Cell fate decisions incorporate distinct and overlapping mechanisms. The activity of caspase 3 was initially understood to be a cell death restricted event, however numerous studies have implicated this enzyme in the regulation of both differentiation and proliferation. How the activity of caspase 3 promotes a non-death cell fate remains unclear. Here we examine the role caspase 3 activity plays during skeletal muscle differentiation; in particular we explore the hypothesis that the mechanism of inducing DNA strand breaks during cell death is also a key feature of differentiation, albeit with a distinctly different outcome. We delineate the transient formation of Caspase 3/Caspase activated DNase (CAD) dependent DNA strand breaks during differentiation. The formation of these breaks is essential for differentiation and the regulation of specific genes. In particular expression of the cell cycle inhibitor p21 is related to the formation of a DNA strand break within the gene’s promoter element. Further, we explored the genome wide association of CAD using Chromatin Immunoprecipitation coupled to high through put sequencing (ChIP-seq). This approach identified a potential role for Caspase3/CAD in regulating the expression of Pax7. Here, a CAD directed DNA strand break in the Pax7 gene is correlated with decreased Pax7 expression, an outcome that has been shown to be critical for progress of the myogenic differentiation program. The regulation of Pax7 expression through a CAD induced DNA strand break raises an intriguing connection between this regulation and oncogenic transformation observed in alveolar rhabdomyosarcoma. The putative site of CAD induced DNA strand breaks that promote decreased Pax7 expression during differentiation corresponds to site of chromosomal translocations responsible for Pax7 fusion events in alveolar rhabdomyosarcoma.

In recent years, the degradation and subsequent loss of tissues is an issue that has affected people worldwide. Although there are treatments addressing the degradation of tissues, such treatments involve complicated and expensive procedures, where full tissue regeneration is not achieved. For these reasons, in recent years, tissue engineering has developed cutting-edge biomaterials capable of inducing effective tissue regeneration both under cellular or acellular conditions. Peptide hydrogels are versatile biomaterials composed of the basic components of life amino acids, which act as building blocks to form hierarchical structures, which subsequently go on to form well-defined scaffolds. Biomaterials have been widely used for the culture of mammalian cells, tissue engineering, regenerative medicine, drug delivery, etc. This is thanks to their capability of providing a three-dimensional architecture to cells, which mimics the natural architecture of the extracellular matrix (ECM). Peptide- based hydrogels can be easily functionalised with active biological cues, which can direct the cellular response. It has been shown that the ionic-complementary FEFEFKFK hydrogel, succeeded to support the culture of mammalian cells such as bovine chondrocytes. In this work, we used the same FEFEFKFK hydrogel to investigate the capability of this hydrogel to support the three-dimensional culture of both human osteoblasts (hOBs), and human mesenchymal stem cells (hMSCs) for bone regeneration applications. To achieve this goal, hOBs were cultured within both FEFEFKFK (non-functionalised) and RGD-FEFEFKFK (functionalised) gels. Then the suitability of the FEFEFKFK gels to induce cellular proliferation, synthesis of bone ECM and mineralisation was explored. In addition, taking advantage of the inherent plasticity of hMSCs, we also investigated the capability of the FEFEFKFK gel to foster the osteogenic differentiation of hMSCs, and subsequently to induce bone mineralisation in 3-D under osteogenic stimulation. Based on the results obtained in this work, the FEFEFKFK gel arises as a promising biomaterial for both bone and dental tissue regeneration applications.

