Aptamers are biomolecular ligands composed of nucleic acids. They can be selected to bind specifically to a range of target molecules and subsequently exploited in a fashion analogous to more traditional biomolecules such as antibodies. In this study a method for selecting new aptamers which specifically bind whole live bacterial cells is described. A non-pathogenic strain of Escherichia coli K12 was used to develop the method. A DNA library containing 100 bases long random nucleotide sequences was produced and the aptamer selection process was repeated nine times. An enzyme-linked technique was first used to detect bound aptamers thereafter fluorimetry and fluorescence microscopy methods were used for the detection. The aptamers were cloned and sequenced and the cloned aptamers produced with fluorescent labels. The E. coli K12-binding aptamers were used to demonstrate the detection of the bacterial cells in a complex food matrix, namely probiotic yogurt, by using fluorescence based detection method. The aptamer selection method with some modifications was also used to select aptamers with specificity for the food pathogens E. coli O157, Listeria monocytogenes, L. innocua, S. typhimurium and S. enteritidis. The aptamers against E. coli O157 and S. typhimurium were cloned and the sequences and the binding properties of these aptamers were analysed. The use of E. coli K12 as a target organism and the aptamer sequences presented in this study, have not previously been published in scientific literature. This is also the first report where the aptamers have been used in detection of live bacterial cells in yogurt.

Citrobacter rodentium is a gut pathogen, which infects the distal colon of mice. It has many similarities to human Enteropathogenic and Enterohemorrhagic E.coli in terms of mechanisms of pathogenicity and methods of transmission. Like many other gram negative bacteria, C. rodentium has developed a complex and highly specialised protein secretion system, known as type three (T3SS), to deliver bacterial proteins into eukaryotic cells. By injecting effector proteins into host cell cytoplasm, the pathogens are able to modulate host cellular functions to facilitate their own survival and replication. There is growing evidence that Attaching Effacing (AE) pathogens can inject effector proteins into gut epithelial cells, which dampen pro-inflammatory responses. There is also evidence that EPEC, Yersinia and Shigella can inject effectors into immune cells and also modulate their function. The objective of this work was to visualise and identify the host cells targeted for type III secretion by C. rodentium, and consequently determine the effect on host immune responses. The method chosen to detect cells targeted for effector protein delivery was the β-lactamase reporter system, where cells loaded with the fluorogenic substrate CCF2-AM emit a green FRET signal upon excitation by UV light, but emit a blue signal when cleaved by β-lactamase. By creating reporter strain of C.rodentium expressing fusion proteins between NleD effector and β-lactamase, I was able to show that C.rodentium is capable of injecting NleD in a wide variety of murine cell lines including Swiss 3T3 fibroblasts, J774 macrophages, CMT93 epithelial cells and BW715 T cells in a dose and time dependent manner in vitro. In addition, I found that C.rodentium has the ability to inject proteins into the cytoplasm of immune cells isolated from mouse lymphoid tissues including the spleen, mesenteric lymph nodes and Peyer's patches. Detailed analysis of the types of cells injected with effectors in vitro showed that NleD- injected cells represented B cells, dendritic cells and T cells. After inoculation of mice with the reporter strain of CitropACYCnleD, the plasmid encoded reporter fusion remained stable throughout infection and was able to inject cells in vitro after passage through the mouse gut. Unfortunately under the conditions described in this study, we were unable to visualise any gut cells targeted for protein delivery by C. rodentium in vivo, thus highlighting the complex nature of the host pathogen relationships in the gut. Although there is a need to develop better strategies to visualise effector translocation in vivo, our study has demonstrated, for the first time, the ability of C. rodentium to target immune cells for effector injection in vitro.

