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New Insights into the Regulation of Lymphocyte Signalling, Centriolar Satellites Proteostasis and Necroptotic Cell Death by Post-translational Modifications / Caractérisation de la régulation de la signalisation lymphocytaire, la protéostase des satellites centriolaire et la mort cellulaire necroptotique par des modifications post-traductionnellesDouanne, Tiphaine 30 October 2019 (has links)
L’ubiquitination est une modification posttraductionnelle (MPT) qui gouverne la plupart des processus cellulaires Eucaryotes. Elle consiste en l’ajout d’une ubiquitine via une liaison covalente à une protéine cible, altérant son devenir (activité, localisation, dégradation). Le LUBAC (linear ubiquitin chain assembly complex) est le seul complexe capable de catalyser une forme énigmatique de cette modification, l’ubiquitination linéaire. Ces dernières années, ce complexe ternaire a été identifié comme un acteur majeur dans l’activation de NF-κB en réponse à de nombreux immunorécepteurs. Le but principal de cette thèse est de caractériser la régulation du LUBAC et ses partenaires dans des systèmes variés et d’identifier d’éventuelles MPT en charge de moduler ce complexe. Dans un premier temps, nous avons montré que la sous-unité du LUBAC HOIL1 est dynamiquement clivée par la paracaspase MALT1 en réponse à l’engagement des récepteurs antigéniques dans les lymphocytes pour pleinement activer NF-κB. HOIL1 est également constitutivement clivée dans un sous-groupe agressif de lymphome diffus à grandes cellules B (DLBCL), qui présentent une activation aberrante de MALT1, dévoilant de potentielles stratégies thérapeutiques. Dans un second temps, nous avons observé qu’une partie du partenaire du LUBAC CYLD est liée aux satellites centriolaires, des structures granulaires qui gravitent autour des centrosomes et orchestrent la ciliogenèse. Nos données suggèrent que CYLD contrôle la protéostase des satellites centriolaires et gouverne ainsi la formation du cil primaire. Enfin, nous avons analysé les phases précoces de la nécroptose, une forme de nécrose sous le contrôle du LUBAC. Nous avons mis en évidence que l’activation des canaux Pannexin-1 restreint la production de cytokines pro-inflammatoires associée à la nécroptose. Ensemble, ces travaux illustrent comment les MPT contrôlent les voies de signalisation cellulaires associées à l’ubiquitine et comment elles peuvent être corrompues dans des conditions pathologiques. / Ubiquitination is a pivotal multifaceted post-translational modification (PTM), which governs most cellular processes in Eukaryotic cells. Ubiquitination consists in the covalent binding of ubiquitin onto a target protein, altering its fate (activity, localisation, degradation). The “linear ubiquitin chain assembly complex” (LUBAC) is the only known complex capable of catalysing the enigmatic “head-to-tail” linear ubiquitination. Over the years, this tertiary complex has been shown to be essential for signalling to the NF-κB transcription factors family in response to the stimulation of various immunoreceptors. The primary goal of this PhD was to better understand how the LUBAC and its partners are modulated in different systems and identify possible PTMs regulating this complex. First, we discovered that the LUBAC subunit HOIL1 is dynamically cleaved by the paracaspase MALT1 upon antigen receptor engagement in lymphocytes to further NF- κB activation. HOIL1 is also constitutively cleaved in a subset of aggressive diffuse large B-cell lymphoma (DLBCL), which displays aberrant activation of MALT1, unveiling potential therapeutic strategies. Second, we observed that a portion of the LUBAC’s partner CYLD is bound to the centriolar satellites, a set of granular structures surrounding centrosomes that orchestrate ciliogenesis. Our data suggests that CYLD governs the proteostasis of centriolar satellites, and thereby the formation of primary cilia. Lastly, we analysed the initial steps of necroptosis, a regulated form of necrosis under the control of the LUBAC. We found that activation of Pannexin-1 channels restrains the production of proinflammatory cytokines associated with necroptosis. Altogether, this work illustrates how PTM finely tune ubiquitin-associated signalling pathways, and how they can be perverted in pathological conditions.
