京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19743号 / 工博第4198号 / 新制||工||1647(附属図書館) / 32779 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 田畑 泰彦, 教授 秋吉 一成, 教授 木村 俊作 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Este trabalho foi realizado com o objetivo de avaliar a influência do volume e da granulometria do substrato comercial, utilizado na produção de mudas em tubetes, sobre o crescimento vegetativo das plantas de café, bem como o tempo de formação das mudas e a estabilidade ao manuseio do conjunto muda-substrato. O experimento foi conduzido no viveiro do Centro de Café do IAC, localizado na Fazenda Santa Elisa, Campinas, SP, utilizando a cultivar Catuaí Vermelho IAC 144 (Coffea arabica L.). Foram adotados nove tratamentos com quatro repetições, com delineamento de blocos ao acaso em esquema fatorial 3 x 3, formado pela combinação de três volumes de substrato (50, 120 e 200 cm3) e três granulometrias proporcionadas pelas seguintes composições granulométricas: 100% de substrato comercial na granulometria original, 100% de substrato comercial finamente moído e pela mistura, em volume, de 50% de substrato na granulometria comercial com 50% de substrato moído. A influência das variáveis (volume e granulometria) do substrato no crescimento das mudas de café foi avaliada por meio das determinações dos parâmetros biométricos vegetativo da parte aérea e raízes como: número de pares de folhas, altura da planta, diâmetro do caule, matéria seca da parte aérea e das raízes, área foliar total, área foliar média, área do 1o par de folhas, comprimento e superfície de raízes. Em complemento aos objetivos do trabalho foram, também, avaliados o tempo de formação das mudas e a estabilidade ao manuseio do conjunto muda-substrato. O crescimento das plantas depende do volume e da granulometria do substrato, sendo maior com a utilização de 200 cm3 de substrato e a diminuição da granulometria pela mistura, em partes iguais, do substrato finamente moído com o substrato comercial na granulometria original. O tempo de formação das mudas correlacionou com o volume de substrato, demandando 134, 124 e 81 dias para a emissão do 4o par de folhas, quando as plantas cresceram nos recipientes com 50, 120 e 200 cm3 de substrato, respectivamente. A estabilidade ao manuseio do conjunto mudasubstrato varia com o tamanho do recipiente, sendo maior nos tubetes com 50 e 120 cm3 de substrato, e a redução parcial da granulometria, pela mistura granulométrica, aumentou a aderência das partículas com as raízes e, portanto, a estabilidade do conjunto. / The objectives of this research are the evaluation of substrate volume and granulometer influence - used in production of coffee seedlings in plastic tubes - on coffee plants growth, the time of seedlings development and also seedling-substrate handling stability. The investigation was carried out in a nursery at Coffee Experimental Center of IAC, SP, Brazil, with the cultivar Catuaí Vermelho IAC 144 (Coffea arabica L.). Nine treatments were tested with 4 replicates and the experimental design used was randomized blocks with 3x3 factorial composed by 3 substrate volumes (50, 120 and 200 cm3) and 3 granulometer substrate levels obtained by the following granulometer compositions: 100% of substrate on original granulometer, 100% of crushed substrate and the mix, in volume, of 50% of substrate on original granulometer and 50% of crushed substrate. The substrate volume and granulometer influence on coffee seedlings growth was evaluated by determination of growth parameters of shoot and root as: number of leaves, seedling height, stem diameter, root and shoot dry matter, total leaf area, average leaf area, first leaf area, root length and surface. In addition to these parameters, the time of seedlings development and seedling-substrate handling stability were also investigated. The seedlings growth depend on substrate volume and granulometer, being higher when 200 cm3 of substrate volume are used keeping an equal proportion of the different substrate granulometers (original and crushed). The time of seedlings growth did show a correlation with the substrate volume demanding 134, 124 and 81 days for developing the 4th leaf pair when the plants developed in 50, 120 and 200 cm3 of substrate, respectivelly. The seedling-substrate handling stability differs with recipient size, while the granulometer reduction increases the seedling-substrate stability.

