No description available.

Glioblastomas are highly vascular brain tumors that are characterized as heterogeneous, comprised of an anatomically and functionally irregular blood brain barrier (BBB) that contributes to variable drug distribution and possibly associated pharmacodynamics (PD) responses. Standard pharmacokinetic (PK) approaches that are based on whole tumor homogenates and accordingly averaged drug concentrations are limited in their ability to depict regional variations in drug concentrations, and could lead to faulty assessments of drug distribution and activity. Given the paucity of quantitative information on intratumoral PK/PD variability of anticancer drugs, the goal of this project was to characterize the regional PK and PD properties of gefitinib, an EGFR inhibitor, in brain tumors, in the context of biological characteristics, and use the information to develop mechanistic PK/PD models that may be valuable to understand why some anticancer drugs are inactive. Towards this end, in vitro cytotoxicity assays and pilot in vivo studies were first conducted and identified U87VIII mutant cell line as a gefitinib sensitive orthotopic brain tumor model with suitable growth characteristics to allow for immunohistochemical (IHC) analysis of tumor biological characteristics for further studies. Subsequently, in vitro PD studies identified phosphorylated-ERK1/2 (pERK) as a PD marker for this cell line. Thereafter, to set up a framework to obtain intratumoral PK/PD information in vivo, a novel tumor sectioning protocol was devised and sensitive and robust PK (LC-MS/MS) and PD (Meso Scale Discovery, MSD, electrochemiluminescence-based assay) methods were developed to enable the use of minimal amounts of tumor samples to assess the intratumoral PK and PD characteristics of gefitinib. Mice bearing orthotopic U87VIII mutant tumors were administered gefitinib at doses of 150 mg/kg and 50 mg/kg orally (p.o.), followed by collection of plasma and tumor samples at various time points, based on a serial sacrifice study design. Serial tumor sections were obtained in four distinct regions according to the aforementioned protocol for PK, PD and IHC measurements. An IHC index called microvessel pericyte index (MPI), a measure of BBB integrity, reflected the variability in gefitinib brain tumor concentrations, and was used to bin the data to generate three intratumoral PK/PD data sets. These data sets were then used to develop a set of hybrid physiologically based PK/PD models accounting for variable BBB permeability within the tumor. Each model consisted of a forcing function describing plasma concentration profile, a tissue compartment to represent the drug disposition within the tumor, and target-response compartments for the PD model. The intratumoral variation in the PKs of gefitinib was accurately described by the MPI classifications and ranged about 2-fold, and was responsible for the associated PD variability. In summary, using a novel tumor sectioning protocol and sensitive analytical methods in an orthotopic glioblastoma (GBM) model, the intratumoral variability of gefitinib PK/PD could be binned according to BBB integrity and enabled the development of a mechanistic hybrid physiologically based PK/PD models, which provides a means to assess the influence of tumor heterogeneity on drug response. / Pharmaceutical Sciences

A lo largo del tiempo, con el incremento de la población mundial y la necesidad de producción de mas energía para satisfacer las necesidades del día a día, ha habido un creciente interés a nivel mundial por encontrar soluciones al problema de la eliminación de óxidos de nitrógeno (NOx), y otros residuos gaseosos, en fuentes móviles y estacionarias ya que estos gases son contaminantes atmosféricos que contribuyen a la formación de la lluvia ácida y de la niebla tóxica fotoquímica. La aplicación de los catalizadores de tres vías ha permitido controlar con éxito la emisión de gases de escape de motores que consumen naftas como combustible. La actual generación de estos catalizadores usa principalmen-te Pd como metal activo para las reacciones de oxidación y reducción, recurriéndose al agregado de promotores para aumentar su eficiencia en la eliminación de NOx en condicio-nes oxidantes y en la operación a baja temperatura. La re-ducción de NOx con CO es una de las principales reacciones que tiene lugar sobre los mencionados catalizadores, recurrién-dose al agregado de óxidos reducibles como Cu, Cr, MoOx, La2O3 y principalmente CeOx para aumentar su efectividad. El notable aumento de la velocidad de reacción con el agregado de CeOx se ha atribuido a un aumento de la capacidad de di-sociación del NO, como así también a la mayor activación del CO, resultante de la interacción Pd-Ce. Por lo tanto, la locali-zación del Ce con relación al Pd, es un tema de considerable importancia. La influencia