La phosphorylation/déphosphorylation des protéines est un mécanisme de signalisation intracellulaire commun. Parmi les kinases végétales, les Mitogen-Activated Protein Kinases (MAPKs) sont impliquées dans de nombreux processus biologiques importants, comme la réponse aux stress biotiques et abiotiques, le développement et la dynamique du cytosquelette. Chez Arabidopsis thaliana et ce malgré de nombreux efforts, les fonctions des kinases impliquées dans les cascades MAPK restent peu inconnues. L'activation des kinases en utilisant des mutations mimant la phosphorylation des sites normalement phosphorylés est une approchequi a fait ses preuves dans le cas de MAP2Ks et a largement contribué à élucider leurs fonctions. Cette stratégie s’est révélée impossible dans le cas des MAPKs, puisque les résidus à muter restent encore à identifier. Pour contourner ce problème, nous avons adapté un crible basé sur la complémentation fonctionnelle d’un mutant MAPK de levure avec des formes aléatoirement mutées de MPK6d’Arabidopsis dans le but d'identifier des mutants présentant une activité constitutive. Nous en avons identifiés plusieurs et avons montré que ces formes constitutivement actives (CA) de MPK6 sont actives sans phosphorylation par les MAP2Ks. Par ailleurs, les mutations des résidus équivalents dans d'autres MAPKs les rendent également hyperactives, ce qui indique que cette stratégie peut être utilisée comme approche générale pour activer les MAPKs afin d’en comprendre les fonctions. L’étude des interactions protéine-protéine et l’analyse des profils dephosphorylation indiquent que les MAPKs CA conservent leur spécificité envers leurs substrats et interacteurs. Comme preuve de concept, nous avons généré des formes actives du MPK4. La MPK4 CA exprimée sous son propre promoteur a parfaitement complémenté le mutant mpk4. La caractérisation des lignées exprimant MPK4 CA confirme le rôle négatif de cette kinase dans les réponses de défense aux pathogènes des plantes que ce soit dans la PTI (PAMP Triggered Immunity) ou dans la ETI (Effector Triggered Immunity). Globalement, ce travail permettra de fournir des informations directes sur les cibles des MAPKs et devrait contribuer à la compréhension globale de la transduction du signal chez les plantes. / Protein phosphorylations and dephosphorylations are common events occurring duringintracellular signaling processes. Among plant kinases, Mitogen-Activated Protein Kinases (MAPKs) are involved in signaling of many important biological processes, including biotic and abiotic stresses, development and cytoskeleton organization. Despite an abundant literature on MAPKs, the exact roles and direct targets of many Arabidopsis thaliana MAPKs are not clear yet. The activation of kinases using phospho-mimicking mutations of the phosphorylated residues was a successful approach in the case of MAP2Ks, helping to elucidate their functions. This strategy failed in the case of MAPKs since the necessary residues to mutate remain unclear. To bypass this problem, we adapted a screen based on the functional complementation of a MAPK yeast mutant with randomly mutated Arabidopsis MPK6 in order to identify the ones mutants showing constitutive activity. We identified several clones and showed that these constitutively active (CA) of MPK6 candidates are indeed active without phosphorylation by MAP2Ks. Interestingly, mutations of the equivalent residues in other MAPKs triggered constitutive activity as well, indicating that this strategy may be used as a general approach to activate MAPKs and identify their functions. Interaction and phosphorylation assays indicatedthat CA MAPKs retain their substrate and interactor specificity. As proof-of-concept, we generated active versions of MPK4. CA MPK4 expressed under itsown promoter successfully complements mpk4 mutant plants. Characterization of CA MPK4 lines further confirmed the negative role of MPK4 in plant pathogen defense responses and its implication in both PTI (PAMP Triggered Immunity) and ETI (Effector Triggered Immunity). Overall, the work will help to provide direct information on all MAPK targets and should be an important contribution to the overall understanding of signal transduction in plants.

