L’athérosclérose et la pathologie anévrysmale sont principalement caractérisées par un remodelage de laparoi artérielle au cours de leur évolution. Ce travail a examiné un aspect de la destruction de la paroiartérielle à travers l’étude de la métalloprotéase MMP-14 au cours de l’athérome et un aspect dereconstruction artérielle à travers l’étude d’une thérapie cellulaire d’un modèle d’Anévrysme de l’AorteAbdominale (AAA) par Cellules Souches Mésenchymateuses (CSMs).En utilisant un modèle de greffe de Moëlle Osseuse (MO) dans des souris Ldlr-/-, nous avons montré que ladélétion d’expression de MMP-14 dans les cellules issues de la MO provoquait une accumulation decollagène interstitiel dans la plaque athéromateuse sans modification de la composition cellulaire nivariation de taille. Une mesure de l’activité collagénolytique par substrat fluorescent a confirmé que ladélétion en MMP-14 chez les macrophages provoquait une baisse de l’activité collagénolytique. Cetteactivité est indépendante de l’activité MMP-2 et MMP-8 et pourrait être médiée partiellement parl’activation de MMP-13. Nous avons mis en évidence la présence de CSMs à la surface luminale de thrombus de AAA et nous avonsmontré une diminution significative des CSMs circulantes chez des patients porteurs de AAA. Nous avonspu stabiliser la croissance de AAA expérimentaux chez le rat à partir de xénogreffe artérielle par perfusionendoluminale de CSMs. La perfusion de CSMs provoquait une diminution de l’inflammation à court termeet favorisait la reconstruction artérielle par accumulation de collagène et d’élastine à moyen terme.En conclusion, l’activité collagénolytique de MMP-14 est un des mécanismes moléculaires possibles del’évolution de la plaque athéromateuse par rupture de plaque. Elle ouvre la perspective d’une nouvelleapproche thérapeutique et pourrait être une cible comme substrat pour une imagerie fonctionnelle de laplaque athéromateuse. L’évolution de la maladie anévrysmale pourrait être secondaire à une altération dessystèmes de réparation tissulaire dont les CSMs seraient des acteurs clé. La perfusion endoluminale desCSMs dans un modèle expérimental a permis la restauration de ces systèmes de réparation tissulaire etouvre la perspective d’un nouvel outil thérapeutique contre les AAA / Pas de résumé anglais

