Orientadores: Marcelo Menossi Teixeira, Rodrigo Franco de Carvalho / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T18:01:39Z (GMT). No. of bitstreams: 1 Modolo_DiegoGrando_D.pdf: 3351414 bytes, checksum: 2f6c8310f0efbb6e6f3d2e48e34de879 (MD5) Previous issue date: 2014 / Resumo: O Brasil é conhecido por ter o segundo maior efetivo bovino e por ser o maior exportador de carne bovina no mundo. Dentre os problemas que afetam a pecuária nacional está a papilomatose bovina, uma doença infecto contagiosa causada pelo papilomavírus bovino (BPV). Diversas doenças de alto impacto econômico não só na pecuária nacional mas também mundial estão associadas ao BPV, sendo que ainda não existe uma vacina que possa prevenir o alastramento da doença e nem métodos eficázes de tramento. A proteína L1 do capsideo do BPV tipo 1 é uma forte candidata para ser utilizada na formulação vacinal por ser altamente imunogênica e ter a capacidade de formar, sozinha, partículas "virus-like" (VLPs). Devido a incapacidade de se multiplicar papilomavírus "in vitro", a utilização de sistemas de produção de proteínas recombinantes é a melhor estratégia para produção de proteínas virais em larga escala que possam ser utilizadas em formulações vacinais ou em testes de diagnósticos. Neste trabalho desenvolvemos uma plataforma de produção e purificação da proteína recombinante L1 em bactéria. A proteína L1 recombinante foi purificada por cromatografia de troca iônica e foi possível obter frações em alto nível de pureza. Também produzimos plantas transgênicas visando a produção da proteína L1. A transformação genética de plantas foi confirmada por PCR e RT-PCR, mas devido a inespecificidade dos anticorpos comerciais disponíveis e a uma possível baixa expressão do gene L1 em plantas, não foi possível confirmar a expressão da proteína recombinante. A proteína L1 obtida em bactérias poderá ser analisada como vacina e bem como na obtenção de anticorpos mais específicos e na produção de testes de diagnósticos. O conhecimento obtido neste trabalho poderá ser adaptado no desenvolvimento de outras vacinas de importância socio-econômica. / Abstract: Brazil is known as the largest exporter of beef and for having the second largest cattle herd in the world. Several illness that affect the national cattle industry are associated to the bovine papillomatosis, an infectious disease caused by Bovine Papillomavirus (BPV). Althought the economic impacts of these diseases affect the livestock at a global scale, there is no BPV vaccine or effective treatment methods available yet. The L1 capsid protein of BPV type 1 is a good candidate to be used in a vaccine formulation due its high immunogenicity and the ability to form virus-like particles (VLPs). Due to the inability to multiply papillomavirus in vitro, the use of recombinant protein production systems is the best strategy to produce viral proteins in large scale that can be used in vaccine formulations or for diagnosis testing. In this work, we developed a bacteria expression system to produce the recombinant L1 protein. The recombinant L1 protein was purified by ion exchange chromatography and highly purified fractions of L1 were obtained. We also obtained transgenic plants to produce the L1 protein. The genetic transformation of plants was confirmed by PCR and RT-PCR. Due to lack of specificity of commercial antibodies used in this study and a possible low expression of the L1 gene in plants, it was not possible to confirm the accumulation of the recombinant protein. The protein obtained in bacteria can be evaluated as vaccine as well as in the production of more specific antibodies and diagnosis tests. The knowledge obtained in this work can be adapted in the development of vaccines for other important socio-economic diseases / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Human papillomavirus infection is the cause of more than 99% of cervical cancer cases. The current vaccine is ineffective therapeutically; highlighting the need for continued papillomavirus research. One avenue that could be explored in this regard is the function of the papillomavirus E2 regulatory proteins. HPV E2 represses expression of the viral E6 and E7 oncoproteins. Reintroduction of E2 into cervical carcinoma cells results in growth arrest and cellular senescence. Understanding the mechanism of how E2 regulates the early promoter may be key to developing new therapeutic and prophylactic vaccines. Here, we describe regulation of E2 through acetylation and possibly through direct interaction with a novel cellular interacting protein, RINT1. Histone acetyltransferase (HAT) proteins have been demonstrated to interact with Bovine Papillomavirus (BPV) and Human Papillomavirus (HPV) E2 proteins as well as enhance E2 dependant transcription luciferase reporter plasmid containing E2 binding sites. We demonstrate that HATs p300, CBP, and pCAF are limiting for E2 dependant transcriptional activation and that each protein functions independently. We have also identified that BPV-1 E2 is a substrate for acetylation by p300. Mutants of E2 that cannot be acetylated on lysines 111 or 112, display abnormal transcriptional phenotypes. Cells deficient in p300 display similar transcriptional defects that are intensified by CBP depletion. We propose that acetylation of BPV-1 E2 is necessary for transcriptional activation. Acetylation generates a binding site through which a co-factor may interact via a bromodomain. Regulation of E2 dependent transcriptional activation through a post-transcriptional modification represents a novel method through which BPV-1 controls gene expression. We also present evidence for a direct interaction between BPV-1 E2 and the cellular factor RINT1. This interaction does not appear to be critical for transcriptional regulation; however, several other functional pathways are indicated by the cellular complexes in which RINT1 functions. Some of these, such as ER/Golgi vesicular transport and hTERT independent telomere maintenance, are pathways in which E2 has no known role. Further investigation into regulation and consequences of E2 acetylation and the biological significance of the interaction between E2 and RINT1 could prove important in understanding the complex role of E2 in papillomavirus infection.