Zellkultursysteme mit Krebszelllinien werden seit Langem zur Untersuchung der Interakti-on zwischen Pathogenen und ihren Wirtszellen eingesetzt. Diese Systeme eignen sich aufgrund der reduzierten Komplexität für die Analyse einzelner Faktoren, spiegeln jedoch nicht den Zustand primärer Zellen oder die komplexe Gewebestruktur wieder. Um die Beschränkungen zu umgehen, wurden in dieser Arbeit neue Modelle etabliert auf der Grundlage von reversibel immortalisierten humanen Primärzellen und ex vivo Kultur von intaktem humanem Eileitergewebe. Infektionen mit dem humanpathogenen Bakterium Chlamydia trachomatis, welches chronische Schmerzen oder Unfruchtbarkeit auslösen kann, wurden in diesen Modellen untersucht. Reversible Immortalisierung wurde mit pri-mären human Eileiterzellen (FT Zellen) und humanen Nabelschnurzellen (HUVEC) durchgeführt. Das System basiert auf lentiviralem Gentransfer und dem Cre-lox-System. HUVEC Zellen wurden mit Kombinationen der Onkoproteine hTERT, SV40T und Bmi1 immortalisiert. Immortalisierung von FT Zellen wurde mit SV40T und Bmi1 erreicht. Eine Analyse der FT Zelllinien zeigte Veränderungen des Karyotyps durch die Immortalisie-rung. Bemerkenswerterweise konnten die Stammzellmarker CD44 und Oct4 in FT Zellen nachgewiesen werden. Ex vivo Gewebekultur humaner Eileiter wurde als stabiles Infekti-onsmodel für Chlamydia trachomatis etabliert. Mittels hochauflösender Konfokal-mikroskopie wurde gezeigt, dass die Infektion mit C. trachomatis tiefgreifende Verände-rungen im Epithel der Mukosa auslöst und zum Verlust der Zelladhäsion und Zellpolarität führt. Ein erhöhter Anteil apoptotischer Zellen wurde nach Infektion mit Serovar D beo-bachtet, einem klinischen Isolat des Genitaltraktes. Dieses Ergebnis steht im Gegensatz zu Infektionen mit dem Laborstamm Serovar L2. Phänotypische Veränderungen in nicht infizierten Zellen weisen auf die Existenz parakriner Signalwege während der akuten In-fektion und Veränderung der epithelialen Homeostase hin. / Cell culture systems with cancer-derived cell lines have long been used to study the interaction between pathogens and their host cells. Due to reduced complexity these systems are convenient for the analysis of single factors; however, they do not represent the condition of primary cells or the complex tissue structure. To circumvent these limitations new models were established in this study on the basis of reversibly immortalized human primary cells and ex vivo culture of intact human fallopian tube tissue. Infections with the human pathogenic bacterium Chlamydia trachomatis, which can lead to chronic pain or infertility, were analyzed in these models. Reversible immortalization was applied to primary human fallopian tube (FT) cells and human umbilical vein cells (HUVEC). This system is based on lentiviral gene transfer and the Cre-lox-system. HUVEC cells were immortalized with a combination of two of the oncoproteins hTERT, SV40T and Bmi1. Immortalization of FT cells was achieved with SV40T and Bmi1. Analysis of FT cell lines revealed changes of the karyotype induced by immortalization. Remarkably, the stem cell markers CD44 and Oct4 were detected in FT cells. Ex vivo tissue culture of human fallopian tubes was established as stable and reliable infection model for Chlamydia trachomatis. Via high resolution confocal analysis the infection with C. trachomatis was discovered to trigger profound changes in the epithelial mucosa, causing loss of cell adhesion and polarity. Interestingly, an increase in the rate of apoptotic cells was observed after infection with serovar D, a clinical genital tract isolate. This finding is in contrast to infections with serovar L2, a laboratory strain. Phenotypic changes in non-infected cells suggest the existence of paracrine signalling during acute infection and change in epithelial homeostasis.

