449 research outputs found

    Quantitative physiology of bacterial survival under carbon starvation and temperature stress

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    A large number of the bacteria on Earth live for long periods in states of very low metabolic activity and little or no growth due to starvation and other environmental stresses. Within millions of years, bacteria have developed several strategies to adapt to many different environments, where they survive and evolve to optimize their fitness and to undergo rapid division cycles when conditions become favourable. However, many of these survival strategies are still a puzzle and relatively little is known about the mechanisms that underpin the dominant modes of bacterial existence. This is particularly alarming, as the growth-arrest phase has become crucial to understand the contribution of microorganisms to human physiology and predisposition to disease as well as microbial tolerance and resistance to antibiotics. The dearth of information is mainly due to the difficulties in defining, reproducing and measuring bacterial behaviours in growth-arrest states, which may often seem erratic and unpredictable, while cell physiology is similarly diverse and often specific to the particular environmental conditions. Thus, determining how molecular contributions affect survival is challenging. This explains why, in the last century, bacteria have been mainly studied during the exponential growth phase, which is, on the contrary, a well-defined and reproducible steady state of constant growth, gene expression and molecular compositions. As a result, an increasing combined use of experiments and predictive models focused on this phase has provided a deep understanding of bacterial physiology and gene regulation during growth. A similar quantitative approach that focuses on the growth-arrest phase is largely missing. In this thesis, we contribute to fill this gap by developing new quantitative approaches to investigate bacterial physiology in hostile environments where stresses, such as lack of nutrients and additional environmental perturbations, like temperature increase, force the cells to activate strategies of survival. To do so, we choose to work with the bacterium Escherichia coli (E. coli) that, among the estimated 10^12 microbial species living in our planet, is one of the most studied thanks to its hardiness, versatility and ease of handling. In Chapter 1, we provide an overview of the physiology of E. coli life cycle and of the main quantitative methods so far used to study it, especially focusing on its behaviour during the growth-arrest phase. In Chapter 2, we establish the missing quantitative approach to study E. coli physiology in the death phase. We show that in carbon starvation, an exponential decay of viability emerges as a collective phenomenon, with viable cells recycling nutrients from dead cells to maintain viability. The observed collective death rate is determined by the maintenance rate of viable cells and the amount of nutrients recovered from dead cells, the yield. Using this relation, we study the cost of a wasteful enzyme during starvation and the benefit of the stress response sigma factor RpoS. While the enzyme activity increases maintenance and thereby the death rate, RpoS improves biomass recycling, decreasing the death rate. Our approach thus enables quantitative analyses of how cellular components affect the survival of non-growing cells. In Chapter 3, we use the quantitative approach developed in the previous chapter to study how survival of E. coli in carbon starvation depends on the previous culture conditions. We show that environments that support only slow growth lead to longer survival in starvation because of a decrease of maintenance rate, meaning that slower growing cells need less energy to survive. Our results suggest a physiological trade-off between the ability to proliferate fast and the ability to survive long that could shed light on the long-standing question of why bacteria outside of laboratory environments are not optimized for fast growth. In Chapter 4, we study E. coli physiology under the combined stresses of carbon starvation and high temperatures, characterizing a thermal fuse that leads to a dormant and antibiotic persistent sub-population. This fuse is implemented by a thermally unstable enzyme, MetA, in the methionine synthesis pathway. The combination of a positive feed-back in the methionine system and a dual-use of methionine for protein synthesis and as a methyl-donor results in the bacterial population splitting into two distinct states at elevated temperatures, growing and dormant. We then reveal that these dormant bacteria not only survive antibiotic treatment, but also heat shocks, suggesting that the thermal fuse has originally evolved as a ''bet-hedging'' strategy to ensure survival in heat shocks. Our findings, summarized in Chapter 5, pave the way for the development of a new theoretical framework and experimental approach to understand bacterial physiology in the growth-arrest phase, by linking phenomenological modeling to molecular mechanisms.Eine große Anzahl der Bakterien auf der Erde lebt ĂŒber große ZeitrĂ€ume in einem Zustand mit sehr geringer StoffwechselaktivitĂ€t und nur geringem oder keinem Wachstum. Ein Grund dafĂŒr sind widrige UmwelteinflĂŒsse und die damit einhergehenden Belastungen wie beispielsweise Ressourcenmangel. Innerhalb von Millionen von Jahren haben Bakterien diverse Strategien zur Anpassung an verschiedene Umgebungen, in denen sie ĂŒberleben und sich weiterentwickeln, entwickelt, um ihre