Chinese hamster ovary cells (CHO) were arrested with hydoxyurea at the beginning of DNA synthesis. Subsequent treatment with caffeine induced cells to bypass S-phase and undergo mitosis with unreplicated genomes (MUG). Treated cells built a normal spindle and distributed unattached kinetochores to daughter cells. To determine if MUG cells obey the metaphase checkpoint, we used immunoflourescence to detect and localize known metaphase checkpoint and motor proteins. In addition, the drug taxol was used to stabilize microtubules in MUG cells. The localization of CENP- E, the presence of anaphase A, taxol arrest, and taxol release acted in a similar manner as in controls. The localization of kinesin differed from the controls and that of MAD2 was inconclusive. These results imply that MUG kinetochores behave similarly to controls and probably have an operational metaphase checkpoint.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第17801号 / 生博第289号 / 新制||生||37(附属図書館) / 30608 / 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 松本 智裕, 教授 石川 冬木, 教授 西田 栄介 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Financial markets have been subjected to numerous regulations during the last two decades. MiFID2/MiFIR and MAD2/MAR are two extensive regulations that will apply on European level during 2016 - 2018. Both these regulations stress areas that are of relevance to trade surveillance. Trade surveillance systems are IT systems applied to the market for financial instruments to identify market abuse or other harmful patterns in participants’ trading activity. The purpose of this report is to map the market of trade surveillance technology in Stockholm, Sweden, and examine the impact on these actors in conjunction with the regulations. Since MiFID2/MiFIR and MAD2/MAR are extensive regulations, these were condensed to key points that were considered as relevant for surveillance.   This research is a qualitative study and data was gathered by interviews with market actors. A pre-study and a literature study were made. These were used as basis to construct an analytical framework for market dynamics, which was used as a descriptive concept to design interview questions, structure data and analyze results. The framework was named Market Dynamics Framework and considered the macro-environmental factors: Technology, Actors’ preferences, Market structure and Regulations.   The market was segmented in order to more accurately examine regulatory impact. Market actors were divided into four groups. The results were analyzed according to the framework and for each of the segmented market actor groups. Preference of surveillance solution was shown to be one distinct difference between every segment. A purchased surveillance system from a vendor was most common, and actors of smaller scale preferred to outsource.   The market is concluded to be prepared in terms of having systems and arrangement for monitoring trades in place. Expected impact is mostly related to new market structures and more detailed data of larger amounts. Increased capacity need for surveillance departments is expected in combination with a need for more advanced technologies; e.g. automatic screening of social media, efficient minimization of false positives, functionality coverage for a broader range of financial instruments.   This research introduces two concepts as descriptive frames, Market Dynamics Framework and a segmentation. These are proposed as methods when conducting a market analysis. A validation study for these methods is suggested as a possible topic for future studies.

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Os fungos dermatófitos são caracterizados pela capacidade de invadir os tecidos queratinizados, usando queratina como principal fonte de nutrientes. Ao invadirem os tecidos hospedeiros eles causam um tipo de micose chamada de dermatofitose. Dentre os dermatófitos, a espécie Trichophyton rubrum é o causador mais comum de tinea pedis, tinea unguium, tinea cruris e tinea corporis, sendo considerado um fungo antropofílico e cosmopolita. Entretanto, o conhecimento da interação deste patógeno com o hospedeiro é escasso. Devido à importância clínica de T. rubrum, este trabalho analisou a composição de aminoácidos de proteínas queratinas oriundas de Homo sapiens e Bos taurus e a expressão de genes envolvidos na degradação de queratina, metabolismo e controle do ciclo celular. O perfil de expressão dos genes sub3 e sub5 que codificam para queratinases, mad2 e mad2B que codificam proteínas envolvidas no controle da mitose, e os genes idh1 (subunidade regulatória da isocitrato desidrogenase - NAD+), idh2 (subunidade catalítica da isocitrato desidrogenase - NAD+), idhp (isocitrato desidrogenase - NADP+), icl (isocitrato liase) e meicl (metilisocitrato liase), que codificam proteínas envolvidas em vias metabólicas, foi analisado após o cultivo de T. rubrum em meios contendo glicose, glicina, glicose com glicina, ou queratina. A atividade queratinolítica foi avaliada após o cultivo de T. rubrum