No description available.

ROGDI is a novel gene which has unknown function. According to GenBank, the gene is located on chromosome 16p13.3 and the size of coding region is 864 bp which encodes 287 amino acids. It was a novel gene isolated from primary human renal epithelial cells in NEDO human cDNA sequencing project (AK026039). The definition of its reference sequence (RefSeq NM_024589) described as Homo sapiens rogdi homolog (Drosophila), ROGDI. By bioinformatic analysis, the gene was predicted as a hydrophilic protein with leucine zipper domain and located in cytoplasm. A partial cDNA of this gene was cloned in our laboratory. For further study of the biological function of the gene, the coding region of this gene was cloned into pGEX-6p and pET-28a vector and expressed in E. coli BL21 (DE3). The fusion protein was partially purified for preparation of polyclonal antibody. Northern blot analysis revealed that the gene was not expressed in all tissues. From the results of RT-PCR and western blot analysis, it can be concluded that the products, both mRNA and protein, of this gene were found in many cancer cell lines, but protein level expression of the gene was much less in normal cell lines. By immunocytochemistry analysis and subcellular localization analysis of GFP-tagged ROGDI, the gene was expressed both in the nucleus and cytoplasm, but expressed more in the nucleus than cytoplasm. In addition, ROGDI was up-regulated in early stages of liver fibrosis of TAA-treated mouse livers. This novel gene may play roles in tumorigenesis and liver fibrosis.

No description available.

No description available.

Immunoassays for 2-methylisoborneol (MIB) and monensin in water were developed, devised and tested to see if the sensitivity could be established and improved. MIB and monensin are hydrophobic haptens with molecular weights of 168 and 671 Da, respectively. Rabbits were immunized with (-) camphor-BSA and (-) borneol-BSA for the production of polyclonal antibodies (pAbs) to MIB. Monoclonal antibodies (mAbs) were produced in Mus musculus using (-) camphor-BSA as immunogen. (+) Bornylamine-thyroglobulin (TG) and MIB-TG were synthesized and used as plate coatings. For the monensin immunoassay, monensin was conjugated to BSA and OVA for immunogen and plate coating, respectively. Several physical parameters that affect the sensitivity of immunoassays including pre-incubation of antibody and antigen, incubation time and temperature, detergent, organic solvents, and ionic strength were evaluated. Improvement of immunoassay sensitivity was also performed by reducing the concentrations of coating antigen and antibodies and using alternative reporter systems such as chemiluminescence (CICL-ELISA), tyramide signal amplification (TSA) and biotin-streptavidin. Different assay formats, i.e., competitive indirect and competitive direct were also compared. Usability of both pAb-based immunoassays for MIB and monensin was evaluated in fortified water samples. A polyclonal-based (pAb) ELISA for MIB had a detection limit of 4.8 ng mL-1 and an IC50 of 105 ng mL-1. Rabbits immunized with (-) camphor-BSA showed a higher immune response than rabbits immunized with (-) borneol-BSA. One clone (i.e., 4F11) of fourteen characterized clones was used to create the monoclonal antibody (mAb)-based ELISA, which had an IC50 of 100.2 ng mL-1 and an LOD of 1.9 ng mL-1. The pAb- and mAb-based CI-ELISA were not specific to MIB alone and cross reacted with camphor and camphor-like compounds. Meanwhile, a pAb-based ELISA for monensin produced a detection limit of 0.1 ng mL-1 and had an IC50 of 1.056-1.090 ng mL-1 with high specificity to monensin. Other reporter systems did not improve the sensitivity of the immunoassays significantly. MIB and monensin polyclonal-based assays showed good correlation to analytical instrumental methods (i.e., GC-MS and LC-MS) in fortified water samples. With a detection limit of ca. 5 ng mL-1 and 0.1 ng mL-1 for MIB and monensin, respectively, both polyclonal-based assays can be used for detection of these analytes in water from different sources and employed as screening tools to complement GC/HPLC-MS instrument methods.

