Thesis (Ph. D.)--John Hopkins University, 1906. / Biography. Bibliography: p. [5].

Thesis (Ph. D.)--John Hopkins University, 1906. / Biography. Bibliography: p. [5].

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 102-113). Also available on the World Wide Web.

La peste est une maladie mortelle qui survient le plus souvent après piqûre de puce et, plus rarement après l’inhalation d’aérosol contaminé. Elle est causée par la bactérie Yersinia pestis. Cette dernière a récemment émergé de Yersinia pseudotuberculosis, une bactérie responsable le plus souvent d’une maladie bénigne du tube digestif. Y. pestis et Y. pseutoduberculosis produisent des protéines homologues à Ivy (inhibitor of vertebrate lysozyme) et MliC (membrane bound lysozyme inhibitor of C-type lysozyme) d’Escherichia coli. Il avait été suggéré qu’Ivy et MliC jouent un rôle dans la virulence bactérienne. Mais, aucune preuve expérimentale n’étayait cette hypothèse avant notre travail. De plus, des études suggéraient que le rôle physiologique d’Ivy serait de contrôler les autolysines bactériennes plutôt que de protéger la bactérie contre le lysozyme rencontré au cours d’une infection. Nous avons montré que Y. pestis (mais pas Y. pseudotuberculosis) nécessite Ivy pour résister au lysozyme et aux polynucléaires neutrophiles lors de l’infection. Par contre, Y. pestis n’a pas besoin de MliC pour produire la peste. Nos conclusions sont fondées sur des données provenant (1) de la quantification de la survie bactérienne au contact au contact du lysozyme, du sérum et de phagocytes ; (2) de la mesure de virulence (en utilisant différent modèles de peste, plusieurs souches et espèce de rongeurs et, des souris neutropéniques ou KO pour la production de lysozyme) ; (3) de l’évaluation de la réponse inflammatoire chez la souris. Notre travail a démontré l’importance des inhibiteurs de lysozyme dans la virulence bactérienne et suggère que l’acquisition verticale d’ivy par Y. pestis a joué un rôle important dans l’émergence de la peste. / Yersinia pestis (the agent of flea-borne plague) harbors genes encoding putative homologues of the respectively periplasmic and membrane-bound vertebrate lysozyme inhibitors Ivy and MliC. Both inhibitors are thought to control autolytic activity rather than protect bacteria against lysozyme molecules encountered during host infection. Here, we show that MliC was not required for lysozyme resistance and the development of plague. In contrast, Y. pestis required Ivy for lysozyme resistance when grown at 37°C but not at 21°C (the optimal temperature for flea-borne transmission). Deletion of ivy did not affect Y. pestis’ ability to grow in human serum or resist human macrophages but it did decrease resistance to human polymorphonuclear neutrophils. Y. pestis lacking Ivy had attenuated virulence in rodent models of bubonic and pneumonic plague but was fully virulent in lysozyme M-deficient or GR1+ cell-depleted mice. Our results demonstrate the importance of vertebrate lysozyme inhibitors in bacterial pathogenesis and support a scenario in which Y. pestis deposited in the dermis produces Ivy to inhibit the lysozyme released by polymorphonuclear neutrophils. Our results also suggest that once Y. pestis is in the flea gut, it resists lysozyme via an Ivy-independent mechanism. Lastly, Yersinia pseudotuberculosis (Y. pestis’ recent ancestor, which causes self-limited bowel disease in humans) did not require Ivy for lysozyme resistance or virulence. Thus, our study also shows that a gene which is not necessary for the virulence of an ancestral bacterium may become essential in the emergence of a new pathogen.

Thesis (M.S.)--University of Wisconsin--Madison, 1949. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [105]-108).

Thesis (M.S.)--University of Wisconsin--Madison, 1963. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [313]-319).

