京都大学 / 新制・課程博士 / 博士(理学) / 甲第23735号 / 理博第4825号 / 新制||理||1690(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)准教授 齊藤 尚平, 教授 依光 英樹, 教授 若宮 淳志 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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in English While axon fasciculation plays a key role in the development of neural networks, very lit- tle is known about its dynamics and the underlying biophysical mechanisms. In a model system composed of neurons grown ex vivo from explants of embryonic mouse olfactory epithelia, we observed that axons dynamically interact with each other through their shafts, leading to zippering and unzippering behaviour that regulates their fasciculation. Taking advantage of this new preparation suitable for studying such interactions, we carried out a detailed biophysical analysis of zippering, occurring either spontaneously or induced by micromanipulations and pharmacological treatments. We show that zippering arises from the competition of axon-axon adhesion and me- chanical tension in the axons. This is upheld on quantitative level by conforming change of network global structure in response to various pharmacological treatments, without active involvement of growth cones. The calibrated manipulations of interacting shafts provide qualitative support for the hypothesis, and also allow us to quantify the mechan- ical tension of axons in our system. Furthermore, we introduce a biophysical model of the zippering dynamics, which efficiently serves the purpose of estimating the magnitude of remaining involved...

La nécessité de délivrer des médicaments directement dans les cellules est grandissante avec le développement des thérapies géniques. Les peptides pénétrants (Cell Penetrating Peptides : CPP) représentent une possibilité pour administrer ces médicaments dans les cellules sans effet délétère sur la membrane. Ce sont des peptides d’une dizaine d’acides aminés, généralement cationiques. Ils sont capables de traverser la membrane cellulaire, et conservent cette propriété lorsqu’une cargaison leur est attachée. Cependant, leurs mécanismes d’entrée ne sont toujours pas tous connus. Nous avons caractérisé quelques aspects des mécanismes permettant aux CPP de traverser directement la membrane plasmique à l’aide de trois techniques biophysiques. i) Nous avons ainsi pu mettre en évidence le rôle des sulfates d’héparane comme partenaire d’adhésion forte du CPP pénétratine, à l’aide d’un outil de mesure de force : le Biomembrane Force Probe. ii) Nous avons montré la possibilité pour la pénétratine de franchir la bicouche lipidique (sans mécanisme cellulaire actif) si celle-ci est suffisamment riche en lipides chargés négativement. Ce passage a été étudié sur bicouches modèles, formées à l’interface entre gouttelettes obtenues par émulsion inverse dans de l’huile en présence de lipides. iii) Pour visualiser la translocation de CPP à l’échelle de la molécule unique nous avons développé un montage original de microscopie à onde évanescente sur bicouche suspendue. / Gene therapy relies on an efficient and specific delivery of drugs into targeted cells. For this purpose, the use of carriers that will help the drugs to cross the membrane, without introducing deleterious effect due to the membrane disruption, are promising. A family of such carriers is known as Cell Penetrating Peptides (CPPs). These peptides are short, about ten amino acids, and often cationic. They are able to translocate through the membrane with different cargos and deliver them into the cytosol. However the mechanisms are still, to a great extent, unknown. We used three biophysical techniques to gain insights into the mechanisms leading to the translocation of a CPP. i) We found the heparan sulfates to be the strongest partner of the CPP penetratin at the cell surface. This adhesion has been pointed out using the Biomembrane Force Probe, a force measuring tool. ii) We evidenced the translocation of penetratin through the lipid bilayer (without any cell mechanism) as long as it contains enough negatively charged lipids. This has been carried out using model bilayers formed at the interface between droplets generated by an inverted emulsion: water in an oil and lipid mixture. iii) To view the translocation of CPPs at the single molecule level we developed a total internal reflection fluorescence microscope (TIRFM) on a suspended bilayer.

Mechanochromic polymers are force-sensitive materials that change their color as a response to mechanical stimuli. This visualization of forces can be used to further optimize polymer-based materials by understanding microscopic force transduction or to display fatigue of material. Most mechanochromic systems rely on bond cleavage, so they can only distinguish between “on” and “off” state without any further correlation of the applied force with the optical signal. Although reversibility to the initial state is possible for most of these systems, it often demands time or input of energy making them rather unsuitable for sensing forces in real-time. In this work, the development and application of mechanochromic donor (D)-acceptor (A) torsional springs is presented as a new concept for mechanochromic materials. The mechanically induced planarization of D and A leads to a continuous red-shift of both absorption and emission color. A suitable DA-system is found in ortho-substituted diphenyldiketopyrrolopyrrole (o-DPP) having the large torsional angle required for the equilibrium geometry and therefore showing blue-shifted optical spectra compared to reference compounds with a smaller torsional angle. The covalent incorporation into tough poly(meta,meta,para-phenylene) (PmmpP) by Suzuki polycondensation enables sufficient force transduction to the DA spring during uniaxial elongation of thin-film specimens. The detected mechanochromic response correlates with the applied stress and shows full reversibility upon stress release. Theoretical experiments based on density functional theory (DFT) confirm the experimental results and offer a detailed explanation of the molecular deformations responsible for the optical shift. In addition, the application as stress sensor was tested investigating the molecular force transduction in glassy PmmpP as a function of the number average molecular weight (Mn) by blending o-DPP-PmmpP probe chains of varying Mn with pristine PmmpP. The distinct mechanochromic response for entangled and non-entangled probe chains, respectively, allows the extraction of the critical molar mass (Mc) that is required for entanglements to become effective. The resulting value for the entanglement molar mass Me ≈ 1/2 Mc is in excellent agreement with the value determined by rheology.

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