Apoptosis-induced compensatory proliferation (AiP) is a mechanism that maintains tissue homeostasis after stress-induced cell death. During AiP, apoptotic cells induce proliferation of the neighboring surviving cells to compensate for tissue loss. AiP is important for wound healing and tissue regeneration in several model organisms. Additionally, AiP is an important feature of tumorigenesis and tumor relapse as it contributes to tumor repopulation following radiation or chemotherapy. Using an overgrowth tumor model (“undead tissue”) in Drosophila melanogaster, we determined that the initiator caspase Dronc promotes generation of extracellular Reactive Oxygen Species (ROS), which drive activation of the stress kinase JNK and downstream mitogens to promote AiP. We also observed increased numbers of Drosophila macrophages, termed hemocytes, which are attracted to undead tissue. However, the specific mechanisms by which macrophages are recruited to undead tissue are still unclear. Here, we report that the tissue microenvironment of the overgrown undead tissue directs macrophage recruitment during AiP. We demonstrate that ROS, JNK, and the matrix metalloproteinase Mmp2 are important for recruiting macrophages. Mechanistically, undead tissue-produced ROS and active JNK damage the basement membrane (BM) surrounding the undead tissue, by upregulating the expression and activity of Mmp2. The damaged BM then recruits macrophages to the undead tissue. Taken together, we propose a model in which the ROS-JNK-Mmp2 signaling axis damages the BM of undead tissue, resulting in changes in the tissue microenvironment that recruit macrophages to the area of damage to promote AiP and overgrowth.

No description available.

Introdução: A correção anatômica da transposição das grandes artérias após o período neonatal demanda a preparação prévia do ventrículo subpulmonar, com bandagem do tronco pulmonar, para induzir a hipertrofia ventricular. Estudos experimentais prévios demonstraram que a sobrecarga sistólica intermitente determina uma hipertrofia ventricular mais eficiente, em relação à bandagem convencional (fixa) do tronco pulmonar. Os mecanismos adaptativos envolvidos no retreinamento do ventrículo subpulmonar ainda não estão completamente estabelecidos, pois se sabe que, além da hipertrofia e hiperplasia das células contráteis, também células do interstício e vasos sofrem alterações fenotípicas. Permanece indefinida a taxa ideal de incremento, tanto da matriz extracelular quanto da vascularização do miocárdio na situação do retreinamento ventricular, para suportar a resistência sistêmica. Objetivo: Avaliar a adaptação do ventrículo subpulmonar no que se refere a apoptose e estímulo à neovascularização miocárdica em resposta à sobrecarga pressórica contínua versus intermitente, obtida pela bandagem ajustável do tronco pulmonar de cabritos jovens. Método: Foram utilizados 21 cabritos hígidos, com idade de 30 a 60 dias e pesos comparáveis, divididos em três grupos: Controle (n = 7, sem sobrecarga sistólica), Contínuo (n = 7, sobrecarga sistólica contínua do VD), Intermitente (n = 7, 12 horas/dia de sobrecarga sistólica intermitente do VD). A sobrecarga sistólica do VD foi mantida por 96 horas no grupo Contínuo e por quatro períodos de 12 horas, alternados com 12 horas de descanso, no grupo intermitente. Os animais do grupo Controle foram submetidos ao implante do dispositivo de bandagem, o qual foi mantido desinsuflado. As medidas hemodinâmicas foram tomadas diariamente, antes e após o ajuste da sobrecarga sistólica. Avaliações ecocardiográficas foram realizadas no pré-operatório e no final do protocolo de estudo. Após 96 horas de estudo, os animais foram mortos para avaliação dos parâmetros morfológicos (peso e conteúdo de água das massas cardíacas e análise imuno-histoquímica da apoptose e da expressão do VEGF). Resultados: Ao final do protocolo, o ecocardiograma revelou uma diferença significativa da espessura do VD no grupo Intermitente (+129,2%), quando comparado ao grupo Contínuo (+58,2%; p < 0,001) e de ambos os grupos de estudo quando comparados ao grupo Controle (p < 0,001). Sob a análise morfológica, ambos os grupos de estudo apresentaram ganho de magnitude semelhante nas massas do VD (Intermitente: + 115,8%; Contínuo: + 90,8%; p < 0,0001) e do septo (Intermitente: +55,8%; Contínuo: + 45,4%; p < 0,047), em relação ao grupo Controle, apesar do menor tempo de sobrecarga pressórica no grupo Intermitente. O protocolo de sobrecarga sistólica do VD não influenciou a massa muscular do VE. Houve um discreto aumento do conteúdo de água do VD (Contínuo: +3,5%, Intermitente: +4,6%) e do septo (ambos os grupos de estudo: +3,5%) em relação ao grupo Controle (p < 0,002). A expressão do VEGF foi maior no VD do grupo Intermitente (2,89% ± 0,41%; p=0,005) em relação ao VD dos demais grupos (Controle: 1,43% ± 0,18%; Contínuo: 1,80% ± 0,19%). A expressão desta molécula no miocárdio do VD do grupo Intermitente foi também maior que o do VE e septo dentro do mesmo grupo (p < 0,050). Não houve diferença na expressão do VEGF das demais massas cardíacas (VE: p > 0,252; Septo: p > 0,740). Em relação à apoptose, não foram observadas diferenças significativas no miocárdio do VD dos três grupos (Caspase: p=0,784; TUNEL: p=0,374). Conclusões: Ambos os grupos de estudo desenvolveram hipertrofia miocárdica do VD, não acompanhada de edema miocárdico importante ou apoptose. No entanto, a sobrecarga sistólica intermitente promoveu maior expressão do VEGF no miocárdio do VD. Esta associação entre sinalização de proliferação vascular e hipertrofia tem implicações importantes quando se objetiva preparar um ventrículo para suportar pressões sistêmicas, uma vez que o desejável é que a proliferação vascular ocorra de forma sustentada, permitindo uma hipertrofia do VD mais eficiente / Introduction: Surgical correction of transposition of the great arteries beyond the neonatal period needs a previous pulmonary artery band to promote left ventricular hypertrophy thereby preparing the ventricle. Experimental studies have demonstrated that intermittent systolic overload causes a more efficient ventricular hypertrophy, as compared to traditional pulmonary artery banding. The adaptive mechanisms involved in the subpulmonary ventricle retraining are not completely established. Nevertheless, besides the hypertrophy and/or hyperplasia of the contractile cardiomyocytes, noncontractile cells (vascular and interstitial) from the stimulated ventricle also present structural phenotype changes. It remains unclear the ideal increasing rate of the myocardial interstitium as well as capillary vessel proliferation in the process of ventricular retraining before undertaking the arterial switch. Objective: This study sought to assess adaptive changes of the subpulmonary ventricle in regards to vascular endothelial growth factor (VEGF) expression and apoptosis in young goats submitted to continuous versus intermittent systolic overload by means of an adjustable pulmonary artery band. Methods: 21 young goats were separated into 3 groups: Control (no systolic overload), Continuous (96-hour continuous systolic overload), and Intermittent (four 12-hour periods of systolic overload paired with a 12-hour resting period). Systolic overload was adjusted to achieve a 0.7 RV / aortic pressure ratio. Hemodynamic evaluations were performed before and after systolic overload every day postoperatively. Echocardiograms were obtained preoperatively and at the end of protocol. After the study period, the animals were humanely killed for morphologic assessment, apoptosis and vascular endothelial growth factor (VEGF) expression. Results: Echocardiography revealed a marked increase in RV wall thickness in the Intermittent group (+129.2%), compared with the Continuous group (+58.2%; p<0.001), as well as both trained groups compared to Control group (p < 0.001). Regardless of the shorter systolic overload exposure of Intermittent group, both study groups had a similar increase in RV mass (Intermittent: + 115.8%; Continuous: +90.8%; p < 0.001), and septal mass (Intermittent: + 55.8%; Continuous: + 45.4%; p < 0.047), compared with the Control group. No significant changes in the left ventricle mass were seen. There was a negligible but significant increase in water content of RV (Continuous: +3.5%, Intermittent: +4.6%) and septal masses (both study groups: +3.5%) compared with that in the Control group (p < 0.002). RV VEGF expression was greater in the Intermittent group (2.89% ± 0.41%) than in the Continuous (1.80% ± 0.19%) and Control (1.43% ± 0.18%) groups (p < 0.023). VEGF expression in the myocardium of the right ventricle in the Intermittent group was also greater than that in the left ventricle and septum within the same group (p < 0.050). There was no significant difference in VEGF expression between the other cardiac sections or within the Control and Continuous groups. Regarding apoptosis, there were no significant changes in the RV myocardium of the three groups (Caspase: p=0,784; TUNEL: p=0,374). Conclusions: Both study groups have developed RV hypertrophy with no apoptosis or relevant myocardial edema. Nevertheless, intermittent systolic overload causes upregulation of VEGF expression in the subpulmonary ventricle, an adaptation that provides a mechanism for increased myocardial perfusion during the rapid myocardial hypertrophy of young goats. The association of the marked increase in RV mass and increased angiogenesis signaling has an important implication on the subpulmonary ventricle retraining protocol by promoting a compensatory growth of the coronary vasculature, allowing for a more efficient hypertrophy

