Le parvovirus adéno-associé (AAV) possède un génome à ADN linéaire simple brin de 4,7kb encadré par deux séquences palindromiques inversées et identiques de 145 nucléotides appelées ITRs, requises en cis pour la réplication et l’encapsidation de l’ADN viral. Dans un AAV recombinant (rAAV), la totalité de la partie codante du génome viral est remplacée par une cassette d’expression et seuls les ITRs sont conservés. Le rAAV constitue un outil de choix pour le transfert de gènes dans diverses applications thérapeutiques. Cependant, dans bon nombre d’entre elles, il est nécessaire de pouvoir moduler l’expression du transgène quantitativement et au cours du temps. Plusieurs systèmes de régulation ont été décrits dont le système d’activation (Tet-On) de l’expression du transgène par la tétracycline et ses analogues (ex : la doxycycline). Le transfert et l’activation de l’expression du transgène par la doxycycline (Dox) nécessite deux vecteurs d’expression, un premier vecteur dans lequel le transactivateur (rtTA) est exprimé à partir d’un promoteur constitutif et un second qui porte le gène d’intérêt sous le contrôle du promoteur tétracycline (Ptet). Le Ptet est constitué du promoteur minimal du cytomégalovirus humain (PhCMVmini) placé en aval d’une répétition de séquences dites "opérateurs" (tetO). En présence de la Dox, le rtTA change de conformation, se lie au tetO et active la transcription du gène d’intérêt à partir du PhCMVmini. Pour le transfert de gène in vivo, il est cependant préférable de disposer d’un vecteur portant les deux cassettes d’expression au sein d’une seule construction (rAAV unique). Toutefois, les ITRs d’AAV d’une part et les séquences "enhancers" du promoteur utilisé pour exprimer le rtTA d’autre part, interfèrent avec le Ptet donnant lieu à une expression du gène d’intérêt à l’état non induit et par conséquent à un faible facteur d’induction. Nous décrivons dans ce travail un vecteur rAAV unique dont l’expression du transgène est activée par la tétracycline après transfert dans le cerveau de rat. En effet, nous avons développé un vecteur autorégulable dans lequel les deux cassettes d’expression sont placées en orientations opposées et la transcription du transgène et du rtTA est initiée à partir d’un promoteur tétracycline bidirectionnel (Ptet-bidi) et terminée par les signaux de polyadénylation bidirectionnels de SV40. Placées à côté de chaque ITR, ces dernières séquences pourraient également servir à arrêter la trancription à partir des ITRs d’AAV en absence de l’inducteur. Les performances de notre vecteur portant le gène rapporteur egfp (rAAV-ptetbidi-EGFP) ont été établies dans diverses lignées cellulaires immortalisées, dans des cultures primaires de cellules de Schwann ainsi que dans le cerveau du rat et des facteurs d’induction allant de 20 à 100 fois ont été observés. Nous avons également évalué la capacité de la minocycline (Mino), un antibiotique de la famille des tétracyclines utilisé pour ses propriétés anti-inflammatoires dans le cerveau, à induire l’expression du transgène à partir du Ptet dans une lignée de cellules U87-MG exprimant de façon stable le plasmide ptetbidi-EGFP. Quoique l’induction maximale de l’expression du transgène par la Mino nécessite des doses plus élevées et un temps plus long de traitement comparée à la Dox, elle apparaît moins toxique à des doses effectrices. Nous avons également évalué la réversibilité du système. Les résultats montrent une extinction plus rapide dans des cellules induites par la Mino comparée à celle obtenue dans des cellules induites par la Dox. Cependant, la cinétique d’induction du rAAV-ptetbidi-EGFP était lente et le niveau basal d’expression était encore élevé. De plus, à l’état induit, le nombre de cellules transduites par ce vecteur in vitro et in vivo reste inférieur à celui obtenu avec un vecteur équivalent portant le transgène sous le contrôle d’un promoteur constitutif. Nous avons réussi