Adult bone marrow contains a population of mesenchymal stem cells capable to self-renew and to differentiate into haematopoietic-supportive stroma, osteo, adipo- and chondrocytes. However, the identity of mesenchymal stem cells still remains uncertain. The complex population of their descendants, bone marrow mesenchymal stromal cells (BM MSCs), represents a model to study the principles of differentiation and commitment into mesodermal lineages. The experiments using BM MSCs are often hampered by their low proliferative capacity in vitro. In the present study, we established conditionally immortalized BM MSCs from tetracycline-regulated SV40 Large T-antigen transgenic mice. The identity of the conditionally immortalized BM MSCs was confirmed by marker expression, ability to support haematopoiesis and differentiation potential. The advantages of the conditional immortalization are encompassed in (1) indefinite expansion of cell populations, (2) possibility to perform cellular cloning and (3) prevention from spontaneous differentiation. We demonstrated the heterogeneity of BM MSCs and identified at least 6 types of progenitors within BM MSCs population based on their differentiation potential (“OAC”, “OA”, “OC”, “AC”, “O”, “A”). A hypothetical model of BM MSC hierarchy and the relationships between the progenitors has been proposed. We observed that the Wnt/β-catenin signaling pathway and GSK3 activity could modulate the efficiency of osteo- and adipogenic differentiation pathways, but we didn’t find evidence that the lineage commitment of BM MSCs is determined by Wnt. We elucidated the mechanism of transcriptional regulation of the adipogenic induction of BM MSCs in vitro. Our data revealed the key regulatory role of PPARγ1 during adipogenesis in BM MSCs. Furthermore, we assume that PPARγ1 is a potential trigger of the adipogenic commitment of the BM MSCs progenitors. Finally, the non-adipogenic BM MSCs progenitors were converted into the adipogenic lineage using ectopical expression of the transcription factors C/EBPα, C/EBPβ and C/EBPδ. Our findings provide a novel insight into the molecular mechanisms of BM MSCs lineage commitment.

La cécité d’origine cornéenne est Ia deuxième cause de cécité dans le monde. Son traitement de choix est la kératoplastie. ll est aujourd’hui nécessaire de réfléchir à de nouvelles solutions pour remplacer la kératoplastie dans sa forme actuelle qui bien que satisfaisante, n’est pas totalement parfaite. En effet, les techniques actuelles de kératoplastie utilisent une allogreffe. Le greffon va subir chez le donneur une diminution de la densité des cellules endothéliales (CE) du greffon au fil du temps. Ce travail présente successivement les solutions abordées par le BiiGC pour produire des CE en masse ainsi que les différents supports en vue d’une kératoplastie lamellaire postérieure. Les solutions envisagées par le laboratoire BiiGC doivent permettre un transfert à l’être humain dans des conditions de sécurité et d’acceptabilité maximales. Les différents contrôles de l’identité des cellules obtenues et les contrôles de sécurité nécessitent une connaissance robuste de la biologie, du morphotype et du phénotype des CE cornéennes. Cette thèse présente les différentes exigences réglementaires et de qualité nécessaires à l’obtention de greffons cornéens bioengineerés. Le choix du BiiGC devant se conformer d’emblée à ces exigences en vue d’un transfert à l’être humain. Dans une première partie, nous présentons une revue de la littérature sur le bioengineering de greffons cornéens. La production de CE et de stroma seront ensuite exposés avec les différentes approches abordées par le BiiGC. Enfin, les différentes techniques de validation morphologiques et fonctionnelles de greffons obtenus seront présentées. / Corneal diseases are the first leading cause of blindness worldwide. Its treatment relies on keratoplasty which in its actual form is a satisfying but not a perfect solution. We have to think about new solutions for corneal graft. Today, corneal graft is often an allograft. With this technique, there is a loss of endothelial cell (EC) density through time. This work presents the solutions which BiiGC laboratory is working on for mass production of EC and different carriers for these cells for posterior keratoplasty. These solutions must be possible on human being with safety conditions. Controls of identity, of obtained cells and security controls need a strong knowledge of biology, morphology and phenotype of EC. This work presents the legal conditions of quality needed to obtain bioengineered corneal grafts. The choice of BiiGC laboratory must involve these conditions for a transfer to human being. In a first part, we present a review of the literature about corneal bioengineering. Production of EC and of corneal stroma are discussed with different approaches by BiiGC laboratory. In the end, the different techniques of morphological controls of corneal grafts are discussed.

