Köln, Universiẗat, Diss., 2002.

M. Tech. / Internationally, 9.2 million new cases and 1.7 million deaths occurred from tuberculosis (TB) in 2006. The vast majority of TB deaths occurred in the developing world, that is, Asia and Africa (WHO, 2008). The risk of infection is worsened by overcrowding of healthcare facilities which share re-circulated air without high efficiency particulate arrestance (HEPA) filtration or effective decontamination devices. With emerging and re-emerging infectious agents, the importance of the microbial influence on indoor air quality is gaining momentum around the world. The transmission of Mycobacterium tuberculosis (MTB) is a recognized occupational hazard and the mode of airborne transmission in risk settings needs to be investigated. The current study examined the efficacy of Polymerase Chain Reaction (PCR) for the early detection of airborne MTB using three types of filters: Polytetrafluoroethylene (PTFE), Polycarbonate (PC) and Gelatine, and a sedimentation gel (impaction method). A total of 520 samples, 68 internal positive controls and 68 internal negative controls were tested using two different PCR detection methods. The four different sampling types were each exposed to samples containing an avirulent strain of MTB (H37Ra) and negative controls exposed to aerosolized distilled water in an uncontrolled environment. The air was filtered at a flow rate of 2.5 L/min for a specified time. The filter membranes and sedimentation gel were removed from their respective holders, washed and analysed using conventional and real time (RT) PCR. An additional step using magnetic bead separation was used to assess its performance in overcoming inhibition. The sampling methods used included an in - house preparation of sedimentation gel. This sampling method was used in a study by Vadrot and colleagues in 2004 for detection of airborne MTB using the PCR method. Commercially available filters that were used as sampling methods included PTFE, PC and Gelatine. The detection methods included conventional PCR which detected the MTB complex in three hours, RT-PCR which detected MTB species in 70 minutes and RT-PCR coupled to magnetic bead separation (RT-PCR M) which detected MTB species in 90 minutes. The magnetic bead separation method purified the nucleic acids in the sample by eliminating the inhibitors that were present. The current study showed that by using the magnetic bead capture assay in conjunction with RT-PCR, gave excellent results of 100% sensitivity and 100% specificity when using PTFE, PC and Gelatine sampling methods. The sedimentation gel showed results of 90% sensitivity and 90% specificity. The Gelatine sampling method results showed 100% inhibition with conventional PCR. In conclusion, the use of conventional PCR is limiting for the detection of airborne MTB, possibly due to inhibition factors. In addition, the PTFE filter demonstrated excellent results for all detection methods used. The sedimentation gel did not perform well with PCR and RT-PCR, however gave excellent results with RT-PCR M. The PC filter can be considered the second sampling method of choice after PTFE filter, showing superior results for all diagnostic parameters using RT-PCR M, followed by RT-PCR and then PCR. The greatest application of using this validated method will be in the area of infection control. Environmental practitioners and occupational hygienists would be able to use this method to evaluate environmental control measures and monitor the air quality in healthcare facilities and other workplaces.