Fitness zu optimieren und bei gĂŒnstigen Bedingungen schnelle Teilungszyklen zu durchlaufen. Viele dieser Überlebensstrategien sind jedoch immer noch ein RĂ€tsel und es ist nur relativ wenig ĂŒber die Mechanismen bekannt, die den dominanten Formen der bakteriellen Existenz zu Grunde liegen. Dies ist von besonderer Bedeutung, da die Phase unterdrĂŒckten Wachstums entscheidend ist, um den Beitrag von Mikroorganismen zur menschlichen Physiologie und AnfĂ€lligkeit fĂŒr Krankheiten, sowie zur mikrobiellen VertrĂ€glichkeit und Antibiotikaresistenz zu verstehen. Der Mangel an Informationen ist hauptsĂ€chlich auf die Schwierigkeiten bei der Definition, Reproduktion und Messung des Verhaltens von Bakterien in ZustĂ€nden des Wachstumsstillstands zurĂŒckzufĂŒhren, die oft unberechenbar und unvorhersehbar erscheinen, wĂ€hrend die Zellphysiologie Ă€hnlich vielfĂ€ltig und oft spezifisch fĂŒr die jeweiligen Umgebungsbedingungen ist. Daher ist es schwierig zu bestimmen, wie sich molekulare Mechanismen auf das Überleben auswirken. Dies erklĂ€rt, warum im letzten Jahrhundert Bakterien hauptsĂ€chlich wĂ€hrend der exponentiellen Wachstumsphase untersucht wurden, die im Gegenteil ein genau definierter und reproduzierbarer Gleichgewichtszustand des konstanten Wachstums, der Genexpression und der molekularen Zusammensetzung ist. Infolgedessen hat eine zunehmende Kombination von Experimenten und Vorhersagemodellen, die sich auf diese Phase konzentrieren, ein tiefes VerstĂ€ndnis der bakteriellen Physiologie und Genregulation wĂ€hrend des Wachstums geliefert. Ein Ă€hnlicher quantitativer Ansatz, der sich auf die Phase der Stagnation konzentriert, fehlt weitgehend. In dieser Doktorarbeit tragen wir dazu bei, diese LĂŒcke durch die Entwicklung neuer quantitativer AnsĂ€tze zur Untersuchung der bakteriellen Physiologie in ungĂŒnstigen Umgebungen zu fĂŒllen, in denen Stressfaktoren, wie beispielsweise NĂ€hrstoffmangel, auftreten und zusĂ€tzliche umweltbedingte Störungen, wie eine Temperaturerhöhung, die Zellen zwingen, Strategien zum Überleben zu aktivieren. Dazu arbeiten wir mit dem Bakterium Escherichia coli (E. coli), das unter den circa 10^12 mikrobiellen Spezies, die auf unserem Planeten leben, wegen seiner WiderstandsfĂ€higkeit, Vielseitigkeit und einfachen Handhabung eines der am besten untersuchten Bakterien darstellt. In Kapitel 1, geben wir einen Überblick ĂŒber die Physiologie des Lebenszyklus von E. coli und ĂŒber die wichtigsten bisher verwendeten quantitativen Methoden, wobei wir uns auf das Verhalten wĂ€hrend der Wachstumsphase konzentrieren. In Kapitel 2, stellen wir den fehlenden quantitativen Ansatz zur Untersuchung der Physiologie von E. coli wĂ€hrend der Sterbephase fest. Wir zeigen, dass bei Kohlenstoffmangel ein exponentieller Zerfall der LebensfĂ€higkeit als kollektives PhĂ€nomen auftritt, wobei lebensfĂ€hige Zellen NĂ€hrstoffe aus toten Zellen recyceln, um die LebensfĂ€higkeit aufrechtzuerhalten. Die beobachtete kollektive Sterberate wird durch die Erhaltungsrate lebensfĂ€higer Zellen und die Menge an NĂ€hrstoffen, die aus toten Zellen als Ertrag gewonnen werden, bestimmt. Unter Verwendung dieser Beziehung untersuchen wir die Kosten einer verschwenderischen EnzymaktivitĂ€t wĂ€hrend des Hungerns und den Nutzen des Sigma Faktors RpoS fĂŒr die Stressreaktion. WĂ€hrend diese AktivitĂ€t die Instandhaltung und damit die Sterblichkeitsrate erhöht, verbessert RpoS das Recycling der Biomasse und senkt die Sterblichkeitsrate. Unser Ansatz ermöglicht daher quantitative Analysen darĂŒber, wie sich zellulĂ€re Komponenten auf das Überleben nicht wachsender Zellen auswirken. In Kapitel 3, verwenden wir den im vorherigen Kapitel entwickelten quantitativen Ansatz, um zu untersuchen, wie das Überleben von E. coli bei Kohlenstoffmangel von den vorherigen Kulturbedingungen abhĂ€ngt. Wir zeigen, dass Umgebungen, die nur langsames Wachstum unterstĂŒtzen, aufgrund einer verringerten Erhaltungsrate zu einem lĂ€ngeren Überleben fĂŒhren, was bedeutet, dass langsamer wachsende Zellen weniger Energie zum Überleben benötigen. Unsere Ergebnisse legen einen physiologischen Kompromiss zwischen der FĂ€higkeit, sich schnell zu vermehren, und der FĂ€higkeit, lange zu ĂŒberleben, nahe, der Auschluss darĂŒber geben könnte, warum Bakterien außerhalb von Laborumgebungen nicht fĂŒr schnelles Wachstum optimiert sind. In Kapitel 4, untersuchen wir die Physiologie von E. coli unter dem kombinierten Stress von Kohlenstoffmangel und hohen Temperaturen und charakterisieren eine thermische Sicherung, die zu einer ruhenden und antibiotisch persistierenden Subpopulation fĂŒhrt. Diese Sicherung wird durch ein thermisch instabiles Enzym, MetA, im Methioninsyntheseweg implementiert. Die Kombination aus einer positiven RĂŒckkopplung im Methioninsystem und einer doppelten Verwendung von Methionin fĂŒr die Proteinsynthese und als Methyldonor fĂŒhrt dazu, dass sich die Bakterienpopulation bei erhöhten Temperaturen in zwei verschiedene ZustĂ€nde aufspaltet, wobei jeweils eine Subpopulation wĂ€chst und die Andere schlĂ€ft. Wir zeigen dann, dass diese ruhenden Bakterien nicht nur eine Antibiotikabehandlung, sondern auch Hitzeschocks ĂŒberstehen, was darauf hindeutet, dass sich die thermische Sicherung ursprĂŒnglich als eine ''bet-hedging'' Strategie entwickelt hat, um das Überleben bei Hitzeschocks sicherzustellen. Unsere Ergebnisse, die in Kapitel 5 zusammengefasst sind, ebnen den Weg fĂŒr die Entwicklung eines neuen theoretischen Rahmens und experimentellen Ansatzes zum VerstĂ€ndnis der Bakterienphysiologie in der Phase des Wachstumsstopps, indem phĂ€nomenologische Modelle mit molekularen Mechanismen verknĂŒpft werden

