Previous work carried out in our laboratory has identified that phase variation of type III R-M systems found in Haemophilus influenzae, Neisseria meningitidis and N. gonorrhoeae is reversible, and occurs at high frequency, as seen both through mod::lacZ fusions, and by measuring changes in repeat tract length. In addition, phase variation of the methyltransferases results in coordinated switching of expression of a distinct group of genes in each of the strains studied so far. WE have termed this phenomenon the PHASEVARION, for phase variable regulon, to identify the set of genes whose expression is affected by moe phase variation. Many of the genes found to be regulated by mod phase variation are known virulence factors and even include some genes investigated as candidates for vaccine development (Srikhanta et al., 2005 and 2009. The aims of this project was to further the investigation of how these R-M systems regulated the expression of genes which hitherto had not been predicted to phase vary. The first step in the process of investigating how phase variable R-M systems influence expression of unrelated genes is to identify the DNA sequences methylated by the methyltransferases of interest. As discussed in Chapter 3, elucidation of the ModA1 methylation target site was in part facilitated by predictions that the phase variable methyltransferase found in H. influenzae strain Rd methylated the same sequence as did HinfIII, isolated from H. influenzae strain Rf. This hypothesis was confirmed by methylation dependent inhibition of digestion, revealing that ModA1 methylates the second A in its recognition sequence, 5’-CGAAT-3’. Once confirmed, the genes found to be regulated by modA1 phase variation in the initial phasevarion study could be investigated for the presence of ModA1 methylation sites within their promoters or upstream of their transcriptional regulators. Two such methylation target sites were located just upstream of the dnaK ORF. Transcriptional start site analysis of the dnaK gene revealed three transcripiotnal start sites, one of which is unduced by heat shock. Exactly 10 nucleotides upstream of this heat shock induced transcriptional start site lies one of these ModA1 methylation target sequences. Ongoing invetigations are looking into the importance of this ModA1 site located within the dnaK promoter, and whether this is the site responsible for ModA1 dependent variations in dnaK expression. Although numerous methods were investigated for their potential to identify all sites methylated by the different modA alleles, the only method which resulted in identification of any methylation target sites was methylation dependent inhibition of restriction. This method allowed us to confirm the ModA1 recongition sequence, and to discover the methylation sequence, and adenine targeted by the modA13 allele, which is found in many clinically relevant N. gonorrhoeae strains. As will be discussed in Chapter 5, ModA13 dependent inhibition of restriction was first observed when the Neisserial plasmid pCmGFP was extracted from modA13 ON and modA13::kan cells, and further investigated and confirmed using a Southern blot approach to determine whether ModA13 dependent inhibtion could be detected as differential methylation of the chromosome. It was found that ModA13 recognised the sequence 5’-AGAAA-3’, with methylation occurring on the second last A. This sequence was mapped not only to the genes found to be regulated by modA13 phase variation, but also to the entire FA1090 chromosome, and this information will be used in future studies to investigate the direct molecular mechanisms by which modA13 phase variation results in subpopulations with different phenotypes in relation to antimicrobial resistance and biofilm/cell invasion.

PCR is a rapid, reproducible method for nucleic acid detection. However, this technology displays significant deficiencies when applied in clinical microbiology. This work’s aim was to improve current diagnostics and provide sensitive and quantitative real-time PCRs. Paper I describes the development of a sensitive and specific quantitative real-time PCR for the detection of Streptococcus pneumoniae, based on the Spn9802 DNA fragment. Applied to nasopharyngeal aspirates from 166 pneumonia patients, Spn9802 PCR had a sensitivity of 94% and a specificity of 98%. In Paper II the performance of a ply gene PCR for identification of pneumococcal lower respiratory tract infection (LRTI) was evaluated on bronchoalveloar lavage fluids. At the detection limit 103 genome copies/mL, 89% sensitivity but only 43% specificity was achieved. Paper III shows that S. pneumoniae DNA is detectable in plasma from acutely febrile patients. Sensitivities were low (26-42%) for detection of pneumococcal pneumonia, for bacteraemic pneumococcal pneumonia they were 60-70%. Paper IV describes evaluation of four PCR targets for Haemophilus influenzae detection. A real-time PCR based on the P6 gene was developed and applied to 166 CAP patients, using cut-off of 104 genome copies/mL the assay had a sensitivity of 97% and a specificity of 96%. In paper V, the two real-time PCRs presented in papers I and IV were combined with a PCR for detection of Neisseriae meningitidis. The analytical sensitivity of this multiplex real-time PCR was not affected by using a mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae) in single tubes. Applied to 156 LRTI patients, this PCR had sensitivities over 90% for S. pneumoniae and H. influenzae, and specificities of 89% and 96%, respectively. In conclusion, real-time PCR assays are useful for the diagnosis of S. pneumoniae and H. influenzae. They enable detection after antibiotic installation, and quantification increases the etiological specificity of pneumonia.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Paediatrics, 2004. / "March 2004" Includes bibliographical references (leaves 213-272).

