No description available.

The alveolar bone repair process may be influenced by several local and systemic factors that include mediators and immune system cells. Among these cells, macrophages are essential to trigger the repair process, and may acquire an inflammatory (M1) or anti-inflammatory and pro-reparative profile (M2). In this context, we evaluated the effects of FTY720 on macrophage polarization towards the M2 profile and its effects on the alveolar bone repair process. In this study, we used 8 weeks old male C57BL / 6 mice (N = 5 / time / group). The animals were divided in FTY720 group receiving the drug orally at a dose of 3mg / kg / 24h during the whole experimental period, and the control group receiving only the equivalent vehicle. All animals were submitted to extraction of the right upper incisor and were evaluated at 0, 1, 3, 7 and 14 days after extraction, followed by computed tomography (CT), histomorphometry, birefringence, immunohistochemical and molecular analyzes (PCRArray). Our results demonstrated that in the 14-day period, the FTY720 group presented higher bone tissue density, higher bone tissue volume (BV), greater tissue volume fraction (BV / TV), greater number and thickness of trabeculae (Tb.1 and Tb.Th, respectively) (p<0.05). In the 14-day period, the FTY720 group had a higher number of osteoblasts and osteoclasts than the control group (p<0.05). Accordingly, the expression of various bone markers such as BMP2, BMP7, ALPL, SOST and RANK had their mRNA expressions increased in the FTY720 group. This increase may be related to the potentiation in the formation of the bone tissue compared to the control group. The levels of FIZZ, ARG2 and IL-10 mRNA increased in the FTY720 group together with the presence of CD206 + cells in the 14 days period, suggesting a participation of M2 macrophages in the potentiation of the alveolar bone repair process. The FTY720 group also showed increased expression levels of CCR2, CCR5, CXCR1, CXCL1, CXCL3, CCL20 and CCL25 mRNA, chemokines and chemokine receptors involved in the recruitment of inflammatory cells and undifferentiated mesenchymal cells (MSCs) most notably was the up CXCL12 up regulation (p<0.05). CXCL12 is responsible in the recruitment of MSCs to the repair site. The increase in CXCL12 expression was accompanied by an increase in CD34 expression over a period of 14 days (p<0.05), indicating a higher presence of MSCs in the repair site. Thus, our results demonstrate that FTY720 favored the process of alveolar bone repair in C57BL / 6 mice, possibly because it increased the expression of markers related to bone tissue development (ALPL, SOST, RANK), tissue repair (CXCL12, CD34) and inflammatory cells (CCR2, CCR5) and apparently in the induction of macrophages to an M2 profile (ARG2, FIZZ). / O processo de reparo ósseo alveolar pode ser influenciado por vários fatores locais e sistêmicos que incluem mediadores e células do sistema imunológico. Dentre essas células, os macrófagos são essenciais para desencadear o processo de reparo, podendo adquirir um perfil inflamatório (M1) ou anti-inflamatório e pró-reparativo (M2). Nesse contexto, avaliamos os efeitos do FTY720 na polarização de macrófagos para o perfil M2 e seus efeitos no processo de reparo ósseo alveolar. Nesse estudo foram utilizados camundongos C57BL/6 (N=5/tempo/grupo), machos, com 8 semanas de idade. Os animais foram divididos em grupo que recebeu o fármaco FTY720 via oral, na dosagem de 3mg/Kg/24h, durante todo o período experimental, e grupo controle que recebeu apenas o veículo em regime equivalente. Todos animais foram submetidos à extração do incisivo superior direito e avaliados nos períodos de 0, 1, 3, 7 e 14 dias pós extração, seguido por análises de tomografia