del Ce y el V en la actividad, selecti-vidad y estabilidad del Pd para la reacción NO + CO a 300 oC ha sido motivo de estudio en esta tesis. Para este trabajo se han preparado catalizadores con cargas de Pd y Ce inferiores al 1 %, agregando el promotor al catalizador Pd/g-Al2O3, mientras que la influencia del V se ha estudiado sobre una muestra donde el Pd fue incorporado a una alúmina dopada previamente con el promotor. En la caracterización de las muestras frescas y usadas hemos aplicado las técnicas de AA, ICP, quimisorción de H2, TEM, XPS, TPR y FTIR de CO y NO adsorbido. En general, los estudios publicados sobre la in-fluencia del Ce en la reacción NO + CO sobre Pd, fueron reali-zados sobre catalizadores con una carga de Ce igual o mayor a la necesaria para formar una monocapa sobre el material soporte, depositada antes del agregado del Pd. También, a diferencia de otros trabajos se han utilizado concentraciones más bajas de NO y CO. Continuando estudios previos de nues-tro laboratorio, se ha investigado el efecto de VOx como óxido reducible, demostrándose que conduce a una disminución de la selectividad a N2 favoreciendo la formación de N2O. Este comportamiento se ha explicado por la existencia de un esta-do de oxidación del Pd en condiciones de reacción, detectado por FTIR y XPS, que no se presenta en ausencia del promo-tor. Si bien la reacción de reducción de NO con CO es aparen-temente simple, desde el punto de vista de las etapas elemen-tales requeridas para la formación de N2, N2O y CO2, a baja temperatura se forman especies adsorbidas estables a partir de las cuales se generan productos secundarios. La identifi-cación de dichas especies y productos, y su influencia en la actividad de los catalizadores se ha logrado mediante la aplicación de espectroscopía FTIR a la caracterización de los catalizadores en condiciones de reacción y el correspondiente análisis de la fase gas. El estudio de las especies adsorbidas en condiciones de reacción se efectuó alimentando a la celda de IR la corriente de salida del reactor a 300 oC, la cual cir-culó en contacto con una pastilla de catalizador. Es bien co-nocido que la reacción NO + CO da lugar a la formación de la especie isocianato, que puede ubicarse sobre el soporte y/o sobre el metal pero su participación en la selectividad de la reacción global es aún motivo de discusión. Como se demues-tra en esta tesis otras especies adsorbidas estables son generadas a partir del isocianato con la participación de los grupos OH del material soporte, como el ácido isociánico (HNCO). La posterior hidrólisis de este ácido da lugar a la formación de NH3, proceso que se intensifica en presencia de H2O. Poniendo especial atención al proceso de formación descomposición de las especies adsorbidas como así también a su reactividad con NO se ha comprobado que dichas especies no participan de la formación de N2O sobre Pd. / During these times, together with the world population increase and the consequently mounting needs of energy production to satisfy the demands of day after day, there has been an upsurge of interest worldwide in finding solutions to the problem of the elimination of gaseous contaminants. Nitrogen oxides (NOx), and other gaseous residues, derived from mobile and stationary sources are atmospheric pollutants that contribute to the formation of the acid rain and the toxic photochemical fog. The application of three way catalysts has allowed us to control successfully the gas emissions of gasoline engines. The current generation of these catalysts uses mainly Pd as active metal for the reactions of oxidation and reduction, with the addition of promoters to increase their efficiency for NOx s elimination under oxidizing conditions in the low temperature range.NOxs inreduction with CO is among the main reactions that take place on the mentioned catalysts, with the addition of reducible oxides as Cu, Cr, MoOx, La2O3 and principally CeOx. The notable increase of reaction rate with CeOx addition has been attributed to a higher NO dissociation activity and to CO activation due to a PdCe interaction. Therefore, Ce location related to Pd is a topic of considerable importance. The influence of Ce and V on the activity, selectivity and stability of Pd for the NO + CO reaction at 300 C has been studied in this thesis. In this work catalysts with Pd and Ce loadings lower than 1 % have been prepared by adding the promoter to the Pd/g-Al2O3 catalyst, whereas V influence has been studied on a sample where Pd was incorporated into an alumina previously doped with the promoter.In the published literature about the influence of Ce on the NO + CO reaction over Pd, the catalysts had a Ce amount higher than the quantity needed to form a monolayer, which was added before Pd. Another difference with other studies is the use of lower concentrations of CO and NO in the reactive