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No. of bitstreams: 1 analuizadasilvaalbertoni.pdf: 1260028 bytes, checksum: cc0af4aab8dc00d61833430e0eb2ce88 (MD5) Previous issue date: 2017-08-14 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A obesidade é uma doença crônica multifatorial caracterizada pelo excesso de gordura corporal e por um estado inflamatório de baixo grau, conhecido como metainflamação. Atualmente, é um dos principais problemas de saúde pública do mundo. Dados da OMS indicam que 13% da população mundial é obesa e no Brasil, o índice subiu para 17,9% da população em 2014. Fatores genéticos, dieta, desordens metabólicas, como intolerância à glicose, dislipidemias, hipertensão arterial sistêmica, desbalanço hormonal e mudanças na microbiota intestinal, são gatilhos da inflamação envolvendo adipócitos. O tecido adiposo obeso é caracterizado pelo aumento da infiltração de macrófagos, sendo estes uma fonte importante de inflamação neste tecido. Fatores transcricionais com propriedades imunorregulatórias estão envolvidos em processos inflamatórios e na adipogênese. PPARγ é um importante receptor ativado por ligantes lipídicos, regulador central da adipogênese, com funções na ativação de células do sistema imune e no metabolismo lipídico. Dados de nosso grupo demonstraram que ativação de PPARγ induz a biogênese de organelas dinâmicas denominadas Corpúsculos Lipídicos (CLs) durante infeção micobacteriana. Estas organelas possuem funções ativas no estoque de lipídios para geração de energia, síntese de membrana, síntese de mediadores inflamatórios, sinalização celular e inflamação. Além disso, os CL em macrófagos funcionam como sítios para sobrevivência de patógenos, como as micobactérias. Apesar do impacto da obesidade em doenças metabólicas e cardiovasculares ser bem compreendido, os mecanismos envolvidos na relação entre adipócitos e macrófagos infectados com patógenos intracelulares não são conhecidos, sendo este esclarecimento o objetivo de nosso estudo. Para isso, diferencianciamos células NIH3T3-L1 em adipócitos e utilizamos o sobrenadante obtido, para estimular macrófagos peritoneais infectados ou não com M. bovis BCG. Nós avaliamos a biogênese de corpúsculos lipídicos, expressão de PPARγ, síntese e secreção de citocinas, adipocinas e NO. Os resultados comprovaram que a diferenciação de células NIH3T3-L1 em adipócitos é um processo eficiente que envolve mudanças na morfologia celular e acúmulo de Corpúsculos Lipídicos. O estímulo de macrófagos com sobrenadante de adipócitos potencializou a biogênese de CLs, assim como a expressão de PPARγ, na presença de infecção micobacteriana. Nos tempos de 6 e 48 horas de estímulo com sobrenadante e infecção, a produção de TNF-α também foi potencializada, porém um decréscimo significativo foi observado no tempo de 24 horas. Quanto aos níveis de IL-10, um aumento foi observado na presença de infecção e estímulo com sobrenadante nos três tempos analisados. Além disso, apenas no tempo de 24 h observamos a secreção de nitrito de modo significativo pelos macrófagos peritoneais infectados, efeito este independente do estímulo com sobrenadante. Quanto às adipocinas, importantes no desenvolvimento da obesidade e síndrome metabólica, analisamos os níveis de leptina produzida pelos macrófagos que não foram significativos, enquanto a produção de adiponectina, apresentou-se aumentada nos macrófagos controles estimulados com sobrenadante, efeito este, que foi inibido durante a infecção por BCG. Assim, nossos resultados sugerem, um efeito modulador de fatores secretados por adipócitos na ativação de macrófagos, atribuídos a formação de corpúsculos lipídicos, expressão de PPARγ, síntese de citocinas como TNF-α e IL-10 e uma modulação negativa da produção de adiponectina durante a infecção por M. bovis BCG. / Obesity is a chronic multifactorial disease characterized by excess of body fat and a low- grade inflammatory state, known as meta-inflammation. It is currently one of the world's leading public health problems. WHO data indicate that 13% of the world population is obese, in Brazil, the obesity index was 17.9% of the population in 2014. In addition to genetic factors, diet and metabolic disorders, such as glucose intolerance, dyslipidemia, systemic arterial hypertension, hormonal imbalance and changes in the intestinal microbiota have been proposed as triggers for inflammation involving adipocytes. Recent studies have shown that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that these are an important source of inflammation in this tissue. Transcriptional factors with immunoregulatory properties are involved in inflammatory processes and adipogenesis. Such as the PPARγ, is a receptor activated by lipid ligands, central regulator of adipogenesis, with functions in the immune cells activation and lipid metabolism. Data from our group, demonstrated that PPARγ activation induces the biogenesis of dynamic organelles, the Lipid Droplets (LD) during mycobacterial infection. These organelles have functions in lipid storage