    Genetic studies on the role of type IA DNA topoisomerases in DNA metabolism and genome maintenance in Escherichia coli

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    Le surenroulement de l’ADN est important pour tous les processus cellulaires qui requiĂšrent la sĂ©paration des brins de l’ADN. Il est rĂ©gulĂ© par l’activitĂ© enzymatique des topoisomĂ©rases. La gyrase (gyrA et gyrB) utilise l’ATP pour introduire des supertours nĂ©gatifs dans l’ADN, alors que la topoisomĂ©rase I (topA) et la topoisomĂ©rase IV (parC et parE) les Ă©liminent. Les cellules dĂ©ficientes pour la topoisomĂ©rase I sont viables si elles ont des mutations compensatoires dans un des gĂšnes codant pour une sous-unitĂ© de la gyrase. Ces mutations rĂ©duisent le niveau de surenroulement nĂ©gatif du chromosome et permettent la croissance bactĂ©rienne. Une de ces mutations engendre la production d'une gyrase thermosensible. L’activitĂ© de surenroulement de la gyrase en absence de la topoisomĂ©rase I cause l’accumulation d’ADN hyper-surenroulĂ© nĂ©gativement Ă  cause de la formation de R-loops. La surproduction de la RNase HI (rnhA), une enzyme qui dĂ©grade l’ARN des R-loops, permet de prĂ©venir l’accumulation d’un excĂšs de surenroulement nĂ©gatif. En absence de RNase HI, des R-loops sont aussi formĂ©s et peuvent ĂȘtre utilisĂ©s pour dĂ©clencher la rĂ©plication de l’ADN indĂ©pendamment du systĂšme normal oriC/DnaA, un phĂ©nomĂšne connu sous le nom de « constitutive stable DNA replication » (cSDR). Pour mieux comprendre le lien entre la formation de R-loops et l’excĂšs de surenroulement nĂ©gatif, nous avons construit un mutant conditionnel topA rnhA gyrB(Ts) avec l’expression inductible de la RNase HI Ă  partir d’un plasmide. Nous avons trouvĂ© que l’ADN des cellules de ce mutant Ă©tait excessivement relĂąchĂ© au lieu d'ĂȘtre hypersurenroulĂ© nĂ©gativement en conditions de pĂ©nurie de RNase HI. La relaxation de l’ADN a Ă©tĂ© montrĂ©e comme Ă©tant indĂ©pendante de l'activitĂ© de la topoisomĂ©rase IV. Les cellules du triple mutant topA rnhA gyrB(Ts) forment de trĂšs longs filaments remplis d’ADN, montrant ainsi un dĂ©faut de sĂ©grĂ©gation des chromosomes. La surproduction de la topoisomĂ©rase III (topB), une enzyme qui peut effectuer la dĂ©catĂ©nation de l’ADN, a corrigĂ© les problĂšmes de sĂ©grĂ©gation sans toutefois restaurer le niveau de surenroulement de l’ADN. Nous avons constatĂ© que des extraits protĂ©iques du mutant topA rnhA gyrB(Ts) pouvaient inhiber l’activitĂ© de surenroulement nĂ©gatif de la gyrase dans des extraits d’une souche sauvage, suggĂ©rant ainsi que la pĂ©nurie de RNase HI avait dĂ©clenchĂ© une rĂ©ponse cellulaire d’inhibition de cette activitĂ© de la gyrase. De plus, des expĂ©riences in vivo et in vitro ont montrĂ© qu’en absence de RNase HI, l’activitĂ© ATP-dĂ©pendante de surenroulement nĂ©gatif de la gyrase Ă©tait inhibĂ©e, alors que l’activitĂ© ATP-indĂ©pendante de cette enzyme demeurait intacte. Des suppresseurs extragĂ©niques du dĂ©faut de croissance du triple mutant topA rnhA gyrB(Ts) qui corrigent Ă©galement les problĂšmes de surenroulement et de sĂ©grĂ©gation des chromosomes ont pour la plupart Ă©tĂ© cartographiĂ©s dans des gĂšnes impliquĂ©s dans la rĂ©plication de l’ADN, le mĂ©tabolisme des R-loops, ou la formation de fimbriae. La deuxiĂšme partie de ce projet avait pour but de comprendre les rĂŽles des topoisomĂ©rases de type IA (topoisomĂ©rase I et topoisomĂ©rase III) dans la sĂ©grĂ©gation et la stabilitĂ© du gĂ©nome de Escherichia coli. Pour Ă©tudier ces rĂŽles, nous avons utilisĂ© des approches de gĂ©nĂ©tique combinĂ©es avec la cytomĂ©trie en flux, l’analyse de type Western blot et la microscopie. Nous avons constatĂ© que le phĂ©notype Par- et les dĂ©fauts de sĂ©grĂ©gation des chromosomes d’un mutant gyrB(Ts) avaient Ă©tĂ© corrigĂ©s en inactivant topA, mais uniquement en prĂ©sence du gĂšne topB. En outre, nous avons dĂ©montrĂ© que la surproduction de la topoisomĂ©rase III pouvait corriger le phĂ©notype Par- du mutant gyrB(Ts) sans toutefois corriger les dĂ©fauts de croissance de ce dernier. La surproduction de topoisomĂ©rase IV, enzyme responsable de la dĂ©catĂ©nation des chromosomes chez E. coli, ne pouvait pas remplacer la topoisomĂ©rase III. Nos rĂ©sultats suggĂšrent que les topoisomĂ©rases de type IA jouent un rĂŽle important dans la sĂ©grĂ©gation des chromosomes lorsque la gyrase est inefficace. Pour Ă©tudier le rĂŽle des topoisomĂ©rases de type IA dans la stabilitĂ© du gĂ©nome, la troisiĂšme partie du projet, nous avons utilisĂ© des approches gĂ©nĂ©tiques combinĂ©es avec des tests de « spot » et la microscopie. Nous avons constatĂ© que les cellules dĂ©ficientes en topoisomĂ©rase I avaient des dĂ©fauts de sĂ©grĂ©gation de chromosomes et de croissance liĂ©s Ă  un excĂšs de surenroulement nĂ©gatif, et que ces dĂ©fauts pouvaient ĂȘtre corrigĂ©s en inactivant recQ, recA ou par la surproduction de la topoisomĂ©rase III. Le suppresseur extragĂ©nique oriC15::aph isolĂ© dans la premiĂšre partie du projet pouvait Ă©galement corriger ces problĂšmes. Les cellules dĂ©ficientes en topoisomĂ©rases de type IA formaient des trĂšs longs filaments remplis d’ADN d’apparence diffuse et rĂ©parti inĂ©galement dans la cellule. Ces phĂ©notypes pouvaient ĂȘtre partiellement corrigĂ©s par la surproduction de la RNase HI ou en inactivant recA, ou encore par des suppresseurs isolĂ©s dans la premiĂšre partie du projet et impliques dans le cSDR (dnaT18::aph et rne59::aph). Donc, dans E. coli, les topoisomĂ©rases de type IA jouent un rĂŽle dans la stabilitĂ© du gĂ©nome en inhibant la rĂ©plication inappropriĂ©e Ă  partir de oriC et de R-loops, et en empĂȘchant les dĂ©fauts de sĂ©grĂ©gation liĂ©s Ă  la recombinaison RecA-dĂ©pendante, par leur action avec RecQ. Les travaux rapportĂ©s ici rĂ©vĂšlent que la rĂ©plication inappropriĂ©e et dĂ©rĂ©gulĂ©e est une source majeure de l’instabilitĂ© gĂ©nomique. EmpĂȘcher la rĂ©plication inappropriĂ©e permet la sĂ©grĂ©gation des chromosomes et le maintien d’un gĂ©nome stable. La RNase HI et les topoisomĂ©rases de type IA jouent un rĂŽle majeur dans la prĂ©vention de la rĂ©plication inappropriĂ©e. La RNase HI rĂ©alise cette tĂąche en modulant l’activitĂ© de surenroulement ATP-dependante de la gyrase, et en empĂȘchant la rĂ©plication Ă  partir des R-loops. Les topoisomĂ©rases de type IA assurent le maintien de la stabilitĂ© du gĂ©nome en empĂȘchant la rĂ©plication inappropriĂ©e Ă  partir de oriC et des R-loops et en agissant avec RecQ pour rĂ©soudre des intermĂ©diaires de recombinaison RecA-dĂ©pendants afin de permettre la sĂ©grĂ©gation des chromosomes.DNA supercoiling is important for all cellular processes that require strand separation and is regulated by the opposing enzymatic effects of DNA topoisomerases. Gyrase uses ATP to introduce negative supercoils while topoisomerase I (topA) and topoisomerase IV relax negative supercoils. Cells lacking topoisomerase I are only viable if they have compensatory mutations in gyrase genes that reduce the negative supercoiling level of the chromosome to allow bacterial growth. One such mutation leads to the production of a thermosensitive gyrase (gyrB(Ts)). Gyrase driven supercoiling during transcription in the absence of topoisomerase I causes the accumulation of hypernegatively supercoiled plasmid DNAs due to the formation of R-loops. Overproducing RNase HI (rnhA), an enzyme that degrades the RNA moiety of R-loops, prevents the accumulation of hypernegative supercoils. In the absence of RNase HI alone, R-loops are equally formed and can be used to prime DNA replication independently of oriC/DnaA, a phenomenon known as constitutive stable DNA replication (cSDR). To better understand the link between R-loop formation and hypernegative supercoiling, we constructed a conditional topA rnhA gyrB(Ts) mutant with RNase HI being conditionally expressed from a plasmid borne gene. We found that the DNA of topA rnhA gyrB(Ts) cells was extensively relaxed instead of being hypernegatively supercoiled following the depletion of RNase HI. Relaxation was found to be unrelated to the activity of topoisomerase IV. Cells of topA rnhA gyrB(Ts) formed long filaments full of DNA, consistent with segregation defect. Overproducing topoisomerase III (topB), an enzyme that can perform decatenation, corrected the segregation problems without restoring supercoiling. We found that extracts of topA rnhA gyrB(Ts) cells inhibited gyrase supercoiling activity of wild type cells extracts in vitro, suggesting that the depletion of RNase HI triggered a cell response that inhibited the supercoiling activity of gyrase. Gyrase supercoiling assays in vivo as well as in crude cell extracts revealed that the ATP dependent supercoiling reaction of gyrase was inhibited while the ATP independent relaxation reaction was unaffected. Genetic suppressors of a triple topA rnhA gyrB(Ts) strain that restored supercoiling and corrected the chromosome segregation defects mostly mapped to genes that affected DNA replication, R-loop metabolism and fimbriae formation. The second part of this project aimed at understanding the roles of type IA DNA topoisomerases (topoisomerase I and topoisomerase III) in chromosome segregation and genome maintenance in E. coli. To investigate the role of type IA DNA topoisomerases in chromosome segregation we employed genetic approaches combined with flow cytometry, Western blot analysis and microscopy (for the examination of cell morphology). We found that the Par- phenotypes (formation of large unsegregated nucleoid in midcell) and chromosome segregation defects of a gyrB(Ts) mutant at the nonpermissive temperature were corrected by deleting topA only in the presence of topB. Moreover, overproducing topoisomerase III was shown to correct the Par- phenotype without correcting the growth defect, but overproducing topoisomerase IV, the major cellular decatenase, failed to correct the defects. Our results suggest that type IA topoisomerases play a role in chromosome segregation when gyrase is inefficient. To investigate the role of type IA DNA topoisomerases in genome maintenance, in the third part of the project, we employed genetic approaches combined with suppressor screens, spot assays and microscopy. We found that cells lacking topoisomerase I suffered from supercoiling-dependent growth defects and chromosome segregation defects that could be corrected by deleting recQ, recA or overproducing topoisomerase III and by an oriC15::aph suppressor mutation isolated in the first part of the project. Cells lacking both type 1A topoisomerases formed very long filaments packed with diffuse and unsegregated DNA. Such phenotypes could be partially corrected by overproducing RNase HI or deleting recA, or by suppressor mutations isolated in the first part of the project, that affected cSDR (dnaT18::aph and rne59::aph). Thus, in E. coli, type IA DNA topoisomerases play a role in genome maintenance by inhibiting inappropriate replication from oriC and R-loops and by preventing RecA-dependent chromosome segregation defect through their action with RecQ. The work reported here reveals that inappropriate and unregulated replication is a major source of genome instability. Preventing such replication will ensures proper chromosome segregation leading to a stable genome. RNase HI and type IA DNA topoisomerases play a leading role in preventing unregulated replication. RNase HI achieves this role by modulating ATP dependent gyrase activity and by preventing replication from R-loops (cSDR). Type IA DNA topoisomerases ensure the maintenance of a stable genome by preventing inappropriate replication from oriC and R-loops and by acting with RecQ to prevent RecA dependent-chromosome segregation defects

