A Chlamydia trachomatis é um patógeno causador de doenças sexualmente transmissíveis (DST). Esse agente afeta significativamente a saúde sexual e reprodutiva de mulheres, estando relacionado à esterilidade em número bastante significativo,sendo também responsável por desfechos em gestantes acometidaspor DSTs no Brasil e no mundo. Apesar da sua alta prevalência, muito pouco se sabe sobre a distribuição de genótipos de Chlamydiatrachomatis. Este estudo teve como objetivo estimar a prevalência e os fatores associados à infecção causada por esse patógeno em gestantes admitidas na Maternidade da Fundação Santa Casa de Misericórdia do Pará. O estudo constou de uma amostra mínima de 363 gestantes atendidas por demanda espontânea, sendo incluídas na amostra o excedente de 32 gestantes totalizando 395 gestantes,em um período da coleta de 3 meses. Foram aplicados testes Qui-quadrado para verificação de associações entre as variáveis selecionadas para ump<0,05 como estatisticamente significativo. A prevalência de infecção porChlamydiafoi de 9,11%. A infecção porChlamydianão se associou à idade(p = 0,826),à realização de consulta pré-natal (p = 0,451),à presença de HIV (p = 0,379)ao exame VDRL(p = 0,344) ou à prematuridade (p = 0,229). O estudo mostra alta prevalência de infecção por Chlamydiatrachomatis em gestantes. A infecção urogenital por Chlamydia trachomatis representa uma importante causa de morbidade perinatal, que pode ser adequadamente tratada por antibioticoterapia durante a gestação. A prevalência da infecção mostrou-se também superior às obtidas em outras populações de gestantes, sendo o Estado do Pará considerado de alta prevalência para a infecção. Fatores de risco para DSTs, como baixa idade, ausência de parceiro fixo e concomitância com outras DSTs apresentam-se importantes também dentro do quadro da infecção por Chlamydia trachomatis. / Chlamydia trachomatis is a pathogen that causes sexually transmitted infections (STIs). This agent significantly affects the sexual and reproductive health of women, and is related to sterility in a rather significant number of cases. It is also responsible for outcomes in pregnant women who have STIs in Brazil and worldwide. Despite its high prevalence, very little is known about the distribution of Chlamydia trachomatis genotypes. The objective of this study was to estimate the prevalence and factors associated with infection caused by this pathogen in pregnant women admitted to the Maternity Department at Fundação Santa Casa de Misericórdia do Pará, in the state of Pará. Sample of 363 pregnant women seen due to spontaneous demand, and the sample included the surplus of 32 pregnant women, to a total of 395 pregnant women in a 3-month collection period. Chi-Square tests were applied to verify the associations between the variables selected for a p < 0.05 as statistically significant. The prevalence of Chlamydia infection was 9.11%. The result of Chlamydiawas not associated with age (p = 0.826), antenatal visit (p = 0.451), presence of HIV (p = 0.379) VDRL test (p = 0.344) and with prematurity (p = 0.229). The study shows a high prevalence of infection due to Chlamydiatrachomatis in pregnant women. Urogenital infection due to Chlamydia trachomatis is a major cause of perinatal morbidity, which can be treated by antibiotics during pregnancy. The prevalence of infection also proved superior to those obtained in other populations of pregnant women, and the state of Pará is considered a place with a high prevalence of the infection. Risk factors for STIs, such as young age, absence of a fixed partner and concomitance with other STIs are also important in the picture of infection by Chlamydia trachomatis.

