Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Könssjukdomen klamydia är bland de vanligaste könssjukdomarna i världen. Könssjukdomen är anmälningspliktig och det ingår sjuksköterskans profession att arbeta preventivt med bland annat provtagning och smittspårning för att ständigt förebygga smittan. Syftet med denna litteraturstudie är att belysa unga vuxnas upplevelser av att testa sig för könssjukdomen. Författarna har analyserat nio vetenskapliga artiklar som resulterat i två huvudkategorier och fem underkategorier. Den första kategorin samt underkategorierna till denna var följande: Vårdrelaterade upplevelser av klamydiatestning; En känsla av okunskap; Känsla av tillit till vårdarna. Kategori nummer två löd: Psykosociala upplevelser av klamydiatestning med underkategorierna; ”Andra” människors problem; Risk för stigmatisering; Ett moraliskt ansvar. Resultatet visade att det hos unga vuxna fanns brist på kunskap kring könssjukdomen klamydia och testning kring denna. Vårdpersonalen hade i samband med testning stor inverkan och kunde påverka unga vuxnas val kring vart testet föredrogs att utföras samt av vilken befattning. Könssjukdomen visade sig också vara stigmatiserad och kunde skada relationer samt väcka känsla av skuld och skam. Litteraturstudien bidrar med att synliggöra unga vuxnas upplevelser kring testning vilket medför möjlighet för vårdpersonal att förbättra bemötandet så att en god vård kan erbjudas.

Antibody combining sites are constructed from limited set of germ-line gene segments, yet are capable of both recognizing a broad range of common epitopes and eliciting an adaptive response to newly encountered pathogens. Carbohydrate antigens generally do not draw T cell help and concomitant affinity maturation in the humoral response. Therefore, anti-carbohydrate responses must rely more heavily on the primary germ-line gene repertoire. Antibodies are usually thought of as highly specific. It has been suggested that polyspecificity and cross-reactivity in germ-line antibodies is necessary to provide the protective mechanisms required to broaden the potential number of antigens recognized; however, the molecular mechanism underlying polyspecificity is poorly understood. To investigate the phenomena of specificity, cross-reactivity and polyspecificity in germ-line antibodies my thesis focuses first on the unique LPS inner core oligosaccharide of Chlamydiaceae, which contains variations within the conserved inner core trisaccharide Kdo(2→8)Kdo(2→4)Kdo (3-deoxy-D-manno-oct-2-ulosonic acid). Antibodies raised against this family-specific trisaccharide showed strong V-region restriction with two sets of heavy and light chain V genes accounting for almost all clones isolated. These groups were named after their prototypic clones as the ‘S25-2 type’ and the ‘S25-23 type’. In contrast to the cross-reactive S25-2 and related antibodies, the S25-23 family of antibodies were shown to be specific for the Chlamydiaceae-specific trisaccharide antigen with no cross-reactivity to Kdo mono or disaccharides or to the Kdo(2→4)Kdo(2→4)Kdo trisaccharide antigen. Interest in S25-23 was sparked by its rare high μM affinity and strict specificity for the family-specific trisaccharide antigen. The structures of the antigen binding fragments of four S25-23-type mAbs have been determined to high resolution in complex with the Chlamydiaceae-specific epitope, revealing the molecular basis for their binding behaviour. The germ-line-encoded paratopes of these antibodies differ significantly from previously characterized S25-2-type mAbs. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germ-line residues to recognize the length of the trisaccharide antigen. Further S25-23-type antibodies are glycosylated on the variable heavy chain. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three-dimensional structure of a Gal(α1→3)Gal containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as it has been implicated in allergic responses in patients receiving therapeutic antibodies. The second focus of my thesis research is the lipid A moiety of LPS, which is involved in septic shock. Though the lipid A epitope appears to be cryptic during infection with Gram-negative bacteria, there have been several reported instances of lipid A specific antibodies isolated from human sera. While these antibodies are strictly selective for lipid A, there are reports of polyspecificity of some anti-lipid A antibodies for single stranded DNA. In such cases, the breakdown of negative selection through polyspecificity has been reported to result in the unfortunate consequences of autoimmune disease. This thesis reports the first crystal structures of antibodies in complex with lipid A and single stranded nucleic acids, elucidating their mechanism for polyspecificity. Perhaps more importantly, the structures may yield clues to the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases. / Graduate / 2020-04-18 / 0487 / 0982