à améliorer l’inductibilité de notre vecteur portant le gène rapporteur egfp ou le gène thérapeutique hgdnf codant pour un facteur neurotrophique ayant un effet neuroprotecteur sur les neurones dopaminergiques mais également des effets non désirés : i) en plaçant, en aval du rtTA, le WPRE, une séquence de régulation post-transcriptionnelle d’origine virale permettant l’accumulation du transactivateur à concentration plus élevée dans les cellules transduites. Il en résulte un démarrage plus rapide et un niveau plus élevé de l’expression du transgène ainsi qu’une augmentation du nombre de cellules transduites dans le striatum de rat en réponse à la Dox; ii) en remplaçant le rtTA par le rtTA2SM2 moins toxique, plus stable et ayant une meilleure affinité de liaison au tetO. L’utilisation du rtTA2SM2 permet une réduction du niveau basal d’expression du transgène et son induction à plus faible dose d’inducteur. La version améliorée de notre vecteur a été ensuite encapsidée dans le sérotype 1 d’AAV, qui, injecté dans le striatum de rat, permet d’améliorer le volume de transduction et d’augmenter le nombre de cellules "GFP-positives" transduites comparé au sérotype 2 couramment utilisé. Un facteur d’induction de l’ordre de 10 fois a été également obtenu au moyen d’un rAAV1-ptet-bidi-hGDNF avec une quantité de GDNF exprimée à l’état induit dans la gamme des concentrations neuroprotectrices (100 pg/mg de tissu).

No description available.

Ten-Eleven-Translocation (TET) proteins form a family of enzymes responsible for active DNA demethylation by oxidation of 5-methylcytosine. TET proteins play a key role in genomic reprogramming in vitro and in vivo. Although TET proteins are expressed in embryonic stem cells (ESCs), their role in regulating pluripotency remains unclear. In addition, the mechanisms by which TET proteins are recruited to chromatin are largely unknown. To visualise TET protein dynamics during pluripotency and differentiation, the endogenous Tet1/2/3 alleles were fused to epitope tags in ESCs using CRISPR/Cas9. Characterisation of these cell lines showed that TET1 is the highest expressed TET protein in both naïve and primed pluripotent cells. In contrast, TET2 is expressed heterogeneously in ESCs and marks cells with a high self-renewal capacity. To assess the function of Tet genes in pluripotent stem cells, the endogenous Tet1/2/3 ORFs were removed using CRISPR/Cas9. Comparative analysis of single and combined Tet gene knockout ESC lines indicated that Tet1 and Tet2, but not Tet3, play redundant roles to promote loss of pluripotency. Furthermore, Tet-deficient cells retained a naïve morphology in differentiating conditions, suggestive of a LIF-independent self-renewal phenotype. To characterise physiological TET1 protein-protein interactions, TET1 protein partners were identified in ESCs by mass spectrometry and co-immuno-precipitations. This revealed that TET1 interacts with multiple epigenetic and pluripotency-related factors in ESCs. Moreover, detailed characterisation of the interaction between TET1 and NANOG identified three regions of TET1 involved in protein-protein interactions that are conserved in evolution. To investigate TET1 chromatin binding in ESCs, both at the molecular and cellular levels, TET1 was characterised by ChIP-seq analysis and live imaging experiments. Interestingly, TET1 is targeted to chromatin by two different mechanisms, involving distinct protein regions. The interaction with multiple protein partners, including NANOG, might enable TET1 to be targeted to specific chromosomal locations. Additionally, TET1 has the unusual ability to bind mitotic chromatin through its N-terminus, independently of its interaction with NANOG. Together these analyses provide a new understanding of the role of TET proteins in pluripotent cells, as well as a detailed map of TET1 residues involved in protein-protein interactions and mitotic chromatin binding.

