Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-11-19T14:09:47Z No. of bitstreams: 2 Dissertação - Fabiana Carmo Approbato - 2012.pdf: 1717961 bytes, checksum: a9307564863c1765301f00c5f05b6d5f (MD5) license_rdf: 23748 bytes, checksum: b92763cfc0af52c7c868455edfaf3266 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-11-20T14:28:48Z (GMT) No. of bitstreams: 2 Dissertação - Fabiana Carmo Approbato - 2012.pdf: 1717961 bytes, checksum: a9307564863c1765301f00c5f05b6d5f (MD5) license_rdf: 23748 bytes, checksum: b92763cfc0af52c7c868455edfaf3266 (MD5) / Made available in DSpace on 2014-11-20T14:28:48Z (GMT). No. of bitstreams: 2 Dissertação - Fabiana Carmo Approbato - 2012.pdf: 1717961 bytes, checksum: a9307564863c1765301f00c5f05b6d5f (MD5) license_rdf: 23748 bytes, checksum: b92763cfc0af52c7c868455edfaf3266 (MD5) Previous issue date: 2012-04-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Chlamydia trachomatis is a common factor of sexually transmitted diseases. If not treated it can induce tubal obstruction, ectopic pregnancy and infertility. In adolescent women, the prevalence can reach 30%. When look for infertility treatment, the women are at older age, so they are at a different prevalence of Chlamydia. Objectives: To evaluate the prevalence of Chlamydia trachomatis by PCR, enzyme immunoassay or indirect immuno fluorescence at infertile patients. Methods: Design: Prevalence study. Diagnoses Test. Setting: Reproductive Laboratory (LABREP) - HC / UFG and Mater Clinic of Obstetrics and Gynecology, Goiânia, Brazil. Patients: One hundred and twenty patients attended from 2011 to 2012, from 20 to 48 years old. Main Outcome Measures: Age frequency histogram, exposition to pregnancy time, Chlamydia prevalence, Odds ratio for tubal obstruction, ectopic pregnancy and others STDs (Sexually Transmitted Diseases). It was calculate the age histogram frequency, and pregnancy exposition time. We used Chi Square statistical for Odds risk to tubal obstruction for serum positive patients; the Odds risk to ectopic pregnancy for serum positive patients and Odds risk to others STDs for serum positive patients. The rejection p was 5 % (p = 0.05). The Statistical Software used was BioEstat® and excel®. The study was submitted and approved by ethics committee of Clinical Hospital of Federal University of Goias State, Brazil. Results: The patients average age was 33.2 years, above of adolescent age prevalence, when chlamydia is frequently found. The average of exposition to pregnancy was 48.6 months. The PCR Chlamydia detection was less than 1 % (0.83 %). The Chlamydia PCR prevalence for serum positive was 2.4 % (one patient). We did not found Positive PCR between serum negative patients. The Odds to tubal obstruction for serum positive patients was 2.5. This was statistically significant. The Odds Ratio to ectopic pregnancy for serum positive patients was 1.31. This was not statistically significant. The Odds Ratio to others STDs for serum positive patients was 4.1 (p = 0.024). The Odds Ratio to ectopic pregnancy for tubal obstruction was 19.1. This was highly significant (p = 0.001). The NNT was 42. Conclusions: We conclude that C. trachomatis has a very low frequency at this population ( < 1 %), but heavy sequels of infection stays. The average age of the infertile patients was 33.2 years, above of the age mean of adolescents. We found association of positive serology screening and statistically significant risk for tubal obstruction, ectopic pregnancy and to others STDs. / A Chlamydia trachomatis é fator etiológico comum de doenças sexualmente transmissíveis. Quando não tratada pode provocar sequelas como obstrução tubária, gravidez ectópica e infertilidade. Entre mulheres adolescentes, a taxa de prevalência pode chegar a 30%. Por outro lado, nas mulheres que procuram tratamento para infertilidade a faixa etária é maior, e a prevalência diferente. Objetivos: Avaliar a prevalência de Chlamydia trachomatis pela técnica de PCR, por enzimaimunoensaio ou imunofluorescência indireta em pacientes com esterilidade feminina. Métodos: Desenho: Estudo de prevalência. Teste diagnóstico. Local: Laboratório de Reprodução Humana (LABREP) - HC / UFG e Mater Clínica de Ginecologia e Obstetrícia, Goiânia. Pacientes: Foram estudadas 120 pacientes atendidas entre 2011 a 2012 com idade entre 20 e 48 anos. Principais resultados medidos: Faixa etária, tempo de exposição à gravidez, prevalência da clamídia, risco de Odds para obstrução tubária, gravidez ectópica e outras DSTs. Foram calculados os histogramas de frequência para idade e tempo de exposição. Foi utilizada a estatística de Qui quadrado para cálculo do risco de Odds de obstrução tubária entre as pacientes soro positivas; o risco de Odds de gravidez ectópica em pacientes soro positivas e o risco de Odds das pacientes soropositivas ter outras DSTs. O nível de significância escolhido foi 5 % (p = 0,05). O programa utilizado foi o BioEstat® e a planilha do Excel®. O projeto foi submetido e aprovado pelo Comitê de Ética do Hospital das Clínicas na Universidade Federal de Goiás. Resultados: A média de idade das pacientes foi 33,2 anos, acima da faixa de prevalência nas adolescentes, quando a presença de clamídia é elevada. A média de tempo de exposição (tempo tentativa de gravidez) foi de 48,6 meses. A prevalência de infecção detectada pelo exame de PCR foi menor do que 1 % (0,83 %). A prevalência da infecção por clamídia pelo exame de PCR entre as pacientes soro positivas foi de 2,4 % (uma paciente). Não encontramos PCR positivo entre as soro negativas. O risco de Odds entre as pacientes soro positivas para obstrução tubária foi de 2,5. Este valor foi estatisticamente significativo. O risco de Odds entre as pacientes soro positivas para apresentar gravidez ectópica foi de 1,31. Este valor não atingiu nível estatístico significativo. O risco de Odds entre as pacientes soro positivas foi de 4,1 para apresentar outras DSTs. Este valor foi estatisticamente significativo. O risco de Odds entre as pacientes com obstrução tubária para apresentar gravidez ectópica foi de 19,1. Este valor atingiu nível estatístico significativo (p = 0,001). Encontramos um NNT de 42 neste trabalho. Conclusões: Neste trabalho, a prevalência da clamídia detectada por PCR foi menor do que 1 % permanecendo as sequelas graves da infecção. Encontramos associação entre a sorologia positiva para Chlamydia trachomatis e obstrução tubária, gravidez ectópica e outras DSTs neste estudo. A média de idade das pacientes foi 33,2 anos, acima da faixa de prevalência nas adolescentes, quando a presença de clamídia é elevada.