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Estudo dos mecanismos de morte celular induzida por fenotiazinas em células hepáticas em culturaFaria, Priscila Afonso de January 2013 (has links)
Orientador:Tiago Rodrigues / Tese (doutorado) - Universidade Federal do ABC. Programa de Pós-Graduação em Ciência e Tecnologia/Química, 2013
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Ativação da heme oxigenase-1 e via da necroptose como mecanismos imunopatogênicos na infecção de macrófagos por Leishmania infantumLuz, Nívea Farias January 2015 (has links)
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Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A Leishmaniose visceral (LV) apresenta ampla distribuição geográfica e é fatal caso não seja
tratada. As manifestações hematológicas são constantes na LV e em casos não tratados os
pacientes evoluem à óbito por sangramento maciço ou anemia grave. Neste cenário,
mecanismos ligados à morte celular, hemólise, metabolismo do heme e atividade da enzima
heme oxigenase podem estar envolvidos na imunopatogênese da LV. A heme oxigenase (HO)
tem importantes propriedades regulatórias e está envolvida em processos fisiológicos e
patofisiológicos como citoproteção e inflamação. Nesse projeto testamos a hipótese de que a
ativação da enzima heme oxigenase-1 (HO-1) favorece a infecção por Leishmania infantum
chagasi, principal agente etiológico da LV humana no Brasil e de que mecanismos de morte
celular inflamatória induzida por heme estão associados com a resistência ao parasita. Nossas
observações nesse trabalho indicam que a enzima HO-1 é induzida em macrófagos durante a
infecção por L. chagasi e que a indução farmacológica da HO-1, pela CoPP aumenta a carga
parasitária de macrófagos infectados por L. chagasi e reduz a produção de mediadores próinflamatórios.
Além disso, a HO-1 favorece um ambiente anti-inflamatório onde prevalece a
presença de IL-10 sobre a de TNF. Macrófagos derivados de medula óssea de camundongos
deficientes no gene HO-1 têm menor carga parasitária, quando infectados por L. chagasi em
comparação aos macrófagos de camundongos selvagens. Além disso, pacientes com LV
apresentam maiores níveis de heme-oxigenase 1 e de heme no soro. Nossas observações
indicam que heme é capaz de induzir necroptose em macrófagos humanos, e de que moléculas
da via da necroptose estão associadas com a resistência na infecção por Leishmania. A
molécula RIPK1 controla a replicação de Leishmania por um mecanismo independente da
produção de IL-1β, enquanto que a molécula PGAM5 depende de IL-1βpara controlar o
crescimento do parasita. Por fim, encontramos que essas proteínas participam do controle da
replicação por Leishmania em um modelo experimental de Leishmaniose cutânea. Esses
achados indicam um potencial deletério para a HO-1 na infecção por L. chagasi, e um papel
protetor da necroptose na infecção porLeishmania. / Visceral leishmaniasis (VL) is a widespread disease and is fatal if left untreated.
Hematological manifestations are common in VL and untreated patients evolve to death from
massive bleeding and severe anemia. In this scenario, mechanisms related to cell death
pathways, hemolysis, heme metabolism and enzymatic activity of heme oxygenase may be
involved in the immunopathogenesis of the disease. Heme oxygenase (HO) has important
regulatory properties and is involved in patho-physiological processes such as cytoprotection
and inflammation. This project tested the hypothesis that heme oxygenase- 1 (HO-1)
activation favors Leishmania infantum chagasi infection, the main etiologic agent of human
VL in Brazil, we also tested whether heme induced inflammatory cell death pathways are
involved in resistance to Leishmania infection. Our observations indicate that HO-1 is
induced in macrophages infected with L. infantum chagasi and pharmacological induction for
HO-1 by CoPP increases parasite load of infected macrophages and reduces production on
inflammatory mediators. In addition, HO-1 contributes to the anti inflammatory pathway that
favors L. chagasi replication through a higher IL-10/TNF-α ratio in macrophages. We also
observed that bone marrow derived macrophages knockout to HO-1 gene have a significant
lower parasite load when infected by L. infantum chagasi than their wild type counterparts.
Beyond this, we found that patients with VL presented higher systemic concentrations of HO-
1 and heme than healthy individuals. We found that heme is able to induce programmed
necrosis “necroptosis” in human cells and that molecular players from necroptosis pathway
contribute to resistance to Leishmania infection. RIPK1 controls Leishmania replication
through a mechanism independent of IL-1β production, while PGAM5 requires IL-1β to
control Leishmania replication. Finally, we found that RIPK1 and PGAM5 play an important
role in controlling Leishmania replication in a cultaneous leishmaniasis experimental model.
