OBJETIVOS: Correlacionar o nível de expressão das proteínas reguladoras do complemento (Cregs) CD55, CD59, CD35 e CD46 nos linfócitos B em uma coorte de pacientes com artrite reumatoide (AR) iniciando terapia com rituximabe (RTX) com a depleção e tempo de repopulação destas células no sangue periférico, associando, ainda, o nível de expressão destas proteínas à resposta clínica conforme os critérios do Colégio Americano de Reumatologia (ACR). MÉTODOS: Dez pacientes com AR receberam duas infusões de RTX 1g separadas por intervalo de 14 dias. Análises imunofenotípicas para detecção de CD19, CD55, CD59, CD35 e CD46 foram realizadas pré-infusão e após 1, 2, 6, 12, 18 e 24 meses ou até recaída clínica. Depleção de linfócitos B no sangue periférico foi definida como valor de CD19 menor que 0,005x109/l no total de leucócitos. Resposta ACR20 em 6 meses foi considerada positiva e recaída clínica foi definida como perda dessa resposta. A não obtenção de ACR20 em 6 meses foi considerada falha de resposta ao tratamento. RESULTADOS: Dez mulheres com mediana de 49 anos e DAS28 basal de 5,6; nove delas soropositivas para fator reumatoide foram acompanhadas. Repopulação de linfócitos B ocorreu em 2 meses em cinco pacientes e em 6 meses nas demais. Houve correlação entre o nível de expressão basal de CD46 com o tempo de repopulação (coeficiente de correlação de -0,733, p=0,016). Tendência semelhante foi detectada com CD35, porém sem significância estatística (coeficiente de correlação de -0,522, p=0,12). Não houve associação entre resposta clínica e expressão das proteínas regulatórias do complemento. CONCLUSÕES: Expressão aumentada de CD46 foi preditora de repopulação mais precoce de linfócitos B em pacientes com AR tratados com RTX. Estudos com amostras maiores serão necessários para avaliar associação das demais Cregs. / OBJECTIVES: To correlate the level of expression of the complement regulatory proteins (Cregs) CD55, CD59, CD35, and CD46 on B cells from a cohort of 10 patients with rheumatoid arthritis (RA) initiating treatment with rituximab (RTX) with the depletion and time of repopulation of these cells in peripheral blood, additionally correlating the level of expression of these proteins to clinical response according to the criteria of the American College of Rheumatology (ACR). METHODS: Ten patients with RA received two 1g RTX infusions within 14 day intervals. Immunophenotype analyses for CD19, CD55, CD59, CD35 and CD46 were performed before the infusion and at 1, 2, 6, 12, 18 and 24 months or until recurrence. Depletion of B cells on peripheral blood was defined as the CD19 count < 0.005x109/l. ACR20 at 6 months was considered a good clinical response and recurrence was defined as loss of this response. RESULTS: Ten women with median age of 49 years and basal DAS28 of 5.6 were monitored; 9 were seropositive for rheumatoid factor. Repopulation of B cells occurred within 2 months in 5 patients and within 6 months in the remaining women. There was correlation between the basal level of CD46 expression and the time to achieve repopulation (correlation coefficient -0.733, p=0.016). A similar trend was observed with the CD35, but without statistical significance (correlation coefficient - 0.522, p=012). There was no association between clinical response and the complement regulatory proteins. CONCLUSIONS: Increased CD46 expression predicted earlier repopulation of B cells in RA patients treated with RTX. Studies with larger samples are necessary to assess the association with the other Cregs.

