Orientador: Eloiza Helena Tajara da Silva / Banca: Dorotéia Rossi Silva Souza / Banca: Fábio César Gozzo / Banca: Victor Evangelista de Faria Ferraz / Banca: Elaine Sbroggio de Oliveira Rodini / Resumo: A Neurofibromatose Tipo 1 (NF1) é uma doença autossômica dominante causada por mutações no gene NF1, responsável pela síntese da proteína neurofibromina. Muitos estudos publicados sobre NF1 têm focado as alterações desse gene e de seu produto em indivíduos afetados, mas as análises de expressão protéica são escassas. No presente estudo, nós investigamos diferenças quantitativas e qualitativas da expressão de proteínas entre amostras de neurofibroma e pele adjacente histologicamente normal, utilizando abordagem proteômica. As proteínas de neurofibroma e pele normal foram separadas por eletroforese bidimensional (2-DE) e identificadas por peptide mass fingerprinting, utilizando espectrometria de massas por dessorção e ionização a laser auxiliada por matriz com base no tempo de vôo (MALDI-TOF). Cinco proteínas foram identificadas: a caspase 14 e a proteína de choque térmico 27/HSP 27, que exibiram expressão reduzida em neurofibromas; a imunoglobulina, a flavina redutase e a proteína de ligação a fosfatidiletanolamina/PEBP, com expressão elevada em neurofibromas. Do nosso conhecimento, este é o primeiro relato de análise comparativa de neurofibromas e pele normal de pacientes com neurofibromatose tipo 1. Das proteínas identificadas, a HSP27 e a PEBP estão conectadas com as vias de sinalização celular p21ras ou cAMP, também relacionadas com a atuação da neurofibromina. A caspase 14 não exibe um elo conhecido com essas cascatas e tal fato pode abrir novos caminhos para o estudo da neurofibromatose. Estudos adicionais ainda são necessários para elucidar o papel dessas proteínas no desenvolvimento da neurofibromatose. Nosso estudo é um passo inicial na descoberta de mecanismos moleculares desta doença e mostra o valor da utilização da análise proteômica na identificação de novos parceiros da neurofibromina relacionados com o desenvolvimento da NF1. / Abstract: Neurofibromatosis Type 1 (NF1) is a common autosomal dominant disorder caused by mutations in the NF1 gene. Many of the studies published on NF1 have focused attention on the gene level, but protein expression analyses are scarce. In the present study, we investigated quantitative and qualitative differences in neurofibroma and histologically normal surrounding skin protein expression of NF1 patients, using a proteomic approach. Proteins from neurofibroma and normal skin were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Five proteins were identified: caspase 14 and heat shock protein 27 kDa protein/HSP 27 (downregulated in neurofibroma), immunoglobulin, flavin reductase and phosphatidylethanolamine binding protein/PEBP (upregulated in neurofibroma). To our knowledge, this is the first report of a comparative analysis of neurofibromas and normal skin from neurofibromatosis type 1 patients. Of the proteins identified, HSP27 and PEBP have a connection with p21ras or cAMP signaling. Caspase 14 has no known link with these pathways and may open a new avenue for studying neurofibromatosis. Further studies are still needed to elucidate the actual roles of the differentially expressed proteins. Our work is an initial step toward uncovering the molecular mechanism of this disease and shows the value of using proteomic analysis to identify novel partners of neurofibromin related to the development of NF1. / Doutor

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type I (NF1) is a common genetic disease that affects over 200,000 patients in North America, Europe, and Japan. Individuals with NF1 display a wide variety of pathologies; importantly, 15-40% of NF1 patients are affected by plexiform neurofibromas. Neurofibromas are complex tumors consisting of tumorgenic Schwann cells surrounded by endothelial cells, fibroblasts, and inflammatory mast cells. These peripheral nerve sheath tumors contribute significantly to the morbidity and mortality associated with NF1. Currently, no medical therapies exist for treating neurofibromas. Recent evidence indicates that the hematopoietic tumor microenvironment carries out a crucial function in the formation of plexiform neurofibromas. Neurofibromatosis is the result of mutations at the NF1 locus, which encodes the GTPase activating protein neurofibromin. Neurofibromin is a negative regulator of the proto-oncogene Ras. Ras hyperactivation is the molecular basis of NF1 associated phenotypes, and it has been demonstrated that restoration of Ras signaling to wild type levels can correct NF1 associated phenotypes in vitro and in vivo. In keeping with the long term goal of detecting potential molecular targets for medical therapies to treat human plexiform neurofibromas, we have identified the kinase Pak1 as a possible downstream intermediary of Ras signaling in NF1 deficient cells. Studies described here utilized murine genetic models to study the effects of genetic inactivation of Pak1 on molecular signaling and cellular functions related to neurofibromas. We demonstrate that inactivation of Pak1 leads to correction of SCF mediated gain-in-function phenotypes seen in Nf1 haploinsufficient mast cells, in vivo and in vitro. However, by using a conditional Nf1 knockout mouse that is a reliable model of plexiform neurofibroma formation, we shown that loss of Pak1 alone in the hematopoeitic compartement is not sufficient to prevent neurofibroma formation. Additionally, we describe a key role for Pak1 in regulating PDGF and TGF-β mediated fibroblast functions, in vitro and in vivo. These studies provide insight into the causes of debilitating tumors related to a common genetic disease, and this research could potentially lead to the development of medical therapies for these tumors, increasing the quality of life for tens of thousands of affected individuals each year.

Made available in DSpace on 2014-06-11T19:32:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-10-07Bitstream added on 2014-06-13T18:43:19Z : No. of bitstreams: 1 marqui_abt_dr_sjrp.pdf: 1283437 bytes, checksum: 0fe3659e1058875d6800b1e4a6048ab1 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A Neurofibromatose Tipo 1 (NF1) é uma doença autossômica dominante causada por mutações no gene NF1, responsável pela síntese da proteína neurofibromina. Muitos estudos publicados sobre NF1 têm focado as alterações desse gene e de seu produto em indivíduos afetados, mas as análises de expressão protéica são escassas. No presente estudo, nós investigamos diferenças quantitativas e qualitativas da expressão de proteínas entre amostras de neurofibroma e pele adjacente histologicamente normal, utilizando abordagem proteômica. As proteínas de neurofibroma e pele normal foram separadas por eletroforese bidimensional (2-DE) e identificadas por peptide mass fingerprinting, utilizando espectrometria de massas por dessorção e ionização a laser auxiliada por matriz com base no tempo de vôo (MALDI-TOF). Cinco proteínas foram identificadas: a caspase 14 e a proteína de choque térmico 27/HSP 27, que exibiram expressão reduzida em neurofibromas; a imunoglobulina, a flavina redutase e a proteína de ligação a fosfatidiletanolamina/PEBP, com expressão elevada em neurofibromas. Do nosso conhecimento, este é o primeiro relato de análise comparativa de neurofibromas e pele normal de pacientes com neurofibromatose tipo 1. Das proteínas identificadas, a HSP27 e a PEBP estão conectadas com as vias de sinalização celular p21ras ou cAMP, também relacionadas com a atuação da neurofibromina. A caspase 14 não exibe um elo conhecido com essas cascatas e tal fato pode abrir novos caminhos para o estudo da neurofibromatose. Estudos adicionais ainda são necessários para elucidar o papel dessas proteínas no desenvolvimento da neurofibromatose. Nosso estudo é um passo inicial na descoberta de mecanismos moleculares desta doença e mostra o valor da utilização da análise proteômica na identificação de novos parceiros da neurofibromina relacionados com o desenvolvimento da NF1. / Neurofibromatosis Type 1 (NF1) is a common autosomal dominant disorder caused by mutations in the NF1 gene. Many of the studies published on NF1 have focused attention on the gene level, but protein expression analyses are scarce. In the present study, we investigated quantitative and qualitative differences in neurofibroma and histologically normal surrounding skin protein expression of NF1 patients, using a proteomic approach. Proteins from neurofibroma and normal skin were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Five proteins were identified: caspase 14 and heat shock protein 27 kDa protein/HSP 27 (downregulated in neurofibroma), immunoglobulin, flavin reductase and phosphatidylethanolamine binding protein/PEBP (upregulated in neurofibroma). To our knowledge, this is the first report of a comparative analysis of neurofibromas and normal skin from neurofibromatosis type 1 patients. Of the proteins identified, HSP27 and PEBP have a connection with p21ras or cAMP signaling. Caspase 14 has no known link with these pathways and may open a new avenue for studying neurofibromatosis. Further studies are still needed to elucidate the actual roles of the differentially expressed proteins. Our work is an initial step toward uncovering the molecular mechanism of this disease and shows the value of using proteomic analysis to identify novel partners of neurofibromin related to the development of NF1.

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La neurofibromatose de type 1 (NF1) est un syndrome de prédisposition tumorale causée par une mutation perte-de-fonction du gène suppresseur de tumeurs NF1. Près de la moitié des patients atteints de NF1 développent un type de tumeurs bénignes des gaines des nerfs périphériques appelés neurofibromes plexiformes. Ces tumeurs sont majoritairement constituées de cellules de Schwann présentant une inactivation somatique du deuxième allèle NF1. Les neurofibromes plexiformes peuvent se transformer en tumeurs malignes dénommées MPNST (Malignant Peripheral Nerve Sheath Tumor) qui sont des sarcomes extrêmement agressifs, résistants aux thérapies actuelles et représentant la première cause de mortalité des patients NF1. A ce jour, les acteurs à l’origine de cette transformation maligne ne sont pas clairement établis. Leur identification représente donc un enjeu majeur pour une prise en charge appropriée des patients et le développement de nouvelles molécules thérapeutiques. Dans ce contexte, le travail mené au cours de ma thèse a eu pour objectifs la recherche et la caractérisation de nouvelles voies de signalisations impliquées dans la tumorigenèse NF1. D’une part, une approche orientée par les travaux antérieurs au laboratoire a permis de montrer l’implication de la voie WNT dans la tumorigenèse NF1. D’autre part, une approche génomique plus large a conduit à la mise en évidence de l’inactivation du répresseur transcriptionnel PRC2 (Polycomb Repressive Complex 2) dans près de la moitié des MPNST. La génération de modèles cellulaires in vitro a facilité l’exploration des gènes surexprimés lors de la perte de fonction du PRC2. Elle a également permis d’entreprendre un crible lentiCRISPR pan-génomique à la recherche des gènes essentiels à la survie des cellules tumorales mutées pour le PRC2. / Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome caused by loss-offunction mutations in the NF1 tumor suppressor gene. Almost half of NF1 patients develop a specific type of benign peripheral nerve sheath tumor called plexiform neurofibromas. These tumors are mainly composed of Schwann cells in which the second NF1 allele is inactivated. Plexiform neurofibromas can give rise to malignant tumors called MPNST that are extremely aggressive sarcomas, resistant to therapy and which represents the first cause of early demise of NF1 patients. The molecular mechanisms underlying this malignant transformation remain enigmatic. Their identification is crucial for appropriate management of NF1 patients and development of new therapies. The goal of my PhD was to identify and characterize new signaling pathways involved in NF1 tumorigenesis. On the one hand, we highlighted the involvement of WNT pathway in NF1 tumorigenesis. On the other hand, a larger genomic approach led to the identification of the transcriptional repressor PRC2 (Polycomb Repressive Complex 2) inactivation in almost half of MPNST. We have generated various cell models, which facilitated the exploration of genes aberrantly expressed consequently to PRC2 loss-offunction. These models also allowed performing a pan-genomic lentiCRISPR screen searching for essential genes for PRC2-mutated tumor cells survival.

Noonan spectrum disorders belong to the RASopathies, a group of clinically related developmental disorders caused by dysregulation of the RAS-MAPK pathway. This thesis describes genetic and clinical investigations of six families with Noonan spectrum disorders. In the first family, the index patient presented with severe Noonan syndrome (NS) and multiple café-au-lait (CAL) spots, while four additional family members displayed multiple CAL spots only. Genetic analysis of four RAS-MAPK genes revealed a de novo PTPN11 mutation and a paternally inherited NF1 mutation, which could explain the atypically severe NS, but not the CAL spots trait in the family. The co-occurrence of two mutations was also present in another patient with a severe/complex NS-like phenotype. Genetic analysis of nine RASopathy-associated genes identified a de novo SHOC2 mutation and a maternally inherited PTPN11 mutation. The latter was also identified in her brother. Both the mother and the brother displayed mild phenotypes of NS. The results from these studies suggest that an additive effect of co-occurring mutations contributes to severe/complex NS phenotypes. The inherent difficulty in diagnosing Noonan spectrum disorders is evident in families with neurofibromatosis-Noonan syndrome (NFNS). An analysis of nine RASopathy-associated genes in a five-generation family with NFNS revealed a novel NF1 mutation in all affected family members. Notably, this family was initially diagnosed with NS and CAL spots. The clinical overlap between NS and NFNS was further demonstrated in three additional NFNS families. An analysis of twelve RASopathy-associated genes revealed three different NF1 mutations, all segregating with the disorder in each family. These mutations have been reported in patients with NF1, but have, to our knowledge, not been associated with NFNS previously. Together, these findings support the notion that NFNS is a variant of NF1. Due to the clinical overlap between NS and NFNS, we propose screening for NF1 mutations in NS patients negative for mutations in NS-associated genes, preferentially when CAL spots are present. In conclusion, this thesis suggests that co-occurrence of mutations or modifying loci in the RAS-MAPK pathway contributes to the clinical variability observed within Noonan spectrum disorders and further demonstrates the importance of accurate genetic diagnosis.

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis Type 1 (NF1) is a highly penetrant autosomal dominant genetic disorder where mutations in the tumor suppressor gene NF1 leads to decreased neurofibromin. The most debilitating manifestation is the presence of complex multilineage Schwann cell-derived plexiform neurofibromas (PN). Historically, little clinical success has been achieved targeting PN through surgery or chemotherapies. I performed an shRNA library screen of patient-derived Schwann cell lines to identify novel therapeutic targets to disrupt PN formation and progression. An shRNA library screen of human kinases and Rho-GTPases was performed in NF1-/- and paired NF1 competent immortalized Schwann cell lines. Following sequencing, candidates were identified. We previously developed a novel mouse model of NF1 wherein a neural crest specific Postncre targeted loxp-flanked Nf1 that replicated the PN found in patients. Additional cohorts of mice were generated with biallelic deletion of Rac1 (Nf1f/fRac1f/f Postn-Cre+; DKO ). Mice were aged for 9 months and peripheral nerves were harvested and fixed in formalin. Peripheral nerve size was measured and tumors were identified through blinded analysis of hematoxylin and eosin and Masson’s Trichrome (collagen) stained slides. Rho family members, including RAC1, were identified as candidates through an shRNA library screen. Genetic disruption of Rac1 in the Schwann cell lineage resulted in the prevention of tumor formation in DKO mice, as observed by peripheral nerve size and histological analysis. I observed an average of 14.8 +/- 2.65 tumors per mouse in the Nf1f/f Postnviii Cre+ cohort compared to 0 tumors in the DKO (p<0.0001). Following an shRNA library screen, RAC1 was identified as a candidate to modulate PN formation. Biallelic deletion of Rac1 in vivo prevented PN formation. I demonstrate that a candidate identified in an shRNA library screen can translate to an biological effect in a mouse model of PN.

No description available.