Introdução: O fígado é um órgão chave na manutenção da homeostasia corpórea e o transplante hepático ainda continua sendo o padrão-ouro no tratamento da insuficiência hepática aguda. A falta de doadores tem favorecido o desenvolvimento da terapia celular. Células derivadas de medula óssea podem se diferenciar em células tipo-hepatócitos em menos de 24 horas e a comunicação através de vesículas extracelulares (VEs) é um dos mecanismos propostos para explicar essa capacidade. Muitos estudos têm demonstrado que as células-tronco mesenquimais (CTMs) da medula tem alta plasticidade para se diferenciar em hepatócitos, mas os protocolos normalmente utilizados levam entre 7 e 28 dias. Objetivo: Analisar a capacidade de diferenciação das CTMs da medula em se tornar uma célula tipo-hepatócito através do mecanismo de comunicação celular por VEs em cultura (6 e 24 horas). Materiais e métodos: Para avaliar o efeito de hepatócitos primários isolados de ratos saudáveis e lesados com CCl4 na diferenciação das CTMs foi utilizado um sistema de co-cultivo com insertos que não permitem o contato entre as células colocando as CTMs na câmera superior e os hepatócitos na câmera inferior do sistema. Meio condicionado de hepatócitos com lesão foi utilizado para avaliar a capacidade das CTMs de capturar VEs e se diferenciar em célula-tipo hepatócito. Os marcadores de célula tipo-hepatócito avaliados foram expressão gênica (alfa fetoproteina, albumina e citoqueratina-18), armazenamento de glicogênio e liberação de ureia. Para rastrear VEs, hepatócitos de ratos lesados foram marcados com PKH-26. As VEs foram obtidas por ultracentrifugação do sobrenadante e analisadas por citometria de fluxo. Hepatócitos e CTMs também foram analisados por citometria de fluxo na busca de marcação positiva. Resultados: CTMs co-cultivadas durante 6 e 24 horas com hepatócitos não apresentaram expressão de genes hepáticos, mesmo quando expostas a um ambiente de lesão. Os ensaios funcionais confirmaram a falta de sinais de diferenciação, sendo que não foi observado armazenamento de glicogênio nem liberação de ureia nas CTMs. Um achado interessante foi que ao analisar o sobrenadante da câmera superior do sistema de co-cultivo, não foram achadas VEs marcadas com PKH-26 nem CTMs com rastros do marcador. Por outro lado, os experimentos utilizando meio condicionado mostraram que as CTMs têm capacidade de capturar VEs. A citometria de fluxo mostrou que às 6 horas e 24 horas respetivamente 2,28% e 3,97% das CTMs eram positivas para o marcador PKH-26. Quando analisadas no microscópio de fluorescência, foram vistos pontos vermelhos nas CTMs alguns dos quais parecem carregar a proteína albumina. Porém a expressão gênica e analise de ureia não se adequaram a um perfil de célula tipo-hepatócito. Conclusões: O sistema de co-cultivo não foi adequado para permitir a transferência e comunicação através de VEs entre hepatócitos e CTMs sendo que as VEs não conseguem atingir a câmara superior. Os experimentos com meio condicionado sugerem que as CTMs têm capacidade de capturar VEs derivadas de hepatócitos, porém a captação não conduz ao desenvolvimento de um perfil de célula tipo-hepatócito em 6 e 24 horas. São necessários mais estudos para esclarecer a dinâmica de transferência das VEs e suas consequências em longo prazo. / Introduction: Liver is a key organ for corporeal homeostasis maintenance and whole organ replacement still remains the gold standard procedure to treat acute liver failure. Shortage of liver donor has promoted the increase on cell-therapy research. Bone marrow (BM) derived cell have shown potential for differentiation into hepatocyte-like cells in a short time and extracellular vesicles communication (EVs) is one of the proposed mechanisms. Plasticity of bone marrow mesenchymal stem cells (BM-MSCs) is extensively supported by scientific literature but protocols applied to differentiation usually take from 7 to 28 days. Objective: To analyze in vitro differentiation potential of BM-MSCs into hepatocyte-like cells through EVs transfer mechanism in 6 