OBJETIVOS: Correlacionar o nível de expressão das proteínas reguladoras do complemento (Cregs) CD55, CD59, CD35 e CD46 nos linfócitos B em uma coorte de pacientes com artrite reumatoide (AR) iniciando terapia com rituximabe (RTX) com a depleção e tempo de repopulação destas células no sangue periférico, associando, ainda, o nível de expressão destas proteínas à resposta clínica conforme os critérios do Colégio Americano de Reumatologia (ACR). MÉTODOS: Dez pacientes com AR receberam duas infusões de RTX 1g separadas por intervalo de 14 dias. Análises imunofenotípicas para detecção de CD19, CD55, CD59, CD35 e CD46 foram realizadas pré-infusão e após 1, 2, 6, 12, 18 e 24 meses ou até recaída clínica. Depleção de linfócitos B no sangue periférico foi definida como valor de CD19 menor que 0,005x109/l no total de leucócitos. Resposta ACR20 em 6 meses foi considerada positiva e recaída clínica foi definida como perda dessa resposta. A não obtenção de ACR20 em 6 meses foi considerada falha de resposta ao tratamento. RESULTADOS: Dez mulheres com mediana de 49 anos e DAS28 basal de 5,6; nove delas soropositivas para fator reumatoide foram acompanhadas. Repopulação de linfócitos B ocorreu em 2 meses em cinco pacientes e em 6 meses nas demais. Houve correlação entre o nível de expressão basal de CD46 com o tempo de repopulação (coeficiente de correlação de -0,733, p=0,016). Tendência semelhante foi detectada com CD35, porém sem significância estatística (coeficiente de correlação de -0,522, p=0,12). Não houve associação entre resposta clínica e expressão das proteínas regulatórias do complemento. CONCLUSÕES: Expressão aumentada de CD46 foi preditora de repopulação mais precoce de linfócitos B em pacientes com AR tratados com RTX. Estudos com amostras maiores serão necessários para avaliar associação das demais Cregs. / OBJECTIVES: To correlate the level of expression of the complement regulatory proteins (Cregs) CD55, CD59, CD35, and CD46 on B cells from a cohort of 10 patients with rheumatoid arthritis (RA) initiating treatment with rituximab (RTX) with the depletion and time of repopulation of these cells in peripheral blood, additionally correlating the level of expression of these proteins to clinical response according to the criteria of the American College of Rheumatology (ACR). METHODS: Ten patients with RA received two 1g RTX infusions within 14 day intervals. Immunophenotype analyses for CD19, CD55, CD59, CD35 and CD46 were performed before the infusion and at 1, 2, 6, 12, 18 and 24 months or until recurrence. Depletion of B cells on peripheral blood was defined as the CD19 count < 0.005x109/l. ACR20 at 6 months was considered a good clinical response and recurrence was defined as loss of this response. RESULTS: Ten women with median age of 49 years and basal DAS28 of 5.6 were monitored; 9 were seropositive for rheumatoid factor. Repopulation of B cells occurred within 2 months in 5 patients and within 6 months in the remaining women. There was correlation between the basal level of CD46 expression and the time to achieve repopulation (correlation coefficient -0.733, p=0.016). A similar trend was observed with the CD35, but without statistical significance (correlation coefficient - 0.522, p=012). There was no association between clinical response and the complement regulatory proteins. CONCLUSIONS: Increased CD46 expression predicted earlier repopulation of B cells in RA patients treated with RTX. Studies with larger samples are necessary to assess the association with the other Cregs.

