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Fungos Presentes Em Sedimentos Marinhos E Lacustres Da Antártica: Taxonomia, Diversidade E Bioprospecção De Metabólitos Bioativos

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Fungos Presentes Em Sedimentos Marinhos E Lacustres Da Antártica: Taxonomia, Diversidade E Bioprospecção De Metabólitos Bioativos Universidade Federal de Minas Gerais Programa de Pós-Graduação em Microbiologia Fungos presentes em sedimentos marinhos e lacustres da Antártica: taxonomia, diversidade e bioprospecção de metabólitos bioativos Mayara Baptistucci Ogaki Belo Horizonte, MG 2019 Universidade Federal de Minas Gerais Programa de Pós-Graduação em Microbiologia Fungos presentes em sedimentos marinhos e lacustres da Antártica: taxonomia, diversidade e bioprospecção de metabólitos bioativos Tese apresentada ao programa de Pós-Graduação em Microbiologia do Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais. Orientador: Dr. Luiz Henrique Rosa Departamento de Microbiologia/ICB, UFMG Belo Horizonte, MG 2019 Colaboradores: Dr. Carlos Augusto Rosa Departamento de Microbiologia/ICB, UFMG Dr. Carlos Leomar Zani Dra. Tânia Maria de Almeida Alves Centro de Pesquisas René Rachou, FIOCRUZ/MG Dra. Rosemary Vieira Dr. Arthur Ayres Universidade Federal Fluminense, RJ Dr. Juan Manoel Lírio Instituto Antartico Argentino, Buenos Aires, Argentina 043 Ogaki, Mayara Baptistucci. Fungos presentes em sedimentos marinhos e lacustres da Antártica: taxonomia, diversidade e bioprospecção de metabólitos bioativos [manuscrito] / Mayara Baptistucci Ogaki. – 2019. 212 f. : il. ; 29,5 cm. Orientador: Dr. Luiz Henrique Rosa. Tese (doutorado) – Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas. Programa de Pós-Graduação em Microbiologia. 1. Microbiologia. 2. Regiões Antárticas. 3. Fungos. 4. Bioprospecção. 5. Biodiversidade. I. Rosa, Luiz Henrique. II. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. III. Título. CDU: 579 Ficha Catalográfica elaborada por Fabiane C. M. Reis – CRB6/ 2680 Dedicatória Aos meus maiores apoios, “minha família e amigos”. Agradecimentos Agradeço a todo corpo docente e funcionários do programa de pós-graduação em Microbiologia e ao apoio financeiro do CNPq e CAPES pela concessão de minha bolsa de estudos e pelos subsídios que permitiram a concretização desta tese. Ao meu orientador Luiz Henrique Rosa pela acolhida, oportunidades, ajuda e compreensão a mim oferecidas durante todo o tempo que permaneci em sua equipe e também por ter proporcionado a maior experiência profissional por mim vivenciada. As minhas amigas de doutorado, Thamar e Mariana, pelo respeito, amor e cuidado que recebo diariamente. A todas as amigas do laboratório pela benevolência, suporte emocional e intelectual nos dias de angústia e também nos de comemoração à vida. Aos meus professores Wagner e Marcus por me fazerem notar a beleza da vida mesmo sob tantas dificuldades humanas. Somente suportei as perdas e aguentei as dores graças à cada palavra despretensiosa de luz e carinho. Aos meus amigos paranaenses Bruna, Eddie, Paloma, Paula, Sara, Renato, Larissa, Angela e Guilherme por todas as risadas, o aconchego e as palavras de incentivo que deram acalento as minhas lamúrias e foram em grande parte responsáveis pela minha permanência na carreira acadêmica até hoje. Aos queridos Olímpio, Diogo, Diego, Laís, Clayton, Marina, Bruno e Rafael por todo abraço fraterno e pela insistência para que eu buscasse ser uma pessoa melhor, mais feliz e atenta a enxergar as dores do mundo com outros olhos. Aos meus pais Maria e Valter, ao meu irmão William e a minha avó Adelaide pelo amor incondicional que sempre me permitiu sonhar e ser capaz mesmo quando eu não acreditava ser possível continuar. Não te rendas, ainda estás a tempo de alcançar e começar de novo, aceitar as tuas sombras enterrar os teus medos, largar o lastro, retomar o voo. Não te rendas que a vida é isso, continuar a viagem, perseguir os teus sonhos, destravar os tempos, arrumar os escombros, e destapar o céu. Mario Benedetti i SUMÁRIO LISTA DE ABREVIATURAS ...................................................................................... IV LISTA DE FIGURAS ..................................................................................................... V RESUMO ....................................................................................................................... VI 5 ABSTRACT ............................................................................................................... VIII 1. RELEVÂNCIA E JUSTIFICATIVA......................................................................... X 2. INTRODUÇÃO .......................................................................................................... 1 2.1 Antártica ................................................................................................................ 1 2.2 Micobioma antártico e estratégias adaptativas.................................................. 