Pattern recognition receptors, including members of the NBD and LLR-containing (NLR) family, are key sensors of infection and injury. Early sensing of pathogen invasion and subsequent activation of pro-inflammatory signaling cascades is essential for controlling infection. However, signaling pathways activated upon pathogen recognition can also contribute to inflammation-mediated tissue damage. The studies detailed in Chapters 3 and 4 are primarily concerned with the roles of two NLR family members, Nlrp12 and Nlrc4, during influenza A virus (IAV) infection. While IAV itself is cytopathic, the immune response is responsible for a great deal of the tissue damage during infection in some contexts. NLR family members are involved in both pathogen sensing and modulation of pro-inflammatory signaling, thus they are perfectly situated to shift the balance between pathogen clearance and immunopathology. Nlrp12 has been implicated in regulation of pro-inflammatory signaling through NFκB family members. In Chapter 3 we report that during IAV infection, we find no differences in those pathways, and instead we describe a novel role for Nlrp12 in regulating transcript stability. Previous work has shown that one of the key differences between lethal and sublethal IAV infections is the early and exaggerated recruitment of neutrophils. Previous studies in our laboratory had established a role for Nlrp12 in CXCL1-mediated neutrophil recruitment during respiratory bacterial infections. We therefore hypothesized that Nlrp12-/- mice would be protected from pathogenic neutrophil recruitment during lethal IAV infection due to decreased CXCL1 production. In Chapter 3 we show that indeed, Nlrp12-/- mice have improved survival, decreased pulmonary microvascular permeability, and decreased necrosis and hemorrhage in their airways compared to WT mice. Nlrp12-/- mice also have fewer neutrophils in their lungs, due to decreased production of CXCL1 by neutrophils, DCs and macrophages. Our data showing decreased Cxcl1 transcript stability in R848-treated Nlrp12-/- BMDCs strongly suggest that the reduction in CXCL1 production by DCs in the Nlrp12-/- lungs is a result of decreased Cxcl1 transcript stability. Nlrc4 is a best known as a member of the Nlrc4 inflammasome, which is activated upon sensing of Gram-negative bacterial pathogens. However, a recent study from our laboratory showed an inflammasome-independent role for Nlrc4 in supporting critical anti-tumor T cell responses. Given that T cells are also critical for successful resolution of IAV infection, we hypothesized that during IAV infection, Nlrc4-/- mice would have compromised IAV-specific T cell responses and therefore poorer survival. Indeed, our studies in Chapter 4 show that in IAV-infected Nlrc4-/- mice, the pulmonary IAV-specific CD4 T cell response is significantly diminished and mortality is significantly increased compared to WT mice. During IAV infection, the blunted CD4 T cell response is a result of increased death of the CD4 T cells, perhaps due to increased expression of FasL on CD11c+ cells in the Nlrc4-/- lung environment.

