Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17556号 / 工博第3715号 / 新制||工||1566(附属図書館) / 30322 / 京都大学大学院工学研究科機械理工学専攻 / (主査)教授 富田 直秀, 教授 井手 亜里, 教授 安達 泰治 / 学位規則第4条第1項該当

Abstract Collagen XIII is a type II transmembrane protein, which has a short intracellular domain and a large, mainly collagenous ectodomain. It is located at many cell-matrix junctions and in focal adhesions in cultured cells and it has a function in cell adhesive processes. Overexpression of collagen XIII molecules with an 83 amino acid deletion in part of the ectodomain leads to fetal lethality in Col13a1del transgenic mice. Doppler ultrasonography was performed at 12.5 days of gestation on fetuses resulting from heterozygous matings and matings between heterozygous and wild-type mice. Some fetuses had atrioventricular valve regurgitation (AVVR) and all of them were transgene positive. In addition, fetuses had pathological changes in functional parameters. Histological analysis showed the trabeculation of the ventricles to be reduced and the myocardium to be thinner in the fetuses with AVVR. Based on in situ hybridization (ISH), collagen XIII mRNA are normal constituents of these structures. Overexpression of mutant collagen XIII results in mid-gestation cardiac dysfunction in fetuses, and these disturbances in cardiac function may lead to death in utero. The heterozygous mice that were initially of normal appearance had an increased susceptibility to develop B cell lymphomas, which originated in the mesenteric lymph node. Collagen XIII protein was not detected in normal lymph nodes or in the lymphomas. The incidence of lymphomas was higher in conventional conditions than in a specific pathogen-free facility. In addition, the expression of collagen XIII was localized in the intestine and the basement membrane was highly abnormal. These findings suggest that collagen XIII is a critical determinant of lymphanogenesis. Using ISH, antibody staining and RT-PCR techniques collagen XIII expression was analyzed during carcinogenesis in mice and in man. Collagen XIII expression increased during carcinogenesis in mice and in man. In the malignant process collagen XIII mRNA localized in the basal epithelium and in the invasive cells. According to antibody staining malignant invasive cells were positive. Results may reflect the disturbed adhesion of epithelial cells and ECM and that may affect the behaviour of the malignant cells, suggesting that collagen XIII has a significant role in the initiation of the invasion.

Abstract Neurofibromatosis type 1 (NF1) is a dominantly inherited neurocutaneous disorder caused by mutations in the NF1 gene. Common clinical manifestations associated with NF1 are neurofibromas, café-au-lait macules (CALM), axillary freckling and Lisch nodules of the iris. Other important manifestations are vasculopathy, a variety of osseous lesions, including short stature, scoliosis and pseudoarthrosis, optic gliomas and an increased risk for certain malignancies. The best characterized function of the NF1 gene is to act as a downregulator of Ras proto-oncogene signalling by accelerating the switch of active Ras-GTP into inactive Ras-GDP. The NF1 gene is considered a tumor suppressor since some malignancies may display a loss of heterozygosity or homozygotic inactivation of the gene. The present study investigated the behaviour and function of the NF1 gene during keratinocyte differentiation, wound healing and psoriasis using human epidermis and epidermal keratinocytes as a model. The NF1 protein was shown to associate with the intermediate filament network during keratinocyte differentiation both in vitro and in vivo, and it is thus suggested to play a role in the cytoskeletal re-organization or in the formation of cell adhesions. NF1 gene expression was also studied in psoriasis, in which keratinocytes are hyperproliferative and cell differentiation is altered. NF1 gene expression was downregulated in psoriatic keratinocytes both in vivo and in vitro, suggesting that the NF1 gene might have role in downregulating keratinocyte proliferation. During epidermal wound healing, NF1 gene expression was increased. However, the process of wound healing showed no apparent differences between NF1 patients and controls. Furthermore, an increased number of cells immunoreactive for active Ras-MAPK was demonstrated in vascular tissues of NF1 patients, but not in epidermal keratinocytes or dermal fibroblasts. The finding suggests that the NF1 protein functions as a Ras-GAP in some, but not all tissues.

