Au cours de la préimplantation, l'embryon de souris produit deux lignages cellulaires, le trophectoderme (TE), et la masse cellulaire interne (MCI) qui elle-même se différencie en épiblaste (Epi) et en endoderme primitif (EPr), caractérisés respectivement par l'expression exclusive de Nanog et de Gata6. La voie FGF/MAPK joue un rôle critique dans l’acquisition de l’identité EPr. J’ai examiné l’expression de pERK, DUSP4 et ETV5 qui permettent de visualiser l'activité des MAPK. Ces analyses ont été effectuées en activant ou inhibant la voie FGF/MAPK, ainsi que dans des embryons mutants pour Nanog et/ou Gata6. Ceci a permis d’observer l’activation de la voie FGF/MAPK dès E3,25. Un autre volet de mon travail a été d'analyser la voie de l’IGF dans les embryons préimplantatoires afin de comprendre l’influence de cette voie dans les différents lignages. J’ai montré que le récepteur activé pIGF1R est exprimé de manière différentielle dans le TE, l’EPr et l’Epi au cours du développement. Une supplémentation d’IGF1 induit une augmentation du nombre de cellules en deux phases, d'abord de l’Epi puis de l’EPr. A l’inverse, une perte de fonction d’IGF1R induit une diminution du nombre de cellules entre E3,75 et E4,25. / During preimplantation, mouse embryo produces two cellular lineages, the trophectoderm (TE), and the inner cell mass (ICM), which differentiates in epiblast (Epi) and primitive endoderm (PrE), characterized respectively by the complementary expression of Nanog and Gata6. FGF/MAPK pathway plays a critical role in the acquisition of a PrE identity. I examined the expression of the markers of MAPK activity pERK, DUSP4 and ETV5. The analyze was performed with activation or inhibition of FGF/MAPK pathway and in mutant embryos for Nanog or Gata6. This showed that FGF/MAPK pathway is activated as soon as E3,25. I have also analyzed the IGF pathway in preimplantation embryos in order to understand the role of this pathway in embryonic lineages. I showed that active receptor pIGF1R is differentially expressed in TE, PrE and Epi during embryonic development. Supplementation with IGF1 induces an increase in cell number in two phases, first in Epi then in PrE. Conversely, loss of function of IGF1R induces a decrease in cell number between E3,75 and E4,25.

Linker histone H1 plays a key role in facilitating folding of higher order chromatin structure. Previous studies have shown that deletion of three somatic H1 subtypes together leads to embryonic lethality and that H1c/H1d/H1e triple knockout (TKO) embryonic stem cells (ESCs) display bulk chromatin decompaction. Following this initial work, we investigated the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, as well as the regulation of Hox genes expression. We find that H1 TKO ESCs are more resistant to spontaneous differentiation, impaired in embryoid body differentiation, and largely blocked in neural differentiation. We present evidence that H1 contributes to efficient repression of the expression of pluripotency factors, Oct4 and Nanog, and participates in establishment and maintenance of DNA methylation and histone modification necessary for silencing pluripotency genes during stem cell differentiation and embryogenesis. In addition, we find reduced expression of a distinct set of Hox genes in embryos and ESCs, respectively. Furthermore, by characterizing H1c−/−; H1d−/−; and H1e−/− single-H1 null ESCs established in this study, we showed that individual H1 subtypes regulated specific Hox genes in ESCs. Finally, we demonstrate that the levels of H3K4me3 were significantly diminished at the affected Hox genes in H1 TKO- and single-H1 KO- ESCs, whereas H3K27me3 occupancy is modestly increased at specific Hox genes. Our results suggest that marked reduction of H1 levels and decondensation of bulk chromatin affect the expression of pluripotency genes and Hox genes in embryos and ESCs, which may be in part mediated through establishment and maintenance of epigenetic marks.