Introdução: A correção anatômica da transposição das grandes artérias após o período neonatal demanda a preparação prévia do ventrículo subpulmonar, com bandagem do tronco pulmonar, para induzir a hipertrofia ventricular. Estudos experimentais prévios demonstraram que a sobrecarga sistólica intermitente determina uma hipertrofia ventricular mais eficiente, em relação à bandagem convencional (fixa) do tronco pulmonar. Os mecanismos adaptativos envolvidos no retreinamento do ventrículo subpulmonar ainda não estão completamente estabelecidos, pois se sabe que, além da hipertrofia e hiperplasia das células contráteis, também células do interstício e vasos sofrem alterações fenotípicas. Permanece indefinida a taxa ideal de incremento, tanto da matriz extracelular quanto da vascularização do miocárdio na situação do retreinamento ventricular, para suportar a resistência sistêmica. Objetivo: Avaliar a adaptação do ventrículo subpulmonar no que se refere a apoptose e estímulo à neovascularização miocárdica em resposta à sobrecarga pressórica contínua versus intermitente, obtida pela bandagem ajustável do tronco pulmonar de cabritos jovens. Método: Foram utilizados 21 cabritos hígidos, com idade de 30 a 60 dias e pesos comparáveis, divididos em três grupos: Controle (n = 7, sem sobrecarga sistólica), Contínuo (n = 7, sobrecarga sistólica contínua do VD), Intermitente (n = 7, 12 horas/dia de sobrecarga sistólica intermitente do VD). A sobrecarga sistólica do VD foi mantida por 96 horas no grupo Contínuo e por quatro períodos de 12 horas, alternados com 12 horas de descanso, no grupo intermitente. Os animais do grupo Controle foram submetidos ao implante do dispositivo de bandagem, o qual foi mantido desinsuflado. As medidas hemodinâmicas foram tomadas diariamente, antes e após o ajuste da sobrecarga sistólica. Avaliações ecocardiográficas foram realizadas no pré-operatório e no final do protocolo de estudo. Após 96 horas de estudo, os animais foram mortos para avaliação dos parâmetros morfológicos (peso e conteúdo de água das massas cardíacas e análise imuno-histoquímica da apoptose e da expressão do VEGF). Resultados: Ao final do protocolo, o ecocardiograma revelou uma diferença significativa da espessura do VD no grupo Intermitente (+129,2%), quando comparado ao grupo Contínuo (+58,2%; p < 0,001) e de ambos os grupos de estudo quando comparados ao grupo Controle (p < 0,001). Sob a análise morfológica, ambos os grupos de estudo apresentaram ganho de magnitude semelhante nas massas do VD (Intermitente: + 115,8%; Contínuo: + 90,8%; p < 0,0001) e do septo (Intermitente: +55,8%; Contínuo: + 45,4%; p < 0,047), em relação ao grupo Controle, apesar do menor tempo de sobrecarga pressórica no grupo Intermitente. O protocolo de sobrecarga sistólica do VD não influenciou a massa muscular do VE. Houve um discreto aumento do conteúdo de água do VD (Contínuo: +3,5%, Intermitente: +4,6%) e do septo (ambos os grupos de estudo: +3,5%) em relação ao grupo Controle (p < 0,002). A expressão do VEGF foi maior no VD do grupo Intermitente (2,89% ± 0,41%; p=0,005) em relação ao VD dos demais grupos (Controle: 1,43% ± 0,18%; Contínuo: 1,80% ± 0,19%). A expressão desta molécula no miocárdio do VD do grupo Intermitente foi também maior que o do VE e septo dentro do mesmo grupo (p < 0,050). Não houve diferença na expressão do VEGF das demais massas cardíacas (VE: p > 0,252; Septo: p > 0,740). Em relação à apoptose, não foram observadas diferenças significativas no miocárdio do VD dos três grupos (Caspase: p=0,784; TUNEL: p=0,374). Conclusões: Ambos os grupos de estudo desenvolveram hipertrofia miocárdica do VD, não acompanhada de edema miocárdico importante ou apoptose. No entanto, a sobrecarga sistólica intermitente promoveu maior expressão do VEGF no miocárdio do VD. Esta associação entre sinalização de proliferação vascular e hipertrofia tem implicações importantes quando se objetiva preparar um ventrículo para suportar pressões sistêmicas, uma vez que o desejável é que a proliferação vascular ocorra de forma sustentada, permitindo uma hipertrofia do VD mais eficiente / Introduction: Surgical correction