Os mecanismos que conferem resistência do Mycobacterium tuberculosis (Mtb) à destruição pelo hospedeiro, além da sua capacidade em permanecer e/ou multiplicar-se no interior das células fagocitárias são ainda pouco compreendidos. Nosso grupo de pesquisa tem contribuído para o entendimento do papel dos mediadores lipídicos, que incluem prostaglandinas (PGs) e leucotrienos (LTs) na tuberculose. PGs inibem a resposta imune celular TH1, a produção de citocinas e a fagocitose, e assim facilita a infecção. LTs estão envolvidos no recrutamento de leucócitos, e na modulação da síntese de citocinas, no aumento da fagocitose e dos mecanismos microbicidas, e assim contribui para a eliminação da micobactéria. Neste projeto, avaliamos in vivo e in vitro a produção dos mediadores lipídicos induzidos por cepas de Mtb isolados de pacientes com tuberculose ativa. Demonstramos neste trabalho que macrófagos alveolares infectados com os bacilos da cepa SV009 levam a maior produção de TNF- e nitrito, do que aqueles infectados com a cepa SV068. Em contraste, macrófagos alveolares infectados com os bacilos da cepa SV068 induzem a produção de muito mais LTB4, quando comparado aos bacilos da cepa SV009. Obtivemos maior recuperação de unidades formadoras de colônia (UFC) de macrófagos alveolares tratados com MK886 e infectados com bacilos da cepa SV068; enquanto que mais UFCs foram recuperadas após o tratamento com ácido caféico e infecção com a cepa SV009. Com relação a formação de corpúsculos lipídicos (CLs), observamos um maior número destes quando macrófagos alveolares foram infectados com bacilos da cepa SV068. Ainda, observamos diminuição de CLs quando tratados com MK886 ou ácido caféico. Os bacilos da cepa SV068 foram mais fagocitados, mas os macrófagos não foram muito eficazes na atividade microbicida dos mesmos. Nos experimentos in vivo vimos que camundongos balb/c infectados com a cepa SV068 morrem mais e o tratamento com MK886 parcialmente os protege e a mortalidade não está relacionada com a maior carga bacilar no pulmão ou baço. Houve aumento no recrutamento de neutrófilos induzido pela infecção especialmente após infecção com os bacilos da cepa SV068, sendo que o tratamento com MK886 inibe significativamente o recrutamento quando comparado à infecção com os bacilos da cepa SV009. Células mononucleares também foram recrutadas e permaneceram aumentadas até o final do período observado, sem muitas diferenças significativas quando comparamos a infecção com os isolados SV009 e SV068. A produção de nitrito também encontrou-se elevada em animais infectados com bacilos da cepa SV068. A análise histopatológica dos pulmões dos animais infectados mostrou intensa reação inflamatória com maior comprometimento do parênquima pulmonar dos camundongos infectados bacilos da cepa SV068, com intensa deposição de colágeno e multiplicação bacilar. Encontramos diferenças significativas em relação à producão de citocinas IL-6, IL-10, IL-1, IFN-, TNF- and IL-12 após infecção de 30 e 60 dias com as cepas SV009 e SV068. Também mostramos que há diferenças na produção de LTB4 e PGE2 após 30 e 60 dias de infecção com as cepas SV009 e SV068 em células do camundongos balb/c. Experimentos com animais 129 e 5LO-/- infectados com as duas cepas também foram realizados, e vimos que os animais 5LO-/- são mais suscetíveis à infecção especialmente quando infectados com a cepa SV068. Sugerimos que as cepas são diferentes, mas dependentes de um conjunto de fatores, e nossos dados sugerem que dentre estes mecanismos a produção de TNF- e também de mediadores lipídicos (LTB4 e PGE2) estão envolvidos. / The mechanisms that confer resistance to Mycobacterium tuberculosis (Mtb) for destruction by the host, in addition to its ability to retain and/or multiply within phagocytic cells are still poorly understood. Our research group has contributed to the understanding of the role of lipid mediators, including prostaglandins (PGs) and leukotrienes (LTs) in tuberculosis. PGs inhibit Th1 cell immune response, cytokine production and phagocytosis, thus facilitating the infection. LTs are involved in the leukocytes recruitment, and modulation of cytokine synthesis, phagocytosis and microbicidal mechanisms enhancement, and contribute to the elimination of the mycobacteria. In this project, we evaluated in vivo and in vitro the lipid mediators production induced by Mtb strains isolated from patients with active tuberculosis. We demonstrated in this study that alveolar macrophages infected with bacilli from SV009 strain lead to an increase of TNF- production and nitrite, than those infected with the strain SV068. In contrast, alveolar macrophages infected with bacilli from SV068 strain induced more LTB4 production when compared to SV009 infection. We obtained higher recovery colony forming units (CFU) of alveolar macrophages treated with MK886 and infected with bacilli from SV068 strain; while more CFUs were recovered after treatment with caffeic acid and infection with bacilli from SV009 strain. Regarding the lipid bodies (LBs) formation, we observed a greater number of these structures, when alveolar macrophages were infected with bacilli from SV068 strain. Still, we observed a decrease of LBs when the macrophages were treated with MK886 and caffeic acid. Bacilli from SV068 strain were more phagocytosed, but macrophages were not very effective in the microbicidal activity. In the in vivo experiments we found that mice infected with SV068 strain die more than the other and MK886 treatment partially protects the mice, besides, the mortality is not related to the higher bacterial load in the lung or spleen. There was an increase in neutrophil recruitment induced after infection, especially after infection with SV068 strain, and treatment with MK886 significantly inhibits recruitment when compared to infection with SV009 strain. Mononuclear cells were also recruited and remained increased until the end of the observed period, without many significant differences when comparing infection with SV009 and SV068 strains. The nitrite production was also found greater in animals infected with bacilli from SV068 strain. Histopathological analysis of the infected mice lungs showed an intense inflammatory reaction with greater impairment of the mice lungs when infected with bacilli from SV068 strain with an intense collagen deposition and multiplication of bacilli. We suggest that the SV068 strain is more virulent and participates of the immune response by lipid mediators dependent mechanisms.

