The chalcone motif is a privileged structure present in an extensive range of biologically active molecules. The chalcone structure can also serve as a versatile starting material for more complex molecules in medicinal chemistry. Eleutherobin, isolated from the Australian coral Eleutherobia and sarcodictyin, isolated from the Mediterranean coral Sarcodictyon roseum are natural products displaying nanomolar cytotoxicity against a range of cancer cell lines including Taxol®-resistant cell lines. Both natural products act as microtubule stabilising agents and will be valuable additions to the clinic, however their limited availability and lengthy total syntheses prevent further development. The urocanic ester side chain present in eleutherobin and sarcodictyin was identified as being critical for biological activity. We discuss the design, synthesis and biological evaluation of fourteen chalcone analogues based on this urocanic motif with the lead chalcone displaying promising antiproliferative activity in a range of cancer cell lines. Combretastatin A-4 is a promising microtubule destabiliser under clinical development. The Z configuration is vital for biological activity, however it can isomerise to the inactive E configuration. We report a library of twenty pyrazolines synthesised from chalcones as “Z restricted” combretastatin analogues with the lead pyrazoline displaying potent antiproliferative activity in cancer cell lines due to the disruption of tubulin. Tissue engineering is a diverse interdisciplinary field that applies engineering principles to the biological sciences with the aim of maintaining or replacing tissue function. Recent developments have revealed metal chelation to be a valuable tool to control the architecture of tissue engineering scaffolds. We report a library of ten novel pyrazolines and their potential as metal chelators. Maltol is a well established Fe3+ chelator with a low toxicity profile. We report a novel maltol hydrazide which can be attached to the cell surface which upon addition of Fe3+ results in cellular aggregation due to metal chelation. Further studies revealed that this process can be applied to form heterocellular aggregates composed of two different cancer types with valuable applications in tissue engineering and cancer research.

Il est maintenant établi que les cellules souches mésenchymateuses (MSC), résident dans la même niche que les cellules souches hématopoïétiques (HSC), au sein de la moelle osseuse (MO). Il est connu que la pression en O2 (pO2) de la niche est inférieure à la normale, soit moins de 5% pO2 contre 12-15 % pO2 dans le sang artériel. Cette hypoxie a des conséquences sur le métabolisme, en protégeant les cellules contre le stress oxydatif et en favorisant leur caractère multipotent. Notre hypothèse est que les MSC cultivées en hypoxie devraient être plus proches de leur condition physiologique, donc plus multipotentes. Des MSC de la MO humaine ont été cultivées en pO2 de 21% et 5%. Leur morphologie, capacité de différenciation en ostéocytes et adipocytes, et transcriptome ont été comparés à différents passages. Nous avons observé un ralentissement de la prolifération à des temps précoces à pO2 5%, caractérisée par une inhibition de l'expression de gènes impliqués dans la réplication et le cycle cellulaire, puis une augmentation à des passages tardifs. Les gènes codant pour des molécules d'adhérence et de la matrice extracellulaire sont stimulés par l'hypoxie. A des temps tardifs, la capacité de différenciation des MSC est stimulée en hypoxie, les cellules présentent un aspect plus immature et une diminution de synthèse des mitochondries. Surtout, l'hypoxie stimule la synthèse de « gènes de la plasticité » suivant le logiciel « Gene Ontology », et de gènes impliqués dans le développement épithélial et neuronal. En conclusion, la culture des MSC de MO en hypoxie semble plus physiologique et pourrait être utile pour des applications en médecine régénératrice / It is now settled that mesenchymal stem cells (MSC), reside in the same microenvironment or niche than hematopoietic stem cells (HSC), within the bone marrow (BM). It is also known that the O2 tension (pO2) of the niche is below 5% as compared to 21% O2 in the air and 12-15% in the arterial blood. As developed in our recent review, this physiological hypoxia protects stem cells from oxidative stress and maintains their multipotential state. Our hypothesis is that MSC cultured in hypoxia should be closer to their physiological condition and therefore more "multipotent". MSC from human BM were cultured et 21% and at 5% pO2. Their morphology, their ability to differentiate into osteocytes and adipocytes, and their transcriptome were compared at different passages. We observed a decrease of proliferation rate in early times in hypoxia, characterized by inhibition of the expression of genes involved in cell replication and cell cycle, and an increase in later passages. Whatever the passage, the genes encoding adhesion molecules and extracellular matrix are stimulated by hypoxia. At later times, the ability of MSC differentiation is stimulated by hypoxia, the cells look to be more immature and show decreased synthesis of mitochondria. Indeed, hypoxia stimulates the synthesis of plasticity genes according to "Gene Ontology" (GO) terms, and of several genes involved in neuronal- and epithelial-cell development. In conclusion, the culture of MSC from BM in hypoxia seems to be more physiological and may be useful for regenerative medicine applications.

