Zugl.: Berlin, Humboldt-Univ., Diss., 2007

Thesis (M.S.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2005. / Vita. Includes bibliographical references.

Thesis (Master of Health Policy & Administration)--Washington State University, May 2006. / Includes bibliographical references (p. 58-62).

Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Proefschrift Universiteit Maastricht. / Met lit. opg. - Met samenvatting in het Nederlands.

TELES, Rosiane Alves de Sousa. Infecção genital por Chlamydia trachomatis em mulheres jovens : prevalência, fatores de risco e achados citopatológicos e colposcópicos associados. 2012. 64 f. Dissertação (Mestrado em Patologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2012. / Submitted by denise santos (denise.santos@ufc.br) on 2013-05-10T15:58:14Z No. of bitstreams: 1 2012_dis_rasteles.pdf: 651513 bytes, checksum: c0f30c61f8be5d8505c06dd8ea0784c5 (MD5) / Approved for entry into archive by Erika Fernandes(erikaleitefernandes@gmail.com) on 2013-05-16T13:40:10Z (GMT) No. of bitstreams: 1 2012_dis_rasteles.pdf: 651513 bytes, checksum: c0f30c61f8be5d8505c06dd8ea0784c5 (MD5) / Made available in DSpace on 2013-05-16T13:40:10Z (GMT). No. of bitstreams: 1 2012_dis_rasteles.pdf: 651513 bytes, checksum: c0f30c61f8be5d8505c06dd8ea0784c5 (MD5) Previous issue date: 2012 / Chlamydia trachomatis (Ct) is the most common bacterial sexually transmitted infection worldwide, but there are few published dates about it in Brazil. The aim of this study was to determinate the prevalence of Chlamydia trachomatis infection and to assess the socialdemographic behavioral, clinical and cytopathological factors associated with this infection among adolescents and young women in a low-income area of Fortaleza - Brazil. A cross-sectional study was conducted in 200 sexually active women aged 12 to 25 years, from august 2011 to august 2012, in gynecology outpatient clinic of the General Hospital District Gonzaga Mota - Barra do Ceará. Personal information and date of gynecogical examination were recorded in a questionnaire and the patients underwent collection of material from the endocervix to hybrid capture test for C. trachomatis and Pap test, followed by colposcopy. Data were analyzed using Graphpad Prism 5.0 software, proceeding to descriptive and analytical analysis using the Student t test for nominal variables and the Fisher exact test for quantitative variables. No association was found between infection and studied the socio-demographic characteristics, sexual habits, signs and symptoms questioned, cylindrical ectropion and colposcopic changes.Among the abnormal cytological atypia, ASC-US was presented in 20,7% of cases and 4,5% of controls (p=0,0067, RR=3,452, IC=1,72-6,89), with a positive association with the infection. G. vaginalis morphotype was identified in 54,8% of infected women and 30,7% of negative patients (p=0,0133, RR=2,305. IC=1,21-4,39), showing a relationship with the infection. It was concluded that infection with C. trachomatis had a high prevalence in the population studied, no association was observed with socio-demographic, biological and clinical findings and colposcopic changes. There was association of ASC-US and G. vaginalis in cytology with the infection studied. / A Chlamydia trachomatis (Ct) é a bactéria de transmissão sexual mais comum em todo o mundo, apesar de existirem poucos dados sobre este agravo na população brasileira. O objetivo da pesquisa foi determinar a prevalência da infecção por Ct, avaliando os fatores sóciodemográficos e achados clínicos, colposcópicos e citopatológicos associados à ocorrência desta infecção em mulheres jovens na periferia de Fortaleza. Foi realizado um estudo de corte transversal em 200 mulheres sexualmente ativas, com idade entre 12 e 25 anos, atendidas no período de agosto de 2011 a agosto de 2012, no ambulatório de ginecologia geral do Hospital Distrital Gonzaga Mota – Barra do Ceará. Informações pessoais e dados do exame ginecológico foram anotados em um questionário e as pacientes submeteram-se à coleta de material da endocérvice para teste de captura híbrida II para Chlamydia trachomatis e para citologia oncótica convencional, seguido de colposcopia. Os dados foram analisados utilizando o software Graphpad Prism 5.0, procedendo-se a análise descritiva e analítica utilizando o teste t de Student para as variáveis nominais e o teste exato de Fisher para as variáveis quantitativas. A prevalência da infecção por Chlamydia trachomatis foi 15,5% (31/200) em mulheres adolescentes e adultas jovens. Não foi encontrada associação entre a infecção estudada e as características sóciodemográficas, hábitos sexuais, sinais e sintomas questionados, ectopia cilíndrica e alterações colposcópicas. Dentre as atipias citológicas, o ASC-US esteve presente em 20,7% dos casos e 4,5% dos controles (p=0,0067, RR=3,452, IC=1,72-6,89), mostrando uma associação positiva com a infecção clamidiana. A G. vaginalis foi encontrada em 54.8% das pacientes infectadas e em 30,7% das pacientes negativas (p=0,0133, RR=2,305. IC=1,21-4,39), mostrando uma relação com a infecção estudada. Concluiu-se que a infecção por C. trachomatis teve uma prevalência alta na população estudada, que não houve associação com fatores de risco sócio-demográfico, biológico, com achados clínicos e/ou colposcópicos. Houve associação de ASC-US e G. vaginalis na citologia oncótica com a infecção estudada.

