La cardiomyopathie dilatée est l'une des principales causes d'insuffisance cardiaque en Europe. Dans le cadre de la cardiomyopathie liée aux mutations du gène LMNA, en dépit des soins médicaux conventionnels, aucun traitement satisfaisant ne permet de pallier à la dilatation cardiaque progressive et à la perte de la contractilité. Le gène LMNA code pour les lamines nucléaires de type A, qui sont les principaux constituants de la lamina nucléaire. De précédents travaux démontrent que l'activation anormale de ERK 1/2 est impliquée dans la pathophysiologie de la cardiomyopathie dilatée liée aux mutations du gène LMNA. L'inhibition de la voie ERK 1/2 ralentit également la progression de la fibrose myocardique, relativement développée chez l'homme, en cas de cardiomyopathie dilatée. Dans le cadre de ma thèse, j’ai suggéré que l'activité aberrante de la voie TGF-β pourrait participer à l’activation anormale de ERK 1/2 et être impliquée dans la physiopathologie de dysfonction contractile ventriculaire gauche dans la cardiomyopathie liée aux mutations du gène LMNA. Les mécanismes moléculaires sous-jacents à la modulation de la signalisation ERK1/2, causée dans le cœur, par les mutations du gène LMNA, restent totalement incertains. De ce fait, j'ai testé l'hypothèse selon laquelle la modulation anormale de ERK1/2 induirait l'altération des cibles cytosoliques et modifierait le réseau du cytosquelette cardiaque. Mon travail a mis en évidence un nouveau partenaire de la forme active (phosphorylée) d’ERK1/2 : cofiline-1. La cofiline induit la déramification des filaments d'actine. Ce projet met en lumière un rôle inattendu joué par la signalisation ERK1/2 dans la dynamique de l'actine et dans le développement de la dysfonction ventriculaire gauche de la cardiomyopathie liée aux mutations du gène LMNA. / Dilated cardiomyopathy is one of the leading causes of heart failure in Europe. Despite of the conventional medical care, there is no definitive treatment for the progressive cardiac dilatation and loss of contractility in LMNA cardiomyopathy often leading to sudden death or heart transplantation. LMNA gene encodes nuclear A-type lamins, which are the main constituents of the nuclear lamina. Previous studies clearly show that the abnormal ERK1/2 activation is involved in the pathophysiology of LMNA dilated cardiomyopathy. However, its role in the development of cardiac dysfunction remains unclear. Inhibition of ERK1/2 signaling also slows progression of myocardial fibrosis, which is prominent in humans with dilated cardiomyopathy. I suggested that aberrant TGF-β signaling activity could participate to the abnormal ERK1/2 activation and be involved is the pathophysiology of left-ventricular contractile dysfunction in LMNA cardiomyopathy. Given that the understanding of molecular and cellular mechanisms underlying the modulation of ERK1/2 signaling in the heart caused by LMNA mutation remains totally unclear, I tested the hypothesis that ERK1/2 abnormal modulation leads to alteration of cytosolic targets and alter cardiac cytoskeleton network. My work highlighted a novel partner of activated (phosphorylated) ERK1/2, ADF/cofilin-1. Cofilin promotes debranching of actin filaments. I showed that disrupted actin dynamics leads to abnormal sarcomere structure. This project unraveled an unexpected role played by ERK1/2 signaling in actin dynamics and in the development of left-ventricular dysfunction in LMNA cardiomyopathy.