mixture. For the characterization of fresh and used samples the techniques of AA, ICP, H2 chemisorption, TEM, XPS, TPR and FTIR of adsorbed CO and NO were used. As a continuation of our previous laboratory studies, the effect of VOx as reducible oxide has been investigated, in order to demonstrate that it leads to a decreasing N2 selectivity, thus favoring the N2O formation. This behavior has been explained by the existence of an oxidized Pd state under reaction conditions, which was detected by FTIR and XPS measurements; this state is not observed if the promoter is absent. Although the reaction of NO reduction with CO is apparently simple from the viewpoint of the elementary steps involved in N2, N2O and CO2s formation, at low temperature adsorbed stable species are formed thus leading to secondary products. The identification of the above mentioned species and products and the determination of its influence in the catalysts activity have been carried out by the application of FTIR spectroscopy for the characterization of the catalysts under reaction conditions together with the corresponding analysis of the gas phase. The study of the species adsorbed under reaction conditions was accomplished by feeding the stream emerging from the reactor at 300o to the IR cell that contained a catalyst disk kept at the same temperature. It is well-known that the NO + CO reaction on supported metals gives place to the formation of isocyanate species, which can be located on the support and/or on the metal although its participation in the selectivity of the global reaction is still under discussion. As demonstrated in this thesis, other adsorbed stable species are generated from the isocyanate by the participation of -OH groups of the material support, like isocyanic (HNCO) acid. The hydrolysis of this acid gives place to NH3 formation, which is a process that is enhanced by the H2O presence. Paying special attention to the process of formationdecomposition of the species adsorbed and also to their reactivity with NO, it was verified that the adsorbed species do not take part in N2O formation on Pd.

The role of Public Diplomacy (PD) in conducting foreign relations has emerged as a critical component for modern statecraft, foreign policy and arguably for the state’s global existence. With the world now more interconnected than ever before, public diplomacy has taken on new meaning and new importance. However, despite these marked shifts, public diplomacy remains under-researched and under-utilised both as a concept and as a vital activity. The complexities that underscore the development and evolution of public diplomacy within a dynamic international milieu therefore warrant renewed attention. The benefits and opportunities that accompany public diplomacy are equally complicated by a number of challenges to its practice. This research identifies and advances a critical understanding of public diplomacy through a theoretically rigorous perspective that accounts for the fluid environment in which it operates, the evolving nature of stakeholders and audiences involved in shaping it, the sweeping impact of global information and communications development, the persistence of cultural divides and conflicts of interests and how they contribute to the lack of strategic frameworks in place to advance public diplomacy practice. This thesis investigates Qatar as a case study due to its international stature and influence despite it being a small geographical state. Renowned for its international role as a leading actor in conflict reconciliation and commended for its contributions to international humanitarianism, the ambitious, forward-looking and steadfast foreign policy of Qatar has faced increased pressure in recent years. Qatar has encountered significant challenges in the form of the Nepali workers’ crisis that ensued following its selection as host for FIFA World Cup 2022 and with the diplomatic siege against it by a number of Gulf Cooperation Council (GCC) states. This study critically analyses Qatar’s public diplomacy practice considering these pressing developments and advances an original strategic public diplomacy framework that can assist Qatar in managing and mitigating the effect of these crises on its global image and reputation. This research contributes to enhancing public diplomacy practice within the diplomatic context of statecraft and foreign policy by developing a nuanced and original framework that can be utilised by Qatar and other states to manage and mitigate modern public diplomacy challenges. The thesis utilises a mixed-methods research approach that includes literature reviews, media analyses, interviews and questionnaires. The study contributes to knowledge and practice by advancing research in an understudied field and by developing and implementing an original strategic PD empowerment framework.