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-02-12T17:38:37Z No. of bitstreams: 2 Dissertação - Rafael Navarrete Fernandez - 2004.pdf: 737923 bytes, checksum: 9f9c7b498798d7f0e65f37b958ef6d70 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-02-12T17:38:59Z (GMT) No. of bitstreams: 2 Dissertação - Rafael Navarrete Fernandez - 2004.pdf: 737923 bytes, checksum: 9f9c7b498798d7f0e65f37b958ef6d70 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-12T17:39:02Z (GMT). No. of bitstreams: 2 Dissertação - Rafael Navarrete Fernandez - 2004.pdf: 737923 bytes, checksum: 9f9c7b498798d7f0e65f37b958ef6d70 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2004-04-20 / Introduction: Chlamydia trachomatis is the bacteria responsible for the most prevalent sexually transmitted disease worldwide. Most of the infections in men and women is asymptomatic and when undiagnosed and untreated may reach the joints causing not only arthritis, but also other acknowledged complications related to the female reproductive system. Objective: To investigate C. trachomatis DNA in the urine and synovial fluid from patients with spondyloarthropathies (SpA) and rheumatoid arthritis (RA) and evaluate serum anti-C. trachomatisIgG and IgM antibodies. Methods: The population consisted of 15 patients with spondyloarthropathies, being nine with undifferentiated spondyloarthropathy (US) and six with reactive arthritis (ReA) (group I), and 15 patients with rheumatoid arthritis (RA) (group II). The chlamydial DNA was assessed in synovial fluid and urine samples of all patients by Amplicor PCR. The anti-chlamydial IgG and IgM antibodies were quantified through indirect imunofluorescence (IIF), while 15 patients of group I were typed for HLA-B27 by the use of flow citometry. Social demographical data and all information on sexual behavior and presence of symptoms were collected through a (questionnaire in the form of) an interview. Results: C. trachomatis DNA was found in only one synovial fluid sample from patient with ReA (6,7%). In two patients with RA, chlamydial DNA was identified in the urine sample (13,3%). The anti-chlamydial IgG antibodies were present in eight patients of the population studied; being three patients from group I (20%), and five from group II (33,3%). The greatest titer of this antibody 1/256 was associated with the presence of chlamydial DNA in a patient from group II. The IgM antibody was not detected in any of the samples from both groups. Four individuals from group II (26,7%) were HLA-B27 positive and its presence was 16 related to sacroiliitis. Conclusion: The results in this study show that in patients with spondyloarthropathies and rheumatoid arthritis, presenting a picture of articular activity one might not exclude C. trachomatisas the triggering agent. / Introdução: A Chlamydia trachomatis é a bactéria responsável pela doença sexualmente transmissível mais prevalente no mundo. A maioria das infecções em homens e mulheres é assintomática e, quando não diagnosticada e tratada, pode alcançar as articulações e causar artrite, isto sem mencionar outras complicações conhecidas relacionadas ao aparelho reprodutor feminino. Objetivos: Pesquisar o DNA de C. trachomatis no líquido sinovial e urina, em pacientes com espondiloartropatias e artrite reumatóide (AR) e avaliar a presença de anticorpos séricos IgG e IgM anti-C. trachomatis nestes dois grupos de doenças. Identificar o antígeno HLA-B27 em pacientes com espondiloartropatias. Metodologia: A população do estudo consistiu de 15 pacientes com espondiloartropatias: nove com espondiloartropatia indiferenciada (EI) e seis com artrite reativa (ARe) (grupo I) e 15 pacientes com AR (grupo II). O DNA clamidial foi pesquisado em amostras de líquido sinovial e urina de todos eles empregandose a PCR (Amplicor Roche). Os anticorpos IgG e IgM anti-clamidiais foram quantificados por imunofluorescência indireta (IFI), enquanto o HLA-B27 foi tipado em 15 pacientes do grupo I por citometria de fluxo. Os dados sócio-demográficos, de comportamento sexual e presença de sintomas foram obtidos através de questionário na forma de entrevista. Resultados: O DNA da C. trachomatis foi evidenciado apenas em uma amostra de líquido sinovial do grupo I (6,7%), sendo o paciente portador de ARe. Em dois pacientes com AR, o DNA de clamidial foi identificado na urina (13,3%). Os anticorpos IgG anti-clamidiais estavam presentes em oito pacientes da população estudada, três do grupo I (20%) e cinco do grupo II (33,3%). O maior título desse anticorpo (1/256) associou-se com a presença do DNA clamidial na urina de um paciente do grupo II. O anticorpo IgM não foi detectado em nenhuma amostra 14 dos dois grupos. O antígeno HLA-B27 foi positivo em quatro indivíduos do grupo II (26,7%) e sua presença relacionou-se com sacroiliite. Conclusões: Os resultados desse estudo indicam que em portadores de espondiloartropatias e artrite reumatóide, com quadro articular em atividade, a C. trachomatisnão pode ser excluída como agente desencadeador.