Leishmania species are vector-borne protozoan parasites that cause a spectrum of human diseases, with an estimated 12 million people infected in 88 countries. Inflammation plays distinct roles in the different clinical syndromes. Visceral leishmaniasis, in which parasites migrate from the site of infection and proliferate in liver and spleen, is accompanied by systemic immune suppression. Cutaneous leishmaniasis, where parasites remain at the site of inoculation and create a long-term ulcer, is associated with vigorous systemic immunity to the parasite. The innate immune sensing pathways responding to Leishmania spp. parasites are not fully described. NLR proteins are a class of structurally related cytosolic proteins. The most well described NLRs form inflammasome complexes that generate strong inflammatory responses to “danger” signals. Other NLRs do not form inflammasomes and have anti-inflammatory functions. While NLR proteins are known to be important in the immune response to many pathogens, the roles NLR proteins in leishmaniasis have only begun to be investigated. We hypothesized that NLR proteins affect the pathogenesis of leishmaniasis through their ability to modulate inflammatory responses. We hypothesized that inflammasome activation in cutaneous leishmaniasis would be detrimental, leading to greater disease pathology, and that the potential anti-inflammatory functions of the non-inflammasome NLRs, NLRP6, NLRP10, and NLRP12, would be protective, reducing tissue damage. In contrast, we hypothesized that in visceral leishmaniasis greater inflammation due to activation of the inflammasome would be protective and control parasite replication, while the anti-inflammatory NLRs would be permissive to parasite replication in the liver and spleen by contributing to the immunosuppressive strategy of the parasite. We used knockout mouse strains lacking the inflammasome adaptor protein ASC, and several non-inflammasome forming NLRs, to investigate NLR proteins in murine models of visceral or cutaneous leishmaniasis. Our data showed that NLR proteins have important functions in visceral leishmaniasis, where they are essential for appropriate parasite homing and replication in the liver and spleen. In cutaneous leishmaniasis, we found that NLRP10 is essential for controlling inflammation in the skin, limiting lesion development and tissue damage at the site of infection. Taken together our findings show important functions for NLR proteins in leishmaniasis, influencing localized tissue specific inflammation, the adaptive immune responses, and clearance or long term residence of the parasite in the infected organs. This research underscores the importance of localized inflammation at the infection site to the pathogenesis and the course of leishmaniasis.

Phytophthora root and stem rot is the most destructive soybean soil-borne disease worldwide and can be managed using soybean cultivars with genes conferring resistance to <i>Phytophthora sojae</i>. Here we show that soybean <i>Rps11</i> is an ~27-kb nucleotide-binding site leucine-rich repeat (NLR) gene that confers broad-spectrum resistance to the pathogen. This giant gene is located in a genomic region containing 12 unusually large NLR genes of a single origin and was formed by rounds of intergenic/intragenic unequal recombination that involves the promoter regions and the LRR regions. Comparison of the genomic region in the Rps11 donor line with its corresponding regions in 29 diverse soybean genomes revealed drastic regional diversification including NLR copy number variation ranging from 5 to 23, and absence of allelic copy of <i>Rps11</i> in all 29 genomes. This study highlights innovative evolution and complexity of an NLR cluster and enables precise selection of <i>Rps11</i> for cultivar improvement.

Active Galactic Nuclei (AGN) are axisymmetric systems to first order; their observed properties are likely strong functions of inclination with respect to our line of sight. However, except for a few special cases, the specific inclinations of individual AGN are unknown. We have developed a promising technique for determining the inclinations of nearby AGN by mapping the kinematics of their narrow-line regions (NLRs), which are easily resolved with Hubble Space Telescope (HST) [O III] imaging and long-slit spectra from the Space Telescope Imaging Spectrograph (STIS). Our studies indicate that NLR kinematics dominated by radial outflow can be fit with simple biconical outflow models that can be used to determine the inclination of the bicone axis, and hence the obscuring torus, with respect to our line of sight. We present NLR analysis of 52 Seyfert galaxies and resultant inclinations from models of 17 individual AGN with clear signatures of biconical outflow. From these AGN, we can for the first time assess the effect of inclination on other observable properties in radio-quiet AGN, including the discovery of a distinct correlation between AGN inclination and X-ray column density.

No description available.