Made available in DSpace on 2014-12-02T11:16:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-09-05Bitstream added on 2014-12-02T11:21:05Z : No. of bitstreams: 1 000777406_20141231.pdf: 641973 bytes, checksum: 588137dce1974a2914edd3aadb3736f1 (MD5) Bitstreams deleted on 2015-01-05T11:00:55Z: 000777406_20141231.pdf,Bitstream added on 2015-01-05T11:01:50Z : No. of bitstreams: 1 000777406.pdf: 2095617 bytes, checksum: c1c25558e3e61efd44b8465985d10f95 (MD5) / A proteína MxA humana é membro da superfamília de GTPases dinaminas. De maneira geral, as chamadas proteínas Mx estão presentes na grande maioria dos organismos vertebrados, estudados até o momento, e possuem dois domínios estruturais, chamados GTPase e CID-GED (stalk), além da capacidade de homooligomerização e associação com membranas intracelulares. Além disso, as proteínas Mx são produzidas unicamente após a sensibilização celular por interferons do tipo I e III. Entre as propriedades funcionais de MxA, destaca-se a sua vasta atividade contra diferentes vírus de RNA e DNA, incluindo o vírus influenza e membros da família dos buniavírus. Além disso, o silenciamento gênico de MxA está associado ao fenótipo de imortalização celular em uma série de neoplasias. Assim, MxA desperta o interesse por ser uma das proteínas chave nas respostas mediadas por interferons e por estar envolvida no controle da proliferação celular. Recentemente, por meio de rastreamento de duplo-híbrido, utilizando uma biblioteca de cDNA preparada a partir de cérebro fetal humano, foi possível mostrar que MxA interage com fatores envolvidos no processo de SUMOilação de proteínas e na formação de corpúsculos nucleares denominados PMLNB e com uma série de proteínas relacionadas com o controle da transcrição e apoptose. Neste estudo, foi investigada a interação entre MxA e as proteínas envolvidas nos processo de SUMOilação. Assim, foi possível confirmar a interação física entre a proteína MxA e os ligantes Ubc9 e SUMO1, por ensaios de coimunoprecipitação. A seguir, através de ensaios de duplo-híbrido, foi possível determinar que a região EIL (E67-interacting Loop) de SUMO1 e o domínio CID-GED de MxA estão envolvidos na interação entre essas proteínas e que esta interação independe de sequências SIM (SUMO-interacting motif) presentes em MxA. Ainda, foi possível determinar que Ubc9 interage diretamente com ... / The human MxA protein is a member of the superfamily of GTPases dynamins. The so called Mx proteins are present in the majority of the vertebrate organisms investigated so far and contain two structural domains, named GTPase and CID-GED (stalk), besides the capabilities of homo-oligomerization and association with intracellular membranes. Moreover, the Mx proteins are strictly produced upon cell sensibilization with type I and III interferons. The vast antiviral activity against RNA and DNA viruses, including the Influenza virus and members of the bunyaviridae family, is among the functional properties of MxA. Moreover, MX1 epigenetic silencing is associated with cellular immortalization in neoplasias. Therefore, the study of MxA is of great interest as it is a key component of the Interferon-mediated pathways and cell proliferation control. Recentely, in a two-hybrid screen using a fetal brain cDNA library, it was possible to reveal that MxA interacts with proteins related to the post-translational modification process named SUMOylation and to the assemble of the nuclear bodies named PML-NBs and with proteins implicated in the control of transcription and apoptose. In this study, it was investigated the interaction between MxA and the components of the protein SUMOylation pathway. It was possible to confirm the physical interaction between MxA and Ubc9 and SUMO1, using co-immunopreciptation assay. Then, using the yeast two-hybrid system, it was possible to determine that the EIL (E67-interacting loop) region on SUMO1 interacts with the CID-GED domain of MxA without the requirement of the SIM sequences (SUMO-interacting motif) present in MxA. Moreover, it was determined that Ubc9 interacts with the GTPase domain of MxA and that MxA homo-oligomerization is important for its interaction with SUMO1 and Ubc9. Also, we were able to demonstrate for the first time that the protein MxA undergoes SUMOylation by SUMO1, SUMO2 and SUMO3. Finally, it ...