of transposition of the great arteries beyond the neonatal period needs a previous pulmonary artery band to promote left ventricular hypertrophy thereby preparing the ventricle. Experimental studies have demonstrated that intermittent systolic overload causes a more efficient ventricular hypertrophy, as compared to traditional pulmonary artery banding. The adaptive mechanisms involved in the subpulmonary ventricle retraining are not completely established. Nevertheless, besides the hypertrophy and/or hyperplasia of the contractile cardiomyocytes, noncontractile cells (vascular and interstitial) from the stimulated ventricle also present structural phenotype changes. It remains unclear the ideal increasing rate of the myocardial interstitium as well as capillary vessel proliferation in the process of ventricular retraining before undertaking the arterial switch. Objective: This study sought to assess adaptive changes of the subpulmonary ventricle in regards to vascular endothelial growth factor (VEGF) expression and apoptosis in young goats submitted to continuous versus intermittent systolic overload by means of an adjustable pulmonary artery band. Methods: 21 young goats were separated into 3 groups: Control (no systolic overload), Continuous (96-hour continuous systolic overload), and Intermittent (four 12-hour periods of systolic overload paired with a 12-hour resting period). Systolic overload was adjusted to achieve a 0.7 RV / aortic pressure ratio. Hemodynamic evaluations were performed before and after systolic overload every day postoperatively. Echocardiograms were obtained preoperatively and at the end of protocol. After the study period, the animals were humanely killed for morphologic assessment, apoptosis and vascular endothelial growth factor (VEGF) expression. Results: Echocardiography revealed a marked increase in RV wall thickness in the Intermittent group (+129.2%), compared with the Continuous group (+58.2%; p<0.001), as well as both trained groups compared to Control group (p < 0.001). Regardless of the shorter systolic overload exposure of Intermittent group, both study groups had a similar increase in RV mass (Intermittent: + 115.8%; Continuous: +90.8%; p < 0.001), and septal mass (Intermittent: + 55.8%; Continuous: + 45.4%; p < 0.047), compared with the Control group. No significant changes in the left ventricle mass were seen. There was a negligible but significant increase in water content of RV (Continuous: +3.5%, Intermittent: +4.6%) and septal masses (both study groups: +3.5%) compared with that in the Control group (p < 0.002). RV VEGF expression was greater in the Intermittent group (2.89% ± 0.41%) than in the Continuous (1.80% ± 0.19%) and Control (1.43% ± 0.18%) groups (p < 0.023). VEGF expression in the myocardium of the right ventricle in the Intermittent group was also greater than that in the left ventricle and septum within the same group (p < 0.050). There was no significant difference in VEGF expression between the other cardiac sections or within the Control and Continuous groups. Regarding apoptosis, there were no significant changes in the RV myocardium of the three groups (Caspase: p=0,784; TUNEL: p=0,374). Conclusions: Both study groups have developed RV hypertrophy with no apoptosis or relevant myocardial edema. Nevertheless, intermittent systolic overload causes upregulation of VEGF expression in the subpulmonary ventricle, an adaptation that provides a mechanism for increased myocardial perfusion during the rapid myocardial hypertrophy of young goats. The association of the marked increase in RV mass and increased angiogenesis signaling has an important implication on the subpulmonary ventricle retraining protocol by promoting a compensatory growth of the coronary vasculature, allowing for a more efficient hypertrophy

Les polymorphonucléaires neutrophiles (PMNs) représentent une arme primordiale dans la défense contre divers agents pathogènes; notamment les bactéries, les champignons, les cellules tumorales de même que les cellules infectées par des virus. Cependant, certaines pathologies reliées à l’inflammation chronique soulèvent l’implication des neutrophiles notamment dans l’arthrite rhumatoïde. La réponse inflammatoire persistante générée par l’activation et la survie des neutrophiles engendre une destruction des tissus environnants suite à la sécrétion non contrôlée de leurs produits cytotoxiques. Même si l’activation chronique des neutrophiles est néfaste dans plusieurs pathologies, elle pourrait s’avérer un bon outil en cas de neutropénie, comme c’est souvent le cas les