The resolution of fluorescence light microscopy was long believed to be limited by the diffraction limit of light of around 200-250 nm described in 1873 by Ernst Abbe. Within the last decade, several approaches, such as structured illumination microscopy (SIM), stimulated emission depletion STED and (direct) stochastic optical reconstruction microscopy (d)STORM have been established to bypass the diffraction limit. However, such super-resolution techniques enabling a resolution <100 nm require specialized and expensive setups as well as expert knowledge in order to avoid artifacts. They are therefore limited to specialized laboratories. Recently, Boyden and colleagues introduced an alternate approach, termed expansion microscopy (ExM). The latter offers the possibility to perform superresolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded. Since its introduction in 2015, expansion microscopy has developed rapidly offering protocols for 4x, 10x and 20x expansion of proteins and RNA in cells, tissues and human clinical specimens. Mitochondria are double membrane-bound organelles and crucial to the cell by performing numerous tasks, from ATP production through oxidative phosphorylation, production of many important metabolites, cell signaling to the regulation of apoptosis. The inner mitochondrial membrane is strongly folded forming so-called cristae. Besides being the location of the oxidative phosphorylation and therefore energy conversion and ATP production, cristae have been of great interest because changes in morphology have been linked to a plethora of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. However, cristae imaging remains challenging as the distance between two individual cristae is often below 100 nm. Within this work, we demonstrate that the mitochondrial creatine kinase MtCK linked to fluorescent protein GFP (MtCK-GFP) can be used as a cristae marker. Upon fourfold expansion, we illustrate that our novel marker enables visualization of cristae morphology and localization of mitochondrial proteins relative to cristae without the need for specialized setups. Furthermore, we show the applicability of expansion microscopy for several bacterial pathogens, such as Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae and Staphylococcus aureus. Due to differences in bacterial cell walls, we reveal important aspects for the digestion of pathogens for isotropic expansion. We further show that expansion of the intracellular pathogens C. trachomatis and S. negevensis, enables the differentiation between the two distinct developmental forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We demonstrate the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside the chlamydial inclusion. Moreover, we show that expansion microscopy enables the investigation of bacteria, herein S. aureus, within LAMP1 and LC3-II vesicles. With the introduction of the unnatural α-NH2-ω-N3-C6-ceramide, we further present the first approach for the expansion of lipids that may also be suitable for far inaccessible molecule classes like carbohydrates. The efficient accumulation and high labeling density of our functionalized α-NH2-ω-N3-C6-ceramide in both cells and bacteria enables in combination with tenfold expansion nanoscale resolution (10-20 nm) of the interaction of proteins with the plasma membrane, membrane of organelles and bacteria. Ceramide is the central molecule of the sphingolipid metabolism, an important constituent of cellular membranes and regulates many important cellular processes such as differentiation, proliferation and apoptosis. Many studies report about the importance of sphingolipids during infection of various pathogens. While the transport of ceramide to Chlamydia has been reported earlier, one of the unanswered questions remaining was if ceramide forms parts of the outer or inner bacterial membrane. Expansion of α-NH2-ω-N3-C6-ceramide enabled the visualization of ceramide in the inner and outer membrane of C. trachomatis and their distance was determined to be 27.6 ± 7.7 nm. / Aufgrund der Beugungseigenschaften des Lichtes wurde bereits 1873 durch Ernst Abbe für die Lichtmikroskopie eine theoretische Auflösungsgrenze von 200-250 nm definiert. Durch die Einführung verschiedener hochauflösender Mikroskopiemethoden, wie beispielsweise SIM-Mikroskopie (structured illumination microscopy), STED-Mikroskopie (stimulated emission depletion) und (d)STORM-Mikroskopie ((direct) stochastic optical reconstruction microscopy), konnte im letzten Jahrzehnt jedoch die Auflösung auf unter 100 nm verbessert werden. Allerdings benötigen solche Hochauflösungstechniken sowohl spezialisierte und kostenintensive Geräte als auch Expertenwissen zur Vermeidung von Artefakten, sodass diese nur in wenigen Laboren angewendet werden können. Ein alternativer Ansatz, die sogenannte Expansionsmikroskopie, wurde kürzlich von der Arbeitsgruppe um Ed Boyden etabliert. Hierbei wird eine Probe mit einem quellfähigen Gel vernetzt, welches daraufhin isotrop expandiert wird, sodass auch an konventionellen konfokalen Mikroskopen Hochauflösung ermöglicht wird. Seit ihrer Einführung im Jahre 2015 hat sich die Expansionsmikroskopie schnell entwickelt und bietet Protokolle für 4-fache, 10-fache oder sogar 20-fache Expansion von Proteinen als auch RNA in Zellen oder sogar komplexen Geweben. Mitochondrien besitzen zwei Membranen und sind für die Zelle von großer Bedeutung, da sie eine Vielzahl wichtiger Aufgaben übernehmen - von der ATP-Produktion durch die oxidative Phosphorylierung über die Produktion vieler wichtiger Metabolite bis hin zur Regulation zellulärer Signalwege. Die innere Mitochondrienmembran ist stark gefaltet und bildet Einstülpungen, die sogenannten Cristae, in welchen die oxidative Phosphorylierung und somit die Energieumwandlung und ATP-Synthese stattfindet. Morphologische Veränderungen der Cristae können sowohl beim Altern von Zellen, als auch bei verschiedenen Infektionen beobachtet werden und können darüber