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 5 uppsatser.

Thesis (M.S.)--University of Missouri-Columbia, 2004. / "December, 2004." The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 78-85).

Made available in DSpace on 2015-07-08T12:28:19Z (GMT). No. of bitstreams: 2 6.pdf: 926522 bytes, checksum: f6e46b30951c9cffb7632bb9dd259770 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / As bactérias do gênero Haemophilus, família Pasteutelleaceae têm ainda na espécie influenzae influenzae (Hi)a mais importante nas infecções humanas. Hi, que inclui os sorotipos capsulares (a-f) e os NT é responsável por diversos quadros infecciosos. Haemophilus influenzae b (Hib) era um dos principais responsáveis pelos casos de meningites em diversos países do mundo. Estava também associado a outras infecções graves como epiglotite, artrite séptica, bacteremia, pneumonia e septicemia, principalmente em crianças. As doenças associadas ao Hib são preveníveis pela vacina conjugada formada do PRP e uma proteína carreadora, incluída no PNI/MS em agosto de 1999. Após a introdução da vacina conjugada contra o Hib houve redução expressiva das doenças causadas pelo Hib, nos diversos países que introduziram a vacina em seus calendários de imunização, porém, os outros sorotipos, inclusive os HiNT passaram a ser isolados com maior frequência como agente infeccioso de meningites sendo também atualmente um dos principais agentes etiológicos da Otite Média Aguda (OMA), o que o torna alvo de importantes pesquisas para novas vacinas. A colonização desta é fundamental para que ocorra a infecção e, crianças de creches e orfanatos apresentam variáveis taxas desta bactéria. A importância deste estudo continuando uma linha de pesquisa sobre o Hi no INCQS-FIOCRUZ, com ênfase no estudo de cepas isoladas após a implantação da vacina está associada à necessidade de verificar a situação de portadores, em uma população fechada (creche) para contribuir com o conhecimento da situação do Hi após treze anos da introdução da vacina conjugada contra o Hib no Brasil, que reduziu, mas não impediu a ocorrência da doença. O objetivo principal deste estudo foi determinar a ocorrência de Hi em crianças frequentadoras de uma creche em Jacobina, Bahia. Realizou-se coleta de material da nasofaringe de 73 crianças. / The Bacterial genus Haemophilus is inclued in the family Pasteutelleaceae and the influenzae specie (Hi) is the most important in human infections. Hi includes capsular serotypes (a-f) and non-capsulated strains (NT), which are responsible for many infections. Haemophilus influenzae b (Hib) has been one of the main cause of meningitis in many countries worldwide. It has been also associated with other severe infections such as epiglottitis, septic arthritis, bacteremia, pneumonia and septicemia, mainly in children. Since 1988 these infections are preventable by Hib conjugate vaccine consisting of PRP and a carrier protein, and it has been included in the national vaccination program (PNI/ MS) in 1999. After the introduction of Hib conjugate vaccine, diseases caused by Hib had significantly decreased in several countries where the vaccine was introduced into their immunization schedules however, other serotypes, including HiNT, has been isolated with greater frequency as agents of infectious meningitis which is currently one of the main etiological agents of Acute Otitis Media (AOM) and the subject of important researches on new vaccines. Colonization is essential to start the infection, and children from daycare centers present variable rates of the bacteria in their nasopharynx. The importance of this study on an ongoing line of research at INCQS-FIOCRUZ with emphasis on the study of strains isolated after the introduction of the vaccine, is associated with the monitor of carriers in a closed population (daycare) to contribute to the knowledge of the circulation of Hi after thirteen years using conjugate vaccine against Hib in Brazil, which reduced but did not prevent the occurrence of the disease. The main objective of this study was to determine the occurrence of Hi in children attending a daycare center in Jacobina, Bahia. For the study we collected material from the nasopharynx of 73 children. We found a colonization rate of 78.08% and all isolates were classified as HiNT. Biotype III was the most prevalent. The study of antimicrobial susceptibility showed ampicillin-resistant strains producing and non-producing β-lactamase. We also observed high rates of resistance to ampicillin (41.8%) and trimethoprim+sulfametoxazol (67.2%). The gene blaTEM, which is primarily responsible for ampicillin resistance was found in 81.8% of the strains. The blaROB gene, was not observed among the strains analyzed. The PBP3 gene of βLNAR samples was sequenced and changes were observed, the major changes were in positions 547V → 547I found in three samples, 422N→ 422S and 273S→ 273F. Strain susceptibility was evaluated against amoxicillin/clavulanic acid, ceftriaxone, rifampicin, chloranphenicol and were susceptible. This study confirms the efficacy of the vaccine against Hib and points to the relevance to vigilance HiNT, as presented high percentage of colonization and resistance to some antimicrobials.