for energy generation, membrane synthesis, inflammatory mediators synthesis, cell signaling and inflammation. Moreover, the LD in macrophages are niches for the pathogens survival. Although the impact of obesity on metabolic and cardiovascular diseases is understood, the mechanisms involved in the interactions between adipocytes and macrophages infected with intracellular pathogens are not known. In this study we differentiated NIH3T3-L1 cells into adipocytes and used the supernatant obtained to stimulate peritoneal macrophages infected or not with M. bovis BCG. We analyze the lipids droplets formation, PPARγ expression, cytokine, adipokines and NO synthesis. The results have shown that NIH3T3-L1 cells differentiation into adipocytes is an efficient process involving changes in cell morphology and LD storage. The macrophages stimulation with adipocyte supernatant was able to potentiated LD biogenesis, as well as, PPARγ activation, during mycobacterial infection. After 6 and 48 hours of stimulation and infection, the TNF-α levels were also potentiated, although a significant decrease was observed after 24 hours of infection. The IL-10 levels, was increase in the presence of infection and supernatant stimulation at 6, 24 and 48 hours after infection. To nitrite analysis, we observe that after 24 h of infection, there was an increase of levels secreted by macrophages, however this effect was independent of the supernatant stimulation. The adipokines, as leptin and adiponectin, are important factors in the obesity and metabolic syndrome development. Therefore, we analyzed the levels of leptin and adiponectin produced by macrophages during BCG infection. The Leptin levels were not detect, while adiponectin production, was increased in the control macrophages in presence of supernatant, an effect that was inhibited during BCG infection. Thus, our results suggest a modulating effect of secreted factors by adipocytes on the macrophages activation, attributed to the lipid droplets formation, PPARγ expression, cytokines synthesis as TNF-α and IL-10 and a dowmodulation of adiponectin during M. bovis BCG, infection.

Le fer est un oligoélément indispensable pour tout organisme vivant. Le taux de fer systémique est régulé par la fixation de l’hepcidine, hormone synthétisée majoritairement par le foie mais également par les macrophages, à la ferroportine seul exporteur du fer. L’expression de ces deux protéines est régulée par le taux de fer et les processus inflammatoires. Des mécanismes d’acquisition et de séquestration du fer sont mis en place respectivement par le pathogène et l’hôte durant l’infection et régulent en parallèle l’expression de l’hepcidine et la ferroportine. Les travaux de recherche effectués dans le cadre de ma thèse ont porté d’une part sur un aspect fondamental à améliorer nos connaissances du mécanisme de régulation de l’axe hepcidine - ferroportine en condition inflammatoire et analyser l’influence du fer sur la réponse immune au niveau des macrophages; d’autre part une deuxième partie de mes recherches s’est orientée vers une étude plus appliquée du rôle du fer dans la réponse immune induite par une infection mycobactérienne. Nous montrons que l’expression de l’hepcidine et de la ferroportine est différentiellement régulée en corrélation avec la polarisation des macrophages via les voies de signalisation intracellulaires PI3K et autres kinases. Le fer influence la polarisation des macrophages et module ainsi la réponse inflammatoire, et représente aussi un signal de danger capable de stimuler une voie MyD88-dépendante. Enfin, la réponse à l’infection Mycobacterium. bovis BCG est modulée par un régime modérément enrichi en fer, réduisant la charge bactérienne et l’inflammation. / Iron is an essential trace element for all organisms. In mammals, systemic iron homeostasis relies on hepcidin, a peptide hormone synthesized by liver but also macrophages with defensing properties, and its target, the cell iron exporter ferroportin. Iron content and inflammation regulate hepcidin and ferroportin expression in mammals. During infection, pathogens develop sophisticated mechanisms for iron acquisition and sequestration. In response, host regulates the bioavailability of iron through hepcidin and ferroportin expression. First, this work contributes to improve our fundamental knowledge on hepcidin and ferroportin regulation during inflammation and analyzes the influence of iron in macrophages immune response. Second, the role of iron in response to mycobacterial infection was investigated. We show that hepcidin and ferroportin expression was regulated differentially in correlation with macrophages polarization through intracellular signaling pathways involving PI3K and others kinases. In addition, iron influenced macrophages polarization leading to a decrease of inflammatory response with a potent effect on MyD88 pathway stimulation. Finally, we showed that moderate iron-rich diet modulated Mycobacterium bovis BCG response reducing the bacterial burden and inflammation.

