Les canaux calciques incluent notamment les canaux dépendants des stocks (ORAI) et dépendants du voltage (Cav) qui sont considérés de première importance pour l'entrée du calcium dans les cellules non-excitables et excitables, respectivement. Les canaux calciques voltage-dépendants tels que Cav1 sont essentiels pour le fonctionnement des cellules excitables, notamment la transmission neuronale, la contraction musculaire ou la sécrétion hormonale. Cependant, de nombreuses études montrent désormais que les canaux Cav1 sont aussi exprimés dans des cellules non excitables, et sont importants pour les fonctions effectrices des lymphocytes T. Les canaux Cav1 sont constitués de la sous-unité a1 formant le pore ionique et des sous-unités auxiliaires ß et a2δ. Ces sous-unités sont importantes pour l'activité électrique du canal, mais aussi pour sa régulation, sa stabilité et son expression à la membrane plasmique dans les cellules excitables. Les travaux de notre groupe ont clairement identifié les sous-unités a1 de Cav1.2 et Cav1.3 comme essentielles pour la fonction des lymphocytes Th2, une sous-population de cellules T responsable des maladies allergiques. L'inhibition pharmacologique et génétique de ces canaux réduit de manière significative l'expression des cytokines dans les lymphocytes Th2 chez la souris, mais pas dans les Th1. Le but de mon travail a été de comprendre si les sous-unités auxiliaires des canaux Cav, et plus particulièrement la sous-unité ß, sont nécessaires au fonctionnement des canaux Cav1 dans les lymphocytes Th2 qui ne sont pas des cellules excitables. Nous avons utilisé des oligonucléotides antisens ciblant toutes les sous-unités ß afin de réduire l'expression de ß1 et ß3, les deux sous-unités exprimées dans des lymphocytes Th2. La transfection de Th2 murines et humaines avec ces oligonucléotides diminue l'influx de calcium dépendant du TCR et l'expression des cytokines. En outre, l'effet des oligonucléotides antisens semble résulter de la perte d'expression de la sous-unité a1 selon un mécanisme similaire à celui décrit dans les neurones. De plus, L'utilisation de shRNA spécifiques de ß1 et ß3 dans les Th2 de souris montre un rôle essentiel la sous-unité ß1 dans la réponse fonctionnelle des lymphocytes Th2. Enfin, les antisens Cavß diminuent l'inflammation dans un modèle d'asthme allergique chez la souris, de même qu'un inhibiteur pharmacologique des sous-unités a2δ. Ce travail a donc permis d'identifier les sous-unités auxiliaires des canaux Cav comme de nouvelles cibles thérapeutiques potentielles dans le cadre des maladies allergiques telles que l'asthme. / Calcium channels include store-operated (ORAI) and voltage-gated (Cav) channels that are considered to be important for calcium entry in non-excitable and excitable cells, respectively. Voltage-gated calcium channels such as Cav1 are essential for excitable cell function, including neuronal transmission, muscle contraction or hormone secretion. However, numerous studies show that Cav1 channels are expressed in non-excitable cells as well, and are important for T cell effector functions. Cav1 channels are composed of the a1 subunit forming the ion pore and auxiliary subunits ß and a2δ. These subunits are important for the electric activity of the channel but also for its regulation, its stability and its expression at the plasma membrane in excitable cells. Our group clearly identified the a1 subunit of Cav1.2 and Cav1.3 channels as essential for the function of Th2 lymphocytes, a T cell subset responsible for allergic diseases. Pharmacological and genetic inhibition of these channels significantly reduces the expression of cytokines in mouse Th2 cells, but not in Th1 cells. The goal of my work was to understand whether the auxiliary subunits of Cav channels, particularly the ß subunit, are necessary for the function of Cav1 channels in Th2 lymphocytes that are not excitable cells. We used antisense oligonucleotides targeting all ß subunits to reduce the expression of ß1 and ß3, the two subunits expressed in Th2 lymphocytes. Transfection of murine and human Th2 with these oligonucleotides decreases TCR-dependent calcium influx and cytokine expression. In addition, the effect of the Cavß antisense oligonucleotides seems to result from the loss of expression of the a1 subunit, as similarly described in neurons. In addition, the use of shRNA specific to ß1 and ß3 in mouse Th2 shows a critical role the ß1 subunit in the functional response of Th2 lymphocytes. Finally, the Cavß antisense oligonucleotides reduce the airway inflammation in an allergic asthma model in mice, as well as a pharmacological inhibitor of a2δ subunits. This work has identified auxiliary subunits of Cav channels as new potential therapeutic targets in allergic diseases such as asthma.