and 24 hours. Materials and Methods: Co-culture system with cell-impermeable inserts and conditioned medium experiments were used to explore the effects of healthy and CCl4-injured hepatocytes, over BM-MSCs differentiation. Assessment of hepatocyte-like cell profile on BM-MSCs was revealed by gene expression (alpha fetoprotein, albumin and cytokeratin-18), glycogen storage and urea release. Hepatocytes from CCl4-injured rats were labeled by PKH-26 to track EVs. Ultracentrifugation was used to isolate EVs from supernatant medium of the two chamber of the co-culture system. PKH-26 positive EVs and PKH-26 positive cells were revealed by flow cytometry analysis and fluorescent microscopy. BM-MSCs cultured with conditioned medium were stained with ALB-FITC antibody. Results: Co-cultured BM-MSCs for 6 and 24 hours, showed no expression of hepatocyte-like genes, even after exposure to damaged microenvironment. Functional assays confirm the lack of differentiation signs there were no glycogen storage or urea release. Interestingly, EVs traffic analysis revealed no PKH-26 positive EVs at the upper chamber of co-culture system and no positive BM-MSCs were found either. On the other hand, conditioned medium experiment showed that BM-MSCs could uptake EVs. Flow cytometry analysis showed positive PKH-26 BM-MSCs at 6 (2.28%) and 24 (3.97%) hours. Flourescence microscopy revealed red points into BM-MSCs and immunofluorescence suggest that some EVs contain albumin. Gene expression and urea assay of BM-MSCs were not in accordance with a hepatocyte-like profile. Conclusions: Co-culture system, by using cell-impermeable membrane, was not adequate to promote EVs transfer between hepatocyte and BM-MSCs since EVs do not pass from the lower to the upper chamber. Conditioned medium experiments can suggest that BM-MSCs could uptake hepatocyte-derived EVs but this not drive to a hepatocyte-like profile in a short period of time. More studies will be necessary to clarify the dynamic of EVs transfer and their long time effects.

Introdução: O fígado é um órgão chave na manutenção da homeostasia corpórea e o transplante hepático ainda continua sendo o padrão-ouro no tratamento da insuficiência hepática aguda. A falta de doadores tem favorecido o desenvolvimento da terapia celular. Células derivadas de medula óssea podem se diferenciar em células tipo-hepatócitos em menos de 24 horas e a comunicação através de vesículas extracelulares (VEs) é um dos mecanismos propostos para explicar essa capacidade. Muitos estudos têm demonstrado que as células-tronco mesenquimais (CTMs) da medula tem alta plasticidade para se diferenciar em hepatócitos, mas os protocolos normalmente utilizados levam entre 7 e 28 dias. Objetivo: Analisar a capacidade de diferenciação das CTMs da medula em se tornar uma célula tipo-hepatócito através do mecanismo de comunicação celular por VEs em cultura (6 e 24 horas). Materiais e métodos: Para avaliar o efeito de hepatócitos primários isolados de ratos saudáveis e lesados com CCl4 na diferenciação das CTMs foi utilizado um sistema de co-cultivo com insertos que não permitem o contato entre as células colocando as CTMs na câmera superior e os hepatócitos na câmera inferior do sistema. Meio condicionado de hepatócitos com lesão foi utilizado para avaliar a capacidade das CTMs de capturar VEs e se diferenciar em célula-tipo hepatócito. Os marcadores de célula tipo-hepatócito avaliados foram expressão gênica (alfa fetoproteina, albumina e citoqueratina-18), armazenamento de glicogênio e liberação de ureia. Para rastrear VEs, hepatócitos de ratos lesados foram marcados com PKH-26. As VEs foram obtidas por ultracentrifugação do sobrenadante e analisadas por citometria de fluxo. Hepatócitos e CTMs também foram analisados por citometria de fluxo na busca de marcação positiva. Resultados: CTMs co-cultivadas durante 6 e 24 horas com hepatócitos não apresentaram expressão de genes hepáticos, mesmo quando expostas a um ambiente de lesão. Os ensaios funcionais confirmaram a falta de sinais de diferenciação, sendo que não