Made available in DSpace on 2014-06-12T23:03:29Z (GMT). No. of bitstreams: 2 arquivo8806_1.pdf: 602566 bytes, checksum: 4d7bc38669878a55e825fd9e78020363 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / A citologia oncótica cérvico-vaginal, além de ser útil na triagem de câncer cervical, também pode ser utilizada na detecção de características sugestivas de infecção pela Chlamydia trachomatis (C. trachomatis).O objetivo deste estudo foi comparar a citologia com a técnica de imunofluorescência direta (IFD) para Chlamydia trachomatis, utilizando 171 esfregaços cérvico-vaginais de mulheres atendidas no setor de coleta preventiva de câncer de colo uterino, no Centro Integrado de Saúde Amaury de Medeiros-CISAM, no Recife-PE. Seis casos (3,5%) foram positivos para C. trachomatis pela IFD. A citologia mostrou inclusões eosinofílicas em células metaplásicas, sendo consideradas sugestivas de infecção por C. trachomatis em apenas um caso (0,6%), confirmado pela IFD. A citologia pode contribuir na detecção da infecção por Chlamydia, desde que sejam levadas em consideração coletas adequadas, com presença de células glandulares endocervicais e/ou metaplásicas, além de se considerar também o conhecimento dos critérios citológicos característicos da infecção, visto que, quando comparada a IFD, mostrou baixa sensibilidade. A IFD deve continuar sendo utilizada na detecção de casos de infecção por Chlamydia, com critério e, se possível, com maior abrangência em serviços públicos de saúde

Chlamydia trachomatis is a leading cause of bacterial genital infection in the US and worldwide. The chlamydial replication cycle is biphasic, meaning it enters the host cell as an infectious elementary body (EB), differentiates into an actively dividing reticulate body (RB) inside of a vacuole-like compartment, called a chlamydial inclusion, and differentiates back into EB form before exiting the host. Studies have demonstrated that estrogen aids Chlamydia infection in human and swine endometrial cells and in guinea pigs. Our prior data showed that antibody blockage of estrogen receptors or exposure to the ERβ antagonist tamoxifen in vitro decreased the development of chlamydial inclusions. Given these observations, we wanted to further examine the role of ER signaling on chlamydial infection in a murine model. We hypothesized that the absence of estrogen receptors would alter the establishment and/or progression of chlamydial infection in mice. To test this hypothesis, we compared C. muridarum infection in wild type (ERαWT or ERβWT) versus knockout (ERαKO or ERβKO) mice. Groups of eight ERαWT, ERβWT, ERαKO, or ERβKO mice were Depo-Provera treated seven days prior to vaginal infection with C. muridarum. Vaginal swabs were taken every three days for 21 days to monitor infection by EB titer analysis. Interestingly, titer data showed that peak EB shedding occurred earlier in the ERαKO mice compared to the ERαWT. At day 3 pi, EB shedding from ERαKO mice was 12-fold greater than shedding from ERαWT mice. On day 6, however, ERαWT mice shed >3-fold more EB than ERαKO mice. Conversely, there was no significant difference observed in ERβKO versus ERβWT mice or in ERαKO versus ERβKO mice. In a subset of experiments, genital tracts were collected on day 9pi and processed for flow cytometry analysis of the immune response to infection. ERαKO mice had significantly more monocytes and macrophages than ERαWT and ERβKO mice, as well as significantly more T cells than ERβKO mice. There was no significant difference in the immune cells in ERβKO and ERβWT mice. Together, these data suggest that: 1) the absence of ERs in mice does not inhibit chlamydial infection as has been observed in human cells in vitro; and 2) C. muridarum infection progression is affected by ERα signaling, possibly via alterations in the immune response.