Τα κύτταρα της νευρικής ακρολοφίας (Neural Crest Cells - NCCs) αποτελούν έναν πολυδύναμο, μεταναστευτικό πληθυσμός βλαστικών κυττάρων τα οποία δίνουν γένεση σε μια πληθώρα κυτταρικών τύπων κατά την ανάπτυξη των σπονδυλωτών, συμπεριλαμβανομένων των νευρώνων και των νευρογλοιακών κυττάρων του περιφερικού νευρικού συστήματος (ΠΝΣ). Η δημιουργία των κυττάρων της νευρικής ακρολοφίας πραγματοποιείται στο στάδιο της γαστριδίωσης μετά την επαγωγή της νευρικής πλάκας. Οι διαδικασίες αυτές επηρεάζονται από σηματοδοτικά μονοπάτια στα οποία εμπλέκονται τόσο μεταγραφικοί παράγοντες όσο και επιγενετικοί τροποποιητές . Το Εντερικό Νευρικό Σύστημα (ΕΝΣ), προέρχεται από τα κύτταρα της νευρικής ακρολοφίας της αυχενικής και της ιερής μοίρας και ελέγχει την ομαλή λειτουργία της γαστρεντερικής οδού. Τα κύτταρα της νευρικής ακρολοφίας της αυχενικής μοίρας αποικίζουν ολόκληρο τον εντερικό σωλήνα και δίνουν γένεση στο ΕΝΣ ξεκινώντας από την 9η εμβρυική ημέρα. Η δημιουργία ενός πλήρως λειτουργικού ΕΝΣ εξαρτάται από την ικανότητα μετανάστευσης, πολλαπλασιασμού και διαφοροποίησης των NCCs. Στόχος της παρούσας διπλωματικής εργασίας ήταν η μελέτη της έκφρασης γονιδίων που ελέγχουν τον πολλαπλασιασμό και τη μετανάστευση τόσο των πρόδρομων κυττάρων της νευρικής ακρολοφίας όσο και των NCCs που έχουν δεσμευτεί προς εντερική μοίρα σε έμβρυα μυός απουσία της πρωτεΐνης Geminin. Η Geminin είναι μια πρωτεΐνη που έχει δειχθεί να επηρεάζει την ισορροπία μεταξύ αυτο-ανανέωσης και διαφοροποίησης, μέσω της αλληλεπίδρασης της με μεταγραφικούς παράγοντες και πρωτεΐνες αναδιαμόρφωσης της χρωματίνης. Με σκοπό να διερευνήσουμε τον in vivo ρόλο της Geminin στα κύτταρα της νευρικής ακρολοφίας, δημιουργήσαμε διαγονιδιακούς μύες από τους οποίους αδρανοποιήσαμε το γονίδιο της Geminin ειδικά στα κύτταρα της νευρικής ακρολοφίας. Τα αποτελέσματα μας έδειξαν ότι η απουσία της Geminin από τα κύτταρα της νευρικής ακρολοφίας, οδηγεί στη δημιουργία εμβρύων με μορφολογικές αλλοιώσεις κατά τα πρώιμα στάδια της ανάπτυξης ενώ σε μεταγενέστερα αναπτυξιακά στάδια χαρακτηρίζονται από σοβαρές κρανιοπροσωπικές δυσμορφίες. Επιπλέον, η ιστοειδική αδρανοποίηση της Geminin οδήγησε σε απορρύθμιση των επιπέδων έκφρασης γονιδίων που επηρεάζουν τόσο την επαγωγή όσο και τη μετανάστευση των NCCs (mChd7, mSnail2, mTwist2,mFoxD3) κατά την 10.5η εμβρυική ημέρα. Μελέτη του κυτταρικού κύκλου των εντερικών κυττάρων νευρικής ακρολοφίας έδειξε ότι η αποσιώπηση της Geminin διαταράσσει το προφίλ του κυτταρικού τους κύκλου καθώς τα κύτταρα αυτά «μπλοκάρουν» κατά τη μετάβαση από την G2 στη Μ φάση (E9.5). Συμπερασματικά, τα αποτελέσματά μας δείχνουν ότι η Geminin συμμετέχει ενεργά στον ρύθμιση της έκφρασης γονιδίων που παίζουν σημαντικό ρόλο στην επαγωγή και τη μετανάστευση των NCCs κατά τα πρώιμα στάδια της εμβρυικής ανάπτυξης (Ε10.5). Τέλος, η Geminin διαδραματίζει σημαντικό ρόλο στον πολλαπλασιασμό των πρόδρομων κυττάρων του εντερικού νευρικού συστήματος (Ε9.5). / Neural Crest cells (NCCs) comprise a multipotent, migratory cell population that generates a diverse array of cell and tissue types during vertebrate development, including neurons and glial cells of Peripheral Nervous System (PNS). The induction of neural crest specification occurs at the end of gastrulation at the neural plate border initiate an epithelial-to-mesenchymal transition (EMT) that transforms these stationary cells into migratory cells. These processes are influenced by changes in the expression of transcription factors and epigenetic regulators. The Enteric Nervous System (ENS), is a subdivision of the PNS that controls the function of the gastrointestinal (GI) tract and is derived from the Vagal and Sacral NCCs. The formation of a fully functional ENS depends on the coordinated