hinaus auch im Rahmen diverser Erkrankungen, wie beispielsweise Krebs, Diabetes oder neurodegenerativen Erkrankungen auftreten. Die Visualisierung der Cristae durch Fluoreszenzmikroskopie ist herausfordernd, da der Abstand zwischen einzelnen Cristae oftmals unter 100 nm beträgt. In der vorliegenden Arbeit wird gezeigt, dass die Expression der mitochondrialen Kreatinkinase gekoppelt an das Fluoreszenzprotein GFP (MtCK-GFP) als Cristaemarker genutzt werden kann. In Kombination mit vierfacher Expansion ermöglicht unser Marker die Untersuchung morphologischer Veränderungen von Cristae, sowie die Lokalisierung mitochondrialer Proteine relativ zu den Cristae. Darüber hinaus wird im Rahmen dieser Arbeit die Anwendbarkeit der Expansionsmikroskopie für mehrere bakterielle Pathogene, und zwar Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae und Staphylococcus aureus, gezeigt. Hierbei verdeutlichen wir wichtige Aspekte für den vollständigen Verdau unterschiedlicher bakterieller Zellwände und somit isotropen Expansion. Die Expansion der intrazellulären Pathogene C. trachomatis und S. negevensis ermöglichte es uns an konventionellen konfokalen Mikroskopen zwischen den zwei verschiedenen Entwicklungsstadien, der katabolisch aktiven Retikulärkörperchen (RBs) und der infektiösen Elementarkörperchen (EBs), zu unterscheiden. Außerdem konnte die Möglichkeit der präzisen Lokalisierung chlamydialer Proteine wie CPAF und Cdu1 innerhalb und außerhalb der chlamydialen Inklusion gezeigt werden und Bakterien, in diesem Fall S. aureus, in LAMP1 und LC3-II Vesikeln visualisiert werden. Mit der Einführung des unnatürlichen α-NH2-ω-N3-C6-Ceramides, präsentieren wir zudem ein erstes Konzept für die Expansion von Lipiden, welches möglicherweise auch für deutlich unzugänglichere Molekülklassen wie beispielsweise Kohlehydrate geeignet ist. Die effiziente Akkumulierung unseres funktionalisierten α-NH2-ω-N3-C6-Ceramides in Zellen sowie Bakterien ermöglicht in Kombination mit zehnfacher Expansion die Untersuchung der Interaktion von Proteinen mit der Zellmembran, Membranen von Organellen und Bakterien mit einer räumlichen Auflösung von 10-20 nm. Ceramid ist das zentrale Molekül des Sphingolipidstoffwechsels, ein wichtiger Baustein zellulärer Membrane und reguliert viele essentielle Prozesse wie die Zelldifferenzierung, die Proliferation als auch die Apoptose. Viele Studien berichten von der Bedeutung der Sphingolipide während der Infektion verschiedener Pathogene. So wurde beispielsweise zuvor berichtet, dass Ceramide aktiv zu Chlamydien transportiert und in deren Membranen eingebaut werden. Hierbei verblieb allerdings die Frage, ob Ceramide in der äußeren oder inneren bakteriellen Membran lokalisiert sind. Die Expansion unseres α-NH2-ω-N3-C6-Ceramides ermöglichte es uns Ceramide in der inneren und äußeren Membran von C. trachomatis zu visualisieren und den Abstand zwischen beiden Membranen auf 27.6 ± 7.7 nm zu bestimmen.

archives@tulane.edu / Background: Chlamydia trachomatis (Ct) is the most common bacterial sexually transmitted infection in the United States. The rate among Black Americans is disproportionately higher than that of White Americans. Ct acquisition is influenced by factors at different levels of the social-ecological model. Methods: Data was collected through Check It, a community venue-based screening study for Black men aged 15-24 who have sex with women, in New Orleans, Louisiana between 05/17/2017-03/16/2020. Latent class analysis identified classes (patterns) of behaviors and relationship traits. Regression mixture modeling assessed associations between covariates and distal outcomes within classes. Path analysis examined mediation of neighborhood factors on Ct by sexual behaviors. Results: At the individual level, five classes of risk behaviors—including substance use, condomless sex, and multiple recent partners—were identified among 1872 men; Ct prevalence ranged from 7.3%-13.6%. Age and education beyond high school were significant risk factors for two classes and health insurance was significantly protective against Ct for two classes. Among 2906 partners reported, five relationship classes emerged with Ct prevalence ranging from 10.1%-18.3%. Community members provided diverse descriptions of the classes. Age, education, substance use, multiple partners, health insurance, and time in a detention facility were predictive of class membership. The effects of three neighborhood and institutional level factors (everyday discrimination, neighborhood safety, and time in a detention facility) on Ct prevalence were mediated by behaviors (substance use, multiple partners, and condomless sex). The three neighborhood and institutional factors were highly interrelated. Conclusion: This work describes the unique characteristics of this population and can be utilized in risk assessment and delivering sexual health services to young Black men who have sex with women. Understanding risk factors across levels of the social-ecological model supports the need for policy changes that address unequal environments and opportunities that increase the risk of Ct acquisition. / 1 / Megan Clare Craig-Kuhn

Chlamydia trachomatis is an important cause of immune-mediated damage to the reproductive tract of infected patients. Certain chlamydial antigens and host genetic factors have been identified as contributing to immunopathological events, but a comprehensive understanding of specific components involved in destructive vs. protective immune responses to chlamydial infections is far from clear. In this study, it is shown that C. trachomatis-infected patients generate antibodies against an iron-responsive chlamydial protein, YtgA. The identity of YtgA was confirmed by mass spectrometry following two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. This finding underscores a necessity to examine patient sera samples to identify chlamydial antigens that are likely encountered and important to the immune response during human infections.