foi observado armazenamento de glicogênio nem liberação de ureia nas CTMs. Um achado interessante foi que ao analisar o sobrenadante da câmera superior do sistema de co-cultivo, não foram achadas VEs marcadas com PKH-26 nem CTMs com rastros do marcador. Por outro lado, os experimentos utilizando meio condicionado mostraram que as CTMs têm capacidade de capturar VEs. A citometria de fluxo mostrou que às 6 horas e 24 horas respetivamente 2,28% e 3,97% das CTMs eram positivas para o marcador PKH-26. Quando analisadas no microscópio de fluorescência, foram vistos pontos vermelhos nas CTMs alguns dos quais parecem carregar a proteína albumina. Porém a expressão gênica e analise de ureia não se adequaram a um perfil de célula tipo-hepatócito. Conclusões: O sistema de co-cultivo não foi adequado para permitir a transferência e comunicação através de VEs entre hepatócitos e CTMs sendo que as VEs não conseguem atingir a câmara superior. Os experimentos com meio condicionado sugerem que as CTMs têm capacidade de capturar VEs derivadas de hepatócitos, porém a captação não conduz ao desenvolvimento de um perfil de célula tipo-hepatócito em 6 e 24 horas. São necessários mais estudos para esclarecer a dinâmica de transferência das VEs e suas consequências em longo prazo. / Introduction: Liver is a key organ for corporeal homeostasis maintenance and whole organ replacement still remains the gold standard procedure to treat acute liver failure. Shortage of liver donor has promoted the increase on cell-therapy research. Bone marrow (BM) derived cell have shown potential for differentiation into hepatocyte-like cells in a short time and extracellular vesicles communication (EVs) is one of the proposed mechanisms. Plasticity of bone marrow mesenchymal stem cells (BM-MSCs) is extensively supported by scientific literature but protocols applied to differentiation usually take from 7 to 28 days. Objective: To analyze in vitro differentiation potential of BM-MSCs into hepatocyte-like cells through EVs transfer mechanism in 6 and 24 hours. Materials and Methods: Co-culture system with cell-impermeable inserts and conditioned medium experiments were used to explore the effects of healthy and CCl4-injured hepatocytes, over BM-MSCs differentiation. Assessment of hepatocyte-like cell profile on BM-MSCs was revealed by gene expression (alpha fetoprotein, albumin and cytokeratin-18), glycogen storage and urea release. Hepatocytes from CCl4-injured rats were labeled by PKH-26 to track EVs. Ultracentrifugation was used to isolate EVs from supernatant medium of the two chamber of the co-culture system. PKH-26 positive EVs and PKH-26 positive cells were revealed by flow cytometry analysis and fluorescent microscopy. BM-MSCs cultured with conditioned medium were stained with ALB-FITC antibody. Results: Co-cultured BM-MSCs for 6 and 24 hours, showed no expression of hepatocyte-like genes, even after exposure to damaged microenvironment. Functional assays confirm the lack of differentiation signs there were no glycogen storage or urea release. Interestingly, EVs traffic analysis revealed no PKH-26 positive EVs at the upper chamber of co-culture system and no positive BM-MSCs were found either. On the other hand, conditioned medium experiment showed that BM-MSCs could uptake EVs. Flow cytometry analysis showed positive PKH-26 BM-MSCs at 6 (2.28%) and 24 (3.97%) hours. Flourescence microscopy revealed red points into BM-MSCs and immunofluorescence suggest that some EVs contain albumin. Gene expression and urea assay of BM-MSCs were not in accordance with a hepatocyte-like profile. Conclusions: Co-culture system, by using cell-impermeable membrane, was not adequate to promote EVs transfer between hepatocyte and BM-MSCs since EVs do not pass from the lower to the upper chamber. Conditioned medium experiments can suggest that BM-MSCs could uptake hepatocyte-derived EVs but this not drive to a hepatocyte-like profile in a short period of time. More studies will be necessary to clarify the dynamic of EVs transfer and their long time effects.

