<i>Rhodococcus equi</i> (<i>R. equi</i>) is an intracellular, gram-positive coccobacillus that causes pneumonia in foals aged 2 to 4 months. Neonatal foals are susceptible to <i>R. equi</i> infection probably due to inefficient Toll-like receptor (TLR)-2 signaling and inability to produce interferon gamma. One of the reasons for inefficient receptor signaling and recognition of <i>R. equi</i> by the foals immune system may be the inefficient sequestration of TLRs in lipid rafts, which act as signaling platforms. However, there are no protocols to isolate lipid rafts from equine cells and, therefore, no data on the association of TLRs with the lipid rafts in the lung cells of normal and infected foals. Because of the clinical importance of the disease, there is considerable interest in developing effective prophylactic methods, which in turn requires a better understanding of fundamental immunology of the foals. In this study, I have examined the effect of <i>R. equi</i> vaccination on the lung inflammation induced following challenge with <i>R. equi</i>. I also developed a protocol to isolate lipid rafts from broncho-alveolar lavage (BAL) cells and investigated the association of lipid rafts with TLRs.<p> In the first study, 15 mixed breed draft-type foals up to 7 weeks of age were studied. The foals were divided into control (n=7) and a vaccinated (n=8). The control foals were given 10 mL phosphate buffered saline intramuscularly while the vaccinated group was vaccinated on day 0 of the study followed by a booster on day 14. All the foals were challenged with <i>R. equi</i> (5x106 cells/mL into the dorso-caudal region of the right lung lobe). BAL was performed on day 14, 28 and 35 and all the foals were euthanized on day 49 of the study.<p> The study design did not leave any non-infected foal at the end of the experiment. Therefore, lung samples were obtained from two untreated control (non-vaccinated non-infected) foals from the Department of Veterinary Pathology, University of Saskatchewan were used. The data showed similar levels of lung inflammation in both the control and vaccinated foal groups based on BAL cytology, gross pathology and histopathology. Gross and histopathological studies indicated that both control and vaccinated foals developed granulomatous lesions. Immunohistology showed increased expression of TLR4, TLR2 and TNF alpha in alveolar septa and in some cases in the vascular endothelium and airway epithelium in the lungs of both groups compared to the untreated control foals. Western blots showed increased expression of TLR2 but not TLR4 in the lung extracts from both the vaccinated and the control foals. Vaccinated foals showed higher concentrations of TNF alpha(p=0.0219) in their BAL on day 28 but lower concentrations of IL-10 (p=0.0172) in their lung extracts collected on day 49 compared to the controls. There were no differences in IFN gamma and protein concentrations between the two groups.<p> To understand the role of lipid rafts in TLR4 and TLR2 signaling, I developed an efficient and simpler protocol to isolate lipid rafts from BAL cells of foals and confirmed their identity by localizing Flotillin-1 and GM-1 (fractions 6-9), which are lipid raft markers, and transferrin receptor (fractions 1-4) which is present in non-lipid raft fractions. Lung macrophages from naïve foals lacked sequestration of Flotillin-1 and GM-1 in the higher fractions compared to the vaccinated foals. Further, the data showed that while TLR4 and TLR2 were localized in most of the fractions (1-9) in control foal BAL collected on day 14 and 28, the TLR4 and TLR2 association was restricted to fractions 6-9 in the lipid rafts isolated from BAL cells of vaccinated foals. These data suggest that BAL cells of neonatal foals may not have effective signaling machinery because of lack of association of TLR2 and TLR4 with lipid rafts.<p> Taken together, the data show similar levels of lung inflammation in the control and vaccinated foals upon infection with <i>R. equi</i>. The vaccination, however, appeared to have some effect on the immunohistologic expression of TLR2, TLR4 and TNFalpha in the lung tissues, and increased association of TLR2 and TLR4 with the lipid raft fractions. Based on the higher expression of TNF alpha and lower expression of IL-10, the vaccinated foals may be more competent to mount an immune response against <i>R. equi</i>.

