Thesis (MHSc--Health Science) -- AUT University, 2008. / Includes bibliographical references. Also held in print (ix, 144 leaves ; 30 cm.) in the Archive at the City Campus (T 613.954 WAE)

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [83]-96).

Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr.: f. [100]-113. Publié aussi en version électronique.

Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr.: f. 62-76. Publié aussi en version électronique.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Universiẗat, Diss., 2005--Jena.

Chlamydia trachomatis (Ct), the most common sexually transmitted bacterium, has been associated with adverse pregnancy outcomes including controversial data on miscarriage, intrauterine growth restriction and low birth weight, however the causative mechanisms are unknown. A successful pregnancy requires normal endometrial stromal cell (ESC) decidualisation and trophoblast invasion, processes that involve chemokine action and lead to successful implantation. My objectives were to determine whether Ct infection impacts upon ESC decidualisation and chemokine secretion on human primary ESC invitro, to investigate the role of Ct infection in pregnancy in-vivo using a murine model of pregnancy and to investigate the role of Ct in miscarriage in a statistically powered case control study. A novel finding is that Ct can infect and proliferate in ESC, resulting in suboptimal decidualisation as measured by decidualisation marker prolactin’s reduced mRNA and protein levels in infected ESC. Furthermore, the altered secreted chemokine profile of decidualised ESC suggests an attenuated innate immune response from infected ESC. Focusing on chemokines C-X-C motif chemokine 12 (CXCL12) and CXCL16, important for trophoblast invasion, decreased mRNA and protein concentrations were detected in infected decidualised cells. From the in-vivo mouse model of past Ct infection in pregnancy, it was demonstrated that Ct infection did neither affect the fertility of the mice, pregnancy or resorption numbers in C3H mice nor alter embryonic and placental weight on e12 embryos. However, Ct infection caused reduction of embryo and placenta weight on e14 embryos. Finally, preliminary data from the case control study indicate that past Ct infection is not associated with miscarriage. Our in house PGP3 ELISA that detects past Ct infection was more sensitive than a commercially available MOMP ELISA. My data suggests that Ct infection affects pregnancy during the implantation stage by impairing decidualisation and altering chemokine secretion predisposing for adverse pregnancy outcomes that include growth restriction during later gestation.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, 2008. / Submitted by Jaqueline Oliveira (jaqueoliveiram@gmail.com) on 2008-12-08T18:17:18Z No. of bitstreams: 1 DISSERTACAO_2008_JoseAugustodeOliveiraBotelho.pdf: 2980105 bytes, checksum: f5b5a96a5b7e8a0bd74ff1e7bbaafbd7 (MD5) / Approved for entry into archive by Georgia Fernandes(georgia@bce.unb.br) on 2009-02-17T18:04:51Z (GMT) No. of bitstreams: 1 DISSERTACAO_2008_JoseAugustodeOliveiraBotelho.pdf: 2980105 bytes, checksum: f5b5a96a5b7e8a0bd74ff1e7bbaafbd7 (MD5) / Made available in DSpace on 2009-02-17T18:04:52Z (GMT). No. of bitstreams: 1 DISSERTACAO_2008_JoseAugustodeOliveiraBotelho.pdf: 2980105 bytes, checksum: f5b5a96a5b7e8a0bd74ff1e7bbaafbd7 (MD5) / Introdução: A infecção em gestantes por Chlamydia trachomatis é um grande problema de saúde pública e que não raramente tem como conseqüência a ocorrência de abortos. Objetivos: Conhecer a prevalência de infecção por Chlamydia trachomatis em gestantes e abortos, por faixa etária e município de residência no Mato Grosso do Sul, de 2005 a 2007. Método: Realizou-se estudo epidemiológico descritivo. Examinaram-se 74.701 cartões do Programa de Proteção à Gestante, juntamente com dados dos resultados de testes laboratoriais (Enzimaimunoensaio) em papel de filtro; nos resultados positivos realizaram-se testes sorológicos confirmatórios. Empregou-se o teste Qui-quadrado (X2), com 95% confiabilidade na análise dos dados. Resultados: Encontrou-se uma prevalência de infecção por C. trachomatis de 6,64% e taxa de infecção na faixa etária de 20 a 35 anos de 6,8% e OR 1,07 (p=0,002). A freqüência de infecção e abortos relatados anteriormente foi de 6,77% com OR 1,2 (p<0,0001). Conclusões: A prevalência de infecção por C. trachomatis nas gestantes pesquisadas no Mato Grosso do Sul está em conformidade com as taxas brasileiras. Verificou-se que existem diferenças na ocorrência de abortos entre os municípios localizados em diferentes áreas do Estado, microrregião de influência de Campo Grande, assentamentos e áreas indígenas. _________________________________________________________________________________________ ABSTRACT / Introduction: The infection in pregnant women for Chlamydia trachomatis is a major public health problem and not rarely has to occur as a result of abortions. Objectives: To know the prevalence of C. trachomatis infection in pregnant women and abortions, by age and city of residence in Mato Grosso do Sul, between 2005 and 2007. Method: There was descriptive epidemiological study. Were examined to 74,701 collection cards of the Pregnancy Protection Program, together with data of the results of laboratory tests (Enzyme immunoassay) in tissue dried blood filter paper; the positive results were confirmed by serological tests. We applied the Chisquare (X2), with 95% confidence in the analysis of data. Results: We found a prevalence of infection by C. trachomatis of 6.64% and rate of infection in age from 20 to 35 years of 6.8%, OR 1.07 (p = 0002). The frequency of infection and abortions reported previously was 6.77% with OR 1.2 (p <0.0001). Conclusions: The prevalence of infection by C. trachomatis in pregnant women searched in Mato Grosso do Sul is in accordance with the Brazilian rates. It was found that there are differences in the occurrence of abortions among cities in different areas of the state, micro regions by influence of Campo Grande, settlements and indigenous areas.

