La scoliose est la déformation de la colonne vertébrale la plus répandue. Elle atteint 3 à 4% de la population pédiatrique et dans 85% des cas, aucune cause n’a été identifiée. Ces cas sont appelés idiopathiques et les symptômes apparaissent durant la puberté; d’où le terme de ‘scoliose idiopathique de l’adolescent (SIA). Cette pathologie atteint le plus souvent les jeunes filles, en nombre et en sévérité. Ces dernières années, plusieurs hypothèses ont été proposées afin d’élucider l’étiologie de cette pathologie. Celles-ci ont mis de l’avant différents facteurs génétiques, biochimiques, mécaniques, neurologiques, musculaires ou hormonaux. Plusieurs études ont rapporté des formes familiales de scoliose, soutenant la thèse d’une prédisposition génétique. Nous avons démontré que les patients souffrant de SIA présentent un défaut de signalisation cellulaire médiée par les protéines Gi et un taux élevé d’ostéopontine (OPN) circulante. En utilisant une approche de type ‘gène candidat’, nous avons montré que la protéine tyrosine phosphatase μ (PTPμ) régule l’activité du complexe d’intégrines α5/β1 (récepteur de l’OPN) via la protéine kinase PIPKIγ. Dans ce but, nous avons utilisé des cultures primaires d’ostéoblastes issues de biopsies de patients et de cas traumatiques comme sujets contrôles. Les biopsies osseuses de patients ont été obtenues lors de l’intervention chirurgicale à partir des vertèbres T3 à L4, selon les différentes procédures. Les biopsies issues de cas traumatiques proviennent d’autres types d’os (tibia, crête iliaque, fémur). Les profils d’expression du gène PTPRM (codant pour la protéine PTPμ) ont été étudiés par PCR quantitative (qPCR). Les taux de protéines PTPμ ont été analysés par immunoprécipitation suivi d’un western blot. Pour évaluer le rôle de cette protéine, nous avons bénéficié d’un modèle murin. Machida et al. ont démontré qu’il existe un taux plus élevé de scoliose parmi les souris C57Bl/6 bipèdes obtenues suite à l’amputation des membres supérieurs, sous anesthésie, cinq semaines après la naissance. Nous avons utilisé des cultures primaires d’ostéoblastes issues de la colonne ii vertébrale de souris C57Bl/6 bipèdes, délétées du gène PTPRM (souris dites ‘KO’), afin d’évaluer le niveau de signalisation cellulaire spécifique des protéines Gi par un test fonctionnel: la technique de spectroscopie cellulaire di-électrique (SCD). Selon nos données, 85% des souris bipédales ‘KO’ pour le géne PTPRM développent une scoliose (modérée à sévère) contre 55% des souris contrôles C57Bl6 bipèdes. De plus, les niveaux de PTPμ exprimée par les ostéoblastes de 34 patients SIA se trouvent diminués par comparaison à 17 sujets contrôles. Nos études de souris bipèdes ont montré que l’inactivation du gène PTPRM augmente l’incidence et la sévérité de la scoliose, sans pour autant affecter les taux circulant d’OPN ou l’expression de ses récepteurs. Par ailleurs, dans ce même contexte, nous avons remarqué une augmentation de l’interaction entre l’OPN et l’intégrine β1 en l’absence du gène PTPRM. Les cellules issues de ces souris bipèdes KO montrent une réduction dans leurs niveaux de signalisation cellulaire médiée par les protéines Gi après stimulation par l’OPN. Cette diminution est en grande partie récupérée après traitement des cellules par un siRNA spécifique de la protéine PIPK1γ, substrat de PTPμ qui favorise la fixation de ligands aux intégrines. Ces études apportent les premières indications que la perte d’expression de PTPμ est impliquée dans le développement de la SIA, en amplifiant probablement l’effet inhibiteur de l’OPN sur la signalisation cellulaire médiée par les protéines Gi. Ces études permettent une meilleure compréhension de l’étiologie de la SIA. Elles pourraient avoir une contribution importante dans le développement futur de méthodes diagnostique et thérapeuthique dans le but d'arrete l’apparition et l’évolution de la maladie chez les enfants atteints. / Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis that affects 3-4% of the global pediatric population. In more than 85% of cases, no specific cause can be identified. Such cases are called idiopathic and occur mostly during adolescence. AIS affects mainly females in number and severity. Over the past years, many hypotheses were proposed to explain the etiology of AIS, including genetic, biochemical, mechanics, neurological, muscular and hormonal factors. Several studies have reported a high incidence of scoliosis in some families, which argues for a genetic cause of this disease. We demonstrated that AIS patients have a Gi protein signaling defect and exhibit high levels of circulating Osteopontin (OPN). The goal of this thesis is to identify the mechanisms regulating OPN signaling activity in AIS patients. We have used a candidate gene driven approach and discovered that protein tyrosine phosphatase μ (PTP μ) regulates α5β1 integrin (a known OPN receptor) through phosphate kinase type 1 gamma (PIPK1γ). To achieve our goal, we have used primary osteoblast cell cultures derived from AIS patients and biopsies from control subjects. Bone specimens were obtained intraoperatively from vertebras (varying from T3 to L4 according to the surgical procedure performed) while with trauma cases used as non-scoliotic controls, bone specimens were obtained from other anatomical sites (tibia, femur or iliac crest). Expression profiles of the RPTPM gene (encoding for PTPμ) were studied by qPCR. On the other hand, PTPμ protein levels were determined by immunoprecipitation followed by western blot. To evaluate the role of this protein in AIS etiopathogenesis, we took advantage of an animal model exhibiting a higher scoliosis incidence when maintained in a bipedal state. [1], [2] Bipedal surgeries were performed on C57Bl/6 mice after weaning (5-weeks after birth) by amputation of the forelimbs and tail under anesthesia as reported by Oyama et al. (2006). [1] We used the same approach with PTPμ knockout mice and primary osteoblast culture were derived from the spine of these mice to assess Gi protein signaling through a functional assay termed Cellular Dielectric Spectroscopy (CDS). Bipedal PTPμ knockout mice develop scoliosis more often (85%) in number and severity, than control C57Bl/6 bipedal mice (55%). Interestingly, functional analysis of osteoblasts derived from PTPμ KO mice by CDS method showed a flaw in the transmission of Gi protein coupled receptor signaling similar to a specific AIS patient subgroup. Furthermore, the clinical relevance of PTPμ was strengthened by the fact that a decrease in the gene expression level of PTPμ was observed in 34 AIS patients when compared to 17 control subjects. Such a decrease was also confirmed at the protein level. We demonstrated that genetic deletion of PTPμ enhances the incidence and severity of scoliosis without affecting plasma levels of OPN or the expression of its receptors. In contrast, increased interaction of OPN with β1 integrin was noticed in cells depleted of PTPμ. Furthermore, reduction of Gi- protein coupled receptor GiPCR signaling by OPN was also enhanced in these cells, while their response to GiPCR stimulation was improved with siRNA of phosphatidylinositol-phosphate kinase type 1 gamma (PIPK1γ), a PTPμ substrate that favours ligand binding to integrins. These studies provide the first indication that the loss of PTPμ influences the nature of idiopathic scoliosis, possibly by amplifying the inhibitory effect of OPN on GiPCR signaling. This study allows a better understanding of AIS etiology and could have an impact for the future development of innovative diagnostic methods and eventual pharmacological approaches in order to prevent AIS and stop its progression in affected children.

L’angiogenèse et l’augmentation de la perméabilité vasculaire sont des éléments clés pour la croissance et la progression tumorale. Par conséquent, de nombreux efforts sont déployés à comprendre les mécanismes moléculaires impliqués dans la formation et le remodelage des vaisseaux sanguins de manière à identifier de nouvelles cibles thérapeutiques potentielles. De cette optique, les travaux de cette thèse se sont concentrés sur la protéine tyrosine phosphatase DEP-1, initialement identifiée comme un régulateur négatif de la prolifération et de la phosphorylation du VEGFR2 lorsque fortement exprimée dans les cellules endothéliales. Toutefois, en utilisant une approche d’ARNi, il a été démontré que via sa capacité à déphosphoryler la tyrosine inhibitrice de Src (Y529), DEP-1 était également un régulateur positif de l’activation de Src dans les cellules endothéliales stimulées au VEGF. Puisque Src joue un rôle central dans la promotion de l’angiogenèse et la perméabilité vasculaire, nous avons en plus démontré que DEP-1 était un promoteur de ces fonctions in vitro et que la tyrosine phosphorylation de sa queue C-terminale, permettant l’interaction et l’activation de Src, était requise. Les travaux de recherche présentés dans cette thèse démontrent dans un premier temps à partir d’une souris Dep1 KO, dont le développement ne présente aucun phénotype apparent, que la perte de l’expression de DEP-1 se traduit en une inhibition de l’activation de Src et de l’un de ses substrats, la VE-Cadherine, en réponse au VEGF chez la souris adulte. Nos résultats démontrent donc, pour la première fois, le rôle primordial de DEP-1 dans l’induction de la perméabilité vasculaire et de la formation de capillaires in vivo. Conséquemment, la croissance tumorale et la formation de métastases aux poumons sont réduites due à une inhibition de leur vascularisation ce qui se traduit par une diminution de la prolifération et une augmentation de l’apoptose des cellules cancéreuses. De façon intéressante, l’expression élevée de DEP-1 dans les vaisseaux sanguins tumoraux de patientes atteintes du cancer du sein corrèle avec une vascularisation accrue de la tumeur. En plus du rôle de DEP-1 dans la réponse angiogénqiue à l’âge adulte, nos travaux ont également démontré le rôle important de DEP-1 lors de la vascularisation de la rétine, un modèle in vivo d’angiogenèse développementale. Dans ce contexte, DEP-1 inhibe la prolifération des cellules endothéliales et limite leur bourgeonnement et la complexification du réseau vasculaire rétinien en permettant l’expression adéquate du Dll4, un régulateur crucial de l’organisation de la vascularisation développementale. Cette expression du Dll4 découlerait de la stabilisation de la β-caténine par l’inactivation de la GSK3β, un régulateur important de la dégradation de la β-caténine, en réponse au VEGF selon la voie de signalisation VEGFR2-Src-PI3K-Akt-GSK3β. Ainsi, ces travaux identifient DEP-1 comme un régulateur important de l’organisation vasculaire rétinienne. Les rôles positifs de DEP-1 dans les cellules endothéliales découlent principalement de sa capacité à lier et activer la kinase Src. En plus de contribuer à la réponse angiogénique, Src est également un oncogène bien caractérisé notamment pour sa contribution au programme invasif des cellules cancéreuses mammaires. Les travaux de cette thèse illustrent que DEP-1 est préférentiellement exprimée dans les cellules cancéreuses mammaires invasives et qu’il régule l’activation de Src, de voies de signalisation invasives et, par le fait même, de l’invasivité de ces cellules in vitro et in vivo. De façon intéressante, ces observations corrèlent avec des données cliniques où l’expression modérée de DEP-1 est associée à un mauvais pronostic de survie et de rechute. Ces résultats démontrent donc, pour la première fois, le rôle positif de DEP-1 dans l’activation de Src au niveau des cellules endothéliales et des cellules cancéreuses mammaires ce qui permet la régulation du bourgeonnement endothélial, de la perméabilité vasculaire, de l’angiogenèse normale et pathologique en plus de l’invasion tumorale. / Angiogenesis and increased vascular permeability are key component of tumor growth and progression. Consequently, numberous efforts are currently deployed to illucidate the molecular mecanisms contributing to the formation and remodelling of blood vessels in oder to identify new potential therapeutic targets. In that thought, the work of this thesis was focused on the protein tyrosine phosphatase DEP-1, initially identified as a negative regulator of proliferation and VEGFR2 phosphorylation when highly expressed in endothelial cells. However, using RNAi, it was shown that through its capacity to dephosphorylate the inhibitory tyrosine of Src (Y529), DEP-1 could also positively regulate Src activation in endothelial cells in response to VEGF. As Src is a central promoter of angiogenesis and vascular permeability, we showed that DEP-1 was a promoter of these vascular functions in vitro and that the tyrosine phosphorylation of its C-terminal tail, allowing interaction and activation of Src, was required. Interestingly, the catalytic inactivation of DEP-1 in mice resulted in increased proliferation in endothelial cells, but also in desorganization of vascular structures which contrast the absence of phenotype in DEP-1 complete knock-out mice (KO). The work of this thesis demonstrates for the first time that DEP-1 deletion causes inhibition of Src and one of its substrate, VE-Cadherin, activation in response to VEGF in Dep-1 KO mice, which develop normally. Our results show the crucial role of DEP-1 in VEGF-induced vascular permeability and capillary formation in vivo. Consequently, tumor growth and lung metastases formation were inhibited due to reduced tumor vascularisation causing reduced proliferation and increased apoptosis of tumor cells. Accordingly, high expression of DEP-1 in tumor-associated blood vessels of breast cancer patients correlates with greater tumor vascularisation. In addition to DEP-1 role in post-natal angiogenic response, our work also demonstrates the important role of DEP-1 during retinal vascularisation, an in vivo developmental angiogenesis model. In this context, DEP-1 inhibits proliferation of endothelial cells and limits their sprouting by allowing adequate β-catenin-dependant expression of Dll4, a Notch ligand regulating developmental vascularisation organisation. DEP-1 allows β-catenin stabilisation via inactivation of GSK3β, an important regulator of β-catenin degradation, in response to VEGF through VEGFR2-Src-PI3K-Akt-GSK3β signaling pathway. Thus, this work identifies DEP-1 as an important regulator of retinal vascular sprouting. The positive roles of DEP-1 in endothelial cells depend on it ability to bind and activate the kinase Src. In addition to its contribution to angiogenic response, Src is also a well-characterized oncogene notably for its contribution to invasive program of mammary cancer cells. This work illustrates that DEP-1 is preferentially expressed in invasive mammary cancer cells and that it regulates Src activation, pro-invasive signaling pathways and, consenquetly, cell invasiveness in vitro and in vivo. All these activities are dependant on DEP-1 catalytic activity and ability to bind Src. Interestingly, these results correlate with clinical data where moderate expression of DEP-1 is associated with poor pornostic of survival and relapse. Collectively, the results presented here demonstrate, for the first time, the crucial role of DEP-1 in Src activation in endothelial and breast cancer cells, leading to endothelial sprouting, vascular permeability, normal or pathological angiogenesis, and breast cancer invasiveness and metastases formation.

Deregulierung des RAS/MAPK Signalwegs führen nicht nur zur Krebsentstehung, sondern sind auch mitverantwortlich für Entwicklungsstörungen, dieals Keimbahnmutationen in Schlüsselregulatoren des MAPK Signalwegs zurückzuführen sind, werden aufgrund überlappender Phänotypen unter dem Begriff RASopathien subsumiert. Obwohl die Inzidenz für solide Tumore bei diesen Patienten gering ist, wird ein Zusammenhang zum Auftreten verschiedener Leukämieformen deutlich. Im Rahmen dieser Arbeit wurden Mutationen zweier Schlüsselregulatoren des MAPK Signalwegs, PTPN11 und BRAF, hinsichtlich ihrer Fähigkeit zur neoplastischen Transformation analysiert. Zur Durchführung funktioneller Assays wurden Zelllinien mit lentiviraler Vektoren mit NS-, LS- oder NS und Leukämie-assoziierten SHP2 oder CFC-assoz. BRAF Mutationen (Mut), generiert. Die Testung des neoplastischen Potentials erfolgte anhand nicht-tumorigenen humanen sowie an 208F Ratten-Zelllinie. SHP2/BRAF-Mutationen haben eine spindel-ähnliche Zellmorphologie, Proliferation sowie das Dichte- und Anker-unabhängiges Wachstum in 208F gefördert. Diese Ergebnisse sprechen dafür, dass RASopathie-assoziierte Mutationen zu einem Transformationsphänotyp führen können, ähnlich wie die klassischen Ras Onkogenen. Um zu testen, ob Mut-SHP2 das in vivo Tumorwachstum beeinflusst, wurden SHP2-208F-Zellen in Nacktmäuse injiziert. Eine Förderung des Tumorwachstums konnte sowohl durch mut- als auch durch wt-SHP2 beobachtet werden. RASopathie-assoziierte mutierte Proteine führten auch zu einer moderaten Aktivierung des MAPK-Signalwegs. Eine erhöhte Bindungsstärke zu GAB1 konnte mittels ein TAP-Assay ermittelt werden. Auf transkriptioneller Ebene konnte einer Gensignatur, die sowohl durch RASopathien als auch der klassischen onkogenen BRAF identifiziert werden.Die Ergebnisse dieser Studie können für ein besseres Verständnis der Downstream-liegenden Mechanismen von RASopathie-bezogenen Signalwegen und ihrer Beteiligung an der Tumorprogression beitragen / Deregulation of the Ras/MAPK signaling is implicated in a variety of human diseases, including cancer and developmental disorders. The RASopathies are characterized by an overlapping phenotype in patients and result from germline mutations in key regulators of the MAPK signaling cascade. Although the incidence of solid tumors is rather low, reports on different leukemia forms have increased. In this work, a group of mutations in the genes PTPN11 and BRAF were selected for expression in cell lines for a comprehensive molecular and phenotypic characterization. Non-tumorigenic human cell lines and the rat 208F fibroblasts were transduced with lentiviral particles with SHP2/BRAF wildtype (wt), Noonan (NS)-, NS- and leukemia- or LS–associated SHP2 mutations (mut) and CFC-associated BRAF mutations to identify their potential roles in neoplastic transformation. Mutations in both genes promoted cell morphology alterations, cell proliferation, density- and anchorage-independent growth in rat fibroblasts. These results suggested that RASopathies-associated mutations in both genes confer a transformation phenotype in vitro similar to the classical oncogenes. To investigate whether mutations in SHP2 contribute to tumor growth in vivo, 208F cells expressing wt/mut SHP2 were injected in nude mice. Both wt/mut SHP2 expressing cells promoted tumor growth. Additionally, RASopathies-associated mutant SHP2 and BRAF proteins constitutively activate the MAPK signaling in a moderate manner compared to oncogenic BRAF. To identify modifications in the protein interaction of mut-SHP2, TAP assays were performed. Mut-SHP2 proteins showed an increased binding strength to GAB1 compared to wt. Finally, a microarray analysis revealed a gene cluster commonly regulated in both RASopathies and the oncogenic BRAF. The findings of this work might be useful for a better understanding of the downstream mechanisms of RASopathies-related signaling and their involvement in cancer progression.

A síndrome de Noonan (SN), caracterizada por baixa estatura, aspectos dismórficos e cardiopatia congênita, foi associada ao gene PTPN11. Estudamos o PTPN11 em pacientes com SN, pais de portadores de mutação e crianças com baixa estatura idiopática (BEI) que apresentam estigmas sugestivos da SN, sem critérios suficientes para o diagnóstico. Encontramos mutações missense em heterozigoze no PTPN11 em 42,3% dos pacientes com SN. Não identificamos alterações nos pais de portadores de mutação no PTPN11 com fenótipo normal tampouco em crianças com BEI. A única diferença estatisticamente significante entre os grupos com e sem mutação foi a resposta em longo prazo ao hGH, melhor no grupo sem mutação / Noonan syndrome (NS), characterized by short stature, dysmorphic facial and thoracic features and congenital heart disease, was associated to PTPN11 gene. We studied the PTPN11 in patients with NS, parents of mutation-positive NS patients and idiopathic short stature children with signs related to NS without fulfilling the diagnostic criteria. We found missense mutations in 42.3% of the NS group. Parents of NS mutation-positive patients did not present mutations, nor did children with short stature. The only statistically significant difference between groups with and without mutations was response to long term use of hGH, better on the mutation-negative group

INTRODUÇÃO: A síndrome de Noonan é uma doença autossômica dominante caracterizada por baixa estatura, dismorfismos faciais (hipertelorismo ocular, inclinação para baixo das fendas palpebrais, ptose palpebral, palato alto e má-oclusão dentária), pescoço curto e/ou alado, defeitos cardíacos, principalmente a estenose pulmonar valvar, deformidade esternal e criptorquia nos pacientes do sexo masculino. O gene PTPN11, localizado no braço longo do cromossomo 12 (12q24.1), é responsável por aproximadamente 50% dos casos de síndrome de Noonan. OBJETIVO: Detectar a freqüência de mutações no gene PTPN11 em uma amostra de pacientes os quais preenchiam os critérios clínicos para a síndrome de Noonan e síndromes Noonan-like e estabelecer uma correlação genótipo-fenótipo. MÉTODOS: Cinqüenta probandos com síndrome de Noonan, 3 com síndrome de LEOPARD, 3 com síndrome de Noonan-like/lesões múltiplas de células gigantes e 2 com neurofibromatose-Noonan foram incluídos nesse estudo. O estudo molecular foi realizado através da técnica da cromatografia líquida de alta precisão desnaturante e, naqueles com um perfil anormal, a técnica do seqüenciamento do éxon em questão foi concretizada. RESULTADOS: Mutações missense no gene PTPN11 foram identificadas em 21 probandos com síndrome de Noonan (42%), em todos os três pacientes com a síndrome de LEOPARD, em um caso com síndrome de Noonan-like/lesões múltiplas de células gigantes e em um paciente com síndrome da neurofibromatose-Noonan. Este último probando também apresentava uma mutação no gene NF1. A única anomalia que atingiu uma diferença estatisticamente significante quando comparados os grupos de pacientes com e sem mutação foi o grupo de distúrbios hematológicos. Um paciente com síndrome de Noonan que apresentou uma doença mieloproliferativa possuía a mutação T73I. CONCLUSÃO: A síndrome de Noonan é uma doença heterogênea, uma vez que mutações no gene PTPN11 são responsáveis por 42% dos casos. Uma correlação genótipo-fenótipo definitiva não foi estabelecida, mas a mutação T73I parece predispor a distúrbios mieloproliferativos. Com relação às síndromes Noonan-like, o gene PTPN11 é o principal responsável pela síndrome de LEOPARD e também desempenha um papel na síndrome da neurofibromatose-Noonan. A síndrome de Noonan-like/lesões múltiplas de células gigantes, a qual faz parte do espectro da síndrome de Noonan, é também uma doença heterogênea. / INTRODUCTION: Noonan syndrome is an autosomal dominant disorder comprising short stature, facial dysmorphisms (ocular hypertelorism, downslanting palpebral fissures, palpebral ptosis, high arched palate and dental malocclusion), short and/or webbed neck, heart defects, mainly valvar pulmonary stenosis, sternal deformity and cryptorchidism in males. The PTPN11 gene, localized in the long arm of chromosome 12 (12q24.1), is responsible for approximately 50% of the cases. OBJECTIVE: To detect the PTPN11 gene mutation rate in a cohort of clinically well-characterized patients with Noonan and Noonan-like syndromes and to study the genotype-phenotype correlation. METHODS: Fifty probands with Noonan syndrome ascertained according to well-established diagnostic criteria, 3 with LEOPARD syndrome, 3 with Noonan-like/multiple giant cell lesion syndrome and 2 with neurofibromatosis/Noonan were enrolled in this study. Mutational analysis was performed using denaturing high-performance liquid chromatography followed by sequencing of amplicons with an aberrant elution profile. RESULTS: Missense mutations in the PTPN11 gene were identified in 21 probands with Noonan syndrome (42%), in all three patients with LEOPARD syndrome, in one case with Noonan-like/multiple giant cell lesion syndrome and in one with neurofibromatosis-Noonan syndrome. This last patient also showed a NF1 gene mutation. The only anomaly that reached statistical significance when comparing probands with and without mutations was the hematological abnormalities. A Noonan syndrome patient presenting a myeloproliferative disorder showed a T73I mutation. CONCLUSION: Noonan syndrome is a heterogeneous disorder, once PTPN11 gene mutations is responsible for 42% of the cases. A definitive genotype-phenotype correlation is not established, but the T73I mutation seems to predispose to a myeloproliferative disorder. Regarding Noonan-like syndromes, the PTPN11 gene is the main one in LEOPARD syndrome and also plays a role in neurofibromatosis-Noonan syndrome. Noonan-like/multiple giant cell lesion syndrome, part of the spectrum of Noonan syndrome, is also heterogeneous.

INTRODUÇÃO: A síndrome de Noonan é uma doença autossômica dominante caracterizada por baixa estatura, dismorfismos faciais (hipertelorismo ocular, inclinação para baixo das fendas palpebrais, ptose palpebral, palato alto e má-oclusão dentária), pescoço curto e/ou alado, defeitos cardíacos, principalmente a estenose pulmonar valvar, deformidade esternal e criptorquia nos pacientes do sexo masculino. O gene PTPN11, localizado no braço longo do cromossomo 12 (12q24.1), é responsável por aproximadamente 50% dos casos de síndrome de Noonan. OBJETIVO: Detectar a freqüência de mutações no gene PTPN11 em uma amostra de pacientes os quais preenchiam os critérios clínicos para a síndrome de Noonan e síndromes Noonan-like e estabelecer uma correlação genótipo-fenótipo. MÉTODOS: Cinqüenta probandos com síndrome de Noonan, 3 com síndrome de LEOPARD, 3 com síndrome de Noonan-like/lesões múltiplas de células gigantes e 2 com neurofibromatose-Noonan foram incluídos nesse estudo. O estudo molecular foi realizado através da técnica da cromatografia líquida de alta precisão desnaturante e, naqueles com um perfil anormal, a técnica do seqüenciamento do éxon em questão foi concretizada. RESULTADOS: Mutações missense no gene PTPN11 foram identificadas em 21 probandos com síndrome de Noonan (42%), em todos os três pacientes com a síndrome de LEOPARD, em um caso com síndrome de Noonan-like/lesões múltiplas de células gigantes e em um paciente com síndrome da neurofibromatose-Noonan. Este último probando também apresentava uma mutação no gene NF1. A única anomalia que atingiu uma diferença estatisticamente significante quando comparados os grupos de pacientes com e sem mutação foi o grupo de distúrbios hematológicos. Um paciente com síndrome de Noonan que apresentou uma doença mieloproliferativa possuía a mutação T73I. CONCLUSÃO: A síndrome de Noonan é uma doença heterogênea, uma vez que mutações no gene PTPN11 são responsáveis por 42% dos casos. Uma correlação genótipo-fenótipo definitiva não foi estabelecida, mas a mutação T73I parece predispor a distúrbios mieloproliferativos. Com relação às síndromes Noonan-like, o gene PTPN11 é o principal responsável pela síndrome de LEOPARD e também desempenha um papel na síndrome da neurofibromatose-Noonan. A síndrome de Noonan-like/lesões múltiplas de células gigantes, a qual faz parte do espectro da síndrome de Noonan, é também uma doença heterogênea. / INTRODUCTION: Noonan syndrome is an autosomal dominant disorder comprising short stature, facial dysmorphisms (ocular hypertelorism, downslanting palpebral fissures, palpebral ptosis, high arched palate and dental malocclusion), short and/or webbed neck, heart defects, mainly valvar pulmonary stenosis, sternal deformity and cryptorchidism in males. The PTPN11 gene, localized in the long arm of chromosome 12 (12q24.1), is responsible for approximately 50% of the cases. OBJECTIVE: To detect the PTPN11 gene mutation rate in a cohort of clinically well-characterized patients with Noonan and Noonan-like syndromes and to study the genotype-phenotype correlation. METHODS: Fifty probands with Noonan syndrome ascertained according to well-established diagnostic criteria, 3 with LEOPARD syndrome, 3 with Noonan-like/multiple giant cell lesion syndrome and 2 with neurofibromatosis/Noonan were enrolled in this study. Mutational analysis was performed using denaturing high-performance liquid chromatography followed by sequencing of amplicons with an aberrant elution profile. RESULTS: Missense mutations in the PTPN11 gene were identified in 21 probands with Noonan syndrome (42%), in all three patients with LEOPARD syndrome, in one case with Noonan-like/multiple giant cell lesion syndrome and in one with neurofibromatosis-Noonan syndrome. This last patient also showed a NF1 gene mutation. The only anomaly that reached statistical significance when comparing probands with and without mutations was the hematological abnormalities. A Noonan syndrome patient presenting a myeloproliferative disorder showed a T73I mutation. CONCLUSION: Noonan syndrome is a heterogeneous disorder, once PTPN11 gene mutations is responsible for 42% of the cases. A definitive genotype-phenotype correlation is not established, but the T73I mutation seems to predispose to a myeloproliferative disorder. Regarding Noonan-like syndromes, the PTPN11 gene is the main one in LEOPARD syndrome and also plays a role in neurofibromatosis-Noonan syndrome. Noonan-like/multiple giant cell lesion syndrome, part of the spectrum of Noonan syndrome, is also heterogeneous.

Although considered as a poison for long time, the importance of selenium as an essential trace element is now well recognized. In proteins, the redox active selenium moiety is incorportated as selenocysteine (Sec), the 21st amino acid. In mammals, selenium exerts its redox activities through several selenocysteine-containing enzymes, which include glutathione peroxidase (GPx), iodothyronine deiodinase (ID) and thioredoxin reductase (TrxR). Although these enzymes have Sec in their active sites, they catalyze completely different reactions and their substrate specificity and cofactor or co-substrate systems are significantly different. The most widely studied selenoenzyme GPx protects various organisms from oxidative stresses by catalyzing the reduction of hydroperoxides by using glutathione (GSH) as cofactor. The chemical aspects of the reduction of hydroperoxide by GPx have been extensively studied with the help of synthetic selenium and tellurium compounds. For example, 2-phenyl, 1, 2-benzoisoselenazol-3(2H)-one, commonly known as ebselen exhibits significant GPx activity by using GSH as cofactor. The anti-inflammatory, antiatherosclerotic and cytoprotective properties of ebselen have led to the design and synthesis of nex GPx mimics for potential therapeutic applications. In the first chapter, the importance of selenium in biochemistry in general and the function of selenoenzyme GPx and its synthetic mimics in particular are discussed. In the second chapter, the importance of ebselen as a GPx mimic and how thiol exchange reaction in the selenenyl sulfide intermediate deactivates its catalytic cycle and the possible ways to overcome thiol exchange reaction are described. The third chapter deals with the first synthetic chemical model that effectively mimics the unusual cyclization of sulfenic acid to a sulfenyl amide in protein Tyrosien Phosphatase 1B(PTP1B). PTP1B is a cysteine containing enzyme where the sulfenic acid (PTP1B-SOH) intermediate produced in response to its oxidation by H2O2 is rapidly converted into a sulfenyl amide species, in which sulfur atom of the catalytic cysteine is covalently bonded to the main chain nitrogen of an adjacent serine residue. This unusual protein modification in PTP1B has been proposed to protect the sulfur centre from irreversible oxidation to sulfinic acid and and sulfonic acids. In the fourth chapter, it is shown that not only the catalytic efficiency of ebselen but also its phosphatase like behavior is important for its antioxidant activity. Ebselen is regenerated from selenenic acid (R-SeOH) under a verity of conditions, which protects its selenium centre from irreversible oxidation and thus reduces its toxicity. The fifth chapter deals with spirodizaselenurane and Spirodiazatellurane. Although the chemistry of spirodioxyselenuranes and spirodiazasulfuranes has been studied extensively due to their interesting structural and stereochemical properties, there is no example of stable spirodiazaselenurane and its tellurium analogues. In the fifth chapter, the synthesis, structure and GPx-like activity of the spirodizzaselenurane and spirodiazatellurane are discussed. In summary, the synthetic sulfenic acids and seleneric acids undergo cyclization to their corresponding sulfenyl amides and selenenyl amides and thus protect their sulfur and selenium centers from irreversible inactivation. We have also observed that selenoxides and telluroxides with nearby amide moieties undergo cyclization to their corresponding cyclic spiro compounds. This unusual transformation of sulfenic acids has been recently discovered in PTP1B. As the redox regulation cycle of PTP1B and the catalytic cycle of GPx are similar we believe that GPx may involve a selenenyl amide intermediate in its catalytic cycle.

H HARP (heparin-affin regulatory peptide), γνωστή και ως πλειοτροπίνη (PTN), είναι ένας 18 kDa αυξητικός παράγοντας, ο οποίος έχει υψηλή συγγένεια για την ηπαρίνη. Η HARP έχει πολλαπλές βιολογικές δράσεις, όπως συμμετέχει στη ρύθμιση του κυτταρικού πολλαπλασιασμού, στη μετανάστευση και τη διαφοροποίηση. Επιπλέον η έκφραση της σχετίζεται με την φυσιολογική και καρκινική αγγειογένεση in vitro και in vivo. Στην παρούσα εργασία μελετήθηκε η έκφραση της HARP και των υποδοχέων της, ALK και RPTPβ/ζ, στις διάφορες ημέρες ανάπτυξης της CAM εμβρύου όρνιθας. Επίσης, μελετήθηκε η μείωση της έκφρασης της ενδογενούς HARP, με πλασμίδιο που φέρει την αντινοηματική αλληλουχία (AS-HARP), στην αγγειογένεση in vivo, στη φωσφορυλίωση των Εrk1,2 και στη λεμφαγγειογένεση της CAM εμβρύου όρνιθας. Ανάλυση κατά Western και RT-PCR στις διάφορες ημέρες ανάπτυξης του εμβρύου έδειξε ότι η έκφραση της HARP συμβαδίζει με τη δημιουργία νέων αγγείων στη CAM, ενώ η έκφραση των υποδοχέων της HARP στην CAM φαίνεται να είναι αυξημένη στα πρώτα στάδια ανάπτυξης του ιστού. Επίσης, η μείωση της έκφρασης της HARP μετά τη χορήγηση του πλασμιδίου AS-HARP, μείωσε τα επίπεδα της πρωτεΐνης, το μήκος των αγγείων και τη φωσφορυλίωση των Erk1/2 στο in vivo μοντέλο της CAM εμβρύου όρνιθας. Αντίθετα, η μείωση της έκφρασης της HARP μετά τη χορήγηση του πλασμιδίου AS-HARP, δεν επηρέασε τη λεμφαγγειογένεση της CAM εμβρύου όρνιθας. Σαν τελικό συμπέρασμα προκύπτει ότι η έκφραση της ενδογενούς HARP στην CAM εμβρύου όρνιθας είναι σημαντική για τη φυσιολογική αγγειογένεση in vivo. / Heparin-affin regulatory peptide (HARP), also known as pleiotrophin or heparin-binding growth-associated molecule, is an 18 kDa growth factor that has a high affinity for heparin. HARP is involved in the control of cellular proliferation, migration and differentiation. Moreover, there is a strong correlation between HARP expression and tumor growth and angiogenesis. In the present work, we studied the expression of HARP and its receptors, ALK and RPTPβ/ζ, during development of the chicken embryo chorioallantoic membrane (CAM), in relation to angiogenesis. By western blot analysis and RT-PCR, it was shown that HARP, ALK and RPTPβ/ζ expression increased at days of on-going angiogenesis and decreased at later time points. Transfection of CAMs with an anti-sense HARP gene construct led to a significant decrease in HARP amounts compared to vector control transfected CAMs, a significant decrease in the length of CAM blood vessels, and a decrease in the phosphorylation of Erk1/2. Contrary, transfection of CAMs with the anti-sense HARP gene construct had no influence in lymphangiogenesis of the chicken embryo chorioallantoic membrane (CAM). These data suggest that endogenous HARP is involved in angiogenesis in vivo.

A síndrome de Noonan (SN), caracterizada por baixa estatura, aspectos dismórficos e cardiopatia congênita, foi associada ao gene PTPN11. Estudamos o PTPN11 em pacientes com SN, pais de portadores de mutação e crianças com baixa estatura idiopática (BEI) que apresentam estigmas sugestivos da SN, sem critérios suficientes para o diagnóstico. Encontramos mutações missense em heterozigoze no PTPN11 em 42,3% dos pacientes com SN. Não identificamos alterações nos pais de portadores de mutação no PTPN11 com fenótipo normal tampouco em crianças com BEI. A única diferença estatisticamente significante entre os grupos com e sem mutação foi a resposta em longo prazo ao hGH, melhor no grupo sem mutação / Noonan syndrome (NS), characterized by short stature, dysmorphic facial and thoracic features and congenital heart disease, was associated to PTPN11 gene. We studied the PTPN11 in patients with NS, parents of mutation-positive NS patients and idiopathic short stature children with signs related to NS without fulfilling the diagnostic criteria. We found missense mutations in 42.3% of the NS group. Parents of NS mutation-positive patients did not present mutations, nor did children with short stature. The only statistically significant difference between groups with and without mutations was response to long term use of hGH, better on the mutation-negative group

Abstract The metabolic syndrome is a cluster of components predisposing to type 2 diabetes and cardiovascular disease. Abdominal obesity and insulin resistance seem to be central in the metabolic syndrome, although no unifying pathophysiological mechanism is available. The aim of this thesis was to determine out how the variation in PTP1B and adiponectin gene as well as variations in the plasma adiponectin concentration contribute to the risk of obesity related diseases. PTP1B is a negative regulator of insulin signalling and therefore considered a candidate gene for type 2 diabetes. In the first study, it was found that three PTP1B polymorphisms studied have not strong impact on type 2 diabetes. However, one SNP may be slightly protective against type 2 diabetes, since it was more frequent in the healthy group compared to group of patients with type 2 diabetes. Another SNP was associated with body mass index (BMI). The combination of certain alleles of PTP1B and LEPR (leptin receptor) genes was also associated to BMI. Adiponectin is an adipocytokine expressed in adipose tissue. It has insulin sensitizing effects in liver and muscle and it has also beneficial effects on cardiovascular health. In the second study, the contribution of adiponectin genotypes with obesity-related phenotypes was studied. In Caucasians, the carriers of rare allele of Tyr111His polymorphism were more insulin resistant and at a higher risk of developing type 2 diabetes. In African-Americans, other polymorphisms were associated with BMI and lipids. Thus, the effects of polymorphisms on obesity related phenotypes seemed to be different between ethnic groups. Plasma adiponectin levels were measured from different study groups. In the third study, it was found out that low plasma adiponectin levels associated with different components of the metabolic syndrome and there was a trend towards reductions in adiponectin with an increasing number of components. Fourth study indicated that baseline low adiponectin level associated with a more than 2-fold risk for developing impaired glucose tolerance or type 2 diabetes in the follow-up study of normoglycemic middle-aged Finnish subjects. In the fifth study, plasma adiponectin levels were measured from postmenopausal women receiving estrogen replacement therapy. We observed a reduction in adiponectin levels in women having peroral estradiol which could be part of the "early harm" profile on cardiovascular risk factors of the peroral estrogen replacement therapy detected in clinical trials. These studies further strengthen the role of plasma adiponectin in the obesity related diseases and bring new information of polymorphisms in the adiponectin and PTP1B genes in different populations. / Tiivistelmä Metabolinen oireyhtymä on kertymä tekijöitä, jotka altistavat tyypin 2 diabetekselle ja sydän- ja verisuonitaudeille. Keskivartalolihavuus ja insuliiniresistenssi, eli insuliinin heikentynyt teho, vaikuttavat olevan keskeisiä metabolisessa oireyhtymässä. Kuitenkaan taustalla olevaa syntymekanismia ei täysin tunneta. Väitöskirjatyön tavoitteena oli tutkia PTP1B- ja adiponektiinigeenin muuntelun sekä plasman adiponektiinitason yhteyttä metaboliseen oireyhtymään, sen osatekijöihin ja seurauksiin. PTP1B on insuliinin toimintaa soluissa estävä molekyyli. Ensimmäisessä tutkimuksessa havaittiin että kolme tutkittua PTP1B-geenin nukleotidimuutosta eivät ole vahvasti yhteydessä tyypin 2 diabetekseen. Eräs nukleotidimuutos saattaisi olla lievästi suojaava tyypin 2 diabetesta vastaan, sillä se oli yleisempi terveillä kuin tyypin 2 diabetesta sairastavilla. PTP1B:n ja leptiinireseptorigeenin eräiden alleelien yhdistelmä oli yhteydessä painoindeksiin. Adiponektiini on rasvakudoksen erittämä hormoni, jolla on suotuisia, insuliinin vaikutusta edesauttavia vaikutuksia elimistössä sekä edullisia vaikutuksia verenkiertoelimistössä. Toisessa työssä havaittiin että Amerikan valkoihoisilla, joilla oli eräs harvinainen adiponektiinigeenin alleeli (Tyr111His), oli heikompi insuliinin teho kuin henkilöillä joilla ei ollut kyseistä muutosta. Tämä alleeli oli yleisempi suomalaisilla tyypin 2 diabetesta sairastavilla kuin terveillä, mikä saattaa tarkoittaa että se liittyy suurentuneeseen riskiin tyypin 2 diabetekselle. Afroamerikkalaisilla taas toiset nukleotidimuutokset olivat yhteydessä lihavuuteen ja plasman rasva-arvoihin. Adiponektiinin pitoisuutta plasmassa mitattiin erilaisissa aineistoissa. Kolmannessa tutkimuksessa havaittiin, että matala pitoisuus oli yhteydessä metabolisen oireyhtymän eri osatekijöihin ja pitoisuus oli sitä matalampi, mitä enemmän osatekijöitä henkilöllä on. Neljännessä tutkimuksessa havaittiin että matala plasman adiponektiinipitoisuus oli yhteydessä suurentuneeseen riskiin saada huonontunut glukoosin sietokyky tai tyypin 2 diabetes tulevaisuudessa. Viidennessä tutkimuksessa adiponektiinitaso määritettiin naisilta jotka olivat ohittaneet vaihdevuodet ja saivat estrogeenikorvaushoitoa. Havaittiin että plasman adiponektiinitaso laski niillä naisilla, jotka saivat korvaushoitoa suun kautta. Tämä saattaisi osittain selittää suun kautta annettavan estrogeenikorvaushoidon epäedullista vaikutusta sydän ja -verisuonitautien riskitekijöihin. Tutkimus vahvistaa edelleen adiponektiinin merkitystä lihavuuteen liittyvissä sairauksissa ja tuo uutta tietoa adiponektiini- ja PTP1B-geenien muuntelun merkityksestä eri väestöissä.