Weltweit kommt es jährlich zu 90 Mio. Neuinfektionen mit dem sexuell übertragbaren Erreger Chlamydia trachomatis. Allerdings sind die Faktoren, die eine erfolgreiche bakterielle Vermehrung ermöglichen, weitgehend unbekannt. Während ihrer obligat intrazellulären Entwicklung sind Chlamydien auf die Errichtung und Erhaltung ihrer Nische, der Inklusion, angewiesen. Durch Interaktionen mit vesikulären Transportwegen der Wirtszelle, welche z.B. Sphingolipide transportieren, sichern die Bakterien ihr Überleben. In der vorliegenden Arbeit konnte gezeigt werden, dass Chlamydien während einer Infektion die Auflösung der Struktur des Golgi-Apparates induzieren. Mit Hilfe der RNA-Interferenz-Technik (RNAi) wurden die Auswirkungen des Verlustes von Golgi-Strukturproteinen auf die bakterielle Vermehrung untersucht. Der funktionelle Ausfall von Golginen, wie z.B. Golgin-84 führte zu einer Fragmentierung des Golgi-Apparates. Diese begünstigte die chlamydiale Produktion neuer infektiöser Partikel, was eine verbesserte Versorgung mit Nährstoffen nahelegt. Im vesikulären Transport von Nährstoffen übernehmen Rab-Proteine eine Schlüsselrolle. Interessanterweise konnte in dieser Arbeit gezeigt werden, dass der Verlust von Rab6 und Rab11 durch RNAi zu einer signifikanten Verringerung der Anzahl infektiöser Nachkommen führte. In diesen Zellen wurde der Golgi-Apparat nicht fragmentiert und der Transport von Sphingolipiden zu den Bakterien war stark vermindert. Untersuchungen nach simultaner Herunterregulation von Golgin-84 und Rab6 oder Rab11 demonstrierten abschließend, dass eine Kontrolle der Golgin-84-induzierten Golgi-Fragmentierung über Rab-Proteine möglich sein könnte. Die Ergebnisse dieser Arbeit offenbaren einen neuen Zusammenhang zwischen der Struktur des Golgi-Apparates und dessen Kontrolle über Rab-Proteine und ermöglichen einen tieferen Einblick in die Funktion des Golgi-Apparates während einer Chlamydien-Infektion. / Worldwide, approximately 90 mio. people are infected with the obligate intracellular bacterium Chlamydia trachomatis. However the factors involved in its successful infection and replication remain unknown. Chlamydia survive and replicate within a membrane bound niche inside host cells, termed the inclusion. To ensure survival, the chlamydial inclusion intercepts vesicular trafficking pathways of the host cell to acquire essential nutrients, such as sphingolipids. However, the exact mechanisms by which Chlamydia acquire these lipids have not been elucidated. The present work established that infection of host mammalian cells with C. trachomatis induced fragmentation of the Golgi-apparatus, but details of the mechanism to the bacterium’s pathogenesis are still required. Using RNA-Interference the role of specific Golgi-apparatus structural proteins in bacterial infectivity was investigated. Knockdown of Golgins in host cells resulted in a fragmented Golgi-apparatus and an associated increase in chlamydial replication, suggesting an enhanced acquisition of nutrients. Since Rab-proteins are known to co-ordinate the intracellular vesicular transport of nutrients, their importance in chlamydial infectivity was also investigated. Interestingly, knock down of Rab6 and Rab11 led to a significant reduction in infectious progeny. Surprisingly, upon knock down of Rab6 or Rab11 the Golgi-apparatus remained intact and sphingolipid transport into the inclusion was severely perturbed. Finally, analysis of cells simultaneously depleted of golgin-84 and Rab6 or Rab11 suggested a possible role of Rab-proteins in the control of golgin-84-induced Golgi fragmentation. These data demonstrate a yet unknown relationship between the structure of the Golgi-apparatus and its regulation and control by Rab-proteins. Furthermore, this work contributes to the existing knowledge regarding the function of the Golgi-apparatus during chlamydial infections.

Chlamydia trachomatis ist ein obligat intrazellulär lebendes Bakterium. Es wird mit einer Vielzahl von Krankheiten in Verbindung gebracht. Das Überleben von Chlamydia trachomatis hängt in nahezu allen Aspekten von der Wirtszelle ab, angefangen bei der Anheftung an die Wirtszelle, über die Invasion, bis zur intrazellulären Replikation. Für ein vollständiges Bild der Pathogenese sind sowohl das Verstehen der dazu beitragenden bakteriellen wie auch Wirtszellfaktoren essenziell. Die vorliegende Arbeit konzentriert sich dabei auf die an der Infektion beteiligten Wirtsfaktoren. Mit Hilfe eines RNA-Interferenz-vermittelten Funktionsverlustscreens wurden 59 Wirtszellgene identifiziert, die einen Einfluss auf die Infektion und Vermehrung