Orientador: Marcelo Lancellotti / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T12:32:22Z (GMT). No. of bitstreams: 1 Cury_GiseleCristianeGentile_D.pdf: 5956781 bytes, checksum: b28217e4d95beb4734f0357ed4181696 (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital quando for liberada / Abstract: The abstract is available with the full electronic document when available / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

No description available.

Orientador: Marcelo Lancellotti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T07:21:21Z (GMT). No. of bitstreams: 1 Varela_JuliaNogueira_M.pdf: 1464845 bytes, checksum: 930cccae996588333b99f1aaa50988c0 (MD5) Previous issue date: 2013 / Resumo: Haemophilus influenzae é uma bactéria causadora de doenças tipicamente associadas ao trato respiratório superior e inferior. Tal bactéria é classificada em linhagens capsuladas e não capsuladas - as não tipadas. As grandes responsáveis por patogenias mais severas são as capsuladas, especialmente as do sorotipo b, a existência de uma vacina para somente esse sorotipo, faz com que ocorra uma emergência de casos com H. influenzae não tipado - NTHi. A crescente resistência a antibióticos dessa bactéria está associada à plasmídios de resistência, bem como sua competência natural. A presença desses patógeno é maior em países nos quais não existe acesso a vacina, devido ao alto custo da mesma, que acabam utilizando antibióticos mais acessíveis como o cloranfenicol no tratamento. Esse trabalho estudou a transferência horizontal do gene ompP2 em diversas cepas de H. influenzae com a ajuda de nanopartículas de óxido de grafeno. Essas nanopartículas mimetizam uma atmosfera rica em partículas suspensas como as grandes cidades e zonas de agricultura precoce, já que, nesses locais ocorrem com maior frequência mutações e adaptações desse patógeno. Quando as nanopartículas encontravam-se no meio de cultura, verificou-se um aumento da taxa de transformação dessas bactérias. Assim como uma modificação no padrão de adesão celular das bactérias mutadas quando comparadas com as selvagens em linhagens celulares distintas e expostas ao antibiótico de resistência, levando a um aumento da taxa de adesão das cepas mutadas com relação às cepas selvagens. Como esse gene é e de possível aquisição entre cepas de H. influenzae em seu ambiente natural seria possível utilizá-lo para obtenção de uma proteína recombinante, com possível antigenicidade. Uma vez que a taxa de adesão aumenta com a presença do mesmo, levando a uma possível nova vacina que também protegeria contra cepas não tipadas e não somente capsuladas / Abstract: Haemophilus influenzae is a bacteria that causes diseases typically associated with the upper and lower respiratory tract. Their strains are divided in capsulated and non-capsulated - the non typable. The major responsible for more severe cases are the capsulated types, specially the b type. The existence of a vaccine for the serotype b, allows the emergence of cases of non typable H. influenzae - NTHi. The growing resistance is associated with resistance plasmids, and with its natural competence, that enables the bacteria to acquire DNA fragments between it's' species. Since this pathogen is common in countries that there is no access to this vaccine, therefore the use of accessible and cheaper antibiotics, such as chloramphenicol for treatment is. This work studied the horizontal transference of the ompP2 gene from multiresistant strains of H. influenzae, with the aid of grafen oxide nanoparticles, that mimesis an atmosphere rich in suspended particles, such as great urban areas and ancient agricultural zones. In these environments a great frequency in mutation and adaptations of these bacteria is verified. When we look at the adhesion patterns of these bacteria we can see that it is modified when they are mutated and exposed to the resistance antibiotic. Leading to an augmentation of the adhesion patterns when we compare to the wild strains. Since this gene was present in all strains and it was of easy acquisition between strains, it would be possible to use it to obtain a recombinant protein with likely antigen properties. Because the adhesion tax enhances with the presence of this gene. Leading to a possible new vaccine target, for NTHi and capsulated strains also / Mestrado / Fármacos, Medicamentos e Insumos para Saúde / Mestra em Biociências e Tecnologia de Produtos Bioativos