A produção in vitro de embriões é uma biotecnologia bastante difundida mundialmente. O Brasil, em 2015, foi responsável por aproximadamente 67% dos embriões bovinos produzidos in vitro no mundo (PERRY, 2014). Embora essa tecnologia seja bastante utilizada, é de grande interesse para o mercado desenvolver estratégias que levem ao maior aproveitamento dos oócitos obtidos e compreender os mecanismos, dentro do ambiente folicular, que determinam a competência oocitária. O microambiente folicular é fundamental para o crescimento e a aquisição da competência oocitária, tornando o oócito apto a desenvolver-se e manter o embrião até a transição materno embrionária. Para tanto, os componentes foliculares - células da granulosa, células do cumulus, oócito e líquido folicular - precisam trabalhar em unidade, visto que possuem uma relação de interdependência. Dentro dos mecanismos de comunicação existentes no ambiente folicular estão as vesículas secretadas por células, chamadas vesículas extracelulares, que foram descritas no líquido folicular, contendo material bioativo como proteínas, lipídios e RNAs, incluindo os microRNAs. Contudo, os componentes do ambiente folicular podem refletir na qualidade do oócito que será produzido e a hipótese geral deste trabalho é de que os miRNAs presentes no ambiente folicular regulam vias de sinalização importantes para o desenvolvimento oocitário e que os miRNAs e ou RNAs mensageiros presentes nas células foliculares podem ser explorados como importantes ferramentas para o diagnóstico da qualidade oocitária. O primeiro estudo determinou perfis transcricionais de miRNAs em células da granulosa, em complexos cumulus-oócito e em vesículas extracelulares derivadas destas células e também do fluido folicular. Além de conhecer a origem e o papel dos miRNAs no ambiente folicular, estes perfis de expressão indicaram a regulação no ambiente folicular da via de sinalização da PI3K-Akt. No segundo estudo, componentes desta via de sinalização foram então determinados em células foliculares associadas a oócitos de alta ou baixa competência ao desenvolvimento. Os resultados demonstram que a ativação da via PI3K-Akt em células foliculares correlaciona-se à maior competência oocitária; de modo oposto, a menor atividade desta via nas células foliculares está relacionada à reduzida competência oocitária. Nos estudos três e quatro, um conjunto de experimentos foram realizados para determinar o perfil de microRNAs e RNAs mensageiros em células do cumulus associadas a oócitos de alta ou baixa competência ao desenvolvimento. Buscou-se esclarecer alguns dos mecanismos responsáveis pela melhor qualidade oocitária e levar a identificação de biomarcadores de qualidade oocitária permitindo o avanço e o desenvolvimento de novas ferramentas para intensificar a produção in vitro de embriões bovinos. Por fim, estes resultados demonstram respostas integradas entre oócitos e células foliculares durante o desenvolvimento folicular e o processo de aquisição de competência oocitária e identificaram marcadores de qualidade oocitária. / In vitro embryo production of is a widespread biotechnology worldwide. In 2015 Brazil was responsible for approximately 67% of bovine embryos produced in vitro in the world (PERRY, 2014). Although widely used, to develop strategies that lead to better use of oocytes and understand the mechanisms (in follicular microenvironment) that determine oocyte competence is of great interest to national market. The follicular microenvironment is fundamental for oocyte growth and acquisition of competence, making the oocyte able to develop and maintain the embryo until the embryonic maternal transition. For this end, the follicular components - granulosa cells, cumulus cells, oocytes and follicular fluid - need to work in unity, since they have arelation of interdependence. Within the mechanisms of communication existing within the follicular environment are vesicles secreted by cells, called extracellular vesicles, which were described in follicular fluid, containing bioactive material such as proteins, lipids and RNAs, including microRNAs. The components of follicular environment may reflect the quality of the oocyte that will be produced and the general hypothesis of this work is that the miRNAs present in the follicular environment regulate signaling pathways important for oocyte development and that the miRNAs and/or messenger RNAs present in the follicular cells can be explored as important tools for the diagnosis of oocyte quality. The first study determined profiles of miRNAs in granulosa cells, cumulus-oocyte complexes and extracellular vesicles derived from these cells and also in follicular fluid. In addition, by knowing the origin and role of miRNAs in the follicular environment, the expression profiles indicated the PI3K-Akt signaling pathway regulation in the follicular environment. In the second study, components of this signaling pathway were then determined in follicular cells associated with oocytes of high or low developmental competence. The results demonstrated that the activation of the PI3K-Akt pathway in follicular cells correlates with increased oocyte competence; conversely, the lower activity of this pathway in follicular cells is related to reduced oocyte competence. In studies three and four, experiments were performed to determine the profile of microRNAs and messenger RNAs in cumulus cells associated with oocytes of high or low developmental competence. Aiming to clarify some of the mechanisms responsible for the best oocyte quality that lead to the identification of oocyte quality biomarkers, allows the advancement and development of new tools to intensify the in vitro production of bovine embryos. Finally, results obtained demonstrate integrated responses between oocytes and follicular cells during follicular development and give new insights to the study of oocyte competence acquisition process and identified oocyte quality markers.

