Interferon regulatory factor-1 (IRF-1) is a transcription factor and tumour suppressor, involved in many diverse cellular processes including immune responses and growth regulation. An interesting feature of IRF-1 is that it can both activate and repress gene expression, possibly by acting with co-activator or co-repressor proteins. In a previous phage display assay, a homologous peptide to the known repressor protein, zinc finger 350 (ZNF350), was found to bind to the C-terminus of IRF-1. ZNF350, also known as ZBRK1 (Zinc finger and BRCA1-interacting protein with KRAB domain-1), is a member of the Krüppel-associated box (KRAB)-containing zinc finger (KZF) proteins, which is a group of the widely distributed transcriptional repression proteins in mammals. ZNF350 has previously been shown to repress the expression of a number of genes including ANG1 and GADD45A, often in complex with other proteins. This study confirms the direct interaction between IRF-1 and ZNF350 and identifies key residues, including the LXXLL repression motif within the C-terminus of IRF-1, necessary for the binding interface. The two proteins have additionally been shown to interact within a cellular environment, shown by using techniques including immunoprecipitation and a proximity ligation assay. In addition, the ZNF350/IRF-1 complex formation appears to occur in the basal state of the cell, as opposed to in response to cellular stress such as viral infection or DNA damage. On the basis of ZNF350 being a negative regulator of transcription, a novel technique was developed to identify putative targets of both ZNF350 and IRF-1. This involved an initial bioinformatics screen using candidate IRF-1 binding site data obtained from CENTIPEDE, an algorithm that combines genome sequence information, with cell-specific experimental data to map bound TF binding sites. This allowed for the identification of novel target genes that contained the ZNF350 consensus binding site, GGGxxCAGxxxTTT, within close proximity to an IRF-1 consensus site, such as the immune response gene IL-12A. Lastly, a peptide phage display screen was combined with high-throughput sequencing to identify other potential binding partners of ZNF350 and perhaps help to understand the mechanism by which transcriptional repression is controlled by complex formation.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22595号 / 生博第428号 / 新制||生||57(附属図書館) / 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 藤田 尚志, 教授 松田 道行, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

Codirecteur de recherche: Dr Sylvain Meloche / Durant ces quatre dernières années, le champ de recherche concernant l’immunité innée a grandement été influencé par la découverte des IKK-related kinases, TBK1 et IKKi, deux kinases régulant l’activité des facteurs de transcription IRF-3/IRF-7 et NF-κB. Les kinases TBK1 and IKKi furent notamment démontrées comme étant responsables de la phosphorylation en C-terminal de IRF-3. Toutefois, l’identité des sites phosphoaccepteurs ciblés par ces kinases restait un sujet de controverse. En combinant la spectrométrie de masse aux essais de phosphorylation in vitro de His-IRF-3 par la kinase recombinante TBK1, nous démontrons que les sérines 396 et 402 sont directement phosphorylées par cette kinase. Nos analyses biochimiques révèlent également que la mutation S396A, localisée dans le cluster II, abolit l’homodimérisation, l’association à CBP et l’accumulation nucléaire de IRF-3. De façon intéressante, la mutation de la sérine 339, impliquée dans la stabilité de IRF-3, provoque également une perte d’association à CBP et de la dimérisation du facteur de transcription sans toutefois affecter la transactivation des gènes antiviraux en autant que la sérine 396 soit disponible pour accepter un événement de phosphorylation. Nos expériences de complémentation de MEFs IRF-3 KO révèlent la présence d’un mécanisme compensatoire impliquant la