Les β-arrestines jouent un rôle important dans la transduction du signal par les récepteurs couplés aux protéines G (RCPG). Nous montrons dans cette thèse que les β-arrestines exercent des régulations plus complexes et subtiles qu'on ne le pensait jusque-là sur la voie AMPc/PKA/CREB qui est activée par les RCPGs couplés à Gs. Nous montrons que les β-arrestines interagissent directement la PKAcat et contribuent à sa translocation nucléaire. De plus, nous mettons en évidence une interaction β-arrestine/CREB qui conduit à la formation d'un complexe transcriptionnellement actif sous l’action de l’agoniste. D’autre part, nous avons constaté que les β-arrestines interagissent directement avec PKAcat, p70S6K et Src via un même site et lesquelles sont donc potentiellement mutuellement exclusives. Nous avons ensuite mesuré l’impact d’une mutation et d’un polymorphisme du R-FSH sur la signalisation dépendante des β-arrestines, notamment grâce à l’utilisation de senseurs FRET et BRET. / Β-arrestins play an important role in G protein-coupled receptor (GPCR)-induced signal transduction. In this thesis, we show here that β-arrestins exert more complex and subtle regulation than previously thought on the cAMP/PKA/CREB pathway which is activated by Gs-coupled GPCRs. We demonstrate that β-arrestins directly interact with PKAcat and promote its translocation to the nucleus. Moreover, we provide evidence that β-arrestins directly interact with CREB thereby forming a transcriptionally active complex upon agonist stimulation. We also found that PKAcat, p70S6K and Src all directly interact with β-arrestins through the same interaction site and are therefore potential mutually exclusive interactions. We then measured the impact of a point mutation and of a polymorphism in the FSH-R on β-arrestin-dependent signaling, in part using FRET and BRET sensors.

Objectifs : Les Récepteurs Couplés aux Protéines G (RCPG) constituent une grande famille ubiquitaire. Leur structure est caractérisée par sept hélices transmembranaires. Les déformations de ces hélices jouent un rôle majeur dans l’activation de ces récepteurs. La plupart de ces déformations sont liées à la présence de prolines conservées. Cependant, les prolines de l’hélice 2 et 5 des RCPG ne sont pas systématiquement présentes. De plus, la position de la proline dans l’hélice 2 est variable (2.58, 2.59 ou 2.60). Nous nous intéressons aux rôles des prolines des hélices 2 et 5 dans l’activation de deux RCPG : le récepteur de la thyrotropine (TSHR) et le récepteur 2 de la vasopressine (V2R). Méthodes : pour le TSHR et le V2R, nous concevons et caractérisons des mutants pour chaque position conservée de la proline dans l’hélice 2 et/ou 5, ainsi que des mutants sans proline. Résultats : Les mutants du TSHR n’ont pas le même comportement en termes d’expression, de glycosylation ou d’activité. La position la mieux tolérée, 2.59, nous permet de rapprocher le TSHR des récepteurs avec une proline en position 2.59 qui possèdent un renflement dans l’hélice 2. Pour l’hélice 5, les données expérimentales couplées à l’analyse des séquences et la modélisation moléculaire suggèrent une structure non renflée. Pour le V2R, le changement de position de la proline de l’hélice 2 est plus délétère que l’absence de proline dans cette hélice. La proline de l’hélice 5 est indispensable pour l’activité de ce récepteur. Conclusion : Les données obtenues sur le TSHR permettent de proposer un modèle avec une hélice 2 renflée et une hélice 5 non renflée et d’améliorer la modélisation de la cavité interne de ce récepteur, ce qui est essentiel pour le drug design. L’étude du V2R permet de proposer un modèle évolutif de ce récepteur et met en évidence sa spécificité par rapport à des récepteurs proches. / Objectives : Class A G-Protein-coupled receptors (GPCRs) constitute a large family of transmembrane receptors. Helical distortions play a major role in the overall fold and in the activation mechanism of these receptors. Most distortions are related to the presence of conserved proline residues. However, in helices TM2 and TM5, the presence of proline is not mandatory and the correlated mutation of these proline residues is observed in several GPCR sub-families. In addition, the position of the TM2 proline is variable (2.58 to 2.60). We are interested in the role of the TM2 and TM5 proline residues in the folding and activation mechanism of two GPCRs : the thyrotropin receptor (TSHR) and the vasopressin receptor type 2 (V2R). Methods : For both receptors, we engineered and characterized mutants with proline residues at different positions in TM2 and/or at position 5.50 in TM5, and without proline. Results : The expression, the glycolysation or the activity of TSHR mutants are differentially altered by changes in the proline pattern. The “best” mutant, TSHR P2.59, is consistent with a bulged structure for TM2. Experimental data in addition to sequences analysis and modeling suggest an unbulged structure for TM5. For V2R, the absence of proline in TM2 is better tolerated than ashift in the position. The TM5 proline is mandatory for the receptor activation. Conclusion : We suggest a model for TSHR with a bulged TM2 and an unbulged TM5. This should improve the modeling of the transmembrane cavity, which is fundamental for drug design. Our results on V2R suggest an evolutionary model for this receptor and enlighten its specificity compared to nearby receptors.

