Les canaux ioniques pentamériques activables par un ligand (pLGIC) sont l'une des principales familles de canaux transmembranaires. Ils permettent la transduction rapide du signal dans le système nerveux central et périphérique via la liaison de neurotransmetteurs. Les pLGIC sont également présents chez les archées et les bactéries. Seuls deux pLGIC bactériens ont été caractérisés biochimiquement et structurellement jusqu'à présent (GLIC et ELIC). Ils servent de modèle d’étude à de nombreux scientifiques et ont été largement étudiés aussi bien au niveau fonctionnel que structural. Dans la première partie de mon travail de thèse, j'ai purifié, cristallisé et résolu la structure cristalline d'un nouveau pLGIC originaire d'un symbiote de gamma-protéobactérie de Tevnia jerichonana (sTeLIC). Des expériences fonctionnelles montrent que sTeLIC est activé par un pH alcalin, est sélectif pour les ions cationiques monovalents et inhibé par les cations divalents. La structure cristalline résolue à pH 8,0 présente un pore largement ouvert qui est le premier de ce type à être caractérisé dans cette famille pLGIC. De plus, nous avons identifié un modulateur fortement positif qui se lie au "site vestibulaire" dans le domaine extracellulaire, et nous avons résolu la structure cristalline de ce complexe. Des expériences fonctionnelles montrent également que sTeLIC partage de nombreuses fonctionnalités avec ELIC. ELIC et sTeLIC constitutent les archétypes d’une nouvelle classe de pLGICs, dont la forme active se caractérise par un pore largement plus ouvert que les autres pLGICs.Dans la deuxième partie de mon travail de thèse, les résidus senseurs de protons dans GLIC ont été cartographiés, afin de déterminer comment la liaison du proton stabilise l'état ouvert de GLIC. Tous les résidus titrables de GLIC ont été cartographiés par mutagenèse dirigée afin de découvrir des capteurs de protons impliqués dans le processus de déclenchement. Nous avons ainsi démontré que la résidu E35 est un résidu clé, dont la forme chargée stabilise l’état de repos, et la forme protonée l'état actif. Nous avons également démontré que la réponse au proton dépend de deux réseaux distincts à l'interface ECD-TMD qui stabilisent l'état ouvert de GLIC. Dans la troisième partie, j'ai cloné, purifié, cristallisé et déterminé les structures cristallines des formes ouvertes et fermées de DeCLIC, un pLGIC de la protéobactérie Desulfofustis. Chaque sous-unité contient un grand domaine additionnel N-terminal constitué de deux sous-domaines (NTD1 et NTD2). Il s’agit de la première structure d’un pLGIC qui contient un domaine supplémentaire extracellulaire non-canonique. / Ligand-gated pentameric ion channels (pLGIC) are one of the major families of transmembrane receptors. They allow rapid signal transduction in the central and peripheral nervous systems via neurotransmitters binding. PLGICs are also present in archaea and bacteria. Only two bacterial pLGICs have been biochemically and structurally characterized so far (GLIC and ELIC). They serve as working models for many scientists and have been extensively studied both at the functional and structural levels. In the first part of my thesis, I purified, crystallized and solved the crystal structure of a new pLGIC from gamma-proteobacterial symbionts of Tevnia jerichonana (sTeLIC). Functional experiments show that sTeLIC is activated by alkaline pH, and is selective for monovalent cationic ions and inhibited by divalent cations. The crystal structure solved at pH 8.0 displays a widely open pore that is the first of this kind to be characterized in the pLGIC family. In addition, we identified a strongly positive modulator that binds to the "vestibule site" in the extracellular domain, and we solved the crystal structure of this complex. Functional experiments show that sTeLIC shares many features with ELIC. ELIC and sTeLIC are the archetypes of a new class of pLGICs, whose active form is characterized by a much more open pore than other pLGICs. In the second part of my thesis, the proton sensor residues in GLIC have been mapped. All titratable GLIC residues were tested by site-directed mutagenesis to discover proton sensors involved in the triggering process. We have demonstrated that the residue E35 is a key residue, whose charged form stabilizes the resting state, and the protonated form the active state. We have also demonstrated that the proton response is dependent on two distinct networks at the ECD-TMD interface, which stabilize the open state of GLIC.In the third part of my thesis, I cloned, purified, crystallized and determined the crystal structures of the open and closed forms of DeCLIC, a pLGIC of Desulfofustis proteobacterium. Each subunit contains a large N-terminal additional domain consisting of two subdomains (NTD1 and NTD2). This is the first structure of a pLGIC which contains a non-canonical additional extracellular domain.

O estudo do domínio público no direito autoral não se resume a analisar os prazos de proteção conferido às obras intelectuais. De tratamento escasso pela doutrina, o tema é bem mais complexo do que aparenta em um primeiro momento, abrangendo diversas áreas do direito e tendo implicações diretas na vida da sociedade. Uma vez que o direito autoral é composto de dois feixes distintos de direitos o patrimonial e o moral compreender o domínio público é, em primeiro lugar, determinar que efeitos decorrem do ingresso de determinada obra em domínio público quanto a cada um de tais grupos de direitos. Além disso, o impacto do domínio público se faz sentir em outras áreas jurídicas, como direito contratual, direito de propriedade, direito do consumidor, direito de família, direito das sucessões. Sem contar com a relação inevitável a aspectos econômicos e sociais relacionados ao uso de obras em domínio público.Esta tese procura determinar a estrutura jurídica do domínio público no direito autoral brasileiro a partir das leis atualmente em vigor, bem como traçar a função do instituto, a fim de dar ao domínio público a importância devida e estimular o desenvolvimento sócio-cultural do país. / The study of public domain in the copyright law is not limited to the analysis of the terms of copyright protection. Being scarcely studied by the doctrine, the issue is more complex than it appears at first, covering different areas of law and having direct implications in society. Since copyright is composed of two distinct bundles of rights - the economic and the moral - to understand the public domain is, first, to determine what effects result from the entry of certain work in public domain on each of these groups of rights. Moreover, the impact of public domain is felt in other legal areas such as contract law, property law, consumer law, family law, law of succession. Apart from the inevitable relation to economic and social aspects related to the use of public domain works. This thesis seeks to determine the legal structure of the public domain in copyright according to Brazilian laws currently in force, as well as tracing the role of the institute in order to give due importance to the public domain and stimulate the social and cultural development of the country.

O estudo do domínio público no direito autoral não se resume a analisar os prazos de proteção conferido às obras intelectuais. De tratamento escasso pela doutrina, o tema é bem mais complexo do que aparenta em um primeiro momento, abrangendo diversas áreas do direito e tendo implicações diretas na vida da sociedade. Uma vez que o direito autoral é composto de dois feixes distintos de direitos o patrimonial e o moral compreender o domínio público é, em primeiro lugar, determinar que efeitos decorrem do ingresso de determinada obra em domínio público quanto a cada um de tais grupos de direitos. Além disso, o impacto do domínio público se faz sentir em outras áreas jurídicas, como direito contratual, direito de propriedade, direito do consumidor, direito de família, direito das sucessões. Sem contar com a relação inevitável a aspectos econômicos e sociais relacionados ao uso de obras em domínio público.Esta tese procura determinar a estrutura jurídica do domínio público no direito autoral brasileiro a partir das leis atualmente em vigor, bem como traçar a função do instituto, a fim de dar ao domínio público a importância devida e estimular o desenvolvimento sócio-cultural do país. / The study of public domain in the copyright law is not limited to the analysis of the terms of copyright protection. Being scarcely studied by the doctrine, the issue is more complex than it appears at first, covering different areas of law and having direct implications in society. Since copyright is composed of two distinct bundles of rights - the economic and the moral - to understand the public domain is, first, to determine what effects result from the entry of certain work in public domain on each of these groups of rights. Moreover, the impact of public domain is felt in other legal areas such as contract law, property law, consumer law, family law, law of succession. Apart from the inevitable relation to economic and social aspects related to the use of public domain works. This thesis seeks to determine the legal structure of the public domain in copyright according to Brazilian laws currently in force, as well as tracing the role of the institute in order to give due importance to the public domain and stimulate the social and cultural development of the country.

Esta pesquisa tem como enfoque as personagens femininas de Graciliano Ramos em suas obras Caetés, São Bernardo, Angústia, Vidas Secas e Infância. Partindo dos conceitos de Antonio Candido sobre estrutura e função, busca-se analisar como a construção literária se relaciona com os dados sociais e históricos da época em que os livros foram escritos e/ou publicados, a década de 1930. A partir das análises feitas, desenvolve-se a ideia da importância dessas personagens que, mesmo deixadas em segundo plano, são essenciais para a construção dos textos literários. / The following research focuses on the female characters of Graciliano Ramos in his works Caetés, São Bernardo, Angústia, Vidas Secas and Infância. Thinking about the concepts of Antonio Candido on structure and function, we analyze how the literary construction relates to the social and historical life in the moment that the books were written, the decade of 1930. From the analyzes made, we realized the importance of these female characters, even when left in the background.

Pentameric ligand-gated ion channels (pLGICs) are membrane bound receptors found in the nervous system. They are responsible for detecting neurotransmitters released from neurons and subsequently mediating responses of the cells on which they are found. Thus, pLGICs play an invaluable role in communication between cells of the nervous system and understanding their function is pivotal to understanding how the nervous system works in general. One factor which is known to mediate pLGIC function is lipids found in the membrane environment in which pLGICs are embedded. This dissertation explores the various ways in which lipids interact with and modulate the function of pLGIC. Potential mechanisms and biological consequences of this modulation will be presented and discussed within the context of our current state of knowledge of pLGIC and nervous system function.

A importância das matas ciliares para a manutenção de diversas funções ecológicas e hidrológicas, reconhecida a ponto de tais formações receberem proteção legal específica, não impediu que estas fossem destruídas indiscriminadamente durante o processo de ocupação do território brasileiro. Estima-se que, só no Estado de São Paulo, cerca de 600 mil hectares de áreas marginais aos corpos d\'água encontram-se ocupados com outros usos que não a vegetação natural, necessitando, portanto, de ações de recuperação. Embora exista um número significativo de trabalhos de revegetação de áreas degradadas, principalmente em matas ciliares, poucos são os resultados de pesquisas sobre o desenvolvimento a longo-prazo de plantios de recomposição de mata ciliar. Nem os plantios mais antigos, aleatórios, e nem os mais recentes, baseados nos processos sucessionais, têm sido submetidos a análises que possibilitem avaliar a eficácia destes métodos de plantio na restauração da mata ciliar com sua forma e funções originais. O objetivo geral desta pesquisa foi o de avaliar a auto-sustentabilidade e as tendências sucessionais de uma mata ciliar implantada, com base em aspectos da estrutura e funcionamento da floresta em trechos de diferentes idades. Neste sentido avaliou-se a floresta existente atualmente, do ponto de vista da composição e estrutura da comunidade; caracterizou-se e quantificou-se o estrato regenerativo da floresta, visando compreender os processos de regeneração natural;caracterizou-se a sazonalidade da queda de folhedo através de sua quantificação periódica e sua contribuição para o retorno de nutrientes ao solo; caracterizou-se o solo sob a floresta, e comparou-se os resultados com área não reflorestada; comparou-se a floresta implantada com florestas naturais através dos parâmetros analisados: riqueza e diversidade florística do estrato arbóreo, densidade absoluta, densidade relativa, área basal, estratificação da floresta, similaridade florística, cobertura das copas, regeneração natural, características químicas do solo e produção e conteúdo de nutrientes no folhedo. Analisando-se as áreas reflorestadas como um todo, constata-se a existência de uma mata ciliar exuberante e fisionomicamente bem estruturada, cujo papel de proteção aos recursos hídricos e ao solo tem sido desempenhado de forma evidente. Alguns resultados obtidos são de extrema importância: no setor de 28 anos, 69% das árvores amostradas surgiram espontaneamente e apenas 31% são indivíduos plantados, demonstrando que está havendo um aumento natural da riqueza florística, o que prova que a natureza encarrega-se de perpetuar a floresta fazendo os \"ajustes\" necessários. Processos ecológicos como a dispersão de sementes e a regeneração natural estão incorporados na dinâmica florestal do local, podendo-se afirmar que a auto-sustentabilidade florestal da comunidade que se formou a partir do plantio de mata ciliar já está garantida. A hipótese deste trabalho foi confirmada pelos resultados obtidos: com o decorrer do tempo a mata ciliar implantada tende a se tornar cada vez mais semelhante à mata ciliar natural, em estrutura e função. / The importance of riparian forests to maintain the many hydrologic and ecological functions, acknowledged to the point of receiving specific legal protection, did not prevent their indiscriminate destruction during the process of land settlement in Brazil. It is estimated that in São paulo state alone about 600 thousands hectares of stream-side areas have been occupied by other uses rather than natural vegetation, therefore, requiring actions of restoration. Although there is a significant number of studies in restoration of degraded areas mainly in riparian forests, the results in research on development of restoration planting of riparian forests in the long term are few. Neither older random plantings nor more recent ones based on sucession processes have been subjected to analysis that would make it possible to evaluate the efficacy of these methods of planting for restoration of riparian forest with its original form and functions. The general aim of this study was to evaluate self-sustainability and sucession tendencies of a planted riparian forest, based on aspects of structure and functioning of the forest in patches of different ages. Thus, the current existing forest was evaluated regarding composition and structure of the community; the regenerative stratum of the forest was characterized and quantified with the purpose of understanding natural regeneration processes; the seasonability of fall foliage through its periodic quantification and itscontribution to the return of nutrients to the soil was characterized; the soil underneath the forest was characterized, and the results in areas that were not restored were compared; planted forests and natural forests were compared through parameters analyzed: richness and floristic diversity of the tree stratum, absolute density, relative density, basal area, forest stratification, floristic similarity, canopy cover, natural regeneration, chemical characteristics of the soil and production and nutrients content in the foliage. By analyzing restored areas as a whole, we can confirm an exuberant and physiognomically well structured riparian forest, whose role of protecting hydric resources and the soil has been easily noticed. Some ecological indicators assessed such as the abundant natural regeneration existing under the planted forest and the spontaneous development of new species show that there has been a natural increase in floristic richness, which proves that nature manages to perpetuate forests by making the necessary adjustments. The hypothesis of this work was confirmed by the results obtained: with time the planted forest tends to become more and more similar to the natural forest, both in structure and function.

While evidence suggests there is indeed a relationship between arterial stiffness and changes in brain structure and function cross-sectionally, the longitudinal relationship between arterial stiffness and changes in brain structure and function is unclear. Also unclear is whether a regional effect of arterial stiffness on brain structure exists, or if the effect is homogenous across brain regions. Using a healthy cohort of the UK Biobank study (N = 1858, meanSD: 61  7 years), we investigated the longitudinal association between changes in arterial stiffness index (ASI) and brain structure (grey matter cortical thickness, whole brain grey matter volume, white matter hyperintensity volume) and function (cognitive performance in 6 tests) over 2.5  1 years. We also examined the association between baseline ASI and all structural and functional brain outcomes 8-11 years post-baseline (N = 630). Prior to post-hoc correction, we observed a significant effect of changes in ASI over 2.5  1 years on grey matter cortical thickness in 11 brain regions contributing to reductions between 0.0004-0.0024mm annually, but none of the 11 regions remained significant post-correction. Following correction there was also no effect of changes in ASI on whole brain grey matter volume (p = 0.76), white matter hyperintensity volume (p = 0.84), or cognitive performance in the domains of interest. Baseline ASI was not associated with regional grey matter cortical thickness, white matter hyperintensity volume, or cognitive function, but did have a significant negative association with whole brain grey matter volume 8.5  1.05 (p = 0.015) years later and 11  1.02 (p = 0.03) years later. Our findings suggest that taken with the effect of age, elevations in ASI may have an additive effect to accelerate changes in brain structure beyond the range that is to be expected as a part of normal aging. Our findings also suggest the relationship between ASI and reductions in whole brain grey matter volume may require long-term exposure to elevations in arterial stiffness in otherwise healthy older adults. / Thesis / Master of Science in Kinesiology / Arterial stiffening both accompanies the normal aging process and can progress due to acquired health conditions. As arteries begin to stiffen the ability to buffer high pressure blood flow is impaired and can put microvasculature at risk of damage. Microvascular damage in the brain can disrupt blood and subsequent oxygen delivery to the brain. When delivery to the brain does not meet the metabolic demand, changes in brain structure brain can occur. Changes in brain structure are associated with impaired brain function, as well as potentially accelerating the progression of neurological diseases. What remains unclear is whether arterial stiffness impacts brain structure differently across regions or all regions homogenously. The purpose of this thesis was to examine the relationship between arterial stiffness and structural and functional changes in the brain over time (objective 1: 2-5 years; objective 2: 8-11 years). Our observations suggest that the progression of arterial stiffness had an effect that was equivalent to approximately 30% of the rate of grey matter tissue loss associated with normal healthy aging (~0.25% reduction in grey matter per year). We found no effect of changes in arterial stiffness on the progression of total grey matter volume, white matter lesions or brain function. We did observe a significant negative relationship between arterial stiffness at baseline and total grey matter volume 8-11 years later. We found no relationship between baseline arterial stiffness and brain structure or function 8–11-years post-baseline. Taken with the effects of normal aging, the loss of tissue in select brain regions associated with changes in arterial stiffness may result in grey matter reductions beyond the range associated with what is considered healthy or normal aging. The association of arterial stiffness and total grey matter volume 8-11 years later suggests that changes in whole brain structure are the product of long-term exposure to arterial stiffness.