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-08-25T16:00:45Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T20:58:38Z (GMT) No. of bitstreams: 2 dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T20:58:46Z (GMT) No. of bitstreams: 2 dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-28T20:58:56Z (GMT). No. of bitstreams: 2 dissertacao_karen_silva_leal.pdf: 538927 bytes, checksum: 188167bf7d281218897286c3f36f6a7f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-03-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O Mycobacterium bovis BCG é utilizado mundialmente como vacina contra tuberculose há mais de meio século, com altos níveis de segurança. O BCG possui características que o tornam um promissor vetor para a produção de vacinas recombinantes multivalentes. Modificações genéticas podem aprimorar as propriedades imunogênicas e adjuvantes do BCG. Uma dessas modificações permite que o BCG escape do endossoma de células apresentadoras de antígenos. Desta maneira, os imunógenos de BCG acessam vias de apresentação cruzada de antígenos, ampliando a sua imunogenicidade. Este trabalho teve como objetivo desenvolver uma cepa de BCG ΔleuD expressando listeriolisina (Hly) de Listeria monocytogenes e avaliar seu potencial citotóxico na linhagem celular 5637 de câncer de bexiga. Para isso, o BCG ΔleuD foi transformado com o plasmídeo recombinante pUP410 contendo o gene hly. A cepa obtida demonstrou um nível de citotoxicidade similar à cepa BCG Pasteur parental, quando avaliada na linhagem celular de câncer de bexiga. Esta cepa necessita ser avaliada em outras linhagens de câncer para comprovar seu possível efeito citotóxico potencializado. / Mycobacterium bovis BCG has been used worldwide as a vaccine against tuberculosis for over fifty years with an outstanding safety record. BCG has features that make it a promising vector for production of recombinant multivalent vaccines. Genetic modification can enhance the adjuvant and immunogenic properties of BCG. One of these modifications would allow BCG to escape the endosome of antigen presenting cells. Therefore, theimmunogens of BCG can access antigens cross priming routes, increasing their immunogenicity. The aim of this work was to develop a strain of BCG ΔleuD expressing listeriolysin (Hly) of Listeria monocytogenes and evaluate its cytotoxic potential on cell line 5637 of bladder cancer. Thus, BCG ΔleuD was transformed with recombinant plasmid pUP410 containing the hly coding sequence. The recombinant BCG strain expressing listeriolysin showed a similar level of cytotoxicity when compared to the parental BCG Pasteur. This strain needs to be evaluated in other cancer lines in order to confirm its possible potentiated cytotoxic effect.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-23T18:00:32Z No. of bitstreams: 1 sabrinapiresmaciel.pdf: 1298915 bytes, checksum: 7f5399c736441ab3197f7480f41e60c3 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-26T20:31:19Z (GMT) No. of bitstreams: 1 sabrinapiresmaciel.pdf: 1298915 bytes, checksum: 7f5399c736441ab3197f7480f41e60c3 (MD5) / Made available in DSpace on 2016-09-26T20:31:19Z (GMT). No. of bitstreams: 1 sabrinapiresmaciel.pdf: 1298915 bytes, checksum: 7f5399c736441ab3197f7480f41e60c3 (MD5) Previous issue date: 2015-04-14 / O câncer de mama é uma neoplasia maligna que se desenvolve no tecido mamário, caracterizado pelo crescimento desordenado das células epiteliais. É o cancro mais freqüente nas mulheres e sofre influência da obesidade. Sendo a obesidade e o câncer condições que direcionam um processo inflamatório crônico, cabe a necessidade de se estudar os mediadores dessa reação e suas possíveis conseqüências para o organismo. Corpúsculos lipídicos (CL) são organelas dinâmicas envolvidas no metabolismo de lipídeos e sinalização intracelular, que aumentam em número e tamanho nos leucócitos envolvidos nos processos inflamatórios, neoplásicos e infecciosos, incluindo infecções intracelulares por Mycobacterium bovis BCG e Trypanosoma cruzi. A lipogênese está associada com o mau prognóstico em diversas neoplasias, sugerindo o papel dos CLs no desenvolvimento do câncer. Diante disso, o presente trabalho estudou a correlação do tumor de mama e da obesidade na resposta inflamatória durante infecções por M. bovis BCG e T. cruzi. Para esse propósito, camundongos Balb/c fêmeas foram tratados com dieta padrão e hiperlipídica por 16 semanas e estimulados com células da linhagem tumoral 4T1 por 14 dias. Macrófagos foram extraídos do tecido adiposo e do peritônio e re-estimulados in vitro com M. bovis BCG e T. cruzi para a análise da formação de CLs,citocinas e eicosanóides. Os resultados demonstraram que macrófagos obtidos de animais obesos estimulados in vivo com células tumorais e in vitro com BCG e T. cruzi, apresentam o número de CLs consideravelmente aumentando, mostrando que a presença das células tumorais e obesidade são capazes de estimular processo inflamatório, tendo em vista a formação destas organelas. Além disso, macrófagos de animais obesos apresentaram maior produção de PGE2 e expressão de COX-2 que os de animais magros. Foi demonstrado também que macrófagos dos animais obesos com tumores quando foram re-estimulados exibiram aumento significativo na síntese