This dissertation contains theoretical investigations on the morphology and statistical mechanics of vesicles. The shapes of homogeneous fluid vesicles and inhomogeneous vesicles with fluid and solid membrane domains are calculated. The influence of thermal fluctuations is investigated. The obtained results are valid on mesoscopic length scales and are based on a geometrical membrane model, where the vesicle membrane is described as either a static or a thermal fluctuating surface. The thesis consists of three parts. In the first part, homogeneous vesicles are considered. The focus in this part is on the thermally induced morphological transition between vesicles with prolate and oblate shape. With the help of Monte Carlo simulations, the free energy profile of these vesicles is determined. It can be shown that the shape transformation between prolate and oblate vesicles proceeds continuously and is not hampered by a free energy barrier. The second and third part deal with inhomogeneous vesicles which contain intramembrane domains. These investigations are motivated by experimental results on domain formation in single or multicomponent vesicles, where phase separation occurs and different membrane phases coexist. The resulting domains differ with regard to their membrane structure (solid, fluid). The membrane structure has a distinct effect on the form of the domain and the morphology of the vesicle. In the second part, vesicles with coexisting solid and fluid membrane domains are studied, while the third part addresses vesicles with coexisting fluid domains. The equilibrium morphology of vesicles with simple and complex domain forms, derived through minimisation of the membrane energy, is determined as a function of material parameters. The results are summarised in morphology diagrams. These diagrams show previously unknown morphological transitions between vesicles with different domain shapes. The impact of thermal fluctuations on the vesicle and the form of the domains is investigated by means of Monte Carlo simulations. / Die vorliegende Arbeit enthält theoretische Untersuchungen zur Morphologie und statistischen Mechanik von Vesikeln. Es wird die Gestalt homogener fluider Vesikel und inhomogener Vesikel mit fluiden und festen Membrandomänen berechnet. Der Einfluss thermischer Fluktuationen wird untersucht. Die erzielten Ergebnisse beziehen sich auf mesoskopische Längenskalen und basieren auf einem geometrischen Membranmodell, in welchem die Vesikelmembran als statische, beziehungsweise thermisch fluktuierende Fläche beschrieben wird. Die Arbeit besteht aus drei Teilen. Im ersten Teil werden homogene fluide Vesikel betrachtet. Das Interesse gilt dem thermisch induzierten Morphologieübergang zwischen prolaten und oblaten Vesikelformen. Mit Hilfe von Monte-Carlo-Simulationen wird ein freies Energieprofil für diese Vesikel ermittelt. Es kann gezeigt werden, dass die Formumwandlung zwischen prolaten und oblaten Formen kontinuierlich verläuft und mit keiner freien Energiebarriere verbunden ist. Der zweite und dritte Teil beschäftigt sich mit inhomogenen Vesikeln, die intramembrane Domänen enthalten. Ausgangspunkt und Motivation der Berechnungen sind experimentelle Studien über Domänbildung in ein- oder mehrkomponentigen Vesikelmembranen, bei denen Phasentrennung stattfindet und unterschiedliche Membranphasen koexistieren. Die dabei auftretenden Domänen unterscheiden sich hinsichtlich ihrer Membranstruktur (fest, fluid). Diese beeinflusst die Form der Domäne und des gesamten Vesikels auf entscheidende Weise. Im zweiten Teil werden Vesikel untersucht, bei denen feste und fluide Membrandomänen koexistieren, Teil drei widmet sich Vesikeln mit zwei koexistierenden fluiden Membranphasen. In Abhängigkeit von Materialparametern werden durch Minimierung der Membranenergie die Grundzustandsformen von Vesikeln mit einfachen und komplexen Domänenformen bestimmt. Die Ergebnisse werden in Morphologiediagrammen zusammengefasst. Dabei werden bisher unbekannte Morphologieübergänge zwischen Vesikeln mit unterschiedlichen Domänformen beobachtet. Die Auswirkungen thermischer Fluktuationen auf die Vesikel und die Form ihrer Domänen werden mittels Monte-Carlo-Simulationen untersucht.

