Recent attention has focused on the use of renewable resources as an alternative to petroleum-based polymers. Fudge et al. (2010) demonstrated that hagfish slime threads, which are composed of “keratin-like” intermediate filament (IF) proteins undergo an α→β transition when strained and when exposed to glutaraldehyde, mechanical properties further improved. Negishi et al. (2012) showed that fibres produced from solubilized hagfish slime threads did not possess comparable mechanical characteristics to native slime threads and were unable to assemble into 10 nm filaments. In this study, fibres were produced from solubilized recombinant vimentin protein and assembled vimentin filaments. Solubilized protein fibres did not display mechanical properties as impressive as fibres made from filaments assembled in the presence magnesium and glutaraldehyde. Additionally, X-ray diffraction analysis of fibres cross-linked with magnesium showed an α→β transition when draw-processed. These data show that fibres produced using IFs can potentially be used for production of sustainable protein polymers.

Implication de SIP1 (Smad Interacting Protein 1) dans la transition épithélio-mésenchymateuse associée à la progression métastatique

The active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when it forms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VN complex serves many functions related to fibrinolysis and cell migration but key to these effects is its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) on activated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternary complexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyi s not well understood. I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaces when microparticle release leads to exposure of vimentin cytoskeleton which can lead to either assembly of the ternary complex or become involved in an autoimmune response specific for vimentin. To follow the intracellular and extracellular fate of PAI-1, I constructed an expression vector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live cell tracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin is expressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentin positive platelets or PMP's and atomic force microscopy was performed to image platelets or PMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression in which the junction of microparticle release results in the exposure of cytoskeletal vimentin on both the cell and the microparticle. This exposed vimentin could potentially induce VN multimerization on the same cell surface leading to incorporation of multiple PAI-1:VN complexes. Finally, I investigated how anti-vimentin antibodies can induce platelet:leukocyte conjugate formation. To achieve this, in vitro tests were performed to determine the binding site of anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, my results suggest that vimentin exposure in our model of microparticle release can lead to ternary complex assembly if suitable quantities of PAI-1 are released during platelet activation. In the setting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes can bind these autoantibodies, which then leads to platelet activation and the formation of platelet:leukocyte conjugates.

No description available.

The active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when it forms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VN complex serves many functions related to fibrinolysis and cell migration but key to these effects is its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) on activated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternary complexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyi s not well understood. I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaces when microparticle release leads to exposure of vimentin cytoskeleton which can lead to either assembly of the ternary complex or become involved in an autoimmune response specific for vimentin. To follow the intracellular and extracellular fate of PAI-1, I constructed an expression vector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live cell tracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin is expressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentin positive platelets or PMP's and atomic force microscopy was performed to image platelets or PMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression in which the junction of microparticle release results in the exposure of cytoskeletal vimentin on both the cell and the microparticle. This exposed vimentin could potentially induce VN multimerization on the same cell surface leading to incorporation of multiple PAI-1:VN complexes. Finally, I investigated how anti-vimentin antibodies can induce platelet:leukocyte conjugate formation. To achieve this, in vitro tests were performed to determine the binding site of anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, my results suggest that vimentin exposure in our model of microparticle release can lead to ternary complex assembly if suitable quantities of PAI-1 are released during platelet activation. In the setting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes can bind these autoantibodies, which then leads to platelet activation and the formation of platelet:leukocyte conjugates.