sérine 339 et la sérine 396 dans l’induction des IFN-stimulated genes (ISGs), ISG56 and ISG54. Globalement, les données présentées dans cette étude nous ont permis de reconsidérer le modèle d’activation du facteur de transcription IRF-3 actuellement proposé et d’y ajouter certaines subtilités. TRAF3 est également un médiateur central impliqué dans l’induction de la réponse interféron de type I. Cette fois, en couplant la spectrométrie de masse à la technique de purification protéique par affinité, nous avons identifié Sec16A et p115, deux protéines du système de transport vésiculaire ER-Golgi , comme étant des nouveaux partenaires protéiques de Flag-TRAF3. Nos expériences démontrent la localisation cellulaire de TRAF3 au niveau du système de transport vésiculaire. De plus, la diminution des niveaux d’expression de p115 ou Sec16A provoque une redistribution cellulaire de TRAF3 et affecte la réponse interféron suivant une stimulation par de l’ARN double brin. Nos résultats démontrent également une colocalisation de TRAF3 et TRADD au niveau du cis-Golgi ainsi qu’une interaction avec la protéine du translocon Sec61β médiée par l’intermédiaire de Sec5. De façon générale, nos données suggèrent que la localisation cellulaire de TRAF3 au niveau des compartiments de transport vésiculaire est requise afin d’obtenir une réponse antiviral optimale par la voie de signalisation cellulaire associée aux RIG-I-like RNA helicases, RIG-I et MDA5. Nos données appuient également le rôle potentiel précédemment suggéré de l’exocyste dans l’établissement d’une réponse antivirale. / Over the past four years, the field of the innate immune response has been highly influenced by the discovery of the IκB kinase (IKK)-related kinases, TBK1 and IKKi, which regulate the activity of IRF-3/IRF-7 and NF-κB transcription factors. The IKK-related kinases, TBK1 and IKKi, were recently shown to be responsible for the C-terminal phosphorylation of IRF-3. However, the identity of the phosphoacceptor site(s) targeted by these two kinases remains unclear. By combining mass spectrometry analysis to in vitro kinase assays using full length His-IRF3 as a substrate, we have demonstrated that serine 402 and serine 396 were directly targeted by TBK1. Analysis of Ser/Thr to Ala mutants revealed that S396A mutation, located in cluster II, abolished IRF-3 homodimerization, CBP association and nuclear accumulation. Interestingly, mutation of serine 339, which is involved in IRF-3 stability, also abrogated CBP association and dimerization without affecting gene transactivation as long as serine 396 remained available for phosphorylation. Complementation of MEFs IRF-3 KO also reveals a compensatory mechanism of serine 339 and serine 396 in the ability of IRF-3 to induce IFN-stimulated genes (ISGs) ISG56 and ISG54 expression. These data lead us to reconsider the current model of IRF-3 activation. TRAF3 is also a central mediator that is important for inducing type I interferon production in response to intracellular double-stranded RNA. By combining Flag-Affinity purification using Flag-TRAF3 as a bait to mass spectrometry, we have identified Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel TRAF3 interactors. We found that TRAF3 localizes to the ER-to-Golgi vesicular pathway and behaves like a cis-Golgi protein. Depletion of p115 or Sec16A disrupts the cis-Golgi cellular localization of TRAF3 and affects type I Interferon response following double-stranded RNA treatment. Furthermore, we demonstrate that TRAF3 colocalizes with TRADD at the cis-Golgi and also interacts with the translocon protein Sec61β in a Sec5 dependent manner. Together, our data suggest that the cellular localization of TRAF3 to the ER-to-Golgi transport compartments is required for an optimal RIG-I-like Helicases (RLH)-Cardif-dependent antiviral immune response. Our findings also highlight the potential role of the exocyst in the innate immune response.

