abstract: The goal of this thesis is to test whether Alzheimer's disease (AD) is associated with distinctive humoral immune changes that can be detected in plasma and tracked across time. This is relevant because AD is the principal cause of dementia, and yet, no specific diagnostic tests are universally employed in clinical practice to predict, diagnose or monitor disease progression. In particular, I describe herein a proteomic platform developed at the Center for Innovations in Medicine (CIM) consisting of a slide with 10.000 random-sequence peptides printed on its surface, which is used as the solid phase of an immunoassay where antibodies of interest are allowed to react and subsequently detected with a labeled secondary antibody. The pattern of antibody binding to the microarray is unique for each individual animal or person. This thesis will evaluate the versatility of the microarray platform and how it can be used to detect and characterize the binding patterns of antibodies relevant to the pathophysiology of AD as well as the plasma samples of animal models of AD and elderly humans with or without dementia. My specific aims were to evaluate the emergence and stability of immunosignature in mice with cerebral amyloidosis, and characterize the immunosignature of humans with AD. Plasma samples from APPswe/PSEN1-dE9 transgenic mice were evaluated longitudinally from 2 to 15 months of age to compare the evolving immunosignature with non-transgenic control mice. Immunological variation across different time-points was assessed, with particular emphasis on time of emergence of a characteristic pattern. In addition, plasma samples from AD patients and age-matched individuals without dementia were assayed on the peptide microarray and binding patterns were compared. It is hoped that these experiments will be the basis for a larger study of the diagnostic merits of the microarray-based immunoassay in dementia clinics. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2011

O metabolismo celular é substancialmente alterado durante a oncogênese, progressão tumoral e outros processos patológicos. Tem sido frequentemente analisado para compreensão dos processos que permitem o crescimento dos tecidos, reprodução, manutenção da homeostase e resposta a sinais extracelulares. Dentre os vários métodos para caracterização de alterações metabólicas, a espectrometria de massas tem contribuído significativamente para a identificação e quantificação de proteínas envolvidas no metabolismo, e também para a caracterização do metaboloma. A análise proteômica baseada em espectrometria de massas permite estudos qualitativos em grande escala, adequados para a busca e descoberta de analitos relevantes. A análise proteômica dirigida complementa esse caráter com qualidade quantitativa para proteínas alvo pré-selecionadas. Neste trabalho foram desenvolvidos métodos de proteômica dirigida para o monitoramento de alterações quantitativas nos níveis de proteínas envolvidas na via glicolítica do metabolismo da glicólise. Para tal peptídeos proteotípicos para cada proteína foram identificados e padronizados utilizando a estratégia de monitoramento de reações múltiplas. O painel foi aplicado para obter resultados das alterações que ocorrem em um modelo de progressão tumoral. Com esta estratégia empregada, foi possível selecionar e utilizar vários peptídeos representativos das enzimas da via glicolítica e também de algumas proteínas relevantes ao câncer. A utilização de peptídeos sintéticos facilitou substancialmente o processo de desenvolvimento do método. Por fim, com a metodologia desenvolvida, foi demonstrado para células MCF7, que o fator EGF alterou a expressão das proteínas da via glicolítica, aumento no fluxo para via das pentoses e capacidade aumentada da respiração celular. Este estudo, portanto, sugere uma nova disposição do metabolismo celular dado o conhecimento estabelecido em relação aos efeitos na respiração, como o efeito Warburg. / Cellular metabolism is altered during ontogenesis, cancer progression and several other pathological events. Because of that, the metabolism is constantly analyzed in order to provide further comprehension of processes that allow tissue growth, reproduction, homeostasis maintenance, and response to extracellular signals. Among the methods great diversity of methods used to characterize metabolic alterations, mass spectrometry has been contributing significantly to identify and quantify proteins involved in a diversity of metabolic pathways, but also to monitor the changes in metabolome. Proteomics based on mass spectrometry allows highthroughput in-depth qualitative resources for discovery phases stages, providing the new relevant candidates for further biochemical characterization. In a complementary way, targeted proteomics allows precise quantitative analyses of such selected protein targets. Here, it was developed a targeted proteomics method for multiplex monitoring glycolytic pathway enzymes and relevant cancer-related proteins. For that, proteotypic peptides representing proteins of interest were selected and studied in detail to be incorporated in a multiple reaction monitoring assay. The developed method was applied to monitor the alterations in the glycolytic pathway in a cancer progression model. Using targeted proteomics strategies, we selected and applied for quantification several proteotypic peptides representing glycolitic enzymes and cancerrelated proteins. The use of synthetic peptides allowed faster method development and more sensitive methods. The application of such methods, demonstrated alterations in glycolytic pathways and cancer-related proteins promoted in MCF7 cells treated with EGF. Also, an activation of pentose-phosphate pathway was suggested and increase in cellular oxygen consumption. This study, therefore, suggests changes in the cellular metabolism that differs from the classic Warburg effect observed during cancer development.

No description available.

Proteomics is an increasingly important area of biological research and has gathered much attention over recent years. Major challenges that make a proteomic analysis difficult are sample complexity, diversity and dynamic range. Progress in the area of proteomics relies heavily on new analytical tools for the sensitive, selective, and high-throughput studies of target analytes. It is estimated that there are several hundred thousand proteins in a human cell. In order to be able to analyze such a complex sample, an analytical method must be capable of separating and detecting many different sample peaks. The complexity of such samples indicates that a single separation method will not be able to provide the needed resolution. If two methods that are orthogonal are combined, then the peak capacity of the combined system is the product of the two individual peak capacities. Development of such systems would cater to the current demands of proteomics studies. Matrix assisted laser desorption/ionization (MALDI) mass spectrometry has evolved into a primary analytical tool for proteomics research. MALDI is fast and efficient and has a high tolerance to non-volatile buffers and impurities. The samples for MALDI are typically applied to solid supports after having been subjected to off-line liquid or gel separations. Several methods have been reported involving various chromatographic or electrophoretic separation methods. However, the current methods often require highly sophisticated sample handling systems, which are often expensive and in need of skilled human resources. The current demands of proteomic analyses require fast, efficient and inexpensive methods for separation to fully harness the capability of MALDI mass spectrometry. In this work a microfluidic device has been designed to perform dynamic isoelectric focusing (DIEF) based protein separation with digital sample deposition directly on a MALDI target for offline analysis. DIEF is related to capillary isoelectric focusing which and can facilitate the interface without the loss of the separation resolution. Compared to traditional capillary isoelectric focusing (cIEF) DIEF uses additional high-voltage power supplies to control the pH gradient by manipulating the electric field. The proteins can be focused at a desired sampling position according to their isoelectric point, to be collected for further analysis by MALDI mass spectrometry. DIEF has a peak capacity of over a thousand and offers an ease of interfacing to other techniques making it a preferred separation method for the interface with mass spectrometric techniques such as MALDI. The design of the microfluidic device is based on a digital droplet fractionation. Multiple fractions of the sample solution from DIEF are generated to retain the resolution and to act as an additional separation mode. The microfluidic device is controlled by actuating pneumatic valves built into the device. The DIEF operational parameters were optimized according to the surface functionality and the design of the microfluidic device. A suitable MALDI sample preparation method was found by studying different existing methods. The methods were studied using test proteins prepared in solutions having the additives used in the experiment. A simple mixture of three proteins was used to demonstrate the application of the developed method. The separation between the proteins insulin, hemoglobin and the myoglobin was demonstrated by varying the separation resolution in three experiments.

Submitted by Luciane Willcox (luwillcox@gmail.com) on 2016-09-12T17:18:24Z No. of bitstreams: 1 Tese_Doutorado_ICC.pdf: 40560568 bytes, checksum: 1e1c7efcf21605b84ddacb307516a772 (MD5) / Approved for entry into archive by Luciane Willcox (luwillcox@gmail.com) on 2016-09-12T18:02:31Z (GMT) No. of bitstreams: 1 Tese_Doutorado_ICC.pdf: 40560568 bytes, checksum: 1e1c7efcf21605b84ddacb307516a772 (MD5) / Made available in DSpace on 2016-09-12T18:02:31Z (GMT). No. of bitstreams: 1 Tese_Doutorado_ICC.pdf: 40560568 bytes, checksum: 1e1c7efcf21605b84ddacb307516a772 (MD5) Previous issue date: 2016-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Programa de Apoio à Pesquisa Estratégica em Saúde (Papes) da Fiocruz, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Microsoft Research. / Instituto Carlos Chagas, Fiocruz-PR, Curitiba, PR, Brasil / O estudo de estruturas e interações proteicas é uma importante área de pesquisa para se entender as funções das proteínas. No entanto, essa é também uma das áreas de grandes desafios experimentais, devido à inerente complexidade atômica de proteínas e peptídeos. Os métodos de elucidação estrutural de alta resolução (e.g. difração de raios-X e RMN) são hoje os considerados “padrões-ouro” para esses tipos de estudos. No entanto, uma grande parte das proteínas e seus respectivos complexos não são passíveis de serem resolvidos por esses métodos, motivando o desenvolvimento de novas técnicas para a caracterização estrutural de proteínas e seus complexos. Neste sentido, a espectrometria de massas acoplada à técnica de crosslinking (XL-MS) é uma grande promessa, devido às suas características intrínsecas, tais como alta sensibilidade e ampla aplicabilidade. Neste trabalho, desenvolveu-se um software com aplicações pioneiras, denominado SIM-XL, capaz de identificar peptídeos covalentemente ligados e analisados por espectrometria de massas, a fim de caracterizar estruturas de proteínas, bem como de complexos proteínas-proteínas e proteína-peptídeo. Esse software faz uso de técnicas de reconhecimento de padrões para resolver um gargalo na modelagem proteica e interação proteína-proteína. Portanto, o algoritmo aqui apresentado, traz benefícios imediatos nas áreas de biologia e biotecnologia e indiretamente, em diversas outras áreas, como por exemplo, no desenvolvimento de novos fármacos. / The study of protein structures and interactions is an important area of development for understanding the function of proteins. However, this is also an area of great experimental challenge, due to the inherent atomic complexity of proteins and peptides. The methods of structural elucidation of high-resolution (e.g. X-ray diffraction and NMR) are currently considered the “gold standard” for these types of studies. However, many proteins are not amendable to being solved by these methods; thus motivating the development of new techniques for structural characterization of proteins and their complexes. In this regard, mass spectrometry coupled by crosslinking technique (XL-MS) poses as a promise to overcome these limitations as it provides a high sensitivity and wide applicability. Here we present SIM-XL, a software pioneer in many ways, capable of identifying cross-linked peptides analyzed by mass spectrometry and thus ultimately aiding in structural characterization and in determining protein-protein interactions. Our software uses pattern recognition strategies to address a bottleneck in protein modeling and protein-protein interaction. As such, various fields related to biology and biotechnology suffer an immediate benefit from this work, and other areas, say, the development of new drugs, are indirectly benefited as well.

Les complexes histones méthyltransférases SET1, hautement conservés de la levure aux mammifères, sont ciblés aux régions promotrices par la protéine CFP1/CXXC, résultant en l’implémentation de la méthylation de la lysine 4 de l’histone H3 (H3K4me), modification post-traductionnelle influençant l’expression des gènes selon le contexte chromatinien. La présence de plusieurs complexes SET1 distincts dans différents systèmes modèles eucaryotes a compliqué l’étude de leurs fonctions dans un contexte développemental. Caenorhabditis elegans contient une seule protéine homologue de SET1, SET-2, et d’uniques homologues des autres sous-unités du complexe, RBBP5, ASH2, WDR5, DPY30 et CFP1. Cependant, la composition biochimique du complexe n’a pas été décrite. En couplant des expériences de co-immunoprécipitation avec des analyses de spectrométrie de masse, j’ai identifié le complexe SET-2/SET1 dans les embryons de C. elegans. D’autre part, j’ai montré que le complexe SET-2/SET1 co-immunoprécipite aussi un autre complexe conservé modifiant la chromatine et j’ai mis en évidence les interactions mises en jeu entre ces deux complexes. Mon analyse génétique a démontré que les mutants de perte de fonction des sous-unités des deux complexes partagent des phénotypes communs, en cohérence avec des fonctions développementales communes. Le laboratoire a également entrepris des expériences de transcriptomique et d’immunoprécipitation de la chromatine montrant un nouveau rôle de CFP-1 dans le recrutement de ce complexe au niveau de sites spécifiques de la chromatine. / The highly conserved SET1 family complexes are targeted by CFP1/CXXC protein to promoter regions through multivalent interactions to implement methylation of histone H3 Ly4 (H3K4me), a modification that correlates with gene expression depending on the chromatin context. The presence of distinct SET1 complexes in multiple eukaryotic model systems has hampered studies aimed at identifying the complete array of functions of SET1/MLL regulatory networks in a developmental context. Caenorhabditis elegans contains one SET1 protein, SET-2, one MLL-like protein, SET-16, and single homologs of RBBP5, ASH2, WDR5, DPY30 and CFP1. The biochemical composition of the complex however, has not been described. Through the use of co-immunoprecipitation coupled to mass spectrometry-based proteomics, I identified the SET-2/SET1 complex in C. elegans embryos. Most importantly, I showed that the SET-2/SET1 complex also co-immunoprecipitates another conserved chromatin-modifying complex and I highlighted the interactions involved between these two complexes. My genetic analysis revealed that loss of function mutants of the two complex subunits share common phenotypes, consistent with common developmental functions. The laboratory has also undertaken transcriptomic and chromatin immunoprecipitation experiments showing that CFP-1 has a role in the binding of this complex at specific chromatin regions.

The PI3K/AKT/mTOR signaling pathway is commonly dysregulated in cancer. The goal of this thesis project was to assess the hypothesis of a strong correlation between PI3K/AKT/mTOR pathway activity and the response to targeted therapies, by using a protein quantitation technique called immuno-matrix assisted laser desorption/ionization (iMALDI). The use of iMALDI as a clinical tool was demonstrated by automating an established iMALDI assay for quantifying plasma renin activity. The results from the automated method gave high correlation coefficients of ≥0.98 with a clinical LC-MS/MS method and could be performed significantly faster than with manual sample preparation. The 7.5-fold faster analysis compared to LC-MS/MS, reduction in human error, and higher throughput, demonstrated the suitability of this assay for clinical use. The automated iMALDI platform was then adapted for use with cancer cell lines and tissue analysis, targeting the kinases AKT1 and AKT2 as surrogate proteins for signaling pathway activity. Using minute amounts (10 µg/capture), AKT1 and AKT2 expression and phosphorylation stoichiometry (PS) were successfully quantified via their C-terminal tryptic peptides, which encompassed key phosphorylation sites. After assay optimization, the assays were analytically validated for linear range, accuracy, and interferences. In addition, PS cut-off values based on measurement errors were established for confident PS quantitation. The functionality of the assay was demonstrated with cell lines, and