Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 23, 2008). Includes bibliographical references.

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

Thesis (Ph.D.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

Submitted by Ligia Souza (ligia@ufscar.br) on 2017-09-21T14:27:46Z No. of bitstreams: 1 TeseMCG.pdf: 3682312 bytes, checksum: fd17a7f18e76507395baaaf315348805 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-09-25T12:32:38Z (GMT) No. of bitstreams: 1 TeseMCG.pdf: 3682312 bytes, checksum: fd17a7f18e76507395baaaf315348805 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-09-25T12:32:44Z (GMT) No. of bitstreams: 1 TeseMCG.pdf: 3682312 bytes, checksum: fd17a7f18e76507395baaaf315348805 (MD5) / Made available in DSpace on 2017-09-25T12:37:57Z (GMT). No. of bitstreams: 1 TeseMCG.pdf: 3682312 bytes, checksum: fd17a7f18e76507395baaaf315348805 (MD5) Previous issue date: 2017-07-31 / Outra / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Infectious pneumonia is a major cause of morbidity/mortality, mainly due to the increasing rate of microorganisms resistant to antibiotics. Photodynamic Inactivation (PDI) is emerging as a promising approach, as effects are based on oxidative stress, preventing the emergence of resistant microorganism strains. In previous studies, the in vitro inactivation of Streptococcus pneumoniae using indocyanine green (ICG) and infrared light source was successful, and achieved reduction of 5 log10 colony-forming units (CFU/mL) with concentration as low as 10 μM ICG. In the present study, a proof-of-principle protocol was designed to treat lung infections by PDI using extracorporeal illumination with a 780 nm laser device and ICG as photosensitizer. In a first row of experiments, hairless mice were infected with S. pneumoniae and PDI was performed two days after infection. For control groups, CFU recovery ranged between 103-104 CFU/mL/mouse. For PDT group, however, no bacteria were recovered in 80% of the animals. Animal survival was evaluated separately over 50 days. No deaths occurred in PDT group, whereas 60% of the control did not survive. Lung injury analyses were performed in BALB/c mice, the bacteria reduction were 2 and 4 log10 in 2 mice (5 in total) and the wet-to-dry ratio showed that PDI did not increase the edema in lungs. The bronchoalveolar lavage data indicated a larger absolute number of cells (mononuclear and polymorphonuclear) in the PDI group in contrast to control group, meaning that the technique could increase the immune system response. In vitro results showed the irradiated ICG could generate aggregates or photoproducts that help PDI to inactivate the bacteria. Our results indicate that extracorporeal PDI has potential for pneumonia treatment, and pulmonary decontamination with PDI may be used as a single therapy or as an antibiotics adjuvant. / A pneumonia infecciosa é uma das principais causas de morbidade/mortalidade no mundo, principalmente devido à taxa crescente de micro-organismos resistentes a antibióticos. A inativação fotodinâmica (IFD) está emergindo como uma abordagem promissora, cujos efeitos são baseados no estresse oxidativo, impedindo o surgimento de cepas de micro-organismos resistentes. Em estudos anteriores, a inativação in vitro de Streptococcus pneumoniae utilizando indocianina verde (ICV) e fonte de luz de infravermelho foi efetiva, inativando 5 log10 (UFC/mL) com apenas 10 μM ICV. Neste estudo, foi avaliado protocolo de prova de princípio para tratar infecções pulmonares por IFD usando irradiação extracorpórea com dispositivo a laser emitindo em 780 nm e ICV como fotossensibilizador (FS). Em uma primeira série de experimentos, camundongos hairless SKH-1 foram infectados com S. pneumoniae e a IFD foi realizada dois dias após a infecção. Para os grupos de controles, a recuperação de UFC variou entre 103-104 UFC/mL/animal. Para o grupo IFD, no entanto, nenhuma bactéria foi recuperada em 80% dos animais. A sobrevivência animal foi avaliada durante 50 dias. Não ocorreram mortes no grupo IFD, enquanto 60% do grupo controle foi a óbito. Foram realizadas análises de lesões pulmonares em camundongos BALB/c, onde a redução bacteriana foi de 2 e 4 log10 em dois animais (total 5) em comparação com o grupo controle, e a proporção de peso úmido e seco mostrou que a IFD não aumentou o edema nos pulmões. Os dados de lavagem broncoalveolar indicaram um aumento no número absoluto de células (mononucleares e polimorfonucleares) no grupo IFD, o que indica que a técnica pode aumentar a resposta do sistema imunológico. Os resultados in vitro mostraram que a ICV irradiada pode gerar agregados ou fotoprodutos que auxiliam a IFD a inativar a bactéria. Os resultados deste estudo indicam que a IFD com irradiação extracorpórea tem potencial para o tratamento de pneumonia, e a descontaminação pulmonar com IFD pode ser usada como terapia ou como adjuvante aos antibióticos. / CAPES: 99999.003154/2015-07

