O presente estudo tem como principal objetivo o emprego da reação em cadeia da polimerase (PCR) na elucidação da etiologia dos derrames pleurais dos pacientes com suspeita de pleurite tuberculosa, comparando-o com as técnicas diagnósticas disponíveis na atualidade. O diagnóstico da tuberculose pleural costuma ser feito por meio dos dados clínicos e radiológicos, teste tuberculínico, exames bioquímicos, microbiológicos, e citológicos do líquido pleural e da histopatologia de fragmento de pleura obtido por punção-biópsia. Ainda assim estes métodos apresentam muitas limitações, dentre as quais a baixa sensibilidade e o longo período necessário para a confirmação do diagnóstico. Estudamos 58 amostras de 45 pacientes. Destes, 16 pacientes tiveram diagnóstico clínico ou laboratorial confirmado de tuberculose, totalizando 22 amostras. Consideramos como caso de tuberculose todos os pacientes com baciloscopia, cultura ou histopatológico positivos no líquido pleural ou outro material biológico, ou que tenham apresentado melhora clínica após a introdução do tratamento empírico. Das 22 amostras com o diagnóstico de tuberculose, a PCR foi positiva em 6 amostras de 6 pacientes. A reação foi positiva em 1 amostra de um paciente em que o diagnóstico de tuberculose foi descartado. A sensibilidade, especificidade e os valores preditivo positivo e negativo nos 16 pacientes com diagnóstico de tuberculose foram 31,3%, 96,6%, 83% e 71% respectivamente. A PCR, apesar de ter apresentado uma baixa sensibilidade, demonstrou uma especificidade ótima em relação aos métodos convencionais. Está técnica pode ter utilidade como método complementar às técnicas disponíveis, na tentativa de obter um diagnóstico mais precoce e preciso nos casos de tuberculose pleural. / The present study evaluates the use of the polimerase chain reaction (PCR) to confirm the etiology of pleural effusions in patients with suspicion of pleural tuberculosis, comparing it to the current available techniques. The diagnosis of pleural tuberculosis is made through the combination of clinical data, radiologic findings, biochemical, microbiological and cytological examination of the pleural fluid and by the pathology of pleural fragment obtained by biopsy. However, these methods present a lot of limitations, including a low sensitivity and a long incubation period to confirm the diagnosis by culture. We studied 58 samples of 45 patients with pleural effusion. Of these, 16 patients had clinical diagnosis or confirmed by laboratory, in a total of 22 samples. We defined as case of tuberculosis all patients with culture or positive pathology in the fluid pleural or other biological material and those with clinical improvement after empirical treatment. Of the 22 samples with the tuberculosis diagnosis PCR was positive in 6 samples of 6 patients. The reaction was positive in a sample of a patient whose diagnosis of tuberculosis was later discarded. The sensitivity, specificity, positive predictive value and negative predictive value in the 16 patients with tuberculosis diagnosis were 31,3%, 96,6%, 83% and 71% respectively. In spite of having presented a low sensitivity, PCR specificity was greater than the conventional methods. This technique can be useful as an additional method to the available techniques, in the attempt of obtaining a more precocious and precise diagnosis in the cases of pleural tuberculosis.