Developing foliar growth of ivy geraniums (Pelargonium peltatum) bleaches white after exposure to temperatures greater than 30°C. This study investigated chloroplast development in ivy geraniums under heat stress comparing a heat sensitive cultivar, Temprano™ Lavender, and a heat tolerant cultivar, Contessa™ Red. Using transmission electron microscopy and spectrophotometry, foliar bleaching under heat stress was found to be due to an absence of developed chloroplasts within the bleached new growth accompanied by lower chlorophyll content. To determine whether heat stress related foliar bleaching could be prevented, cytokinin and gibberellins were applied in combination, at different rates before, during or after a heat stress event. Applying 50 to 100 ppm gibberellins before heat stress reduced bleaching in new growth. Gibberellins applied at 50 ppm within a week of a heat stress event decreased bleaching. Net photosynthesis and chlorophyll fluorescence was greater in non-heat stressed plants than heat stressed plants.

The development of whitening of the youngest leaves of actively growing ivy geranium (Pelargonium peltatum L.) has been observed as the season changes from late spring to summer. This study was conducted to determine the specific environmental causes of whitening, if micronutrients deficiencies cause similar whitening, whether low night temperatures can reverse whitening, and whether salicylic acid affects growth and whitening in ivy geraniums. Two cultivars, ‘Beach’ and ‘Butterfly’, with different susceptibility to whitening were chosen for this study. Elevated air temperature, but not elevated root-zone temperature, was found to be the environmental cause of whitening in ivy geranium. Elevated air temperatures severely reduced plant growth, leaf area, fresh weight, and dry weight in both cultivars. Elevated air temperature reduced photosynthetic pigments and their ratios in ivy geranium. Carotenoids and pheophytins decreased in ‘Butterfly’ at elevated air temperature. Foliar total Fe levels indicated no inhibition of Fe-uptake at elevated temperatures. Applications of Fe-chelate at elevated temperatures helped chlorophyll synthesis in ivy geraniums. Deficiency treatments of all micronutrients, Fe, Mn, Zn, S or Mg did not result in whitening in either cultivar of ivy geraniums. Salicylic acid did not affect whitening of ivy geraniums. It did not affect growth, leaf area, fresh or dry (stem, leaf or total) weight, fresh: dry weight ratio, leaf area ratio, specific leaf area or foliar nutrient (Fe, Mn, Zn, Mg and S) content in either cultivar. Cultivars varied in their response to low night temperature. ‘Beach’ reduced its plant growth and fresh to dry weight ratio whereas ‘Butterfly’ did not. Fe-chelate application did not reduce growth, leaf area, fresh weight, dry weight or fresh:dry weight ratio of either cultivar. Although Fe-application did not reduce whitening in ivy geraniums, it helped to preserve chlorophyll, particularly chlorophyll b as indicated by Chl a:b ratio in ‘Beach’. Whitening in ivy geranium is a heat stress response initially exhibited by young, developing leaves and is caused by elevated air temperatures. Whitening is the result of impaired photosynthetic pigments synthesis and/or degradation.