Oral and oropharyngeal squamous cell carcinoma (OOSCC) is a malignant neoplasm of epithelial origin that accounts for 90 to 95% of tumors in oral cavity. Alcohol and tobacco consumption are main risk factors, but the formation of reactive oxygen species (ROS) and presence of chronic inflammatory processes have been shown to favor the carcinogenesis process. Human serum paraoxonase 1 (PON1) is a protein with an important antioxidant action and prevents oxidative stress induced by ROS, in turn, high levels of tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) are commonly found in inflammatory processes, and changes in these proteins have been related to development of different neoplasms. Present study aims to investigate the prognostic value of functional polymorphisms in PON1, TNF-α and TGF-β genes in OOSCC. This is a prospective cohort study with patients attended at the Advanced Oncology Center of the North-Riograndense League against Cancer. Data collection of clinical variables was done through the form: gender, age, tumor site, TNM (primary tumor, regional nodule and distant metastasis) classification, clinical stage; and through interview: smoking habits and alcohol intake by the patient. A total of 163 samples from patients with OOSCC and 146 control samples were genotyped by real-time PCR. It was observed that 76.1% of the population was males, 84% older than 52 years, with a more frequent intra-oral clinical presentation (53.4%), in the tongue region (21.5%), tumors greater than 4cm (56.45%), presence of nodal involvement (58.9%) and stages III and IV (79.15%). There was a positive association between drinking and smoking habits in patients with OOSCC and between clinical stage and tumor site (p <0.05). The polymorphisms were in Hardy-Weinberg equilibrium, with exception of rs662 of PON1. TNF-α wild-type GG homozygous genotype (rs1800629) was associated with intra-oral lesions, clinical stage of the most advanced disease (III and IV), and decreased overall disease survival, whereas the polymorphic AA genotype was associated with lip lesion, clinical stage I and II and a longer overall disease survival (p <0.05). It was observed association of TGF-β polymorphic AG and AA genotypes (rs1800469) with larger diameter tumors (T3 and T4) (p <0.05). Finally, the polymorphic TT genotype of PON1 (rs662) in recessive model was associated with a shorter disease survival time within threshold of significance (p = 0.05). In view of the findings, it is suggested that wild-type GG homozygous genotype of TNF-α rs1800629 and TGF-β rs1800469 polymorphic AG and AA genotypes may exert an influence on more aggressive biological behavior of OOSCC and that AG genotype of TNF-α rs1800629 and TT genotype of PON1 rs662 could be prognostic markers in OOSCC. In the clinical practice of oncology, these genotypes can be used to perform early diagnosis, knowledge of the biological behavior of the tumor and choice of appropriate individualized therapy / O carcinoma de células escamosas oral e orofaríngeo (CCEO) é uma neoplasia maligna de origem epitelial que representa 90 a 95% dos tumores da cavidade oral. O consumo de álcool e de tabaco são os principais fatores de risco, mas a formação de espécies reativas de oxigênio (EROs) e a presença de processos inflamatórios crônicos têm-se mostrado favorável ao processo de carcinogênese. A paraoxonase de soro humano 1 (PON1) é uma proteína com importante ação antioxidante e previne o estresse oxidativo induzido pelas EROs, por sua vez, elevados níveis do fator de necrose tumoral-alfa (TNF-α) e do fator de crescimento transformante-beta (TGF-β) são comumente encontrados em processos inflamatórios, e alterações nessas proteínas têm sido relacionadas com o desenvolvimento de diferentes neoplasias. O presente estudo tem por objetivo investigar o valor prognóstico dos polimorfismos funcionais nos genes da PON1, TNF-α e TGF-β no CCEO. Trata-se de um estudo coorte prospectivo com pacientes atendidos no Centro Avançado de Oncologia da Liga Norte-Riograndense contra o Câncer. Realizou-se a coleta de dados das variáveis clínicas por meio de formulário: gênero, idade, localização do tumor, classificação TNM (Tumor primário, nódulo regional e metástase à distância), estadiamento clínico (EC); e por meio de entrevista: hábitos de fumo e ingestão alcoólica pelo paciente. Foram genotipadas 163 amostras de pacientes com CCEO e 146 amostras controle por meio de PCR em tempo real. Observou-se que 76,1% da população era do gênero masculino, sendo 84% com mais de 52 anos, com apresentação clínica mais frequente intra-oral (53,4%), na região da língua (21,5%), tumores maiores que 4cm (56,45%), presença de envolvimento nodal (58,9%) e em estágios III e IV (79,15%). Evidenciou-se associação positiva entre os hábitos de beber e fumar com pacientes portadores de CCEO e entre o EC e a localização do tumor (p<0,05). Os polimorfismos encontravam-se em equilíbrio de Hardy-Weinberg, com exceção do rs662 da PON1. O genótipo homozigoto selvagem GG do TNF-α (rs1800629) associou-se com lesões intra-orais, EC da doença mais avançado (III e IV) e menor sobrevida global da doença, enquanto o genótipo AA polimórfico associou-se a lesão de lábio, EC I e II e maior sobrevida global da doença (p<0,05). Observou-se associação dos genótipos AG e AA polimórfico do TGF-β (rs1800469) com tumores de maior diâmetro (T3 e T4) (p < 0,05). Por fim, o genótipo TT polimórfico da PON1 (rs662) no modelo recessivo apresentou associação com menor tempo de sobrevida da doença dentro do limiar de significância (p=0,05). Diante dos achados, sugere-se que o genótipo GG selvagem do rs1800629 do TNF-α e os genótipos AG e AA polimórfico do rs1800469 do TGF-β podem exercer influência no comportamento biológico mais agressivo do CCEO e que o genótipo AG do rs1800629 do TNF-α e o genótipo TT polimórfico do rs662 da PON1 poderiam ser marcadores com valor prognóstico no CCEO. Na prática clínica da oncologia, esses genótipos podem ser utilizados para realização de diagnóstico precoce, conhecimento do comportamento biológico do tumor e escolha da terapêutica individualizada adequada. / Lagarto, SE