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-08-21T14:02:39Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Kely M. B. Pinto.pdf: 5466832 bytes, checksum: 967bb4c1f999d5dcdfb6ecc4a68f6116 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-08-21T14:03:11Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Kely M. B. Pinto.pdf: 5466832 bytes, checksum: 967bb4c1f999d5dcdfb6ecc4a68f6116 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-08-21T14:03:24Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Kely M. B. Pinto.pdf: 5466832 bytes, checksum: 967bb4c1f999d5dcdfb6ecc4a68f6116 (MD5) / Made available in DSpace on 2017-08-21T14:03:24Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Kely M. B. Pinto.pdf: 5466832 bytes, checksum: 967bb4c1f999d5dcdfb6ecc4a68f6116 (MD5) Previous issue date: 2017-04-17 / Tuberculosis is a disease caused by Mycobacterium tuberculosis that remains one of the deadliest diseases in the world, being necessary efforts to combat it. The conventional light bacilloscopy is a sputum smear microscopic examination to diagnose tuberculosis. Automated applications in microscopic images, such as automated bacilloscopy, face a big problem: blurriness in the images acquired. Thus, it is emphasized the importance of using multi-focus image fusion methods to overcome this problem. In this context, this work aims to propose a method to multi-focus image fusion applied to sputum smear microscopy images in order to obtain images where the greatest number of bacilli is in focus and application of sharpness enhancement to the resulting images. This factor has great importance to enabling automated sputum smear microscopy. To find out existing schemes, we performed simulations and analyzes in some of the techniques in the literature. When it was analized the performance of these techniques through the quality metrics Multichanel Edge Information Preservation and Variance, it was possible to propose four new methods based on the best techniques in the literature, among which, the method with the best performance was choose to investigate configurations that would guarantee the best quality of processed. Has also been proposed the use of an unsharp masking to the post-processing of the new methods, and is shown through examples the effectiveness of this process in increased sharpness and visual appearance. As future work, it is suggested to evaluate this method using grayscale images to verify the impact on the processing time and the quality of the fused images, also the evaluation of the applicability of the image registration. / A tuberculose é uma doença causada pelo Mycobacterium tuberculosis que persiste como uma das doenças transmissíveis mais mortais do mundo, sendo necessários esforços para combatê-la. A baciloscopia de campo claro é um exame utilizado para diagnosticar a tuberculose. Aplicações automatizadas em imagens microscópicas, como a baciloscopia de campo claro automatizada, enfrentam um grande problema: o efeito borrado nas imagens adquiridas. Dessa forma, ressalta-se a importância da utilização de técnicas de fusão de imagens multifocais para contornar este problema. Neste contexto, este trabalho tem o objetivo de propor um método para fusão de imagens multifocais aplicados a imagens baciloscópicas de campo claro, visando a obtenção de imagens onde o maior número possível de bacilos esteja em foco, e emprego de realce às imagens resultantes. Este fator é de grande importância para viabilização da baciloscopia automatizada. Para levantamento de esquemas de fusões existentes, foram feitas simulações e análises com algumas das técnicas presentes na literatura. Ao analisar o desempenho destas técnicas através das métricas de qualidade Multichanel Edge Information Preservation e Variância, foi possível propor quatro novos métodos baseados nas melhores técnicas presentes na literatura, dentre os quais, o método de melhor desempenho foi escolhido para investigação de configurações que garantissem a melhor qualidade das imagens processadas. Foi proposto ainda a utilização da máscara de nitidez como pós-processamento adicional, sendo mostrado através de exemplos a eficiência deste processo no aumento de nitidez e aspecto visual. Como trabalhos futuros, sugere-se avaliação do referido método utilizando imagens em escala de cinza para verificar o impacto no tempo de processamento e a qualidade das imagens, além da avaliação da aplicabilidade do registro das imagens multifocais.

