The Fragile X-related mental retardation 1 (FXR1) is an N6-Methyladenosine reader involved in mRNAs metabolism like mRNA splicing, stability, transport, and miRNA regulation. It is also important in transcription, cell proliferation, differentiation, translation, polysome assembly and stress granule assembly. The protein is present in all eukaryotic cells, but so far it has been specifically essential for correct neural function. Until now, FXR1 has not been investigated in the concept of Human Adenovirus infection but we have observed an upregulation of FXR1 during the late phase of the Human Adenovirus 5 (HAdV-5) infection and an upregulation of some late HAdV-5 proteins in HeLa cells overexpressing FXR1. Our results furthermore showed that a FXR1 knockdown resulted in a reduced level of some HAdV-5 proteins at the same time as HAdV-5 mRNA were stabilized, indicating that FXR1 might be involved in translation of HAdV-5 late genes. Further investigation of the mechanism behind FXR1 mediated translation, a Death-associated protein 5 (DAP5) was founded to have an overall effect on the translation of HAdV-5 late proteins. / Part of a post-doctoral research

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-26T10:51:28Z No. of bitstreams: 2 Dissertação - Thainara Calixto da Paz - 2016.pdf: 1536674 bytes, checksum: 9a527593418c3d06cd5b9029999ee4b3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-26T10:52:00Z (GMT) No. of bitstreams: 2 Dissertação - Thainara Calixto da Paz - 2016.pdf: 1536674 bytes, checksum: 9a527593418c3d06cd5b9029999ee4b3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-26T10:52:00Z (GMT). No. of bitstreams: 2 Dissertação - Thainara Calixto da Paz - 2016.pdf: 1536674 bytes, checksum: 9a527593418c3d06cd5b9029999ee4b3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-12-05 / Human adenoviruses (HAdVs) may cause several clinical syndromes, and are a major cause of respiratory and acute gastroenteritis (AGE), especially among children. However, data on viral load, in more than one type clinical sample obtained from the same child, are still scarce. The aims of the present study were to evaluate the frequency of the HAdV, to determine the load viral in clinical samples, and to proceed molecular characterization of positive samples from children up to five years of age in association with symptomatology. For this, 200 children attended at Hospital Materno Infantil in Goiânia, Goiás; between March 2014 ad July 2015. One fecal and one nasopharyngeal swab sample was obtained from each child. The clinical samples (fecal and nasopharyngeal swabs) were submitted the DNA extraction by a commercial kit (Qiagen-Hilden, Alemanha), and screened by RT-qPCR (TaqMan) assay, with specific primers and probe targeting the hexon region of HAdV genome. The global frequency of HAdVs was 21% (42/200). Positivity in swabs was 9.5% (19/200), and in fecal samples 16% (32/200). Among the symptomatic children (n=129), 21% were positive in fecal samples (22/105) and 9.2% (10/108) in swab samples. Futhermore, 4.5% (9/200) were positive in both clinical samples. High viral loads were observed in both fecal and nasopharyngeal swab samples from symptomatic and asymptomatic children, and major positivity was found in symptomatic children with high load viral. High viral loads were observed in samples from symptomatic and asymptomatic children. Major positivity and load viral was found betwenn symptomatic children. HAdV types 3 of species B and 41 of HadV F species were detected. We hope that the data obtained can help in a better understanding of the pathogenesis of HAdV in children. / Os adenovírus humanos (HAdVs) são importantes agentes causadores de gastroenterite aguda e doença respiratória, principalmente entre as crianças menores de cinco anos de idade. Dados sobre a carga viral, em mais do que um tipo de amostra clínica obtida de um mesmo indivíduo durante a infecção, ainda são escassos. Os objetivos do presente estudo foram avaliar a frequência do HAdVs, determinar a carga viral em amostras clínicas, e proceder a caracterização molecular de amostras positivas de crianças até aos cinco anos de idade, em associação com a sintomatologia. Foram incluídas no estudo amostras de 200 crianças atendidas no Hospital Materno Infantil de Goiânia, Goiás; entre março de 2014 e julho de 2015. Uma amostra fecal e um swab nasofaringeano foram obtidos de cada criança. As amostras foram submetidas a extração, utilizando kit comercial (RNA mini-kit, Qiagen), e triadas por ensaio RT-qPCR (TaqMan, Life Technologies), com iniciadores e sonda específicos para a região codificadora do hexon. Foi observado índice global de positividade para HAdVs de 21% (42/200), o índice de positividade nas fezes foi 16% (32/200) e em swab foi de 9,5% (19/200). Entre as crianças sintomáticas (n=129), 21% foram positivas em amostras fecais (22/105) e 9,2% (10/108) em amostras de swabs. Ainda, 4,5% (9/200) foram positivas em ambas as amostras clínicas. Cargas virais elevadas foram observadas em amostras de crianças sintomáticas e assintomáticas, maior positividade foi encontrada em crianças sintomáticas com maiores cargas virais. Foram detectados HAdV tipos 3 da espécie B e 41 da espécie F de HadV. Esperamos que os dados obtidos possam auxiliar em um melhor entendimento da patogenia dos HAdV na população infantil.