Usability has long been considered an important component of an innovation (Norman, 2002), evidenced by the fact that usability research has dominated innovation design efforts for a number of years. However, recent research has shown that satisfying usability is not sufficient for the successful diffusion and adoption of an innovation (Karsh, 2004). To develop an useful innovation, one must understand the mechanisms by which people choose to adopt and use an innovation, as well as how an innovation fits different levels of a socialtechnical system (Karsh, Escoto, Beasley, & Holden,(2006). The goal of this research, therefore, was to develop an innovation analytic and design framework that would enable designers to design a more likely adopted innovation and to validate it through the design and evaluation of a fall-protection training intervention for residential roofing subcontractors. The proposed innovation analytic and design framework was based on the traditional systems-engineering process: Requirement Analysis, Prototype Development, and Summative Evaluation. Rogers’ Theory of Innovation Diffusion and Adoption, as well as Participatory Design, were utilized to obtain a holistic view of technology-adoption challenges and opportunities. The requirement analysis involved the development and use of a questionnaire and semi-structured interviews to identify the contributors of safety technology adoption in small roofing companies, as well as to understand the practices of safety technology adoption and fall-protection training. One hundred and four questionnaires from workers in North Carolina and Virginia were collected, and 29 workers received the follow-up semi-structured interview. Results showed that (1) social influence had a significant impact on the diffusion and adoption of safety technology; (2) workers’ satisfaction with existing safety performance standards/practices, as well as disengagement during available safety training, caused difficulties in implementing regular safety training; (3) management commitment and presentation of good/bad consequences of unsafe behavior were expected to facilitate the rate of adoption of safety technology. Results also identified specific recommendations for a fall-protection training intervention. The prototype development was performed by a six-member Participatory Design (PD) team in a PD workshop, who used the results of the questionnaire and semi-structured interviews to develop a training intervention. Four PD approaches (PICTIVE, Inspiration Card Workshop, Scenario Building, and Future Workshop) were employed in the development of a Personal Fall Arrest System (PFAS) as an industry-specific training intervention. This research also used summative comparative evaluation to assess the developed PFAS training intervention against a standard PFAS training intervention with respect to (1) adoption propensity, (2) expected adoption outcome, and (3) results demonstrability. Eighteen roofing workers were recruited to evaluate and compare the two interventions. The standard PFAS training intervention was developed by two experts using the safety manual published by the National Roofing Contractor Association. Results suggested that (1) the developed PFAS training intervention was more likely to be adopted and easier to diffuse among roofing subcontractors than the standard PFAS training intervention, and (2) use of the developed PFAS training intervention would better improve company's safety performance in comparison to the standard training intervention. Results of the evaluations confirmed the efficacy of the proposed innovation analytic and design framework in designing a more likely adopted innovation. / Ph. D.

Colloidal nanoparticles are widely used for industrial and scientific purposes in many fields, including catalysis, biosensing, drug delivery, and electrochemistry. It has been reported that most of the functional properties and performance of the nanoparticles are highly dependent on the particle size and morphology. Therefore, controlled synthesis of nanomaterials with desired size and structure is greatly beneficial to the application. This dissertation presents a systematic study on the effect of ligands on the colloidal Pd nanoparticle synthesis mechanism, kinetics, and final particle size. Specifically, the research is focused on investigating how the ligand bindings to different metal species, i.e., metal precursor and nanoparticle surface, affect the nucleation and growth pathways and rates and connecting the binding thermodynamics to the kinetics quantitatively. The first part of the work (Chapters 4 and 5) is establishing isothermal titration calorimetry (ITC) methodology for obtaining the thermodynamic values (Gibbs free energy, equilibrium constant, enthalpy and entropy) of the ligand-metal precursor binding reactions, and the simultaneous metal precursor trimer dissociation. In brief, the binding products and reactions were characterized by nuclear magnetic resonance (NMR), and an ITC model was developed to fit the unique ITC heat curve and extract the thermodynamic properties of the reactions above. Furthermore, in Chapter 6, the thermodynamic properties, especially the entropy trend changing with the ligand chain length was investigated on different metal precursors based on the established ITC methodology, showing that the entropic penalty plays a significant role in the binding equilibrium. The second part of the dissertation (Chapter 7 and 8) presents the kinetic and mechanistic study on size-tuning of the colloidal Pd nanoparticles only by changing different coordinating solvents as ligands together with the trioctylphosphine ligand. In-situ small angle X-ray scattering was applied to characterize the time evolutions of size, size distribution, and particle concentration using