Submitted by Lúcia Brandão (lucia.elaine@live.com) on 2015-12-11T19:01:49Z No. of bitstreams: 1 Dhêmerson - Dhêmerson Souza de Lima.pdf: 2134258 bytes, checksum: 027d2a19329c1030eb634da31db37ae6 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-01-19T14:57:53Z (GMT) No. of bitstreams: 1 Dhêmerson - Dhêmerson Souza de Lima.pdf: 2134258 bytes, checksum: 027d2a19329c1030eb634da31db37ae6 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-01-19T15:02:05Z (GMT) No. of bitstreams: 1 Dhêmerson - Dhêmerson Souza de Lima.pdf: 2134258 bytes, checksum: 027d2a19329c1030eb634da31db37ae6 (MD5) / Made available in DSpace on 2016-01-19T15:02:05Z (GMT). No. of bitstreams: 1 Dhêmerson - Dhêmerson Souza de Lima.pdf: 2134258 bytes, checksum: 027d2a19329c1030eb634da31db37ae6 (MD5) Previous issue date: 2015-05-08 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Tuberculosis (TB) is a serious public health problem, considered as a priority by the government of Brazil since 2003. This disease is endemic in low and middle-income countries, mainly affecting the population living in urban peripheries when associated with poor living conditions, malnutrition and drug abuse. Brazil is one of 22 countries contributing to the global high TB burden; it is on 16th position in number of TB cases. In 2014, Amazonas state had the highest national incidence rate of TB (68.4 per 100.000 inhabitants). Poverty, social exclusion, operational difficulties for diagnosis and treatment of TB are crucial to maintaining the high rate of incidence. In addition, the host immunogenic factors are associated with TB. Human Leukocyte Antigen (HLA) genes are associated with susceptibility or resistance to TB. This study verifies the frequency of HLA-DRB1*04 allele and subtypes in 622 subjects (316 patients with pulmonary TB and 306 controls). HLA-DRB1*04 was more frequent in TB patients (187/316; 59,2%) than control group (101/306; 33,0%). In this study, nine subtypes of HLA-DRB1*04 were identified. The subtype HLA-DRB1*04:11:01 was associated with susceptibility to pulmonary TB (p = 0.0019; OR = 2.23; 95% CI = 1.34 to 3.70), while HLA-DRB1*04:07:01 was associated with protection (p < 0.0001; OR = 0.02; 95% CI = 0.001 to 0.33) and HLA-DRB1*04:92 was associated with transmission to pulmonary TB disease (p = 0.0112; OR = 8.62; 95% CI = 1.63 to 45.5). These results suggest three subtypes of HLA-DRB1*04 as potential immunogenetic markers in TB and can help in better understanding of mechanisms involved in disease, as well as the reasons for high rates of TB incidence in Amazonas state. / A tuberculose (TB) é um grave problema de saúde pública, considerada como prioridade pelo governo do Brasil desde 2003. A doença é endêmica nos países de baixa e média renda, acometendo principalmente a população que reside nas periferias urbanas (favelas e invasões), estando associada às más condições de moradia, à alimentação, ao saneamento básico e ao uso de drogas. O Brasil é um dos 22 países que contribuem com as maiores cargas de TB no mundo, ocupando a 16.a posição em número absoluto de casos. O Estado do Amazonas, em 2014, apresentou o maior coeficiente de incidência nacional de casos de TB (68,4 por 100 mil habitantes). Miséria, exclusão social, dificuldades operacionais de diagnósticos e tratamento da TB são preponderantes para manutenção da alta taxa de incidência. Além disso, os fatores imunogenéticos do hospedeiro são descritos e associados à suscetibilidade ou à resistência da doença. Dentre os genes mais fortemente associados à TB, estão os do Antígeno Leucocitário Humano (HLA). No presente estudo, foi investigada a frequência do alelo HLA-DRB1*04 em 622 indivíduos, 316 pacientes com TB pulmonar e 306 controles. O HLA-DRB1*04 foi frequente nos pacientes (187/316; 59,2%), quando comparado aos dados dos controles (101/306; 33,0%). Neste estudo, foram identificados nove subtipos de HLADRB1*04; destes, o HLA-DRB1*04:11:01 foi associado à suscetibilidade (p = 0,0019; OR =2,23; IC 95% = 1,34 – 3,70) e à transmissão de TB pulmonar, enquanto o HLA-DRB1*04:07:01 foi associado à proteção (p < 0,0001; OR = 0,02; IC 95% = 0,001 – 0,33), e o HLA-DRB1*04:92 foi associado à transmissão da doença (p = 0,0112; OR = 8,62; IC 95% = 1,63 – 45,5). Esses resultados sugerem três subtipos do HLA-DRB1*04 como potenciais marcadores imunogenéticos na TB, os quais podem ajudar na compreensão dos mecanismos envolvidos na doença, bem como esclarecer os motivos das altas taxas de incidência de TB no Estado do Amazonas.