von Chlamydia trachomatis hatten. Unter Zuhilfenahme von bioinformatischen Signaltransduktionsweganalyse-Programmen konnten einige der Hits bekannten zellulären Signalnetzwerken, unter anderem dem Ras/Raf/Mek/Erk-Signalweg, zugeordnet werden. Insbesondere der Funktionsverlust zweier validierter Targets, Ras und Raf1, erhöhte das Chlamydien-Wachstum und deren Vermehrung. Bisher wurde angenommen, dass die bei der Chlamydien-Infektion beobachtete Aktivierung der Kinase Erk, die mit der Aktivierung der Phospholipase cPLA2, der Induktion des Interleukins 8 sowie der Stabilisierung des antiapoptotischen Faktors Mcl-1 in Verbindung steht, über den Ras/Raf/Mek-Signalweg vermittelt wird. Ich konnte jedoch zeigen, dass die Chlamydien-induzierte Erk-Aktivierung unabhängig von Ras und Raf1 stattfindet. Vielmehr wird während der Chlamydien-Infektion, Raf1, abhängig von der Kinase Akt, durch Phosphorylierung an Serin259 inaktiviert. Zudem wird das inaktivierte Raf1 zur bakteriellen Inklusion rekrutiert. Dies lässt vermuten, dass das Überleben derChlamydien und deren Wachstum nicht nur von der Erk-Aktivierung und dessen Substrate, sondern auch von der Inaktivierung von Raf1 und dessen Rekrutierung zur Inklusion abhängt. / Chlamydia trachomatis is a Gram-negative obligate intracellular bacterial pathogen with a major impact on human health. As an obligate intracellular pathogen, Chlamydia rely on host cell for all aspects of their survival. Here we present RNAi screen that identified 59 host cell genes influencing C. trachomatis infection and infectivity. Network analysis of hits revealed several prominent signaling networks, including Ras-Raf-MEK-ERK pathway. Knockdown of Ras and Raf1 components of the aforesaid pathway led to increased Chlamydial growth and survival. In Chlamydia infections, ERK activation is believed to be activated through upstream kinases Ras-Raf-MEK and involved in activation of cPLA2, induction of IL8 and stabilization of the anti-apoptotic Mcl-1. However, I could show that ERK activation after Chlamydia infection is independent of Ras and Raf1. Moreover, I also showed that Raf1 is inactivated by phosphorylation at Ser259 in an Akt dependent manner. Consequently, the Ser259 phosphorylated Raf1 was recruited to the Chlamydia inclusion in an Akt and 14-3-3β dependent manner. This strongly suggests that Chlamydia survival and replication in the host cell depends not only on the activation of ERK and its downstream targets such as cPLA2, but also on the inactivation of Raf1 by phosphorylation and recruitment to the inclusion.

C. trachomatis est une bactérie Gram-négative intracellulaire obligatoire et un pathogène humain. Première cause de maladie sexuellement transmissible d'origine bactérienne, elle est également responsable, dans les pays en développement, d'infections oculaires pouvant conduire à la cécité (trachome). Son cycle de développement bi-phasique a lieu au sein d'un compartiment appelé inclusion. Grâce à un système de sécrétion de type 3 (SST3), Chlamydia sécrète des protéines dans le cytosol de la cellule afin de promouvoir sa survie et sa multiplication. Ces protéines sont désignées sous le terme d'effecteurs. / C. trachomatis is an obligate intracellular Gram-negative bacteria and a human pathogen. It is the most prevalent cause of sexually transmitted diseases of bacterial origin and a leading cause of preventable blindness in the developing world. During their biphasic developmental cycle the bacteria remains in a membrane-bounded cellular compartment called an inclusion. Using a type 3 secretion system (T3SS) they translocate effector proteins inside the cytosol of the cell to promote its survival and multiplication.The aim of the PhD was to study the function of CT622, a hypothetic protein from C. trachomatis. We showed that CT622 is an effector protein from the T3SS and that it is secreted early during the infection. We identified a bacterial protein that binds to CT622, and we showed that it acts as a chaperone, stabilizing CT622 and enhancing its secretion. We obtained bacteria lacking CT622 expression, thus demonstrating that CT622 is not essential for bacterial growth in vitro. However, preliminary studies indicate that in the absence of CT622 bacterial development is delayed and T3SS is defective.We identified several molecules interacting with CT622: geranylgeranyl diphosphate, Rab39 and Atg16L1 proteins. Future work will aim at understanding how these identified interactions, or other bacterial or cellular partners still to be discovered, contribute to the establishment of a niche favorable to bacterial development.