Our findings argue that HO-1 has a critical role in L. chagasi replication and necroptosis
pathway is involved in resistance against Leishmania infection.
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Análise citotóxica e caracterização química de frações do extrato hidroalcoólico da semente de Euterpe Oleracea mart / CITOTOXIC ANALYSIS AND CHEMICAL CHARACTERIZATION OF FRACTIONS OF THE HYDROALCOOLIC EXTRACT OF THE SEED OF Euterpe oleracea MARTFreitas, Dayanne da Silva 14 March 2016 (has links)
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Previous issue date: 2016-03-14 / The Euterpe oleracea Mart seed. (Acai) has demonstrated different biological activities, such as antioxidant, anti-inflammatory, antihypertensive, antinociceptive and antineoplastic. In this new study, the aim was to analyze the antineoplastic activity of fractions derived from hydroalcoholic extract of Euterpe oleracea Mart. seed in cell line MCF-7, and to identify the compounds responsible for the antineoplastic action by mass spectrometry using electrospray ionization source, and a ciclotrônico analyzer coupled to a Fourier transform (ESI-FT-ICR MS). The MCF-7 cell line was treated with 10, 20, 40 and 60 μg L-1 with fractions hexane (FH), chloroform (FC) and ethyl acetate (FAE) of the hydroalcoholic extract acai seed for 24, 48 and 72 hours. After treatment the cell viability was measured using the assay with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) and the cell type of death was assessed using annexin - Iodide propidium (PI) assay. Data were analyzed statistically by analysis of variance (ANOVA) or Student's t test, us appropriate. It was observed that all the fractions caused significant reduction of cell viability of MCF-7, but the FAE was the most cytotoxic (p <0.001). In the test Annexin-PI, there was no significant labeling annexin but increased PI staining was significant (p <0.001). This study showed that the FAE was more effective in reducing cell viability, following necroptose mechanism in MCF-7 cell. The FAE is composed of epicatechin, proanthocyanidin A2 and trimeric and tetrameric procyanidins. / A semente de Euterpe oleracea Mart. (açaí) tem demonstrado diferentes atividades biológicas, tais como: antioxidante, anti-inflamatória, anti-hipertensiva, antinociceptiva e antineoplásica. Neste estudo, o objetivo foi analisar a atividade antineoplásica de frações do extrato hidroalcoólico da semente de Euterpe oleracea Mart. na linhagem celular MCF-7, assim como identificar os compostos responsáveis pela ação antineoplásica por espectrometria de massas utilizando fonte de ionização por eletrospray e um analisador ciclotrônico acoplado a uma transformada de Fourier (ESI- FT-ICR MS). A linhagem MCF-7 foi tratada com 10, 20, 40 e 60 μg L-1 com as frações hexânica (FH), clorofórmica (FC) e de acetato de etila (FAE) do extrato hidroalcoólico da semente do açaí por 24, 48 E 72 horas. Após o tratamento a viabilidade celular foi mensurada usando o ensaio com 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) e o tipo de morte celular foi avaliado com o ensaio Anexina - Iodeto de propídeo (PI). Os dados foram analizados estatisticamente por análise de variância (ANOVA) ou por teste T student, quando apropriado. Foi observado que todas as frações causaram redução significativa da viabilidade celular da MCF-7, porém a FAE foi a mais citotóxica (p < 0,001). No ensaio de anexina-pi, não foi observado marcação significativa para anexina porém o aumento da marcação de PI foi significativo (p<0,001). O presente estudo demonstrou que a FAE foi a mais eficaz na redução da viabilidade celular, seguindo mecanismo de necroptose na célula MCF-7. A FAE é composta por epicatequina, proantocianidina A2 e procianidinas trimérica e tetramérica.