The human pathogen Chlamydia trachomatis is the main cause of sexually transmitted infections worldwide. The obligate intracellular bacteria are the causative agent of several diseases that reach from conjunctivitis causing trachoma and blindness as well as salpingitis and urethritis which can lead to infertility if left untreated. In order to gain genetically engineered Chlamydia that inducible knock down specific gene expression, the CRISPRi system was established in C. trachomatis. In a proof of principle experiment it was shown that C. trachomatis pCRISPRi:gCdu1III target ChlaDUB1 expression and reduce the protein amount up to 50 %. Knock-down of the DUB did not influence protein levels of anti-apoptotic Mcl-1 and did not make cells susceptible for apoptosis. However, reduced dCas9 protein size, bacterial growth impairment and off target effects interfering with the GFP signal, form obstacles in CRISPRi system in Chlamydia. For routinely use of the CRISPRi method in C. trachomatis further investigation is needed. Since the bacterial life cycle includes two morphological and functional distinct forms, it is essential for chlamydial spread to complete the development cycle and form infectious progeny. Therefore, Chlamydia has evolved strategies to evade the host immune system in order to stay undetected throughout the developmental cycle. The bacteria prevent host cell apoptosis via stabilization of anti-apoptotic proteins like Mcl-1, Survivin and HIF-1α and activate pro-survival pathways, inhibiting invasion of immune cells to the site of infection. The host cell itself can destroy intruders via cell specific defense systems that involve autophagy and recruitment of professional immune cells. In this thesis the role of the chlamydial deubiuqitinase ChlaDUB1 upon immune evasion was elucidated. With the mutant strain Ctr Tn-cdu1 that encodes for a truncated DUB due to transposon insertion, it was possible to identify ChlaDUB1 as a potent opponent of the autophagic system. Mutant inclusions were targeted by K48 and K63 chain ubiquitination. Subsequently the inclusion was recognized by autophagic receptors like p62, NBR1 and NDP52 that was reversed again by complementation with the active DUB. Xenophagy was promoted so far as LC3 positive phagosomes formed around the inclusion of Ctr Tn-cdu1, which did not fuse with the lysosome. The detected growth defect in human primary cells of Chlamydia missing the active DUB was not traced back to autophagy, but was due to impaired development and replication. It was possible to identify Ankib1, the E3 ligase, that ubiquitinates the chlamydial inclusion in a siRNA based screen. The activating enzyme Ube1 and the conjugating enzyme Ube2L3 are also essential in this process. Chlamydia have a reduced genome and depend on lipids and nutrients that are translocated from the host cell to the inclusion to proliferate. Recruitment of fragmented Golgi stacks to the inclusion surface was prevented when ChlaDUB1 was inactive, probably causing diminished bacterial growth. Additionally, the modification of the inclusion by Ankib1 and subsequent decoration by autophagic markers was not only present in human but also murine cells. Comparison of other Chlamydia strains and species revealed Ankib1 to be located at the proximity of the inclusion in C. trachomatis strains only but not in C. muridarum or C. pneumoniae, indicating that Ankib1 is specifically the E3 ligase of C. trachomatis. Moreover, the role of ChlaDUB1 in infected tissue was of interest, since ChlaDUB1 protein was also found in early EB stage and so might get in contact with invading immune cells after cell lysis. While bacteria spread and infect new host cells, Chlamydia can also infect immune cells. Infection of human neutrophils with Ctr Tn-cdu1 shows less bacterial survival and affirms the importance of the DUB for bacterial fitness in these cells. / Chlamydia trachomatis ist weltweit der häufigste Auslöser von sexuell übertragenen Krankheiten. Das obligat intrazelluläre Bakterium manifestiert sich in diversen Krankheitsbildern, darunter Konjunktivitis, die zu einem Trachom oder sogar Erblindung führen kann und Salpingitis oder Urethritis, die