During starch synthesis, knowledge of how the supra molecular organization of starch granules occur is still unknown. This dissertation begins exploring the changes to wheat starch morphology, structure and organization during granule development. Starches from wheat seeds harvested at different days after anthesis were isolated and studied for their molecular organization at different scales. The interaction of granular starch to iodine vapor was used to reveal information about the physical state of the polymers in the granules. Both large and small granule populations initiated as spherical shaped granules, but large granules changed into lenticular shape, while small granules remained spherical. Blocklet, lamellae, and crystalline structures were well developed already at seven days of maturity, suggesting their presence at the center of mature granules. Furthermore, (i) the size of blocklets decreased from center to the periphery in both granule populations, but at all developmental stages, they were larger in small granules than in large granules; (ii) Lamellae repeat distance was shorter close to the center of granules; (iii) B-type crystallites were mixed with A-type crystallites at the center of granules from both populations, but the proportion of B-type crystallites was higher in large granules, which correlated with a higher proportion of amylopectin long chains in these granules; (iv) The relative crystallinity (RC) of starches from both populations was higher at the center of the granule than that at the periphery. RC was lower in large granules than in small granules suggesting influence of the higher amylose content in large granules, which introduces more defects reducing RC. AFM visualized surfaces with features different from the regular type of surface among immature starch granules, which was interpreted as the amorphous growth ring of the starch granule. Exposure of starches to iodine vapor demonstrated different levels of molecular organization between them and revealed possible interaction of inter-cluster chain segments and spacer segments of amylopectin with iodine. In-situ AFM imaging of the granule surface exposed to iodine vapor under humid conditions revealed that glucan polymers or polymer segments in between and on top of blocklets have the ability to interact with iodine.

Synapse is the fundamental unit of synaptic transmission. Learning, memory and neurodegenerative diseases of the brain are attributed to the maintenance and alteration in synaptic connections. The efficiency for synaptic transmission depends on how well the post synapse receives the signals from the presynapse; this in turn depends on the receptors present in the post synaptic density (PSD). PSD is present in the post synapse right opposite to the neurotransmitter release site in presynapse (active zone) is an indispensable part of the synapse. The PSD is comprised of receptors and scaffold proteins, which is ultimately supported by the actin cytoskeleton of the dendritic spines. Cytoskeletal dynamics is shown to influence the structural plasticity of spine and also PSD, but how it regulates the dynamicity of the synaptic transmission is not completely understood. Here we studied the influence of actin depolymerisation on sub synaptic organization of an excitatory synapse. In order to study the organization of the synapse at molecular resolution, the conventional microscopy cannot be employed due to the limit of diffraction. Super resolution microscopy circumvents this diffraction limitation. In this study we have used custom built fluorescence microscope with Total Internal Reflection Fluorescence (TIRF) modality to observe the nanometre sized structures inside spines of mouse hippocampal primary neurons. The setup was integrated with Metamorph imaging software for both operating the microscope and imaging acquisition purpose with a separate appropriate laser system. This setup was successful in achieving the lateral resolution of ~30nm and axial resolution of ~51nm. Over all we were able to observe the loss of spines and significant reduction in area of nanometer sized protein clusters in postsynaptic density with in the spines of latrunculin A treated mouse hippocampal primary neurons compared to the native neurons. Along with the morphological alterations in neurons we also observed the changes in nanoscale organization of few key molecules in the postsynaptic density.

La microscopie non-linéaire résolue en polarisation est un outil puissant pour accéder à des informations structurelles dans les assemblages biomoléculaires. Les interactions non-linéaires entre matière et lumière induisent des processus complexes où des champs électromagnétiques cohérents interagissent avec les dipôles de transitions moléculaires. Le contrôle de la polarisation des champs électromagnétiques excitateurs et l’étude des réponses non-linéaires induites procurent de riches informations sur la distribution angulaire des molécules présentes dans le volume focal de l’objectif du microscope. Dans cette thèse, nous appliquons cette sensibilité à la polarisation à plusieurs modalités de microscopie cohérentes sans marquage (diffusion cohérente Raman anti-Stokes (CARS), diffusion Cohérente stimulée (SRS)) et à la fluorescence à deux photons (2PEF) afin d’obtenir des informations quantitatives sur la forme de la distribution moléculaire et l’orientation des lipides dans les membranes artificielles, ainsi que dans les membranes biologiques telles que la myéline des tissus de la moelle épinière. Avec cette technique, nous adressons une question fondamentale sur le comportement des ensembles lipidiques dans les membranes et sur l’effet d’autres molécules telles que le cholestérol et les marqueurs fluorescents. Nous démontrons que le CARS résolu en polarisation permet d’accéder à de fines informations sur l’organisation des lipides dans les membranes de la myéline, en deçà de la limite de diffraction. / Polarization resolved nonlinear microscopy is a powerful tool to image structural information in biomolecular assemblies. Nonlinear interaction between light and matter lead to complex processes where coherent combinations of optical fields couple to assemblies of molecular transition dipoles. Controlling polarized optical fields and monitoring nonlinear induced signals in a medium can nevertheless bring rich information on molecular orientational organization within the focal spot of a microscope objective. In this PhD thesis we apply this polarization sensitivity to different label-free optical coherent techniques (coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS)) and to two-photon fluorescence (2PEF) to retrieve quantitative information on the static molecular distribution shape and orientation of lipids in model membranes and biological membranes such as myelin sheaths in spinal cord tissues. With this technique, we address fundamental questions about lipid packing behavior in membranes, and how it can be affected by other molecules such as cholesterol and the insertion of fluorescent lipid probes. We demonstrate that polarization resolved CARS give access to fine details on lipids arrangement in myelin sheaths, at a sub-diffraction scale. In the context of experimental autoimmune encephalomyelitis disease (EAE) we show, that even at the stage of disruption of the myelin envelope during the demyelination process, lipids multilayers reveal strong capability to preserve their macroscopic self-assembly into highly organized structures, with a degree of disorganization occurring only at the molecular scale.

This diploma thesis deals with the conformation and molecular organization of humic acids in aqueous solutions. Humic substances have heterogeneous and polydisperse nature, therefore their secondary chemical structure has not yet been defined properly, neither has their conformational arrangement. The conformation of humic substances in the solutions is mainly stabilized by weak disperse forces, such as Van der Waals, -, CH- interactions and hydrogen bonds. Humic substances in the solutions tend to form aggregates that are held together mostly by the intermolecular hydrophobic interactions. Concentration series of humic acids were prepared in three different mediums of constant ionic strength: NaOH and NaCl (prepared either by neutralization NaOH by HCl or direct dilution by solution of NaCl). Several analytical methods have been used to determine conformation and molecular organization of humic acids: potentiometric determination of pH, direct conductometry, ultraviolet and visible spectroscopy, density measurement, dynamic light scattering, laser Doppler velocimetry and high resolution ultrasound spectrometry. It was confirmed that the conformation and molecular organization of humic acids in aqueous solutions depend not only on pH of medium, but they also depend on chemical nature and concentration with the same pH. Results showed that hydrodynamic diameter of particles is significantly increasing in NaCl medium prepared by neutralization NaOH by HCl, particularly at low concentrations, which corresponds to higher values of zeta potential obtained. Concentration dependencies of ultrasonic velocity and compressibility also indicate the changes in conformation and molecular organization corresponding with results from other methods used.