computadorizada (CT), histomorfométrica, birrefringência, imuno-histoquímica e molecular (PCRArray). Nossos resultados demonstraram que no período de 14 dias, o grupo FTY720 apresentou maior densidade de tecido ósseo, maior volume de tecido ósseo (B.V), maior volume de fração de tecido ósseo pelo tecido total (BV/TV), maior número e espessura de trabéculas (Tb.1 e Tb.Th, respectivamente) (p<0.05). Ainda no período de 14 dias, o grupo FTY720 apresentou maior número de osteoblastos e osteoclastos em relação ao grupo controle (p<0.05). Em concordância, a expressão de vários marcadores de tecido ósseo como, BMP2, BMP7, ALPL, SOST e RANK, tiveram suas expressões de mRNA aumentadas no grupo FTY720. Esse aumento pode estar relacionado com a potencialização na formação do tecido ósseo comparado ao grupo controle. Os níveis de mRNA de FIZZ, ARG2 e IL-10, sofreram aumento no grupo FTY720 em conjunto com a presença de células CD206+ no período de 14 dias, podendo sugerir uma participação dos macrófagos M2 na potencialização do processo de reparo ósseo alveolar. O grupo FTY720 também mostrou aumento nos níveis de expressão de mRNA de CCR2, CCR5, CXCR1, CXCL3, CCL20 e CCL25, quimicionas e receptores de quimiocinas envolvidos no recrutamento de células inflamatórias e células mesenquimais indiferenciadas (MSCs) com destaque para o aumento da expressão de CXCL12 (p<0.05), quimiocina responsável no recrutamento de MSCs para o local de reparo. O aumento na expressão de CXCL12 foi acompanhado pelo aumento na expressão de CD34 no período de 14 dias (p<0.05) podendo indicar maior presença de MSCs no sitio de reparo. Assim, os nossos resultados demonstram que o FTY720 favoreceu o processo de reparo ósseo alveolar em camundongos C57BL/6, possivelmente por ter aumentado a expressão de marcadores relacionados com o desenvolvimento do tecido ósseo(ALPL, SOST, RANK), no reparo tecidual (TGF-1, IL-10), no recrutamento de células indiferenciadas (CXCL12, CD34) e células inflamatórias (CCR2, CCR5) e aparentemente na indução de macrófagos para um perfil M2 (ARG2, FIZZ).

Nach Ischämie und Reperfusion des Pankreas kommt es zu einer Mikrozirkulationsstörung sowie zu einer T-Zell-induzierten subakuten entzündlichen Reaktion mit daraus resultierender Zellzerstörung. Ziel dieser tierexperimentellen Arbeit war es, sowohl den Einfluss des S1PStrukturanalogons FTY720 auf zirkulierende T-Lymphozyten und deren Endothel- Interaktion in den postkapillären Venolen des Pankreas zu beobachten sowie die Mikrozirkulation und damit das Ausmaß des Ischämie-Reperfusionsschadens nach einer 60-minütigen Pankreasischämie durch die Gabe von FTY720 zu beurteilen. Hierfür erfolgte eine reversible Okklusion der zuführenden Gefäße des Pankreas an Ratten (48 Wistar-Ratten, 300 - 350 g KG) für 60 Minuten zur Induktion einer normothermen Pankreasischämie. Untersucht wurden folgende Gruppen: I Scheinoperation ohne Therapie, II Scheinoperation und Therapie, III Ischämie ohne Therapie, IV Ischämie und Therapie mit dem Sphingosin-1-Phosphat Strukturanalogon FTY720 (1 mg/kg KG i.v., Substitution 10 Minuten vor Reperfusionsbeginn). Mit Hilfe der invivo-Fluoreszenzmikroskopie wurden die Parameter der Mikrozirkulation (Zellzahl, Flussgeschwindigkeit, Kapillardurchmesser, Funktionelle Kapillardichte, T-Lymphozyten-Endothel-Interaktion und Leukozyten-Endothel- Interaktion) 10 Minuten nach Substitution der markierten CD4+-T-Lymphozyten beurteilt. Des Weiteren wurde der Blutparameter Lipase bestimmt. Die Studie erbrachte folgende Ergebnisse: 1. Die intravenöse Therapie mit FTY720 als Sphingosin 1-Phosphat-Rezeptor- Agonist 15 Minuten vor Reperfusion führte zu einer partiellen Verbesserung der Mikrozirkulation des Pankreas mit signifikant erhöhten Kapillardurchmesser und Funktioneller Kapillardichte. 