Les capacités de guérison humaine étant limitées et grandement associées à la fibrose, la possibilité de régénérer tous tissus contribueraient grandement à l’amélioration de la santé des patients. Dans le cadre de ce projet de doctorat, nous avons publié un article montrant les limitations de certains modèles de recherche en ce qui a trait à la guérison des plaies. Ces limitations sont d’autant plus importantes lorsque la recherche traite de régénération tissulaire. Aussi, cette publication positionne l’axolotl (Ambystoma mexicanum) comme un excellent modèle pour étudier le processus de régénération épimorphique ainsi que l’importance de la signalisation TGF-β. La cytokine multifonctionnelle TGF-β est impliquée dans la guérison, l’induction des cicatrices, la différenciation, la croissance et la migration cellulaire. Cette cytokine est responsable de la guérison quasi parfaite des muqueuses buccales chez les mammifères, mais est aussi liée à la cicatrisation de plusieurs autres types tissulaires. La famille des TGF-β est aussi impliquée dans la régénération épimorphique chez l’échinoderme, ainsi que dans la régénération hépatique (hyperplasie compensatoire), ce qui confirme son rôle régulateur de la guérison parfaite. Des travaux précédents ont montré que l’utilisation d’un inhibiteur spécifique de la signalisation des TGF-β (SB-431542) empêche la régénération (Lévesque et al., 2007). Comme la voie canonique de signalisation de TGF-β s’opère via les protéines Smads (Smad2 & 3), l’étude de ces deux protéines est au cœur du second article. Lors du processus de régénération, Smad2 est phosphorylé entre 6h et 48h post-amputation (pa), ce qui correspond à la phase de migration cellulaire et au début de la prolifération. D’un autre côté, Smad3 est phosphorylé plus tôt, entre 3h et 6h pa, alors que la quantité de protéine totale diminue lors de la phase de préparation. L’administration de l’inhibiteur SB-431542 au moment de l’amputation bloque l’activation de Smad2 et de Smad3. Aucun blastème ne se forme, bien que la plaie ferme normalement. L’utilisation des inhibiteurs SIS3 et Naringenin (spécifique à Smad3) réduisent la phosphorylation de Smad3 d’environ 50 % (lorsque mesurée par immunobuvardage). Le processus de régénération ne semble toutefois pas affecté. La régulation différentielle des Smads est donc centrale au processus de régénération de l’axolotl. Dans le cadre de ce projet, nous avons aussi tenté de bloquer spécifiquement l’expression, ainsi que l’activation de Smad2. J’ai premièrement établi que Smad2 et Smad3 étaient présents dans la lignée cellulaire AL-1 et qu’ils peuvent être phosphorylés. J’ai ensuite tenté, par différentes techniques, de réduire l’activation spécifique de Smad2, sans succès. D’autre part, plusieurs expériences complémentaires confirment que l’activation de Smad3 est difficilement détectable et est peu importante pour la formation du blastème. La capacité exceptionnelle de régénération de l’axolotl est intimement liée à une activation différentielle des protéines Smad2 et Smad3. L’activation de Smad2 est associée à une prolifération cellulaire importante. D’autre part, l’absence de fibrose est potentiellement due à la faible activation de Smad3 au cours du processus de régénération. / Since wound healing in human is imperfect and associated with fibrosis, understanding how regeneration works would be a great asset to improve patient’s health. During this PhD project, we have published a paper exposing the weaknesses of certain research models when studying wound healing. Those limitations are even more striking when studying regeneration. This publication sets the stage for the use of the axolotl (Ambystoma mexicanum) as an excellent model to study regeneration and the importance of TGF- for the process. The multifunctional cytokine TGF-β is involved in healing, scarring, cellular differentiation, growth and migration. This cytokine is associated with the near perfect healing of oral tissues in humans, but is also associated with scarring of multiple tissue types. TGF-β is also associated with epimorphic regeneration in echinoderm and liver hyperplasia. Previous work had shown that treatment of regenerating axolotl limbs with a specific inhibitor of TGF-β canonical signalling (SB-431542) prevents regeneration (Lévesque et al., 2007). Since canonical signaling goes through Smad2 and Smad3, those two proteins are at the center of the second publication. During limb regeneration, Smad2 is phosphorylated at 6h-48h post-amputation (pa), which corresponds to the cellular migration phase and the beginning of the proliferative phase. On the other hand, Smad3 phosphorylation happens earlier (3h-6h pa), while the total protein expression is lower. Treatment with SB-421543 blocks the phosphorylation of both Smad2 and Smad3. No blastema is formed, but the wound closes at the same rate. Treatment with other inhibitors, SIS3 or Naringenin (specifically targeting Smad3), blocks approximately 50% of Smad3 phosphorylation (as determined by western blotting), but regeneration is not affected. Differential regulation of Smads is essential for proper regeneration to occur. Lastly, we have tried multiple approaches to diminish specifically the activation of Smad2. Using the only axolotl cell line available (AL-1), we have tried inhibition with LNA molecules, long antisense and overexpression of a competitor. None of these approaches specifically reduced the levels of Smad2. In addition, other experiments confirmed that activation of Smad3 during the regeneration process is limited. The extraordinary ability to regenerate that the axolotl possesses is tightly linked to a differential activation of Smad2 and Smad3 proteins. Smad2 phosphorylation is associated with cellular proliferation and migration, hence blastema formation, while the apparent lack of Smad3 activity might partly explain why these animals do not form scar tissues.

