The current proliferation of connected displays and mobile devices from smart phones and tablets to wall-sized displays presents a number of exciting opportunities for information visualization and visual analytics. When a user employs heterogeneous displays collaboratively to achieve a goal, they form what is known as a display ecology. The display ecology enables multiple displays to function in concert within a broader technological environment to accomplish tasks and goals. However, since information and tasks are scattered and disconnected among separate displays, one of the inherent challenges associated with visual analysis in display ecologies is enabling users to seamlessly coordinate and subsequently connect and integrate information across displays. This research primarily addresses these challenges through the creation of interaction and visualization techniques and systems for display ecologies in order to support sensemaking with visual analysis. This dissertation explores essential visual analysis activities and design considerations for visual analysis in order to inform the new design of display ecologies for visual analysis. Based on identified design considerations, we then designed and developed two visual analysis systems. First, VisPorter supports intuitive gesture interactions for sharing and integrating information in a display ecology. Second, the Spatially Aware Visual Links (SAViL) presents a cross-display visual link technique capable of guiding the user's attention to relevant information across displays. It also enables the user to visually connect related information over displays in order to facilitate synthesizing information scattered over separate displays and devices. The various aspects associated with the techniques described herein help users to transform and empower the multiple displays in a display ecology for enhanced visual analysis and sensemaking. / Ph. D.

No description available.

Small stable protein domains are attractive scaffolds for engineering affinity proteins due to their high tolerance to mutagenesis without loosing structural integrity. The albuminbinding domain is a 5 kDa three-helix bundle derived from the bacterial receptor Protein G with low-nanomolar affinity to albumin. In this thesis, the albumin-binding domain is explored as a scaffold for engineering novel affinity proteins with the possible benefit of combining a prolonged serum half-life with specific targeting in a single small scaffold protein. Previously, a library was created by randomizing surface-exposed residues in order to engineer affinity to a new target antigen in addition to the inherent albumin affinity. Here, phage display selections were separately performed against the tumor antigens ERBB2 and ERBB3. The ERBB3 selection resulted in a panel of candidates that were found to have varying affinities to ERBB3 in the nanomolar range, while still retaining a high affinity to albumin. Further characterization concluded that the clones also competed for binding to ERBB3 with the natural activating ligand Heregulin. The selections against ERBB2 resulted in sub-nanomolar affinities to ERBB2 where the binding site was found to overlap with the antibody Trastuzumab. The binding sites on ABD to albumin and either target were found in both selections to be mutually exclusive, as increased concentrations of albumin reduced the level of binding to ERBB2 or ERBB3. An affinity-matured ERBB2 binder, denoted ADAPT6, which lacked affinity to albumin was evaluated as a radionuclide-labeled imaging tracer for diagnosing ERBB2-positive tumors. Biodistribution studies in mice showed a high renal uptake consistent with affinity proteins in the same size range and the injected ADAPT quickly localized to the implanted tumor. High contrast images could be generated and ERBB2-expressing tissue could be distinguished from normal tissue with high contrast, demonstrating the feasibility of the scaffold for use as diagnostic tool. In a fourth study, affinity maturation strategies using staphylococcal cell-surface display were evaluated by comparing two replicate selections and varying the stringency. A sub-nanomolar target concentration was concluded to be inappropriate for equilibrium selection as the resulting output was highly variable between replicates. In contrast, equilibrium sorting at higher concentrations followed by kinetic-focused off-rate selection resulted in high output overlap between attempts and a clear correlation between affinity and enrichment. / <p>QC 20160510</p>

