Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/01/15). Includes bibliographical references.

Orientador: Antonio Carlos Pizzolitto / Banca: Isabel Yoko Ito / Banca: Sérgio Aparecido Torres / Banca: Maria de Lourdes Ribeiro de Souza / Banca: Clarice Queico Fujimura Leite / Resumo: Os Staphylococcus coagulase-negativa, particularmente, os Staphylococcus epidermidis são a causa mais freqüente de infecções relacionadas ao cateter por sua habilidade em aderir a uma superfície e entre si (aderência intercelular) formando biofilme em multicamadas sobre superfícies de polímeros. O objetivo do presente estudo foi avaliar cepas hospitalares de Staphylococcus coagulasenegativa isoladas de cateteres intravenosos, quanto à resistência a oxacilina, produção de slime, aderência ao poliestireno, habilidade de formar biofilme sobre superfícies abióticas (cateter esterilizado) e a presença de genes icaAD. Na presente pesquisa, a presença de icaA e icaD foi determinada pelo método PCR, em uma coleção de 27 amostras Staphylococcus coagulase-negativa (10 Staphylococcus epidermidis, 4 S. haemolyticus, 2 S. hominis, 2 S. lugdunensis, 1 S. saprophyticus, 1 S. schleiferi, 2 S. xylosus e 4 S. warneri). Os genes icaAD foram detectados em dez cepas S. epidermidis... (Resumo completo, clicar acesso eletrônmico abaixo) / Abstract: Coagulase-negative Staphylococcus, particularly, Staphylococcus epidermidis are frequent cause of infections associated with catheters and is attributed to the attachment ability on a surface and each other (intercellular adhesion) forming a multilayered biofilm on polymeric surfaces. The objective of the present study was to evaluate coagulase-negative Staphylococcus strains isolated from intravenous catheters by oxacillin resistance, slime production (qualitative method) and spectrophotometric assay (quantitative method), ability to form biofilm on abiotic surfaces (steriled catheter) and the presence of icaAD genes. In the present study icaA and icaD were determined by PCR method, in a collection of 27 coagulasenegative Staphylococcus (10 Staphylococcus epidermidis, 4 S. haemolyticus, 2 S. hominis, 2 S. lugdunensis, 1 S. saprophyticus, 1 S. schleiferi, 2 S. xylosus and 4 S. warneri). The icaA genes were detected in nine S. epidermidis and icaD in ten. The slime-producing ability was determined by culture on Congo red agar plates in which slime-producing strains formed black colonies in 10 S. epidermidis, 4 S.haemolyticus, 4 S. warneri, 2 S. xylosus and 1 S. chromogenes, while nonslimeformingones develop red colonies. The quantitative assay of coagulase-negative Staphylococcus was observed in 19 strains, including: 10 S. epidermidis, 3 S.haemolyticus, 3 S. warneri, 2 S. xylosus, 1 S. chromogenes. The ability of coagulasenegative Staphylococcus to form biofilm embedded in an amorphous substance wasobserved by scanning electronic microscope on abiotic surface in 10 S. epidermidis,3 S. haemolyticus, 2 S. hominis, 2 S. lugdunensis, 1 S. saprophyticus, 1 S. schleiferi,2 S. xylosus and 3 S. warneri. The oxacillin resistance was observed in 9 strains S.epidermidis, 3 S. haemolyticus, 3 S. warneri, 1 S. xylosus and 1 S. chromogenes. All strains of staphylococci were susceptible... (Complete abstract, click eletronic address below) / Doutor

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Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 170-192).