Les protéines du groupe Polycomb (PcG) ont initialement été identifiées chez la Drosophile, en tant que facteurs nécessaires au maintien de l’expression spatio-temporelle de gènes homéotiques le long de l’axe antéro-postérieur. Depuis, leur rôle en tant que régulateurs du développement a été mis en évidence chez la plupart des métazoaires ainsi que chez les plantes, chez lesquelles elles orchestrent les transitions développementales, l’organogenèse et la différenciation cellulaire. Les protéines PcG sont nécessaires au maintien de la répression transcriptionnelle de gènes cibles, par la régulation de leur structure chromatinienne via des modifications post-traductionnelles des histones. Elles forment des complexes multiprotéiques, notamment les Complexes Répressifs Polycomb PRC1 et PRC2. PRC2 est responsable de la tri-méthylation de la lysine 27 de l’histone H3 (H3K27me3) et est constitué de 4 sous-unités principales qui, pour la plupart, sont présentes sous forme de familles multigéniques dans le génome d’Arabidopsis thaliana. Ainsi, il existe plusieurs complexes PRC2 constitués de combinaisons alternatives de ces sous-unités, qui sont potentiellement présents au sein d’une même cellule et dont les rôles sont considérés comme partiellement redondants. En utilisant des approches moléculaires, génétiques et génomiques, nous avons analysé le rôle des sous-unités PRC2 exprimées dans la pointe racinaire d’Arabidopsis. Nous avons montré que l’interaction entre différents PRC2 est nécessaire pour réguler l’activité du méristème, le déroulement temporel de la différenciation cellulaire, ainsi que pour le maintien de l’identité des cellules matures. De plus, notre travail montre que les complexes PRC2 contenant l’une ou l’autre des deux méthyltransférases, CLF et SWN, régulent des groupes de gènes communs ainsi que distincts, à travers des mécanismes différents incluant une fonction non-canonique. Par ailleurs, nos résultats indiquent que les différences fonctionnelles entre CLF-PRC2 et SWN-PRC2 reposent, au moins en partie, sur les sous-unités non-catalytiques avec lesquelles la méthyltransférase interagit. Pour identifier les gènes régulés dynamiquement par PRC2 durant la différenciation cellulaire, nous avons développé des approches permettant d’accéder à la résolution des types cellulaires afin d’analyser les états chromatiniens à l’intérieur de la niche de cellules souches et de la zone de maturation de la racine. Nos données suggèrent que PRC2 participe au maintien de l’identité du Centre Quiescent (QC) en réprimant des voies de signalisations spécifiques. De plus, la différenciation cellulaire en direction de la zone de maturation est accompagnée par un accroissement du répertoire des cibles PRC2, incluant des régulateurs méristématiques ainsi que des gènes spécifiquement exprimés dans différents types cellulaires. Enfin, nos résultats suggèrent qu’une proportion significative des cibles PRC2 sont présentes sous la forme de domaines bivalents H3K27me3-H3K4me3 dans les cellules souches végétales, cette proportion étant moins importante que celle décrite chez les cellules souches embryonnaires de mammifères. Dans l’ensemble, ce travail apporte une vue intégrée de la fonction, la dynamique et la multiplicité de l’activité PRC2 au cours du processus de différenciation cellulaire, dans le contexte d’un organe en développement. Nos résultats mettent en évidence le rôle de PRC2 en tant que régulateur majeur de la différenciation cellulaire, qui apporte à la fois robustesse et plasticité aux programmes transcriptionnels qui sous-tendent l’acquisition spatio-temporelle et le maintien de l’identité cellulaire. / The Polycomb group (PcG) proteins were originally identified in Drosophila as factors required for maintaining the spatio-temporal expression of homeotic genes along the head-to-tail axis. Since then, their role as developmental regulators has been highlighted in most metazoans as well as plants, in which they orchestrate developmental transitions, organogenesis and cell differentiation. PcG proteins are required to maintain the transcriptional repression of target genes by regulating their chromatin structure via post-translational histone modifications. They are found in multiprotein complexes, including Polycomb Repressive Complexes PRC1 and PRC2. PRC2 is responsible for the trimethylation of histone H3 at lysine 27 (H3K27me3) and consists of four core subunits, most of which are represented by multigene families in Arabidopsis thaliana. Thus, distinct PRC2 complexes formed by alternative subunit combinations exist, possibly in the same cell, and are thought to play partly overlapping roles. By combining molecular, genetic and genomic approaches, we have analyzed the role of the PRC2 subunits expressed in the Arabidopsis root tip used as a model. We show that the interplay between distinct PRC2s is necessary to regulate the activity of the meristem and the timing of cell differentiation, as well as the maintenance of cell identity. In addition, our work reveals that PRC2 complexes containing either of the two related methyltransferases CLF or SWN regulate common as well as specific sets of genes through distinct mechanisms, including a non-canonical