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Id: biblio-839393
Autor: Yu, Yi; Chang, De; Xu, Huiwen; Zhang, Xuelin; Pan, Lei; Xu, Chou; Huang, Bing; Zhou, Hong; Li, Jia; Guo, Jun; Liu, Changting.
Título: The virulence of Streptococcus pneumoniae partially depends on dprA
Fonte: Braz. j. microbiol;48(2):225-231, April.-June 2017. graf.
Idioma: en.
Projeto: National Basic Research Program of China; . National Major Scientific and Technological Special Project for \"Significant New Drugs Development\"; . National Natural Science Foundation of China; . Basic Research Program of General Hospital of Chinese People's Armed Police Forces.
Resumo: Abstract Streptococcus pneumoniae is one of the most frequent opportunistic pathogens worldwide. DNA processing protein A (DprA) is an important factor involved in bacterial uptake and DNA integration into bacterial genome, but its role in S. pneumoniae virulence remains unclear. The aim of this study was to characterize the effects of the pneumococcal dprA gene on the pathogenesis of S. pneumoniae. To construct a dprA-deficient pneumococcal strain, the dprA gene of the S. pneumoniae strain D39 was inactivated. The virulence of this dprA-deficient strain, designated ΔD39, was compared with that of the wild-type strain by evaluating their respective capabilities to adhere to human pulmonary epithelial cells (PEC-A549) and by analyzing their choline-binding protein expression levels. In addition, the expression profiles of genes associated with virulence and host survival assays were also conducted with the mutant and the wild-type strain. Our results indicate that the capability of ΔD39 to adhere to the PEC-A549 airway cells was significantly lower (p < 0.01) compared with D39. Additionally, the 100-KD choline-binding protein was not detected in ΔD39. The addition of competence-stimulating peptide (CSP) lead to a significantly reduction of psaA mRNA expression in the dprA-deficient mutant and an increased level of psaA transcripts in the wild-type strain (p < 0.01). The median survival time of mice intraperitoneally infected with ΔD39 was significantly higher (p < 0.01) than that of mice infected with D39. The results of this study suggest that DprA has a significant effect on virulence characteristics of S. pneumoniae by influencing the expression of choline-binding protein and PsaA.
Descritores: Infecções Pneumocócicas/patologia
Streptococcus pneumoniae/patogenicidade
Proteínas de Bactérias/metabolismo
Aderência Bacteriana
Fatores de Virulência/análise
Proteínas de Membrana/metabolismo
-Infecções Pneumocócicas/microbiologia
Streptococcus pneumoniae/genética
Proteínas de Bactérias/análise
Proteínas de Bactérias/genética
Análise de Sobrevida
Linhagem Celular
Fatores de Virulência/genética
Modelos Animais de Doenças
Células Epiteliais/microbiologia
Técnicas de Inativação de Genes
Proteínas de Membrana/genética
Camundongos
Limites: Seres Humanos
Animais
Responsável: BR1.1 - BIREME


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Id: biblio-1021652
Autor: Wu, Yan; Li, Tao; Cao, Qinghua; Li, Xuedan; Zhang, Yizheng; Tan, Xuemei.
Título: RecET recombination system driving chromosomal target gene replacement in Zymomonas mobilis
Fonte: Electron. j. biotechnol;30:118-124, nov. 2017. tab, ilus, graf.
Idioma: en.
Projeto: National Key Technology R&D Program.
Resumo: Background: Zymomonas mobilis is a Gram-negative microaerophilic bacterium with excellent ethanol-producing capabilities. The RecET recombination system provides an efficient tool for direct targeting of genes in the bacterial chromosome by PCR fragments. Results: The plasmids pSUZM2a-RecET and pSUZM2a-RecE588T were first developed to co-express RecE or RecE588 and RecT for homologous recombination. Thereafter, the PCR fragments of the tetracycline resistance marker gene flanked by 60 bp of adhA (alcohol dehydrogenase I) or adhB (alcohol dehydrogenase II) homologous sequences were electroporated directly into ZM4 cells harboring pSUZM2a-RecET or pSUZM2a-RecE588T. Both adhA and adhB were replaced by the tetracycline resistance gene in ZM4, yielding two mutant strains, Z. mobilis ZM4 ΔadhA and Z. mobilis ZM4 ΔadhB. These two mutants showed varying extent of reduction in ethanol production, biomass generation, and glucose metabolism. Furthermore, enzyme activity of alcohol dehydrogenase II in Z. mobilis ZM4 ΔadhB exhibited a significant reduction compared to that of wild-type ZM4. Conclusion: This approach provided a simple and useful method for introducing mutations and heterologous genes in the Z. mobilis genome.
Descritores: Zymomonas/genética
Recombinação Homóloga
-Plasmídeos
Recombinação Genética
Álcool Desidrogenase/metabolismo
Zymomonas/enzimologia
Eletroporação
Etanol/metabolismo
Técnicas de Inativação de Genes
Mutação
Responsável: CL1.1 - Biblioteca Central