proliferation and differentiation decisions of NCCs. Vagal NCCs emigrate from the vagal region of the neural tube (somites 1-7), in a rostal to caudal direction and enter the foregut at embryonic day E9-9.5 (in mice), generating the ENS. The formation of a fully functional ENS depends on proliferation and differentiation decisions of NCCs. The aim of our work was to study the expression of genes that control self-renewal decisions and migration ability of the NCCs in mouse embryos in the absence of Geminin. Towards this direction we studied the in vivo role of Geminin in the early stages of NCCs development and in the developing ENS. Geminin is a Protein that has been shown to affect the balance between self-renewal and differentiation through multiple interactions with transcription factors and chromatin remodeling proteins. In order to further elucidate the in vivo role of Geminin in NCCs, we generated transgenic mice lacking Geminin expression specifically in NCCs. We have shown that the deletion of Geminin in NCCs, causes severe morphological and craniofacial malformation during embryonic development. The conditional inactivation of Geminin resulted in the deregulation of various genes that affect the induction and migration of NCCs (mChd7, mSnail2, mTwist2, mFoxD3) at E10.5. Examination of the cell cycle profile of enteric NCCs showed that the deletion of Geminin disrupted the proliferation of NCCs as cells are blocked at the transition from G2 to M phase (E9.5). In conclusion, our results highlight Geminin as an important molecule during the induction and migration of NCCs at the early stages of mouse embryonic development (10.5). Finally, Geminin plays an important role for the proliferation of ENS progenitor cells (E9.5).

Unravelling the genetic architecture of common diseases is a continuing challenge in human genetics. While genome-wide association studies (GWAS) have proven to be successful in identifying many new disease susceptibility loci, the extension of these studies beyond single-SNP methods of analysis has been limited. The incorporation of multi-locus methods of analysis may, however, increase the power of GWAS to detect genes of smaller effect size, as well as genes that interact with each other and the environment. This investigation carried out large-scale simulations of four multi-locus model selection techniques; namely forward and backward selection, Bayesian model averaging (BMA) and least angle regression with a lasso modification (lasso), in order to compare the type I error rates and power of each method. At a type I error rate of ~5%, lasso showed the highest power across varied effect sizes, disease frequencies and genetic models. Lasso penalized regression was then used to perform three different types of analysis on GWAS data. Firstly, lasso was applied to the Wellcome Trust Case Control Consortium (WTCCC) data and identified many of the WTCCC SNPs that had a moderate-strong association (p<10-5) type 2 diabetes (T2D), as well as some of the moderate WTCCC associations (p<10-4) that have since been replicated in a large-scale meta-analysis. Secondly, lasso was used to fine-map the 17q21 childhood asthma risk locus and identified putative secondary signals in the 17q21 region, that may further contribute to childhood asthma risk. Finally, lasso identified three potential interaction effects potentially contributing towards coronary artery disease (CAD) risk. While the validity of these findings hinges on their replication in follow-up studies, the results suggest that lasso may provide scientists with exciting new methods of dissecting, and ultimately understanding, the complex genetic framework underlying common human diseases.