Les vésicules extracellulaires (VE), incluant exosomes et microparticules (MP), vecteurs d’information biologique, peuvent moduler la fonction de cellules cibles. Une élévation du taux de VE circulantes est observée dans les pathologies cardiovasculaires dont l’obésité est l’un des facteurs de risque majeur. Cependant, il existe peu de données concernant la production de VE adipocytaires et leur capacité à moduler le métabolisme des adipocytes. Tout d’abord, nous avons caractérisé de manière morphologique et biochimique les MP et les exosomes adipocytaires. Nous montrons une production accrue de ces VE dans un contexte d’obésité murine. L’analyse protéomique des VE adipocytaires révèle un enrichissement spécifique des MP et des exosomes en protéines clé du métabolisme énergétique et de l’inflammation, respectivement. Dans une seconde partie, nous avons étudié l’effet de MP lymphocytaires portant le morphogène Hedgehog (MPHh+) sur la différenciation adipocytaire. A l’instar d’une activation classique de la voie de signalisation Hh, les MPHh+ inhibent l’adipogenèse. Bien que dépendant du récepteur Smoothened (Smo), cet effet inhibiteur est indépendant des facteurs de transcription Gli. Nous montrons que les MPHh+ activent un axe anti-adipogénique Smo/Lkb1/Ampk pouvant être stimulé par un nouvel agoniste de Smo, le GSA-10. Nos résultats démontrent, d’une part, la capacité des adipocytes à sécréter des VE, et d’autre part, le potentiel fonctionnel des MPHh+ à inhiber l’adipogenèse par une voie de signalisation Hhnon-canonique. Les VE pourraient contribuer aux dysfonctions métaboliques associées à l’obésité en véhiculant des messages métaboliques à l’échelle de l’organisme. / Extracellular vesicles (EV), including microparticles (MP) and exosomes, are able to modulate target cell function through exchange or transfer of biological material. Although EV are present in the blood of healthy individuals, an elevated quantity of circulating EV is associated with cardiovascular diseases, which obesity is a major cardiovascular risk factor. Nevertheless, few studies have reported the ability of adipocytes to release EV and their implication in adipose tissue metabolism. First of all, we could determine morphological and biochemical features of adipocyte-derived exosomes and MP through a combination of methods. We were able to demonstrate an increase in adipocyte EV production in a murine model of obesity. Proteomic analysis of adipocyte EV further revealed a specific enrichment of proteins crucial for glucose and lipid metabolism and related to inflammation in MP and exosomes respectively. We then evaluated the ability of lymphocytes-derived MP harboring the Sonic Hedgehog morphogen to control adipocyte differentiation. Activation of the Hedgehog canonical pathway inhibited adipogenesis, as did MPHh+. Surprisingly, although Smo dependent, inhibitory potential of such MP did not involve the Gli transcription factors. We show that MPHh+ inhibit adipocyte differentiation through a Smo/Lkb1/Ampk axis as does a new agonist of Smo, GSA-10. Our results demonstrate, on one hand, the ability of adipocyte to release EV and on the other hand, the capacity of MPHh+ to control adipogenesis through a non-canonical Hh signaling pathway. In conclusion, EV might contribute to obesity related metabolic dysfunctions through systemic regulation of metabolic pathways.

No description available.

