京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24270号 / 生博第484号 / 新制||生||64(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 原田 浩, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Yes / The microstructure and molecular orientation distribution over both the length and the thickness of microinjection‐molded linear low‐density polyethylene with a weld line were characterized as a function of processing parameters using small‐angle X‐ray scattering and wide‐angle X‐ray diffraction techniques. The weld line was introduced via recombination of two separated melt streams with an angle of 180° to each other in injection molding. The lamellar structure was found to be related to the mold temperature strongly but the injection velocity and the melt temperature slightly. Furthermore, the distributions of molecular orientation at different molding conditions and different positions in the cross section of molded samples were derived from Hermans equation. The degree of orientation of polymeric chains and the thickness of oriented layers decrease considerably with an increase of both mold temperature and melt temperature, which could be explained by the stress relaxation of sheared chains and the reduced melt viscosity, respectively. The level of molecular orientation was found to be lowest in the weld line when varying injection velocity, mold temperature, and melt temperature, thus providing an effective means to identify the position of weld line induced by flow obstacles during injection‐molding process. / Jilin Scientific and Technological Development Program. Grant Number: 20180519001JH; National Key R&D Program of China. Grant Number: 2018YFB0704200; National Natural Science Foundation of China. Grant Numbers: 21674119, 21790342; Newton Advanced Fellowship of Royal Society. Grant Number: NA150222

Yes / In polymer melt processing, the heat transfer coefficient (HTC) determines the heat flux across the interface of the polymer melt and the mould wall. The HTC is a dominant parameter in cooling simulations especially for microinjection moulding, where the high surface to volume ratio of the part results in very rapid cooling. Moreover, the cooling rate can have a significant influence on internal structure, morphology and resulting physical properties. HTC values are therefore important and yet are not well quantified. To measure HTC in micromoulding, we have developed an experimental setup consisting of a special mould, and an ultra-high speed thermal camera in combination with a range of windows. The windows were laser machined on their inside surfaces to produce a range of surface topographies. Cooling curves were obtained for two materials at different processing conditions, the processing variables explored being melt and mould temperature, injection speed, packing pressure and surface topography. The finite element package Moldflow was used to simulate the experiments and to find the HTC values that best fitted the cooling curves, so that HTC is known as a function of the process variables explored. These results are presented and statistically analysed. An increase in HTC from the standard value of 2500 W/m2C to values in the region 7700 W/m2C was required to accurately model the observations. / EPSRC

Yes / Polyetheretherketone (or PEEK) is a thermoplastic polymer known for its high plasticity and toughness and has been widely employed as a material for a variety of load-bearing medical devices ranging from trauma implants to interspinal spacers and femoral stems. While being inherently chemically inert and therefore biocompatible and having very short lived post-radiation free radicals, PEEK presents different mechanical properties depending on its degree of crystallinity. It can be processed via extrusion, injection or compression moulding. However, these techniques do not allow high precision control over the fine morphological structure that strongly influences mechanical properties. Microinjection moulding, in contrast, makes it possible to produce fine details of medical implants with high precision and accuracy. Another advantage of this method is the controlled production of the material with heterogeneous structure due to variations in crystallinity. Having stiffness in the middle of the sample different from that at the edges enables a structure that mimics the bone/cartilage parts of an implant. This paper reports on the manufacturing of PEEK components by microinjection moulding, and their characterisation by physico-chemical (XRD, SAXS, TEM, FTIR, POM) and mechanical (tensile testing) means, in order to assess the suitability of use for biomedical application, such as spinal implants. We discuss the influence of such parameters as mould temperatures, injection speeds and hold pressures on the crystallinity and mechanical properties of the material. / Science Bridges: Bradford-China Programme for Pharmaceutical Sciences and Medical Technology, EP/G042365/1

Yes / Polypropylene (PP) reinforced with 2 and 4 wt% of multi-walled carbon nanotubes (MWNT) were melt-blended in twin screw extruder and then molded by compression or micromolding process. The impact of injection speed on the surface morphology, rheological and tensile characteristics was investigated by using a scanning electron microscope, parallel plate rheometry, and tensiometry. Results showed that the tensile properties of micro-molded specimens were remarkably higher than those of the compression molded sheets. Compared to compression molded sheets, micromolded specimens demonstrated up to 40 and 244% higher tensile stiffness and yield strength, respectively, most likely due to the alignment of polymer chain segments in the flow direction induced during the micromolding process. It was observed that the fast filling speed caused a drop in the tensile properties of the nanocomposites and polymer. Rheological examination revealed that the presence of a rheological percolation network in the nanocomposites produced by micromolding and the fast injection speed was beneficial for establishing the percolated network. Morphological examination revealed that the size of nanotube agglomerations that appeared in micromolded specimens was up to five times smaller than in compression molded sheets and the agglomeration size decreased with the increase of the injection speed.