Mycobacterium tuberculosis complex (MTBC) is a causative agent of tuberculosis (TB) in humans and animals. The burden of tuberculosis in South Africa is worsened by the concurrent epidemic of HIV. The dynamic of TB epidemics has been investigated and yet little data has been given about the Eastern Cape, particularly Port Elizabeth. The study aimed to investigate the prevalence of drug resistant MTBC and to determine the mutations causing resistance in Port Elizabeth. One hundred and ninety (190) DNA samples isolated from sputum specimen in humans suspected of having TB were amplified using the Seeplex® MTB Nested ACE detection assay. To differentiate Mycobacterium tuberculosis complex (MTBC) members for surveillance purposes a multiplex polymerase chain reaction (PCR) method was done based on genomic regions of differences such as RD1, RD1mic, RD2seal, RD4, RD9 and RD12. Target genes known to confer resistance to first and second-line drugs were amplified and the amplicons sequenced using Big Dye Terminator DNA sequencing kit v3.1 (Applied Biosystems, UK). The patient’s demographic profiles were obtained from the National Health Laboratory Service (NHLS). All hundred and ninety DNA samples tested positive for MTBC using the Seeplex® MTB Nested ACE assay. Results show a high prevalence of extensive drug resistant TB in Port Elizabeth, Eastern Cape Province. One hundred and eighty four (184) DNA isolates were used in the identification of different MTBC species. We ended up working with 184 DNA isolates because we ran out of DNA, and we could not go back to isolate DNA from the affected individuals due to the fact that some patients died, while some have been released to go to their homes. From the 184 DNA isolates 45 (24.5%) isolates were identified to be M. tuberculosis, 94 isolates (51.1%) to be M. bovis BCG and 3 isolates (1.6%) to be M. cannetti. Sequencing results show the position of mutation in each DNA isolate; however in the study we got resistance to MDR to be 100% and 42% pre-XDR while 58% was XDR. These results raise an alarm for the prevalence MDR in MTBC from Port Elizabeth. This is a serious health concern which calls for a need to strategise on the identification of extensive drug resistant TB patients from multi-drug resistant TB patients and ensure monitoring of their treatment.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) kills 1.6 million people worldwide every year, and there is an urgent need for targeting host-pathogen interactions as a strategy to reduce mycobacterial resistance to current antimicrobials. Non-coding RNAs are emerging as important regulators of numerous biological processes and avenues for exploitation in host-directed therapeutics. Although long non-coding RNAs (lncRNAs) are abundantly expressed in immune cells, their functional role in gene regulation and bacterial infections remains under-studied. Here, we identify an immunoregulatory, lincRNA-MIR99AHG, which is upregulated in macrophages upon IL-4/IL-13 stimulation and downregulated after Mtb infection and in active TB patients. To evaluate the functional role of lincRNA-MIR99AHG, we employed antisense GapmeR-mediated lncRNA knockdown experiments. Knockdown of lincRNA-MIR99AHG with LNA-GapmeRs significantly reduced intracellular Mtb growth in mouse and human macrophages and reduced proinflammatory cytokine production. In addition, in vivo treatment with MIR99AHG LNA-GapmeRs reduced the mycobacterial burden in the lung and spleen. In vivo LNA-GapmeR treatment experiments demonstrated a role of lincRNA-MIR99AHG as a regulator of macrophage polarization and a host-mediated response post Mtb infection. Further, lincRNA-MIR99AHG translocated to the nucleus and interacts with a high affinity to hnRNPA2/B1 following IL-4/IL-13 stimulation and Mtb infection. Together, these findings identify lincRNA-MIR99AHG as a positive regulator of inflammation to promote Mtb growth and a possible for host-directed targeting or for adjunctive therapeutics against TB.

Thesis (M.S.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Pathology. Includes bibliographical references.

