La contractilité cellulaire, un phénomène orchestrée par le système d'actomyosine, est un régulateur critique d'une large gamme de processus cellulaires, y compris l'établissement de la polarité cellulaire, la migration cellulaire, l'intégrité des tissus au cours de la morphogenèse ou du développement. Une simple perturbation de la génération de la force et des propriétés mécaniques des cellules peut affecter leurs fonctions physiologiques et par conséquent peut conduire à des défauts pathologiques y compris le cancer.Cependant, les mécanismes qui contrôlent la production de la force par le système acto-myosine et leurs modes de régulation dans les cellules ne sont pas pleinement compris. Au cours de ma thèse, j'ai utilisé un système biomimétique fait d'un ensemble minimal de protéines purifiées pour étudier les propriétés contractiles du système actomyosin.L'objectif était de comprendre comment l'architecture des filaments d'actine peut modifier la réponse contractile. A cet effet, j'étais d'abord intéressée par la construction d'une variété d'organisation de l'actine qui servira après comme substrat pour les moteurs moléculaires (la myosine) lors de la contraction.Afin de comprendre les principes généraux qui dictent l'assemblage de l'actine, nous avons développé un modèle numérique qui nous a permis d'identifier les paramètres clés, y compris l'interaction entre les filaments d'actine, les propriétés mécaniques de ces filaments et l'activation par contact entre une région de nucléation et les filaments d'actine qui poussent à partir d'un motif adjacent. Ce modèle a été utilisé en premier lieu pour implémenter les propriétés reliées à l'actine et en second lieu pour évaluer la réponse contractile des structures d'actine induite par la myosine.Durant ma thèse, j'ai pu démontrer que le niveau de connectivité module la déformation du réseau d'actine induite par la myosine, selon leur architecture. J'ai montré aussi que les protéines de pontages des filaments d'actine sont nécessaires pour effectuer une déformation et générer des forces au niveau des réseaux d'actine dynamiques en présence de la myosine. De plus, nous avons développé les simulations numériques dans le but de relier la déformation macroscopique des structures d'actines due à la myosine avec le mécanisme microscopique sous-jacent.Ce travail a révélé comment la variété des réseaux d'actine contracte d'une façon différente même en respectant les mêmes conditions biochimiques et a démontré l'importance de l'effet du réarrangement dynamique des structures d'actine sur la modulation de sa contractilité. / Cellular contractility – the internal generation of force by a cell orchestrated by theactomyosin machinery – is a critical regulator of a wide range of cellular processes includingthe establishment of cell polarity, cell migration, tissue integrity or morphogenesis duringdevelopment. Disruptions of the force generation and of mechanical properties of living cellsaffect their physiological functions and consequently can lead to pathological defectsincluding cancer. However, the parameters or mechanisms that drive force production by theactin-myosin system and their mode of regulation in cells are not fully understood. During myPhD, I used biomimetic system made of a minimum set of proteins to study the properties ofactomyosin contractile systems. The goal was to understand how/if the actin architecture canmediate the contractile response. For this purpose, I was first interested in building a varietyof actin organization that will serve next as substrate for myosin during contraction. Tounderstand the general principles that dictate geometrically-controlled actin assembly, wedeveloped a model that allowed us to identify key parameters including filaments/filamentsinteraction, filament mechanical property and contact activation between actin filamentsgrowing from the adjacent pattern and the nucleation area. These actin templates were usedthen to evaluate the response of oriented actin structures to myosin-induced contractility. Idemonstrated that crosslinking level modulates the myosin-induced deformation of actinnetworks according to their architecture. I showed also that crosslinkers are necessary tosustain myosin-driven deformation and force production of dynamic actin networks. Inaddition, we developed numerical simulation in order to relate the observed myosin-drivenactin deformation with the underlying microscopic mechanism. This work revealed howdiverse cellular actin networks contract differently to a define set of biochemical conditionsand hence how dynamic rearrangements can modulate network contractility