The purpose of this study was to investigate the association between Chlamydia trachomatis reinfection rates of Puerto Rican adolescents and failure to follow the retesting protocol, failure of sexual partners to receive treatment, and failure to participate in the sexual orientation program about risk factors. Secondary data analysis, from a historical prospective study from the Health Department of Puerto Rico, was used in this study. Data analysis was restricted to adolescents 15 to 19-years-old who had a positive chlamydia result and reinfection pattern since January 2008 through December 2012. Multiple logistic regression analyses were run to predict Chlamydia trachomatis reinfection. Results showed a statistically significant association association between Chlamydia trachomatis reinfection and not having followed the retesting protocol (OR=1.243, 95% CI 1.089-2.930, p-value 0.038). A statistically significant association association was found between Chlamydia trachomatis reinfection and sexual partners having not received treatment (OR=1.713, 95% CI 0.761-2.024, p-value 0.029). A statistically significant association was found between Chlamydia trachomatis reinfection and having not participated in the Puerto Rico Department of Health's sexual orientation program (OR=1.243, 95% CI 0.762-2.026, p-value 0.034). The contribution to social change is identifying factors significantly associated with Chlamydia trachomatis reinfection. Study findings provide useful guidance for clinicians and public health professionals on how to reduce Chlamydia trachomatis reinfection rates among at risk Puerto Rican adolescents.

Good sexual and reproductive health is fundamental to the overall health status of Māori communities. In 2001, the Ministry of Health reported that New Zealand was facing a Chlamydia epidemic. This epidemic has not abated as rates of Chlamydia have increased significantly in the past five years, with disproportionately high rates in young Māori women compared to non-Māori women. Despite significant sexual health disparities, young Māori have had limited opportunities to participate in research focussed on sexual health and voice their opinions and concerns on sexual health issues. This qualitative descriptive study has used a Māori inquiry paradigm and approached the research from a Kaupapa Māori perspective. The primary research question asked what the knowledge, attitudes and reported behaviours of young urban Māori women were in regards to sexual health and in particular, the sexually transmitted infection Chlamydia. Secondary aims were to ascertain the sources of information used and accessibility of sexual health services, as well as identifying resilience factors associated with protection against Chlamydia infection. The data was collected from semi-structured interviews with 16 young Māori women living in the Auckland region. Data analysis involved the inductive approach of categorical content analysis to identify major categorical themes to answer the specific questions posed. The main conclusion was that there are a number of barriers to accessing quality sexual health information and services for young Māori women. Participants’ level of knowledge varied reflecting their personal experiences and many were unaware that Chlamydia is asymptomatic and that delayed diagnosis can lead to fertility problems. The main sources of sexual health information accessed were whānau, peers, school, and contact with health professionals. Racism was identified as one of the barriers to sexual healthcare services. The young women expressed a clear preference for sexual health services to be delivered by Māori. Most importantly, for sexual health interventions to be successful it is essential that Māori communities, including young people and their whānau, are an integral part of creating positive solutions. Resilience factors that may help protect young women from contracting chlamydia characteristic of this group were having a strong connection with a caring adult or friend and parents who viewed sexuality as a normal part of adolescent development. In addition, having a positive Māori cultural identity with an ability to understand bicultural differences was strongly associated with participants accessing sexual healthcare services despite identified barriers.