Le « Brain-Derived Neurotrophic Factor » (BDNF) est la neurotrophine la plus abondante et la plus répandue dans le cerveau humain. De nombreuses études se sont intéressées à son rôle dans la survie neuronale, la croissance et la plasticité synaptique. La signalisation BDNF est dépendante de deux récepteurs, le récepteur tyrosine kinase (TrkB) et le récepteur neurotrophine p75 (p75NTR). Il est bien établi que le rôle trophique du BDNF est assuré via son récepteur de haute-affinité TrkB, alors que la forme précurseur proBDNF active p75NTR vers la voie d'apoptose. Cette double signalisation est physiologiquement contrôlée par un équilibre entre les différentes voies. Les résultats obtenus à partir des études cliniques et des modèles animaux suggèrent un rôle de la signalisation BDNF dans les tauopathies, caractérisées par l'existence de dépôts intracérébraux de protéine tau, une caractéristique commune à certaines maladies neurodégénératives, notamment la maladie d'Alzheimer (MA). Cependant, aucune investigation n'a été menée jusqu'à présent sur les modifications que pouvaient induire les tauopathies dans la signalisation BDNF et si une dérégulation de l'expression du BDNF pouvait affecter ses propres récepteurs TrkB et p75NTR.Dans ce travail de thèse, nous avons utilisé une lignée de poisson-zèbre transgénique portant la mutation humaine TAUP301L retrouvée notamment dans le démence fronto-temporale. Nous avons mesuré l'expression de BDNF et de ses deux récepteurs au niveau transcriptionnel et protéique. Nous n'avons observé aucune modification des taux d'expression de BDNF et de TrkB, en revanche, nous avons noté une augmentation significative de p75NTR. A l'aide de la même lignée transgénique, nous avons induit une baisse d'expression de BDNF via la micro-injection de morpholinos. De manière remarquable, la baisse d'expression de BDNF affecte de façon différentielle TrkB et p75NTR. En effet, nous avons observé une diminution de l'expression de TrkB et parallèlement une augmentation de p75NTR. De plus, la baisse d'expression de BDNF aggrave la neurotoxicité associée au développement de la tauopathie ce qui se traduit par une augmentation de la mort neuronale et de l'hyperphosphorylation de tau, cette dernière étant concommittante à une activation de la Glycogen Synthétase Kinase 3 beta (GSK3beta).Une diminution de l'effet neuroprotecteur de BDNF à travers un déséquilibre de ces récepteurs de signalisation a été également montré en étudiant le rôle de BDNF au cours du développement de la ligne latérale postérieure (PLL). Ce système est considéré comme un modèle d'étude particulièrement pertinent pour évaluer différents processus biologiques comme la migration cellulaire collective ou la régénération cellulaire. Nous avons détecté l'expression de BDNF dans plusieurs structures de la PLL. La diminution d'expression de BDNF conduit à un défaut de migration du primordium de la PLL, associé à une augmentation de la mort cellulaire. De plus, nous avons observé une réduction de la prolifération cellulaire et un défaut de repousse axonale du nerf, ce qui conduit à des anomalies de régénération à la fois du nerf de la PLL et des cellules ciliées. Nos résultats suggèrent que le BDNF joue un rôle essentiel au cours du développement de la PLL et démontrent la pertinence du système de la ligne latérale en tant que modèle d'étude des fonctions de BDNF.En conclusion, notre étude représente la première analyse du rôle in vivo de BDNF et de ses 2 récepteurs de signalisation. Nous avons ainsi montré les répercussions d'une dérégulation des voies de signalisation du BDNF. Un équilibre entre ces deux voies est essentiel pour le développement et la survie cellulaire, ce qui fait de BDNF non seulement une cible thérapeutique potentielle, mais également une neurotrophine clé pouvant activer plusieurs circuits de signalisation, potentialisant ainsi son rôle protecteur. / Brain-derived neurotrophic factor (BDNF) is the most abundant secreted and widely distributed neurotrophin in human brain. It has been extensively studied for its role in neuronal survival, growth and synaptic plasticity. BDNF signaling mediated through tryosine receptor kinase B (TrkB) and p75NTR neurotrophin receptor (p75NTR). It is well established that BDNF beneficial actions are mediated by it is high-affinity TrkB, whereas pro-BDNF activates p75NTR towards apoptosis. This diverse dual signaling is normally under a tight balance regulation. Based on clinical and animal studies, it has been suggested that BDNF signaling is involved in tauopathy, which is a pathological hallmark in several neurodegenerative diseases, including Alzheimer's disease (AD). However, what changes tauopathy may induce on BDNF signaling, and whether BDNF deregulation could affect its two signaling receptors (TrkB, p75NTR), and eventually tauopathy pathogenesis, have not been investigated. In this study we used a transgenic zebrafish line for human Tau-P301L tauopathy, and measured transcriptional and protein levels of BDNF and of its two signaling receptors. We found no modification of BDNF and TrkB expression levels, but a significant up-regulation of p75NTR. We then used the same transgenic line to generate BDNF knockdown using morpholino microinjection technique. Interestingly, BDNF knockdown differentially affects TrkB and p75NTR; we observed a reduction of TrkB expression and an increase in p75NTR expression. In addition, BDNF knockdown aggravates tauopathy-associated toxicity; we found an increase in neuronal cell death and tau hyperphosphorylation, the latter was accompanied by an activation of tau glycogen synthase kinase 3beta (GSK3beta). Attenuation of BDNF neuroprotective effects through imbalance of its signaling receptors was further highlighted through studying BDNF role in the development of zebrafish posterior lateral line system (PLL). This system has recently emerged as a powerful tool to study several dynamic biological processes, including collective cell migration and nerve/hair cells regeneration. We detected BDNF expression in different PLL components. BDNF knockdown led to an impairment of the PLL primordium migration due to concomitant increase in cell death rate. In addition, reduced cell proliferation and defect in axonal re-growth were observed , which led to major defects of PLL nerve/hair cells regeneration, respectively. These findings suggest that BDNF has an essential role in PLL development, but more important they introduce PLL as research model to study BDNF functions. This is the first study to provide a detailed in vivo analysis of BDNF and its two signaling receptors. Our findings highlight several implications of BDNF signaling deregulation. Balanced signaling clearly has essential roles in survival and development, in addition to being a therapeutic target, BDNF can itself activate diverse molecular pathways, thus setting up a potential circuitry that could enhance its protective role.