As grandes mudanças para os pesquisadores da área de vacinas sao a optimizaçao das vacinas de DNA para uso em humanos ou animais de grande porte e criar vacinas efetivas de uma única dose usando sistemas de liberação controlada apropriados. O plasmídeo de DNA codificando a proteína de heatshock 65 (hsp65) (DNA-hsp65) foi capaz de induzir resposta imune protetora e terapêutica no modelo murino da tuberculose (TB). Apesar do sucesso da vacina baseada na DNA-hsp65 em solução para proteger camundongos contra TB, esta requer múltiplas doses de grande quantidade de DNA para imunização efetiva. No intuito de otimizar esta vacina e simplificar o protocolo de vacinação, nós co-encapsulamos DNA-hsp65 e o adjuvante dimicolato de trealose (DMT) em microesferas biodegradáveis de ácido polilático-poliglicólico (PLGA) para uma única administração. Além disso, foi também desenvolvida uma formulação prime-boost da vacina de dose única baseada na mistura de duas diferentes microesferas de PLGA, apresentando liberação rápida e lenta, de DNAhsp65 e proteína hsp65 recombinante, respectivamente. Essas formulações foram testadas em cobaias pela comparação da eficácia protetora (components efetores da imunidade protetora) e toxicidade (componentes efetores da patologia) induzida pela preparação da preparação do DNA em solução ou BCG. A formulação de dose única prime-boost nitidamente apresentou maior eficácia e reduziu a patologia nos pulmões de cobaias. / The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly(DLlactide- co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in guinea pigs by comparison with the efficacy (effector components of protective immunity) and toxicity (effector components of pathology) induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in guinea pigs.

Anotação proteogenômica é uma abordagem que une a análise proteômica com a anotação genômica. O intuito de tal abordagem é prover uma anotação mais detalhada ao gene. Intuito esse, que nem sempre é possível quando se trata apenas de genes, uma vez que produtos gênicos, com funções importantes preditas, somente passam a ter papel na fisiologia do organismo quando expressos e traduzidos. Com todo o avanço atual de estudos na área proteogenômica, a geração de dados tem crescido de modo exponencial e, com esse crescimento, nota-se a necessidade cada vez maior da criação de sistemas capazes de processar, armazenar e gerenciar essas novas informações produzidas. Assim, é descrito nesse trabalho o desenvolvimento do ProGen AP , sendo constituído de uma interface web construída em HTML/PHP5, um banco de dados cujo SGBD é o mySQL e de módulos de processamento de dados proteômicos, neste caso o LabKey (com o core Xtandem!) e o QuickMod. Todos os módulos são open source e comunicam entre si através de scripts PERL. Nesse sistema, o pesquisador fornece dados de experimentos proteômicos e o sistema, então, os processa e retorna ao usuário informações sobre o gene expresso, a localização dos peptídeos dentro do gene aos quais pertencem e, ainda, informações quantitativas sobre o peptídeo e a proteína identificados. Além disso, o uso de um processamento esquematizado reduz a possibilidade de erro de entrada/saída de dados nos módulos intermediários do processamento. Aqui, o ProGen AP foi aplicado no estudo proteômico do Mycobacterium tuberculosis (MTb). Na literatura, o genoma do MTb cepa H37Rv contém apenas 4062 open reading frames (ORFs) preditos e o complemento funcional desse genoma, o proteoma, ainda não está totalmente elucidado. A análise do proteoma do MTb, com o uso do ProGen AP, resultou em uma lista total de 154.982 identificações de peptídeos, representando um total de 147.334 peptídeos únicos. Até o momento, foram identificadas 2.369 proteínas, cobrindo aproximadamente 58% de todo o genoma do MTB. É importante ressaltar que, dentre todas as proteínas identificadas até o momento, a maioria delas está anotada como proteinas hipotéticas em seu genoma, e, por consequência, os resultados obtidos nesse projeto confirmam e validam a existência de tais produtos gênicos. Além disso, 567 peptídeos foram identificados como N-terminal e 1229 como C-terminal, o que indica a correta predição do início e do término da tradução de tais genes. Todos esses resultados positivos confirmam que a abordagem utilizada no ProGen AP é eficiente e pode ser usada em vários outros organismos de interesse do pesquisador. / Proteogenomic annotation is an approach that combines proteomic analysis and genomic annotation. The aim of this approach is to provide a more detailed annotation, which is not possible in most of the times when dealing mostly with genes, once that genomic products, with