A Chlamydia trachomatis é um patógeno causador de doenças sexualmente transmissíveis (DST). Esse agente afeta significativamente a saúde sexual e reprodutiva de mulheres, estando relacionado à esterilidade em número bastante significativo,sendo também responsável por desfechos em gestantes acometidaspor DSTs no Brasil e no mundo. Apesar da sua alta prevalência, muito pouco se sabe sobre a distribuição de genótipos de Chlamydiatrachomatis. Este estudo teve como objetivo estimar a prevalência e os fatores associados à infecção causada por esse patógeno em gestantes admitidas na Maternidade da Fundação Santa Casa de Misericórdia do Pará. O estudo constou de uma amostra mínima de 363 gestantes atendidas por demanda espontânea, sendo incluídas na amostra o excedente de 32 gestantes totalizando 395 gestantes,em um período da coleta de 3 meses. Foram aplicados testes Qui-quadrado para verificação de associações entre as variáveis selecionadas para ump<0,05 como estatisticamente significativo. A prevalência de infecção porChlamydiafoi de 9,11%. A infecção porChlamydianão se associou à idade(p = 0,826),à realização de consulta pré-natal (p = 0,451),à presença de HIV (p = 0,379)ao exame VDRL(p = 0,344) ou à prematuridade (p = 0,229). O estudo mostra alta prevalência de infecção por Chlamydiatrachomatis em gestantes. A infecção urogenital por Chlamydia trachomatis representa uma importante causa de morbidade perinatal, que pode ser adequadamente tratada por antibioticoterapia durante a gestação. A prevalência da infecção mostrou-se também superior às obtidas em outras populações de gestantes, sendo o Estado do Pará considerado de alta prevalência para a infecção. Fatores de risco para DSTs, como baixa idade, ausência de parceiro fixo e concomitância com outras DSTs apresentam-se importantes também dentro do quadro da infecção por Chlamydia trachomatis. / Chlamydia trachomatis is a pathogen that causes sexually transmitted infections (STIs). This agent significantly affects the sexual and reproductive health of women, and is related to sterility in a rather significant number of cases. It is also responsible for outcomes in pregnant women who have STIs in Brazil and worldwide. Despite its high prevalence, very little is known about the distribution of Chlamydia trachomatis genotypes. The objective of this study was to estimate the prevalence and factors associated with infection caused by this pathogen in pregnant women admitted to the Maternity Department at Fundação Santa Casa de Misericórdia do Pará, in the state of Pará. Sample of 363 pregnant women seen due to spontaneous demand, and the sample included the surplus of 32 pregnant women, to a total of 395 pregnant women in a 3-month collection period. Chi-Square tests were applied to verify the associations between the variables selected for a p < 0.05 as statistically significant. The prevalence of Chlamydia infection was 9.11%. The result of Chlamydiawas not associated with age (p = 0.826), antenatal visit (p = 0.451), presence of HIV (p = 0.379) VDRL test (p = 0.344) and with prematurity (p = 0.229). The study shows a high prevalence of infection due to Chlamydiatrachomatis in pregnant women. Urogenital infection due to Chlamydia trachomatis is a major cause of perinatal morbidity, which can be treated by antibiotics during pregnancy. The prevalence of infection also proved superior to those obtained in other populations of pregnant women, and the state of Pará is considered a place with a high prevalence of the infection. Risk factors for STIs, such as young age, absence of a fixed partner and concomitance with other STIs are also important in the picture of infection by Chlamydia trachomatis.

Arginine uptake and degradation systems are common throughout bacteria and archaea. The genome of human pathogen Chlamydia pneumoniae encodes three proteins now called AaxA, AaxB, and AaxC which function together to take up arginine, decarboxylate it, and expel the decarboxylation product, agmatine. AaxB is the previously characterized pyruvoyl-dependent arginine decarboxylase, AaxC is an inner membrane amino acid transport protein that functions as an arginine-agmatine antiporter, and AaxA is an outer membrane porin, which facilitates the uptake of arginine and also functions as a general porin with broad specificity. C. pneumoniae is a non-typical Gram negative bacteria and an obligate intracellular parasite