Assembly of branched actin filaments produces dynamic structures required during membrane associated processes including cell motility and endocytosis. The Actin Related Protein 2/3 (Arp2/3) complex is the only known regulator capable of nucleating actin branches. To specify the sub cellular localization and timing of actin assembly the complex is tightly regulated. Canonical activation of the Arp2/3 complex by Wiskott-Aldrich Syndrome proteins (WASP), requires preformed actin filaments, ensuring the complex nucleates new actin filaments off the sides of preformed filaments. WASP proteins can therefore propagate branch formation but cannot initiate a Y-branch without performed filaments. A key question, then, is what is the source of preformed filaments that seed branched actin network formation in cells? It is unclear how activation of Arp2/3 by multiple regulators is balanced to specify actin filament architectures that are productive in vivo. In this dissertation, we identified WISH/DIP1/SPIN90 (WDS) family proteins as activators of the Arp2/3 complex that do not require preformed filaments, and evaluated whether WDS proteins seed branching nucleation. In chapter II, we dissected the biochemical properties of WDS proteins and found they activate the Arp2/3 complex using a non-WASP like mechanism. Importantly, we discovered WDS-mediated Arp2/3 activation produces linear, unbranched filaments, and this activity is conversed from yeast to mammals. These observations highlight that WDS proteins have the biochemical capacity to seed actin branches. In chapter III, we observed WDS-generated linear filaments can seed WASP-mediated branching directly using single molecule microscopy with fluorescently labeled Dip1. We find that WDS-mediated nucleation co-opts features of branching nucleation. In chapter IV, we investigated how WDS activity is balanced with WASP. We discovered WDS proteins use a single turnover mechanism to activate Arp2/3 and this is conserved during endocytosis. In contrast, WASP-mediated activation is multi-turnover, highlighting a crucial difference between WDS proteins and WASP. Our observations explain how Arp2/3 may limit linear filament production to initiate networks and favor branches during network propagation. Finally, we use fission yeast to show that increasing Dip1 is sufficient to cause defects in actin assembly and the timing of actin patches at sites of endocytosis.

Burkholderia pseudomallei is a Gram-negative intracellular bacterium that causes melioidosis, a serious disease of humans and animals in tropical countries. This pathogen can subvert the host cell actin machinery by a process known as actibased motility, for promoting its movement both within and between cells. The bacterial factor required for this process is known as BimA (Burkholderia intracellular motility A). Intracytoplasmic bacterial pathogens use distinct mechanisms for actin-based motility, hijacking host cytoskeletal proteins for their benefit. However, the molecular mechanism by which BimA subverts the cellular actin machinery is ill-defined. From an affinity approach coupled with mass spectrometry to identify cellular proteins recruited to BimA-expressing bacteria under conditions that promote actin polymerisation, a group of cellular proteins that are recruited to the B. pseudomallei surface in a BimA-dependent manner was identified. A subset of these proteins was independently validated with specific antisera including IQ motif containing GTPase activating protein 1 (IQGAP1). IQGAP1 is a ubiquitous scaffold protein that integrates several key cellular signalling pathways including those involved in actin dynamics. Previous studies demonstrated IQGAP1 was targeted by pathogens to regulate the actin cytoskeleton, for example promoting Salmonella invasion into epithelial cells or supporting cell attachment and pedestal formation of Enteropathogenic Escherichia coli. The aim of this study is to explore the roles of IQGAP1 in the intracellular life of B. pseudomallei. This present study revealed that IQGAP1 was recruited to B. pseudomallei actin tails in infected HeLa cells. This protein has not previously been associated with actin-based motility of other intracellular pathogens. To examine the effect on actibased motility of B. pseudomallei, siRNA was utilised to knockdown IQGAP1 in HeLa cells. After optimisation of siRNA transfection, IQGAP1 expression in HeLa cells was suppressed by approximately 70% as assessed by IQGAP1 immunoblotting. The siIQGAP1 knockdown cells were infected with B. pseudomallei. The bacteria could still form actin tails in the knockdown cells, however, the data showed a statistically significant increase in overall tail length with a concomitant decrease in actin density, compared with the tails formed by B. pseudomallei in control cells. Actin-based motility is essential in the life cycle of several cytoplasmic bacterial pathogens, particularly in cell-to- cell spread. After entry into the host cell cytosol, B. pseudomallei polymerises actin