important predicted functions are only important in the organism physiology when they are expressed and translated. There have been occurring several advances in proteogenomic studies and the generation of new data sets has been growing in an exponential wave. With all this growth, the creation of systems able to storing, processing and analyzing all the new knowledge produced is eminent. This study presents the deployment of ProGen AP, a system built with a HTML/PHP5 web interface, a mySQL data management system to store the data and two processing modules (LabKey, with core X!Tandem and QuickMod). In this system, the researcher provides a data set from a proteomic experiment and then the system processes it and returns to the researcher information about the expressed gene, the peptides localization inside the gene that they belong and, also, quantitative information about the peptide and the protein that were identified. Also, the use of an automated pipeline reduces the possibility of making mistakes in input/output of the data when using the intermediate modules. Here, the ProGen AP were applied to perform a proteogenomic annotation of Mycobacterium tuberculosis (MTb). In literature, the MTb genome, strain H37RV, have only 4062 predicted open reading frames (ORFs) and the functional complement of this genome is not completely known. The MTb analysis using ProGen AP, resulted in a list of 154.982 peptides identification, representing a total of 147.334 single peptides. Until now, were identified 2.369 proteins, covering nearly of 58% of the whole MTb genome. Is very important to highlight that, among all the identified proteins until now, most of them are annotated as hypothetical proteins in the MTb genome, so can be affirmed that the results of this project can confirm and validate the existence of all these genomic products. Beside this, 567 peptides were identified as been an N-terminal peptide and 1229 were identified as been a C-terminal, this fact indicates that the prediction of the beginning and the end of translation of those genes are right. All these positive results corroborate that the approach utilized in the ProGen AP is efficient and can be used in studies of other organisms.

A tuberculose, causada por Mycobacterium tuberculosis, ainda é uma emergência de saúde pública global. O surgimento das cepas multirresistentes (MDR) e das cepas extensivamente resistentes (XDR) agravam a situação, diminuindo o número de fármacos disponíveis para o tratamento. Embora a isoniazida seja uma das primeiras moléculas introduzidas no tratamento da tuberculose, diferentes mecanismos de resistência têm sido propostos e o tema ainda não foi totalmente esclarecido. Neste trabalho foi realizada a caraterização estrutural e biofísica de 7 mutantes da proteína InhA identificadas a partir de isolados clínicos de M. tuberculosis resistentes à isoniazida. Os ensaios de calorimetria de titulação isotérmica (ITC) mostram diminuições nos valores da constante de dissociação (Kd) dos mutantes para os NADH em aproximadamente cinco vezes quando comparado com a proteína selvagem. As estruturas cristalográficas dos mutantes de InhA mostram novas moléculas de água que parecem estar envolvidas nas variações entrópicas e entálpicas observadas em dados calorimétricos. Estes resultados corroboram e sugerem que a diminuição na afinidade pelo NADH e a desestabilização do tetrâmero de InhA podem ser fenômenos associados a resistência à isoniazida. / Tuberculosis, caused by the infection of Mycobacterium tuberculosis, still remains as a global health emergency. The emergence of multidrug-resistant strains (MDR) and extensively drug-resistant strains (XDR) strains further aggravates the crisis, reducing the limited number of drugs available for the treatment of the disease. Even though isoniazid was one of the first drugs introduced in the antitubercular therapy, many resistance mechanisms were proposed and the subject is still not clear. In this work, a structural and biophysical characterization of seven mutant InhA proteins identified in clinical M. tuberculosis strains resistant to isoniazid were performed. Isothermal titration calorimetry (ITC) assays showed a decrease in the dissociation constant (Kd) values of the InhA mutants by up to almost five-fold when compared to the wild-type protein. Crystallographic structures of InhA mutants showed new water molecules that appear to be involved in the entropic and enthalpic variations described by the thermodynamic assays. These results corroborate and suggest that the decrease in affinity for NADH and the destabilization of the InhA tetramer may be the phenomena associated to isoniazid resistance.

