The problem of this investigation is to prepare a merchandise display manual that can be used by the teacher-coordinator, students of distributive education, other trainees, training sponsors, and salespeople.

Le travail de thèse présente une approche innovante pour la construction d’une bibliothèque de protéines basées sur l’ossature protéique. L’objectif est de générer une source de biodiversité artificielle permettant la création de nouvelles capacités d’interaction avec des cibles d’intérêts. Une banque, basée sur une ossature protéique bactérienne avait déjà été construite dans l’équipe, mais elle nécessitait d’être optimisée. L’étape initiale a été d’explorer les raisons de l’instabilité des protéines de la banque de première génération, ceci par des approches d’étude de la structure in silico suivie d’une stratégie de mutagenèse dirigée. Des positions déstabilisantes existant dans la première banque ont donc été remplacées dans la banque de deuxième génération. La deuxième étape a eu pour objectif de diminuer le nombre de positions diversifiées et de simplifier le schéma de diversification des variants de la banque. Puis un procédé de filtration et de shuffling de ces variants a été mis au point pour augmenter la proportion de séquences codantes correctes. Une nouvelle stratégie de filtration basée sur la technique d’exposition sur phage a été élaborée, en exploitant le fait que la protéine matrice de la banque, l'ossature protéique a un partenaire biologique capable d’interagir sur la zone « constante », non modifiée par le schéma de diversification. Ainsi les variants de la banque exposés dans une conformation correcte à la surface des phages ont pu être capturés par ce partenaire. Ensuite, les séquences correspondant à ces variants ont été recombinées entre elles pour recréer une plus grande diversité utile. Une bibliothèque optimisée composée de 2.8 x 108 protéines indépendantes a ainsi été obtenue. Cette nouvelle banque optimisée a permis de sélectionner par Phage display, des interacteurs contre plusieurs cibles de structures différentes. Ces nouveaux interrupteurs sont spécifiques de leurs cibles et présentent des affinités de l’ordre du μM. Une approche de séquençage à haut débit a également été entreprise pour réaliser une analyse plus approfondie des séquences de cette bibliothèque et de notre processus de sélection. Cette approche nous a apporté une nouvelle dimension pour la caractérisation des banques construites au laboratoire notamment concernant la diversité réelle de ces banques. Pour le suivi de sélections, nous avons appréhendé le séquençage haut débit comme un moyen d’identifier les interacteurs spécifiques d’une cible par l’analyse exhaustive des séquences issues des sélections. L’objectif est ici de mettre au point un protocole utilisant l’approche NGS pour identifier les interacteurs spécifiques isolées par Phage Display. / Here new methods to build a library of artificial proteins based on a new protein framework have been developed. The objective is to generate a source of artificial biodiversity allowing the creation of new interaction capacities with various specific targets. A first-generation library was previously built with this scaffold, but it needed to be optimized. The first step was to explore the reasons of the instability of the first generation proteins library through an in silico approaches followed by a site-directed mutagenesis strategy. The second step was to reduce the number of diversified positions and simplify the randomization scheme of the variants of the library. Then a method of filtering and shuffling of the variants of the bank was elaborated. To increase the proportion of correct coding sequences, a new filtration strategy based on the phage display technique has been developed, exploiting the fact that the scaffold of the librarie. This scaffold has a particularly interesting biological partner able to interact on the "constant" zone. This filtration made it possible to recover a set of well-folded clones. Then, DNA sequences corresponding to these clones were recombined with each other to recreate a greater useful diversity. An optimized library of 2.8 x 108 independent proteins was obtained. This new optimized library has enabled us to select, by a phage display approach, binders against several targets of different structures. These new binders are specific for their targets and have affinities in the μM range. A high throughput sequencing (NGS) approach was also undertaken to further analyse the library sequences and the selection process. This approach offers a new dimension for the characterization of the library built in the laboratory, especially concerning it actual diversity. To follow the selections, we have considered the NGS as a way to identify the target-specific binders through exhaustive analysis of the sequences obtained from selections. The objective here is to develop a general protocol using the NGS approach to identify specific binders isolated by Phage Display.