Energy functions are found to be a key of protein structure prediction. In this work, we propose a novel 3-dimensional energy function based on hydrophobic-hydrophilic properties of amino acid where we consider at least three different possible interaction of amino acid in a 3-dimensional sphere categorized as hydrophilic versus hydrophilic, hydrophobic versus hydrophobic and hydrophobic versus hydrophilic. Each of these interactions are governed by a 3-dimensional parameter alpha used to model the interaction and 3-dimensional parameter beta used to model weight of contribution. We use Genetic Algorithm (GA) to optimize the value of alpha, beta and Z-score. We obtain three energy scores libraries from a database of 4332 protein structures obtained from Protein Data Bank (PDB) server. Proposed energy function is found to outperform nearest competitor by 40.9% for the most challenging Rosetta decoy as well as better in terms of the Z-score based on Moulder and Rosetta decoy sets.

FGF2 (Fibroblast Growth Factor 2), o fundador da família FGF, tem funções regulatórias na mitogênese, diferenciação, morfogênese e reparo tecidual. Diversas espécies moleculares de FGF2 compartilham uma seqüência C-terminal comum de 155 aminoácidos, pois se originam de diferentes sítios de iniciação de leitura de um único mRNA. O menor, o FGF2-18kDa, é liberado extracelularmente para se ligar a receptores específicos (FGFRs) para disparar as funções parácrinas e autócrinas pelas quais este fator é conhecido. Por outro lado, as espécies maiores (FGF2-21, 22, 22,5 e 34kDa) são intracelulares se ligam a parceiros moleculares desconhecidos para exercer funções intrácrinas ainda indefinidas. O objetivo desta tese foi produzir espécies recombinantes do FGF2-18 e FGF2-22,5, na forma de proteínas de fusão, para analisar funções biológicas e mecanismos de sinalização. Nas células malignas Y1 de camundongo, os recombinantes de FGF2-18kDa (FGF2-18, His-FGF2-18 e His-FGF2-18-ProA) dispararam uma resposta antagônica estimulando as vias de sinalização mitogênica, mas bloqueando o ciclo celular. Nos fibroblastos não tumorigênicos Balb3T3, estes mesmos recombinantes de FGF2-18kDa dispararam apenas a resposta mitogênica clássica. Todos os efeitos biológicos destes recombinantes de FGF2-18kDa foram bloqueados pelo inibidor específico da proteína quinase de tirosina dos FGFRs, PD173074, demonstrando que são respostas intermediadas pelos FGFRs. Portanto, os domínios estruturais adicionados aos recombinantes de FGF2-18kDa não impediram que estas proteínas se ligassem e ativassem os FGFRs. Por outro lado, o recombinante His-FGF2-22,5 dispara apenas as vias de sinalização mitogênica em ambas as células Y1 e 3T3, mas este efeito biológico não é inibido por PD173074. Estes resultados sugerem que a seqüência N-terminal de 55 resíduos, rica em aminoácidos básicos, impede que o FGF2-22,5kDa se ligue e/ou ative os FGFRs. Entretanto, o recombinante His-FGF2-22,5ProA dispara a resposta antagônica característica do FGF2-18kDa. As implicações destes últimos resultados é que o domínio de ProA adicionado ao C-terminal torna o FGF2-22,5kDa um bom ligante dos FGFRs. A interação física entre ligante e receptor das formas recombinantes His-FGF2-18kDa (ou His-FGF2-18ProA) e FGF2-22,5kDa com os putativos FGFRs foi analisada através da técnica de SPR e os resultados mostram KDs aproximados (Kd18=21, 488.10-9 e Kd22,5=20,70393.10-9), enquanto que o número de sítios ligantes em vesículas microssomais das células é significantemente inferior para o