2. Es zeichneten sich bei allen weiteren Parametern nur Tendenzen einer Verbesserung unter Substitution von FTY720 ab (Flussgeschwindigkeit, Lipase). 3. Eine Aussage zur T-Lymphozyten-Endothel-Interaktion konnte aufgrund der geringen Anzahl von „rollern“ und „stickern“ nicht gemacht werden. 4. Eine quantitative Auswertung der zirkulierenden Leukozyten war nicht möglich. Das vorliegende Bildmaterial war jedoch in allen Gruppen identisch, sodass hier kein Effekt von FTY720 auf zirkulierende Leukozyten nachweisbar war. 5. Zu einer signifikanten Zunahme der T-Lymphozyten-Zahl kam es in der Gruppe IV (Ischämie und Therapie mit FTY720). Die Ergebnisse dieser tierexperimentellen Untersuchung zeigen, dass die intraoperative Gabe des Immunmodulators FTY720 nur partiell eine Verbesserung der Mirkozirkulation und daraus resultierend eine Verminderung des Ischämie-Reperfusionsschadens bewirkt. Nach den Ergebnissen dieser Studie und dem aktuellen Stand der Literatur wäre somit für ein optimales Wirkmaximum die präoperative Gabe von FTY720 erforderlich. Die Studie zeigt ebenfalls, dass die Substitution der markierten CD4+-T-Lymphozyten zu einem früheren Zeitpunkt erfolgen muss, um eine Wirkung des FTY720 auf diese T-Zellen nachweisen zu können.

Despite advances in bone grafting technology for musculoskeletal injury, re-injury or incomplete healing persists. Efforts to modify bone allografts sing proteins and growth factors show improvement in wound healing outcomes. We use FTY720, an agonist of S1P receptors 1 and 3, to improve bone graft integration through bone regeneration and vascularization. Four methods of delivering FTY720 into a bone defect are described: 1) FTY720 loaded onto a PLAGA-coated bone allograft and implanted in a critical size rat cranial defect; 2) FTY720 loaded onto a PLAGA-coated bone allograft and implanted in a rat tibial defect; 3) FTY720 loaded into a Matrigel plug and injected into a mouse tibial fracture; and 4) FTY720 directly adsorbed to human bone xenografts and implanted in a critical size rat cranial defect. In each of these models, FTY720 release was characterized, and bone regeneration and vascularization was monitored within the defect. Additionally, local tissue composition and immune response was evaluated. The results presented here indicate that FTY720 released locally into the bone defect improved new bone formation and vascularization, promoting improved graft integration.

CCR5 et CXCR4 sont les corécepteurs d'entrée du VIH utilisés par le virus in vivo en plus du récepteur principal CD4 pour infecter les cellules. Au début et tout le long de l'infection, on retrouve chez les patients infectés par le VIH, des virions R5 utilisant le corécepteur CCR5 pour infecter les cellules. Dans les stades tardifs de l'infection et chez environ la moitié des personnes infectées par le VIH, on observe en plus de ces souches R5, l'apparition de souches X4, utilisant le corécepteur CXCR4 pour infecter les cellules. Cette apparition de souches X4 est un facteur d'aggravation de la maladie. Les causes de cette commutation de R5 vers X4 sont mal définies. Le but de mon travail de thèse a été de trouver de nouvelles stratégies thérapeutiques visant l'un ou l'autre de ces corécepteurs. La première partie de mon travail compare les deux isoformes de CXCR4 en tant que corécepteurs du VIH. Ces deux isoformes, CXCR4-A et CXCR4-B, diffèrent de 9 acides aminés en NH2 terminal suite à un épissage alternatif. Nous avons montré que l'isoforme CXCR4-B est la plus performante en tant que corécepteur du VIH mais que ces deux variants sont équivalents pour la migration vers leur ligand commun SDF-1. Ainsi, nous proposons qu'en ciblant exclusivement l'isoforme B qui est