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-12-01T16:24:52Z No. of bitstreams: 2 Dissertação- Weslley José Moreira Garcia - 2013.pdf: 1042957 bytes, checksum: a4d228204aeaaf723a235c9bd9929256 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-12-04T14:30:35Z (GMT) No. of bitstreams: 2 Dissertação- Weslley José Moreira Garcia - 2013.pdf: 1042957 bytes, checksum: a4d228204aeaaf723a235c9bd9929256 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-12-04T14:30:35Z (GMT). No. of bitstreams: 2 Dissertação- Weslley José Moreira Garcia - 2013.pdf: 1042957 bytes, checksum: a4d228204aeaaf723a235c9bd9929256 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-01-23 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Brazil was the first country to introduce the pneumococcal conjugate 10valent vaccine into the National Immunization Program for infants, in 2010. The nasopharyngeal colonization by Streptococcus pneumoniae occurs early in life. It is the first step for the development of invasive diseases. So far no study has evaluated the impact of vaccination on the reduction on pneumococcal carriage. The evaluation of the impact of vaccination should based on technologies with high accuracy. In this investigation we applied molecular technologies, recently developed, to ascertain pneumococcal nasopharyngeal colonization and serotypes. Objectives: (i) to compare the prevalence of S. pneumoniae nasopharyngeal colonization by using real-time PCR (RT-PCR) and multiplex PCR, and culture (“gold standard”) in children residing in Goiania municipality; (ii) to evaluate the simultaneous colonization by different serotypes by using the multiplex PCR technique. Methods: A household populationbased survey was carried out between October/2010 and March/2011 by using a systematic sampling, weighted by census tract. Based on previous studies, the sample size was calculated taking into account an estimated 50% of pneumocococcal carriage. A total of 1,437 nasopharyngeal swabs were collected from children less than 24 months of age. Broth-enriched culture of nasopharynx specimens followed by pneumococcal isolation by both, culture and RT-PCR targeting the lytA gene (S. pneumoniae) were performed. Pneumococcal carriage was defined for RT-PCR Cycle threshold (Ct) < 35.0, and therefore all samples were submitted to multiplex PCR to detect serotypes. ROC curve (Receiver Operating Characteristics) were built up to identify Ct values predicted of S. pneumoniae positive culture. Results: The prevalence of pneumococcal carriage by RT-PCR (56.9%) was statistically higher (p< 0,001), compared to that obtained by culture (39.3%), regardless of the vaccination status. Among the 818 positive children/samples by RT-PCR, in 54.2% of them it was possible to detect the serotype. Simultaneous colonization by different types was found in 6.9% of the children. Ct values Ct33.0 showed the best accuracy (91.4%) to predict positive pneumococcal culture (Sensitivity=88% and Specificity=81.2%). When using Ct values  32.0 we found the best accuracy of multiplex PCR in detecting serotypes (Sensitivity =90% and Specificity =84,7%). Conclusion: Our findings suggest that RT-PCR and multiplex PCR techniques showed great potential to be used in evaluating the vaccination impact. Further studies are needed to evaluate the cost-effectiveness of using these technologies on a large scale. / O Brasil foi o primeiro pais a introduzir a vacina pneumocócica conjugada, 10-valente (PCV10), no Programa de Imunização infantil, em 2010. A colonização nasofaringeana pelo Streptococcus pneumoniae ocorre na infância e é etapa obrigatória para desenvolvimento da doença invasiva. Até o momento nenhum estudo avaliou o impacto da vacinação na redução do estado de portador. Para avaliação do impacto de vacinas deve-se utilizar tecnologias de alta acurácia. Este estudo utiliza técnicas moleculares, recentemente desenvolvidas, para detecção de pneumococo e sorotipos de secreção nasofaringeana.Objetivos: (i) Comparar a prevalência de colonização nasofaringeana por S. pneumoniae pelas técnicas de PCR em tempo real (RT-PCR) e cultura (―padrão-ouro‖) em crianças residentes em Goiânia no primeiro ano de introdução da PCV10; (ii) avaliar a colonização simultânea por pneumococo por diferentes sorotipos por meio da reação de PCR multiplex. Métodos: Um inquérito populacional domiciliar foi conduzido de outubro/2010 a março/2011, com coleta de 1.437 swabs nasofaríngeos de crianças < 24 meses de idade. A amostragem foi sistemática, ponderada por setor censitário, com tamanho da amostra calculado para prevalência esperada de 50% de portador. O isolamento do pneumococo foi realizado a partir do caldo enriquecido (meio STGG). A cultura foi realizada pela semeadura do caldo em placas de Agar sangue de carneiro. A RT-PCR foi direcionada para o gene lytA do pneumococo, utilizando como positividade valores do ciclo da PCR (Ct-Cycle threshold) <35,0. A reação de PCR multiplex para sorotipagem foi realizada para amostras com valores de Ct<35,0. Foram construídas curvas ROC (Receiver Operating Characteristics) para identificação de valores de Ct preditivos de cultura positiva e de tipo capsular. Resultados: A prevalência de pneumococo obtida pela RT-PCR foi de 56,9%, estatisticamente maior do que a prevalência de 39,3% obtida pela cultura (p< 0,001), independente da situação vacinal da criança. Dentre as 818 crianças positivas pela RT-PCR, em 54,2% delas foi possível detectar-se o tipo capsular. Cocolonização por diferentes sorotipos foi encontrada em 6,9% (100/1.437) das crianças. Valores de Ct33,0 apresentaram a melhor acurácia (91,4%) na predição de cultura positiva para pneumococo (sensibilidade/S=88% e especificidade/E=81,2%). Para detecção de sorotipos a melhor acurácia da PCR multiplex foi para valores de Ct32,0 (S=90% e E=84,7%). Conclusão: Os resultados sugerem que as técnicas de PCR em tempo real e multiplex apresentam grande potencial para serem utilizadas em estudos de avaliação de impacto da vacinação, respectivamente no portador e nos sorotipos vacinais. Estudos deverão ser conduzidos para se avaliar o custo-benefício da utilização desta tecnologia em larga escala.