Made available in DSpace on 2016-04-28T20:17:28Z (GMT). No. of bitstreams: 1 2008 - Cleia Maria Monteiro da Cunha.pdf: 485275 bytes, checksum: a4db2d06d84a6f336975955e4fb1bd45 (MD5) Previous issue date: 2008-10-09 / Streptococcus agalactiae, also referred as group B streptococci (GBS) are commensals microorganisms adapted to asymptomatic colonization of the mammalians gut and genitourinary tract. Initially, this specie was recognized as a major etiologic agent for bovine mastitis but has becoming a leading cause of invasive infections in human neonates. The reasons behind the prompt and persistent emergence of GBS neonatal disease have not been completely elucidated, once human and bovine GBS populations are assumed be distinct and unrelated by divergence on their own physiological characters. Aiming to contribute for the characterization of the two S. agalactiae sub-populations that are in a proximal coexistence in the Rio de Janeiro state, this study evaluated phenotypic and genotypic diversity aspects of regional groups of GBS. The first made up of 50 isolates obtained from human specimens whilst the other group was constituted of 36 isolates from milk of dairy cows presenting clinical or sub clinical mastitis. Phenotypic characterization was based on physiological and serological tests, antimicrobial susceptibility assays were carried out by the disk standard procedure and microdilution method. The genetic aspect was assessed by PCR for detection of genes associated with resistance to tetracycline. According to the results of physiologic tests, β-hemolysis was a faculty shared by about 28% of bovine isolates and 100% of human isolates. GBS bovine isolates also shows different profile of sensitivity to bacitracin, only 33% of them were susceptive to the antibiotic, regardless of the whole human isolates set had demonstrated a 100% susceptive pattern to this substance. A 100% sensitivity percentual to penicillin was shared by all isolates assayed in this study corroborating the general procedure for antibiotic therapy of GBS infection. Otherwise, and in an overall view, bovine isolates showed higher resistance rates to a set of antibiotics, including cephoxitin, erytromicin, clyndamicin, sulphamethoxazole, azithromycin and ciprofloxacin than in their human counterparts. In a similar sense, it was observed in this study that 13,9% of the animal GBS isolates expressed cMLSB and 2,8 % M phenotypes. The M phenotype was expressed in 6% of the human related isolates as the unique MLSB parameter. Genetical assays performed detected 13,8% (5/36) and 14% (7/50) for tet (M), and 30,5% (11/36) and 10% (5/50) for de tet (O), respectively, in bovine and human isolates. These genes are implicated in tetracycline resistance by ribosome protection mechanism through enzymatic structural modification. / Os Streptococcus agalactiae, tamb?m designados como estreptococos do grupo B (EGB), s?o microrganismos comensais adaptados para fazer a coloniza??o assintom?tica do tubo digestivo, e do trato geniturin?rio de mam?feros. Inicialmente, reconhecida como um dos mais importantes agentes etiol?gicos da mastite bovina, esta esp?cie foi tamb?m implicada como uma das principais causas de infec??es invasivas em rec?m-nascidos humanos. As raz?es para a r?pida e consistente evolu??o do EGB como importante agente causal de infec??es neonatais ainda n?o foram completamente elucidadas, uma vez que as subpopula??es de EGB nos isolados de humanos e bovinos s?o independentes e distintas com base no reconhecimento da diversidade de suas caracter?sticas fisiol?gicas. Com o objetivo de contribuir para a caracteriza??o das duas subpopula??es de S. agalactiae que coexistem no Estado do Rio de Janeiro, este estudo avaliou aspectos da diversidade gen?tica e fenot?pica de dois grupos de EGB regionais, sendo o primeiro composto por 50 isolados obtidos a partir de esp?cimes cl?nicos humanos, e o segundo constitu?do por 36 isolados a partir de leite de vacas leiteiras com ind?cios de mastite cl?nica ou subcl?nica. A caracteriza??o fenot?pica dos isolados foi baseada em testes sorol?gicos e fisiol?gicos, testes de suscetibilidade antimicrobiana realizados com t?cnica padronizada para utiliza??o de disco de difus?o e pelo m?todo de microdilui??o. O aspecto gen?tico foi avaliado pela aplica??o de PCR para detec??o de genes associados ? resist?ncia ? tetraciclina. Os testes fisiol?gicos demonstraram que a capacidade de promover β-hem?lise era uma caracter?stica partilhada por cerca de 28% dos isolados a partir do material de natureza bovina, mas que manifestava-se em todos os isolados de origem humana. Os isolados de EGB bovinos