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Porphyromonas gingivalis à um patÃgeno extremamente associado com a etiologia da periodontite crÃnica e agressiva. O objetivo deste estudo foi avaliar atravÃs de reaÃÃo em cadeia da polimerase em tempo real (Real Time-PCR) a presenÃa de Porphyromonas gingivalis (Pg) e dos genÃtipos fimA II e IV em indivÃduos com periodontite agressiva generalizada (PAG). Quarenta indivÃduos com PAG (29,7  8,1 anos) foram analisados clinicamente - Ãndice de Placa (IP), Ãndice Gengival (IG), profundidade de sondagem (PS), nÃvel de inserÃÃo clÃnico (NIC) - e microbiologicamente, atravÃs de Real Time PCR, quanto à presenÃa de Pg e dos genÃtipos fimA II e IV. Amostras de biofilme subgengival foram colhidas do sÃtio proximal com maior PS e maior NIC. MÃdias de PS e NIC desses sÃtios foram respectivamente: 9,5  2,2 mm e 10,2  2,8 mm. P. gingivalis foi observado em 26 (65%) dos indivÃduos. O genÃtipo fimA II foi verificado em 16 (61,53%) enquanto o genÃtipo fimA IV em 7 (26,92%) dos que apresentaram P. gingivalis. Entretanto, nÃo foi observada diferenÃa estatÃstica entre os parÃmetros clÃnicos dos indivÃduos que apresentaram ou nÃo o microrganismo ou seus respectivos genÃtipos. TambÃm nÃo foi verificada associaÃÃo entre a presenÃa dos genÃtipos e idade ou gÃnero dos pacientes. Os dados sugerem uma associaÃÃo entre genÃtipos fimA II de Porphyromonas gingivalis quando da ocorrÃncia deste microrganismo em indivÃduos com periodontite agressiva generalizada. / Porphyromonas gingivalis is a pathogen strongly associated with the etiology of chronic and aggressive periodontitis. The purpose of this study was to evaluate by Real-Time polymerase chain reaction (Real Time-PCR) the presence of Porphyromonas gingivalis (Pg) and fimA genotypes type II and type IV in patients with generalized aggressive periodontitis (GAgP). Forty individuals with aggressive periodontitis (AgP) (29.7  8.1 years) were clinical analyzed through plaque index (PI), gingival index (GI), probing depth (PD), clinical attachment level (CAL) and microbiologically, by Real Time-PCR for the presence of Pg and fimA genotypes type II and type IV. Subgingival biofilm samples were collected from the interproximal periodontal sites (> PD and > CAL). The PD and CAL average of this sites were respectively: 9,5  2,2 mm e 10,2  2,8 mm. P. gingivalis was observed in 26 (65%) of individuals. FimA genotypes type II was detected in 16 (61,53%) while fimA genotypes type IV in 7 (26,92%) of those with P. gingivalis. However, no differences were observed between the clinical parameters of patients who presented or not the organism or its genotypes. There was also no association between the presence of genotypes and age or gender of patients. The data suggest an association between P. gingivalis fimA genotypes upon the occurrence of this microorganism in patients with generalized aggressive periodontitis.