This thesis presents data on two separate areas relevant to the prevention of HIV and sexually transmitted infection (STI) transmission in homosexual men. These data arise from the community-based Health in Men (HIM) cohort of HIV-negative homosexual men in Sydney. First, the association of circumcision status with HIV and STIs was examined. Older age, ethnicity and country of birth were demographic factors independently associated with circumcision status. Self-report was a valid measure of circumcision status in this population. Overall, being circumcised was associated with a non-significant reduced risk of HIV seroconversion in the HIM cohort (HR 0.76, 95% CI 0.41-1.41, p=0.381). Among the one-third of participants predominantly practising the insertive role in anal intercourse (AI), being circumcised was associated with a significantly reduced risk of HIV infection (HR 0.15, 95% CI 0.03-0.80, p=0.026). Circumcised HIM participants also had a lower risk of incident syphilis (HR 0.35, 95% CI 0.15-0.84, p=0.019), however circumcision status had no significant effect on the remainder of prevalent and incident STIs examined. Second, risk factors for pharyngeal gonorrhoea and chlamydia were investigated. The BD ProbeTec nucleic acid amplification test (NAAT) had a positive predictive value (PPV) for pharyngeal gonorrhoea diagnosis of only 30.4% (95% CI 25.2-36.1%) when compared to a previously validated NAAT targeting the gonococcal porA pseudogene. Pharyngeal gonorrhoea was common in HIM, mostly occurred without concurrent anogenital infection and may frequently spontaneously resolve. Infection was independently associated with younger age (p-trend=0.001), higher number of male partners (p-trend=0.002), contact with gonorrhoea (p<0.001) and insertive oro-anal sex with casual partners (p-trend=0.044). Pharyngeal chlamydia was less common but a high prevalence/incidence ratio suggested that infection may persist in the pharynx for long periods. Pharyngeal chlamydia was independently associated with receptive penile-oral sex with casual partners (p-trend=0.009). In conclusion, circumcision may have a role as an HIV prevention intervention among the subgroup of homosexual men who predominantly practise insertive rather than receptive AI. Regular screening of the pharynx including a validated supplemental NAAT for gonorrhoea diagnosis may prevent much transmission to anogenital sites, whereas chlamydia occurs too infrequently in the pharynx to recommend routine screening in homosexual men.

Amino acid biosynthesis is an essential process in living organisms. Certain amino acids can be synthesized by some organisms but not by others. L-Lysine is one of the essential amino acids that bacteria can synthesize but humans cannot. This is somewhat inconvenient for humans as much of their L-lysine must come from their diet. However, the lack of the lysine biosynthetic pathway in humans makes the bacterial enzymes within the pathway attractive drug targets. Recently, a novel lysine biosynthetic pathway was discovered in plants, Chlamydiae and some archaea. It is called the diaminopimelate aminotransferase (DAP-AT) pathway. In this pathway, LL-DAP-AT plays a key role by directly converting L-tetrahydrodipicolinate to LL-DAP in a single step. This is a quite interesting characteristic of LL-DAP-AT as the above conversion takes three sequential enzymatic steps in the previously known lysine biosynthetic pathways. Due to its absence in humans, LL-DAP-AT would be an attractive target for the development of novel antibiotics. In order to understand the catalytic mechanism and substrate recognition of LL-DAP-AT, the structural characterization of LL-DAP-AT is of paramount importance. In this thesis, the overall architecture of LL-DAP-AT and its substrate recognition mechanism revealed by the crystal structures of LL-DAP-AT from Arabidopsis thaliana and Chlamydia trachomatis will be discussed. The crystal structure of the native LL-DAP-AT from A. thaliana (AtDAP-AT) presented in this thesis is the first structure of LL-DAP-AT to be determined. This structure revealed that LL-DAP-AT forms a functional homodimer and belongs to the type I fold family of PLP dependent aminotransferases. The subsequent determination of the substrate-bound AtDAP-AT structure showed how the two substrates, (LL-DAP and L-Glu) significantly different in size, are recognized by the same set of residues without significant conformational changes in the backbone structure. In addition, the LL-DAP-bound AtDAP-AT structure shows that the C-amino group of LL-DAP is recognized stereospecifically by the active site residues that are unique to the family of LL-DAP-AT enzymes. Lastly, the chlamydial LL-DAP-AT presented in this thesis shows a new open conformation for LL-DAP-AT. The implications of the conformational flexibility of CtDAP-AT on the differences in substrate specificities among LL-DAP-AT are discussed.

