Thesis (Ph. D.)--Indiana University, 2008. / Title from screen (viewed June 24, 2009). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Maureen Harrington. Includes vita. Non-Latin script record. Includes bibliographical references (leaves 126-132).

Activated islet specific T cells are central to the destructive autoimmune response observed in type 1 diabetes (T1D). Not surprisingly, intense focus is placed on understanding how autoreactive T cell responses arise and contribute to disease pathology in the hope of using this information to develop novel therapeutic strategies for treatment of T1D. Here we investigate the mechanisms underlying defective c-Maf binding to the IL-4 promoter in T cells from diabetes prone mice and identify the mechanisms responsible for suppression of T cells by the inhibitory receptor Ly49A. It is not clear why development of protective Th2 cells is poor in T1D. c-Maf transactivates the IL-4 gene promoting Th2 cell development; therefore abnormalities in c-Maf may contribute to reduced IL-4 production by CD4 cells from nonobese diabetic (NOD) mice. Here we demonstrate that, despite normal expression, c-Maf binds poorly to the IL-4 promoter (IL-4p) in NOD CD4 cells. Immunoblots demonstrate that c-Maf can be modified at lysine 33 by small ubiquitin-like modifier-1 (SUMO-1). Sumoylation is facilitated by direct interaction with the E2 conjugating enzyme Ubc9 and increases following T cell stimulation. In addition, c-Maf physically interacts with p65/RelA. This interaction is dependent on the DNA binding domain of c-Maf and phosphorylation of p65 at serine 536. In transfected cells, overexpression of SUMO-1 or p65 decreases c-Maf transactivation of IL-4p-driven luciferase reporter activity, reduces c-Maf binding to the IL-4p in chromatin immunoprecipitation (ChIP) assays and enhances c-Maf localization into promyelocytic leukemia nuclear bodies (PML-NBs) or nucleoli, respectively. Sumoylation of c-Maf and phosphorylation of p65 are increased in NOD CD4 cells compared to CD4 cells from diabetes-resistant B10.D2 mice, suggesting that increased c-Maf sumoylation and interaction with p65 contribute to immune deviation in T1D by reducing c-Maf access to and transactivation of the IL-4 gene. Islet specific CD4 cells expressing inhibitory receptors may be a useful therapeutic tool for treating T1D. Engagement of transgenic Ly49A inhibits CD4 cell activation and delays onset of T1D in mice. However, in vitro studies suggest the inhibitory effect of Ly49A is incomplete. Here we report that following simultaneous T cell receptor (TCR) and Ly49A engagement, phosphorylation of Zap70, Erk1/2 and c-Jun were significantly diminished. Kinetic studies indicated that Ly49A did not simply delay activation but had a long-lasting effect. In contrast, when only costimulatory signals were provided through CD28, Ly49A engagement did not block p38 MapK or Akt phosphorylation. Likewise, expression of the downstream targets Bcl-xl and Baff were unaffected. Together these data suggest that engagement of Ly49A selectively inhibits signals downstream of the TCR but spares those unique to CD28. These results suggest that when considering its use as an immunotherapy, the potency of inhibitory receptors such as Ly49A may be further improved by pairing them with costimulatory blockade. Take together, these studies suggest that abnormal post-translational regulation of c-Maf function is a novel marker of altered T cell function in T1D and use of inhibitory receptors such as Ly49A may be optimized combining this approach with other complementary therapies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22357号 / 医博第4598号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 伊佐 正, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le cancer de la prostate (CaP) est le plus diagnostiqué chez les hommes au Canada et représente le troisième cancer le plus meurtrier au sein de cette population. Malgré l’efficacité des traitements de première ligne, de nombreux patients finiront par développer une résistance et, le cas échéant, verront leur CaP progresser vers une forme plus agressive. Plusieurs paramètres, essentiellement cliniques, permettent de prédire la progression du CaP mais leur sensibilité, encore limitée, implique la nécessité de nouveaux biomarqueurs afin de combler cette lacune. Dans cette optique nous nous intéressons au facteur de transcription NF-κB. Des études réalisées au laboratoire et ailleurs, associent RelA(p65) à un potentiel clinique dans le CaP, soulignant ainsi l’importance de la voie classique NF-κB. L’implication de la voie alternative NF-κB dans la progression du CaP a aussi été suggérée dans une de nos études illustrant la corrélation entre la distribution nucléaire de RelB et le score de Gleason. Alors que la voie classique est largement documentée et son implication dans la progression du CaP établie, la voie alternative, elle, reste à explorer. La présente thèse vise à clarifier l’implication de la voie alternative NF-κB dans le CaP et répond à deux objectifs fixés dans ce