Candida albicans is the most prevalent human fungal pathogen. The research described in this thesis has focused on the identification and characterization of the regulatory pathways in this pathogen controlling white-opaque switching, mating and biofilm formation as well as the relationship between them. White-opaque switching and mating in C. albicans are under the repression of the a1-α2 complex. Based on this, a chromatin immunoprecipitation-microarray analysis of the a1-α2 target genes was conducted to search for the master switch locus. The result identified TOS9 (WOR1) as a master regulator gene, and overexpression of TOS9 resulted in a switch en masse from white to opaque. In 2006, a novel form of communication was demonstrated between white and opaque cells in C. albicans. It was shown that minority opaque cells through the release of pheromone signaled majority white cells of the opposite mating type to become cohesive, adhesive and form enhanced biofilms. These biofilms in turn facilitated opaque cell chemotropism required for opaque cell mating. To identify the pathway regulating the white cell pheromone response, deletion mutants were generated for select genes mediating the opaque cell mating response. It was demonstrated that the pathways regulating the white and opaque cell responses to the same pheromone share the same upstream components, including receptors, heterotrimeric G protein, and mitogen-activated protein kinase cascade, but they use different downstream transcription factors that regulate the expression of genes specific to the alternative responses. This configuration, although found in higher, multicellular systems, is uncommon in fungi and suggests that it may be an antecedent to multicellularity in higher eukaryotes. In addition, it was found that a C. albicans-specific 55-amino-acid region of the first intracellular loop, IC1, of the α-pheromone receptor, is required for the α-pheromone response of white cells, but not that of opaque cells. Finally, to test the generality of the white cell pheromone response, evidence was presented that the response occurs in all tested media and in all of the 27 tested strains, including a/a and α/α strains, derivatives of the common laboratory strain SC5314, and representatives from all of the five major clades. The white cell response to pheromone, therefore, proved to be a general characteristic of MTL-homozygous strains of C. albicans.

In this dissertation I explore the application of two novel modeling techniques for improving risk analysis of vector-borne disease and discuss their potential use in integrating environmental risk assessment that guides environmental and public health decisions. Techniques for analyzing risk have been considered inadequate due to a lack of understanding of the problem and an appropriate analytic-deliberative process clarifying the meaning of analytic findings and uncertainty (National Research Council (NRC), 1996; Peterman and Anderson, 1999). Thus, new integrative risk analysis tools are needed that are responsive to more complex environmental problems. In this work, I develop a qualitative community model that combines a conventional biomathematical model of vector-borne disease transmission with recent developments in community modeling. My procedure predicts the change in risk of vector-borne disease from press perturbations, a disturbance that results in a permanent change in a growth parameter. I also use a Relational Bayesian Modeling technique to exploit existing data to determine plausible mechanisms and geospatial and temporal patterns of disease spread. I apply these tools to Lyme disease and West Nile Encephalitis as examples of two different vector-borne diseases associated with complex ecological communities. Both the qualitative modeling and Bayesian methods provide an integrated risk analysis framework that identifies relationships important in the system and thus, guide the application of quantitative models or provide sufficient information for management decisions. / Graduation date: 2003

Chemical investigation of the transformation of the crucifer phytoalexins brassinin, brassilexin and camalexin by the phytopathogenic fungus <i>Alternaria brassicicola</i> was carried out. The objectives of this study included:<p> 1) the isolation and characterization of the metabolites of biotransformation of brassinin, brassilexin and camalexin by <i>A. brassicicola</i>;<p> 2) determination of the antifungal activity of these phytoalexins and their metabolites against <i>A. brassicicola.</i><p> The phytoalexins were synthesized and characterized using HPLC retention time tR, 1H NMR, 13C NMR, LC-MS and HRMS-ESI data. The metabolites of the biotransformation were also synthesized and characterized similarly. The metabolism of each phytoalexin and their metabolites was studied by analyzing broth extracts by HPLC. The percent inhibition of growth of <i>A. brassicicola</i> was determined by radial growth mycelial assays.<p> The biotransformation of brassinin by <i>A. brassicicola</i> afforded Nb-acetyl-3-indolylmethylamine via indole-3-methylamine intermediate. Brassilexin was metabolized to 3-(amino)methyleneindoline-2-thione by the reduction of the isothiazole ring. Camalexin did not appear to be metabolized or the metabolism was very slow. The results of biotransformation and bioassay studies established that the metabolism of brassinin by <i>A. brassicicola</i> was a detoxification process. However, these studies using brassilexin did not provide a rigorous conclusion. Camalexin showed strong inhibition of growth against <i>A. brassicicola</i> suggesting its importance in defense against this pathogen.