with a unique 2-phase life cycle. The role of this system for arginine-agmatine exchange has yet to be determined but it may function to deplete host cell arginine as a means of inactivating host inducible nitric oxide synthase (iNOS), a molecule used in the innate immune response that has been shown to have an inhibitory affect on the growth of C. pneumoniae in cell culture. AaxB and AaxC are able to complement the loss of extreme acid-resistance in E. coli mutants that lack their own system for arginine-agmatine exchange, making pH homeostasis another possible role for this system. The porin AaxA is able to enhance arginine-agmatine exchange by AaxB and AaxC in E. coli mutants as well as by the native arginine decarboxylase AdiA and the native arginine-agmatine antiporter AdiC in wild type E. coli. AaxA is not an arginine-specific porin and instead acts as a general porin with a broad specificity. AaxA discriminates only against large and negatively charged solute molecules, and therefore it may have a broad role in the uptake of various biomolecules essential for chlamydial growth in addition to its role as part of a system for arginine-agmatine exchange. / text

Chlamydia is an obligate intracellular bacterial pathogen that causes a wide spectrum of diseases worldwide. At present, there are no vaccines to prevent chlamydial infections due to poor understanding of how anti-chlamydial immunity ensues. In this study, we employed a variety of in vitro and in vivo systems, including knockout (KO) mice and adoptive transfer, to investigate the role of lung dendritic cells (LDCs) and their relationship with innate lymphocytes, natural killer (NK) and invariant NKT (iNKT) cells, in host defense against chlamydial lung infections in mice. We found that iNKT cells altered the phenotype and cytokine production pattern of LDCs following C. pneumoniae infection. Adoptive transfer of LDCs from infected Jα18-KO mice, which lack iNKT cells, into naïve wild-type (WT) mice promoted Th2 (IL-4) immunity following infection challenge, whereas the transfer of LDCs from the infected WT mice induced protective Th1/Tc1 (IFN-γ) immunity. On the other hand, upon adoptive transfer, LDCs from C. muridarum-infected NK-cell-depleted mice (NK-LDCs) conferred reduced protection after chlamydial challenge than the recipients of LDCs from infected sham-treated mice (NK+LDCs). NK+LDC recipients exhibited an enhanced Th1/Th17, in contrast to Th2, response compared to the NK-LDC recipients. In coculture experiments, NK cells isolated from the infected mice promoted IL-12p70, IL-6, and IL-23 production by LDCs through NKG2D receptor signaling. These findings indicate that iNKT and NK cells condition LDCs to confer protective Th1/Tc1/Th17 immunity against chlamydial lung infection. We also analyzed the contribution of major LDC subsets, CD103+ and CD11bhi LDCs, in host defense against C. muridarum infection. We found that CD103+ and CD11bhi LDC subsets expanded following chlamydial infection. CD103+ LDCs showed higher expression of costimulatory molecules and greater production of Th1- and Th17-inducing cytokines (IL-12, IL-6 and IL-23) than CD11bhi LDCs. Coculture of Chlamydia-specific CD4+ T cells with LDC subsets revealed that the T cells cultured with CD103+ LDCs produced larger amounts of IFN-γ and IL-17 compared to those with CD11bhi LDCs. To test their function in vivo, we isolated CD103+ and CD11bhi LDC subsets from infected mice and transferred them into naïve syngeneic mice that received chlamydial challenge. CD103+ LDC-recipients showed better protection, as evidenced by their reduced body weight loss, bacterial burden and lung pathology, than CD11bhi LDC recipients. Mice that received CD103+, compared to CD11bhi, LDCs produced enhanced Th1/Th17 cytokines (IFN-γ and IL-17) in the lung and the MLNs. In conclusion, these findings demonstrate that CD103+ LDCs are more efficient in inducing Th1/Th17 immunity to chlamydial infection than CD11bhi LDCs. Taken together, our findings have provided direct in vivo evidence on the role of LDCs and their conditioning by iNKT and NK cells in generating mucosal T-cell immunity against a bacterial lung infection. The findings have added new knowledge to the field of lung immunology, which have implications for developing prophylactic and/or therapeutic strategies against respiratory diseases. / October 2015

No description available.