La pollution atmosphérique est un problème de santé publique. En 2013, le Centre International de Recherche sur le Cancer a classé la pollution de l'air, ainsi que les particules fines (PM₂.₅), de taille inférieure à 2,5 µm, comme cancérogène de groupe I pour l'homme. Les PM₂.₅ ont la capacité de pénétrer en profondeur dans l'appareil respiratoire. En se déposant au niveau des alvéoles pulmonaires, elles entraînent une réponse inflammatoire importante notamment via la libération de médiateurs de l'inflammation et des vésicules extracellulaires (EV) par les cellules immunitaires infiltrantes ou résidentes. Dans ce contexte, ce projet de thèse comportait deux objectifs majeurs : i) déterminer les caractéristiques chimiques des PM₂.₅ collectées en milieu industrialo-urbain, en identifier les origines et la variabilité saisonnière de leur composition chimique ; ii) étudier la réponse et les EV produites par les macrophages suite à l'exposition aux PM₂.₅ et observer les effets des EV sur les cellules pulmonaires.Pour cela, des macrophages primaires ont été exposés aux PM₂.₅ prélevées à Dunkerque et leur réponse cellulaire (stress oxydant, inflammation, polarisation, miARN) a été mesurée. Les EV produites en réponse à cette exposition ont été isolées et caractérisées. Enfin, des cellules épithéliales pulmonaires, les BEAS-2B, ont été exposées aux EV libérées en réponse à l'exposition aux PM₂.₅ et les effets de cette exposition (inflammatoire, stress oxydant, miARN) ont été mesurés. Nous avons mis en évidence une inflammation et une réponse anti-oxydante dans les macrophages, ainsi qu'une modification de leur polarisation. Les PM₂.₅ entrainent une libération plus importante d'EV. / Air pollution is a major public health problem. In 2013, The International Agency for Research on Cancer classified air pollution and fine particle (PM₂.₅), with size lower than 2.5 µm, as carcinogenic to humans (group I). PM₂.₅ are able to penetrate deeply in lungs. When PM₂.₅ settle in pulmonary alveolar, they lead to strong inflammatory response, with inflammatory mediators ans extracellular vesicles release by infiltrating or resident immune cells. In this context, this thesis included two major aims : i) evaluate the physico-chemical characteristics of PM₂.₅ sampled in an industrial-urban site, identify their origin and study the seasonal variability of their composition ; ii) investigate the cellular response and EV produced by macrophages in response to PM₂.₅ and study EV's effects on epithelial cells. To achieve this, macrophages are exposed to PM₂.₅ collected in Dunkerque, and cellular response (oxidative stress, inflammation, polarization, miRNA) was quantified. EV released in response to PM₂.₅ exposition was isolated and characterized. Finally, epithelial cells, BEAS-2B, are exposed to EV released by exposed and non exposed macrophages to evaluate effects from this exposure (inflammation, oxidative stress, miRNA). We observed inflammation and anti-oxidant response in macrophages after PM₂.₅ exposure, as well as polarization modification. PM₂.₅ lead to increased number of EV by macrophages.

Small extracellular vesicles (small EVs) are endogenous membrane-enclosed nanocarriers released from essentially all cells. They have been shown to carry proteins, lipids, nucleic acids to transmit biological signals throughout the body, including to the brain. Some evidence has suggested that small EVs can cross the blood-brain barrier (BBB), moving from the peripheral circulation to the central nervous system (CNS). The BBB is a dynamic barrier that regulates molecular trafficking between the peripheral circulation and the CNS. As a result, small EVs have attracted attention for their potential as a novel delivery platform for nucleic acid-based therapeutics across the BBB. Silencing RNAs (siRNAs) are a potent drug class but using “naked” siRNA is not feasible due to their short half-life, vulnerability to degradation and low penetration in cells. Despite the excitement for the development of small EV-based therapeutics, their clinical development is hampered by the lack of reliable methods for packing therapeutics into them. Reshke et al. has shown that cells can be genetically engineered to produce customizable small EVs packaged with siRNA against any protein by integrating the siRNA sequence into the pre- miR-451 structure. Mounting evidence has established that in a misfolded state, α-synuclein becomes insoluble and phosphorylated to form intracellular inclusions in neurons (known as Lewy bodies) which leads to Parkinson’s disease (PD) pathogenesis. Given that increased α-synuclein expression causes familial and idiopathic PD, decreasing its synthesis by using siRNA is an attractive therapeutic strategy. Here, we genetically engineered cells to produce small EVs packaged with siRNA against α-synuclein integrated in the pre-miR451 backbone, tested their ability to cross an in vitro BBB, and deliver its cargo to silence endogenous α-synuclein in neuron- like cells. The therapeutic potential of α-synuclein siRNA delivery by these small EVs was demonstrated by the strong mRNA (60-70%) and protein knockdown (43%) of α-synuclein in neuron-like cells. We also demonstrated that approximately at 4% and 2%, respectively of small EVs-derived from human brain endothelial cells (hCMEC/D3) and human embryonic kidney (HEK293T) were transported cross the in vitro BBB model. Interestingly, we observed that small EVs-derived from HEK293T deliver their cargo to induced brain endothelial cells (iBECs) (~74% α-synuclein mRNA reduction) but their rate of transport across BBB was lower and did not reduce α-synuclein mRNA expression in neuron-like cells, seeded on the far side of the BBB. Small EVs- derived from hCMEC/D3 reduced α-synuclein mRNA (40%) in neuron-like cells across the BBB model. This finding suggests that small EVs derived from different cell sources can undergo different intracellular trafficking routes, providing various opportunities to influence the efficiency of delivery and fate of intracellular cargo. Using small EVs-derived from hCMEC/D3, two different routes of administration, a single bolus intravenous (IV) or intra-carotid (ICD) injection, showed small EVs largely accumulated in the liver, spleen, small intestines and kidneys; and only a small amount of small EVs were detected in the brain. These results indicate that human brain endothelial cells may serve as a promising cell source for CNS treatments based on small EVs.

