Fibroblast Growth Factor 19 (FGF19 chez l’humain ; FGF15 chez la souris) est un régulateur central du métabolisme hépatique. Cette molécule a un impact important au niveau de la différentiation neurologique et de l’oreille interne au stade foetal. À l’âge adulte, le patron d’expression est restreint au système gastro-intestinal. Contrairement aux autres membres de la superfamille des FGFs, FGF19/15 agit de manière endocrine car il n’est pas retenu par la matrice extracellulaire et peut rejoindre la circulation sanguine. L’expression de FGF19/15 est induite par les acides biliaires au niveau de l’intestin grêle, plus précisément l’iléon. Les acides biliaires lient le récepteur nucléaire Farnesoid-XReceptor (FXR) qui peut ensuite s’hétérodimériser avec Retinoid-X-Receptor (RXR) pour se lier au promoteur de FGF19/15, ce qui enclenche son expression. Une fois dans le sang, l’hormone rejoint le foie et son action est médiée par le complexe de récepteur Fibroblast Growth Factor Receptor 4 (FGFR4) et β-Klotho (BKL). Une fois les récepteurs activés, FGF19/15 module la glycémie en inhibant la néoglucogenèse hépatique et en activant la synthèse du glycogène, le flux protéique en activant eIF4B et la lipémie en inhibant les enzymes clefs de la lipogénèse. FGF19/15 joue aussi un rôle majeur au niveau du métabolisme biliaire. Ce dernier permet de réduire la production d’acide biliaire en inhibant la Cholesterol 7-α oxygenase (CYP7A1). Les travaux présentés dans ce mémoire portent dans un premier temps sur la caractérisation des conséquences amenées par l’infection sur l’axe endocrine FGF15/FGFR4. Un premier manuscrit traite de l’expression des différents gênes clefs du système et de leur perte lors d’une infection à Salmonella typhimurium, l’agent pathogène causant la fièvre typhoïde chez la souris, et des conséquences sur l’homéostasie biliaire. Il est possible de remarquer une perte de l’expression de FGF15 au niveau de l’intestin et de FGFR4 et β-Klotho au niveau du foie, en plus de plusieurs transporteurs responsables d’amener différents composants clefs de la bile à la vésicule biliaire ou à la circulation sanguine. Le deuxième volet du travail consistait à déterminer le mécanisme derrière la perte du complexe de récepteurs FGFR4/β-Klotho. Les résultats préliminaires démontrent que la perte de β-Klotho semble être médiée seulement par le processus inflammatoire normal et la perte de FGFR4 semble être Salmonella dépendante, par le biais de la voie c- Jun N-terminal kinases (JNK) et le facteur de transcription Hepatic Nuclear Factor 1 alpha (HNF1α)

No description available.

No description available.

Depuis quelques années, il y a émergence de souches de Salmonella enterica sérovar Typhimurium multirésistantes causant une septicémie et la mort chez le porc. Ceci constitue un problème majeur pour l’industrie porcine et possiblement pour la santé publique. L’objectif de ce projet était de comparer et de caractériser une souche capable de causer une septicémie chez le porc et une souche commensale, en observant l’interaction avec des cellules épithéliales, des macrophages humains et d’identifier des gènes exprimés par les souches septicémiques et les souches commensales. Tout d’abord, l’infection de cellules épithéliales permet d’observer l’adhérence et l’invasion des bactéries, pour ainsi mettre en évidence la capacité des souches à coloniser le tractus gastro-intestinal. La souche commensale possède un pouvoir d’adhésion supérieur à la souche septicémique. Par la suite, l’infection de macrophages permet de caractériser le niveau de phagocytose et de survie. L’importance de la survie dans les macrophages pourrait permettre de faire un lien avec la septicémie. Toutefois, aucune différence n’est observable dans les conditions qui ont été testé. Ensuite, la technique SCOTS (Selective Capture of Transcribed Sequences) est utilisée pour capturer des gènes uniques à la souche septicémique et un autre SCOTS est fait pour capturer