but. Le premier objectif fut d’évaluer l’impact de l'activation de la voie alternative NF-κB sur la biologie des cellules cancéreuses prostatiques. L’étude de la surexpression de RelB a souligné les effets de la voie alternative NF-κB sur la prolifération et l'autophagie. Étant ainsi impliquée tant dans la croissance tumorale que dans un processus de plus en plus associée à la progression tumorale, quoique potentiellement létal pour les cellules cancéreuses, son impact sur la tumorigénèse du CaP reste encore difficile à définir. Il n'existe, à ce jour, aucune étude permettant de comparer le potentiel clinique des voies classique et alternative NF-κB. Le second objectif de ce projet fut donc l'analyse conjointe de RelA(p65) et RelB au sein de mêmes tissus de patients atteints de CaP afin de déterminer l'importance clinique des deux signalisations NF-κB, l'une par rapport à l'autre. Le marquage immunofluorescent de RelA(p65) et RelB en a permis l'analyse quantitative et objective par un logiciel d'imagerie. Nos travaux ont confirmé le potentiel clinique associé à RelA(p65). La variable RelA(p65)/RelB s’est, elle, avérée moins informative que RelA(p65). Par contre, aucune corrélation entre RelB et les paramètres cliniques inclus dans l'étude n’est ressortie. En définitive, mon projet de thèse aura permis de préciser l'implication de la voie alternative NF-κB sur la biologie du CaP. Son impact sur la croissance des cellules cancéreuses prostatiques ainsi que sur l'autophagie, dénote l’ambivalence de la voie alternative NF-κB face à la tumorigénèse du CaP. L’étude exhaustive de la signalisation NF-κB souligne davantage l'importance de la voie classique dont l’intérêt clinique est principalement associé au statut de RelA(p65). Ainsi, bien que RelB n’affiche aucun potentiel en tant que biomarqueur exploitable en clinique, l’analyse de l’intervention de la voie alternative NF-κB sur la biologie des cellules cancéreuses prostatiques reste d’intérêt pour la compréhension de son rôle exact dans la progression du CaP. / Prostate cancer (PCa) is the most frequently diagnosed cancer and represents the third cause of cancer-death in Canadian men. Despite effective first-line therapies, many patients experience disease recurrence where PCa progresses toward a more aggressive form. Several parameters, largely clinical, have been used to predict the progression of PCa but their accuracy is still limited and implies the need for new biomarkers to fill this gap. Previous research from the laboratory has demonstrated that the transcription factor NF-B, and its nuclear localization, could be such a prognostic biomarker. Studies in our laboratory and elsewhere have correlated RelA(p65) with a clinical progression in PCa, underlining the importance of the classical NF-B pathway. The involvement of the alternative NF-B pathway in the progression of PCa was also suggested in one of our studies showing the correlation between the nuclear distribution of RelB and Gleason score. While the classical pathway is well documented and its involvement in the PCa progression established, the alternative NF-B pathway remains largely unexplored. This thesis describes two research objectives that aims to clarify the involvement of the alternative NF-B pathway in PCa. The first objective assessed the impact of the alternative NF-B pathway activation on the biology of PCa cells. RelB overexpression in 22RV1 PCa cells highlighted an effect of the alternative NF-B pathway on cell proliferation and autophagy. Its dual role in cell growth and a form of cell death requires further study to understand the balance of these in PCa tumorigenesis. To date no study has addressed the comparative prognostic potential of both the classical and alternative NF-B pathways simultaneously. Therefore the second objective of this research project was to analyze both RelA(p65) and RelB at a cellular level in the same tissue of patients with PCa to determine their unique and combined contribution to predicting biochemical recurrence in patients. This analysis was possible through immunofluorescent labeling of RelA (p65) and RelB, and was followed by a quantitative and objective analysis using an appropriate software. Our work confirmed the predictive value of RelA(p65) for biochemical recurrance. Combining RelA(p65) with RelB weakened the association, and RelB on its own was not found to predict biochemical recurrance. Ultimately, the research presented here has clarified the involvement of the alternative NF-B pathway on the biology of PCa. Its impact on the growth of PCa cells as well as autophagy reveals the dual role of the alternative NF-B pathway in PCa tumorigenesis. This exhaustive study of NF-B in PCa tissues further underscores the importance of the classical pathway whose clinical interest is mainly associated with RelA(p65) status. Thus, although RelB shows no potential as a clinically exploitable biomarker, further studies are needed to determine whether RelB contributes, either positively or negatively, and in a temporal fashion, to PCa progression.