泛基因组的概念来源于比较分析同一微生物物种的多个基因组。泛基因组分析已经被用于研究病原微生物基因组的变化，并且揭示了与菌株进化和宿主适应相关的特异基因。目前泛基因组研究主要集中在估计不同物种的泛基因组大小。但是对泛基因组的结构进行进化分析的生物信息方法还有待开发，以便研究不同基因在不同菌株的进化，比如基因的获得或者丢失，或者共同进化的基因簇。这样的分析方法可以把基因和菌株之间的表型关联起来，为进一步的生物学实验提供线索。 / 为了研究结核分枝杆菌种和分枝杆菌属泛基因组进化的规律并揭示其生物学意义，本论文开发了两种泛基因组数据分析的生物信息学方法。第一个是基于局部最大简约计算的祖先状态重构算法。它被用来分析结核分枝杆菌北京型全基因组水平插入／缺失序列（indels）的进化。分析表明基因组退化不仅塑造了该物种不同亚种的形成，并且也塑造了同一亚种不同亚型的分化，比如北京型。该分析还找出了北京型全基因组水平的RD区域和各种被中断的基因，这些基因可能同北京型的毒力进化相关。同时，该算法提供了另一个理解简约分析的视角；该视角可以把统计分析引入到该算法中。本论文提出的第二个模型是基于泛基因组进化的基因聚类模型。通过计算泛基因组中不同基因家族的分布频率，结合基于图论的聚类算法，该模型可以找出泛基因组中共同进化的基因聚类。对分枝杆菌属的泛基因组进行聚类分析发现了不同类别的基因簇，它们与不同分枝杆菌种的表型进化相关。这些结果说明了，一方面结核分枝杆菌在进化过程中丢失大量环境相关的基因；另一方面，它可能通过水平基因转移获得一些基因，特别是PE/PPE基因家族。因此，结核分枝杆菌可能是通过不断的基因组收缩，从一个环境菌种进化为与宿主共进化的病原菌。 / 总地来说，上面的两种方法能够被有效地用于结核分枝杆菌种和分枝杆菌属的泛基因组分析。 将来的工作可以考虑进一步引进随机模型；同时需要建立分枝杆菌的泛基因组数据库，以面对大规模测序的需求。 / Comparative analysis of multiple genomes of the same microbial species has led to the concept of pangenome to characterize the variations of gene content in different strains and to study their relationship to strain phenotype variations. Pangenome studies of microbial pathogens have identified strain-specific genes that may play roles in the evolution and adaptation of the pathogens. In previous studies, much attention was paid to estimate the size of the pangenome of different microbial species. But it is also important to develop bioinformatic methods for analyzing the evolution of the pangenome of a species, such as gene gain and loss or coevolution of clusters of genes, which may help to associate genotype variations with phenotype variations of a microbial species, and thus provides biological insights for further studies. / In this thesis, to analyze the pangenome consisting of complete mycobacterial genomes from public database and additional five Mycobacterium tuberculosis (MTB) Beijing genotype genomes sequenced by our own project, two bioinformatic approaches have been developed. The first is a local parsimony ancestral state reconstruction method, which was used to analyze genome-wide indels evolution of the MTB Beijing genotype. The key finding was that reductive evolution shaped the formation of not only different MTB species, but also different subspecies or genotypes, such as the Beijing genotype, for which genome-wide deletions of large RDs and disruption of individual genes were identified. This finding might have implications for the virulence evolution of the Beijing genotype. The method also provides an alternative perspective to understand parsimony analysis in phylogenetics, which can be used to incorporate statistical analysis into the method. / The second approach developed is a pangenome phyletic model for analyzing the coevolution of genes in the pangenome of a microbial species. This phyletic model calculates coevolution scores of gene frequencies in a pangenome. And graph-based clustering is used to identify coevolved clusters of genes. Applying this method to the genus Mycobacterium helped us to identify various gene clusters, from conserved core clusters of housekeeping genes to species-specific clusters, including genes related to pathogenesis. The key finding was that different MTB species have arose from their mycobacterial ancestor mainly by loss of many environmental related genes. On the other hand, gain of genes has also occurred within the MTB genomes, especially the clusters of the PE/PPE genes. This finding implied that the MTB species have undergone reductive evolution from an environmental species to adapt to and coevolve with their specific hosts. / In conclusion, the two methods were shown to be powerful in analyzing the pangenome of the MTB species and also of the Mycobacterium genus, and have provided useful insights into their genome and virulence evolution for further studies, including both pathogenesis related genes and genotyping genetic markers. Future works in that direction is to introduce stochastic models of gene evolution into these two methods. Finally, this work indicated that pangenome modeling is critical and can provide a good starting point for comprehensive pangenome sequencing of mycobacteria. Therefore, a database of Mycobacterial genomes for integrative pangenome annotation and evolutionary analysis should be developed. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhou, Haokui. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 121-135). / Abstracts also in Chinese.

Orientador: Fernando Rogério Pavan / Banca: Patricia Bento da Silva / Banca: Jean Leandro dos Santos / Resumo: A tuberculose (TB) é uma doença infecciosa que tem como principal patógeno o Mycobacterium tuberculosis e continua sendo um importante problema de saúde pública mundial, exigindo o desenvolvimento de estratégias para o seu controle. Em 2011 foram notificados 8,7 milhões de casos da doença no mundo. Ao longo dos anos o cenário da doença não tem se mostrado otimista, devido ao aumento de número de casos de TB multi resistente a fármacos (TB-MDR) e o surgimento de cepas de resistência estendida (TB-XDR). A pesquisa de novos fármacos, em um contexto geral, apresenta-se como um enorme desafio científico para a era moderna. Neste sentido, a Química Inorgânica Medicinal tem se mostrado uma ferramenta bastante promissora. Este trabalho objetivou a caracterização da atividade anti- M. tuberculosis intra e extracelular e a citotoxicidade de 158 compostos de coordenação com metais. A citotoxicidade usando linhagens celulares de macrófago (J774A.1) e células epiteliais (VERO) também foi investigada. Diante Os resultados demonstraram que 16 compostos apresentaram uma alta seletividade, ou seja, alta atividade contra o bacilo da tuberculose e baixa citotoxicidade frente às linhagens testadas. Quatro desses 16 compostos selecionados foram analisados quanto a atividade intracelular; dos quais 2 compostos de coordenação de Co (cobalto) mostraram-se promissores quanto a esta atividade. Com base nos resultados encontrados mais estudos serão realizados a fim de garantir a eficácia e segurança desses novos compostos de coordenação candidatos à fármacos para tratamento da tuberculose / Abstract: Not available / Mestre

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 161-176). Also available on the World Wide Web.