synthesis reactor connected to a capillary flow cell. From the real-time kinetic measurements, the nucleation and growth rates were calculated and correlated with the thermodynamics, i.e., Gibbs free energies of solvent-ligand-metal precursor reactivity and ligand-nanoparticle surface binding which were modified by the coordination of different solvents. Higher reactivity leads to faster nucleation and high nanoparticle concentration, and stronger solvent/ligand-particle coordination energy results in higher ligand capping density and slower growth. The interplay of both effects reduces the final particle size. Furthermore, because of the significance of the ligand-metal interactions, the synthesis temperature and ligand to metal precursor ratio were systematically to modify the relative binding between the ligand and precursor, and the ligand and nanoparticle, and determine the effect on the nucleation and growth rates. The results show that the relative rates of nucleation and growth is critical to the final size. A methodology for using the in-situ measurements to predict the final size by developing a kinetic model based is discussed. / Doctor of Philosophy / Metal nanoparticles dispersed in solution phase, i.e., colloidal nanoparticles, are of great scientific interests due to their unique properties different from bulk metal materials. The size, shape and other morphology features can largely affect the nanomaterial properties and functional performances. Therefore, a successful synthesis of nanoparticles with desired structures is highly beneficial to the development of their application. Ligands, which are long-chain molecules that can cap on the surface of the nanoparticles, have been known as stabilizers of the nanoparticles in the solution phase. Whereas in recent studies, it has been found that changing the ligand type and concentration in the synthesis can result in different sizes and shapes of nanomaterials, which indicates that the ligands are playing critical roles in the synthesis mechanisms to control the kinetics. To have a better understanding on the control effects of the ligands, systematic studies were conducted on the ligand interactions (bindings) between the ligand-metal compound (as the metal source and initial agent in the nanomaterial synthesis) and ligand-nanoparticle surface, of which both can be quantified by thermodynamics. Using isothermal titration calorimetry, the ligand-metal precursor binding strength was measured and found to be dependent on ligand chain length and the metal precursors, which further affects the reactivity of the metal precursor based on the results of density functional theory calculations. On the other hand, the ligand-nanoparticle surface binding strength was found to affect the capping density of the ligands on the nanoparticle surface. In order to connect the thermodynamics to the kinetics, namely the nucleation (formation of new particles) and growth (particle size increase) rates, small angle X-ray scattering (SAXS) characterization was performed in real time during the synthesis on the nanoparticles. This technique allows the capture of the size, size distribution and concentration of nanoparticles changing with time, and the nucleation and growth rates were further calculated from the SAXS data. By changing solvents with the same functions of ligands but of different coordinating abilities, a correlation between the kinetics and thermodynamics was observed. The nucleation rate increases with the metal precursor reactivity, which corresponds to stronger solvent binding to the precursor. On the other hand, the stronger ligand-nanoparticle binding slows down the growth by lowering the surface capping density. To go deeper into the ligand-metal binding and kinetics correlation, the binding properties were tuned by changing other synthesis conditions, i.e., different temperatures and ligand to metal ratios (ligand concentration), and a qualitative discussion was given on the effects of these conditions on the synthesis kinetics and final particle size.

In this work, we first analyze the behavior of the acoustic wave from the theoretical point of view using a simplified 1-dimensional model. The model was developed based on the conservation of mass, the conservation of momentum, and the state equation; in addition, the fluid medium obeys Stokes assumption and it is homogeneous, adiabatic and isentropic. Experiment and simulation results show consistency to theoretical calculation. The second part of this thesis focuses on the PD signal analysis from an on-site PD measurement of the in-house design fiber optic sensors (by Virginia Tech, Center for Photonics Technology). Several commercial piezoelectric transducers (PZTs) were also used to compare the measurement results. The signal analysis employs the application of wavelet-based denoising technique to remove the noises, which mainly came from vibration, EMI, and light sources, embedded in the PD signal. The denoising technique includes the discrete wavelet transform (DWT) decomposition, thresh-holding of wavelet coefficients, and signal recovery by inverse discrete wavelet transform. Several approaches were compared to determine the optimal mother wavelet. The threshold limits are selected to remove the maximum Gaussian noises for each level of wavelet coefficients. The results indicate that this method could extract the PD spike from the noisy measurement effectively. The frequency of the PD pulse is also analyzed; it is shown that the frequencies lie in the range of 70 kHz to 250 kHz. In addition, with the assumed acoustic wave propagation delay between PD source and sensors, it was found that all PD activities occur in the first and third quadrant in reference to the applied sinusoidal transformer voltage. / Master of Science

No description available.