in a BimA-dependent manner and propels itself within and between cells. This is accompanied by cell fusion which generates multi-nucleated giant cells (MNGCs), a process mediated by a Type 6 Secretion System that is co-regulated with BimA. To gain an understanding of the impact of IQGAP1 on the intracellular life of B. pseudomallei, IQGAP1 was successfully knocked-out from HeLa cells using CRISPR-Cas9 technique. Interestingly, Burkholderia invasion was not affected in HeLa cells lacking IQGAP1. However, the bacteria showed a defect in intracellular survival in IQGAP1 knockout cells that was revealed after 6 hours post-infection. Moreover, there was no difference in the proportion of bacteria associated with actin in the control and knockout cells at 16 hours post-infection, although the bacteria formed longer actin tails in control cells with similar actin density. Consequently, the number of MNGCs decreased dramatically in the cells lacking IQGAP1, which was indicated by the absence of plaque formation. Another element of this study was to determine whether BimA and IQGAP1 are direct interacting partners. Using either an in vitro pulldown assay or in vivo yeast two-hybrid system, a direct interaction between these proteins could not be detected. It is, therefore, likely that IQGAP1 is recruited to B. pseudomallei actin tails through its intrinsic ability to interact with F-actin. Despite the lack of a direct interaction between these two proteins, an N-terminal IQGAP1 fragment significantly augmented BimA-mediated actin polymerisation in vitro. Taken together, this study provides the first evidence of the presence of IQGAP1 in B. pseudomallei actin tails and presents the importance of IQGAP1 in actin-based motility and intracellular life of this bacterium. Understanding the mechanism of B. pseudomallei actin-based motility is useful to gain insights into host cell actin dynamics and its role in pathogenesis. Targeting host cellular proteins that are required for the intracellular life of pathogens are a topical area of research, with the potential to be useful alternatives to classic antibiotic therapy. Indeed, IQGAP1 could be a potential novel therapeutic target to develop drugs for treating B. pseudomallei infection.

Prions are infectious protein entities capable of self-replication. Prions are the causal agents behind the transmissible spongiform encephalopathies causing neurodegeneration and death in affected organisms. Prions have been identified in yeast with the best-characterized prions being [PSI+] and [PIN+], whose respective native proteins are the Sup35 translation termination factor and Rnq1 (function unknown). Autophagy is a cellular housekeeping mechanism mediating the degradation of damaged proteins and superfluous organelles. It is a highly sequential process regulated by autophagy related genes (ATGs). Autophagy has also been implicated in the clearance of amyloidogenic proteins including prions. However, the mechanistic basis underlying this activity is poorly understood, and a key objective of this project was to characterize how autophagy prevents spontaneous prion formation. Our study found that the deletion of core ATGs correlated with an increase in de novo [PSI+] and [PIN+] formation as well as Sup35 aggregation. Enhancement of autophagic flux through spermidine treatment attenuated the increased levels of de novo [PSI+] formation in mutants that normally show elevated levels of [PSI+] formation. Defective autophagy correlated with increased oxidatively damaged Sup35 in an atg1 mutant whereas anaerobic growth abrogated the increased [PSI+] formation in the atg1 mutant to wild-type levels. Our data suggest that autophagy serves a protective role in the clearance of oxidatively damaged Sup35 proteins that otherwise has a higher propensity towards [PSI+] prion formation. We also investigated the role of prion formation and autophagy during yeast chronological ageing which is the time that non-dividing cells remain viable. Prion diseases are associated with advanced age which correlates with a decline in cellular protective mechanisms including autophagy. Our study found an age dependent increase in the frequency of de novo [PSI+] formation with chronological age of yeast cells, more so in an atg1 mutant relative to the wild-type. Autophagy competent cells carrying the [PSI+] and [PIN+] prions also had improved chronological lifespan relative to prion free cells and atg1 cells. Cells carrying the [PSI+] prion elicited elevated autophagic flux that may promote improved lifespan thus suggesting a beneficial role of the [PSI+] prion during chronological ageing. The actin cytoskeleton provides the structural framework essential for a multitude of cellular processes to occur. We investigated the role of the Arp2/3 complex responsible for branching of actin filaments towards prion formation. Knockout mutants of the nucleation promoting factors of the Arp2/3 complex, in particular the abp1 mutant, showed reduced de novo [PSI+] formation and Sup35 aggregation under basal and oxidative stress conditions. Similarly, treatment with latrunclin A, an actin monomer-sequestering drug also abrogated de novo [PSI+] formation. Colocalization studies revealed that Sup35 often does not colocalize with Rnq1, a marker for the insoluble protein deposit (IPOD) in an abp1 mutant. This suggests a role for the Abp1 protein in the efficient transport of Sup35 molecules to the IPOD that may facilitate de novo [PSI+] prion formation under vegetative states and oxidant challenges.

