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Mon projet de thèse s’inscrit dans le cadre des études visant à comprendre comment les cellules embryonnaires intègrent les différents signaux auxquels elles sont exposées pour s’engager dans une voie de différenciation définie. Il est plus particulièrement centré sur le rôle des protéines Smad dans ces processus et peut se diviser en deux axes de recherche. Le premier a trait au rôle de Mad (Smad1) dans les interactions entre signaux Wnt (Wg) et BMP chez la drosophile. Nous avons pu démontrer que la forme Mad non phosphorylée par le récepteur BMP se lie au complexe transcriptionnel ß-catenin/dTCF et est requise pour le signal Wnt canonique. La phosphorylation de Mad par le récepteur BMP dirige Mad vers la voie BMP, créant la possibilité d’une compétition entre ces deux classes de signaux. Le second axe de recherche concerne le facteur de transcription Smad4 qui est requis pour la transduction des signaux TGF-ß et BMP. J’ai pu identifier trois sites potentiels de phosphorylation par la kinase GSK3 dans la séquence primaire de Smad4. En utilisant de nombreuses techniques de biochimie, j’ai pu montrer que Smad4 est phosphorylé par la kinase Erk, puis par GSK-3 en réponse à un signal FGF. Lorsque Smad4 est doublement phosphorylé, il est reconnu par une E3-ligase, beta-TrCP, ce qui entraine sa polyubiquitination et sa dégradation. La voie Wnt étant capable d’inhiber GSK-3, j’ai pu montrer que Smad4 est stabilisé par des signaux Wnt. Ce mécanisme augmente la sensibilité des cellules aux signaux TGF-beta lorsqu’elles reçoivent également un signal Wnt. / During my PhD I studied how cells receive and integrate multiple signals from the extracellular milieu. I focused on Smad proteins and my project can be divided into two parts. My first project was centered on the transcription factor Mad (Smad1) and its requirement for the BMP and Wg pathways. Using a combination of genetic and biochemistry experiments, we showed that Mad is required for Wg signaling both in Tcf reporter gene assays and in vivo in Drosophila. We found that the choice for Mad to transduce Dpp or Wg signals is controlled by C-terminal phosphorylations so that Mad binds to Pangolin and participates in Wg target genes transcription only when not phosphorylated at its C-terminus. This results in a competition between Dpp and Wg controlled by the phosphorylation state of Mad. My second project was focused on the tumor suppressor Smad4. When I first joined the lab, I identified three new potential GSK3 phosphorylation sites in Smad4 primary sequence. I used a home-made phospho-specific antibody to demonstrate that FGF or EGF stimulation trigger Erk-mediated phosphorylation of Smad4 which primes subsequent GSK3 phosphorylations. These phosphorylations regulate a transcription activation domain located in Smad4 linker region and generate a Wnt-regulated phosphodegron recognized by the E3 ligase beta-TrCP. This mechanism provides a means of integrating distinct pathways which would otherwise remain insulated, allowing cells to sense FGF and Wnt inputs and adapt TGF-beta outcome to their context. It provides a molecular explanation of the long-standing mystery of the “competence modifier” effect of Wnt on Nodal signals discovered 20 years ago.