iii Abstract Glycobiology represents a very progressive subject of cell biology. Protein-saccharide interactions play not only supporting and cell organization role, but they also represent medium for information storage and its decoding. Galectins, group of animal lectins (saccharide-binding proteins), which have selective affinity to ß-galactosides, are multifactorial molecules. They participate in cell-cell and cell-matrix interaction, transmembrane signaling, apoptosis, pre- mRNA splicing and are also present in various types of carcinomas. High expression of galectin-1 has been detected in cancer stroma originated from squamous cell epithelium. In the previous study we established that the fibroblasts - myofibroblasts transition, apart from the known TGF- beta, is also induced by galectin-1. We compared relationship between galectin-1 expression, presence of myofibroblasts and gene expression in tissue samples from patients with head and neck squamous cell carcinoma. Cancer stroma with myofibroblasts was rich in galectin-1 expression in comparison with stroma without myofibroblasts. Moreover, we used microarray analysis (ILLUMINA) to compare the whole genome transcriptome from samples with and without presence of galectin-1. High expression of galectin-1 in tissue samples corresponded with expression...

Les fibroblastes activés qui composent les stromas tumoraux sont des acteurs majeurs des interactions tumeur-stroma impliquées dans la croissance et la dissémination des cellules tumorales. Ce processus d'activation des fibroblastes est caractérisé par l'expression de marqueurs protéiques spécifiques parmi lesquels figure l'alphaSMA et FAPalpha;. Le TGFbeta;, cytokine secrétée massivement par les cellules tumorales, est un des éléments impliqués dans l'activation des fibroblastes et la formation du stroma tumoral qui en résulte. L'EMMPRIN, glycoprotéine transmembranaire surexprimée dans les cellules tumorales est également un médiateur des interactions tumeur-stroma puisqu'il a la capacité d'induire la synthèse des MMPs par les fibroblastes péri-tumoraux accroissant ainsi la propagation des cellules tumorales à travers l'organisme. Nos travaux identifièrent que le TGFbeta secrété par les cellules tumorales induisait la synthèse du marqueur FAPalpha par les fibroblastes. L'EMMPRIN stromal apparaît comme récepteur de ces signaux tumoraux et est nécessaire à la synthèse du marqueur FAPalpha; par les fibroblastes. L'EMMPRIN participe donc à l'activation TGFbeta; dépendante des fibroblastes. Son inhibition dans ces cellules conduit à un dysfonctionnement de la signalisation médiée par les protéines Smad2/Smad3 aboutissant à une diminution de la synthèse du marqueur alphaSMA ainsi que de certaines protéines matricielles induites par le TGFbeta. L'étude du mécanisme d'action de l'EMMPRIN dans ce processus a permis d'identifier l'EMMPRIN comme nouvelle protéine chaperonne du récepteur de type I au TGFbeta;. / Tumor stroma activated fibroblasts are major actors of tumor stroma interactions taking to tumor growth and spreading. Activated fibroblasts are characterized by the expression of specific markers including alphaSMA and FAPalpha;. The TGFbeta;, a cytokine highly secreted by tumor cells, is one of the key factors involved in fibroblast activation and tumor stroma formation. EMMPRIN, a transmembrane glycoprotein overexpressed in tumor cells, is also a mediator of tumor-stroma interactions by its ability to induce the synthesis of MMPs by peri-tumor fibroblasts enhancing then tumor cells dissemination across the organism.Here, we demonstrate that TGFbeta; secreted by tumor cells is the tumor factor involved in the synthesis of FAPalpha; by fibroblasts. Stromal EMMPRIN appeared to be the receptor of these tumor-stroma interactions and is required for the synthesis of FAPalpha; by fibroblasts. EMMPRIN was also evidenced to take part in TGFbeta;-dependent fibroblast activation. Its inhibition in these cells correlate to a dysfunction in Smad2/Smad3 signaling leading to a decrease in the expression of alphaSMA and matrix proteins induced by TGFbeta;. The study of the mechanism used by EMMPRIN in this process evidenced this protein as a new chaperone for the type I TGFbeta; receptor.

