Browsing by Author "Goulart Filho, Luiz Ricardo"

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    High-through identification of T cell-specific phage-exposed mimotopes using PBMCs from tegumentary leishmaniasis patients and their use as vaccine candidates against Leishmania amazonensis infection.
    (2019) Carvalho, Gerusa Brandão de; Costa, Lourena Emanuele; Lage, Daniela Pagliara; Ramos, Fernanda Fonseca; Santos, Thaís Teodoro de Oliveira; Ribeiro, Patrícia Aparecida Fernandes; Dias, Daniel Silva; Salles, Beatriz Cristina Silveira; Lima, Mariana Pedrosa; Carvalho, Lívia Mendes; Dias, Ana C. S.; Alves, Patrícia Terra; Franklin, Michelle Lucrécio; Silva, Renata A. M.; Duarte, Mariana Costa; Souza, Daniel Menezes; Roatt, Bruno Mendes; Chávez Fumagalli, Miguel Angel; Goulart Filho, Luiz Ricardo; Teixeira Junior, Antonio Lucio; Coelho, Eduardo Antônio Ferraz
    In the current study, phage-exposed mimotopes as targets against tegumentary leishmaniasis (TL) were selected by means of bio-panning cycles employing sera of TL patients and healthy subjects, besides the immune stimulation of peripheral blood mononuclear cells (PBMCs) collected from untreated and treated TL patients and healthy subjects. The clones were evaluated regarding their specific interferon-γ (IFN-γ) and interleukin-4 (IL-4) production in the in vitro cultures, and selectivity and specificity values were calculated, and those presenting the best results were selected for the in vivo experiments. Two clones, namely A4 and A8, were identified and used in immunization protocols from BALB/c mice to protect against Leishmania amazonensis infection. Results showed a polarized Th1 response generated after vaccination, being based on significantly higher levels of IFN-γ, IL-2, IL-12, tumour necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF); which were associated with lower production of specific IL-4, IL-10 and immunoglobulin G1 (IgG1) antibodies. Vaccinated mice presented significant reductions in the parasite load in the infected tissue and distinct organs, when compared with controls. In conclusion, we presented a strategy to identify new mimotopes able to induce Th1 response in PBMCs from TL patients and healthy subjects, and that were successfully used to protect against L. amazonensis infection
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    Identification and analysis of seven effector protein families with different adaptive and evolutionary histories in plant-associated members of the Xanthomonadaceae.
    (2017) Assis, Renata de Almeida Barbosa; Polloni, Lorraine Cristina; Patané, José Salvatore Leister; Thakur, Shalabh; Felestrino, Érica Barbosa; Diaz Caballero, Julio; Digiampietri, Luciano Antonio; Goulart Filho, Luiz Ricardo; Almeida Junior, Nalvo Franco de; Nascimento, Rafael; Dandekar, Abhaya M.; Zaini, Paulo Adriano; Setubal, João Carlos; Guttman, David S.; Moreira, Leandro Marcio
    The Xanthomonadaceae family consists of species of non-pathogenic and pathogenic γ-proteobacteria that infect different hosts, including humans and plants. In this study, we performed a comparative analysis using 69 fully sequenced genomes belonging to this family, with a focus on identifying proteins enriched in phytopathogens that could explain the lifestyle and the ability to infect plants. Using a computational approach, we identified seven phytopathogen-enriched protein families putatively secreted by type II secretory system: PheA (CM-sec), LipA/LesA, VirK, and four families involved in N-glycan degradation, NixE, NixF, NixL, and FucA1. In silico and phylogenetic analyses of these protein families revealed they all have orthologs in other phytopathogenic or symbiotic bacteria, and are involved in the modulation and evasion of the immune system. As a proof of concept, we performed a biochemical characterization of LipA from Xac306 and verified that the mutant strain lost most of its lipase and esterase activities and displayed reduced virulence in citrus. Since this study includes closely related organisms with distinct lifestyles and highlights proteins directly related to adaptation inside plant tissues, novel approaches might use these proteins as biotechnological targets for disease control, and contribute to our understanding of the coevolution of plant-associated bacteria.
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    Peptídeos ligantes de células tumorais e de imunoglobulinas g específicos do câncer de mama.