Les glioblastomes (GBMs) sont les tumeurs cérébrales primaires les plus courantes chez l’adulte avec un pronostic fatal dans les douze mois suivant le diagnostic. De nouvelles avancées dans la connaissance de la pathologie moléculaire des GBMs et de leurs régulateurs clés sont indispensables à l’émergence de nouvelles pistes thérapeutiques. Inositol Requiring Enzyme 1 α (IRE1α) est une protéine résidente du réticulum endoplasmique (RE) agissant en tant que détecteur proximal de l’Unfolded Protein Response (UPR) en conditions physiologiques ou pathologiques. IRE1α est une enzyme bivalente possédant une activité Ser/Thr kinase et endoribonucléasique (RNase). Récemment, des mutations ponctuelles dans le gène ERN1/IRE1α ont été détectées dans les cancers chez l’homme, (en particulier dans les GBMs), et IRE1α a été proposé comme un régulateur majeur de la progression tumorale parmi les protéines kinases. Dans ce travail, nous avons montré que le blocage des deux activités Set/thr kinase et RNase dans les cellules U87-MG réprime fortement l’angiogenèse tumorale, la perfusion des vaisseaux et l’expression de facteurs pro-angiogéniques dans des modèles tumoraux de xénogreffes. Ce changement phénotypique est accompagné d’une réponse dite« d’échappement » des cellules tumorales. Celles-ci envahissent le tissu cérébral sain par migration de long des vaisseaux (mécanismes appelé co-option vasculaire). De plus, ce phénotype a été montré comme étant fonctionnellement associé au processus de transition mésenchymateuse. Par mutagenèse dirigée, nous avons montré qu’IRE1α module les processus d’angiogenèse et d’invasion par ces deux domaines catalytiques : l’activité Ser/Thr kinase d’IRE1α est essentielle pour l’angiogenèse alors que le domaine RNase d’IRE1α contrôle le phénotype invasif et co-opté. IRE1α est ainsi identifié comme un régulateur clé de la croissance du glioblastome, agissant au carrefour de signalisations majeures dans le contrôle de l’adaptation de la cellule tumorale à son micro-environnement. / Glioblastomas (GBMs) are the most common primary brain tumors in humans and remain essentially incurable. New advances in the knowledge of GBM molecular pathology and their key regulators are crucial to identify new putative ways for GBM therapy. Inositol Requiring Enzyme 1 α (IRE1α) is a transmembrane Endoplasmic Reticulum (ER)-resident protein acting as proximal sensor of the Unfolded Protein Response (UPR) in both physiological and pathological situations. IRE1α is a bivalent enzyme, displaying Ser/Thr kinase and endoribonuclease (RNase) activities in its cytosolic side. Recently, single mutations in IRE1α gene were detected in human cancers, including GBM, and IRE1α was proposed as a major contributor to tumor progression among protein kinases. In this work, we have shown that blockade of both IRE1α Ser/Thr kinase and RNase activities in U87-MG cells highly repressed tumor angiogenesis, blood perfusion and the expression of pro-angiogenic factors in human xenograft tumor models. This phenotypic change is adversely associated to the so-called "evasive response" of tumors cells. The cells began to migrate along pre-existing brain capillaries and invade healthy tissue (a process named blood vessel co-option). Moreover, this phenotype was shown to be functionally linked to the mesenchymal differentiation process. By using site-directed mutagenesis, we demonstrated that IRE1α protein modulates both angiogenesis and invasive processes through its two catalytic domains: IRE1α Ser/Thr kinase domain was essential for IRE1-mediated angiogenesis, whereas IRE1's RNase domain drove the invasive, co-opted phenotype. IRE1α is therefore identified as a key regulator of glioma progression, acting at the crossroads of major signaling networks in the control of tumor cell adaptation to its microenvironment.

Osteogenic differentiation of mesenchymal stem cells (MSCs) would be possible to induce by creating of a cell bioactive scaffold that mimic the properties of bone extracellular matrix (ECM). This induction will be not only due to the addition of osteogenic supplements, but also due to the addition of differentiation peptides. These peptides activate signaling pathways leading to cell differentiation. The aim of this study was to evaluate the effect of selected peptides on adhesion, metabolic activity, proliferation and osteogenic differentiation of porcine MSCs. Four peptides with amino acid sequences of DGEA, IAGVGGEKSGGF, GQGFSYPYKAVFSTQ and KIPKASSVPTELSAISTLYL were selected. These peptides were derived from receptor binding sequences of collagen I, collagen III, BMP-7 and BMP-2 respectively. Scaffolds were prepared from a biocompatible and biodegradable poly-ε-caprolactone (PCL) polymer, suitable for cell cultivation. Cells were cultured on scaffolds for three weeks. Various concentration of differentiation peptides were added to the culture medium. As observed in the experiment of cells cultured in basal medium supplemented with differentiation peptides no effect on adhesion, proliferation or metabolic activity of porcine MSCs was observed. In groups treated with peptides derived from BMP-2...

Mesenchymal stem cells (MSC) represent a heterogenous population of nonhematopoietic stem cells with multipotent differential potential. MSC can be isolated from various tissues of organism, the most common tissue are bone marrow or adipose tissue. MSC are good candidates for treatment of autoimmune diseases and possess the ability to prevent graft rejection or graft versus host disease. The immunosuppressive drugs are currently used for inhibition of unwanted immune reaction but they exhibit serious side effects. The use of MSC in therapy can reduce doses of immunosuppressive drugs and eliminate side effects. The study of MSC and immunosuppressant interactions should be detected before MSC can be used for clinical application. We aimed to analyze the interaction between MSC and immunosuppressive drugs and their effect on immune cells. Cyclosporine A and mycophenolate mofetil were used in our research. We demonstrated changes in the expression of surface molecules, production of interleukin 6 and in metabolic activity of MSC after treatment with immunosuppressive drugs. MSC are in organism, in cooperation with the number of other cells. Therefore we studied MSC cocultured with splenocytes in the presence of immunosuppressive drugs. Our results show the effect of MSC and immunosuppressive drugs on different...