    Extracellular Vesicle-based Nano/Microparticles for Novel Vaccination Approaches

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    Pneumococcal infections cause many fatalities worldwide. Growing resistance to antibiotics and incomplete coverage of available vaccines against all serotypes made the search for novel vaccination approaches a global necessity. Extracellular membrane vesicles are secreted nanostructures, which are physiologically secreted from seemingly all living cells and harbor their virulence factors and immunogenic components. In this work, several research objectives were explored: (i) Isolation and characterization of pneumococcal vesicles. (ii) Biocompatibility and uptake with cell lines and primary human cells. (iii) Yield Enhancement of pneumococcal vesicles. (iv) Immunostimulation of immune cells by pneumococcal vesicles. (v) Formulation of spray-dried vaccine microparticles for pulmonary immunization. The isolated vesicles exhibited excellent biocompatibility with several cell lines and primary cells, without cytotoxic effects. Pneumococcal vesicles demonstrated rapid uptake into immune cells and stimulated the release of pro-inflammatory cytokines. We successfully formulated spray-dried vaccine microparticles with enhanced stability and increased cytokine release for pulmonary delivery. Our findings confirm the strong potential of pneumococcal membrane vesicles as vaccine candidates, and provide a sound basis for further translation and scale-up for pulmonary delivery and immunization.Pneumokokken-Infektionen fĂŒhren weltweit zu zahlreichen TodesfĂ€llen. Die zunehmende Resistenz gegen Antibiotika und die unvollstĂ€ndige Abdeckung durch verfĂŒgbare Impfstoffe gegen alle Serotypen machen die Suche nach neuen ImpfansĂ€tzen eine globale Notwendigkeit. ExtrazellulĂ€re Membranvesikel sind sezernierte Nanostrukturen, die von ziemlich allen Zellen ausgeschieden werden und ihre Virulenzfaktoren und immunogenen Komponenten beherbergen. In dieser Arbeit wurden mehrere Forschungsziele verfolgt: (i) Isolierung und Charakterisierung von Pneumokokken-Vesikeln. (ii) BiokompatibilitĂ€t und Aufnahme gegenĂŒber Zelllinien und primĂ€ren menschlichen Zellen. (iii) Erhöhung der Ausbeute. (iv) Immunstimulation von Immunzellen durch Pneumokokken-Vesikel. (v) Formulierung von sprĂŒhgetrockneten Impfstoff-Mikropartikeln fĂŒr die Immunisierung der Lunge. Die isolierten Vesikel zeigten eine ausgezeichnete BiokompatibilitĂ€t mit verschiedenen Zelllinien und PrimĂ€rzellen. Pneumokokken-Vesikel zeigten eine schnelle Aufnahme in Immunzellen und stimulierten die Freisetzung von proinflammatorischen Zytokinen. SprĂŒhgetrocknete Impfstoff-Mikropartikel wurden mit verbesserter StabilitĂ€t und erhöhter Zytokinfreisetzung fĂŒr die pulmonale Verabreichung formuliert. Unsere Ergebnisse bestĂ€tigen das große Potenzial von Pneumokokken-Membranvesikeln als Impfstoffkandidaten und bilden eine wichtige Grundlage fĂŒr die weitere Umsetzung und das Scale-up fĂŒr die pulmonale Verabreichung und Immunisierung.PhD fellowship (German-Egyptian Research Long-term Scholarship) from the German Academic Exchange Service (DAAD) and Egyptian Ministry of Higher Education. NanoMatFutur grant from the Federal Ministry of Education and Research

    Protein-protein interactions of the cold shock protein CspE of salmonella typhimurium