Les cellules souches mésenchymateuses (CSM) issues de la gelée de Wharton (GW) du cordon ombilical sont immuno-privilégiées et immunosuppressives. Dans le cadre d’une utilisation en ingénierie vasculaire, la persistance de ces propriétés après différenciation endothéliale a été explorée. La différenciation a été induite par ensemencement des CSM-GW sur des films multicouches de polyélectrolytes en présence du milieu EGM-2. L’immunogénicité des cellules endothéliales-« like » (CEL) a été vérifiée en évaluant l’expression de 2 marqueurs immunologiques HLA-DR et CD86. Afin d’évaluer l’effet immunosuppresseur des CSM-GW avant et après différenciation, celles-ci ont été cultivées en premier lieu en présence de cellules mononucléées stimulées en contact cellulaire direct ou séparées, puis avec des cellules Natural Killer (NK) et lymphocytes T (LT). Les résultats ont montré que les CEL expriment les marqueurs endothéliaux, n’expriment pas HLA-DR et CD86 et qu’elles inhibent la prolifération des différentes populations immunitaires selon un mécanisme dépendant de facteurs solubles tels que IDO, les IL-6 et -1β et de la PGE2, et de contacts cellulaires. La co-culture de LT avec les CEL a induit l’apparition d’une population de LT régulateurs de manière similaire aux CSM-GW. La co-culture des cellules NK en présence de CEL a induit une diminution du récepteur activateur NKG2D et a aussi induit un transfert du CD73 à leur surface, suggérant leur capacité à produire de l’adénosine, un puissant immunosuppresseur. Les CEL maintiennent le caractère immunoprivilégié et immunosuppresseur des CSM-GW, suggérant leur possible utilisation en ingénierie vasculaire / Umbilical cord Wharton’s jelly mesenchymal stem cells (WJ-MSC) are immune-privileged and immunosuppressive. Our study sought to determine the effect of endothelial differentiation on the immunomodulatory capacities of WJ-MSC. Endothelial-like cells (ECL) differentiation was performed by seeding MSC on polyelectrolyte multilayer films as substrate and stimulating them by EGM-2 culture medium. The expression of two immunological markers HLA-DR and CD86 was followed during the differentiation time. The effect of co-culture with ELC or MSC either in contact or separated by a transwell on different immune cells (peripheral blood mononuclear cells, T Lymphocytes (LT), Natural Killer (NK) cells) was assessed by evaluating immune cells proliferation. The results showed that ELC expressed endothelial markers, expressed low level of HLA-DR and CD86 and inhibited the proliferation of the different immune cell populations, and this inhibition is thought to be mediated by soluble factors as IDO, IL-6, IL-1β and PGE2, and by direct cellular contact. We reported also the capacity of ELC to generate a population of regulatory T cells and to decrease the expression of activating receptor NKG2D by NK cells. Moreover, we demonstrated that co-cultures with ELC induce CD73 expression on NK cells, a mechanism that may induce adenosine (a potent immunosuppressor) secretion by NK cells. Thus, ELC maintain the hypo-immunogenic and immunosuppressive characters of WJ-MSC suggesting their possible use in vascular engineering

Les cellules souches mésenchymateuses (CSM) de la moelle osseuse ont été intensivement étudiées pour leur capacité de régénération et leurs propriétés immunomodulatrices. Beaucoup d’études ont montré que la thérapie qui utilise les CSM améliore les fonctions de tissu ostéo-articulaire particulièrement le cartilage en vue de leur capacité de différenciation en chondrocytes. Les CSM présentent un certain nombre d'avantages pour la médecine régénérative, ces cellules peuvent être facilement isolées et multipliées en culture pour obtenir un nombre approprié pour la thérapie cellulaire. De plus, elles ont une faible immunogénicité, ce que les rende aptes à la transplantation allogénique. Depuis les années 1960, de nombreuses études ont souligné les propriétés médicinales des polysaccharides notamment les β-glucanes qui ont une place particulière du fait de leurs effets immunostimulants. L’objectif de notre travail était de mettre en évidence les capacités d’un β-glucane particulier, la laminarine, sur la prolifération et la différenciation des CSM dans la perspective d’applications