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Id: biblio-841772
Autor: Villela, Anne Drumond; Rodrigues Junior, Valnês da Silva; Pinto, Antônio Frederico Michel; Wink, Priscila Lamb; Sánchez-Quitian, Zilpa Adriana; Petersen, Guilherme Oliveira; Campos, Maria Martha; Basso, Luiz Augusto; Santos, Diógenes Santiago.
Título: Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis
Fonte: Mem. Inst. Oswaldo Cruz;112(3):203-208, Mar. 2017. graf.
Idioma: en.
Projeto: CNPq; . CNPq; . CNPq; . CNPq; . BNDES; . FAPERGS-CAPES; . CNPq.
Resumo: BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.
Descritores: Macrófagos/microbiologia
Mycobacterium tuberculosis/crescimento & desenvolvimento
Mycobacterium tuberculosis/enzimologia
Mycobacterium tuberculosis/genética
N-Glicosil Hidrolases/genética
-Técnicas de Inativação de Genes
Genes Bacterianos
Limites: Seres Humanos
Responsável: BR1.1 - BIREME


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Id: biblio-847147
Autor: Nagai, Maíra Harume.
Título: RIC-8B, uma GEF de sistema olfatório, é essencial para o desenvolvimento do sistema nervoso / RIC-8B, an olfactory GEF, is essential for the development of the nervous system.
Fonte: São Paulo; s.n; 2014. 133 p. tab, graf, ilus.
Idioma: pt.
Tese: Apresentada a Universidade de São Paulo. Instituto de Química para obtenção do grau de Doutor.
Resumo: RIC-8B é um fator trocador de nucleotídeo de guanina (GEF) predominantemente expresso em neurônios olfatórios maduros de camundongos adultos. Trabalhos desenvolvidos em nosso laboratório mostraram que RIC-8B interage com Gαolf e Gγ13, duas subunidades de proteína G que estão enriquecidas nos cílios dos neurônios olfatórios, onde participam da transdução do sinal de odorantes. In vitro, RIC-8B é capaz de amplificar a sinalização de receptores olfatórios através de Gαolf, no entanto, seu papel fisiológico ainda é desconhecido. Para determinar a função desempenhada por essa proteína in vivo, nós utilizamos a tecnologia de Gene Trap com o objetivo de produzir um camundongo knockout para Ric-8B. Apesar de a expressão de Ric-8B ser restrita a poucos tecidos no camundongo adulto, descobrimos que homozigotos para a mutação em Ric-8B são inviáveis e morrem por volta do dia embrionário E10,5. Além disso, são menores e apresentam evidente falha no fechamento do tubo neural na região cranial (exencefalia). Utilizamos o gene repórter ß-galactosidase expresso pelo alelo mutado para determinar o padrão de expressão de Ric-8B em embriões durante o desenvolvimento. Observamos que, no estágio E8,5, Ric-8B é expresso nas pregas neurais da região cefálica e na notocorda. De E9,5 a E12,5, a expressão de Ric-8B é detectada predominante no assoalho da placa. Esse padrão de expressão se assemelha ao de outro gene importante para a embriogênese, Sonic hedgehog (Shh). SHH é um morfógeno diretamente responsável pela padronização dorsoventral do sistema nervoso central e sua sinalização depende de cílio primário. Cílio primário é uma organela baseada em microtúbulos que se projeta da superfície da maioria das células de mamíferos e funciona como um centro de sinalização intracelular. Nossos dados mostram que fibroblastos embrionários Ric-8B-/- formam cílios primários, assim como alguns tecidos do embrião. Além disso, não encontramos alterações na sinalização de Shh em embriões homozigotos mutantes. No entanto, observamos que esses embriões apresentam apoptose aumentada em células migratórias da crista neural cranial. Shh é importante para a sobrevivência de células da crista neural migratória, sugerindo um possível papel para Ric-8B a downstream da sinalização de SHH