<i>Rhodococcus equi</i> (<i>R. equi</i>) is an intracellular, gram-positive coccobacillus that causes pneumonia in foals aged 2 to 4 months. Neonatal foals are susceptible to <i>R. equi</i> infection probably due to inefficient Toll-like receptor (TLR)-2 signaling and inability to produce interferon gamma. One of the reasons for inefficient receptor signaling and recognition of <i>R. equi</i> by the foals immune system may be the inefficient sequestration of TLRs in lipid rafts, which act as signaling platforms. However, there are no protocols to isolate lipid rafts from equine cells and, therefore, no data on the association of TLRs with the lipid rafts in the lung cells of normal and infected foals. Because of the clinical importance of the disease, there is considerable interest in developing effective prophylactic methods, which in turn requires a better understanding of fundamental immunology of the foals. In this study, I have examined the effect of <i>R. equi</i> vaccination on the lung inflammation induced following challenge with <i>R. equi</i>. I also developed a protocol to isolate lipid rafts from broncho-alveolar lavage (BAL) cells and investigated the association of lipid rafts with TLRs.<p> In the first study, 15 mixed breed draft-type foals up to 7 weeks of age were studied. The foals were divided into control (n=7) and a vaccinated (n=8). The control foals were given 10 mL phosphate buffered saline intramuscularly while the vaccinated group was vaccinated on day 0 of the study followed by a booster on day 14. All the foals were challenged with <i>R. equi</i> (5x106 cells/mL into the dorso-caudal region of the right lung lobe). BAL was performed on day 14, 28 and 35 and all the foals were euthanized on day 49 of the study.<p> The study design did not leave any non-infected foal at the end of the experiment. Therefore, lung samples were obtained from two untreated control (non-vaccinated non-infected) foals from the Department of Veterinary Pathology, University of Saskatchewan were used. The data showed similar levels of lung inflammation in both the control and vaccinated foal groups based on BAL cytology, gross pathology and histopathology. Gross and histopathological studies indicated that both control and vaccinated foals developed granulomatous lesions. Immunohistology showed increased expression of TLR4, TLR2 and TNF alpha in alveolar septa and in some cases in the vascular endothelium and airway epithelium in the lungs of both groups compared to the untreated control foals. Western blots showed increased expression of TLR2 but not TLR4 in the lung extracts from both the vaccinated and the control foals. Vaccinated foals showed higher concentrations of TNF alpha(p=0.0219) in their BAL on day 28 but lower concentrations of IL-10 (p=0.0172) in their lung extracts collected on day 49 compared to the controls. There were no differences in IFN gamma and protein concentrations between the two groups.<p> To understand the role of lipid rafts in TLR4 and TLR2 signaling, I developed an efficient and simpler protocol to isolate lipid rafts from BAL cells of foals and confirmed their identity by localizing Flotillin-1 and GM-1 (fractions 6-9), which are lipid raft markers, and transferrin receptor (fractions 1-4) which is present in non-lipid raft fractions. Lung macrophages from naïve foals lacked sequestration of Flotillin-1 and GM-1 in the higher fractions compared to the vaccinated foals. Further, the data showed that while TLR4 and TLR2 were localized in most of the fractions (1-9) in control foal BAL collected on day 14 and 28, the TLR4 and TLR2 association was restricted to fractions 6-9 in the lipid rafts isolated from BAL cells of vaccinated foals. These data suggest that BAL cells of neonatal foals may not have effective signaling machinery because of lack of association of TLR2 and TLR4 with lipid rafts.<p> Taken together, the data show similar levels of lung inflammation in the control and vaccinated foals upon infection with <i>R. equi</i>. The vaccination, however, appeared to have some effect on the immunohistologic expression of TLR2, TLR4 and TNFalpha in the lung tissues, and increased association of TLR2 and TLR4 with the lipid raft fractions. Based on the higher expression of TNF alpha and lower expression of IL-10, the vaccinated foals may be more competent to mount an immune response against <i>R. equi</i>.

published_or_final_version / Public Administration / Master / Master of Public Administration