la plus favorable à l'infection, via par exemple des siRNA, il serait possible de limiter les infections par des souches X4 tout en gardant une partie des fonctions essentielles de ce récepteur dans l'organisme, assurées par l'isoforme A. Nos résultats suggèrent également que l'infection par des souches R5 augmente le ratio en ARNm CXCR4-B / CXCR4-A dans des PBMC, et que ce ratio est en partie responsable de la commutation de R5 vers X4 associée à une aggravation de la maladie. Cibler cette isoforme CXCR4-B pourrait donc se révéler bénéfique. La deuxième partie de cette thèse étudie la modulation de la fonction de corécepteur du VIH de CCR5 par S1P1, un autre membre de la famille des récepteurs couplés aux protéines G qui permet la remise en circulation des lymphocytes après leur séjour dans les organes lymphoïdes secondaires par chimiotactisme vers son ligand S1P, abondant dans le sang. Nous montrons que S1P1 interagit physiquement avec CCR5 et gêne l'entrée des virus R5 dans la cellule-hôte. A l'inverse, S1P1 active les étapes post-entrée du cycle viral, notamment l'expression génique virale. La résultante de ces effets opposés est une augmentation de la production virale par des cellules infectées in vitro. Ce projet a également montré que l'utilisation de FTY720, un antagoniste fonctionnel de S1P1, diminue l'infection de cellules dendritiques par des virus HIV-R5 in vitro, ainsi que la virémie dans un modèle de souris SCID infectées après reconstitution immunologique. La mise en évidence des interactions entre CCR5 et S1P1 ouvre donc des perspectives thérapeutiques. / CCR5 and CXCR4 are the two HIV entry coreceptors used by the virus in addition to the main receptor CD4 in vivo to infect cells. R5 virions, that use CCR5 as a coreceptor to infect cells, are detected in most HIV patients. At late stages of infection and in about half of HIV infected persons, there is an emergence of X4 virions that use CXCR4 as a coreceptor, in addition to R5 virions. This emergence is associated with an increase in disease progression. The reasons for this R5 to X4 switch are poorly understood. The goal of my PhD work was to find new therapeutic strategies that target these coreceptors.The first part of this work compares the two CXCR4 isoforms as HIV coreceptors. Those two isoforms, CXCR4-A and CXCR4-B, differ by 9 amino acids at their NH2 terminal extremity as a consequence of an alternative splicing. We have shown that CXCR4-B isoform is more efficient as an HIV coreceptor but that those two variants are equivalent in terms of chemotaxis toward their common ligand SDF-1. Thus, we propose that by targeting specifically the B isoform that supports infection, via siRNA by example, it is possible to limit X4 development while keeping essential functions of this receptor. Our results also suggest that R5 infection increases CXCR4-B / CXCR4-A mRNA ratio in PBMC and that this ratio is in part responsible for R5 to X4 switch. Thus, targeting CXCR4-B isoform could be beneficial.The second part of this PhD thesis studies the effect on CCR5 coreceptor function of S1P1, another G protein-coupled receptor that enables lymphocytes egress from lymph nodes by chemotaxis toward its ligand S1P that is abundant in blood. We have shown that S1P1 physically interacts with CCR5 and blocks R5 virus entry. On the other hand, S1P1 activates post-entry steps of the viral cycle, in particular gene expression. The resulting effect is an increase in viral production by infected cells in vitro. We also showed that the use of FTY720, a S1P1 functional antagonist, decreases dendritic cell infection by R5 viruses in vitro, and in vivo infection in a SCID mouse model. The emphasis of CCR5 and S1P1 interactions opens new therapeutic strategies.

