Histophilus somni virulence factors include expression and antigenic variation of lipooligosaccharide (LOS). Phosphorylcholine (ChoP) is often expressed on H. somni LOS and also undergoes antigenic variation. In this study, five genes that play a role in expression and antigenic variation of ChoP, lic1ABCD and glpQ, were identified in the genome sequence of H. somni through sequence homology with Haemophilus influenzae genes. The open reading frame (ORF) of lic1A contained a variable number of tandem repeats of the tetranucleotide unit 5'-AACC-3'. Slipped strand mispairing in the repeat region during replication leads to shifting the downstream reading frame in and out of frame with the start codon, thus controlling phase variation of lic1A expression. Removal of the repeats from lic1A, cloning the gene in E. coli, and performing a functional assay on the product indicated that lic1A encodes a choline kinase and that the repeats were not required for expression of a functional gene product. Variation in the number of repeats in lic1A correlated with the antigenic variation of ChoP expression in strain 124P, but not in strain 738. This result supported previous findings that antigenic variation of ChoP expression in strain 738 is controlled through extension/truncation of the LOS outer core. Therefore, these results indicated that the lic1ABCD and glpQ genes control expression and antigenic variation of ChoP on the LOS of H. somni and that there are two possible mechanisms for ChoP antigenic variation. The role of H. somni expression of ChoP in colonization of the host respiratory tract was also examined. Experimental infection in the natural host showed that the population of H. somni that expresses ChoP was enriched in the bacteria that colonized the respiratory tract. In addition, bacteria expressing ChoP were able to aggregate bovine platelets through binding to the platelet activating factor receptor (PAF-R), which is also present on epithelial and endothelial cells. These results indicated that ChoP may play a role in the process of colonization and subsequent systemic invasion of host tissues, which may occur through binding of ChoP to PAF-R. Bacteria that did not express ChoP were more prevalent in systemic sites, indicating that ChoP expression may be disadvantageous for the organism during systemic dissemination. / Ph. D.

Ovine (strains 9L and 3384Y) and bovine (strains 649, 2336 and 8025) isolates of Histophilus somni were investigated for their ability to acquire iron from haemoglobin (Hb). Bovine isolates were capable of utilizing bovine, but not ovine, porcine or human Hb as a source of iron. Ovine isolates could not obtain iron from Hb. Bovine isolates bound bovine, ovine, and human Hbs by means of the same iron-repressible receptor(s) and produced a ~120-kDa iron-repressible, outer membrane protein. Using PCR approaches, an iron-regulated operon containing hugX and hugZ homologues and a gene (hgbA) that encodes a TonB-dependent, Hb-binding proteins were identified in strains 649, 9L and 3384Y. In strains 9L and 3384Y, HgbA is truncated offering a possible explanation for their lack of utilization of Hb as an iron source. In strains 2336 and 8025, expression of HgbA was also subject to a form of phase variation.

