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Neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease are linked to mitochondrial dysfunction and the underexpression of TOM40, a protein with chaperone-like qualities that is responsible for transporting precursor proteins into the mitochondria. Overexpression of TOM40 is reported to partially restore mitochondrial dysfunction and decrease the accumulation of neurotoxic aggregates of α-synuclein. Our goal is to develop an effective method for delivery of TOM40 protein to the brain. Previous studies have used lentiviruses to carry TOM40 into the hippocampus of α-synuclein transgenic mice. The disadvantage of lentiviral transfection is the random insertions of the target gene into the host genome, which could cause toxic effects. Synthetic phospholipid vesicles containing TOM40 were considered as an alternative delivery method, but these "liposomes" elicit not only toxicity, but also an immune response. Thus, development of a safer delivery method of TOM40 protein is needed. We investigated exosomes, which are extracellular vesicles originating from multivesicular endosomes filled with protein, lipid, or RNA cargoes for cell-cell communication. Since exosomes are created from host cells, they are non-immunogenic and may be a more desirable method. Expression constructs have been made for the production of TOM40 protein within or on the surface of exosomes. In order to target the delivery of TOM40 to the brain, we attached peptides to the surface of the exosomes, which specifically interact with receptors on neural cells. We attempted to confirm the functionality of the expression constructs through immunocytochemistry followed by flow cytometry and Western blotting.

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Cardiovascular disease remains the leading cause of morbidity and mortality worldwide and Myocardial Infarction (MI) and subsequent heart failure remains the leading cause for death. Despite the improvement in prognosis and treatment of acute MI patients, the underlying causes including loss of cardiomyocytes and microvasculature remain potential risk and lack proper and efficient solutions. Stem cell-based therapies for repair and regeneration have evolved and have been applied in clinical trials. Different types of stem cells, including Endothelial progenitor cell (EPC), Mesenchymal Stem Cell (MSC), induced Pluripotent Stem Cell (iPSC) and cardiac progenitor cells etc. have been used for potential long term recovery and cardiac regeneration. However, results from the clinical trials have been largely disappointing and improvement in cardiac functions have been modest likely due to the limitations of cell therapy including low integration in myocardium, poor survival, cellular dysfunction and limited differentiation ability. It is therefore necessary and urgent to develop cell free alternatives as next generation regenerative therapies. There is a consensus that the beneficial effect of stem cell therapy is largely due to paracrine effects. Exosomes have recently emerged as important functional units mediating stem cell paracrine effects. Exosomes are the family of extracellular vesicles (EV) which are 30-150nm in size, secreted by almost all types of cells and responsible for cell-cell communication via delivering their cargo including RNAs and proteins to host cells. Studies from our and other labs have shown that exosomes mimic parental stem cell in improving post-MI functions. The essential feature of exosome is decided by their cargo including RNA and protein, which are subject to dynamic changes depending on the environment of parental cells. Our studies were focused on Endothelial Progenitor Cell (EPC)-derived exosomes. EPCs are generated in bone marrow, and home to the site of tissue injury and orchestrate neovascularization and tissue repair. Patients with ischemic heart disease, are usually accompanied with comorbidities such as systemic inflammation, aging, diabetes, etc. which are known to compromise EPC functions. We hypothesized that EPCs under inflammatory stress produce dysfunctional exosomes with altered RNA and protein content, leading to impaired cardiac reparative properties. We chose interleukin-10 knockout (IL-10KO) mice as a model of systemic inflammation. EPCs were isolated from IL-10KO and wild-type (WT) mice, and their exosomes (Exo) were compared for their reparative properties both in vitro and in vivo. Our in vitro studies showed WT-EPC-Exo treatment attenuated recipient cell apoptosis, enhanced cell mobilization