Equine herpesvirus-1 (EHV-1) is one of the most important and prevalent viral pathogens of horses causing a major threat to the equine industry throughout most of the world. EHV-1 primarily causes respiratory disease but viral spread to distant organs enables the development of more severe sequelae; abortion and neurologic disease. In order to produce disease, EHV-1 has to overcome the innate barrier of the type-I interferon (IFN) system in host cells. However, the underlying mechanisms employed by EHV-1 to circumvent the type-I IFN response in host cells are not well understood. In this project study, using molecular techniques, we explored how EHV-1 is able to escape the type-I IFN response in host cells during infection. We also investigated whether EHV-4, a closely related but less pathogenic virus, has similar effects on type-I IFN as a clue to understanding how widespread IFN suppressive function is found among equine alphaherpesviruses. Our data showed that inhibition of the type-I IFN response in host cells is not a function of neuropathogenicity of EHV-1 strains. However, a reduced type-I IFN response correlated with pathogenicity as EHV-4, unlike EHV-1, was unable to down-regulate the type-I IFN response in equine endothelial cells (EECs). Investigation of the mechanisms employed by EHV-1 to suppress type-I IFN revealed that the virus sequentially prevented outside-in signaling events that lead to type-I IFN production. Specifically, EHV-1 blocked the expression of Toll-like receptors (TLR) 3 and TLR4 at 6 hours post-infection (hpi) and 12 hpi. EHV-1 also prevented the transcription of IRF7 and IRF9 at different time-points during infection. The virus also perturbed the JAK-STAT signaling pathway by negatively regulating the cellular levels of TYK2 and phosphorylation-mediated activation of STAT2 molecules. Immunofluorescence data revealed that during infection, EHV-1 was able to sequester STAT2 molecules from nuclear translocation. This may be a limiting step preventing the formation of interferon- stimulated gene factor 3 (ISGF3) whose nuclear translocation is required to transactivate interferon-stimulated genes (ISGs) including IRF7. Further investigation showed that unlike EHV-1, EHV-4 only interfered with phosphorylation-mediated activated STAT1 and STAT2 molecules at 3 and 6 hpi. EHV-4 was unable to block TLR3/4 and IRF7/9 mRNA expression at any time-point. Intriguingly, while viral late gene of EHV-1 mediates inhibition of STAT phosphorylation, our data showed that for EHV-4, a virus late gene did not mediate the inhibition of STAT phosphorylation. The findings from this study help illuminate how EHV-1 strategically interferes with limiting steps required for type-I IFN response in host cells to promote pathology. Our data also strengthen the hypothesis that the ability to shut off host factors required for type-I IFN production might be directly related to the degree of pathogenicity of the EHV subtypes.

The estimation of the Pareto distribution can be computationally expensive and the method is badly biased. In this work, an improved Least Squares derivation is used and the estimation will be less biased. Numerical examples and figures are provided so that one may observe the solution more clearly. Furthermore, by varying the different methods of estimation, a comparing of the estimators of the parameters is given. The improved Least Squares derivation is confidently employed for it is economic and efficient.

La protection contre certains agents infectieux ainsi que le traitement de cancers nécessite l'induction d'une réponse cellulaire. Le développement de vaccins induisant une réponse T CD8 efficace est donc essentiel. La présentation croisée de l'antigène est importante pour l'activation de lymphocytes T CD8 spécifiques et de nombreux facteurs participent au développement d'une réponse lymphocytaire T efficace. Nous nous sommes intéressés à deux d'entre eux : la voie d'immunisation et la séquence d'aministration de l'antigène et d'un adjuvant. Nous avons développé une technique d'enrichissement des lymphocytes T CD8 spécifiques d'un antigène, ce qui a permis une étude précise de la réponse T CD8 endogène. Nous avons ensuite étudié l'influence de la voie d'immunisation sur l'efficacité de la réponse T CD8. Nous avons observé que l'injection intradermique d'un antigène cellulaire induit une réponse T CD8 plus tardive, comparée à une administration par voie systémique. Cependant, la réponse T CD8 induite par une injection locale de l'antigène est plus efficace. Puis, nous avons évalué l'influence de l'administration d'un adjuvant - le poly I:C connu pour induire la production d'interférons (IFN) de type I - en parallèle de celle de l'antigène. Nous avons montré que le moment optimal d'administration de l'adjuvant dépend de la voie d'immunisation. De plus, il existe une durée limitée durant laquelle l'adjuvant induit des effets positifs sur l'activation des lymphocytes T CD8. Nous avons identifié plusieurs effets du poly I:C et des IFN de type I sur les cellules du système immunitaire