Ric-8B is a guanine nucleotide exchange factor (GEF) which is predominantly expressed in mature olfactory sensory neurons in adult mice. We have previously shown that RIC-8B interacts with both Gαolf and Gγ13, two G protein subunits, which are enriched in olfactory cilia and are required for odorant signal transduction. In vitro, RIC-8B is able to amplify odorant receptor signaling through Gαolf, however, its physiological role remains unknown. To determine the role played by RIC-8B in vivo we used the Gene trap technology to generate a Ric-8B knockout mouse. We found that, despite the limited distribution of Ric-8B gene expression in adult mice, Ric-8B homozygous mutants are not viable and die around the E10,5 stage. Mutant embryos are also smaller and fail to close the neural tube at the cranial region (exencephaly). We used the activity of the ß-galactosidase reporter gene to determine the pattern of expression of the Ric-8B gene in heterozygous embryos. At E8,5 the Ric-8B gene is expressed in the notochord and neural folds of the cephalic regions. From E9,5 to E12,5 Ric-8B is predominantly expressed in the floor plate, in a pattern that strongly resembles the one shown by Sonic hedgehog (Shh). SHH is a morphogen directly responsible for the dorsoventral patterning of the central nervous system and its signaling depends on primary cilia. Primary cilia are microtubule-based organelles that protrude from the surface of mammalian cells and act as a signaling center. We show that Ric-8B-/- embryonic fibroblasts and some embryonic tissues grow primary cilia normally. In addition, we did not find alterations in the SHH signaling of homozygous mutants. Instead, we found an increased apopotosis in migratory cells of the cranial neural crest in these embryos. Shh is an important factor to survival of neural crest cells, suggesting a role for RIC-8B downstream of the SHH signaling
Descritores: Fatores de Troca do Nucleotídeo Guanina/análise
Sistema Nervoso/crescimento & desenvolvimento
Olfato
-Desenvolvimento Embrionário
Técnicas de Inativação de Genes/métodos
Biologia Molecular
Defeitos do Tubo Neural
Limites: Animais
Masculino
Feminino
Camundongos
Tipo de Publ: Técnicas In Vitro
Estudos de Avaliação
Responsável: BR40.1 - DBD - Divisão de Biblioteca e Documentacão do Conjunto das Químicas
BR40.1; T 574.88, N147r. 30100025393-Q


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Id: biblio-846935
Autor: Gutiyama, Luciana Mayumi.
Título: RIC-8B, um fator trocador de nucleotídeo guanina (GEF), é essencial para a embriogênese / RIC-8B, a guanine nucleotide exchange factor (GEF), is essential for embryogenesisPalavras-chave em inglês Cilia.
Fonte: São Paulo; s.n; 2013. 113 p. tab, graf, ilus.
Idioma: pt.
Tese: Apresentada a Universidade de São Paulo. Instituto de Química para obtenção do grau de Doutor.
Resumo: RIC-8B é uma proteína que apresenta, in vitro, atividade de fator de troca de nucleotídeos guanina (GEF). No entanto, seu papel in vivo não é conhecido. Dados anteriores do nosso laboratório demonstraram que essa proteína interage especificamente com Gαolf, que é uma proteína G exclusiva do sistema olfatório, presente nos cílios dos neurônios olfatórios, onde ocorre a transdução de sinal ativada pelos odorantes. No camundongo adulto verificou-se, por meio de ensaios de hibridização in situ, que RIC-8B está presente somente em regiões de expressão de Gαolf: no epitélio olfatório maduro e no núcleo estriado do sistema nervoso central. Para avaliar a função fisiológica de RIC-8B in vivo, resolvemos gerar uma linhagem de camundongo knockout para Ric-8B. Verificamos que a linhagem é inviável devido à letalidade dos embriões já em fases precoces do desenvolvimento (por volta de E8,5 e E9,0). A coloração de embriões com X-gal mostra que RIC-8B é especificamente expressa em regiões que darão origem ao sistema nervoso, como na região ventral do tubo neural, e em regiões cefálicas. Interessantemente, mostramos que RIC-8B é expressa na placa do assoalho do tubo neural, de uma maneira muito semelhante ao padrão de expressão de Sonic Hedgehog (SHH), que apresenta um papel fundamental para a organização do sistema nervoso, entre outras funções. Nossos resultados indicam, portanto, que RIC-8B desempenha um papel crucial durante a embriogênese, e que este papel pode estar relacionado com o papel exercido por SHH. Além disso, como a via de sinalização de SHH ocorre em cílios primários nas células alvo, nossos dados levantam a interessante possibilidade de que RIC-8B apresenta funções relacionadas a cílios, tanto no camundongo adulto (neste caso nos cílios dos neurônios olfatórios) como no embrião (neste caso nos cílios primários)