Abstract Genomic clones covering the entire mouse type XIII collagen gene (Col13a1) were isolated, and the complete exon-intron organization was characterized. The gene was found to be about 135 kb in size and to locate in the mouse chromosome 10. Comparison of gene structures and promoter regions between man and mouse indicated high conservation between the two species. In order to understand the biological function of type XIII collagen, a mouse line that expresses type XIII collagen with replacement of the cytosolic and transmembrane domains by a short, non-descript sequence was generated using homologous recombination. Expression of this aminoterminally altered type XIII collagen led to mild but progressive muscular atrophy in mice. The integrity of muscle cells was disturbed and the basement membrane showed areas of detachment from the sarcolemma as well as clearly altered structure at myotendinous junctions. These phenotypical changes were, nevertheless, local, since the majority of the muscle was intact. The results show the importance of the membrane anchorage of the type XIII collagen protein in adhesion and, consequently in the maintenance of muscle integrity. To study the significance of various regions of type XIII collagen, wild-type and mutant forms of the protein were produced recombinantly in insect cells. The transmembrane domain and the adjacent region of ectodomain were found to be crucial for the formation of type XIII collagen molecules with all of the three collagenous domains in trimeric conformation. A previously characterized conserved membrane-proximal region of the ectodomain was predicted to harbour a coiled-coil conformation. This was suggested to begin in the transmembrane domain of type XIII collagen and in several other collagenous transmembrane proteins. Type XIII collagen lacking this coiled-coil sequence was correctly folded with respect to its central COL2 and carboxylterminal COL3 domains. Between them, in the NC3 domain, a second coiled-coil sequence was found, and this was suggested to function as a second association region. The second coiled-coil sequence was found to be conserved in the two other type XIII collagen-like molecules as well. To obtain precise information about the location and level of type XIII collagen expression, a reporter mouse line synthesizing a recombinant protein with the cytoplasmic and transmembrane portions of type XIII collagen linked in-frame with the β-galactosidase enzyme was generated. The reporter mice showed high expression of type XIII collagen at neuromuscular junctions and in the periosteum of bone. Interestingly, the growth of the reporter mice was reduced at puberty. Their long bones showed a decreased diameter and impaired mechanical properties. In addition, their peripheral nerves showed areas of detachment from muscle cells at neuromuscular junctions. These results provide further evidence for the role of type XIII collagen in cell adhesion. They also show the importance of proper adhesion conducted by type XIII collagen in signaling between the extracellular matrix and cells and in the cellular response.

Abstract Epithelial cell-cell adhesions have a critical role in morphogenesis, establishment and maintenance of tissue architecture, cell-cell communication, normal cell growth and differentiation. These adhesions are disrupted during malignant transformation and tumour cell invasion. Several kinases, phosphatases and small GTPases regulate cell-cell contacts. In the present work we investigated the dynamics of cell-cell adhesion structures after microinjection of fluorophore tagged vinculin, during transformation caused by an active Src tyrosine kinase and during Helicobacter pylori infection. The regulatory role of Rac GTPase as well as the behaviour of actin and cadherin were analysed in all these conditions. Microinjection of vinculin into bovine kidney epithelial MDBK cells induced release of actin, cadherin and plakoglobin to cytoplasm of the cells, caused disruption of protein complexes at adherens and tight junctions that finally led to formation of polykaryons. Activated Rac GTPase, in turn, enhanced accumulation of cadherin to membranes and thereby diminished the formation of polykaryons, whereas inactive Rac removed cadherin from membranes. Incorporation of vinculin to lateral membranes took place also in acidifying and depolarising conditions where cell fusions were prevented. Thus, the membrane potential seemed to control fusion ability. In src-MDCK cells, activation of Src kinase led to disintegration of adherens junctions. Clusters of junctional components and bundles of actin were seen at the basal surface already within 30 min after Src activation. p120ctn was the only component of adherens junction whose relocation correlated to its phosphorylation. Inhibition of Src by a specific inhibitor PP2 restored the cubic morphology of the cells and accumulated cadherin back to lateral walls. Still p120ctn remained in cytoplasm and thereby was not responsible for the epithelial phenotype. Activation of Rac GTPase by Tiam1 also increased the amount of cadherin at lateral membranes and maintained the morphology of src-MDCK cells practically normal after activation of Src kinase. In the same way, actin cytoskeleton was reorganised in gastric carcinoma cells in response to infection with H. pylori via activation of Rac signalling pathway. Hence, Rac and cadherin seem to be the major players in the maintenance of epithelial cell morphology.