<p>Ribosomes are the universal ribonucleoprotein organelles that translate the genetic message from mRNA to protein. In prokaryotes, the ribosomal subunits are 30S and 50S subunit, which bind together during the translation process forming 70S ribosome. The ribosome is a highly dynamic structure, and acts as a working platform for the different factors involved in the process of converting the genetic information into protein.</p><p>Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3D) reconstruction at molecular resolution with a close-to-native preservation of the specimen. Here, we have applied this method to reconstruct rifampicin-treated <i>Escherichia coli</i> individual 30S subunits in vitro and in situ, and individual 50S subunits in situ. In the 30S subunit, the head, the platform and the body show large conformational movements relative to each other. The particles are grouped into three conformational groups according to the width/height ratios. Also, an S15 fusion protein derivative has been used as a physical reporter to localize S15 in the 30S subunit. In the 50S subunit, the L1 stalk, the L7/L12 stalk, the central protuberance (CP), and the peptidyl transferase center (PTC) cleft are the most dynamic and flexible parts in the reconstructed structures with clear movements indicated. Different locations of the tunnel in the central cross-sections through the in situ 50S subunits indicate a flexible pathway inside the large subunit. In addition, gross morphological changes were also been observed in our reconstructions. Our results demonstrate a considerable conformational flexibility among individual ribosomal subunits, both in vitro and in situ.</p><p>Translation is an essential process for all cells and organisms. Translation initiation is the rate-limiting step and the most highly regulated phase of translation process. Several regions along the mRNA have been reported to influence translation initiation. The Shine-Dalgarno (SD) sequence located 5-9 bases upstream of the initiation codon supports translation initiation by complementary binding to the Anti-Shine-Dalgarno (ASD) sequence on the 16S rRNA.</p><p>We have here compared how an SD⁺ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD⁺ is confirmed. A downstream SD⁺ gives decreased gene expression. If an SD⁺ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD⁺ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD⁺ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD⁺ located between them. A minor positive contribution to upstream initiation resulting from 3’ to 5’ ribosomal diffusion along the mRNA is suggested. Since the location of SD⁺or SD-like sequences can strongly influence gene expression, this should be of significant evolutionary importance.</p>

Ribosomes are the universal ribonucleoprotein organelles that translate the genetic message from mRNA to protein. In prokaryotes, the ribosomal subunits are 30S and 50S subunit, which bind together during the translation process forming 70S ribosome. The ribosome is a highly dynamic structure, and acts as a working platform for the different factors involved in the process of converting the genetic information into protein. Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3D) reconstruction at molecular resolution with a close-to-native preservation of the specimen. Here, we have applied this method to reconstruct rifampicin-treated Escherichia coli individual 30S subunits in vitro and in situ, and individual 50S subunits in situ. In the 30S subunit, the head, the platform and the body show large conformational movements relative to each other. The particles are grouped into three conformational groups according to the width/height ratios. Also, an S15 fusion protein derivative has been used as a physical reporter to localize S15 in the 30S subunit. In the 50S subunit, the L1 stalk, the L7/L12 stalk, the central protuberance (CP), and the peptidyl transferase center (PTC) cleft are the most dynamic and flexible parts in the reconstructed structures with clear movements indicated. Different locations of the tunnel in the central cross-sections through the in situ 50S subunits indicate a flexible pathway inside the large subunit. In addition, gross morphological changes were also been observed in our reconstructions. Our results demonstrate a considerable conformational flexibility among individual ribosomal subunits, both in vitro and in situ. Translation is an essential process for all cells and organisms. Translation initiation is the rate-limiting step and the most highly regulated phase of translation process. Several regions along the mRNA have been reported to influence translation initiation. The Shine-Dalgarno (SD) sequence located 5-9 bases upstream of the initiation codon supports translation initiation by complementary binding to the Anti-Shine-Dalgarno (ASD) sequence on the 16S rRNA. We have here compared how an SD+ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD+ is confirmed. A downstream SD+ gives decreased gene expression. If an SD+ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD+ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD+ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD+ located between them. A minor positive contribution to upstream initiation resulting from 3’ to 5’ ribosomal diffusion along the mRNA is suggested. Since the location of SD+ or SD-like sequences can strongly influence gene expression, this should be of significant evolutionary importance.