Les maladies des plantes causées par les champignons sont un problème majeur en agriculture. Pour les contrôler, les gènes de résistance (R) qui permettent de développer des variétés de plantes résistantes sont des éléments clés. La majorité des gènes R codent pour des protéines NLRs caractérisées par la présence d'un domaine de liaison aux nucléotides (NB-ARC) et un domaine de répétitions riches en leucines (LRR). Ces protéines agissent comme des récepteurs immunitaires intracellulaires et reconnaissent des facteurs de virulence des agents pathogènes appelés effecteurs. Les champignons phytopathogènes possèdent de vastes répertoires d'effecteurs qui contiennent centaines de protéines sécrétés, de petites tailles et sans similarités de séquence entre elles.La première question abordée dans ma thèse concerne l’origine de l'immense diversité des effecteurs fongiques. Une analyse structurale a identifié une famille d’effecteurs de séquences différentes mais qui possèdent une structure conservée. Cette famille a été appelée MAX-effectors (Magnaporthe Avrs and ToxB like) et elle est particulièrement importante chez Magnaporthe oryzae, l'agent causal de la pyriculariose du riz. Par des analyses d'expression, j'ai confirmé que la majorité des effecteurs MAX de M. oryzae sont spécifiquement exprimés durant la phase précoce de l'infection, suggérant une fonction importante durant la colonisation de la plante. Les effecteurs MAX constituent la première famille d'effecteurs fongiques définis par leur structure. Cette étude apporte donc de nouvelles pistes pour l'identification d'effecteurs chez les champignons et contribue à une meilleure compréhension de l'évolution des effecteurs. En effet, le scénario observé chez les effecteurs MAX suggère que beaucoup d’effecteurs fongiques appartiennent à un nombre restreint de familles d'effecteurs définies par leur structure. La seconde question que j’ai abordée durant ma thèse est le mécanisme moléculaire de la reconnaissance des effecteurs par les NLRs. J'ai abordé cette question en étudiant la reconnaissance de l'effecteur AVR-Pia par le couple de NLRs RGA4/RGA5. Des travaux précédents ont montré que RGA5 agit comme récepteur et se lie directement à AVR-Pia tandis que RGA4 agit comme élément de signalisation constitutivement actif, qui, en absence de l’agent pathogène, est réprimé par RGA5. Un domaine de RGA5, normalement absent chez les protéines NLR et similaire à la chaperonne du cuivre ATX1 (domaine RATX1), interagit physiquement avec AVR-Pia. Il a été suggéré que ce domaine RATX1 puisse agir comme un leurre de la cible de virulence d’AVR-Pia. Ce leurre, intégré dans la structure de RGA5, permettrait de « piéger » l’effecteur par interaction directe et jouerait donc un rôle crucial dans sa reconnaissance spécifique. Grâce à une analyse structurale détaillée d’AVR-Pia j’ai pu confirmer le rôle central de l'interaction AVR-Pia-RATX1 dans la reconnaissance de cet effecteur ce qui conforte le modèle du « leurre intégré ». De plus, j’ai caractérisé la surface d'interaction avec laquelle AVR-Pia lie le domaine RATX1. De plus, j'ai détecté des interactions entre AVR-Pia et d'autres parties de RGA5, indépendantes du domaine RATX1, notamment les domaines NB-ARC et LRR. Ceci a permis de développer un modèle qui explique comment la liaison d’un effecteur à un récepteur NLR comportant un leurre intégré par différentes interactions indépendantes conduit à une reconnaissance très sensible et spécifique qui est peu affectée par des mutations ponctuelles de l’effecteur. En résumé, cette étude a produit des connaissances nouvelles sur la fonction des récepteurs des plantes de type NLRs et sur leur capacité à reconnaitre des effecteurs. Ceci contribue à une meilleure compréhension du système immunitaire des plantes, ce qui est un élément important pour l’obtention de cultures durablement résistantes aux maladies / Plant diseases caused by fungi constitute a worldwide threat to food security and disease resistance (R) genes that allow to breed resistant crops are key elements for efficient disease control. The vast majority of R genes code for NLR multi domain proteins characterized by nucleotide-binding and leucine-rich repeat domains and acting as intracellular immune receptors for pathogen-secreted virulence factors termed effectors. Phytopathogenic fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The first question I addressed in my PhD thesis is: how is the tremendous diversity of fungal effectors generated? A structural analysis had identified the family of sequence-unrelated but structurally conserved MAX-effectors (Magnaporthe Avrs and ToxB like) that has expanded specifically in Magnaporthe oryzae the causal agent of rice blast disease. By expression analysis, I confirmed that the majority of M. oryzae MAX-effectors are expressed specifically during early infection suggesting important functions during host colonization. MAX effectors are the first structurally defined family of effectors in fungi and this study gives therefore news clues for the identification of candidate effectors in fungi and constitutes a crucial step towards a better understanding of effector evolution. In fact, the scenario observed for MAX-effectors leads to the hypothesis that the enormous number of sequence-unrelated fungal effectors belong in fact to a restricted set of structurally conserved effector families.The second question I investigated in my PhD thesis is: what are the molecular mechanisms of effector recognition by NLR immune receptors? I addressed this question by studying recognition of the M. oryzae effector AVR-Pia by the rice NLR pair RGA4/RGA5. Previous work has shown that RGA5 acts as a receptor that binds directly to AVR-Pia while RGA4 acts as a constitutively active signaling protein that is, in the absence of pathogen, repressed by RGA5. This functional interaction involves formation of an RGA4/RGA5 receptor complex. By protein-protein interaction studies, I showed that complex formation involves interactions between the RA4 and RGA5 NB-ARC and LRR domains, in addition to previously identified interactions between the coiled-coil domains. AVR-Pia recognition seems not to induce dissociation of the RGA4/RGA5 complex but a ternary RGA4/RGA5/AVR-Pia complex could also not be detected consistently. How effector recognition is translated into receptor complex activation remains therefore to be elucidated in more detail in the future. Previous work has shown that a domain of RGA5 normally not present in NLRs and related to the copper chaperone ATX1 (RATX1 domain) interacts physically with AVR-Pia and may be crucial for effector recognition. The RATX1 domain was hypothesized to mimic the true host targets of AVR-Pia leading to the development of the ‘integrated decoy’ model that states that unconventional domains in NLRs act as decoys in the recognition of effector proteins. By detailed structure-informed analysis of AVR-Pia, I could confirm the pivotal role of the AVR-Pia-RATX1 interaction for effector recognition lending important support to the integrated decoy model. In addition, I could precisely characterize the interaction surface with which AVR-Pia binds to the RGA5 RATX1 domain. Finally, I detected interactions of AVR-Pia with other parts of RGA5, in particular the NB-ARC and the LRR domains. Based on these results, I developed a model that explains how such binding to several independent sites in NLRs leads to high overall affinity and robust effector recognition that is resilient to effector mutations. Taken together, this study provides important novel insight into NLR function and effector recognition and contributes by this to a better understanding of plant immunity which is crucial for generating durable disease resistance in crops.