O estudo pretendeu abordar a imunidade humoral na Tuberculose. Foi um estudo de teste diagnóstico que avaliou um antígeno específico, constituinte da parede celular da micobactéria, denominado LIPOARABINOMANNAN (LAM), proveniente de Cambridge, MA, USA da Companhia Dyna-Gen. O objetivo principal do estudo foi a detecção de anticorpos IgG anti-LAM em casos de tuberculose pulmonar, extrapulmonar e formas combinadas da doença. A casuística total compreendeu 173 pacientes portadores de tuberculose, sendo a mesma confirmada por métodos bacteriológicos e/ou anatomopatológicos de biópsias de diversos órgãos em 114 casos (65,8%). Em 46 casos (26,5%) a doença se confirmou por rigorosos critérios clínicos, radiológicos e de seguimento após tratamento adequado. Cento e quinze pacientes eram do sexo masculino (66,5%) e 58 do sexo feminino (33,5%). Cento e trinta e um eram brancos (75,7%), 24 negros (13,9%) e 18 mistos (10,4%). O total de formas pulmonares foi de 88 casos (51%), sendo 81 (46,8%) formas bacilíferas e 7 casos (4,0%) não-bacilíferas. Dos casos com baciloscopia direta negativa, 3 apresentaram culturas positivas, 2 culturas negativas e em 2 casos a mesma não foi realizada. Formas extrapulmonares compreenderam 71 casos (41%) com predomínio de forma miliar, ganglionar, pleural e do SNC. A combinação de ambas as formas ocorreu em 14 casos (8,1%). Radiologicamente, houve predomínio de lesões escavadas (30,1%), consolidação (13,9%), padrão miliar (11%) e exame radiológico normal (11%), além de outros achados. Da série, 118 pacientes eram HIV negativos (68,2%) e 55 eram HIV positivos (31,8%). As principais comorbidades associadas foram Diabetes Melittus (DM), Alcoolismo, Cardiopatia e Neoplasia, entre outras. Exames culturais foram realizados em 145 pacientes, sendo que em 72 casos a cultura foi positiva (41,6%) e foi negativa em 10 casos (5,8%). Dos 72 exames culturais positivos, o teste do MycoDot foi positivo em 47 casos (65,2%) e negativo em 25 (34,7%). Em 10 exames culturais negativos, o mesmo foi positivo em 6 casos (60%) e negativo em 4 casos (40%). Em 63 exames culturais não realizados, o teste do MycoDot foi positivo em 46 casos e negativos em 17. Os resultados do teste MycoDot na série total foram: positivos em 120 pacientes (69,4%) e negativos em 53 pacientes (30,6%). As formas pulmonares bacilíferas, não bacilíferas, extrapulmonares e combinadas apresentaram sensibilidade de 74,1%, 85,7%, 63,4% e 64,3% respectivamente. O grupo controle foi de 77 indivíduos assim distribuídos: 41 sadios, 16 portadores de lesões residuais de tuberculose, 6 sadios com BCG prévia, 6 sadios sem BCG prévia e 8 com outras comorbidades. O resultado do teste MycoDot foi negativo em 73 casos (94,8%) e positivo em 4 casos (5,2%). A sensibilidade da casuística total foi de 69,4%, a especificidade foi de 94,8%, o valor preditivo positivo (VPP) foi de 96,8% e o valor preditivo negativo (VPN) foi de 57,9%. Dos pacientes HIV positivos a sensibilidade foi de 61,8% e a especificidade foi de 100%. Nos pacientes HIV negativos a sensibilidade foi de 72,9% e a especificidade foi de 94,7%. Concluiu-se que o Teste MycoDot é de fácil realização, baixo custo, podendo ser útil como uma ferramenta adicional para o diagnóstico da tuberculose. / This study evaluated the humoral immunity in tuberculosis. It was a study of diagnostic test using a specific antigen, designed Lipoarabinomannan (LAM) provenient of Cambridge, MA, USA, Dyna-Gen Company. This antigen is a cell wall component of mycobacteria. The major objective was to study anti-LAM IgG antibodies in cases of pulmonary, extrapulmonary and associated forms of tuberculosis. The casuistic was 173 patients with tuberculosis, confirmed by bacteriologic and/or anatomopathologic methods in 114 cases (68,8%). In the other cases, the tuberculosis was confirmed by clinical, radiological features and follow up of the patients after chemoterapy. The series presented 115 males, 58 females; 131 was caucasian, 24 black and 18 was non-caucasian. The total pulmonary forms was 88 cases (51%), 81 with positive AFB in sputum and 7 with negative AFB in sputum. Three have positive culture, two with negative cultures. Extrapulmonary tuberculosis was found in 71 cases (41%). The predominant forms of X-ray of the thorax was miliar and necrositing lesions. HIV-positive patients were 118 and negative 55. The MycoDot test results were: positive in 120 patients (69,4%) and negative in 53 patients (30,6%). In control group, MycoDot was negative in 73 cases (94,8%) and positive in 4 cases (5,2%). The final results were: sensitivity of 69,4%, specificity of 94,8%, the positive predictive value was 96,8% and the negative predictive value was 57,9%. In HIV-positive patients, the sensibility was 61,8% and the specificity was 100%. The conclusions were the MycoDot test is easy and ideal for use in the diagnosis of active tuberculosis in conjunction with the other methods.

Submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry, Durban University of Technology, Durban, South Africa, 2017. / In drug discovery and development of anti-tubercular therapeutics, it is necessary to study the physiology and genetics of the molecular mechanisms present in the Mycobacterium tuberculosis. The virulence of M. tuberculosis is attributed to its unique genome, which contains a high frequency of glycine-rich proteins and genes involved in the metabolism of the fatty acids. Consequently, the presence of a diversity of the pathogenic pathways such as acid tolerance and drug resistance mechanisms in M. tuberculosis makes the treatment of Tuberculosis (TB) challenging. However, the molecular basis of the virulence factors involved in the pathogenesis is not fully understood. Accordingly, the current study focuses on better understanding of the pathogenic proteins present in this bacterium using available computational techniques. In South Africa, there is an alarming increase in the drug-resistant TB in HIV co-infected patients, which is one of the biggest challenges to the current anti-tubercular therapies. An extensive literature search showed that the mutations in the virulent proteins of M. tuberculosis resulted in the development of drug tolerance in the pathogen. The molecular and genetic studies identified frequently occurring point mutations associated with the drug resistance in proteins of M. tuberculosis. Despite the efforts, TB infection is still increasing because different pathogenic pathways in the bacterial system are still undiscovered. Therefore, this study involves an in silico approach aimed at the identification of novel drug resistance implicated point mutations. The site- directed mutations leading to the development of resistance against four first-line drugs (Ethambutol, Isoniazid, Rifampicin, and Streptomycin) were studied extensively. In the primary investigation, pathogenic mutational landscapes were classified in the sequences of the studied proteins. The effects of these mutations on the stability of the proteins were studied using diverse computational techniques. The structural basis of the point mutations with the highest destabilizing effects was analyzed using the principles of the Density Functional Theory (DFT), molecular docking and molecular dynamics (MD) simulation studies. The varied conformational behavior resulted from these predicted substitutions were compared with the experimentally derived mutations reported in the literature. The outcome of this study enabled the identification of the novel drug resistance-associated point mutations which were not previously reported. Furthermore, a detailed understanding of the conformational behavior of diverse virulent proteins present in M. tuberculosis was also generated in this study. Literature study showed that inside the host’s macrophage cells, the virulent proteins such as isocitrate lyase, lipase lipF, magnesium transporter MgtC, porin protein OmpATb, a protein of two component systems PhoP, Rv2136c and Rv3671c have an established role in the development of the acid tolerance. On the other hand, information regarding their role in the acid resistance is scarce. Accordingly, the structural basis of their role in acid resistance was analyzed using constant pH based MD simulations. In the studied proteins, the lipF and PhoP showed highest structural stability in highly acidic conditions throughout the course of MD simulations. Therefore, these proteins may play a primary role in the process of resistance. In addition to these pathogenic proteins, there is a need to identify new undiscovered virulent proteins in the genome of M. tuberculosis, which increases the efficiency of the current therapy. The knowledge generated by the analyses of the proteins involved in resistance and pathogenic mechanisms of M. tuberculosis forms the basis for the identification of new virulence factors. Therefore, an in silico protocol was used for the functional annotations and analyses of the virulence characteristics. M. tuberculosis contains 1000 Hypothetical Proteins (HPs), which are functionally uncharacterized proteins and their existence was not validated at the biochemical level. In this study, the sequences of the HPs were extensively analyzed and the functions of 662 HPs were successfully predicted. Furthermore, 483 HPs were classified in the category of the enzymes, 141 HPs were predicted to be involved in the diverse cellular mechanisms and 38 HPs may function as transporters and carriers proteins. The 307 HPs among this group of proteins were less precisely predicted because of the unavailability of the reliable functional homologs. An assessment of the virulence characteristics associated with the 1000 HPs enabled the classification of 28 virulent HPs. The structure of six HPs with highest predicted virulence score was analyzed using molecular modelling techniques. Amongst the predicted virulent HPs, the clone for Rv3906c purchased from the DNASU repository because of the ease of its availability. The gene of Rv3906c was isolated and cloned into a pET-21c expression vector. The analyses of the nucleotide sequence showed that Rv3906c gene (500 bp) encodes a 169 amino acid protein of molecular weight 17.80 kDa (~18.0 kDa). The sequence analyses of Rv3906c showed that the HPs showed high similarities with pullulanase, a thermophilic enzyme. The stability profile at different temperatures for Rv3906c generated using MD simulations showed that Rv3906c maintained its structural identity at higher temperatures. It is expected that this study will result in the design of better therapeutic against the infection of M. tuberculosis, as novel undiscovered virulence factors were classified and analyzed in addition to the conformational profiles of the virulent proteins involved in the resistance mechanisms. / M