Orientador: Sandro Roberto Valentini / Co-orientador: Cleslei Fernando Zanelli / Banca: Alexandra Ivo de Medeiros / Banca: Andréia Machado Leopoldino / Banca: Mari Cleide Sogayar / Banca: Érico Tosoni Costa / Resumo: A proteína MxA humana é membro da superfamília de GTPases dinaminas. De maneira geral, as chamadas proteínas Mx estão presentes na grande maioria dos organismos vertebrados, estudados até o momento, e possuem dois domínios estruturais, chamados GTPase e CID-GED (stalk), além da capacidade de homooligomerização e associação com membranas intracelulares. Além disso, as proteínas Mx são produzidas unicamente após a sensibilização celular por interferons do tipo I e III. Entre as propriedades funcionais de MxA, destaca-se a sua vasta atividade contra diferentes vírus de RNA e DNA, incluindo o vírus influenza e membros da família dos buniavírus. Além disso, o silenciamento gênico de MxA está associado ao fenótipo de imortalização celular em uma série de neoplasias. Assim, MxA desperta o interesse por ser uma das proteínas chave nas respostas mediadas por interferons e por estar envolvida no controle da proliferação celular. Recentemente, por meio de rastreamento de duplo-híbrido, utilizando uma biblioteca de cDNA preparada a partir de cérebro fetal humano, foi possível mostrar que MxA interage com fatores envolvidos no processo de SUMOilação de proteínas e na formação de corpúsculos nucleares denominados PMLNB e com uma série de proteínas relacionadas com o controle da transcrição e apoptose. Neste estudo, foi investigada a interação entre MxA e as proteínas envolvidas nos processo de SUMOilação. Assim, foi possível confirmar a interação física entre a proteína MxA e os ligantes Ubc9 e SUMO1, por ensaios de coimunoprecipitação. A seguir, através de ensaios de duplo-híbrido, foi possível determinar que a região EIL (E67-interacting Loop) de SUMO1 e o domínio CID-GED de MxA estão envolvidos na interação entre essas proteínas e que esta interação independe de sequências SIM (SUMO-interacting motif) presentes em MxA. Ainda, foi possível determinar que Ubc9 interage diretamente com ... / Abstract: The human MxA protein is a member of the superfamily of GTPases dynamins. The so called Mx proteins are present in the majority of the vertebrate organisms investigated so far and contain two structural domains, named GTPase and CID-GED (stalk), besides the capabilities of homo-oligomerization and association with intracellular membranes. Moreover, the Mx proteins are strictly produced upon cell sensibilization with type I and III interferons. The vast antiviral activity against RNA and DNA viruses, including the Influenza virus and members of the bunyaviridae family, is among the functional properties of MxA. Moreover, MX1 epigenetic silencing is associated with cellular immortalization in neoplasias. Therefore, the study of MxA is of great interest as it is a key component of the Interferon-mediated pathways and cell proliferation control. Recentely, in a two-hybrid screen using a fetal brain cDNA library, it was possible to reveal that MxA interacts with proteins related to the post-translational modification process named SUMOylation and to the assemble of the nuclear bodies named PML-NBs and with proteins implicated in the control of transcription and apoptose. In this study, it was investigated the interaction between MxA and the components of the protein SUMOylation pathway. It was possible to confirm the physical interaction between MxA and Ubc9 and SUMO1, using co-immunopreciptation assay. Then, using the yeast two-hybrid system, it was possible to determine that the EIL (E67-interacting loop) region on SUMO1 interacts with the CID-GED domain of MxA without the requirement of the SIM sequences (SUMO-interacting motif) present in MxA. Moreover, it was determined that Ubc9 interacts with the GTPase domain of MxA and that MxA homo-oligomerization is important for its interaction with SUMO1 and Ubc9. Also, we were able to demonstrate for the first time that the protein MxA undergoes SUMOylation by SUMO1, SUMO2 and SUMO3. Finally, it ... / Doutor