Frente ao custo social dos quadros de abuso e dependência de substâncias psicoativas, diversos modelos explicativos têm sido propostos, sejam eles baseados em preceitos morais, religiosos, experiências de grupos de autoajuda ou na pesquisa científica. As Comunidades Terapêuticas (CTs) são uma modalidade de atendimento a usuários abusivos e dependentes de substâncias psicoativas que se baseiam em vários desses modelos, que se mostram bastante diversas em sua organização e nos serviços oferecidos, mas relativamente coesas em termos dos seus princípios. Diversos estudos têm tentado explicar os aspectos relacionados a esse modelo de tratamento, sugerindo sua eficácia. Apesar disso, existem poucas pesquisas compreensivas acerca das mudanças psicológicas que ocorrem nas pessoas que passam por esse tipo de instituição. No Brasil, a escassez de trabalhos acadêmicos sobre as CTs aponta para a necessidade de estudos sobre seus componentes terapêuticos, a fim de conhecer melhor os alcances e limites desse tipo de tratamento. A presente investigação visou estudar as mudanças psicológicas de pessoas que passaram por tratamento numa CT da região de Ribeirão Preto (SP), por meio de entrevistas de avaliação no início e no final de sua internação. Os participantes do estudo foram sete homens entre 21 e 35 anos de idade, nível socioeconômico médio baixo e dependentes de crack (predominantemente), cocaína e álcool. Para a realização das avaliações, foram utilizados um roteiro de entrevista semiestruturado, o Inventário Multifásico Minnesota de Personalidade, Improved Readability Form (MMPI-IRF) e cartões selecionados do Teste de Apercepção Temática (TAT). Os dados obtidos foram categorizados e analisados a partir de dimensões e domínios do processo de mudança na CT, conforme embasamento teórico de autores da área. O conjunto de dados foi interpretado a partir de teorias psicodinâmicas de personalidade. Os resultados mostram que o tratamento promoveu melhoras no funcionamento psicológico em graus diferentes para dois subgrupos de participantes, em todas as dimensões do processo de mudança. Os resultados são discutidos em termos das diferenças entre os subgrupos em aspectos como a estrutura e o nível de funcionamento da personalidade e qualidade da introjeção das experiências durante o tratamento. Ainda, são feitas considerações acerca do método de pesquisa adotado e dos alcances e limites do tratamento em CTs e sugestões para estudos futuros no Brasil. / Considering the high social cost of substance abuse and dependence, several models have been proposed, based on moral and/or religious principles, self-help groups experiences or scientific research. The Therapeutic Community (TC) is a treatment modality for substance abusers based in many of the models above, which shows a variety in terms of its organization and services offered, as well as a relatively cohesion of principles along these institutions. Several studies have been trying to explain the aspects related to such treatment modality, suggesting its efficacy. In spite of this, there are few comprehensive studies regarding the psychological changes people treated in the CT go through. In Brazil, the lack of academic studies on the TC suggests the need of comprehending its therapeutic components, in order to point out both flaws and successful points of such treatment modality. The present study aimed to study the psychological changes of people treated in a TC in the region of Ribeirão Preto (State of São Paulo Brazil), who were interviewed both in the beginning and the end of their stay in the institution. The participants were seven men, aged between 21 and 36 years old, average-low socioeconomic level who were predominantly crack abusers, with cocaine and alcohol dependents also included. In order to perform the assessment, the instruments used were a semi-structured interview guide, the Minnesota Multiphasic Personality Inventory Improved Readability Form (MMPI-IRF) and selected cards of the Thematic Apperception Test (TAT). All data was categorized and analyses using the dimensions and domains of the change process in the TC, according to the theoretical frame of authors in this field. Still, the data set was analyzed from a psychodynamic theoretical frame. The results show that the treatment promoted improvements in psychological functioning in different degrees for two subgroups of participants, along all dimensions of the change process. These results are discussed in terms of the differences observed in terms of level of psychological functioning and structure of the personality, as well as the introjection quality of treatment-related experiences. Still, considerations are made regarding the research method adopted and both high points and flaws of the treatment on the TC and the status of directions for future research in Brazil.

Den här uppsatsen behandlar det kommunikativa klassrummet och vad det har för eventuella fördelar. Uppsatsen utreder begreppen bundet och fritt tal och hur de påverkar elevers lärande. En annan punkt som utreds är talångest och vilken påverkan det kan ha på eleverna i det kommunikativa klassrummet. Resultatet visar vilka fördelar ett kommunikativt klassrum har. Genom det kommunikativa klassrummet blir eleverna mer aktiva och motiverade. Fokus ligger på eleverna eftersom klassrummet blir mer elevstyrt. Det fria talet, som låte releverna styra talet, är bättre för elevers lärande än det bundna där läraren styr. Ett problem som kan förhindra elevers lärande i det kommunikativa klassrummet är elevers talångest. Talångest påverkas av olika faktorer såsom rädslan att göra fel, dåligt självförtroende, kulturella faktorer och lärarens påverkan på eleverna. Detta kan motarbetas genom att läraren inte hakar upp sig på elevers misstag och att läraren kan skapa ett tryggt klassrum för eleverna.Genom att arbeta med ett kommunikativt klassrum öppnas nya vägar upp för eleven och fokus läggs mer på elevernas tal än på att läsa och skriva.