De 50 à 100 millions de cas de maladie de dengue sont recensés chaque année dans le monde. Le virus de la dengue présente aujourd’hui un problème de santé publique avec son émergence en Europe et particulièrement en France. L’infection par le virus peut être asymptomatique ou être responsable de trois pathologies: l’une avec des symptômes grippaux (DF), une autre avec des hémorragies modérées (DHF) et une dernière avec des hémorragies sévères entraînant un syndrome de choc (DSS).Les facteurs de l’hôte jouent un rôle important dans le développement de formes sévères mais les facteurs viraux impliqués restent peu décrits. Le but de ce travail de thèse était de mieux comprendre ces facteurs viraux au travers de l’étude des dynamiques de circulation de souches de dengue 3 génotype III en Afrique et de la caractérisation de trois souches de dengue de sérotype I du Cambodge. Ce travail nous a permis de mettre en évidence la circulation de variants pendant les épidémies, permettant de supposer que la présence de variants permet une meilleure dissémination, ainsi que des caractéristiques génotypiques et phénotypiques particulières in vitro aux souches associées aux formes hémorragiques et aux formes avec syndrome de choc chez l’homme. Ces travaux ont été complétés par le développement d’un système original de détection du virus de la dengue et des autres virus du genre Flavivirus. Ce travail de thèse a permis d’identifier de potentiels facteurs de virulence propres au virus, ouvrant la voie à la recherche sur le rôle de certaines protéines virales dans la pathogénicité. / From 50 to 100 million cases of dengue illness occurred every year in the world. Today, dengue virus is a public health problem with its emergence in Europe, particularly in France. DENV infection can be asymptomatic or be responsible for three distinct pathologies: one with flu-like symptoms (DF), another with moderate hemorrhage (DHF) and the last one with severe bleeding leading to shock syndrome (DSS). Host factors have an important role in the development of severe forms but implicated viral virulence factors stay not well described. The aim of this research work was to better understand these viral factors through study of dengue serotype 3 genotype III dynamics of circulation in Africa and through the characterization of three dengue serotype 1 strains in Cambodia. This work highlighted the circulation of variants during epidemics, allowing us to suppose that the presence of variants permits a better dissemination, as well as specific phenotypic and genotypic characteristics in vitro of strains associated with hemorrhagic forms or forms with shock syndrome in humans. These works were completed by the development of an original system of detection of dengue virus and other viruses of genus Flavivirus. This research work allowed identifying potential virulence factor specific to virus, opening the way for research on the role of certain viral proteins in pathogenicity.

Orientador: Antonio Olavo Cardoso Jorge / Banca: Luciane Dias de Oliveira / Banca: Mariella Vieira Pereira Leão / Resumo: O objetivo foi avaliar as interações entre Candida albicans (ATCC 18804) com Streptococcus mitis (49456) e Streptococcus sanguinis (10556) in vitro e in vivo avaliando-se a possível influência destas associações, na expressão de genes, na filamentação e formação de biofilme de Candida albicans. A formação de biofilme, foi realizado mono e multiespécie em placa de 96 poços por 48 h à 37 ºC com 5% CO2. Os biofilmes foram desagregados e diluídos para semeadura em ágar e após incubação as UFC/mL foram contadas. A filamentação de C. albicans, in vitro foi realizada em placas de 24 poços e in vivo em Galleria mellonella, com análise histológica e contagem de UFC/mL. A avaliação da expressão gênica de ALS1, ALS3, BRC1, CPH1, EFG1 e HWP1, foi realizada por PCR em tempo real utilizando o gene normalizador ACT1. Os resultados da UFC/mL (p < 0.05), demonstrou que o biofilme de C. albicans monoespécie apresentou maior crescimento, quando comparado com o biofilme associado com S. mitis (p = 0,001) ou com S. sanguinis (p = 0,001). A filamentação in vitro demonstrou que a interação com S. mitis inibiu a filamentação de C. albicans (p = 0,0006), entretanto, a interação com S. sanguinis não inibiu (p = 0,1554). Os genes ALS1, ALS3 e HWP1 foram super expressos na interação com S. mitis. A interação com S. sanguinis, promoveu super expressão dos genes ALS3 e HWP1. Os genes BRC1, CPH1 e EFG1 foram super expressos na interação com S. mitis e sub expressos, na interação com S. sanguinis. Não houve... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract :The objetive was to evaluate the interactions between Candida albicans (ATCC 18804) with Streptococcus mitis (49456) and Streptococcus sanguinis (10556) in vitro and in vivo evaluating the possible influence of these associations, in the expression of genes, in the filamentation and biofilm formation of Candida albicans. Biofilm formation was performed mono- and multispecies in 96-well plate for 48 h at 37 ºC with 5% CO2. Biofilms sonicated and diluted for sowing on agar and after incubation the colonies counted to obtain the colony forming units (CFU/mL). The filamentation in vitro was performed in 24-well plate and in vivo Galleria mellonella, with histological and CFU/mL. The evaluation of gene expression ALS1, ALS3, BRC1, CPH1, EFG1 and HWP1 was performed by real-time PCR using the normalizing gene ACT1. The results of CFU/ml (p<0.05), found that C. albicans biofilm monoespécie showed increased growth, as compared to the biofilm associated with S. mitis (p = 0.001) and S. sanguinis (p = 0.001). The filamentation in vitro demonstrated that the interaction with S. mitis inhibited C. albicans filamentation (p = 0.0006), interaction with S. sanguinis could not (p = 0.1554). The ALS1, ALS3, HWP1 gene, were superexpressed in S. mitis interaction. Interaction with S. sanguinis, promoted overexpression of ALS3 and HWP1 genes. The BRC1 genes, CPH1 and EFG1 were super expressed in interaction with S. mitis and sub expressed when there was interaction with S. sanguinis. There was no statistical difference in the in vivo studies of filamentation and CFU/mL. It follows that in vitro, S. mitis and S. sanguinis were able to inhibit the formation of C. albicans biofilms. Like the interaction with S. mitis inhibited its filamentation. The interaction with S. mitis appears to increase the virulence factors of C. albicans. ALS1, ALS3, HWP1 as the ...(Resumo completo, clicar acesso eletrônico abaixo). / Mestre

