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Id: biblio-1128856
Autor: Rodrigues, P. R. C; Cunha, R. C; Santos, F. D. S; Gonçalves, V. S; Albuquerque, P. M. M; Santos Júnior, A. G; Lima, M; Leite, F. P. L.
Título: Expressão e caracterização da glicoproteína D do herpesvírus equídeo 1 em Pichia pastoris / Expression and characterization of equid herpesvirus 1 glycoprotein D in Pichia pastoris
Fonte: Arq. bras. med. vet. zootec. (Online);72(3):703-710, May-June, 2020. ilus, graf.
Idioma: pt.
Resumo: O herpesvírus equídeo 1 (EHV-1) apresenta distribuição mundial e causa graves prejuízos à equideocultura. É agente de surtos de doença respiratória, reprodutiva e neurológica, em equídeos jovens e adultos. A glicoproteína D (gD) do envelope viral é essencial para ligação e penetração em células permissivas e direcionamento do sistema imunológico do hospedeiro, induz respostas imunes humorais e celulares, sendo um antígeno apropriado para ser utilizado em vacinas e imunodiagnóstico. O objetivo deste trabalho foi expressar e caracterizar a gD do EHV-1 em Pichia pastoris para posterior utilização como antígeno em técnicas de imunodiagnóstico e formulação de vacinas recombinantes. Uma sequência de DNA que codifica uma forma truncada da gDEHV-1 foi clonada no vetor pPICZαA de expressão em P. pastoris. Obteve-se uma proteína de ~41 kDa, como esperado. A proteína apresentou glicosilação entre 4 kDa e 16 kDa, demonstrada por deglicosilação enzimática. A proteína recombinante foi caracterizada antigenicamente e imunogenicamente por Western blot, utilizando-se anticorpos policlonais equinos anti-EHV-1, e por ELISA indireto em modelo murino, demonstrando que a gD recombinante manteve epítopos similares aos da proteína nativa. Esses resultados sugerem que a gDEHV-1 é um antígeno promissor para uso como imunobiológico no controle do EHV-1.(AU)

Equine herpesvirus 1 (EHV-1) has a worldwide distribution and causes serious damage to horse breeding. It is an agent of respiratory, reproductive and neurological disease outbreaks in young and adult equids. Viral envelope glycoprotein D (gD) is essential for binding and penetration into permissive cells and targeting the host immune system, inducing humoral and cellular immune responses, and is an appropriate antigen for use in vaccines and immunodiagnostics. The objective of this work was to express in Pichia pastoris and to characterize EHV-1 gD for later use as an antigen in immunodiagnostic techniques and formulation of recombinant vaccines. A DNA sequence encoding a truncated form of gDEHV-1 has been cloned into the P. pastoris expression vector pPICZαA. A protein of ~41 kDa was obtained as expected. The protein presented glycosylation between 4 kDa and 16 kDa, demonstrated by enzymatic deglycosylation. The recombinant protein was antigenically and immunogenically characterized by Western blot using equine polyclonal anti-EHV-1 antibodies, and by indirect ELISA in a murine model, demonstrating that the recombinant gD maintained epitopes similar to those of the native protein. These results suggest that gDEHV-1 is a promising antigen for use as an immunobiological in the control of EHV-1.(AU)
Descritores: Pichia/isolamento & purificação
Glicoproteínas
Herpesvirus Equídeo 1/isolamento & purificação
-Doenças Respiratórias/veterinária
Cavalos/virologia
Limites: Animais
Responsável: BR68.1 - Biblioteca Virginie Buff D'Ápice


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Berne, Maria Elisabeth Aires
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Id: lil-750757
Autor: Pinheiro, Amanda Fernandes; Borsuk, Sibele; Berne, Maria Elisabeth Aires; Pinto, Luciano da Silva; Andreotti, Renato; Roos, Talita; Roloff, Barbara Couto; Leite, Fábio Pereira Leivas.
Título: Use of ELISA based on NcSRS2 of Neospora caninum expressed in Pichia pastoris for diagnosing neosporosis in sheep and dogs / Utilização de um ELISA baseado em NcSRS2 de Neospora caninum expressa em Pichia pastoris para diagnóstico de neosporose em ovinos e cães
Fonte: Rev. bras. parasitol. vet;24(2):148-154, Apr-Jun/2015. graf.
Idioma: en.
Projeto: CAPES.
Resumo: Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.

