamics calculations. Different conformations of the protein associated with the catalysis process of TrpRS, as captured through experiments, are discussed in the context of conformational sampling. High temperature simulations are carried out to explore the larger conformational space. Chapter 4 is focused on the results obtained from the MD simulation of human Tryptophanyl‐tRNA synthetase (hTrpRS). The structure of human TrpRS bound to the activated ligand (TrpAMP) and the cognate tRNA(tRNATRP) is modeled since no structure in the presence of both TrpAMP and tRNATRP is available. MD simulations on these modeled as well as other complexes of hTrpRS are performed to capture the dynamical process of ligand induced conformational changes (Hansiaetal., communicated). Both the local and the global changes in the protein conformation from the protein structure network (PSN) of MD snapshots are analyzed. Several important information such as the ligand induced correlation between different residues of the protein, asymmetric binding of the ligands to the two subunits of the protein, and the path of communication between the anticodon region and the aminoacylation site are obtained. Also, the role of the dimmer interface, from a dynamic perspective, is obtained for the first time. The interface dynamics which stabilize different quaternary structures of lectins (with high sequence and structure similarity) were investigated in a collaborative work (Hansiaetal.,2007). The lectin peanut agglutinin (PNA) is a tetramer with three different types of interfaces. The interface dynamics of this protein in the presence and in the absence of metal ions was investigated and the paper reporting the results from this study is included as appendix in this thesis. Chapter 5 deals with high level ab initio quantum chemical calculations on tri‐ and diphosphate fragments of adenosine triphosphate (ATP). Pyrophosphate prototypes such as methyl triphosphate and methyl diphosphate molecules in their different protonation states have been investigated at high levels of calculations (Hansiaetal., 2006a). The optimized geometries, the thermochemistry of the hydrolysis and the molecular orbitals contributing to the high energy of these compounds have been analyzed. These investigations provide insights into the‘‘highenergy’’character of ATP molecule. Further, the dependence of vibrational frequencies on the number of phosphate groups and the charged states has also been presented. These results aid in the interpretation of spectra obtained by experiments on complexes containing pyrophosphate prototypes. Hydrogen bonding is fundamental in understanding the structure and properties of molecules of biological interest including proteins. A recent analysis carried out in our lab showed that a significant number of short hydrogen bonds (SHB) are present in proteins (Rajagopal and Vishveshwara, 2005). Chapters 6 and 7 elucidate the results obtained from ab initio quantum chemical calculations on some of these SHBs to get aquantitative estimation of their geometry and strength. In chapter 6, asystematic analysis of the geometries and the energetics of possible SHB systems, which are frequently encountered in proteins, are presented at different levels of theory (HF,DFTandMP2). It is found that the SHBs involving both charged residues in the proteins are intrinsic in nature. However, two neutral residues form a SHB in the protein crystal structures either due to geometric constraints or due to the environment of these residues. This analysis enables one to distinguish SHBs which are formed because of geometric constraints from those which are formed because of the inherent property of the chemical groups involved in the hydrogen bonding. These results are useful in refining protein structures determined by crystallographic or NMR methods. In addition, sulfur atom of methionine and cysteinein proteins also participate in SHBs, which are not so well characterized. Chapter 7 presents the similar analysis carried out on short hydrogen bonds in proteins involving sulfur atom. A detailed analysis of SHBs of sulfur containing groups in a data set of proteins has been carried out. Some of the residue pairs from this analysis were considered for ab initio calculations. However, the optimization of these examples resulted in breaking of the hydrogen bonds involving sulfur atoms and formation of new hydrogen bonds with oxygen and/or nitrogen atoms. Hence model systems, which mimic the real examples, were designed to carry out ab initio studies and to investigate the short hydrogen bonds involving sulfur atoms. Another study on the protein‐water interaction, which does not fall under the realm of the main objective of the thesis, is discussed in Chapter 8. Protein–water interaction is crucial for accomplishing many biological functions of proteins. In the recent past, natural probe tryptophan, located at the protein surfaces, has been extensively investigated using femtosecond spectroscopy experiments to understand salvation dynamics (Peonetal.,2002). In this chapter a method is described to follow up the molecular events of the protein–water interactions in detail. Tryptophan–water interaction in the protein Monellin is investigated in order to get the atomic level insights into the hydration dynamics, by carrying out MD simulations on Monellin (Hansiaetal.,2006b). The results are compared with those obtained from femtosecond resolved fluorescence spectroscopy. The time constants of the survival correlation function match well with the reported experimental values.This validates the procedure, adapted here for Monellin, to investigate the hydration dynamics in general. The last chapter (Chapter9) summarizes the results obtained from various studies and discusses the future directions. First part of this thesis aims to present the analysis by carrying out MD simulations on monomeric and dimeric TrpRS protein in order to understand the two steps of the aminoacylation reaction: activation of the aminoacid Trp in the first step and the transfer of the activated amino acid in the next step. In the second part, quantitative estimation of the geometry and the strength of pyrophosphate bond and short hydrogen bonds in proteins are reported in detail by subjecting the systems to high levels of quantum mechanical calculations(QM). The use of ab initio QM/MM calculations by combining the quantum mechanics(QM) with the molecular mechanics(MM) in order to study the enzymatic reactions is discussed as the future