de leptina e das citocinas TNF-α, TGF-β em comparação com os macrófagos de animais magros com tumores. Como conclusão, nossos resultados sugerem que o desenvolvimento tumoral, em conjunto com a obesidade, potencia a formação dos CLs e a síntese de mediadores inflamatórios durante infecções por patógenos intracelulares, como BCG e T. cruzi, sugerindo o papel dos CLs como alvo para futuras intervenções terapêuticas no sentido de influenciar o balanço das interações entre o patógeno e o hospedeiro. / Breast cancer is a malignant neoplasm that develops in the breast tissue, characterized by the uncontrolled growth of epithelial cells. It’s the most frequent cancer in women and is influenced by obesity. As obesity and cancer conditions that drive to a chronic inflammatory process, have a need to study the mediators of this reaction and possible consequences for the organism. Lipid bodies (LB) are dynamic organelles involved in lipid metabolism and intracellular signaling, which is increase in number and size in leukocytes involved in the inflammatory, neoplastic and infectious processes, including intracellular Mycobacterium bovis BCG and Trypanosoma cruzi. The lipogenesis is associated with poor prognosis in several cancers, suggesting the role of LB in cancer development. Therefore, this study presents the correlation of breast tumor and obesity in the inflammatory response during infection by M. bovis BCG and T. cruzi. For this purpose, Balb/ c female mice were treated with standard and fat diet for 16 weeks and stimulated with tumor cells 4T1 for 14 days. Macrophages were extracted from adipose tissue and peritoneum and restimulated in vitro with M. bovis BCG and T. cruzi for analysis of formation of LBs, cytokines, and eicosanoids. The results showed that macrophages obtained from obese animals stimulated with tumor cells in vivo and with BCG and T. cruzi in vitro, LBs showed a number considerably increased, showing that the presence of tumor cells and obesity are capable of stimulating inflammatory process, having for training these organelles. Moreover, macrophages from obese animals had higher production of PGE2 and expression of COX-2 than in thin animals. It has also been shown that macrophages from obese animals when restimulated with tumors exhibited a significant increased synthesis of leptin and cytokine TNF-α, TGF-β compared to macrophages from thin animals with tumors. In conclusion, our results suggest that tumor growth, together with obesity, enhances the formation of LBs and the synthesis of inflammatory mediators during infections by intracellular pathogens such as BCG and T. cruzi, suggesting the role of target LBs for future therapeutic interventions to influence the balance of the interactions between the pathogen and the host.

No description available.

Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Author: Marianna Šamajová Supervisor: RNDr. Klára Konečná, Ph.D. Consultant: plk. gšt. doc. RNDr. Zuzana Kročová, Ph.D. Title of diploma thesis: Entry of bacteria Mycobacterium bovis BCG into B lymphocytes Background: The objective of this work was to evaluate the entry of bacterium Mycobacterium bovis BCG into B lymphocytes and the role of selected receptors in this process. Methods: Peritoneal cell suspensions with unblocked and/or blocked receptors on BALB/c mouse B lymphocytes we infected by bacterium M. bovis BCG-GFP unopsonized and/or opsonized by fresh murine serum ("complement") or immune serum ("antibodies"). Using flow cytometry we evaluated the entry of bacterium M. bovis BCG-GFP into B lymphocytes and their subpopulations B1a, B1b and B2. Results: M. bovis BCG-GFP actively enters into B lymphocytes. Depending on the subpopulation, it most infects B1a, less B1b and at least B2 lymphocytes. Only the subpopulation B2 responds significantly to the opsonization by complement. Opsonization by antibodies had no significant effect on the infection. Entry into CD19+ cells is mediated through the BCR receptor, especially in subpopulations B1a and B1b. Under the opsonized conditions, the CR1/2 complement receptor is...

Die Familie der Mykobakterien setzt sich aus pathogenen und apathogenen Vertretern zusammen. In dieser Arbeit wurden 3 Mitglieder dieser Familie für Untersuchungen herangezogen: ihr prominentester pathogener Vertreter Mycobacterium tuberculosis, der Erreger der Tuberkulose, das als Impfstoff eingesetzte Mycobacterium bovis BCG, das durch Attenuierung aus dem Rindertuberkulose-Erreger Mycobacterium bovis hervorging und das apathogene Bodenbakterium Mycobacterium smegmatis. Ein Schlüssel zum Verständnis der Mykobakterien und speziell ihrer Widerstandsfähigkeit ist die Kenntnis ihrer komplexen Zellwand. Peptidoglycan als deren Bestandteil und insbesondere der mittels Undecaprenyl-Monophosphat bewerkstelligte Transport von Peptidoglycan-Vorläufern aus dem Cytoplasma an die Zelloberfläche steht dabei im Zentrum der Zellwandbildung. In M. tuberculosis, M. bovis BCG und M. smegmatis wurden Deletionsmutanten für die Undecaprenyl-Phosphokinase (Upk) hergestellt. Für M. smegmatis wurde gezeigt, daß die delta upk Deletionsmutante, in Übereinstimmung mit Deletionsmutanten homologer Gene in anderen Bakterien, eine erhöhte Sensitivität gegenüber dem die