Abstract New rapid methods for sensitive and specific detection of pneumococci are not only needed to improve the diagnosis of pneumococcal disease but are also essential for vaccine and carriage studies. The purpose of this study was to develop sensitive PCR methods for the detection and quantification of S. pneumoniae and to study the applicability of these methods to detecting pneumococci in clinical samples. A previously described PCR method was first developed further by introducing a Europium-labelled hybridisation probe for the detection of amplification products. The hybridisation method was easy to use and improved the specificity of the PCR assay. The developed PCR assay was established as a sensitive method for detecting pneumococcal DNA when the presence of pneumococcal DNA in over 2500 middle ear fluid (MEF) samples of children with acute otitis media (AOM) was studied by using the method. Pneumococcal findings increased by 76% when using PCR detection in addition to culture, compared to using culture alone. However, the PCR-positive, culture-negative AOM events represented a less severe type of disease compared to the culture-positive events. A positive PCR finding seems to indicate the presence of viable, although often non-culturable pneumococci within the middle ear cleft. To be able to rapidly detect and quantify the initial numbers of pneumococcal genome copies in clinical samples, a real-time PCR method for the detection and quantification of pneumococcal DNA was developed. In real-time PCR, amplification and detection of amplification products occur simultaneously, which makes it possible to monitor the phase of the reaction at a particular stage or continuously. The method developed here was applied to the analysis of MEF samples and to investigating the nasopharyngeal carriage of pneumococcus. The sensitivities of bacterial culture and real-time PCR in detecting pneumococci were also compared. The real-time PCR assay was found to be rapid and sensitive and to provide information about the differences between the numbers of bacteria in samples. However, the quantitative results were shown to be dependent on the DNA extraction method applied. The real-time PCR method developed appears to be a good aid in research where an accurate and sensitive pneumococcal diagnosis is needed.