function. Furthermore, our results indicate that the functional differences between CLF-PRC2 and SWN-PRC2 rely, at least in part, on the non-catalytic subunit they are interacting with. To identify the genes dynamically regulated by PRC2 during cell differentiation, we have developed cell type-specific approaches to analyze chromatin marks in cell populations within the stem cell niche and the maturation zone of the root. Our data suggest that PRC2 participates in the maintenance of the quiescent center (QC) identity by repressing specific signaling pathways. In addition, cell differentiation towards the maturation zone is accompanied by an increase of the repertoire of PRC2 targets including stem cell and meristem regulators, as well as cell type-specific genes. Finally, our findings suggest that bivalent H3K27me3-H3K4me3 domains in the QC represent a significant, though smaller proportion of PRC2 targets in plant stem cells compared to what has been described in mammalian embryonic stem cells. Overall, this work provides an integrated view of the function, dynamics and multiplicity of PRC2 activity during the cell differentiation process, in the context of a developing organ. Our results highlight the role of PRC2s as major regulators of cell differentiation that provide both robustness and plasticity to the transcriptional programs underlying cell fate acquisition and identity maintenance.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22830号 / 医博第4669号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 江藤 浩之, 教授 武藤 学, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23383号 / 医博第4752号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 髙折 晃史, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les gliomes sont les tumeurs cérébrales les plus fréquentes chez l’adulte. Il s’agit d’un groupe de tumeurs hétérogène pouvant être classées dans différents sous-groupes selon des critères moléculaires et histopathologiques, donnant une indication sur leur agressivité. Les p21-activated kinase (PAK) sont des sérine-thréonine kinases effectrices des GTPases Rac et Cdc42 et font ainsi partie de nombreuses voies de signalisation. Elles régulent de nombreuses voies d’intérêt dans la biologie des cancers, comme les voies Mek/Erk, PI3K/Akt et Wnt/b-caténine. PAK1 est fréquemment surexprimé et/ou suractivé dans plusieurs cancers, notamment du sein, des ovaires de la prostate et du cerveau, alors que PAK3 induit la sortie du cycle cellulaire et la différenciation des cellules au cours de la neurogenèse dans plusieurs modèles animaux. Durant ma thèse, j’ai pu observer que l’expression de PAK3 était de bon pronostic pour les patients atteints de gliomes, et était plus élevée dans un sous-type de tumeurs caractérisé par la codélétion 1p/19q. In vitro, l’expression de PAK3 était plus élevée dans une lignée de cellules de gliome de signature proneurale, non tumorigène après xenogreffe chez la souris. Au cours de la différenciation des cellules de gliome, l’augmentation de l’expression de PAK3 est associée avec des marqueurs de lignage neural et neuronal. L’inhibition de l’expression de PAK3 entraîne l’augmentation de la capacité d’auto-renouvellement et de la tumorigénicité d’une lignée de cellules de gliome, et favorise la différenciation gliale des cellules. / Gliomas are the most common and lethal adult primary brain tumors. Their complex heterogeneity is evidenced by numerous genomic studies showing distinct molecular entities in glioma. P21-activated-kinases (PAK) are serine threonine kinases involved in multiple signal transduction pathways as downstream effectors of Rac and Cdc42. They regulate several key cancer-relevant pathways like cell division and movement. PAK1 and PAK3 are highly expressed in the brain; PAK1 is frequently overexpressed and/or over-activated in several human cancers whereas PAK3 is involved in neural differentiation and the developmental proneural pathway. The role of these two kinases in brain tumor pathophysiology is unknown. We have observed that PAK3 expression was associated with a longer survival for patients with glioma and was higher in 1p/19q gliomas. In vitro, PAK3 was highly expressed in a glioma cell line with a proneural signature that did not induce tumor after xenograft. Its increasing expression upon a set of differentiation paradigms was correlated with those of neural and/or neuronal markers in glioma cell lines. Inhibition of PAK3 expression increased cell renewal and tumorigenicity. It impaired cell differentiation, promoting the glial lineage.