Zellwandsynthese hemmenden Antibiotikum Bacitracin aufwies. Überraschenderweise zeigte M. tuberculosis delta upk diesen Phänotyp nicht. Weiterhin ließ sich für M. smegmatis delta upk im Vergleich zum M. smegmatis Wildtyp Peptidoglycan an der Zelloberfläche in geringerem Maße nachweisen. Eindrucksvoll zeigte sich die Bedeutung der Undecaprenyl Phosphokinase in der gestörten Entwicklung von Biofilmen im Falle der M. smegmatis delta upk Mutante. Dies galt sowohl für in vitro Bedingungen als auch für ein, im Rahmen dieser Arbeit, neu entwickeltes in vivo Modell. Vergleiche von M. tuberculosis Wildtyp und M. tuberculosis Mutante auf der Ebene von Proteom- und Transkriptom-Analysen führten zur Identifikation eines zum mykobakteriellen Fettsäure-Synthese II (FASII) System gehörenden Operons, das im Falle der upk-Deletion verstärkt exprimiert wurde und damit möglicherweise einen Kompensationsmechanismus für die fehlende Phosphokinase darstellt. Eine reduzierte Persistenz von M. smegmatis delta upk in infizierten Makrophagen legte nahe, daß Upk bei mykobakteriellen Infektionen eine entscheidende Rolle für das Überleben der Bakterien und ihre Virulenz spielt. Dies konnte erstmals für M. tuberculosis im Rahmen von Maus-Infektionsversuchen gezeigt werden. M. tuberculosis delta upk ließ sich als neues Mitglied in eine Reihe von als growth in vivo (giv) klassifizierten Mutanten einreihen. Die Herstellung von Deletionsmutanten wird als Möglichkeit betrachtet, verbesserte Impfstoffe herzustellen. Die physiologische Konsequenz der Deletion sollte bestenfalls neben einer Attenuierung des Ausgangsbakteriums (gilt besonders für M. tuberculosis) eine Überexpression protektionsrelevanter Antigene zur Folge haben. Im Vergleich zum bestehenden Impfstoff M. bovis BCG führte die Impfung von Mäusen mit M. bovis BCG delta upk sowohl zu geringerer bakterieller im Anschluß an die Vakzinierung als auch zu einer verbesserten Langzeit-Protektion gegen Tuberkulose. / The family of mycobacteria is composed of pathogenic and apathogenic bacteria. This study was performed with 3 members of this family, the most prominent pathogenic member, Mycobacterium tuberculosis, the causative agent of tuberculosis, the vaccine strain Mycobacterium bovis BCG which was developed by attenuation of the bovine tuberculosis agent Mycobacterium bovis, and Mycobacterium smegmatis which is apathogenic and widely distributed in soil. A key to understanding mycobacteria and, especially, their resistance is to understand the complexity of their cell wall. Peptidoglycan is a major component of the cell wall and the transport of peptidoglycan precursors out of the cytoplasm to the bacterial surface by undecaprenyl monophosphate is central to cell wall synthesis. Therefore, deletion mutants of the undecaprenyl phosphokinase gene (upk) were generated in M. tuberculosis, M. bovis BCG, and M. smegmatis. In the case of M. smegmatis it was shown that a delta upk deletion mutant, as with deletion mutants of homologous genes in other bacteria, exhibited an increased sensitivity to the antibiotic bacitracin, indicating that cell wall synthesis was hampered. Surprisingly, M. tuberculosis delta upk did not exhibit this phenotype. Furthermore, a lower level of peptidoglycan was detected on the cell surface of an M. smegmatis delta upk mutant compared to M. smegmatis wildtype. Relevance of the undecaprenyl phosphokinase was demonstrated by impaired biofilm development in the case of the M. smegmatis delta upk mutant. This was observed in vitro as well as in vivo using an animal model which was newly developed in this thesis. A fatty acid synthase II (FASII) system related operon revealed by comparative proteome- and transcriptome-analyses comparing M. tuberculosis wildtype and M. tuberculosis delta upk mutant, and may reflect a compensatory mechanism for the loss of upk. Reduced persistence of M. smegmatis in infected macrophages suggested a decisive role of Upk in mycobacterial infection concerning survival and virulence of bacteria. This was later demonstrated to be true for M. tuberculosis in a mouse model. M. tuberculosis delta upk was, therefore, classified as a new member of the group of growth in vivo (giv) mutants. Construction of deletion mutants is a strategy to identify improved vaccines. Ideally, the physiologic consequences of a gene deletion would result in attenuation of the modified bacterium (especially in the case of M. tuberculosis) and overexpression of antigens relevant for protection. Compared to the existing vaccine M. bovis BCG, vaccination of mice with M. bovis BCG delta upk exhibited a lower bacterial load upon vaccination as well as an improved long-lasting protection against M. tuberculosis infection.