OBJETIVOS: Correlacionar o nível de expressão das proteínas reguladoras do complemento (Cregs) CD55, CD59, CD35 e CD46 nos linfócitos B em uma coorte de pacientes com artrite reumatoide (AR) iniciando terapia com rituximabe (RTX) com a depleção e tempo de repopulação destas células no sangue periférico, associando, ainda, o nível de expressão destas proteínas à resposta clínica conforme os critérios do Colégio Americano de Reumatologia (ACR). MÉTODOS: Dez pacientes com AR receberam duas infusões de RTX 1g separadas por intervalo de 14 dias. Análises imunofenotípicas para detecção de CD19, CD55, CD59, CD35 e CD46 foram realizadas pré-infusão e após 1, 2, 6, 12, 18 e 24 meses ou até recaída clínica. Depleção de linfócitos B no sangue periférico foi definida como valor de CD19 menor que 0,005x109/l no total de leucócitos. Resposta ACR20 em 6 meses foi considerada positiva e recaída clínica foi definida como perda dessa resposta. A não obtenção de ACR20 em 6 meses foi considerada falha de resposta ao tratamento. RESULTADOS: Dez mulheres com mediana de 49 anos e DAS28 basal de 5,6; nove delas soropositivas para fator reumatoide foram acompanhadas. Repopulação de linfócitos B ocorreu em 2 meses em cinco pacientes e em 6 meses nas demais. Houve correlação entre o nível de expressão basal de CD46 com o tempo de repopulação (coeficiente de correlação de -0,733, p=0,016). Tendência semelhante foi detectada com CD35, porém sem significância estatística (coeficiente de correlação de -0,522, p=0,12). Não houve associação entre resposta clínica e expressão das proteínas regulatórias do complemento. CONCLUSÕES: Expressão aumentada de CD46 foi preditora de repopulação mais precoce de linfócitos B em pacientes com AR tratados com RTX. Estudos com amostras maiores serão necessários para avaliar associação das demais Cregs. / OBJECTIVES: To correlate the level of expression of the complement regulatory proteins (Cregs) CD55, CD59, CD35, and CD46 on B cells from a cohort of 10 patients with rheumatoid arthritis (RA) initiating treatment with rituximab (RTX) with the depletion and time of repopulation of these cells in peripheral blood, additionally correlating the level of expression of these proteins to clinical response according to the criteria of the American College of Rheumatology (ACR). METHODS: Ten patients with RA received two 1g RTX infusions within 14 day intervals. Immunophenotype analyses for CD19, CD55, CD59, CD35 and CD46 were performed before the infusion and at 1, 2, 6, 12, 18 and 24 months or until recurrence. Depletion of B cells on peripheral blood was defined as the CD19 count < 0.005x109/l. ACR20 at 6 months was considered a good clinical response and recurrence was defined as loss of this response. RESULTS: Ten women with median age of 49 years and basal DAS28 of 5.6 were monitored; 9 were seropositive for rheumatoid factor. Repopulation of B cells occurred within 2 months in 5 patients and within 6 months in the remaining women. There was correlation between the basal level of CD46 expression and the time to achieve repopulation (correlation coefficient -0.733, p=0.016). A similar trend was observed with the CD35, but without statistical significance (correlation coefficient - 0.522, p=012). There was no association between clinical response and the complement regulatory proteins. CONCLUSIONS: Increased CD46 expression predicted earlier repopulation of B cells in RA patients treated with RTX. Studies with larger samples are necessary to assess the association with the other Cregs.

Chez les mammifères l’initiation de la traduction et, plus particulièrement, la formation du complexe eIF4F, est principalement régulée par la protéine kinase TOR (Target of rapamycin). Cette voie de régulation fait intervenir les protéines 4E-BP (eIF4E-binding proteins) dont l’activité est modulée par la phosphorylation par TOR. Sous leur forme non-phosphorylée, les 4E-BP se lient au facteur d’initiation eIF4E, empêchent son recrutement dans le complexe eIF4F et inhibent ainsi l’initiation de la traduction. Phosphorylées par TOR, les 4E-BP perdent leur affinité pour eIF4E et sont remplacées par eIF4G ce qui active la traduction. La régulation de l’initiation de la traduction par TOR via 4E-BP a été bien décrite dans plusieurs modèles eucaryotes, tels que la levure, les insectes et les mammifères, mais reste encore obscure chez les plantes. Les recherches réalisées au cours de ma thèse ont permis l’identification de deux protéines homologues de 4E-BP chez Arabidopsis. Ces protéines, que nous avons appelées ToRP1 et ToRP2 (TOR Regulatory Proteins), sont caractérisées par la présence d’un motif