The signal transduction pathway of halophilic archaea remains a fascinating example of adaptation to extreme environments. Despite similarities with bacterial taxis systems, its structural and dynamics patterns during signal relay remain debatable. The currently investigated SRII/HtrII phototaxis system of Natronomonas pharaonis shows remarkable similarities with chemoreceptors in its membrane and HAMP domains functioning design. By combining site-directed spin labeling (SDSL) with electron paramagnetic resonance (EPR) spectroscopy we investigate the kinase control domain (i.e. signaling domain) of NpSRII/HtrII both in terms of dynamic and structural properties. Our data, as provided by continuous wave and pulse (DEER) EPR techniques, builds on current dynamics based signaling models for HAMP domains (such as the “frozen–dynamic” or two-state equilibrium models). We present an expanded mechanism for signal propagation throughout the signaling domain, where salt and temperature variations trigger subtle shifts in dynamics. Extreme dynamics motional ranges (compact or highly-dynamic) associate with a specific flagellar signaling state, here the kinase-off response, where a more moderate dynamics motion (dynamic) associates with the kinase-on response. Structurally, we reference our data on PML and ND reconstituted NpSRII/HtrII to the EcTsr crystal structure and the NpHtrII homology model. We show that, despite a difference in packing, NpHtrII oligomerizes in a similar manner as EcTsr, even in the absence of stabilizing structures such as the CheA/CheW baseplate. The presence of trimers-of-dimers but also dimers-of-dimers in membrane sheet samples exposes the high affinity with which NpHtrII signaling domains interact. We hope our structural and dynamics details will push further not just drug design but also environmental preservation efforts where taxis systems drive colonization and virulence of pathogens in plants, animals and humans alike.

Sjökaptensstudenter på Sjöfartshögskolan i Kalmar genomgår praktik under sammanlagt tolv månader i form av fartygsförlagd utbildning indelad i tre olika perioder under deras fyraåriga utbildning. Praktikperioderna ombord på fartyg ger studenterna möjligheten att lära sig yrket på ett praktiskt sätt, inte bara från läroboken. Ombord på fartyg används härdplaster i en stor utsträckning och som student på Sveriges två sjöfartshögskolor går man ut på fartygsförlagda praktikperioder med olika grundkunskaper inom härdplaster. Syftet med arbetet var att undersöka hur sjökaptensstudenter förhåller sig till härdplaster genom en kvalitativ metod med semistrukturerade intervjuer där sex avgångsstudenter från Sjökaptensprogrammet i Kalmar deltog. Resultatet visar att alla respondenter hanterade härdplaster under sina praktikperioder på fartyg, i form av tvåkomponentsfärger med olika utsträckningar av förståelse angående hälsorisker och PSU som ska användas för att minska hälsorisker. Studien visade också att faktorer som ålder, erfarenheter samt hur de ombordanställdas attityd kring hantering och användning av härdplaster spelar en stor roll på hur studenter förhåller sig till härdplaster. / Sea captain students at the Kalmar School of Maritime Capital undergo internship for a total of twelve months in the form of onboard education divided into three different periods during their four-year training. Practice periods onboard ships give students the opportunity to learn the profession in a practical way, not just from the textbook. Onboard ships, hardener is used extensively and as a student at Sweden’s two maritime universities goes out on onboard training with different basic knowledge about hardeners. The aim of the work was to examine how sea captain students relate to hardener through a qualitative method of semistructured interviews where six last year students from the sea captain Program in Kalmar participated. The result shows that all respondents handled hardener during their onboard training in the form of two-component paint with different extensions of understanding regarding health risks and PPE to be used to reduce its risks. The study also showed that factors such as age, experience and how the onboard attitude regarding handling and use of hardener play a big role in how students handle hardener by themselves.

