La floculation est un procédé que l’on peut retrouver dans plusieurs applications au niveau industriel comme la fabrication du papier ou le traitement de l’eau. Il est nécessaire de comprendre la formation et la structure des agrégats floculés pour maîtriser et optimiser un tel procédé. La plupart des études sur la floculation implique des particules sphériques, impliquant ainsi un manque de connaissance sur la floculation de particules anisotropes comme par exemple des plaquettes de colloïdes argileux. C’est pour cela que dans cette étude, j’ai étudié la floculation d’une suspension d’argile type Montmorillonite dans l’eau par des polyélectrolytes cationiques modèles appelés ionènes. En utilisant ce système modèle, il est possible d’étudier l’effet de la densité de charges des ionènes, la taille des plaquettes d’argile ou encore la longueur des chaînes sur le mode de floculation et l’arrangement des chaînes et des plaquettes au sein des agrégats. Dans un premier temps, l’effet de la densité de charge des polyélectrolytes sur l’efficacité de floculation a été étudié en combinant des mesures de turbidité et de potentiel zêta. Ces mesures ont permis de montrer que la densité de charge des ionènes influence le mode de floculation et les conditions de floculation optimale. La conformation des chaînes dépend de la densité de charge des ionènes et c’est pour cela que j’ai poursuivi l’étude par des mesures de diffusion de rayonnement aux petits angles afin de pouvoir corréler l’effet de la conformation des chaînes avec la structure des agrégats à différentes échelles. La diffusion des rayons X aux petits angles a permis de montrer la présence d’organisation des plaquettes sous forme d’empilement face-face. Ensuite, cette technique a permis de souligner l’effet de la densité de charge sur la distance entre plaquettes dans les empilements. Pour les ionènes les moins chargés, la formation de boucles de chaînes des ionènes entre les faces d’argile induit les distances entre plaquettes les plus grandes au sein des agrégats. Les différences de conformations des différents ionènes ont été confirmées par diffusion de neutrons aux petits angles. Cette technique, sous la condition de variation de contraste, permet de sonder uniquement les chaînes dans les agrégats. J’ai également étudié le comportement des différents ionènes en solution aqueuse en absence des particules d’argile par diffusion de neutrons aux petits angles et RMN. Dans le domaine des hautes concentrations, ces systèmes montrent clairement des différences d’interaction entre les chaînes en fonction de la nature des contre-ions. L’évolution de ces interactions suit parfaitement la série de Hofmeister. / Flocculation is a key process in numerous environmental and industrial technologies such as purification of waste-water or paper making. It is necessary to understand the formation and structure of the aggregates to control and optimize such a process. Most of the studies on flocculation involve spherical particles, but there is a clear need to understand the flocculation of anisotropic particles such as clay colloids, which are platelets. I studied the flocculation of montmorillonite clay suspensions in water by well-defined cationic polyelectrolytes called ionenes. By using this model system, it is possible to study the effect of relative charge densities and sizes of ionene chains and clay platelets, on the mode of flocculation and their arrangement inside the aggregates formed. Initially, I studied the effect of charge density of polyelectrolytes on the flocculation efficiency of clay platelets, by combining measurements of turbidity and zeta potential. While the aggregates are always ionene-deficient, the charge density of ionenes leads to variations in the mode of flocculation and in the optimum flocculation conditions. The conformation of the ionene chains inside the aggregates depends on the charge density of ionenes and this is why I continued the study by small-angle scattering experiments in order to correlate the conformation of the chains with the structure of aggregates on different spatial scales. Small-angle X-ray scattering showed the presence of platelet stacks in a face-face configuration, demonstrating itself as a clear stacking peak in the scattering spectra. Further, this technique allowed to highlight the effect of the charge density on the interlamellar spacing inside these stacks. For the most weakly charged ionenes, loops of the ionene chains between the faces of adjacent platelets induce a higher interlamellar spacing inside the stacks. The differences of conformation of ionenes are confirmed by small-angle neutron scattering. This technique, under the condition of contrast matching, allows to probe only ionene chains within the aggegates aggregates. On a larger spatial scale, I have observed that by matching closely the charge density on ionene chains and on clay platelets the most compact aggregates are produced. I have also studied the behaviour of the different ionenes in aqueous solution in absence of clay particles by small-angle neutron scattering and NMR. At high chain concentrations, these systems clearly show differences in inter-chain interactions, depending on the nature of their counterions. The evolution of these interactions follows closely the Hofmeister series.