consensus indispensable pour la liaison à eIF4E, et qui existe chez les protéines 4E-BP des mammifères ainsi que chez eIF4G et eIFiso4G d’Arabidopsis. La protéine ToRP1 est capable d’interagir spécifiquement avec eIF4E, mais aussi avec TOR via son extrémité N-terminale en système double-hybride de levure. ToRP1 et ToRP2 ont également été caractérisées comme étant des cibles directement phosphorylées par TOR chez Arabidopsis. Deux sérines, en position 49 et 89 dans la protéine ToRP1, ont été identifiées comme des sites potentiels de cette phosphorylation. De plus, l’état de phosphorylation de ces sites affecte l’interaction avec eIF4E en système double-hybride de levure. Par ailleurs, des plants d’Arabidopsis déficients en ToRP1 et ToRP2 renforcent la traduction strictement coiffe-dépendante de l’ARNm CYCB1;1, alors que la surexpression de ToRP1 ou de ToRP2 réprime sa traduction. Ces résultats suggèrent donc que les protéines ToRP, identifiées chez Arabidopsis, sont de nouvelles cibles directes de TOR, qui, par leur phosphorylation, régule l’initiation de la traduction coiffe-dépendante. / The target of rapamycin (TOR) is an evolutionarily conserved kinase that is a critical sensor of nutritional and cellular energy and a major regulator of cell growth. TOR controls cap-dependent translation initiation, in particular the assembly of the eIF4F complex, by modulating the activity of eIF4E-binding proteins (4E-BPs). In their unphosphorylated state 4E-BP proteins sequester eIF4E and repress translation. Upon phosphorylation by TOR, 4E-BPs have a low affinity binding to eIF4E and are replaced by eIF4G thus activating translation initiation. 4E-BPs have been discovered in yeast and mammals but remain to be obscure in plants. Here, we identified and characterized two Arabidopsis proteins termed TOR Regulatory Proteins (ToRPs 1 and 2) that display some characteristics of mammalian 4E-BPs. ToRP1 and ToRP2 contain a canonical eIF4E-binding motif (4E-BM) found in mammalian 4E-BPs and Arabidopsis eIF4G and eIFiso4G. ToRP1 interacts with eIF4E, and, surprisingly, the N-terminal HEAT domain of TOR in the yeast two-hybrid system. ToRP1 and ToRP2 are highly phosphorylated at several phosphorylation sites in TOR-dependent manner in planta. Two of these phosphorylation sites have been identified as—S49 and S89—their phosphorylation status modulates ToRP1 binding to eIF4E in the yeast two-hybrid system. In plant protoplasts, ToRP2 can function as translation repressor of mRNAs that are strictly cap-dependent. Our results suggest that ToRPs can specifically bind the Arabidopsis cap-binding proteins (eIF4E/eIFiso4E) and regulate translation initiation under the control of TOR

Thesis (Ph. D.)--University of Oklahoma. / Includes bibliographical references.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-15T14:17:14Z No. of bitstreams: 1 Jaqueline Goes de Jesus. Estudo...2014.pdf: 4655999 bytes, checksum: 99ee2ef801cc69dd80d0a344e8f01be2 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-15T14:18:33Z (GMT) No. of bitstreams: 1 Jaqueline Goes de Jesus. Estudo...2014.pdf: 4655999 bytes, checksum: 99ee2ef801cc69dd80d0a344e8f01be2 (MD5) / Made available in DSpace on 2016-02-15T14:18:33Z (GMT). No. of bitstreams: 1 Jaqueline Goes de Jesus. Estudo...2014.pdf: 4655999 bytes, checksum: 99ee2ef801cc69dd80d0a344e8f01be2 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / As ORFs I e IV do genoma do HTLV-1 codificam, respectivamente, as proteínas p12/p8 (acessória) e Tax (regulatória). p12/p8, de 99 aminoácidos, pode ser clivada em sua extremidade amino terminal gerando a proteína p8. A primeira clivagem proteolítica de p12 remove o sinal de retenção ao RE, enquanto a segunda clivagem, gera o produto de 8kDa, referido como p8. p12 localiza-se no sistema de endomembranes, residindo em RE e aparato de Golgi, enquanto p8 dirige-se para a membrana plasmática, onde é recrutada para a sinapse imunológica, através da ligação com o receptor de células T (TCR), além de participar da sinapse virológica e da formação de conduítes. A proteína Tax, por outro lado, atua como transativador transcricional do HTLV-1, sendo referida também na indução da expressão de diversos genes celulares, aumentando a proliferação e a migração das células infectadas. Na via de transporte de vesículas secretórias, vesículas produzidas como pós-Golgi são transportadas ao longo do citoesqueleto por motores celulares. A Miosina-Va, um motor não convencional, transporta diversos cargos, incluindo vesículas secretórias, vesículas sinápticas e de retículo endoplasmático. Outra proteína relacionada ao citoesqueleto é a Paxilina, que atua como