les gènes spécifiques à la souche commensale. Finalement, les gènes sont clonés, leur spécificité face aux souches est analysé par dot blot et ils sont identifiés par séquençage suivient d’une analyses bioinformatiques. Les gènes identifiés par SCOTS, lors des captures pour la souche septicémique et la souche commensale, se trouvent à être des gènes communs aux Salmonella. Toutefois, la différence de pathologie causée par les deux souches, n’est peut-être pas l’acquisition de nouveaux gènes, mais plutôt une différence d’expression entre les deux souches. / For many years, there has been an emergence of new multi-drug resistant Salmonella enterica serovar Typhimurium strains responsible for high mortality rates in regards to swine infections. This represents a major problem for the swine industry and also for public health. The objective of this project was to characterize an isolate capable of creating swine septicemic illness and another which is naturally present in this host’s intestinal tract. The study conducted here includes observations of bacteria-epithelial cell interactions, bacteria-macrophage interactions and identification of genes expressed during different epithelial cell infections. Bacterial adherence and invasion of cells were observed after epithelial cell infection. This type of assay underlines the ability of the isolates to colonize the intestinal tract. The commensal strain has a better adherence then the other. Macrophage infections allowed characterization of bacterial phagocytosis and intracellular survival. Survival within macrophages reflects the capacity of the bacterial isolates to infect and survive in the host. There’s no difference between the two strains with the condition we test. A technique termed SCOTS (Selective Capture of Transcribed Sequences) was used to capture unique transcripts from the isolate which caused illness during swine infection. SCOTS was also used to find unique genes expressed by the commensal isolate. The captured fragments were cloned, their strain specificity was analyzed by dot blot and the genes transcribed were identified by direct sequencing followed by bioinformatic analysis. SCOTS carried out with the septicemic and the commensal isolates was done. During the SCOTS conducted with the strain isolated from a sick swine or with the isolate from healthy swine, common Salmonella genes were found. Thus, even though these two isolates cause different pathologies, virulence of the strains does not rely on the acquisition of new genes, but perhaps on differential gene expression among both.

Schlagwörter: Salmonella , Salmonella enterica , Salmonella typhimurium , Salmonellose , Escherichia coli , Shigella , Infektion , Bakterielle Infektion , Zellkultur , HeLa-Zelle , Apoptosis , Metabolismus , Stoffwechsel , Glucose , Glucosetransport , Glucosestoffwechsel , Katabolismus , Kohlenstoff , Kohlenstoffbedarf , Kohlenstoffhaushalt , Kohlenstoffstoffwechsel , Kohlenstoff-13 , Kohlenstoffisotop Salmonella Typhimurium und enteroinvasive E. coli (EIEC) sind fakultativ intrazelluläre Bakterien aus der Familie der Enterobacteriaceae. Während erstere sich nach der Internalisierung durch eukaryotische Zellen normalerweise in einem spezialisierten Phagosom, der Salmonella-enthaltenden Vakuole (SCV), vermehren, replizieren EIEC im Zytoplasma der Wirtszellen. In der vorliegenden Arbeit wurde zunächst durch Mikroinjektion die Fähigkeit von S. Typhimurium 14028s untersucht, ebenfalls im Zytoplasma von Caco-2-Zellen replizieren zu können. Dabei wurde festgestellt, daß ein früher als S. Typhimurium 14028s WT bezeichneter Stamm eine Insertion eines Desoxythymidins an Position 76 des offenen Leserasters von rfbP trägt, einem Gen, dessen Protein an der LPS-Synthese beteiligt ist. Weiterhin synthetisierte dieser Stamm ein rauhes LPS. Aufgrund von Agglutination konnte der Rauh-Stamm nur mit geringem Erfolg mikroinjiziert werden. Hingegen lag 5 h nach der Mikroinjektion einer nicht