O carcinoma espinocelular é uma neoplasia maligna que representa aproximadamente 94% de todas as ocorrências presentes em boca e uma das suas principais características celulares é a migração de suas células para formar metástases. A adesão celular é considerada um dos eventos determinantes da migração celular. Para as células formarem uma estrutura tecidual tridimensional as adesões entre células e entre células e matriz extracelular são de grande importância. As junções de adesão celulares surgem, caracteristicamente, pela interação entre receptores adesivos, vias de sinalização e elementos do citoesqueleto. A proteína E-caderina está presente em adesões entre células no tecido epitelial. A proteína FAK está envolvida na maioria dos eventos relacionados à adesão celular estimulada por integrinas. A Vinculina é uma proteína de adesão que se liga ao citoesqueleto de actinomiosina como uma proteína de adesão focal através das integrinas. Estudos recentes sugerem que há alteração na expressão e atividade de proteínas de adesão em tumores malignos. O objetivo deste trabalho foi descrever o padrão de expressão e de regulação da atividade de proteínas de adesão em amostras de tumores de carcinoma espinocelular. Foram realizadas reações de imunoistoquímica para verificar o padrão de distribuição das proteínas E-caderina, Vimentina e FAK-y397 em amostras de tumores de carcinoma espinocelular oral. Verificou-se a diminuição da expressão de E-caderina e de Vinculina em regiões de adesão célula-célula e em contrapartida constatou-se aumento na marcação citoplasmática de Vinculina bem como na marcação de FAK-y397 em todas as amostras de tumores. Apesar dos avanços, ainda são necessários mais estudos observacionais que averiguem não apenas o grau de expressão dessas proteínas de adesão, mas também o seu nível de regulação. A partir dos resultados deste estudo, pode-se sugerir que o controle do nível de expressão e de atividade da adesão celular podem ser considerados como potenciais alvos para a aplicação de terapias coadjuvantes que visam a diminuir ou impedir a progressão tumoral, bem como o desenvolvimento de metástases. / Squamous cell carcinoma is a malignant neoplasm that accounts for approximately 94% of all occurrences present in mouth and one of its main characteristics is the cellular migration of its cells to form metastases. Cell adhesion is considered one of the defining events of cell migration. For a three-dimensional tissue structure, adhesions between cells and between cells and the extracellular matrix is of great importance. Cell adhesion junctions arise characteristically by interaction between adhesive receptors, signaling pathways and cytoskeletal elements. The protein E-cadherin is present in cells in the adhesion between epithelial tissue. The Focal Adhesion Kinase (FAK) protein is involved in most events related to cell adhesion stimulated by integrins. The vinculin is an adhesion protein that binds cytoskeletal protein through integrins activaion. Recent studies suggest that there are alterations in the expression and activity of adhesion proteins in malignant tumors. The aim of this study was to describe the pattern of expression and regulation of the activity of adhesion proteins in tumor samples of squamous cell carcinoma. Immunohistochemical reactions were performed to check the distribution pattern of the protein E-cadherin, vimentin and FAK-y397 in tumor samples of oral squamous cell carcinoma. There was a decrease in the expression of E-cadherin and vinculin in regions of cell-cell adhesion but, on the other hand, it was found to increase in cytoplasmic as well as unscheduled vinculin FAK-y397 in all tumor samples. Despite progress, it is necessary more observational studies that examine not only the degree of expression of these adhesion proteins, but also its level of regulation. From the results of this study it is suggested that the control of the expression level and activity of cell adhesion may be considered as potential targets for application adjuvant therapies that aim to reduce or prevent tumor progression and the development metastases.

