Regulation of the bacterial second messenger cyclic-di-GMP in Salmonella Typhimurium allows it to delicately alter phenotypes to optimize invasion and survive intracellularly in epithelial cells and macrophages to become virulent and cause infection. Cyclic-di-GMP concentration is regulated by the presence of external stimuli, sensory diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), and cyclic-di-GMP binding effectors. Previous studies established that maintenance of low cyclic-di-GMP concentrations is required for survival in macrophages, and that deletion of 3 active PDEs reduces this survival. Here I showed that these 3 PDEs also influenced the infection of epithelial cells. Further studies re-established the decreased survival in an immortalized macrophage cell line and determined that cyclic-di-GMP-binding cellulose synthase BcsA was responsible for the decreased survival in macrophages. Finally, I also identified an active DGC whose deletion within the 3xKO restores survival levels, suggesting that this enzyme is responsible for the synthesis of cyclic-di-GMP during macrophage infection.

Acinetobacter baumannii is an increasingly multidrug-resistant pathogen contributing to hospital-acquired infections necessitating the discovery of novel treatments. A bacterial second messenger, cyclic diguanosine monophosphate (cyclic di-GMP), can regulate various persistence factors that are potentially advantageous for survival in hospital environments. Cyclic di-GMP–modulating enzymes and cyclic di-GMP–binding effectors predictively are encoded in the Acinetobacter baumannii genome. I hypothesized that cyclic di-GMP controls motility, biofilm formation, and desiccation tolerance in Acinetobacter baumannii. Disrupting cyclic di-GMP–modulating enzymes or cyclic di-GMP–binding effectors should alter the regulatory effectiveness of these phenotypes. I tested the multidrug-resistant isolate Acinetobacter baumannii strain AB5075 and identified several transposon mutants that altered twitching motility, biofilm formation, and desiccation tolerance; these results suggest that cyclic di-GMP plays a role during these three responses in Acinetobacter baumannii AB5075. Inhibiting these cyclic di-GMP signaling pathways could produce novel mechanisms to combat this pathogen in the hospital environment.

Aufgrund ihrer niedrigen Stoffwechselrate zeigen Puppen des Atlasspinners Attacus atlas diskontinuierlichen Gasaustausch (DGC) mit drei deutlich getrennten Phasen. Andererseits sind einzelne Stigmen entweder vollständig offen oder vollständig geschlossen. In dieser Arbeit wurden Attacus Puppen mit künstlichen Gasmischungen perfundiert, um das Steady-State Verhalten der Stigmen zu ermitteln. Es konnte gezeigt werden, daß die Flutterphase des DGC kein Gleichgewichtsverhalten darstellt. Weiterhin deuteten die Ergebnisse der Perfusion auf eine mögliche Interaktion zweier unabhängiger Regelkreise hin. Zwei mathematische Modelle untersuchten daraufhin mögliche theoretische Mechanismen, welche die beobachteten Muster erzeugen könnten. Die Modelle erlaubten die Erzeugung der Gasaustauschmuster ohne komplexe neurale Integration und deuten auf eine rein passive Erzeugung des DGC. Die Diskussion erläutert mögliche biologische Mechanismen, welchen den abstrakten Beschreibungen der Modelle zu Grunde liegen könnten. Sowohl die Perfusion als auch die Modelle sind dabei mit der ''Emergent Property Hypothesis'' der DGC Entstehung vereinbar. Zusätzlich wird dargelegt, wie alternative Hypothesen widerspruchslos in das sich abzeichnende Bild eingefügt werden können. / While having low metabolic activity, pupae of the giant silk moth Attacus atlas show triphasic discontinuous gas exchange cycles (DGC). However, a single spiracle is either fully open or fully closed. In this study moth pupae have been perfused to assess the steady state behavior of the spiracles in response to the endotracheal gas composition. It could be shown that the flutter phase of DGC is not a steady state behavior. Moreover, the results from the perfusion hint to a possible interaction between two independent spiracle control mechanisms. Two mathematical models then explore possible mechanisms of generating the observed respiratory patterns during DGC. The models indicate that DGC might be generated without complex neural integration and might thus be a purely ''passive'' pattern. The discussion focuses on biological processes and mechanisms that may back the abstract descriptions in the models. Both, the perfusion and the two mathematical models, are consistent with the emergent property hypothesis of DGC origin. However, it is discussed that other existing hypotheses are not mutually exclusive and that they can be subsumed using the non-adaptive emergent property hypothesis as a framework.

No description available.