Made available in DSpace on 2016-12-23T13:56:05Z (GMT). No. of bitstreams: 1 Marcelo Joaquim Barbosa.pdf: 493310 bytes, checksum: 1cc616c331737b5497291791ef93fb83 (MD5) Previous issue date: 2010-07-12 / Objetivos: Determinar a prevalência de Neisseria gonorrhoeae (NG) e Chlamydia trachomatis (CT) e identificar fatores demográficos, comportamentais e clínicos correlacionados a essas infecções em homens atendidos em seis clínicas de DST no Brasil. Métodos: Estudo multicêntrico, em corte transversal realizado em homens que procuraram atendimento em clínicas de DST. O estudo incluiu clínicas de DST em seis cidades distribuídas nas cinco macrorregiões do Brasil, 2004/2005. Metodologia: Coletou-se 20 ml do primeiro jato de urina para testar NG e CT por DNA-PCR. Resultados: Um total de 767 (92,9%) homens foi incluído no estudo. A mediana de idade foi de 24 (DIQ21 30) anos. A prevalência de infecção por CT foi 13,1% (IC95% 10,7%-15,5%) e por NG 18,4% (IC95% 15,7%-21,1%). A prevalência de coinfecção foi 4,4% (IC95% 2,95%-5,85%). Os fatores identificados como sendo independentemente associados com a infecção por clamídia no modelo final de regressão logística foram: ser jovem (15-24 anos) [OR=1,4 (IC95% 1,01-1,91)], apresentar corrimento uretral ao exame [OR=4.8 (IC95% 1,52-15,05)], verrugas genitais [OR=3,0 (IC95% 1,49-5,92)] e história prévia de corrimento uretral [OR=2,4 (IC95% 1,11-5,18)]. As variáveis associadas com gonorréia foram: ser jovem (15-24 anos) [OR=1,5 (IC95% 1,09-2,05)], apresentar corrimento uretral ao exame [OR=9,9 (IC95% 5,53-17,79)], verrugas genitais [OR=18,3 (IC95% 8,03-41,60)] e úlcera ao exame clínico [OR=4,9 (IC95% 1,06-22,73)]. Conclusões: Estes resultados mostram implicações importantes na realização de diagnóstico e tratamento precoces para evitar a transmissão, complicações e implementação de medidas de educação em saúde direcionadas aos homens e deve ser estimulado o oferecimento de testes para DST na rotina dos nos serviços. / Objectives: To determine the prevalence of Neisseria gonorrhoeae(NG) and Chlamydia trachomatis (CT) infections and identified demographic, behavioral, and clinical correlates of these infections in men attending six STI clinics in Brazil. Methods: Multicentric, cross-sectional study performed among men attending STI clinics in Brazil. The study included STD clinics in six diverse cities in the five geographic regions of Brazil in 2005. Patients provided 20 ml of first catch urine for testing for NG and CT by DNA-PCR. Results: A total of 767 (92.9%) men were included in the study. The median age was 24 (IQR 21 30) years old. Prevalence of Chlamydia infection was 13.1% (95%CI 10.7%-15.5%) and gonorrhea was 18.4% (95%CI 15.7%-21.1%). Coinfection prevalence was 4.4% (95%CI 2.95%-5.85%) in men who sought assistance in STI clinics. Factors identified as being independently associated with Chlamydia trachomatis in the final multiple logistic model were being younger (15-24) [OR=1.4 (95%CI 1.01-1.91)], present urethral discharge [OR=4.8 (95%CI 1.52-15.05)], genital warts [OR=3.0 (95%CI 1.49-5.92)] and previous history of urethral discharge [OR=2.4 (95%CI 1.11-5.18)]. Variables associated with gonorrhea were being younger (15 to 24) [OR=1.5 (95%CI 1.09-2.05)], presence of urethral discharge [OR=9.9 (95%CI 5.53-17.79)], genital warts [OR=18.3 (95%CI 8.03-41.60)] and ulcer in clinical examination [OR=4.9 (95%CI 1.06-22.73)]. Conclusions: These findings have important implications for implementing education and prevention efforts directed toward men at risk of HIV/STD. A venue-based approach to offer routinely testing to men in STD clinic should be stimulated.

Made available in DSpace on 2016-12-23T13:56:06Z (GMT). No. of bitstreams: 1 Dissertacao de Marcelo Joaquim Barbosa.pdf: 493316 bytes, checksum: 05c09f00c2dba0603712d1b6f468b750 (MD5) Previous issue date: 2010-07-12 / Objetivos: Determinar a prevalência de Neisseria gonorrhoeae (NG) e Chlamydia trachomatis (CT) e identificar fatores demográficos, comportamentais e clínicos correlacionados a essas infecções em homens atendidos em seis clínicas de DST no Brasil. Métodos: Estudo multicêntrico, em corte transversal realizado em homens que procuraram atendimento em clínicas de DST. O estudo incluiu clínicas de DST em seis cidades distribuídas nas cinco macrorregiões do Brasil, 2004/2005. Metodologia: Coletou-se 20 ml do primeiro jato de urina para testar NG e CT por DNA-PCR. Resultados: Um total de 767 (92,9%) homens foi incluído no estudo. A mediana de idade foi de 24 (DIQ21 30) anos. A prevalência de infecção por CT foi 13,1% (IC95% 10,7%-15,5%) e por G 18,4% (IC95% 15,7%-21,1%). A prevalência de coinfecção foi 4,4% (IC95% 2,95%-5,85%). Os fatores identificados como sendo independentemente associados com a infecção por clamídia no modelo final de regressão logística foram: ser jovem (15-24 anos) [OR=1,4 (IC95% 1,01-1,91)], apresentar corrimento