Embryonic stem (ES) cell pluripotency is sustained by a network of transcription factors centred on Oct4, Sox2 and Nanog. Whilst Oct4 and Sox2 expression is relatively uniform, ES cells fluctuate between states of high Nanog expression possessing high self-renewal efficiency, and low Nanog expression exhibiting increased differentiation propensity. Moreover, modulation in the level of Nanog expression determines the efficiency of ES cell self-renewal. To identify genes regulated by Nanog, genome-wide transcriptional profiling was performed on ES cells expressing different Nanog levels and Nanog-null ES cells expressing a Nanog-ERT2 fusion protein in which nuclear Nanog activity can be regulated by tamoxifen. Surprisingly, only a minor fraction of the genes to which Nanog binds showed significant changes in response to Nanog induction. Prominent amongst Nanog-responsive genes is Estrogen-related receptor b (Esrrb). Nanog binds directly to Esrrb, enhances binding and pause-release of RNAPolII from the Esrrb promoter and stimulates Esrrb transcription. Consistent with these findings, elevation of Nanog produces a cell population that expresses uniformly high Esrrb levels. Moreover, double fluorescent reporter lines show that Esrrb and Nanog levels are strongly correlated in individual cells. Loss of Nanog is required for downregulation of Esrrb, which coincides with commitment to differentiate. Esrrb overexpression results in LIF independent self-renewal, and blocks neural differentiation, even in the absence of Nanog. Cell fusion experiments between ES and neural stem (NS) cells show that elevated Esrrb levels allow the reprogramming of the NS cell genome in the absence of Nanog. Esrrb can rescue stalled reprogramming during the derivation of Nanog-/- induced pluripotent stem (iPS) cells. Moreover, targeted knock-in of Esrrb at the Nanog locus rescues the ability of Nanog null ES cells to maintain germ cell development beyond E12. Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent selfrenewal to ES cells. Together these data identify Esrrb as a critical downstream mediator of Nanog function.

Les gliomes sont les tumeurs cérébrales primitives les plus fréquentes de l’adulte. Le glioblastome (grade IV) en est la forme la plus agressive, caractérisé par sa résistance aux traitements actuels (chirurgie, chimiothérapie et radiothérapie). La mortalité de cette pathologie est quasi constante (survie médiane de 15 mois), ce qui justifie l’importance de découvrir de nouvelles cibles thérapeutiques. Le challenge est d'arriver à identifier des marqueurs spécifiques pour proposer un schéma thérapeutique alignant des stratégies de thérapies ciblées qui vont améliorer la prise en charge clinique, la survie globale et la survie sans progression des patients atteints de ces pathologies. Deux axes sont au centre des recherches fondamentales, translationnelles et cliniques. Le premier axe se définit autour du développement de molécules inhibitrices des voies de signalisation et le second autour du concept de cellules souches tumorales (CST) de glioblastomes (GBM) découvertes récemment dans le cerveau et qui révolutionnent la conception de la transformation tumorale.ASPM (Abnormal Spindle Like Microcéphaly Associated) est une cible candidate pertinente susceptible de participer au développement des gliomes (Horvath et al., 2007 ; Hagmann et al., 2008). Cette protéine régule la prolifération des neuroblastes, elle est fortement exprimée au stade embryonnaire, mais, reste faiblement exprimée dans le cerveau adulte. Par ailleurs, ASPM est impliquée dans divers processus de cancérisation (surexprimée dans les cancers du sein, du foie et du cerveau…), toute fois, le mécanisme responsable de cette dérégulation n’est pas encore bien caractérisé.Nos études menées sur une série de 169 gliomes humains, sélectionnés à partir de notre cohorte de patients, montrent que le gène ASPM est un marqueur de la progression vers la malignité, les grades les plus élevés exprimant le plus fortement