La maladie de la pourriture noire des Brassicacées est causée par la colonisation du système vasculaire des plantes par Xanthomonas campestris pv. campestris (Xcc). Cette bactérie, largement retrouvée sur les cinq continents, peut infecter de nombreuses espèces d'intérêts agronomiques et la plante modèle Arabidopsis. Malgré l'importance de cette maladie, la génétique et les bases moléculaires de la résistance à Xcc et de la résistance vasculaire en général restent méconnues et ont été le sujet de mes travaux de thèse. Des approches génétiques chez Arabidopsis ont permis d'identifier trois gènes (ZAR1, PBL2, RKS1) requis pour la résistance à Xcc et la reconnaissance de l'effecteur de type III XopAC de Xcc. Ces trois gènes codent respectivement une protéine de résistance canonique (famille des NLR) et deux kinases de la famille des RLCK. En collaboration avec l'équipe de J-M Zhou (Pékin), nous avons élucidé le mécanisme moléculaire de reconnaissance de XopAC: Un complexe de résistance préformé ZAR1-RKS1 reconnaît spécifiquement l'uridylylation de la kinase PBL2 par XopAC et induit la résistance à Xcc. Contrairement aux données existantes, la spécificité de reconnaissance du complexe de résistance n'est pas portée par la NLR mais par la kinase adaptateur RKS1. Ces résultats constituent la première description des mécanismes moléculaires sous tendant la résistance des brassicacées à Xcc et pourront servir de bases pour la mise en place de programmes rationnels d'amélioration variétale chez les Brassicacées cultivés. / Black rot disease of Brassicaceae is caused by the colonization of plant vasculature by Xanthomonas campestris pv. campestris (Xcc). This worldwide-distributed bacterium causes serious losses in brassica crops and also infects Arabidopsis. Despite of the economic importance of this disease, genetics and molecular bases of resistance to Xcc and vascular resistance in general is poorly understood. This topic was thus selected as my thesis project. Genetic approaches in Arabidopsis identified three genes (ZAR1, PBL2, RKS1) required for resistance to Xcc and the recognition of the Xcc type III effector XopAC. These three genes code for a canonical resistance protein of the NLR family and two kinases of the RLCK family, respectively. In collaboration with the group of J-M Zhou (Beijing), we uncovered the molecular mechanism of XopAC recognition: a preformed ZAR1-RKS1 resistance complex specifically recognizes PBL2 uridylylated by XopAC and triggers resistance to Xcc. In contrast to existing knowledge, the recognition specificity of the resistance complex is not conferred by the NLR but by the adaptor kinase RKS1. These results are the first description of the molecular mechanisms underlying Brassicaceae resistance to Xcc and pave the avenue for the rationale breeding of resistance in Brassica crops.