dans l’arthrose. Les CSM ont été cultivés dans les milieux de croissance et de différenciation chondrocytaire. La viabilité et l'apoptose des cellules ont été explorées par le comptage, les tests MTT et la coloration à l'annexine V. En outre, l'analyse des protéines spécifiques de la prolifération a été effectuée par le western blott. De plus, l'expression des marqueurs spécifiques des CSM et des chondrocytes a été étudiée à l'aide de la RT-qPCR et de l’immunofluorescence. Nos résultats ont démontré que la stimulation des CSM à la laminarine avec la dose de 1 mg/ml soit en condition de culture de croissance basique ou en chondrogenèse a inhibé la prolifération des cellules sans induire leur apoptose. Encore, dans les conditions de culture chondrogénique, la laminarine à une dose similaire a empêché la différenciation des CSM en chondrocytes. / Mesenchymal stems cells (MSCs) are a population of multipotent cells residing in several readily available adult tissue compartments, thus allowing for their ex vivo expansion. MSCs have a reliable potential for differentiation (plasticity) into cells of the mesodermal lineage (chondrocytes, osteoblasts, adipocytes). Bone marrow-derived MSCs have been a focus of stem cell research in light of their relative ease of isolation and expansion and of their high potential for differentiation. Herein, the aim of the present PhD is to explore the potential of a β-glucan (laminarin) on Mesenchymal stem cell proliferation and differentiation for future benefit for osteoarthritis treatment. MSCs were cultured in MSC growth and chondrogenic differentiation mediums. Cells viability and apoptosis were explored by cell count, MTT assays and Annexin V staining. In addition, Analysis of the specific protein of cell proliferation was performed by western blott. Furthermore, mRNA and protein expression of specifics markers for MSCs and chondrocytes were studied using qPCR and immunofluorescence. Our results demonstrated that stimulation of MSC with laminarin at a dose of 1 mg/ml in either basic growth culture or chondrogenesis inhibited cell proliferation without inducing their apoptosis. Furthermore, under chondrogenic culture conditions, laminarin at a similar dose prevented the differentiation of MSC into chondrocytes.

Les lésions cartilagineuses survenant majoritairement dans un contexte de traumatisme, ne se réparent pas spontanément. Les traitements chirurgicaux et les techniques d'ingénierie cellulaire, utilisés en clinique, donnent des résultats perfectibles. L'ingénierie tissulaire du cartilage fait l'objet de nombreux travaux dans le but de produire au sein de la lésion un tissu de réparation dont les caractéristiques structurales et fonctionnelles sont similaires à celles du cartilage natif. Le diagnostic précoce de ces lésions et l'évaluation du tissu de réparation sont également des enjeux majeurs en orthopédie. L'IRM est une technique d'imagerie non invasive et à haute résolution permettant une évaluation du cartilage tant d'un point de vue architectural que biochimique en utilisant des séquences dédiées. En raison de leurs potentialités chondrogéniques, les cellules souches, provenant de différents tissus, sont une piste encourageante pour induire la régénération du cartilage lésé par des traumatismes articulaires. Des cellules provenant de différents tissus (moelle osseuse, membrane synoviale) ainsi que des chondrocytes dédifférenciés ont été ensemencés dans une structure tridimensionnelle poreuse à base de collagène I (éponge de collagène I) et soumis à un environnement chondrogénique afin de produire un implant fonctionnalisé. L'évaluation de la qualité de la synthèse matricielle in vitro dans l'implant a démontré le potentiel chondrogénique des différents contingents. La faisabilité d'un marquage de cellules souches mésenchymateuses (CSM) par des particules d'oxyde de fer superparamagnétiques (SPIO), diminuant le signal en IRM a été démontrée in vitro à 3 Teslas (3T) et 7 Teslas (7T). Des concentrations inférieures à 25 µg Fe/mL peuvent être utilisées sans endommager massivement la synthèse d'une matrice cartilagineuse par des CSM osté-médullaires. L'implantation des biomatériaux fonctionnalisés en site ectopique chez la souris nude a conduit à une dérive