The ribosome is one of the most universal molecular machinery, synthesizing proteins in all living systems. The small ribosomal subunit plays a crucial role in decoding the messenger RNA during translation. We propose and validate a new architectural model of the ribosomal small subunit, with broad implications for function, biogenesis and evolution. We define an rRNA domain: compact and modular, stabilized by self-consistent molecular interactions, with ability to fold autonomously when it is isolated from surrounding RNA or protein. Each rRNA helix must be allocated uniquely to a single domain. These criteria identify a core domain of small subunit rRNA (domain A), which acts as a hub, linking to all other domains by A-form helical spokes. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2’OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with autonomous folding, and therefore classification as a domain. We show that the domain concept is applicable and useful for understanding the small ribosomal subunit. Our results support the utility of the concept of the domain as applied to at least some RNAs, the interdependence of the elements of domain A, and its ability to fold autonomously. Moreover, domain A, which exhibits elements of tRNA mimicry, is the essential core of the small ribosomal subunit. Understanding the structure and dynamics of domain A will provide valuable insight into the translational machinery.

The immunoproteasome, known to play an important role in MHC class I antigen processing and presentation, have been linked to neurodegenerative diseases and hematological cancers. However, the pathophysiological functions of the immunoproteasome in these diseases are still not very well established. This can be attributed mainly to the lack of appropriate molecular probes that selectively target the immunoproteasome catalytic subunits. Herein, we report the development of a small molecular inhibitor (AM) that selectively targets the major catalytic subunit, LMP2, of the immunoproteasome. We show that the compound covalently modifies the LMP2 subunit with high specificity in human prostate cancer cell. AM was also shown to selectively inhibit the chymotrypsin-like activity of LMP2 subunit. More importantly, the anti-proliferative activity of AM is more pronounced in prostate cancer cells that highly express LMP2 without inducing toxicity in normal cells. These results implicate an important role of LMP2 in regulating cell growth of malignant tumors that highly express LMP2. Subsequently, the modes of action of AM were investigated. Prostate cancer cells that highly express LMP were shown to induce G2/M cell cycle arrest and apoptosis via PARP cleavage when treated with the compound. Similar to epoxomicin, the treatment of AM induced the accumulation of poly-ubiquitination in prostate cancer cells, which indicates the inhibition of proteolysis. However, unlike epoxomicin, the treatment of AM did not appear to inhibit the activation of inflammation. In conclusion, these results suggest that the LMP2 inhibitor, AM, may induce cytotoxicity prostate cancer cells that highly express LMP2 catalytic subunit in similar modes of action as epoxomicin but it does not involve the inflammatory pathway.

Les récepteurs du glutamate de type AMPA (rAMPA) sont les récepteurs ionotroniques responsables de la majeure partie des courants excitateurs rapides lors de la transmission synaptique dans le système nerveux central. Le nombre de rAMPA stabilisés à la synapse est responsable en partie de l’intensité de la transmission synaptique et de nombreux phénomènes de plasticité synaptique. Les rAMPA se répartissent en trois populations en équilibre dynamique: les récepteurs intracellulaires, les récepteurs extra-synaptiques, et les récepteurs synaptiques stabilisés au niveau de la densité post-synaptique. L’implication des protéines transmembranaires régulatrices des rAMPA (TARP) dans la stabilisation des rAMPA est établie, et repose au moins en partie sur la liaison de la protéine TARP γ-2 avec la protéine d’échafaudage PSD-95. Dans l’hippocampe, siège de nombreux phénomènes de plasticité, l’isoforme γ-8 est particulièrement enrichie. La TARP γ-8 a pour particularité de posséder un domaine C-terminal plus long que son homologue γ-2 et de s’exprimer au niveau synaptique et extra-synaptique. Mon travail de thèse à consisté à étudier les mécanismes moléculaires mis en jeu dans la régulation de la liaison des protéines TARP γ-2 et γ-8 avec la protéine PSD-95, ainsi que l’implication respective des deux isoformes dans la régulation de la mobilité latérale des rAMPA. Les résultats majeurs de cette étude sont : a) l’interaction entre γ-2 et PSD-95 est régulée par la longueur apparent du domaine C-terminal de γ-2 modulée par la phosphorylation; b) γ-8 lie PSD-95 dans les compartiments