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DISSERTAÇÃO NINA VERSÃO FINAL.Pdf UNIVERSIDADE FEDERAL DE MINAS GERAIS INSTITUTO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE GENÉTICA, ECOLOGIA E EVOLUÇÃO PROGRAMA DE PÓS GRADUAÇÃO EM GENÉTICA DISSERTAÇÃO DE MESTRADO Prospecção de bactérias lácticas para uso como probióticos e avaliação do efeito protetor em modelo murino de mucosite intestinal induzida por 5-FU ORIENTADA: Nina Dias Coelho Rocha ORIENTADOR: Prof. Dr. Vasco Ariston de Carvalho Azevedo CO-ORIENTADORA: Dra. Pamela Del Carmen Mancha Agresti BELO HORIZONTE 2020 I Nina Dias Coelho Rocha Prospecção de bactérias lácticas para uso como probióticos e avaliação do efeito protetor em modelo murino de mucosite intestinal induzida por 5-FU Versão final Dissertação de Mestrado apresentada ao Programa de Pós-Graduação em Genética da Universidade Federal de Minas Gerais como requisito parcial para obtenção do título de Mestre em Genética. Orientador: Prof. Dr. Vasco Ariston de Carvalho Azevedo Coorientadora: Dra. Pamela Del Carmen Mancha Agresti Belo Horizonte 2020 II III UNIVERSIDADE FEDERAL DE MINAS GERAIS Programa de Pós-Graduação em Genética Instituto de Ciências Biológicas ATA DE DEFESA DE DISSERTAÇÃO / TESE ATA DA DEFESA DE DISSERTAÇÃO Nº 303/2020 Entrada 1º/2019 Nina Dias Coelho Rocha CPF: 134.972.596-08 Às quatorze horas do dia 18 de dezembro de 2020, reunidos remotamente devido a pandemia do novo coronavírus (COVID 19) a Comissão Examinadora de Dissertação, indicada pelo Colegiado do Programa, para julgar, em exame final, o trabalho intitulado: "Prospecção de bactérias lácticas para uso como probióticos e avaliação do efeito protetor em modelo murino de mucosite intestinal induzida por 5- FU", requisito para obtenção do grau de Mestre em Genética. Abrindo a sessão, o Presidente da Comissão, Dr. Vasco Ariston de Carvalho Azevedo, após dar a conhecer aos presentes o teor das Normas Regulamentares do Trabalho Final, passou a palavra à candidata, para apresentação de seu trabalho. Seguiu- se a arguição pelos Examinadores, com a respectiva defesa da candidata. Logo após, a Comissão se reuniu, sem a presença da candidata e do público, para julgamento e expedição de resultado final. Foram atribuídas as seguintes indicações: Prof./Pesq. Instituição CPF Indicação Dr. Vasco Ariston de Carvalho Azevedo UFMG 283.171.225-49 APROVADA Dr. Rodrigo Dias de Oliveira Carvalho UFMG 080.173.536-00 APROVADA Dr. Álvaro Cantini Nunes UFMG 678.159.606-34 APROVADA Dr. Flaviano dos Santos Martins UFMG 043.684.026-00 APROVADA Pelas indicações, a candidata foi considerada: APROVADA O resultado final foi comunicado publicamente à candidata pelo Presidente da Comissão. Nada mais havendo a tratar, o Presidente encerrou a reunião e lavrou a presente ATA, que será assinada por todos os membros participantes da Comissão Examinadora. Belo Horizonte, 18 de dezembro de 2020. IV Dr. Vasco Ariston de Carvalho Azevedo – Orientador Dr. Rodrigo Dias de Oliveira Carvalho Dr. Alvaro Cantini Nunes Dr. Flaviano dos Santos Martins Assinatura dos membros da banca examinadora: Documento assinado eletronicamente por Flaviano dos Santos Martins, Professor do Magistério Superior, em 18/12/2020, às 16:21, conforme horário oficial de Brasília, com fundamento no art. 5º do Decreto nº 10.543, de 13 de novembro de 2020. Documento assinado eletronicamente por Alvaro Cantini Nunes, Membro, em 18/12/2020, às 16:24, conforme horário oficial de Brasília, com fundamento no art. 5º do Decreto nº 10.543, de 13 de novembro de 2020. Documento assinado eletronicamente por Rodrigo Dias de Oliveira Carvalho, Usuário Externo, em 18/12/2020, às 16:26, conforme horário oficial de Brasília, com fundamento no art. 5º do Decreto nº 10.543, de 13 de novembro de 2020. Documento assinado eletronicamente por Vasco Ariston de Carvalho Azevedo, Professor do Magistério Superior, em 18/12/2020, às 17:15, conforme horário oficial de Brasília, com fundamento no art. 5º do Decreto nº 10.543, de 13 de novembro de 2020. A autenticidade deste documento pode ser conferida no site https://sei.ufmg.br/sei/controlador_externo.php? acao=documento_conferir&id_orgao_acesso_externo=0 , informando o código verificador 0483824 e o código CRC F61F362D. Referência: Processo nº 23072.247734/2020-64 SEI nº 0483824 V UNIVERSIDADE FEDERAL DE MINAS GERAIS Instituto de Ciências Biológicas Programa de Pós-Graduação em Genética FOLHA DE APROVAÇÃO "Prospecção de bactérias lácticas para uso como probióticos e avaliação do efeito protetor em modelo murino de mucosite intestinal induzida por 5-FU" NINA DIAS COELHO ROCHA Dissertação aprovada pela banca examinadora constituída pelos Professores: Prof. Vasco Ariston de Carvalho Azevedo - Orientador UFMG Rodrigo Dias de Oliveira Carvalho UFMG Álvaro Cantini Nunes UFMG Flaviano dos Santos Martins UFMG Belo Horizonte, 18 de dezembro de 2020. Documento