Bilateral breast augmentation is one of the most common cosmetic surgical procedures carried out on women in the western world. Breast augmentation involves increasing the volume of a woman‘s breasts through surgery by placing a silicone implant in the subglandular or subpectoral cavity. Although a capsule forms inevitably around breast implants as a natural part of healing, it can cause significant morbidity if the capsule becomes firm and contracted, a condition known as breast capsular contracture (BCC). The aetiology of BCC remains unknown however it is characterised by dense fibrocollagenous connective tissue with a local inflammatory response. Host response is influenced by several factors including implant surface texture, chemistry and interactions between cells and the extracellular matrix. Texturing holds the implant in place, thus preventing micromotion at the host prosthesis interface. While in smooth surfaces, the implant moves inside the breast, making the fibroblasts repeatedly produce collagen in response to this host-prosthesis shearing motion. In this thesis, the effect of surface characteristics and specific coatings on the cell-surface interaction has been examined on smooth compared to textured surfaces using commercially available breast implants. The properties of breast implants shells have been characterised using confocal laser microscopy, contact angle measurements, confocal Raman spectroscopy and tensile testing. Confocal laser microscopy was used to evaluate the topographical features and surface roughness of the implant surfaces. Contact angle measurements were carried out to determine the hydrophobicity of the implant surfaces. Chemical characterisation was carried out recording Raman images and spectra of the implants using confocal Raman spectrometer. The mechanical properties of the breast implant shells were measured via tensile testing. Adhesive interactions of breast-derived fibroblasts with breast implant surfaces were examined in-vitro. For this purpose, the effect of four molecule coatings (aggrecan, collagen I, fibronectin, and hyaluronic acid) was evaluated on fibroblast attachment, proliferation, fibroblast morphology, spreading, cytotoxicity and gene expression. Results from in-vitro assays demonstrated cell susceptibility to topography and protein coatings and further showed cytoskeletal re-organisation and modification with specific cell adhesion patterns. Combination of diverse topographies and specific coatings induced differential regulation of the expression of adhesion related genes, such as focal adhesion kinase, paxillin, vinculin, and α-actinin on breast fibroblasts. In conclusion, this thesis has demonstrated the extent and strength of cell adhesion and subsequent cell proliferation and differentiation. This is based on the physical interactions between cells and the extracellular environment in the form of topography and on the chemical interactions mediated by specific coatings. Precise characterisation of the silicone breast implant surfaces was achieved. This may play an important role in the development of improved breast implant surfaces with improved qualities leading the development of surfaces that may be less prone to capsular contracture.

Hydroxyapatite (HA) has received wide attention in orthopedics, due to its biocompatibility and osseointegration ability. Despite these advantages, the brittle nature and low fracture toughness of HA often results in rapid wear and premature fracture of implant. Hence, there is a need to improve the fracture toughness and wear resistance of HA without compromising its biocompatibility. The aim of the current research is to explore the potential of nanotubes as reinforcement to HA for orthopedic implants. HA- 4 wt.% carbon nanotube (CNT) composites and coatings are synthesized by spark plasma sintering and plasma spraying respectively, and investigated for their mechanical, tribological and biological behavior. CNT reinforcement improves the fracture toughness (>90%) and wear resistance (>66%) of HA for coating and free standing composites. CNTs have demonstrated a positive influence on the proliferation, differentiation and matrix mineralization activities of osteoblasts, during in-vitro biocompatibility studies. In-vivo exposure of HA-CNT coated titanium implant in animal model (rat) shows excellent histocompatibility and neobone integration on the implant surface. The improved osseointegration due to presence of CNTs in HA is quantified by the adhesion strength measurement of single osteoblast using nano-scratch technique. Considering the ongoing debate about cytotoxicity of CNTs in the literature, the present study also suggests boron nitride nanotube (BNNT) as an alternative reinforcement. BNNT with the similar elastic modulus and strength as CNT, were added to HA. The resulting composite having 4 wt.% BNNTs improved the fracture toughness (~85%) and wear resistance (~75%) of HA in the similar range as HA-CNT composites. BNNTs were found to be non-cytotoxic for osteoblasts and macrophages. In-vitro evaluation shows positive role of BNNT in osteoblast proliferation and viability. Apatite formability of BNNT surface in ~4 days establishes its osseointegration ability.