RIC-8B is a protein that, in vitro, acts as a guanine nucleotide exchange factor (GEF). However, its role in vivo remains unknown. Previous data from our laboratory demonstrated that this protein is able to interact specifically with Gαolf, a G protein found only in the olfactory system. This G protein is located in the cilia from olfactory neurons, where odorant signaling occurs. In situ hybridization experiments showed that RIC-8B, in adult mice, is expressed only in regions where Gαolf is expressed, such as the olfactory epithelium and the nucleus striatum in the central nervous system. In order to determine the function of RIC-8B in vivo, we decided to generate a knockout mouse strain for Ric-8B. We found that this strain is not viable due to the lethality of embryos in the early stages of development (around days E8.5 and E9.0). X-gal staining of embryos shows that RIC-8B is specifically expressed in regions that originate the nervous system, such as the ventral neural tube and also cephalic regions. Interestingly, we show that RIC-8B is restrictedly expressed in the floor plate of the neural tube, in a pattern that is very similar to the one shown by Sonic Hedgehog (SHH). The SHH protein plays a fundamental role in the organization of the nervous system, among other functions. Therefore, our results indicate that RIC-8B plays an essential role during the embryogenesis, and that this role can be related to the role played by SHH. Furthermore, because the SHH signaling pathway occurs in primary cilia in the target cells, our data raise the interesting possibility that the role played by RIC-8B is related to ciliary functions, both in adult mice (in this case, in olfactory cilia), and in the embryo (in this case, in primary cilia)
Descritores: Desenvolvimento Embrionário/genética
Técnicas de Inativação de Genes
Olfato/fisiologia
-Proteínas de Ligação ao GTP
Nucleotídeos
Neurônios Receptores Olfatórios
Fenótipo
Proteínas/análise
Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
Pesquisa com Células-Tronco
Limites: Animais
Masculino
Feminino
Camundongos
Tipo de Publ: Estudos de Avaliação
Responsável: BR40.1 - DBD - Divisão de Biblioteca e Documentacão do Conjunto das Químicas
BR40.1; T 574.88, G984r. 30100020448


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Id: lil-727661
Autor: de Araújo, E.S.S.; Vasques, L.R.; Stabellini, R.; Krepischi, A.C.V.; Pereira, L.V..
Título: Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line
Fonte: Braz. j. med. biol. res = Rev. bras. pesqui. méd. biol;47(12):1029-1035, 12/2014. graf.
Idioma: en.
Projeto: FAPESP.
Resumo: DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.
Descritores: Metilação de DNA/genética
Repressão Epigenética/genética
Genoma Humano
Genoma/genética
Impressão Genômica/genética
Fator de Crescimento Insulin-Like II/genética
RNA Longo não Codificante/genética
-Azacitidina/administração & dosagem
Azacitidina/análogos & derivados
/genética
DNA (CYTOSINE-ABDOMEN-)-METHYLTRANSFERASE/genética
Metilação de DNA/efeitos dos fármacos
Técnicas de Inativação de Genes
Genoma Humano/efeitos dos fármacos
HCTACETYLATION CELLS
Hibridização in Situ Fluorescente/métodos
Análise em Microsséries
Polimorfismo de Nucleotídeo Único
Proteínas/metabolismo
RNA Longo não Codificante/metabolismo
Reação em Cadeia da Polimerase em Tempo Real/métodos
Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
Limites: Seres Humanos
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: BR1.1 - BIREME