invasiven Mutante von Salmonella mit vollständigem LPS der Anteil an Caco-2-Zellen, die mehr als 32 Bakterien enthielten, bei etwa 30 %. Der Anteil war 2-3 mal höher als bei früheren Mikroinjektionen in HeLa-Zellen. Daher wurde das Verhalten von HeLa-Zellen nach einer Infektion durch S. Typhimurium ΔsifA - einer Mutante, die aus der SCV ins Zytoplasma entkommt - untersucht. Dabei wurde festgestellt, daß die sifA-Mutante 10 h nach der Infektion die Aktivität der Caspasen 9 und 3 in HeLa-Zellen, aber nicht in Caco-2-Zellen induziert. In weiteren Versuchen wurde die Bedeutung von Glukose, Glukose-6-phosphat und Mannose als Kohlenstoffquellen für die extra- und intrazelluläre Replikation zweier Isolate enteroinvasiver E. coli und eines S. Typhimurium Stammes analysiert. Zu diesem Zweck wurden zunächst definierte Mutanten in den beiden wichtigsten Phosphoenolpyruvat-abhängigen Phosphotransferasesystemen (PTS) für die Aufnahme von Glukose und Mannose, ptsG und manXYZ, sowie im Antiporter für die Aufnahme von Glukose-6-phosphat, uhpT, konstruiert. Bei Wachstum im Minimalmedium mit Glukose als einziger C-Quelle waren die Generationszeiten aller ΔptsG- und ΔptsG, manXYZ-Mutanten im Vergleich zu den Wildstämmen deutlich verlängert. Ebenso wuchsen ΔmanXYZ-Mutanten bzw. ΔuhpT-Mutanten deutlich langsamer auf Mannose bzw. Glukose-6-phosphat. Jedoch ergaben sich hierbei Stamm-spezifische Unterschiede. So erreichte EIEC 4608-58 ΔuhpT in der stationären Phase eine ähnliche Zelldichte wie der Wildstamm in Gegenwart von Glukose-6-phosphat und eine ΔptsG, manXYZ-Mutante von S. Typhimurium 14028s konnte immer noch effizient mit Glukose wachsen. Infektionsversuche mit Caco-2-Zellen zeigten weiterhin, daß die Deletion von ptsG zu einer signifikanten Erhöhung der Adhärenz und Invasivität von EIEC 4608-58 führt, während sich die intrazellulären Generationszeiten aller hier untersuchten Mutanten kaum veränderten. Selbst die ΔptsG, manXYZ, uhpT-Dreifachmutanten der drei hier verwendeten Enterobakterien und die ΔptsG, manXYZ, glk-Mutante von S. Typhimurium 14028s konnten immer noch in Caco-2-Zellen replizieren, wenn auch mit Stamm-spezifisch verringerten Geschwindigkeiten. 13C-Markierungsexperimente mit [U-13C6]-Glukose als Substrat ergaben jedoch, daß in der Tat alle hier untersuchten enterobakteriellen Wildstämme Glukose während der Replikation in Caco-2-Zellen unter Zellkulturbedingungen verwerten. Glukose-6-phosphat, Glukonat oder Fettsäuren konnten dagegen als wichtigste Kohlenstoffquellen für das intrazelluläre Wachstum ausgeschlossen werden. EIEC 4608-58 metabolisierte Glukose jedoch weniger effizient als EIEC HN280 und schien zudem noch zusätzlich C3-Substrate aus der Wirtszelle aufzunehmen. Das Markierungsmuster zeigte einen Stamm-spezifischen Kohlenstofffluß durch Glykolyse und/oder Entner-Doudoroff-Weg, Pentosephosphatzyklus, Citratzyklus und den anaplerotischen Reaktionen zwischen PEP und Oxalacetat. Mutanten mit Deletionen in ptsG und manXYZ konnten auf alternative C3-Substrate wechseln und glichen dies durch eine erhöhte Aufnahme von Aminosäuren aus den Wirtszellen aus. / Salmonella Typhimurium and enteroinvasive E. coli (EIEC) are facultative intracellular bacteria belonging to the family of Enterobacteriaceae. After internalisation by eukaryotic cells Salmonella normally resides inside a specialised phagosome called Salmonella-containing vacuole (SCV) whereas EIEC replicates inside the cytosol of host cells. In this study the ability of S. Typhimurium 14028s to replicate inside the cytosol of Caco-2 host cells was investigated by microinjection. It was thereby observed that a formerly used strain also called S. Typhimurium 14028s WT harboured an insertion of one deoxythymidin at position 76 of the rfbP open reading frame, a gene whose protein is involved in the LPS biosynthesis. Furthermore this strain expressed a rough LPS. Due to agglutination the microinjection procedure of the rough strain had only little success. But the