A neosporose é uma doença causada pelo protozoário Neospora caninum que leva a perdas econômicas importantes em muitos países. No presente estudo, é descrita a utilização da proteína recombinante NcSRS2 de N. caninum expressa em Pichia pastoris em um ensaio imunoenzimático indireto (ELISA) para o diagnóstico de infecção por Neospora em ovelhas e cães. Observou-se, que utilizando-se um ELISA, o teste produziu uma especificidade de 94,5% e uma sensibilidade de 100% para ovinos; e uma especificidade de 93,3% e sensibilidade de 100% para cães. Uma maior sensibilidade foi observada em relação à IFI que foi confirmada por Western blot. Os resultados deste estudo sugerem que a proteína recombinante expressa em P. pastoris é bom antígeno para ser utilizado no diagnóstico imunológico para detectar N. caninum em duas espécies importantes expostas a esta parasitose.
Descritores: Infecções Protozoárias em Animais/sangue
Doenças dos Ovinos/sangue
Ensaio de Imunoadsorção Enzimática
Proteínas de Protozoários/sangue
Neospora/imunologia
Doenças do Cão/parasitologia
Doenças do Cão/sangue
Antígenos de Protozoários/sangue
Antígenos de Superfície/sangue
-Pichia/metabolismo
Infecções Protozoárias em Animais/diagnóstico
Doenças dos Ovinos/diagnóstico
Doenças dos Ovinos/parasitologia
Ovinos
Proteínas de Protozoários/biossíntese
Proteínas de Protozoários/imunologia
Doenças do Cão/diagnóstico
Antígenos de Protozoários/biossíntese
Antígenos de Protozoários/imunologia
Antígenos de Superfície/biossíntese
Antígenos de Superfície/imunologia
Limites: Animais
Cães
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: BR1.1 - BIREME


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Id: biblio-1053200
Autor: Guerrero, Karlo; Arancibia, Alejandra; Caceres, Manuel; Aroca, German.
Título: Release of formaldehyde during the biofiltration of methanol vapors in a peat biofilter inoculated with Pichia pastoris GS115
Fonte: Electron. j. biotechnol;40:10-16, July. 2019. tab, ilus, graf.
Idioma: en.
Projeto: National Comission for Science and Technology (CONICYT) Chile.
Resumo: Background: Methanol can be effectively removed from air by biofiltration. However, formaldehyde is one of the first metabolic intermediates in the consumption of methanol in methylotrophic microorganisms, and it can be released out of the cell constituting a secondary emission. Results: The total removal of methanol was achieved up to input loads of 263 g m−3 h−1 and the maximum elimination capacity of the system was obtained at an empty bed residence times of 90 s and reached 330 g m− 3 h−1 at an input methanol load of 414 g m−3 h−1 and 80% of removal efficiency. Formaldehyde was detected inside the biofilter when the input methanol load was above 212 g m−3 h−1 . Biomass in the filter bed was able to degrade the formaldehyde generated, but with the increase of the methanol input load, the unconsumed formaldehyde was released outside the biofilter. The maximum concentration registered at the output of the system was 3.98 g m−3 when the methanol load was 672 g m−3 h−1 in an empty bed residence times of 60 s. Conclusions: Formaldehyde is produced inside a biofilter when methanol is treated in a biofiltration system inoculated with Pichia pastoris. Biomass present in the reactor is capable of degrading the formaldehyde generated as the concentration of methanol decreases. However, high methanol loads can lead to the generation and release of formaldehyde into the environment
Descritores: Pichia/metabolismo
Metanol/metabolismo
Formaldeído/metabolismo
-Biomassa
Poluentes Atmosféricos
Meio Ambiente
Filtração
Responsável: CL1.1 - Biblioteca Central