patients ayant reçu des traitements de chimiothérapie. Ce projet fait suite aux travaux doctoraux de Lagraoui (1999). Il vise à identifier le(s) facteur(s) du liquide synovial qui augmente la survie des neutrophiles ainsi que le mécanisme d’action impliqué dans ce processus. Similairement au facteur semi-pur isolés par Lagraoui (1999), le milieu conditionné concentré (MCC) augmente la survie des PMNs de 75% (39% ± 9.5 vs 68% ± 2.5, p<0.01). Suivant le séquençage du MCC parallèlement au facteur semi-pur actif, deux protéines ont été identifiées à la fois dans le MCC et dans le facteur semi-pur soient : l’albumine et la fétuine. Notre projet vise donc à comparer les effets de l’albumine et de la fétuine à ceux du GM-CSF dans l’optique d’une thérapie alternative au GM-CSF en tant qu’adjuvant de chimiothérapie. La présence d’albumine, de fétuine ou de GM-CSF chez les PMNs incubés 24 heures avec la Mutamycin® induit une diminution du nombre de cellules en apoptose par rapport à la Mutamycin® (Ctrl : 43% ± 10; A : 74% ± 3; F : (82% ± 6 et GM : 74% ± 7; p<0.01). L’effet de l’albumine dépend de la voie de la kinase PI3 mais également celle la kinase ERK, alors que celle de la fétuine dépend de la kinase PI3. Similairement l’EPO, l’albumine et la fétuine supporte la différentiation des HSCs en précurseurs érythrocytaires de type BFU-E. Dans un modèle murin de chiomioprotection, l’albumine augmente la concentration cellulaire rapport au groupe contrôle des leukocytes de la rate (66 ±8 x106c/ml vs 81 ±16 x106c/ml) et du sang (3.6 ±0.4 x106c/ml vs 5.7 ±2.3 x106c/ml). Donc, in vitro, l’albumine et la fétuine sont comparables au GM-CSF au niveau fonctionalité et mécansimes d’action. Cependant, vu leur manque de spécificité, l’application thérapeutique en tant qu’adjuvant de chiomiothérapie de l’albumine et la fétuine est peu prometteuse. Par contre, les maladies dégénératives et les évènements ischémiques pourraient s’avérer de bonnes cibles thérapeutiques, principalement pour l’albumine. / Circulating polymorphonuclear neutrophils (PMN) possess a short half-life and are constantly renewed by the bone marrow to ensure the first-line of defense. Therefore, homeostasis must be maintained through a well-regulated process of apoptosis. Survival of PMN can be regulated by several cytokines as well as conditioned media (CM). Although PMN are crucial for protection against microorganisms, activated neutrophils can lead to severe tissue damage in diseases characterized by chronic inflammation. Indeed, in rheumatoid arthritis (RA), activated PMN contribute to tissue damage by releasing a number of destructive agents. On the other hand, chronic activation of PMN could prevent opportunistic infections present in immunosuppressed patients. This project addresses the isolation and mechanism of action of synovial liquid components on the survival of neutrophils based on previous work (Lagraoui, 1999). Following tangential flow filtration (MW cut off: 30 and 50 kDa), concentrated CM enhanced the viability (75%) of 24-hour cultured human neutrophils isolated from peripheral blood of healthy volunteers (39% ± 9.5 vs 68% ± 2.5, p<0.01) as seen in Lagraoui (1999) previous work. N-terminal protein sequence analysis of the concentrated CM and fractionated conditioned media from previous work revealed 2 known proteins contained in both analysis: albumin, and fetuin. In view of the importance of neutrophiles in immune defense, we compared the benefits of albumin and fetuin to those of granulocytes macrophages-colony stimulating factor (GM-CSF), a growth factor used as an adjunct to cancer chemotherapy. Albumin and fetuin were tested by the AnnexinV-FITC/7-AAD method and displayed an inhibition of neutrophil apoptosis of two to three folds relative to control value. Moreover, albumin (A : 200μM) and fetuin (F : 200μM) rescue human PMN from mutamycin-induced apoptosis, comparable to GM-CSF (GM : 10ng/ml); (Ctrl : 43% ± 10; A : 74% ± 3; F : (82% ± 6 et GM : 74% ± 7; p<0.01). Albumin also induces cellular signaling pathways activation via PI3-K and ERK, whereas fetuin acts through PI3-K pathway only. They induce the differentiation of HSCs into erythrocytes progenitors BFU-E. In immunosuppressed mice, albumin protects white blood cells depletion induced by cytotoxic agent from spleen and blood. Considering all the benefits of albumin and fetuin, their targeting as an adjunct to cancer chemotherapy could be disappointing in view of their lack of specificity. On the other hand, their multiple benefits could have a major impact on neurodegenerative disorders and ischemic events.