Este trabalho foi realizado com o objetivo de avaliar a influência do volume e da granulometria do substrato comercial, utilizado na produção de mudas em tubetes, sobre o crescimento vegetativo das plantas de café, bem como o tempo de formação das mudas e a estabilidade ao manuseio do conjunto muda-substrato. O experimento foi conduzido no viveiro do Centro de Café do IAC, localizado na Fazenda Santa Elisa, Campinas, SP, utilizando a cultivar Catuaí Vermelho IAC 144 (Coffea arabica L.). Foram adotados nove tratamentos com quatro repetições, com delineamento de blocos ao acaso em esquema fatorial 3 x 3, formado pela combinação de três volumes de substrato (50, 120 e 200 cm3) e três granulometrias proporcionadas pelas seguintes composições granulométricas: 100% de substrato comercial na granulometria original, 100% de substrato comercial finamente moído e pela mistura, em volume, de 50% de substrato na granulometria comercial com 50% de substrato moído. A influência das variáveis (volume e granulometria) do substrato no crescimento das mudas de café foi avaliada por meio das determinações dos parâmetros biométricos vegetativo da parte aérea e raízes como: número de pares de folhas, altura da planta, diâmetro do caule, matéria seca da parte aérea e das raízes, área foliar total, área foliar média, área do 1o par de folhas, comprimento e superfície de raízes. Em complemento aos objetivos do trabalho foram, também, avaliados o tempo de formação das mudas e a estabilidade ao manuseio do conjunto muda-substrato. O crescimento das plantas depende do volume e da granulometria do substrato, sendo maior com a utilização de 200 cm3 de substrato e a diminuição da granulometria pela mistura, em partes iguais, do substrato finamente moído com o substrato comercial na granulometria original. O tempo de formação das mudas correlacionou com o volume de substrato, demandando 134, 124 e 81 dias para a emissão do 4o par de folhas, quando as plantas cresceram nos recipientes com 50, 120 e 200 cm3 de substrato, respectivamente. A estabilidade ao manuseio do conjunto mudasubstrato varia com o tamanho do recipiente, sendo maior nos tubetes com 50 e 120 cm3 de substrato, e a redução parcial da granulometria, pela mistura granulométrica, aumentou a aderência das partículas com as raízes e, portanto, a estabilidade do conjunto. / The objectives of this research are the evaluation of substrate volume and granulometer influence - used in production of coffee seedlings in plastic tubes - on coffee plants growth, the time of seedlings development and also seedling-substrate handling stability. The investigation was carried out in a nursery at Coffee Experimental Center of IAC, SP, Brazil, with the cultivar Catuaí Vermelho IAC 144 (Coffea arabica L.). Nine treatments were tested with 4 replicates and the experimental design used was randomized blocks with 3x3 factorial composed by 3 substrate volumes (50, 120 and 200 cm3) and 3 granulometer substrate levels obtained by the following granulometer compositions: 100% of substrate on original granulometer, 100% of crushed substrate and the mix, in volume, of 50% of substrate on original granulometer and 50% of crushed substrate. The substrate volume and granulometer influence on coffee seedlings growth was evaluated by determination of growth parameters of shoot and root as: number of leaves, seedling height, stem diameter, root and shoot dry matter, total leaf area, average leaf area, first leaf area, root length and surface. In addition to these parameters, the time of seedlings development and seedling-substrate handling stability were also investigated. The seedlings growth depend on substrate volume and granulometer, being higher when 200 cm3 of substrate volume are used keeping an equal proportion of the different substrate granulometers (original and crushed). The time of seedlings growth did show a correlation with the substrate volume demanding 134, 124 and 81 days for developing the 4th leaf pair when the plants developed in 50, 120 and 200 cm3 of substrate, respectivelly. The seedling-substrate handling stability differs with recipient size, while the granulometer reduction increases the seedling-substrate stability.