A Distrofia Muscular de Duchenne (DMD) tem como característica marcante a substituição do músculo pelo tecido fibroso, sendo este um dos maiores obstáculos para a eficácia de terapias para a distrofia muscular. Intervenções para preveni-la provavelmente poderão ser necessárias como parte de um tratamento eficaz. Correlações significativas entre fibrose e a expressão do TGF-beta, uma citocina fibrogênica multifuncional nas distrofias musculares têm sido relatadas, enfatizando o papel dessa citocina no desenvolvimento da fibrose muscular e sugerindo-a como alvo para terapias antifibróticas. Nesse estudo avaliamos o efeito do Losartan sobre o desenvolvimento da fibrose na musculatura esquelética do modelo canino Golden Retriever Muscular Dystrophy (GRMD). Foi realizado previamente um estudo piloto com um cão distrófico para estipular dosagem e eventuais efeitos colaterais ao medicamento. Foram utilizados cinco cães adultos, sendo dois machos e duas fêmeas e um animal controle. Utilizou-se a dose de 50mg de Losartan, administrada via oral, uma vez ao dia. Os exames clínicos e laboratoriais não evidenciaram reação adversa durante o período do experimento, portanto, o Losartan mostrou-se como uma terapia segura. Os fragmentos da biopsia muscular retirados antes de iniciar com o Losartan (T0) e após (Tf) foram utilizados para histologia e imuno-histoquimica do TGF-beta1 para comparação destes dois tempos. As avaliações de goniometria e perimetria juntamente com os resultados de imuno-histoquimica e quantificação do colágeno ajudaram a inferir sobre o efeito do Losartan na fibrose do músculo distrófico. Não foi encontrada diferença significativa para os valores de goniometria e perimetria. Já a porcentagem da área de deposição de colágeno dos animais nos Tf foi estatisticamente menor do que o T0. A diminuição da presença do TGF-beta1 evidenciada nas imagens de imuno-histoquimica, com a diminuição do depósito de colágeno, após o período de uso do Losartan, sugerem um efeito inibitório do medicamento sobre esta citocina nos músculos dos cães GRMD estudados. / Duchenne Muscular Dystophy (DMD) has the substitution of the muscle by connective tissue as its most relevant characteristic. Once fibrotic proliferation is a major obstacle to the efficacy of therapies for muscular dystrophies, early interventions to prevent it will probably be necessary as part of an effective treatment. A significant correlation between fibrosis and the expression of TGF-beta 1, a multifunctional cytokine, in Duchenne muscular dystrophies has been reported, emphasizing the role of this cytokine in the development of muscle fibrosis, and suggesting it as target for fibrotic therapies. In this study we evaluated the effect of Losartan over the development of the connective tissue on the skeletal musculature of the canine model GRMD. One dystrophic dog was previously used in the pilot study to estipulate the dosage and any side effects caused by Losartan. Five adults dystrophics dogs, two male and two female and one control animal were used in the experiment. A dose of 50mg of Losartan was orally given once a day. The clinical and laboratorial exams did not show any adverse effect through the experimental period, therefore Losartan utilization showed to be a safe therapy. Muscle biopsy fragments have been removed before starting Losartan (T0) and after (Tf) were used for histology and TGF-beta1 imunohistochemistry to compare this two times. The evaluations range of motion and limb circumference measures within imunohistochemistry and colagen quantification results helped to infer about Losartan effect in the dystrophic muscle fibrose. Range of motion and limb circumference values did not show statistical difference. Although the percentage of connective tissue deposition area in the animals in Tf was statistically lower than T0. The decrease of TGF-beta1 signalization showed in imunohistochemistry pictures within the decrease of connective tissue deposition, after Losartan, suggest an inhibitory effect of this medication through this cytokine in the studied GRMD muscle.

A transição endotélio-mesenquimal (EndMT) é uma forma especializada da transição epitéliomesenquimal (EMT) e é caracterizada pela alteração da morfologia celular para um formato fibroblastoide, perda da expressão dos marcadores endoteliais e ganho da expressão dos marcadores mesenquimais, bem como a aquisição de propriedades invasivas e migratórias. Entretanto, o mecanismo molecular envolvido nesse processo ainda não está totalmente elucidado. O objetivo desse trabalho foi avaliar os fatores indutores da EMT em células endoteliais (CEs) de fontes distintas por meio da superexpressão do fator de transcrição SNAIL e do tratamento com TGF-?2, bem como identificar os mecanismos moleculares envolvidos nesse processo. Para tal, as linhagens de CE da artéria pulmonar (HPAEC), pool de CE primária de veia de cordão umbilical (PHUVEC), CE da aorta (PAEC) e CE da artéria coronária (CAEC) foram induzidas em três condições distintas: I) TGF-?2; II) superexpressão do fator de transcrição SNAIL; III) superexpressão do fator de transcrição SNAIL associado ao tratamento com TGF-?2 (SNAIL+TGF-?2). Após a indução, a expressão dos genes relacionados com a EndMT foi analisada por PCR em tempo real (qPCR) e as CAECs foram as células que apresentaram maior mudança no perfil de expressão gênica, no qual o grupo SNAIL+TGF-?2 apresentou um aumento dos marcadores mesenquimal FN1, SM22, CNN1 e CD90. O grupo SNAIL+TGF-?2 também mostrou uma diminuição dos marcadores endoteliais CD31 e CDH5 por Western blot. Em seguida, a técnica de microarray foi realizada nas CAECs induzidas à EndMT e as análises revelaram um dendrograma cujo perfil mostrou que SNAIL e SNAIL+TGF-?2 se agrupam separadamente das outras condições. Os dados de microarray resultaram em uma rede na qual os genes mesenquimais COL1A1, COL1A2, FN1 e CNN1 estavam aumentados no grupo SNAIL+TGF-?2 comparado com o grupo controle. Os genes diferencialmente expressos entre a análise CT vs. SNAIL+TGF-?2 foram analisados quanto a participação em vias canônicas e a via de regulação da EMT foi uma das mais representadas, a qual inclui a via de sinalização Notch e Wnt. Nos dados de microarray, NOTCH3 e WNT5B estavam superexpressos no grupo SNAIL+TGF-?2 comparado com o controle. Sabendo que Wnt5b pode inibir a via ?-catenina, a expressão de NOTCH3, WNT5B e ?-CATENINA foi avaliada por qPCR e a expressão de NOTCH3 e WNT5B confirmou os dados do microarray e nenhuma diferença estatística foi observada na expressão de ?- CATENINA. Ainda, as CAECs induzidas foram submetidas ao ensaio de migração e de capacidade de formação de estruturas semelhantes a capilares. Foi observado que as CAECSNAIL+ TGF-?2 migraram significativamente comparadas com as outras condições e nenhuma das células induzidas (TGF-?2, SNAIL e SNAIL+TGF-?2) foram capazes de formar estruturas semelhantes a capilares. Alguns microRNAs foram selecionados e avaliados por qPCR. O miR-let7a foi significativamente expresso no grupo SNAIL e SNAIL+TGF-?2. O ensaio de perda e ganho de função do miR-let7a foi realizado, entretanto, a repressão ou a indução do miR-let7a não alterou a EndMT. Esses resultados sugerem que as CEs de fontes anatômicas distintas apresentam respostas diferentes quando estimuladas a sofrerem EndMT. Ademais, a associação entre SNAIL+TGF-?2 é um potente indutor para EndMT e essa indução pode ser mediada pelas vias de sinalização Notch e Wnt não canônica. / Endothelial-mesenchymal transition (EndMT) is a specialized form of epithelialmesenchymal transition (EMT) which is characterized by changes in cell morphology as a fibroblastoid conversion, expression of endothelial markers decreased, expression of mesenchymal markers increased and acquirement of invasive and migratory properties. However, the molecular mechanism associated with this process is not completely elucidated. The aim of this study was to evaluate the EMT-inducing factors in the endothelial cells (ECs) from different sources through the overexpression of the transcription factor SNAIL and through the treatment with TGF-?2, as well as to identify the molecular mechanisms involved in EndMT. For this purpose, primary pulmonary artery EC (HPAEC), primary pooled umbilical vein EC (PHUVEC), primary aortic EC (PAEC), primary coronary artery EC (CAEC) lineages were induced under three distinct conditions: I) TGF-?2; II) ectopic expression of SNAIL; III) ectopic expression of SNAIL associated with TGF-?2 (SNAIL+TGF- ?2). After the EndMT induction, the expression of the genes associated with EndMT was analyzed by Real time PCR (qPCR) and CAECs showed the most prominent alterations on their gene expression profile which showed that SNAIL+TGF-?2 group presented an increase of mesenchymal markers FN1, SM22, CNN1, and CD90 expression. CAEC-SNAIL+TGF-?2 group also showed a decrease of endothelial markers CD31 and CDH5 by western blot. Then, microarray was performed in CAECs after EndMT induction and hierarchical clustering analysis showed that the ectopic expression of SNAIL and SNAIL+TGF-?2 clustered separately from the other conditions. Microarray data resulted in a network which presented an upregulation of the mesenchymal genes such as COL1A1, COL1A2, FN1, and CNN1 in the CAEC-SNAIL+TGF-?2 compared to control cells. We analyzed the canonical pathways related to the differentially regulated genes between CAEC- SNAIL+TGF-?2 and control cells and the regulation of EMT pathways was the most represented, which includes Notch and Wnt signaling pathway. In the microarray data, NOTCH3 and WNT5B were overexpressed in CAEC-SNAIL+TGF-?2 compared to control. It is known that Wnt5b might inhibit the ?- catenin pathway. Therefore, NOTCH3, WNT5B and ?-CATENIN gene expression were analyzed by qPCR. NOTCH3 and WNT5B gene expression confirmed the microarray data and no statistical difference were observed in ?-CATENIN expression. Moreover, all the CAECs conditions were subjected to scratch migration assay and the formation of capillary-like structures assay. CAEC-SNAIL+TGF-?2 had a significant migration compared to other conditions and the three EndMT inductions (TGF-?2, SNAIL, and SNAIL+TGF-?2) were not able to form capillary-like structures. Some microRNAs were selected and evaluated by qPCR. The miR-let7a was significantly expressed in the SNAIL and SNAIL+TGF-?2 groups. The assay of gain or loss of function of miR-let7a was realized; however, the repression or induction of miR-let7a did not change the EndMT. These results suggest that endothelial cells from distinct anatomical sources have different responses when stimulated to undergo the EndMT. Moreover, the association between SNAIL+TGF-?2 is a potent inductor for EndMT and this induction can be mediated by Notch and non-canonical Wnt signaling pathway activation.

L’atteinte musculaire peut être un facteur aggravant du pronostic dans certaines pathologies. Durant cette thèse nous nous sommes intéressés à des médiateurs de l’inflammation, tels que les cytokines, et leurs effets sur la fonction du muscle cardiaque et des muscles striés squelettiques. Dans la première étude, nous avons montré que le TNF-α entraîne une diminution de la force contractile cardiaque, via l’activation des PKC, mais que l’excitabilité membranaire n’est pas altérée. Dans la seconde étude nous montrons que l’IL-13 augmente la force de contraction du coeur, via l’activation de la voie β2 adrénergique - PKA, et permet également d’augmenter l’excitabilité membranaire des cardiomyocytes en augmentant le nombre de canaux sodiques potentiel dépendants. Néanmoins après un sepsis chronique l’IL-13 perd ses effets sur le coeur. Dans la troisième étude, nous démontrons qu’une chimiothérapie, le docétaxel, est capable d’induire une atrophie musculaire associée à la libération de cytokines pro-inflammatoires et de diminuer la force de contraction du muscle strié squelettique. Néanmoins, l’ajout de dantrolène, un inhibiteur de canaux calciques, permet de prévenir ces effets délétères. Enfin dans la dernière étude, nous avons montré que l’inhibition de la voie des TGF-β peut avoir un effet bénéfique sur la dysfonction diaphragmatique lors du sepsis, en restaurant à la fois la masse musculaire et la force de contraction. / Muscular dysfunction is associated with a worsen outcome for different pathologies. During this thesis, we have focused on mediators of the inflammation, such as cytokines, and their effects on heart and striated muscle function. In the first study, we have shown that the TNF-α decreased the heart contractile force by PKC pathway activation, but that the membrane excitability remained unchanged. In the second study, we showed that IL-13 increased the heart contraction, by the activation of β2 adrenergic - PKA pathway, and increased the membrane excitability of cardiomyocytes associated to an increase of the number of channels at the membrane level. Nevertheless, IL-13 lost its effect on septic heart. In the third study, we showed that the chemotherapy, docetaxel, was able to induce muscle atrophy associated to the release of pro-inflammatory cytokines, and a decrease of the contractile force of the skeletal muscle. However, the addition of dantrolene, an inhibitor of calcium channels, can prevent these deleterious effects. For the last study, we showed that the inhibition of TGF-α pathway can have beneficial effects on diaphragm dysfunction during sepsis, by preserving both muscle mass and muscle contractile force.

Les cellules NK sont des lymphocytes de l'immunité innée impliqués dans la reconnaissance et l'élimination de cellules tumorales ou infectées par des pathogènes intracellulaires. La biologie des cellules NK est régulée par des facteurs intrinsèques comme les facteurs de transcription ainsi que par des facteurs environnementaux comme les cytokines, produites en condition homéostatique ou inflammatoire. Certaines cytokines, comme l'IL-15, l'IL-12 ou l'IL-18 sont connues pour potentialiser les fonctions effectrices des cellules NK. L'IL-15, en activant la voie STAT5 permet, d'une part, d'assurer la survie des cellules NK et, d'autre part via la kinase mTOR, d'induire leur prolifération, d'augmenter leur métabolisme ainsi que leurs fonctions effectrices. D'autres cytokines comme le TGF-ß sont connues pour inhiber les fonctions des cellules NK. Le TGF-ß1 est une des cytokines les plus immunosuppressives du système immunitaire et, en étant secrété´ par différents types de cancers, il participe a` l'échappement tumoral. Depuis longtemps, les effets du TGF-ß in vitro sont connus pour contrer ceux de l'IL-15. L'objectif de ce travail a été´ d'étudier les effets du TGF-ß sur la biologie des cellules NK. Nous avons observé´ que l'ajout de TGF-ß, in vitro, induit un blocage rapide de l'activation de la voie mTOR par l'IL-15, que le TGF-ß a des effets très proches de ceux de la rapamycine, un inhibiteur spécifique de mTOR et que, in vivo chez la souris, l'activation constitutive des voies de signalisation activées par le TGF-ß induit un phénotype proche de celui de la délétion de mTOR dans les cellules NK / NK cells are innate lymphocytes involved in the recognition and elimination of tumor or infected cells. The biology of NK cells is regulated by intrinsic factors such as transcription factors but also by cytokines produced at steady state or under inflammatory conditions. Some of these cytokines like IL-15, IL-12 or IL-18 are known to increase NK cells functions. IL-15 allows NK cell survival via STAT5 and, via mTOR, increase NK cell proliferation, metabolism and acquisition of functions. In the other hand, cytokines like TGF-ß are known to inhibit NK cell function. TGF-ß1 is a major immunosuppressive cytokine, often secreted by tumor cells and participates to tumor escape. The inhibitory effects of TGF-ß in vitro on IL-2/15 mediated NK cell activation have long been shown, but the mechanism remains unknown. The objective of this work was to characterize the effects of TGF-ß at a molecular level. We have observed that TGF-ß induces a rapid blockade of IL-15 induced mTOR activation, in vitro. TGF-ß and the mTOR inhibitor rapamycin have similar effects. Finally, using genetic mouse models in vivo, constitutive TGF-ß signaling or mTOR deletion results in similar developmental arrests in NK cells

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-12T15:08:18Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação_Luana Prado_digital.pdf: 1685713 bytes, checksum: e1e056c8dd45056eda6306999941d592 (MD5) / Made available in DSpace on 2017-07-12T15:08:18Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação_Luana Prado_digital.pdf: 1685713 bytes, checksum: e1e056c8dd45056eda6306999941d592 (MD5) Previous issue date: 2016-03-15 / CAPES / As úlceras maleolares (UMs) são manifestações cutâneas frequentes na anemia falciforme (AF). Cursam com alta taxa de recorrência, retardo na cicatrização e maior probabilidade de tornarem-se crônicas. Os mecanismos desencadeantes envolvem episódios hemolíticos e vaso-oclusivos, e mais recentemente, foram apontados polimorfismos em genes regulatórios da via do TGF-β como contribuintes deste processo. Polimorfismos nos genes que regulam esta via mostram-se alvos moleculares promissores na elucidação da fisiopatologia das UMs. Com isso, nosso objetivo foi investigar a relação de polimorfismos nos genes SMAD7 e SMURF1 na ocorrência de UMs em pacientes com AF. O estudo foi realizado em duas coortes independentes, composta por 331 pacientes de Pernambuco (131 com UM e 200 sem UM) e 197 pacientes do estado do Rio de Janeiro (108 com UM e 89 sem UM). As genotipagens dos polimorfismos SMAD7 C>T (rs736839) e SMURF1 C>G) (rs219825) foram realizadas por PCR em tempo real. Empregando o modelo de análise recessivo, foi encontrada associação entre os homozigotos variantes (TT; GG) e a maior ocorrência de UM nos pacientes das duas coortes avaliadas, para SMAD7 e SMURF1 em Pernambuco (P=0.001 e P=0.050) e Rio de Janeiro (P=0.029 e P=0.006). Ademais, avaliando a coorte de Pernambuco os pacientes com genótipo TT apresentaram uma maior taxa de desenvolvimento das UMs (70%) em relação aos com genótipos CC + CT (45%) (P<0,0001) para a SMAD7, e os pacientes com genótipo GG para SMURF1 apresentaram uma maior taxa de desenvolvimento das UMs (65%) em relação aos genótipos CC+CG (46%) (P=0.009). Sumariamente, nossos resultados mostram que os polimorfismos estudados estão envolvidos na ocorrência das UMs nas coortes de pacientes com anemia falciforme, mostrando-se como potenciais moduladores fenotípicos na doença falciforme. / Leg ulcers are one of the most common clinical manifestations of sickle cell anemia (SCA). They present a high rate of reoccurrence, delayed wound healing and a higher probability of becoming chronical. The triggering mechanisms involve hemolytic and vaso-occlusive episodes, and polymorphisms in regulatory genes of the TGF-β pathway were identified recently as contributors of this process. Polymorphisms in genes that regulate this pathway are considered as promising molecular targets in the elucidation of the pathophysiology of leg ulcers. Therefore, our aim was to investigate the relationship of the polymorphisms in SMAD7 and SMURF1 genes with the occurrence of leg ulcers in patients with SCA. The study was conducted in two independent cohorts consisting of 331 patients from Pernambuco (131 with leg ulcer and 200 without leg ulcer) and 197 patients from Rio de Janeiro (108 with leg ulcer and 89 without leg ulcer). The genotyping of SMAD7 C>T (rs736839) and SMURF1 C> G (rs219825) were performed by real time PCR. Using the recessive model of analysis, we found an association between homozygous variants (CC; GG) and the higher incidence of leg ulcers in patients from the two evaluated cohorts for SMAD7 and SMURF1 in Pernambuco (P = 0.001 and P = 0.050) and Rio de Janeiro (P = 0.029 and P = 0.006). Moreover, assessing the cohort of Pernambuco, patients with CC genotype had a higher rate of leg ulcers development (70%), compared to those with CC + CT genotypes (45%) (P <0.0001) for SMAD7, and patients with GG genotype for SMURF1 showed a higher rate of development of leg ulcers (65%) compared to CC + CG genotype (46%) (P = 0.009). In summary, our results demonstrated that the studied polymorphisms are involved in the occurrence of leg ulcers in cohorts of patients with sickle cell anemia, showing up as potential phenotypic modulators of sickle cell disease.

Le monoxyde d’azote (NO) est une molécule gazeuse synthétisée par les NO Synthases à partir de L-arginine. NO est une puissante molécule de signalisation dans de nombreux processus physiologiques comme la vasodilatation et la neurotransmission. Il module l’activité de multiples protéines (ex : guanylate cyclase soluble et ribonucléotide réductase) grâce à la nitrosylation de groupements thiol ou de métaux de transition. En tant que radical libre, NO peut réagir avec de nombreuses espèces comme l’oxygène moléculaire, et ainsi former des dérivés réactifs. Grâce à ces propriétés, NO est un acteur majeur de l’immunité innée et de l’inflammation. Les phagocytes produisent de grandes quantités de NO en réponse à des stimuli proinflammatoires, via l’activité NO Synthase inductible (iNOS). En raison des effets délétères des dérivés de NO, l’activité iNOS doit être finement régulée. Le suppresseur de tumeur p53 est capable de réprimer l’expression du gène Nos2 après avoir été lui-même activé en réponse à une accumulation de NO. La protéine p73 est un homologue de p53 encodé par un gène qui génère à la fois des isoformes actives (TAp73) et des isoformes qui sont dépourvues du domaine de transactivation N-terminal et exercent un effet dominant négatif (ΔNp73). Cette étude se focalise sur le rôle des isoformes TAp73 dans la régulation de l’expression de la iNOS. Nous démontrons que les isoformes TAp73 régulent négativement l’expression de la iNOS aux niveaux transcriptionnel et post-traductionnel en potentialisant l’effet répresseur du TGF-β, ce qui résulte en une forte surexpression de la iNOS dans les cellules TAp73-/-. Ces résultats confortent le rôle de la famille p53 comme un réseau essentiel de protéines régulatrices des fonctions du TGF-β. / Nitric oxide (NO) is a gaseous molecule synthesized from L-arginine by Nitric Oxide Synthases. NO acts as a potent signaling molecule in various physiological processes like vasorelaxation and neurotransmission. It modulates the activity of many proteins (e.g. soluble guanylate cyclase and ribonucleotide reductase) through nitrosylation of thiol moieties or transition metal ions. As a free radical, NO can also react with a number of cellular species, notably molecular oxygen, to form reactive oxygen species and reactive nitrogen species. Thanks to these properties, NO appears as a major component of innate immune response and inflammation. Phagocytes produce large amounts of NO in response to proinflammatory through inducible Nitric Oxide Synthase (iNOS) activity. Because of the harmful effects of NO derivatives on cellular components, iNOS activity needs to be tightly regulated. The p53 tumor suppressor has been shown to repress Nos2 after being activated by NO itself. The p73 protein is an homologous encoded by the TP73 gene that generate transcriptionally active TAp73 isoforms and ΔNp73 isoforms that lack the transactivation domain and exert a dominant negative effect. This study focuses on the role of TAp73 isoforms in regulation of iNOS expression. We demonstrate that TAp73 isoforms potentiate the repressive effect of TGF-β on iNOS expression at transcriptional and post-traductional levels, resulting in a substantial iNOS overexpression in TAp73-/- cells. These results emphasize the emerging role of p53 family as a master regulator of TGF-β functions.

Les maladies fibroprolifératives se caractérisent par l’accumulation de constituants de la matrice extracellulaire en réponse à une agression chronique et répétée conduisant à la destruction de l’architecture tissulaire. Les myofibroblaste, sous l’influence du TGFβ, représentent les cellules effectrices majoritaires dans ce processus. Les miARN, régulateurs négatifs de l’expression génique, interviennent dans de nombreux mécanismes physio-pathologiques dont la fibrose tissulaire mais leur mécanismes d’action et la synergie potentielle entre miARN co-régulés au sein d’un même cluster restent mal compris. Nos travaux ont consisté à caractériser l’implication du cluster miR-199/214 généré à partir du LncARN DNM3os, dans la fibrose pulmonaire. Nous avons montré que les miARN de ce cluster participent à l’activation et à la différenciation des fibroblastes en myofibroblastes via la régulation des voies canoniques et non canoniques du TGF-β. Ce cluster agit également en tant qu’inhibiteur de la réparation épithéliale. In vivo, l’inhibition de l’un de ces « fibromiRs », a permis, dans un modèle murin, de réduire significativement les lésions de fibrose. Par ailleurs, de par leur présence et leur stabilité dans les fluides biologiques, les miARN représentent également une nouvelle classe de biomarqueurs diagnostiques ou pronostiques non invasifs. Nous avons ainsi montré que les niveaux sériques de miR-21-5p étaient augmentés chez les patients présentant une fibrose rénale sévère. Ces travaux soulignent l’importance des miARN dans la pathogénèse des maladies fibroprolifératives et montrent qu’ils représentent de nouvelles cibles thérapeutiques ou des biomarqueurs non invasifs. / Fibrotic diseases are characterized by the accumulation of extracellular matrix components in response to chronic aggression leading to the destruction of tissue architecture. Myofibroblasts, controlled by TGFβ, are central effectors in this process. MiRNAs, negative regulator of gene expression, are involved in many patho-physiological mechanisms, including tissue fibrosis but their mechanisms of action and the potential synergistic activity between co-regulated miRNAs within the same cluster remain poorly understood. Our work consisted in characterizing the involvement of the miR-199/214 cluster, generated from LncRNA DNM3os, in pulmonary fibrosis. We have shown that these miRNAs are involved in the activation and the differentiation of fibroblasts to myofibroblasts through the regulation of canonical and non-canonical TGF-β pathways. This cluster also acts as an inhibitor of epithelial repair. In vivo, the inhibition of one of this “FibromiR”, significantly decrease fibrosis, in a murine lung fibrosis model. In addition, because of their presence and their stability in biological fluids, miRNAs also represent a new class of non-invasive diagnostic or prognostic biomarker. We showed that serum levels of miR-21-5p were increased in patients with severe kidney fibrosis. These studies highlight the importance of miRNAs in pathogenesis of fibrotic disorders and show that they represent new therapeutic targets or non-invasive biomarkers.

BAKGRUND: I Sverige är cancer en av de vanligaste sjukdomarna. Bröstcancer är den vanligaste cancersjukdomen hos kvinnor. Bröstcancer kan sprida sig i kroppen och bilda en sekundär cancer som kallas metastas. Transformerande tillväxtfaktor-β-receptorer (TGF-β-receptorer) har en viktig roll i tumörutveckling. SYFTE: Syftet med denna studie är att studera rollen av TGF-β-receptorer i cancer, särskild bröstcancer med fokus på metastaserna. METOD: Den är en litteraturstudie, som är baserad på sex vetenskapliga artiklar. Dessa artiklar hämtas från databas PubMed genom användning av olika specifika sökord. RESULTAT: Resultat visar att TGF-β-receptorer har två motsatta rollar i cancerutveckling. Första fungerar TGF-β-receptorer som en tumörundertryckare i början av cancerutveckling. Däremot bidrar TGF-β-receptorer till utveckling av maligna celler lite senare under cancerutveckling. Det gör det genom att förbättra metastatiska potential samt undertrycka antitumörimmunitet. Metastaser bildas via Epithelial to mesenchymal transition med hjälp av vissa andra faktorer såsom cancerassocierade fibroblaster. Epithelial to mesenchymal transition och andel av cancerassocierade fibroblaster kan stimuleras av TGF-β-receptorer, som kan i sin tur leda till ökning av metastaseringen. Ett exempel på metastas är skelettcancer. DISKUSSION: Transformerande tillväxtfaktor-β receptorer, som kan påverka cancer, betraktas som ett bra fynd i läkemedelsutveckling mot cancer. TGF-β-receptorer har en viktig roll i tumörutveckling, särskilt sekundär cancer. Sekundär cancer/metastas är farligare än primär cancer, därför är det bättre att försöka förstå hela mekanismen bakom TGF-β-receptorer på ett tydligt sätt, för att kunna behandla patienter i god tid. SLUTSATS: Transformerande tillväxtfaktor- β -receptorer är en av de viktigaste parametrarna för att få den optimala effekten av medicineringen mot cancer.