Le cancer de la prostate (CaP) est le plus diagnostiqué chez les hommes au Canada et représente le troisième cancer le plus meurtrier au sein de cette population. Malgré l’efficacité des traitements de première ligne, de nombreux patients finiront par développer une résistance et, le cas échéant, verront leur CaP progresser vers une forme plus agressive. Plusieurs paramètres, essentiellement cliniques, permettent de prédire la progression du CaP mais leur sensibilité, encore limitée, implique la nécessité de nouveaux biomarqueurs afin de combler cette lacune. Dans cette optique nous nous intéressons au facteur de transcription NF-κB. Des études réalisées au laboratoire et ailleurs, associent RelA(p65) à un potentiel clinique dans le CaP, soulignant ainsi l’importance de la voie classique NF-κB. L’implication de la voie alternative NF-κB dans la progression du CaP a aussi été suggérée dans une de nos études illustrant la corrélation entre la distribution nucléaire de RelB et le score de Gleason. Alors que la voie classique est largement documentée et son implication dans la progression du CaP établie, la voie alternative, elle, reste à explorer. La présente thèse vise à clarifier l’implication de la voie alternative NF-κB dans le CaP et répond à deux objectifs fixés dans ce but. Le premier objectif fut d’évaluer l’impact de l'activation de la voie alternative NF-κB sur la biologie des cellules cancéreuses prostatiques. L’étude de la surexpression de RelB a souligné les effets de la voie alternative NF-κB sur la prolifération et l'autophagie. Étant ainsi impliquée tant dans la croissance tumorale que dans un processus de plus en plus associée à la progression tumorale, quoique potentiellement létal pour les cellules cancéreuses, son impact sur la tumorigénèse du CaP reste encore difficile à définir. Il n'existe, à ce jour, aucune étude permettant de comparer le potentiel clinique des voies classique et alternative NF-κB. Le second objectif de ce projet fut donc l'analyse conjointe de RelA(p65) et RelB au sein de mêmes tissus de patients atteints de CaP afin de déterminer l'importance clinique des deux signalisations NF-κB, l'une par rapport à l'autre. Le marquage immunofluorescent de RelA(p65) et RelB en a permis l'analyse quantitative et objective par un logiciel d'imagerie. Nos travaux ont confirmé le potentiel clinique associé à RelA(p65). La variable RelA(p65)/RelB s’est, elle, avérée moins informative que RelA(p65). Par contre, aucune corrélation entre RelB et les paramètres cliniques inclus dans l'étude n’est ressortie. En définitive, mon projet de thèse aura permis de préciser l'implication de la voie alternative NF-κB sur la biologie du CaP. Son impact sur la croissance des cellules cancéreuses prostatiques ainsi que sur l'autophagie, dénote l’ambivalence de la voie alternative NF-κB face à la tumorigénèse du CaP. L’étude exhaustive de la signalisation NF-κB souligne davantage l'importance de la voie classique dont l’intérêt clinique est principalement associé au statut de RelA(p65). Ainsi, bien que RelB n’affiche aucun potentiel en tant que biomarqueur exploitable en clinique, l’analyse de l’intervention de la voie alternative NF-κB sur la biologie des cellules cancéreuses prostatiques reste d’intérêt pour la compréhension de son rôle exact dans la progression du CaP. / Prostate cancer (PCa) is the most frequently diagnosed cancer and represents the third cause of cancer-death in Canadian men. Despite effective first-line therapies, many patients experience disease recurrence where PCa progresses toward a more aggressive form. Several parameters, largely clinical, have been used to predict the progression of PCa but their accuracy is still limited and implies the need for new biomarkers to fill this gap. Previous research from the laboratory has demonstrated that the transcription factor NF-B, and its nuclear localization, could be such a prognostic biomarker. Studies in our laboratory and elsewhere have correlated RelA(p65) with a clinical progression in PCa, underlining the importance of the classical NF-B pathway. The involvement of the alternative NF-B pathway in the progression of PCa was also suggested in one of our studies showing the correlation between the nuclear distribution of RelB and Gleason score. While the classical pathway is well documented and its involvement in the PCa progression established, the alternative NF-B pathway remains largely unexplored. This thesis describes two research objectives that aims to clarify the involvement of the alternative NF-B pathway in PCa. The first objective assessed the impact of the alternative NF-B pathway activation on the biology of PCa cells. RelB overexpression in 22RV1 PCa cells