percentage of Caco-2 cells that harboured more than 32 bacteria was about 30 % 5 h after injection of a non invasive mutant of Salmonella expressing full-length LPS chains. This was 2-3 times higher than the results observed before using HeLa cells. Therefore, the behaviour of HeLa cells infected by S. Typhimurium ΔsifA - a mutant that escapes from the SCV into the cytosol - was studied. The results showed that the sifA mutant strain induced the activity of caspases 9 and 3 in HeLa cells 10 h after infection but not in Caco-2 cells. In further experiments the contribution of glucose, glucose 6-phosphate and mannose as carbon sources for extra- and intracellular growth of two enteroinvasive E. coli isolates and one S. Typhimurium strain was analysed. Therefore, defined mutants of the most important phosphoenolpyruvate-dependent phosphotransferase systems (PTS) taking up glucose or mannose, i.e. ptsG and manXYZ, were constructed as well as mutants carrying a deletion of uhpT the antiporter for uptake of glucose 6-phosphate. During growth of the resulting ΔptsG and ΔptsG, manXYZ mutants in minimal medium with glucose as sole carbon source considerably longer generation times were observed. Likewise, ΔmanXYZ mutants and ΔuhpT mutants grew significantly slower on mannose or glucose 6-phosphate, respectively. But there were also strain specific differences. EIEC 4608-58 ΔuhpT reached a similar cell density as the wild-type strain during stationary phase when grown in the presence of glucose 6-phosphate and S. Typhimurium ΔptsG, manXYZ could still grow efficiently on glucose. Infections of Caco-2 cells showed that the deletion of ptsG increased the ability of EIEC 4608-58 significantly to adhere and invade these cells. But the intracellular generation times of all mutants under study were hardly changed. Even the triple mutants ΔptsG, manXYZ, uhpT of all three enterobacterial strains and the ΔptsG, manXYZ, glk mutant of S. Typhimurium 14028s were still able to replicate in Caco-2 cells, albeit at strain specific lower rates. 13C-Isotopologue profiling using [U-13C6]glucose as precursor revealed that in deed all analysed enterobacterial wild-type strains utilised glucose for their replication in Caco-2 cells under the applied conditions. Glucose 6-phosphate, gluconate and fatty acids could be ruled out as main carbon sources for intracellular growth. EIEC 4608-58 metabolised the applied glucose less efficiently than EIEC HN280 and seemed to take up C3-compounds from the host cells in addition to glucose. The labelling patterns reflected strain specific carbon fluxes via glycolysis and/or Entner-Doudoroff pathway, pentose phosphate pathway, citric acid cycle and the anaplerotic reactions between PEP and oxaloacetate. Mutants carrying deletions in ptsG and manXYZ switched to alternative C3-substrates and counterbalanced this by an increased uptake of amino acids from the host cells.

Food borne pathogens that cause systemic disease must cross the intestinal barrier. Many of these pathogens, eg Salmonella typhimurium and Shigella flexneri, use M cells, found only within the follicle associated epithelium (FAE) that overlies Peyer’s patches and other lymphoid follicles, to enter the host. This study is primarily an investigation into the interaction of S. typhimurium and Listeria monocytogenes with the intestinal epithelium, representing the early stage of an infection. / Alle in Nahrungsmitteln vorkommenden Pathogene, die eine systemische Infektion auslösen, müssen die Darmwand überwinden. Für die Invasion bevorzugtes Ziel vieler Lebensmittelpathogene, wie z.B. Salmonella typhimurium und Shigella flexneri, sind M-Zellen, die nur innerhalb des Follikel-assoziierten Epithels (FAE) über den Peyer’schen Plaques und anderen Lymphoid-Follikel vorkommen. In dieser Arbeit wurde in erster Linie die Interaktion von S. typhimurium und Listeria monocytogenes mit dem FAE untersucht, welche die frühe Phase einer Infektion repräsentiert.