uretral ao exame [OR=4.8 (IC95% 1,52-15,05)], verrugas genitais [OR=3,0 (IC95% 1,49-5,92)] e história prévia de corrimento uretral [OR=2,4 (IC95% 1,11-5,18)]. As variáveis associadas com gonorréia foram: ser jovem (15-24 anos) [OR=1,5 (IC95% 1,09-2,05)], apresentar corrimento uretral ao exame [OR=9,9 (IC95% 5,53-17,79)], verrugas genitais [OR=18,3 (IC95% 8,03-41,60)] e úlcera ao exame clínico [OR=4,9 (IC95% 1,06-22,73)]. Conclusões: Estes resultados mostram implicações importantes na realização de diagnóstico e tratamento precoces para evitar a transmissão, complicações e implementação de medidas de educação em saúde direcionadas aos homens e deve ser estimulado o oferecimento de testes para DST na rotina dos nos serviços / Objectives: To determine the prevalence of Neisseria gonorrhoeae(NG) and Chlamydia trachomatis (CT) infections and identified demographic, behavioral, and clinical correlates of these infections in men attending six STI clinics in Brazil. Methods: Multicentric, cross-sectional study performed among men attending STI clinics in Brazil. The study included STD clinics in six diverse cities in the five geographic regions of Brazil in 2005. Patients provided 20 ml of first catch urine for testing for NG and CT by DNA-PCR. Results: A total of 767 (92.9%) men were included in the study. The median age was 24 (IQR 21 30) years old. Prevalence of Chlamydia infection was 13.1% (95%CI 10.7%-15.5%) and gonorrhea was 18.4% (95%CI 15.7%-21.1%). Coinfection prevalence was 4.4% (95%CI 2.95%-5.85%) in men who sought assistance in STI clinics. Factors identified as being independently associated with Chlamydia trachomatis in the final multiple logistic model were being younger (15-24) [OR=1.4 (95%CI 1.01-1.91)], present urethral discharge [OR=4.8 (95%CI 1.52-15.05)], genital warts [OR=3.0 (95%CI 1.49-5.92)] and previous history of urethral discharge [OR=2.4 (95%CI 1.11-5.18)]. Variables associated with gonorrhea were being younger (15 to 24) [OR=1.5 (95%CI 1.09-2.05)], presence of urethral discharge [OR=9.9 (95%CI 5.53-17.79)], genital warts [OR= 8.3 (95%CI 8.03-41.60)] and ulcer in clinical examination [OR=4.9 (95%CI 1.06-22.73)]. Conclusions: These findings have important implications for implementing education and prevention efforts directed toward men at risk of HIV/STD. A venue-based approach to offer routinely testing to men in STD clinic should be stimulated

Made available in DSpace on 2014-12-17T14:10:29Z (GMT). No. of bitstreams: 1 PauloAFM_DISSERT.pdf: 770018 bytes, checksum: c4b92c68824123455aa7a5f94bd77119 (MD5) Previous issue date: 2013-08-08 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Genital infection with Chlamydia trachomatis is now recognized as one of the most prevalent sexually transmitted infections (STDs). Despite major advances in laboratory diagnosis techniques, primarily the character of asymptomatic chlamydial infection in both men and in women constitutes the basis for the formation of reservoirs that perpetuate transmission and acquisition of this and other STDs. The asymptomatic in women favors the rise of infection to the upper genital tract, causing injuries that can result in infertility. An examination of population screening for early detection and treatment of asymptomatic infections is the key step in combating this major public health problem. The present study aimed to evaluate the prevalence of infection by C. trachomatis in sexually active women attended the screening program for cervical cancer of the uterus in health facilities in municipalities in different regions of the State of Rio Grande do Norte, and identify factors that may contribute to the spread of this pathogen and its relationship with the lesions of the uterine cervix. It is a cross-sectional study aimed at detecting the presence of genital tract infection by C. trachomatis either in isolated form or in association with human papilloma virus (HPV) infection in asymptomatic women. Were included in this study, a total sample of 1,134 women aged 13-76, mean 34.4 years, from March 2008 to September 2012. Specimens containing exfoliated cells of the epithelium of the uterine cervix were analyzed by examining Pap cytology for the detection of possible injuries, and the polymerase chain reaction (PCR) for detection of plasmid DNA from C. trachomatis and HPV. Infection with C. trachomatis was detected with overall prevalence rate of 8.1% in the isolated form and 2.8% in co-infection with HPV. The infection was detected in 7.4% of women with normal cytology 11.5% of those with atypical cells of undetermined significance (ASC-US) and 16.7% of those with low-grade squamous intraepithelial lesion (LSIL). We observed an association between C. trachomatis and incidence of low-grade squamous intraepithelial lesion (LSIL). The genital tract infection by C. trachomatis alone was associated with education level, ethnicity