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A vacina anti-diftérica de uso corrente no Brasil (DTP), embora de alta eficácia na prevenção da difteria, está associada com episódios de toxicidade e reatogenicidade no recipiente vacinal, resultantes de proteínas residuais derivadas do processo de produção ou detoxificação. Estratégias para o desenvolvimento de vacinas menos reatogênicas e ao mesmo tempo mais eficazes e economicamente viáveis contra a difteria têm sido alvo de intensa investigação. A alternativa proposta por nosso grupo é a utilização da vacina contra a tuberculose (Mycobacterium bovis BCG sub-cepa Moreau), como vetor do gene que codifica o fragmento B da toxina diftérica (dtb) de 58,3 kDa. Neste trabalho o dtb foi clonado no vetor micobacteriano bifuncional (pUS977) de expressão citoplasmática e os clones recombinantes (pUS977dtbPW8), após a transformação do BCG, foram testados com relação a expressão do DTB em BCG e quanto a antigenicidade frente a anticorpos policlonais anti-toxóide diftérico por Immunobloting. A integridade do gene dtb e a identidade das sequências de DNA da construção plasmidial pUS977dtbPW8 foram confirmadas por sequenciamento de DNA e análise de similaridade. A imunogenicidade do BCGr pUS977dtbPW8 expressando o DTB foi investigada em camundongos BALB/c, os resultados obtidos revelaram uma soroconversão específica (IgG). A infectividade e atividade microbicida do BCGr pUS977dtbPW8 no ambiente intracelular foi avaliada através da infecção de linhagens de células de monócitos humano (THP-1), os dados obtidos indicaram que houve sobrevivência intracelular em até 12 dias. Nesse contexto, esplenócitos dos camundongos imunizados com 30 e 60 dias foram extraídos, mostrando que o BCGr pUS977dtbPW8 persistiu até 60 dias na ausência de pressão seletiva e a viabilidade celular não sofreu alteração significativa durante o período testado. Por outro lado, o BCGr pUS977dtbPW8, quando submetido a seis sub-cultivos consecutivos in vitro não apresentou diferença significativa na capacidade de expressar o DTB, demonstrando portanto a persistência da estabilidade funcional da linhagem recombinante. A estabilidade estrutural da construção pUS977dtbPW8 também foi avaliada por PCR confirmando a presença do gene dtb em colônias do BCGr pUS977dtbPW8 . Adicionalmente, foi possível avaliar preliminarmente in vitro a capacidade soroneutralizante dos soros de camundongos imunizados com BCGr pUS977dtbPW8 após 30 e 60 dias em células VERO. A ação citotóxica da toxina diftérica entre as diluições de 1/4 e 1/16 foram neutralizadas com o pool de soros imunes com 60 dias. Finalmente, em nosso estudo foi possível avaliar o potencial da vacina BCG como vetor de expressão de um antígeno de Corynebacterium diphtheriae in vitro e in vivo. / The diphtheria vaccine currently used in Brazil (DTP), despite its history of high efficacy in the prevention of diphtheria, is associated with episodes of toxicity and vaccine reactogenicity in the vaccinee, resulting from the presence in the vaccine of residual proteins derived from the production process or detoxification. Strategies for the development of new vaccines more effective and economically viable against diphtheria have been the subject of intense investigation. The alternative proposed by our group is the use of the vaccine against tuberculosis (Mycobacterium bovis BCG Moreau sub strain) as a vector for the gene that encodes the 58.3 kDa fragment B of the diphtheria toxin (DTB). In our project the dtb gene was cloned into the bifunctional vector pUS977 for cytoplasmic expression and recombinant BCG (rBCG) clones, selected after transformation of BCG, were tested for expression of the DTB polypeptide and antigenicity against polyclonal antibodies anti- diphtheria toxoid by immunoblotting. The integrity and identity of the DNA sequence encoding the dtb gene carried by the plasmid construct pUS977dtbPW8 was confirmed by DNA Sequencing and Analysis of Similarity. The immunogenicity of the rBCG expressing the DTB was investigated in BALB/c mice and the results revealed a specific seroconversion (IgG). Also, infectivity and microbicidal activity were analyzed in the intracellular environment by infecting human monocytes (THP-1 cell line) with rBCG. The data obtained indicated intracellular survival within 12 days. In this context, splenocytes collected from mice at days 30 and 60 after immunization were removed and assayed for live bacteria. The results showed that rBCG persisted viable up to 60 days in the absence of selective pressure and cell viable counts did not change significantly during testing. Additionally, the rBCG subjected to six consecutive sub-cultures in vitro showed no significant difference in the ability to express the DTB, thus demonstrating the functional stability of the recombinant vaccine. The structural stability of the construct pUS977dtbPW8 was also confirmed by PCR detection of the dtb gene in rBCG colonies. Also, it was possible to have a preliminary evaluation of the neutralizing capacity of sera from mice immunized with BCGr 30 and 60 days after immunization. The cytotoxic action of diphtheria toxin, between dilutions 1/ 4 and 1/16, was neutralized by mice sera in an in vitro assay using VERO cells. Finally, in our study it was possible to evaluate the potential of BCG as a vector for expression of an antigen of Corynebacterium diphtheriae in vitro and in vivo.