>Magister Scientiae - MSc / Insect-derived baculoviruses have been used extensively as a safe and versatile research model for transgenic protein expression. Preclinical studies have revealed the promising potential of Baculoviruses as a delivery vector for a variety of therapeutic applications, including vaccination, tissue engineering and cancer treatments. Coronaviruses are enveloped viruses containing linear, non-segmented ribonucleic acid. Human coronavirus NL63 was first discovered in the Netherlands in January 2004, where a 7-month-old girl presented with an acute respiratory tract infection that was later established to predominantly infect infants, the elderly and immunocompromised individuals. In addition to the known non-structural and structural proteins of coronaviruses, an accessory protein known as open reading frame 3 which is conserved in the Coronaviridae family has not been extensively researched. Open reading frame 3 encodes a putative membrane-bound protein. This study cloned the open reading frame 3 viral gene of 741 base pairs into the baculovirus expression construct via competent bacterial cell lines. Open reading frame 3-Baculovirus particles were generated in Spodoptera frugiperda insect cells. Recombinant cells containing the viral protein gene were used to infect healthy Spodoptera frugiperda 9 cells at varying ratios of multiplicity of infection over a fixed time-course. The open reading frame 3 viral protein was not detected by quantification methods at a molecular weight of 26 kilo Dalton, due to polyclonal antibody degradation.

O Tomato chlorosis virus (ToCV) é uma espécie do gênero Crinivirus que causa danos, principalmente na cultura do tomateiro (Solanum lycopersicum). Foi primeiramente isolado e descrito em 1998, nos Estados Unidos, e em seguida foi reportado em doze países. No Brasil, foi constatado primeiramente no Estado de São Paulo, na região de Sumaré, em 2008, e posteriormente nos Estados da Bahia, Espírito Santo, Goiás, Minas Gerais e Rio de Janeiro. Há evidência da sua presença também nos Estados do Paraná e Santa Catarina. O ToCV pode infectar outras solanáceas além do tomateiro e, recentemente, foi observado infectando plantas de batata (Solanum tuberosum) no Brasil. Esse crinivirus é transmitido no Brasil principalmente pelo aleirodídeo (mosca branca) Bemisia tabaci MEAM1. Considerando o patossistema batateira/ToCV, não há estudos sobre a ocorrência, sintomatologia em diferentes variedades e danos provocados por esse crinivirus. Também não há antissoro policlonal para o isolado brasileiro do ToCV para uso na diagnose da doença em solanáceas. Esse trabalho teve por objetivos: purificar o ToCV e produzir antissoro policlonal; avaliar a reação de genótipos de batateira à infecção com o ToCV; avaliar o dano provocado por esse vírus em duas variedades de batateira. A purificação do vírus a partir de folhas de tomateiro e a produção de antissoro policlonal em coelho foram satisfatórias. No entanto, o antissoro não foi eficiente em ELISA, mas sim em dot-blot e somente na diluição de 1:20. Foi avaliada a reação de 21 genótipos de batateira à infecção com o ToCV, por meio da inoculação com B. tabaci MEAM1, com chance de escolha do vetor. Nenhum genótipo exibiu resistência à infecção; enquanto a variedade Camila foi assintomática e não apresentou alteração na fotossíntese. Plantas de batateira das variedades Ágata e Asterix sadias foram inoculadas com o ToCV, por meio da B. tabaci MEAM1 e ao final foram avaliadas a massa fresca da parte aérea, peso e número dos tubérculos colhidos. Em dois experimentos independentes, as reduções médias no peso fresco da parte aérea foram de 60,1% para Ágata e 46% para Asterix. Porém, as reduções nas produções dessas variedades, no primeiro experimento