No description available.

In open angle glaucoma, dysfunction of the trabecular meshwork (TM) results in impaired aqueous humour outflow leading to an elevated intraocular pressure (IOP) that underlies optic nerve damage and irreversible blindness. Currently, no curative treatment is available for the disease. Indeed, most pharmacological and surgical interventions usually provide only temporary relief from elevated IOP while little progress has been made in targeting the root cause of this disease: correcting the dysfunctional TM. In this context, we hypothesized that regeneration/refunctionalization of the TM may represent an effective therapeutic option to halt disease progression or even reverse the pathologic process. We previously demonstrated in a rat model of glaucoma that the injection of mesenchymal stromal cells (MSCs) cultured under hypoxic conditions or their conditioned media (MSC-CM) into laser-damaged TM area results in tissue regeneration. Injection of MSC or conditioned media in our glaucoma model led to activation and proliferation of ocular progenitor cells culminating in TM regeneration and a decrease in IOP. However, the mechanistic basis for this regenerative process remained elusive. Thus, the aim of this thesis is to elucidate the mechanistic basis of MSC secretome-mediated TM regeneration and the subsequent decrease in IOP. We now demonstrate that injection of hypoxic MSC-CM into laser-induced glaucomatous eyes resulted in massive immune cell recruitment. We also demonstrate that these hypoxic MSC-CM conditioned cells produced pro-regenerative factors in vitro and in vivo. Next, employing a proteomic approach, we identified and verified the pro-regenerative effect of several factors secreted by hypoxic MSC-CM recruited cells, which in turn induced the activation/proliferation of ocular progenitor cells leading to TM regeneration and decreased IOP. Upon individual injection of the purified factors into glaucomatous rat eyes, we observed a partial and delayed but significant decrease in IOP that correlated with an increase in the activation and proliferation of neuronal progenitor cells in the TM area. The co-injection of these factors resulted in a significant decrease in IOP compared with individual factor injection. Importantly, this drop in IOP was associated with restoration of retinal functionality, thus demonstrating the importance of these factors in the TM regeneration process and disease control. The findings presented in this thesis provide a novel acellular therapeutic approach for glaucoma treatment via in situ TM regeneration. Moreover, the knowledge gained here could have a lasting impact on how we induce tissue regeneration in other degenerative diseases and lead to novel therapeutic advances in regenerative medicine. / Dans le glaucome à angle ouvert, le dysfonctionnement du trabéculum (TM), un tissu nécessaire à la filtration de l'humeur aqueuse, entraîne une élévation de la pression intraoculaire (PIO). Ceci cause des lésions au niveau du nerf optique et une cécité irréversible. Présentement, aucun traitement curatif n'a été développé pour cette maladie. Nous émettons l'hypothèse que la régénération et re-fonctionnalisation du trabéculum peut représenter une option thérapeutique efficace pour arrêter ou inverser la progression de la maladie dans de nombreux cas de glaucome. Nous avons précédemment démontré les effets régénérateurs des cellules mésenchymateuses (MSCs) et de leurs milieux conditionnés par l'hypoxie (MSC-CM) dans la régénération du TM suite à un dommage par laser. Ce processus a conduit à l'activation et à la prolifération des cellules progénitrices oculaires résultant en une diminution de la PIO dans un modèle de glaucome induit par laser chez le rat. Cependant, la base mécanistique de ce processus de régénération reste encore inconnue. Ainsi, le but de cette thèse de recherche est d'élucider cette base mécanistique de la régénération du TM médiée par le sécrétome des MSC et la diminution subséquente de la PIO. À cette fin, l'injection de MSC-CM hypoxique dans les yeux glaucomateux induits par laser a entraîné un important recrutement de cellules immunitaires. Sous l’action du MSC-CM, ces cellules produisent des facteurs pro-régénératifs in vitro et in vivo. Ensuite, nous avons utilisé une approche protéomique et vérifié l'effet pro-régénératif des facteurs sécrétés par ces cellules exposées au MSC-CM hypoxique, sur l'activation et la