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A vacina anti-diftérica de uso corrente no Brasil (DTP), embora de alta eficácia na prevenção da difteria, está associada com episódios de toxicidade e reatogenicidade no recipiente vacinal, resultantes de proteínas residuais derivadas do processo de produção ou detoxificação. Estratégias para o desenvolvimento de vacinas menos reatogênicas e ao mesmo tempo mais eficazes e economicamente viáveis contra a difteria têm sido alvo de intensa investigação. A alternativa proposta por nosso grupo é a utilização da vacina contra a tuberculose (Mycobacterium bovis BCG sub-cepa Moreau), como vetor do gene que codifica o fragmento B da toxina diftérica (dtb) de 58,3 kDa. Neste trabalho o dtb foi clonado no vetor micobacteriano bifuncional (pUS977) de expressão citoplasmática e os clones recombinantes (pUS977dtbPW8), após a transformação do BCG, foram testados com relação a expressão do DTB em BCG e quanto a antigenicidade frente a anticorpos policlonais anti-toxóide diftérico por Immunobloting. A integridade do gene dtb e a identidade das sequências de DNA da construção plasmidial pUS977dtbPW8 foram confirmadas por sequenciamento de DNA e análise de similaridade. A imunogenicidade do BCGr pUS977dtbPW8 expressando o DTB foi investigada em camundongos BALB/c, os resultados obtidos revelaram uma soroconversão específica (IgG). A infectividade e atividade microbicida do BCGr pUS977dtbPW8 no ambiente intracelular foi avaliada através da infecção de linhagens de células de monócitos humano (THP-1), os dados obtidos indicaram que houve sobrevivência intracelular em até 12 dias. Nesse contexto, esplenócitos dos camundongos imunizados com 30 e 60 dias foram extraídos, mostrando que o BCGr pUS977dtbPW8 persistiu até 60 dias na ausência de pressão seletiva e a viabilidade celular não sofreu alteração significativa durante o período testado. Por outro lado, o BCGr pUS977dtbPW8, quando submetido a seis sub-cultivos consecutivos in vitro não apresentou diferença significativa na capacidade de expressar o DTB, demonstrando portanto a persistência da estabilidade funcional da linhagem recombinante. A estabilidade estrutural da construção pUS977dtbPW8 também foi avaliada por PCR confirmando a presença do gene dtb em colônias do BCGr pUS977dtbPW8 . Adicionalmente, foi possível avaliar preliminarmente in vitro a capacidade soroneutralizante dos soros de camundongos imunizados com BCGr pUS977dtbPW8 após 30 e 60 dias em células VERO. A ação citotóxica da toxina diftérica entre as diluições de 1/4 e 1/16 foram neutralizadas com o pool de soros imunes com 60 dias. Finalmente, em nosso estudo foi possível avaliar o potencial da vacina BCG como vetor de expressão de um antígeno de Corynebacterium diphtheriae in vitro e in vivo. / The diphtheria vaccine currently used in Brazil (DTP), despite its history of high efficacy in the prevention of diphtheria, is associated with episodes of toxicity and vaccine reactogenicity in the vaccinee, resulting from the presence in the vaccine of residual proteins derived from the production process or detoxification. Strategies for the development of new vaccines more effective and economically viable against diphtheria have been the subject of intense investigation. The alternative proposed by our group is the use of the vaccine against tuberculosis (Mycobacterium bovis BCG Moreau sub strain) as a vector for the gene that encodes the 58.3 kDa fragment B of the diphtheria toxin (DTB). In our project the dtb gene was cloned into the bifunctional vector pUS977 for cytoplasmic expression and recombinant BCG (rBCG) clones, selected after transformation of BCG, were tested for expression of the DTB polypeptide and antigenicity against polyclonal antibodies anti- diphtheria toxoid by immunoblotting. The integrity and identity of the DNA sequence encoding the dtb gene carried by the plasmid construct pUS977dtbPW8 was confirmed by DNA Sequencing and Analysis of Similarity. The immunogenicity of the rBCG expressing the DTB was investigated in BALB/c mice and the results revealed a specific seroconversion (IgG). Also, infectivity and microbicidal activity were analyzed in the intracellular environment by infecting human monocytes (THP-1 cell line) with rBCG. The data obtained indicated intracellular survival within 12 days. In this context, splenocytes collected from mice at days 30 and 60 after immunization were removed and assayed for live bacteria. The results showed that rBCG persisted viable up to 60 days in the absence of selective pressure and cell viable counts did not change significantly during testing. Additionally, the rBCG subjected to six consecutive sub-cultures in vitro showed no significant difference in the ability to express the DTB, thus demonstrating the functional stability of the recombinant vaccine. The structural stability of the construct pUS977dtbPW8 was also confirmed by PCR detection of the dtb gene in rBCG colonies. Also, it was possible to have a preliminary evaluation of the neutralizing capacity of sera from mice immunized with BCGr 30 and 60 days after immunization. The cytotoxic action of diphtheria toxin, between dilutions 1/ 4 and 1/16, was neutralized by mice sera in an in vitro assay using VERO cells. Finally, in our study it was possible to evaluate the potential of BCG as a vector for expression of an antigen of Corynebacterium diphtheriae in vitro and in vivo.