and parity, revealing that women with higher education, those of non-white ethnicity and those who had three or more pregnancies were more likely to acquire infection. Levels very close to statistical significance were observed for chronological age, age at first sexual intercourse and first pregnancy. There was no association with marital status, number of sexual partners. Co-infection with C. trachomatis and HPV was detected in 2.3% of women with normal cytology, who had 5.1% in ASC-US and 10.4% in those with LSIL. No association was found between infection C. trachomatis and increased risk of HPV infection, but women with simultaneous infection by both pathogens showed greater risk for LSIL. Co-infection was more prevalent among single women, who had in the first sexual intercourse under 18 years and those who had two or more sexual partners over a lifetime / A infec??o genital por Chlamydia trachomatis ? reconhecida atualmente como uma das mais prevalentes infec??es sexualmente transmiss?veis (IST). Apesar dos grandes avan?os das t?cnicas de diagn?stico laboratorial, o car?ter primariamente assintom?tico da infec??o clamidial tanto em homens, quanto em mulheres constitui-se na base para forma??o de reservat?rios que perpetuam a transmiss?o e a aquisi??o desta e de outras IST. A forma assintom?tica em mulheres favorece a ascens?o da infec??o para o trato genital superior, ocasionando agravos que podem resultar em infertilidade. O exame de triagem populacional para a detec??o precoce e o tratamento das infec??es assintom?ticas ? o procedimento chave no combate a este importante problema de sa?de p?blica. O presente estudo teve como objetivo avaliar a preval?ncia da infec??o por C. trachomatis em mulheres sexualmente ativas atendidas pelo programa de rastreamento do c?ncer de colo do ?tero em unidades de sa?de de munic?pios das diferentes regi?es do Estado do Rio Grande do Norte, bem como identificar fatores que podem contribuir para a dissemina??o desse pat?geno e sua rela??o com as les?es da c?rvice uterina. Trata-se de um estudo transversal que visa detectar a presen?a de infec??o do trato genital por C. trachomatis seja na forma isolada, ou em associa??o com o v?rus do papiloma humano (HPV) em mulheres assintom?ticas. Foram estudadas 1.134 mulheres com idade variando de 13 a 76, m?dia de 34,4 anos, no per?odo de mar?o de 2008 a setembro de 2012. Esp?cimes contendo c?lulas descamadas do epit?lio da c?rvice uterina foram analisados por meio do exame citol?gico de Papanicolau para a detec??o de poss?veis les?es, e pela rea??o em cadeia da polimerase (PCR) para detec??o do DNA plasmidial da C. trachomatis e do HPV. A infec??o por C. trachomatis foi detectada com taxa de preval?ncia global de 8,1%, na forma isolada e de 2,8% em co-infec??o com HPV. A infec??o foi detectada em 7,4% das mulheres com citologia normal, 11,3% daquelas com c?lulas at?picas de significado indeterminado (ASC-US) e 16,7% das que tinham les?o intraepitelial de baixo grau (LSIL). Observou-se associa??o entre C. trachomatis e ocorr?ncia de les?o intraepitelial de baixo grau (LSIL). A infec??o do trato genital por C. trachomatis sozinha mostrou-se associada com escolaridade, etnia e n?mero de gesta??es, revelando que as mulheres com maior escolaridade, as de etnia n?o branca e aquelas que tiveram tr?s ou mais gesta??es apresentaram maiores chances de adquirir infec??o. N?veis muito pr?ximos da signific?ncia estat?stica foram observados, para idade cronol?gica, idade do primeiro intercurso sexual e da primeira gesta??o. N?o se observou associa??o com situa??o conjugal e n?mero de parceiros sexuais. A co-infec??o por C. trachomatis e HPV foi detectada em 2,3% das mulheres com citologia normal, 5,1% nas que tinham ASC-US e 10,4% naquelas com LSIL. N?o se observou associa??o entre a infec??o C. trachomatis e aumento de risco de infec??o pelo HPV, mas as mulheres com infec??o simult?nea pelos dois pat?genos apresentaram maior risco de ter LSIL. A co-infec??o foi mais prevalente nas mulheres solteiras, nas que tiveram o primeiro intercurso sexual com menos de 18 anos e nas que tiveram dois ou mais parceiros sexuais ao longo da vida

Orientadores: Paulo César Giraldo, Ana Katherine da Silveira Gonçalves / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T20:57:25Z (GMT). No. of bitstreams: 1 Amaral_RoseLuceGomesdo_D.pdf: 1220448 bytes, checksum: d6f8f0907e1226cc41668603ab978683 (MD5) Previous issue date: 2010 / Resumo: O uso de duchas vaginais é comum em diferentes povos e culturas. Entre as mulheres que a praticam, a maioria a faz após a menstruação, antes ou após a relação sexual, com a finalidade de evitar o odor desagradável ou para eliminar corrimentos, refrescar a genitália ou prevenir a gravidez. Não está claro na literatura se as duchas poderiam causar problemas às usuárias. Objetivo: Verificar se o uso habitual de duchas vaginais associa-se à infecção