The inability of the myocardium to regenerate after injury plus the inadequate number of available hearts for transplantation have drawn attention to the creation of functional tissue constructs for implantation within the injured heart. In addition, there is an increasing interest in developing in vitro models to study heart physiology and pathology as well as to evaluate drug efficacy. Formation of these in vitro models and tissue constructs requires highly specific conditions to mimic the normal environment of cells in the body. Firstly, in this study, plasma lithography patterning of elastomeric substrates is exploited for creating microtissues composed of neonatal cardiomyocytes, and investigating their development in different mechanical microenvironments. Immunofluorescence microscopy and force spectroscopy show that the size and shape of the cardiomyocyte clusters, as well as the sarcomere length, fiber alignment, and beating amplitude and frequency of the cardiomyocytes, are regulated by microenvironmental cues. Computational analysis reveals that the mechanical stress at the cluster-substrate interface strongly correlates with the aforementioned characteristics of the cardiomyocytes. Taken together, our results underscore a collective mechanoadaptation scheme in cardiac development. Secondly, a silicone substrate with tunable elasticity is characterized for biological studies. Uniaxial tensile testing and microindentation show that these substrates could cover the biological range of stiffness for normal and pathological conditions. Spectrophotometry demonstrates that the transmittance of these substrates is comparable to those of glass and Sylgard 184. Atomic force microscopy shows that the surface roughness of samples is lower than that of widely-used Sylgard 184. Contact angle measurements before and after exposure to air plasma indicate that these samples are compatible with plasma lithography patterning. Thirdly, a new technique for cell patterning is developed which utilizes selective plasma lithography to modify protein adhesion on the substrate. This approach is based on controlling the conformation of Pluronic F-127 layer adsorbed on the surface by modifying surface wettability. Contact angle measurements show that both PDMS and plastic petri dish are compatible with this technique. X-ray photoelectron spectroscopy and atomic force microscopy confirm the adsorption of PF-127 layers with controlled conformation. Fluorescent and bright-field microscopy demonstrate selective adhesion of proteins and attachment of cells merely on plasma-treated areas. Finally, micropillar arrays are employed to determine the effects of two proteins associated with regulation of thin filament length, i.e. Lmod2 and Tmod1, on contractile force generation at the cellular level. Our results demonstrate that the contractile force of single isolated Lmod2-KO cardiomyocytes decreases compared to the wildtype control. Transduction of Lmod2 in the knockout cardiomyocytes restores their contractile force to the level of their WT counterparts, verifying that the observed contractile dysfunction is specific to the loss of Lmod2. Our data demonstrate that overexpression of Tmod1 in cardiomyocytes decreases their contractile force compared to the WT cells and confirm the effects of Lmod2 knockout on contractile force generation.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20701号 / 工博第4398号 / 新制||工||1683(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 小寺 秀俊, 教授 中部 主敬, 教授 安達 泰治, 准教授 横川 隆司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Encore en 2015, un grand nombre d’individus décèdent de pathologies du rythme cardiaque non contrôlées ou d’un manque de disponibilité de donneurs d’organes compatibles. Le génie tissulaire en créant, réparant ou améliorant la fonction des tissus est une option prometteuse afin de diminuer la mortalité associée à ces pathologies. L’objectif global de mon projet de recherche était de développer des outils et d’étudier l’impact fonctionnel des différents stimuli (mécanique et électrique) de l’environnement cardiaque dans le but de définir des conditions optimisées de culture pour la fabrication de tissu de remplacement par génie tissulaire. Cette thèse présente le développement d’un bioréacteur; un système qui optimise les conditions pour la culture cellulaire. L’efficacité du bioréacteur est validée par des expériences de culture cellulaire qui se concentrent sur la prolifération cellulaire, l’organisation cellulaire, l’expression génique et protéique de même que sur l’activité contractile spontanée. En premier lieu, nos résultats montrent, bien que la fréquence de contraction moyenne mesurée reste inchangée, une augmentation significative du nombre de cas de réentrées pour les cultures sur verre comparativement aux cultures sur Polydimethylsiloxane. Une augmentation de l’instabilité spatiotemporelle a été démontrée lorsque les cardiomyocytes étaient déposés sur un support de Polydimethylsiloxane et cette dernière corrèle avec une diminution non-significative de l’ARNm de la connexine-43 et une augmentation significative de l’ARNm pour CaV3.1 et HCN2. La culture sur Polydimethylsiloxane est également associée avec une plus forte réponse à l’isoprotérénol (β-adrénergique) et à l’acétylcholine (parasympathique). En second lieu, nous présentons les résultats du développement de notre bioréacteur