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    Despite their name, a number of the cold shock proteins are expressed during normal growth, and not just during cold shock, in several species. The function of these constitutively expressed CspA paralogues is unclear. In Salmonella Typhimurium (a major worldwide cause of gastrointestinal disease) they have been linked to various stress responses and the establishment of virulence. Study of the cold shock proteins as gene regulators is therefore of great interest, and they also have potential as targets for antimicrobial development. CspE in Salmonella Typhimurium is constitutively expressed during normal growth. In order to determine its function, attempts were made to identify the interactions it forms with other cellular proteins. Initially, a proteomic investigation attempted to identify proteins which complex with CspE by in vivo cross-linking and affinity purification followed by mass spectrometry. Although no defined complex was consistently identified, the results suggested a handful of proteins which might interact with CspE in a weak or transient manner. These proteins included many from the nucleoid and ribosomal entry site, hinting at CspE’s cellular localisation. In order to investigate these transient interactions, a bacterial two-hybrid system was employed. Interactions between CspE and HupA, a nucleoid protein identified in the proteomic analysis, were probed, as were interactions between CspE and CsdA, an RNA helicase thought to function co-operatively with CspE. The twohybrid system also allowed investigation of CspE dimerisation, which has been reported in vitro but not investigated in vivo until this study. CspE appears not to interact significantly with either HupA, CsdA, or itself at 37oC. Finally in a further attempt to identify interactions of CspE, a genomic library was created to test CspE interactions by two-hybrid assay with random peptides derived from the whole Salmonella genome. The library was successfully created and screened for evidence of interaction, and revealed an association between CspE and a transcriptional repressor, DeoT. DeoT is a repressor of several genes for catabolic processes, suggesting a role for CspE in the regulation of central metabolism. The findings of this work present a number of novel discoveries and several interesting opportunities for further studies

    Type 2 Diabetes and Bone: The Interactions Between Glucose and Bone-forming Osteoblasts

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    Bone is an active tissue that undergoes constant remodelling. Bone-forming osteoblasts use various energy sources to meet their energy demand, and one of the main energy sources of the cells is glucose. Glucose is transferred into the cell via passive transportation through the protein family of glucose transporters. In disorders of glucose metabolism, such as type 2 diabetes, bone metabolism is disturbed, and patients with type 2 diabetes have an increased risk of fragility fractures. Diabetes is characterized by elevated blood glucose levels, hyperglycaemia, and long-term hyperglycaemia impairs the functions of osteoblasts. The mechanisms responsible for these changes are complicated and not yet fully understood. In this thesis, multiple glucose transporters were shown to be expressed and have unique functions in rat osteoblasts and their precursors, mesenchymal stromal cells, in vitro. Further, short- and long-term exposures to hyperglycaemia were shown to have different responses in the osteoblast transcriptome. Long-term hyperglycaemia decreased the proliferation of osteoblasts, whereas short-term exposure to hyperglycaemia increased the expression of genes related to osteoblast differentiation and function. In addition, a new recombinant antibody-based immunoassay was developed to measure osteoblast-specific, non-carboxylated form of osteocalcin in human blood samples. Negative associations between type 2 diabetes, blood glucose levels and bone formation were demonstrated with this assay. In summary, osteoblasts rely on several glucose transporters to ensure sufficient energy, and long-term exposure to high glucose decreases the functions of osteoblasts. A proper balance in glucose metabolism is necessary for proper bone formation.Tyypin 2 diabetes ja luu – glukoosin ja luuta muodostavien osteoblastien vĂ€liset vuorovaikutukset Luu on aktiivinen kudos, joka kĂ€y lĂ€pi jatkuvaa uudismuodostusta. Luuta muodostavat osteoblastit kĂ€yttĂ€vĂ€t luunmuodostuksessa eri energianlĂ€hteitĂ€ ja yksi pÀÀenergianlĂ€hteistĂ€ on glukoosi. Glukoosi siirtyy soluun passiivisesti glukooosinkuljettajaproteiinien kautta. Glukoosiaineenvaihdunnan sairauksissa, kuten tyypin 2 diabeteksessa, luun metabolia on hĂ€iriintynyt ja diabeetikoilla on lisÀÀntynyt luunmurtumisriski. Diabeteksessa veren glukoosipitoisuus on kohonnut ja pitkĂ€aikaisen kohonneen verensokerin eli hyperglykemian onkin todettu heikentĂ€vĂ€n osteoblastien toimintaa. NĂ€iden muutosten taustalla olevat mekanismit ovat monimutkaisia, eikĂ€ niitĂ€ vielĂ€ tĂ€ysin tunneta. TĂ€ssĂ€ vĂ€itöskirjassa osoitettiin rotan osteoblastien ja niiden esiasteiden, mesenkymaalisten stroomasolujen, kĂ€yttĂ€vĂ€n useita glukoosinkuljettajaproteiineja turvaamaan glukoosinsaantinsa ja kullakin kuljettajaproteiinilla olevan oma merkityksensĂ€ solun toiminnassa. LisĂ€ksi osoitettiin saman osteoblastisolumallin avulla ja RNA-sekvensointia hyödyntĂ€mĂ€llĂ€, kuinka lyhyt- ja pitkĂ€aikaisella hyperglykemialla on erilainen vaikutus osteoblastien geeni-ilmentymiseen. PitkĂ€aikainen altistus heikensi osteoblastien kasvua, kun taas lyhytaikainen altistus puolestaan lisĂ€si osteoblastien erilaistumiseen ja toimintaan liittyvien geenien ilmentymistĂ€. LisĂ€ksi vĂ€itöskirjassa kehitettiin uusi vasta-aineisiin perustuva mÀÀritysmenetelmĂ€ erÀÀn osteoblastiperĂ€isen proteiinin, ei-karboksyloidun osteokalsiinin, mittaamiseksi ihmisten verinĂ€ytteistĂ€. MÀÀrityksen avulla todettiin tyypin 2 diabeteksella sekĂ€ veren glukoosipitoisuudella olevan negatiivinen yhteys luun muodostuksen kanssa. Yhteenvetona voidaan todeta glukoosin olevan osteoblastien toiminnalle elintĂ€rkeÀÀ, mutta pitkĂ€aikainen, liian korkea glukoosipitoisuus kasvuelinympĂ€ristössĂ€ heikentÀÀ osteoblastien toimintaa. Glukoosinaineenvaihdunnan tasapaino onkin vĂ€lttĂ€mĂ€töntĂ€ normaalin luun muodostumisen turvaamiseksi