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Id: lil-716313
Autor: Alcantara, Monica Visnieski; Fragoso, Stenio Perdigão; Picchi/, Gisele Fernanda Assine.
Título: Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture
Fonte: Mem. Inst. Oswaldo Cruz;109(4):511-513, 03/07/2014. graf.
Idioma: en.
Resumo: Gene knockout is a widely used approach to evaluate loss-of-function phenotypes and it can be facilitated by the incorporation of a DNA cassette having a drug-selectable marker. Confirmation of the correct knockout cassette insertion is an important step in gene removal validation and has generally been performed by polymerase chain reaction (PCR) assays following a time-consuming DNA extraction step. Here, we show a rapid procedure for the identification of Trypanosoma cruzi transfectants by PCR directly from liquid culture - without prior DNA extraction. This simple approach enabled us to generate PCR amplifications from different cultures varying from 106-108 cells/mL. We also show that it is possible to combine different primer pairs in a multiplex detection reaction and even to achieve knockout confirmation with an extremely simple interpretation of a real-time PCR result. Using the “culture PCR” approach, we show for the first time that we can assess different DNA sequence combinations by PCR directly from liquid culture, saving time in several tasks for T. cruzi genotype interrogation.
Descritores: Trypanosoma cruzi/genética
-Primers do DNA/genética
DNA de Protozoário/genética
Técnicas de Inativação de Genes
Genótipo
Reação em Cadeia da Polimerase
Transfecção
Responsável: BR1.1 - BIREME


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Id: lil-614553
Autor: Borsuk, Sibele; Seixas, Fabiana Kommling; Ramos, Daniela Fernandes; Mendum, Tom; McFadden, Johnjoe; Dellagostin, Odir.
Título: Rational design of diagnostic and vaccination strategies for tuberculosis
Fonte: Braz. j. infect. dis;16(1):68-73, Jan.-Feb. 2012. ilus.
Idioma: en.
Resumo: The development of diagnostic tests which can readily differentiate between vaccinated and tuberculosis-infected individuals is crucial for the wider utilization of bacillus Calmette-Guérin (BCG) as vaccine in humans and animals. BCG_0092 is an antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both animals and humans infected with the tubercle bacilli. We carried out bioinformatics analyses of the BCG_0092 and designed a diagnostic test by using the predicted MHC class I epitopes. In addition, we performed a knockout of this gene by homologous recombination in the BCG vaccine strain to allow differentiation of vaccinated from infected individuals. For that, the flanking sequences of the target gene (BCG_0092)were cloned into a suicide vector. Spontaneous double crossovers, which result in wild type revertants or knockouts were selected using SacB. BCG_0092 is present only in members of the Mycobacterium tuberculosis complex. Eight predicted MHC class I epitopes with potential for immunological diagnosis were defined, allowing the design of a specific diagnostic test. The strategy used to delete the (BCG_0092) gene from BCG was successful. The knockout genotype was confirmed by PCR and by Southern blot. The mutant BCG strain has the potential of inducing protection against tuberculosis without interfering with the diagnostic test based on the use of selected epitopes from BCG_0092.
Descritores: Adjuvantes Imunológicos
Epitopos de Linfócito T/imunologia
Mycobacterium tuberculosis/imunologia
Tuberculose/diagnóstico
Tuberculose/imunologia
-Vacina BCG/imunologia
Biologia Computacional
Epitopos de Linfócito T/análise
Técnicas de Inativação de Genes
Antígenos de Histocompatibilidade Classe I/imunologia
Hipersensibilidade Tardia/imunologia
Mycobacterium bovis/genética
Mycobacterium bovis/imunologia
Mycobacterium tuberculosis/genética
Vacinas contra a Tuberculose/imunologia
Tuberculose/prevenção & controle
Limites: Seres Humanos
Responsável: BR1.1 - BIREME



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