Thesis (Ph. D.)--Washington State University, December 2008. / Title from PDF title page (viewed on July 6, 2009). "School of Molecular Biosciences." Includes bibliographical references.

A tuberculose (TB) é uma séria doença infecciosa causada por Mycobacterium tuberculosis, e constitui um importante problema de saúde pública em todo o mundo. Novas drogas e vacinas de segunda geração são urgentemente necessárias para o controle da TB, mas a complexa biologia de M. tuberculosis tem dificultado o desenvolvimento de estratégias terapêuticas inovadoras. A manipulação genética de M. tuberculosis também é complicada, mas, atualmente, técnicas novas e mais eficientes de troca alélica permitem o estudo detalhado de diversos genes micobacterianos. No presente trabalho, os genes da corismato mutase (CM) e fosforilase de nucleosídeos purínicos (PNP) de M. tuberculosis e Mycobacterium smegmatis foram estudados. A CM catalisa a conversão de corismato em prefenato na rota de biossíntese dos aminoácidos aromáticos fenilalanina e tirosina em bactérias, fungos e plantas. Dois genes de CMs monofuncionais (aroQ e *aroQ) foram identificados, clonados, expressos e bioquimicamente caracterizados em ambas as espécies de micobactérias. Esses genes também foram investigados usando uma metodologia de recombinação homóloga e ensaios de atividade promotora. Os resultados indicam que os genes aroQ são provavelmente essenciais ao crescimento in vitro de M. tuberculosis e M. smegmatis, enquanto uma linhagem mutante para o gene *aroQ de M. smegmatis pôde ser obtida. A PNP catalisa a interconversão e reciclagem de bases, nucleosídeos e nucleotídeos purínicos na via de salvamento das purinas. A construção de mutantes para o gene deoD (que codifica a PNP) de M. tuberculosis e M. smegmatis, usando um método eficiente de troca alélica em duas etapas, não foi possível. Assim, sugere-se que, nas condições testadas, o gene deoD seja essencial ao crescimento in vitro dessas micobactérias. A identificação de genes essenciais é de fundamental importância, pois indica tanto a relevância biológica dos mesmos como, no caso de M. tuberculosis, que seus produtos constituem alvos moleculares interessantes para o desenvolvimento de novas drogas antimicobacterianas. / Tuberculosis (TB), a serious infectious disease caused by Mycobacterium tuberculosis, still remains a public health problem in the world. New drugs and second generation vaccines are urgently required to control TB, but the complex biology of M. tuberculosis has hindered the development of novel therapeutic tools. Genetic manipulation of M. tuberculosis is also difficult, but currently new, more efficient gene replacement techniques have allowed detailed studies of many mycobacterial genes. In the present work, the chorismate mutase (CM) and purine nucleoside phosphorylase (PNP) genes from M. tuberculosis and Mycobacterium smegmatis were studied. CM catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway that forms phenylalanine and tyrosine in bacteria, fungi, and plants. Two monofunctional CM genes (aroQ and *aroQ) were identified, cloned, expressed, and biochemically characterized in both mycobacteria. Those genes were also investigated by homologous recombination methods and promoter activity assays. AroQ genes seem to be essential for the growth of M. tuberculosis and M. smegmatis, while an *aroQ mutant strain could be generated in M. smegmatis. PNP promotes the interconversion and recycling of purine bases, nucleosides, and nucleotides in the purine salvage pathway. Construction of deoD (which codes for PNP) deletion mutants of M. tuberculosis and M. smegmatis using an efficient two-step gene replacement technique was not possible. Thus, it is suggested that deoD is also essential for mycobacterial growth under the conditions tested. The identification of essential genes is of great importance, both because it indicates their biological significance, and because, with pathogens like M. tuberculosis, these may also indicate attractive molecular targets for the development of new antimycobacterial drugs.