O sistema NADPH oxidase humano é responsável pela geração de reativos intermediários do oxigênio e defeitos neste sistema resultam na Doença Granulomatosa Crônica (DGC). Nesta tese de doutorado, investigamos o efeito do IFN-g sobre eventos pós-transcricionais em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. Os dados obtidos sugerem que o uso do IFN-g in vitro interfere no processamento da mensagem causando aumento da expressão de transcritos do gene CYBB e NCF1 em células B-EBV de indivíduos sadios e pacientes DGC analisados. Observamos também que o IFN-g dimunui a expressão dos genes THOC4 NONO, SF3A1, SRRM1 e UPF3A e promove aumento de expressão de SRSF10, SNRPA1 e C2 em células B-EBV de paciente X-DGC secundária a defeitos de splicing. Identificamos que o IFN-g e o TNF-α aumentam a expressão das proteínas envolvidas no processo do splicing. Concluímos que o IFN-g aumenta a expressão de genes importantes para uma resposta eficiente do sistema imunológico, incluindo os do sistema NADPH oxidase, além de promover aumento da expressão de genes e de proteínas relacionados ao processo de splicing, que podem estar relacionados aos efeitos benéficos observados no uso do IFN-g em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. / The human phagocyte NADPH oxidase is responsible for the generation of reactive oxygen intermediates and defects in this system result in Chronic Granulomatous Disease (CGD). In this PhD Thesis, we investigated the effect of IFN-g on post-transcriptional events in normal individuals and patients with X-linked CGD, caused by splicing defects. The obtained data suggests that the use of IFN-g in vitro interferes in the message processing causing an increase of expression of CYBB and NCF1 gene transcripts in B-EBV cells of healthy individuals and analyzed CGD patients. We also observed that IFN-g decreases the expression of THOC4, NONO, SF3A1, SRRM1 and UPF3A, and increases the expression of SRSF10, SNRPA1 and C2 genes in cells from X-CGD patients, due to splicing defects. We identified that IFN-g and TNF-α induce expression of proteins involved in the splicing process. We conclude that IFN-g increases the expression of important genes for an effective immune response, including the NADPH oxidase system genes, and promotes augment of gene and protein expression related to the splicing process, which may be related to the beneficial effects related to the use of IFN-g in CGD patient caused by splicing defects.

O sistema NADPH oxidase humano é responsável pela geração de reativos intermediários do oxigênio e defeitos neste sistema resultam na Doença Granulomatosa Crônica (DGC). Nesta tese de doutorado, investigamos o efeito do IFN-g sobre eventos pós-transcricionais em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. Os dados obtidos sugerem que o uso do IFN-g in vitro interfere no processamento da mensagem causando aumento da expressão de transcritos do gene CYBB e NCF1 em células B-EBV de indivíduos sadios e pacientes DGC analisados. Observamos também que o IFN-g dimunui a expressão dos genes THOC4 NONO, SF3A1, SRRM1 e UPF3A e promove aumento de expressão de SRSF10, SNRPA1 e C2 em células B-EBV de paciente X-DGC secundária a defeitos de splicing. Identificamos que o IFN-g e o TNF-α aumentam a expressão das proteínas envolvidas no processo do splicing. Concluímos que o IFN-g aumenta a expressão de genes importantes para uma resposta eficiente do sistema imunológico, incluindo os do sistema NADPH oxidase, além de promover aumento da expressão de genes e de proteínas relacionados ao processo de splicing, que podem estar relacionados aos efeitos benéficos observados no uso do IFN-g em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. / The human phagocyte NADPH oxidase is responsible for the generation of reactive oxygen intermediates and defects in this system result in Chronic Granulomatous Disease (CGD). In this PhD Thesis, we investigated the effect of IFN-g on post-transcriptional events in normal individuals and patients with X-linked CGD, caused by splicing defects. The obtained data suggests that the use of IFN-g in vitro interferes in the message processing causing an increase of expression of CYBB and NCF1 gene transcripts in B-EBV cells of healthy individuals and analyzed CGD patients. We also observed that IFN-g decreases the expression of THOC4, NONO, SF3A1, SRRM1 and UPF3A, and increases the expression of SRSF10, SNRPA1 and C2 genes in cells from X-CGD patients, due to splicing defects. We identified that IFN-g and TNF-α induce expression of proteins involved in the splicing process. We conclude that IFN-g increases the expression of important genes for an effective immune response, including the NADPH oxidase system genes, and promotes augment of gene and protein expression related to the splicing process, which may be related to the beneficial effects related to the use of IFN-g in CGD patient caused by splicing defects.