Limit Cycle Oscillation is a type of aircraft wing structural vibration caused by the non-linearity of the system. The objective of this thesis is to provide a numerical study of this aeroelastic behavior. A CFD solver is used to simulate airfoils displaying such an aeroelastic behavior under certain airflow conditions. Two types of airfoils are used for this numerical study, including the NACA64a010 airfoil, and the supercritical NLR 7301 airfoil. The CFD simulation of limit cycle oscillation (LCO) can be obtained by using published flow and structural parameters. Final results from the CFD solver capture LCO, as well as flutter, behaviors for both wings. These CFD results can be obtained by using two different solution schemes, including the Roe and Zha scheme. The pressure coefficient and skin friction coefficient distributions are computed using the CFD results for LCO and flutter simulations of these two airfoils, and they provide a physical understanding of these aeroelastic behaviors.

No description available.

Bakgrund: Användningen av nanopartiklar ökar varje dag men mycket är oklart i vilkenutsträckning nanopartiklar har hälsopåverkan hos människor. I studier har det visats attnanopartiklar under 100 nm kan passera cellmembranet och under 40 nm kan passeranuklearmembran. I dagsläget används guldnanopartiklar (GNP) inom medicin för visualisering, diagnostisering och läkemedelsbärare. En av biverkningar som GNP kan ha är inflammation. Det finns flera studier som försökt redogöra GNP:s effekter samt hur de uppkommer men det är intehelt klarlagt. Vidare är det inte helt tydligt vilka egenskaper hos GNP som står för de olikaeffekterna. Syfte: Syftet med studien är att undersöka rollen som GNP:s storlekar och former har i NLRfamily pyrin domain containing 3 (NLRP3) inflammasomens interleukin-1β-frisättning hoshuman leukemia monocytic cell line (THP1-celler). Metod: Studien är en experimentell studie där THP1-celler differentieras och exponeras för GNPi olika storlekar, former och koncentrationer. LUMIT immunoassay används sedan för att erhållade olika interleukin-1β-koncentrationerna (IL-1β). Resultat: I datan som erhölls efter att experimenten utförts observerades det att GNP 30 nm i 40μg/mL gav störst inflammasominduktion med en IL-1β-koncentration på 645 ±388 pg/mL.Nästhögst IL-1β-koncentration gav GNP 100 nm i 40 μg/mL på 592 ±270 pg/mL. Slutsats: Nakna GNP kan inducera inflammasomaktivering och på så sättIL-1β-koncentrationshöjningar. De “gold urchins” som undersökts visade sig ha svagarepåverkan på cellerna jämfört med de sfäriska GNP.