A general aim was to combine molecular typing methods with clinical background information to increase epidemiological knowledge about Chlamydia trachomatis infections. An outbreak of Lymfogranuloma venereum (LGV), caused by a more invasive variant of C. trachomatis, was reported from the Netherlands in 2003 among men who have sex with men (MSM). All Chlamydia positive specimens from a venereal disease clinic for MSM in Stockholm during one year were genotyped. No spread of LGV was found, apart from three symptomatic cases. The same ompA genotypes were found among MSM in Melbourne, but the genotype distribution was different compared to findings among the heterosexual population in Sweden. The standard method for genotyping of Chlamydia is ompA-sequencing, but it has low resolution because one genotype predominates. A multilocus sequence typing (MLST) system based on five targets was developed. In a sample of 47 specimens, 32 variants were found with MLST, but only 12 variants with ompA-sequencing. The polymorphisms in the hctB gene, one MLST target, are caused by an element of 108 bp that is present in two to four repetitions and in different variants. Although the DNA-binding function of Hc2 that is encoded by hctB has been studied, our findings of a considerable size variation show that new studies are needed. In 2006, specimens with a 377 bp deletion in the cryptic plasmid covering the target region for diagnostic test systems from Abbott and Roche were discovered in Sweden. Applying MLST to these specimens indicated that there was a single clone, denoted nvCT. The proportion of nvCT in all detected Chlamydia cases was higher (20% to 65%) in counties using Abbott/Roche compared to counties using the BectonDickinson test system (7% to 20%). The proportions of nvCT converge in counties with high or low levels when detection systems were adjusted to detect nvCT.

<p><italic>Chlamydia<italic> <italic>trachomatis<italic> remains a highly relevant clinical pathogen as it is the causative agent of the most commonly reported sexually transmitted disease in the western hemisphere, and the most common cause of infectious blindness in the developing world. As an obligate intracellular pathogen, <italic>Chlamydia<italic> employs a vast assay of virulence proteins to hijack and remodel the host cellular machinery to facilitate its growth and dissemination. Besides delivering effector proteins into the host cytoplasm via a conserved type III secretion machinery, Chlamydia encodes components of multiple secretion systems, such as type II and IV. Chapter 3 of this document describes the secretion, processing and localization of two putative autotransporters (Pls1 and Pls2) and their involvement in inclusion expansion.</p><p> </p><p>In recent years, many new chlamydial effector proteins have been described. CPAF (Chlamydial Protease-like Activity Factor) is a secreted serine protease that is emerging as a central virulence protein: it is proposed to play a central role in <italic>Chlamydia<italic> pathogenesis by cleaving proteins involved in antigen-presentation, apoptosis and cytoskeletal re-arrangements. However, the functional significance of CPAF remains elusive due to the lack of specific inhibitors and <italic>Chlamydia<italic> mutants. The body of work presented herein demonstrates that in addition to targeting host proteins, CPAF cleaves a subset of early chlamydial effector proteins, including Inc-proteins that reside on the nascent pathogenic vacuole ("inclusion"). The design and development of a CPAF-specific inhibitory peptide demonstrates that these chlamydial effector proteins are true targets of CPAF. This peptide reversed the cleavage of bacterial targets by CPAF both in an in vitro cleavage assay and during infection, indicating that these effectors are bona fide targets. Inhibition of CPAF activity also revealed that this protease regulates multiple facets of chlamydial pathogenesis. CPAF inhibition in infected epithelial cells led to the complete dismantling of the inclusion, secretion of pro-inflammatory cytokines and engagement of an inflammasome-dependent programmed cell death pathway. While fibroblasts defective in various inflammasome components were resistant to <italic>Chlamydia<italic>-induced cell death, inclusion integrity and bacterial replication was still compromised upon CPAF inhibition, indicating that loss of inclusion integrity was not a consequence of caspase-1 activation. Overall, these findings revealed that CPAF, in addition to regulating host function, directly modulates the activity of secreted effectors and early Inc-proteins. Furthermore, we establish that CPAF is an essential virulence factor that is required to maintain the integrity of the inclusion and prevent the engagement of innate immune programmed cell death pathways in infected epithelial cells. CPAF activity thus remains a compelling mechanism by which intracellular pathogens employ proteolytic events to modify the host environment.</p> / Dissertation