phénotypique ostéoïde de l'implant. En revanche, en site articulaire, chez le rat nude, les implants induisent la production d'un tissu de réparation comblant l'intégralité de la lésion et présentant des caractéristiques proche du cartilage sain environnant / Cartilaginous lesions mainly occur from a traumatic background and do not heal spontaneously. The chirurgical treatment and the cellular engineering techniques, usually used in clinic, produce perfectible results. Cartilage tissue engineering is the subject of many works in order to produce a repair tissue into the lesion. This repair tissue aims to have the same structural an functional characteristics as the native cartilage. In orthopaedic field, the early diagnostic of chondral lesions and the evaluation of the repair tissue are major issues. MRI is a high resolution and non invasive imaging technique that could be used to evaluate the architectural and biochemical structures of the cartilage by using dedicated sequences. Because of their chondrogenic capacities, stem cells provide a promising avenue to regenerate damaged cartilage in articular traumas. The stem cells from various origins (bone marrow, synovium) and the dedifferentiated chondrocytes were seeded into a porous 3D scaffold in collagen I (collagen I sponge). These cells were cultivated in chondrogenic conditions to produce a functionalized implant. The quality of the matrix synthesis was evaluated in vitro and demonstrated the chondrogenic potential of these various cell types. Superparamagnetic iron oxid particle (SPIO) labelling of the mesenchymal stem cells (MSC) is feasible in vitro at 3 Teslas (3T) and 7 Teslas (7T). A SPIO concentration lower than 25 ?g Fe/mL could be used without reducing the cartilaginous matrix synthesis by bone marrow MSC. The functionalized biomaterial implantation in an ectopic site in a nude mouse model showed an osseous split. However, in articular site in a nude rat model, the implants produced a repair tissue filling the totality of the lesion. This tissue seems to have similar characteristics of the surrounding healthy cartilage

Variation at regulatory elements, identified through hypersensitivity to digestion by Deoxyribonuclease I (DNase I), is believed to contribute to variation in complex traits, but the extent and consequences of this variation are poorly characterized. To investigate the relationship between sequence variation, and the functional consequences of variation in chromatin accessibility, genome-wide DNase I hypersensitive sites (DHS) of terminally differentiated erythroblasts were studied in eight inbred strains of mice studied (A/J, AKR/J, BALBc/J, C3H/HeJ, C57BL/6J, CBA/J, DBA/2J, and LP/J). These strains were selected because of the availability of their genome sequence and quantitative trait loci (QTL) data. After confirming that next generation sequencing could identify DNase I hypersensitive sites with high sensitivity and specificity, and that differential peaks could be found, an automated peak calling pipeline was developed and optimized. 36,693 DHS peaks were identified covering 9.1 Mb (0.29%) of mouse genome. There was no indication of within strain variation. Between strains reproducible variation was observed at approximately 5% of DNase hypersensitive sites (1,397 DHSs). Variable DHSs were more likely to be enhancers than promoters and less likely to occur at conserved regions of the genome. Only 36% of such variable DHSs contain a sequence variant predictive of site variation and 12% contain at least one variant that disrupts transcription factor binding sites. The majority (86%) of variable DHSs differ in size/shape and the remaining 14% demonstrate discrete variation in single peak or cluster of peaks. Sequence variants within variable DHS are more likely to be associated with complex traits than those in non-variant DHS, and variants associated with complex traits preferentially occur in enhancer-like elements. Changes at a small proportion (7%) of discretely variable DHS are associated with changes in nearby transcriptional activity. Our results show that whilst DNA sequence variation is not the major determinant of variation in open chromatin, where such variants exist they are likely to be causal for complex traits.