The obligate intracellular bacterium Chlamydia trachomatis requires iron in order to complete its developmental cycle. Addition of an iron-chelating drug, Desferal (deferoxamine mesylate), to infected cell culture causes Chlamydia to enter persistence. Here, we explore the ability of a stably-transfected cell line with inducible over-expression of the eukaryotic iron efflux protein ferroportin to starve C. trachomatis serovar E for iron. Ferroportin-induced iron removal is perhaps a more direct method of removing iron from the intracellular compartment versus exposure to an exogenous chemical chelator. Following induction, ferroportin-green fluorescent protein (Fpn-GFP) was detected in the plasma membrane, and cells expressing Fpn-GFP remained viable throughout the timescale required for Chlamydia to complete its developmental cycle. Following Fpn-GFP induction in infected cells, chlamydial infectivity remained unchanged, indicating chlamydiae were not in persistence. Ferritin levels indicate only a small decrease in cellular iron following Fpn-GFP expression relative to cultures exposed to Desferal. These data indicate that expression of Fpn-GFP in chlamydiae-infected cells is not capable of reducing iron below the threshold concentration needed to cause chlamydiae to enter persistence.

The WHO-designated neglected-disease pathogen Chlamydia trachomatis (CT) is a gram-negative bacterium responsible for the most frequently diagnosed sexually transmitted infection worldwide. CT infections can lead to infertility, blindness and reactive arthritis, among others. CT acts as an infectious agent by its ability to evade the immune response of its host, which includes the impairment of the NF-κB mediated inflammatory response and the Mcl1 pro-apoptotic pathway through its deubiquitylating, deneddylating and transacetylating enzyme ChlaDUB1 (Cdu1). Expression of Cdu1 is also connected to host cell Golgi apparatus fragmentation, a key process in CT infections. Cdu1 may this be an attractive drug target for the treatment of CT infections. However, a lead molecule for the development of novel potent inhibitors has been unknown so far. Sequence alignments and phylogenetic searches allocate Cdu1 in the CE clan of cysteine proteases. The adenovirus protease (adenain) also belongs to this clan and shares a high degree of structural similarity with Cdu1. Taking advantage of topological similarities between the active sites of Cdu1 and adenain, a target-hopping approach on a focused set of adenain inhibitors, developed at Novartis, has been pursued. The thereby identified cyano-pyrimidines represent the first active-site directed covalent reversible inhibitors for Cdu1. High-resolution crystal structures of Cdu1 in complex with the covalently bound cyano-pyrimidines as well as with its substrate ubiquitin have been elucidated. The structural data of this thesis, combined with enzymatic assays and covalent docking studies, provide valuable insights into Cdu1s activity, substrate recognition, active site pocket flexibility and potential hotspots for ligand interaction. Structure-informed drug design permitted the optimization of this cyano-pyrimidine based scaffold towards HJR108, the first molecule of its kind specifically designed to disrupt the function of Cdu1. The structures of potentially more potent and selective Cdu1 inhibitors are herein proposed. This thesis provides important insights towards our understanding of the structural basis of ubiquitin recognition by Cdu1, and the basis to design highly specific Cdu1 covalent inhibitors. / Der Krankheitserreger Chlamydia trachomatis (CT) - ein gramnegatives Bakterium - ist verantwortlich für die häufigste sexuell übertragene Infektionskrankheit weltweit, die CT basierte Chlamydiose. Sie wird von der Weltgesundheitsorganisation zu den vernachlässigten Krankheiten gezählt. CT Infektionen können unter anderem zu Unfruchtbarkeit, Erblindung und reaktiver Arthritis führen. CT agiert als Krankheitserreger mittels seiner Fähigkeit, die Immunantwort des Wirts zu umgehen. Dies umfasst unter anderem die Schwächung und Störung der NF-κB vermittelten Entzündungsantwort und des Mcl1 pro-Apoptoseweges über ihr deubiquitinierendes, deneddylierendes und trans-acetylierendes Enzym ChlaDub1 (Cdu1). Die Expression von Cdu1 ist aber auch mit der Fragmentierung des Golgi-Apparates des Wirtes verknüpft, ein Schlüsselprozess bei Infektionen mit CT. Cdu1 ist daher vermutlich ein attraktives Zielprotein für die Entwicklung von Wirkstoffen, um CT Infektionen zu behandeln. Eine Leitstrukturverbindung zur Entwicklung neuer wirksamer Inhibitoren war bislang jedoch noch nicht bekannt. Sequenzvergleiche und phylogenetische Untersuchungen verorten Cdu1 im CE Clan der Cysteinproteasen. Die Adenovirus-Protease (Adenain) gehört ebenfalls diesem Clan an und besitzt strukturelle Ähnlichkeit mit Cdu1. Unter Ausnutzung der topologischen Ähnlichkeiten der aktiven Zentren von Cdu1 und Adenain wurde ein Target-Hopping Ansatz mit einem klar definierten und fokussierten Satz von bei Novartis entwickelten Adenain-Inhibitoren verfolgt. Die hierbei identifizierten Cyano-Pyrimidine stellen die ersten kovalenten Inhibitoren von Cdu1 dar, die an das aktive Zentrum von Cdu1 binden und es direkt adressieren. Hochauflösend wurden Kristallstrukturen sowohl von Komplexen von Cdu1 mit kovalent gebundenen Cyano-Pyrimidinen als auch mit Cdu1’s natürlichem Substrat Ubiquitin bestimmt. Die Kristallstrukturdaten dieser Doktorarbeit in Kombination mit Enzymassays und kovalenten Docking-Studien liefern wertvolle Hinweise bezüglich der Aktivität des Enzyms, der molekularen Substraterkennung, der Flexibiliät der Proteintasche rund um das aktive Zentrum und potentielle Hotspots für die Wechselwirkung mit Liganden. Ein strukturbasiertes Wirkstoffdesign erlaubte die Optimierung des Cyano-Pyrimidin-basierten Molekülgerüstes, die zu der Entwicklung der HJR108 Verbindung führte. Es ist das erste Molekül seiner Art, das speziell dazu entworfen wurde Cdu1 zu inhibieren. Strukturen potentiell noch wirksamerer und selektiver Cdu1 Inhibitoren werden in dieser Arbeit vorgeschlagen. Diese Dissertationsschrift liefert somit wertvolle Beiträge zum Verständnis der strukturellen Grundlagen der molekularen Erkennung von Ubiquitin durch Cdu1 und Hinweise, die die Entwicklung hoch-spezifischer kovalenter Cdu1 Inhibitoren erlauben sollten.