Infecções por Chlamydia trachomatis estão entre as mais freqüentes doenças sexualmente transmissíveis (DST) em todo o mundo, apresentando grande importância epidemiológica. A identificação deste patógeno pode ser difícil e um método de detecção baseado na amplificação de ácidos nucleicos é altamente desejável, por sua acurácia e rapidez. O presente estudo avaliou a acurácia, sensibilidade e especificidade de uma reação em cadeia da polimerase (PCR) in “house” em amostras de urina de homens com e sem sintomas de DST comparados a um teste comercial, o COBAS Amplicor CT/NG (Roche, Suiça). Foram utilizados primers específicos para amplificar um segmento do plasmídio críptico de C. trachomatis gerando um fragmento de 201 pb, cuja seqüência foi confirmada por clivagem enzimática e seqüenciamento automático. A especificidade analítica dos primers foi confirmada frente ao DNA de diferentes microrganismos patogênicos e não patogênicos da microbiota urogenital masculina. A detecção limite do DNA clamidial pela PCR in house após hibridização (Southern blot), foi de 1 pg. O COBAS Amplicor CT/NG foi considerado o teste de referência por ser automatizado e conter um programa de controle interno da reação para identificar inibição da DNA polimerase. Entre as 37 amostras testadas positivas para C. trachomatis pelo COBAS Amplicor, 33 foram confirmadas positivas pela PCR in house. Foram detectados inibidores da reação nas 4 amostras que apresentaram resultados falso-negativos, utilizando-se a técnica de contaminação da reação com o DNA clamidial (spiked) e um controle interno da reação com primers que amplificam a β-globina do DNA humano. Após congelamento e descongelamento para eliminar prováveis substâncias inibidoras lábeis, as 4 amostras foram re-testadas, resultando na positividade de mais 2 amostras, completando 35 amostras positivas pela PCR in house. Entre as 74 amostras testadas negativas para C. trachomatis pelo COBAS Amplicor, 72 foram confirmadas negativas pela PCR in house. Das 2 amostras prováveis falso-positivas, apenas 1 permaneceu positiva, após re-teste. Dos 111 pacientes analisados, 108 apresentaram resultados idênticos nos dois testes, o equivalente a uma acurácia = 97,3%, sensibilidade = 94,6% e especificidade = 98,6%. A alta sensibilidade e especificidade apresentada pela PCR in house, demonstra a possibilidade de triar e diagnosticar C. trachomatis em urina de homens, importante reservatório da infecção clamidial para as mulheres. / Chlamydia trachomatis infections are among the most commum sexually transmitted diseases and of great epidemiological importance world-wide. Identification of this pathogen can be difficult, and it is highly desirable to have a rapid and accurate nucleic acid amplification based detection method. The present study was designe to evaluate the accuracy, sensitivity and specificity of a in house plasmid-based polymerase chain reaction (PCR) and hybridization on first void urine specimens from symptomatic and asymptomatic men, compared with a commercial test the PCR COBAS Amplicor CT/NG (Roche, Switzerland), for detection of C. trachomatis (CT). Specific primers for the cryptic plasmid of C. trachomatis were designed to amplify a 201 bp fragment confirmed by enzymatic cleavage and automatic sequencing. The analytic specificity was determined by submitting to the intended protocol, the DNA of normal and pathogenic urogenital microbiota, the specific primers anneling was confirmed. The detection limit of the PCR and the Southern blot hibridization, was mesured in 1 pg of chlamydial DNA. The COBAS Amplicor CT/NG was considered the reference test, it contains a internal control (IC) programme to identify DNA polymerase inhibition. Among 37 positive specimens tested by COBAS Amplicor, 33 were confirmed positive by in house PCR and inhibitors of PCR were demonstrated in the 4 possibly false-negative samples performing DNA chlamydial spiking experiments and using a positive internal control of human β-globin DNA. The specimens were freezedthowed and re-tested to remove labile inhibitors, but 2 samples remained negative. Among 74 negative specimens by COBAS Amplicor, 72 were confirmed negative by in house PCR. The 2 problaby false-positive samples were re-tested and just one remained positive. The final results of the two tests were identical for 108 of the 111 pacients, accuracy = 97,3% ; sensitivity = 94,6% and specificity = 98,6%. This study shows that the in house plasmid-based PCR is fasible for detection of Chlamydia trachomatis in male urine specimens. This test presented high accuracy, sensitivity and specificity, offering great potencial for the screening of men, an important reservoir of infection chlamydial for women.