The goal of the research is to devise an equitable road pricing system which would leave the majority of routes free of tolls, so that low income people would suffer no cash loss although they would probably suffer loss of time. The aims of the dissertation are twofold. The first is to provide a numerical analysis of how urban commuters with differing abilities to pay would respond to additional road user charges. The welfare implications of such differential responses are examined and their policy implications analysed. The second aim is to develop a practical framework to model congestion pricing policies in the context of heterogeneous users. To achieve these aims, the following objectives have been set: (a) Using a simple network with two parallel competing routes, determine both welfare maximising and revenue maximising tolls under the constraint that only one route can be priced. In this setting, determine the allocation of traffic between the alternative routes, the efficiency gain, the revenue, the changes in travel cost and the distributional effects. (b) Establish a realistic model of an actual urban area to examine the impacts of selectively tolling congestible routes. As in the simple network case, assess the effects of toll policy on traffic distribution, network efficiency, revenues, and the welfare of the individual consumer and society. (c) Evaluate whether the non-identical treatment of users will enhance the acceptability of congestion pricing as a transport policy. Results from the simulations indicate that non-identical treatment of drivers? responses to toll charges provides better understanding of the differential impacts of various pricing policies. Allowing for heterogeneity in time valuation provides a better assessment of the efficiency of pricing policies and of the welfare impacts of toll charges, as it is able to capture their differential effects. More importantly, it shows that low-income commuters may not be significantly worse off with pricing especially when there is a free alternative route. This research demonstrates the need to adopt appropriate analytical techniques and assumptions when modelling the traffic equilibrium in a network with tolls. These include relaxing the homogeneity assumption, examining sensitivity to supply function parameter values and to the effect of vehicle operating cost, and using a route rather than link based measure of consumer surplus

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / "May 2008" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Thesis (M.A.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 57-58).

Zugl.: Berlin, Humboldt-Univ., Diss., 2008

Thesis (M.A.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 57-58). Also available in print.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 101-125).