ASPM. En outre, nous avons également montré que le niveau des transcrits d’ASPM est augmenté dans les récidives de gliomes et qu’en in vitro, ASPM contrôle la formation des gliomasphères (CST de GBM) avec une augmentation de l’expression de ses transcrits dans les cultures in vitro au fil des passages. En continuité de ces observations, nous avons alors développé un sh-miR-RNA spécifique d’ASPM permettant l’extinction post-transcriptionnelle de ce gène. Les résultats obtenus in vitro montrent que la perte d’expression d’ASPM conduit à un arrêt de la prolifération et aboutit à une mort cellulaire massive.Actuellement, des modèles de greffe de gliomasphères chez la souris (orthotopique) sont en cours de développement pour confirmer les effets observés in vitro et vérifier in vivo la validité de notre approche thérapeutique. En perspective, nous tenterons d’étudier les effets du silencing d’ASPM sur la voie de signalisation la plus dérégulée (pRB / E2F ou PI3K / AKT). Enfin, nous étudierons le rôle potentiel de cette protéine dans le contrôle du cycle cellulaire, et, in fine la mise en évidence de ses partenaires… / Glioblastoma (GBM) is the most frequent and aggressive form of primary brain tumors in adults; it is characterized by its resistance to current treatments (surgery, chemotherapy and radiotherapy). The prognosis is grim with a median survival of only 15 months underlining the importance to develop new therapeutic strategies. The recent development of the “tumor stem cell” (TSC) concept in hemopathies has been secondarily applied to gliomas with the identification of subpopulations of GBM cells which express neural stem cell markers and fulfill the criteria for stemness. Some evidences also suggest that this subpopulation could play a primary role in resistance to radio- and chemotherapy.ASPM (Abnormal Spindle Like Microcephaly Associated) is a protein regulating the proliferation of neuroblasts, highly expressed in the embryonic stage but weakly expressed in the adult brain. Preliminary reports suggesting that it could be involved in the development of gliomas (Horvath et al., 2007, Hagemann et al., 2008) prompted us to analyze further the role of this protein, focusing on its potential as a relevant candidate therapeutic target. In a series of 175 gliomas samples of various grades, we found that ASPM mRNA expression was strongly correlated with increasing tumor grade. We also found that ASPM expression increased at recurrence when compared to the initial lesion. Subsequently, we could demonstrate in vitro and in vivo that ASPM expression also increased over serial passages in gliomaspheres and in a mouse glioma xenograft model. In a therapeutic perspective, the effect of lentivirus-mediated shRNA post-transcriptional silencing of ASPM was evaluated in two different gliomasphere models and a dramatic proliferation arrest and cell death was observed. Taken together, these data suggest that ASPM is involved in the malignant progression of gliomas, possibly through expansion of a cancer stem cell compartment, and could be an attractive therapeutic target in glioblastoma multiforme.Another potential candidate tumor stem cell target in glioma is the sonic hedgehog pathway (hedgehog-Gli) which is required for GBM growth and stem cell expansion. In a collaborative study, it was found that NANOG, a transcription factor critically involved with self-renewal of undifferentiated embryonic stem cells, modulates gliomasphere clonogenicity, CD133+ stem cell behavior and proliferation. NANOG was regulated by hedgehog-Gli signalling and was essential for GBM tumourigenicity in orthotopic xenografts suggesting that it could also be a useful potential therapeutic target.Conclusions: Accumulating evidences suggest that tumor stem cells play an important role in the oncogenesis of gliomas and in their resistance to treatment. Our data support this concept and suggest that specific stemness markers may become useful targets to improve treatment of this devastating disease.