La bioimpression assistée par laser (LAB) permet de positionner des gouttesde cellules avec une précision micrométrique. Il est ainsi possible de donner uneorganisation initiale aux cellules au sein d’une structure tissulaire 3D. Notre objectif estd’utiliser le LAB pour reproduire l’histo-architecture du stroma cornéen. Le stroma cornéenest un assemblage transparent de lamelles d’une épaisseur totale de 500 μm. Au sein dechaque lamelle, les fibres de collagène ont une même direction, un même diamètre et sontrégulièrement espacées grâce à la présence de protéoglycanes spécifiques du stromacornéen. Pour reproduire cette organisation, nous avons fait l’hypothèse qu’en alignant desfibroblastes du stroma sur un hydrogel de collagène à l’aide du LAB, il serait possibled’aligner les fibres de collagène dans la même direction. Du fait que les cellules impriméessont vivantes et dynamiques, le motif cellulaire initialement imprimé est soumis à desprocessus d’auto-organisation. Il a donc fallu déterminer les paramètres, à la foisd’impression et de culture, permettant d’obtenir de façon reproductible des alignements decellules stables dans le temps. Grâce à la microscopie à génération de secondeharmonique, le remaniement des fibres de collagène par les fibroblastes cornéens a pu êtreobservé. La direction des fibres de collagène correspond à celle de l’alignement cellulaire.En imprimant les fibroblastes de cornée sur des couches successives de collagène, noussommes parvenus à reproduire les variations de direction des fibres de collagène d’unelamelle à l’autre qui sont observées dans le stroma cornéen natif. / Laser-Assisted Bioprinting allows positioning of cell droplets with amicrometric precision. It is thus possible to give an initial organization to the cells within a3D tissue structure. Our objective is to use LAB to reproduce the corneal stroma histoarchitecture.The corneal stroma is a transparent assembly of lamellae with a totalthickness of 500 μm. Within each lamella, collagen fibers have the same direction, thesame diameter, and a regular spacing thanks to the presence of proteoglycans which arespecific from the corneal stroma. To reproduce this organization, we make the hypothesisthat through corneal fibroblasts alignment, using LAB, on a collagen hydrogel, it would bepossible to align collagen fibers in the same direction. Because printed cells are alive anddynamic, the cell pattern initially printed is subjected to self-organization processes. It isthus necessary to determine the printing and culture parameters that promote reproducibleand stable cell alignments. By using second harmonic generation microscopy, collagenfiber reorganization by corneal fibroblasts has been observed. Collagen fiber direction ismatching with cell alignment. Corneal fibroblasts have been printed on successive collagenlayers; it allows reproducing the variations in collagen fiber direction from one lamella toanother that are observed in the native corneal stroma.

L'élastine est la protéine de la matrice extracellulaire responsable de l'élasticité des tissus. Elle est synthétisée en abondance dans les tissus soumis à de fortes contraintes mécaniques tels que la peau, les poumons, et les artères. Ce biopolymère présente une demi-vie longue de 70 ans et est par conséquent sujet aux altérations liées à l'âge. De plus, l'élastine est dégradée lors de la progression tumorale, notamment celle du mélanome. Sa dégradation entraîne la libération de peptides dérivés de l'élastine (EDP) dotés d'activités biologiques propres et pouvant être impliqués dans l'invasion tumorale (prolifération, angiogenèse, sécrétion de protéases, invasion, survie). Nous avons été les premiers à démontrer que les EDP sont capables de fortement promouvoir le développement de mélanome in vivo dans un modèle murin au travers de l'augmentation de la prolifération et de l'invasion des cellules tumorales impliquant la collagènase-1 ou MMP-1. Ces données suggèrent ainsi que le récepteur de ces peptides, le complexe récepteur de l'élastine (CRE), pourrait constituer une cible thérapeutique innovante afin de lutter contre ce cancer pour lequel un manque flagrant de thérapies efficientes prévaut à l'heure actuelle. Dans le cadre de cette étude, nous avons développé différentes stratégies thérapeutiques basées sur le ciblage du récepteur CRE. Nous démontrons pour la première fois que le ciblage thérapeutique du CRE permet de limiter de manière efficace la progression du mélanome in vitro et in vivo dans un modèle murin. De plus, le vieillissement étant un des facteurs de risque majeur du mélanome, nous avons évalué l'impact de l'âge du stroma sur les effets des EDP et le développement tumoral associé. / Elastin is the extracellular matrix protein responsible for tissue elasticity. It is abundant in tissues experiencing important mechanical constraints such as skin, lungs and arteries. This biopolymer possesses a half-life of 70 years and, consequently, it is subjected to age-related alterations. Moreover, elastin is degraded during tumor progression, notably that of melanoma. Its degradation results in the release of elastin-derived peptides (EDP) which possess their own biological activities that can be linked to tumor invasion (proliferation, angiogenesis, proteases secretion, invasion, survival). We have been the first to show that EDP can promote melanoma development in an in vivo mouse model by raising the proliferation and invasion of tumor cells through collagenase-1 (MMP-1) up-regulation. These data thus suggest that the receptor for these peptides, the elastin receptor complex, could constitute a novel therapeutic target to fight against this type of cancer for which no therapies are efficient now. In this study, we have developed therapeutic strategies targeting the ERC complex. We show for the first time that the therapeutic targeting of the ERC could efficiently limit melanoma progression in an in vitro and in vivo murine model. Additionally, as aging is one of melanoma major risk factors, we have evaluated the impact of stromal age on EDP effects and the associated tumor development.