highlighted an effect of the alternative NF-B pathway on cell proliferation and autophagy. Its dual role in cell growth and a form of cell death requires further study to understand the balance of these in PCa tumorigenesis. To date no study has addressed the comparative prognostic potential of both the classical and alternative NF-B pathways simultaneously. Therefore the second objective of this research project was to analyze both RelA(p65) and RelB at a cellular level in the same tissue of patients with PCa to determine their unique and combined contribution to predicting biochemical recurrence in patients. This analysis was possible through immunofluorescent labeling of RelA (p65) and RelB, and was followed by a quantitative and objective analysis using an appropriate software. Our work confirmed the predictive value of RelA(p65) for biochemical recurrance. Combining RelA(p65) with RelB weakened the association, and RelB on its own was not found to predict biochemical recurrance. Ultimately, the research presented here has clarified the involvement of the alternative NF-B pathway on the biology of PCa. Its impact on the growth of PCa cells as well as autophagy reveals the dual role of the alternative NF-B pathway in PCa tumorigenesis. This exhaustive study of NF-B in PCa tissues further underscores the importance of the classical pathway whose clinical interest is mainly associated with RelA(p65) status. Thus, although RelB shows no potential as a clinically exploitable biomarker, further studies are needed to determine whether RelB contributes, either positively or negatively, and in a temporal fashion, to PCa progression.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Obesity is associated with chronic inflammation and increased risk of developing cardiovascular disease (CVD). Obesity is also associated with nitric oxide (NO)-mediated vascular endothelial dysfunction (VED), an independent predictor of increased CVD risk in humans. Pro-inflammatory cytokines secreted by the adipose tissue, such as TNF-Α, may contribute to VED through promotion of insulin resistance or directly through a reduction in endothelial NO synthase (eNOS) expression and/or phosphorylation. Sodium salicylate (Na-Sal) is a non-acetylated aspirin that inhibits the pro-inflammatory transcription factor nuclear factor-ΚB (NF-ΚB) and activates the cellular metabolism regulator AMP-activated protein kinase (AMPK). AMPK is a known activator of eNOS. We tested the hypothesis that Na-Sal increases eNOS expression/phosphorylation in TNFΑ-stimulated endothelial cells through an AMPK-dependent mechanism. Human aortic endothelial cells (HAECs) incubated in vitro with TNF-Α (10 ng/ml, 2 hrs) demonstrated decreased (vs. control) expression (via Western blotting) of eNOSser1177 phosphorylation (n=8; PThr172 phosphorylation (n=8, Pser1177 phosphorylation (vs. control, n=7; P=0.14) and AMPKThr172 phosphorylation (vs. control, n=9; P=0.42). The AMPK activator AICAR prevented eNOSser1177 phosphorylation down-regulation by TNF-Α in a manner similar to Na-Sal (n=2, P=0.839). Co-treatment with the AMPK inhibitor compound C (10 μM, 30 min) abolished the ability of Na-Sal to prevent down-regulation of eNOSser1177 phosphorylation by TNF-Α (vs. control, n=3; Pser1177 in endothelial cells in part through AMPK.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The innate and adaptive immune responses are critical for host survival. The TNFα/NF-κB signaling pathway is a major regulator of the immune response. The TNFα/NF-κB signaling pathway has also been proposed to play a role in the regulation of hematopoiesis. In the TNFα signaling pathway, full induction of NF-κB (specifically the p65 subunit) dependent transcription is regulated by a co-activator SIMPL. The biological significance of SIMPL in TNFα dependent responses is poorly understood. To study SIMPL in vitro and in vivo in mammalian cells, a knockdown system utilizing shRNA (short hairpin RNA) was used. Analysis of hematopoietic progenitor cells infected with a retrovirus encoding the SIMPL shRNA was used to study the role of SIMPL in hematopoiesis. The ability of progenitor cells lacking SIMPL to grow and differentiate was not compromised. In contrast in the progenitors cells lacking SIMPL, TNFα mediated inhibition of colony formation was significantly enhanced. These growth inhibitory effects of SIMPL were not due to an increase in apoptosis. The enhanced inhibitory affects were specific for TNFα and not found in other common hematopoietic inhibitors (TGF-β1 and IFNγ). Results of this work reveal that SIMPL is a component of the hematopoiesis that is required for TNFα dependent effects upon myeloid progenitors.

No description available.