Propone un nuevo conjunto de 10 polimorfismos de un solo nucleótido (SNPs) utilizando un total de 249 genomas de Mycobacterium tuberculosis (MTB), que por primera vez se seleccionaron basándose en el principio de inclusión/exclusión (IE) utilizando datos de Spoligotyping y filogenia, seguido de la selección de SNPs no sinónimos presentes en clúster de grupos ortólogos (COG) más conservados de cada genotipo de MTB. La asignación del genotipo utilizando el nuevo conjunto de 10 SNPs fue validado con 34 genomas de MTB adicionales y los resultados mostraron una correlación del 100% con sus genotipos ya conocidos. Nuestro conjunto de 10 SNPs no ha sido reportado previamente y abarca los genotipos de MTB que son más prevalentes en todo el mundo. Este conjunto de SNPs proporciona una resolución superior a las técnicas actuales y podría ser utilizado en epidemiología molecular con marcadores resistentes a fármacos, determinando los factores de riesgo en la transmisión de la tuberculosis en diferentes poblaciones para desarrollar estrategias para el control de esta enfermedad. / Tesis

The World Health Organization regards tuberculosis as a world pandemic disease. There is increased demand for new drugs to tackle this threat. This threat has been further elevated with the emergence of drug resistant strains of the causative pathogen, Mycobacterium tuberculosis (Mtb), thereby increasing the urgency for development of novel anti-tubercular drugs. Success in whole genome sequence determination of Mtb revealed a large cohort of cytochrome P450 (CYP) enzymes. Research on these Mtb P450s has shown that several of them are critical to the survival of the pathogen. CYP121A1 and CYP128A1 have been demonstrated to be essential using knockout experiments. CYP125A1 and CYP142A1 have been shown to play crucial roles in bacterial catabolism of host steroids, with CYP125A1 also shown to be located within a gene cluster highly important for bacterial virulence and infectivity. CYP144A1 was shown to be one of the genes whose expression is elevated when Mtb was exposed to macrophage-like conditions, and gene knockout studies using the H37Rv virulent strain of Mtb indicated the ΔCYP144A1 mutant to be more sensitive to the clotrimazole antifungal. CYP126A1 was shown to be located within a cluster of genes highly important for the de novo synthesis of purines in Mtb. These and other data suggested these enzymes to be important to the growth process of Mtb and thus potential drug targets for developing novel therapeutics. Findings in this PhD have revealed that many characteristics of CYP144A1 and CYP126A1 are comparable to previous Mtb P450s reported to date. CYP144A1 is highly conserved within the Mycobacterium genus and specifically within pathogenic species. Transcriptomic analysis has revealed an alternative truncated transcript leading to the production of two physiologically relevant versions of CYP144A1. Our comparative biophysical characterization of both versions (CYP144A1-FLV and -TRV) show both enzymes to be similar in their binding tightly to azole antifungals. EPR and DSC studies show that the 30 amino acid truncation (to form CYP144A1-TRV) does not affect the heme electronic environment and the overall thermal stability of the enzymes. X-ray crystallography was utilized to determine the first crystal structure of a Mtb CYP144 family enzyme. The structure reveals that CYP144A1 possesses a large hydrophobic active site primed for accommodating large hydrophobic substrates. Further chemoproteomic profiling identified novel compounds, which bind in both inhibitor-like and substrate-like modes to CYP144A1, resulting in the development of novel CYP144A1 compounds for use as chemical probes for this P450. Fragment and compound screening identified several ligands with varying binding affinities for CYP126A1, suggesting that this P450 is capable of binding and catalyzing reactions with a wide range of substrates. Turnover experiments proved catalytic activity of CYP126A1 on one of these compounds (Compound 4). Crystallization of CYP126A1 with various compound “hits” (compounds 1 and 7, the azole drug ketoconazole) revealed involvement of several important residues within the active site of CYP126A1 in interactions with these molecules, thus providing important information for designing inhibitors for this enzyme. Both CYP144A1 and CYP126A1 display important characteristics that contribute to our general understanding of cytochromes P450 as a whole, and of Mtb P450s in particular. This PhD project has established the first instance of leaderless transcripts in Mtb P450s and has presented the first crystal structures of both CYP144A1 and CYP126A1, as well as identifying novel, useful chemicals that can be used as mechanistic probes for these enzymes as well as providing the basis for Mtb P450 isoform-specific inhibitors.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Objectives: Diagnosis of pleural tuberculosis (PT) is still a challenge, particularly in resource-constrained settings. Alternative diagnostic tools are needed. We aimed at evaluating the utility of Clinical Prediction Rules (CPRs) for diagnosis of pleural tuberculosis in Peru. Methods: We identified CPRs for diagnosis of PT through a structured literature search. CPRs using high-complexity tests, as defined by the FDA, were excluded. We applied the identified CPRs to patients with pleural exudates attending two third-level hospitals in Lima, Peru, a setting with high incidence of tuberculosis. Besides pleural fluid analysis, patients underwent closed pleural biopsy for reaching a final diagnosis through combining microbiological and histopathological criteria. We evaluated the performance of the CPRs against this composite reference standard using classic indicators of diagnostic test validity. Results: We found 15 eligible CPRs, of which 12 could be validated. Most included ADA, age, lymphocyte proportion and protein in pleural fluid as predictive findings. A total of 259 patients were included for their validation, of which 176 (67%) had PT and 50 (19%) malignant pleural effusion. The overall accuracy of the CPRs varied from 41% to 86%. Two had a positive likelihood ratio (LR) above 10, but none a negative LR below 0.1. ADA alone at a cut-off of ≥40 IU attained 87% diagnostic accuracy and had a positive LR of 6.6 and a negative LR of 0.2. Conclusion: Many CPRs for PT are available. In addition to ADA alone, none of them contributes significantly to diagnosis of PT.

