Bacterial cell division relies on the cell division septum to form at the mid-cell position. In gram negative bacteria, this is mediated by three proteins, MinC, MinD and MinE. Together these proteins interact with each other and the membrane in a dynamic, oscillating process which prevents cell division septum formation at the cell poles. The early phase of this process involves MinD binding to the membrane, which is triggered upon binding of ATP. Subsequent interactions with MinE result in stimulation of the ATPase activity of MinD. After hydrolysis, MinD is released from the membrane and diffuses to a new binding site. Many in silico models have been constructed of the Min system in an attempt to describe its self-organizing behaviour. A limitation of these models is that, in order to prevent rapid re-binding of MinD to the membrane after hydrolysis of ATP, the exchange of bound ADP for ATP is assumed to be a slow process, on the order of 1/s . In order to provide experimental evidence of the rate of nucleotide binding, we performed a series of triple-resonance NMR experiments to complete a partial assignment of backbone atom resonances, which required the application of deuterium labelling and amino acid-specific selective unlabelling. After the introduction of ATP, it was discovered that no dimerization had been induced, in contrast with existing literature. It was proposed that MinD from N. gonorrhoeae only forms a dimer in the presence of a membrane, while literature with MinD from E. coli shows it does not have this requirement. Interestingly while dimerization had not been induced, there was a persistent population of dimeric species even in the absence of nucleotide. This was discovered to be the result of disulfide formation, likely an artifact of established purification protocols. Binding of both ADP and ATP to MinD were studied by titration using NMR, with the relative affinity of both nucleotides to MinD being indistinguishable. By analyzing peak coalescence in the half-bound condition, a maximum rate was determined for nucleotide binding, with the lifetime being on the order of 170ms. Results from this experiment support models requiring a slow nucleotide binding step, and help enhance understanding of how Min proteins sustain oscillations required for normal cell division.

Neisseria gonorrhoeae is an obligate human pathogen that requires iron for its survival within the host. N. gonorrhoeae expresses high-affinity iron acquisition systems to acquire iron from host iron binding proteins. The gonococcal transferrin-iron uptake system is composed of two transferrin binding proteins, TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter, while TbpB is a surface-exposed lipoprotein. Unlike TbpA, TbpB is not required for transferrin utilization, but makes the process more efficient. The precise mechanism by which TbpA and TbpB function to mediate transferrin-iron uptake has not been fully characterized. However, the mechanism of iron acquisition from transferrin is distinct from characterized TonB-dependent ferric-siderophore uptake systems. The transferrin-iron uptake system is unique in two ways: the involvement of the TbpB lipoprotein component and the process of iron acquisition and internalization. Unlike siderophore transporters, the transferrin-iron uptake system requires the removal of iron from transferrin for its subsequent internalization. Based on analogy with characterized TonB-dependent transporters, TbpA is proposed to consist of two distinct domains: a b-barrel and plug domain. Previous studies suggest that the plug domain has a specific role in iron internalization and this study addresses the role of the plug domain in transferrin-iron acquisition. It is thought that the TbpA plug domain facilitates iron removal from transferrin and subsequent iron binding and transport. To analyze this, iron binding by the TbpA plug domain was performed and site-directed substitution mutagenesis of putative iron-coordinating residues was carried out. From these analyses, it can be concluded that the plug domain binds iron and likely plays an active role in the process of iron internalization. Mutagenesis revealed specific residues of the plug domain critical for transferrin-iron uptake, but defects imparted by these mutations were compensated for by TbpB. Thus, this study also attempts to characterize the compensatory function provided by TbpB. Through mutagenesis, critical domains involved in the efficiency of transferrin-iron acquisition were identified. One additional study describes and characterizes a novel mechanism of TonB-independent transferrin-iron acquisition. Overall, these studies further elucidate mechanisms utilized by Neisseria gonorrhoeae in the process of iron acquisition from human transferrin.

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2015-10-27T17:20:50Z No. of bitstreams: 2 Dissertação - Fernanda Alves de Brito e Cardoso - 2010.pdf: 3862835 bytes, checksum: bac9d567036b30752cd19dc0042891c0 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-28T14:25:41Z (GMT) No. of bitstreams: 2 Dissertação - Fernanda Alves de Brito e Cardoso - 2010.pdf: 3862835 bytes, checksum: bac9d567036b30752cd19dc0042891c0 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-28T14:25:41Z (GMT). No. of bitstreams: 2 Dissertação - Fernanda Alves de Brito e Cardoso - 2010.pdf: 3862835 bytes, checksum: bac9d567036b30752cd19dc0042891c0 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2010-12-14 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The introduction of the nucleic acid amplification tests (NAATs) was a major breakthrough in the screening for the sexually transmitted diseases (STD) caused by Chlamydia trachomatis and Neisseria gonorrhoeae because they are highly sensitive and they can be used with noninvasive specimens, such as urine. The use of urine has made it far easier to test asymptomatic individuals and has also made it possible to perform epidemiological studies in places other than clinical settings. Many studies have shown also that vaginal swab can be used for detection of both infections, however, just the