Abstract Dentin formation in physiological and pathological conditions has been widely studied, but the events and regulation are still not completely understood. Odontoblasts, terminally differentiated post-mitotic cells located in a single cell layer around pulp tissue, synthesize and mineralize dentin organic matrix. Growth factors, such as TGF-β1 and BMP-2, have been implicated in the regulation of the responses of odontoblasts and pulp tissue to external irritation. Matrix metalloproteinases (MMPs), a family of 28 endopeptidases collectively capable of degrading virtually all extracellular matrix components, and their specific tissue inhibitors (TIMPs) participate in the organo- and morphogenesis, physiological tissue turnover and pathological tissue destruction in many tissues, but very little is known about their presence, function, and regulation in the dentin-pulp complex cells and tissues. The aim of the work presented in this thesis was to analyze the expression and regulation of collagens, MMPs and TIMPs by TGF-β1 and BMP-2 in mature human odontoblasts and pulp tissue. Odontoblasts synthesize and secrete type I and type III collagens, with no clear effect of TGF-β1 on their expression levels. MMP-1, -2, -8, -9, -10, -11, -14, -15, -16, -19 and TIMP-1, -2, -3 and -4 were expressed by both odontoblasts and pulp tissue. MMP-3 and MMP-12 were not expressed in native odontoblasts or pulp tissue, and MMP-7, -24, and -25 were expressed only in odontoblasts. MMP-2, -10, -14, -20 and -23 were expressed more abundantly in odontoblasts, whereas pulp tissue expressed more MMP-13 and MMP-17. Growth factors differentially regulated the expression of different MMPs and TIMPs within and among the cells and tissues studied. In odontoblasts, MMP-1, -8 and -14 were down-regulated, but MMP-7, MMP-9, TIMP-1 and TIMP-3 up-regulated, by either TGF-β1 or BMP-2, alone or in combination. In pulp tissue, growth factors up-regulated the expression of MMP-1, -2, -10, -13, -17 and TIMP-3, but down-regulated TIMP-4. The widespread of expression of MMPs and TIMPs by mature human odontoblasts and pulp tissue suggests that they may participate in dentin matrix organization prior to mineralization, and that growth factors may further control dentin matrix modeling, not by regulating the synthesis of type I or III collagens as previously believed, but rather by differentially regulating each MMPs and TIMPs.

Transforming growth factor-β (TGF-β) superfamily of cytokines comprises of several members, which can broadly be sub-divided into three classes [TGF-βs, Activin/Nodal, and Bone morphogenetic proteins (BMPs)]. Most members of this family play critical roles during embryo development differentiation and regulation of homeostasis. In mammals there are three TGF-β isoforms, TGF-β1, 2 and 3. All the three TGF-β isoforms have important roles in embryo development as revealed by mouse knock-out models. TGF-β has also been associated with several pathological conditions such as inflammation, Fibrosis, and cancer. In cancers, TGF-β plays both tumor suppressive and tumor promoting roles depending upon the context. TGF-β has growth inhibitory effect on epithelial cells which is essential to maintain tissue homeostasis. TGF-β induces the expression of several cyclin dependent kinase inhibitors such as p21Cip1, p15Ink4b while down-regulating the expression of cMYC in the epithelial cells. In lieu of its tumor suppressive role, several cancers harbor mutations in the components of the TGF-β signaling axis such as receptors and effector molecules called SMADs. Interestingly various cancers also show hyper activation of TGF-β signaling. It has been suggested that cancer cells become unresponsive to the growth inhibitory effects of TGF-β by losing the expression of p21Cip1, and p15INK4b. Oncogenic transformation of cancer cells can override the growth inhibitory effects of TGF-β. While the loss of growth inhibitory effects by TGF-β are seen in the tumor cells, several tumor promoting actions are also observed in these cells such as induction of EMT. TGF-β activates mesenchymal cells leading to the formation of a reactive stroma in tumors and TGF-β suppresses almost all types of cells of the immune system causing a local immune-suppressive environment. TGF-β also recruits mesenchymal stem cells into the stroma which secrete several cytokines. The sum total of all these effects is pro-angiogenic, pro-infiltrative and pro-metastatic. In the canonical TGF-β signaling pathway, ligands bind to the hetero-tetrameric receptor complex of TGFβR1 and TGFβR2 leading to activation of the TGFβR1 by TGFβR2. Activated TGFβR1 then phosphorylates and activates R-SMAD molecules (SMAD2, SMAD3) which complexes with the co-SMAD (SMAD4) and translocate into the nucleus to effect transcriptional changes. Non-canonical TGF-β signals are many and almost all the known signaling pathways like MAPK, WNT, PI3K-AKT, NOTCH, Integrin, Hedgehog, Hippo etc. have been shown to be activated by TGF-β in different contexts. The canonical TGF-β/SMAD pathway has been shown to be essential for both tumor suppressive and tumor promoting actions of TGF-β. Although the non-canonical signalling pathways have been shown to be context dependent, the exact mechanisms have not been elucidated. In previous studies, we have shown the importance of non-canonical TGF-β signaling in normal vs. carcinoma cells. However, there has been no study that addressed the differential effects of TGF-β on cells of connective tissue origin. To throw light on such questions we have undertaken this study with the following objectives: 1) Whole genome expression profiling of TGF-β targets in normal fibroblasts, transformed fibroblasts and sarcoma cells 2) Elucidation of non-canonical signaling pathways differentially regulated by TGF-β 3) Identification and characterization of novel TGF-β targets The cell-lines chosen for the study are: 1) hFhTERT (human foreskin fibroblasts immortalized with human terminal telomerase); 2) hFhTERT-LTgRAS (hFhTERT transformed with SV40 large T antigen and activated RAS); and 3) HT1080 (fibrosarcoma). We performed whole genome expression profiling using 4×44K Agilent Human Whole Genome Oligonucleotide Arrays. Analysis of the microarray results revealed that TGF-β regulated a large number of genes in all the three cell-lines but few targets were found to be commonly regulated between any two or all the three cell-lines. 5291 genes were differentially regulated by TGF-β between hFhTERT and hFhTERT-LTgRAS and 2274 genes were differentially regulated by TGF-β between hFhTERT and HT1080 cells. Gene set enrichment analysis (GSEA) of these two gene lists revealed enrichment of similar gene sets in the HT1080 and hFhTERT-LTgRAS cells compared to the hFhTERT cells. MAPK signaling pathway components were enriched in the hFhTERT cells. Closer inspection revealed that several upstream regulators of the MAPK pathway were in fact down-regulated by TGF-β in these cells compared to both hFhTERT-LTgRAS and HT1080 cells suggesting a depression of the MAPK pathway by TGF-β in the hFhTERT cells. Assessment of the phosphorylation status of ERK1/2 and p38 MAPK proteins after TGF-β treatment showed that both ERK1/2 and p38 MAPK pathways were not activated in response to TGF-β in the hFhTERT cells. On the other hand in hFhTERT-LTgRAS and HT1080 cells, both ERK1/2 and p38 MAPK were activated post TGF-β treatment. Activity of the AP1 and SMAD responsive p3TP-lux reporter plasmid was dependent on only the SMAD pathway in hFhTERT cells while in the hFhTERT-LTgRAS and HT1080 cells both MAPK and SMAD pathway were found to regulate the expression of the p3TP-lux reporter. This suggests activation of MAPK and SMAD pathways in transformed and tumor cells while there is no activation of MAPK in normal cells of mesenchymal origin. Components of the WNT signaling pathway such as WNT ligands WNT4, and WNT11, frizzled receptors, FZD4, FZD8 and FZD9, regulators like SFRP1, SFRP2, AXIN2 and several targets of the WNT-β-catenin pathway were regulated by TGF-β in the hFhTERT cells but not in the hFhTERT-LTgRAS and HT1080 cells suggesting a positive regulation of the pathway by TGF-β in the hFhTERT cells. Indeed, TGF-β induced the activity of the WNT responsive reporter, pTOP-FLASH in the hFhTERT cells but not in the hFhTERTLTgRAS and HT1080 cells. WNT4 and WNT11 were two of the novel targets of TGF-β identified in hFhTERT cells. Further experiments suggested that TGF-β conferred regulation of these genes was specific to the fibroblast cells since induction of these genes by TGF-β was not observed in any of the cancer cell lines or in HaCaT cells. Some recent studies have demonstrated remodelling of cytoskeleton in epithelial cells by the non-canonical WNT ligands such as WNT5a, WNT4 and WNT11. WNT4 has also been shown to be required for the maintenance of α-SMA levels in smooth muscle cells. In this study we have shown that WNT4 can induce α-SMA in the hFhTERT cells leading to their activation. TGF-β conferred activation of these cells was also found to be dependent on the presence of WNT4. In brief, our study identified differentially activated pathways by TGF-β in immortal and transformed fibroblasts. WNT4 was identified as a crucial molecule required for the TGF-β conferred activation of fibroblasts.

Activin B, a member of the transforming growth factor-β superfamily, is ubiquitously expressed in diverse tissues and is a regulator of reproduction, embryonic development, and adult tissue homeostasis. We aimed to determine whether activin B is involved in skeletal muscle injury and if selective inhibition of activin B would provide a regenerative benefit. The local introduction of activin B into normal skeletal muscle increased the expression of inflammatory and muscle atrophy genes TWEAK, TNFα, GDF3 and TRIM63, by 2-, 10-, 10-, and 4-fold, respectively. The data indicate a sensitive response of skeletal muscle to activin B. Six hours after cardiotoxin-induced skeletal muscle damage, circulating activin B protein expression in serum increased by 9-fold and InhβB gene expression increased by 30-fold in muscle. After cardiotoxin-induced skeletal muscle damage, activin B protein expression in muscle was significantly increased at 48- and 120-hours by 1.5 and 2-fold, respectively. Muscle histopathological features showed that activin B antibody–treated mice displayed a reduction in necrotic debris, with a concomitant reduction in intramyocellular space at 9-days after injury. Activin B treated C2C12 myoblasts also displayed a dose-dependent reduction in active myogenesis. Furthermore, the increased presence of activin B early in muscle injury impedes muscle repair and remodeling. In summary, acute muscle injury leads to increases in activin B levels and when selectively neutralized with a monoclonal antibody, there is augmented skeletal muscle repair.

Atherosclerosis, is a chronic inflammatory disease, characterized by the narrowing of the arteries resulting from the formation of intimal plaques in the wall of arteries. Yet the molecular mechanisms responsible for maintaining the development and progression of atherosclerotic lesions have not been fully defined. In this study, we show that TGF-β activates the endothelial-to-mesenchymal transition (EndMT) in cultured human aortic endothelial cells (HAECs) and this transition is dependent on the key executor of the Wnt signaling pathway . This study presents the first evidence describing the mechanistic details of the TGF-β-induced EndMT signaling pathway in HAECs by documenting the cellular transition to the mesenchymal phenotype including the expression of mesenchymal markers α-SMA and PDGFRα, and the loss of endothelial markers including VE-cadherin and CD31. Furthermore, a short hairpin RNA (shRNA) screening revealed that Wnt2 signaling is required for TGF-β-mediated EndMT of HAECs. Also, we found that LDLR mice fed on a high-fat western-type diet (21% fat, 0.2% cholesterol) expressed high levels of Wnt2 protein in atherosclerotic lesions, confirming that this signaling pathway is involved in atherosclerosis . These findings suggest that Wnt2 may contribute to atherosclerotic plaque development and this study will render Wnt2 as a potential target for therapeutic intervention aiming at controlling atherosclerosis.

no / This study aimed at determining the role of the transforming growth factor-beta (TGF-β) isomers and their combinations in bone cell behaviour using MG63 cells. The work examined how TGF-β1, 2 and 3 and their solvent and carrier (HCl and BSA, respectively) effected cell morphology, cell proliferation and integrin expression. This study also aimed at examining how the TGF-βs and their solvent and carrier influenced wound closure in an in vitro wound closure model and how TGF-βs influence extracellular matrix (ECM) secretion and integrin expression. The wound healing response in terms of healing rate to the TGF-βs and their solvent/carrier was investigated in 300 μm ± 10–30 μm SD wide model wounds induced in fully confluent monolayers of MG63 bone cells. The effect of different TGF-β isomers and their combinations on proliferation rate and cell length of human bone cells were also assessed. Immunostaining was used to determine if TGF-βs modifies integrin expression and ECM secretion by the bone cells. Imaging with WSPR allowed observation of the focal contacts without the need for immunostaining. The wound healing results indicated that TGF-β3 has a significant effect on the wound healing process and its healing rate was found to be higher than the control (p < 0.001), TGF-β1 (p < 0.001), TGF-β2 (p < 0.001), BSA/HCl (p < 0.001) and HCl (p < 0.001) in ascending order. It was also found that TGF-β1 and TGF-β2 treatment significantly improved wound closure rate in comparison to the controls (p < 0.001). All TGF-β combinations induced a faster healing rate than the control (p < 0.001). It was expected that the healing rate following treatment with TGF-β combinations would be greater than those healing rates following treatments with TGF-β isomers alone, but this was not the case. The results also suggest that cell morphological changes were observed significantly more in cells treated with TGF-β(2 + 3) and TGF-β(1 + 3) (p < 0.001). Any cell treated with TGF-β1, TGF-β(1 + 2) and TGF-β(1 + 2 + 3) showed significantly less elongation compared to the control and other TGF-β isomers. In terms of proliferation rate, TGF-β3 and TGF-β(2 + 3) increased cell numbers more than TGF-β1, TGF-β2 and other combinations. TGF-β1 and its combinations did not show significant proliferation and attachment compared to the control. Immunostaining indicated that treatment with TGF-β3 significantly enhanced the secretion of collagen type I, fibronectin and integrins α3 and β1. The WSPR experiments also indicated that TGF-βs influenced the distribution of focal contacts. In conclusion, combining TGF-β3 with any other TGF-β isomer resulted in a faster model wound closure rate (p < 0.001), while treatment with TGF-β1 in any TGF-β combination reduced the healing rate (p < 0.001). It can therefore be concluded that the presence of TGF-β1 has an inhibitory effect on bone wound healing while TGF-β3 had the opposite effect and increased the rate of wound closure in a 2 dimensional cell culture environment. / Emailed Mansour for final draft 27/06/2016

Les phénomènes cicatriciels et de carcinogenèse partagent des points communs et peuvent être définis comme des processus complexes et adaptatif régulés par les interactions entre l'hôte et le microenvironnement tissulaire. Après la chirurgie, la radiothérapie est la seconde modalité la plus efficace dans le traitement du cancer et une approche thérapeutique multimodale impliquant chirurgie, radiothérapie et chimiothérapie est aujourd’hui classiquement utilisée. Des résultats récents suggèrent que, en plus des effets létaux sur les cellules tumorales, la radiothérapie modifie le microenvironnement tissulaire. Ces modifications affectent le phénotype cellulaire, le métabolisme des tissus, et les événements de signalisation entre les cellules.Les interactions complexes entre les cellules stromales et les cellules cancéreuses suscitent beaucoup d’intérêt et dans la première partie de ma thèse, j’ai exploré les interactions entre stroma et cellules de carcinome en réponse à la radiothérapie par modulation génétique du stroma après irradiation. J’ai constaté que les fibroblastes, indépendamment de leur statut RhoB, ne modulaient pas la radiosensibilité intrinsèque des TC- 1, mais produisaient des facteurs diffusibles capables de modifier le devenir des cellules tumorales. Ensuite, j’ai constaté que fibroblastes sauvages et RhoB déficients stimulaient la migration des TC-1 par des mécanismes distincts, impliquant respectivement TGF- β1 et MMP. J’ai également constaté que la co-irradiation, des fibroblastes et des TC- 1, abrogait le phénotype pro-migratoire des TC-1 par répression de la sécrétion du TGF- β et des MMP. Alors que le protocole de co-irradiation utilisé mime la situation clinique, mes résultats sont en désaccord avec les publications récentes et suggèrent que le stroma irradié ne renforce pas la migration des cellules tumorales mais au contraire pourrait être manipulé pour promouvoir une réponse immunitaire anti-tumorale.Deuxièmement, mes expériences in vivo, semblent confirmer les données obtenues in vitro et montrent que l’irradiation préalable du lit tumoral ne stimule ni croissance de la tumeur et ni sa dissemination. Nos résultats semblent montrer que l’irradiation du stroma ne favorise pas la migration des cellules de carcinome et ceci indépendamment de leur génotype. La Troisième Partie De Mon Projet, a été consacrée à l’étude des cellules tumorale circulantes (CTC) après la radiothérapie. En accord avec les résultats rapportés après la chirurgie, le nombre de CTC augmente dans la circulation sanguine après radiothérapie probablement à cause des lésions vasculaires radio-induites ou/et par induction d’EMT dans les cellules tumorales. Néanmoins ces CTC semblent être piégées dans la cavité cardiaque. La signification de la présence de ces CTC pour le développement métastatique n’est pas élucidée mais on peut suspecter un effet promoteur de métastase. Ainsi le microenvironnement pourrait avoir des effets antagonistes promoteurs ou inhibiteurs de malignité. / Wound healing and carcinogenesis are defined as complex, adaptive processes which are controlled by intricate communications between the host and the tissue microenvironment. A number of phenotypic similarities are shared by wounds and cancers in cellular signaling and gene expression. Radiotherapy is the second most effective modality of cancer treatment after surgery and can be used, either alone or in combination with chemotherapy. Recent findings suggest that radiotherapy apart from tumor cell death also rapidly and persistently modifies the tissue microenvironment. These modifications affect cell phenotype, tissue metabolism, bidirectional exchanges and signaling events between cells. The complex interactions between stromal cells and cancer cells are of immense interest and in The First Part of My Thesis, I tried to explore the crosstalk between stromal and carcinoma cells in response to radiotherapy by genetic modulation of the stroma and irradiation. We found that fibroblasts, irrespective of their RhoB status, do not modulate intrinsic radiosensitivity of TC-1 but produce diffusible factors able to modify tumor cell fate. Then we found that Wt and RhoB deficient fibroblasts stimulated TC-1 migration through distinct mechanisms respectively, TGF-β1 and MMP-mediated. We also found that co-irradiation of fibroblasts and TC-1 abrogated the pro-migratory phenotype by repression of TGF-β and MMP secretion. This result is highly relevant to the clinical situation and suggests that conversely to, the current view; irradiated stroma would not enhance carcinoma migration and could be manipulated to promote anti-tumor immune response. Secondly, our in vivo experiments, tends to confirm the in vitro data showing that irradiated tumor bed does not stimulate tumor growth and escape. Our results also challenges the view that irradiated stroma would promote migration of carcinoma cells as we show that independently from their genotype co-irradiation of fibroblasts and carcinoma cells repressed carcinoma cell migration and confirmations studies are currently performed in vivo. The Third Part of My Project, was dedicated to investigate the effect on CTC release after radiotherapy. Consistently with the results reported after surgery , the number of CTC increases in the blood stream after radiotherapy probably due to radiation-induced vascular injury induced or/and by EMT induction in tumor cells but these cells seemed to be entrapped into the cardiac cavity. The significance of these CTC to metastatic development is still under investigation but there is evidence for a metastasis-promoting effect of RT from animal studies.Thus the microenvironment can exert antagonist stimulatory or inhibitory effects on malignant cells.

The amphibian species Xenopus laevis, along with mouse and chicken is a very important model system, used widely to dissect the molecular intricacies of various aspects of vertebrate development. Study with Xenopus has clear advantages in terms of various technical considerations including the ease of handling early stage of embryos and due to the remarkable documentation of several early molecular events during development. The concept of inductive interactions between various cell types during early development was first revealed by the studies performed in Xenopus, and among the various factors proposed for mesoderm induction, the members of transforming growth factor-β (TGF- β) superfamily have been considered to be the most probable candidates. About forty different members of the TGF-β superfamily have been cloned and characterized from various organisms. The superfamily members like activins and BMPs have been studied extensively with respect to their functional role during development. While BMPs were assigned as candidates for inducing ventral mesoderm, activins oppose the role of BMPs by inducing dorsal mesoderm. Studies that helped in delineating their roles were performed using three approaches that utilized the ligands, receptors or down stream signaling components (Smads). All the three components were studied with respect to their endogenous expression pattern and effects of ectopic expressions of the wild type or dominant negative mutants. These approaches led to the accumulation of evidences supporting the importance of these signaling molecules. All the above mentioned studies were only possible due to the cloning and characterization of cDNAs of the various proteins involved in the signaling pathway including the ligands. TGF-β2 and 5 are the two isoforms of TGF-β cloned from the amphibian system. We have earlier cloned and characterized the promoter for TGF-β5 gene, which suggested possible regulation of this factor by tissue specific transcription factors. Messenger RNA in situ hybridization analysis to study the TGF-β5-expression pattern during Xenopus development, showed spatial and temporal expression pattern. The expression was confined to specific regions that include notochord, somites, and tail bud among others, in the various stages analyzed. This suggested a possible role for TGF-β5 in organogenesis during the amphibian development. To better understand the role of TGF-β in Xenopus development, studies to examine the specific receptor expression pattern for this growth factor is very essential. With the lack of any reports on cloning of TGF-β receptors from this system, the aim of the present study was to isolate and characterize the receptors for TGF-β from Xenopus laevis. PCR cloning using degenerate primers based on the conserved kinase domains of this class of receptors, coupled to library screenings enabled the identification of two novel receptor cDNAs of the TGF-β receptor superfamily. Characterization of the isolated cDNAs suggested that one of them codes for a type II receptor for TGF-β. Further the cDNAs were found to be ubiquitously expressed during development, as judged by RT-PCR analysis. The cloned cDNAs can now be employed as tools, to study the expression pattern by means of mRNA in situ hybridization, on the various developmental stage embryos and to perform studies using antisense and dominant negative mRNA injection experiments in vivo. Such studies will greatly assist in delineating the role of TGF-β ligands and receptors during amphibian development.