Poison ivy (Toxicodendron radicans (L.) Kuntze) is a native perennial liana widely recognized for the production of urushiol, and the associated contact dermatitis it causes in humans. Poison ivy is predicted to become both more prevalent and more noxious in response to projected patterns of global change. Moreover, poison ivy is an important food source for avian species, and urushiol has numerous applications as a high-value engineering material. Thus, this curious weed has many avenues for future concern, and promise. Here, I address gaps in knowledge about poison ivy ecology and physiology so that we may better understand its weediness and utilize its benefits. I address three core areas: poison ivy establishment patterns; biotic interactions with multiple taxa; and the development of molecular tools for use in poison ivy. I found that the early life stage of seedling emergence is a critical linchpin in poison ivy establishment due largely to herbivore pressure from large grazers. I also describe the multifaceted relationship between poison ivy and avian frugivores that not only disperse the drupes of poison ivy but also aid in reduction of fungal endophytic phytopathogens. A survey of poison ivy urushiols yielded that while variation in urushiol congeners was high across individuals, relative congener levels were stable within individuals over a two month period. Lastly I demonstrate best practices for introducing and transiently expressing recombinant DNA in poison ivy as a step towards future reverse genetic procedures. / Doctor of Philosophy / Poison ivy is a native plant best known for its capacity to cause allergenic skin reactions in humans due to the chemical urushiol, which is found in all parts of the plant. While most people prefer to avoid this plant, poison ivy is an important food source for birds. In addition, urushiol has numerous applications as an engineered material. Despite these positive aspects, poison ivy is among those plants that are responding well to global change, such as increasing CO₂ levels and habitat fragmentation. Poison ivy has been shown to increase in size and produce more allergenic forms of urushiol under elevated CO₂ levels and there are concerns that poison ivy prefers the disturbed areas created by habitat fragmentation. These attributes suggest that poison ivy will become more prevalent and more noxious in the coming years. Thus, this curious weed has many avenues for both future concern and promise. To aid in our ability to manage poison ivy in the future, I used a combination of field, greenhouse, and laboratory studies to study the ecology of poison ivy. I investigated the early stages of the poison ivy life cycle, and the relationship between poison ivy and the animals that interact with it. I found that the earliest life stages of poison ivy are a critical linchpin for poison ivy survival due largely to large animals like deer eating the seedlings. I also describe the multifaceted relationship between poison ivy and birds, which not only disperse the seeds of poison ivy but also aid in reducing pathogens associated with the seeds. I surveyed the amounts and types of urushiols that poison ivy produces and found them to be highly variable from plant to plant, but relatively stable over time within a plant. Lastly, I demonstrate best practices for transient transgene expression in poison ivy leaves as a step towards future genetic studies. These studies help expand our understanding of a problematic weed, and pave the way for future studies in weed ecology and in the utilization of urushiol in positive applications, showing that even poison ivy can be of benefit to the environment and humans.