3 10 2.3 Fungos em ambientes aquáticos .......................................................................... 5 2.3.1 Fungos de ambientes marinhos da Antártica .............................................. 7 2.3.2 Fungos em ambientes lacustres da antártica .............................................. 9 2.4 Bioprospecção de metabólitos bioativos de fungos extremófilos ..................... 11 3. OBJETIVOS ............................................................................................................. 18 15 3.1 Objetivo Geral .................................................................................................... 18 3.2 Objetivos Específicos .......................................................................................... 18 4. MATERIAIS E MÉTODOS .................................................................................... 19 4.1 Fungos em sedimentos marinhos ...................................................................... 19 4.1.1 Coleta e processamento das amostras de sedimento marinho ..................... 19 20 4.2 Fungos em sedimentos lacustres ........................................................................ 20 4.2.1 Coleta e processamento das amostras de sedimentos lacustres ............... 20 4.2.2 Isolamento de fungos em sedimentos lacustres ......................................... 23 4.3 Preservação dos fungos ...................................................................................... 24 4.4 Identificação dos fungos ..................................................................................... 24 25 4.4.1 Identificação de fungos filamentosos ......................................................... 24 4.4.1.1 Extração de DNA total ..................................................................................... 25 4.4.1.2 Amplificação utilizando o iniciador (GTG)5 .................................................... 25 ii 4.4.1.3 Amplificação da região ITS .............................................................................. 26 4.4.1.4 Amplificação parcial do gene da β-tubulina .................................................... 26 4.4.1.5 Amplificação da RNA polimerase II ................................................................. 27 4.4.1.6 Caracterização morfofisiológica das espécies do gênero Penicillium ............. 27 5 4.4.2 Identificação de leveduras .......................................................................... 28 4.4.2.1 Extração de DNA total ..................................................................................... 28 4.4.2.2 Amplificação do domínio D1/D2 ...................................................................... 28 4.4.3 Purificação dos amplicons........................................................................... 29 4.4.4 Sequenciamento ........................................................................................... 29 10 4.4.4.1 Reações de sequenciamento ............................................................................. 29 4.4.4.2 Precipitação da reação de sequenciamento ..................................................... 30 4.4.4.3 Análise computacional das sequências e análise filogenética ......................... 30 4.5 Avaliação da diversidade da comunidade fúngica ........................................... 31 4.6 Cultivo dos fungos e preparo dos extratos para os ensaios biológicos ........... 32 15 4.6.1 Preparo dos extratos de fungos marinhos ................................................. 32 4.6.2 Preparo dos extratos de fungos lacustres .................................................. 33 4.7 Ensaios biológicos ............................................................................................... 33 4.7.1 Ensaio antimicrobiano contra Cladosporium sphaerospermum ............... 33 4.7.2 Ensaio in vitro com a forma amastigota-like de Leishmania (Leishmania) 20 amazonenses .......................................................................................................... 34 4.7.3 Ensaio in vitro com formas tripomastigotas e formas amastigotas intracelulares de Trypanosoma cruzi e ensaio citotóxico in vitro de extratos tripanossomicidas ................................................................................................. 35 4.7.4 Manutenção e ensaios esquizonticidas in vitro de formas sanguíneas de 25 Plasmodium falciparum ........................................................................................ 36 4.7.5 Triagem da atividade antiviral de extratos de plantas e fungos contra o vírus da dengue sorotipo 2 (DENV2) ............................................................................ 37 4.7.6 Determinação de atividade herbicida ........................................................ 38 4.7.7 Determinação da atividade nematicida ....................................................
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