L’infection par le VIH-1 est caractérisée par une déplétion progressive des cellules T CD4+ ainsi que par un dysfonctionnement des cellules T qui, en l’absence de traitements anti-rétroviraux, conduit inéluctablement à la progression de la maladie vers le stade SIDA. Certains des mécanismes impliqués dans ce dysfonctionnement de la réponse cellulaire T ont été élucidés et ont révélé un rôle important de la molécule PD-1 dans l’exhaustion des cellules T en phase chronique de l’infection. En effet, des niveaux élevés de PD-1 ont été associés à une charge virale élevée ainsi qu’à une diminution de la production de cytokines et de la capacité de proliférer des cellules T spécifiques du virus. De plus, bloquer in vitro l’interaction de PD-1 avec son ligand PD-L1 en utilisant un anticorps bloquant rétabli la fonction de ces cellules. De façon intéressante, notre groupe ainsi que d’autres équipes, ont montré que l’expression de PD-1 était non seulement augmentée sur les cellules spécifiques de l’antigène mais aussi sur les cellules T totales. Cependant, peu de choses sont connues quant à l’impact de l’expression de PD-1 sur le renouvellement et la différenciation des cellules T qui expriment PD-1, et ce au cours de l’infection. L’expression de PD-1 n’a notamment pas été étudiée en phase aigue de l’infection. Nous montrons clairement que, aussi bien chez les individus en phase aigue qu’en phase chronique de l’infection, l’expression de PD-1 est augmentée sur toutes les sous-populations T, y compris les cellules naïves. Nous avons également mis en relief une distribution anormale des sous-populations T, ces cellules ayant un phénotype plus différencié, et ce à tous les stades de la maladie. Dans cette thèse, nous discutons le rôle possible de PD-1 dans l’homéostasie des cellules T chez les individus infectés par le VIH-1. En étudiant la transition de la phase aigue à la phase chronique de l’infection, nous avons trouvé que les sous-populations T CD8+ des individus récemment infectés exprimaient moins de PD-1 que celles des individus à un stade plus avancé de la maladie. Ces niveaux plus élevés de PD-1 sur les cellules T CD8+ en phase chronique sont associés à des niveaux réduits de prolifération in vivo – comme mesuré par l’expression de Ki67 – suggérant que l’expression de PD-1 est partiellement impliquée dans cette perte de fonction des cellules T CD8+. De plus, les cellules naïves s’accumulent en fréquence lors de la transition de la phase aigue à la phase chronique de l’infection. Considérant que les cellules naïves expriment déjà des hauts niveaux de PD-1, nous avons émis l’hypothèse que l’activation initiale des cellules T chez les individus chroniquement infectés est affectée. En résumé, nous proposons un modèle où des hauts niveaux d’expression de PD-1 sont associés à (1) un dysfonctionnement de la réponse cellulaire T CD8+ et (2) un défaut d’activation des cellules naïves ce qui contribue non seulement à la progression de la maladie mais aussi ce qui va limiter l’efficacité de potentiels vaccins dans l’infection par le VIH-1 en empêchant toute nouvelle réponse d’être initiée. Afin de mieux disséquer la réponse immunitaire mise en place lors d’une infection comme celle du VIH-1, nous avons développé un outil qui permet de détecter les cellules T CD4+ i.e. des tétramères de CMH de classe II. Ces réactifs ont pour but d’augmenter l’avidité du CMH de classe II pour son ligand et donc de détecter des TCR de faible affinité. Dans cette thèse, nous décrivons une méthode originale et efficace pour produire diverses molécules de HLA-DR liant de façon covalente le peptide antigénique. Mieux déterminer les mécanismes responsables de l’exhaustion des cellules T dans l’infection par le VIH-1 et de la progression de la maladie, ainsi que développer des outils de pointe pour suivre ces réponses T, est central à une meilleure compréhension de l’interaction entre le virus et le système immunitaire de l’hôte, et permettra ainsi le développement de stratégies pertinentes pour lutter contre l’infection par le VIH-1. / HIV-1 infection leads to a progressive CD4+ T cell depletion and T cell dysfunction, which in the absence of successful anti-retroviral therapy, results in individuals progressing to AIDS. Some of the underlying mechanisms for this T cell dysfunction have been elucidated and reveal an important role for the inhibitory receptor program death-1 (PD-1) in T cell exhaustion during chronic HIV-1 infection. Indeed, PD-1 up regulation correlates with increased viral load as well as decreased cytokine production and proliferative capacity of HIV-1 