and tube formation, whereas IL-10KO-EPC-Exo were functionally deficient or even had detrimental effects. We used MI mouse model to compare the in vivo function of two groups of exosomes and found WT-EPC-Exo treatment significantly improved left ventricular (LV) cardiac function, inhibited cell death, promoted angiogenesis and attenuated cardiac remodeling; while these cardioprotective effects were lost in IL-10KO-EPC-Exo treated group. Both in vitro and in vivo studies proved that even the same progenitor cell type (EPCs), under inflammatory stimulus (IL-10KO), secretes exosomes with different reparative properties. Next, we explored whether the observed difference in exosome function is caused by altered exosome content. Using Next Generation RNA Sequencing (NGS RNAseq) and mass spectrometry we found RNA and protein expression patterns were drastically different in wild type and IL-10 knockout EPC derived exosomes. This evidence leads to the conclusion that alteration in exosome content is fundamental for exosome function. We picked two candidates that are highly enriched in IL-10KO-EPC-Exo for further study, miR-375 and Integrin-Linked Kinase (ILK). We treated IL-10KO-EPC with anti-miR against miR-375 and siRNA against ILK separately, and successfully decreased the expression of miR-375 and ILK in both EPCs and EPC derived exosomes. Then we explored the function of those miR and protein ‘modified exosomes’ with similar in vitro and in vivo experiments as previously described. Compared to IL-10KO-EPC-Exo, miR-375 knockdown exosomes showed enhanced angiogenesis and inhibited cell apoptosis, while ILK knockdown in exosomes rescued functions in both in vitro and in vivo experiments. These results suggested the possibility that exosome manipulation of identified factors may partially rescue their reparative functionality. In summary, our studies revealed that stem cell derived exosomes are capable for independent cardiac repair in ischemic heart disease, however, parental stem cells under pathological stimulus secrete dysfunctional exosomes with altered RNA and protein content. Exosome function can be rescued or enhanced through RNA and protein content modification. / Biomedical Sciences

The fields of biotechnology and biomedical sciences are rapidly evolving and involve the constant growth of knowledge. As a consequence, engineering design has to also remain at the cutting edge in order to not inhibit the growth of these fields. This study focuses on engineering design and analysis as it pertains to the field of biotechnology, at every step of the engineering process. More specifically, how the engineering design and analysis approach can assist in solving medical problems relating to bone diseases and biomaterials. The first part of the study focuses on a project to design and manufacture a novel exosome isolation device, with the primary purpose of creating an affordable and accessible method of isolating exosomes for the testing and diagnosis processes in the Biomaterials & Nanoscience laboratory. The second part of the study focuses on the design and analysis of biodegradable bone implants, before, during, and after implantation. Together, these projects aim to show the engineering processes of design and analysis and serve to provide insight as to how engineering principles can be applied to the medical field.

La synthèse du ribosome est un processus compliqué, très hiérarchisé et essentiel à toutes les cellules vivantes. La complexité de ce processus tient notamment au fait que les différentes étapes de la biogenèse du ribosome eucaryote sont temporellement et spatialement organisées dans des compartiments cellulaires différents (le nucléole, le nucléoplasme et le cytoplasme). Il est toutefois connu que le pré-ARNr 35S (le précurseur de trois des quatre ARNr, les ARNr 18S, 5.8S et 25S) est pris en charge dès sa synthèse par des facteurs impliqués dans sa maturation. Ainsi, la formation d’un ribosome requiert l’association, sur le transcrit naissant, des facteurs de synthèse, au nombre de 400. Ces facteurs essentiels interagissent transitoirement avec l’ARNr et ne font pas partie des particules ribosomiques matures impliquées dans la traduction. Leur rôle est d’assister le remodelage constant du pré-ribosome et le processus d’assemblage des sous-unités.