Made available in DSpace on 2014-07-29T15:26:22Z (GMT). No. of bitstreams: 1 Michelle Cristina.pdf: 992743 bytes, checksum: 88514e8f0562bfda3227e0f6ecd44957 (MD5) Previous issue date: 2011-07-07 / Tuberculosis (TB) affects millions of people every year and it is estimated that one third of world‟s population is infected with Mycobacterium tuberculosis (Mtb). The identification of infection and monitoring of close contacts of TB patients would contribute to disease dissemination control. In the present study the epidemiological characteristics that may facilitate contact‟s infection were evaluated. Additionally, the antibody profile against Hspx from Mtb, among individuals that were in frequent contact of pulmonary tuberculosis (PTB) patients, was investigated as a marker for risk of active disease development. All participants were submitted to tuberculin skin test (TST) and their blood were harvested to determine the antibody profile (IgM/IgG - ELISA). Forty-five PTB patients and 177 contacts were followed for one year. During the follow-up period, only daily contact with patient was associated with positive TST at the moment of diagnosis. Two TST positive contacts developed active PTB, eight contacts converted their TST to positive, 6 individuals presented the booster effect, 44 remained TST negative and 20 dropped out of the study. The IgM-Hspx levels were 0,841±0,40 for TST positive contacts, 0,807±0,32 for TST negative, 0,732±0,218 for TST converted and 0,961±0,48 to contacts that boosted their TST. The IgG-Hspx levels were 0,242±0,10 for TST positive contacts, 0,237±0,10 for TST negative, 0,140±0,02 for TST converted and 0,255±0,22 for contacts that boosted their TST, with significant statistical difference (p=0,019). Our data show that daily contact is associated with TST positivity and the risk of disease development and antibodies against Hspx (both, IgM and IgG) were not associated among recent latently infected contacts of PTB patients. / A Tuberculose (TB) atinge milhões de pessoas anualmente e aproximadamente um terço da população mundial está infectado pelo Mycobacterium tuberculosis (Mtb). A detecção da infecção e o monitoramento dos contatos de TB com alto risco de adoecimento contribuiriam para o controle da disseminação da doença. Propõe-se neste estudo a avaliação de características epidemiológicas que podem contribuir para a infecção e o uso da resposta imune humoral contra o antígeno recombinante Hspx de Mtb para avaliação do risco de adoecimento em indivíduos recém infectados numa coorte de contatos de pacientes com tuberculose pulmonar. Os indivíduos incluídos foram submetidos à prova tuberculínica (PT) e coleta de sangue para dosagem de anticorpos (IgM/ IgG - ELISA). Quarenta e cinco pacientes com TBP e 177 contatos foram recrutados nos Centros de Assistência Integral a Saúde (CAIS) da região metropolitana de Goiânia de 2008 a 2009 e acompanhados por um (1) ano. Durante o acompanhamento, apenas a frequência do contato (diário) com o paciente foi associada ao status da PT no momento do diagnóstico (p=0,006). Dois (2) contatos PT positiva adoeceram, oito (8) contatos converteram a PT para positiva, seis (6) sofreram booster, 44 permaneceram PT negativa e 20 desistiram do estudo. Os níveis de IgM-Hspx encontrados foram de 0,841±0,40 para os contatos PT positivos, de 0,807±0,32 para os PT negativos, de 0,732±0,21 para os PT convertidos e de 0,961±0,48 para os que sofreram booster. Os níveis de IgM-Hspx dos contatos que adoeceram permaneceram inalterados. As dosagens de IgG-Hspx foram de 0,242±0,10 nos contatos PT positiva, de 0,237±0,10 nos PT negativa, de 0,140±0,02 nos PT convertidos e de 0,255±0,22 nos que sofreram booster, sendo significativa a diferença entre as médias (p=0,019). Os resultados obtidos mostraram que contato diário com o paciente está relacionado à PT positiva e que não houve associação entre a presença de anticorpos (IgM/IgG) e risco de adoecimento em contatos recém infectados de pacientes com tuberculose pulmonar.