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Id: lil-757775
Autor: Terrazas, W. D. M; Aizemberg, R; Gattás, E. A. L.
Título: Using Pichia pastoris to produce recombinant glycerol kinase / Produção de glycerol quinase recombinante utilizando o sistema Pichia pastoris
Fonte: Rev. ciênc. farm. básica apl;35(2):279-284, jun. 2014.
Idioma: pt.
Resumo: The methylotrophic yeast Pichia pastoris has been developed into an efficient expression system for the production of recombinant protein under the tight control of the methanol-induced alcohol oxidase promoter (pAOX1). In this study, a 2.5-liter culture system was developed for the growth of a P. pastoris strain bearing the GUT1 gene from Saccharomyces cerevisiae for the expression of recombinant glycerol kinase (GK). The best culture conditions to produce high levels of secreted GK were investigated by growing the recombinant strain of P. pastoris in shake flasks and a fermenter. Cell growth and enzyme production were found to be optimal after two days of growth. Enzyme production was affected by the nitrogen source, Difco peptone being the most appropriate for this purpose. Three different rates of air flow (1 to 3 L/min) were tested to observe their effect on cell growth and the secretion of GK into a medium containing 1% methanol as the sole carbon source. Increasing the rate of air bubbling in the culture medium enhanced both cell growth and GK activity, reaching a dry biomass of 7.84 mg/mL, cell viability of 98.4% and a maximal GK activity of 1.57 U/mL, at a flow rate of 2.0 L/minute, at 30° C and pH 6.0. Moreover, the enzyme activity in the P. pastoris culture medium was 2.3 times higher under these conditions than in the shake-flask culture, demonstrating the significant influence of aeration on biomass production and GK activity secreted by P. pastoris...

A levedura metilotrófica Pichia pastoris possui um sistema de expressão eficiente para a produção de proteínas recombinantes. A indução da produção da proteína de interesse é feita com metanol, que é capaz de ativar a transcrição do gene de interesse clonado sob controle do promotor do gene AOX1. Um meio de cultura de 2.5 litros foi elaborado para o crescimento da cepa Pichia pastoris construída com o gene GUT1 de Saccharomyces cerevisiae para expressar a enzima recombinante glicerol quinase (GK). As condições ideais de cultura, para alcançar altos níveis de expressão de GK foram investigados em crescimentos realizados em frascos e fermentador. Crescimento celular e produção de enzima atingiram valores ótimos em dois dias de cultura. A produção enzimática foi afetada pela fonte de nitrogênio no meio. Peptona da marca Difco foi a fonte de nitrogênio mais adequada para a expressão destaenzima. Três diferentes concentrações (1-3 L / min) defluxo de ar foram analisados em ensaios de crescimento celular e secreção da GK, no meio contendo 1 % demetanol como única fonte de carbono. O aumento do fluxo do ar no meio de cultura produziu melhores resultados para o crescimento celular e atividade da GK, atingindo 7,84 mg / mL de biomassa seca e 98,4%de viabilidade. A máxima atividade de GK foi de 1,57U / mL, com a concentração de fluxo de ar de 2,0 L/ minuto a 30 ° C e pH 6.0. O aumento da atividade enzimática foi 2,3 vezes maior no meio de cultura da Pichia pastoris nestas condições, revelando a influência deste parâmetro na produção de biomassa e atividade da GK...
Descritores: Glicerol Quinase
Pichia/crescimento & desenvolvimento
-Biomassa
Limites: Humanos
Responsável: BR1.1 - BIREME