unbehandelt unfruchtbar macht. Das CRISPRi System wurde in C. trachomatis etabliert, um genetisch veränderte Bakterien zu bekommen, in denen induzierbar die spezifische Genexpression herunter gefahren werden kann. Es wurde gezeigt, dass in C. trachomatis pCRISPRi:gCdu1III, einem Stamm, der mit der Genexpression von ChlaDUB1 interferiert, die Menge an ChlaDUB1 um bis zu 50 % reduziert ist. Die Sensitivität für Apoptose durch sinkende Mcl-1 Proteinmengen wurde dadurch jedoch nicht wieder hergestellt. Das verkürzte dCas9 Protein, vermindertes bakterielles Wachstum, sowie Effekte auf andere Genexpressionen, wie z.B. das GFP Signal zeigen die Problematik des CRISPRi Systems in C. trachomatis. Um CRISPRi als Routinemethode für genetische Transformation in Chlamydien zu etablieren, stehen noch weitere Untersuchungen an. Der Lebenszyklus von Chlamydien zeichnet sich durch zwei morphologisch und funktionell unterschiedliche Stadien aus, weshalb die Vollendung des Lebenszyklus und die Produktion infektiöser Partikel essenziell sind. Daher haben die Pathogene Strategien entwickelt, um dem Immunsystem des Wirts zu entgehen und sich unerkannt in der Zelle zu entwickeln. Die Bakterien verhindern Apoptose infizierter Zellen durch die Stabilisierung von anti-apoptotischen Proteinen wie Mcl-1, Survivin und HIF-1α und aktivieren Überlebens-Signalwege, die die Invasion von Immunzellen in das infizierte Gewebe unterdrücken. Die Wirtszelle selbst ist in der Lage bakterielle Eindringlinge durch die eigenen Abwehrmechanismen wie Autophagie und die Rekrutierung von professionellen Immunzellen zu zerstören. In dieser Arbeit wurde die Rolle der chlamydiellen Deubiquitinase ChlaDUB1 auf die Vermeidungsstrategien vor dem Immunsystem untersucht. Mit Hilfe der Mutante Ctr Tn-cdu1, die durch Insertion eines Transposons für eine verkürzte und inaktive Deubiquitinase codiert, konnte gezeigt werden, dass ChlaDUB1 ein Gegenspieler des Autophagiesystems ist. Die Inklusionen der Mutante wurden mit K48 und K63 Ubiquitinketten modifiziert, was die Rekrutierung von Autophagiemarkern wie p62, NBR1 und NDP52 zur Folge hatte. Die Rekomplementierung mit aktivem ChlaDUB1 Protein hob die Modifikation der Inklusion wieder auf. Jedoch wurde die Xenophagie so weit vorangetrieben, bis sich LC3 positive Phagosomen um die Inklusionen von Ctr Tn-cdu1 bildeten, die allerdings nicht mit dem Lysosom verschmolzen. Das beobachtete Wachstumsdefizit in Chlamydien, die keine funktionelle Deubiquitinase exprimieren, konnte nicht auf die Autophagie zurückgeführt werden, sondern war voraussichtlich aufgrund verlangsamter Entwicklung und Replikation entstanden. In einem siRNA basierten Experiment konnte die E3 Ligase Ankib1 für die Ubiquitinierung der Ctr Tn-cdu1 Inklusion identifiziert werden. Des Weiteren sind das Ubiquitin aktivierende Enzym Ube1 und das Ubiquitin konjugierende Enzym Ube2L3 essentiell für die Modifikation der Inklusion. Da Chlamydien ein reduziertes Genom haben und nicht für alle Enzyme selbst kodieren, sind sie auf Lipide und Metabolite der Wirtszelle für ihr Wachstum angewiesen. Die Rekrutierung der fragmentierten Glogi-Membranen zur Inklusionsoberfläche wurde durch inaktives ChlaDUB1 Protein verhindert, das wahrscheinlich die bakterielle Entwicklung negativ beeinflusst. Des Weiteren ubiquitinierte Ankib1 nicht nur Inklusionen in humanen, sondern auch in murinen Zellen, was auch hier die Bindung von Autophagiemarkern zur Folge hatte. Der Vergleich unter verschiedenen chlamydiellen Serotypen und Arten zeigte, dass Ankib1 nur an Inklusionen von C. trachomatis zu finden war, nicht aber für C. muridarum oder C. pneumoniae. Des Weitern wurde die Rolle von ChlaDUB1 in infiziertem Gewebe genauer betrachtet, da die Protease auch während frühen EB Phasen nachgewiesen wurde, in denen sie Kontakt zu immigrierenden Immunzellen haben könnte. Während der Zelllyse werden Bakterien frei gesetzt, die neue Wirtszellen, aber auch Immunzellen, infizieren können. Die Infektion von humanen Neutrophilen mit Ctr Tn-cdu1 zeigte vermindertes bakterielles Wachstum und verdeutlicht die Bedeutung von ChlaDUB1 für das Überleben in diesen Immunzellen.

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