The involvement of mitochondrial signaling in mammalian PCD regulation has been examined extensively via biochemical analyses and cellular studies in vitro. However there still exist considerable gaps in our knowledge regarding its contribution in specific tissues and cell types during mammalian development in vivo. In addition, given the numerous pathologic conditions associated with aberrant PCD, modulation of this signaling process represents an attractive target for therapeutic intervention. In this thesis I have therefore examined the regulation of mitochondrion-mediated PCD signaling as it pertains to several forms of developmental and injury-induced cell death. In the first component of the thesis I have examined the differential sensitivity of Bcl2 on the survival of motor neuron populations from two distinct developmental origins (alpha and gamma motor neurons), demonstrating that gamma motor neurons are preferentially affected in Bcl2 null mice. Thus, Bcl-2 plays a critical in vivo in regulating subtype-specific motor neuron survival during development. In the second study I have demonstrated that a major portion of the neuroprotective effect exerted by the immunophilins cyclosporin A and FK-506 are mediated through calcineurin signaling; rather than MOMP-mediated events as previously held. Additional findings of this study demonstrated the first neuroprotective effects of the pyrethroid insecticide cypermethrin and calcineurin-mediated control of Bad phosphorylation. Such findings establish a link between calcineurin signaling and mitochondrion-mediated cell survival. The above studies established critical features of mitochondrion-mediated PCD in regulating survival of several neuronal subpopulations. I therefore followed these studies with an examination of how post-mitochondrial PCD signaling is regulated following MOMP permeabilization. Specifically I examined regulation of the Smac-IAP-caspase axis, investigating how combinatorial deletion of Casp3 and Diablo alter PCD progression in mouse embryonic fibroblasts. Using a series of injury stimuli in the context of biochemical and cellular analyses I have developed a model of how endogenous Smac/DIABLO regulates executioner caspase activity. Collectively these studies elucidate key aspects of mitochondrial signaling during both developmental and injury-induced PCD in vivo.

The involvement of mitochondrial signaling in mammalian PCD regulation has been examined extensively via biochemical analyses and cellular studies in vitro. However there still exist considerable gaps in our knowledge regarding its contribution in specific tissues and cell types during mammalian development in vivo. In addition, given the numerous pathologic conditions associated with aberrant PCD, modulation of this signaling process represents an attractive target for therapeutic intervention. In this thesis I have therefore examined the regulation of mitochondrion-mediated PCD signaling as it pertains to several forms of developmental and injury-induced cell death. In the first component of the thesis I have examined the differential sensitivity of Bcl2 on the survival of motor neuron populations from two distinct developmental origins (alpha and gamma motor neurons), demonstrating that gamma motor neurons are preferentially affected in Bcl2 null mice. Thus, Bcl-2 plays a critical in vivo in regulating subtype-specific motor neuron survival during development. In the second study I have demonstrated that a major portion of the neuroprotective effect exerted by the immunophilins cyclosporin A and FK-506 are mediated through calcineurin signaling; rather than MOMP-mediated events as previously held. Additional findings of this study demonstrated the first neuroprotective effects of the pyrethroid insecticide cypermethrin and calcineurin-mediated control of Bad phosphorylation. Such findings establish a link between calcineurin signaling and mitochondrion-mediated cell survival. The above studies established critical features of mitochondrion-mediated PCD in regulating survival of several neuronal subpopulations. I therefore followed these studies with an examination of how post-mitochondrial PCD signaling is regulated following MOMP permeabilization. Specifically I examined regulation of the Smac-IAP-caspase axis, investigating how combinatorial deletion of Casp3 and Diablo alter PCD progression in mouse embryonic fibroblasts. Using a series of injury stimuli in the context of biochemical and cellular analyses I have developed a model of how endogenous Smac/DIABLO regulates executioner caspase activity. Collectively these studies elucidate key aspects of mitochondrial signaling during both developmental and injury-induced PCD in vivo.