Pluripotent embryonic stem cells (ESCs) possess an unlimited capacity for self-renewal. This property of ES cells is both defining and unique. Harnessing this potential of ESCs would provide tremendous opportunity in the field of regenerative medicine and its attempts to combat degenerative diseases such as Parkinson’s, muscular dystrophy, etc. In 2006, Shinya Yamanaka was able to demonstrate that the ectopic expression of four proteins could reverse the process of differentiation and provide somatic cells with the characteristics ESCs. One year later, James Thompson’s group proved the same feat could be accomplished in human somatic cells using a different set of four proteins, including Nanog. The prospect of converting one’s own cells into a stem cell which could subsequently differentiate and repopulate an area of the body afflicted by gross degeneration was revolutionary. In the years following Yamanaka’s and Thompson’s discoveries, however, there has been little insight gained into how these proteins are regulated post-translationally. In this study, four proteins which had previously been identified by Yamanaka as being ‘pluripotency factors’ were used as baits in order to ascertain a protein-protein interaction network. This network was subsequently interrogated using various chemical compounds and small molecules in order to dissect the signal transduction pathways feeding into pluripotency, as well as, post-translational modifications regulating the factors themselves. In this way, the chemical inhibitor H89 was found to decrease the presence of Nanog phosphorylation and possibly its dimerization resulting in the Nanog protein being destabilized and targeted for degradation. Inversely, the pan-cullin inhibitor MLN4924 was identified to increase the abundance of both phosphorylated Nanog and total Nanog protein. In an attempt to identify the Cullin Ring Ligase (CRL) responsible for the degradation of Nanog protein in ESCs, each cullin identified in the protein interaction network was inhibited using specific shRNAs. Quantitative fluorescence microscopy was performed and identified that inhibition of CUL3 increases Nanog protein levels, suggesting that a CUL3-based CRL may be responsible for the post-translation regulation of Nanog. Additionally, the quantitation of Sox2 protein levels in CUL4B shRNA cell line demonstrates that Sox2 protein levels may be regulated by a CUL4B-based CRL. Further studies will reveal whether or not CUL4A depletion also results in elevated Sox2 protein levels. If not, this would include the pluripotency factor Sox2 among the recently identified CUL4B-isoform-specific substrates for degradation and possibly provide the basis for a hypothesis of developmentally regulated substrate specificity. In addition to MLN4924, several other small molecules were identified as being able to increase phospho-Nanog protein levels in this study. Among them were the cell permeable peptides Ht-31 and PKI (14-22) amide. These peptides were found to both stabilize phospho-Nanog and produce ES cell colonies that uniformly express the Nanog protein. The development of a growth medium containing these peptides in order to maintain homogeneous pluripotent ES cells is currently in progress and received backing for a patent application by the University of Edinburgh on February 23, 2012.

Made available in DSpace on 2014-12-17T14:13:52Z (GMT). No. of bitstreams: 1 JailmaAL_TESE.pdf: 4285605 bytes, checksum: 24785ef0d4160d79d6624877783e325f (MD5) Previous issue date: 2014-03-21 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Fucanas s?o polissacar?deos sulfatados encontrados em algas marrons e equinodermos. Tem sido demonstrado que uma fucana denominada de fucana A, obtida da alga marrom Spatoglossum schr?ederi, apresenta uma s?rie de efeitos biol?gicos, em particular, a atividade antitumoral. Com intuito de se potencializar essa atividade, foram adicionados grupamentos ti?is a estrutura da fucana A. Posteriormente, os nanog?is foram sintetizados pela forma??o de nanocomplexos entre a fucana A tiolada e o polietileno glicol (PEG) em v?rias rela??es 2.5, 5.0, 10, 15 e 30. Os nanog?is com as rela??es de 10 e 15 (FucA:PEG10 e FucA:PEG15) foram os que se apresentaram com os menores tamanhos, mais esf?ricos, com di?metro em torno de 186,95 ? 