tamb?m mostraram um perfil diferente quanto ? sensibilidade ? bacitracina, uma vez que apenas 33% delas se revelaram suscet?veis a esse antibi?tico contra 100% de sensibilidade para os isolados de origem humana. O percentual de 100% de sensibilidade ? penicilina demonstrado por todos os isolados analisados neste estudo, tamb?m corrobora a import?ncia do uso desse antibi?tico como procedimento geral na terapia de infec??es por EGB. De uma forma geral, neste estudo foi observado que os isolados originados de material bovino demonstraram percentuais de resist?ncia ao conjunto de antibi?ticos analisados (cefoxitina, eritromicina, clindamicina, sulfametoxazol, azitromicina e ciprofloxacina) superiores aos observados em isolados de material humano. Foi tamb?m observado que 13,9% dos isolados de EGB animais examinados expressaram o fen?tipo cMLSB e 2,8% o fen?tipo M. O fen?tipo M foi o ?nico par?metro MLSB expresso entre os isolados de S. agalactiae humanos, com um percentual de 6%. Quanto ? presen?a de genes de resist?ncia a tetraciclina entre as subpopula??es de EGB, detectou-se percentuais de 13,8% (5/36) e 14% (7/50) para tet(M), e 30,5% (11/36) e 10% (5/50) para tet(O), respectivamente, nos isolados bovinos e humanos avaliados. Estes genes est?o implicados na resist?ncia ? tetraciclina por um mecanismo prote??o ribossomal, pela altera??o estrutural mediada por a??o enzim?tica. Palavras-chave: Streptococcus agalactiae, caracteriza??o fenot?pica, gene tet

Mammalian DNA methylathion is a chemical reaction catalyzed by DNA methyltransferases (DNMTs) and involves the addition of a methyl group from the methyl donor SAM to the carbon 5 position of cytosine (C) in a CpG dinucleotide. Specifically, DNA methylation is essential for normal development and is involved in numerous key mechanisms such as genomic imprinting, X-chromosome inactivation, suppression of repetitive elements and may be involved in the regulation of single-copy gene expression. In the human genome the majority of CpGs are methylated whereas regions with high density of CpG sites, termed CpG islands and often co-localized within gene promoters, are typically free of this mark. Recently, a new modified cytosine, 5-hydroxymhetylcytosine (5-hmC), was identified and found at significant levels in mouse brain and both mouse and human embryonic stem (ES) cells. The conversion of 5-mC to 5-hmC is catalyzed by the ten-eleven translocation (TET) proteins of the 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase superfamily. Many studies were conducted since the identification of 5-hmC and significant levels of 5-mC hydroxylation were found in many other mouse and human tissues. Importantly, many of the techniques used for 5-mC detection, such as bisulphite sequencing and methyl-sensitive restriction digestion, are incapable of distinguishing between 5mC and 5hmC implying the necessity not only to develop techniques specific for 5-hmC characterization but also reevaluation of previously published 5mC data. The biological function of 5-hmC is unknown however many recent studies have suggested a role for 5-hmC as an intermediate of either passive or active demethylation. The majority of studies of 5- hmC and TETs have used mouse ES cells as model system. Therefore, very little is known about 5-hmC patterns and TET expression within and between normal tissues. During my PhD, I used the recently developed 5-hmC-specific antibody for tiling microarrays and 5hmC-qPCR to examine both global 5hmC content and locus-specific patterns of 5hmC in several normal human tissues and breast cancer. I found that global 5-hmC content is highly variable between tissues compared to global 5-mC content. Moreover, TETs genes are highly expressed in most of tissues tested. Importantly, both global 5-hmC content and TETs genes are rapidly and significantly reduced as consequence of adaptation of cells from normal human tissue to cell culture. Using the 5hmC-specific antibody for tiling microarrays and 5-hmC-qPCR to profile locus-specific patterns of 5hmC, I found that 5-hmC patterns are tissue-specific in human samples. In addition, comparing array data to RNA-seq data, 5- hmC was found to co-localize at gene bodies of active genes. Moreover, despite the global 5-hmC reduction in cell lines, 5-hmC content remains enriched in some specific loci. In summary, my results show that tissue type is a major modifier of both global and locus-specific 5hmC at genes in normal human tissues. Furthermore, I also show that both TET gene expression and 5hmC content are significantly reduced and 5-hmC profiles reprogrammed during the passage from tissues to cell culture.