Com o objetivo de estudar a interação do HAdV-41 com células permissivas, primeiramente foi observada a cinética de infecção do HAdV-41 em células HEK-293, durante 7 dias. A seguir, as culturas foram analisadas por MCVL e por MET. O HAdV-41 apresentou um ciclo replicativo lento com liberação da progênie viral por mecanismo não lítico. A seguir, com o intuíto de verificar a participação da proteina FC do HAdV-41 nas etapas de entrada nas células HEK-293 e CaCo2, obteve-se os dodecaedros recombinantes (DR) em células High five (base-Ad3, base-Ad3+FC-Ad41, base-Ad3+FL-Ad41, baseRGEHS-Ad3+FCAd41 e base-Ad3+FAd3). Esses dodecaedros foram inoculados em células HEK-293 e CaCo2. Após a análise por MCVL, observou-se que a proteína FC talvez não desempenhe função na entrada do DR nas células estudadas. A seguir, uma alíquota do DR base-Ad3+FC-Ad41 foi digerida com a enzima pepsina e analisada por WB. Notou-se que a FC sofreu proteólise. Acredita-se que essa proteólise seja necessária para o reconhecimento de receptores no trato gastro-intestinal. Esses resultados fornecerão subsídeos para o desenvolvimento de vetores de terapia gênica direcionada para o epitélio intestinal e vetores vacinais administrados por via oral. / Our objective was study the interaction between HAdV-41 and permissive cells. First, it was observed the kinetic of infection between HAdV-41 and HEK-293 cells, for 7 days. Second, the cultures were analyzed by CLSM and by TEM. The HAdV-41 showed a slower replicative cycle with release of viral progeny by non-lytic mechanisms. In order to verify the participation of SF protein of the HAdV-41 during the phases of entry in HEK-293 and CaCo2 cells, we producted recombinant dodecahedrons (DR) in high five cells (base-Ad3, base-Ad3+SF-Ad41, base-Ad3+SF-Ad41, baseRGEHS-Ad3+SFAd41 and base-Ad3+FAd3). These decahedrons were inoculated in HEK-293 and CaCo2 cells. After analysis with CLSM, observed that SF protein may not have a role in dodecahedron entry in the cells studied. Next, recombinant dodecahedrons base-Ad3+SF-Ad41 and base-Ad3 were digested with pepsin and analyzed by WB. We observed proteolysis of the SF. We believe that this proteolysis may be necessary for the recognition of receptors in intestinal cells. The results obtained will be the base for the development of gene-therapy vectors directed to intestinal epithelium, as well as orally administered vaccine vectors.

Com o objetivo de estudar a interação do HAdV-41 com células permissivas, primeiramente foi observada a cinética de infecção do HAdV-41 em células HEK-293, durante 7 dias. A seguir, as culturas foram analisadas por MCVL e por MET. O HAdV-41 apresentou um ciclo replicativo lento com liberação da progênie viral por mecanismo não lítico. A seguir, com o intuíto de verificar a participação da proteina FC do HAdV-41 nas etapas de entrada nas células HEK-293 e CaCo2, obteve-se os dodecaedros recombinantes (DR) em células High five (base-Ad3, base-Ad3+FC-Ad41, base-Ad3+FL-Ad41, baseRGEHS-Ad3+FCAd41 e base-Ad3+FAd3). Esses dodecaedros foram inoculados em células HEK-293 e CaCo2. Após a análise por MCVL, observou-se que a proteína FC talvez não desempenhe função na entrada do DR nas células estudadas. A seguir, uma alíquota do DR base-Ad3+FC-Ad41 foi digerida com a enzima pepsina e analisada por WB. Notou-se que a FC sofreu proteólise. Acredita-se que essa proteólise seja necessária para o reconhecimento de receptores no trato gastro-intestinal. Esses resultados fornecerão subsídeos para o desenvolvimento de vetores de terapia gênica direcionada para o epitélio intestinal e vetores vacinais administrados por via oral. / Our objective was study the interaction between HAdV-41 and permissive cells. First, it was observed the kinetic of infection between HAdV-41 and HEK-293 cells, for 7 days. Second, the cultures were analyzed by CLSM and by TEM. The HAdV-41 showed a slower replicative cycle with release of viral progeny by non-lytic mechanisms. In order to verify the participation of SF protein of the HAdV-41 during the phases of entry in HEK-293 and CaCo2 cells, we producted recombinant dodecahedrons (DR) in high five cells (base-Ad3, base-Ad3+SF-Ad41, base-Ad3+SF-Ad41, baseRGEHS-Ad3+SFAd41 and base-Ad3+FAd3). These decahedrons were inoculated in HEK-293 and CaCo2 cells. After analysis with CLSM, observed that SF protein may not have a role in dodecahedron entry in the cells studied. Next, recombinant dodecahedrons base-Ad3+SF-Ad41 and base-Ad3 were digested with pepsin and analyzed by WB. We observed proteolysis of the SF. We believe that this proteolysis may be necessary for the recognition of receptors in intestinal cells. The results obtained will be the base for the development of gene-therapy vectors directed to intestinal epithelium, as well as orally administered vaccine vectors.