Adult bone marrow contains a population of mesenchymal stem cells capable to self-renew and to differentiate into haematopoietic-supportive stroma, osteo, adipo- and chondrocytes. However, the identity of mesenchymal stem cells still remains uncertain. The complex population of their descendants, bone marrow mesenchymal stromal cells (BM MSCs), represents a model to study the principles of differentiation and commitment into mesodermal lineages. The experiments using BM MSCs are often hampered by their low proliferative capacity in vitro. In the present study, we established conditionally immortalized BM MSCs from tetracycline-regulated SV40 Large T-antigen transgenic mice. The identity of the conditionally immortalized BM MSCs was confirmed by marker expression, ability to support haematopoiesis and differentiation potential. The advantages of the conditional immortalization are encompassed in (1) indefinite expansion of cell populations, (2) possibility to perform cellular cloning and (3) prevention from spontaneous differentiation. We demonstrated the heterogeneity of BM MSCs and identified at least 6 types of progenitors within BM MSCs population based on their differentiation potential (“OAC”, “OA”, “OC”, “AC”, “O”, “A”). A hypothetical model of BM MSC hierarchy and the relationships between the progenitors has been proposed. We observed that the Wnt/β-catenin signaling pathway and GSK3 activity could modulate the efficiency of osteo- and adipogenic differentiation pathways, but we didn’t find evidence that the lineage commitment of BM MSCs is determined by Wnt. We elucidated the mechanism of transcriptional regulation of the adipogenic induction of BM MSCs in vitro. Our data revealed the key regulatory role of PPARγ1 during adipogenesis in BM MSCs. Furthermore, we assume that PPARγ1 is a potential trigger of the adipogenic commitment of the BM MSCs progenitors. Finally, the non-adipogenic BM MSCs progenitors were converted into the adipogenic lineage using ectopical expression of the transcription factors C/EBPα, C/EBPβ and C/EBPδ. Our findings provide a novel insight into the molecular mechanisms of BM MSCs lineage commitment.

iii Abstract Glycobiology represents a very progressive subject of cell biology. Protein-saccharide interactions play not only supporting and cell organization role, but they also represent medium for information storage and its decoding. Galectins, group of animal lectins (saccharide-binding proteins), which have selective affinity to ß-galactosides, are multifactorial molecules. They participate in cell-cell and cell-matrix interaction, transmembrane signaling, apoptosis, pre- mRNA splicing and are also present in various types of carcinomas. High expression of galectin-1 has been detected in cancer stroma originated from squamous cell epithelium. In the previous study we established that the fibroblasts - myofibroblasts transition, apart from the known TGF- beta, is also induced by galectin-1. We compared relationship between galectin-1 expression, presence of myofibroblasts and gene expression in tissue samples from patients with head and neck squamous cell carcinoma. Cancer stroma with myofibroblasts was rich in galectin-1 expression in comparison with stroma without myofibroblasts. Moreover, we used microarray analysis (ILLUMINA) to compare the whole genome transcriptome from samples with and without presence of galectin-1. High expression of galectin-1 in tissue samples corresponded with expression...

The thymic stroma primarily comprises epithelial, mesenchymal and endothelial cells, interspersed with those of haematopoietic origin. Thymic epithelial cells (TECs) are highly heterogeneous, but can be divided into two broad lineages, cortical and medullary, based on phenotype, functionality and location. A population of Plet1+ TEC progenitors have been identified which, when isolated from mouse E12.5 or E15.5 fetal thymus, reaggregated, and grafted, can produce a functional thymus. However, the potential of individual progenitors to form cortex and/or medulla is undefined. The main aim of this thesis was to use retrospective clonal analysis to ascertain the point during thymus ontogeny at which the cortical and medullary lineages diverge. To this end, I used transgenic mice carrying a ubiquitous ROSA26laacZ reporter gene (where a duplication within lacZ encodes non-functional b-galactosidase). Here, rare, random laacZ-lacZ genetic recombinations result in heritable expression of functional b-gal, producing labelled clones. As this occurs at a known frequency, determination of TEC numbers would enable calculation of the expected number of TEC clones present throughout ontogeny. Due to the lack of quantitative data on all thymic cell populations, I determined the size not only of TEC (lin-EpCAM+), but also haematopoietic (CD45+), mesenchymal (lin-PDGFRa+ and/or lin-PDGFRb+) and endothelial (lin-CD31+) populations from E12.5 until E17.5. I then showed that the absolute number of Plet1+ TECs remains constant during this time, although the proportion of Plet1+ cells in cycle decreases. From these collective data, I propose a model for the role of the Plet1+ population in thymus development, in which Plet1+ cells continually give rise to Plet1- TECs in a self-renewing manner. Finally, I present a ‘dual origin coefficient’ strategy for analysis of a library of prospective TEC clones. I calculated the number of TEC lacZ+ clones expected to be present throughout thymus ontogeny, selecting an appropriate developmental stage for analysis. Although I observed several clones of apparent mesenchymal origin, supporting a single origin for intrathymic and capsular mesenchyme at E15.5, I observed no TEC clones in this extensive analysis. The CpG content of the ROSA26 promoter suggests a possibility of methylation-induced silencing brought about by de novo methylation of the lacZ reporter gene.

Orientador: Julio Lopes Sequeira / Resumo: A interação entre células neoplásicas e estroma influencia na gênese, amplificação/ inibição tumoral e cinética das drogas antineoplásicas. O fator de crescimento do endotélio vascular (VEGF) é uma citocina envolvida na angiogênese tumoral, relacionada a sobrevida dos pacientes. Dentre os componentes celulares, os fibroblastos associados ao câncer têm papel fundamental na progressão tumoral e secretam diversas citocinas, incluindo o VEGF. Assim, os objetivos deste trabalho foram caracterizar a distribuição, arranjo e quantidade das fibras de colágeno e reticulina no estroma dos linfomas caninos através dos métodos de Picrosirius Red e reticulina, avaliação imuno-histoquímica dos colágenos tipo I e III, do VEGF e α-SMA. Sessenta linfomas foram agrupados de acordo com o imunofenótipo e grau. O colágeno tipo III foi o predominante. As porcentagens e escores de marcação da reticulina, colágeno e VEGF foram diferentes entre os grupos e imunofenótipos, sendo que o linfoma T de alto grau exibiu os maiores níveis tanto de colágeno, reticulina e VEGF. Houve correlação entre expressão de VEGF e índice proliferativo. A expressão de α-SMA foi semelhante entre os grupos, imunofenótipos e graus. Como conclusão, a maior densidade de componentes fibrosos e níveis de expressão de VEGF nos linfomas T poderia, ao menos em parte, contribuir para o pior prognóstico apresentado por pacientes com esse tipo de tumor. Ainda, o VEGF tem potencial como marcador prognóstico no linfoma canino e o uso de ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Neoplastic cells interaction with stroma influences tumorigenesis, amplification or inhibition of tumor progression and anticancer drugs kinetics. Vascular endothelial growth factor (VEGF) is a crucial cytokine implicated in tumoral angiogenesis and survival and among cellular components cancer associated fibroblasts play a role on tumour progression, secreting various cytokines, including VEGF. Therefore, the aim of this study was to characterize fibres distribution, arrangement and amount on stroma evaluated in Picrosirius Red and reticulin stained slides as well as in type I and III immunolabelled sections of canine lymphoma, in addition to the immunoexpression of VEGF and α-SMA on neoplastic lymphoma cells and stroma, respectively. Sixty lymphomas were divided into four groups according to immunophenotype and grade. Type III was the preponderant collagen. Staining percentage and scores of reticulin and collagen, as well as scores of VEGF immunolabelling, were significantly different between groups and immunophenotypes, with high-grade T-cell lymphomas exhibiting the largest quantities. Additionally, VEGF was positively correlated with proliferation index. There was no difference regarding α-SMA expression within groups, immunophenotypes or grades. In conclusion, the greater density of fibrous components on T-cell lymphomas, combined with bigger VEGF levels, could, at least partially, explain the poorer prognosis for patients presenting those tumours. Furthermore, VEGF has... (Complete abstract click electronic access below) / Doutor