Injury, aging, and congenital disabilities of the muscular and neural systems impose a significant burden on patients and their families. Due to the tissue’s limited regenerative capacity, effective treatment interventions for restoring progressive damage is still lacking. Cell replacement therapy is primarily limited by the restricted supply of viable donor cells and variable graft survival. For addressing these limitations, we propose new strategies to obtain a target cell of interest from an autologous cell source. Herein, we engineer cell fate decisions by 1) harnessing host microenvironment and the CRISPR/dCas9-mediated transcriptional activation system to promote myogenesis of human endothelial progenitor cells (EPCs); and 2) employing substrate-mediated biophysical cues with soluble factors (biochemical cues) to drive cell commitment to neuronal lineages. For the first strategy, we hypothesized that therapeutic cells could be obtained in situ by employing the CRISPR/dCas9 system to engineer cell fate in the host tissue. Using this system, we transactivated MYOD1, a master regulator for myogenesis, to directly reprogram primary EPCs to skeletal myoblasts (SkMs). EPCs were chosen as a cell source for their easy accessibility, high proliferation, and potential contribution to regenerate vasculature and musculature tissue. The early myogenic commitment of EPCs was confirmed in vitro by MYOD1 expression, which yielded a 230-fold higher induction than the original EPCs. These cells were then transplanted for assessing their therapeutic efficacy in myotoxin-induced muscle injury model in immunodeficient mice. A one-month post-injury study resulted in the integration of induced SkMs to the injured host tissue, promotion of neoangiogenesis, and reduction in fibrotic scar formation. These findings indicate that CRISPR/Cas9-mediated target gene activation can be achieved in situ to accelerate muscle regeneration after myotoxin-induced damage. For the second strategy, we utilized both soluble and insoluble factors to convert the cell fate of neural stem/progenitor and somatic cells to various neuronal lineages, including motor neurons (MNs) and dopaminergic (DA) neurons. For soluble factors, cells were exposed to various biochemical factors, inspired by the neuronal niche environs during the natural developmental process. For insoluble factors, the conductive graphene substrate was used to support the endogenous electrical signal between neurons for enhancing the neuronal phenotypes and their functionality. We postulated that exposing the cells to these collective stimuli in vitro can alter their intrinsic signaling pathway to tailor their fate to neuronal lineages. To test the hypothesis, neural stem/progenitor and somatic cells were cultured on various substrates with or without electroactive graphene and aligned patterns. After two weeks to one month of cell fate induction in the chemically defined conditions, our results implied that cell adhesion, survival, neurite outgrowth, and maturation were facilitated on the electroactive substrates with aligned patterns compared to the control platforms. Taken together, our results in this dissertation demonstrate the feasibility of tailoring the donor cell fates within or across the germ layers. We achieved this by employing a transcriptional gene activation system and tunable microenvironmental cues elicited by soluble (chemical and growth factors) and insoluble (physical cues from the substrate) factors. Utilizing such strategies hold great promise for elucidating the optimal conditions to guide cell fate to target lineages. This work provides a rational basis for establishing a robust protocol and an in vitro culture platform to module cell fate decisions that could help realize the autologous cell-based therapy for muscular and neurodegenerative diseases.

During regeneration, cells must coordinate proliferation and differentiation to rebuild tissues that are lost. Understanding how source cells execute the regeneration process has been a longstanding goal in regenerative biology with implications in wound healing and cell replacement therapies. Melanocytes are pigment-producing cells in the skin of vertebrates that can be lost during hair graying, injury and disease-related depigmentation. Melanoma is an aggressive skin cancer that develops from melanocytes, and it is hypothesized that melanoma cells have properties that are similar to melanocyte stem cells. To gain insight into melanocyte regeneration we set out to identify the source of regeneration melanocytes in adult zebrafish and the path through which progenitor cells reconstitute the pigment pattern. Using targeted cell ablation and single cell lineage-tracing analyses we identified that a majority of regeneration melanocytes arise through direct differentiation of mitfa-expressing progenitor cells. Concurrently, other mitfa-expressing cells divide symmetrically to generate additional mitfa-positive progenitors, thus maintaining regeneration capability. Using reporter assays and drug studies, we found that Wnt signaling gets turned on in progenitor cells during regeneration and Wnt inhibition after melanocyte ablation blocks regeneration. Based on our finding that Wnt signaling is active in differentiated melanocytes but not in the progenitor cells, we explored the role of Wnt signaling in tumor initiation. We found that approximately half of the melanomas are Wnt silent, and overexpression of dkk1b, a negative regulator of canonical Wnt signaling, accelerates melanoma onset. This work defines an unappreciated contribution by direct differentiation in melanocyte regeneration and suggests a broader role for this process in the maintenance of epithelial sheets. This study also identifies a shared pathway between melanocyte progenitors and melanoma cells, which could be applicable to other cancers.