Nach Ischämie und Reperfusion des Pankreas kommt es zu einer Mikrozirkulationsstörung sowie zu einer T-Zell-induzierten subakuten entzündlichen Reaktion mit daraus resultierender Zellzerstörung. Ziel dieser tierexperimentellen Arbeit war es, sowohl den Einfluss des S1PStrukturanalogons FTY720 auf zirkulierende T-Lymphozyten und deren Endothel- Interaktion in den postkapillären Venolen des Pankreas zu beobachten sowie die Mikrozirkulation und damit das Ausmaß des Ischämie-Reperfusionsschadens nach einer 60-minütigen Pankreasischämie durch die Gabe von FTY720 zu beurteilen. Hierfür erfolgte eine reversible Okklusion der zuführenden Gefäße des Pankreas an Ratten (48 Wistar-Ratten, 300 - 350 g KG) für 60 Minuten zur Induktion einer normothermen Pankreasischämie. Untersucht wurden folgende Gruppen: I Scheinoperation ohne Therapie, II Scheinoperation und Therapie, III Ischämie ohne Therapie, IV Ischämie und Therapie mit dem Sphingosin-1-Phosphat Strukturanalogon FTY720 (1 mg/kg KG i.v., Substitution 10 Minuten vor Reperfusionsbeginn). Mit Hilfe der invivo-Fluoreszenzmikroskopie wurden die Parameter der Mikrozirkulation (Zellzahl, Flussgeschwindigkeit, Kapillardurchmesser, Funktionelle Kapillardichte, T-Lymphozyten-Endothel-Interaktion und Leukozyten-Endothel- Interaktion) 10 Minuten nach Substitution der markierten CD4+-T-Lymphozyten beurteilt. Des Weiteren wurde der Blutparameter Lipase bestimmt. Die Studie erbrachte folgende Ergebnisse: 1. Die intravenöse Therapie mit FTY720 als Sphingosin 1-Phosphat-Rezeptor- Agonist 15 Minuten vor Reperfusion führte zu einer partiellen Verbesserung der Mikrozirkulation des Pankreas mit signifikant erhöhten Kapillardurchmesser und Funktioneller Kapillardichte. 2. Es zeichneten sich bei allen weiteren Parametern nur Tendenzen einer Verbesserung unter Substitution von FTY720 ab (Flussgeschwindigkeit, Lipase). 3. Eine Aussage zur T-Lymphozyten-Endothel-Interaktion konnte aufgrund der geringen Anzahl von „rollern“ und „stickern“ nicht gemacht werden. 4. Eine quantitative Auswertung der zirkulierenden Leukozyten war nicht möglich. Das vorliegende Bildmaterial war jedoch in allen Gruppen identisch, sodass hier kein Effekt von FTY720 auf zirkulierende Leukozyten nachweisbar war. 5. Zu einer signifikanten Zunahme der T-Lymphozyten-Zahl kam es in der Gruppe IV (Ischämie und Therapie mit FTY720). Die Ergebnisse dieser tierexperimentellen Untersuchung zeigen, dass die intraoperative Gabe des Immunmodulators FTY720 nur partiell eine Verbesserung der Mirkozirkulation und daraus resultierend eine Verminderung des Ischämie-Reperfusionsschadens bewirkt. Nach den Ergebnissen dieser Studie und dem aktuellen Stand der Literatur wäre somit für ein optimales Wirkmaximum die präoperative Gabe von FTY720 erforderlich. Die Studie zeigt ebenfalls, dass die Substitution der markierten CD4+-T-Lymphozyten zu einem früheren Zeitpunkt erfolgen muss, um eine Wirkung des FTY720 auf diese T-Zellen nachweisen zu können.