10,62 nm e carga de superf?cie ligeiramente negativa. Ap?s a s?ntese dos nanog?is, estes foram submetidos aos ensaios antiproliferativos com c?lulas da linhagem tumoral 786-0 nas concentra??es 8,0 a 64 μg/mL. As c?lulas foram analisadas durante um per?odo de 24, 48 e 72 horas. Os dados mostraram que em todas as concentra??es de nanog?is de fucana A, a atividade antiproliferativa foi tempo e dose dependente, o mesmo n?o sendo observado para a fucana A avaliada isoladamente. O nanogel de FucA:PEG15 tamb?m induziu apoptose por mecanismos dependentes e independentes de caspases. Posteriormente, FucA:PEG15 tamb?m foi marcado com FITC sendo completamente incorporado pelas c?lulas 786-0 ap?s 1 hora. Quando a endocitose celular foi parada, o FucA:PEG15 teve o seu efeito antiproliferativo reduzido. Apesar de FucA:PEG15 n?o possuir efeito anticoagulante por aPTT e PT (at? 100 μg/mL), ele apresesentou efeito antioxidante e angiog?nico. Esses dados mostram que o nanogel de fucana A exibe v?rias efeitos (antiproliferativa, antioxidante e antiangiog?nica) e, portanto, o seu potencial para a terapia do c?ncer deve ser investigada

A live birth from a somatic cell nuclear transfer (SCNT) embryo represents a small percentage of donor cells that survived the reprogramming gauntlet. The inability to reprogram histone modifications in the donor cell line could add to the reprogramming deficiencies associated with SCNT. The effects of two histone modifications associated with transcriptional activation (H3K4m3 and H4K16ac) and two histone modifications associated with repressing transcription (H3K9m2 and H3K27me3) were evaluated in the context of their association to three genes known to contribute to maintaining totipotency: Nanog, Oct4, and Sox2. A µChIP assay was utilized using antibodies specific for each histone modification followed by real time PCR (qPCR) analysis to quantify the percentage of each gene associated with each particular histone modification. Gene expression analysis was followed by immunofluorescence and protein analysis. Results of these analyses suggest that gene association to certain histone modifications did not accurately predict gene expression in bovine blastocyst embryos. Of the three genes studied, only Oct4 expression differed significantly between in vitro fertilized (IVF; control) and SCNT blastocysts. Protein levels detected through immunofluorescence correlated directly with the gene expression analysis. Nanog and Sox2 expression profiles of IVF and SCNT bovine blastocysts are similar, yet the histone modification profiles associated with all three genes differ significantly. Altered expression levels in developmentally important genes will likely result in abnormal activity of the associated cellular pathway. Aberrant histone modifications, along with abnormal Oct4 expression, may contribute to the low percentage of SCNT embryos that result in live offspring.

Matrin3 (MATR3) is an RNA binding protein involved in many roles in the nucleus, such as chromatin architecture and gene expression regulation, modulating transcriptional and post-transcriptional processes as RNA splicing and mRNA stabilization. Nevertheless, some functions of MATR3 within the cells are not entirely clear. MATR3 has been associated with Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that damages motor neuron (MN) cells and leads to progressive muscle paralysis and respiratory failure. A better understanding of MATR3 activity within cell physiology could represent an essential breakthrough for studying MATR3-associated pathologies. Using MATR3-silenced human pluripotent stem cell (hiPSC) line model, we collected data on the MATR3 role in the pluripotency and in the neural induction and differentiation. We found that the downregulation of MATR3 alters the expression level of crucial self-renewal factors such as OCT4, NANOG, KLF4, and LIN28A. We observed MATR3 acts at multiple levels of the gene expression, i.e. regulating YTHDF1 expression, and in RNA metabolism, having a role in mRNA stabilization and translation. The reduction of stemness potential caused by MATR3 downregulation creates a defect during the neurodifferentiation process, which does not arrest motor neurons formation but induces selective alterations that may affect motor neurons functionality. Indeed, several morphological and molecular abnormalities were observed during the neuronal differentiation, such as the alterations of the formation of neuroepithelial rosettes that arise in a reduction of neurite lengths and arborization in neuronal cells. On this basis, we investigated neuronal differentiation in the brain organoids grown from iPSCs derived from ALS patients fibroblasts. We show, for the first time, that MATR3 is a critical factor in orchestrating the stemness network through transcriptional, post-transcriptional, and translational regulation, therefore affecting the differentiation of mature neurons.