Mes travaux de thèse ont pour but d’évaluer l’approche « épitope display » pour sa capacité d'induire les réponses cellulaires. Ad à capside modifiée par insertion de différents épitopes T issus de la ovalbumine ont étés produit. Après administration à des souris C57BL/6, j'ai mis en évidence l'induction de la réponse cellulaire dirigée contre les épitope insérés (grâce aux techniques ELISPOT, tétramères, ou bien en quantifiant la production d’IFNg par les splénocytes restimulés in vitro). J’ai démontré que ces réponses sont limités chez des souris préalablement immunisées avec Ad. Des manipulations en course ont pour but de confirmer ces résultats et d'évaluer la cinétique de ces réponses. Mon 2ème objectif est de comprendre les paramètres qui contrôlent l’immunogénicité des Ad présentant des épitopes. Ainsi, j’ai montré que l’ablation des interactions des Ad porteurs d’un épitope issu de l’ovalbumine avec les récepteurs/facteurs (intégrines, facteur X de la coagulation) impliqués dans l’entrée de l’Ad dans les cellules ne modifiait pas leur capacité à induire une réponse humorale contre l’ovalbumine. Ces résultats suggèrent que le processus d’infection virale n’est pas requis pour l’induction d’une réponse humorale par les Ad porteurs d’épitopes. / Recombinant adenoviruses (Ad) have recently been employed for a wide range of vaccination strategies. Unfortunately, highly prevalent pre-existing neutralizing antibodies (Abs), reduce their ability to trigger transgene expression. To avoid the step of gene transfer a new vaccination strategy has been proposed based on the use of Ad displaying epitopes inserted into their capsid proteins. Using an ovalbumin-derived B cell epitope, our group demonstrated that vaccination efficiency depends on both the site of peptide insertion and the host immune status towards Ad (Lanzi et al; 2011). The present work aims at (1) evaluating the potency of Ad displaying T-cell epitopes from ovalbumin to elicit cellular responses and (2) understanding the molecular bases controlling the efficacy of this vaccination strategy. 1) Ad displaying T-cell epitopes from ovalbumin were constructed, produced and characterized in vitro. First in vivo experiments in naive mice showed induction of cellular responses, assessed with techniques like ELISPOT, tetramer staining and in vitro splenocyte restimulation. Subsequent experiments showed that pre-exisitng anti-vector immunity is hampering the potent induction of anti-epitope cellular responses. Current work is aiming at confirming the obtained results as well as at evaluating the kinetics of cellular responses induced upon "epitope display" vaccination. 2)First, the influence of interactions of Ad (displaying OVA peptide) with their natural receptors was investigated. Different detargeted Ads were produced and characterized in vitro. Upon mice immunization these vectors led to unmodified anti-epitope humoral responses, suggesting that their efficacy does not depend on the ability to transduce cells. In parallel we sought to evaluate the impact of innate immunity on the outcome of anti-epitope adaptive immune responses. Upon immunization of WT and MyD88-/- mice with Ad displaying OVA epitope we observed that cellular responses induced in MyD88-/- mice are significantly diminished while humoral responses were not altered. These results remain to be confirmed but question the role of other innate immunity sensors in the immunogenicity of Ad-based vaccines. Altogether, our work is expected to provide the foundations for the development of Ad-based vaccines with minimized side effects and unaltered adjuvant properties.

This thesis improves on the design of the Optical Trap Display (OTD), presented in 2018 [1]. Contributions include: real time animation; single beam, multiparticle suspension, point primitive anisotropic scattering, and virtual image approximation. First, real time animation was demonstrated on the OTD for the first time in full color at up to 30Hz refresh. Second, multi-particle systems allow for scaling of the display by a multiplicative factor, potentially up to orders of magnitude greater than the first OTD. Third, anisotropic scattering of point primitives was shown for individual suspended particles and multiple simultaneously suspended particles. Fourth, virtual images have been previously considered impossible in volumetric displays but by using perspective projections we have shown in simulation and experiment for the first time that an effect similar to a virtual image can be created.