Au cours de sa vie, 1 femme sur 9 sera atteinte du cancer du sein, 1 sur 27 sera emportée par cette maladie et 10 à 15 % des patientes développeront des métastases dans les trois années suivant le diagnostic. Le diagnostic précis et personnalisé du cancer du sein ainsi que l'évaluation de son potentiel métastatique est donc un enjeu majeur. Une analyse plus précise des caractéristiques moléculaires d'une tumeur primaire devrait conduire à une médecine personnalisée, un traitement et un suivi plus efficace. La détection de biomarqueurs sériques serait un moyen de diagnostiquer un cancer métastatique. Dans le but de découvrir de nouveaux marqueurs, l'analyse protéomique d'échantillons de patient a un fort potentiel mais souffre de limitations techniques, incluant le manque d'anticorps stables reconnaissant des marqueurs tumoraux d'intérêt. Par l'utilisation de fragments d'anticorps aux propriétés remarquables nommé single domain antibody (sdAb), et grâce à la mise au point d'une stratégie innovante de phages display, ce travail apporte d'importantes réponses en termes de disponibilité d'anticorps, d'analyse spécifique d'échantillon et de découverte de nouvelles cibles. Nous avons élaboré une stratégie permettant la découverte de biomarqueurs et l'isolement des anticorps correspondants. Après la construction de banques de sdAb à partir de lamas immunisés par des biopsies, une nouvelles stratégie de sélection in vitro par phage display, la sélection masquée, nous a permis d'isoler des anticorps spécifiques du cancer du sein. / In a lifetime, 1 in 9 women will develop breast cancer, 1 of 27 will be swept away by the disease and from 10 to 15% of patients will develop metastases within three years of diagnosis. Accurate and personalized diagnosis of breast cancer and the detection of its metastatic potential is a major challenge. It is essential to develop new analytical methods allowing an effective monitoring of breast cancer. A closer analysis of the molecular characteristics of a primary tumor should lead to more effective personalized medicine, treatment and monitoring. The efficient detection of serum biomarkers would be a way to diagnose metastatic cancer and to modify treatment based on these results. Toward this goal, the proteomic analysis of patient samples has great potential but suffers from technical limitations, including the lack of a wide variety of antibodies and tumor marker. By the use of innovative antibody fragments with remarkable properties named single domain antibody (sdAb), and through the development of a new innovative strategy of phage display, this work provides important answers in terms of availability of antibody, specific proteomic analysis of sample and new target discovery. We have developed a strategy allowing the simultaneous discovery of new biomarkers and the isolation of corresponding antibodies. After the construction of sdAb libraries from llamas immunized with biopsies, and using a new in vitro selection strategy by phage display named masked selection, we have isolated breast cancer-specific antibodies.

Actuellement, 60% des médicaments sur le marché ont pour cible des protéines membranaires. Toutefois, l'étude de ces protéines membranaires reste un challenge de par leur structure particulière (domaines transmembranaires hydrophobes et domaines extra- et intra-cellulaires hydrophiles), mais également par leur faible expression sur les cellules.L'entreprise Ciloa, dans laquelle j'ai effectué ma thèse, a développé une technologie brevetée, qui permet d'exprimer à la surface des exosomes, des vésicules membranaires de tailles comprises entre 30 et 100nm, des protéines membranaires natives, grâce à un peptide d'adressage, le DCTM. Cette technologie possède de nombreux domaines d'applications, comme le criblage de médicaments, le développement de vaccins ou encore