Advances in several areas of scientific research is currently hampered by the slow progress in developing a non-viral, high precision technique capable of safely and efficiently injecting targeted single cells with impermeable molecules. To date, one of the most promising techniques employs the laser to temporarily create a pore in the cell membrane to allow the entry of exogenous molecules. This technique has potentially wide applications. In this thesis, I utilised the precision of laser transfection, also known as optoporation, to deliver two histone demethylase inhibitors (8-hydroxyquinoline and FMF1293) of the JmjC-domain protein JMJD3 into vital cells. The enzyme, JMJD3, demethylates histone H3 lysine K27, the methylation state of which has been shown in previous studies to regulate genes in such a way as to play a key role in the formation of tumours and even maintenance of stem cell pluripotency. The research here shows proof of principle that optoporation can be employed to quickly screen and test the efficacy of novel drugs by delivering them into cells at significantly low concentrations while still maintaining inhibition activity. I also used optoporation to deliver relatively large proteins such as bovine serum albumin (BSA), phalloidin and novel synthetic antibodies into living cells without fixatives. This offers the possibility of using reporter systems to monitor living cells over time. Finally, an attempt was made to generate iPS colonies by optoporating plasmid DNA into somatic cells, however, I find that this technique was unable to efficiently transfect and reprogram primary cells. Two automated image-based systems that can be integrated into existing microscopes are presented here. First, an image processing algorithm that can quickly identify stem cell colonies non-invasively was implemented. When tested, the algorithm’s resulting specificity was excellent (95 – 98.5%). Second, because optoporation is a manual and time consuming procedure, an algorithm to automate optoporation by using image processing to locate the position of cells was developed. To my knowledge, this is the first publication of a system which automates optoporation of human fibroblasts in this way.

L'utilisation grandissante des cellules souches mésenchymateuses (CSM) en ingénierie tissulaire augmente la nécessité d'améliorer leur expansion. Ces travaux ont concerné l'étude d'un procédé performant d'expansion de CSM porcines en mode agité. Tout d'abord, un milieu de culture a été adapté aux CSM porcines multipotentes. Puis, différents modes d'expansion en conditions agitées ont été évalués avec les cellules fixées sur des microporteurs. La culture sur le microporteur Cytodex 1 a permis d'atteindre une vitesse spécifique de croissance de 0,54 j-1, supérieure à celle observée en flacon statique (0,31 j-1), avec les mêmes conditions de culture. En parallèle, une méthode de comptage innovante a été proposée pour le dénombrement automatique des cellules cultivées sur Cytodex 1, sans passer par une étape de trypsination. Enfin, les conditions opératoires du procédé d'expansion ont été étudiées. En comparaison d'une culture de CSM sur Cytodex 1 sans agitation, une agrégation des cellules et une baisse apparente de la concentration cellulaire ont été observées à 25 et 75 rpm. Par ailleurs, l'ajout de microporteurs au cours d'une culture de 300 h, réalisée dans un système de culture agité à 25 rpm et dans un volume de 200 mL, a permis de prolonger la prolifération cellulaire en évitant l'agrégation tout en maintenant la multipotence des CSM. Une concentration cellulaire de 3 x 105 cellules/mL a été obtenue, au lieu de 1,2 x 105 cellules/mL en flacons statiques avec les mêmes conditions de culture. Un procédé performant d'expansion de CSM porcines en conditions agitées a ainsi pu être proposé / The extensive use of mesenchymal stem cells (MSC) in tissue engineering increases the necessity to improve the expansion performance. This work aimed at studying an efficient expansion process for porcine MSC in agitated mode. First, a culture medium was adapted to the multipotent porcine MSC. Then, various expansion modes and agitation conditions were evaluated with the cells fixed on microcarriers. Cultures on the Cytodex 1 microcarrier enabled to reach a specific growth rate of 0.54 d-1, which was higher than the one observed in static T-flasks (0.31 d-1), with the same culture conditions. In parallel, an innovative counting method was proposed for the automatic enumeration of cells cultivated on Cytodex 1, without passing by a trypsination step. Finally, the operating conditions of the expansion process were studied. Compared to a culture of MSC on non-agitated Cytodex 1 microcarriers, cell aggregation occurred and an apparent decrease in the cell concentration was observed at an agitation rate of 25 and 75 rpm. Moreover, the addition of microcarriers during a 300 h culture, performed in an agitated culture at 25 rpm and in a volume of 200 mL enabled to prolong the cell proliferation without any aggregation, while maintaining the multipotency of the cells. A cell concentration of 3 x 105 cells/mL was obtained, instead of the 1.2 x 105 cells/mL in static flasks with the same culture conditions. An efficient expansion process for porcine MSC under agitated conditions has therefore been proposed