<p>Parmi ces facteurs de synthèse, nous avons caractérisé en détail, chez la levure et chez l’homme, la protéine Las1 impliquée dans la maturation des deux extrémités de l’ITS2, séquence qui sépare les ARNr 5.8S et 25S/28S. Chez la levure, en absence de la protéine Las1, les analyses de profils de polysomes révèlent un déficit de sous-unité 60S et l’apparition d’« halfmères ». Les techniques de purification d’affinité et de gradient de sédimentation nous indiquent que Las1 est associée aux pré-ribosomes 60S et qu’elle interagit avec de nombreux facteurs de synthèse de la petite, de la grande sous-unité ou des deux. De plus, Las1 copurifie avec des pré-ribosomes qui contiennent aussi les exoribonucléases 5’-3’ Rat1/Rai1 et Xrn1. Rai1 coordonne la maturation aux deux extrémités de l’ARNr 5.8S. Nous suggérons que Las1 appartient à un macrocomplexe connectant spatialement des sites de clivages éloignés sur la séquence primaire du pré-ARNr qui seraient rapprochés suite au reploiement de l’ITS2.<p>Un autre aspect de ce travail de thèse consiste en l’étude de l’assemblage des particules ribonucléoprotéiques et plus spécifiquement du pré-ribosome et des sous-unités ribosomiques eucaryotes. Nous avons utilisé la technique d’immunoprécipitation de chromatine (Ch-IP) pour caractériser l’assemblage d’une structure appelée le « SSU processome ». Celui-ci correspond à un pré-ribosome en formation ainsi que l’assemblage des protéines ribosomiques sur l’ARNr naissant.<p>Enfin, nous avons étudié le rôle d’une plateforme d’activation de méthyltransférases d’ARN et de protéines, la protéine Trm112 dans la ribogenèse. Nous avons montré que chez la levure, Trm112 est impliquée dans la synthèse du ribosome et dans la progression de la mitose. En absence de cette protéine, les pré-ARNr sont dégradés par un mécanisme de surveillance. Trm112 copurifie avec plusieurs facteurs de synthèse du ribosome dont la méthyltransférase Bud23, impliquée dans la modification post-transcriptionnelle de l’ARNr18S. Trm112 est requise pour cette méthylation et nous postulons que la protéine Bud23 est incapable de se lier aux pré-ribosomes en l’absence de Trm112.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

L’ischémie/reperfusion (I/R) occurrente à toutes transplantations d’organe solide, constitue un stimulus pro-autophagique/pro-apoptotique pour les cellules endothéliales. Nous avons récemment démontré que les cellules endothéliales apoptotiques (CEapo) sécrètent des vésicules apoptotiques exosome like (ApoExo) qui induisent une réponse auto-immune et accélèrent le rejet vasculaire dans un modèle de greffe aortique chez la souris. Ces ApoExo qui diffèrent des corps apoptotiques classiques par leur structure et leur contenu protéique contiennent le fragment C-terminal du perlécan, LG3. Le LG3, un auto-antigène d’importance en transplantation, favorise le remodelage vasculaire et est augmenté en circulation chez les patients greffés rénaux atteints d’un rejet vasculaire. De plus, la présence d’anticorps anti-LG3 avant la transplantation est associée à un risque plus élevé de développer un rejet vasculaire chez des patients greffés du rein et à la perte du greffon à long terme. Il est connu que la génération du fragment LG3 implique la protéolyse du perlécan par la cathepsine L, une protéase lysosomiale, mais le mécanisme de transport de ce fragment au sein des ApoExo est encore inconnu. Nous avons émis l’hypothèse que l’activité lysosomiale et l’autophagie jouent un rôle important dans la maturation et le chargement du LG3 dans les vésicules ApoExo sécrétées par les CEapo. Une étude longitudinale de microscopie électronique chez les cellules endothéliales humaines carencées en sérum pendant 1h, 2h et 3h, nous a révélé la présence du perlécan / fragment LG3 au sein de compartiments autophagiques (autophagosomes et autophagolysosomes) à différentes étapes du processus autophagique. Après 3 heures de carence en sérum, nous avons identifié le fragment LG3 dans des vésicules membranaires situées au sein de grands réseaux vacuolaires s’apparentant à des autophagolysosomes. L’inhibition de la cathepsine L, de l’acidification du lysosome et de l’autophagie diminuent la présence du fragment LG3 dans les vésicules ApoExo sans affecter la sécrétion de vésicules démontrant donc le rôle de l’autophagie dans l’intégration du fragment LG3 au sein des ApoExo. Toutefois, l’injection de vésicules ApoExo LG3-, provenant de cellules murines aortiques traitées à la bafilomycine, dans un modèle murin de greffe aortique induit une réponse auto-immune anti-LG3 ainsi qu’un remodelage vasculaire à des niveaux similaires aux souris ayant reçu des ApoExo véhicule. Une analyse protéomique de ces vésicules ApoExo LG3-, nous a révélé que la bafilomycine modifie le contenu protéique des ApoExo en induisant une augmentation de la présence