En tant que constantes de la littérature de tous les peuples et de tous les temps l’alba et le somni (l’aube et le rêve) participent par leur réalisations typiques de l’époque au jeu poétique de la poésie romane qui se concrétise, sur le modèle des schémas déjà préétablis, entre imitation, variation et transgression. Les réalisations possibles, inhérentes aux motifs et thèmes d’alba et de somni (comme p. ex. les adieux des amoureux à l’aube, la souffrance et la maladie amoureuse, le rêve comme compensation ou acte de la création poétique, le moment magique de la transition), sont accentuées et développées par rapport à une aire culturelle donnée, dont témoignent les exemples des troubadours et des trouvères, les cantigas galego-portugais et la poésie italienne du Duecento. Le jeu des variations de ces constantes poétiques se réalise dans un contexte d’un réseau de genres, dans lequel les textes singuliers interagissent et forment un système complexe de références intertextuelles. Cette étude sur les variations de l’alba et du somni romanes se considère comme contribution à la mise en valeur des structures et de la poétique inhérente de la poésie médiévale. Elle montre non seulement comment la perspective comparatiste peut contribuer à la compréhension de la richesse des facettes de la poésie médiévale et des chemins et des formes de la réception et de la fécondation mutuelles, mais aussi quel rôle peut jouer l’examen des dictatz no principals encore négligés. L’étude de ces genres « mineurs » thématiques n’a pas seulement d’intérêt en soi, car ces dictatz indiquent généralement les sujets centraux et les points culminants de la poésie, mais aussi parce qu’elle peut contribuer à l’éclaircissement ou à une meilleure compréhension des modèles déjà connus et étudiés. / Being constants of the literature of all nations and ages, alba and somni (dawn and dream) participate in the poetic play of the Romanic poetry by means of their typical realisations characteristic of a time, that is concretised, on the basis of preformed patterns, between imitation, variation and transgression. The development and accentuation of possible realisations inherent to the motifs and themes of alba and somni (such as e. g. the parting at dawn, suffering and lovesickness, dreaming as compensation or act of poetic creation, the magical moment of transition) differ depending on the cultural area as is indicated by the examples of the troubadours and trouvères, the Galician-Portuguese cantigas and the Italian poetry of the Duecento. The play of variations of these poetical constants is realised in the context of a network of genres, in which the individual texts interact and form a complex system of intertextual references. This study of the Romanic variations of alba and somni is considered as a contribution to an enlightenment of the structures and poetics inherent to medieval poetry. It does not only show how the comparative perspective can contribute to the comprehension of the abundance of facets of medieval poetry and of the ways and forms of mutual reception and fecundation, but moreover point out the role that can be assigned to the study of the dictatz no principals which is still and often neglected. The study of these “minor” genres of thematic kind is not only interesting as such, for the dictatz generally indicate the central subjects and climactic points of poetry, but also because it can contribute to an elucidation or a better comprehension of the patterns already established and studied.

No description available.

Incorporation of N-acetyl neuraminic acid (NANA), or sialic acid, onto lipooligosaccharide (LOS) enhances the virulence of several bacterial species. In the present study, we assessed the effect of sialylation of Histophilus somni LOS on complement-mediated killing, binding of complement factor H (which converts C3b to inactive C3b (iC3b) and inhibit the alternative complement pathway) to the bacteria, complement activation by the LOS, and phagocytosis and killing of the bacteria by bovine polymorphonuclear leukocytes (PMN). Killing of H. somni by alternative complement pathway was measured by incubation of sialylated or non-sialylated H. somni with antibody-free precolostral calf serum (PCS) followed by viable plate count. A complement dose-dependent response to killing of non-sialylated H. somni by PCS was observed. However, sialylated H. somni were significantly (P = 0.001) more resistant to killing at any of the concentrations of PCS used. Sialylated H. somni LOS activated (P = 0.025) and consumed (P = 0.001) less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized sheep red blood cells or rabbit red blood cells, and by western blotting of C3 activation products. Sialylated H. somni bound more factor H than non-sialylated bacteria (determined by enzyme-linked immunosorbent assay) (P = 0.004), supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni inhibited both PMN phagocytosis of 3H-thymidine-labelled bacteria (P = 0.004) and intracellular killing of the bacteria (P = 0.0001), compared to non-sialylated bacteria. Therefore, sialylation of the LOS results in enhanced binding of complement factor H to the bacteria, resulting in diminished complement activation, resistance to complement-mediated lysis, and PMN phagocytosis and killing. / Master of Science