specific T cells. Moreover, blocking in vitro the interaction of PD-1 with its counter-receptor PD-L1 using antibodies restores HIV-1 specific T cell effector functions. Interestingly, our group and others have shown that levels of PD-1 during chronic HIV-1 infection are not only up regulated on virus-specific T cells but also on the total pool of CD4+ and CD8+ T cells. However, little is known about the impact of PD-1 expression on the turnover and maturation status of the PD-1 expressing cells during the course of the disease. Of note, PD-1 expression has never been investigated in acute HIV-1 infection. In this thesis, we clearly show that, in both acutely and chronically HIV-1 infected individuals, PD-1 is up regulated on all T cell subsets, including naïve T cells. We also uncovered an abnormal distribution of T cell subsets toward a more differentiated phenotype at all stages of the disease. In this thesis, we discuss the possible role of PD-1 in the homeostasis breakdown observed in HIV-1 infected individuals. More interestingly, if we focus on the transition from the acute to the chronic phase of the infection, we found that PD-1 is expressed at much lower levels on total CD8+ T cell subsets from acutely infected individuals than chronically infected individuals. These augmented PD-1 expression levels on CD8+ T cell in chronic infection are associated with reduced levels of in vivo cell proliferation - as monitored by Ki67 expression - suggesting that PD-1 expression may be partially responsible for the loss of CD8+ T cell function. In addition, naïve T cells accumulate in frequency during the transition from the acute to the chronic phase of the infection. Considering that naïve T cells already express high levels of PD-1, we hypothesize that priming of T cell might be impaired in chronically infected individuals. Altogether, we propose a model where high PD-1 expression is associated with (1) impaired CD8+ T cell function in chronic HIV-1 infection and suggest that lower levels of PD-1 may partially preserve the CD8+ T cell function and (2) impaired priming of T cells contributing to the progressive immunodeficiency in HIV-1 infection but also limiting the effectiveness of vaccine strategies by preventing any new responses to be triggered. To better understand immune responses in infection such as HIV-1 disease, we next developed multimeric reagents for the detection of CD4+ T cells, namely tetramers of HLA-DR molecules. These reagents aim at increasing the overall avidity of peptide-MHC class II complexes to detect low affinity TCRs. In this thesis, we describe a versatile and efficient method to produce different soluble HLA-DR molecules covalently linked to antigenic peptides. Gaining further insights into mechanisms underlying T cell exhaustion and disease progression, in addition to the development of state-of-the-art immune monitoring tools, will be crucial in better understanding of the interplay between the virus and the host immune system, leading to rational strategies in the fight against the AIDS epidemic.

La sclérose en plaques (SEP) est une maladie inflammatoire démyélinisante et neurodégénérative du système nerveux central (SNC). Les cellules T activées qui expriment le PD-1 sont inhibées via l’interaction avec l’un des ligands: PD-L1 ou PD-L2. Des études effectuées chez le modèle murin de la SEP, l’encéphalomyélite auto-immune expérimentale (EAE), ont démontré que l’interaction du PD-1 avec ses ligands contribue à atténuer la maladie. Toutefois, le rôle du PD-1 et de ses ligands dans la pathogenèse de la SEP chez l’humain et dans le modèle murin n’a pas été complètement élucidé. Nous avons déterminé que plusieurs cellules du SNC humain peuvent exprimer les ligands du PD-1. Les astrocytes, les microglies, les oligodendrocytes et les neurones expriment faiblement le PD-L1 dans des conditions basales mais augmentent de façon significative cette expression en réponse à des cytokines inflammatoires. Le blocage de l’expression du PD-L1 par les astrocytes à l’aide de siRNA spécifiques mène à l’augmentation significative des réponses des cellules T CD8+ (prolifération, cytokines, enzymes lytiques). Nos résultats établissent ainsi que les cellules gliales humaines peuvent exprimer des niveaux suffisants de PD-L1 en milieu inflammatoire pour inhiber les réponses des cellules T CD8+. Notre analyse de tissus cérébraux post-mortem par immunohistochimie démontre que dans les lésions de la SEP les niveaux de PD-L1 sont significativement plus élevés que dans les tissus de témoins; les astrocytes et les microglies/macrophages