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-17T14:40:56Z No. of bitstreams: 1 anaclaudiacarvalhogouveia.pdf: 2060044 bytes, checksum: a2757483182ff953fdffbf86f346380b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-28T14:35:33Z (GMT) No. of bitstreams: 1 anaclaudiacarvalhogouveia.pdf: 2060044 bytes, checksum: a2757483182ff953fdffbf86f346380b (MD5) / Made available in DSpace on 2016-06-28T14:35:33Z (GMT). No. of bitstreams: 1 anaclaudiacarvalhogouveia.pdf: 2060044 bytes, checksum: a2757483182ff953fdffbf86f346380b (MD5) Previous issue date: 2012-08-30 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A asma alérgica é uma doença inflamatória crônica das vias aéreas, caracterizada por uma resposta de hipersensibilidade imediata, obstrução brônquica, inflamação pulmonar e níveis elevados de IgE. A doença é mediada principalmente por uma resposta imunológica alérgeno-específica tipo Th2. Nas últimas décadas, a prevalência da asma alérgica tem aumentado significativamente, sobretudo nos países desenvolvidos. A Hipótese da Higiene atribui este aumento a uma menor exposição a determinados microrganismos durante a infância, quando o amadurecimento adequado do sistema imunológico requer estímulos que induzam respostas imunológicas de perfil Th1, fundamentais para o equilíbrio de respostas Th2 exacerbadas. Diversos trabalhos epidemiológicos parecem comprovar esta hipótese, evidenciando a existência de uma relação inversa entre o contato com microrganismos indutores de uma resposta Th1 e o desenvolvimento de asma alérgica. Paralelamente, estudos em modelos murinos constataram que o tratamento com Mycobacterium bovis BCG (BCG) reduz respostas Th2 alérgenoespecíficas. No entanto, os mecanismos pelos quais a micobactéria inibe o desenvolvimento da resposta alérgica são ainda pouco conhecidos. Este estudo avaliou o efeito da administração do BCG sobre a resposta imunológica ocorrida na alergia pulmonar em camundongos BALB/c previamente sensibilizados e desafiados com OVA. Vinte e quatro horas após o último desafio, o sangue e o lavado broncoalveolar foram coletados para análises de imunoglobulinas e contagem de células, respectivamente. Adicionalmente, os pulmões foram submetidos à análise histológica, avaliação da atividade de EPO e dosagens de citocinas e quimiocinas, assim como avaliação da expressão de CTLA-4, Foxp3 e IL-10 por citometria de fluxo. Os resultados obtidos indicam que o tratamento com BCG melhorou o processo alérgico através da redução dos principais parâmetros relacionados à resposta Th2, como o infiltrado eosinofílico pulmonar, a atividade de EPO, IL-4, IL-13, CCL11, além de IgE e IgG1 específicas anti-OVA. Por outro lado, a administração da micobactéria aumentou os níveis de IFN-γ, IL-10 e TGF-β, além das expressões de Foxp3 e CTLA-4 pelos linfócitos T CD4+. Paralelamente, houve um aumento na produção de IL-10 pelos linfócitos T CD8+. Esses dados sugerem que, além da indução de uma resposta imune Th1, a ação imunomoduladora do BCG está relacionada também à indução de mecanismos reguladores. / Atopic asthma is a chronic respiratory disease characterized by airway hyperresponsiveness, reversible airway obstruction, lung inflammation, and high levels of allergen-specific IgE, driven by allergen-specific Th2 cells. The increasing prevalence of allergic diseases, particularly in industrialized countries, has led to the hygiene hypothesis, which states that the newborn infant’s immune system is skewed toward Th2 responses and needs timely and appropriate environmental stimulus to create a balanced immune response. Supporting this hypothesis, epidemiological and experimental evidence has shown an inverse correlation between Th1-induced microbial infections and atopic asthma. Similarly, some animal studies have demonstrated that exposure to Mycobacterium tuberculosis or to environmental mycobacteria is able to protect against the development of allergic responses. However the exact mechanism underlying this inhibition still remains poorly understood. This study aimed to evaluate the ability of BCG to suppress an established allergic response in a mouse model of OVA-induced airway inflammation. To achieve this, OVA sensitized and challenged BALB/c mice were twice treated with BCG via nasal and 21 days after the first treatment, mice were rechallenged with OVA. Twenty-four hours after the last challenge, blood samples were collected to detect anti-OVA immunoglobulin isotypes, and bronchoalveolar lavage (BAL) was harvested for cell count. Additionally, lungs were collected for histological analysis, detection of EPO activity and measurement of cytokines and chemokines. The expression of CTLA-4, Foxp3 and IL-10 was also determined in lung tissue by flow cytometry. The data indicated that BCG treatment was able to inhibit an established allergic Th2-response by decreasing the allergen-induced eosinophilic inflammation, EPO activity, levels of IL-4, IL-13, CCL11 and serum levels of IgE and IgG1. Mycobacteria treatment increased lung levels of IFN-γ, IL-10 and TGF-β, and expressions of Foxp3 and CTLA-4 in CD4+T cells. Additionally, an increased production of IL-10 by CD8+ T cells was observed, even though no detectable changes in CD4+IL-10+ was noticed. Altogether, these results suggest that the mechanism underlying the down-regulatory effects of BCG on OVA-induced airway inflammation appear to be associated with the induction of both Th1 and T regulatory immune responses.