Virulent strains of the bovine opportunistic pathogen Haemophilus somnus (Histophilus somni) cause multi-systemic diseases in cattle. One of the reported virulence factors that H. somnus may use to persist in the host is resistance to intracellular killing. It is reported in this dissertation that H. somnus significantly (P <0.001) inhibited production of superoxide anion (O2-) by bovine mammary and alveolar macrophages as well as by polymorphonuclear leukocytes. Inhibition of O2- production was time- and dose-dependent and did not occur after incubation with Escherichia coli, H. influenzae, or Brucella abortus. Non-viable H. somnus, purified lipooligosaccharide (LOS), or cell-free supernatant from mid-log phase cultures did not inhibit O2- production, indicating that O2- inhibition required contact with live H. somnus. Commensal isolates of H. somnus were less capable or incapable of inhibiting macrophage O2- production compared to isolates tested from disease sites. H. somnus shares conserved epitopes in its LOS with Neisseria gonorrhoeae, N. meningitidis, and H. influenzae, and can also undergo structural phase variation of these LOS epitopes. Sialylation of the terminal galactose of H. somnus LOS is another reported virulence mechanism. Current sequencing of the genomes of H. somnus strains 2336 (pathogenic) and 129Pt (commensal) has enabled in silico identification of three open reading frames (ORFs) involved in sialylation. The ORFs-1 (hsst-I) and -2 (hsst-II) had BLASTx homology to sialyltransferases, while ORF-3 (neuAhs) had BLASTx homology to CMP-sialic acid synthetases. These ORFs were amplified by PCR and cloned into the expression vector pCWOri+. Thin layer chromatography of the hsst-I gene product showed this sialyltransferase exhibited preference for sialylation of terminal N-acetyllactosamine (LacNAc, beta-Gal-[1,4]-beta-GlcNAc-R). However, Hsst-II preferentially sialylated lacto-N-biose (LNB, beta-Gal-[1,3]-beta-GlcNAc-R). In this study, phase variation of the terminal linkage in isolate 738 from a 3 linked galactose (LNB) to a 4 linked galactose (LacNac) was demonstrated. Such variation of a glycose linkage appears to be a novel mechanism of LOS phase variation. Furthermore, the ability of sialylated strain 738 LOS vs de-sialylated strain 738 LOS to induce Toll-like receptor 4 signaling was decreased by 28%, as determined by ELISA for Macrophage Inflammatory Protein-2. Therefore, sialylated LOS may aid H. somnus to avoid host innate immunity. / Ph. D.

H. somni is capable of forming a prominent biofilm, and luxS is known to play an important role in biofilm formation through quorum sensing, but has also been postulated to function in gene regulation. In order to further study the function of H. somni LuxS, mutants 2336::TnluxS and 2336::TnuspE were identified from a bank of mutants generated with EZ-Tn5 <KAN-2>Tnp transposome (EpiCentre). The 2336::TnluxS and 2336::TnuspE mutants were highly attenuated in mice, but only 2336::TnuspE was deficient in biofilm formation. However, the electrophoretic profiles of the LOS and serum sensitivity of both mutants were substantially altered compared to the parent strain, but exopolysaccharide production during biofilm formation also only decreased in 2336::TnuspE. The altered phenotypes were partially restored in complemented recombinant clones obtained using shuttle vector pHS649S. To clarify whether luxS regulates the expression of various virulence genes, mRNA from both the parent strain and 2336::TnluxS was sequenced. It was determined that the transcription level of 53 genes in 2336::TnluxS and 42 genes in 2336::TnuspE in planktonic form were changed. In biofilm, 320 genes in 2336::TnluxS and 230 genes in 2336::TnuspE were differentially regulated compared to biofilm formed by strain 2336. The immunogloblin binding protein A (IbpA) of H. somni is known to be cytotoxic to phagocytic cells. In this study, we found that strains with a mutation in ibpA were less capable of early replication in monocytes. The IbpA protein concentrated from the culture supernatant of strain 2336 facilitated the intracellular survival of strain 129Pt, which lacks IbpA. However, the ability of several ibpA mutants to resist intracellular killing was not significantly impaired by 48 h post-infection. Two transposon mutants 2336::TnluxS and 2336::TnuspE replicated in monocytes in a similar manner as the ibpA mutants. Confocal microscopy revealed that the intracellular-replicable strains (2336, 64Vc, 2336::TnluxS, 2336::TnuspE and the ibpA mutants) prevented the acidification of the bacterial-containing phagosome and the expression of lysosome marker LAMP-2, which may facilitate survival of H. somni in monocytes. An enzyme-linked immunosorbent assay was developed to detect bovine antibodies to the H. somni exopolysaccharide that is formed during biofilm formation. When an index value of 0.268 was used the sensitivity of the assay for experimentally- and naturally-infected calves was 90.5% at 3 weeks post-infection, and the specificity of the assay for healthy calves was 92.5%. The EPS ELISA may aid in identifying calves with diseases due to H. somni. / Ph. D.