    The Histoplasma Capsulatum DDR48 Gene is Required for Survival Within Macrophages and Resistance to Oxidative Stress and Antifungal Drugs

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    Histoplasma capsulatum(Hc)is a systemic, dimorphic fungal pathogen that affects upwards of 500,000 individuals in the United States annually. Hc grows as a multicellular mold at environmental temperatures; whereas, upon inhalation into a human or other mammalian host, it transforms into a unicellular, pathogenic yeast. The research presented in this dissertation is focused on characterizing the DNA damage-responsive gene HcDDR48. HcDDR48was originally isolated via a subtractive DNA library enriched for transcripts enriched in the mold-phase of Hcgrowth. Upon further analysis we found that HcDDR48is not just expressed in the mold morphotype, but both growth programs dependent upon the environment. Since the yeast phase of Hcis the phase that interacts with the host, the research in this dissertation focused solely on HcDDR48’s involvement in yeast-phase Hc. We found that HcDDR48is involved in oxidative stress response, antifungal drug response, and survival within resting and activated macrophages. Growth of ddr48Dyeasts was severely decreased when exposed to the reactive oxygen species generator paraquat, as compared to wildtype controls. We also found that ddr48Dyeasts were 2-times more sensitive to the antifungal drugs amphotericin b and ketoconazole. To testHcDDR48’s involvement in vivo, we infected resting and activated RAW 264.7 murine macrophages with Hcyeasts and measured yeast survival 24-hours post-infection. We observed a significant decrease in yeast recovery in the ddr48Dstrain compared to wildtype Hclevels. Herein, we demonstrate the importance of maintaining a functional copy of HcDDR48in order for Hcyeasts to sense and response to numerous environmental and host-associated stressors

    Investigating the molecular basis of cold temperature and high pressure adapted growth in photobacterium profundum SS9

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    Photobacterium profundum SS9 is a γ-proteobacterium which grows optimally at 15°C and 28 MPa (a psychrophilic piezophile) and can grow over a range of temperatures (2-20oC) and pressures (0.1-90 MPa). Previous research had demonstrated that P. profundum SS9 adapts its membrane proteins and phospholipids in response to growth conditions. In this study, methodology was developed for growing P. profundum SS9 under cold temperatures and high pressures in both liquid and solid cultures. The effect of changing growth conditions on cell envelope polysaccharides was then investigated. The lipopolysaccharide (LPS) profile of a rifampicin resistant P. profundum SS9 derivative, SS9R, was shown to change at 0.1 MPa with respect to temperature and at 15°C with respect to pressure. Compositional analysis showed that the LPS was almost entirely composed of glucose. This provides evidence that, under these conditions, the major polysaccharide produced by P. profundum SS9 is a glucan. Two putative polysaccharide mutants, FL26 & FL9, were previously isolated from a screen for cold-sensitive mutants of P. profundum SS9R. Both mutants displayed an increased sensitivity to cold temperatures on solid medium and were unaffected in their growth at high pressure. FL26 was found to exhibit an LPS alteration similar to previously published O-antigen ligase mutants, providing evidence that this mutant is likely to lack O-antigen ligase. Interestingly, FL26 was also shown to have a reduced ability to form biofilms and had increased swimming motility. This suggests that there are a number of changes which occur in FL26 in the absence of O-antigen. FL9 was found to have an altered LPS and capsular polysaccharide (CPS), similar to an E. coli wzc mutant. In E. coli, Wzc is involved in the polymerisation and transport of CPS, disruption of which can also lead to LPS alterations. The LPS and CPS alterations may lead to the cold-sensitivity phenotype, either individually or in combination. In conclusion, alterations in the cell envelope polysaccharides were shown to affect cold temperature sensitivity on solid agar. Cold-sensitivity is most likely directly related to the LPS alterations and stability of the membrane under cold temperatures. Exopolysaccharides (EPS) have previously been shown to affect desiccation and freezethaw resistance, making it is possible that the CPS plays a similar role in this case

    Functionalization of Nanoparticles with Tyrosine Hydroxylase : Biotechnological and Therapeutic Implications