de protéines lysosomiales et de la matrice extracellulaire dont le perlécan. Ceci suggère que la présence du motif LG3 au sein du perlécan natif non clivé dans les ApoExo LG3- pourrait être responsable de la mise en place de la réponse anti-LG3 observées chez les souris. Les travaux de ce doctorat ont permis de mettre en évidence que l’autophagie dans les cellules endothéliales productrices d’ApoExo ne régule pas l’immunogénicité des ApoExo. Cependant, elle régule au sein des cellules endothéliales apoptotiques le transport et le clivage du perlécan qui lui est immunogène. En effet, l’autophagie module les différentes formes de perlécan natif et clivé sécrétées dans les ApoExo. L’étude chez la souris greffée nous a donc permis de considérer non plus l’implication du fragment LG3 mais l’implication du motif LG3 présent au sein du perlécan natif non clivé et de ses différentes formes intermédiaires dans la réponse auto-immune anti-LG3 et le rejet vasculaire. La modulation de la sécrétion du perlécan et du LG3 dans les ApoExo constitue une cible thérapeutique potentielle afin de diminuer la réponse auto-immune pouvant augmenter le dommage vasculaire après la greffe / Ischemia/reperfusion (I/R) occurring in all solid organ transplantation, constitute a proautophagic / pro-apoptotic stimulus on endothelial cells. We recently demonstrated that apoptotic endothelial cells (CEapo) secrete apoptotic exosome-like vesicles (ApoExo) that induce autoimmune response and accelerate vascular rejection in a mouse aortic transplant model. These ApoExo, that differ from classical apoptotic bodies in structure and protein content, contain the C-terminal fragment of perlecan, LG3. LG3, an autoantigen of importance in transplantation, promotes vascular remodeling and is increased in circulation in renal transplant patients undergoing vascular rejection. In addition, the presence of anti-LG3 antibodies prior to transplantation is associated with a higher risk of developing vascular rejection in kidney transplant patients and long-term graft loss. It is known that the generation of LG3 fragment involves the proteolysis of perlecan by cathepsin-L, a lysosomal protease, but the mechanism of export of this fragment within ApoExo is still unknown. We hypothesized that lysosomal activity and autophagy play an important role in the maturation and the secretion of LG3 in ApoExo vesicles secreted by CEapo. Longitudinal electron microscopy study after 1h, 2h and 3h in serum starved endothelial human cells revealed the presence of perlecan / LG3 fragment within autophagic compartments (autophagosomes and autophagolysosomes) at different stages of the autophagic process. After 3 hours of serum starvation, we identified LG3 fragment in membrane vesicles located within large vacuolar networks reminiscent of autophagolysosomes. Inhibition of cathepsin-L, of lysosomal acidification and of autophagy decrease the presence of LG3 fragment in ApoExo vesicles without affecting vesicle secretion thus demonstrating the role of autophagy in the secretion of LG3 fragment within ApoExo. However, Injection of ApoExo LG3- vesicles from bafilomycin-treated aortic murine cells into a murine aortic transplant model induces autoimmune anti-LG3 response and vascular remodeling at levels similar to the ApoExo vehicle mice control group. Proteomic analysis of ApoExo from bafilomycin-treated aortic murine cells has demonstrated that bafilomycin modifies ApoExo protein content by inducing an increase of the presence of lysosomal proteins and the extracellular matrix, including perlecan. This suggests that the presence of LG3 motif in uncleaved native perlecan in ApoExo LG3- could be responsible for the establishment of the anti-LG3 response as well as the vascular remodeling observed in mice. Collectively, these results demonstrate that autophagy in endothelial cells producing ApoExo does not regulate the immuogenicity of ApoExo. However, it regulates within apoptotic endothelial cells the processing and cleavage of perlecan who is immunogenic. Indeed, autophagy modulates the different forms of native and cleaved perlecan secreted in ApoExo. Grafted mice study thus allowed us to consider neither the involvement of the LG3 fragment but the implication of the LG3 motif present in the uncleaved native perlecan and its intermediate forms in the anti-LG3 autoimmune response and vascular rejection. Modulating perlecan and LG3 secretion in ApoExo vesicles is a potential therapeutic target to reduce the autoimmune response that can increase vascular damage after transplantation.