La miniaturisation des pièces est une étape importante pour la progression de la microtechnologie dans plusieurs domaines (connectique, médical, optique, microsystèmes mécaniques). Pour cela, le moulage par microinjection, semble être la solution clé pour la production à grande échelle de micro-composants de polymères. Pour les polymères semi-cristallins, la cristallisation, sous fort taux de cisaillement et sous des vitesses de refroidissement élevées (about 100 K/s), induit des morphologies et des propriétés spécifiques. Elle prend donc une importance considérable dans le processus de microinjection par rapport au moulage par injection classique où les épaisseurs injectées sont généralement supérieures à 1 mm. Ces microstructures ont une grande influence sur les propriétés mécaniques du produit final. La prédiction de ces propriétés à partir de la description de la microstructure est un défi technique et scientifique. Durant cette thèse, deux polymères semi-cristallins ont été microinjectés, le polyéthylène haute densité et le polyamide 12. Les analyses obtenues par la microscopie otiques montrent que les morphologies cristallines varient entre les micro- et les macro-pièces. Tandis que la morphologie de ‘peau-cœur' est présente dans les macropièces, les micropièces présentent une morphologie plutôt particulière. Les analyses combinées de diffusion et de diffraction des rayons X (SAXS et WAXS) avec un microfaisceau synchrotron, nous ont permis de déterminer la microstructure induite par le processus de microinjection dans toute l'épaisseur des pièces. Nous avons constaté que la morphologie et les orientations cristallines induites sont très dépendantes des conditions d'injection ou de microinjection. Une diminution de l'épaisseur, de la vitesse et de la température du moule, augmente l'orientation cristalline en limitant la relaxation des chaînes de polymères. / The components miniaturization is an important step in the evolution of micro technology in several domain (connectivity, medical, optical, mechanical, microsystems). For this purpose, the micro-injection molding seems to be the key solution for the large-scale micro-polymer components production.The crystallization of the semi-crystalline polymers under high shear and cooling rates (about 100 K / s), induces specific properties and morphologies, consequently, it takes a substantial importance in the process of micro-injection compared to conventional injection molding where the usually injected thicknesses is over 1 mm. These micro-structures have a great influence on the mechanical propertie of the final product. The prediction of the final product's properties based on the illustration of the micro-structure is a technical and scientific challenge. In this thesis, two semi-crystalline polymers were micro-injected, the high density polyethylene and the polyamide 12. The obtained analyzes with the use of an optical microscope showed that the Morphology of Crystals vary between micro-and macro-pieces. While the morphology of 'peau-cœur' is present in the macro-pieces, the micro-parts have a particular morphology. The combined analysis of diffusion and X-ray diffraction (WAXS and SAXS) along with the synchrotron microbeam, has allowed us to determined the micro-structure induced by the micro-injection process throughout the thickness of the pieces.We have identified that the morphology and the induced crystal's orientation are very dependent on the conditions of injection or micro-injection. The decrease of the thickness,speed and temperature of the mold will increase the crystal orientation by limiting the relaxation of the polymer chains.