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Análise citotóxica e caracterização química de frações do extrato hidroalcoólico da semente de Euterpe oleracea Mart. / Cytotoxic analysis and chemical characterization of fractions derived hydroalcoholic Euterpe oleracea Mart seedFreitas, Dayanne da Silva 14 March 2016 (has links)
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Previous issue date: 2016-03-14 / The Euterpe oleracea Mart seed. (Acai) has demonstrated different biological activities, such as antioxidant, anti-inflammatory, antihypertensive, antinociceptive and antineoplastic. In this new study, the aim was to analyze the antineoplastic activity of fractions derived from hydroalcoholic extract of Euterpe oleracea Mart. seed in cell line MCF-7, and to identify the compounds responsible for the antineoplastic action by mass spectrometry using electrospray ionization source, and a ciclotrônico analyzer coupled to a Fourier transform (ESI-FT-ICR MS). The MCF-7 cell line was treated with 10, 20, 40 and 60 μg L-1 with fractions hexane (FH), chloroform (FC) and ethyl acetate (FAE) of the hydroalcoholic extract acai seed for 24, 48 and 72 hours. After treatment the cell viability was measured using the assay with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) and the cell type of death was assessed using annexin - Iodide propidium (PI) assay. Data were analyzed statistically by analysis of variance (ANOVA) or Student's t test, us appropriate. It was observed that all the fractions caused significant reduction of cell viability of MCF-7, but the FAE was the most cytotoxic (p <0.001). In the test Annexin-PI, there was no significant labeling annexin but increased PI staining was significant (p <0.001). This study showed that the FAE was more effective in reducing cell viability, following necroptose mechanism in MCF-7 cell. The FAE is composed of epicatechin, proanthocyanidin A2 and trimeric and tetrameric procyanidins. / A semente de Euterpe oleracea Mart. (açaí) tem demonstrado diferentes atividades biológicas, tais como: antioxidante, anti-inflamatória, anti-hipertensiva, antinociceptiva e antineoplásica. Neste estudo, o objetivo foi analisar a atividade antineoplásica de frações do extrato hidroalcoólico da semente de Euterpe oleracea Mart. na linhagem celular MCF-7, assim como identificar os compostos responsáveis pela ação antineoplásica por espectrometria de massas utilizando fonte de ionização por eletrospray e um analisador ciclotrônico acoplado a uma transformada de Fourier (ESI- FT-ICR MS). A linhagem MCF-7 foi tratada com 10, 20, 40 e 60 μg L-1 com as frações hexânica (FH), clorofórmica (FC) e de acetato de etila (FAE) do extrato hidroalcoólico da semente do açaí por 24, 48 E 72 horas. Após o tratamento a viabilidade celular foi mensurada usando o ensaio com 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) e o tipo de morte celular foi avaliado com o ensaio Anexina - Iodeto de propídeo (PI). Os dados foram analizados estatisticamente por análise de variância (ANOVA) ou por teste T student, quando apropriado. Foi observado que todas as frações causaram redução significativa da viabilidade celular da MCF-7, porém a FAE foi a mais citotóxica (p < 0,001). No ensaio de anexina-pi, não foi observado marcação significativa para anexina porém o aumento da marcação de PI foi significativo (p<0,001). O presente estudo demonstrou que a FAE foi a mais eficaz na redução da viabilidade celular, seguindo mecanismo de necroptose na célula MCF-7. A FAE é composta por epicatequina, proantocianidina A2 e procianidinas trimérica e tetramérica.