Les polymorphonucléaires neutrophiles (PMNs) représentent une arme primordiale dans la défense contre divers agents pathogènes; notamment les bactéries, les champignons, les cellules tumorales de même que les cellules infectées par des virus. Cependant, certaines pathologies reliées à l’inflammation chronique soulèvent l’implication des neutrophiles notamment dans l’arthrite rhumatoïde. La réponse inflammatoire persistante générée par l’activation et la survie des neutrophiles engendre une destruction des tissus environnants suite à la sécrétion non contrôlée de leurs produits cytotoxiques. Même si l’activation chronique des neutrophiles est néfaste dans plusieurs pathologies, elle pourrait s’avérer un bon outil en cas de neutropénie, comme c’est souvent le cas les patients ayant reçu des traitements de chimiothérapie. Ce projet fait suite aux travaux doctoraux de Lagraoui (1999). Il vise à identifier le(s) facteur(s) du liquide synovial qui augmente la survie des neutrophiles ainsi que le mécanisme d’action impliqué dans ce processus. Similairement au facteur semi-pur isolés par Lagraoui (1999), le milieu conditionné concentré (MCC) augmente la survie des PMNs de 75% (39% ± 9.5 vs 68% ± 2.5, p<0.01). Suivant le séquençage du MCC parallèlement au facteur semi-pur actif, deux protéines ont été identifiées à la fois dans le MCC et dans le facteur semi-pur soient : l’albumine et la fétuine. Notre projet vise donc à comparer les effets de l’albumine et de la fétuine à ceux du GM-CSF dans l’optique d’une thérapie alternative au GM-CSF en tant qu’adjuvant de chimiothérapie. La présence d’albumine, de fétuine ou de GM-CSF chez les PMNs incubés 24 heures avec la Mutamycin® induit une diminution du nombre de cellules en apoptose par rapport à la Mutamycin® (Ctrl : 43% ± 10; A : 74% ± 3; F : (82% ± 6 et GM : 74% ± 7; p<0.01). L’effet de l’albumine dépend de la voie de la kinase PI3 mais également celle la kinase ERK, alors que celle de la fétuine dépend de la kinase PI3. Similairement l’EPO, l’albumine et la fétuine supporte la différentiation des HSCs en précurseurs érythrocytaires de type BFU-E. Dans un modèle murin de chiomioprotection, l’albumine augmente la concentration cellulaire rapport au groupe contrôle des leukocytes de la rate (66 ±8 x106c/ml vs 81 ±16 x106c/ml) et du sang (3.6 ±0.4 x106c/ml vs 5.7 ±2.3 x106c/ml). Donc, in vitro, l’albumine et la fétuine sont comparables au GM-CSF au niveau fonctionalité et mécansimes d’action. Cependant, vu leur manque de spécificité, l’application thérapeutique en tant qu’adjuvant de chiomiothérapie de l’albumine et la fétuine est peu prometteuse. Par contre, les maladies dégénératives et les évènements ischémiques pourraient s’avérer de bonnes cibles thérapeutiques, principalement pour l’albumine. / Circulating polymorphonuclear neutrophils (PMN) possess a short half-life and are constantly renewed by the bone marrow to ensure the first-line of defense. Therefore, homeostasis must be maintained through a well-regulated process of apoptosis. Survival of PMN can be regulated by several cytokines as well as conditioned media (CM). Although PMN are crucial for protection against microorganisms, activated neutrophils can lead to severe tissue damage in diseases characterized by chronic inflammation. Indeed, in rheumatoid arthritis (RA), activated PMN contribute to tissue damage by releasing a number of destructive agents. On the other hand, chronic activation of PMN could prevent opportunistic infections present in immunosuppressed patients. This project addresses the isolation and mechanism of action of synovial liquid components on the survival of neutrophils based on previous work (Lagraoui, 1999). Following tangential flow filtration (MW cut off: 30 and 50 kDa), concentrated CM enhanced the viability (75%) of 24-hour