The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Cell adhesion is the first step of cell response to materials and the extracellular matrix (ECM), and is essential to all cell behaviours such as cell proliferation, differentiation, migration and apoptosis for anchor-dependent cells. Therefore, studies of cell attachment have important implications to control and study cell behaviours. During many developed techniques for cell attachment, the manipulation of surface chemistry is a very important method to control initial cell attachment. To control cell adhesion on a two-dimensional surface is a simple model to study cell behaviours, and is a fundamental topic for cell biology, tissue engineering, and the development of biosensors. From the engineering point of view, the preparation of a material with controllable surface chemistry can help studies of cell behaviours and help scientists understand how surface features and chemistry influence cell behaviours. During the fabrication, the challenge is to create a surface with heterogeneous surface properties in the micro scale and subsequently to guide cell initial adhesion. In order to control cell adhesion in a spatial and temporal manner, a photochemical method to control surface chemistry was employed to control the surface property for cell adhesion in this project. Two photocleavable derivatives of the nitrobenzyl group were tried on two types of surfaces: a model self-assembled monolayer (SAM) with alkanethiol-gold surface and biodegradable chitosan. Reactive functional groups on two different surfaces can be inactivated by covalent binding with these photocleavable molecules, and light can be further introduced into the system as a stimulus to recover their reactivity. By simply applying a photomask with diffe

A Bacteroides gingivalis adhesin mediating attachment to red blood cells and buccal epithelial cells was isolated, cloned and characterized. The isolation procedure involved gentle stirring of the cells followed by ammonium sulphate precipitation, ion-exchange and gel chromatography. The native molecule had a Mr in excess of 10⁶ kDa and was made up of subunits with an Mr of 43 kDa. Antisera raised to the adhesin and its subunits reacted with antigens on the surface of B. gingivalis cells. No reaction with fimbriae was seen. The IgG fractions from these antisera inhibited the adherence of B. gingivalis to host tissue. Proteolytic enzymes destroyed binding capability of whole cells and of the purified adhesin but the molecular weight of the haemagglutinin was not altered. A genomic library of B. gingivalis DNA was created in E. coli JM83. 5500 colonies were screened by a colony immunoassay with anti-S. gingivalis serum and by a direct haemagglutinating assay. 337 clones tested positive by the immunoassay and two clones, 1-3,and 1-49 tested positive for haemagglutinating activity. Both haemagglutinating positive recombinants had inserts of 3.2 kb. One clone, 1-49 was chosen for further characterization. E. coli 1-49 expressed a protein of 43 kDa that was not present in E. coli JM 83 control as seen by SDS-PAGE and Western blot analysis. Anti-1-49 serum inhibited the haemagglutinating activity of B. gingivalis and E. coli 1-49. This serum reacted with surface molecules on B. gingivalis and E. coli 1-49 as seen by immunogold electron microscopy and immunofluorescence, and to the purified haemagglutinin by Western blot analysis. Like the haemagglutinin on B. gingivalis, the haemagglutinating activity of E. coli 1-49 was destroyed by heating and proteolytic enzymes but the apparent size of the molecule as determined by SDS-PAGE was not affected. A bacterial coaggregating adhesin from B. gingivalis was isolated and characterized. The isolation procedure involved adsorption of the solubilized adhesin on S. mitis followed by elution with glycine buffer. SDS-PAGE of the boiled adhesin revealed a protein with an Mr of 46 kDa. Proteolytic digestion destroyed all bacterial aggregating activity and hydrolysed the 46 kd protein. Antisera raised to the 46 kDa protein reacted with surface molecules on all strains of B. gingivalis tested. This antiserum inhibited the coaggregation reaction between B. gingivalis and other bacteria. Vesicles produced by B. gingivalis were found to enhance the binding of S. sanguis to serum coated hydroxy apatite (SeHA). Maximum vesicle mediated binding took place at 37°C and was destroyed by heating. The lipopolysaccharide from several black pigmented bacteroides were isolated and characterized physically, chemically and immunologically. All of the LPS were of the smooth type and contained the sugars rhamnose, glucose, galactose, glucosamine and galactosamine; no KDO or heptose were found. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate