Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that induces apoptosis in a number of cell systems, including osteoblasts. Transforming growth factor β1 (TGF-β1) is an abundant growth factor that is known to stimulate bone formation. This study was designed to examine the role of TGF-β1 on TNF-α-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 20 ng/ml of TNF-α, 10 ng/ml of TGF-β1, or combination, for 6 h. TNF-α exerted a variety of effects on the apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that TNF-α upregulated the mRNA levels of caspase-1, -7, -11, -12, and FAS. Western blot analysis showed enhanced processing of caspase-1, -7, -11, and -12, with the appearance of their activated enzymes 24 h after TNF-α treatment. In addition, caspase-3-like activity was significantly activated following TNF-α treatment. Levels of cleaved poly(ADP-ribose) polymerase and FAS protein were also elevated by TNF-α. Finally, Hoechst staining, terminal deoxynucleotidyl-transferase nick-end labeling (TUNEL) assay, and oligonucleosome ELISA all indicated that TNF-α induced apoptosis. In contrast, the addition of TGF-β1 attenuated all of the aforementioned effects of TNF-α. Our results demonstrate that TGF-β1 can decrease TNF-α-induced apoptosis in murine osteoblasts at least in part by attenuating TNF-α-induced caspase gene expression.

No description available.

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

<p dir="ltr"><i>Legionella pneumophila</i> strains harboring wild-type <i>rpsL</i> such as Lp02<i>rpsL</i>_WT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The mechanism of this unique infection-induced cell death remains unknown. Using a genome-wide CRISPR/Cas9 screening, we identified <i>Hmg20a </i>and <i>Nol9</i> as host factors important for restricting strain Lp02<i>rpsL</i>_WT in BMDMs. Depletion of <i>Hmg20a</i> protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by <i>Hmg20a</i> was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02<i>rpsL</i>_WT in BMDMs. Our results establish that <i>L. pneumophila</i> exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.</p>

A lesão granulomatosa é caracterizada como um agregado compacto de fagócitos maduros formado em resposta à um estímulo persistente. Os mediadores pró-inflamatórios da família da IL-1, ao promoverem a ativação da imunidade inata e o remodelamento tecidual descontrolados, geram a fisiopatologia da paracoccidioidomicose, doença pulmonar granulomatosa causada pelo fungo P. brasiliensis. A principal via inflamatória envolvida na secreção de IL-1?, IL-18 e IL-1? é a ativação dos inflammasomas, complexos protéicos conhecidos pela sua capacidade de ativar proteoliticamente a enzima caspase-1. Neste estudo abordamos os mecanismos subjacentes às vias canônica e não canônica do inflamassoma, avaliando a importância funcional das caspases 1, 11 e 8 na resistência do hospedeiro durante a infecção por P. brasiliensis. Demonstramos que a resposta imunológica protetora mediada pelo IFN-?, após a liberação de IL-18 pela via da caspase-1, é o principal mecanismo responsável pelo controle fúngico quando o dano celular desencadeado pelo fungo é reconhecido pelo inflamassoma de NLRP3. No entanto, apesar do papel importante da caspase-1 na maturação da IL-1?, a falta de caspase-1 apenas reduz parcialmente os níveis de IL-1? durante a infecção por P. brasiliensis. Ao contrário do esperado, a deficiência de caspase-11 não prejudicou a produção de IL-1?, mas em vez disso, preveniu, na célula, a morte por piroptose e a secreção IL-1?, citocina importante para restringir o crescimento fúngico através da síntese de IL-17. Por fim, observamos que a ativação de caspase-8 pela sinalização de dectina-1 / Syk, além de mediar a maturação da IL-1?, que acontece de maneira independente de caspase-1 e 11, é necessária também para o funcionamento eficiente da via canônica de caspase-1, demonstrando uma rede interligada entre as vias canônica de caspse-1 e não canônica de caspase-8 para coordenar o processamento da IL-1?. Em conjunto, nossos resultados mostram contribuições distintas das vias canônica e não canônica do inflamassoma na produção de citocinas da família da IL-1, enfatizam a versatilidade desta plataforma em recrutar várias proteínas efetoras para adequar a resposta imunológica antifúngica e evidenciam a complexidade envolvida nas interações patógeno-hospedeiro. / Granuloma is a lesion characterized by a compact aggregate of mature phagocytes arising in response to a persistent stimulus. By driving uncontrolled innate immunity and tissue remodelling, IL-1 family pro-inflammatory mediators govern the pathophysiology of paracoccidioidomycosis, a granulomatous lung disorders caused by Paracocccidioides brasiliensis. A major inflammatory pathway involved in IL-1?, IL-18 e IL-1? secretion is the activation of inflammasomes, large multimolecular complexes best known for their ability to control activation of the proteolytic enzyme caspase-1. In this study we addressed the mechanisms that underlie canonical and non-canonical inflammasome pathways, assessing the functional importance of caspase-1, caspase-11 and caspase-8 in the regulation of inflammasome-mediated host resistance during P. brasiliensis infection. We found that IFN-?-mediated protective immune response following by caspase-1- dependent IL-18 release after is the key mechanism responsible for the fungal control after P. brasiliensis-induced cell damage recognition by NLRP3 canonical inflammasome pathway. Nonetheless, despite the important role of caspase-1 in the IL-1? maturation, the lack of caspase-1 only partially reduced IL-1? levels during P. brasiliensis infection. Unlike caspase-1, caspase-11 deficiency did not impair IL-1? production, cytokine strictly secreted by canonical caspase-1 inflammasome pathway. Instead, P. brasiliensis-triggered caspase-11 activation in an ill-defined manner leads to a rapid pore-mediated cell lysis and is required for IL-1? production during P. brasiliensis infection. IL-1?, in turn, is important for promoting the restriction of fungal growth trough IL-17-based inflammation. Finally, the caspase-8 induction by dectin-1/Syk signaling besides playing a role in mediating the caspase-1/11-independent IL-1? maturation is also required to efficient canonical caspase-1 inflammasome pathway, demonstrating a connected network between non-canonical caspase-8 and canonical caspase-1 inflammasome pathways to coordinate IL-1?. Taken together, our results revealed distincts contributions of both canonical and non-canonical inflammasome pathways in IL-1 family cytokine production and emphasizes the versatility of this platform to recruit several effector proteins to tailor the antifungal immune response, sheding new light on the complexity of this hostpathogen interaction.

A lesão granulomatosa é caracterizada como um agregado compacto de fagócitos maduros formado em resposta à um estímulo persistente. Os mediadores pró-inflamatórios da família da IL-1, ao promoverem a ativação da imunidade inata e o remodelamento tecidual descontrolados, geram a fisiopatologia da paracoccidioidomicose, doença pulmonar granulomatosa causada pelo fungo P. brasiliensis. A principal via inflamatória envolvida na secreção de IL-1?, IL-18 e IL-1? é a ativação dos inflammasomas, complexos protéicos conhecidos pela sua capacidade de ativar proteoliticamente a enzima caspase-1. Neste estudo abordamos os mecanismos subjacentes às vias canônica e não canônica do inflamassoma, avaliando a importância funcional das caspases 1, 11 e 8 na resistência do hospedeiro durante a infecção por P. brasiliensis. Demonstramos que a resposta imunológica protetora mediada pelo IFN-?, após a liberação de IL-18 pela via da caspase-1, é o principal mecanismo responsável pelo controle fúngico quando o dano celular desencadeado pelo fungo é reconhecido pelo inflamassoma de NLRP3. No entanto, apesar do papel importante da caspase-1 na maturação da IL-1?, a falta de caspase-1 apenas reduz parcialmente os níveis de IL-1? durante a infecção por P. brasiliensis. Ao contrário do esperado, a deficiência de caspase-11 não prejudicou a produção de IL-1?, mas em vez disso, preveniu, na célula, a morte por piroptose e a secreção IL-1?, citocina importante para restringir o crescimento fúngico através da síntese de IL-17. Por fim, observamos que a ativação de caspase-8 pela sinalização de dectina-1 / Syk, além de mediar a maturação da IL-1?, que acontece de maneira independente de caspase-1 e 11, é necessária também para o funcionamento eficiente da via canônica de caspase-1, demonstrando uma rede interligada entre as vias canônica de caspse-1 e não canônica de caspase-8 para coordenar o processamento da IL-1?. Em conjunto, nossos resultados mostram contribuições distintas das vias canônica e não canônica do inflamassoma na produção de citocinas da família da IL-1, enfatizam a versatilidade desta plataforma em recrutar várias proteínas efetoras para adequar a resposta imunológica antifúngica e evidenciam a complexidade envolvida nas interações patógeno-hospedeiro. / Granuloma is a lesion characterized by a compact aggregate of mature phagocytes arising in response to a persistent stimulus. By driving uncontrolled innate immunity and tissue remodelling, IL-1 family pro-inflammatory mediators govern the pathophysiology of paracoccidioidomycosis, a granulomatous lung disorders caused by Paracocccidioides brasiliensis. A major inflammatory pathway involved in IL-1?, IL-18 e IL-1? secretion is the activation of inflammasomes, large multimolecular complexes best known for their ability to control activation of the proteolytic enzyme caspase-1. In this study we addressed the mechanisms that underlie canonical and non-canonical inflammasome pathways, assessing the functional importance of caspase-1, caspase-11 and caspase-8 in the regulation of inflammasome-mediated host resistance during P. brasiliensis infection. We found that IFN-?-mediated protective immune response following by caspase-1- dependent IL-18 release after is the key mechanism responsible for the fungal control after P. brasiliensis-induced cell damage recognition by NLRP3 canonical inflammasome pathway. Nonetheless, despite the important role of caspase-1 in the IL-1? maturation, the lack of caspase-1 only partially reduced IL-1? levels during P. brasiliensis infection. Unlike caspase-1, caspase-11 deficiency did not impair IL-1? production, cytokine strictly secreted by canonical caspase-1 inflammasome pathway. Instead, P. brasiliensis-triggered caspase-11 activation in an ill-defined manner leads to a rapid pore-mediated cell lysis and is required for IL-1? production during P. brasiliensis infection. IL-1?, in turn, is important for promoting the restriction of fungal growth trough IL-17-based inflammation. Finally, the caspase-8 induction by dectin-1/Syk signaling besides playing a role in mediating the caspase-1/11-independent IL-1? maturation is also required to efficient canonical caspase-1 inflammasome pathway, demonstrating a connected network between non-canonical caspase-8 and canonical caspase-1 inflammasome pathways to coordinate IL-1?. Taken together, our results revealed distincts contributions of both canonical and non-canonical inflammasome pathways in IL-1 family cytokine production and emphasizes the versatility of this platform to recruit several effector proteins to tailor the antifungal immune response, sheding new light on the complexity of this hostpathogen interaction.

Alzheimer's disease is the most common type of dementia in the elderly; it is characterized by early deficits in learning and memory formation and ultimately leads to a generalised loss of higher cognitive functions. While amyloid beta (Aβ) and tau are traditionally associated with the development of Alzheimer disease, recent studies suggest that other factors, like the intracellular domain (APP-ICD) of the amyloid precursor protein (APP), could play a role. In this study, we investigated whether APP-ICD could affect synaptic transmission and synaptic plasticity in the hippocampus, which is involved in learning and memory processes. Our results indicated that overexpression of APP-ICD in hippocampal CA1 neurons leads to a decrease in evoked AMPA-receptor and NMDA-receptor dependent synaptic transmission. Our study demonstrated that this effect is specific for APP-ICD since its closest homologue APLP2-ICD did not reproduce this effect. In addition, APP-ICD blocks the induction of long term potentiation (LTP) and leads to increased of expression and facilitated induction of long term depression (LTD), while APLP2-ICD shows neither of these effects. Our study showed that this difference observed in synaptic transmission and plasticity between the two intracellular domains resides in the difference of one alanine in the APP-ICD versus a proline in the APLP2-ICD. Exchanging this critical amino-acid through point-mutation, we observed that APP(PAV)-ICD had no longer an effect on synaptic plasticity. We also demonstrated that APLP2(AAV)-ICD mimic the effect of APP-ICD in regards of facilitated LTD. Next we showed that the full length APP-APLP2-APP (APP with a substitution of the Aβ component for its homologous APLP2 part) had no effect on synaptic transmission or synaptic plasticity when compared to the APP-ICD. However, by activating caspase cleavage prior to induction of LTD or LTP, we observed an LTD facilitation and a block of LTP with APP-APLP2-APP, effects that were not seen with the full length APLP2 protein. APP is phosphorylated at threonine 668 (Thr668), which is localized directly after the aforementioned critical alanine and the caspase cleavage site in APP-APLP2-APP. Mutating this Thr668 for an alanine abolishes the effects on LTD and restores LTP induction. Finally, we showed that the facilitation of LTD with APP-APLP2-APP involves ryanodine receptor dependent calcium release from intracellular stores. Taken together, we propose the emergence of a new APP intracellular domain, which plays a critical role in the regulation of synaptic plasticity and by extension, could play a role in the development of memory loss in Alzheimer’s disease. / La maladie d’Alzheimer est la forme la plus commune de démence liée au vieillissement ; elle est caractérisée par des déficits précoces d’apprentissage et de mémorisation et entraîne à terme une perte généralisée des fonctions cognitives supérieures. Alors que l’amyloïde-bêta (Aβ) et la protéine tau sont traditionnellement associées au développement de la maladie d’Alzheimer, des études récentes suggèrent que d’autres facteurs, tels que le domaine intracellulaire (APP-ICD) du précurseur de la protéine amyloïde (APP), pourraient jouer un rôle. Dans notre étude, nous avons testé si l’APP-ICD pourrait affecter les mécanismes de transmission ou de plasticité synaptique dans l’hippocampe, qui sous-tendent les processus d’apprentissage et de mémorisation. Nos résultats ont indiqué que la surexpression de l’APP-ICD dans des neurones du CA1 de l’hippocampe entraînait une diminution de la transmission synaptique dépendante des récepteurs AMPA et NMDA. Notre étude a montré que cet effet était spécifique de l’APP-ICD puisque son plus proche homologue l’APLP2-ICD n’a pas eu cet effet. De plus, l’APP-ICD entraînait un blocage de la potentialisation à long terme (LTP), une augmentation de l’expression et une facilitation de l’induction de la dépression à long terme (LTD), mais l’APLP2-ICD n’a eu aucun de ces effets. Notre étude a montré que cette différence observée en transmission et en plasticité synaptique entre les deux peptides réside dans le changement d’une seule alanine dans l’APP-ICD pour une proline dans l’APLP2-ICD, et que l’APP(PAV)-ICD n’avait pas d’effet sur la plasticité synaptique. Nous avons aussi démontré que l’APLP2(AAV)-ICD mimait l’effet de l’APP-ICD pour la facilitation de la LTD. Ensuite nous avons montré que la longue forme APP-APLP2-APP (APP avec un changement de la séquence de l’Aβ pour celle homologue de l’APLP2) ne montrait pas d’effet en comparaison avec l’APP-ICD, ni sur la transmission synaptique ni sur la plasticité synaptique. Cependant, en activant le clivage par les caspases préalablement à l’induction de la LTD ou la LTP, nous avons observé une facilitation de la LTD et un iii blocage de la LTP avec l’APP-APLP2-APP, des effets que nous n’avons pas reproduit avec la longue forme APLP2. La thréonine 668 phosphorylable se trouve immédiatement après l’alanine et le site de clivage par les caspases dans l’APP-APLP2-APP. La mutation de la Thr668 pour une alanine a aboli son effet sur la LTD et restauré la LTP. Finalement, nous avons montré que la facilitation de la LTD par l’APP-APLP2-APP dépendait de la libération de calcium intracellulaire par les récepteurs ryanodines. En conséquence, nous proposons l’émergence d’un nouveau domaine de l’APP jouant un rôle critique, en plus de l’Aβ, dans les processus à la base de l’apprentissage et qui en conséquence pourrait jouer un rôle dans le développement de la maladie d’Alzheimer.

Type 2 diabetes mellitus represents a metabolic disease reaching epidemic dimensions in the 21st century. Fatty acid-induced apoptosis of pancreatic β-cells significantly contributes to its pathogenesis. Saturated fatty acids (FAs) are strongly cytotoxic for β-cells, whereas unsaturated FAs are well tolerable by β-cells, they are even able to inhibit proapoptotic effects of saturated FAs when co-incubated. According to recent studies, FAs-induced apoptosis in pancreatic β-cells is partly regulated by autophagy, a catabolic process involved in the degradation and recyclation of cell components in lysosomes. The aim of this diploma thesis was to contribute to the clarification of the role of autophagy in FAs-induced apoptosis regulation. We induced apoptosis in human pancreatic β- cell line NES2Y by 1 mM stearic acid (SA) and inhibited it with 0.2 mM oleic acid (OA) co- incubated with SA. We revealed, that the saturated SA used in apoptosis-inducing concentration simultaneously inhibits the autophagic flux in pancreatic NES2Y cell line. When SA is co- incubated with unsaturated OA in concentration sufficient for inhibition of proapoptotic effect of SA, OA is also able to inhibit the block of autophagy induced by the effect of SA. Application of unsaturated OA alone in this concentration did not...

Promastigotas de Leishmania são encontradas no trato digestivo do inseto vetor e podem ser cultivados em alguns meios de cultura, onde ocorre a diferenciação regulada geneticamente entre procíclicos e metacíclicos infectivos. Amastigotas são intracelulares e irão se estabelecer e se multiplicar dentro dos vacúolos parasitóforos dos macrófagos. Os metacíclicos e os amastigotas possuem capacidade de evasão do sistema de defesa do hospedeiro vertebrado e conseguem estabelecer uma relação parasita/hospedeiro bem-sucedida. A apoptose, em organismos multicelulares, é uma forma controlada geneticamente de suicídio celular. A eliminação das células apoptóticas é feita pelos fagócitos sem que seja induzida uma resposta inflamatória. Em Leishmania, os eventos da apoptose parecem seguir os mesmos dos encontrados em metazoários, tais como: queda do potencial da mitocôndria, ativação de caspases, clivagem de substratos naturais de caspases, além da externalização de fosfatidilserina (PS) e seu papel na modulação da fagocitose. Nossos dados demonstraram que Leishmania é capaz de mimetizar e utilizar um dos fenômenos observados na apoptose em metazoários, a exposição de PS, como um mecanismo adaptativo para se estabelecer como um parasita de mamíferos. / Promastigotes of Leishmania are found in the midgut environment of the vector insect and can be grown in some culture medium. In culture the genetically regulated differentiation from procyclic to infective metacyclic can be observed. Amastigotes are intracellular parasites and which multiply inside phagolysosomes of macrophages. Metacyclics and amastigotes are endowed with the capacity to evade from the innate and adaptive immune system of the vertebrate host and to establish successful infections. Apoptosis, in multicellular organisms, is a form genetically controlled cellular suicide. The elimination of apoptotic cells is made by phagocytes without initiating an inflammatory event. In Leishmania, the events of apoptosis are similar to those observed in multicellular organisms such as: decrease in mitochondrial transmembrane potential, caspase activation, cleavage of caspase substrates besides exposure of phosphatidylserine (PS) and its role in the modulation of phagocytosis. Our data showed that Leishmania is capable of mimicking one of the features of apoptosis, mainly PS exposure, and to use as an adaptive mechanism for survival in mammalian hosts.

La maladie de Parkinson (MP) est un syndrome neurodégénératif, caractérisé par une dégénérescence spécifique des neurones dopaminergiques de la substance noire, associée à la présence d'inclusions cytoplasmiques appelées corps de Lewy. Les formes familiales résultent de mutations portées par: la parkine (PK), DJ-1, PINK1, l'-synucléine, l'UCHL1, et LRRK2. Ces mutations s'accompagnent d'un dysfonctionnement du système ubiquitine-protéasome, de la mitochondrie ainsi que d'une augmentation du stress oxydatif induisant une mort neuronale par apoptose. Au cours de mon travail de thèse, je me suis intéressée à la synphiline-1, un partenaire de l'-synucléine, et à deux protéines impliquées dans les formes récessives de la MP, DJ-1 et la PK.<br />Je me suis consacrée à l'étude de leurs fonctions physiologiques, ainsi qu'à leurs implications dans les processus apoptotiques. Ainsi, j'ai déterminé que ces protéines réduisent l'activité de la caspase-3 induite par différents stimuli. Ce rôle protecteur passe par la régulation de la voie dépendante de p53. De plus, j'ai identifié DJ-1 et la synphiline-1 comme étant substrats des caspases, mais aussi que les fragments C-terminaux issus de ce clivage portent leurs activités biologiques.<br />Parallèlement, j'ai étudié des aspects de la régulation transcriptionnelle de DJ 1 et de la PK par le facteur de transcription p53. J'ai mis en évidence une boucle de régulation entre p53, DJ-1 et la PK. J'ai montré que ces deux protéines sont capables de réguler l'expression de p53. De plus, j'ai déterminé que la PK régule positivement DJ-1, et que ce contrôle s'effectue via la régulation transcriptionnelle de DJ-1 par p53.