During an adenovirus infection the accumulation of alternatively spliced mRNAs is subjected to a tight temporal regulation. The IIIa protein is a structural protein expressed exclusively late after infection. To study the significance of the restricted IIIa protein expression we used a Tet-ON regulated adenoviral vector to overexpress the IIIa protein during the early phase of infection. The results show that unregulated IIIa protein expression caused a reduction in late viral protein accumulation and a slight block of viral DNA replication. Further, the results indicate that IIIa splicing might be subjected to a regulation via a feed back loop stimulating its own expression. To improve the efficacy of vectors for regulated transgene expression, we constructed binary adenoviral vectors based on the Tet-ON and Tet-OFF systems. These vectors encode both the transcriptional activator and the tetracycline-regulated expression cassette from the same viral unit, ensuring that each infected cell will express both the activator and the reporter gene. In model experiments this system was shown to result in a tight control of gene expression with no detectable background expression of the transgene and induction levels reaching 500-600 fold. Introduction of dsRNA into a cell will induce a sequence specific degradation of the homologous mRNA via a mechanism named RNA interference (RNAi). The adenovirus VA RNAs are short highly structured RNAs that are expressed in large amounts late during an adenovirus infection. Here we showed that both VA RNAI and VA RNAII functions as virus-encoded suppressors of RNAi, by interfering with the activity of Dicer, the enzyme that cleaves the initial dsRNA to short-interfering RNAs (siRNAs) that mediate RNAi. Further, the VA RNAs themselves are substrates for Dicer and are cleaved into siRNAs in vivo that are incorporated into active RNA-induced silencing complexes. There is a great interest in developing novel therapeutic strategies based on RNAi. We constructed adenoviral vectors that express short hairpin RNAs, which in vivo will be cleaved to siRNAs that induce sequence-specific RNAi. We compared the efficiency of RNAi induced by vectors based on the viral VA RNAI and the human U6 promoters. Our results suggest that under conditions where the recombinant virus does not replicate, the VA RNA promoter is more effective in down regulating target gene expression, whereas the U6 promoter was more effective under replicative conditions.

AbstractThe Iosipescu shear test, also known as asymmetric four point bending of a V-notched beam,is frequently used for measuring in-plane shear properties of composites. The ASTM standard(ASTM D-5379-05) regulates how the test is to be performed. It prescribes a notch openingangle of 90° independently of the material tested, although this has proven to produceinhomogenous strain distributions in the test region (between the notches) for orthotropicmaterials. Commonly, strain gauges are attached in the center of the test region where thedeviation from average strain is high. Thus, systematic errors in the measurement in the rangeof 10% or more may be introduced. The modified Iosipescu shear test, presented in this thesis, uses a variable notch opening angledepending on the material orthotropy and orientation to accomplish even stress- and strainfields in the test region. The variable notch opening angle accommodates both anisotropicmaterials and their orientation. Based on an elastic rescaling theory for orthotropic materials,the geometry was rescaled to recreate the same stress distribution in the test region as forisotropic materials. Specifically the notch opening angle was rescaled depending on theorthotropic ratio, the ratio of the two in-plane principal stiffnesses (Ex/Ey), to obtain theoptimal notch geometry. The rescaling procedure has been verified numerically with FEsimulationsand experimentally for several materials of different orthotropic ratio showingthat this was a very feasible method. Using a whole field optical measurement system duringtesting, significantly more homogenous strain fields were observed than for the standardspecimen geometry. Thus, there is no longer any need for correction factors, relying on FEsimulation,to obtain correct shear moduli. Constitutive shear properties and strength can