Lanthanide chelates are effective agents for improving contrast in MR images. Optimizing the relaxation of inner sphere water molecules is a common focus of research in this field. However, the efforts to design an optimal contrast agent have commonly over-looked the relationship of water position and water exchange kinetics. This work explores structural conformation, the impact of very fast water exchange kinetics on hydration, and differing tumbling rates for regioisomers of a number of lanthanide chelates. We have grown crystals of LnDOTMA and obtained structural data by X-ray diffraction that provide a picture of the chelate during water exchange and demonstrate that chelate conformation is associated with water position. We observe increased population of the major isomer with increased water exchange rates in variable temperature 1H NMR studies of HoDOTMA. This suggests that water position and water exchange rates are linked. We therefore recommend that accurate water exchange data be included in the application of the SBM equations when interpreting experimental data. As further support of this recommendation, we measured water exchange kinetics with 17O NMR for the rigid GdNB-DOTMA chelates. These results were used in the fitting of 1H NMRD profiles to establish tumbling parameters. Similar results were also observed in the less rigid GdNB-DOTA, establishing the first identification of regioisomers in these chelates and their biphenyl derivatives. Binding studies of GdBP-DOTA indicate that the side isomer is a more effective agent, but it is the minor species in solution. Our work herein shows that predicting efficacy of contrast agents with SBM equations requires a more complete consideration of chelate hydration (q/r6).

Accumulation and aggregation of the 42-residue amyloid-[beta] (A[beta]) protein fragment, which originates from the cleavage of amyloid precursor protein by beta and gamma secretase, correlates with the pathology of Alzheimer's disease (AD). Possible therapies for AD include peptides based on the A[beta] sequence, and recently identified small molecular weight compounds designed to mimic these, that interfere with the aggregation of A[beta] and prevent its toxic effects on neuronal cells in culture. Here, we use molecular dynamics simulations to compare the mode of interaction of an active (LPFFD) and inactive (LHFFD) [beta]-sheet breaker peptide with an A[beta] fibril structure from solid state NMR studies. We found that LHFFD had a weaker interaction with the fibril than the active peptide, LPFFD, from geometric and energetic considerations, as estimated by the MM/PBSA approach. Cluster analysis and computational alanine scanning identified important ligand-fibril contacts, including a possible difference in the effect of histidine on ligand-fibril [pi]-stacking interactions, and the role of the proline residue establishing contacts that compete with those essential for maintenance of the inter-monomer [beta]-sheet structure of the fibril. Our results show that molecular dynamics simulations can be a useful way to classify the stability of docking sites. These mechanistic insights into the ability of LPFFD to reverse aggregation of toxic A[beta] will guide the redesign of lead compounds, and aid in developing realistic therapies for AD and other diseases of protein aggregation. We have also performed long explicit solvent MD simulations of unliganded amyloid fibril in three putative protonation states, in order to better understand the energetic and mechanical features of the fibril receptor. Over 100 ns MD simulations, the trajectories where fibril has Glu11 and Glu22 side-chains protonated exhibit the least deviation from the initial solid state NMR structures. Free energy calculations on these rajectories suggest that the weakest fibril interface lies in the lateral rather than transverse direction and that there is little dependence on whether the lateral interface is situated at the edge or middle of the fibril. This agrees with recent reported steered molecular dynamics calculations. Secondly, in an effort to improve the ability of atomistic simulation techniques to directly resolve protein tertiary structure from primary amino acid sequence, we explore the use of a molecular dynamics technique based on swarm intelligence, called SWARM-MD, to identify the native states of two peptides, polyalanine and AEK17, as well as Trp-cage miniprotein. We find that the presence of cooperative swarm interactions significantly enhanced the efficiency of molecular dynamics simulations in predicting native conformation. However, it also is evident that the presence of outlying simulation replicas can adversely impact correctly folded replica structures. By slowly removing the swarm potential after folding simulations, the negative effect of the swarm potential can be alleviated and better agreement with experiment obtained.

O objetivo deste trabalho foi avaliar alguns aspectos reprodutivos de éguas Crioulas a fim de verificar a influência destes nos índices de prenhez: idade das éguas, status reprodutivo, uso do cio do potro, presença de líquido intra-uterino (LIU), eficiência dos tratamentos pós-cobertura, presença de cistos endometriais, realização de vulvoplastia, incidência de ovulações duplas e gestações gemelares. Foram utilizados 517 ciclos de 406 éguas Crioulas de um rebanho comercial, com idades entre dois e vinte e dois anos. O índice de prenhez por ciclo foi de 80,5% aos 12 dias, 76,5% aos 42 dias e a mortalidade embrionária foi de 3,9%. O índice de prenhez foi influenciado pela idade das éguas (P < 0,01). O índice de prenhez no cio do potro foi de 85,4%, melhor que o índice obtido pelas éguas em que não se utilizou este cio e que foram cobertas no subseqüente (P = 0,07). O método de cobertura influenciou a prenhez, sendo a inseminação superior à monta natural (P = 0,01). A idade influenciou a presença de LIU sendo a incidência em éguas com mais de 12 anos maior que nas mais jovens (P < 0,01). A presença de cistos endometriais influenciou negativamente a taxa de prenhez aos 12 e aos 42 dias (P = 0,00). A correção cirúrgica da região perineal melhorou os índices de prenhez aos 12 e aos 42 dias (P < 0,05). A taxa de ovulações duplas foi de 4,84% e não influenciou os índices de prenhez aos 12 e aos 42 dias (P > 0,5). Em 6 ciclos (1,16%) de 5 éguas solteiras foram observadas gestações gemelares. Concluímos que a fertilidade das éguas Crioulas é influenciada pela idade das éguas, pela conformação vulvar, pela presença de cistos endometriais e pelo status reprodutivo. O acúmulo de LIU é um fator importante na reprodução da égua Crioula e deve ser observado e devidamente tratado para a obtenção de melhores índices de prenhez. / The aim of this study was to evaluate the reproductive aspects of Crioulo mares to verify the influence of the following aspects on the pregnancy rates: mare’s age, reproductive status, use of foal heat, presence of intrauterine fluid (IUF), efficiency of post-breeding treatments, presence of endometrial cysts, incidence of double ovulations and twin pregnancies. There were used 517 cycles of 406 Crioulo mares of a commercial herd, with ages varying from 2 to 22 years old. Pregnancy rate per cycle was 80.5% at 12 days, 76.5% at 42 days and embryo mortality was 3.9%. The pregnancy rate was influenced by the age of the mares (P < 0.01). Pregnancy rate on foal heat was 85.4%, better than the rate obtained by the mares which foal heat was not used and the subsequent cycle was (P = 0.07). The breeding method influenced pregnancy, being artificial insemination superior to natural breeding (P = 0.01). Age influenced the presence of IUF, once the incidence was higher in mares with more than 12 years old than in younger mares (P < 0.01). The presence of endometrial cysts negatively influenced the pregnancy rate at 12 and 42 days (P = 0.00). Surgical repair of perineal region improved the pregnancy rates at 12 and 42 days (P < 0.05). The rate of double ovulations was of 4.84% and did not influence the pregnancy rates either at 12 and 42 days (P > 0.5). In 6 cycles (1.16%) of 5 single mares there were observed twin pregnancies. We concluded that the fertility of Crioulo mares is influenced by the age of the mares, vulvar conformation, presence of endometrial cysts and reproductive status. The accumulation of IUF is an important factor on the reproduction of Crioulo mares and must be observed and correctly treated in order to obtain better pregnancy rates.

Ca2+ is an important messenger that affects almost all cellular processes. Ca2+ signaling involves events that happen at various time-scales such as Ca2+ diffusion, trans-membrane Ca2+ transport and Ca2+-mediated protein-protein interactions. In this work, we utilized multi-scale computational methods to quantitatively characterize Ca2+ diffusion efficiency, Ca2+ binding thermodynamics and molecular bases of Ca2+-dependent protein-protein interaction. Specifically, we studied 1) the electrokinetic transport of Ca2+ in confined sub-µm geometry with complicated surfacial properties. We characterized the effective diffusion constant of Ca2+ in a cell-like environment, which helps to understand the spacial distribution of cytoplasmic Ca2+. 2) the association kinetics and activation mechanism of the protein phosphatase calcineurin (CaN) by its activator calmodulin (CaM) in the presence of Ca2+. We found that the association between CaM and CaN peptide is diffusion-limited and the rate could be tuned by charge density/distribution of CaN peptite. Moreover, we proposed an updated CaM/CaN interaction model in which a secondary interaction between CaN’s distal helix motif and CaM was highlighted. 3) the roles of Mg2+ and K+ in the active transport of Ca2+ by sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. We found that Mg2+ most likely act as inhibitor while K+ as agonist in SERCA’s transport process of Ca2+. Results reported in this work shed insights into various aspects of Ca2+ signaling from molecular to cellular level.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21819号 / 農博第2332号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5191(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三上 文三, 教授 植田 充美, 教授 宮川 恒 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

L’organisation nucléaire de la chromatine n’est pas aléatoire. Sa structure est parfaitement contrôlée, suivant un modèle hiérarchique avec différents niveaux d’organisation et de compaction. A large échelle, chaque chromosome occupe son propre espace au sein du noyau. A plus fine résolution, un chromosome est subdivisé en compartiments actifs ou répressifs, caractérisés par un état de la chromatine plus ou moins compact. A l’échelle du méga-base, cette organisation hiérarchique peut encore être divisée en domaines topologiques (ou TADs), jusqu’à la caractérisation de boucle d’ADN facilitant les interactions entre promoteurs et régions régulatrices. Très brièvement, et bien que les méchanismes exactes restent à déterminer, il a récemment été démontré que l’organisation spatiale de la chromatine dans une cellule normale joue un rôle primordial dans la régulation et l’expression des gènes. L’organisation en domaines topologiques implique la présence de complexes protéiques insulateurs tel que CTCF/cohésine. Ces facteurs jouent un rôle de barrière en restreignant et favorisant les interactions entre éléments régulateurs et gènes à l’intérieur d’un domaine, tout en limitant les interactions entre domaines. De cette façon, deux régions appartenant au même domaine topologique pourront fréquemment interagir, alors que deux régions appartenant à des domaines distincts auront une très faible probabilité d’interaction. Dans la cellule cancéreuse, l’implication de l’épigénome et de l’organisation spatiale de la chromatine dans la progression tumorale reste à ce jour largement inexplorée. Certaines études récentes ont toutefois démontré qu’une altération de la conformation de l’ADN pouvait être associée à l’activation de certains oncogènes. Même si les mécanismes exacts ne sont pas encore connus, cela démontre que l’organisation de la chromatine est un facteur important de la tumorigenèse, permettant, dans certains cas, d’expliquer les méchanismes moléculaires à l’origine de la dérégulation de certains gènes. Parmi les cas rapportés, une alération des régions insulatrices (ou frontières) entre domaines topologiques permettrait à des régions normalement éloignées spatialement de se retrouver en contact, favorisant ainsi l’activation de certains gènes. Une caractérisation systématique de la conformation spatiale des génomes cancéreux pourrait donc permettre d’améliorer nos connaissances de la biologie des cancers. Les techniques haut-débit d’analyse de la conformation de la chromatine sont actuellement largement utilisées pour caractériser les interactions physiques entre régions du génome. Brièvement, ces techniques consistent à fixer, digérer, puis liguer ensemble deux régions du génome spatialement proches. Les fragments d’ADN chimériques ainsi générés peuvent alors être séquencés par leurs extrémités, afin de quantifier le nombre de fois où ces régions ont été trouvées en contact. Parmi les différentes variantes de ces techniques, le Hi-C associé à un séquençage profond permet l’exploration systématique de ces interactions à l’échelle du génome, offrant ainsi une vue détaillée de l’organisation tri-dimensionnelle de la chromatine d’une population cellulaire. / The chromatin is not randomly arranged into the nucleus. Instead, the