le développement d'anticorps monoclonaux.L'objectif de ma thèse a été, dans un premier temps, de mettre en place l'outil exosomes recombinants grâce à la technologie de Ciloa et dans un deuxième temps, d'utiliser ces outils pour le développement d'anticorps, grâce aux exosomes recombinants.Ainsi, j'ai d'abord mis au point différentes techniques de caractérisation des exosomes recombinants (ELISA), et également participé à la mise en place de différents protocoles de production et de purification, en fonction leur utilisation. Une fois ces outils optimisés, j'ai pu les utiliser pour le développement d'anticorps. J'ai testé en parallèle deux méthodes de production d'anticorps, une méthode classique, l'hybridation lymphocytaire après immunisation de souris BALB/c, et une méthode plus récente, le criblage d'une banque de scFvs par phage display.L'hybridation lymphocytaire a permis la production d'hybridomes, dont les anticorps ont été criblés sur exosomes, par ELISA. Dans le cadre du criblage par phage display, j'ai participé au développement d'une banque de scFvs, basée sur le modèle du 13R4, dont nous avons modifié les longueurs de boucles des différents CDRs, notamment le CDRH3, afin de cibler les épitopes faiblement accessibles des protéines membranaires. Les sélections de scFvs ont été effectuées sur exosomes recombinants, exprimant des protéines membranaires. / Nowadays, more than 60% of marketed drugs target membrane proteins. However, their study still represents a challenge, essentially due to their particular 3D-structure (hydrophobic transmembrane domains and hydrophilic extra- and intra-cellular domains), but also to their low expression level in cells.Ciloa, the start-up company in which I realized my PhD, has developed a patented technology that enables to express native membrane proteins on exosomes, membrane vesicles of 30 to 100nm, using a pilot peptide called DCTM (for Cytosolic Domain of TransMembrane). This technology displays a lot of different applications, in different domains such as drug screening, vaccines development or monoclonal antibodies (mAbs) development.The purpose of my PhD research was, first, to set up the recombinant exosomal tool using Ciloa's innovative technology, and then to use this tool to develop monoclonal antibodies.Thus, at the beginning of my PhD, I set up exosomal characterization technics, such as ELISA, and I also took part in the setup of several production and purification protocols, depending of the use of exosomes. Once these tools had been optimized, I was able to use them to develop mAbs. I tested two methods, one classical, the generation of hybridoma after Balb/c mice immunizations, and a more recent technology, the screening of scFvs library by phage display.Therefore, I obtained hybridoma and was able to screen the derived antibodies by ELISA on exosomes. Concerning the phage display technology, I took part in the development of a new scFvs library, based on the 13R4 scaffold, of which we changed the CDRs lengths, mostly the CDRH3, in order to target epitopes with low accessibility, such as the one of membrane proteins. The library screening was realized on recombinant exosomes.

Adenocarcinomas e distroglicanopatias são doenças graves que estão associadas a quadros de hipoglicosilação de mucinas tumorais, como a MUC1 (transmembrane glycoprotein Mucin 1) e de mucinas de ?-distroglicana (?-DG). Um dos mais importantes desafios associados à terapia anti-câncer refere-se ao desenvolvimento de estratégias terapêuticas que permitam o direcionamento da ação de drogas anti-tumorais para a célula cancerosa com o objetivo de evitar o acometimento de células saudáveis. Nessa linha, é crucial a construção de sistemas de liberação de medicamentos sítio específicos por meio de marcadores tumorais. Quanto ao diagnóstico das distroglicanopatias, atualmente este se baseia principalmente na observação de manifestações clínicas, biópsias musculares e medidas enzimáticas, sendo que os anticorpos monoclonais disponíveis no mercado não são específicos para a condição do músculo distrófico. Dessa forma, mucinas tumorais e mucinas de ?-DG modificadas tem sido consideradas potenciais alvos para o desenvolvimento de novas estratégias diagnósticas e/ou terapêuticas aplicáveis a estas doenças. Para este trabalho, foram sintetizados, em fase sólida, glicopeptídeos miméticos de MUC1 e ?-DG hipoglicosilados, os quais foram utilizados como ferramenta de busca por novos anticorpos recombinantes. Estes antígenos foram imobilizados em uma placa e sobre eles foi aplicada uma biblioteca de fragmentos de anticorpos (Fabs) humanos recombinantes para o desenvolvimento do processo de seleção pela tecnologia de Phage Display. Após quatro rounds consecutivos de seleção, os genes codificadores dos Fabs da biblioteca não selecionada e selecionada foram sequenciados e analisados in silico na plataforma ATTILA. Esta análise permitiu rastrear o enriquecimento dos domínios VH e VL durante a seleção, além de possibilitar a escolha de inúmeros clones para produção. Para este trabalho, quatro fragmentos de anticorpos scFvs recombinantes inéditos para a mucina tumoral MUC1 e ?-DG hipoglicosilados foram desenhados e clonados em vetor de expressão pET29(a) contendo um marcador de identificação (peptídeo FLAG) e outro de purificação (cauda de histidina). A expressão de um scFv recombinante anti-MUC1 foi realizada em E. coli BL21-DE3 pela adição de 0,5mM de IPTG com indução a 20ºC por 16 horas. A purificação foi realizada por cromatografia de afinidade em resina de níquel, seguida de gel filtração, sendo estas etapas monitoradas por SDS-PAGE. A identificação imunoquímica da proteína recombinante foi confirmada por Western Blot, utilizando o anticorpo anti-FLAG. Entende-se que este trabalho, por meio da produção de novas ferramentas biotecnológicas, poderá cooperar para o desenvolvimento de novas formas abordagens diagnósticas e/ou terapêuticas para tumores e distroglicanopatias. / Adenocarcinomas and dystroglycanopathies are serious diseases associated with hypoglycosylation of tumoral mucins, such as MUC1 (transmembrane glycoprotein Mucin 1) and ?-dystroglican mucins (?-DG). One of the most important challenges associated with anti-cancer therapy is the development of therapeutic strategies that allow the targeting of anti-tumor drugs to the cancer cell in order to avoid the involvement of healthy cells. In this regard, the construction of site-specific drug delivery systems by tumor markers is crucial. The diagnosis of dystroglicanopathies are currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic muscle condition. Thus, tumoral mucins and modified ?-DG mucins have been considered potential targets for the development of new diagnostic and/or therapeutic strategies applicable to these diseases. For this work, glycoproteins MUC1 and ?-DG hypoglycosylated mimetics were synthesized by solid phase reaction, and were used as a search tool for new recombinant antibodies. These antigens were immobilized in a plate and a library of recombinant human antibody (Fabs) fragments was applied thereon for the development of the screening process by Phage Display technology. After four consecutive rounds of selection, the Fabs coding genes from the unselected and selected library were sequenced and analyzed in silico on ATTILA platform. This analysis allowed us to track the enrichment of the VH and VL domains during selection process, and also presented several option of clones to choose for production. For this work, four novel fragments of recombinant scFvs antibodies specific for tumoral mucin MUC1 and ?-DG hypoglycosylated were designed and cloned into pET29 (a) expression vector containing an identification marker (FLAG peptide) and a purification tag (histidine tail). Expression of a recombinant anti-MUC1 scFv was performed on E. coli BL21-DE3 by the addition of 0.5 mM of IPTG with induction at 20°C for 16 hours. Purification was performed by affinity chromatography on nickel resin, followed by gel filtration, these steps being monitored by SDS-PAGE. Immunochemical identification of the recombinant protein was confirmed by Western Blot, using the anti-FLAG antibody. It is understood that this work, through the production of new biotechnological tools, could cooperate for the development of new forms of diagnostic and/or therapeutic approaches for tumors and dystroglicanopathies.