Over the last decade, a significant amount of research has been done on sphingosine-1-phosphate (S1P). S1P regulates survival, proliferation and migration of breast cancer cells45, as well as playing an important but still unknown role in ovarian46, prostate, and glioma47 cell carcinomas. S1P plays a significant role in the regulation, proliferation and angiogenesis, and is therefore an area of considerable interest for cancer research. Previous research in our group has shown that two stilbenoid sphingosine analogs (1 & 2) were effective at sphingosine kinase (SphK) inhibition in addition to showing down-regulation of Vascular Endothelial Growth Factor (VEGF), a novel and distinctly different mechanism of action compared to reported sphingosine kinase inhibitors. Both compounds showed novel toxicity toward the parasite Toxoplasma gondii. T. gondii causes toxoplasmosis; a disease carried by up to 1/3rd of the world’s population and one that has been implicated in schizophrenia. Unfortunately, use of 1 & 2 as fluorescent probes is limited; their emission wavelengths fall between 360 and 380 nm, within the range of background fluorescence. The synthesis of five fluorescent sphingosine analogs is described herein, three of which have emission wavelengths above 420 nm. These compounds show single digit µM cytotoxicity towards T. gondii. The second generation synthesis of Fingolimod (FTY720) is also described. The process was optimized for large-scale production. Improvements to the synthesis included increased atom economy, purification and overall reaction efficiency. Yield was increased to 36%, from 33% in the first generation synthesis. Lastly, a synthesis of p-divinylbenzene is described, using “green” solvents and easy sample work-up. The product contains no meta impurities, an issue that arises when p-divinylbenzene is purchased commercially. / Thesis / Candidate in Philosophy

Research Doctorate - Doctor of Philosophy (PhD) / Reversible protein phosphorylation plays a central role in the regulation of intracellular signalling, and is controlled by the opposing activities of protein kinases and phosphatases. Deregulation of these mechanisms can result in increased proliferation and enhanced survival, which is a hallmark feature of malignant transformation. For example, over 90% of chronic myeloid leukaemia (CML) patients express the BCR/ABL oncoprotein, which exhibits unrestrained tyrosine kinase activity. In addition, activating mutations within the receptor tyrosine kinase, c-KIT, contribute to the pathogenesis of gastrointestinal stromal tumours (GIST), systemic mastocytosis, acute myeloid leukaemia (AML), testicular seminoma and melanoma. The advent of small molecule tyrosine kinase inhibitors, such as imatinib, has revolutionised the treatment of malignancies driven by these oncogenic kinases. However, a proportion of patients are either unresponsive or develop resistance, and as such, relapse and disease progression is a major clinical problem. In order to improve the treatment outcome for these patients, a greater understanding of the signalling pathways regulated downstream of BCR/ABL and c-KIT is required. The data presented in this thesis indicates that oncogenic BCR/ABL and mutant c-KIT both require inhibition of the tumour suppressor, protein phosphatase 2A (PP2A), to induce tumourigenesis. PP2A is a large family of serine/threonine phosphatases that provide the fine control on signalling pathways by governing the rate and duration of phosphorylation. The heterotrimeric PP2A enzyme is comprised of a structural subunit (PP2A Aα and Aβ), a catalytic subunit (PP2Acα and cβ) and a regulatory subunit, which consists of three unrelated families: B55 (α, β, γ, δ), B56 (α, β, γ, δ, ε) and B" (PR72/130 / PR70/48). Binding of the regulatory subunit to the core PP2A AC dimer directs both the substrate specificity and cellular localisation of the enzyme. The combinatorial assembly of these individual components permits the formation of distinct complexes which have been implicated in numerous cellular functions such as proliferation, survival and mitosis. In particular, important roles for PP2A in various aspects of malignant transformation are beginning to emerge. Recent work demonstrates that PP2A is functionally inactivated by BCR/ABL in myeloid progenitor cells. Using the mouse myeloid progenitor cell line, FDC-P1, these observations were confirmed in the current study. Detailed investigation into the underlying mechanisms have demonstrated for the first time that active BCR/ABL increases the expression of the PP2A structural and certain regulatory subunits. This alters the PP2A holoenzyme composition and results in the abundance of complexes containing B55α and B56α. Consequently, B56γ, a known tumour suppressive subunit, appears to be simultaneously displaced. To investigate which subunits are functionally important for BCR/ABL-mediated leukaemogenesis, individual PP2A subunits were targeted with shRNA sequences in WT BCR/ABL FDC-P1 cells. Subsequent evaluation identified B56α as a key player which facilitates the leukaemic phenotype. In accordance with an