Philosophiae Doctor - PhD / Supervised machine learning approaches have been applied successfully to the prediction of protein-protein interactions (PPIs) within a single organism, i.e., intra-species predictions. However, because of the absence of large amounts of experimentally validated PPIs data for training and testing, fewer studies have successfully applied these techniques to host-pathogen PPI, i.e., inter-species comparisons. Among the host-pathogen studies, most of them have focused on human-virus interactions and specifically human-HIV PPI data. Additional improvements to machine learning techniques and feature sets are important to improve the classification accuracy for host-pathogen protein-protein interactions prediction. The primary aim of this bioinformatics thesis was to develop a binary classifier with an appropriate feature set for host-pathogen protein-protein interaction prediction using published human-Hepatitis C virus PPI, and to test the model on available host-pathogen data for human-Bacillus anthracis PPI. Twelve different feature sets were compared to find the optimal set. The feature selection process reveals that our novel quadruple feature (a subsequence of four consecutive amino acid) combined with sequence similarity and human interactome network properties (such as degree, cluster coefficient, and betweenness centrality) were the best set. The optimal feature set outperformed those in the relevant published material, giving 95.9% sensitivity, 91.6% specificity and 89.0% accuracy. Using our optimal features set, we developed a neural network model to predict PPI between human-Mycobacterium tuberculosis. The strategy is to develop a model trained with intra-species PPI data and extend it to inter-species prediction. However, the lack of experimentally validated PPI data between human-Mycobacterium tuberculosis (Mtuberculosis), leads us to first assess the feasibility of using validated intra-species PPI data to build a model for inter-species PPI. In this model we used human intra-species PPI combined with Bacillus anthracis intra-species data to develop a binary classification model and extend the model for human-Bacillus anthracis inter-species prediction. Thus, we test our hypotheses on known human-Bacillus anthracis PPI data and the result shows good performance with 89.0% as average accuracy. The same approach was extended to the prediction of PPI between human-Mycobacterium tuberculosis. The predicted human-M-tuberculosis PPI data were further validated using functional enrichment of experimentally verified secretory proteins in M-tuberculosis, cellular compartment analysis and pathway enrichment analysis. Results show that five of the M-tuberculosis secretory proteins within an infected host macrophage that correspond to the mycobacterial virulent strain H37Rv were extracted from the human-M- tuberculosis PPI dataset predicted by our model. Finally, a web server was created to predict PPIs between human and Mycobacterium tuberculosis which is available online at URL:http://hppredict.sanbi.ac.za. In summary, the concepts, techniques and technologies developed as part of this thesis have the potential to contribute not only to the understanding PPI analysis between human and Mycobacterium tuberculosis, but can be extended to other pathogens. Further materials related to this study are available at ftp://ftp.sanbi.ac.za/machine learning. / National Research Foundation (NRF) and SANBI

Chez la bactérie Escherichia coli, SecB appartient au réseau de chaperons moléculaires qui assistent le repliement et l'adressage des protéines nouvellement synthétisées. SecB est connu pour faciliter l'export en interagissant avec les pré-protéines sous forme non-native et les adressant au translocon Sec via une interaction directe avec le moteur ATPase SecA. SecB possède aussi une activité de chaperon générique et est capable d'assister le repliement de certaines protéines cytosoliques en absence des chaperons majeurs DnaK et Trigger factor. Alors que le translocon Sec est universellement conservé, le chaperon SecB est retrouvé principalement chez les protéobactéries. Cependant, de plus en plus de séquences de type SecB sont retrouvées dans d'autres groupes de la taxonomie bactérienne, comme par exemple chez le pathogène humain majeur Mycobacterium tuberculosis. Chez cette bactérie, une séquence de type SecB appelée Rv1957 est présente en association avec un système toxine-antitoxin (TA) appartenant à la famille HigBA. Généralement, les TA sont des systèmes à deux composants qui modulent la croissance en réponse à des conditions de stress spécifiques, favorisant ainsi l'adaptation et la persistance. Dans le cas de ce système atypique toxine-antitoxine-chaperon (TAC), Rv1957 interagit avec l'antitoxine HigA et la protège à la fois de