Leptospirose é uma doença infecciosa emergente cujos agentes etiológicos são espécies patogênicas do gênero Leptospira. Leptospiras patogênicas possuem inúmeros genes específicos codificando proteínas com funções desconhecidas, sugerindo que as leptospiras apresentam fatores de virulência únicos. Adesinas bacterianas são importantes fatores de virulência e, assim, a identificação de adesinas conservadas em espécies patogênicas de Leptospira pela construção de bibliotecas genômicas expostas na superfície de bacteriófagos (shotgun phage display), seguida por seleção em células e/ou componentes da matriz extracelular (biopanning), pode revelar novos antígenos e alvos para o tratamento e prevenção da leptospirose. Bibliotecas foram construídas com o DNA genômico de L. interrogans fragmentado e o fagomídeo pG8SAET, sendo testadas algumas abordagens para clonagem como a ligação entre extremidades cegas (blunt-end) e técnicas baseadas em ligação entre extremidades coesivas, incluindo a obtenção de ORESTES e a utilização de adaptadores em grampo. Apesar de serem encontradas algumas limitações, a clonagem por ligação blunt-end se mostrou a mais eficiente para a construção de bibliotecas, sendo adotada para a construção de três bibliotecas em maior escala. A seleção de novas possíveis adesinas a partir das bibliotecas construídas foi realizada em células eucarióticas através da metodologia BRASIL. A primeira biblioteca (BBT1) exibiu 106 clones totais, a partir da qual foram selecionados quatro proteínas em fase apenas com a proteína VIII do fago (pVIII). No entanto, nenhuma delas seria exposta por programas de predição na bactéria. Outras duas bibliotecas foram construídas (BBT2 e BBT3), as quais obtiveram um número ideal de clones para uma ampla cobertura do genoma (>2x107 clones). Por apresentar maior proporção de clones válidos, a BBT2 foi utilizada para a seleção de adesinas, resultando em onze clones em fase com pVIII e/ou sequência sinal do fago. Análises por programas de predição revelaram três proteínas hipotéticas, denominadas LepA962, LepA069 e LepA388, as quais poderiam estar expostas ou ser secretadas pela bactéria, sugerindo uma possível função de adesina. O estudo da proteína LepA388 levou ao reconhecimento de outras doze proteínas semelhantes e pertencentes a uma família paráloga contendo um domínio denominado DUF_61, motivo de função desconhecida presente em proteínas compartilhadas somente entre as espécies patogênicas mais virulentas de Leptospira. Por esta razão, a proteína LepA388 foi a mais estudada. A clonagem de três porções da proteína (LepA388P, LepA388NR e LepA388F) para expressão heteróloga resultou em proteínas recombinantes insolúveis e, considerando a riqueza em resíduos de cisteína presente em sua estrutura, não foi possível renaturá-las adequadamente. Diante dos obstáculos encontrados, apenas a porção contendo a sequência apresentada pelo fago (LepA388P) foi utilizada para obtenção de antissoros em camundongos, os quais apresentaram altos títulos, demonstrando a alta imunogenicidade da proteína LepA388P. O reconhecimento de proteínas nativas da família paráloga DUF_61 em extratos de diferentes sorovares de Leptospira não foi observado, assim como sua expressão in vitro a partir de bactérias em diferentes condições de cultivo. Estudos adicionais sobre a expressão in vivo e funções dos membros desta família são necessários para uma compreensão mais ampla de seu papel na biologia de leptospiras e, possivelmente, na patogênese da leptospirose. / Leptospirosis is an emerging infectious disease whose etiologic agents are pathogenic species of the genus Leptospira. Pathogenic leptospires have countless specific genes encoding proteins with unknown functions, suggesting that leptospires have unique virulence factors. Bacterial adhesins are important virulence factors and so the identification of conserved adhesins in pathogenic Leptospira species from shotgun phage display libraries, followed by selection (biopanning) in cells and/or extracellular matrix components, can reveal new antigens and strategies for leptospirosis treatment and prevention. Libraries were constructed using fragmented genomic DNA from L. interrogans and pG8SAET phagemid vector. Cloning approaches included blunt-end ligation and techniques based in cohesive-end ligation, such as ORESTES strategy and hairpin linkers. Despite some limitations, cloning by blunt-end ligation was the most efficient for library construction, being adopted for the construction of three libraries on a larger scale. Selection of new possible adhesins was performed by biopanning of the libraries in eukaryotic cells through BRASIL methodology. The first library called BBT1 exhibited approximately 106 total clones, and its biopanning resulted in four proteins fused to phage protein VIII, but none of them were expected to be exposed by the bacteria. Other libraries were built (BBT2 and BBT3) which reached the expected number of clones to obtain a larger genome representation (> 2x107 clones). Since it showed the highest proportion of positive clones, BBT2 was selected to perform a second biopanning, resulting in eleven proteins fused to phage protein VIII and/or signal peptide. In silico analysis revealed three hypothetical proteins, named LepA962, LepA069 and LepA388, that would be exposed or secreted by the bacteria, suggesting a possible adhesin function. The study of LepA388 protein led to the recognition of twelve other similar proteins belonging to a paralogous family that contains a domain called DUF_61, domain of unknown function that is present in proteins shared only among the most virulent pathogenic species of Leptospira. For this reason, the LepA388 protein was the most studied. The cloning of three portions of the protein (LepA388P, LepA388NR and LepA388F) for heterologous expression resulted in insoluble recombinant proteins, and given the presence of many cysteine residues in its structure, it was not possible to renature them appropriately. In face of the imposed obstacles, only the portion containing the sequence presented by the bacteriophage (LepA388P) was used to obtain antisera in mice, which showed high titers, demonstrating the high immunogenicity of the protein LepA388P. Recognition of native DUF_61 paralogous family proteins in extracts from distinct Leptospira serovars was not observed, as well as its in vitro expression from bacteria cultured in different conditions. Additional studies on the in vivo expression and functions of members of this family are needed for a broader understanding of their role in leptospiral biology and possibly in the pathogenesis of leptospirosis.

Leishmania spp. são protozoários parasitas transmitidos a vertebrados pela picada de insetos flebotomíneos fêmeas. Apresentam duas formas principais em seu ciclo de vida: promastigotas e amastigotas. Os promastigotas vivem no intestino do hospedeiro invertebrado. Os amastigotas vivem preferencialmente no interior de macrófagos, fagócitos profissionais capazes de destruir patógenos. Para isso, são capazes de burlar a atividade microbicida desses macrófagos, sobreviver e se multiplicar em seu interior. Essa capacidade se deve a diversas moléculas produzidas pelo parasita, denominadas fatores de virulência, que ajudam em sua internalizarão e proliferação na célula hospedeira. A proteína LaLRR17 de Leishmania (L.) amazonensis recebeu esse nome porque contém em sua região central seis repetições ricas em leucina (LRRs) e por seu gene se localizar no cromossomo 17. Os motivos LRR de diversos organismos estão normalmente envolvidos em interações proteicas. A LaLRR17 é expressa em promastigotas e amastigotas, e foi detectada no citoplasma do macrófago infectado. Parasitas superexpressores dessa proteína apresentaram aumento da infectividade in vitro. Acreditamos que a proteína LaLRR17 participa de interações com moléculas de macrófagos, e que essas interações estão associadas à virulência dessa proteína. No entanto, não eram conhecidas moléculas do macrófago interagiam com a LaLRR17. O Phage Display é uma técnica baseada na expressão de proteínas sintéticas nos capsídeos de fagos que tem sido utilizada para identificação de ligantes de proteínas. A aplicação do Phage Display sobre a LaLRR17 recombinante permitiu identificar potenciais ligantes dessa proteína no macrófago, e aliada a cromatografia de afinidade, apontou diversos candidatos que podem participar do aumento de virulência conferido pela LaLRR17, entre eles o mais promissor, GRP78 do macrófago, que tem papel efetivo na fagocitose da Leishmania mediada pela LaLRR17. / Leishmania spp. are protozoan parasites transmitted to vertebrates by the bite of female phlebotomine insects. They present two main forms in their life cycle: promastigotes and amastigotes. Promastigotes live in the gut of the invertebrate host. The amastigotes live preferably in macrophages, \"professional phagocytes\" capable of destroying pathogens. They are able to circumvent the microbicidal activity of macrophages, survive and multiply inside them. This ability is due to several molecules produced by the parasite, called virulence factors, which help their internalization and proliferation inside the host cell. The protein LaLRR17 of L. (L) amazonensis was named because contains in its central region six leucine-rich repeats (LRRs) and its gene locates on chromosome 17. LRR motifs of various organisms are usually involved in protein-protein interactions. LaLRR17 is expressed in promastigotes and amastigotes, and was detected in the cytoplasm of the infected macrophage. Parasites overexpressing this protein showed increased infectivity in vitro. We believe that LaLRR17 participates in interactions with macrophages molecules, and that these interactions are associated with the virulence this protein. However, we do not know which molecules from the macrophage interact with LaLRR17. Phage Display is a technique based on the expression of synthetic proteins in phage capsids that has been used to identify protein binders. The application of Phage Display on recombinant LaLRR17 allowed the identification of potential ligands of this protein in the macrophage, and allied to affinity chromatography, pointed out several candidates that could participate in the increased virulence conferred by LaLRR17, among them the most promising macrophage GRP78, which plays an effective role in LaLRR17-mediated Leishmania phagocytosis.