increase in PP2A activity, knockdown of B56α significantly inhibited the cellular growth and reduced the clonogenic potential of BCR/ABL⁺ myeloid progenitors. Furthermore, suppression of the B56δ subunit in WT BCR/ABL FDC-P1 cells appears to delay progression through the cell cycle. Together, these findings provide new insights into the biology of PP2A and begin to define the precise mechanisms by which BCR/ABL induces leukaemogenesis via PP2A in CML. Investigation of the regulation of PP2A was also extended to the oncogenic tyrosine kinase, c-KIT. Using FDC-P1 cells expressing imatinib-sensitive (V560G) or –resistant (D816V) mutant c-KIT, this work demonstrates for the first time that constitutive activation of c-KIT impairs the activity of PP2A, and this is essential for tumourigenesis. Pharmacological reactivation of PP2A with FTY720 significantly reduced the proliferation, impaired the clonogenic potential and induced apoptosis of oncogenic c-KIT cells, whilst having no effect on empty vector controls or WT c-KIT cells stimulated with stem cell factor (SCF). These cytotoxic effects of FTY720 are mediated, in part, by the rapid dephosphorylation, and hence inactivation, of oncogenic c-KIT receptors. These promising in vitro findings were translated into an in vivo model, where the daily administration of FTY720 significantly delayed the growth of mutant c-KIT⁺ tumours. Furthermore, FTY720 markedly prevented the infiltration of D816V c-KIT tumour cells into secondary lymphoid organs, such as the spleen and bone marrow. As a result, the survival of FTY720-treated mice was significantly prolonged compared to saline-treated controls. Overall, this body of work greatly enhances our understanding of PP2A function and identifies the complex mechanisms of PP2A regulation by the oncogenic tyrosine kinases, BCR/ABL and c-KIT. Taken together, the data suggests that inhibition of PP2A may represent a general mechanism employed by constitutively active kinases to facilitate tumour growth. As such, this work supports the future application of PP2A-activating agents in a broad range of human malignancies.

No description available.

Les lymphomes non hodgkiniens (LNH) regroupent un panel hétérogène de pathologies originaires de cellules lymphatiques. Parmi les LNH à cellules B matures, la leucémie lymphoïde chronique (LLC) constitue la forme de leucémie de l’adulte la plus fréquente en Occident. La physiopathologie des LNH à cellules B matures est marquée par l’inhibition des mécanismes de la mort cellulaire, notamment via la surexpression de la protéine MCL-1. Une première partie de ce travail de thèse a été de déterminer quelles pouvaient être les enzymes de déubiquitination (DUBs) impliquées dans la survie des LNH à cellules B matures et la stabilisation de MCL-1. Notre étude a permis d’identifier la DUB USP14, qui est liée au système ubiquitine-protéasome, comme capable de réguler MCL-1 et la survie cellulaire. Nos travaux montrent également pour la première fois que l’activité DUB des cellules, ainsi que l’activité d’USP14, sont directement régulées par la signalisation du BCR via l'activité de la tyrosine kinase SYK. Le FTY720, un analogue de la sphingosine utilisé comme immunosuppresseur dans la sclérose en plaques, a montré un effet cytotoxique dans des hémopathies malignes sans toutefois que son mécanisme d’action soit clairement expliqué. Une deuxième partie de ce travail de thèse a été de caractériser la mort induite par le FTY720. Notre étude montre que la caractérisation de la morphologie cellulaire et des marqueurs induits par la mort due au FTY720 dans les LLC correspond en fait à une nécrose cellulaire programmée indépendante de RIPK1, mais dépendante d'une enzyme régulatrice de la fission mitochondriale, DRP1. / Non-Hodgkin lymphoma (NHL) include a diverse range of pathologies originate from the lymphatic cells. Among the mature B-cell NHL, chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries. The pathophysiology of mature B-cell NHL is marked by the inhibition of cell death mechanisms, particularly through the overexpression of MCL-1 protein. The first part of this thesis was to determine which deubiquitinating enzymes (DUBs) are involved in the survival of mature B-cell NHL and in the stabilization of MCL-1. Our study identified the DUB USP14, which is linked to the ubiquitin-proteasome system, as able to regulate MCL-1 and cell survival. Our work also shows for the first time that the DUB activity of the cells and the activity of USP14 are directly regulated by BCR signaling through the activity of the SYK tyrosine kinase. FTY720, a sphingosine analog used as an immunosuppressive drug in multiple sclerosis, showed a cytotoxic effect in hematological malignancies but its mechanism of action is not well understood. A second part of this thesis was to characterize the death induced by FTY720. Our study shows that the characterization of the cellular morphology and markers induced by death due to FTY720 in the LLC corresponds in fact to a programmed RIPK1-independent necrosis cell death, but dependent on DRP1, a regulatory enzyme of the mitochondrial fission.