La gélification est un phénomène courant dans lequel une grande quantité de solvant est immobilisée dans un réseau constitué de relativement petites quantités de substrat. Avec des propriétés à la fois solides et liquides, un gel est un état unique. L'étude des propriétés et du mécanisme de la gélification attire l'attention des chercheurs du monde entier. Cependant, de nombreuses questions restent en suspens, telles que le processus d'auto-assemblage et les interactions moléculaires dans le système de gel, la relation entre les solutions, les gels et les cristaux et l'organisation moléculaire dans le réseau de gel. L'exploration de ces questions fournira des connaissances sur le mécanisme de gélification et contribuera à la conception et à la fabrication de nouveaux gels aux applications diverses. Cette thèse décrit notre étude des gels et de leur relation avec les solutions et les cristaux à l'aide de sels biliaires, qui sont des molécules amphiphiles naturelles abondantes. La rigidité de la partie stéroïde et l'hydrophobie variable des sels biliaires facilitent l'étude du processus d'autoassemblage. La recherche est présentée à travers trois articles publiés ou soumis au cours de mon programme de doctorat. Le premier article explore les interactions moléculaires qui se produisent dans la formation d'hydrogels moléculaires fabriqués à partir de mélanges de désoxycholate de sodium et d'acide formique. La spectroscopie de résonance magnétique nucléaire fournit de nouvelles informations sur la transition gel-sol au niveau moléculaire, l'interaction entre les espèces libres/gélifiées et l'interaction des régions hydrophobes des sels biliaires avec le réseau de gel. Le deuxième article résume notre exploration de la relation entre les gels et les cristaux, en particulier la façon dont les composants moléculaires sont organisés. Les sels d'ammonium d'acide lithocholique produisent différents modèles d'auto-assemblage, tels que des gels, des fibres et des cristaux, avec divers anions d'ammonium. L'organisation moléculaire de l’acide lithocholique dans différentes conditions est remarquablement cohérente, indiquant qu'il existe une relation intime entre la gélification et la cristallisation dans ce système. Les résultats ont également mis en lumière la question de longue date de l'agencement des molécules dans les fibres de gel. Le troisième article décrit notre étude systématique de la gélification et de la cristallisation en utilisant une gamme plus large de sels biliaires. Généralement, avec l'augmentation de iii l'hydrophobie des sels biliaires, la préférence pour la formation de solutions est progressivement remplacée par une tendance à produire des gels et finalement des cristaux. Une association bord à bord d'anions biliaires est également observée dans différents types de sels biliaires. Les résultats renforcent notre conclusion selon laquelle les structures moléculaires internes des fibres dans les gels et dans les cristaux sont étroitement liées. / Gelation is a common phenomenon in which a large amount of solvent is immobilized in a network made up of relatively small amounts of substrate. With properties of both solid and liquid, a gel is a unique state. Gelation draws attention from researchers worldwide to study its properties and mechanism. However, many questions are still unraveled, such as the self-assembly process and molecular interactions in the gel system, the relationship between solutions, gels, and crystals, and the molecular organization in the gel network. Exploring these questions will provide knowledge about the mechanism of gelation and contribute to the design and fabrication of new gels for different applications. This thesis describes our study of gels and their relationship with solutions and crystals using bile salts, which are abundant natural amphiphiles. The rigid steroid moiety and the variable hydrophobicity of the bile salts facilitate the study of the self-assembly process. The research is presented through three articles published or submitted during my Ph.D. program. The first paper probes the molecular interactions that occur in the formation of molecular hydrogels made from mixtures of sodium deoxycholate and formic acid. Nuclear magnetic resonance spectroscopy provides new information about the gel-sol transition on the molecular level, the interaction between free/gelated species, and the interaction of hydrophobic regions of bile salts with the gel network. The second paper summarizes our exploration of the relationship between gels and crystals, especially how the molecular components are organized. Ammonium salts of lithocholic acid produce different patterns of self-assembly, such as gels, fibers, and crystals, with various ammonium anions. The molecular organization of lithocholates under different conditions is remarkably consistent, indicating that there is an intimate relationship between gelation and crystallization in this system. The results also shed light on the long-existing question of how molecules are arranged in gel fibers. The third paper describes our systematic study of gelation and crystallization using a broader range of bile salts. Generally, with increasing hydrophobicity of the bile salts, the preference to form solutions is gradually superseded by a trend to produce gels and finally crystals. An edge-to-edge association of bile anions is also observed in different kinds of bile salts. The results strengthen our conclusion that the internal molecular structures of fibers in gels and in crystals are closely related.