prolifération des cellules progénitrices oculaires et la PIO. Lors de l'injection de ces facteurs chez le rat glaucomateux, nous avons observé une augmentation significative de l'activation et de la prolifération des cellules progénitrices neuronales présentes dans la zone du TM, résultant en une diminution de la PIO. De plus, l’injection combinée de ces facteurs résulte en une diminution synergique importante de la PIO. Cette baisse de la PIO était associée à une restauration de la fonction rétinienne, démontrant ainsi l'importance de ces facteurs dans le processus de régénération du TM et de contrôle de la maladie. Les résultats présentés dans cette thèse pourraient amener à une nouvelle approche thérapeutique acellulaire pour le traitement du glaucome via la régénération du TM. De plus, les connaissances acquises au cours de cette thèse pourraient avoir un impact durable sur la manière d’aborder la régénération tissulaire dans d'autres maladies dégénératives et amener des avancées thérapeutiques nouvelles en médecine régénératrice

Le vieillissement est l'un premier facteur de risque pour plusieurs maladies telles que l’athérosclérose, la fibrose, l’Alzheimer, le diabète de type 2 et le cancer. L'accumulation de cellules sénescentes avec l'âge contribue au développement de maladies liées à l'âge en induisant une inflammation chronique causée par le phénotype sécrétoire associé à la sénescence (SASP). Il y a également une augmentation de la sécrétion de vésicules extracellulaires (EV) lors de la sénescence. Les EV sont des structures à bicouche lipidique permettant le transport de molécules actives vers des cellules réceptrices. De plus, les EV participent aux effets pathologiques des cellules sénescentes. Ainsi, nous proposons que les macrophages sénescents participent au développement de maladies liées à l’âge en induisant l'inflammation par l’action combinée des facteurs solubles du SASP et des EV. Tout d'abord, nous avons établi un modèle de macrophages sénescents induit par l’oncogène Raf-1. Une analyse transcriptionnelle de notre modèle a démontré un profil inflammatoire régulé par Nf-κB. La sécrétion d'EV est également augmentée par les macrophages sénescents. En outre, les EV dérivées de macrophages sénescents sont enrichies en miARN, tels que miR-21, miR-155 et miR-132, ainsi qu'en protéines ribosomiques, qu'en protéine Alix et qu'en protéine Mvp. Les sécrétions des macrophages sénescents induisent un échappement de la sénescence chez les cellules MEF, probablement par l'action combinée des molécules solubles du SASP et des EV. Nous concluons que les macrophages sénescents sécrètent des signaux prolifératifs et inflammatoires dans les cellules réceptrices, ce qui suggère leur rôle potentiel dans le développement de cancer. Le traitement avec le navitoclax élimine les macrophages sénescents et pourrait prévenir leurs effets pathologiques. / Aging is the first risk factor for several diseases such as atherosclerosis, fibrosis, Alzheimer’s, type 2 diabetes and cancer. The accumulation of senescent cells with age contributes to development of age-related diseases by inducing chronic inflammation. This inflammation is induced by the senescence-associated secretory phenotype (SASP). During senescence, there is also an increase of extracellular vesicles (EV) secretion. EVs are lipid bilayer structures that allow the transport of active molecules to recipient cells. In addition, EVs participate in pathologic effects of senescent cells. Thus, we propose that senescent macrophages participate in development of age-related diseases by inducing inflammation through the combined effect of SASP soluble factors and EV. First, we established a model of senescent macrophages induced by the oncogene Raf-1. Transcriptional analysis of our model demonstrated an Nf-κB-regulated inflammatory profile. EV secretion is also increased by senescent macrophages. Moreover, EVs derived from senescent macrophages are enriched in miRNA, such as miR-21, miR-155 and miR-132, as well as ribosomal proteins, Alix protein an Mvp protein. Secretion of senescent macrophages induce senescence escape in MEF cell, probably through the combined action of SASP soluble factor and EV. We conclude that senescent macrophages secrete proliferative and inflammatory signals in recipient cell, suggesting their potential role in cancer development. Treatment with navitoclax eliminates senescent macrophages and may prevent their pathological effects.