thusbe more accurately measured, more completely and with fewer sources of error. Notablyhigher shear strengths at larger strains were also recorded compared to standard testing.The function of the new fixture was evaluated and compared with the standard Wyomingfixture. Combined in-situ 3D deformation measurements of both the new fixture and thespecimen showed that out of plane specimen deformation was very low and substantiallylower than the Wyoming fixture. Thus considerably lower parasitic stresses are introducedwith the new fixture. Recommendations regarding fastening of the specimen were determined based on simpleanalysis combined with FE-calculations and experiments. For both isotropic and orthotropic itwas found favorable if the clamp load used to hold the specimen and the expected net peakload and were set about equal. This reduces the risk of failure outside the test region bycrushing, brushing, splitting and etc. The same effects as shown in the FE-simulations werealso observed experimentally and of similar relative magnitude.Problems with differences in strains arising on the front and back face of the specimen duringtesting have been frequently reported in the literature. This is believed to stem from deviationsfrom nominal specimen geometry such as non-parallel and/or non-perpendicular boundingsurfaces. Three types of these combinations were evaluated numerically and the two mostsignificant were confirmed experimentally. The most critical geometrical deviation assessedwas a specimen with slightly conical cross section in the gripping region. For both isotropicand orthotropic materials, very small deviations from nominal geometry, caused unacceptablylarge errors in measurements of constitutive behavior / QC 20100827

Two common strategies used to identify specific genes that influence aging in Drosophila melanogaster are overexpression screens and candidate gene approaches. Both of these strategies rely on gene expression systems. A very popular gene expression system in Drosophila is the bipartite UAS/GAL4 system, where binding of GAL4 to a UAS sequence can direct the expression of a UAS-linked transgene in a pattern determined by GAL4. Although the UAS/GAL4 system allows for spatial regulation of transgene expression, it does not allow researchers to control when transgene expression will occur. This is an important consideration since aging research is primarily interested in identifying genes that influence aging during adulthood, therefore requiring that transgene expression be effectively blocked during pre-adult stages. Both the Gene-Switch and the Tet-Off/GAL80 systems are attempts to establish temporal control over GAL4 activity. The Gene-Switch system is based on a modified form of GAL4 whose transcriptional activity can be controlled through the antiprogestin molecule RU486. The Tet-Off/GAL80 system, where expression of GAL80 (a negative regulator of GAL4) is under the control of a tetracycline sensitive expression system, allows regulation of GAL4 activity through the antibiotic tetracycline. Characterization of these systems reveals that although neither system can completely repress leaky transgene expression, the Tet-Off/GAL80 system is much better at preventing unwanted transgene expression at most stages of the fly life cycle. Furthermore, comparison of muscle specific GAL4 and Gene-Switch strains revealed that upon treatment with their respective inducers, the Tet-Off/GAL80 system allows for GAL4 activity in the muscles, while the Gene-Switch system results in GAL4 activity in other tissues in addition to the muscles. In other characterized Gene-Switch strains, GAL4 activity is achieved only in a subset of the cells of the targeted tissue, suggesting that the Gene-Switch system may be ill-suited for aging studies. These findings, along with the fact that the Tet-Off/GAL80 but not the Gene-Switch system is compatible with the hundreds of characterized GAL4 lines presently available which allows transgene expression to be targeted to most tissues, indicate that the Tet-Off/GAL80 system is the best-suited for aging studies in Drosophila at present. / Thesis (Ph.D, Biology) -- Queen's University, 2008-12-22 17:13:42.089

Thesis (Ed. D.)--State University of New York at Binghamton, School of Education, 2007. / Includes bibliographical references.