Salmonella enterica is an intracellular bacterial pathogen of humans and the causative agent of the acute gastrointestinal disease, salmonellosis, and the chronic systemic infection, typhoid fever. Sensor proteins convert environmental signals, including signals detected within the host environment, into biochemical signals to control cellular responses. It has been previously established that the two component regulatory system SsrA-SsrB, consisting of the integral membrane sensor kinase protein SsrA and the cytoplasmic DNA-binding response regulator SsrB are essential for regulation of bacterial factors during systemic intracellular infection. The first chapter of this thesis describes characterization of the sensor kinase SsrA. The structure of the periplasmic sensor domain is modeled and evidence is presented that it is involved in enhancing signaling activity in response to environmental acidification encountered within the intracellular environment. A mechanism whereby protonation of histidine residues within this region in response to acidification drives conformational strain and thereby signaling is proposed. The second chapter describes identification of the DNA-binding motif of SsrB within regulated promoters as well as its regulon. Integration of experimental data with comparative genomics data resulted in identification of the palindromic heptameric DNA recognition motif of SsrB as well as identification of novel SsrB-regulated promoters. In addition, a DNA microarray analysis is described wherein the complete SsrB regulon is identified. Finally, the third chapter describes regulatory input of SsrB to the S. enterica type VI secretion system. This chapter also describes the contribution of this system to systemic dissemination of S. enterica during host infection. Altogether, these data advance understanding of how Salmonella controls factors essential for disease in response to the host environment during infection. / Thesis / Doctor of Philosophy (PhD)

Phosphate is an essential nutrient for all forms of life. Escherichia coli has a PhoR/PhoB two component regulatory system that controls the expression of various genes whose products allow the cell to thrive in low phosphate environments. The signaling mechanism of the PhoR/PhoB system has been studied and the phosphorylation cascade that controls gene expression is well understood. What is still unknown is how PhoR senses the phosphate level of the environment. The PstS, PstC, PstA, PstB, and PhoU proteins play a role in this signal sensing. This work confirms the hypothesis that the PstSCAB complex senses the environmental phosphate and that phosphate signal is passed through PhoU to PhoR. Further, this work characterizes residues important for interaction on PhoU and PhoR and identifies a structural model for interaction. This model points to a potential mechanism for PhoU mediated signaling to PhoR. We tested this model with direct coupling analysis and obtained further confirmation. Further use of these techniques may elucidate more of the interactions necessary for proper phosphate signaling.

No description available.

No description available.

Les souches d’Escherichia coli pathogènes aviaires (APEC) sont responsables d’infections respiratoires et de septicémies chez la volaille. Le régulon Pho est contrôlé conjointement par le système à deux composantes PhoBR et par le système de transport spécifique du phosphate (Pst). Afin de déterminer l’implication de PhoBR et du système Pst dans la pathogenèse de la souche APEC O78 χ7122, différentes souche mutantes phoBR et pst ont été testées pour divers traits de virulence in vivo et in vitro. Les mutations menant à l’activation constitutive du régulon Pho rendaient les souches plus sensibles au peroxyde d’hydrogène et au sérum de lapin comparativement à la souche sauvage. De plus, l’expression des fimbriae de type 1 était affectée chez ces souches. L’ensemble des mutants Pho-constitutifs étaient aussi significativement moins virulents que la souche sauvage dans un modèle de coinfection de poulet, incluant les souches avec un système Pst fonctionnel. De plus, l’inactivation du régulateur PhoB chez un mutant Pst restaure la virulence. Par ailleurs, l’inactivation de PhoB n’affecte pas la virulence de la souche χ7122 dans notre modèle. De manière intéressante, le degré d’atténuation des souches mutantes corrèle directement avec le niveau d’activation du régulon Pho. Globalement, les résultats indiquent que l’activation du régulon Pho plutôt que le transport du phosphate via le système Pst joue un rôle majeur dans l’atténuation des APEC. / Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections and septicemia in poultry. The Pho regulon is jointly controlled by the two-component regulatory system PhoBR and by the phosphate-specific transport (Pst) system. To determine the involvement of the PhoBR regulon and the Pst system in the pathogenesis of the APEC O78 strain χ7122, different phoBR and pst mutant strains were tested for in vivo and in vitro virulence traits. Mutations resulting in constitutive activation of the Pho regulon rendered strains more sensitive than the wild-type to hydrogen peroxide and to the bactericidal effects of rabbit serum. In addition, production of type 1 fimbriae was also impaired in these strains. Using a chicken competitive infection model, all PhoB constitutive mutants were out-competed by the wild-type parent, including strains containing a functional Pst system. In addition, cumulative inactivation of the Pst system and the PhoB regulator resulted in a restoration of virulence. Loss of the PhoB regulator alone did not affect virulence in the chicken infection model. Interestingly, the level of attenuation of the mutant strains correlated directly with the level of activation of the Pho regulon. Overall, results indicate that activation of the Pho regulon rather than phosphate transport by the Pst system plays a major role in the attenuation of APEC.