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Id: lil-409564
Autor: Lozano M., Adelina; Caycho O., Jorge; Antunez de Mayolo, Antonio; Vera, Dolly; Posadas R., David.
Título: Inmunogenicidad y eficacia de una nueva vacuna de DNA recombinante de Hepatitis B en el Perú / Inmunogenicity and efficacy of a new recombinant DNA vaccine for Hepatitis B virus in Peru
Fonte: Rev. gastroenterol. Perú;23(4):259-264, oct.-dic. 2003. tab, graf.
Idioma: es.
Resumo: Aproximadamente 350 millones de personas están infectadas con hepatitis B en el mundo. El Perú es uno de los países con alta prevalencia de Hepatitis B en América Latina. La hepatitis B causa hepatitis crónica, cirrosis y carcinoma hepatocelular. La vacunación es el único método que ha demostrado ser eficaz en el control de la enfermedad. Este es un estudio prospectivo, aleatorio, conducido con el propósito de evaluar la inmunogenicidad y seguridad de la vacuna Hepavax-Gene, una nueva vacuna de DNA recombinante, en personas sanas mayores de 10 años de edad en el Perú. La vacuna fue administrada vía intramuscular en el músculo deltoides en dosis 10 microgramos a personas de 10 a 19 años y de 20 microgramos a mayores de 19 años, con el esquema de vacunación de tres dosis: el día O, a los 30 días y a los 180 días. 67 personas terminaron el estudio de un total de 188 personas admiitdas inicialmente. La inmunogenicidad fue satisfactoria: 100 por ciento de los participantes en el Grupo A (10 a 19 años) 100 por ciento de los participantes del Grupo B (mayores de 19 años). Con una media aritmética de 28,583.92 mLU/mL y una media geométrica de 5,754.32 mIU/mL en el grupo A. y con una media aritmética de 8,708.3 mIU/mL 2 y una media geométrica de 2,179.32 mIU/mL en el Grupo B. Los eventos adversos fueron de 4.69 por ciento. Ninguno fue severo. La vacuna de DNA recombinante derivada de H. Polymorpha es inmunogénica y segura en peruanos sanos mayores de 10 años aplicada en tres dosis a los días 0, 30 y 180.
Descritores: Pichia
Vacinas contra Hepatite B
Hepatite B
Anticorpos
-Estudos Prospectivos
Limites: Humanos
Masculino
Adolescente
Adulto
Feminino
Criança
Responsável: PE1.1 - Oficina Universitária de Biblioteca


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Beçak, W
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Id: lil-606544
Autor: Coimbra, E. C; Gomes, F. B; Campos, J. F; D’arc, M; Carvalho, J. C; Mariz, F. C; Jesus, A. L. S; Stocco, R. C; Beçak, W; Freitas, A. C.
Título: Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector
Fonte: Braz. j. med. biol. res = Rev. bras. pesqui. méd. biol;44(12):1209-1214, Dec. 2011. ilus, tab.
Idioma: en.
Resumo: Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.
Descritores: Alphapapillomavirus/imunologia
Proteínas do Capsídeo/biossíntese
Proteínas Oncogênicas Virais/biossíntese
Pichia/metabolismo
-Alphapapillomavirus/genética
Anticorpos Antivirais/imunologia
Proteínas do Capsídeo/genética
Transformação Celular Viral/fisiologia
Eletroforese em Gel de Poliacrilamida
Regulação Viral da Expressão Gênica
Proteínas Oncogênicas Virais/genética
Vacinas contra Papillomavirus/imunologia
Pichia/genética
Pichia/virologia
Reação em Cadeia da Polimerase Via Transcriptase Reversa
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: BR1.1 - BIREME