en mettant l’emphase sur les caractéristiques (composantes accessibles, étirement uniaxial, électrode de carbone, stimulation biphasique) tout en validant notre approche pour optimiser les conditions de culture et améliorer la rentabilité des étapes de production du tissu de remplacement. Pour finir, nous partageons une nouvelle approche d’évaluation des caractéristiques contractiles de cellules cardiaques en culture. Nous avons développé des algorithmes qui utilisent les données de vidéomicroscopie pour valider l’impact de stimuli, évaluer l’hétérogénéité du signal enregistré et détecter des conditions favorables au développement d’arythmies. / In 2015, there are still a large number of people who die due to diseases of uncontrolled heart rhythm or due to lack of availability of compatible donor organs. Tissue engineering aim to create, repair or improve the function by different techniques. Tissue engineering is a viable option to reduce the mortality associated with many heart conditions. The overall goal of my PhD research was to study the functional impact of different stimuli in cardiac environment (mechanical and electrical stimulation) on cardiac cell cultures. This, in order to define optimized culture conditions for the production of replacement tissue using tissue engineering. This thesis presents the stages of creation and development of a bioreactor; a system that permits the culture of cardiac cells by integrating various stimuli. The optimization of culture conditions by using the bioreactor was confirmed by cell culture experiments that focus on cell proliferation, cell organization, gene and protein expression as well as on spontaneous activity. In the first place, our results show that although mean frequency of spontaneous activity remained unaltered, incidence of reentrant activity was significantly higher in samples cultured on glass compared to PDMS substrates. Higher spatial and temporal instability of the spontaneous rate activation was found when cardiomyocytes were cultured on PDMS, and correlated with decreased connexin-43 (unsignificant) and a significant increased CaV3.1 and HCN2 mRNA levels. Compared to cultures on glass, cultures on PDMS were associated with the strongest response to isoproterenol (β-adrenergic) and acetylcholine (parasympathetic). Secondly, we present the design of our bioreactor with an emphasis on its characteristics and by putting in perspective the relevance of our approach to optimize culture conditions and to improve profitability culture experiences and production stages of replacement heart tissue. Finally, a new approach is proposed to evaluate the characteristics of the contractile cells in culture which allows to validate the functional impact of stimuli, evaluate the heterogeneity in the beating behavior of the cells and to detect localized abnormal activity that could favour arrhythmia.

Cette thèse s’est articulée autour de l’élaboration de revêtements surfaciques à base de polymères et de composants naturels. Dans un premier projet, des surfaces mécano-sensibles pour des applications de libération de molécules bioactives ont été élaborées. Des films de multicouches polyélectrolytes constitués d'une strate « réservoir » permettant le chargement d’une molécule bioactive, le paclitaxel, et d'une strate « barrière » mécano-sensible recouvrant ce réservoir et confinant le paclitaxel ont été élaborés. Lors de la mise sous étirement du film, la barrière est rendue perméable vis-à-vis d'une enzyme présente dans le surnageant. Cette enzyme induit ensuite la dégradation enzymatique du « réservoir » et la libération du paclitaxel. Dans un second projet, des substrats cellulaires nourriciers ont été réalisés à partir de films minces de gélatine réticulés mimant la matrice extracellulaire. Ces films peuvent être chargés: 1) en facteurs de croissance, ce qui permet de s'affranchir ensuite de l'ajout de ces molécules dans le milieu de culture; 2) en nanoparticules afin de moduler les propriétés mécaniques des films; 3) en agents antimicrobiens pour assurer une stérilité de la culture cellulaire. Ainsi, ces substrats aux propriétés biochimiques et biophysiques modulables permettent un contrôle précis du microenvironnement cellulaire. / This PhD work is about designing surface coatings with polymers and natural compounds. In the first project, mechanosensitive surfaces have been developed for drug release applications. Polyelectrolyte multilayer films have been designed with i) one reservoir strata for the loading of a bioactive molecule, paclitaxel, and ii) one mechanosensitive barrier strata on top of the reservoir to confine the molecule. When a mechanical stretch is applied on the structure, the barrier becomes permeable and enables the diffusion of an enzyme within the film.This enzyme degrades the reservoir strata and triggers the release of paclitaxel. In a second project, ECM-mimicking feeder substrate has been developed with crosslinked gelatin thin films. These films can be loaded with: i) growth factors to prevent any further addition of these compounds in the culture medium; ii) nanoparticles to modulate mechanical properties of the substrate; iii) antimicrobial agents to ensure sterility during cell culture experiments. Finally, these substrates have some biochemical and biophysical tunable properties that enable the precise control of cell microenvironment.