The expanding field of tissue engineering provides a new approach to regenerative medicine for common ailments such as cardiovascular disease and type-I diabetes. Biomaterials can be administered as a delivery vehicle to introduce therapeutic cells to sites of damaged or diseased tissue. A specific class of biomaterials, termed hydrogels, is suitable for this application as they can provide a biocompatible, biodegradable scaffold that mimics the physical properties of the native soft tissue. Injectable hydrogels are increasingly being developed for biomedical applications due to their ability to be delivered in a minimally invasive manner. One potential use for such materials is in the delivery of therapeutics such as cells or growth factor-releasing particles. In this study, the first aim was to determine the interactive effects between collagen-based hydrogels and additives (cells and microspheres) for cardiac regeneration. The results demonstrated that the addition of either cells or microspheres to a collagen-based hydrogel decreased its gelation time and increased its viscosity. Increased cross-linker concentrations resulted in lower cell viability. However, this cell loss could be minimized by delivering cells with the cross-linker neutralizing agent, glycine. As a potential application of these materials, the second aim of this study was to develop a hydrogel for use as an ectopic islet transplant site. Specifically, collagen-chitosan hydrogels were synthesized and characterized, with and without laminin, and tested for their ability to support angiogenic and islet cell survival and function. Matrices synthesized with lower chitosan content (20:1 collagen:chitosan) displayed greater cell compatibility for both angiogenic cells and for islets and weaker mechanical properties, while matrices with higher chitosan content (10:1 collagen:chitosan) had the opposite effect. Laminin did not affect the physical properties of the matrices, but did improve angiogenic cell and islet survival and function. Overall the proposed collagen-based hydrogels can be tailored to meet the physical property requirements for cardiac and islet tissue engineering applications and demonstrated promising cell support capabilities.

B virus (Cercopithecine herpesvirus 1), which causes an often fatal zoonotic infection in humans, shares extensive homology with human herpes simplex virus type 1 (HSV-1). The ƒ×134.5 gene of HSV-1 plays a major role in counteracting dsRNA-dependent protein kinase (PKR) activity. HSV-1 Us11 protein, if expressed early as a result of mutation, binds to PKR and prevents PKR activation. The results of experiments in this dissertation revealed that although B virus lacks a ƒ×134.5 gene homolog, it is able to inhibit PKR activation, and subsequently, eIF2ƒÑ phosphorylation. The initial hypothesis was that B virus Us11 protein substitutes for the function of ƒ×134.5 gene homolog by blocking cellular PKR activation. Using western blot analysis, Us11 protein (20 kDa) of B virus was observed early following infection (3 h post infection). Expression of B virus Us11 protein was not blocked by phosphonoacetic acid (PAA), an inhibitor of DNA replication, confirming Us11 is not a ¡§true late¡¨ gene of B virus as it is in HSV-1. Analysis of these results suggested that B virus Us11 protein compensates for the lack of the ƒ×134.5 gene homolog and prevents PKR activation. Next, the results demonstrated that B virus Us11 recombinant protein prevented PKR activation by dsRNA in vitro. A B virus Us11 protein stable expression cell line (U373-BVUs11) was established to investigate whether Us11 protein inhibited PKR activation in vivo. Experiments revealed that B virus Us11 protein stably expressed in U373 cells prevented PKR activation and subsequent eIF2ƒÑ phosphorylation induced by the infection of these cells with ƒ´ƒ×134.5 of HSV-1. As the consequence of preventing PKR activation and subsequent eIF2ƒÑ phosphorylation, B virus Us11 protein complemented ƒ´ƒ×134.5 HSV-1 in U373 cells as evidenced by restoration of virus protein synthesis and replication in U373 cells. Furthermore, pull-down assays showed that B virus Us11 protein binds to PKR. In addition, the results demonstrated that B virus Us11 protein stably expressed in U373 cells counteracted the inhibiting effect of IFN-ƒÑ on HSV-1 replication by preventing PKR activation. These data suggested that B virus and HSV-1, two closely related viruses, use different mechanisms to counteract PKR activity.