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T13:56:09Z No. of bitstreams: 2 Dissertação - Kelly Deyse Segati - 2012.pdf: 1381150 bytes, checksum: 6c27c539f7ddade59c13124dfc688935 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T13:57:32Z (GMT) No. of bitstreams: 2 Dissertação - Kelly Deyse Segati - 2012.pdf: 1381150 bytes, checksum: 6c27c539f7ddade59c13124dfc688935 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-09T13:57:32Z (GMT). No. of bitstreams: 2 Dissertação - Kelly Deyse Segati - 2012.pdf: 1381150 bytes, checksum: 6c27c539f7ddade59c13124dfc688935 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2012-12-18 / Infection with Chlamydia trachomatis (CT) may be associated with persistent carcinogenic HPV types and the development of cervical neoplasia. There are indications that detection of CT serum antibodies rather than DNA is a better measure of cumulative exposure to CT or of exposure occurring several years prior to the development of cervical disease. The objective of this study was to compare the positivity for CT by ELISA and PCR and to correlate with the severity of cervical neoplasia in women with abnormal cervical smear. Between February 2007 and March 2009, 136 women were referred to the colposcopy clinic at the Santa Casa de Misericordia in Goiânia-GO. HPV DNA was detected by the polymerase chain reaction (PCR) and genotyping was performed by reverse line-blot hybridization assay. CT seropositivity was tested by ELISA for the detection of IgG antibodies and the detection of CT was done by PCR to amplify a sequence in the cryptic plasmid generating a fragment of 512 base pairs. The total prevalence of HPV infection was 85.2%. Seropositivity for CT was 26%. Thirty-one women 26.7 were tested positive for CT antibodies and HPV-DNA. Of these 10.3% had diagnosis of cervical intraepithelial neoplasia grade 1 (CIN1) or cervicitis, while 16.3% had histological diagnosis of CIN2 worse diagnosis. When employed PCR test positivity was found to be 8.8%. Eleven women 9.48% were tested positive for CT and HPV DNA. Of these 5.1% had diagnosis of cervicitis or CIN1 and 4.3% had a diagnosis of CIN2 or worse diagnosis. The agreement between serology and PCR tests for CT was considered poor (kappa=0.10 IC 95% 0.69-7.9). Taking as reference the cases negatives for HPV and CT, a positivity for HPV and CT seropositivity was significantly associated with a diagnosis of CIN2 or worse diagnosis, for all HPV types (OR=11.9 IC=2.00-91.5 p=0.0009) and types 16 and 18 (OR=7.50 IC=0.91-76.28 p=0.02). Significant association was observed after adjustment for HPV. A Borderline significance was observed considering other HPV types (OR=7.50 IC=0.91-76.28 p=0.02). CT seropositivity was associated with CIN2 worse diagnosis in women infected by HPV, mainly when the types 16 and 18 were involved. This study did not show any association between CT infection detected by PCR and CIN2 or worse diagnosis. These data support the hypothesis that seropositivity for CT compared to PCR positivity in HPV positive women, especially for types 16 and 18, is a better measure of previous exposure, which reflects a higher probability of association with the severity of cervical neoplasia. / A infecção por Chlamydia trachomatis (CT) pode estar associada com a persistência dos tipos de Papilomavírus humano (HPV) oncogênicos e desenvolvimento da neoplasia cervical. Há indicações de que a detecção de CT por sorologia seja uma melhor medida de exposição cumulativa ou da exposição passada quando comparada a detecção pela reação da polimerase em cadeia (PCR). O objetivo deste estudo foi comparar a positividade para CT pelos métodos de ELISA e PCR e relacionar com a gravidade da neoplasia cervical em mulheres com anormalidades citológicas. Entre fevereiro de 2007 e março de 2009, 136 mulheres, foram encaminhadas à Clínica de Colposcopia na Santa Casa de Misericórdia em Goiânia-GO por exame citológico alterado. A detecção de DNA do HPV foi realizada por PCR utilizando os iniciadores PGMY09/PGMY11, e a genotipagem foi realizada por hibridização reversa em pontos. A positividade para CT foi avaliada por ELISA para detecção de anticorpos IgG e por PCR empregando iniciadores cujo alvo é uma região de plasmídeo críptico, gerando um fragmento de aproximadamente 512 pares de bases. A prevalência total da infecção por HPV foi 85,2%. A positividade para CT por sorologia foi de 25%. Trinta e uma amostras 26,7% foram positivas para HPV e CT. Destas 10,3% tinham diagnóstico de neoplasia intraepitelial cervical grau 1 (NIC1) ou cervicite, enquanto 16,3% tinham diagnóstico histológico de NIC 2 ou pior diagnóstico. Quando empregado o teste de PCR a positividade encontrada foi de 8,8%. Onze amostras 9,48% foram positivas para HPV e CT por PCR, sendo que 5,1% das pacientes apresentavam diagnóstico de NIC1 ou cervicite e 4,3% tinham diagnóstico de NIC 2 ou pior diagnóstico. A concordância entre os testes de sorologia e PCR para CT foi considerado ruim (kappa=0,10 IC 95% 069-7.9). Tomando como referência casos negativos para HPV/CT, a positividade para HPV/CT por sorologia foi significantemente associada com diagnóstico de NIC2 ou pior diagnóstico, para todos os tipos de HPV (OR=11.9 IC=2.00-91.5 p=0.0009) e para os tipos 16 e 18 (OR=16.25 IC=2.28-148.57 p=0.0005). Uma associação limítrofe foi observada considerando outros tipos de HPV (OR=7.50 IC=0.91-76.28 p=0.02). Houve associação estatisticamente significante após o ajustamento para infeção por HPV entre as infecções pelos tipos 16 e 18 e soropositividade para CT com a gravidade da neoplasia cervical. Quando empregado o teste de PCR, não houve associação entre a coinfecção HPV/CT e a gravidade da neoplasia cervical. Estes dados reforçam a hipótese de que a soropositividade para CT quando comparada a positividade por PCR em mulheres HPV positivas, especialmente para os tipos 16 e 18, é uma melhor medida de exposição anterior, o que reflete maior probabilidade de associação com a gravidade da neoplasia cervical.