molécula adaptadora nas adesões focais e cuja expressão está aumentada em indivíduos TSP-HAM. Na tentativa de compreender se Tax influencia no aumento da expressão de Paxilina e, paralelamente, se p8 trafega a partir de Golgi em direção à membrana, de maneira dependente de Miosina-Va, células de linhagem foram transfectadas, com o plasmídeo que expressa Tax ou com plasmídeos que expressam variantes da proteína p12 (pMEp12) fusionada a um tag de HA (hemaglutinina de influenza) e que expressam porções da Miosina-Va, incluindo a cauda completa neuronal conjugada com GFP (MyoVa FTNeu-eGFP), que funciona como dominante negativo e compete com a Miosina-Va constitutiva pelos seus ligantes intracelulares. A localização intracelular das proteínas foi realizada por ensaio de imunofluorescência indireta utilizando anticorpos contra a Paxilina ou contra o tag de HA e a cauda medial da Miosina-Va. Técnicas de microscopia confocal e obtenção de imagens foram realizadas utilizando o microscópio Zeiss LSM 780 (Carl Zeiss Optical, Chester, Va.) e o software Adobe Photoshop CC. Surpreendentemente, nas células que expressavam Tax, a expressão de Paxilina, avaliada por imunofluorescência, foi menor, necessitando de novos ensaios para confirmação dos resultados. Em relação à p12/p8, foi observada a sua sub-localização celular como já descrito na literatura, apresentando-se na região perinuclear (RE e aparato de Golgi), e co-localização entre p12/p8 e Miosina-Va, embora apenas quando o dominante negativo MyoVa FTNeu-eGFP foi expresso simultaneamente com as variantes de p12, a localização de p12/p8 mostrou-se alterada, de pontos dispersos por todo o citoplasma e superfície celular para apresentar-se em forma de grumos agregados independentemente da variante de p12 expressa, sugerindo que a Miosina-Va desempenha um importante papel no tráfego de p8 partindo de Golgi até a superfície celular. / HTLV-1 ORFs I and IV encode respectively p12/p8 (accessory protein) and Tax (regulatory protein). The 99 amino acid p12 protein can be proteolytically cleaved at the amino terminus to generate the p8 protein. The first proteolytic cleavage removes the ER retention/retrieval signal at the amino terminus of p12, while the second cleavage generates the p8 protein. The p12 protein localizes to cellular endomembranes, within the ER and Golgi apparatus, while p8 traffics to lipid rafts at the cell surface and is recruited to the immunological synapse upon T-cell receptor (TCR) ligation, virological synapse and conduits. Tax on the other hand acts as viral transactivator and induces expression of many cellular genes, increasing proliferation and migration of infected cells. In secretory vesicle transport, vesicles produced as post-Golgi are moved along the cytoskeleton by motor proteins. The unconventional myosin motor, Myosin-Va, moves several cargoes including secretory vesicles, synaptic vesicles, and the endoplasmic reticulum. Another cytoskeleton associated protein is Paxillin, an adapter on focal adhesions which expression is increased in TSP-HAM patients. To understand if Tax play a role on increased expression of Paxillin and parallel if p8 traffics from Golgi apparatus to cell surface on a myosin-Va dependent manner, lineage cells were transfected with Tax plasmids or pMEp12 plasmids which express variants (p12WT, p12Δ29 and p12G29S) of the fusion protein of HTLV-1 p12 tagged with the influenza hemagglutinin (HA1) tag and with the Myo-Va plasmids including full-tail neuronal-eGFP conjugated (MyoVa FTNeu-eGFP) plasmid which expresses a negative dominant of myosin Va and competes for intracellular ligands with cellular putative myosin. Proteins intracellular localization were analyzed by indirect immunofluorescence assay using antibodies against Paxillin or the HA-tag and the Myo-Va protein. Confocal microscopy and image collection was performed by using a Zeiss LSM 780 microscope (Carl Zeiss Optical, Chester, Va.) with Adobe Photoshop CC software. Surprisingly in Tax-expressing cells Paxillin fluorescence was decreased requiring another assay to confirm this find. It was reported that p12 expression in Jurkat T, as previous described, was shown in perinuclear region which might be RE and Golgi apparatus and that p12/p8 and MyoVa proteins colocalizes in lineage cells, however only when MyoVa FTNeu-eGFP was simultaneously expressed with pMEp12 plasmids, p12/p8 localization showed to be altered from dots dispersed all over cytoplasm and cell surface to form cytoplasmic aggregates independently on variant of p12 expressed, suggesting that myosin Va plays an important role on traffics of p8 from Golgi to cell surface.