FGF2-22,5kDa. Estes resultados são compatíveis com a existência de receptores diferentes para FGF2-18kDa e FGF2-22,5kDa, uma hipótese ainda a ser definitivamente corroborada. Em conclusão, o FGF2-18kDa, mesmo em formas recombinantes como proteína de fusão, dispara todos os efeitos biológicos descritos para FGF2, através dos FGFRs. Diferentemente, o FGF2-22,5kDa, como fator parácrino, só desencadeou a resposta mitogênica clássica de FGF2, provavelmente através de receptores diferentes dos FGFRs. Os resultados e conclusões desta tese têm um potencial indiscutivelmente relevante para a biologia molecular do câncer, com implicações possíveis em terapia oncológica / FGF2 (Fibroblast Growth Factor 2), the founder of the FGF family, has regulatory functions in mitogenesis, differentiation, morphogenesis and tissue repair. Multiple FGF2 molecular species, sharing a C-terminal sequence of 155 amino acids, are translated from different iniciation sites of the same mRNA. The smaller, the FGF2-18kD, is extracellularly released to bind to specific membrane receptors (FGFRs), performing paracrine and autocrine functions. On the other hand, the larger FGF2s (21, 22, 22.5 and 34kDa) are intracellular species that bind to unknown partners to play still undefined intracrine roles. The aim of this thesis was to produce recombinant species of FGF2-18kDa and FGF2-22,5kDa, in the form of fusion proteins, to analyze functions and signaling mechanisms. In mouse Y1 malignant cells, FGF2-18kD recombinants (FGF2-18kDa and His-FGF2-18kDaProA) triggered an antagonistic response activating mitogenic signaling pathways, but blocking the cell cycle. However, in non tumorigenic Balb3T3 fibroblasts, these same FGF2-18kD recombinants only elicited the classical mitogenic response. All biological effects of these FGF2-18kD recombinants were blocked by the specific inhibitor of FGFR-protein-tyrosine-kinases, PD173074, demonstrating that these responses are mediated by FGFRs. Therefore, the new peptide domains added to FGF2-18kD did not prevent these recombinant fusion proteins to bind and activate FGFRs. Conversely, the recombinant His-FGF2-22,5kDa triggered only mitogenic signaling pathways in both Y1 and Balb3T3 cells, a biological effect not inhibited by PD173074. These results suggested that the additional basic-rich N-terminal sequence of 55 amino acid residues, found in FGF2-22,5kDa, prevents this FGF2 species from binding and / or activate FGFRs. However, surprisingly, the recombinant His-FGF2-22kDaProA triggered the antagonistic response characteristic of FGF2-18kDa. These results imply that the ProA-domain added to the C-terminal end rendered the FGF2-22,5kDaProA a good ligand of FGFRs. The physical interaction between recombinants of both His-FGF2-18kD and His-FGF2-22kDa with putative FGFRs, analyzed by SPR, yielded close KD values (KD18=21, 5.10-9 e K D22,5=20,7.10-9), while the number of binding sites in cell microsomal vesicles were significantly lower for the His-FGF2-22,5kDa. These results are consistent with the existence of different receptors for FGF2 and FGF2-18kD-22,5kDa, a hypothesis that has yet to be definitively confirmed. In conclusion, FGF2-18kD, even as recombinant fusion proteins, triggered all biological effects of FGF2, through FGFRs. Conversely, the FGF2-22, 5kDa only triggered the classical mitogenic response, probably via receptors other than FGFRs. The results and conclusions of this thesis are potentially of great interest in cancer molecular biology, with implications in oncologic therapy.

