FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O trabalho mostra o tratamento e os efeitos do l-deprenil (DEP), no aprendizado, na memÃria e na peroxidaÃÃo lipÃdica em cÃrebros de ratos submetidos à isquemia cerebral transitÃria (ICT). Os animais (ratos Wistar, fÃmeas, 200-250g) foram submetidos à isquemia cerebral transitÃria pela oclusÃo de ambas as artÃrias carÃtidas durante 20 minutos e tratados durante 5 dias com DEP (5 e 10 mg/kg). A temperatura retal foi monitorada e mantida em torno de 37ÂC atravÃs de uma luz incandescente. O mesmo procedimento foi feito no grupo controle + salina, Falso-operado + salina (FO) com exceÃÃo do clampeamento das artÃrias carÃtidas. No 6 dia apÃs a induÃÃo da isquemia, os animais foram submetidos aos testes de atividade locomotora e memÃria (esquiva passiva, labirinto em T elevado e labirinto aquÃtico de Morris), a seguir foram sacrificados e os cÃrebros dissecados sobre gelo (hipocampo e cÃrtex temporal) para as determinaÃÃes de MDA, nitrito/nitrato e atividade da catalase e atividade da protease caspase-3. No protocolo de avaliaÃÃo da Ãrea total do infarto encontramos apÃs 1 hora de ICT uma Ãrea de infarto 38,01  3,44% da Ãrea total do cÃrebro, e apÃs 24 horas de ICT uma Ãrea de infarto 22,00  2,90% da Ãrea total do cÃrebro. Os parÃmetros fisiolÃgicos estudados nÃo mostraram alteraÃÃes entre os grupos ICT e FO. Nenhuma alteraÃÃo na atividade locomotora foi detectada nos grupos FO, ICT, Dep 10 + ICT. PorÃm, um aumento na atividade locomotora foi observado no grupo Dep 5 + ICT (7,37  1, 77, p< 0,02) quando comparado com o grupo FO, tratado com salina, (4,66  1,54). No teste do Labirinto em T elevado (T Maze) a ICT afetou os processos de aquisiÃÃo e retenÃÃo de memÃria quando os animais foram testados no mesmo dia (esquiva 1 e 2) quando comparados com o grupo controle (FO). O teste de Kruskall-Wallis mostrou alteraÃÃo significativa na latÃncia da esquiva inibitÃria (esquiva 1 e 2 quando comparados com o treino) no falso-operado (FO - treino: 20,34  3,43 s; esquiva 1 - 231,6  34,81 s; esquiva 2 â 247,8  27,25 s; KW = 19,62, p< 0,001), e no grupo l-deprenil (5 e 10 mg/kg) + isquemia (Dep 5 â treino: 110,8  56,16 s; esquiva 1 - 299,8  0,16 s; esquiva 2 â 260  40,00 s; KW = 9,16, p< 0,01. Dep 10 â treino: 29,15  8,64 s; esquiva 1 â 299,80  0,25 s; esquiva 2 299,8  0,25 s; KW = 6,98, p< 0,05). Isto indica uma boa aquisiÃÃo de memÃria. Portanto, o resultado do grupo ICT + salina indicou um dÃficit da memÃria (ICT â treino: 37,75  11,52 s; esquiva 1 â 116,30  65,46 s; treino 2 â 195,00  64,10 s; KW = 3,90, p< 0,141). AlÃm disso, existiu uma diferenÃa significativa (Teste Mann-Whitney) entre os grupos na esquiva 3 (retenÃÃo) quando comparados com o grupo ICT (Dep 5, MW (3) = 18,483, p< 0,0003, Dep 10, MW (3) = 18,483, p< 0,003) significando que a retenÃÃo da memÃria foi aumentada pelo tratamento com a droga. No teste da esquiva passiva os animais do grupo controle (FO + salina) apresentaram uma boa retenÃÃo da memÃria, tanto na fase imediata (memÃria recente - MR), quanto na fase de consolidaÃÃo (memÃria tardia - MT), quando comparadas ao treino (ANOVA) (FO + salina (n-7)- treino - 15,94  4,40 s, MR - 138,84  34,60 s, MT - 196,32  34, 71, p< 0,006). Por outro lado, os animais que sofreram ICT nÃo apresentaram diferenÃa no tempo de latÃncia de entrada no lado escuro quando comparado com o treino, significando um dÃficit na aprendizagem e memÃria (ICT (n-7)- treino - 34,37  10,16 s, MR - 105,54  35,21 s, MT - 96,20  33, 44, p< 0,33), e, portanto dano na aquisiÃÃo e retenÃÃo da memÃria. Comparando os tratamentos observamos um aumento significativo, no tempo de latÃncia de entrada no lado escuro do aparelho, nos ratos tratados com l-deprenil 5 mg/kg quando avaliados na MR (FO + salina- 138,84  34,60s; ICT - 105,54  35,21; ICT + Dep 5- 198,88  38,42s; ICT + Dep 10- 188,06  34,60s; Kruskall-Wallis, KW-9,66, p<0,05, Mann-Whitney, Dep 5 vs ICT, p<0,05), enquanto na MT foi observada uma diminuiÃÃo significativa, no tempo de latÃncia de entrada no lado escuro do aparelho, nos ratos tratados com l-deprenil (5 e 10 mg/kg) (FO + salina- 196,32  34,71s; ICT - 96,20  33,44s; ICT + Dep 5- 299,83  0,16s; ICT + Dep 10- 264,70  35,28 s; Kruskall-Wallis, KW-14,57, p<0,05, Mann-Whitney, Dep 5 e Dep 10 vs ICT, p<0,05), significando melhora no aprendizado do animal fazendo-o lembrar o choque recebido durante o treino e indicando uma reversÃo da lesÃo sofrida com a ICT. No teste do Labirinto AquÃtico (Water Maze) a ICT promoveu um dano da retenÃÃo na memÃria dos animais em relaÃÃo ao grupo controle (FO), porÃm o l-deprenil conseguiu reverter o dano na aquisiÃÃo da memÃria induzida pela ICT em ambas as doses (5 e 10 mg/kg), observamos tambÃm que o grupo Dep 5 obteve um melhor desempenho na aquisiÃÃo da memÃria quando comparado com o grupo Dep 10. (FO (n-10): 5,4  0,84s; FO + DEP 10 (n-10): 9,7  2,28s; ICT (n-9): 32,44  2,95s; ICT + DEP 5 (n-8): 12,88  1,4s; ICT + DEP 10 (n-8): 4,5  0,70s; Kruskall-Wallis, KW-29,07, p<0,05, Mann-Whitney, FO + DEP 10, Dep 5 e Dep 10 vs ICT, p<0,05). Os ratos submetidos a ICT mostraram um aumento de 71% nos nÃveis de MDA no hipocampo quando comparados com o grupo controle (FO), e o tratamento com l-deprenil reverteu significativamente este efeito (p<0,05). Os valores dos nÃveis de MDA foram trazidos prÃximos aqueles valores do grupo controle (FO) em relaÃÃo aos grupos (ICT + DEP 5 e ICT + DEP 10, 34 e 38%, respectivamente) com ambas as doses de l-deprenil mais ICT (Hipocampo - FO (n-7): 45,4  4,45; ICT (n-7): 77,6  8,97; ICT + DEP 5 (n-7): 51,2  1,68; ICT + DEP 10 (n-7): 48,5  6,70 nmoles/g; p<0,05, ANOVA e Teste de Tukey). No cÃrtex temporal, a ICT nÃo aumentou os nÃveis de MDA quando comparados com o grupo controle. Portanto, os ratos submetidos a ICT e tratados com altas doses de l-deprenil (10 mg/kg) apresentaram nÃveis de MDA 30% menor que aqueles mostrados por ambos os grupos FO e ICT (CÃrtex temporal - FO (n-7): 46,8  4,36; ICT (n-7): 48,7  1,33; ICT + DEP 5 (n-7): 52,5  3,74; ICT + DEP 10 (n-7): 33,4  2,98 nmoles/g; p<0,05, ANOVA e Teste de Tukey). No hipocampo, os nÃveis de nitrito foram significativamente aumentados apÃs a ICT quando comparados com o grupo controle FO (82% aumento). O DEP 10 reverteu este efeito e os valores foram trazidos para aqueles do controle. Por outro lado, a isquemia nÃo afetou os nÃveis de nitrito no cÃrtex, entretanto o DEP 5 diminui significativamente os nÃveis de nitrito quando comparados com os grupos controle e ICT. A ICT mostrou um aumento em 50 % da atividade da protease caspase-3 no hipocampo; e o tratamento com l-deprenil (10 mg/kg) reverteu este efeito trazendo os valores prÃximos aos do grupo controle (FO), porÃm o tratamento com DEP 5 nÃo mostrou o mesmo (Valor da AbsorbÃncia: FO â 0,083  0,006; ICT - 0,124  0,017; ICT + DEP 10 â 0,080  0,007; ICT + DEP 5 â 0,125  0,007), porÃm nos animais controle que receberam tratamento com DEP 10 (FO + DEP 10) a atividade da caspase â 3 diminui em 99% em relaÃÃo ao grupo ICT. Em conclusÃo mostramos que a administraÃÃo do l-deprenil diariamente por 5 dias melhorou os danos da memÃria observados apÃs a isquemia cerebral transitÃria em ratos. A droga protegeu o cÃrebro contra a hiperperoxidaÃÃo e formaÃÃo de radicais livres observados apÃs o dano isquÃmico, como diminui a atividade da caspase â 3. Pelo menos parte desses efeitos à devido ao efeito antioxidante e conseqÃentemente inibiÃÃo da ativaÃÃo da produÃÃo de radicais livres pelo l-deprenil. / The present work shows the effects of l-deprenyl (DEP, 5 and 10 mg/kg, po) on memory, as well as on rat brain free radical formation after transient cerebral ischemia (TCI). Wistar rats were anesthetized and submitted to TCI by occlusion of both carotid arteries for 20 minutes. In another experiment, animals were submitted to surgery without ischemia (sham-operated). After surgery, ischaemic rats were treated with DEP (DEP, 5 and 10 mg/kg, po) once and daily for 5 days. One group of animals was left untreated (controls). The parameters studied were, memory acquisition and memory retention, locomotor activity and tiobarbituric acid reactive substances, as an index of lipid peroxidation. After treatment all, animals were submitted to passive avoidance test, water maze test and elevated T maze test, and 24 h later sacrificed and their hippocampi and temporal cortex dissected for evaluation of lipid peroxidation and used for catalase activity determinations. The protein concentration was measured according to the method described by Lowry (1951). In another set of experiments the animals were sacrificied forty eight hours after ischemia for caspase activity evaluation. Results show that DEP significantly reversed ischaemia-induced memory deficits. l-Deprenyl treatment significantly improved memory deficits as compared to ischemic group as measured by The elevated T maze and Water maze tests. A similar result was observed on the passive avoidance test where l-deprenyl improved late but not early memory as compared to the ischemic group. Except for an increased locomotor activity observed in the group treated with 5 mg/kg, no other alteration was detected in this behavioral test. Rats submitted to transient cerebral ischemia (and without l-deprenyl) showed an increase im MDA levels in the hippocampus and the treatment with l-deprenyl (5 and 10 mg/kg) significantly reversed this effect bringing values close to those of the sham-operated controls. A similar profile was observed with nitrite levels. Rats submitted to transient cerebral ischemia show an increase in caspase activity in the hippocampus and the treatment with l-deprenil (10 mg/kg) significantly reversed this effect bringing values close to those of the sham-operated controls. Moreover catalase activity in the hippocampi was not altered by ischemia. In conclusion, the work showed a signifant protective effect of l-deprenyl on memory deficits and lipid hyperperoxidation observed after cerebral ischemia. Possibly, the drug is acting at least in part through its antioxidant and antiapoptotic activities.

O presente estudo teve por objetivo estudar a participação da apoptose na carcinogênese hepatocelular, quantificando os corpos apoptóticos imunomarcados por caspase-3 clivada em amostras de carcinoma hepatocelular (CHC) em pacientes com ou sem cirrose, comparando também estes achados com amostras correspondentes de fígado não tumoral. Visou também à análise semi-quantitativa da imuno-expressão da proteína p53, Bax e Citocromo-C, relacionadas à via mitocondrial da apoptose em busca de eventuais relações com as variáveis clínicopatológicas dos carcinomas hepatocelulares. A análise comparativa da distribuição das diversas proteínas aqui estudadas foi ainda efetuada, com vistas à possível demonstração de sua interação no processo de apoptose em CHC. Amostras selecionadas de 79 casos de CHC foram distribuídas em micromatriz tecidual e submetidas a pesquisa imuno-histoquímica com amplificação por polímeros curtos de dextran ligados a peroxidase. IA foi maior nos CHC que nas amostras não-neoplásicas, mostrando ainda tendência a associação com o grau histológico do CHC .A imuno-expressão de p53 foi maior nos CHC em fígado cirrótico (CHC-C), em casos com invasão vascular, e nos graus histológicos altos. Houve maior imunoexpressão de citocromo c em CHC-C, sendo importante sua associação com p53. Bax mostrou apenas tendência a associação com o tamanho do CHC. Essas evidências contribuem para a compreensão da importância da via mitocondrial da apoptose mediada pela proteína p53 no CHC, destacando também prováveis diferenças do mecanismo carcinogenético na presença ou não de cirrose / This study aimed at the assesment of aspects of the role of apoptosis in hepatocellular carcinogenesis, quantifying apoptotic bodies immunomarked by cleaved caspase-3 in samples of hepatocellular carcinoma (HCC) in patients with or without cirrhosis, further comparing these findings to those from samples in non-tumoral areas of these livers. We also aimed herein to semiquantitate the immunoexpression of p53, Bax, Cytochrome-C, participants of the mitochondrial pathway of apoptosis, searching for possible relations with clinico-pathological variables in HCC. Samples from 79 cases of HCC were arranged in tissue microarrays were and submitted to immunohistochemical reaction with signal amplification achieved by the short-polymer-peroxidase system. Apoptotic index measured by immunoexpression of cleaved-caspase 3 was higher in HCC than in samples from non-neoplastic areas. p53 immunoexpression was higher in HCC occurring in cirrhotic livers, (HCC-C), in cases with vascular invasion and in higher histological grades. Cytochrome-c immunoexpression was also higher in HCC-C and, interestingly, was directly related to p53. Bax immunoreactivity showed only a trend for a relation with the size of HCC. The evidences from the present study further demonstrate the importance of p53-mediated pathway of apoptosis in HCC, and also point for possible differences in carcinogenesis in cirrhotic versus non-cirrhotic livers

L'inflammasome est une plateforme moléculaire composée d'un récepteur de l'immunité innée tels que NLRP3 ou NLRP1b, de la protéine adaptatrice ASC et de la caspase-1. Il joue un rôle essentiel dans le déclenchement de la réponse inflammatoire via l'activation de la caspase-1 qui mène à la sécrétion de cytokines pro-inflammatoires telle que l'IL-1ß. Dans un premier axe de recherche, nous avons mis en évidence que les macrophages de souris déficientes pour ASC traités avec l'activateur de l'inflammasome NLRP1b, la toxine létale de bacillus anthracis, étaient capables de sécréter la forme mature de l'IL-1ß en absence de clivage de la caspase-1 pourtant décrit comme un événement indispensable à son activité. En reconstituant des macrophages caspase-1 KO avec une forme mutante non-clivable de la caspase-1, nous avons démontré que la forme entière de la caspase-1 est capable d'induire la sécrétion d'IL-1ß en réponse à la toxine létale alors qu'elle n'est pas fonctionnelle au sein de l'inflammasome NLRP3.Dans un second axe de recherche, mon travail de thèse s'est intéressé à comprendre le rôle de l'inflammasome au cours de la progression tumorale. En effet, l'IL-1ß est une cytokine fréquemment retrouvée dans le microenvironnement tumorale mammaire suggérant donc l'activation de l'inflammasome au sein des tumeurs. Au moyen d'un modèle de tumeurs in vivo, nous avons montré que l'absence de la caspase-1 et de ASC dans les cellules immunitaires chez la souris conduit à une réduction de la croissance tumorale. De plus, l'absence de ces deux protéines provoque également un plus fort recrutement et une meilleure activité des cellules NK au sein des tumeurs.En plus d'identifier un nouveau mécanisme d'activation de la caspase-1, mon travail de thèse a permis de mettre en évidence le rôle de l'inflammasome dans l'altération des cellules NK au cours de la progression tumorale mammaire. Ces données permettent d'envisager l'inflammasome comme une cible thérapeutique dans le cancer / The inflammasome is a molecular platform composed of an innate immune receptor such as NLRP3 or NLRP1b, of the adaptor protein ASC and of the caspase-1. It plays an essential role in triggering inflammatory response through the activation of caspase-1 that leads to the secretion of pro-inflammatory cytokines such as IL-1ß.In a first research axis, we showed that ASC deficient mice macrophages treated with an NLRP1b inflammasome activator, the lethal toxin of Bacillus anthracis, were able to secrete the mature form of IL-1ß in absence of any cleavage of caspase-1 previously described as an essential event for its function. By reconstituting caspase-1 KO macrophages with an uncleavable mutant form of caspase-1, we showed that the entire form of the protein is able to induce IL-1ß secretion upon lethal toxin treatment but is nonfunctional upon NLRP3 inflammasome activation.In a second research axis, my PhD work focused on underlying the inflammasome role during tumor progression. Indeed, IL-1ß is frequently found within breast tumor microenvironment suggesting that inflammasome is activated in tumors. Using in vivo tumor model, we showed that the absence of caspase-1 of ASC in immune cells lead to a delay tumor growth. In addition, the absence of these two proteins also causes a stronger recruitment and an enchanced activity of NK cells within mammary tumors

Des études menées au laboratoire avaient démontré un rôle critique de l’inflammasome NLRP3 dans l’asthme allergique en réponse à l’ovalbumine en absence d’adjuvant. Mes travaux de thèse ont porté sur le rôle de NLRP3 et de la caspase-1 dans un modèle murin d’inflammation pulmonaire induite par l’allergène d’acarien HDM. Nous avons montré un rôle régulateur de la caspase-1 dépendant de l’inflammasome NLRP3 et la molécule adaptatrice ASC mais pas de l’inflammasome NLRC4. Cette régulation de la réponse allergique se caractérise par une augmentation de l’infiltration des éosinophiles, de l’hyperréactivité bronchique et de la production des cytokines de type Th2 telles que l’IL-4, l’IL-5, l’IL-13 et l’IL-33 dans les poumons. Nous avons montré que les mécanismes responsables de cette régulation sont associés à l’IL-33 produite par les macrophages et que la neutralisation de l’IL-33 par administration locale de la protéine de fusion au récepteur ST2 (muST2-Fc) atténue les caractéristiques de l’asthme allergique. Ces résultats suggèrent que l’activation de la caspase-1 réduit la production d’IL-33 in vivo et régule ainsi la réponse l’inflammation pulmonaire induite par HDM et la réponse Th2. D’autre part, nous nous sommes intéressés au rôle de la Protéine Kinase C thêta (PKC-θ) dans ce même modèle d’inflammation pulmonaire. Nous avons démontré que PKC-θ joue non seulement un rôle protecteur dans l’asthme allergique mais également un rôle critique pour la prolifération et l’activation des cellules lymphoïdes innées (ILC2). D’autre part, l’inhibition de PKC-θ in vivo par administration orale de son inhibiteur spécifique C20 (BIX02656) atténue l’inflammation pulmonaire et la production d’IL-5 et d’IL-13. Nous suggérons que PKC-θ est impliquée dans la différenciation des Th2 et des ILC2 via un mécanisme dépendant des facteurs de transcription IRF4 et NFAT-1. Au total, mes travaux de thèse mettent en exergue deux molécules IL-33 et PKC-θ qui pourraient constituer des cibles thérapeutiques potentielles. / Studies from our laboratory have shown a critical role of NLRP3 inflammasome in response to ovalbumin allergen. In the present study we investigate the role of NLRP3 and caspase-1 in a mouse model of pulmonary inflammation induced by HDM. We have shown a regulatory role of caspase-1 dependant of the NLRP3 inflammasome and the adaptator molecule ASC but not NLRC4. The regulation of the allergic response is characterized by an increase of eosinophilia, bronchial hyperreactivity and Th2 cytokines production (IL-4, IL-5, IL-13 and IL-33) in lungs. We have shown that mechanisms responsible of this regulation are associated with IL-33 production by macrophages and that neutralization of IL-33 by local administration of a fusion protein of the ST2 receptor (muST2-Fc) reduce characteristics of asthma. These results suggest that caspase-1 activation reduce IL-33 production in vivo regulating lung inflammation and Th2 response induced by HDM. Moreover, we investigate the role of the Protein Kinase C theta (PKC-θ) in allergic airway inflammation. We have demonstrated that PKC-θ plays a protective role in allergic asthma but is critical for the activation and proliferation of innate lymphoid cells (ILC2). In addition, in vivo inhibition by oral administration of PKC-θ specific inhibitor C20 (BIX02656) reduces pulmonary inflammation with IL-5 and IL-13 production. We suggest that PKC-θ is implicated in Th2 and ILC2 differenciation by a mechanism dependant on transcription factors IRF4 and NFAT-1. Finally, my thesis projects describe IL-33 and PKC-θ as potential therapeutic targets for allergic lung inflammation.

La Caspase-2 (CASP-2) est singulière de par ses multiples rôles physiologiques et par son implication dans les processus neurodégénératifs aigus et chroniques. Dans ce contexte, des études récentes ont contribué à sa validation en tant que cible thérapeutique potentielle de la maladie d’Alzheimer. Par conséquent, le développement d’inhibiteurs spécifiques constituerait des outils pharmacologiques qui permettraient de mieux appréhender ses rôles dans la physiologie et pathologie du neurone. Les inhibiteurs de Caspases actuels sont majoritairement des séquences tétra ou pentapeptidiques qui reproduisent les motifs préférentiellement reconnus par ces enzymes. Dans le cadre de ce travail de thèse, trois stratégies d’identification d’inhibiteurs ont été suivies ; (i) une approche de conception rationnelle de peptides ciblant le site actif, (ii) conception in silico de peptides de l’interface de dimérisation, (iii) criblage aléatoire et rationnel de petites molécules organiques. Parmi ces stratégies, l’inhibition du site actif s’est révélée être la plus fructueuse. Nous avons ainsi pu démontrer que des variations du résidu Alanine en P2 sur un motif VDVAD permettaient d’améliorer les paramètres de sélectivité et d’efficacité. Sur ce constat une série de peptides « LJ » avec des mécanismes d’inhibition variés a été développée. Deux composés LJ2 et LJ3, ont démontré d’excellents paramètres d’inactivation et de sélectivité envers la CASP-2. Dans des réseaux de neurones reconstruits in vitro, LJ2 et LJ3 présentent un effet synaptoprotecteur. Ces travaux de thèse ouvrent donc le champ à de nouvelles perspectives sur le plan fonctionnel ainsi que sur le plan thérapeutique. / Caspase-2 (CASP-2) is unique among Caspases since its involved in a plethora of physiological processes and in severe and chronic neurodegenerative processes. In this context, recent studies have indicated that CASP-2 is a potential therapeutic target for Alzheimer’s disease. It is therefore necessary to develop specific inhibitors which would constitute pharmacological tools to better understand the role of this protease in the physiology and pathology of the neuron. The current Caspases inhibitors are mostly tetra or pentapeptide sequences that reproduce the patterns preferentially recognized by these enzymes. During this thesis project, we used three identification strategies ; (i) a rational design approach of peptides targeting the active site, (ii) in silico design of peptides of the dimerization interface, (iii) rational an random screening of small organic molecules. Among these strategies, inhibition of the active site has been shown to be the most productive one. We have been able to demonstrate that the variation of the Alanine residue in P2 on the pattern VDVAD increased efficiency and selectivity parameters. Based on this observation, a serie of peptides “LJ” with various inhibitory mechanisms has been developed. Two compounds LJ2 and LJ3, demonstrated excellent inactivation and selectivity parameters toward CASP-2. In neuronal networks reconstructed in vitro, LJ2 and LJ3 protect against synapse loss. This thesis project opens the field to new perspectives on the functional level as well as on the therapeutic plan.

No description available.

Blood cells or hemocytes play important roles in immunity. They are a major source of many immune-related molecules such as antibodies in adaptive immunity of vertebrates and prophenoloxidase (proPO) in invertebrates. In the crayfish Pacifastacus leniusculus, the proPO-system has been reported to be an important component of immune responses against microorganisms. In this study, several mutant strains of Aeromonas hydrophila were used to reveal that LPS (lipopolysaccharide) is an important factor for the pathogenicity of A. hydrophila, strongly inducing the proPO system and melanization. This proPO activating system is a multistep process, which has to be tightly controlled to avoid the harmful side effects of toxic intermediates. Many regulating factors have been reported to fine-tune the proPO-system. In this study, the cleavage of caspase-1-like activity was shown to be a novel negative regulator of PO activity in crayfish. Moreover, the fragments obtained by cleavage of proPO by the proPO-activating enzyme and caspase-1-like protein increased bacterial clearance. Thus, the peptides generated also have important biological functions. In addition to being a source of immune proteins, hemocytes also participate in phagocytosis, encapsulation, and nodulation. An infection normally causes a reduction of hemocyte numbers. Consequently, hemocyte homeostasis is important for maintaining appropriate hemocyte numbers in the circulation of the animal. This study shows that the reactive oxygen species level in the anterior proliferation center of crayfish hematopoietic tissue (HPT), together with cell proliferation, was increased during infection. Pl-β-thymosins were proposed to be involved in hemocyte homeostasis by increasing stem cell migration and thus increasing the circulating hemocyte number. Crayfish hemocyte numbers, as well astakine (Ast1 and Ast2) expression in hemocytes and HPT, were previously shown to be under circadian regulation. Here, we show that Ast1, Ast2, and proPO exhibit rhythmic expression in the crayfish brain similarly to their orthologs, prokineticin 1, prokineticin 2 and tyrosinase, respectively, in the zebrafish brain. Tyrosinase expression was detected in zebrafish brain cells while PO-positive cells were identified as hemocytes that had infiltrated into the crayfish brain. Therefore, this information suggests a close relationship between crayfish hemocytes and the crayfish brain as well as vertebrate neurons.

<p>With the prevalence of obesity and metabolic syndrome rising sharply world-wide, it has become increasingly important to define the molecular mechanisms underlying the pathogenesis and progression of diseases associated with lipid-induced cytotoxicity. Cardiovascular disease, type-2 diabetes mellitus, and nonalchoholic fatty liver disease (NAFLD) have all recently gained recognition as diseases that are exacerbated by lipoapoptosis. In this dissertation, we demonstrate a novel role for caspase-2 as an initiator of lipoapoptosis. Using an unbiased metabolomics approach, we discovered that the activation of caspase-2, the initiator of apoptosis in Xenopus egg extracts, is associated with an accumulation of long-chain fatty acid (LCFA) metabolites. Metabolic treatments that block the buildup of LCFAs potently inhibit caspase-2, while add-back of a saturated LCFA restores caspase activation in the extract setting. Extending these findings to mammalian cells, we show that caspase-2 is engaged and activated in response to treatment with the saturated LCFA, palmitate. Down-regulation of caspase-2 significantly impairs cell death induced by saturated LCFAs, revealing a conserved, critical role for caspase-2 in mediating LCFA-induced lipoapoptosis. </p><p> Since lipoapoptosis has been implicated as a key driver of the progression of NAFLD, we aimed to determine the therapeutic significance of our findings by evaluating the importance of caspase-2 in an in vivo model of this disease. We subjected wild-type and caspase-2 knockout mice to a diet which induces severe liver steatosis and the development nonalcoholic steatohepatitis (NASH), the most advanced stage of NAFLD characterized by liver fibrosis. Interestingly, we observed an increase in caspase-2 protein levels in the livers of wild-type mice fed a NASH-inducing diet. These findings were of particular importance, since caspase-2 expression was also significantly elevated in patients diagnosed with NASH. Most importantly, we demonstrated that caspase-2 knockout mice are protected from apoptosis and fibrosis when fed a NASH-inducing diet, suggesting that caspase-2 is major regulator of hepatocyte lipoapoptosis. Together, these findings reveal a previously unknown role for caspase-2 as an initiator of lipoapoptosis and suggest that caspase-2 may be an attractive therapeutic target for inhibiting pathological lipid-induced apoptosis.</p> / Dissertation

Anti-tumor therapeutic strategies based on combinations of chemotherapeutic agents with a death inducing ligand such as TNF-α Related Apoptosis Inducing Ligand (TRAIL), are directed towards selective and effective cancer cell apoptosis and enhanced therapeutic response. We previously demonstrated that proteasome inhibition sensitizes TRAIL resistant prostate cancer cells to TRAIL-mediated apoptosis via stabilization of the active p18 subunit of initiator caspase-8. The present study investigated the functional link between caspase-8 and the proteasome, by analyzing the impact of caspase-8 ubiquitination and proteasomal degradation on the outcomes of the extrinsic apoptosis pathway in cancer cells. Caspase-8 ubiquitination status was assessed by polyubiquitin immunoprecipitation (IP) and fluorescent microscopy. Apoptosis induction in response to death receptor stimuli or proteasome inhibitor was evaluated using the Annexin V/Propidium iodide staining (PI). To determine the consequences of proteasome inhibition on caspase-8 stability, trafficking, and activity following death receptor activation, we used the TRAIL-resistant human prostate cancer LNCaP cells, and the caspase-8 deficient Neuroblastoma 7 (NB7) cells, as cellular models for reconstituting the non-cleavable mutant forms of caspase-8. Our findings demonstrate that the non-cleavable forms of caspase-8 are capable of inducing apoptosis comparably to wild-type caspase-8 upon treatment with proteasome inhibitor and GST-TRAIL. Furthermore, caspase-8 processing into its active subunits preceded caspase-8 polyubiquitination, implicating caspase-8 processing as a potential regulatory mechanism, rather than a requirement for caspase-8 activation in apoptosis induction. The mechanistic control of caspase-8 by ubiquitination in cancer cells may have significant significance in bypassing mechanisms of therapeutic resistance in human tumors and optimization of anti-cancer treatment strategies in human tumors and optimization of anti-cancer treatment strategies.

Pour mieux comprendre la signification des marqueurs d'apoptose dans les spermatozoïdes éjaculés humains, l'objectif de notre étude était de mesurer ces marqueurs dans les spermatozoïdes prélevés à différents niveaux du tractus génital masculin dans différentes situations physiopathologiques. Pour évaluer la qualité fonctionnelle de ces spermatozoïdes, des relations ont été recherchées entre l'expression de ces marqueurs et les résultats obtenus en assistance médicale à la procréation. Les marqueurs analysés sont des facteurs mis en jeu dans l'initiation et l'activation de l'apoptose (poly-caspases, caspase-3, -8 ou -9 activée(s)), et des signes précoces (externalisation de la phosphatidylsérine, PS) ou tardifs (fragmentation de l'ADN) de l'apoptose. Pour certains échantillons, une analyse ultrastructurale des spermatozoïdes a aussi été réalisée. la mesure de l'expression des caspases activées a fait l'objet d'une mise au point compte-tenu de l'hétérogénéité des populations spermatiques et de la faible quantité de spermatozoïdes disponibles. Finalement, nous avons retenu une mesure par double marquage associant un inhibiteur fluorescent vert des caspases activées et un colorant fluorescent rouge (Propidium Iodide) avec une détection soit en cytométrie en flux soit en microscopie à fluorescence selon la nature des spermatozoïdes analysés. Chez des patients présentant une agénésie bilatérale des canaux déférents, la proportion de spermatozoïdes vivants ou morts exprimant des caspases activées est plus élevée dans les spermatozoïdes testiculaires que dans les spermatozoïdes épididymaires suggérant une initiation du processus apoptique dans les testicules et une incapacité des spermatozoïdes épididymaires à initier l'apoptose. Dans ces conditions, en ICSI, le risque d'injecter un spermatozoïde apoptique dans un ovocyte est plus élevé avec les spermatozoïdes testiculaires et pourrait expliquer pour une part, les résultats de moins bonne qualité avec ces spermatozoïdes testiculaires qu'avec les spermatozoïdes épididymaires. Chez des patients infertiles, porteurs d'une translocation chromosomique réciproque ou Robertsonienne autosomique, il existe une expression plus importante des modifications ultrastructurales et des marqueurs biochimiques d'apoptose (caspases activées, fragmentation de l'ADN, externalisation de la PS) associée à des signes d'immaturité ultrastructurale, comparé aux spermatozoïdes d'hommes fertiles. Ces résultats pourraient expliquer que dans l'éjaculat de ces patients, il existe une prédominance de gamètes équilibrés sur le plan chromosomique. En effet, les gamètes présentant un déséquilibre auraient été éliminées préférentiellement par apoptose. En conclusion, les marqueurs d'apoptose exprimés par les spermatozoïdes éjaculés seraient le reflet d'une altération de la spermatogenèse avec une apoptose initiée et avortée dans le testicule associée à des anomalies de maturation et différentiation. La mesure des marqueurs d'apoptose dans les spermatozoïdes apporterait une aide dans la compréhension et la prise en charge de l'infertilité masculine, en particulier en assistance médicale à la procréation.