Durant ces quatre dernières années, le champ de recherche concernant l’immunité innée a grandement été influencé par la découverte des IKK-related kinases, TBK1 et IKKi, deux kinases régulant l’activité des facteurs de transcription IRF-3/IRF-7 et NF-κB. Les kinases TBK1 and IKKi furent notamment démontrées comme étant responsables de la phosphorylation en C-terminal de IRF-3. Toutefois, l’identité des sites phosphoaccepteurs ciblés par ces kinases restait un sujet de controverse. En combinant la spectrométrie de masse aux essais de phosphorylation in vitro de His-IRF-3 par la kinase recombinante TBK1, nous démontrons que les sérines 396 et 402 sont directement phosphorylées par cette kinase. Nos analyses biochimiques révèlent également que la mutation S396A, localisée dans le cluster II, abolit l’homodimérisation, l’association à CBP et l’accumulation nucléaire de IRF-3. De façon intéressante, la mutation de la sérine 339, impliquée dans la stabilité de IRF-3, provoque également une perte d’association à CBP et de la dimérisation du facteur de transcription sans toutefois affecter la transactivation des gènes antiviraux en autant que la sérine 396 soit disponible pour accepter un événement de phosphorylation. Nos expériences de complémentation de MEFs IRF-3 KO révèlent la présence d’un mécanisme compensatoire impliquant la sérine 339 et la sérine 396 dans l’induction des IFN-stimulated genes (ISGs), ISG56 and ISG54. Globalement, les données présentées dans cette étude nous ont permis de reconsidérer le modèle d’activation du facteur de transcription IRF-3 actuellement proposé et d’y ajouter certaines subtilités. TRAF3 est également un médiateur central impliqué dans l’induction de la réponse interféron de type I. Cette fois, en couplant la spectrométrie de masse à la technique de purification protéique par affinité, nous avons identifié Sec16A et p115, deux protéines du système de transport vésiculaire ER-Golgi , comme étant des nouveaux partenaires protéiques de Flag-TRAF3. Nos expériences démontrent la localisation cellulaire de TRAF3 au niveau du système de transport vésiculaire. De plus, la diminution des niveaux d’expression de p115 ou Sec16A provoque une redistribution cellulaire de TRAF3 et affecte la réponse interféron suivant une stimulation par de l’ARN double brin. Nos résultats démontrent également une colocalisation de TRAF3 et TRADD au niveau du cis-Golgi ainsi qu’une interaction avec la protéine du translocon Sec61β médiée par l’intermédiaire de Sec5. De façon générale, nos données suggèrent que la localisation cellulaire de TRAF3 au niveau des compartiments de transport vésiculaire est requise afin d’obtenir une réponse antiviral optimale par la voie de signalisation cellulaire associée aux RIG-I-like RNA helicases, RIG-I et MDA5. Nos données appuient également le rôle potentiel précédemment suggéré de l’exocyste dans l’établissement d’une réponse antivirale. / Over the past four years, the field of the innate immune response has been highly influenced by the discovery of the IκB kinase (IKK)-related kinases, TBK1 and IKKi, which regulate the activity of IRF-3/IRF-7 and NF-κB transcription factors. The IKK-related kinases, TBK1 and IKKi, were recently shown to be responsible for the C-terminal phosphorylation of IRF-3. However, the identity of the phosphoacceptor site(s) targeted by these two kinases remains unclear. By combining mass spectrometry analysis to in vitro kinase assays using full length His-IRF3 as a substrate, we have demonstrated that serine 402 and serine 396 were directly targeted by TBK1. Analysis of Ser/Thr to Ala mutants revealed that S396A mutation, located in cluster II, abolished IRF-3 homodimerization, CBP association and nuclear accumulation. Interestingly, mutation of serine 339, which is involved in IRF-3 stability, also abrogated CBP association and dimerization without affecting gene transactivation as long as serine 396 remained available for phosphorylation. Complementation of MEFs IRF-3 KO also reveals a compensatory mechanism of serine 339 and serine 396 in the ability of IRF-3 to induce IFN-stimulated genes (ISGs) ISG56 and ISG54 expression. These data lead us to reconsider the current model of IRF-3 activation. TRAF3 is also a central mediator that is important for inducing type I interferon production in response to intracellular double-stranded RNA. By combining Flag-Affinity purification using Flag-TRAF3 as a bait to mass spectrometry, we have identified Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel TRAF3 interactors. We found that TRAF3 localizes to the ER-to-Golgi vesicular pathway and behaves like a cis-Golgi protein. Depletion of p115 or Sec16A disrupts the cis-Golgi cellular localization of TRAF3 and affects type I Interferon response following double-stranded RNA treatment. Furthermore, we demonstrate that TRAF3 colocalizes with TRADD at the cis-Golgi and also interacts with the translocon protein Sec61β in a Sec5 dependent manner. Together, our data suggest that the cellular localization of TRAF3 to the ER-to-Golgi transport compartments is required for an optimal RIG-I-like Helicases (RLH)-Cardif-dependent antiviral immune response. Our findings also highlight the potential role of the exocyst in the innate immune response. / Codirecteur de recherche: Dr Sylvain Meloche

Frente ao custo social dos quadros de abuso e dependência de substâncias psicoativas, diversos modelos explicativos têm sido propostos, sejam eles baseados em preceitos morais, religiosos, experiências de grupos de autoajuda ou na pesquisa científica. As Comunidades Terapêuticas (CTs) são uma modalidade de atendimento a usuários abusivos e dependentes de substâncias psicoativas que se baseiam em vários desses modelos, que se mostram bastante diversas em sua organização e nos serviços oferecidos, mas relativamente coesas em termos dos seus princípios. Diversos estudos têm tentado explicar os aspectos relacionados a esse modelo de tratamento, sugerindo sua eficácia. Apesar disso, existem poucas pesquisas compreensivas acerca das mudanças psicológicas que ocorrem nas pessoas que passam por esse tipo de instituição. No Brasil, a escassez de trabalhos acadêmicos sobre as CTs aponta para a necessidade de estudos sobre seus componentes terapêuticos, a fim de conhecer melhor os alcances e limites desse tipo de tratamento. A presente investigação visou estudar as mudanças psicológicas de pessoas que passaram por tratamento numa CT da região de Ribeirão Preto (SP), por meio de entrevistas de avaliação no início e no final de sua internação. Os participantes do estudo foram sete homens entre 21 e 35 anos de idade, nível socioeconômico médio baixo e dependentes de crack (predominantemente), cocaína e álcool. Para a realização das avaliações, foram utilizados um roteiro de entrevista semiestruturado, o Inventário Multifásico Minnesota de Personalidade, Improved Readability Form (MMPI-IRF) e cartões selecionados do Teste de Apercepção Temática (TAT). Os dados obtidos foram categorizados e analisados a partir de dimensões e domínios do processo de mudança na CT, conforme embasamento teórico de autores da área. O conjunto de dados foi interpretado a partir de teorias psicodinâmicas de personalidade. Os resultados mostram que o tratamento promoveu melhoras no funcionamento psicológico em graus diferentes para dois subgrupos de participantes, em todas as dimensões do processo de mudança. Os resultados são discutidos em termos das diferenças entre os subgrupos em aspectos como a estrutura e o nível de funcionamento da personalidade e qualidade da introjeção das experiências durante o tratamento. Ainda, são feitas considerações acerca do método de pesquisa adotado e dos alcances e limites do tratamento em CTs e sugestões para estudos futuros no Brasil. / Considering the high social cost of substance abuse and dependence, several models have been proposed, based on moral and/or religious principles, self-help groups experiences or scientific research. The Therapeutic Community (TC) is a treatment modality for substance abusers based in many of the models above, which shows a variety in terms of its organization and services offered, as well as a relatively cohesion of principles along these