Any metal in excess can be toxic; therefore, metal homeostasis is critical to bacterial survival. Bacteria have developed specialized metal import and export systems for this purpose. For broadly toxic metals such as copper, bacteria have evolved only export systems. The copper export system (cop operon) usually consists of the operon repressor, the copper chaperone, and the copper exporter. In Streptococcus pneumoniae, the causative agent of pneumonia, otitis media, sepsis, and meningitis, little is known about operon regulation. This is partly due to the S. pneumoniae repressor, CopY, and copper chaperone, CupA, sharing limited homology to proteins of putative related function and confirmed established systems. In this study, we examined CopY metal crosstalk, CopY interactions with CupA, and how CupA can control the oxidation state of copper. We found that CopY bound zinc and increased the DNA-binding affinity of CopY by roughly an order of magnitude over that of the apo form of CopY. Once copper displaced zinc in CopY, resulting in operon activation, CupA chelated copper from CopY. After copper was acquired from CopY or other sources, if needed, CupA facilitated the reduction of Cu2+ to Cu1+, which is the exported copper state. Taken together, these data show novel mechanisms for copper processing in S. pneumoniae. IMPORTANCE As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae, remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu2+ to Cu1+. This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria.

För att utreda anledningen till bland annat otit och sinuit tas prov från nasofarynx, som enligt nuvarande metod utodlas på två separata plattor med blod- och hematinagar för att identifiera patogena luftvägsbakterier. Diagnostiska antibiotikadiskar används för att förenkla differentiering mellan olika bakterier. Odling på blodagar med optochin-disk är en tillförlitlig metod för identifiering av Streptococcus pneumoniae. Metoden är tidskrävande och agglutinationstest är ett alternativ för mer tidseffektiv artidentifiering. Syftet med arbetet var dels att validera en ny provsättningsmetod för nasofarynx-prov samt att utvärdera ett nytt agglutinationstest för artidentifiering av S. pneumoniae. För att validera provsättningsmetoden jämfördes den tidigare metoden med en ny där analyserna kombineras på samma platta (biplate) (N=165). För validering av nytt agglutinationstest inokulerades hästblod och bakterieisolat i blododlingsflaskor. Agglutinationstest utfördes därefter med två kommersiella kit, Dryspot, som idag används på laboratoriet, och Immulex. Fynden från nasofarynx efter odling på nya kombinerade plattor bestod av S. pneumoniae, Haemophilus influenzae och Moraxella catarrhalis. Överensstämmelsen mellan odlingsmetoderna var god för nasofarynxodlingar. Vid mätning av bakteriefri zon kring optochin-disk erhölls 18 – 31 mm för biplates respektive 16 – 24 mm för originalmetoden med odling på två separata plattor. För två prov var den bakteriefria zonen på biplates ej mätbar. Vid agglutinationstest för identifiering av S. pneumoniae erhölls agglutination inom respektive tidsgräns för samtliga referensisolat. Ett α-streptokock- och ett enterokock-isolat agglutinerade vid test med Immulex. Slutsatsen av studien är att biplates bör fungera som rutinmetod för odling av nasofarynxprov. Däremot verkar agglutinationstest med Immulex vara mindre pålitligt än Dryspot som används idag. / To investigate the cause of otitis and sinusitis, samples are taken from nasopharynx. To find airway pathogens samples are cultivated on blood agar and hematin agar, for further differentiation diagnostic antibiotic disks are used. Cultivation on blood agar with optochin disks is a reliable method to identify Streptococcus pneumoniae, although it is time-consuming. Agglutination test is a rapid method for identification of Streptococcus pneumoniae. The aim of this study was to validate a new method for cultivation of nasopharynx-samples. Moreover a new agglutination test for identification of S. pneumoniae was evaluated. To validate the new method of cultivation, the original method was compared with the new where blood and hematin agar are combined on the same plate (biplate) (N=165). To validate the new agglutination test, blood culture bottles were inoculated with blood and suspensions of relevant bacterial species. Agglutination tests were performed using Dryspot and Immulex commercial kits. Bacterial findings from nasopharynx after cultivation on the new combined plates included S. pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. The correlation between the methods was good for nasopharynx-cultivations. The inhibitory zones around the optochin disk were 18 – 31 mm for biplates and 16 – 24 mm for the original method. All S. pneumoniae agglutinated within the time limits when agglutination test were performed. One α-Streptococcus and one Enterococcus agglutinated when tests were performed with Immulex, hence giving false positive results. In conclusion, the new cultivation method of nasopharynx samples proved functional.  Agglutination test with the kit presently used, Dryspot, performed better than Immulex.