NAAT Aptima Combo 2 has been cleared by Food and Drug Administration for this specimen use. In Brazil, the most widely used NAAT for the diagnosis of chlamydia and neisseria is the kit Amplicor CT/NG (Roche) and, up to date, there isn’t any study which evaluates the use of vaginal swabs. Objectives: To evaluate the performance of the kit AMPLICOR CT/NG (Roche) in the diagnosis of C. trachomatis and N. gonorrhoeae using urine, endocervical and vaginal swabs and to analyze the agreement of results between the different biological specimens. Methods: The target population was sexually active adolescents and young women between 15 and 24 years from Inhumas, Goias. Socio-demographic and sexual behavior were obtained through a face-to-face interview. The diagnosis was performed by PCR using the AMPLICOR CT/NG (Roche) assay in urine, vaginal swab (VS) and endocervical swab (ES) specimens. For the performance evaluation were calculated the sensitivity, specificity, positive predictive value and negative predictive value. The kappa coefficient was calculated to assess agreement between the samples. It was considered a true-positive result when at least two of three biological samples from the same patient were positive for chlamydia and/or gonococcus. Results:Among the 428 participants the mean age was 19,4 years. The three biological specimens were collected from 309 adolescents (72.2%). Among these, the prevalence rates were 8.7% (IC95% 5,8-12,4) for C. trachomatis and 2.3% (IC95% 0,9-4,6) for N. gonorrhoeae.For chlamydia the sensitivities observed with the different samples were above 80% and specificities exceeding 97% with positive predictive values (PPV) between 78.8% and 84.6% and negative predictive values (VPNs) >98%. For the gonococcus the sensitivities were 42.8% for urine, 71.4% for ES and 100% for VS with specificities >96% for the three samples. The two types of swab showed low PPVs for gonococcus (≈40%) and urine showed PPV of 100%. VPNs were >98%. The agreement of results between specimens was around 94% for the detection of both infections. However, the values of kappa (κ) coefficient ranged from 0.68 to 0.73 for chlamydia, which means substantial agreement between samples. For gonococcal infection, the agreement was slight or fair with κ coefficients ranging from 0.13 to 0.33. Conclusions:The performances of the specimens and the κ values suggest that the vaginal swab appears to be equivalent to urine and endocervical swab for detection of chlamydia and may be suitable for screening studies. The three samples showed different performance in the detection of gonococcus and did not present good agreement of results, suggesting that they are not equivalent in the diagnosis of this infection with the PCR kit used. / A introdução dos testes de amplificação de ácidos nucléicos (NAATs) foi um grande avanço no rastreamento das doenças sexualmente transmissíveis (DST) causadas por Chlamydia trachomatis e Neisseria gonorrhoeae, pois apresentam alta sensibilidade e permitem a utilização de amostras de coleta não invasiva, como a urina. O uso da urina facilitou o diagnóstico em indivíduos assintomáticos e possibilitou a realização de estudos epidemiológicos em locais fora do ambiente clínico. Vários estudos mostram que o swab vaginal também pode ser utilizado na detecção de ambas as infecções, porém a Food and Drug Administration restringe o seu uso apenas pelo NAAT Aptima Combo 2. No Brasil, o NAAT mais utilizado no diagnóstico de clamídia e neisseria é o kit Amplicor CT/NG (Roche) e, até o momento, nenhum estudo avaliou a utilização do swab vaginal. Objetivos:Avaliar o desempenho do kit AMPLICOR CT\NG (Roche) no diagnóstico de C. trachomatis e N. gonorrhoeae empregando urina, swabs endocervical e vaginal e analisar a concordância de resultados entre as diferentes amostras biológicas. Métodos: A população alvo foi constituída de adolescentes e jovens sexualmente ativas, com idade entre 15 e 24 anos, residentes em Inhumas, Goiás. Os dados sócio-demográficos e de comportamento sexual foram obtidos através de entrevista. O diagnóstico foi realizado empregando o kit Amplicor CT/NG (Roche) em amostras de urina, swab endocervical (SE) e swab vaginal (SV). Para avaliação do desempenho foram calculadas a sensibilidade, especificidade, valor preditivo positivo e negativo dos testes. O coeficiente kappa foi calculado para avaliar a concordância entre as amostras. Considerou-se um resultado como verdadeiro-positivo quando pelo menos duas das três amostras biológicas da mesma paciente fossem positivas para clamídia e/ou gonococo. Resultados: Entre as 428 participantes a média de idade foi de 19,4 anos. Os três espécimes biológicos foram coletados de 309 adolescentes (72,2%). Entre estas, as prevalências foram de 8,7% (IC95% 5,8-12,4)para C. trachomatis e de 2,3% (IC95% 0,9-4,6) para N. gonorrhoeae. Para clamídia as sensibilidades observadas com as diferentes amostras foram superiores a 80% e as especificidades superiores a 97%, com valores preditivos positivos (VPPs) entre 78,8% e 84,6% e valores preditivos negativos (VPNs) >98%. Para o gonococo as sensibilidades foram de 42,8% na urina, 71,4% no SE e de 100% no SV com especificidades >96% nas três amostras. Os dois tipos de swab apresentaram baixos VPPs para o gonococo (≈ 40%) e a urina apresentou VPP de 100%. Os VPNs foram >98%. A concordância de resultados da PCR empregando as diferentes amostras foi de cerca de 94% para ambas as infecções. Entretanto, os valores do coeficiente kappa (κ) variaram de 0,68 a 0,73 para clamídia, o que significa concordância substancial entre as amostras. Para a infecção gonocócica, a concordância foi fraca ou razoável com valores de κ variando de 0,13 a 0,33. Conclusões: Os desempenhos das amostras e os valores do κ sugerem que o swab vaginal parece ser equivalente à urina e ao swab endocervical na detecção da clamídia podendo ser recomendado para estudos de triagem. As três amostras diferiram quanto ao desempenho na detecção da infecção gonocócica e não apresentaram boa concordância de resultados sugerindo que não são equivalentes no diagnóstico desta infecção com o kit de PCR utilizado.