This thesis is focused on determining the structures of two anti-carbohydrate antibodies to understand how they achieve their specificity toward antigen. First, the structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide (LPS) has been determined by x-ray diffraction to 2.6 Å resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo (3-deoxy-α-D-manno-oct-2-ulopyranosonic acid) residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment. The new light chain V gene segment codes for a combining site that displays greater avidity, different specificity, and allows a novel antigen conformation that brings a greater number of antigen residues into the combining site than possible in S25-2. Further, while antibodies in the S25-2 family use complementarity determining region (CDR) H3 to discriminate among antigens, S64-4 achieves its specificity via the new light chain V gene segment and resulting change in antigen conformation. These structures reveal an intriguing parallel strategy where two different combinations of germline-coded V gene segments can act as starting points for the generation of germline antibodies against chlamydial antigens and show how anti-carbohydrate antibodies can exploit the conformational flexibility of this class of antigens to achieve high avidity and specificity independently of CDR H3. Second, the structure of a rabbit, single chain variable fragment against terminal mannose-6-phosphate (Man6P) residues, termed scFv M6P-1, has been determined by x-ray diffraction to 2.7 Å resolution with Man6P in the binding site. The Man6P pathway is the predominant pathway that transports acid hydrolases from the trans-Golgi to endosomes. Newly synthesized hydrolases first require the generation of Man6P markers before they can be transported. Maintaining a full complement of hydrolases within lysosomes is essential as failure to do so results in a number of different lysosomal storage diseases. Due to its specificity, scFv M6P-1 is able to diagnose lysosomal storage diseases mucolipidosis II and mucolipidosis III. scFv M6P-1 is also able to purify Man6P containing proteins which may be useful for enzyme replacement therapies. Additionally, scFv M6P-1 is one of the first structures of an antibody fragment that exhibits high specificity for a single carbohydrate residue and is one of the first structures of a rabbit antibody fragment. The specificity of scFv M6P-1, which gives it these unique attributes, is revealed in the structure where multiple hydrogen bonds are seen between the antibody’s heavy chain and the mannose ring while two salt bridges are observed between the antibody’s light chain and the phosphate moiety. Finally, scFv M6P-1 binds in such a way as to allow binding to proteins possessing terminal Man6P residues. Crystallographic challenges that arose during this research included poor crystal growth as well as twinning and these are explored while the structure of scFv M6P-1 complex with Man6P is analysed. / Graduate / 0487 / 0982 / 0307 / dyl.w.evans@gmail.com

Genital chlamydia is the most commonly diagnosed sexually transmitted infection. In August 2008, the Scottish government directed its health boards to involve community pharmacies in providing chlamydia testing and treatment for young people. Lothian Health Board envisaged a pharmacy-based chlamydia testing and treatment (CT&T) service to be able to reach deprived population. This research project set out to evaluate the implementation of the CT&T in Lothian, Scotland. However, the Lothian CT&T service suffered from setbacks such as; implementation delays, minimal advertising of the service, low uptake, withdrawal by central government of specific funding to support the service costs, and subsequent termination of the service in Lothian by March 2011. As it turned out, the CT&T service ran in Lothian for only 10 months. As events unfolded, the aims of the PhD research were successively revised so as to undertake an integrated set of studies that provide important insights and generalizable knowledge about the rationale for such a service, the process of implementation, including barriers and facilitators, and the potential to utilise routine data to assess the impact of a new service. An additional component was added, in that I undertook an analysis of an anonymous routine data on chlamydia testing obtained from the microbiology reference laboratory of Lothian to describe the epidemiology of chlamydia in Lothian (2006-2010) and to report an impact of recent policy changes (2008-2009) on chlamydia surveillance activity. Methods The Centre for Disease Control (CDC) framework for programme evaluation was used to guide design the evaluation of the CT&T service, and a subset of ‘strategic’ stakeholders for the service was involved throughout. Four studies were undertaken towards the evaluation, and these employed diverse methods, as follows: (i) A training need survey of pharmacists and their support staff was undertaken in 166 community pharmacies in Lothian, to inform the training session held prior to the CT&T service launch. (ii) A survey of 33 strategic stakeholders in Lothian was undertaken to provide input to the evaluation objectives and to identify their perceptions and concerns in relation to the CT&T initiative and its evaluation. (iii) A survey of potential service users, young people aged 15-24 years, was carried out at the Genito-Urinary Medicine (GUM) clinic and two other sexual health drop-in clinics in Lothian. The survey ascertain their preferences regarding specific aspects of the CT&T service, and their views on issues identified in previous literature as facilitators or barriers with regard to utilising of such a service. (iv) In order to understand the service provider’s perspective on setting up and delivering of the CT&T service, in-depth interviews were undertaken with participating and nonparticipating pharmacists. Eleven pharmacists were purposively sampled from 66 pharmacies invited by NHS Lothian to pilot the service roll-out. Finally, after the Lothian CT&T service had been terminated, 3 strategic stakeholders for Lothian, and a Scottish Government representative were contacted by email, to elicit their views on factors contributing to policy decisions regarding pharmacybased CT&T services. Results The analysis of disaggregated (individual) routine laboratory data showed that age, gender, year of testing and deprivation were associated with the chlamydia testing outcome measures. The before-and-after analysis, with respect to recent major policy/guidance changes (that is, publication of the sexual health service standards for Scotland in 2008, and of SIGN guidelines for chlamydia in 2009), showed that surveillance activity for chlamydia increased only transiently (i.e. in 2009 only). The annual surveillance target for women aged 15-24 years, of 300 tests / 1000 population, was achieved in 2009 only, but targets for males aged 15-24 years (of 100 tests / 1000 population) were not achieved. With respect to the evaluation studies, the training needs survey (i) had a 53% pharmacy response rate from the pharmacies comprised 41% pharmacists, 32% technicians and 26% counter assistants. The survey showed differences in selfassessed training needs between pharmacy staff groups (pharmacists/ technicians/ counter staff). With regard to pharmacist-only competencies, the highest rates of substantial training needs were for clarity regarding the medico-legal aspects (Fraser guidelines), taking a sexual history, criteria for referral and reviewing own and staff competencies for the CT&T service (83% to 77%). With respect to all staff competencies, the greatest self-reported training need was for inter-communicative aspects of providing the service – for respondents overall, 56% to 83% across competencies within this domain. For the stakeholder survey (ii), the response rate was 52% (n=17). Sixteen stakeholders indicated their strong or moderate concern regarding young peoples’ knowledge about the service. The stakeholders also acknowledged the difficulty inherent in promoting the service to those who might benefit from using it. A view commonly expressed by respondents was that sexual health counselling concomitant with testing would be difficult to deliver through the CT&T service, due to: the difficulty in achieving privacy; a busy retail environment; and pharmacists tending not to have the necessary skills. However, they also acknowledged that chlamydia service delivery is problematic everywhere and not just in pharmacies. The key benefits of the service suggested for young people included increase accessibility, normalization of chlamydia testing and its ability of provision of sexual health service to hard-to-reach population. Such a service was perceived to enhance the role of pharmacist in public health provision. The survey also sought input of strategic stakeholders to ensure that the evaluation questions of most importance to them were included. All the proposed evaluation questions were marked as important. Some suggested questions such as client’s satisfaction with the service or related to the service logistical planning could not be incorporated in the later components of the intended evaluation as the service uptake was too low to answer those questions. The survey of potential service users (iii) had an overall response rate of 20% (n=78). Young people who responded indicated that they felt confident that a pharmacy would offer complete confidentiality for testing, provide reliable test results and have knowledgeable staff to provide the service (90% to 93%). That said, these respondents indicated a preference to be tested in GUM clinic (32%) or drop-in clinics (34%), with only 11% indicating a preference for being tested in a pharmacy. Those who had not previously been tested for chlamydia placed more importance on a toilet facility in a pharmacy they would chose for chlamydia testing, whereas younger respondents (≤ 19 years) placed more importance on a less busy pharmacy. Analysis of in-depth interviews with pharmacists (iv) comprising interviews with 11 lead pharmacists (4 respondents from pilot pharmacies and 7 from non-pilot pharmacies) found that pharmacists were enthusiastic about their newly developed public health role. The respondents foresee a shift to pharmacies for being a first port of call for clients. They were also generally positive about the anticipated attitude of general practitioners and pharmacy support staff towards their provision of chlamydia service. From a pharmacist’s perspective, barriers to delivering the CT&T service were identified as workload and lack of adequate physical infrastructure within a pharmacy such as a consultation room and a toilet facility. On the other hand, the assurance of financial incentives for providing the service was a facilitator.