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 98-113).

The pulmonary infection with Pseudomonas aeruginosa is considered as one of the main causes of health deterioration in cystic fibrosis patients (CF). Efficient management of P. aeruginosa in CF remains difficult mainly with the emergence of multidrug-resistant strains leading ultimately to death. There is a pressing need for new approaches to control these Pseudomonal infections. Current studies on the antimicrobial efficacy of liposomal antibiotics have shown conflicting results. We sought to assess whether the incorporation of clarithromycin into liposomes could improve its antibacterial activity against clinical isolate of Pseudomonas aeruginosa from CF patients. Different formulations of liposomal clarithromycin were prepared, characterized and their antibacterial activities against resistant strains of P. aeruginosa were investigated. These formulations reduced the biofilm formation, the virulence factors production and the bacterial motilities compared to free drug. The therapeutic importance of liposome containing macrolides in the management of experimental pseudomonal lung infection in animals is warranted.

Cholera, an acute diarrheal diseases caused by the intestinal infection of the pathogenic bacterium Vibrio cholerae, continues to be a global killer in the world today. PrtV, a secreted zinc metalloprotease, is a potent cytotoxic virulence factor of V. cholerae. The 102 kDa full length multi-domain PrtV protein undergoes several N and C terminal modifications before being secreted as a 81 kDa pro-protein. The activation of the pro-protein is calcium dependent. The removal of calcium triggers auto-proteolysis to give a stable active protease with the catalytic zinc binding domain. The aim of the thesis was to study the structure and function of the PrtV protein. The results from paper I, identified the end product of the maturation of PrtV as the stable 37 kDa M6 active domain, and not a 55 kDa complex as reported earlier. Results also showed the this 37 kDa active M6 domain alone was sufficient for catalytic activity. A revised model for the maturation of PrtV was proposed. Individual domains were isolated from the PrtV protein by domain phasing methods. This included the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25 kDa fragment (residues 589-839). The isolated domains were recombinantly over expressed as fusion proteins to increase expression and solubility. The PKD1 domain was purified to homogeneity and crystallized. The structure of the PKD1 domain reported in paper II, was solved by X-ray crystallography at an atomic resolution of 1.1 Å. From the structure, a previously unknown calcium binding site was identified at the N-terminal of the PKD1 domain. The structure also revealed two conformations for the PKD1 domain depending on free or bound calcium. From the structure, a function of the PKD1 domain as a protector of the cleavage site in the linker region between the M6 domain and the PKD1 domain in the presence of calcium was elucidated. A new model for the activation of PrtV was given. In paper III, the structure of the N-terminal domain solved by NMR spectroscopy was reported. The structure revealed two well defined helices but a third predicted helix was found to be unstructured.