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Id: biblio-1087710
Autor: Guerrero, Karlo; Arancibia, Alejandra; Caceres, Manuel; Aroca, German.
Título: Release of formaldehyde during the biofiltration of methanol vapors in a peat biofilter inoculated with Pichia pastoris GS115
Fonte: Electron. j. biotechnol;44:58-59, Mar. 2020. ilus.
Idioma: en.
Resumo: BACKGROUND: Methanol can be effectively removed from air by biofiltration (Shareefdeen et al., 1993; Babbitt et al., 2009 [1,2]). However, formaldehyde is one of the first metabolic intermediates in the consumption of methanol in methylotrophic microorganisms (Negruta et al., 2010 [3]), and it can be released out of the cell constituting a secondary emission. RESULTS: The total removal of methanol was achieved up to input loads of 263 g m−3 h−1 and the maximum elimination capacity of the system was obtained at an empty bed residence times of 90 s and reached 330 g m−3 h−1 at an input methanol load of 414 g m−3 h−1 and 80% of removal efficiency. Formaldehyde was detected inside the biofilter when the input methanol load was above 212 g m−3 h−1 . Biomass in the filter bed was able to degrade the formaldehyde generated, but with the increase of the methanol input load, the unconsumed formaldehyde was released outside the biofilter. The maximum concentration registered at the output of the system was 3.98 g m−3 when the methanol load was 672 g m−3 h−1 in an empty bed residence times of 60 s. CONCLUSIONS: Formaldehyde is produced inside a biofilter when methanol is treated in a biofiltration system inoculated with Pichia pastoris. Biomass present in the reactor is capable of degrading the formaldehyde generated as the concentration of methanol decreases. However, high methanol loads can lead to the generation and release of formaldehyde into the environment.
Descritores: Pichia/química
Metanol/química
Formaldeído/análise
-Volatilização
Filtros Biológicos
Biomassa
Reatores Biológicos
Meio Ambiente
Responsável: CL1.1 - Biblioteca Central


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Id: biblio-974317
Autor: Zepeda, Andrea B; Pessoa Jr, Adalberto; Farías, Jorge G.
Título: Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast
Fonte: Braz. j. microbiol;49(supl.1):119-127, 2018. tab, graf.
Idioma: en.
Projeto: Comisión Nacional de Investigación Científica y Tecnológica de Chile; . AZP; . FAPESP.
Resumo: Abstract Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production.
Descritores: Pichia/genética
Proteínas Recombinantes/biossíntese
Proteínas Recombinantes/genética
Carbono/metabolismo
Regiões Promotoras Genéticas
-Pichia/crescimento & desenvolvimento
Pichia/metabolismo
Temperatura
Microbiologia Industrial
Responsável: BR1.1 - BIREME


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Id: biblio-974294
Autor: Zepeda, Andrea B; Figueroa, Carolina A; Pessoa, Adalberto; Farías, Jorge G.
Título: Free fatty acids reduce metabolic stress and favor a stable production of heterologous proteins in Pichia pastoris
Fonte: Braz. j. microbiol;49(4):856-864, Oct.-Dec. 2018. graf.
Idioma: en.
Projeto: FAPESP.
Resumo: ABSTRACT The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14 ºC + 1%M provided the most stable viability. By contrast, the temperature of 18 ºC + 1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids.
Descritores: Pichia/metabolismo
Ácidos Graxos não Esterificados/metabolismo
-Pichia/crescimento & desenvolvimento
Pichia/genética
Temperatura
Proteínas Recombinantes/genética
Meios de Cultura/metabolismo
Meios de Cultura/química
Metanol/metabolismo
Fermentação
Glicerol/metabolismo
Responsável: BR1.1 - BIREME


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Id: biblio-839389
Autor: Eissazadeh, Samira; Moeini, Hassan; Dezfouli, Maryam Ghandizadeh; Heidary, Somayyeh; Nelofer, Rubina; Abdullah, Mohd Puad.
Título: Production of recombinant human epidermal growth factor in Pichia pastoris
Fonte: Braz. j. microbiol;48(2):286-293, April.-June 2017. tab, graf.
Idioma: en.
Resumo: Abstract This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27 µg/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60 h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.
Descritores: Pichia/metabolismo
Proteínas Recombinantes/metabolismo
Fator de Crescimento Epidérmico/metabolismo
-Pichia/genética
Proteínas Recombinantes/genética
Ensaio de Imunoadsorção Enzimática
Expressão Gênica
Clonagem Molecular
Meios de Cultura/química
Fator de Crescimento Epidérmico/genética
Fermentação
Concentração de Íons de Hidrogênio
Limites: Humanos
Responsável: BR1.1 - BIREME



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