nuclear organization is tightly controlled following different organization levels. Recent studies have explored how the genome is organized to ensure proper gene regulation within a constrained nuclear space. However, the impact of the epigenome, and in particular the three-dimensional topology of chromatin and its implication in cancer progression remain largely unexplored. As an example, recent studies have started to demonstrate that defects in the folding of the genome can be associated with oncogenes activation. Although the exact mechanisms are not yet fully understood, it demonstrates that the chromatin organization is an important factor of tumorigenesis, and that a systematic exploration of the three-dimensional cancer genomes could improve our knowledge of cancer biology in a near future. High-throughput chromosome conformation capture methods are now widely used to map chromatin interaction within regions of interest or across the genome. The Hi-C technique empowered by next generation sequencing was designed to explore intra and inter-chromosomal contacts at the whole genome scale and therefore offers detailed insights into the spatial arrangement of complete genomes. The aim of this project was to develop computational methods and tools, that can extract relevant information from Hi-C data, and in particular, in a cancer specific context. The presented work is divided in three parts. First, as many sequencing applications, the Hi-C technique generates a huge amount of data. Managing these data requires optimized bioinformatics workflows able to process them in reasonable time and space. To answer this need, we developped HiC-Pro, an optimized and flexible pipeline to process Hi-C data from raw sequencing reads to normalized contact maps. HiC-Pro maps reads, detects valid ligation products, generates and normalizes intra- and inter-chromosomal contact maps. In addition, HiC-Pro is compatible with all current Hi-C-based protocols.

Hedgehog (Hh) est un morphogène secrété qui contrôle la croissance et la différentiation cellulaire chez les métazoaires. La dérégulation de son activité entraine des maladies développementales et de nombreux cancers chez l’adulte. Chez la drosophile, la transduction du signal Hh est initiée par la fixation de Hh sur son récepteur Patched (Ptc), conduisant à la stabilisation de la protéine membranaire Smoothened (Smo) et à l’activation du complexe de transduction composé de 5 protéines : les kinases Fused (Fu), PKA, GprK2, la kinésine Costal 2 (Cos2), et le facteur de transcription Cubitus Interruptus (Ci). Ma thèse a porté sur l’étude de la régulation et des interactions moléculaires entre les composants du complexe de transduction. Par des approches complémentaires, j’ai montré qu’en absence d’Hh, les protéines PKA et Fu interagissent du côté C-terminal de Ci, alors que la présence d’Hh induit leur relocalisation vers le domaine N-terminal de Ci. J’ai pu prouver que l’élément déclencheur de ce remaniement protéique est Smo. En présence d’Hh, Smo s’incorpore dans le complexe de transduction, conduisant à l’activation et au déplacement de Fu vers la région N-terminale de Ci. Ce remaniement entraine la phosphorylation et l’activation de Ci. Ma thèse révèle l’importance des changements de conformation au sein du complexe de transduction de la voie Hh. Le mécanisme de transduction étant conservé entre invertébrés et invertébrés, mon doctorat apporte des éléments de recherche pour mieux comprendre le fonctionnement normal et pathologique des cellules. / Hedgehog (Hh) is a secreted morphogen that controls growth and differentiation in both vertebrates and invertebrates. The dysregulation of its activity leads to severe developmental defects, and the onset of cancer in adults. In Drosophila, the Hh signal transduction is initiated by the binding of Hh to its receptor Patched (Ptc). This induces the stabilization of the transmembrane protein Smoothened (Smo) and the subsenquent activation of a transduction complex consisting of 5 proteins: the kinases Fused (Fu), PKA and Gprk2, the kinesin Costal2 (Cos2), and the transcription factor of the pathway Cubitus Interruptus (Ci). The aim of my thesis was to study the regulation and molecular interactions between the different components of the transduction complex. Thanks to complementary techniques, I have shown that in absence of Hh the proteins Fu and PKA interact in C-terminal part of Ci, whereas on the presence of Hh induces their relocalization toward the N-terminal domain of Ci. I have proved that the trigger element of this moving is Smo. In presence of Hh, Smo goes into transduction complex, allowing the activation and the moving of Fu toward N-terminal domain of Ci. This relocalization is responsible of Ci phosphorylation and activation. My thesis reveals the importance of conformational changes inside the transduction complex of Hh pathway. As the mechanism of transduction is conserved between species, my PhD provides research elements in order to better understand the normal and abnormal functioning of cells.