synaptiques et extra-synaptiques, toutefois cette interaction n’est pas corrélée avec une immobilisation des rAMPA. Ces résultats suggèrent que γ-2 et γ-8 jouent des rôles bien distincts dans l’adressage des rAMPA à la synapse. / AMPA type glutamate receptors (AMPARs) are ionotropic receptors responsible for most excitatory transmission in the central nervous system. The number of stabilized AMPARs in front of glutamate release sites determines in large part the strength of synaptic transmission and variation in this number is thought to underlie numerous forms of synaptic plasticity. AMPARs are present in three main subcellular pools between which they are in a dynamic equilibrium by processes of trafficking: intracellular receptors, extrasynaptic receptors, and synaptic receptors stabilized at the postsynaptic density (PSD). Transmembrane AMPAR regulatory proteins (TARPs) are known to be implicated in AMPAR stabilization at the synapse through the interaction of TARP γ-2/8 with the scaffolding protein PSD-95. In the hippocampus, a structure exhibiting various synaptic plasticity patterns, γ-8 is the most abundant TARP. This isoform is characterized by a longer C-terminal fragment than γ-2 and a synaptic and extrasynaptic localization. During my Ph.D, I studied the molecular mechanisms involved in the regulation of TARP γ-2 and γ-8 binding to PSD-95 and their respective roles in regulating AMPAR lateral mobility. The main results are: a) γ-2 interaction with PSD-95 is regulated by the apparent length of its C-terminus domain that is modulated by phosphorylation; b) γ-8 binds PSD-95 in synaptic and extrasynaptic compartment however this interaction is not correlated with AMPAR immobilization. Altogether, those results suggest that those two TARP isoforms have independent functional roles.

Duikers are a species rich subfamily of threatened African antelope whose recent origin poses a challenge to the molecular identification of taxa and estimation of their phylogeny. I test the ability of DNA barcodes to identify all taxa within this group. I then use mitochondrial and nuclear genes to estimate a multi-locus species tree and to date divergence times. DNA barcodes are unable to distinguish many sister taxa, calling into question the utility of barcodes for the regulation of duiker trade or in identification of field-collected feces. The multi-locus phylogeny provides support for the relationships among major duiker lineages and placement of two problematic taxa, but challenges the validity of the savanna genus and identifies hybridization between taxa. This study reveals that most duikers diverged during the Pleistocene, meriting further inquiry into the role that Pleistocene glacial cycling played in the diversification and population structuring of duikers.

Dans les cellules eucaryotes, il existe 3 ARN Polymérases nucléaires (Pol I, II et III), chacune ayant une fonction de synthèse d'ARN qui lui est propre. L'ARN polymérase I (Pol I) est responsable de la synthèse du précurseur des grands ARN ribosomiques (ARNr), ce qui correspond à une activité de transcription massive dans la cellule. Des données structurales sur cette enzyme de 14 sous-unités sont disponibles. Ceci permet une meilleure compréhension de son mode de fonctionnement, et a confirmé que l'ARN Pol I possède 3 sous-unités spécifiques, aussi appelées "Built-in Transcription Factors", responsables d'activités régulatrices. Deux d'entres elles, Rpa49 / Rpa34, forment un hétérodimère structuralement proche des facteurs de transcription TFIIF et TFIIE de la Pol II, impliqués tant dans l'initiation que dans l'élongation de la transcription. La dernière, Rpa12, est connue pour avoir un rôle dans la stabilité de l'ARN Pol I et l'activité de clivage du transcrit de la polymérase en cours de pause (via son extrémité C-terminale), comme son homologue TFIIS, facteur de transcription de l'ARN Pol II. Nous avons effectué des études génétiques sur des mutants de l'ARN Pol I dépourvus de la sous-unité Rpa49 (rpa49Δ). Cette délétion est viable mais entraîne des problèmes d'initiation et élongation. Nous étudions dans ce travail le clonage et la caractérisation de "suppresseurs" extragéniques correspondant à des mutations ponctuelles de trois sous-unité de la Pol I qui rétablissent une synthèse d'ARNr en l'absence de la sous-unité Rpa49. Toutes ces mutations suppressives identifiées ont été localisées premièrement dans les deux grandes sous-unités Rpa190 et Rpa135, structuralement très proches de Rpa12, mais également au sein même de Rpa12, indiquant une possible interaction entre les sous-unités Rpa49 et Rpa12 ou la région autour. Notamment, un élément spécifique de l'ARN Pol I dans Rpa190, le "DNA Mimicking Loop", est structuralement très proche de la région où l'on trouve ces mutations suppresseur. Les caractérisations