assinado eletronicamente por Flaviano dos Santos Martins, Professor do Magistério Superior, em 18/12/2020, às 16:21, conforme horário oficial de Brasília, com fundamento no art. 5º do Decreto nº 10.543, de 13 de novembro de 2020. Documento assinado eletronicamente por Alvaro Cantini Nunes, Membro, em 18/12/2020, às 16:24, conforme horário oficial de Brasília, com fundamento no art. 5º do Decreto nº 10.543, de 13 de novembro de 2020 Documento assinado eletronicamente por Rodrigo Dias de Oliveira Carvalho, Usuário Externo, em 18/12/2020, às 16:26, conforme horário oficial de Brasília, com fundamento no art. 5º do Decreto nº 10.543, de 13 de novembro de 2020. Documento assinado eletronicamente por Vasco Ariston de Carvalho Azevedo, Professor do Magistério Superior, em 18/12/2020, às 17:15, conforme horário oficial de Brasília, com fundamento no art. 5º do Decreto nº 10.543, de 13 de novembro de 2020. A autenticidade deste documento pode ser conferida no site https://sei.ufmg.br/sei/controlador_externo.php? acao=documento_conferir&id_orgao_acesso_externo=0 , informando o código verificador 0483899 e o código CRC CD0CEDC2. Referência: Processo nº 23072.247734/2020-64 SEI nº 0483899 VI Aos meus pais, Levindo e Margarida, que sempre colocaram a minha educação acima de tudo e sempre me deram apoio incondicional na trajetória emocionante da Biologia. VII AGRADECIMENTOS Ao meu pai, Levindo, agradeço pelo conforto que sempre me passa acima de qualquer pessoa e pela paciência de me levar na UFMG para a realização dos experimentos, incluindo sábados e domingos. À minha mãe, Margarida, por todas as orações que me ajudaram a chegar nesse momento e pelo orgulho que tem da “filha cientista". À minha irmã, Milla, pelas orações, pela companhia e por ser exemplo de perseverança e determinação para mim. Às minhas tias, tios, avós e primos que tanto me motivam a seguir com honra e ética na profissão. Aos meus amigos de infância do Sagrado que nunca saíram do meu lado, sempre me dando suporte e ao meu namorado, Diego, que foi companhia fundamental nos dias que mais precisei. Meus sinceros agradecimentos ao Professor Vasco pelo acolhimento, em especial na reta final do mestrado. Obrigada pela confiança e por acreditar no meu trabalho e no meu potencial mesmo em épocas que eu estava desmotivada. A todos do grande grupo do LGCM que me ajudaram ativamente nos experimentos. Ao Luis, meu amigo e confidente, a Vivi, Laísa e ao Tales que foram meus companheiros de caminhada, passando as dificuldades e comemorando as vitórias juntos. Aos ICS que me deram a oportunidade de poder ensinar o que sei e que me ajudaram de uma maneira muito importante na condução dos experimentos. Foram 5 anos neste laboratório e levarei para a vida cada ensinamento. Muito obrigada! Aos meus companheiros felinos: Syd, Lucy e Maria Rita. O ser humano só saberá o que é compaixão quando aprender a olhar todos os seres a sua volta como iguais. Agradeço sim aos animais, que são meu refúgio mental e que me dão as maiores lições de lealdade e de um amor paciente. Dedico grande parte da minha saúde mental a eles. Sendo assim, meu reconhecimento também aos animais que me permitiram realizar este experimento. Todo cientista deve valorizar o impacto que vidas tão pequenas trazem para o conhecimento. Sonho pelo dia em que a pesquisa se refinará a ponto de não precisarmos utilizar destes meios para obtermos resultados aplicáveis. E é isso que me faz lutar a cada dia por uma ciência mais justa para todos. A CAPES agradeço pela concessão da bolsa que permitiu me dedicar integralmente a condução deste trabalho. A UFMG, minha segunda casa e ao Programa de Pós Graduação em Genética. VIII RESUMO Os benefícios exercidos pelos probióticos têm impulsionado uma série de pesquisas em busca de novas linhagens com este potencial. Nesse contexto, este estudo visou identificar e caracterizar 21 novas linhagens provenientes de ambientes diversos baseando-se em testes para avaliação de probiose sugeridos por diretrizes de agências reguladoras para posterior verificação do potencial protetor em modelo murino de mucosite intestinal induzida por 5-fluourouracil (5-FU). A identificação por MALDI-TOF revelou que todas as linhagens pertencem ao grupo das bactérias láticas (BLs), sendo relatados os gêneros Lactobacillus, Lactiplantibacillus, Lacticaseibacillus, Pediococcus e Enterococcus. Foram realizados testes in vitro de segurança por meio da análise da resistência à antibióticos, degradação da mucina e potencial hemolítico e de eficácia e funcionalidade, por meio da avaliação da resistência das linhagens aos sais biliares, tolerância ao pH ácido, produção de substâncias antimicrobianas, adesão e imunoestimulação em células Caco-2. Nenhum destes critérios foram excludentes quanto ao enquadramento das linhagens como probióticos,
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