The solvent-solute interface of a biomolecule is a dynamic but yet highly structured domain that links a chemically diverse solute surface to the chemically homogeneous bulk aqueous phase. The role of the resulting intermediate domain, i.e. the "hydration shell", in regulating DNA structure and recognition has been addressed here by time-resolved infrared spectroscopy. A highly reproducible automated hydration pulse regime was established and implemented for attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy to monitor the structural response of DNA to an incremental growth of its hydration shell on its intrinsic time scale of seconds. The transition from the crystallographically defined BI to the BII substate of B-DNA was found to be driven by the increase of water disorder upon growth of the hydration shell, derived from the water OH-stretching absorption frequency and band width changes. 2D correlation analysis was used to identify different water clusters from the temporal behaviour of their water OH stretching frequencies. The results show that BII-stabilizing structural constraints are exerted by strong water-DNA H-bonds in the grooves of B-DNA and are relieved when the groove-bound water merges into a contiguous hydration shell with the less H-bonded PO2- -solvation sphere at ~14 water molecules per DNA phosphate. The H-bond imbalance at the disjunct hydration sites is split symmetrically around the average H-bond strength of bulk water. Thus, merging into a contiguous hydration shell proceeds at little enthalpic cost and homogeneous connectivity to the outer bulk-like H-bond network, such that alteration in the network distant from the DNA can regulate the BI-BII transition in a cooperative manner. The water connectivity is disrupted by DNA-binding peptides. Remarkably, the data show that the replacement of hydration shell water upon ligand biding is crucial in conferring substate specific recognition by peptides that have little intrinsic structural preference. The antibacterial peptide indolicidin secreted from bovine neutrophils dehydrates the non-PO2--bound hydration sites, thereby rendering the unstructured peptide highly specific for the BI state with vibrational signature almost identical to the bacterial minor groove binder netropsin. The proposed dominant role of hydration shell water for DNA conformation was challenged by studying the competing effect of structured water in the coordination-shell of the lanthanide Eu3+ on water structure in the DNA hydration shell. Whereas no effect is seen at low hydration, a hydrogen-like phase is formed at a stoichiometric ratio of Eu3+ :DNA:H2O of 1:10:140, characterized by a strong increase of the molar volume of hydration water. This novel phase appears attractive for lanthanide and possibly actine separation approaches based on biomolecular coordination.

The Distal-less homeobox (Dlx) genes encode a group of transcription factors that are involved in various developmental processes including forebrain development. Dlx genes are arranged in convergently transcribed bigene clusters with enhancer sequences located in the intergenic region of each cluster. The expression patterns of Dlx1/Dlx2 and of Dlx5/Dlx6 are attributed in part to the activity of I12a/I12b and I56i/I56ii intergenic enhancers, respectively. In an effort to determine how Dlx intergenic enhancers interact with the promoter regions of each cluster, I employed the Chromosome Conformation Capture (3C) technique on developing forebrain at E13.5 and E15.5. My 3C analysis provided potential enhancer-promoter interaction, in cis, that are consistent with previously known regulatory mechanisms. Furthermore, trans interactions may exist between Dlx1/Dlx2 and Dlx5/Dlx6 clusters in the developing forebrain at E13.5, thus providing a possible novel cross-regulatory mechanism between these two loci. I have also investigated the phenotypic consequences of Dlx enhancer deletion(s) on forebrain development by characterizing mice with I56ii and I56ii/I12b enhancer deletions. Enhancer deletions significantly impair Dlx expression as well as that of Evf2, Gad2 and of the striatal markers Islet1 and Meis2. Enhancer deletion(s) also reduce the expression of ISLET1 and CTIP2 proteins and Semaphorin 3A, Slit1 and Ephrin A5 that are thought to provide guidance cues in the corridor cells. Overall, these changes may disrupt the guidance of the thalamocortical axons. The data presented here further our understanding of the interactions between Dlx intergenic enhancers and promoter regions. Enhancer deletion(s) furthers our understanding of Dlx regulatory networks necessary that ensure proper Dlx expression, which, in turn may be involved in a genetic pathway underlying the synthesis of GABA, which may be further essential in maintaining the GABAergic phenotype.