genital por Chlamydia trachomatis/ Neisseria gonorrhoeae e Papilomavírus humano (HPV) em mulheres profissionais do sexo. Sujeitos e Métodos: Estudo de corte transversal avaliou 200 mulheres - 111 profissionais do sexo (PS) e 89 não profissionais do sexo (NPS) - assistidas em uma Unidade Básica de Saúde de Campinas, São Paulo, Brasil. Todas as mulheres foram entrevistadas e examinadas por um único pesquisador. A anamnese abordou os antecedentes demográficos, higiênicos, sexuais e médicos, como idade, cor, paridade, escolaridade, tabagismo, estado civil, uso de duchas vaginais, idade da primeira relação sexual, número de parceiros sexuais, número de coitos por semana, práticas sexuais, uso de preservativo e lubrificante. Amostras de células cervicais foram coletadas para testes de captura híbrida objetivando Chlamydia trachomatis/Neisseria gonorrhoaea e HPV. Na análise estatística usou-se o teste exato de Fisher ou X2 para as variáveis discretas, e Mann-Whitney para as variáveis não-paramétricas. O estudo foi aprovado pelo Comitê de Ética em Pesquisa (no 902/2009) e contou com a colaboração da Secretaria de Saúde do Estado de São Paulo e Laboratório Digene. Resultado: Cerca de 40% das mulheres investigadas praticavam duchas vaginais três ou mais vezes por semana (61,7% das PS e somente 14,6% das NPS). A infecção por CT/NG foi positiva em 10,5% do total dos casos, 17 casos (15,3%) em PS e em quatro casos (4,5%) das NPS (p=0,01), porém não houve diferenças significativas entre usuárias de duchas vaginais (14,81%) e não usuárias (7,6%), (p=ns). As PS não usuárias de duchas vaginais tiveram quase o dobro de infecção CT/NG que as NPS usuárias de duchas vaginais (13,9% vs 7,7%). O HPV foi positivo em 40,5% dos casos, sendo 55,8% da PS e em 21,3% das NPS (p=0,001), contudo DNA-HPV não foi significativamente diferente (p=0,47) entre PS usuárias de duchas vaginais (54,4%) e em não usuárias (58,1%). HPV de alto risco foi positivo em 16,2% e 11,6% (p=ns) e o HPV de baixo risco em 23,5% e 30,2% (p=ns) em usuárias e não usuárias de duchas vaginais respectivamente. Os HPV de alto e baixo riscos foram encontrados simultaneamente em 14,7% e 16,2% das usuárias e não usuárias de duchas vaginais, respectivamente (p=ns). Conclusão: O uso de duchas vaginais não se associou às infecções genitais por CT/NG e HPV de alto ou baixo grau nas mulheres estudadas (profissionais do sexo e não profissionais do sexo) / Abstract: The use of vaginal douching (VD) is widespread around the world, and is more common than is to be expected. The majority of women douche after menses, before or after sexual intercourse to prevent odor, to alleviate vaginal symptoms, or to prevent pregnancy, however it is not clear in the literature if this habit can cause damages for women. Objective: Establish whether high frequency VD favors Chlamydia trachomatis (CT)/Neisseria gonorrhoeae (NG) and Papilomavírus humano (HPV) infection in female sex workers (SW). Subjects and Methods: A clinical cross-sectional study involving 200 women, 111 SW and 89 non-sex workers (NSW) in a Health Center in Brazil. The subjects were submitted to an interview and examined by a single researcher. A questionnaire was filled out with data that included, medical and demographic history (age, race, parity, education, smoking, marital status), hygiene (use of VD, frequency and the solution used) and sexual behaviour (first sexual relation, number of sexual partners, frequency, sexual practices, homosexual relationships, use of condom and lubricant). Cervical samples were collected for CT and NG testing by hybrid capture 2 assay. Statistical analysis used the Fisher's exact test or qui square for discrete variables and Mann-Whitney test for nonparametric variables. The study was approved by Committee the Ethics in Research (no 902/2009) and received the cooperation of São Paulo Secretariat of Health and Digene laboratory. Results: Approximately 40% of women practiced douche three or more times per week (61.7% of SW and only 14.6% of the NSW). Infection with CT / NG was positive in 10.5% of the total cases, 17 cases (15.3%) in SW and in four cases (4.5%) of the NSW (p = 0.01). However, It was in 14.81% of D and but in and 7.6% of ND (p = ns). SW douchers had almost double CT and NG infections than NSW douchers (13.9% vs 7.7%). HPV infection was detected in 40.5% of cases, 55.8% of SW and 21.3% of NSW of (p = 0.001), despite of DNA HPV was not different (p = 0.47) between SW douchers (54.4%) and non-douchers (58.1%). High-risk DNA-HPV was positive in 16.2% and 11.6% (p = ns) and low-risk DNA-HPV in 23.5% and 30.2% (p = ns) in douchers and non-douchers respectively. High and low risk DNA-HPV were found simultaneously in 14.7% and 16.2% of douchers and non-douchers respectively (p = ns). Conclusion: The use of VD is not a causal factor for cervical CT/NG and High-/low risk HPV infection in women studied (SW and NSW) / Doutorado / Tocoginecologia / Doutor em Tocoginecologia