A proof-of-concept for a novel microarray used to study protein-ligand interaction in real-time using label-free detection is presented. Many of todays commercially available instruments lack the ability to immobilize membrane proteins. At the same time, the pharmaceutical industry develops drugs directed towards membrane-bound receptors. The need to study drug-target kinetics and to be able to screen for new medical substances is high. To study the biomolecular interactions in real-time, imaging surface plasmon resonance (iSPR) is used. A patterned sensor surface with hydrophobic barriers assisting in the piezodispensing of NeutrAvidin with complex-bound biotin-ssDNA is created. Histidine-tagged proteins are immobilized at the vesicle surface using divalent nitrilotriacetic acid. The concept of the vesicle immobilization, the protein-binding to vesicles and the protein-ligand interaction is initially studied using a Biacore instrument. The dissociation of the ligand IFNα2 from its receptor ifnar-2 (wt) are in accordance with the literature. In the imaging SPR experiments, it is found that the dissociation of IFNα2 from the ifnar-2 (wt) receptor is slower than expected, probably due to rebinding of the ligand. It is also found that imidazole is needed to avoid vesicle-vesicle interaction. The immobilization of proteins had to be done on-line i.e. when the vesicles were bound to the surface. Depending on the mixture of receptors at the vesicle surface the affinity for the ligand was changed. The results achieved were reproducible.

No description available.

Stuttgart, Univ., Diss., 2008.

Pour effectuer des opérations de régulation et de signalisation, les systèmes naturels mettent à profit des processus ioniques et photoniques. Certain aspects de ces processus peuvent être reproduits dans des systèmes artificiels, en combinant senseurs et récepteurs moléculaires photoactifs dans des nanocapsules ou des membranes auto-assemblées. Une série de molécules photoactives amphiphiles et non-amphiphiles a été synthétisée, notamment pour la complexation et la libération d'ions calcium. Ces derniers processus ont été étudiés à l’aide de différentes spectroscopies incluant la fluorimétrie et l’infrarouge. Le couplage du processus photomodulé d'éjection d'ion et sa détection, dans des domaines nanoscopiques et à l'interface membrane / liquide, a été étudié pour connaître l'efficacité du transfert d'ion en solution et milieu organisé, notamment au sein de vésicules de tailles différents. / Natural systems combine ionic and photonic processes in order to control, for example,signalling and regulation. Certain aspects of these processes can be achieved in artificialsystems, on combining photoactive molecular sensors / switches, molecular receptors andself-assembled nanocapsules or membranes. A range of novel amphiphilic andnon-amphiphilic synthetic photoactive molecules and molecular systems are reported, notablyfor the complexation and liberation of calcium ions. These processes are studied using a rangeof spectroscopies including fluorimetry, microscopy and IR-techniques. Coupling processes ofphotocontrolled ion ejection and detection in nanoscopic compartments and at liquid /membrane interfaces has been studied in order to assess the relative efficiency ofintermolecular ion transfer in solution and organized media, notably in vesicle nanodomains.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18431号 / 生博第311号 / 新制||生||41(附属図書館) / 31289 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 豊島 文子, 教授 藤田 尚志, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

No description available.

Changes in the biomechanical properties of cells accompanying the development of various pathological conditions have been increasingly reported as biomarkers for various diseases and as a predictor of disease progression stages. For instance, cancer cells have been found to be less stiff compared to their healthy counterparts due to the proteomic and lipidomic dysregulations conferred by the underlying pathology. The separation and selective recovery of cells or extracellular vesicles secreted from such cells that have undergone these changes have been suggested to be of diagnostic and prognostic value. This dissertation first describes the implementation of a stiffness-based separation of phosphatidylcholine-based vesicles using a method first introduced based on the research in this work and was dubbed thermally-assisted acoustophoresis, or thermo-acoustophoresis. By tuning the temperature, we achieved the separation of vesicles of the same size, shape, and charge but with different stiffness values. It was observed that at a specific transition point, the acoustic contrast factor of vesicles changed sign from positive to negative. This change was mainly due to change in the compressibility of the vesicles, which is inversely proportional to stiffness. The acoustic contrast temperature (Tϕ), corresponding to the temperature at which the contrast factor switches sign, was determined to be unique to the composition of the vesicles. This unique temperature signature allowed us to develop this separation method of vesicles with distinct membrane stiffness with target outlet purities exceeding 95%. We have further explored the functionality of this method by experimenting with cholesterol-containing vesicles. In cells, the cholesterol content plays a crucial role in determining stiffness. Changes in the cholesterol content in cellular membranes can be an indication of pathological disorders. We evaluated the Tϕ of vesicles at different cholesterol molar ratios (Xchol) and developed a multi-stage lab-on-a-chip method to accomplish for the first time the separation of a three-vesicle mixture. Using Xchol = 0.1, 0.2, and 0.3 vesicles, we obtained efficiencies exceeding 93%. The simplicity, rapidity, and label-free nature of this approach holds promise as a diagnostic and separation tool for cells affected by diseases that affect the stiffness and extracellular vesicles such as exosomes and microvesicles.

No description available.