The active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when it forms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VN complex serves many functions related to fibrinolysis and cell migration but key to these effects is its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) on activated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternary complexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyi s not well understood. I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaces when microparticle release leads to exposure of vimentin cytoskeleton which can lead to either assembly of the ternary complex or become involved in an autoimmune response specific for vimentin. To follow the intracellular and extracellular fate of PAI-1, I constructed an expression vector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live cell tracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin is expressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentin positive platelets or PMP's and atomic force microscopy was performed to image platelets or PMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression in which the junction of microparticle release results in the exposure of cytoskeletal vimentin on both the cell and the microparticle. This exposed vimentin could potentially induce VN multimerization on the same cell surface leading to incorporation of multiple PAI-1:VN complexes. Finally, I investigated how anti-vimentin antibodies can induce platelet:leukocyte conjugate formation. To achieve this, in vitro tests were performed to determine the binding site of anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, my results suggest that vimentin exposure in our model of microparticle release can lead to ternary complex assembly if suitable quantities of PAI-1 are released during platelet activation. In the setting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes can bind these autoantibodies, which then leads to platelet activation and the formation of platelet:leukocyte conjugates. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18431号 / 生博第311号 / 新制||生||41(附属図書館) / 31289 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 豊島 文子, 教授 藤田 尚志, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Previous research in our laboratory found that inhibiting expression of vimentin, a marker of epithelial-to mesenchymal transition, inhibited cell growth and motility in vitro and in vivo. Tumors derived from vimentin knockdown cells showed features of epithelial redifferentiation and increased expression of differentiation-specific keratins. It is unknown what causes re-expression of keratins when vimentin is inhibited. Although, canonical Wnt signaling may activate NF-κB and repress of keratin and/or induce vimentin expression through β-catenin. We hypothesize that downregulation of differentiation-specific keratins contributes to tumor progression, mediated directly or indirectly by expression of vimentin. Vimentin-negative HN4 cells were transfected with plasmids encoding wild-type, PKCε-phosphomimetic, or unphosphorylatable versions of vimentin. Expression of vimentin was confirmed by western blot and immunofluorescence. Effects on cell growth and motility were determined using MTT, cell proliferation, and wound-closure assays. These results indicate that mutation of vimentin PKCε-phosphorylation sites cause changes in proliferation and filament assembly. Treatment of cells with an NF-κB inhibitor or 5-Aza-C, which allows re-expression of the Wnt inhibitor DKK3, led to a decrease in proliferation. These results suggest that inhibiting Wnt signaling removes the inhibition on GSK-3β and prevents activation of NF-κB, which decreases proliferation.

The Apelin-APJ system is transiently up-regulated in murine models of cardiac dysfunction. We have previously shown that Apelin-deficient mice subjected to aortic constriction suffer from severe fibrosis. In turn, we hypothesized that Apelin deficiency will also exaggerate the fibrosis phenotype post experimental myocardial infarction, associated with changes in fibroblast cell activity. Apelin-deficient and wildtype mice were randomly subjected to sham operation or left coronary artery ligation. Apelin deficiency worsened cardiac functionality, enhanced fibrosis-related gene expression and morphology, and enhanced vimentin intermediate filament expression, which may be involved in increasing fibroblast proliferation. MicroRNA target prediction softwares predict that apelin and vimentin 3 ’UTRs are potential targets of microRNA-378 regulation, and were confirmed with Luciferase reporter assays and western blot. Apelin up-regulation may be a useful strategy for attenuating unfavorable fibrosis through down-regulating vimentin-mediated adverse fibroblast activity. MicroRNA-378 regulation may be partly responsible for changes in apelin and vimentin expression.

The Apelin-APJ system is transiently up-regulated in murine models of cardiac dysfunction. We have previously shown that Apelin-deficient mice subjected to aortic constriction suffer from severe fibrosis. In turn, we hypothesized that Apelin deficiency will also exaggerate the fibrosis phenotype post experimental myocardial infarction, associated with changes in fibroblast cell activity. Apelin-deficient and wildtype mice were randomly subjected to sham operation or left coronary artery ligation. Apelin deficiency worsened cardiac functionality, enhanced fibrosis-related gene expression and morphology, and enhanced vimentin intermediate filament expression, which may be involved in increasing fibroblast proliferation. MicroRNA target prediction softwares predict that apelin and vimentin 3 ’UTRs are potential targets of microRNA-378 regulation, and were confirmed with Luciferase reporter assays and western blot. Apelin up-regulation may be a useful strategy for attenuating unfavorable fibrosis through down-regulating vimentin-mediated adverse fibroblast activity. MicroRNA-378 regulation may be partly responsible for changes in apelin and vimentin expression.