cultured human neutrophils isolated from peripheral blood of healthy volunteers (39% ± 9.5 vs 68% ± 2.5, p<0.01) as seen in Lagraoui (1999) previous work. N-terminal protein sequence analysis of the concentrated CM and fractionated conditioned media from previous work revealed 2 known proteins contained in both analysis: albumin, and fetuin. In view of the importance of neutrophiles in immune defense, we compared the benefits of albumin and fetuin to those of granulocytes macrophages-colony stimulating factor (GM-CSF), a growth factor used as an adjunct to cancer chemotherapy. Albumin and fetuin were tested by the AnnexinV-FITC/7-AAD method and displayed an inhibition of neutrophil apoptosis of two to three folds relative to control value. Moreover, albumin (A : 200μM) and fetuin (F : 200μM) rescue human PMN from mutamycin-induced apoptosis, comparable to GM-CSF (GM : 10ng/ml); (Ctrl : 43% ± 10; A : 74% ± 3; F : (82% ± 6 et GM : 74% ± 7; p<0.01). Albumin also induces cellular signaling pathways activation via PI3-K and ERK, whereas fetuin acts through PI3-K pathway only. They induce the differentiation of HSCs into erythrocytes progenitors BFU-E. In immunosuppressed mice, albumin protects white blood cells depletion induced by cytotoxic agent from spleen and blood. Considering all the benefits of albumin and fetuin, their targeting as an adjunct to cancer chemotherapy could be disappointing in view of their lack of specificity. On the other hand, their multiple benefits could have a major impact on neurodegenerative disorders and ischemic events.

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The aim of this thesis is the characterization of a protein involved in apoptosis. Our group has identified an early step common to different forms of intrinsic apoptosis stimuli. This step requires de novo synthesis of a novel Cyclin, Cyclin O, that upon apoptosis induction in lymphoid cells forms active complexes, primarily with Cdk2. Cyclin O expression precedes glucocorticoid and gamma radiation-induced apoptosis in vivo in mouse thymus and its overexpression induces apoptosis in cultured cells. Knocking down the endogenous expression of Cyclin O by shRNA leads to the inhibition of glucocorticoid and DNA damage-induced apoptosis while leaving CD95 death receptor mediated apoptosis intact. This data demonstrates that apoptosis induction in lymphoid cells is one of the physiological roles of Cyclin O and it does not act by perturbing a normal cellular process such as the cell cycle. In addition we have identified c-Myb a substrate of Cdk2-Cyclin O complexes and we show that c-Myb is downregulated during apoptosis of lymphoid cells. / L'objectiu d'aquesta tesi és la caracterització d'una proteïna involucrada en l'apoptosi. El nostre grup ha identificat un pas primerenc comú en diversos estímuls apoptòtics de la ruta intrínseca. Aquest pas requereix la síntesi de novo d'una nova Ciclina, Ciclina O, que quan s'indueix apoptosi en cèl·lules limfoides forma complexes actius majoritàriament amb Cdk2. L'expressió de la Ciclina O és prèvia a l'apoptosi induïda per glucocorticoids i radiació gamma i la seva sobreexpressió indueix apoptosi en cultius cel·lulars. La baixada dels nivells d'expressió de la Ciclina O endògena amb shRNA provoca una inhibició de l'apoptosi induïda per glucocorticoids o agents que danyen el DNA, mentre que l'apoptosi mediada pel receptor CD95 es manté intacta. Aquests resultats demostren que la inducció d'apoptosi en cèl·lules limfoides és una de les funcions fisiològiques de la Ciclina O i que no es deu a una pertorbació de processos cel·lulars normals com ara el cicle cel·lular. A més a més, hem identificat c-Myb com a substrat dels complexes Cdk2-Ciclina O i demostrem que els nivells de c-Myb baixen durant l'apoptosis de cèl·lules limfoides.