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-29T11:10:52Z No. of bitstreams: 2 Dissertação - Hugo César Pereira Santos - 2015.pdf: 3535564 bytes, checksum: 4fc7d88b1da6008bc42c01f46e4f8d7a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-29T11:20:29Z (GMT) No. of bitstreams: 2 Dissertação - Hugo César Pereira Santos - 2015.pdf: 3535564 bytes, checksum: 4fc7d88b1da6008bc42c01f46e4f8d7a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-29T11:20:29Z (GMT). No. of bitstreams: 2 Dissertação - Hugo César Pereira Santos - 2015.pdf: 3535564 bytes, checksum: 4fc7d88b1da6008bc42c01f46e4f8d7a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-19 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The human adenoviruses (HAdV) infect people of all ages worldwide, causing a wide range of clinical syndromes, depending on the viral type. In immunocompromised hosts, as transplanted patients, HAdV infection can result in a bad prognosis. Some aspects of viral pathogenesis, such as the association between viremia and/or viral load with disseminated disease and the optimal moment to start the therapy, are still not well established in adult patients that have undergone allogeneic hematopoietic stem cell transplantation (ASCT). Therefore, the main objective of this study was to monitor ASCT recipients for HAdV occurrence, and also correlate viral positivity, viral load and molecular variant with clinical symptoms and patients’ prognosis. For this, stool and serum from 21 patients were monitored in a 2 years period (from October/2012 to October/2014). Serum and fecal samples were screened by Nested-PCR, using primers targeting a partial region of the hexon gene (143bp). Fecal samples were further screened by a commercial enzyme immunoassay (EIA). In total, 57% of the patients had at least one positive sample (serum or stool) for HAdV. Patients presented high viral load (varying from 7,7x103 to 2x108 copies/mL), with a higher viral load in stool when compared to serum. Positive samples were submitted to genomic sequencing, revealing the occurrence of HAdV from C, D and F species. The main clinical symptom presented by infected patients was diarrhea, and graft-versus-host disease was the main intercurrence; however it was not possible to directly associate viral positivity to cause of death. We hope to contribute for a better understanding of the HAdV infection pattern in patients submitted to ASCT. Our data highlights the importance of the inclusion of HAdV testing in the routine laboratory exams of this group of patients. / Os adenovírus humanos (HAdV) podem infectar pessoas de todas as idades, causando uma ampla gama de quadros clínicos. Em pacientes imunocomprometidos, como os indivíduos que receberam transplante, a infecção por esses vírus pode resultar em pior prognóstico para o paciente. Determinados aspectos da patogenia viral, como a associação entre viremia e/ou carga viral com a doença disseminada, bem como o melhor momento para o início da terapia, não estão ainda bem esclarecidos em pacientes que foram submetidos ao transplante de células progenitoras hematopoiéticas (TACPH), principalmente em indivíduos adultos. Dessa forma, o principal objetivo deste estudo foi realizar o monitoramento da infecção por HAdV e a caracterização molecular das variantes virais em pacientes submetidos ao TACPH, bem como determinar a carga viral e correlacionar a infecção com o quadro clínico e prognóstico dos pacientes. Foram analisadas amostras de soro e fezes de 21 pacientes submetidos ao TACPH no período de dois anos (de outubro/2012 a outubro/2014). As amostras de fezes foram triadas por ensaio imunoenzimático e por Nested-PCR, enquanto as amostras de soro foram triadas somente por Nested-PCR de uma região parcial do gene hexon (produto esperado de 143 pb). Foram detectadas amostras positivas de soro e/ou fezes de 57% dos pacientes. De forma geral, os pacientes do presente estudo apresentaram elevadas cargas virais (variando de 7,7x103 à 2x108 CG/mL), que foram mais elevadas nas fezes. As amostras positivas foram submetidas ao sequenciamento genômico e resultados revelaram a ocorrência de adenovírus das espécies C, D e F. O principal quadro clínico apresentado pelos pacientes foi a diarreia e a principal intercorrência observada, a doença do enxerto contra o hospedeiro (DECH). Dez pacientes foram a óbito durante o período de estudo, entretanto, não foi possível associar a infecção por HAdV diretamente à causa mortis. Esperamos que os dados obtidos possam auxiliar no melhor entendimento do padrão da infecção dos HAdVs em pacientes submetidos ao TACPH, de forma a contribuir para que a pesquisa de adenovírus seja incluída na rotina de exames desses pacientes.