nesses meios e também em unha humana. Além disto, o efeito do antifúngico terbinafina na atividade queratinolítica e na expressão dos genes sub3 e sub5 foi analisado em meios contendo unha humana ou queratina. Após o cultivo de T. rubrum a atividade das enzimas IDH e IDHP componentes do ciclo de Krebs, ICL do ciclo do glioxilato e MeICL do ciclo do metilcitrato também foram avaliadas. Essas análises revelaram que a variação da fonte nutricional, do pH do meio de cultivo, e a presença/ausência do fator de transcrição PacC, proporcionam às diferentes linhagens de T. rubrum utilizadas neste trabalho (CBS, H6 e pacC-1) uma modulação diferenciada do acúmulo de transcritos dos genes, bem como da atividade queratinolítica e atividade das enzimas IDH, IDHP, ICL e MeICL. Outro fenômeno verificado foi que o antifúngico terbinafina afeta o acúmulo de transcritos dos genes sub3 e sub5, e a atividade queratinolítica de acordo com a fonte nutricional e a linhagem de T. rubrum. Além disso, em condições de estresse por pH alcalino a enzima IDHP é preferencialmente requisitada para manutenção do ciclo de Krebs, sendo que a linhagem mutante pacC-1 requisita em maior proporção as vias anapleróticas do que a linhagem selvagem H6. É importante enfatizar que a enzima ICL de T. rubrum possui atividade enzimática regulada por fosforilação, sendo sua atividade reduzida por esta modificação pós traducional. A identificação deste mecanismo de regulação da ICL por fosforilação e as análises dos transcritos de icl sugerem que em T. rubrum a regulação do fluxo de carbono no ciclo do glioxilato ocorra tanto em nível transcricional como pós-traducional. Os resultados obtidos possibilitaram ainda propor o primeiro modelo para T. rubrum que relaciona vias metabólicas, amparado por dados experimentais obtidos após o cultivo deste dermatófito em queratina. / Dermatophytes are fungi characterized by the ability to invade keratinized tissues using keratin as a major source of nutrients. When they invade host tissues they cause a type of ringworm called dermatophytosis. Among the dermatophytes, the species Trichophyton rubrum is the most common cause of tinea pedis, tinea unguium, tinea cruris, and tinea corporis, being considered an anthropophilic and cosmopolitan fungus. However, the knowledge of the interaction of this pathogen with the host is scarce. Due to the clinical importance of T. rubrum, this study analyzed the amino acid composition of keratin proteins derived from Homo sapiens and Bos taurus, and the expression of genes involved in keratin degradation, metabolism, and cell cycle control. The expression profile of the sub3 and sub5 genes, encoding keratinases, mad2 and mad2B, encoding proteins involved in the control of mitosis, and the genes idh1 (regulatory subunit of NAD+ -isocitrate dehydrogenase), idh2 (catalytic subunit of NAD+ -isocitrate dehydrogenase), idhp (NADP+ - isocitrate dehydrogenase), icl (isocitrate lyase), and meicl (metilisocitrate lyase), which encode proteins involved in metabolic pathways, was analyzed after cultivation of T. rubrum in media containing glucose, glycine, glucose and glycine, or keratin. The keratinolytic activity was evaluated after cultivation of T. rubrum in these media and also in human nail. Furthermore, the effect of the antifungal agent terbinafine in keratinase activity and sub3 and sub5 gene expression was analyzed in media containing human nail or keratin. After culturing T. rubrum the enzyme activity of IDH and IDHP, components of the Krebs cycle, ICL of the glyoxylate cycle, and MeICL of the metilcitrato cycle were also evaluated. These analyses revealed that the variation in the nutritional source, the pH of the medium, and the presence/absence of the transcription factor PacC, provide to the different T. rubrum strains used in this study (CBS, H6 and pacC-1) differential modulation of transcripts accumulation of the genes, as well as keratinolytic activity and enzymatic activities of IDH, IDHP, ICL and MeICL. Another phenomenon observed was that the antifungal terbinafine affects the accumulation of transcripts of the genes sub3, sub5, and the keratinolytic activity according to nutritional source and T. rubrum strain. In addition, in stress conditions by alkaline pH the IDHP enzyme is preferably required for maintenance of the Krebs cycle, and the mutant pacC-1 requests the anaplerotic pathways in greater proportion than the wild type strain H6. It is important to emphasize that T. rubrum ICL enzyme has its enzymatic activity regulated by phosphorylation, being reduced by this post translational modification. The identification of this regulation mechanism by phosphorylation of ICL and icl transcripts analysis suggest that in T. rubrum the carbon flux regulation in the glyoxylate cycle occurs at both transcriptional and post-translational levels. The results also made possible the proposition of the first model for T. rubrum correlating metabolic pathways, supported by experimental data obtained after cultivation of this dermatophyte in keratin.