Background: Increased numbers of intestinal E. coli are observed in inflammatory bowel disease, but the reasons for this proliferation and it exact role in intestinal inflammation are unknown. Aim of this PhD-project was to identify E. coli proteins involved in E. coli’s adaptation to the inflammatory conditions in the gut and to investigate whether these factors affect the host. Furthermore, the molecular basis for strain-specific differences between probiotic and harmful E. coli in their response to intestinal inflammation was investigated. Methods: Using mice monoassociated either with the adherent-invasive E. coli (AIEC) strain UNC or the probiotic E. coli Nissle, two different mouse models of intestinal inflammation were analysed: On the one hand, severe inflammation was induced by treating mice with 3.5% dextran sodium sulphate (DSS). On the other hand, a very mild intestinal inflammation was generated by associating interleukin 10-deficient (IL-10-/-) mice with E. coli. Differentially expressed proteins in the E. coli strains collected from caecal contents of these mice were identified by two-dimensional fluorescence difference gel electrophoresis. Results DSS-experiment: All DSS-treated mice revealed signs of a moderate caecal and a severe colonic inflammation. However, mice monoassociated with E. coli Nissle were less affected. In both E. coli strains, acute inflammation led to a downregulation of pathways involved in carbohydrate breakdown and energy generation. Accordingly, DSS-treated mice had lower caecal concentrations of bacterial fermentation products than the control mice. Differentially expressed proteins also included the Fe-S cluster repair protein NfuA, the tryptophanase TnaA, and the uncharacterised protein YggE. NfuA was upregulated nearly 3-fold in both E. coli strains after DSS administration. Reactive oxygen species produced during intestinal inflammation damage Fe-S clusters and thereby lead to an inactivation of Fe-S proteins. In vitro data indicated that the repair of Fe-S proteins by NfuA is a central mechanism in E. coli to survive oxidative stress. Expression of YggE, which has been reported to reduce the intracellular level of reactive oxygen species, was 4- to 8-fold higher in E. coli Nissle than in E. coli UNC under control and inflammatory conditions. In vitro growth experiments confirmed these results, indicating that E. coli Nissle is better equipped to cope with oxidative stress than E. coli UNC. Additionally, E. coli Nissle isolated from DSS-treated and control mice had TnaA levels 4- to 7-fold higher than E. coli UNC. In turn, caecal indole concentrations resulting from cleavage of tryptophan by TnaA were higher in E. coli Nissle- associated control mice than in the respective mice associated with E. coli UNC. Because of its anti-inflammatory effect, indole is hypothesised to be involved in the extension of the remission phase in ulcerative colitis described for E. coli Nissle. Results IL-10-/--experiment: Only IL-10-/- mice monoassociated with E. coli UNC for 8 weeks exhibited signs of a very mild caecal inflammation. In agreement with this weak inflammation, the variations in the bacterial proteome were small. Similar to the DSS-experiment, proteins downregulated by inflammation belong mainly to the central energy metabolism. In contrast to the DSS-experiment, no upregulation of chaperone proteins and NfuA were observed, indicating that these are strategies to overcome adverse effects of strong intestinal inflammation. The inhibitor of vertebrate C-type lysozyme, Ivy, was 2- to 3-fold upregulated on mRNA and protein level in E. coli Nissle in comparison to E. coli UNC isolated from IL-10-/- mice. By overexpressing ivy, it was demonstrated in vitro that Ivy contributes to a higher lysozyme resistance observed for E. coli Nissle, supporting the role of Ivy as a potential fitness factor in this E. coli strain. Conclusions: The results of this PhD-study demonstrate that intestinal bacteria sense even minimal changes in the health status of the host. While some bacterial adaptations to the inflammatory conditions are equal in response to strong and mild intestinal inflammation, other reactions are unique to a specific disease state. In addition, probiotic and colitogenic E. coli differ in their response to the intestinal inflammation and thereby may influence the host in different ways. / Hintergrund: Chronisch entzündliche Darmerkrankungen zeichnen sich unter anderem durch eine starke Proliferation intestinaler E. coli aus. Unbekannt ist jedoch, ob diese Vermehrung eine Ursache oder eine Folge der Erkrankung darstellt. Ziel der vorliegenden Doktorarbeit war es daher, E. coli-Proteine