L'utilisation des ressources en fer et les variations du potentiel redox sont des processus impliqués dans la prolifération et la différenciation cellulaire. Ils participent à l'hématopoïèse normale et leur dérégulation peut être associée à des conditions pathologiques. Les hémopathies, telles que la leucémie aiguë myéloïde (LAM), témoignent du lien entre disponibilité en fer, signalisation redox et leucémogenèse. La déplétion en fer induit un arrêt de la prolifération suivi de la mort cellulaire, et pour des cellules primaires leucémiques (blastes) de patients LAM, elle peut conduire à un réengagement de la différenciation vers la lignée monocytaire. Cependant, les besoins en fer des clones leucémiques restent mal définis. Dans les cellules animales, le cœur du réseau de régulation du fer est organisé à travers le système régulateur IRE-IRP. Les Iron Regulatory Proteins (IRP), agissent sur la traduction de nombreuses protéines impliquées dans la gestion du fer par interaction avec les Iron Responsive Elements (IRE) localisés sur les régions non codantes des ARN messager (ARNm) régulés. A partir de lignées cellulaires leucémiques (KG1, K562), de blastes de patients LAM et de progéniteurs CD34+ contrôles issus de sang de cordon et de moelle osseuse de donneurs sains, le statut du système de gestion cellulaire du fer a été caractérisé pour les premières étapes de l'hématopoïèse normale et pathologique. A travers la manipulation des apports cellulaires en fer, notamment par l'utilisation de chélateurs à usage thérapeutique, la réponse du système homéostatique a été suivie. Nos données soulignent les faibles besoins en fer des progéniteurs hématopoïétiques, et d'autres cellules, pour proliférer. Dans les lignées cellulaires le régulateur IRP est en excès par rapport à ses cibles IRE, ce qui pourrait être une caractéristique générale du contrôle de la traduction pour des ARNm spécifiques par fixation de régulateurs translationnels. La régulation semble exclusivement le fait d' IRP1, puisqu' IRP2 n'a pas été détecté dans les progéniteurs hématopoïétiques, qu'ils soit pathologiques ou non. De subtiles différences ont été identifiées dans les quantités des composants du réseau gérant le fer dans les cellules leucémiques en comparaison des cellules saines témoins, ainsi que des capacités différentes à croître dans un milieu minimal comportant des concentrations en fer précisément définies. Les informations obtenues à travers ce travail pourraient bénéficier à l'élaboration de protocoles thérapeutiques, incluant notamment la manipulation du fer, dans les LAM ou d'autres pathologies. / Use of iron resources and variations of the redox balance are processes involved in cell proliferation and differentiation. They participate to normal hematopoiesis and their disturbance may be associated with pathological conditions. Hematological neoplasms, such as acute myeloid leukemia (AML), provide clinical evidence of the link between iron availability, redox signaling, and malignancy. Stringent iron depletion induces arrest of proliferation followed by cell death, and deprived primary leukemic cells of AML patients (blasts) have been previously shown to engage into the monocytic lineage. Yet, the iron needs of leukemic clones are unknown. The core network of cellular iron regulation in mammals is organized around the IRE-IRP system. The Iron Regulatory Proteins (IRP) act on the translation of many proteins involved in iron management by interacting with Iron Responsive Elements (IRE) located on the untranslated regions of messenger RNA (mRNA) coding these proteins. Using leukemic cell lines (KG1, K562), blasts of AML patients and CD34+ progenitors isolated from cord blood or the bone marrow of healthy donors, the status of the iron management system was established in the first stages of normal and pathological hematopoiesis. The response of the homeostatic system upon manipulation of iron provision, including with clinically implemented chelators, has been monitored. Our data emphasize the weak iron requirements of hematopoietic progenitors, and other cells, to proliferate. In cell lines the IRP regulator is in excess of its IRE targets, which may be a general feature of translational control for specific mRNA. The regulation seems exclusively mediated by IRP1, as the IRP2 regulator has not been detected in normal or malignant hematopoietic progenitors. Subtle differences have been found in the iron handling system of leukemic cells as compared to normal cells, together with different abilities to grow on a minimal medium containing precisely defined iron concentrations. The design of improved therapeutic regimens including iron manipulation, in AML and other pathologies, may benefit from considering the information obtained in this work.