The obligate intracellular gram-negative bacterium, Chlamydophila pneumoniae (Cpn), has a significant impact as an acute and chronic disease-causing pathogen. Its potential to undergo persistent infections has been linked to chronic diseases. Several in vitro cell culture models are used to study persistent conditions, mainly IFN_ stimulation, treatment with antibiotics and iron depletion. Little is known about changes in the Cpn transcriptome during the acute and persistent infection. Therefore, the Cpn transcriptome during its acute developmental cycle and iron depletion-mediated persistence was examined in this study. Based on expression profiles, genes with similar expression changes formed 12 clusters using the self-organizing map algorithm. While other studies define genes based on their onset of transcription, here the important feature for clustering was the expression profile. This turned out to be more appropriate for comparing the time specific relevance of a certain cluster of genes to their proposed functions in the cycle. The Cpn clusters were grouped into the 'Early', 'Mid' and 'Late' classes as described for Ctr. Additionally, a new gene expression class containing genes with steadily increasing expression at the end of the developmental cycle was defined and termed 'Tardy' class. Comparison of the Cpn clusters to published proteomics data showed that genes encoding elementary body (EB) proteins peaked in the 'Late' gene cluster. This indicated that genes of the ‘Late’ and ‘Tardy’ class have different roles in RB to EB re-differentiation. Moreover, using lexical comparison the EB mRNA profile was significantly linked to the ‘Tardy’ cluster class. This provided evidence that initial translation in the cycle might be directed from stable transcripts present in the infectious EB form. Based on these criteria the novel ‘Tardy’ class was separated from the ‘Late’ class. The gene ontologies were used to identify specific pathways and physiological functions active during the different phases of development. Additionally, the transcriptome of Cpn in the persistent stage was compared to that of the acute developmental cycle. The Cpn transcriptome was altered in the iron-depletion mediated persistence. Genes upregulated were linked to clusters at the beginning of the developmental cycle, and genes down-regulated were linked to clusters at the end of the developmental cycle. These data provided strong evidence that the Cpn transcriptome during persistence is a gene expression arrest in mid-development. In early acute infection convergently or divergently oriented gene pairs preferentially had an antagonistic expression profile, whereas tandemly oriented gene pairs showed a correlated expression profile. This suggests that the Cpn genome is organized mainly in tandemly arranged operons and in convergently or divergently oriented genes with favored antagonistic profiles. The microarray studies done with the Cpn strain CWL029 also showed expression signals for several genes annotated only for the Cpn strains AR39 and J138. BLAST comparison verified that these genes are also coded in the CWL029 genome. Several of these genes were convergently arranged with their neighboring gene and shared overlapping genome information. Among these were parB, involved in DNA segregation and rpsD, an alternative sigma factor responsible for the transcription at late stages of the developmental cycle. Both genes have been described to have major roles in the chlamydial cycle. These genes had an antagonistic expression profile at the beginning of the acute developmental cycle and in persistence, as described before to be predominant for convergently oriented genes. Real time RT-PCR analysis showed that full-length rpsD mRNA transcripts were down-regulated, whereas short-length rpsD mRNA transcripts were up-regulated during the persistent infection. This demonstrated that the rpsD promoter is activated during the persistent infection and that because of the collision of the RNA polymerases full length transcripts were down-regulated. This sigma factor-independent mechanism is known as ‘Transcriptional Interference’. This is the first description on how the alternative sigma factor rpsD might be down-regulated during persistent infections. Finally, the host cell transcriptome was analyzed in the acute and persistent infection mediated by the depletion of iron. Cpn infection triggered the upregulation of relB, involved in an alternative NF-KB signaling pathway. Several genes coding for cell cycle proteins were triggered, including cyclin G2 and cyclin D1 and inhibitors of CDK4. Taken together, this work provides insights into the modulation of the pathogen and the host transcriptome during the acute infection and the iron mediated persistent infection. / Das obligat intrazelluläre, gram-negative Bakterium Chlamydophila pneumoniae (Cpn) wird mit akuten und chronischen Krankheiten in Verbindung gebracht. Besonders sein Potential persistente Infektionen zu durchlaufen ist mit chronischen Krankheiten korreliert worden und deshalb von besonderem Interesse. Verschiedene in vitro Zellkulturmodelle werden verwendet um persistente Infektionen zu untersuchen, darunter IFN_ Stimulation, Antibiotika Behandlung und Eisenmangel. Über die Genregulation von Cpn als auch der Wirtzelle in der akuten und persistenten Infektion ist jedoch wenig bekannt. In dieser Arbeit wurde das Cpn Transkriptom als auch das von epithelialen Wirtszellen in der akuten und in der durch Eisenmangel induzierten Infektion untersucht. Mittels eines Algorithmuses für ‚selbstorganisierende Netzwerke’ (SOM) wurden signifikant regulierte Gene aufgrund ihres Expressionsprofiles in 12 Cluster gegliedert. Diese 12 Cluster wurden wiederum in die Klassen der ‘Frühen’ (engl.: ‘Early’), ‘Mittleren’ (engl.: ‘Mid’) und ‘Späten’ (engl.: ‘Late’) Gene eingeteilt. Diese Unterteilung lehnt sich an schon beschriebenen Genexpressionsstudien für Ctr an. Weiterhin wurde die neue Klasse der ‘Verspäteten’ (engl.: ‘Tardy’) Gene eingeführt. Diese hatten am Ende des Entwicklungszykluses ein kontinuierlich ansteigendes Expressionsprofil. Mit publizierten Proteinen aus chlamydialen Elementarkörperchen (EK) korrelierten vor allem Gene aus den ‘Späte’ jedoch nicht aus den ‘Verspätete’ Klassen. Gene dieser beiden Klassen müssen also eine unterschiedliche Rolle im EK Redifferenzierungsprozess spielen. Weiterhin waren überdurchschnittlich viele mRNA Transkripte aus der Klasse der ‘Verspäteten’ Gene in den EK vorhanden. Dies führte zu der Annahme, daß ein Teil der initiale Proteinexpression von stabilen mRNA-Transkripten aus der infektiösen EK Form erfolgt. Anschließend wurden, Gene, die für spezifische Signalwege und physiologische Funktionen von Cpn kodieren, basierend auf der ‚Gene Ontology’ während des Entwicklungszykluses untersucht. Weiterhin wurde das Transkriptom von Cpn in der Persistenz mit dem Transkriptom der akuten Infektion verglichen. Unter Persistenzbedingungen zeigte Cpn ein verändertes Expressionsprofil. Hochregulierte Gene konnten akuten Clustern am Anfang des akuten Entwicklungszykluses und herunterregulierte Gene Clustern am Ende des akuten Entwicklungszykluses zugeordnet werden konnten. Dies legt nahe, daß es sich bei der Persistenz nicht um ein neues Transkriptionsprofil handelt, sondern eher um eine Arretierung des Transkriptomes in der Mitte des akuten Entwicklungszykluses. Weiterhin zeigten konvergent und divergent orientierte Gene am Anfang des Zyklus bevorzugt ein antagonistisches Expressionsprofil, während in Reihe angeordnete (‘tandem’) Gene ein korreliertes Expressionsprofil aufwiesen. Bei den mit dem Cpn Stamm CWL029 durchgeführten Mikroarrayexperimenten konnten auch Expressionswerte für einige ausschließlich für die Stämme AR39 und J138 beschriebenen Gene gemessen werden. Ein Vergleich mittels BLAST zeigte, daß diese Gene auch im CWL029 Genom kodiert sind. Dazu gehörten mehrere Gene, welche konvergent zu ihren Nachbargenen orientiert waren und eine Sequenzüberlappung mit diesen aufwiesen. Darunter fielen parB, welches eine Rolle für die Trennung der DNA in der Zellteilung spielt, und rpsD, ein alternativer Sigma-Faktor, der für die Transkription in der späten Phase des Entwicklungszyklus verantwortlich ist. Für beide Genpaare konnte in der frühen akuten und in der persistenten Infektion ein antagonistisches Expressionsprofil beobachtet werden, wie es bei konvergent orientierten Genpaaren überwiegt. Mittels quantitativer qRT-PCR wurde für rpsD gezeigt, dass vollständige mRNA-Fragmente in der Persistenz herunterreguliert, während kurze mRNA-Fragmente hochreguliert waren. Als Erklärung für diesen Effekt dient ein Modell, welches auf einer Kollision der RNA Polymerasen basiert. Dieser Sigma-Faktor unabhängige Mechanismus ist in der Literatur als ‘Transkriptionelle Interferenz’ bekannt und führt so trotz einer Promoteraktivierung zu einer verminderten Anzahl an vollständigen mRNA Transkripten. Die Herunterregulation von RpsD auf Proteinebene in der Cpn Persistenz ist beschrieben worden. Im letzten Teil dieser Arbeit wurde das Wirtszelltranskriptom in der akuten und persistenten Infektion untersucht.Infektion mit Cpn führte zu einer Hochregulation von relB, welches an alternativen NF-KB Signalwegen beteiligt ist und das anti-apoptotische Potential verstärkt. Weiterhin waren Gene differentiell exprimiert, welche für die Zellzyklusproteine Cyclin-G2 und Cyclin-D1 sowie Inhibitoren von CDK4 kodieren. Zusammenfassend gibt diese Arbeit gibt einen Einblick sowohl in das Transkriptom des Pathogens als auch der Wirtszelle während der akuten und durch Eisenmangel ausgelösten persistenten Infektion als auch potentiellen Mechanismen zu Persistenzentstehung auf der Ebene der Genregulation.