Tie1 é um receptor tirosina quinase expresso em células endoteliais importante em angiogênese, formação de vasos sanguíneos a partir de vasos pré-existentes. Este receptor pertence a uma família pequena composta por apenas dois membros (Tie1 e Tie2) para os quais angiopoietinas foram identificadas como ligantes de Tie2. No entanto, Tie1 continua a ser um receptor órfão, sem ligantes identificados até o momento. Sendo assim, é difícil compreender completamente as propriedades biológicas de Tie1 e seus mecanismos moleculares em angiogênese sem um ligante identificado. Entretanto, como sugerido através de estudos de deleção gênica, este receptor é uma molécula essencial na angiogênese, apresentando um papel importante no desenvolvimento da vascularização da retina e desenvolvimento de tumores. O nosso objetivo foi estudar a participação do domínio extracelular de Tie1 na neovascularização e, no processo, identificar possíveis ligantes para este receptor. Através da tecnologia de phage display, identificamos um peptídeo específico e seletivo para Tie1, sugerindo a existência de um sítio de ligação único neste receptor. Mostramos que este peptídeo é capaz de inibir a proliferação de células endoteliais induzida por Ang1, um ligante bem caracterizado de Tie2 que também modula a atividade de Tie1. Além disso, também mostramos que este peptídeo inibe a angiogênese in vivo num modelo animal bastante relevante para estudo de doenças humanas, o modelo da retinopatia induzida por oxigênio. Uma vez que este peptídeo liga-se a um sítio único e seletivo para Tie1, hipotetizamos que o mesmo poderia mimetizar possíveis ligantes naturais deste receptor. Para identificá-los, proteínas com mimetopo cruzado com este peptídeo foram identificadas em extrato proteico de diferentes linhagens celulares. Tais proteínas são possíveis candidatos a interação com Tie1. Em resumo, demonstramos que o domínio extracelular de Tie1 é importante para a angiogênese patológica e identificamos proteínas como possíveis ligantes deste receptor, o que poderá contribuir para um melhor entendimento da participação de Tie1 na formação de vasos. O peptídeo aqui identificado poderá ser ainda uma ferramenta útil para o desenvolvimento de novas terapias anti-angiogênicas com importantes aplicações à saúde humana. / Tie1 is a tyrosine kinase receptor expressed by endothelial cells and important in angiogenesis, the formation of new blood vessels from pre-existing ones. This receptor belongs to a small family of receptors composed of two members only (Tie1 and Tie2) to which angiopoietins have been identified as ligands for Tie2. On the other hand, Tie1 is still an orphan receptor with no ligand identified to date. Thus, it is difficult to assess Tie1 mechanism of action in neovascularization without a known ligand. Nevertheless, gene deletion studies have shown that Tie1 is essential in angiogenesis, and plays an important role in retinal and tumoral vascularization. The aim of our study was to evaluate the participation of Tie1 extracellular domain in angiogenesis, and in the process, to identify putative ligands for this receptor. Utilizing phage display, we have identified and characterized a Tie1 specific and selective ligand peptide, which suggests the existence of a binding site unique to this receptor and not shared by other family members. We show that this peptide prevents endothelial cells proliferation, induced by angiopoetin-1, a ligand for Tie2 but which also modulates Tie1 activity. Using a well-accepted mouse model for human diseases, the oxygen induced retinopathy model, we show that this peptide inhibits angiogenesis in vivo. Since this peptide maps to a unique binding site in Tie1, we hypothesized that it might mimic a natural ligand for this receptor. To identify them, proteins with cross reactive epitopes with an anti-peptide sera were identified by proteomic approaches. These proteins are thus possible ligands for Tie1. In summary, we have shown that Tie1 extracellular domain is important in angiogenesis and we have identified putative ligand for this receptor, which might contribute to a better understanding of the molecular mechanisms associated with Tie1 in blood vessel formation. The peptide here characterized may also be an important tool for the development of novel anti-angiogenesis therapeutic approaches for disesase with an angiogenic component.