by Kwong-Kwok Wong. / Thesis (Ph.D)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 171-183. / TITLE PAGE --- p.I / ABSTRACT --- p.II / STATEMENT --- p.V / ACKNOWLEGEMENTS --- p.VI / ABBREVIATIONS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XIII / LIST OF FIGURES --- p.XVI / Chapter Chapter 1. --- Introduction and Literature Review --- p.1 / Chapter I . --- Introduction --- p.1 / Chapter A. --- General introduction --- p.1 / Chapter B. --- Purpose of study --- p.5 / Chapter II. --- Literature review --- p.6 / Chapter A. --- Global control of aerobic-anaerobic shift --- p.6 / Chapter B. --- Identified anaerobiosis-inducible genes --- p.8 / Chapter C. --- Genetics of anaerobic regulation --- p.15 / Chapter i. --- Redox control --- p.15 / Chapter ii. --- DNA conformation --- p.15 / Chapter iii. --- fnr (oxrA) regulatory gene --- p.16 / Chapter iv. --- narL gene --- p.18 / Chapter v. --- Other regulatory genes --- p.19 / Chapter vi. --- Proposed FNR and NarL recognition sequences --- p.20 / Chapter D. --- future prospect --- p.23 / Chapter Chapter 2. --- Isolation of Anaerobiosis-inducible Promoters --- p.25 / Chapter I . --- Introduction --- p.25 / Chapter A. --- Properties of promoter-probe plasmid pKK232.8 --- p.26 / Chapter B. --- Properties of promoter-probe plasmid pFZYl --- p.28 / Chapter II. --- Materials and methods --- p.30 / Chapter A. --- Bacterial strains and plasmids --- p.30 / Chapter B. --- Media --- p.30 / Chapter C. --- Solutions --- p.31 / Chapter D. --- Small scale prepartaion of plasmid DNA --- p.32 / Chapter E. --- Large scale preparation of plasmid DNA --- p.33 / Chapter F. --- Digestion of DNA with restriction endonucleases --- p.35 / Chapter G. --- Analysis of DNA samples with agarose gel electrophoresis --- p.36 / Chapter H. --- Dephosphorylation of DNA fragments --- p.37 / Chapter I. --- Partial digestion of Chromosomal DNA with restriction enzyme Sau3A. --- p.37 / Chapter J. --- Ligation of DNA --- p.38 / Chapter K. --- Preparation of competent cells --- p.38 / Chapter L. --- Transformation --- p.40 / Chapter M. --- Chloramphenicol resistance levels test for promoter clones with plasmid pKK232.8 --- p.41 / Chapter N. --- Preparation of crude cell extract for chloramphenicol acetyltransferase (CAT) assays --- p.41 / Chapter O. --- CAT assay --- p.42 / Chapter P. --- Protein assay --- p.42 / Chapter Q. --- β-galactosidase assay --- p.43 / Chapter III . --- Results --- p.45 / Chapter A. --- Molecular cloning of anaerobiosis-inducible promoters with promoter-probe plasmid pKK232.8 --- p.45 / Chapter B. --- Molecular cloning of anaerobiosis-inducible promoters with promoter-probe plasmid pFZYl --- p.54 / Chapter IV. --- Summary and Discussion --- p.70 / Chapter A. --- Cloning with promoter-probe plasmid pKK232.8 --- p.70 / Chapter B. --- Cloning with promoter-probe plasmid pFZYl --- p.71 / Chapter C. --- Number of anaerobiosis inducible promoters --- p.73 / Chapter Chapter 3. --- Subcloning and Sequencing --- p.74 / Chapter I . --- Introduction --- p.74 / Chapter II. --- Materials and methods --- p.74 / Chapter A. --- Bacterial strains and bacteriophages --- p.74 / Chapter B. --- Preparation of M13mp RF plasmid --- p.75 / Chapter C. --- DNA sequencing by the chain termination method --- p.75 / Chapter D. --- Polymerase chain reaction (PCR) for the amplification of DNA fragments cloned in plasmid pFZYl --- p.79 / Chapter E. --- Using Exonuclease III to construct unidirectional deletions to generate nested clones --- p.80 / Chapter F. --- Direct gel electrophoresis --- p.81 / Chapter G. --- C-testiscreening for the orientation of insert in M13 phage --- p.81 / Chapter III . --- Results --- p.82 / Chapter A. --- Subcloning and sequencing of pFE29 and pFE117 --- p.82 / Chapter B. --- Subcloning and sequencing of pHSKl --- p.90 / Chapter C. --- Subcloning and sequencing of pHSK8 --- p.100 / Chapter D. --- "Subclonig and sequencing of pFSl, pFS22 and pFS3 4" --- p.109 / Chapter IV. --- Summary and Discussion --- p.113 / Chapter A. --- Trimming