The scope and application of high throughput techniques has expanded from studying a single genome, transcriptome or proteome to understanding complex environments at a greater resolution with the help of novel computational frameworks. Comprehensive structural annotation i.e. description of all functional elements in the genome, is required for measuring genome response accurately, using high throughput methods. Annotation of genome sequences using high throughput data from RNA-seq and proteomics experiments complement computational methods for identifying functional elements and can help validate existing in silico annotation, correct annotation errors, and could potentially identify novel functional elements. Re-annotation studies in recent times have revealed shortcomings of automated methods and the necessity to validate existing annotations using experimental data. This dissertation elucidates re-annotation of Mannheimia haemolytica, Pasteurella multocida and Histophilus somni, bacterial pathogens associated with bovine respiratory disease in cattle. Experimental re-annotation of these bacterial genomes using RNA-seq and proteomics enabled the validation of existing annotation and discovery of novel functional elements that can be utilized in future functional genomics studies. We also addressed the need for developing an automated bioinformatics workflow that is broadly applicable for bacterial genome re-annotation, by developing open source Perl pipeline that can use RNA-seq and proteomics data as input. Simultaneous analysis of host and pathogen gene expression profiling using metatranscriptomics approaches is necessary to improve our understanding of infectious diseases. Traditional methods for analysis of RNA-seq data do not address the impact of cross-mapping of reads to multiple genomes for data originating from a metatranscriptomic study. Analysis of sequence conservation between species can help determine a metric for cross mapping to correct for signal vs. noise. We generated artificial RNA-seq data and evaluated the impact of read length and sequence conservation on cross-mapping. Comparative genomics was used to identify a core and pan-genome for quantifying gene expression. Our results show that cross mapping between genomes can directly be related to evolutionary distance between these genomes and that an increase in RNA-seq read length tends to negate cross mapping.

Esta tesis doctoral surge a partir del hecho de que, aunque los estudios hispánicos son muy conocidos hoy en Grecia, la literatura catalana medieval no resulta particularmente reconocible, lo cual sorprende si tenemos en cuenta, por un lado, la presencia de los catalanes en Grecia y las relaciones culturales en el siglo XIV, y por otro, el alto nivel de la producción literaria en aquella lengua. Aquí presentamos traducida al griego la considerada principal obra medieval en catalán, Lo somni (1399) de Bernat Metge, diálogo que atrae con particularidad al entorno helenístico por tratarse de un importante representante de la tradición del Humanismo mediterráneo y por suponer la introducción del movimiento humanista en la península Ibérica. Acompañan a esta traducción, en primer lugar, un estudio histórico-literario y una investigación detallada de las fuentes, y a continuación, unas notas aclaratorias y unos apéndices para facilitar su uso.

L'efecte hipnòtic en el cinema postmodern desenvolupa una aproximació al discurs postmodern a través de la categoria estètica d'allò sinistre (das unheimliche), com el camí que senyala l'ombra i el fantasma, el desplaçament del subjecte i la representació de l'inconscient, el real del desig, per mitjà de la metàfora de la hipnosi aplicada al cinema.Hypnotic effect in postmodern cinema develops an approach to postmoden theories throught the sinister aesthetic category (das unheimliche), understood as the way that shows the shadow and the ghost, the subject displacement and the unconsciousness representation, the real of the desire, by the metaphor of hypnosis applied to cinema.