<p>When bacterial pathogens infect their hosts, they illicit responses intended on containing and eliminating these invaders. This happens not only on the organismal level, but also on the cellular level. When a cell detects that it has been infected by an intracellular pathogen, it triggers a set of internal signaling events intended to contain the intruder. These events may allow the cell to produce antimicrobial agents or may help recruit members of the immune system to help fight the infection. In the case of closely evolved pathogens, such cell signaling events can be co-opted by the invading bacteria to its advantage. One example of this is infection with the gram-negative bacteria Chlamydia trachomatis. Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase Ras and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA2 are activated and are important for the onset of inflammatory responses during chlamydial infection. In this study we tested if activation of ERK and cPLA2 occurred as a result of Ras signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. we provide genetic and pharmacological evidence that Ras, ERK and, to a lesser extent, cPLA2 activation are uncoupled during infection, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA2 signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells cPLA2, and to a lesser extent ERK, signaling played a significant protective role against C. trachomatis. we determined that cPLA2-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of &#946; interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Overall, these findings define a previously unrecognized role for cPLA2 in the induction of autonomous innate immune responses to Chlamydia infections.</p> / Dissertation

Due to its relative avascularity, low cellularity and lack of an undifferentiated cell reservoir, articular cartilage has a limited capacity for self-repair when damaged through trauma or disease. Articular cartilage impairment and the resultant reduced joint function affects millions of people at a substantial cost. In the U.S. alone, over 20 million adults are afflicted with osteoarthritis, costing more than $65 billion per year in health care and lost wages. Surgical techniques have been developed to address small, focal lesions, but more critical sized defects remain without a viable solution. Tissue engineering strategies produce cartilage-like constructs in vitro containing living cells in the hope of replacing damaged cartilage and restoring joint function. However, these constructs lack both sufficient integration into the surrounding tissue following implantation and the mechanical properties capable of withstanding the demanding and complex in vivo loading environment. Our central hypothesis is that exposure of engineered cartilage to fluid-induced shear stress increases the collagen content and mechanical properties (tensile and compressive). The overall objective of this project is to modulate the matrix composition and mechanical properties of engineered cartilage to be more like native tissue using a novel bioreactor. Improving the matrix components and mechanical stability of the tissue to be more similar to that of native tissue may aid in integration into a defect in vivo. The central hypothesis was proven in that shear stress potently altered the matrix composition, gene expression and mechanical properties of both thick and thin engineered cartilage. Modulation was found to be highly dependent on shear stress magnitude, duration, and waveform and affected different matrix constituents and mechanical properties in disparate ways. Our overall objective was satisfied on the basis that the bioreactor created stronger engineered tissues, but with the caveat that the tissues showed an increase in presence of type I collagen. Such an effect would be undesirable for articular cartilage engineered tissues, but could be very beneficial in fibrocartilaginous tissues such as that found in the temporomandibular joint. In conclusion, the novel bioreactor system provides a flexible platform technology for the study of three-dimensional engineered tissues, not just articular cartilage.

Taiwan public museums and print medias hold many ¡§blockbusters¡¨ one after another since 1990. This trend started from US then spread to all over the world. The considerable amount of visitors not only solved the financial problem, but also built the field for print media chasing the cultural capital and developing new business model. We can generalize this trend to three concepts, first, Issues of the Administrative Corporation of National Museum, second, the trend of museum marketing, third, new museology. Those concepts break the ossified museum organization system and the icy impression. However, a stream of people and money will bring some doubts, such like exhibition quality goes down, over commercialize, and unprofessional. Most of exhibition assessments about museum are overall assessments which include a wide range. But it's not applicable to blockbuster that happens only once, tours around, and needs inter-organizational cooperation. This study is a case study of ¡§Da Vinci Travelling Exhibition¡¨ which is held by United Daily News Group, National Chiang Kai-shek Memorial Hall, and National Science and Technology Museum. The research design in this study is based on ¡§Conceptual Model of Service Quality¡¨ and ¡§Extended Model of Service Quality¡¨ which was brought out by Parasuraman, Zeithmal, & Berry. This study counts out the coefficients which existed in the service quality gap theory, then, establish regression. With the regression, we can judge which service gap character will effect customer¡¦s satisfaction of service quality. So that we can point out the way how to improve blockbuster's service quality and build an exhibition assessment driving from customer. This study also investigated the relation among demographic variables and customer¡¦s satisfaction of exhibition service quality. Three findings are revealed. First, demographic variables have significant effect on customer¡¦s attitude toward blockbuster service quality. Second, except for gap character 1(managerial perception), gap character 2(managerial standard)¡Bgap character 3(service performance) and gap character 4(external communication) has a function relationship to the gap 5 was confirmed. Third, Use service gap theory to establish an exhibition service quality assessment is possible.

Reproduction de : Thèse doctorat : Médecine. Biochimie et biologie moléculaire : Reims : 2005. / Titre provenant de l'écran-titre. Bibliogr. p 205-227.

Reproduction de : Thèse doctorat : Médecine. Biochimie et biologie moléculaire : Reims : 2007. / Titre provenant de l'écran-titre. Bibliogr.