Mesenchymal stem cells (MSCs) represent a heterogeneous population of stromal cells with a pluripotent differentiation potential. They can be isolated from multiple tissues of mesodermal origin, such as bone marrow, adipose tissue, umbilical cord blood and peripheral blood and afterwards externally expanded according their adherence to the plastic surfaces. These cells show remarkable immunomodulatory properties, suppressing T-, B- and NK-cell functions, and also modulating dendritic cell activities and influencing immune responses during tissue repair and recovery. MSCs have been shown to possess ability to migrate to sites of inflammation and tissue injury. All these properties make MSCs a promising tool for clinical application. Our primary goal was to identify processes that may influence immunoregulatory effects of MSCs. In order to promote immunossupressive qualities of MSCs we established the scheme comprising MSCs precultivated with various cytokines and Toll-like receptors (TLR) ligands in vitro, with the final aim to improve the therapeutic effect of MSCs on wound healing in vivo. We studied modulation of MSCs properties and consequently the effect of influenced MSCs on cells of the immune system. The immunosuppression is mainly mediated through secreted factors that MSCs produced after...

Electrospinning is widely used technique to produce nanoscale constructs for tissue engineering. This technique can be used to spin wide range of polymers. One of them is polyvinyl alcohol (PVA), which has very good properties for use in this field. PVA is nontoxic, has good mechanical strength and it's degradable and biocompatible. Electrospun PVA nanofibers have limitations because of their -OH side groups. These groups cause solubility of PVA in water. The solubility can be adjusted with crosslinking techniques, but PVA still remains very hydrophilic, which is causing low adhesion of cells. In recent research we decided to reduce the hydrophilicity of PVA using plasma modification. Polymer modification with cold plasma is an economic and quite simple process to change the surface chemistry without side effects that come with conventional chemical treatment. With radical, formed by discharge, we have deposited hydrocarbons on the PVA surface and we rapidly increased hydrophobicity of the polymer surface. The change of surface chemistry has only a little effect on the fiber morphology. The increase of hydrophobicity allowed better adhesion of mesenchymal stem cells on plasma modified PVA as compared to non-modified PVA and a huge change in cell morphology was observed. These changes suggest that we...

Résumé non transmis / Summary not transmitted

Regenerative medicine is an emerging technology and with hundreds of cell therapies currently in clinical trials there is a need to expand the limited knowledge related to their storage, shipment and preservation. The most widely used medium for human cell cryopreservation is 10%wt dimethyl sulfoxide (DMSO) in serum. However given its potential toxicity, DMSO usage is a key issue in cryopreservation. Methods specify the need to reduce cell exposure time to DMSO above 0°C as much as possible but the maximum amount of time cells can be exposed to DMSO to prevent a detrimental effect needs to be clarified. There are also regulatory issues and concerns with the xenotoxicity, ethics and supply of the other core component in the standard cryomedia formulation: Foetal Bovine Serum (FBS). Developing a viable alternative to FBS is crucial. In cryobiology literature thawing appears poorly understood. A stable process is as vital as freezing to prevent injury to cells. Protocols are currently too vague for cell therapy regulation and need improvement. The time dependent DMSO cytotoxicity was evaluated by overexposing cells to DMSO during and/or after cryopreservation. A broad investigation found that after 1 hour overexposure post thaw viability of human mesenchymal stem cells (hMSCs) was reduced from 96.3±0.6% to 74.1±4.0% and the co-expression of five key hMSC markers was changed from 97.9±1.3% to 68.3±2.6%. This significant change could cause indicate a change in product efficacy and affect patient health, to prevent this, DMSO exposure must be kept to below 1 hour. A range of alternative vehicle solutions were screened and human platelet lysate (hPL) investigated as an alternative. In depth experimentation with hPL as a cryopreservation vehicle solution and culture supplement (in place of FBS) found it to be a worthy, statistically similar alternative. With no xenological or ethical concerns, lower costs than other serum-free alternatives hPL could allow for a move away from xenological components. A heat transfer model was developed and determined that 720J is required to thaw a vial. Using the heat transfer model and additional factors such as pre-thaw stabilisation and on thaw dilution, a two-stage experiment found that the current standard process (warming in a 37°C waterbath) within the current paradigm of a 1.8mL cryovial is optimal but further work is required to define the process for scaled-up product.