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    Tyrosine hydroxylase (TH) er et viktig enzym for nervesystemet, fordi det katalyserer det fĂžrste steget i syntesen av dopamin, noradrenalin og adrenalin. NivĂ„et pĂ„ TH og dopamin synker hos Parkinsons pasienter pga. den gradvise celledĂžden i den hjernedelen som heter substantia nigra. Vanlig behandling gĂ„r ut pĂ„ Ă„ ta levodopa som er forlĂžperen til dopamin, men effekten avtar etter hvert, og pasientene fĂ„r alvorlige bivirkninger ved hĂžye doser. Det trengs altsĂ„ et bedre behandlingstilbud. En mulighet kan vĂŠre Ă„ tilfĂžre mer TH vha. en enzymerstatningsterapi som ogsĂ„ vil gjenopprette dopaminnivĂ„et. HovedformĂ„let med denne avhandlingen er Ă„ finne ut hvordan TH kan bli brukt som biologisk medisin. Vi har derfor utviklet forskjellige nanopartikkel-baserte formuleringer som kan stabilisere og levere TH, og evaluert det terapeutiske potensiale til TH-lastede nanopartikler. Vi begynte med Ă„ produsere TH sammen med forskjellige fusjonspartnere og fikk et stabilt enzym som vi mĂ„lte strukturen av. SĂ„ valgte vi porĂžst silisium og maltodekstrin nanopartikler som mulige bĂŠrere av TH. De fĂžrste forsĂžkene med porĂžst silisium viste at det er en sammenheng mellom fotoluminescensen og frigjĂžringen av et modellprotein som kan vĂŠre nyttig i sporing av legemiddelleveringen i kroppen. Videre fant vi at TH kunne bindes i nanopartikler av porĂžst silisium, men at dette fĂžrte til aggregering av TH. Maltodekstrin-nanopartikler kunne derimot absorbere store mengder TH samtidig som de forhindret eller forsinket TH aggregeringen. Vi observerte at disse nanopartiklene kunne levere TH til nerveceller og hjernevev og dermed Ăžkte den intracellulĂŠre TH aktiviteten. Alt i alt har denne avhandlingen gitt et godt innblikk i de strukturelle mekanismene og de funksjonelle forutsetningene som trengs for Ă„ kunne lage vellykkede nanopartikkel-baserte formuleringer av TH. TH-lastede nanopartikler har muligheten til Ă„ bli videreutviklet til enzymerstatningsterapi for sykdommer hvor det er for lite aktivt TH, som f.eks. ved Parkinsons sykdom.Tyrosine hydroxylase (TH) is important for neuronal function as it is the rate-limiting enzyme in the synthesis of dopamine, noradrenaline, and adrenaline. In Parkinson’s disease, the levels of TH and dopamine decrease, due to progressive loss of the dopaminergic neurons in a part of the midbrain called substantia nigra. Treatment is typically with the dopamine precursor, levodopa, but its pharmacological effect wears off, and the patients develop serious side effects, so there is a need for better treatment options. One alternative could be to replace the lacking TH with an enzyme replacement therapy and thereby restore the dopamine levels. The main goal of this thesis has been to investigate how TH can be pharmacologically developed into a potential biological drug. We have therefore studied different nanoparticle (NP)-based formulations to stabilize and deliver TH and evaluated the therapeutic potential of TH-loaded NPs. We started out by using fusion tags in the preparation of TH to obtain a stable enzyme of which we determined the full-length solution structure. Then we selected porous silicon and maltodextrin NPs as potential carriers of TH. Initial characterization revealed that the photoluminescent properties of porous silicon can be tuned to correlate with the release of a model protein, which can be useful in tracking of drug delivery. Furthermore, we found that TH loading in porous silicon NPs occurred through electrostatic interactions, but that it also induced TH aggregation. On the other hand, maltodextrin NPs absorbed large amounts of TH while preventing or delaying its aggregation. We observed functional delivery of TH by these NPs to neuronal cells and tissue, which significantly increased the intracellular TH activity. All in all, this thesis has given insights into the structural mechanisms and functional prerequisites necessary for successful formulations of TH with NPs, which shows the therapeutic potential of enzyme replacement therapy with TH-loaded NPs.Doktorgradsavhandlin

    Effects of heat stress or ketosis on metabolism and inflammatory biomarkers in ruminants and monogastrics

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    There are several constraints to of the sustainability of the livestock industries in different areas of the world. The two most obvious hurdles to high dairy production are diseases during the transition period and heat stress (HS). Despite decades of research, the actual pathologies of ketosis and HS remain poorly understood. In the US, it was estimated that the economic losses from ketosis is 360/cow/cycle,andtoHSwas 360/cow/cycle, and to HS was ~ 900 million per year. Thus, both severely jeopardize the competitiveness of animal agriculture. Regardless of the herd size, HS and ketosis affect every dairy region in the country. The biological investigations of ketosis and HS have been studied for more than 50 years, but the negative impacts of both are as severe today as they were 30 years ago. In the current dissertation, ketosis in dairy cows and HS in pigs and steers were investigated to better understand the biology and etiology of both disorders. Study 1 (Chapter 2) was conducted to characterize biomarkers of inflammation during the transition period in healthy and clinically diagnosed ketotic cows. The results indicated that circulating NEFA and BHBA were increased and milk yield was decreased in ketotic relative to healthy cows. In addition, pre-calving circulating LPS was increased twofold in cows that were diagnosed with ketosis post-calving compared to healthy cows, but no LPS differences were detected post-partum. Post-calving LPB, SAA and Hp were increased when compared with their healthy counterparts. Our data suggest the development of ketosis may be intimately linked to inflammation and our selection criteria suggest that intestinal permeability may be the origin of maladaptation to lactation. In Study 2 (Chapter 3) we investigated the effects of a mineral supplement (zinc amino acid complex) on temporal biomarkers of intestinal integrity and intestinal morphology in heat-stressed steers. As expected, HS increased thermal indices and decreased feed intake. However, steers supplemented with zinc amino acid complex had decreased rectal temperature, improved biomarkers of leaky gut (haptoglobin, and LBP), altered intestinal morphology (decreasing duodenum villi width, increasing jejunum villi height and jejunum and ileum villi height:crypt depth), and improvement in some of the blood gas variables relative to steers supplemented with zinc sulfate. Altogether, the findings of Study 2 demonstrated that a Zn-amino acid complex may mediate some of the negative effects of HS in a growing ruminant model. Study 3 (Chapter 4) investigated the temporal pattern of metabolic variables and biomarkers associated with intestinal barrier dysfunction during recovery from HS in pigs. Similar to Study 2, HS increased thermal indices and decreased feed intake. Circulating glucose decreased during HS and remained low for 3 d following HS. The insulin:feed intake tended to be increased during HS, and LBP increased linearly during HS recovery. In addition, HS decreased villous height in both jejunum and ileum but intestinal morphology mostly returned to normal following 3 days of recovery. The results of this study confirmed the negative effects of HS on thermal indices, inflammatory biomarkers, and intestinal morphology; however, it suggests that intestinal integrity was restored fairly quickly, but the acute phase protein response increased with time following HS exposure. In conclusion, the results of the aforementioned studies indicate a negative association of impaired gut integrity with the incidence of ketosis in transitioning dairy cows and performance in heat-stressed steers and pigs. A better understanding of the relative contribution of the intestinal barrier dysfunction to ketosis and heat-induced effects on metabolism and gut morphology is a prerequisite for designing targeted strategies to alleviate the negative consequences of ketosis and HS on farm animals’ productivity
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