Although once considered a simple energy storage depot, the adipose tissue is now known to be a powerful regulator of whole body insulin sensitivity and energy metabolism. This metabolically dynamic organ functions to safely store excess fatty acid as triglyceride, thereby preventing lipotoxicity in peripheral tissues and the development of insulin resistance. In addition, the adipose tissue acts as an endocrine organ and secretes factors, called adipokines, which influence whole body insulin sensitivity and glucose homeostasis. Therefore, understanding adipose tissue development and biology is essential to understanding whole body energy metabolism. A master regulator of adipose tissue development and whole body insulin sensitivity is the nuclear receptor, PPARγ. Due to the importance of this nuclear receptor in maintaining adipocyte function, disruptions in PPARγ activity result in severe metabolic abnormalities, such as insulin resistance and type 2 diabetes. Conversely, PPARγ activation by synthetic agonists ameliorates these conditions, demonstrating the potent control this nuclear receptor has on whole body metabolism. Therefore, understanding how PPARγ expression and activity are regulated, particularly in the adipose tissue, is paramount to understanding the pathogenesis of type 2 diabetes. While there are several synthetic PPARγ agonists available, identifying the endogenous ligand or ligands is still an area of intense investigation. Since fatty acids can induce PPARγ activation, in the first part of this thesis, I screened several fatty acid metabolizing enzymes present in the adipocyte to identify novel modulators of PPARγ activity. These studies revealed that the fatty acid Δ9 desaturase, Stearoyl CoA Desaturase 2 (SCD2), is absolutely required for 3T3-L1 adipogenesis and to maintain adipocyte-specific gene expression in fully differentiated cells. Although SCD2 does not appear to regulate PPARγ ligand production, it does potently regulate PPARγ activity by maintaining the synthesis of PPARγ protein. Surprisingly, this effect was found only with SCD2 and not with the highly homologous protein, SCD1. Therefore, these findings identify separate cellular functions for these SCD isoforms and reveal a novel and essential role for fatty acid desaturation in the adipocyte. Equally important to understanding PPARγ regulation is identifying the downstream mechanisms by which PPARγ activation improves insulin sensitivity. Evidence suggests that the PPARγ target gene, Cidea, is involved in mediating insulin sensitivity by binding to lipid droplets and promoting lipid storage in the adipocyte. Therefore, the second part of thesis provides mechanistic detail into Cidea function by showing that the carboxy terminal 104 amino acids is necessary and sufficient for lipid droplet targeting and the stimulation of triglyceride storage. However, these studies also identified a novel function for Cidea, which requires both the carboxy and amino termini: to induce larger and fewer droplets from smaller dispersed droplets, indicating the possible fusion of droplets. Perhaps this striking change in lipid droplet morphology allows tighter packing and more efficient storage of triglyceride and identifies a novel role for Cidea in lipid metabolism. The results presented in this thesis elucidate key aspects of lipid metabolism that maintain adipocyte function: SCD2 is required to maintain PPARγ protein expression in the mouse; Cidea is a downstream effector of PPARγ activity by promoting efficient triglyceride storage. Therefore, these findings enhance our understanding of adipocyte biology.