foram de 99,5% e 98,1%, respectivamente; enquanto no segundo os valores foram de 82,3% e 56,2%, respectivamente. / Tomato chlorosis virus (ToCV) is a species of the genus Crinivirus, which is causing considerable losses mainly on tomato crop (Solanum lycopersicum). It was first isolated and described on 1998 in the United States and subsequently reported in twelve countries. In Brazil, it was first reported in São Paulo State, in Sumaré region in 2008, and after that on the states of Bahia, Espírito Santo, Goiás, Minas Gerais and Rio de Janeiro. There is evidence of the presence of ToCV on the states of Paraná and Santa Catarina. ToCV can also infect other solanaceae and more recently, it was reported infecting potato plants (Solanum tuberosum) in Brazil. This crinivirus is transmitted by Bemisia tabaci MEAM1. Considering the patosystem potato/ToCV, there are no studies on the occurrence, symptomatology in different varieties, and damages caused by this crinivirus. In addition, there is no polyclonal antiserum for the Brazilian isolate of ToCV for use in diagnosis. The objectives of the present work were: to purify the virus and produce a polyclonal antiserum; to evaluate the reaction of potato genotypes to ToCV infection; to evaluate the yield loss caused by this crinivirus on two potato cultivars. The virus purification from tomato leaves and the production of polyclonal antiserum in rabbit were satisfactorily accomplished. However, the antiserum was not efficient on ELISA test, but in dot-blot, only when diluted 1:20. The reaction of 21 potato genotypes to infection with ToCV was evaluated by inoculation with B. tabaci MEAM1, with chance of choice for the vector. All genotypes were infected with ToCV and Camila was the only one asymptomatic. Plants of cultivars Ágata and Asterix were inoculated with ToCV, by means of viruliferous vector, and at the end were evaluated for the fresh mass of the aerial part, weight and number of harvested tubers. In two independent experiments, average reductions in aerial fresh weight were 60.1% for Ágata and 46% for Asterix. However, reductions in yield of these varieties in the first experiment were 99.5% and 98.1%, respectively; while in the second the values were 82.3% and 56.2%, respectively.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-03-08T19:19:08Z No. of bitstreams: 1 117110029.pdf: 698994 bytes, checksum: ceaed51c4f43c77fa5dcfffa7ec011b5 (MD5) / Made available in DSpace on 2015-03-08T19:19:08Z (GMT). No. of bitstreams: 1 117110029.pdf: 698994 bytes, checksum: ceaed51c4f43c77fa5dcfffa7ec011b5 (MD5) Previous issue date: 2013-08-09 / Os vírus são importantes agentes patogênicos de várias espécies animais, entre elas bovinos. No Brasil, diversos agentes virais foram descritos causando infecções no rebanho bovino, e produzindo perdas econômicas significativas. A identificação dos animais infectados por um vírus pode ser realizada de diferentes formas; no entanto, a confirmação definitiva requer a demonstração do agente ou da resposta imune. Para isto, vários métodos com capacidade de detectar a partícula viral, atividade biológica, genoma, antígenos virais, ou então a resposta imune específica foram desenvolvidos. Os imunoensaios são testes amplamente utilizados na rotina laboratorial para detecção de antígenos virais em amostras clínicas ou de pesquisa. Estes ensaios apresentam boa sensibilidade, especificidade e facilidade de execução. A metodologia dos imunoensaios tem como base, o emprego de anticorpos monoclonais ou policlonais específicos para os antígenos virais. Assim sendo, o objetivo do presente estudo foi produzir anticorpos policlonais para alguns vírus bovinos, e avaliar a reatividade destes em testes de imunofluorescência, imunoperoxidase e slot blot. Para isto, cepas e/ou isolados do herpesvírus bovino tipo 1 (BoHV-1), herpesvírus bovino tipo 2 (BoHV-2), herpesvírus bovino tipo 5 (BoHV-5), herpesvírus bovino tipo 5 gE