flash-frozen and FFPE tissue lysates, with, on average, lower AKT1/AKT2 measurements obtained from FFPE samples. The developed assays were sensitive and precise enough to detect differences between matched normal and adjacent tumor tissues. To answer the hypothesis, patient-derived xenograft (PDX) mouse-model tumors treated with Herceptin, Everolimus, a combination of both (E+H), or with no treatment, were assessed for molecular patterns linked to tumor response. One mouse from the E+H group showed a partial response, with elevated total and phosphorylated AKT1/AKT2. Unfortunately, overlapping values between treatment groups were obtained in this study, and the large within-group spread and the low number of biological replicates made it difficult to confirm a definite correlation between PI3K/AKT/mTOR pathway activity and response to treatment. A follow-up study with additional protein targets, a larger number of samples, and serial biopsies will be required to determine if there is, in fact, a correlation between PI3K/AKT/mTOR pathway activity and response to treatment. / Graduate / 2019-10-05

Made available in DSpace on 2015-12-10T14:22:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-26. Added 1 bitstream(s) on 2015-12-10T14:28:25Z : No. of bitstreams: 1 000850009_20170226.pdf: 1029581 bytes, checksum: 69e812dad32c76a53500967a80a8c187 (MD5) Bitstreams deleted on 2017-03-03T11:01:36Z: 000850009_20170226.pdf,. Added 1 bitstream(s) on 2017-03-03T11:02:43Z : No. of bitstreams: 1 000850009.pdf: 2777836 bytes, checksum: 2500fb48a204b51b3ba5201d7d6a82c9 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Desde o advento da nova era da abordagem proteômica, a identificação de assinaturas moleculares individuais em vários sistemas biológicos pela pesquisa básica tem impulsionado a descoberta de biomarcadores diretamente relacionados a aplicações terapêuticas, promovendo desenvolvimento de testes diagnósticos clínicos mais específicos. Neste contexto, a necessidade da aplicação clínica estimula a pesquisa de bancada, sendo que novas tecnologias permitem novas descobertas e podem se transformar em prognósticos efetivos, medicamentos inéditos ou mais eficazes e/ou testes diagnósticos mais específicos. Os bubalinos tem sido alvo de muitas iniciativas econômicas e de pesquisas translacionais. Uma vez que, seu plasma sanguíneo é rico em fibrinogênio, búfalos da espécie Bubalus bubalis raça Murrah, se tornaram doadores primordiais de matéria-prima para o desenvolvimento de insumos biológicos, como por exemplo, o selante de fibrina derivado do veneno de serpentes e do sangue de bubalinos produzido pelo Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP). Para tal, se fez necessário melhor compreender a composição do plasma sanguíneo e do crioprecipitado destes bubalinos, a fim de dar suporte à pesquisas futuras que envolverão a identificação de marcadores moleculares de doenças infecciosas que podem acometer estes animais doadores. Este trabalho foi dividido em duas partes: a primeira destaca-se os achados prévios e significância da bubalinocultura, a utilização do sangue de animais para o desenvolvimento de bioinsumos na saúde animal e humana, a produção e aplicação de diversos selantes de fibrina, o uso da abordagem proteômica como estratégia investigativa para a prospecção de biomarcadores moleculares de doenças infecciosas e uma breve descrição das principais técnicas analíticas empregadas pela abordagem proteômica. A segunda parte deste trabalho, destaca-se a caracterização proteica do... / Since the advent of the new era of proteomics approach, the identification of individual molecular signatures in various biological systems for basic research has driven the discovery of biomarkers directly related to therapeutic applications, promoting the development of more specific clinical diagnostic tests. In this context, the need for clinical application stimulates the bench study, which new technologies and new discoveries can become effective prognostic, or novel drugs more effective and/or more specific diagnostic tests. The bubaline have been targets of several economic initiatives and translational research. Since its plasma is rich in fibrinogen, Bubalus bubalis water buffalos (Murrah) have became main donors of raw material for the development of biological insumes, as example, the fibrin sealant from snake venoms and water buffalos blood produced by The Center for the Studies of Venoms and Venomous Animals (CEVAP) at