Background: Chlamydia trachomatis is the commonest curable sexually transmitted infection in the UK. The prevalence is shared equally by men and women. A National Chlamydia Screening Programme (NCSP) has been introduced in England, supported by advances in testing technologies which enable non-invasive sampling methods to be used in non-healthcare settings. The NCSP tests nearly twice as many women as men and is more likely to test men in non-healthcare settings. Men are seen as an important, but difficult to reach group. Little is known about where men prefer to access testing and whether or not nontraditional settings, such as football clubs, are acceptable. Methods: 1) A national stratified random probability sample survey of men aged between 18 and 35 years resident in Great Britain, exploring attitudes to self-collected testing for Chlamydia, acceptability of venues to collect testing kits, health seeking and sexual risk behaviours. 2) Qualitative interviews with men who play amateur football. It explores the acceptability of three different, club-based, testing pathways; Health-care professional promoted; Peer-led promoted; and poster-led promoted. Results: Men are well engaged with existing health services and find selfcollected testing kits for Chlamydia highly acceptable. Healthcare settings are the most acceptable venues to access testing although sports settings are acceptable to a minority. Attitudes to testing in football clubs are influenced by factors relating to men’s characteristics, promoter characteristics and the impact of testing on time and effort involved. Conclusions: Whilst non-healthcare settings can be used to reach some men for Chlamydia testing, existing services are already well accessed and offer considerable opportunities to test more men. More should be done to ensure men are able to access testing within the context of daily living, without significantly impacting on the time needed to pursue their main interests.

Chlamydia trachomatis is the most common bacterial agent of sexually transmitted infections worldwide and a common co-infection in AIDS patients. Chlamydial genital tract infections are often asymptomatic; therefore many infections go untreated and result in complications like chronic inflammation, ectopic pregnancy, and pelvic inflammatory disease. Chlamydia share a unique developmental cycle and under stress, can enter a state known as persistence, in which the bacteria are noninfectious but still viable. Removal of the stressor allows the chlamydiae to re-enter and complete the developmental cycle. Exposure to low-dose quinolones can cause the chlamydiae to enter persistence and halt the developmental cycle. Notably, 1 in 20 people living with HIV/AIDS also suffers from chlamydial infections. Since the anti-HIV drug Elvitegravir (EVG) is a quinolone derivative, we hypothesized that EVG exposure would inhibit chlamydial development. To ascertain whether EVG affects chlamydial development, HeLa cells were infected with C. trachomatis or C. muridarum and then either mock treated or treated with EVG. The percent infectivity and production of infectious progeny were determined by immunofluorescence assay and chlamydial titer assay, respectively. Transmission electron microscopy (TEM) was used to examine chlamydial morphology and determine whether EVG caused Chlamydia to become persistent. Though percent infectivity and chlamydial morphology were similar between treated and untreated Chlamydia-infected cells, the production of infectious progeny was significantly decreased in EVG-exposed Chlamydia-infected cells. These data indicate that EVG is not a persistence-inducer, but does inhibit chlamydial development in vitro. In other studies, we tested chlamydial polymorphic membrane protein (PMP) expression in chlamydia/HSV co-infected cells by immunofluorescence staining. Since penicillin-induced persistence decreases the expression of some chlamydial PMPs, we hypothesized that expression of PMP-A and PMP-B would be decreased by HSV-induced persistence. The results indicated that there was no significant difference in expression of PMP-A or PMP-B in co-infected versus C. trachomatis singly-infected cells. These data suggest that PMP expression is not a good indicator of chlamydial persistence when induced by HSV.