institutions. Several studies have been trying to explain the aspects related to such treatment modality, suggesting its efficacy. In spite of this, there are few comprehensive studies regarding the psychological changes people treated in the CT go through. In Brazil, the lack of academic studies on the TC suggests the need of comprehending its therapeutic components, in order to point out both flaws and successful points of such treatment modality. The present study aimed to study the psychological changes of people treated in a TC in the region of Ribeirão Preto (State of São Paulo Brazil), who were interviewed both in the beginning and the end of their stay in the institution. The participants were seven men, aged between 21 and 36 years old, average-low socioeconomic level who were predominantly crack abusers, with cocaine and alcohol dependents also included. In order to perform the assessment, the instruments used were a semi-structured interview guide, the Minnesota Multiphasic Personality Inventory Improved Readability Form (MMPI-IRF) and selected cards of the Thematic Apperception Test (TAT). All data was categorized and analyses using the dimensions and domains of the change process in the TC, according to the theoretical frame of authors in this field. Still, the data set was analyzed from a psychodynamic theoretical frame. The results show that the treatment promoted improvements in psychological functioning in different degrees for two subgroups of participants, along all dimensions of the change process. These results are discussed in terms of the differences observed in terms of level of psychological functioning and structure of the personality, as well as the introjection quality of treatment-related experiences. Still, considerations are made regarding the research method adopted and both high points and flaws of the treatment on the TC and the status of directions for future research in Brazil.

No description available.

Submitted by Tommaso Momoli (tommaso.momoli@gmail.com) on 2017-03-29T04:51:52Z No. of bitstreams: 1 Tommaso.Momoli Thesis FGV.pdf: 2459609 bytes, checksum: 56072be31042eb761414eba91a983961 (MD5) / Approved for entry into archive by Josineide da Silva Santos Locatelli (josineide.locatelli@fgv.br) on 2017-03-29T11:04:04Z (GMT) No. of bitstreams: 1 Tommaso.Momoli Thesis FGV.pdf: 2459609 bytes, checksum: 56072be31042eb761414eba91a983961 (MD5) / Made available in DSpace on 2017-03-29T12:16:39Z (GMT). No. of bitstreams: 1 Tommaso.Momoli Thesis FGV.pdf: 2459609 bytes, checksum: 56072be31042eb761414eba91a983961 (MD5) Previous issue date: 2017-01-25 / This is a study regarding the impact of the index investments in the Commodity Future Market. The models applied, focus on the Causal Analysis and the Impulse Response Function through an orthogonalisation of the Vector of Auto Regression (SVAR), this allow to extract lead/lag correlation between the Index and First nearby Return for different Futures Sectors and in addition response to shocks in different equation. The study is divided in three different period, to reflect before and after the Financialization and then after the introduction in the market of the new generation of commodity Indexes. The results show a different behaviors of the parameters throughout time with a particular emphasis for the most traded Commodities to lead the others. / Trata-se de um estudo sobre o impacto dos investimentos em índices no mercado futuro de commodities. Os modelos aplicados, enfocam a Análise Causal e a Função de Resposta ao Impulso através de uma ortogonalização do Vetor de Auto Regressão (SVAR), permitindo extrair a correlação lead / lag entre o Índice e o Primeiro Retorno próximo para diferentes Setores Futuros e, A choques em diferentes equações. O estudo é dividido em três períodos diferentes, para refletir antes e depois da Financialização e, em seguida, após a introdução no mercado da nova geração de índices de commodities. Os resultados mostram um comportamento diferente dos parâmetros ao longo do tempo com uma ênfase particular para os Commodities mais negociados para liderar os outros.