Among heat shock proteins, Hsp90 is unusual because it is not required for the proper folding of most cellular proteins but rather is disproportionally linked to the activation of signal transduction proteins including over forty kinases and many steroid hormone receptors. Mutated forms of many Hsp90 clients are causative agents in cancer, making Hsp90 a promising pharmacological target. Many small molecular inhibitors have been identified that competitively bind to the ATP binding site of Hsp90, some of which are in clinical trials as anticancer agents. Although the activation of kinase and hormone receptor clients by Hsp90 and its co-chaperones has been extensively studied, the molecular mechanism of client protein activation is poorly understood. Hsp90 is a dimeric chaperone containing three domains: the N-terminal (N) and middle (M) domains contribute directly to ATP binding and hydrolysis and the C-terminal (C) domain mediates dimerization. At physiological concentration, Hsp90 predominantly forms dimers, but the possibility that full-length monomers might also function in cells has not been tested. In Chapter 3, we used a single-chain strategy to design a full-length Hsp90 monomer (NMCC). The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. NMCC Hsp90 was also defective at ATP hydrolysis and the activation of kinase and steroid hormone receptor clients in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric. After finding that a full-length Hsp90 monomer containing only one ATPase site was unable to support yeast viability or activate Hsp90 clients, we set out to further explore the role of ATPase activity in client protein activation. Approximately 10 % of the yeast proteome binds to Hsp90 making it important to study Hsp90 function in the cellular environment where all binding partners are present. In Chapter 4, we observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered super-stabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. We utilized these super-stabilized dimers to analyze the ability of ATPase mutant homodimers to activate known Hsp90 client proteins in yeast cells. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of the glucocorticoid hormone receptor (GR) and v-src confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo. In addition to its role in the activation of signal transduction client proteins, Hsp90 has been shown to suppress the in vitro aggregation of numerous hard-to-fold proteins. In Chapter 5, we examine the role of charge in Hsp90 anti-aggregation activity. The charge on Hsp90 is largely concentrated in two highly acidic regions. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. Addition of an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct rescued the anti-aggregation activity of Hsp90 indicating that the net charge contributes to its anti-aggregation activity. The in vitro anti-aggregation activity of Hsp90 studied in Chapter 5 occurs in the absence of ATP. However, all of the biologically important functions of Hsp90 in cells identified to date, including the maturation of kinases and nuclear steroid hormone receptors, clearly require ATP hydrolysis. Why does Hsp90 robustly hinder the aggregation of hard-to-fold proteins without ATP in vitro, but in vivo uses ATP hydrolysis for all of its essential functions? By utilizing separation of function Hsp90 variants (that specifically lack in vitro anti-aggregation activity) we have begun to address this question. We find that anti-aggregation deficient Hsp90 is unable to support yeast growth under stressful conditions, potentially due to reduced cellular expression. Interestingly, the ATP-independent anti-aggregation activity of Hsp90 has no measureable impact on cellular function. Thus, hindering the aggregation of most hard-to- fold proteins by Hsp90 (independent of ATP hydrolysis) does not appear to be important for cell function. These results suggest a cellular model where the Hsp40/60/70 machinery is responsible for hindering the aggregation of most hard-to-fold proteins while Hsp90 assists in the maturation of a select set of clients in an ATP-dependent fashion, potentially aided by its inherent anti-aggregation properties.

Establishing highly sophisticated polymer films for delivery systems in a biological environment and bioanalytical tasks, the formation, thickness, swelling behavior, and (physiological) stability of highly biocompatible polyelectrolyte multilayers (PEMs) are described. These PEMs are composed of the very weak polycation maltose-modified hyperbranched poly(ethyleneimine) (PEI-Mal), strongly polyanion heparin sodium salt (HE − Na +) or weakly charged polyanion hyaluronic acid sodium salt (HA-Na+) deposited on Si wafer substrates. Two different glyco architectures for PEI-Mal are used, characterized by two different degrees of maltose decoration on a PEI scaffold. Using three pH-dependent deposition approaches for optimizing the (physiological) PEM stability and swelling, PEMs are characterized by (in situ) ellipsometry, atomic force microscopy (AFM), and (in situ) attenuated total reflection-Fouriertransform infrared (ATR-FTIR). Thus, PEMs reveal significantly different thicknesses, growth mechanisms (linear versus exponential), and swelling behavior in dependence of both the polycation architectures and the deposition protocol. These PEMs will allow the study of their complexation and release properties as preswollen PEMs against anionic drug molecules, adenosine triphosphate sodium salt (ATP), especially under physiological conditions for future drug delivery coatings.

Establishing highly sophisticated polymer films for delivery systems in a biological environment and bioanalytical tasks, the formation, thickness, swelling behavior, and (physiological) stability of highly biocompatible polyelectrolyte multilayers (PEMs) are described. These PEMs are composed of the very weak polycation maltose-modified hyperbranched poly(ethyleneimine) (PEI-Mal), strongly polyanion heparin sodium salt (HE − Na +) or weakly charged polyanion hyaluronic acid sodium salt (HA-Na+) deposited on Si wafer substrates. Two different glyco architectures for PEI-Mal are used, characterized by two different degrees of maltose decoration on a PEI scaffold. Using three pH-dependent deposition approaches for optimizing the (physiological) PEM stability and swelling, PEMs are characterized by (in situ) ellipsometry, atomic force microscopy (AFM), and (in situ) attenuated total reflection-Fouriertransform infrared (ATR-FTIR). Thus, PEMs reveal significantly different thicknesses, growth mechanisms (linear versus exponential), and swelling behavior in dependence of both the polycation architectures and the deposition protocol. These PEMs will allow the study of their complexation and release properties as preswollen PEMs against anionic drug molecules, adenosine triphosphate sodium salt (ATP), especially under physiological conditions for future drug delivery coatings.

No description available.