Neospora caninum é um protozoário pertencente ao filo Apicomplexa que atinge, dentre diversos hospedeiros intermediários, principalmente bovinos e tem emergido como um importante causador de problemas reprodutivos e abortos em rebanhos de corte e leiteiro. Organismos do filo Apicomplexa são parasitas intracelulares obrigatórios que, para locomoverem-se e realizarem a invasão das células hospedeiras, utilizam um mecanismo próprio de locomoção ativa impulsionada pelo motor actina/miosina denominado motilidade por deslizamento (gliding motility), cujo complexo motor localiza-se entre a membrana plasmática e o complexo de membrana interno do parasita. A investigação a respeito do funcionamento desse mecanismo de locomoção e invasão vem sendo realizada principalmente em Toxoplasma gondii e Plasmodium spp., entretanto não há nenhuma publicação envolvendo actina em N. caninum. Esse trabalho envolveu a clonagem e expressão da sequência NcAct201-310 e deu início a caracterização da actina de N. caninum (NcAct). A sequência NcAct foi obtida em banco de dados ToxoDB, e uma comparação por alinhamento entre as actinas de Apicomplexas relacionados revelou que NcAct é idêntica à TgACT1 (100% identidade). Com outras espécies, a NcAct tem maior identidade/similaridade com a actina de Eimeria tenella (97%/99%), seguida da actina de Plasmodium falciparum PfACT1 (93%/97%), da actina de Babesia bovis (86%/94%) e PfACT2 (80%/92%). Quando localizada com anticorpo anti-?-actina C4, NcAct apresenta-se em duas bandas de 43 e 45 kDa em gel de acrilamida 1D e em nove isoformas em gel de acrilamida 2D. Todas as identidades das bandas e spots foram confirmados por espectrometria de massas (MS/MS). Além disso, NcAct localiza-se, em sua maioria, na região periférica do taquizoíta de N. caninum e sua distribuição é alterada após incubação dos taquizoítas com 5 ?M de jasplakinolida (JAS) ou 2 ?M de citocalasina D (CytD). Por fim, por meio de ensaio de fracionamento de actina monomérica (actina-G) e filamentosa (actina-F), demonstramos que a JAS é capaz de aumentar a quantidade de actina-F em taquizoítas de N. caninum. / Neospora caninum is an Apicomplexan protozoan that infects, among a whole range of intermediate hosts, bovine where it is emerging as a relevant cause of reproductive problems and abortion in dairy and beef cattle. As obligatory intracellular organisms, parasites from Apicomplexa Phylum use their own active locomotion system to move and invade host cells. This mechanism is driven by the actin/myosin motor known as gliding motility, localized between the plasma and the inner membrane complex. Studies involving this locomotion and invasion system have been conducted mainly in Toxoplasma gondii and Plasmodium spp. To our knowledge there is no publication involving actin in N. caninum, so this work was outlined and involved the cloning and expression of the sequence NcAct201-310, initiating the characterization of actin of N. caninum (NcAct). The sequence NcAct was obtained from the Database ToxoDB, and a comparison of actins from Apicomplexa-related revealed total identity of NcAct with TgACT1 (100% identity). With other species, NcAct has higher identity/similarity with Eimeria tenella actin (97%/99%), followed by Plasmodium falciparum actin PfACT1 (93%/97%), Babesia bovis actin (86%/94%) and PfACT2 (80%/92%). When localized with the antibody anti-?-actin C4, NcAct is presented as two bands of 43 and 45 kDa in 1D acrylamide gel and as nine isoforms in 2D acrylamide gel. All these findings were confirmed by mass spectrometry (MS/MS). Moreover, NcAct localizes predominantly in the peripheric region of N. caninum tachyzoites. This distribution is altered after incubation of the tachyzoites with 5 ?M of jasplakinolide (JAS) or 2 ?M of cytochalasin D (CytD). Finally through fractionating assay of monomeric (actin-G) and filamentous (actin-F), we demonstrated that JAS is capable of increasing the quantity of actin-F in N. caninum tachyzoites.

Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.

Cell migration is required for a number of physiological processes and is implicated in pathologies such as tumor metastasis. Cell motility is dependent upon dynamic actin reorganization, and is regulated by the Rho family of small GTPases. Rho GTPases are molecular switches that cycle between their active and inactive conformations. The best-studied members of this family are Rac1, RhoA, and Cdc42. Each is responsible for the formation of specific actin structures. Diacylglycerol kinases (DGKs) act at the membrane to convert diacylglycerol (DAG) and phosphatidic acid (PA), maintaining the balance of these two lipid second messengers. Previous studies from our lab have demonstrated that the ζ isoform of DGK facilitates the release of Rac1 and RhoA from their inhibitor, RhoGDI. Here we studied a closely related isoform, DGKι, using mouse embryonic fibroblasts (MEFS) in which the gene for DGKι had been deleted. Aberrations in cell morphology, spreading, and migration were identified in DGKι-null MEFs. We showed that the activity of Rac1 and RhoC, but not RhoA, was impaired in the absence of DGKι, yet only RhoC protein levels were affected. Reduced activation of these Rho GTPases was accompanied by defects in Rac1- and RhoC- related actin structures. These data demonstrate that DGKι, in addition to DGKζ, contributes to the regulation of GTPase activation and remodeling of the actin cytoskeleton.

Cells are dynamic systems that generate and respond to forces through the complex interplay between biochemical and mechanical regulations. Since cellular processes often happen at the molecular level and are challenging to be observed under in vivo conditions due to limitations in optical microscopy, multiple analysis tools have been developed to gain insight into those processes. One of the ways to characterize these mechanical properties is by measuring their persistence length, the average length over which filaments stay straight. There are several approaches in the literature for measuring the persistence length of the filaments, including Fourier analysis of images obtained using fluorescence microscopy. Here, we show how curvature can be used to quantify local deformations of cell shape and cellular components. We develop a novel technique, called curvature analysis, to measure the stiffness of bio-filaments from fluorescent images. We test our predictions with Monte-Carlo generated filaments. We also apply our approach to microtubules and actin filaments obtained from in vitro gliding assay experiments with high densities of non-functional motors. The presented curvature analysis is significantly more accurate compared to existing approaches for small data sets. To study the effect of motors on filament deformations and velocities observed in gliding assays with functional and non-functional motors, we developed Langevin dynamics simulations of on glass and lipid surfaces. We found that generally the gliding velocity increases with an increase in motor density and a decrease in diffusion coefficient, and that motor density and diffusion coefficient have no clear effect on filament curvatures, except at a very low diffusion coefficients. Finally, we provide an ImageJ plugin to make curvature and persistence length measurements more accessible to everyone.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23110号 / 医科博第121号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 濵﨑 洋子, 教授 竹内 理, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM