Submitted by Luis Guilherme Macena (guilhermelg2004@gmail.com) on 2013-04-08T15:15:58Z No. of bitstreams: 1 Joyce Cantoni.pdf: 528656 bytes, checksum: b67e6826616a4089c8c4f4677f9606de (MD5) / Made available in DSpace on 2013-04-08T15:15:58Z (GMT). No. of bitstreams: 1 Joyce Cantoni.pdf: 528656 bytes, checksum: b67e6826616a4089c8c4f4677f9606de (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto Fernandes Figueira. Departamento de Ensino. Programa de Pós-Graduação em Saúde da Criança e da Mulher. Rio de Janeiro, RJ, Brasil / O objetivo geral foi o de analisar o entendimento de pacientes com Neurofibromatose tipo1 (NF1) sobre sua doença, considerando especialmente o fato desta ser de etiologia genética, sendo objetivos específicos: identificar o que os pacientes pensam sobre ter uma doença genética; captar o entendimento que os pacientes têm a respeito do padrão de herança da doença e a importância dada ao aconselhamento genético; avaliar compreensão que possuem acerca da condição crônica da doença; analisar como os pacientes vêem e quais são as implicações práticas da doença nas suas vidas, considerando principalmente a necessidade de tratamento regular, consultas com especialistas e realização periódica de exames; conhecer as fontes de informação que levam os pacientes a construir seu entendimento sobre a doença. Para atingir estes objetivos, adotou-se a abordagem qualitativa, através de entrevistas temáticas com quatro pacientes, procedendo-se, então, à transcrição do material gravado e à subsequente análise de conteúdo deste. Esta análise evidenciou que a etiologia genética era conhecida por todos, mas as implicações decorrentes não se mostraram motivo de maior preocupação. Evidenciou-se também que manifestações correlatas e a etiologia se confundem. Todos foram diagnosticados tardiamente, acima dos 15 anos de idade, o que merece ser alvo de mais reflexão acerca da premência de se diagnosticar precocemente doenças com a NF 1, para melhor controlá-las. Os entrevistados tinham noção que sua doença era progressiva, crônica, mas este fator isoladamente não foi referido como contribuindo para o acompanhamento regular. Todos reportaram ter nascido, ou ter desde a infância, manchas café leite e “tumores” espalhados pelo corpo (os neurofibromas), entretanto negam ter sofrido qualquer preconceito, porém é evidente que sua aparência incomoda, por isso acabam por relatar como essas características fenotípicas são notadas pelos outros. Um deles, por ser rapaz e adolescente, mostra-se mais centrado na ginecomastia que o acomete, sem estabelecer qualquer tipo de associação entre ela e a NF 1. Quanto ao aconselhamento genético, dois entrevistados reconheceram sua importância, enquanto dois outros se mostraram indiferentes. Só uma paciente recorreu espontaneamente a outras fontes de informação, especificamente a Internet, porém declarou-se assustada com o que viu e revoltada com o tom geral de “vitimização”. Os outros declaram não ter procurado nada na rede por dificuldade de acesso ou por falta de interesse. A exceção de um entrevistado, os demais demonstram estarem desinformados sobre genes, cromossomos e mutações. A doença genética, para todos, é algo que está no sangue, sendo transmitida verticalmente dos pais para os filhos. Conclui-se que diante dos avanços da genômica faz-se necessário preparar os profissionais de saúde, mais especificamente, aqueles que estão na ponta da assistência aos pacientes, fazendo-os entender que se todos estão imersos numa mesma macro-cultura, entretanto, ao nível micro as diferença de hábitos, valores, conhecimentos, linguagens, histórias individuais, emoções e personalidades precisam ser consideradas quando se deseja uma compreensão dos elementos envolvidos numa doença de origem genética, cuja evolução implica em comorbidades que demandam intervenções constantes e cuja etiologia envolve padrões de herança autossômica dominante. / The objective of this study was to evaluate the knowledge of patients with Neurofibromatosis 1 about the disease and its genetic etiology. The specific objectives were: identify what they think about “having” a genetic disease; identify what they think about the inheritance of the disease, and how important they consider the genetic counseling; evaluate how and what they think about living with a chronic disease and about the practical consequences of the disease on their everyday life, with the need for several and regular medical appointments, and complementary exams; try to investigate if they searched for more information about their condition, and where; and how this search helped them to reach the knowledge they have about the NF1. Thematic interviews were applied to four patients in order to accomplish these objectives. Content analysis was then applied in a qualitative approach. All patients were conscious of the genetic etiology, but the consequence of that was not matter of concern for all of them. Also they mixed the etiology with the clinical features, and associated diseases. All of then were diagnosed over 15 years of age. This fact deserves more attention because as more precocious the diagnosis of conditions like the NF 1 is done, better control of the disease becomes possible. Even though all of them knew about that the disease was chronic and progressive, this was not enough to warrant a regular treatment. Although all of them report having the café au lait spots since birth or early childhood, and neurofibromas over their bodies, they denied being victims of prejudice, but, in different ways, they revealed their shame about their physical appearances, being aware that they were noticed by others. For one of them, the youngest, a 19 years male, the main problem was a gynecomastia. Two of them recognized the importance of genetic counseling but for the others, it is completely indifferent. Only one of the interviewed searched by herself information in other sources, mainly in the Internet, what made her scared. Also she refers she did not like the general note of victimization she have found there. The others did not make any kind of research, by lack of interest or because they have not known how to do it. Three of the patients had any kind of knowledge about genes, chromosomes and mutations. For all of them, genetic disease is something in the blood, transmitted from parents to the offspring. With the advances in genomics, health professionals, specially those which deal directly with patients, need to be prepared, educated to understand that in spite of all we live in one and same macro culture, although aspects of the micro level such as values, languages, individual experiences, emotions, and others have to be considered in order to achieve a comprehension of what it is involved in a genetic disease, with autossomic dominant pattern of inheritance, and that can present many comorbities that demand frequent interventions, and has to be carefully and regularly “spyed” by health professionals to assure better conditions of life for those affected.

Neurofibromatosis type 2 (NF2) is an inherited, multiple tumour disease caused by loss of the tumour suppressor protein, Merlin. There are several tumours associated with NF2 including; ependymomas, meningiomas and schwannomas. Merlin loss can also occur sporadically in all of these tumours and is associated with upregulation of various growth factor receptors and their relevant signalling pathways. At present the only treatment options for NF2 are surgery or radiosurgery, both of which incur serious morbidity and are unable to prevent recurrence of tumours. Either new drug treatments, or re-profiling of other drugs already commercially available, are urgently needed to improve outcome for NF2 patients. Cellular prion protein (PrPC), encoded by PRNP gene, is involved in tumour development by altering proliferation, adhesion, and survival in some cancers via focal adhesion kinase (FAK) /Src/ NFκB, cyclin D1 and p53 -proteins. Our group previously showed a strong elevation of PRNP gene activity in schwannoma. I hypothesise that PrPC may contribute to schwannoma development. To study the role of PrPC in schwannoma development I have used the well-established in vitro model of schwannoma that comprises primary human Schwann and schwannoma cells. I show that PrPC is upregulated in schwannoma as well as in Merlin-deficient meningiomas and human malignant mesotheliomas. In schwannoma PrPC is released both via exosomes and by α-cleavage which forms biologically active N- and C-terminal portions of the protein. PrPC contributes to pathological proliferation, adhesion and survival of schwannoma cells by activating ERK1/2, PI3K/AKT, cyclin D1, FAK, p53 pathways via the 37/67kDa non-integrin laminin receptor (LR/37/67kDa) and CD44. Furthermore, schwannoma cells appear to be intrinsically drug-resistant due to upregulation of MDR1 protein p-glycoprotein (p-gp) expression. P-gp expression is dependent on PrPC thus, inhibiting PrPC may be a good potential new therapeutic option for schwannoma patients, either alone or in combination with Sorafenib and p-gp inhibitor Valspodar (PSC833). An inhibitor of LR/37/67kDa/PrP interaction, NSC47924, or Bortezomib, a proteasome/NFκB inhibitor which has been approved for the treatment of multiple myeloma, could also be of beneficial therapeutic effect and is something to investigate in future work. I conclude that PrPC is an interesting new therapeutic target through its involvement with schwannoma patholgenesis and resistance to drug treatments PrPC may prove to be a good therapeutic target in other NF2-related tumours like meningiomas and schwannomas.

Instituto Dr. Josà Frota / A meta da presente pesquisa foi apresentar tÃcnicas cirÃrgicas associadas ou isoladas, na tentativa de, utilizando cirurgias menos traumÃticas e mais eficientes, proporcionar resultados menos estigmatizantes e deformantes para dar aos portadores da neurofibromatose tipo 1 (NF1) uma qualidade de vida mais digna e melhor integraÃÃo social. Estudou-se neste trabalho 30 pacientes portadores de NF1 por um perÃodo de seis anos, durante os quais se comparou a eficiÃncia de diversas tÃcnicas cirÃrgicas em lesÃes localizadas em vÃrias regiÃes anatÃmicas do corpo e cujas ressecÃÃes atingiram dimensÃes que variaram entre 3 e 51cm. A utilizaÃÃo do tratamento cirÃrgico mostrou ser a maneira mais simples, eficaz e rÃpida para a soluÃÃo de afecÃÃo tÃo traumÃtica para os pacientes. Mesmo quando da utilizaÃÃo de retalhos cutÃneos com comprometimento residual da pele o seguimento operatÃrio mostrou que a doenÃa nÃo evoluiu e nÃo houve qualquer sinal de malignizaÃÃo. Das opÃÃes mais empregadas foram os retalhos cutÃneos loco-regionais, as que melhores resultados proporcionaram, quer do ponto de vista funcional como estÃtico, e os que causaram menor nÃmero de seqÃelas. Vinte e um pacientes se beneficiaram de excisÃes e reparaÃÃes com retalhos e os demais com procedimentos mais simples, o que lhes permitiu uma melhor qualidade de vida e melhor aceitaÃÃo social. Os retalhos cutÃneos loco-regionais foram aqueles que possibilitaram o reparo das maiores Ãreas cruentas e a ressecÃÃo dos mais volumosos tumores, com uma mÃdia de 16,4 x 8,1 cm e um peso mÃdio de 373 g. O âSâ itÃlico com uma mÃdia de 8,5 x 5,5 cm e 135 g e a âZâ plÃstia com 8,5 x 4,8 cm e 82,8 g mostraram-se eficazes para a reparaÃÃo de lesÃes de mÃdio porte onde aparecem como uma das opÃÃes para reparaÃÃo imediata das ressecÃÃes da NF1.ExcisÃo e Sutura com 4,9 x 2,8 cm e 33,8 g e pequenos enxertos de pele estÃo indicados nas lesÃes de dimensÃes menores e de localizaÃÃes especiais, como a face e nariz. NÃo foram detectadas malignizaÃÃes nas 52 peÃas encaminhadas ao laboratÃrio de histopatologia.

Abstract Neurofibromatosis type 1 syndrome (NF1) is caused by mutations of the NF1 gene. The NF1 protein (neurofibromin) contains a domain which is related to the GTPase-activating protein (GAP) and accelerates the switch of active Ras-GTP to inactive Ras-GDP. The NF1 protein has been referred to as a tumor suppressor, since the cells of malignant schwannomas of NF1 patients may display a loss of heterozygosity of the NF1 gene. Psoriasis is characterized by hyperproliferation of the epidermis and by down-regulated levels of NF1 mRNA and protein. Ca2+ is an universal signal transduction element modulating cell growth and differentiation. Many cell types coordinate their activities by transmitting waves of elevated intracellular calcium levels from cell to cell. The propagation of calcium waves had not been studied previously in human keratinocytes. Thus, the aim of the present study was to find out which pathways may play a role in Ca2+ signaling at different extracellular calcium concentrations in NF1 and and psoriatic keratinocytes versus normal control keratinocytes. The results demonstrated that NF1 and psoriatic keratinocytes have a tendency to form cultures characterized by altered Ca2+-mediated cell signaling compared to normal keratinocytes. Specifically, the main route of calcium-mediated signaling was gap-junctional in normal keratinocytes. In contrast, ATP-mediated calcium signaling predominated and capacitative calcium influx was defective in NF1 and psoriatic keratinocytes. The results of the present study suggest that mutations of the NF1 tumor suppressor gene or lowered levels of NF1 protein/mRNA may eventually lead to altered intercellular communication.

Abstract Neurofibromatosis type 1 (NF1) is a dominantly inherited neurocutaneous disorder caused by mutations in the NF1 gene. Common clinical manifestations associated with NF1 are neurofibromas, café-au-lait macules (CALM), axillary freckling and Lisch nodules of the iris. Other important manifestations are vasculopathy, a variety of osseous lesions, including short stature, scoliosis and pseudoarthrosis, optic gliomas and an increased risk for certain malignancies. The best characterized function of the NF1 gene is to act as a downregulator of Ras proto-oncogene signalling by accelerating the switch of active Ras-GTP into inactive Ras-GDP. The NF1 gene is considered a tumor suppressor since some malignancies may display a loss of heterozygosity or homozygotic inactivation of the gene. The present study investigated the behaviour and function of the NF1 gene during keratinocyte differentiation, wound healing and psoriasis using human epidermis and epidermal keratinocytes as a model. The NF1 protein was shown to associate with the intermediate filament network during keratinocyte differentiation both in vitro and in vivo, and it is thus suggested to play a role in the cytoskeletal re-organization or in the formation of cell adhesions. NF1 gene expression was also studied in psoriasis, in which keratinocytes are hyperproliferative and cell differentiation is altered. NF1 gene expression was downregulated in psoriatic keratinocytes both in vivo and in vitro, suggesting that the NF1 gene might have role in downregulating keratinocyte proliferation. During epidermal wound healing, NF1 gene expression was increased. However, the process of wound healing showed no apparent differences between NF1 patients and controls. Furthermore, an increased number of cells immunoreactive for active Ras-MAPK was demonstrated in vascular tissues of NF1 patients, but not in epidermal keratinocytes or dermal fibroblasts. The finding suggests that the NF1 protein functions as a Ras-GAP in some, but not all tissues.

In an effort to improve the auditory brainstem implant, a prosthesis in which user outcomesare modest, infrared neural stimulation (INS) was applied to the cochlear nucleus in a ratanimal model. Pulsed INS, delivered to the surface of the cochlear nucleus via an opticalfibre, evoked auditory brainstem responses (ABR) and generated broad neural activation inthe inferior Colliculus (IC). Varying the parameters of the laser stimulation revealed laserpeak power to be the dominating parameter for both ABR and IC responses. Strongestresponses were recorded when the fibre was placed at lateral positions on the cochlearnucleus, close to the temporal bone. After deafening by auditory nerve section, ABR andIC responses to INS disappeared, consistent with a reported "optophonic" effect, a laser-inducedacoustic artifact. Thus, for deaf individuals who use the auditory brainstemimplant, INS alone does not appear promising as a new approach.

La neurofibromatose de type 1 (NF1) représente la maladie génétique autosomale dominante la plus fréquente en France, après la mucovisidose, avec une incidence de 1 individu sur 3500. Il s'agit d'une pathologie multi-systémique présentant un tableau clinique varié. Parmi la pléthore de symptôme, sont comptées des manifestations neurocutanées telles que les taches « café-au-lait » (zone d'hyperpigmentation localisée) et les neurofibromes (tumeurs bénignes de la gaine périphérique de la myéline) mais également des défauts osseux et des troubles cognitifs. La pénétrance de NF1 est complète mais la manifestation et la sévérité des symptômes peuvent varier d'un individu à l'autre. Le gène NF1 responsable de la maladie, localisé sur le chromosome 17, est un gène suppresseur de tumeur qui code pour la neurofibromine. Dans le but de développer un modèle cellulaire humain pertinent pour l'étude des défauts osseux associés à NF1, nous avons utilisé des cellules souches induites à la pluripotence porteuse de la mutation causale NF1 (hiPS-NF1). Dans ces travaux, nous avons montré qu'une perte d'expression de la neurofibromine dans les ostéoblastes dérivés de hiPS-NF1 reproduisait le phénotype d'ostéogénèse réduite et qu'il était possible d'améliorer la capacité des hiPS-NF1 à se différencier en ostéoblaste à l'aide de molécules pharmacologiques. Toujours dans le but de proposer un modèle cellulaire humain le plus relevant possible, nous avons développé des lignées isogéniques avec la technologie CRISPR/Cas 9 afin d'étudier l'impact d'une perte partielle ou totale de l'expression de la neurofibromine sur le phénotype osseux. En parallèle, afin de pouvoir étudier un autre phénotype associé à NF1, un protocole de différenciation de cellules de Schwann sous culture définie en utilisant des facteurs de croissance et des molécules de signalisation, a partir des hiPS a été développé. Des cellules de Schwann-like ont été obtenues en 30 jours en engagement la différenciation des cellules souches vers la crête neurale afin d'induire l'émergence de précurseurs de cellules de Schwann par l'action de molécules telles que le récepteur de type I du TGF-ß (SB431542), l'hereguline ß1, l'IGF1, le FGF2 et un activateur de WNT3a (CHIR99021). L'analyse par q-PCR montre une augmentation des marqueurs de différents stades de différenciation de la cellule de Schwann : crête neurale (SOX10, ERBB3), cellules de Schwann précurseurs (MPZ, CAD19) et cellules de Schwann immatures (S100) au bout de 30 jours de différenciation. Ces résultats ont été complétés par l'analyse protéique des cellules différenciées et par la mise en co-culture de ces cellules avec des motoneurones différenciés à partir d'hiPS. L'ensemble de ce travail a permis de valider la pertinence de l'utilisation des cellules souches pluripotentes porteuses de mutation dans la modélisation de pathologie génétique. Permettant à plus long terme la recherche de molécules actives par des approches de de criblage pharmacologique ou par des approches de thérapie cellulaire. / Neurofibromatosis type 1 (NF1) is an autosomal genetic disease with an incidence of 1 in 3,500 individuals. This is a multi-systemic disorder with a plethora of various symptoms. Among with we find neurocutaneous manifestations such as "café-au-lait" spots (zone of localized hyperpigmentation) and neurofibromas (benign tumors of the peripheral sheath of myelin), but also bone defects and cognitive disorders. The penetrance of NF1 is complete but the manifestation and severity of symptoms may vary from one individual to another. The NF1 gene responsible for the disease, located on chromosome 17, is a tumor suppressor gene that encodes neurofibromin. In order to develop a relevant human cellular model for the study of bone defects associated with NF1, we used pluripotency-induced stem cells that carry the NF1 causal mutation (hiPS-NF1). In this work, we have shown that loss of neurofibromin expression in osteoblasts derived from hiPS-NF1 reproduces the reduced osteogenesis phenotype. We have also shown that pharmacological molecules can improve the ability of hiPS-NF1 to differentiate in osteoblast. In order to propose the most relevant human cell model, we have developed isogenic lines with CRISPR / Cas 9 technology to study the impact of a partial or total loss of neurofibromin on the bone phenotype. Simultaneously, a Schwann cells differentiation protocol from hiPS was developed under culture defined using growth factors and signaling molecules. Schwann-like cells were obtained in 30 days by the use of molecules such as the type I receptor TGF-β (SB431542), heregulin β1, IGF1, FGF2 and activator of WNT3a (CHIR99021). The q-PCR analysis shows an increase in Schwann cell markers: neural crest (SOX10, ERBB3), precursor Schwann cells (MPZ, CAD19) and immature Schwann cells (S100). These results were confirmed by protein analysis of the differentiated cells and by the co-culture analysis of these cells with differentiated motoneurons from hiPS. All of this work validated the relevance of pluripotent stem cells in the modeling of genetic pathology.

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.

Neurofibromatosis type 2 (NF2) is a benign tumor disease of the nervous system. Development of bilateral vestibular schwannomas is characteristic of NF2; however patients frequently present schwannomas on other nerves, as well as meningiomas and ependymomas. Currently, there are no drug therapies for NF2. There is an urgent need for development of NF2 therapeutics and this dissertation presents two independent potential therapeutic targets. The disease is caused by mutations in the NF2 gene that encodes a tumor suppressor called merlin. Loss of merlin function is associated with increased activity of Rac and p21-activated kinases (PAK) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrates for Cdc42/Rac-PAK, and modulate actin dynamics by phosphorylating cofilin, an actin severing and depolymerizing agent. LIMKs also translocate into the nucleus and regulate cell cycle progression. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2ΔEx2) exhibited increased levels of LIMK1, LIMK2, and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared to wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared to normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2ΔEx2 MSC reduced LIMK1 and LIMK2 levels. Pharmacological inhibition of LIMK with BMS-5, decreased the viability of Nf2ΔEx2 MSCs in a dose-dependent manner, but did not affect viability of iv control MSCs. Similarly, LIMK knockdown decreased viability of Nf2ΔEx2 MSCs. The decreased viability of Nf2ΔEx2 MSCs was due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrest cells in early mitosis by decreasing Aurora A activation and cofilin phosphorylation. To increase the search for NF2 therapeutics, we applied an alternative approach to drug discovery with an unbiased pilot high-throughput screen of the Library of Pharmacologically Active Compounds. We assayed for compounds capable of reducing viability of Nf2ΔEx2 MSC as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2, a mammalian sirtuin, is a NAD+ -dependent protein deacetylase. We show that Nf2ΔEx2 MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases Nf2ΔEx2 MSC viability in a dose dependent manner without substantially reducing wildtype MSC viability. Inhibition of SIRT2 activity in Nf2ΔEx2 MSC causes cell death accompanied by release of the necrotic markers lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. Overall this work uncovered two novel potential therapeutic targets, LIMK and SIRT2 for NF2 and tumors associated with merlin deficiency.

No description available.