Chlamydia trachomatis is a Gram-negative, obligate intracellular pathogen that is the causative agent of sexually transmitted infections and the ocular disease trachoma. Chlamydia trachomatis undergoes a biphasic developmental cycle differentiating between the infectious elementary body (EB) and the replicative reticulate body (RB). Under certain stress conditions, C. trachomatis can stall its developmental cycle and enter an aberrant state termed persistence. While in a persistent state, C. trachomatis is refractory toward antibiotics, can evade the host immune response, and becomes undetectable using standard clinical detection methods. Environmental and other pathogenic microbes are known to utilize partner switching mechanisms (PSM) to regulate sigma factors used to initiate a stress response. For this reason, this study focuses on the chlamydial PSM, its role in regulating the availability of the housekeeping sigma factor σ66, and its role in the developmental cycle and stress response of C. trachomatis. The chlamydial PSM is composed of five known proteins: the anti-sigma factor RsbW, two anti-anti-sigma factors RsbV1 and RsbV2, a regulatory phosphatase RsbU, and a second phosphatase-like protein CTL0852. In order to test the role of the PSM in the chlamydial stress response, a panel of C. trachomatis rsbV1 mutants were generated, persistence inducing iron starvation and tryptophan starvation cell culture conditions were optimized, and growth of the rsbV1 mutants under iron starvation conditions were assayed. No significant differences were seen between rsbV1 mutants under iron starvation nor recovery conditions as determined by progeny production and inclusion size analysis. Furthermore, this study generated PSM protein producing Escherichia coli strains for in vitro protein work and performed operon mapping of the PSM genes of C. trachomatis to help aid in future studies of the chlamydial PSM by facilitating the development of new chlamydial PSM mutants. This study gives phylogenetic support to the classification of ctl0852 as a chlamydial PSM gene by comparing relative mutations rates of PSM genes across chlamydial species.