Les douleurs viscérales chroniques font partie des effets secondaires des patients traités pour des cancers de la zone pelvienne. Ces douleurs peuvent affecter grandement la qualité de vie de ces patients. Dans les cas les plus graves, il n'existe pas de traitement analgésique efficace. Ainsi le développement de nouvelles stratégies thérapeutiques efficaces constitue un enjeu majeur. Au sein de notre laboratoire, le potentiel réparateur et immuno-modulateur des cellules stromales mésenchymateuses (CSM) a déjà été démontré dans un modèle d'ulcération colorectale radio-induite chez le rat. Dans ce contexte, l'objectif de ma thèse était d'évaluer d'abord la pertinence de l'utilisation de ce modèle pour l'étude de l'hypersensibilité viscérale persistante radio-induite, puis, le bénéfice thérapeutique de l'utilisation des CSM comme agent antinociceptif. Nous avons dans un premier temps démontré, dans ce modèle, le maintien au cours du temps d'une hypersensibilité viscérale associée à une sensibilisation centrale persistante après irradiation, validant ainsi le modèle. Nous avons ensuite montré l'implication des mastocytes (MC) et suggéré l'implication du neuromédiateur NO dans les mécanismes de la sensibilisation périphérique sous-tendant une telle hypersensibilité. Nous avons enfin mis en évidence que le traitement par des CSM permettait la réduction de l'hypersensibilité viscérale radio-induite persistante. La capacité des CSM à moduler l'activation des MC et/ou leurs interactions avec les fibres nerveuses pourrait être impliquée dans leur action antinociceptive. En conclusion, ce travail a permis d'élargir le spectre d'action thérapeutique des CSM dans notre modèle d'étude. / Patients who undergo pelvic radiotherapy may develop significant incidence of undesirable chronic gastrointestinal complications resulting from radiation-induced damages around the tumour. Chronic visceral pain is one of the radiation-induced side effects that greatly affects the quality of life of “cancer survivors”. The lack of effective analgesic treatment highlights the importance of novel and effective therapeutic strategies. In our laboratory, mesenchymal stromal cell (MSC) based approach showed beneficial immunomodulatory and regenerative effects in a rat model of irreversible radiation-induced colonic ulcers. The goal of my work was to assess the relevance of this model to study radiation-induced visceral persistent hypersensitivity and its modulation by MSC treatment. We first demonstrated that this model is associated with long-lasting visceral hypersensitivity and central neuronal sensitization. In this context we showed then that mast cells (MC) are involved in the mechanism of peripheral sensitization. Moreover, we suggested the implication of the neuromediator NO• in the pathophysiology of persistent radiation-induced visceral hypersensitivity. We also suggested that MSC treatment reversed radiation-induced hypersensitivity by a mechanism that in part may involve the modulation of MC activation and/or the decrease in the number of MC and nerve fiber interactions. In addition, MSC treatment reduced the percentage of nitrinergic neurons, increased after irradiation, and restored colonic muscular contractibility. Such processes may promote the therapeutic benefit of MSC observed in our study. In conclusion, this work provided new insights on the therapeutic benefit of MSC in our study model and a new argument in favour of their use in a future clinical trial to cure abdominopelvic radiotherapy side effects.