The major human pathogen Streptococcus pneumoniae is a unique model for the study of eukaryotic-type serine/threonine protein kinases and its cognate phosphatases in bacteria, since it encodes only a single signaling pair composed of the StkP protein kinase and PhpP phosphatase. This signaling pair plays a role in several cellular processes, mainly in cell wall biosynthesis and cell division. StkP and PhpP proteins with a pleiotropic effect appear to regulate a complex signaling cascade by phosphorylation of many substrates. However, only a few have been characterized so far. Using MS analysis, we have identified about 90 phosphopeptides that are potential substrates for the StkP kinase and PhpP phosphatase. This diploma thesis is focused on the characterization of the new substrate Spr0929 and its role in pneumococcal physiology. One of the objectives was to investigate cell morphology of strains carrying deletion of the spr0929 gene in different genetic backgrounds. It turned out that the role of Spr0929 in cell morphology is strain specific. The growth curves of strains with this deletion were compared to that of the wild type in various physiological conditions as well. As Spr0929 contains a nucleoid-associated domain called NdpA, determination of its cell localization was an important...

La enfermedad causada por el Streptococcus pneumoniae en niños, es responsable de más de 800,000 muertes en el mundo cada año, una fracción importante puede reducirse con la administración de vacuna conjugada neumocócica, existen 2 vacunas indicadas en población menor de 5 años de edad: la vacuna conjugada neumococica 10 valente (10 serotipos) y vacuna conjugada neumocócica 13 valente (13 serotipos), que se diferencian por el número de antígenos incluidos en ellas y la proteína transportadora. Por ello se determina la intervención más costo beneficio para el Perú en relación a su impacto económico en el costo de las neumonías neumocócicas en población menor de 5 años de edad en el Perú, se utilizó el indicador costo de cada AVP evitado en relación al PBI per cápita. Comparativamente se encontró que PCV 13 es una intervención más costosa que PCV10; sin embargo, evita mayor número de casos de neumonía y menor costo de cada AVP evitado en relación al PBI per cápita. Realiza un estudio fármaco económico que estima el beneficio de dos vacunas neumocócicas conjugadas: 10valente y 13valente en su impacto sobre las neumonías en la población menor de 5 años de edad para el Perú 2015. Encuentra que el costo de la implementación de la propuesta de la vacuna conjugada neumocócica a PCV 13, incrementa en 9.6% el costo en comparación a usar PCV 10. Sin embargo, se obtiene mayor número de neumonías evitadas (3025 con PCV13 en comparación con 1782 con PCV 10), mayor número de muertes prematura evitadas (442 con PCV 13 en comparación con 260 con PCV 10) y por otro lado el costo de cada AVP evitado es 55% más bajo con PCV 13, adicionalmente la relación costo de cada AVP evitado en relación con el PBI per cápita es 54.8% más bajo. Concluye que para el Perú, la estrategia de vacunación con PCV 13 es más costo efectivo que la estrategia de vacunación con PCV 10, para evitar neumonías y AVP por neumonías en población menor de 5 años, para el año 2015. / Tesis