Submitted by Alisson Mota (alisson.davidbeckam@gmail.com) on 2015-07-08T20:15:35Z No. of bitstreams: 1 Tese - Norma Suely de Lima Freitas.pdf: 8476174 bytes, checksum: b8e0fdb20a7b419aea33bc83321484fa (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-09T13:56:33Z (GMT) No. of bitstreams: 1 Tese - Norma Suely de Lima Freitas.pdf: 8476174 bytes, checksum: b8e0fdb20a7b419aea33bc83321484fa (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-09T13:54:31Z (GMT) No. of bitstreams: 1 Tese - Norma Suely de Lima Freitas.pdf: 8476174 bytes, checksum: b8e0fdb20a7b419aea33bc83321484fa (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-09T14:05:09Z (GMT) No. of bitstreams: 1 Tese - Norma Suely de Lima Freitas.pdf: 8476174 bytes, checksum: b8e0fdb20a7b419aea33bc83321484fa (MD5) / Made available in DSpace on 2015-07-09T14:05:09Z (GMT). No. of bitstreams: 1 Tese - Norma Suely de Lima Freitas.pdf: 8476174 bytes, checksum: b8e0fdb20a7b419aea33bc83321484fa (MD5) Previous issue date: 2012-08-28 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Despite the high prevalence and the risks associated with Chlamydia trachomatis in Brazil and other countries, little is known about the distribution of genotypes in Brazil and the biological variability of this important transmitting agent of sexually transmitted diseases (STDs). The absence of an inquiry routine for C. trachomatis and effective treatments can cause serious complications and consequences for individuals as pelvic inflammatory disease, infertility, ectopic pregnancy and neonatal infections. Currently, C. trachomatis presents 19 serotypes A-C associated with trachoma, D-K responsible for urogenital infections and L1, L2 and L3, agents responsible for lymphogranuloma venereum syndrome. The MOMP, which is recognized as the immunodominant antigen encoded by the ompA gene displays a large area of nucleotide variation of four variable domains (VDI to VDIV). Studies suggest that mutations occur frequently among genotypes and that these mutations may indicate differences between the immunogenic MOMP genotypes of C. trachomatis. The present work aims to identify the genotypes from samples positive by PCR for C. trachomatis in women diagnosed with infertility and with pregnant women in the city of Manaus, Amazonas - Brazil, in addition to sequence and analyze the ompA gene. The study population consisted of 96 infertile women and 53 pregnant women. Genotyping was performed by the technique of Polymerase Chain Reaction (PCR) from the ompA gene sequence and the following genotypes were identified in pregnant women: D [50.0%], E [25.0%] and F [12.5%] and I [12.5%]. In infertile women genotypes were identified: E [16.7%] F [16,7%] and K [66,7%]. The frequency of genotype K and D found in this study are considered high (66,7%) and (50,0%) and for phylogenetic analysis, we found that the genotypes analyzed shares the same ancestor. It is suggested that these variations in the sequences of genotypes identified arise from point mutations, or possibly by VD recombination in MOMP. The VDII region showed to be the most variable in the sequences analyzed. / Apesar da alta prevalência e dos riscos associados à Chlamydia trachomatis no Brasil e outros países do mundo, pouco se conhece sobre a distribuição dos genótipos no Brasil e a variabilidade biológica deste importante agente transmissor de doenças sexualmente transmissíveis (DST). A ausência de uma investigação rotineira para C. trachomatis e tratamentos efetivos pode originar sérias complicações e conseqüências para os indivíduos como doença inflamatória pélvica, infertilidade, gravidez ectópica e infecções neonatais. Atualmente, a C. trachomatis apresenta 19 sorotipos: A-C associados ao tracoma, D-K responsáveis por infecções urogenitais e L1, L2 e L3, agentes responsáveis pela síndrome do linfogranuloma venéreo. A MOMP, reconhecida como o antígeno imunodominante codificado pelo o gene ompA, exibe uma extensa área de variação nucleotídica sendo por sua vez, conferido por quatro domínios variáveis (VDI a VDIV). Estudos sugerem que as mutações ocorrem frequentemente entre os genótipos e que essas mutações podem indicar diferenças imunogênicas entre as MOMP de genótipos de C. trachomatis. O presente trabalho tem por objetivo identificar os genótipos a partir de amostras positivas por PCR para C. trachomatis de mulheres com diagnóstico de infertilidade e em gestantes na cidade de Manaus, Amazonas – Brasil, além de sequenciar e analisar o gene ompA. A população de estudo consistiu de 96 mulheres inférteis e 53 mulheres gestantes. A genotipagem foi feita pela a técnica de Reação em Cadeia de Polimerase (PCR) a partir da seqüência do gene ompA e os seguintes genótipos foram identificados em gestantes: D [50,0%]; E [25,0%]; F [12,5%] e I [12,5%]. Em mulheres inférteis os genótipos identificados foram: E [16,7%], F [16,7,%] e K [66,7%]. A freqüência de genótipo K e D encontrada neste estudo são consideradas elevadas (66,7%) e (50,0%) e quanto à análise filogenética, verificamos que os genótipos analisados compartilham do mesmo ancestral. Sugere-se que as variações encontradas nas sequências dos genótipos identificados surgem dos pontos de mutação ou possivelmente pela recombinação dos VD na MOMP. A região VDII foi que mais apresentou variações nas seqüências analisadas.