Recent advances in the clinical diagnosis and treatment of diseases using cell transplantation have emphasized the urgent need to cryopreserve many types of cells. In transfusion medicine, red blood cell (RBC) transfusions are used to treat anemia and inherited blood disorders, replace blood lost during or after surgery and treat accident victims and mass casualty events. In regenerative medicine, mesenchymal stem cell (MSC) therapy offers promising treatment for tissue injury and immune disorders. Current cryoprotective agents (CPAs) utilized for RBCs and MSCs are 40% glycerol and 10% dimethyl sulfoxide (DMSO), respectively. Although glycerol is required for successful cryopreservation of RBCs, it must be removed from RBCs post-thaw using costly and time-consuming deglycerolization procedures to avoid intravascular hemolysis. Unfortunately, while DMSO prevents cell damage and increases post-thaw MSC viability and recovery, recent reports have suggested that MSCs cryopreserved in DMSO display compromised function post-thaw. As a result, improvements to the current cryopreservation protocols such as reducing post-thaw RBC processing times and improving MSC function post-thaw are necessary in order to meet the increasing demands of emerging cellular therapies. Ice recrystallization has been identified as a significant contributor to cellular injury and death during cryopreservation. Consequently, the ability to inhibit ice recrystallization is a very desirable property for an effective CPA, unlike the conventional CPAs such as DMSO and glycerol that function via a different mechanism and do not control or inhibit ice recrystallization. Over the past few years, our laboratory has reported several different classes of small molecules capable of inhibiting ice recrystallization such as lysine-based surfactants, non-ionic carbohydrate-based amphiphiles (alkyl and aryl aldonamides) and O-linked alkyl and aryl glycosides. The use of these small molecule ice recrystallization inhibitors (IRIs) as novel CPAs has become an important strategy to improve cell viability and function post-thaw. With the overall goal to identify highly effective inhibitors of ice recrystallization, the first part of this thesis examines the IRI activity of three diverse classes of small molecules including carbohydrate-based surfactants bearing an azobenzene moiety, fluorinated aryl glycosides and phosphate sugars. While the majority of the carbohydrate-based surfactants and fluorinated aryl glycosides were not effective inhibitors of ice recrystallization, this work revealed that monosaccharides possessing a phosphate group could be effective IRIs. Our laboratory has previously demonstrated that small molecule IRIs β-PMP-Glc and β-pBrPh-Glc can protect human RBCs from cellular injury during freezing using reduced concentrations of glycerol (15% w/v). This was significant as reducing the concentration of glycerol can drastically decrease deglycerolization times. Consequently, structure- function studies were conducted on β-PMP-Glc and β-pBrPh-Glc to elucidate key structural features that further enhance their IRI activity and may increase their cryoprotective ability. In particular, the influence of an azido moiety on the IRI activity of β-PMP-Glc and β-pBrPh-Glc was investigated and it was determined that the position of the azide substituent on the pyranose ring is crucial for effective inhibition of ice recrystallization. Furthermore, the presence of an azido group at C-3 was found to increase the IRI activity of β-PMP-Glc and β-pBrPh-Glc. Despite the discovery that β-PMP-Glc and β-pBrPh-Glc are beneficial additives for the freezing of RBCs, a significant amount of cellular damage occurred during deglycerolization, resulting in very low cell recoveries. Thus, IRI active azido aryl glucosides were explored for their cryopreservation potential in RBCs to determine whether they could function as effective additives that reduce cellular damage post-thaw and improve cell recovery. One of the most significant results of this thesis is the discovery that azido aryl glucosides can successfully cryopreserve RBCs in the presence of 15% glycerol with significantly improved cell recovery. This thesis also explores the use of small molecule IRIs to improve the cryopreservation of MSCs. In particular, the addition of an N-aryl-aldonamide (2FA) to the standard 10% DMSO solution was found to enhance the proliferative capacity of MSCs post-thaw. Lastly, the ability of small molecule IRIs to cross the cell membrane and behave as permeating CPAs was evaluated in two different cell models, RBCs and human umbilical vein endothelial cells (HUVECs). These studies demonstrated that small molecule IRIs are capable of permeating the cell membrane and controlling intracellular ice recrystallization.