zu identifizieren, welche der Anpassung an die entzündlichen Bedingungen im Darmtrakt dienen und unter Umständen einen Effekt auf den Gesundheitszustand des Wirtes haben. Weiterhin sollten die molekularen Ursachen für stammesspezifische Unterschiede zwischen probiotischen und gesundheitsschädlichen E. coli näher untersucht werden. Methoden: In den tierexperimentellen Analysen wurden keimfreie Mäuse entweder mit dem probiotischen E. coli Nissle oder dem adhärent-invasiven E. coli UNC monoassoziiert und in zwei verschiedenen Entzündungsmodellen näher untersucht. Einerseits wurde eine starke Darmentzündung durch die Gabe von 3,5% Natrium-Dextransulfat (DSS) ausgelöst. Andererseits wurde in Interleukin 10-defizienten (IL-10-/-) Mäusen eine sehr milde Form der Entzündung durch Besiedlung mit E. coli induziert. Die E. coli Bakterien wurden am Ende der Versuche aus den Caecuminhalten der Mäuse isoliert und die bakterielle Proteinexpression wurde mittels zwei-dimensionaler Gelelektrophorese analysiert. Ergebnisse des DSS-Versuchs: Alle Tiere des DSS-Versuchs entwickelten unabhängig vom E. coli Stamm, mit dem sie besiedelt waren, eine moderate Entzündung im Caecum und eine starke im Colon, wobei die Entzündungsreaktion durch die Monoassoziation mit E. coli Nissle leicht abgeschwächt wurde. In beiden E. coli Stämmen führte die Darmentzündung zu einer verringerten Expression von Enzymen des Kohlenhydratabbaus und der Energiegewinnung. In Folge dessen waren die intestinalen Konzentrationen bakterieller Fermentationsprodukte in den entzündeten Tieren geringer als in den gesunden Kontrolltieren. Weitere differentiell exprimierte Proteine umfassen das Fe-S- Cluster Reparaturprotein NfuA, die Tryptophanase TnaA und das uncharakterisierte Protein YggE. In beiden E. coli Stämmen, welche aus den DSS-Tieren isoliert wurden, war das NfuA Protein dreifach höher exprimiert. Eine Darmentzündung führt zu einer vermehrten Bildung reaktiver Sauerstoffspezies, welche die Fe-S-Cluster in Eisen-Schwefel-Proteinen zerstören und damit zu einer Inaktivierung dieser Proteine führen. In vitro Untersuchungen bestätigten, dass die Reparatur der Eisen-Schwefel-Proteine durch NfuA ein wichtiger Mechanismus ist um oxidativem Stress entgegenzuwirken. Das YggE Protein, welches laut Literaturangaben einen hemmenden Einfluss auf die Bildung reaktiver Sauerstoffspezies hat, war in E. coli Nissle 4- bis 8-fach erhöht (verglichen mit E. coli UNC unter Kontroll- und Entzündungsbedingungen). In vitro Versuche bestätigten diese Daten und zeigten, dass E. coli Nissle im Vergleich zu E. coli UNC eine erhöhte Resistenz gegenüber oxidativem Stress aufweist. Außerdem wurde im Vergleich E. coli Nissle vs. E. coli UNC (unter Entzündungs- und Kontrollbedingungen) ein 4- bis 7-fach erhöhter TnaA-Gehalt nachgewiesen. Indol, das Produkt der TnaA-katalysierten Tryptophanspaltung wurde in erhöhten Mengen im Intestinaltrakt E. coli Nissle-assoziierter Kontrolltiere detektiert. Seit längerem werden entzündungshemmende Eigenschaften für Indol postuliert, die aufgrund der Ergebnisse dieser Doktorarbeit nun auch mit den gesundheitsfördenden Eigenschaften von E. coli Nissle in Zusammenhang gebracht werden können. Ergebnisse des IL-10-/-- Versuchs: Nach einer 8-wöchigen Assoziationsdauer wurde nur in den mit E. coli UNC besiedelten IL-10-/- Tieren eine schwache Entzündungsreaktion nachgewiesen. Bedingt durch diese sehr schwach ausgeprägte Entzündungsantwort waren auch die Veränderungen im bakteriellen Proteom von E. coli UNC nur gering. Wie im DSS-Versuch waren Proteine des bakteriellen Energiestoffwechsels reprimiert, allerdings wurde keine Induktion von NfuA beobachtet. Daher scheint die Induktion von NfuA nur der Anpassung an eine starke Entzündung zu dienen. Weiterhin wurde nachgewiesen, dass E. coli Nissle aus IL-10-/- Tieren den Hemmer für das vertebrate C-Typ Lysozym (Ivy) sowohl auf mRNA- als auch auf Proteinebene stärker exprimiert als E. coli UNC. Überexpression von Ivy unter in vitro Bedingungen zeigte, dass es an der erhöhten Lysozymresistenz von E. coli Nissle beteiligt ist und somit eine Rolle als möglicher Fitnessfaktor von E. coli Nissle spielt. Schlussfolgerungen: In dieser Doktorarbeit wurde gezeigt, dass Darmentzündungen die Proteinexpression eines im Darm lebenden Bakteriums beeinflussen. Einige der aufgedeckten bakteriellen Anpassungsreaktionen werden sowohl bei einer starken als auch bei einer schwachen Entzündung ausgelöst; andere wiederum sind spezifisch für nur einen dieser Entzündungszustände. Weiterhin wurde deutlich, dass sich E. coli-Stämme hinsichtlich ihrer Reaktion auf eine Darmentzündung unterscheiden und damit möglicherweise den Wirt beeinflussen.