Os casos de tuberculose vêm crescendo, coincidindo com o aumento dos casos de AIDS, sendo uma das doenças que causa maior número de mortes no mundo. Entretanto, a busca por novos fármacos cresce de forma lenta, principalmente pelo baixo interesse da indústria farmacêutica, uma vez que a tuberculose ocorre predominantemente em países em desenvolvimento. Tiossemicabazonas têm atraído a atenção de muitos pesquisadores por suas propriedades biológicas e farmacológicas. Esta classe de ligantes é biologicamente ativa contra Mycobacterium tuberculosis, agente causador da tuberculose. Este trabalho apresenta a síntese e caracterização de uma série de complexos de metais da primeira série de transição com ligantes tridentados do tipo 2-acetilpiridina-N(4)-R-tiossemicarbazona (Hatc-R). A caracterização dos compostos envolveu diversas técnicas, como análise elementar, espectroscopia na região do infravermelho e do UV-Vis, condutimetria, ressonância magnética nuclear (1H RMN), voltametria cíclica, voltametria de pulso diferencial, medidas de susceptibilidade magnética e difração de raios X em monocristais. Baseando-se nos resultados de caracterização, verificou-se a formação de complexos de geometria octaédrica do tipo [M(atc-R)2], onde M = Mn(II) (R = hidrogênio, metil, etil, fenil, ciclohexil e morfolinil), Ni(II) (R = etil) e Zn(II) (R = etil e fenil); [M(atc-R)2]X, onde M = Fe(III) (R = etil e fenil, X = HSO4-) e Co(III) (R = etil e fenil, X = Cl-), e complexos de cobre (II) com geometrias quadrática-plana [CuCl(atc-Me)] e pirâmide de base quadrada [Cu2(µ-atc-Me)2µ-SO4]. Com isto, foi possível avaliar a influência dos metais, da geometria formada e dos grupos periféricos (R) dos ligantes na atividade anti-Mycobacterium tuberculosis, bem como na citotoxicidade dos complexos obtidos, de modo a potencializar a atividade desta classe de ligantes e obter um complexo com alto índice de seletividade, com potencial para ser futuramente utilizado na terapia da tuberculose. Dentre os 15 complexos obtidos neste trabalho, destacaram-se os compostos [Mn(atc-Ph)2], [Co(atc-Et)2]Cl e [Co(atc-Ph)2]Cl com valores de IS igual à 50. Estes resultados são comparáveis ou melhores que alguns fármacos de primeira e segunda linha usados atualmente no tratamento de tuberculose, tornando estes complexos fortes candidatos à futuros fármacos. / Tuberculosis cases have been rising, coinciding with the increase of AIDS cases, becoming one of the diseases with highest mortality worldwide. However, the research for new drugs grows slowly, mainly due to the low interest of the pharmaceutical industry, once tuberculosis usually occurs in developing countries. Thiosemicarbazones have attracted the attention of many researchers due to their biological and pharmacological properties. This class of ligands is biologically active against Mycobacterium tuberculosis, the pathogenic agent of tuberculosis. This work presents the synthesis and characterization of a set of complexes of the first transition metals series with tridentate ligands of the type 2-acetylpyridine-N(4)-R-thiosemicarbazone (Hatc-R). The characterization involved many techniques such as elemental analyses, infrared and UV-Vis spectroscopies, conductometry, nuclear magnetic resonance (1H NMR), cyclic voltammetry, differential pulse voltammetry, magnetic susceptibility measurement and X-ray diffraction on single crystals. Based on the results of the characterization it was observed the formation of complexes in octahedral geometry of the type [M(atc-R)2], where M = Mn(II) (R = hydrogen, methyl, ethyl, phenyl, ciclohexyl and morpholinyl), Ni(II) (R = ethyl) and Zn(II) (R = ethyl and phenyl); [M(atc-R)2]X, where M = Fe(III) (R = ethyl and phenyl, X = HSO4-) and Co(III) (R = ethyl and phenyl, X = Cl-), and copper (II) complexes with square-planar [CuCl(atc-Me)] and square pyramidal [Cu2(µ-atc-Me)2µ-SO4] geometries. So, it was possible to evaluate the influence of the metals, of the generated geometry and of the peripheral groups (R) of the ligands on the anti-Mycobacterium tuberculosis activity, as well as the citotoxicity of the obtained complexes, in order to enhance the activity of the class of ligands and to obtain a complex with high selectivity index, with potential to be futurely used in tuberculosis therapy. Among the 15 complexes obtained in these work, the most selective compounds were [Mn(atc-Ph)2], [Co(atc-Et)2]Cl and [Co(atc-Ph)2]Cl with values of SI equal to 50. These results are comparable or better than some drugs of first and second line for the currently treatment of tuberculosis, making these complexes strong candidates for future drugs.