down size of DNA fragments to smaller fragments which still contained anaerobiosis-inducible promoters --- p.113 / Chapter B. --- Nucleotide sequencing --- p.113 / Chapter C. --- Sucloning and sequencing strategy --- p.115 / Chapter Chapter 4. --- Expression of Anaerobiosis-inducible Promoters --- p.120 / Chapter I . --- Introduction --- p.120 / Chapter II. --- Materials and methods --- p.122 / Chapter A. --- Bacterial strains and phages --- p.122 / Chapter B. --- Media --- p.125 / Chapter C. --- Transformation in Salmonella typhimurium --- p.125 / Chapter D. --- Genetic techniques --- p.126 / Chapter III. --- Results --- p.129 / Chapter A. --- Expression of Escherichia coli qlpT promoter --- p.129 / Chapter B. --- "Expression of Salmonella typhimurium anaerobiosis-inducible promoters cloned in pHSK8, pFS22 and pFS34" --- p.134 / Chapter IV. --- Summary and Discussion --- p.137 / Chapter A. --- A pair of divergent promoters were both regulated by anaerobiosis and glucose. --- p.137 / Chapter B. --- fnr(oxrA) dependent and independent promoters --- p.137 / Chapter C. --- Effect of nitrate on anaerobiosis expression. --- p.138 / Chapter Chapter 5. --- Analysis of Anaerobiosis-inducible Promoter-containing DNA sequences and Final Discussion --- p.141 / Chapter I. --- Analysis of anaerobiosis-inducible promoter-containing DNA sequences --- p.141 / Chapter A. --- "Search for initiation codon, conserved ""-10"" and ""-35"" regions" --- p.141 / Chapter B. --- Search for FNR binding sites and NarL binding sites. --- p.151 / Chapter C. --- Homology search among the promoter sequences of all anaerobiosis-inducible genes. --- p.156 / Chapter II. --- Final Discussions. --- p.164 / Chapter A. --- Summary of the properties of the sequenced and characterized promoters cloned in this study --- p.164 / Chapter B. --- Further studies. --- p.167 / REFERENCES --- p.168

Salmonella enterica serovar Typhimurium is a common cause of salmonellosis among humans and animals worldwide. In Australia, Typhimurium is responsible for over half of the salmonellosis cases. The Anderson phage-typing scheme is the primary means of long-term surveillance of Typhimurium outbreak isolates, and has played an important role in epidemiology. However, there exist quite a number of strains of Typhimurium that cannot be defined by the phage-typing scheme. Furthermore, the knowledge of evolutionary relationships among isolates of different phage types is still very limited and the genetic basis of phage type variation remains largely unknown. To address these issues, this study focused on molecular typing and evolution of Typhimurium. Fluorescent amplified-fragment length polymorphism (AFLP) was applied to 46 Typhimurium isolates comprising nine phage types in Australia using the restriction enzymes MseI and EcoRI and MseI +1 / EcoRI +1 primer pair combinations. The selected phage types, DT9, DT135, DT64, DT44, DT126, DT12a, DT1, DT141 and DT108, have been dominant or frequent phage types in animal and human infections in Australia in recent years. AFLP in the present study showed a very good discrimination power with Simpson index of diversity of 0.98, 35 different AFLP patterns were observed in the 46 isolates studied. The tree based on AFLP patterns showed good correlation with phage type, grouped most Typhimurium isolates by phage type, and differentiated all nine phage types. Furthermore, 84 phage-type specific polymorphic AFLP fragments, for which presence or absence correlated with phage type (including 25 with one exception to phage-type specificity) were observed in the 46 strains studied. Eighteen phage-type specific AFLP fragments were cloned and sequenced. Sixteen are of known genes or have a homologue in the databases. It was found a predominance of phage and plasmid genes rather than mutational changes in the AFLP fragments studied. Of the 18 cloned and sequenced AFLP fragments, only four relate to mutational changes in the S. enterica chromosome, the other 14 comprise DNA of mobile elements: nine are phage related, three are plasmid related and two are gain of DNA from unknown origin. Twelve of the 18 sequenced phage-type specific AFLP markers are polymorphic because the DNA is present or absent as indicated by Southern hybridization. Two of these markers were successfully used in preliminary PCR-based typing of 30 DT9 and 29 DT135 isolates from worldwide collections. 