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Objetivos: determinar a prevalÃncia de infecÃÃo genital por Chlamydia trachomatis em gestantes, comparando o subgrupo com diagnÃstico positivo com o de diagnÃstico negativo quanto aos fatores bio-sÃcio-demogrÃficos, histÃria ginecolÃgica e exame fÃsico ginecolÃgico, avaliando-se os fatores associados à presenÃa de infecÃÃo genital por Chlamydia trachomatis. Sujeitos e mÃtodos: submeteram-se ao teste de captura hÃbrida para Chlamydia trachomatis 446 gestantes do ambulatÃrio de prÃ-natal do Hospital Geral Dr CÃsar Cals, da Secretaria Estadual de SaÃde-CearÃ, no perÃodo de Agosto de 2003 a Maio de 2004. A idade mÃdia do grupo foi de 25,98 anos, idade gestacional mÃdia de 19 semanas. Aplicou-se questionÃrio diretamente Ãs gestantes, independente da idade gestacional e de estarem sintomÃticas ou nÃo, excluindo-se aquelas que tivessem feito uso de antibiÃticos ou de qualquer substÃncia quÃmica intravaginal nos quinze dias anteriores à coleta, ou que tivessem mantido relaÃÃes sexuais nos dois dias anteriores à consulta de prÃ-natal, com coleta de swab endocervical para realizaÃÃo de teste de captura hÃbrida II, com material colhido em tubo com soluÃÃo conservadora utilizando o sistema de micro placa, conforme procedimento descrito pelo fabricante. Os dados foram analisados utilizando o software STATA 13.0, procedendo-se anÃlise descritiva e analÃtica atravÃs do teste de qui-quadrado e regressÃo logÃstica, subtraindo-se variÃveis. Resultados: A prevalÃncia de Chlamydia trachomatis entre as gestantes foi de 2.91%. Identificou-se como fatores de risco independentemente associados à infecÃÃo genital por Chlamydia trachomatis a histÃria de dor pÃlvica ou doenÃa inflamatÃria pÃlvica, presenÃa de corrimento vulvar ao exame fÃsico e nÃo uso de preservativo com parceiro eventual. Calculou-se o Odds-ratio (OR), para cada um destes fatores, com respectivos intervalos de confianÃa. ConclusÃes: O subgrupo com rastreamento positivo para Chlamydia trachomatis caracterizou-se por apresentar uma faixa etÃria e renda familiar menor que o subgrupo com sorologia negativa, alÃm de apresentar maior frequencia de pacientes separadas, que usam menos preservativos com parceiros eventuais e com mais antecedentes de corrimento genital e dor pÃlvica. A OR para presenÃa de infecÃÃo genital por Chlamydia trachomatis foi de 1,7 para aquelas que nÃo usam preservativos e foi de 0,10 e 0,17, respectivamente, para ausÃncia de dor pÃlvica/DIP e corrimento vulvar. / Objectives: To estimate the prevalence of Chlamydia trachomatis in pregnant women, comparing the positive group to the negative one in respect to socio-demographic factors, gynecologic history and exam, evaluating the risk factors associated to Chlamydia trachomatis genital infection. Subjects and methods: Hybrid capture test for Chlamydia trachomatis was performed in 446 pregnant women at Hospital Geral Dr CÃsar Cals, from the Health Secretary of the State of CearÃ, from August, 2003 to May, 2004. Medium age in the group was 25.98 years and 19 weeks was the medium age of pregnancy. A structured questionnaire was applied, no matter the age of pregnancy, whether they were or not symptomatic, excluding those who had used antibiotics or any other substance into the vagina, during the previous fifteen days or who had kept sexual relationship until two days before the consultation, with a endocervical swab being performed, in order to have a hybrid capture test for the presence of Chlamydia trachomatis, as indicated by the manufacturer. Data were analysed by STATA 13.0, performed by means of the qui-square and logistic regression tests with descriptive and analytic presentation. Results: The prevalence of Chlamydia trachomatis among the pregnant women was 2.91%. Risk factors independently associated to Chlamydia trachomatis genital infection were history of pelvic pain or pelvic inflammatory disease, vulvar discharge and not using condom with an eventual sex partner. Respective odds ratio and confidence intervals were calculated to these variable. Conclusions: The positive group was younger, had smaller salaries and presented a greater frequency of divorced women, with less preservative use and more positive history of genital discharge and pelvic pain in the past. The OR to the presence of Chlamydia trachomatis genital infection was 1, 7 for those women not using condom and 0, 10 and 0, 17, respectively for a negative history of pelvic pain / PID, and the absence of vulvar discharge