Hon, Ching Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 144-157). / Abstracts in English and Chinese. / Examination Committee List --- p.i / Acknowledgements --- p.ii / Abstract --- p.iv / 摘要 --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xiv / List of Figures --- p.xv / List of Abbreviations --- p.xvii / List of Chemicals --- p.xx / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Mycobacterium tuberculosis --- p.1 / Chapter 1.2 --- Tuberculosis --- p.2 / Chapter 1.3 --- Pathogenesis of TB --- p.3 / Chapter 1.3.1 --- Mycobacterial entry to the host macrophage --- p.3 / Chapter 1.3.2 --- Modulation of the host endocytic pathway --- p.4 / Chapter 1.3.2.1 --- The fusion between lysosome and mycobacterial phagosomeis blocked --- p.4 / Chapter 1.3.2.2 --- The endosomal pH of mycobacterial phagosome is preserved --- p.5 / Chapter 1.3.2.3 --- Mtb successfully mediates host cell apoptosis inhibition --- p.6 / Chapter 1.3.3 --- Cell migration and granuloma formation --- p.7 / Chapter 1.4 --- Insights from the complete genome sequence of Mtb H37Rv --- p.9 / Chapter 1.4.1 --- PE protein family --- p.9 / Chapter 1.4.2 --- PPE protein family --- p.10 / Chapter 1.5 --- Interaction between PE and PPE proteins --- p.11 / Chapter 1.5.1 --- Integrated bioinformatics prediction --- p.11 / Chapter 1.5.2 --- In vitro interaction studies of PE25/PPE41 protein complex --- p.12 / Chapter 1.5.3 --- Structural characterization of PE/PPE complex as revealed by PE25/PPE41 crystal structure --- p.14 / Chapter 1.6 --- Biological roles of PE and PPE proteins --- p.15 / Chapter 1.6.1 --- Immunological roles of PE family proteins --- p.15 / Chapter 1.6.2 --- Immunological roles of PPE family proteins --- p.16 / Chapter 1.6.3 --- Immunological roles of PE/PPE protein complex --- p.16 / Chapter 1.7 --- Sub-cellular localization of PE and PPE proteins --- p.17 / Chapter 1.8 --- PE and PPE as exported proteins --- p.18 / Chapter 1.8.1 --- Association of Mtb secreted proteins to pathogenesis of tuberculosis --- p.18 / Chapter 1.8.2 --- Exported PE and PPE proteins --- p.19 / Chapter 1.9 --- Differential expression of PE and PPE genes --- p.20 / Chapter 1.10 --- Objective of project --- p.21 / Chapter CHAPTER 2 --- "CLONING, EXPRESSION, PURIFICATION AND VERIFICATION OF PE15 AND PPE20 AS COMPLEX" --- p.24 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Bacterial expression plasmids for expression of PE or PPE proteins --- p.24 / Chapter 2.1.2 --- E. coli strains --- p.27 / Chapter 2.1.3 --- Reagents and buffers for molecular cloning --- p.27 / Chapter 2.1.4 --- Reagents and buffers for E. coli protein expression --- p.29 / Chapter 2.1.5 --- Buffers for protein purification --- p.30 / Chapter 2.2 --- Methods --- p.31 / Chapter 2.2.1 --- Molecular cloning --- p.31 / Chapter 2.2.1.1 --- Primers --- p.31 / Chapter 2.2.1.2 --- Gene Amplification by Polymerase Chain Reaction (PCR) --- p.33 / Chapter 2.2.1.3 --- Agarose gel electrophoresis --- p.34 / Chapter 2.2.1.4 --- Extraction and purification of DNA from agarose gel by QIAquick Gel Extraction Kit --- p.34 / Chapter 2.2.1.5 --- Restriction digestion of DNA --- p.35 / Chapter 2.2.1.6 --- Purification of DNA by QIAquick PCR Purification Kit --- p.36 / Chapter 2.2.1.7 --- Ligation of DNA and expression vector to produce recombinant plasmid --- p.37 / Chapter 2.2.1.8 --- Transformation of plasmid into E. coli competent cell --- p.38 / Chapter 2.2.1.9 --- PCR Screening of recombinant clones --- p.39 / Chapter 2.2.1.10 --- Plasmid DNA purification using the QIAprep Spin Miniprep Kit --- p.40 / Chapter 2.2.1.11 --- DNA sequencing --- p.41 / Chapter 2.2.2 --- Expression of PE15 and PPE20 proteins --- p.41 / Chapter 2.2.2.1 --- Small scale protein expression of PE 15 and PPE20 proteins --- p.41 / Chapter 2.2.2.2 --- Small scale co-expression of PE 15 and PPE20 proteins --- p.42 / Chapter 2.2.2.3 --- Protein solubility analysis --- p.43 / Chapter 2.2.2.4 --- Large scale expression of PE and PPE proteins --- p.43 / Chapter 2.2.3 --- SDS-PAGE --- p.44 / Chapter 2.2.4 --- Bradford assay --- p.45 / Chapter 2.2.5 --- Protein purification of PE15 --- p.46 / Chapter 2.2.5.1 --- Sonication and extraction of proteins --- p.46 / Chapter 2.2.5.2 --- Protein purification of PE15 by gutathione-sepharose affinity chromatography --- p.46 / Chapter 2.2.5.3 --- Protein purification of PE15 by re-binding to glutathione-sepharose --- p.47 / Chapter 2.2.5.4 --- Protein purification of PE15 by size exclusion chromatography --- p.47 / Chapter 2.2.5.5 --- Protein purification of GST-PE15 --- p.48 / Chapter 2.2.6 --- Protein purification of PPE20(PPE) --- p.49 / Chapter 2.2.6.1 --- Sonication and extraction of proteins --- p.49 / Chapter 2.2.6.2 --- Protein purification of PPE20(PPE) by glutathione-sepharose affinity chromatography --- p.49 / Chapter 2.2.6.3 --- Protein purification of PPE20(PPE) by re-binding to glutathione-sepharose --- p.49 / Chapter 2.2.6.4 --- Protein purification of PPE20(PPE) by size exclusion chromatography --- p.50 / Chapter 2.2.6.5 --- Protein purification of GST-PPE20(PPE) --- p.50 / Chapter 2.2.7 --- Verification of PEPPE protein complex --- p.50 / Chapter 2.2.7.1 --- Size exclusion chromatography --- p.50 / Chapter 2.2.7.2 --- Cross-linking --- p.50 / Chapter 2.2.7.3 --- Pull-down assay --- p.51 / Chapter 2.3 --- Results --- p.52 / Chapter 2.3.1 --- Construction of bacterial expression plasmids for PE15 and PPE20 genes --- p.52 / Chapter 2.3.2 --- Expression of PE15 and PPE20 proteins --- p.54 / Chapter 2.3.2.1 --- Small scale protein expression of PE15 --- p.55 / Chapter 2.3.2.2 --- Small scale protein expression of PPE20 --- p.56 / Chapter 2.3.2.3 --- Small scale co-expression of PE15 and PPE20 proteins --- p.60 / Chapter 2.3.3 --- Large scale purification of PE15 and PPE20(PPE) proteins --- p.66 / Chapter 2.3.3.1 --- Large scale purification of PE15 --- p.66 / Chapter 2.3.3.2 --- Large scale purification of GST-PE15 --- p.68 / Chapter 2.3.3.3 --- Large scale purification of PPE20(PPE) --- p.69 / Chapter 2.3.3.4 --- Large scale purification of GST-PPE20(PPE) --- p.70 / Chapter 2.3.4 --- Verification of PE15/PPE20 complex --- p.71 / Chapter 2.3.4.1 --- Size exclusion chromatography --- p.71 / Chapter 2.3.4.2 --- Cross-linking study --- p.74 / Chapter 2.3.4.3 --- Pull-down assay --- p.77 / Chapter 2.4 --- Discussion --- p.79 / Chapter 2.4.1 --- Expression of PE15 and PPE20 proteins --- p.79 / Chapter 2.4.2 --- Co-expression of PE15 and PPE20 proteins --- p.81 / Chapter 2.4.3 --- Prediction of PE/PPE interaction --- p.82 / Chapter 2.4.4 --- Study ofPE15 and PPE20(PPE) interaction --- p.83 / Chapter CHAPTER 3 --- PRELIMINARY X-RAY ANALYSIS OF PPE20(PPE) PROTEIN CRYSTAL --- p.86 / Chapter 3.1 --- Materials --- p.86 / Chapter 3.1.1 --- Crystallization screening kits --- p.86 / Chapter 3.1.2 --- Crystallization chemicals --- p.86 / Chapter 3.2 --- Methods --- p.87 / Chapter 3.2.1 --- Crystallization screening using Phoenix´ёØ RE --- p.87 / Chapter 3.2.2 --- Optimization of PPE20(PPE) crystals by grid screening --- p.88 / Chapter 3.2.3 --- Optimization using Additive screen for PPE20(PPE) --- p.88 / Chapter 3.2.4 --- X-ray diffraction and data collection --- p.88 / Chapter 3.3 --- Results --- p.89 / Chapter 3.3.1 --- Crystallization screening --- p.89 / Chapter 3.3.2 --- Crystallization optimization --- p.90 / Chapter 3.3.3 --- Preliminary X-ray diffraction analysis --- p.92 / Chapter 3.3.4 --- Attempts to solve the phase by molecular replacement --- p.96 / Chapter 3.4 --- Discussion --- p.97 / Chapter CHAPTER 4 --- ISOLATION OF INTERACTING PARTNERS OF PE15 AND PPE20(PPE) FROM HUMAN MACROPHAGE U-937 --- p.99 / Chapter 4.1 --- Materials --- p.99 / Chapter 4.1.1 --- Mammalian cell line --- p.99 / Chapter 4.1.2 --- Mammalian cell growth medium --- p.99 / Chapter 4.1.3 --- Reagents and buffers for mammalian cell culture --- p.100 / Chapter 4.1.4 --- Reagents and buffers for mass spectrometry sample preparation --- p.100 / Chapter 4.2 --- Methods --- p.101 / Chapter 4.2.1 --- U-937 cell culturing --- p.101 / Chapter 4.2.1.1 --- Thawing U-937 cells --- p.101 / Chapter 4.2.1.2 --- Monitoring cell growth --- p.102 / Chapter 4.2.1.3 --- Cell differentiation --- p.102 / Chapter 4.2.1.4 --- Cell Harvesting --- p.103 / Chapter 4.2.2 --- In-vitro pull-down to identify interacting partners of PE15 or PPE20(PPE) --- p.103 / Chapter 4.2.2.1 --- Preparation of cellular proteins from U-937 cells --- p.103 / Chapter 4.2.2.2 --- Pre-clearing of U-937 supernatant --- p.104 / Chapter 4.2.2.3 --- Pull-down of U-937 cellular proteins with immobilized GST-PE15 --- p.104 / Chapter 4.2.2.4 --- Pull-down of U-937 cellular proteins with immobilized GST-PPE20(PPE) --- p.106 / Chapter 4.2.2.5 --- SDS-PAGE analysis --- p.106 / Chapter 4.2.2.6 --- Silver staining --- p.106 / Chapter 4.2.3 --- Mass- Spectrometry --- p.107 / Chapter 4.2.3.1 --- De-staining of silver stained gel spots --- p.107 / Chapter 4.2.3.2 --- Trypsin digestion --- p.108 / Chapter 4.2.3.3 --- Peptide Extraction --- p.108 / Chapter 4.2.3.4 --- Desalting and concentration of peptide mixture --- p.109 / Chapter 4.3 --- Results --- p.110 / Chapter 4.3.1 --- U-937 differentiation --- p.110 / Chapter 4.3.2 --- In-vitro pull-down --- p.113 / Chapter 4.3.2.1 --- Pull-down with immobilized GST-PE15 --- p.113 / Chapter 4.3.2.2 --- Pull-down with immobilized GST-PPE20(PPE) --- p.116 / Chapter 4.3.2.3 --- Mass spectrometry identification of protein --- p.120 / Chapter 4.4 --- Discussion --- p.122 / Chapter 4.4.1 --- Differentiation of U-937 --- p.122 / Chapter 4.4.2 --- Isolation of PE15 and PPE20(PPE) interacting partners from U-937 --- p.125 / Chapter CHAPTER 5 --- CONCLUSION AND FUTURE PERSPECTIVES --- p.133 / Chapter 5.1 --- Conclusion --- p.133 / Chapter 5.2 --- Future perspectives --- p.137

Leung, Siu Sing. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 92-97). / Abstracts also in Chinese. / Title from PDF title page (viewed on 25, October, 2016).