Made available in DSpace on 2014-07-22T16:59:27Z (GMT). No. of bitstreams: 4 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 69450.pdf: 2409202 bytes, checksum: abdfee4e0aec1730b503aba8f1bb3000 (MD5) 69450.pdf.txt: 208978 bytes, checksum: 6549c0a44ac6d8e45b4998656b86fa86 (MD5) 69450.pdf.jpg: 1330 bytes, checksum: e4f720b175db53a9cb553b3e194b93d6 (MD5) Previous issue date: 2014-05-07 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, Brasil / Em 2011 foram notificados quase 9 milhões de novos casos de tuberculose (TB), culminando em aproximadamente 2 milhões de mortes. A vacina atual contra tuberculose (BCG) apresenta eficácia variável na proteção de adultos contra a tuberculose pulmonar (0% - 80%), com isso destacamos a necessidade de maiores investigações sobre a vacina. A cepa da vacina utilizada no Brasil é a BCG Moreau. Este trabalho incide sobre a caracterização de Rv3405c, um regulador transcricional (repressor) presente em todas as cepas de BCG, porém truncado em BCG Moreau devido à perda da região genômica RD16. Analisamos por RT-PCR a expressão de genes adjacentes a rv3405c em BCG Pasteur e Moreau, comprovando que sua perda funcional afeta a expressão de rv3406, que codifica uma proteína envolvida no metabolismo do enxofre. O aumento da expressão de rv3406 em BCG Moreau pode representar um ganho funcional, devido à perda desse repressor transcricional (Rv3405) A fim de caracterizar melhor a sua função, rv3405c de BCG Pasteur e rv3406 de BCG Moreau foram clonados e expressos em E. coli. O soro policlonal produzido em camundongos imunizados contra a proteína purificada rRv3406 foi utilizado para analisar sua expressão sob diferentes condições de crescimento. Rv3406 é encontrada na fração intracelular e na superfície de BCG Moreau, não tendo sido detectado em BCG Pasteur. Rv3405c recombinante purificada foi usada no ensaio de retardo em gel (EMSA), confirmando a sua capacidade para se ligar a um motivo de DNA conservado, identificado na região intergênica entre rv3405c-rv3406, sugerindo seu papel na regulação destes genes e justificando a expressão constitutiva de rv3406 em BCG Moreau. Além disso, a adição de bile e seus componentes provoca o deslocamento da proteína desfazendo sua interação com DNA. A caracterização de rv3405c e suas funções é importante para uma análise de genômica funcional comparativa entre BCG Moreau e Pasteur. Este trabalho contribui para uma melhor compreensão da vacina brasileira contra a tuberculose / Tuberculosis (TB) was responsible for almost 9 million new cases in 2011, culminating in approximately 2 million deaths. The current approved TB vaccine (BCG) presents variable efficacy in adult protection against pulmonary TB (0%-80%), highlighting the need for further investigations on this vaccine. The vaccine strain used in Brazil is BCG Moreau. This work focuses on the characterization of Rv3405c, a transcriptional regulator (repressor) present in all BCG strains but truncated in BCG Moreau due to the loss of genomic region RD16. We have analysed the expression of adjacent genes in both BCG Moreau and Pasteur by RT-PCR finding that loss of Rv3405c impacts the expression of rv3406, encoding a protein involved in sulfur metabolism. Its increased expression in BCG Moreau may represent a functional gain through the loss of a transcription repressor. In order to better characterize their function, rv3405c from BCG Pasteur and rv3406 from BCG Moreau were cloned and expressed in E. coli. The polyclonal sera produced in mice immunized with purified rRv3406 were used to follow the expression of this protein under different growth conditions. Rv3406 is found in the intracellular fraction and the surface of BCG Moreau, and was not detected in BCG Pasteur. Purified recombinant Rv3405c was used in electrophoretic mobility shift assays (EMSA), confirming its ability to bind a conserved DNA motif identified in the intergenic region between rv3405c-rv3406, suggesting its role in regulating these genes and justifying the constitutive expression of rv3406 in BCG Moreau. Furthermore, the addition of bile and its components is able to displace Rv3405c from DNA. The characterization of rv3405c and its functions is important for the comparative functional genomics analysis of BCG Moreau and Pasteur. This work contributes to a better understanding of the Brazilian vaccine against TB.