Chlamydien sind Gram-negative, obligat-intrazelluläre Bakterien, die für ein weites Spektrum an relevanten Krankheiten verantwortlich sind. Auf Grund ihres zweiphasigen Entwicklungszyklusses sind Chlamydien von einer intakten Wirtszelle abhängig, um sich erfolgreich vermehren und im Organismus ausbreiten zu können. Daher haben Chlamydien anspruchsvolle Strategien entwickelt, um das Immunsystem des Wirtes auszuschalten oder den programmierten Zelltod ihrer Wirtszelle zu verhindern. In der vorliegenden Arbeit wurde untersucht, ob eine Infektion mit C. trachomatis einen Einfluss auf die zelluläre Antwort auf dsRNA nehmen kann. Die Synthese von dsRNA ist ein charakteristisches Merkmal der Replikation von Viren, welche sowohl die Apoptose induzieren als auch das Immunsystem aktivieren kann. Um eine chlamydiale und virale Co-Infektion zu simulieren, wurden Chlamydien-infizierte Epithelzellen mit der synthetischen dsRNA Polyinosin-Polycytidinsäure (polyI:C) transfiziert. Im ersten Teil der Arbeit wurde untersucht, ob Chlamydien die durch dsRNA eingeleitete Apoptose verhindern können. Eine signifikante Reduktion der dsRNA-induzierten Apoptose konnte in infizierten Zellen beobachtet werden. Es zeigte sich, dass die Prozessierung der Initiator-Caspase-8 in infizierten Zellen unterblieb. Dies war von der frühen bakteriellen Proteinsynthese abhängig und für die dsRNA-vermittelte Apoptose spezifisch, da der durch TNFalpha bewirkte Zelltod nicht auf der Ebene der Caspase-8 verhindert werden konnte. Die Aktivierung von zellulären Faktoren, die bei der Apoptoseinduzierung eine wichtige Rolle spielen, beispielsweise PKR und RNase L, war in infizierten Zellen jedoch unverändert. Stattdessen konnte durch RNA Interferenz-vermittelte Depletion gezeigt werden, dass der zelluläre Caspase-8-Inhibitor cFlip eine entscheidende Rolle bei der chlamydialen Blockierung der dsRNA-vermittelten Apoptose spielt. Mittels Co-Immunopräzipitation konnte ein erster Hinweis darauf gefunden werden, dass C. trachomatis eine Anreicherung von cFlip im dsRNA-induzierten Komplex von Caspase-8 und FADD bewirkt. Im zweiten Teil der Arbeit wurde untersucht, ob Chlamydien die Immunantwort auf virale Infektionen beeinflussen, welche vor allem die Expression von Interferonen und Interleukinen beinhaltet. Es stellte sich heraus, dass die Aktivierung des Interferon regulatory factor 3 (IRF-3) und des zur Familie von NF-kappaB Trankriptionsfaktoren gehörenden p65, zwei zentralen Regulatoren der Immunantwort auf dsRNA, in infizierten Epithelzellen verändert war. Die Degradation von IkappaB-alpha, des Inhibitors von NF-kappaB, war in infizierten Zellen beschleunigt, begleitet von einer Veränderung der Translokation des Transkriptionsfaktors in den Zellkern. Im Gegensatz dazu wurde die nukleäre Translokation von IRF-3 durch die Infektion signifikant verhindert. Die hier vorgestellten Daten zeigen erstmals, dass eine Infektion mit C. trachomatis die zelluläre Antwort auf dsRNA signifikant verändern kann und implizieren einen Einfluss von chlamydialen Infektionen auf den Ausgang von viralen Superinfektionen. / Chlamydia are Gram-negative obligate intracellular bacteria responsible for a wide spectrum of relevant diseases. Due to their biphasic developmental cycle Chlamydia depend on an intact host cell for replication and establishment of an acute infection. Chlamydia have therefore evolved sophisticated strategies to inhibit programmed cell death (PCD) induced by a variety of stimuli and to subvert the host immune system. This work aimed at elucidating whether an infection with C. trachomatis can influence the cellular response to double-stranded RNA (dsRNA). The synthesis of dsRNA is a prominent feature of viral replication inside infected cells that can induce both PCD and the activation of a cellular innate immune response. In order to mimic chlamydial and viral co-infections, Chlamydia-infected cells were transfected with polyinosinic:polycytidylic acid (polyI:C), a synthetic dsRNA. In the first part of this work it was investigated whether C. trachomatis-infected host cells could resist apoptosis induced by polyI:C. A significant reduction in apoptosis, determined by PARP cleavage and DNA fragmentation, could be observed in infected cells. It could be shown that processing of the initiator caspase-8 was inhibited in infected host cells. This process was dependent on early bacterial protein synthesis and was specific for dsRNA because apoptosis induced by TNFalpha was not blocked at the level of caspase-8. Interestingly, the activation of cellular factors involved in apoptosis induction by dsRNA, most importantly PKR and RNase L, was not abrogated in infected cells. Instead, RNA interference experiments revealed the crucial role of cFlip, a cellular caspase-8 inhibitor, for chlamydial inhibition of dsRNA-induced apoptosis. First data acquired by co-immunoprecipitation experiments pointed to an infection-induced concentration of cFlip in the dsRNA-induced death complex of caspase-8 and FADD. In the second part of this work, the chlamydial influence on the first line of defense against viral infections, involving expression of interferons and interleukins, was examined. Activation of the interferon regulatory factor 3 (IRF-3) and the NF-kappaB transcription factor family member p65, both central regulators of the innate immune response to dsRNA, was altered in Chlamydia-infected epithelial cells. polyI:C-induced degradation of IkappaB-alpha, the inhibitor of NF-kappaB, was accelerated in infected cells which was accompanied by a change in nuclear translocation of the transcription factor. Translocation of IRF-3, in contrast, was significantly blocked upon infection. Together the data presented here demonstrate that infection with C. trachomatis can drastically alter the cellular response to dsRNA and imply an impact of chlamydial infections on the outcome of viral super-infections.