Le virus de l'hépatite B (HBV) infecte chroniquement près de 240 millions d'individus dans le monde. L'infection chronique par HBV est un souci de santé publique majeur puisque l'infection peut évoluer au cours du temps vers la cirrhose et/ou l'hépatocarcinome (CHC). Malgré l'existence de traitements efficaces à base d'analogues de nucléos(t)ides permettant de diminuer la charge virale chez les patients, ceux-ci nécessitent une prise médicamenteuse à vie. En effet, malgré la diminution importante du risque de développer un cancer du foie, ces traitements ne permettent pas l'élimination définitive du virus. Les cellules infectées par HBV sont les hépatocytes du foie, qui remplissent la majorité des rôles vitaux de cet organe. La formation d'un minichromosome viral au sein de ces cellules infectées appelés ADNccc (pour ADN circulaire-covalemment-clos), est majoritairement responsable de la persistance du HBV. Les traitements actuels utilisés sont principalement des analogues de nucléos(t)ides et ceux-ci n'ont pas ou peu d'effets sur l'ADNccc. La nécessité de développer de nouvelles stratégies antivirales visant à éliminer définitivement HBV a donc conduit de nombreux laboratoires, dont le nôtre, à étudier l'utilisation de stratégies immuno-thérapeutiques incluant des stimulateurs de l'immunité innée (agonistes de TLR7, TLR8, RIG-1.) dans le cadre d'infections chroniques. De nombreuses études ont démontré que l'utilisation de ligands stimulant les récepteurs de l'immunité innée promouvait un fort effet antiviral, médié par la production endogène et locale de cytokines pro-inflammatoires et l'induction de gènes régulés par l'interféron (1SG). Dans ce but, nous nous sommes intéressés plus particulièrement aux potentiels effets antiviraux de l'agonisation des senseurs de l'immunité innée les plus connus, les Toll-like récepteurs (TLR), dans le cadre de l'infection par HBV dans les cellules hépatiques. La stratégie immuno-thérapeutique envisagée, vise à stimuler aussi bien les cellules immunitaires que les hépatocytes infectés. La caractérisation de l'expression de différents senseurs de l'immunité innée, d'une part dans les cellules primaires isolées du foie et d'autre part dans certaines lignées cellulaires correspondantes, nous a permis d'avoir une vue d'ensemble 1) des récepteurs exprimés par les différentes cellules du foie notamment dans les hépatocytes (TLR2/TLR3/TLR4/TLR5) ; 2) d'évaluer la fonctionnalité de ceux-ci pour la production de cytokines (IL-6 ; IP-10) lors de leur agonisation 3) d'évaluer les modèles disponibles parmi les lignées cellulaires les plus proches immunologiquement des cellules hépatiques. Les cellules HepaRG et une nouvelle lignée dérivée des macrophages du foie les iKC par exemple sont plus proches respectivement des hépatocytes et des macrophages primaires hépatiques et sont donc des modèles relevant pour les études immuno-thérapeutiques. L'utilisation de ligands de TLR2 et TLR3 sur des hépatocytes infectés chroniquement par HBV, a montré le plus fort effet antiviral (incluant une médiation par la sécrétion de cytokines et l'induction d'1SG) aussi bien sur la réplication d'HBV que sur l'ADNccc. De plus, cet effet semble stable au cours du temps sans résurgence massive de productions virales. Cette stratégie cible non seulement les hépatocytes infectés, mais également les cellules immunitaires dont les productions cytokiniques ont également un fort effet antiviral. Bien que l'effet in vivo, dans un modèle murin, ait été plus modeste, un ajustement des doses d'agonistes utilisées ainsi qu'un meilleur moyen de délivrance au foie de ligands de TLR2 ou TLR3 pourraient être une stratégie immuno-thérapeutique intéressante. Enfin nous nous sommes intéressés au cas particulier de l'agonisation du TLR9 en présence d'HBV… [etc] / HBV chronically infects 240 million peoples around the world. HBV chronic infection is a major public health problem and can lead to cirrhosis or/and hepatocarcinoma (HCC). Even if some efficient treatments are already available, based in particular on the use of nucleos(t)ides analogues that induce a decrease of viral load in patients, these drugs do not lead to a definitive HBV cure They enable an important decrease of liver cancer risk but need to be taken life-long. HBV infects hepatocytes the major liver cells which are involve in many vital mechanisms into the organism. The HBV minichromosome, which is formed into infected cells also called cccDNA (i.e., covalently-closed-circular DNA), is not affected by nucleos(t)ides treatments and thus is responsible for HBV persistence. The use of immune receptors (e.g. Toll-like receptors/TLR) agonists can lead to 1) an important cytokines/interferon (IFN) secretion; 2) promote immune cells activation/recruitment and 3) induction of many Interferon-Stimulated Genes (ISG). These mechanisms could lead to a greater viral clearance by cccDNA degradation or silencing. The need for new strategies to permanently eliminate HBV infection led many laboratories, including ours, to explore the use of immunotherapeutic treatments in a context of chronic infection, including innate immune stimulators (e.g. TLR7, TLR8 or RIG-I agonist are under clinical trials). To this end, we got interested on the potential anti-HBV effects of many TLR agonists in liver cells. Our strategy is to stimulate both infected hepatocytes and immune cells. We first characterized the expression of innate immune sensors in primary liver cells as well as in some liver cell lines. This allowed us to: 1) identify which sensors are expressed by liver cells, especially in hepatocytes (TLR2, TLR3, TLR4, TLR5); 2) evaluate their ability to produce cytokines (IL-6, IP-10) upon agonisation; 3) evaluation of cell lines model which are immunologically closed to the primary liver cells. HepaRG and a new liver macrophage cell line call iKC are immunologically close to their primary cells and appear to be relevant models for immune-therapeutics studies. The use of TLR2 and TLR3 agonists on HBV chronically infected hepatocytes showed a strong antiviral effect (i.e., decrease of HBV replication and cccDNA level) mediated directly by NF- kB-inducible and ISG genes activation and indirectly by cytokines secretion. Furthermore, this effect was shown stable over time without any viral replication rebound. This strategy targets not only infected hepatocytes but also immune cells, whose cytokines production also has a strong antiviral effect. Despite a weak in vivo effect in mice, a tuning in agonist doses used and better liver delivery could be an interesting immune-therapeutic strategy. Finally, we were investigated the particular case of TLR9 agonisation in presence of HBV. We showed an interaction between synthetic or not DNA ligands such as CpG ODN and HBV particles. This interaction leads in one hand, to HBV entry inhibition in hepatocytes, on the other hand, to a blockage of ligand delivery to TLR9 in pDC, which is not due to an inhibition of the TLR9 pathway, but to a lack of access of the ligand to its receptor. These two mechanisms are responsible for a decrease of viral infection during its establishment and a decrease in IFN synthesis by pDC, respectively. A decrease in IFN production, which this time was linked to a bona fide inhibition of the TLR9 pathway, in the presence of the sub-viral particles HBsAg was still observed, without retention of TLR9 ligand of the latter. It would seem, therefore, that use of TLR agonists represent an interesting strategy in setting up new anti-HBV immune-therapeutic approaches. However, their improvement will depend on the evaluation of viro-induced inhibitory mechanisms as well as better ways of in vivo delivering these ligands