Es gibt tausende regulatorische Proteine in Eukaryoten, die spezifische cis-regulatorischen Elemente von Genen und/oder RNA-Transkripten binden und die Genexpession koordinieren. Auf DNA-Ebene modulieren Transkriptionsfaktoren (TFs) die Initiation der Transkription, während auf RNA-Ebene RNA-bindende Proteine (RBPs) viele Aspekte des RNA-Metabolismus und der RNA-Funktion regulieren. Für hunderte dieser regulatorischer Proteine wurden die gebundenen Gene beziehungsweise RNA-Transkripte, sowie deren etwaige Sequenzbindepräferenzen mittels in vivo oder in vitro Hochdurchsatz-Experimente bestimmt. Zu diesen Methoden zählen unter anderem Chromatin-Immunpräzipitation (ChIP) gefolgt von Sequenzierung (ChIP-seq) und Protein Binding Microarrays (PBMs) für TFs, sowie Cross-Linking und Immunpräzipitation (CLIP)-Techniken und RNAcompete für RBPs. In vielen Fällen kann die zum Teil hohe Bindespezifität für ein zumeist sehr kurzes Sequenzmotiv regulatorischer Proteine nicht allein durch die gebundene Primärsequenz erklärt werden. Um besser zu verstehen, wie verschiedene Proteine ihre regulatorische Spezifität erreichen, haben wir zwei Computerprogramme entwickelt, die zusätzliche Informationen in die Analyse von experimentell bestimmten Bindestellen einbeziehen und somit differenziertere Bindevorhersagen ermöglichen. Für Protein-DNA-Interaktionen untersuchen wir die Bindungsspezifität paraloger TFs (d.h. Mitglieder der gleichen TF-Familie). Mit dem Fokus auf der Unterscheidung von genomischen Regionen, die in vivo von Paaren eng miteinander verwandter TFs gebunden sind, haben wir ein Klassifikationsframework entwickelt, das potenzielle Co-Faktoren identifiziert, die zur Spezifität paraloger TFs beitragen. Für Protein-RNA-Interaktionen untersuchen wir die Rolle von RNA-Sekundärstruktur und ihre Auswirkung auf die Auswahl von Bindestellen. Wir haben einen Motif-Finding-Algorithmus entwickelt, der Sekundärstruktur und Primärsequenz integriert, um Bindungspräferenzen der RBPs besser zu bestimmen. / There are thousands of eukaryotic regulatory proteins that bind to specific cis regulatory regions of genes and/or RNA transcripts and coordinate gene expression. At the DNA level, transcription factors (TFs) modulate the initiation of transcription, while at the RNA level, RNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. The DNA or RNA targets and/or the sequence preferences of hundreds of eukaryotic regulatory proteins have been determined thus far using high-throughput in vivo and in vitro experiments, such as chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq) and protein binding microarrays (PBMs) for TFs, or cross-linking and immunoprecipitation (CLIP) techniques and RNAcompete for RBPs. However, the derived short sequence motifs do not fully explain the highly specific binding of these regulatory proteins. In order to improve our understanding of how different proteins achieve their regulatory specificity, we developed two computational tools that incorporate additional information in the analysis of experimentally determined binding sites. For protein-DNA interactions, we investigate the binding specificity of paralogous TFs (i.e. members of the same TF family). Focusing on distinguishing between genomic regions bound in vivo by pairs of closely-related TFs, we developed a classification framework that identifies putative co-factors that provide specificity to paralogous TFs. For protein-RNA interactions, we investigate the role of RNA secondary structure and its impact on binding-site recognition. We developed a motif finding algorithm that integrates secondary structure together with primary sequence in order to better identify binding preferences of RBPs.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 5 uppsatser.