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O v?rus sincicial respirat?rio induz NETose cl?ssica ROS-dependente atrav?s da ativa??o de PAD4 e das vias de necroptoseMuraro, Stefanie Primon 16 March 2018 (has links)
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Previous issue date: 2018-03-16 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Respiratory syncytial virus (RSV) is a major cause of diseases of the respiratory tract in humans being mainly associated with bronchiolitis, chronic obstructive pulmonary disease (COPD) and asthma exacerbation. RSV infection occurs primarily in pulmonary epithelial cells and, once infection is established, an innate immune response is triggered and mainly neutrophil recruitment is induced. Neutrophils can extrude neutrophil extracellular traps (NETs) capable of entrapping and inactivate a multitude of microorganisms because of its composition and due to the stringy nature of DNA fibers. Recently, was demonstrated that RSV particles and its fusion (F) protein were able to induce the release NETs coated with neutrophil elastase and myeloperoxidase, both antimicrobial peptides. Also, was observed that the excessive formation of NETs can have negative consequences to the host, such as airway obstruction during RSV infection. Therefore, the aim was to evaluate the mechanisms involved in NET formation induced by RSV infection of neutrophils, alveolar epithelial cells (A549) or lung fibroblasts (MRC5). Human neutrophils were infected with RSV and were able to induce NETs release only after 3 hours of stimulation indicating classical NETosis. Next was characterized NETs formation during infection associating DNA extrusion with MPO, NE and F protein of RSV. Was also observed NADPH oxidase and PAD4 dependence and PI3K/AKT, ERK and p38 MAPK pathways during infection. The inhibition of these signaling pathways, PAD4 and ROS production abolished NET formation. Considering a possible involvement of necroptosis during NETs production, were tested MLKL and RIPK inhibitors and evaluated LDH release in the supernatant of infected neutrophils. Neutrophils released LDH and depend on necroptosis induction to produce NETs. Likewise, neutrophils were co-cultured with A549 or MRC5 cells infected with RSV. Both A549 and MRC5 cells triggered NET release by human neutrophils in a virus concentration-dependent manner, the opposite occurs when used UV-inactivated virus. Briefly, RSV induces the classical/ROS-dependent NETosis by human neutrophils, and this effect relies on specific kinases activity. Furthermore, neutrophils are able to recognize pulmonary cells infected by RSV, releasing NETs. Thus, NETs release control could be crucial for minimizing tissue inflammation caused by RSV infection. / O v?rus sincicial respirat?rio (VSR) ? uma das principais causas de doen?as do trato respirat?rio em humanos sendo associado principalmente com bronquiolite, doen?a pulmonar obstrutiva cr?nica (DPOC) e exacerba??o de asma. O VSR infecta principalmente c?lulas epiteliais pulmonares e, uma vez que a infec??o ? estabelecida, uma resposta imune inata ? desencadeada e ocorre o recrutamento de c?lulas do sistema imune, principalmente neutr?filos. Os neutr?filos podem liberar redes extracelulares de neutr?filos (NETs) capazes de capturar e inativar uma grande quantidade de microrganismos devido ? sua composi??o e natureza fibrosa das fibras de DNA. Recentemente, foi demonstrado que part?culas do VSR al?m da prote?na de fus?o (F) do v?rus foram capazes de induzir a libera??o de NETs revestidas com elastase neutrof?lica e mieloperoxidase, ambos pept?deos com atividade antimicrobiana. Al?m disso, observou-se que a forma??o excessiva de NETs pode ter consequ?ncias negativas para o hospedeiro, como a obstru??o das vias a?reas durante a infec??o por VSR. Portanto, o objetivo foi avaliar os mecanismos envolvidos na forma??o de NET induzida pela infec??o por RSV em neutr?filos humanos, c?lulas epiteliais alveolares (A549) ou fibroblastos pulmonares (MRC5). Neutr?filos humanos foram infectados com VSR e foram capazes de induzir a libera??o de NETs somente ap?s 3 horas de infec??o, indicando uma NETose cl?ssica. Em seguida, foi caracterizada a forma??o de NETs durante a infec??o associando a extrus?o de DNA com as prote?nas MPO, NE e com a prote?na F do VSR. Tamb?m se observou a depend?ncia de NADPH oxidase e PAD4 e das vias de sinaliza??o PI3K / AKT, ERK e p38 MAPK durante a infec??o. A inibi??o dessas vias de sinaliza??o, da produ??o de PAD4 e de EROs aboliu a forma??o de NET. Considerando um poss?vel envolvimento da necroptose na produ??o de NETs, foram utilizados inibidores de MLKL e RIPK1 e foi avaliada a libera??o de LDH no sobrenadante de neutr?filos infectados. Os neutr?filos liberaram LDH e dependeram da ativa??o da necroptose para produzir NETs. Do mesmo modo, os neutr?filos foram co-cultivados com c?lulas A549 ou MRC5 infectadas com VSR. Ambas as c?lulas A549 e MRC5 desencadearam a libera??o de NET por neutr?filos humanos de uma maneira dependente da concentra??o de v?rus, o oposto ocorreu quando usado um v?rus UV-inativado. Resumidamente, o VSR induz a NETose cl?ssica / dependente de EROs em neutr?filos humanos, e este efeito depende de atividade espec?fica de quinases. Al?m
disso, os neutr?filos s?o capazes de reconhecer c?lulas pulmonares infectadas pelo VSR, induzindo a libera??o NETs. Assim, o controle de libera??o de NETs pode ser crucial para minimizar a inflama??o do tecido causada pela infec??o por VSR.
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