27 of the 30 DT9 isolates and all DT135 isolates tested were correctly categorized. The results implied a good potential to use the sequence of these fragments as the basis for a multiplex PCR or a microarray based molecular �phage� typing method for Typhimurium. This thesis also studied the molecular evolutionary relationships among the same set of 46 Typhimurium isolates using mutational changes detected by AFLP, or analysis of intergenic regions and their flanking genes in genome sequences. The complete genome sequence of Typhimurium LT2 was analysed by computer modelled AFLP. The polymorphic AFLP fragments, which matched with the modelled LT2 AFLP fragments, were amplified and sequenced by LT2 genome based primers to determine the changes. Forty-nine intergenic regions with higher pairwise differences between LT2 and Typhi CT18 were amplified and sequenced using LT2 genome based primers for one isolate of each phage type. 51 polymorphic sites were detected consisting of 18 in AFLP fragments and 33 in intergenic regions or their flanking genes. PCR-RFLP (restriction fragment length polymorphism) and SNaPshot were used to further investigate the distribution of the single nucleotide polymorphisms (SNPs) detected in intergenic regions in all isolates studied. Of the 18 mutational changes detected in AFLP fragments, eight were indels (insertions / deletions) and ten single base substitutions. Of the eight indels, four were in genes, three in intergenic regions, and one covered adjacent intergenic and coding regions. The four indels in genes all caused frameshift mutations, including three single base indels and one 19 bp deletion. Of the ten substitutions, one was in an intergenic region and nine in genes comprising three synonymous and six non-synonymous substitutions. Of the 33 polymorphic sites detected from sequences of 23 intergenic regions and their flanking genes, one was IS200 insertion and 32 single nucleotide polymorphisms (SNPs), of which 30 were single base substitutions and two were single base indels. Nine of the 33 variations were found in the flanking genes, which were all single base substitutions comprising four synonymous, four non-synonymous substitutions and one non-sense mutation. More non-synonymous than synonymous substitutions were found for those in coding regions within Typhimurium, indicating that slightly deleterious intraspecies mutations can be fixed within clones, such as various lineages of Typhimurium. The 51 polymorphic sites, which were inferred from sequences of both mutation related AFLP fragments, and intergenic regions and their flanking genes, gave a single phylogenetic tree of the 46 Typhimurium isolates studied. All sequences involved were compared with the homologous sequences in the available S. enterica genome sequences for serovars Typhi, Paratyphi A, Gallinarum, Enteritidis and Pullorum and this enabled the determination of the direction of the mutational changes in the isolates studied and the root of the phylogenetic tree. There were only two events inferred to have occurred twice, the remaining 49 polymorphisms can be explained by a single event. The data indicated that Typhimurium has a very strong clonal structure with a very low level of recombination over the time for diversification of Typhimurium as majority of clonal variations are from point mutations rather than recombination. The phylogenetic tree based on mutational changes showed that most Typhimurium isolates of a given phage type are in the same evolutionary group, but that some phage types appear to have arisen more than once. Comparison of the phylogenetic tree with AFLP