Vascular smooth muscle cells (SMCs) regulate vessel contraction but during diseases including atherosclerosis, SMCs undergo functional changes that contribute to pathology. Chronic inflammation in the vasculature exacerbates disease progression. Acute phase serum amyloid A (SAA) is up-regulated during inflammation and expressed in atherosclerotic lesions. Previous work in our laboratory demonstrated that SAA activates secretory phospholipase A2 group IIA (sPLA2), whose products impact cellular cholesterol homeostasis. It was hypothesized that SAA promotes cholesterol trafficking from the plasma membrane to the endoplasmic reticulum (ER) in an sPLA2-dependent manner. SAA induced SMC cholesterol accumulation in the ER. Levels of plasma membrane cholesterol decreased, confirming that cholesterol moved from the plasma membrane to the ER. Another family member, (cytosolic phospholipase A2, group IV), was also required for SAA-induced sPLA2 activation and cholesterol mobilization. SAA activated neutral sphingomyelinase and blocking this activity inhibited cholesterol trafficking. These studies show that SAA activated sPLA2 which activated neutral sphingomyelinase. As a result, sphingomyelin was cleaved, which liberated cholesterol for movement to the ER. Additional studies demonstrated that SAA repressed expression of SMC contractile markers including Acta2 and Myh11. Toll-like receptor 2 (TLR2) is an SAA receptor implicated in atherogenesis and it was hypothesized that TLR2 plays a role in SAA-mediated phenotype/gene changes. The TLR2 ligands, FSL and Pam3CSK4, down-regulated SMC contractile marker expression. Knockdown of TLR2 demonstrated that SAA-mediated phenotype modulation was TLR2-dependent. SAA, FSL, and Pam3CSK4 also induced mRNA expression of pro-inflammatory and adhesion genes, changes inhibited by TLR2 knockdown. SAA repressed activity of the αSMA promoter, demonstrating transcriptional regulation. Myocardin, a transcription factor required to drive expression of SMC contractile genes, was down-regulated by SAA and FSL. Myocardin overexpression abrogated SAA- and FSL-mediated repression of the αSMA and SM22α promoters. These studies demonstrate that SAA promoted a phenotypic switch through activation of TLR2 and down-regulation of myocardin expression. Taken together, novel SAA- and TLR2-mediated mechanisms of cholesterol trafficking and phenotypic modulation in SMCs are shown. Importantly, this work uncovers previously unknown effects of TLR2 signaling on vascular SMCs and provides a context by which TLR2 activation and lesion-associated SAA may promote atherosclerosis. / 2017-12-01T00:00:00Z

Orientador: Gisele Faria / Resumo: As cistatinas são inibidores naturais de cisteíno proteases e desempenham um papel crítico no controle da degradação de proteínas. As cistatinas de plantas, dentre elas a cistatina produzidas de forma recombinante a partir da Citrus sinensis (CsinCPI-2), têm mostrado potencial para serem usadas em diferentes abordagens terapêuticas. Os objetivos deste estudo foram avaliar a citocompatibilidade e o efeito da fitocistatina CsinCPI-2 sobre a proliferação, migração e diferenciação osteogênica de células da polpa dental humana (hDPCs). Previamente à realização dos ensaios, as hDPCs foram caracterizadas quanto a expressão de marcadores de células tronco mesenquimais por meio de citometria de fluxo. hDPCs expostas à CsinCPI-2 e não expostas (controle) foram avaliadas quanto à viabilidade celular por meio dos ensaios de metiltiazol tetrazólio (MTT) e alamar blue, apoptose por meio de citometria de fluxo, atividade da fosfatase alcalina (ALP) por meio do cálculo da liberação de timolfitaleína, produção de nódulos mineralizados por meio de coloração com vermelho de alizarina, migração celular pelo ensaio de transwell, proliferação por meio de ensaio de incorporação de bromodeoxiuridina (BrdU) e expressão gênica de proteína morfogenética óssea 2 (BMP-2), fator de transcrição osteogênica (RUNX2), fosfatase alcalina (ALP), osteocalcina (OC), sialoproteína óssea (BSP) e proteína da matriz da dentina (DMP-1) por meio da reação da polimerase em cadeia em tempo real quantitativa (qPCR).Os dad... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cystatins are natural inhibitors of cysteine proteases and play a critical role in controlling protein degradation. Plant cystatins, among them a cystatins recombinantly produced from Citrus sinensis (CsinCPI-2), have shown potential to be used in different therapeutic approaches. The objectives of this study were to evaluate the cytocompatibility and effect of phytocystatin CsinCPI-2 on the proliferation, migration and osteogenic differentiation of human dental pulp cells (hDPCs). Previously to the performing the assays, hDPCs were characterized for the expression of mesenchymal stem cell markers by flow cytometry. hDPCs exposed to CsinCPI-2 and unexposed (control) were evaluated from cell viability by the methylthiazole tetrazolium (MTT) and alamar blue assays, apoptosis by flow cytometry, alkaline phosphatase (ALP) activity by calculation of thymolphtalein release, production of mineralized nodules by alizarin red staining, migration cells by Transwell assay, proliferation by bromodeoxyridine (BrdU) incorporation assay and gene expression of bone morphogenetic protein 2 (BMP-2), transcription factor (RUNX2), alkaline phosphatase (ALP), osteocalcin (OC), bone sialoprotein (BSP) and dentine matrix protein -1 (DMP-1) by quantitative real time PCR (qPCR). The data were analyzed by one-way analysis of variance (ANOVA) and Turkey post-test, two-way ANOVA and Bonferroni post-test or t-test (α = 0.05). hDPCs used in assays showed positive marking for mesenchymal stem cells (CD105,... (Complete abstract click electronic access below) / Doutor