Objetivo: este estudo teve como objetivo avaliar as infecções por papilomavírus humano (HPV) e Chlamydia trachomatis (CT) em pacientes com artrite reumatoide (AR) pré e pós-terapia anti-TNF. Método: foram avaliadas 50 pacientes do sexo feminino com AR (preenchiam os critérios do Colégio Americano de Reumatologia) que eram elegíveis para terapia anti-TNF. Cinquenta pacientes foram incluídas no estudo de forma prospectiva. Destas, 45 pacientes foram reavaliadas após 6 meses de terapia anti-TNF. Inicialmente as 50 pacientes com AR foram comparadas com 50 controles saudáveis pareadas por idade. Foram avaliados dados demográficos, avaliação ginecológica (citologia cervical e avaliações histológicas), função sexual, parâmetros de doença e tratamento atual da AR. Os testes para detecções dos DNAs do HPV e CT nas espécimes cervicais foram realizados utilizando a captura híbrida II. Resultados: na avaliação inicial, a mediana da idade das pacientes com AR e controles foi de 49 (18-74) vs. 49 (18-74) anos, p = 1,0. Observou-se uma tendência de menor frequência de infecção por HPV nas pacientes com AR pré anti-TNF em relação aos controles (14% vs. 30%, p = 0,054). Adicionalmente, realizou-se avaliação das pacientes com AR com infecção positiva e negativa por HPV antes da terapia anti-TNF que demonstrou que o primeiro grupo apresentou maior frequência de relações sexuais (100% vs. 48%, p = 0,014), maior número de parceiros sexuais [1 (1-1) vs. 0 (0-1), p = 0,032] e maior frequência de citologia cervical anormal (43% vs. 7%, p = 0,029). A idade atual, a duração da doença, os parâmetros da doença e os tratamentos foram semelhantes em ambos os grupos (p > 0,05). Após 6 meses de terapia anti-TNF, a infecção por HPV permaneceu inalterada em cinco pacientes, enquanto que dois tornaram-se negativos e uma paciente adicional tornou-se positiva (p = 1,0). A infecção por CT foi uniformemente negativa nas pacientes com AR pré e pós-TNF, assim como nas controles. Conclusões: a infecção por HPV observada nas pacientes sexualmente ativas com AR antes da terapia anti-TNF foi leve, sem evidência de infecção por CT. A terapia anti-TNF não aumentou o risco de exacerbação e/ou progressão das infecções por HPV e CT em pacientes com AR / Objective: to evaluate human papillomavirus (HPV) and Chlamydia trachomatis (CT) infections in rheumatoid arthritis (RA) patients pre- and post-TNF blocker. Methods: fifty female RA patients (American College of Rheumatology criteria), who were eligible to anti-TNF therapy, [n = 50 at baseline (BL) and n = 45 after 6 months of treatment (6M)] and 50 agematched healthy controls were prospectively enrolled. They were assessed for demographic data, gynecologic, sexual, cervical cytology and histological evaluations, disease parameters and current treatment. HPV DNA and CT DNA testing in cervical specimens were done using Hybrid Capture II assays. Results: at BL, the median current age of RA patients and controls was 49(18-74) vs. 49(18-74) years, p = 1.0. A trend of lower frequency of HPV infection was observed in AR patients pre anti-TNF compared to controls (14% vs. 30%, p = 0.054). Further evaluation of AR patients with and without HPV infection before anti-TNF therapy showed that the former group had higher frequency of sexual intercourses (100% vs. 48%, p = 0.014), higher median number of sexual partners [1(1-1) vs. 0(0-1), p=0.032] and higher frequency of abnormal cervical cytology (43% vs. 7%, p = 0.029). Current age, disease duration, disease parameters and treatments were alike in both groups (p > 0.05). At 6M after TNF blockage, HPV infection remained unchanged in five patients, whereas two became negative and one additional patient turn out to be positive (p = 1.0). CT infection was uniformly negative in RA patients pre- and post-TNF blockage and in controls. Conclusions: anti-TNF does not seem to increase short-term risk of exacerbation and/or progression of HPV and CT infections in RA patients

<p>Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Although the temporal manner in which effectors are secreted is important for the proper manipulation of host cell functions, the mechanism remains a mystery. In this study, we provide several lines of evidence that T3S chaperones may impart coherence to effector secretion. In addition, we identified a new early T3S effector in Chlamydia. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. By defining proteins that associate with the three most abundant T3S chaperones, Slc1, Scc2 and Mcsc in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry, we identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form stable complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses and lack of C. trachomatis-induced morphological changes. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia.</p> / Dissertation