deletado (BoHV-5 gEΔ), vírus da diarreia viral bovina (BVDV), vírus respiratório sincicial bovino (BRSV), vírus da língua azul (BTV) e vírus da vaccínia (VACV) foram amplificados em cultivo celular e o sobrenadante utilizado para imunizar coelhos. Os animais foram imunizados cinco vezes pela via subcutânea, e cinco dias após o último reforço coletou-se sangue. O soro foi separado do sangue por centrifugação. O soro foi diluído em PBS (1:100 a 1:204.800) e utilizado como anticorpo primário nos ensaios de imunofluorescência, imunoperoxidase e slot blot. A diluição de trabalho foi selecionada pela diluição que produziu reação específica nas células infectadas, e sinal fraco ou ausente nas células controle. Os antissoros apresentaram maior reatividade na técnica de imunoperoxidase do que na imunofluorescência e slot blot. Ainda, para os antissoros do BoHV-1, BoHV-5, BVDV e BRSV demonstrou-se a reatividade com amostras heterólogas nos ensaios de imunofluorescência e imunoperoxidase. Conclui-se que os anticorpos policlonais produzidos em coelhos possuem elevadas concentrações de anticorpos específicos, o que foi detectado pela reatividade nos ensaios de imunofluorescência, imunoperoxidase e slot blot. Desta maneira, estes reagentes podem ser considerados uma importante ferramenta para a detecção e caracterização de vários vírus bovinos na rotina de diagnóstico e pesquisa. / The viruses are significant important pathogenic agents of several animal species, including cattle. In Brazil, several viral agents causing infections have been described in cattle and they produce significant economic losses. The identification of animals infected by a virus can be performed in different ways; however, definitive confirmation requires demonstration of the agent or immune response. For this purpose, various methods with the capacity to detect the viral particle, biological activity, genome, viral antigens, or specific immune response have been developed. Immunoassays are widely used in laboratory routine for detection of viral antigens in clinical or research. These assays exhibit good sensitivity, specificity and easy for implantation. The immunoassay methodologies are based on the employment of monoclonal or polyclonal antibodies specific to the viral antigens. Therefore, the aim of this study was to produce polyclonal antibodies for some bovine virus, and evaluate their reactivity in immunofluorescence, immunoperoxidase and slot blot tests. For this purpose, strains and/or isolates of bovine herpesvirus type 1 (BoHV-1), bovine herpesvirus type 2 (BoHV-2), bovine herpesvirus type 5 (BoHV-5), bovine herpesvirus type 5 gE deleted (BoHV-5 gEΔ), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bluetongue virus (BTV), and vaccinia virus (VACV) were amplified in cell culture and the supernatant were used to immunize rabbits. The animals were immunized five times by the subcutaneous route, and five days after the last boost the blood was collected. The serum was obtained by centrifugation. The serum was diluted (1:100 a 1:204.800) and used as primary antibodies in the immunofluorescence, immunoperoxidase and slot blot assays. The working dilution was selected among those produced specific reaction with infected cells and absent or weak background in control cells. The antiserum showed higher reactivity in immunoperoxidase technique than the immunofluorescence and slot blot. The antiserum of the BoHV-1, BoHV-5, BVDV and BRSV presented the reactivity when tested with eterologous isolates in immunofluorescence, immunoperoxidase assays. In summary, that the polyclonal antibodies raised in rabbits have high concentrations of specific antibodies, which were demonstrated by the reactivity in immunofluorescence, immunoperoxidase and slot blot assays. Additionally, these reagents can be considered an important tool for the detection and characterization of various bovine viruses in diagnostic and research routine.