A plant's response to pathogen infection is tailored dependent on infection strategy. Successful plant pathogens employ various infection strategies to avoid or reduce plant defense responses for the establishment of host compatibility. Autophagy is a non-selective degradation pathway conserved in eukaryotic organisms, which has been implicated in the regulation of cell survival or cell death, depending on cell type and stimulus. In <i>Arabidopsis thaliana</i>, an autophagic response has been reported to be activated during nutrient deprivation. Cellular contents, such as cytoplasm and organelles, are sequestered into double-membraned autophagosomes and delivered to the vacuole for degradation; degradative products, such as amino acids, are released back into the cell and reutilized to maintain cellular function. In this study, the response of the autophagy pathway was investigated in <i>A. thaliana</i> leaf tissues upon biotrophic <i>Erysiphe cichoracearum</i> and hemibiotrophic <i>Colletotrichum higginsianum</i> infections. Expression of some autophagy genes was induced in <i>A. thaliana</i> at 9 days post infection with <i>E. cichoracearum</i> and, 3 and 5 days post infection with <i>C. higginsianum</i>. Using a transgenic <i>A. thaliana</i> plant line over expressing autophagosome associated protein autophagy-8e (<i>ATG8e</i>) conjugated to green fluorescent protein (GFP) (<i>ATG8e-GFP</i>), confocal analysis revealed that autophagosomes specifically accumulated at the infection sites during <i>E. cichoracearum</i> and <i>C. higginsianum</i> invasions. These results indicate that the plant autophagic pathway responds to an interaction between <i>A. thaliana</i> and fungal pathogens. None of the defense signaling molecules including salicylic acid, jasmonic acid, ethylene, hydrogen peroxide and nitric oxide consistently triggered expression of autophagy genes. The insensitivity to defense signaling molecules and the delayed induction of autophagy genes compared to expression of pathogenesis-related genes suggest that the activation of this pathway does not contribute to host resistance responses during the infection process. In <i>A. thaliana</i> mutants, <i>atg4a/b, atg5-1, atg9-1</i> and <i>atg9-6</i> deficient for the autophagic response, virulence of <i>E. cichoracearum</i> was retarded whereas pathogenesis of <i>C. higginsianum</i> was accelerated. Taken together, these data suggest that the autophagy pathway is a potential host susceptibility factor for pathogen infection, possibly involved in establishing/facilitating biotrophy in <i>A. thaliana</i>.

Daphne is a widely dispersed genus with large variation in morphology, native habitats, and use. Unfortunately, broader acceptance of Daphne in the ornamental trade has been limited due to Daphne Sudden Death Syndrome (DSDS), a disease that kills the plant without warning. The results of this research identified Thielaviopsis basicola (Berk. etBr.) Ferr. as the causal agent for this disease. Pure cultures of the pathogen were developed and used in a germ plasm screen. To evaluate Daphne germ plasm in vitro, species-specific protocols were developed that alleviated two common problems in Daphne micropropagation, browning and hyperhydricity. Optimizing the concentrations of both PGRs and charcoal was able to control these problems. Selected species were evaluated for resistance against Thielavipsis basicola in both, in vivo and in vitro, conditions. The results of both methods displayed a strong correlation and indicated significant differences among the taxa. However, there were differences in disease progression rates. Typically, the in vitro challenge produced a comparable level of disease as the in vivo challenge but in two to three weeks less time. Across both screening methods, the most resistant species evaluated were D. tangutica and D. retusa, while D. cneroum was the most susceptible. Based on ITS sequences, phylogenetic relationships among selected Daphne species were established and associated with their resistance against T basicola. The phylogeny indicated that Daphne is possibly a monophyletic group. However, placement of D.genkwa remained problematic. The analysis of ITS sequences data resulted in a parsimony consensus tree with two well-supported major clades and one Glade with less support. In general, the evolutionary tree for resistance, inferred from the phylogenetic data and the results of the screening project, indicate that resistance is a derived character and that plants recently evolved this ability.