l'agrégation et de la dégradation, et est donc strictement requis pour permettre l'inactivation de la toxine par l'antitoxine. La première partie de ce travail avait pour but de reconstruire l'histoire évolutive de ce nouveau système TAC. Pour cela nous avons procédé à une recherche de systèmes similaires dans l'ensemble des génomes disponibles qui a révélé que la présence de systèmes TAC n'est pas limitée aux mycobactéries et que ces systèmes semblent s'être répandus dans la taxonomie par le biais de transferts horizontaux de gènes. Nos résultats suggèrent que les chaperons des systèmes TAC sont évolutivement apparentés au chaperon d'export solitaire SecB et ont divergé pour devenir spécialisés vis-à-vis de leurs antitoxines partenaires. Nous avons ensuite étudié ce phénomène de spécialisation par une approche d'évolution dirigée du chaperon d'export SecB d'E. coli. Nous avons mis en évidence que des substitutions uniques dans SecB sont suffisantes pour améliorer sa capacité à contrôler spécifiquement HigBA du système TAC, et que ces mutations résultaient généralement en une meilleure interaction avec l'antitoxine HigA. Remarquablement, environ la moitié des mutants identifiés sont affectés dans leur activité de chaperon générique en l'absence des chaperons DnaK et Trigger factor, suggérant un conflit entre spécialisation du chaperon et ses fonctions génériques. La plupart des résidus identifiés se trouvent dans une région de SecB non caractérisée et proche du site proposé d'interaction avec le substrat. Des expériences de cross-link à des positions spécifiques ont révélé que cette région interagit directement avec l'antitoxine HigA. Enfin, nous avons montré que HigA est capable d'entrer en compétition avec la fonction d'export d'un SecB spécialisé plus efficacement que pour la version sauvage de SecB, illustrant la potentielle connexion entre les fonctions de type SecB dans l'export et le contrôle d'un système TA. / SecB is part of the intricate network of chaperones that assist folding and targeting of newly synthesized polypeptides in Escherichia coli. SecB is known to interact with nonnative precursor proteins and address them to the Sec translocon via direct interaction with the SecA motor component, thus facilitating their export. SecB is also able to act as a generic chaperone, by assisting the folding of certain cytosolic proteins when the major DnaK/Trigger Factor chaperone pathway is disrupted. While the Sec translocon is universally conserved, the SecB chaperone is mainly found in proteobacteria. However, an increasing number of SecB-like sequences have been found in unusual groups of bacteria and especially in the major human pathogen Mycobacterium tuberculosis. In this bacterium, a SecB-like sequence, Rv1957, is present in association with a toxin-antitoxin (TA) system belonging to the HigBA family. Usually, TA modules are two-component systems that modulate growth in response to specific stress conditions, thus promoting adaptation and persistence. In the case of this atypical toxin-antitoxin-chaperone (TAC) system, Rv1957 interacts with the HigA antitoxin and protects it from both aggregation and degradation, and is thus strictly required for neutralization of the toxin by the antitoxin. The first aim of this work was to reconstruct the evolutionary history of the newly discovered TAC system. We performed a large-scale genome screening and found that TAC is not restricted to mycobacteria and seems to have disseminated in the taxonomy by horizontal gene transfer. Our results suggest that TAC chaperones are evolutionarily related to the solitary export chaperone SecB and have diverged to become specialized towards their cognate antitoxins. Next, we investigated such chaperone specialization event through directed evolution of the E. coli export chaperone SecB. We found that single amino-acid substitutions within SecB were sufficient to improve its ability to specifically control HigBA from TAC, and that these mutations mainly resulted in an increased binding to the HigA antitoxin. Strikingly, about half of the mutants identified were affected in their ability to perform SecB generic chaperone functions in the absence of both DnaK and TF chaperones, suggesting a conflict between specialization and generic chaperone functions. Most of the residues identified are located within a previously uncharacterized region of SecB which is close to the proposed substrate binding site. Further in vitro site-specific cross-linking experiments revealed that this region directly interacts with the HigA antitoxin. Finally, we show that the HigA antitoxin can compete with the export function of specialized SecB more efficiently than it does with wild type SecB, thus illustrating the potential interplay between SecB-like chaperone export functions and TA activation.