Angiogênese é a formação de novos vasos sanguíneos a partir de vasos já existentes e é importante em processos fisiológicos, que em adultos é restrita ao reparo tecidual e ao ciclo reprodutivo feminino. Entretanto, doenças, como câncer ou retinopatias, induzem a formação da angiogênese patológica, necessária para a progressão destas patologias. Anticorpos monoclonais constituem uma das classes de biofármacos que mais cresce e com impacto importante nas doenças dependentes de angiogênese. Entre as diversas metodologias para a identificação de anticorpos monoclonais contra alvos terapêuticos, está o phage display. Por causa do fator de crescimento endotelial vascular (VEGF) ser o principal fator responsável pela formação de novos vasos, o principal biofármaco anti-angiogênico disponível na clínica atualmente é um anticorpo monoclonal (bevacizumab) direcionadas contra o VEGF. Embora terapias anti-VEGF sejam eficazes, ainda não são ideais devido a efeitos colaterais indesejados e a resistência medicamentosa. Novas alternativas são necessárias a fim de aperfeiçoar as terapias angiogênicas. O objetivo do nosso estudo é identificar novos alvos moleculares e desenvolver novos agentes terapêuticos para doenças dependentes de angiogênese. Para atingirmos nossa meta escolhemos o sistema de phage display para selecionarmos anticorpos com propriedades angiogênicas. Uma biblioteca de de anticorpos foi desenvolvida em nosso laboratório, dirigida contra a molécula VEGF, em particular uma de suas isoformas. Os animais imunizados desenvolveram anticorpos específicos, detectados por ELISA e Western-blot. A amplificação do pool de genes das cadeias leve e pesada de imunoglobulinas foi realizada para produzir os fragmentos single-chain (ScFv) que foram então clonados no vetor para a construção da biblioteca. A biblioteca de display de anticorpos ScFv será, portanto, analisada em plataformas angiogênicas para isolar anticorpos específicos contra isoformas de VEGF e novos marcadores moleculares de superfície celular expressos por células endoteliais ativadas. / Angiogenesis is the formation of new blood vessels from pre-existing ones and is an important physiological process, which in adults is mostly restricted to wound healing or the female reproductive cycle. However, different illnesses, such as cancer or retinopathies, induce the formation of pathological angiogenesis, necessary for disease progression. Monoclonal antibodies are one of the fastest growing class of biopharmaceuticals with important implications in angiogenesis dependent diseases. Among various methods for the identification of monoclonal antibodies against therapeutic targets is phage display technology. Because the vascular endothelial growth factor (VEGF) is the main molecular factor responsible for the formation of new blood vessels, the major anti-angiogenic drug available in the clinic today is a monoclonal antibody (bevacizumab) directed against VEGF. However, although anti-VEGF therapies are effective, they are not yet ideal due to undesirable side effects and drug resistance. Novel alternatives are necessary to improve on angiogenic therapies. The aim of our study is to identify novel molecular targets and to develop new therapeutic agents for angiogenic dependent diseases. To achieve our goal we have chosen the phage display system in order to select for antibodies with angiogenic properties. An antibody phage library has been developed in our laboratory, directed against VEGF molecule, particularly one of it isoforms. The animals were immunized and developed specific antibodies, detected by ELISA and Western-blot. Amplification of the pool of light and heavy chain Ig genes was performed to produce the single chain (ScFv) fragments for library construction. The ScFv antibody display libraries will be then screened in angiogenic settings to isolate antibodies against specific VEGF isoforms and novel cell surface molecular markers expressed by activated human endothelial cells