FGFs compreendem um grande família de 24 proteínas, participando de processos chaves nos mais variados tecidos, tendo funções parácrina, autócrina e intrácrina, regulando mitogênese, diferenciação celular, morfogênese e cicatrização. Mas, a relação estrutura-função dos FGFs é pobremente entendida. O membro protótipo desta família é o FGF-2, que apresenta quatro isoformas moleculares incluindo a forma de 18 kDa que é secretada e se liga aos receptores específicos (FGFRs) e dispara uma complexa sinalização. As outras isoformas, de alto peso molecular (21, 22 e 22,5 kDa) são expressas por códons alternativos (CUG) e permanecem no interior da célula interagindo com parceiros moleculares desconhecidos. Para antecipar mecanismos e parceiros do FGF-2 HMW foi realizada modelagem molecular desta isoforma que mostrou: uma estrutura do N-terminal da proteína com motivo β→α&#8594β e manutenção do barril β. A busca por parceiros intracelulares, foi realizada através da técnica do duplo hibrido de levedura, usando um biblioteca de cDNA de cérebro de rato. Foram encontrados 4 possíveis parceiros: BRD2, UBE2I, BRPF1, PC4. Todas essas interações foram confirmadas através do crescimento da levedura em meio sem histidina, produção de β-galactosidase e ensaios de \"pull-down\" com GST. Analises por FACS confirmam que FGF2 não causa apoptose em células adrenais tumorais Y1 de camundongo, mas promovem um acumulo de células na fase S com bloqueio do ciclo celular e da proliferação, configurando uma forma de senescência. Resultados com as células humanas HEK-ER:Ras permitem fazer a seguinte generalização: FGF2 induz senescência em células malignas transformadas pelos oncogenes raso A superexpressão da proteína de fusão FGF-2(18kDa):protA, mas não a da FGF-2(22,5 kDa):protA, protege a célula Y1 da senescência induzida por FGF-2. Por outro lado, a superexpressão destas mesmas isoformas de FGF-2 fusionadas à proteína A em células imortalizadas Balb3T3 não causou transformação celular e nem alterou a resposta mitogênica destas células ao FGF-2 recombinante adicionado ao meio de cultura. Células Y1 quando tratadas com FGF-2 recombinante produz ROS intracelular e libera anions superóxido no meio extracelular. Além disso, o anti-oxidante NAC protege estas células da indução de senescência induzida por FGF-2, sugerindo que ROS pode ser intermediário no disparo de senescência por FGF-2. / FGFs comprise a large fami1y of 24 proteins that play key roles in a number of tissues as local paracrine, autocrine and intracrine regulators of mitogenesis, cellular differentiation, organ morphogenesis and tissue repair. Structure-function relationship among FGFs is still poorly understood. FGF-2, the fami1y prototype member, exists as four molecular species. The 18 kDa form is released to the extracellular milieu and binds to specific receptors (FGFR), initiating a complex array of signals. Other isoforms of higher molecular weights (21, 22 and 22,5 kDa) are translated from alternative codons (CUG) and remain inside of the cell interacting with unknown partners. Aiming to anticipate mechanisms and partners, we modeled the FGF2-HMW molecule, showing that the protein displays β→α&#8594β motif in the N-terminal region and maintains the β-barrel structure common to ali FGFs. By the yeast two-hybrid method, using a cDNA rat brain library, we found four possible partners for FGF2-HMW: BRD2, UBE2I, BRP1 and PC4. Ali partners were confirmed by yeast growth without histidine, production of β-galactosidase and \"pull-down\" assays with GST. FACS analyses confirmed that FGF2 does not cause apoptosis in mouse Y1 adrenal tumor cells. But, FGF2 inhibited S phase progression blocking cell cycle and proliferation, characterizing a form of senescence. In addition, results obtained with the human HEK-ER:Ras cells support the following general statement: FGF2 triggers senescence in malignant cells transformed by ras oncogenes. Ectopic expression of the fusion protein FGF-2(18 kDa):protA, but not of FGF-2(22,s kDa):protA, protected Y1 cells senescence induced by FGF-2. On the other hand, ectopic expression of FGF-2 isoforms fusioned to protA in Balb3T3 immortalized cells did not cause transformation and neither modified the mitogenic response of this cell to recombinant FGF2. Recombinant FGF-2 stimules Y1 cells to produce intracellular ROS and to release superoxide anions into intracellular medium. Moreover, the ROS scavenger NAC protect Y1 cells from senescence induced by FGF-2, suggesting that ROS may be mediate senescence triggering induced by FGF-2.