data gave examples of unrelated isolates of a given phage type having common AFLP fragments comprising plasmid or phage genes, supporting the view that phage type can be determined by presence of specific phages or plasmids. The mutation-based tree showed that six of the nine phage types studied appeared to have a single origin, at least for the isolates studied. It also found that DT1 and DT44 had two independent origins even for the limited set of strains used. The distribution of DT12a isolates into two groups could be explained that the group of three DT12a isolates were derived from the other group of four DT12a isolates, where the root of the tree might be. The data also confirmed that DT64 arose from DT9. The phylogenetic tree that was generated based on essentially mutational changes provides clear relationships of the closely related Typhimurium isolates with high level of consistency and reasonable confidence. This study provided one of the few analyses of relationships of isolates within a clone. Matching actual AFLP with computer modeled AFLP and sequencing intergenic regions provide very good new strategies to identify mutational polymorphisms and to study the molecular evolutionary relationships in the closely related isolates.

Thesis (M.S.)--University of Missouri--Rolla, 2007. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed December 5, 2007) Includes bibliographical references (p. 55-60).

Au niveau de la santé publique, l'impact meurtrier des infections au rotavirus humain (RVH) et les limites d'application des vaccins disponibles déterminent l'urgence et la nécessité de développer de nouvelles approches pour immuniser une grande partie de la population mondiale sans effets secondaires néfastes. Pour pallier à l'énorme demande en médicaments et en vaccins, plusieurs groupes de recherche tentent de développer de nouvelles méthodes de production à base de plantes transgéniques. La production de médicaments et vaccins chez les végétaux possèdent des avantages inhérents dont une absence de pathogènes humain, une inoculation facile, de faibles coûts de production à grande échelle et la production d'inocula bruts stockables. Le succès des systèmes d'expression à base de plantes justifie le développement de plants qui produisent beaucoup de biomasse et se transforment génétiquement de façon courante. Le travail présenté ici démontre le potentiel du système d'expression transitoire par agroinfiltration de Nicotiana benthamiana pour produire les antigènes VP7 et VP4∆ du RVH ainsi que l'adjuvant protéinique fljB de Salmonella typhimurium. Le clonage des séquences codantes des antigènes VP7, VP4∆ et fljB a permis de générer de multiples constructions simple, double et triple. Toutes les contructions ont été exprimées dans les feuilles agroinfiltrées à l'exception de VP4∆::VP7 qui n'a pas été détectée par immunobuvardage. Le rendement de protéines d'intérêt dans les extraits végétaux se situe entre 0,85 et 31,97 µg de protéine recombinante par gramme de feuilles fraîches. Plusieurs facteurs dont la toxicité et la stabilité des différentes constructions dans les plantes peuvent expliquer cet écart. Dans nos conditions expérimentales, toutes les constructions contenant le fragment fljB ont permis la production d'anticorps spécifiques à fljB dans les souris immunisées avec les extraits végétaux. Par contre, aucune construction n'as permis la production d'anticorps spécifiques à VP7 ou VP4. Ces résultats montrent que l'expression transitoire dans Nicotiana benthaminana permet de produire rapidement de multiples antigènes et que les protéines fortement immunogéniques dans les extraits végétaux induisent une réponse humorale chez les souris. Plusieurs applications de cette technologie sont envisageables dans la cadre du contrôle endémique du RVH ou de Salmonella typhimurium. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Rotavirus humain, VP7, VP4, Salmonella typhimurium, fljB, Expression transitoire, Vaccins à base de plantes, Nicotiana benthaminana.