expriment le PD-L1. Cependant, plus de la moitié des lymphocytes T CD8+ ayant infiltré des lésions de SEP n’expriment pas le récepteur PD-1. Au cours du développement de l’EAE, les cellules du SNC augmentent leur niveau de PD-L1. Le PD-1 est fortement exprimé par les cellules T dès le début des symptômes, mais son intensité diminue au cours de la maladie, rendant les cellules T insensibles au signal inhibiteur envoyé par le PD-L1. Nous avons observé que les cellules endothéliales humaines formant la barrière hémato-encéphalique (BHE) expriment de façon constitutive le PD-L2 mais pas le PD-L1 et que l’expression des deux ligands augmente dans des conditions inflammatoires. Les ligands PD-L1 et PD-L2 exprimés par les cellules endothéliales ont la capacité de freiner l’activation des cellules T CD8+ et CD4+, ainsi que leur migration à travers la BHE. L’endothélium du cerveau des tissus normaux et des lésions SEP n’exprime pas des taux détectables de PD-L1. En revanche, tous les vaisseaux sanguins des tissus de cerveaux normaux sont positifs pour le PD-L2, alors que seulement la moitié de ceux-ci expriment le PD-L2 dans des lésions SEP. Nos travaux démontrent que l’entrée des cellules T activées est contrôlée dans des conditions physiologiques grâce à la présence du PD-L2 sur la BHE. Cependant, l’expression plus faible du PD-L2 sur une partie des vaisseaux sanguins dans les lésions SEP nuit au contrôle de la migration des cellules immunes. De plus, une fois dans le SNC, les cellules T CD8+ étant dépourvues du PD-1 ne peuvent recevoir le signal inhibiteur fourni par le PD-L1 fortement exprimé par les cellules du SNC, leur permettant ainsi de rester activées. / Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS). Responses of activated T cells are suppressed upon engagement of the receptor programmed cell death-1 (PD-1) with its ligands (PD-L1 and PD-L2). Experiments using the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), have demonstrated that the PD-1/PD-Ls interaction contributes to attenuate disease severity. However, the expression and the role of PD-1 and PD-Ls have been partially documented in inflammatory murine models and human CNS data are still incomplete. We determined that primary cultures of human astrocytes, microglia, oligodendrocytes, or neurons expressed low or undetectable PD-L1 levels under basal conditions, but inflammatory cytokines significantly induced such expression, especially on astrocytes and microglia. Blocking PD-L1 expression in astrocytes using specific siRNA in co-culture led to significantly increased CD8 T cell responses (proliferation, cytokines, lytic enzyme). Thus, our results establish that inflamed human glial cells can express sufficient and functional PD-L1 to inhibit CD8 T cell responses. Extensive immunohistochemical analysis of post-mortem brain tissues demonstrated a significantly greater PD-L1 expression in MS lesions compared to control tissues, which co-localized with astrocyte and microglia/macrophage cell markers. However, more than half of infiltrating CD8 T lymphocytes in MS lesions did not express PD-1, the cognate receptor. Similar results were obtained in EAE mice. Even though CNS cells expressed PD-L1 at the peak of the disease, PD-1 intensity on infiltrating T cells decreased throughout EAE disease development. This reduction of PD-1 level on activated T cells prevented these cells to receive PD-L1 inhibitory signal. We also investigated whether human brain endothelial cells (HBECs), which form the blood brain barrier (BBB), can express PD-L1 or PD-L2 and thereby modulate T cells. HBECs expressed PD-L2 under basal conditions, whilst PD-L1 was not detected. Both ligands were up-regulated under inflammatory conditions. Blocking PD-L1 and PD-L2 led to increased transmigration and enhanced responses by human CD8 T cells in co-culture assays. Similarly, PD-L1 and PD-L2 blockade significantly increased CD4 T cell transmigration. Brain endothelium in normal tissues and MS lesions did not express detectable PD-L1; in contrast, all blood vessels in normal brain tissues were PD-L2-positive, while only about 50% expressed PD-L2 in MS lesions. Therefore, our results demonstrate that under basal conditions, PD-L2 expression by HBECs impedes the migration of activated immune T cells through the BBB, and inhibits their activation. However, such impact is impaired in MS lesions due to down-regulation of PD-L2 levels on the endothelium. The majority of infiltrating CD8 T cells is devoid of PD-1, thus insensitive to PD-L1 inhibitory signal providing by CNS cells once they have entered the CNS.