Resistance to chemotherapeutic drugs is a major obstacle in the treatment of leukemia and multiple myeloma. We have previously found that myeloma and leukemic cells in transition from low-density log phase conditions to high-density plateau phase conditions exhibit a substantial export of endogenous topoisomerase II alpha from the nucleus to the cytoplasm. In order for topoisomerase-targeted chemotherapy to function, the topoisomerase target must have access to the nuclear DNA. Therefore, the nuclear export of topoisomerase II alpha may contribute to drug resistance, and defining this mechanism may lead to methods to preclude this avenue of resistance. In the current report, we have defined nuclear export signals for topoisomerase II alpha at amino acids 1017-1028 and 1054-1066, using FITC labeled BSA-export signal peptide conjugates microinjected into the nuclei of HeLa cells. Functional confirmation of both signals (1017-1028 and 1054-1066) was provided by transfection of human myeloma cells with plasmids containing the gene for a full-length human FLAG-topoisomerase fusion protein, mutated at hydrophobic amino acid residues in the export signals. Of the six putative export signals tested, the two sites above were found to induce export into the cytoplasm. Export by both signals was blocked by treatment of the cells with leptomycin B, indicating that a CRM-1 dependent pathway mediates export. Site-directed mutagenesis of two central hydrophobic residues in either export signal in full-length human topoisomerase blocked export of recombinant FLAG-topoisomerase II alpha, indicating that both signals may be required for export. Interestingly, this pair of nuclear export signals (1017-1028 and 1054-1066) also defines a dimerization domain of the topoisomerase II alpha molecule.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that establishes life-long latent infection in sensory neurones. This makes it potentially useful as a gene therapy vector to target neuronal cells. HSV-1 enters cells by membrane fusion, the viral envelope and most tegument proteins dissociate, and the capsid is transported to the cell nucleus to establish infection. There is increasing evidence that the retrograde transport of HSV-1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. It is a large complex molecule, with heavy chains providing motility, while intermediate and light chains are involved in specific cargo binding. A library of HSV-1 capsid and tegument structural genes was constructed and tested for interaction with dynein subunits in a yeast two-hybrid system. A strong interaction was demonstrated between the HSV-1 outer capsid protein VP26 (UL35), as well as the tegument protein VP11/12 (UL46), with the homologous 14 kDa dynein light chains rp3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to rp3, Tctex1 and cytoplasmic dynein complexes. Recombinant HSV-1 capsids +/- VP26 were used in similar pull-down assays. Only VP26+ capsids bound to rp3. Recombinant HSV-1 capsids were microinjected into living cells and incubated at 37�C. After 1 h capsids were observed to co-localise with rp3, Tctex1 and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, while VP26- capsids remained in a random distribution. Our results suggest that the HSV-1 outer capsid protein VP26 mediates binding of incoming capsids to the retrograde motor cytoplasmic dynein during cellular infection, through interactions with dynein light chains. It is hoped that these findings will help in the development of a synthetic viral vector, which may allow targeted gene therapy in patients with neurological diseases.

L'addition de nanotubes de carbone multiparois (MWNT) dans une matrice de polyamide 12 (PA 12), électriquement isolante, permet d'augmenter les propriétés électriques vers un comportement conducteur. Cette modification est influencée par l'arrangement des MWNT en chemins de conduction qui permettent le transfert des charges électriques entre deux électrodes. La conductivité électrique des nanocomposites isotropes atteint une valeur asymptote (~10-2 S.m-1) pour des teneurs supérieures à 1,2% en masse (seuil de percolation électrique). En microinjection, les nanocomposites sont soumis à des taux de cisaillement très élevés (~104 s-1) et des gradients de températures extrêmes, qui conditionnent fortement la microstructure et les propriétés électriques de pièces mises en forme par ce procédé. Cette thèse a eu pour but d'expliquer l'influence de la vitesse de cisaillement (0,02 s-1 - 1 s-1) et la vitesse de refroidissement (3 °C.min-1) sur l'évolution des propriétés électriques du nanocomposite PA12/MWNT. L'analyse de ces propriétés a permis de déduire, à l'état fondu, l'évolution de l'arrangement de MWNT dans cette fenêtre de conditions. Dans les pièces microinjectées, nous constatons une perte complète du comportement conducteur dans la direction normale au plan d'écoulement et une chute de la conductivité dans les directions d'injection et transverse. Ces faits suggèrent alors un arrangement en forme d'agrégats faiblement orientés dans le plan d'écoulement, qui est corroboré par la très large distribution d'orientation déterminée par l'analyse en spectroscopie Raman des pièces micro-injectées. Lors du procédé de microinjection, les agrégats de MWNT seraient alors cassés dans des agrégats plus petits, mais fortement déconnectés les uns des autres, expliquant ainsi la chutedes propriétés électriques mais aussi l'observation d'une microstructure quasi isotrope à l'échelle macro et micro.