UNESP. Then, it was necessary to better understand the composition of the blood plasma and cryoprecipitate these buffalo, in order to support future research will involve the identification of molecular markers of infectious diseases that can affect these donor animals. This study was divided into two parts: the first stands up the previous findings and significance of buffalo, the use of animal blood for developing bioinsumes in animal and human health, the production and application of several fibrin sealants, the use proteomics approach as a research strategy for the discovery of molecular biomarkers of infect diseases and a brief description of the analytical methods used by proteomics approach. The second part of this work brings the protein characterization of blood plasma and cryoprecipitate of B. bubalis healthy buffalo, where have evidenced 17 groups of major proteins involved in the signaling pathway of the complement system and the cascade coagulation

Made available in DSpace on 2014-06-11T19:27:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-11-28Bitstream added on 2014-06-13T18:55:58Z : No. of bitstreams: 1 pitangui_ns_me_arafcf.pdf: 11259600 bytes, checksum: 17b255999c9caee5aaf2d3e40ea94a70 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Histoplasma capsulatum var. capsulatum é um fungo dimórfico que causa a histoplasmose, uma micose respiratória e sistêmica. H. capsulatum é primariamente adquirido após a exposição ao aerosol, pela inalação de microconídeos ou fragmentos de hifas. A evolução da doença respiratória depende da capacidade da levedura de Histoplasma em sobreviver e se replicar dentro dos macrófagos alveolares. Neste contexto, é conhecido que a adesão às células do hospedeiro constitui o primeiro passo para a colonização e é essencial para o estabelecimento da infecção. Em vista disso, alguns microrganismos aderem às superfícies biológicas e não biológicas formando biofilmes. Biofilmes são comunidades dinâmicas de microrganismos, que aderidos às superfícies, produzem uma matriz exopolimérica e essa formação representa um estado que permite que as células microbianas sobrevivam em ambientes hostis e dispersem para colonizar novos nichos. Com base na importância potencial de biofilmes para sua sobrevivência no hospedeiro humano e na natureza, este trabalho foi desenvolvido para investigar pela primeira vez a formação de biofilme por H. capsulatum correlacionando com sua capacidade em aderir às células epiteliais, bem como definir particularidades da interação entre macrófagos e H. capsulatum, incluindo análise de fragmentação nuclear pelo ensaio do cometa e TUNEL. Os ensaios foram realizados in vitro com duas cepas clínicas de H. capsulatum, uma isolada no México e a outra no Brasil. Além disso, foi realizada análise proteômica diferencial do fungo em biofilme e sob a condição planctônica. Os resultados mostraram que H. capsulatum é capaz de formar biofilmes e ambos os isolados selecionados tem uma alta capacidade em aderir aos pneumócitos (A549). Através de análise proteômica, o isolado EH-315 exibiu diferentes perfis protéicos, com aproximadamente... / Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a respiratory and systemic mycosis. H. capsulatum is primarily acquired after exposure to the aerosol, by microconideas or hyphal fragments inhalation. The development of a respiratory disease depends on the ability of Histoplasma yeast to survive and replicate within alveolar macrophages. In this context, it is known that the adhesion to host cells is the first step in the colonization and is essential for the establishment of the infection. As a result, some microorganisms adhere to biological and non-biological surfaces producing biofilms. Biofilms are dynamic communities of microorganisms that adhere to surfaces producing an exopolimeric matrix, and this formation is a state that allows microbial cells to survive in hostile environments and to disperse to colonize new niches. Based on the potential importance of biofilms to survive in the human host and in nature, this study was performed to investigate, for the first time, the biofilm formation by H. capsulatum correlating with their ability to adhere to epithelial cells, as well as defining features of the interaction between macrophages and H. capsulatum, including analysis of nuclear fragmentation by comet assay. In addition, we performed differential proteomic analysis of the fungus in biofilm and under planktonic conditions. The results demonstrated that H. capsulatum is able to form biofilms and both selected isolates have a high ability to adhere to pneumocytes (A549). Through proteomic analysis, the EH-315 isolate exhibited different protein profiles, with approximately 250 proteins exclusively expressed in the biofilm format and others with different levels of expression when compared to the fungus in planktonic growth. The sequencing by MALDI-TOF/TOF revealed that most proteins are nuclear or are involved in the metabolism... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:27:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-05Bitstream added on 2014-06-13T20:35:40Z : No. of bitstreams: 1 marcos_cm_me_arafcf.pdf: 2508470 bytes, checksum: bc1a1f1c4040c405fe6c122e87b067ff (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Paracoccidioides brasiliensis é um importante patógeno humano que causa a paracoccidioidomicose (PCM), uma micose sistêmica com ampla distribuição na América Latina. A adesão e invasão de células são eventos cruciais envolvidos na infecção e disseminação do patógeno. Além disso, patógenos utilizam suas moléculas de superfície para se ligar aos componentes da matriz extracelular para estabelecer a infecção. Uma proteína antigênica de P. brasiliensis foi isolada de géis de eletroforese bidimensional do cell-free do fungo e caracterizada. Peptídeos foram obtidos da proteína de 54 kDa e pI 5,6 e mostraram homologia com enolase de Paracoccidioides brasiliensis e outros fungos. A proteína foi purificada através de eletroeluição e utilizada para a produção de anticorpo policlonal em coelho. Por microscopia de fluorescência não foi possível observar a localização exata desta proteína, apenas que se encontra aparentemente distribuídapor todo o fungo, foi possível verificar alterações no citoesqueleto de pneumócitos durante a infecção por P. brasiliensis. A localização foi confirmada por microscopia imunoeletrônica a presença de enolase foi detectada principalmente na parede celular de leveduras de P. brasiliensis e também no citoplasma, ela se demonstrou mais expressa quando este fungo foi cultivado em meio onde houve acréscimo de sangue de carneiro e durante a situação de infecção a pneumócitos. A enolase purificada foi capaz de se ligar a fibronectina, fibrinogênio, laminina, plasminogênio, colágenos tipo I e IV. Foi confirmado que a ligação desta proteína à pneumócitos é influenciada pela sequência de aminoácidos Arg-Gly-Asp contida provavelmente nos receptores da matriz extracelular presentes na célula do hospedeiro. Essas informações indicam que a enolase possivelmente contribui para a adesão do microrganismo aos tecidos do... / Paracoccidioides brasiliensis is an important human pathogen that causes paracoccidioidomycosis (PCM), a systemic mycosis with a wide distribution in Latin America. The ability of P. brasiliensis to cause not only human diseases but also mycoses with a variety of clinical manifestations from localized forms to the disseminated disease progressing to lethality, probably depends on the relationship between the virulence of the fungus, its ability to interact with and to invade the surface structures of the host and the immune response of the host. The adhesion of the pathogen leads to the recognition of carbohydrate and protein ligands on the surface of the host cell or proteins of the extracellular matrix (ECM). The large number of tissue types that fungi can colonize and infect suggests that they use a variety of surface molecules for adhesion.Understanding the interactions between P. brasiliensis and the host tissue depends on the study of the different steps of the process of colonization, especially adhesion, in which the pathogen recognizes ligands on the surface of host cells. This study aimed to verify the role of enolase in the host cell-fungus interaction and the ability of enolase to bind to extracellular matrix components, to determine its subcellular localization. The data revealed that fibronectin is the major ligand of enolase. Evaluation of the location of enolase at an ultrastructural level revealed that it is distributed in various cellular compartments, but at a high level in the cell wall. This suggests that enolase performs additional functions related to the glycolytic pathway and also plays a role of adhesion in P. brasiliensis. Therefore, this study increases the knowledge about the characteristics of enolase and its influence on the binding process of P. brasiliensis