    (Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto., 2011) Maia, Yara Cristina de Paiva; Goulart Filho, Luiz Ricardo
    Até o momento, não existem biomoleculares que sejam efetivamente utilizados no diagnóstico do câncer de mama. Uma alternativa para auxiliar a busca por marcadores moleculares é baseada na seleção de ligantes de alta afinidade por meio de Phage Display (PD). O principal objetivo desta tese foi o desenvolvimento de novos biomarcadores envolvidos no câncer de mama selecionados contra uma linhagem celular tumoral altamente agressiva e contra IgG purificada específica de tecido mamário de pacientes. Após a seleção PD em biblioteca aleatória de peptídeos, todos os clones imunorreativos foram caracterizados por sequenciamento, deduzidos in vitro e submetidos a análises in silico. Validações posteriores foram conduzidas com ELISA e análise histopatológica. Apesar de terem sido relatadas diversas variações da metodologia, a maior parte dos processos de seleção usou ligantes obtidos depois de três ciclos de seleção. Os marcadores relatados na literatura foram pouco explorados devido à complexidade dos processos de validação. Este trabalho demonstrou que as seleções do primeiro e segundo ciclos são viáveis e podem ser utilizadas para aumentar o número de potenciais marcadores a serem testados. Os resultados com linhagem de células tumorais identificaram três potenciais marcadores no terceiro ciclo de seleção, dos quais foi selecionado aquele com melhor relação de absorbância tumor/controle no ELISA para síntese química. O peptídeo sintético foi associado com uma marcação menos intensa em tumores triplo negativos (Receptor de Estrógeno, Receptor de Progesterona e ErbB2). Outros peptídeos do segundo ciclo também demonstraram especificidade a tecidos tumorais. Para a estratégia de mapeamento de antígenos contra IgG purificada específica de tecido mamário, um peptídeo (F4) pode reconhecer tanto tecidos tumorais de mama quanto IgG circulante. Ambos clone F4 e peptídeo sintético SF4 alcançaram diagnóstico de alta acurácia, mas surpreendentemente, o clone F4 apresentou a melhor sensibilidade e especificidade (77,8% e 85,7%, respectivamente), sugerindo que pode ser utilizado no diagnóstico para triagem precoce de câncer de mama, anterior às análises por imagem e patológicas. A identificação e caracterização do biomarcador putativo está em investigação por meio de seleção combinatorial de anticorpos scFv contra o fago F4 e o peptídeo SF4, os quais serão utilizados para captura imunológica do antígeno verdadeiro e para análise por espectrometria de massas. Um sensor eletroquímico de baixo custo utilizando o peptídeo SF4 também foi desenvolvido.
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    PLA-PEG nanospheres decorated with phage display selected peptides as biomarkers for detection of human colorectal adenocarcinoma.
    (2020) Souza, Aline Maria Arlindo de; Borges, William de Castro; Andrade, Milton Hércules Guerra de; Maia, Yara Cristina de Paiva; Goulart Filho, Luiz Ricardo; Lanna, Elisa Gomes; Brito, Ana Carolina Ferreira de; Barboza, Ana Paula Moreira; Mosqueira, Vanessa Carla Furtado; Rúbio, Karina Taciana Santos
    Peptide-mediated targeting to colorectal cancer can increase selectivity and specificity of this cancer diagnosis acting as biomarkers. The present work aimed to select peptides using the phage display technique and associate the peptides with polymeric nanospheres in order to evaluate their cytotoxicity and selectivity during cell interaction with Caco-2 human colon tumor cell line. Two peptides identified by phage display (peptide-1 and peptide-2) were synthesized and exhibited purity higher than 84%. Poly(lactic acid)-block-polyethylene glycol nanospheres were prepared by nanoprecipitation and double emulsion methods in order to load the two peptides. Nanoparticles ranged in size from 114 to 150 nm and peptide encapsulation efficiency varied from 16 to 32%, depending on the methodology. No cytotoxic activity was observed towards Caco-2 tumor cell line, either free or loaded peptides in concentrations up to 3 μM at incubation times of 6 and 24 h, indicating safety as biomarkers. Fluorescein isothiocyanate–labeled peptides allowed evaluating selective interactions with Caco-2 cells, where peptide-1 entrapped in nanospheres showed greater intensity of co-localized cell fluorescence, in comparison to peptide-2. Peptide-1 loaded in nanospheres revealed promising to be investigated in further studies of selectivity with other human colon rectal cells as a potential biomarker.