A plant's response to pathogen infection is tailored dependent on infection strategy. Successful plant pathogens employ various infection strategies to avoid or reduce plant defense responses for the establishment of host compatibility. Autophagy is a non-selective degradation pathway conserved in eukaryotic organisms, which has been implicated in the regulation of cell survival or cell death, depending on cell type and stimulus. In <i>Arabidopsis thaliana</i>, an autophagic response has been reported to be activated during nutrient deprivation. Cellular contents, such as cytoplasm and organelles, are sequestered into double-membraned autophagosomes and delivered to the vacuole for degradation; degradative products, such as amino acids, are released back into the cell and reutilized to maintain cellular function. In this study, the response of the autophagy pathway was investigated in <i>A. thaliana</i> leaf tissues upon biotrophic <i>Erysiphe cichoracearum</i> and hemibiotrophic <i>Colletotrichum higginsianum</i> infections. Expression of some autophagy genes was induced in <i>A. thaliana</i> at 9 days post infection with <i>E. cichoracearum</i> and, 3 and 5 days post infection with <i>C. higginsianum</i>. Using a transgenic <i>A. thaliana</i> plant line over expressing autophagosome associated protein autophagy-8e (<i>ATG8e</i>) conjugated to green fluorescent protein (GFP) (<i>ATG8e-GFP</i>), confocal analysis revealed that autophagosomes specifically accumulated at the infection sites during <i>E. cichoracearum</i> and <i>C. higginsianum</i> invasions. These results indicate that the plant autophagic pathway responds to an interaction between <i>A. thaliana</i> and fungal pathogens. None of the defense signaling molecules including salicylic acid, jasmonic acid, ethylene, hydrogen peroxide and nitric oxide consistently triggered expression of autophagy genes. The insensitivity to defense signaling molecules and the delayed induction of autophagy genes compared to expression of pathogenesis-related genes suggest that the activation of this pathway does not contribute to host resistance responses during the infection process. In <i>A. thaliana</i> mutants, <i>atg4a/b, atg5-1, atg9-1</i> and <i>atg9-6</i> deficient for the autophagic response, virulence of <i>E. cichoracearum</i> was retarded whereas pathogenesis of <i>C. higginsianum</i> was accelerated. Taken together, these data suggest that the autophagy pathway is a potential host susceptibility factor for pathogen infection, possibly involved in establishing/facilitating biotrophy in <i>A. thaliana</i>.

Chemical investigation of the transformation of the crucifer phytoalexins brassinin, brassilexin and camalexin by the phytopathogenic fungus <i>Alternaria brassicicola</i> was carried out. The objectives of this study included:<p> 1) the isolation and characterization of the metabolites of biotransformation of brassinin, brassilexin and camalexin by <i>A. brassicicola</i>;<p> 2) determination of the antifungal activity of these phytoalexins and their metabolites against <i>A. brassicicola.</i><p> The phytoalexins were synthesized and characterized using HPLC retention time tR, 1H NMR, 13C NMR, LC-MS and HRMS-ESI data. The metabolites of the biotransformation were also synthesized and characterized similarly. The metabolism of each phytoalexin and their metabolites was studied by analyzing broth extracts by HPLC. The percent inhibition of growth of <i>A. brassicicola</i> was determined by radial growth mycelial assays.<p> The biotransformation of brassinin by <i>A. brassicicola</i> afforded Nb-acetyl-3-indolylmethylamine via indole-3-methylamine intermediate. Brassilexin was metabolized to 3-(amino)methyleneindoline-2-thione by the reduction of the isothiazole ring. Camalexin did not appear to be metabolized or the metabolism was very slow. The results of biotransformation and bioassay studies established that the metabolism of brassinin by <i>A. brassicicola</i> was a detoxification process. However, these studies using brassilexin did not provide a rigorous conclusion. Camalexin showed strong inhibition of growth against <i>A. brassicicola</i> suggesting its importance in defense against this pathogen.