Two-component signal systems serve as basic stimulus-response coupling mechanism to allow organisms (predominantly bacteria) to sense and respond to changes in many environmental conditions. The prototypical system consists of two proteins, namely a histidine kinase, containing a sensor domain and catalytic kinase core, and a response regulator protein (RR protein). Extracellular stimuli are sensed by a histidine kinase sensor domain. Then ATP is bound to the catalytic kinase core and the γ-phosphoryl group is transferred to the conserved histidine residue. This phosphoryl group is subsequently transferred to a conserved aspartate residue within the RR protein. Phosphotransfer to the RR protein results in activation of a downstream effector domain that elicits the specific response (usually it is transcription activity, but a few RR proteins function as enzymes). The histidine kinase sensor domain is designed for specific ligand interactions. This master thesis focused on the unique histidine kinase containing a sensor domain with a globine structure, which coordinates a heme molecule, namely globin-coupled histidine kinase from Anaeromyxobacter sp. Fw 109-5 (AfGcHK) and its appropriate RR protein. The aim of this thesis was to study and characterize the phosphorylation activity of AfGcHK and RR...

Nesta dissertação nós revisamos a teoria básica que descreve a junção Josephson bosônica para uma e duas espécies partindo do modelo de Bose-Hubbard. Em seguida explicamos como é possível gerar campos de calibe artificiais em um sistema de átomos neutros, como é o caso do condensado de Bose-Einstein. Finalmente, utilizando os conhecimentos teóricos desenvolvidos anteriormente nós buscamos os estados estacionários de um sistema de pseudospin 1/2 submetido a um campo de calibre não-Abeliano artificial, que torna a dinâmica da junção muito mais complexa e rica. Nós também exploramos um novo desbalanceamento de população que surge no sistema, devido a presença do campo de calibre, com características similares as do Macroscopic Quantum Self-Trapping. / In this dissertation we review the basic theory that describes the bosonic Josephson junction for one and two species using the Bose-Hubbard model. Afterwards, we explain how it is possible to generate artificial gauge fields for neutral atoms, like a Bose-Einstein condensate. Finally, using this theoretical background we search for stationary states of a pseudospin 1/2 system subject to a non-Abelian artificial gauge field which turns the dynamic of the junction much more complex and rich. We also explore a possible new populational imbalance that appears on the system due to the presence of the gauge field, with similar features as the Macroscopic Quantum Self-Trapping.

Streptococcus pneumoniae est la cause principale de pneumonies, otites, méningites et septicémies. La transformation génétique naturelle constitue l’élément clé de son adaptation aux changements environnementaux. Elle s’effectue par intégration d’ADN d’origine externe dans le chromosome de la bactérie, et a lieu pendant un état physiologique particulier appelé compétence.Mon travail de thèse a consisté à étudier les acteurs principaux de la régulation de la compétence (ComD, ComE) et les protéines impliquées dans la prise en charge, le traitement de l’ADN transformant et la recombinaison (DprA, RecA). J’ai notamment résolu la structure du facteur de transcription ComE par cristallographie aux rayons X, et réalisé une étude fonctionnelle de sa fixation sur un de ses promoteurs. Les résultats obtenus ont permis de proposer un mécanisme selon lequel la dimérisation induite par la phosphorylation de ComE, couplée à sa fixation sur la séquence promotrice d’ADN, provoquerait une courbure de l’ADN. Cette courbure permettrait la fixation de l’ARN polymérase, activant ainsi la transcription des gènes nécessaires à la mise en place de la compétence. / Streptococcus pneumoniae is the leading cause of community-acquired infections worldwide. The natural genetic transformation is the key to its adaptation to environmental changes. It takes place with the integration in its chromosome of exogenous DNA, during a physiological state called competence.During my thesis I have focused on the main actors of competence regulation (ComD, ComE) and on proteins involved in exogenous DNA processing and recombination (DprA, RecA). In particular, I have solved the structure of the transcriptional activator ComE by X-ray crystallography, and carried out a functional study of its binding to its promoter. The results obtained allowed us to propose a mechanism regarding the transcriptional activation by ComE of the genes necessary for the set up of the competence : the phosphorylation-induced dimerization, coupled to the binding of ComE to its DNA promoter, would curve the DNA and allow the binding of the RNA polymerase.