génétiques, structurales, mais aussi biochimiques et fonctionnelles de ces suppresseurs nous permettent de proposer des hypothèses sur les rôles de Rpa49 et de Rpa12, mais aussi de cette petite région de Rpa190, en l'initiation de la transcription, ce qui n'a jamais été mis en évidence auparavant. / In eukaryotes cells, there are 3 nuclear RNA Polymerases (Pol I, II and III), each having a particular RNA synthesis function. The RNA Polymerase I (Pol I) produces a single transcript: the precursor of the large ribosomal RNA (rRNA), which correspond to a massive transcription activity in the cell. Structural data of this 14-subunits enzyme is now available. This allows a better understanding of its operating mode, and confirmed that the Pol I has 3 specific subunits, also called "Built-in transcription factors", capable of regulatory activities. Two of them, Rpa49/Rpa34, are forming a heterodimer structurally related to the Pol II transcription factors TFIIF and TFIIE, implicated in both initiation and elongation of the transcription. The last one, Rpa12, is known to have a role in Pol I stability and the cleavage activity of the paused Pol I (via its C-terminus part), like its homologous TFIIS in the Pol II. We performed extensive genetic studies of Pol I mutants lacking one of these subunits: Rpa49 (rpa49Δ). This depletion is viable, but results in initiation and elongation problems. Here, we report the cloning and characterization of extragenic suppressors mapping point mutations in three subunits of Pol I restoring efficient Pol I activities in absence of Rpa49. All suppressor mutations identified were structurally mapped firstly in the two largest subunits Rpa190 and Rpa135 very closed to Rpa12, and then in Rpa12 itself, indicating a possible interplay between the Rpa49 and Rpa12 subunits, and the area around. Notably, a RNA pol I specific element in Rpa190, called "DNA Mimicking Loop", is structurally very closed to the region in which we find all of our suppressor mutations. The genetic, structural but also biochemical and functional characterizations of these suppressors allow us to propose roles of these Rpa49 and Rpa12 subunits, but also of the small area of Rpa190, which has never been highlighted before.

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-08-29T11:52:09Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_thais_larre_oliveira.pdf: 616408 bytes, checksum: 404ac55ee19b90246334aac10658a361 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-29T19:49:36Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_thais_larre_oliveira.pdf: 616408 bytes, checksum: 404ac55ee19b90246334aac10658a361 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-29T19:49:43Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_thais_larre_oliveira.pdf: 616408 bytes, checksum: 404ac55ee19b90246334aac10658a361 (MD5) / Made available in DSpace on 2017-08-29T19:49:54Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_thais_larre_oliveira.pdf: 616408 bytes, checksum: 404ac55ee19b90246334aac10658a361 (MD5) Previous issue date: 2014-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A leptospirose é uma doença infecciosa emergente causada por espiroquetas patogênicas do gênero Leptospira, com complicações humana e veterinária. Essa doença é transmitida através do contato direto com um animal reservatório ou com o ambiente contaminado com a urina destes animais. O desenvolvimento de vacinas seguras e multivalentes contra leptospirose, que substituam as bacterinas existentes, permanece um desafio. Bacterinas são reatogênicas e conferem imunidade sorovar-específica e de curta duração. Esforços para o desenvolvimento de vacinas recombinantes tem focado em proteínas de membrana externa (OMPs). A proteína de membrana externa OmpL37 é conservada entre diferentes sorovares de Leptospira e atua na adesão aos tecidos do hospedeiro. Neste estudo, nós relatamos pela primeira vez, a avaliação do potencial imunoprotetor de OmpL37 como antígeno vacinal em hamsters, sob diferentes estratégias: vacina de subunidade, vacina de DNA e prime-boost. A caracterização da resposta imune através de ELISA indireto e qRT-PCR demonstrou que os maiores níveis de IgG foram estimulados pela vacina de subunidade, a qual também induziu resposta inflamatória. A vacina de DNA falhou em induzir imunidade humoral, porém também estimulou TNF-α. Apesar da resposta induzida, nenhuma formulação protegeu significativamente os animais contra a doença. / Leptospirosis is an emerging infectious disease caused by pathogenic spirochetes of Leptospira genus, of human and veterinary concern. This infectious disease is transmitted through direct contact with an animal reservoir or an environmental contaminated with their urine. The development of safe and multivalent vaccines against leptospirosis, which replace existing bacterins, remains