Mycobacterium tuberculosis est la bactérie responsable de la tuberculose (TB), une infection pulmonaire grave. La TB est un problème majeur de santé publique mondial ; un tiers de la population mondiale est porteur de M. tuberculosis et l'émergence de formes de TB résistantes aux antibiotiques confirme la nécessité de développer de nouvelles approches thérapeutiques. Pour atteindre cet objectif, il est crucial de mieux comprendre les mécanismes infectieux de M. tuberculosis. L'équipe de C. Guilhot s'intéresse aux lipides de l'enveloppe bactérienne impliqués dans la virulence de M. tuberculosis, en particulier lors de l'interaction du bacille avec les macrophages de l'hôte. Un type de lipide en particulier s'est démarqué pour son rôle crucial dans cette interaction : les Dimycocérosates de Phthiocérol (DIM). Malgré leur importance, le mécanisme d'action moléculaire des DIM n'est toujours pas connu. Les objectifs de ma thèse ont été 1) d'étudier le rôle des DIM dans le trafic intracellulaire de M. tuberculosis au sein du macrophage, et 2) de comprendre les mécanismes d'action des DIM dans la virulence. Par une combinaison de méthodes biologiques et biophysiques, nous avons montré que les DIM contribuent à l'induction de la rupture du phagosome et de l'apoptose du macrophage infecté par M. tuberculosis, en collaboration avec un autre facteur majeur de virulence bactérien, ESAT-6. Au niveau moléculaire, nous avons confirmé que les DIM sont transférés dans la membrane du macrophage au contact avec la bactérie, et y induisent une rigidification membranaire locale. Nous avons aussi montré que les DIM dans une membrane sont capables de potentialiser l'activité membranolytique d'ESAT-6 et d'autre(s) facteur(s) encore non identifiés. Les DIM ont un rôle pléiotropique dans l'interaction de M. tuberculosis avec le macrophage. Leur mécanisme d'action impliquerait des modifications des propriétés biophysiques membranaires, modifiant l'activité des effecteurs membranaires bactériens et potentiellement de l'hôte. Ces travaux ouvrent la voie à l'étude du mécanisme d'action d'autres lipides de virulence dont certains pourraient également s'insérer dans la membrane du macrophage. / Mycobacterium tuberculosis is the bacterium responsible for tuberculosis (TB), a severe respiratory disease. TB is a major public health threat; one third of the world's population is latently infected by M. tuberculosis, and the emergence of antibiotic-resistant forms of TB confirms that there is a need to develop new therapeutic approaches to control the spread of TB. However, in order to attain that goal, it is crucial to decipher the infectious mechanisms of M. tuberculosis. Research in the team of C. Guilhot focuses on lipids from the envelope of M. tuberculosis which are involved in virulence, in particular those implicated in the interaction of the bacteria with host macrophages. One of these lipids stands out for its crucial role in this interaction: Phthiocerol Dimycocerosate (DIM). Despite its importance, the molecular mechanism of action of DIM is still unknown. The objectives of my PhD were 1) to study the role of DIM in the intracellular trafficking of M. tuberculosis in macrophages, and 2) to decipher the molecular mechanism of action of DIM. By a combination of biological and biophysical techniques, we showed that DIM contributes to phagosomal rupture and induction of apoptosis in macrophages infected with M. tuberculosis, in collaboration with another major virulence factor: ESAT-6. At the molecular level, we confirmed that DIM is transferred to the membrane of the macrophage on contact with M. tuberculosis and induces a local membrane rigidification around the point of contact with the bacterium. We observed that DIM incorporated in membranes is able to promote the membranolytic activity of ESAT-6, and other yet unidentified bacterial factor(s). DIM has a pleiotropic role in the interaction between M. tuberculosis and macrophages, presumably through alterations of the membrane's biophysical properties that influence the activity of membrane effectors from both the bacteria and the host. Thus, this work paves the way for the study of the mechanisms of action of other virulence lipids, some of which could also be inserted in the macrophage membrane.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 19, 2008). Includes bibliographical references.