Os fungos dermatófitos são caracterizados pela capacidade de invadir os tecidos queratinizados, usando queratina como principal fonte de nutrientes. Ao invadirem os tecidos hospedeiros eles causam um tipo de micose chamada de dermatofitose. Dentre os dermatófitos, a espécie Trichophyton rubrum é o causador mais comum de tinea pedis, tinea unguium, tinea cruris e tinea corporis, sendo considerado um fungo antropofílico e cosmopolita. Entretanto, o conhecimento da interação deste patógeno com o hospedeiro é escasso. Devido à importância clínica de T. rubrum, este trabalho analisou a composição de aminoácidos de proteínas queratinas oriundas de Homo sapiens e Bos taurus e a expressão de genes envolvidos na degradação de queratina, metabolismo e controle do ciclo celular. O perfil de expressão dos genes sub3 e sub5 que codificam para queratinases, mad2 e mad2B que codificam proteínas envolvidas no controle da mitose, e os genes idh1 (subunidade regulatória da isocitrato desidrogenase - NAD+), idh2 (subunidade catalítica da isocitrato desidrogenase - NAD+), idhp (isocitrato desidrogenase - NADP+), icl (isocitrato liase) e meicl (metilisocitrato liase), que codificam proteínas envolvidas em vias metabólicas, foi analisado após o cultivo de T. rubrum em meios contendo glicose, glicina, glicose com glicina, ou queratina. A atividade queratinolítica foi avaliada após o cultivo de T. rubrum nesses meios e também em unha humana. Além disto, o efeito do antifúngico terbinafina na atividade queratinolítica e na expressão dos genes sub3 e sub5 foi analisado em meios contendo unha humana ou queratina. Após o cultivo de T. rubrum a atividade das enzimas IDH e IDHP componentes do ciclo de Krebs, ICL do ciclo do glioxilato e MeICL do ciclo do metilcitrato também foram avaliadas. Essas análises revelaram que a variação da fonte nutricional, do pH do meio de cultivo, e a presença/ausência do fator de transcrição PacC, proporcionam às diferentes linhagens de T. rubrum utilizadas neste trabalho (CBS, H6 e pacC-1) uma modulação diferenciada do acúmulo de transcritos dos genes, bem como da atividade queratinolítica e atividade das enzimas IDH, IDHP, ICL e MeICL. Outro fenômeno verificado foi que o antifúngico terbinafina afeta o acúmulo de transcritos dos genes sub3 e sub5, e a atividade queratinolítica de acordo com a fonte nutricional e a linhagem de T. rubrum. Além disso, em condições de estresse por pH alcalino a enzima IDHP é preferencialmente requisitada para manutenção do ciclo de Krebs, sendo que a linhagem mutante pacC-1 requisita em maior proporção as vias anapleróticas do que a linhagem selvagem H6. É importante enfatizar que a enzima ICL de T. rubrum possui atividade enzimática regulada por fosforilação, sendo sua atividade reduzida por esta modificação pós traducional. A identificação deste mecanismo de regulação da ICL por fosforilação e as análises dos transcritos de icl sugerem que em T. rubrum a regulação do fluxo de carbono no ciclo do glioxilato ocorra tanto em nível transcricional como pós-traducional. Os resultados obtidos possibilitaram ainda propor o primeiro modelo para T. rubrum que relaciona vias metabólicas, amparado por dados experimentais obtidos após o cultivo deste dermatófito em queratina. / Dermatophytes are fungi characterized by the ability to invade keratinized tissues using keratin as a major source of nutrients. When they invade host tissues they cause a type of ringworm called dermatophytosis. Among the dermatophytes, the species Trichophyton rubrum is the most common cause of tinea pedis, tinea unguium, tinea cruris, and tinea corporis, being considered an anthropophilic and cosmopolitan fungus. However, the knowledge of the interaction of this pathogen with the host is scarce. Due to the clinical importance of T. rubrum, this study analyzed the amino acid composition of keratin proteins derived from Homo sapiens and Bos taurus, and the expression of genes involved in keratin degradation, metabolism, and cell cycle control. The expression profile of the sub3 and sub5 genes, encoding keratinases, mad2 and mad2B, encoding proteins involved in the control of mitosis, and the genes idh1 (regulatory subunit of NAD+ -isocitrate dehydrogenase), idh2 (catalytic subunit of NAD+ -isocitrate dehydrogenase), idhp (NADP+ - isocitrate dehydrogenase), icl (isocitrate lyase), and meicl (metilisocitrate lyase), which encode proteins involved in metabolic pathways, was analyzed after cultivation of T. rubrum in media containing glucose, glycine, glucose and glycine, or keratin. The keratinolytic activity was evaluated after cultivation of T. rubrum in these media and also in human nail. Furthermore, the effect of the antifungal agent terbinafine in keratinase activity and sub3 and sub5 gene expression was analyzed in media containing human nail or keratin. After culturing T. rubrum the enzyme activity of IDH and IDHP, components of the Krebs cycle, ICL of the glyoxylate cycle, and MeICL of the metilcitrato cycle were also evaluated. These analyses revealed that the variation in the nutritional source, the pH of the medium, and the presence/absence of the transcription factor PacC, provide to the different T. rubrum strains used in this study (CBS, H6 and pacC-1) differential modulation of transcripts accumulation of the genes, as well as keratinolytic activity and enzymatic activities of IDH, IDHP, ICL and MeICL. Another phenomenon observed was that the antifungal terbinafine affects the accumulation of transcripts of the genes sub3, sub5, and the keratinolytic activity according to nutritional source and T. rubrum strain. In addition, in stress conditions by alkaline pH the IDHP enzyme is preferably required for maintenance of the Krebs cycle, and the mutant pacC-1 requests the anaplerotic pathways in greater proportion than the wild type strain H6. It is important to emphasize that T. rubrum ICL enzyme has its enzymatic activity regulated by phosphorylation, being reduced by this post translational modification. The identification of this regulation mechanism by phosphorylation of ICL and icl transcripts analysis suggest that in T. rubrum the carbon flux regulation in the glyoxylate cycle occurs at both transcriptional and post-translational levels. The results also made possible the proposition of the first model for T. rubrum correlating metabolic pathways, supported by experimental data obtained after cultivation of this dermatophyte in keratin.