Cardiovascular diseases are among the leading causes of death worldwide and particularly in the developed World. The search for new therapeutic approaches for improving the functions of the damaged heart is therefore a critical endeavour. Myocardial infarction, which can lead to heart failure, is associated with irreversible loss of functional cardiomyocytes. The loss of cardiomyocytes poses a major difficulty for treating the damaged heart since terminally differentiated cardiomyocytes have very limited regeneration potential. Currently, the only effective treatment for severe heart failure is heart transplantation but this option is limited by the acute shortage of donor hearts. The high incidence of heart diseases and the scarcity donor hearts underline the urgent need to find alternative therapeutic approaches for treating cardiovascular diseases. Pluripotent embryonic stem (ES) cells can differentiate into functional cardiomyocytes. Therefore the engraftment of ES cell-derived functional cardiomyocytes or cardiac progenitor cells into the damaged heart to regenerate healthy myocardial tissues may be used to treat damaged hearts. Stem cell-based therapy therefore holds a great potential as a very attractive alternative to heart transplant for treating heart failure and other cardiovascular diseases. A major obstacle to the realisation of stem cell-based therapy is the lack of donor cells and this in turn is due to the fact that, currently, the molecular mechanisms or the regulatory signal transduction mechanisms that are responsible for mediating ES cell differentiation into cardiomyocytes are not well understood. Overcoming this huge scientific challenge is absolutely necessary before the use of stem cell-derived cardiomyocytes to treat the damaged heart can become a reality. Therefore the aim of this thesis was to investigate the signal transduction pathways that are involved in the differentiation of stem cells into cardiomyocytes. The first objective was the establishment and use of cardiomyocyte differentiation models using H9c2 cells and P19 stem cells to accomplish the specific objectives of the thesis. The specific objectives of the thesis were, the investigation of the roles of (i) nitric oxide (ii) protein kinase C (PKC), (iii) p38 mitogen-activated protein kinase (p38 MAPK) (vi) phosphoinositide 3-kinase (PI3K) and (vi) nuclear factor-kappa B (NF-kB) signalling pathways in the differentiation of stem cells to cardiomyocytes and, more importantly, to identify where possible any points of convergence and potential cross-talk between pathways that may be critical for differentiation to occur. P19 cells were routinely cultured in alpha minimal essential medium (α-MEM) supplemented with 100 units/ml penicillin /100 μg/ml streptomycin and 10% foetal bovine serum (FBS). P19 cell differentiation was initiated by culturing the cells in microbiological plates in medium containing 0.8 % DMSO to form embryoid bodies (EB). This was followed by transfer of EBs to cell culture grade dishes after four days. H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Differentiation was initiated by incubating the cells in medium containing 1% FBS. In both models, when drugs were employed, they were added to cells for one hour prior to initiating differentiation. Cell monolayers were monitored daily over a period of 12 or 14 days. H9c2 cells were monitored for morphological changes and P19 cells were monitored for beating cardiomyocytes. Lysates were generated in parallel for western blot analysis of changes in cardiac myosin heavy chain (MHC), ventricular myosin chain light chain 1(MLC-1v) or troponin I (cTnI) using specific monoclonal antibodies. H9c2 cells cultured in 1% serum underwent differentiation as shown by the timedependent formation of myotubes, accompanied by a parallel increase in expression of both MHC and MLC-1v. These changes were however not apparent until 4 to 6 days after growth arrest and increased with time, reaching a peak at day 12 to 14. P19 stem cells cultured in DMSO containing medium differentiated as shown by the timedependent appearance of beating cardiomyocytes and this was accompanied by the expression of cTnI. The differentiation of both P19 stem cells and H9c2 into cardiomyocytes was blocked by the PI3K inhibitor LY294002, PKC inhibitor BIM-I and the p38 MAPK inhibitor SB2035800. However when LY294002, BIM-I or SB2035800 were added after the initiation of DMSO-induced P19 stem cell differentiation, each inhibitor failed to block the cell differentiation into beating cardiomyocytes. The NF-kB activation inhibitor, CAPE, blocked H9c2 cell differentiation into cardiomyocytes. Fast nitric oxide releasing donors (SIN-1 and NOC-5) markedly delayed the onset of differentiation of H9c2 cells into cardiomyocytes while slow nitric oxide releasing donors (SNAP and NOC-18) were less effective in delaying the onset of differentiation or long term differentiation of H9c2 cells into cardiomyocytes. Akt (protein kinase B) is the key downstream target of PI3K. Our cross-talk data also showed that PKC inhibition and p38 MAPK inhibition respectively enhanced and reduced the activation of Akt, as determined by the phosphorylation of Akt at serine residue 473. In conclusion, PKC, PI3K, p38 MAPK and NF-kB are relevant for the differentiation of stem cells into cardiomyocytes. Our data also show that the PKC, PI3K and p38 MAPK signalling pathways are activated as very early events during the differentiation of stem cells into cardiomyocytes. Our data also suggest that PKC may negatively regulate Akt activation while p38 MAPK inhibition inhibits Akt activation. Our fast NO releasing donor data suggest that nitric oxide may negatively regulate H9c2 cell differentiation.