More and more novel functions are being integrated into the vehicle infotainment system to allow individuals to perform secondary tasks with high accuracy and low accident risks. Mid-air gesture interactions are one of them. This thesis designed and tested a novel interface to solve a specific issue caused by this method of interaction: visual distraction within the car. In this study, a Heads-Up Display (HUD) was integrated with a gesture-based menu navigation system to allow drivers to see menu selections without looking away from the road. An experiment was conducted to investigate the potential of this system in improving drivers’ driving performance, situation awareness, and gesture interactions. The thesis recruited 24 participants to test the system. Participants provided subjective feedback about using the system and objective performance data. This thesis found that HUD significantly outperformed the Heads-Down Display (HDD) in participants’ preference, perceived workload, level 1 situation awareness, and secondary-task performance. However, to achieve this, the participants compensated by having poor driving performance and relatively longer visual distraction. This thesis will provide directions for future research and improve the overall user experience while the driver interacts with the in-vehicle gesture interaction system. / M.S. / Driving is becoming one of the essential daily activities. Unless a fully autonomous vehicle is made, driving will remain as the primary task when operating the vehicle. However, to improve the overall experience during traveling, drivers are also required to perform secondary tasks such as changing the AC, switching the music, navigating the map, and other functions. Nevertheless, car accidents may happen when drivers are performing secondary tasks because those tasks are considered a distraction from the primary task, which is driving safely. Many novel interaction methods have been implemented in a modern car, such as touch screen interaction, voice interaction, etc. This thesis introduces a new gesture interaction system that allows the user to use mid-air gestures to navigate through the secondary task menus. To further avoid visual distraction caused by the system, the gesture interaction system integrated a head-up display (HUD) to allow the user to see visual feedback on their front windshield. The HUD will let the driver use the system without looking in the other directions and keep peripheral vision on the road. The experiment recruited 24 participants to test the system. Each participant provided subjective feedback about their workload, experience, and preference. In the experiment, driving simulator was used to collect their driving performance. The eye tracker glasses were used to collect eye gaze data, and the gesture menu system was used to collect gesture system performance. This thesis expects four key factors to affect the user experience: HUD vs. Heads-Down Display (visual feedback types), with sound feedback vs. without sound feedback. Results showed that HUD helped the driver perform the secondary task faster, understand the current situation better, and reduce workload. Most of the participants preferred using the HUD over using HDD. However, there are some compensations that drivers needed to make if they use HUD: focusing on the HUD for more time while performing secondary tasks and having poor driving performance. By analyzing result data, this thesis provides a direction for conducting HUD or in-vehicle gesture interaction research and improving the users’ performance and overall experience.

This study investigated automobile drivers' ability to localize auditory and haptic (touch) alarms in terms of speed and accuracy. Thirty-two subjects, balanced across age (20-30 years old and 60-70 years old) and gender, participated in the study. Subjects were screened for minimum hearing of 40 dB for 500 Hz through 4000 Hz auditory tones, and maximum bilateral hearing differences of 10 dB. The experiment consisted of subjects identifying the target location of an alarm while driving a 2001 Buick LeSabre at 55 mph in light traffic. Four alarm modes were tested: 1) an auditory broadband alarm, 2) a haptic seat, 3) a combination of the haptic and the auditory alarm modes, and 4) a combination of the haptic alarm mode with a non-directional auditory alarm played from the front speakers of the vehicle. The alarms were evoked from eight target locations: the front-left, front, front-right, right, back-right, back, back-left, and left. The target locations of the auditory alarm mode existed around the interior of the car cabin using the vehicle's stock sound system speakers. The haptic alarm target locations existed in the bottom of the driver seat using an eight-by-eight grid of actuators. The experimenter evoked the alarms while subjects drove along a two-lane highway, and the alarms were not associated with any actual collision threat. Subjects were instructed to quickly identify the location of the alarm by calling them out, while being as correct as possible. Their choice response time and target location selection was recorded. The alarms were presented approximately every minute during fifteen-minute intervals over the duration of two and a half hours. Subjects completed questionnaires regarding their preference to the alarm modes. Under the conditions investigated, subjects localized the haptic alarm mode faster and more accurately than the auditory alarm mode. Subjects performed equally well with the haptic alarm mode and the two auditory and haptic combination alarm modes in terms of speed and accuracy in identifying their location. Subjects did express a preference for the addition of the auditory component to the haptic alarm mode, perhaps owing to a heightened sense of urgency. However, subjects preferred the haptic alarm mode on its own in response to hypothetical false alarm questions, perhaps because it was less annoying. Alarm mode discriminability was believed to affect localization accuracy and response time owing to its effect on the likelihood of correctly identifying a target location and the attention resources required to differentiate adjacent target locations. / Master of Science

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