a challenge. Bacterins are reactogenic and afford serovar specific and short-term immunity. Efforts to develop recombinant vaccines against leptospirosis have focused on outer membrane proteins (OMPs). The outer membrane protein OmpL37 is conserved among different Leptospira serovars and plays a role in adherence to host tissues. In this study we report for the first time, the evaluation of OmpL37 immunoprotective potential as a vaccine antigen in hamsters, under different strategies: subunit vaccine, DNA vaccine and prime-boost. The characterization of the immune response by indirect ELISA and qRT-PCR showed that higher levels of IgG were stimulated by the vaccine subunit, which also induced an inflammatory response. The DNA vaccine failed to induce humoral immunity, but also stimulated TNF-α. Despite the induced response, no formulation significantly protected the animals against the disease.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-09-23T13:40:05Z No. of bitstreams: 2 Sousa, Eduardo Martins.pdf: 2216793 bytes, checksum: b28a546dbb9489fa820abe22bc2b6109 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-09-23T15:17:05Z (GMT) No. of bitstreams: 2 Sousa, Eduardo Martins.pdf: 2216793 bytes, checksum: b28a546dbb9489fa820abe22bc2b6109 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-09-23T15:17:05Z (GMT). No. of bitstreams: 2 Sousa, Eduardo Martins.pdf: 2216793 bytes, checksum: b28a546dbb9489fa820abe22bc2b6109 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-03-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Tuberculosis is a re-emerging infectious disease that remains a major public health problem worldwide. Although there is the BCG vaccine that is effective against severe forms of childhood TB, in adults its efficacy is variable (0-85%). In this context there is a need to develop new vaccines to control the spread of TB. This thesis proposes the development of a new recombinant fusion M. tuberculosis protein (Ag85C-MPT51-HspX) by molecular cloning, expression in E. coli with a histidine tag and purified by ion exchange chromatography. The fusion protein was constructed successfully, expressed in E. coli BL21 and purified. Tests in mice were performed to evaluate the immunogenicity of the recombinant fusion protein Ag85C-MPT51-HspX of M. tuberculosis. Mice were immunized three times with the protein Ag85C-MPT51-HspX formulated with CpG-DNA encapsulated in liposomes, CpG-DNA encapsulated in liposomes, liposome or saline as negative control and the humoral and cellular immune response was evaluated. The immunization with the vaccine formulation induced the production of high titers of specific anti-fusion protein Ag85C-MPT51-HspX IgG1 = 3.08 ± 0.04; IgG2a = 3.10 ± 0.03) and, favored the increase of specific CD4+ IFN-γ (2.14% ± 0.17), CD4+ TNF-α (2.16 ± 0.34%). The recognizing of this protein by seric IgG and IgM discriminated patients with active TB infection from healthy individuals. We conclude that CMX protein has potential to be used for the development of vaccine against M. tuberculosis as well also for TB diagnostic kits. / A tuberculose é uma doença infecciosa re-emergente que permanece como um dos maiores problemas de saúde pública mundial. Embora exista a vacina BCG que é eficiente contra formas graves de TB na infância, em adultos a eficácia é variável (0 a 85%). Nesse contexto existe a necessidade do desenvolvimento de novas vacinas para controlar a disseminação da TB. O presente trabalho teve como objetivo desenvolver uma nova proteína de fusão recombinante (Ag85C-MPT51-HspX) de M. tuberculosis a partir da clonagem molecular, expressão em E. coli com uma cauda de histidina e purificação através de cromatografia de troca iônica. A proteína de fusão foi construída com sucesso expressa em E. coli BL21 e purificada. Ensaios em camundongos foram realizados para avaliar a imunogenicidade da proteína recombinante de fusão Ag85C-MPT51-HspX de M. tuberculosis. Os camundongos foram imunizados três vezes com a proteína Ag85C-MPT51-HspX formulada com CpG-DNA encapsulada em lipossoma, CpG-DNA encapsulado em lipossoma, lipossoma ou salina como controle negativo e a resposta imune humoral e celular foi avaliada . A imunização com a formulação vacinal induziu a produção de altos títulos de anticorpos específicos anti-proteína de fusão Ag85C-MPT51-HspX (IgG1 =3,08±0,04; IgG2a=3,10±0,03), bem como, favoreceu o aumento de células T CD4+IFN-γ (2,14%±0,17), CD4+TNF-α (2,16%±0,34) específicas. A avaliação do reconhecimento desta proteína de fusão tanto por IgM quanto IgG humana sérica permitiu discriminar pacientes com tuberculose ativa de controles saudáveis, demonstrando a antigenicidade desta molécula em humanos. Conclui-se que a proteína CMX poderá ser testada tanto como vacina, assim como para o desenvolvimento de testes de diagnóstico para a tuberculose.