Vehicular communications are a major subject of research and policy activity in industry, government, and academia. Dedicated Short-Range Communications (DSRC) is currently the main protocol used for vehicular communications, and it operates in the 5.9 GHz band. In addition to DSRC radios, other potential uses of this band include Wi-Fi, LTE-V, and communication among unlicensed devices. This dissertation presents an architecture and a feasibility analysis including field measurements and analysis for vehicle-to-train (V2T) communications, a safety-critical vehicular communication application. The dissertation also presents a survey of research relevant to each of several possible combinations of radio-spectrum and vehicular-safety regulations that would affect use of the 5.9 GHz band, identifies the most challenging of the possible resulting technical challenges, and presents initial measurements to assess feasibility of sharing the band by DSRC radios and other devices that operate on adjacent frequencies using different wireless communication standards. Although wireless technology is available for safety-critical communications, few applications have been developed to improve railroad crossing safety. A V2T communication system for a safety warning application with DSRC radios can address the need to prevent collisions between trains and vehicles. The dissertation presents a V2T early warning application architecture with a safety notification time and distance. We conducted channel measurements at a 5.86–5.91-GHz frequency and 5.9-GHz DSRC performance measurements at railroad crossings in open spaces, shadowed environments, and rural and suburban environments related to the presented V2T architecture. Our measurements and analyses show that the DSRC protocol can be adapted to serve the purpose of a V2T safety warning system. The 5.9 GHz band has been sought after by several stakeholders, including traditional mobile operators, DSRC proponents, unlicensed Wi-Fi proponents and Cellular-Vehicle-to-Everything (C-V2X) proponents. The FCC and National Highway Traffic Safety Administration (NHTSA), the two major organizations that are responsible for regulations related to vehicular communications, have not finalized rules regarding this band. The relative merits of the above mentioned wireless communication standards and coexistence issues between these standards are complex. There has been considerable research devoted to understanding the performance of these standards, but in some instances there are gaps in needed research. We have analyzed regulation scenarios that FCC and NHTSA are likely to consider and have identified the technical challenges associated with these potential regulatory scenarios. The technical challenges are presented and for each a survey of relevant technical literature is presented. In our opinion for the most challenging technical requirements that could be mandated by new regulations are interoperability between DSRC and C-V2X and the ability to detect either adjacent channel or co-channel coexisting interference. We conducted initial measurements to evaluate the feasibility of adjacent channel coexistence between DSRC, Wi-Fi, and C-V2X, which is one of the possible regulatory scenarios. We set DSRC at Channel 172, Wi-Fi at Channel 169 for 20 MHz bandwidth and at Channel 167 for 40 MHz, and C-V2X at Channel 174 with almost 100% spectrum capacity. From the measurements, we observed almost no effects on DSRC performance due to adjacent channel interference. Based on our results, we concluded that adjacent channel coexistence between DSRC, C-V2X, and Wi-Fi is possible. DSRC systems can provide good communication range; however, the range is likely to be reduced in the presence of interference and / or Non-Line-of-Sight (NLoS) conditions. Such environmental factors are the major influence on DSRC performance. By knowing the relationship between DSRC and environmental factors, DSRC radios can be set up in a way that promotes good performance in an environment of interest. We chose propagation channel characteristics to generate DSRC performance modelling by using estimation methods. The conducted DSRC performance measurements and propagation channel characteristics are independent; however, they share the same distance parameters. Results of linear regression to analyze the relationship between DSRC performance and propagation channel characteristics indicate that additional V2T measurements are required to provide data for more precise modeling. / PHD / Researchers and regulators in industry, government, and academic institutions are interested in vehicular communications. Dedicated Short-Range Communications (DSRC) is currently the standard protocol for communication between vehicles, including for safety applications, and operates in the band of radio frequencies near 5.9 GHz. In addition to operators of DSRC radios, other potential users are interested in using the 5.9 GHz band. This dissertation presents an architecture and a feasibility analysis including field measurements for vehicle-to-train (V2T) communications, a safety-critical vehicular communication application. The dissertation also identifies major technical challenges that could become important in the future for users of the 5.9 GHz band. The challenges will be different depending on what decisions government regulators make about the types of radios and communication protocols that are allowed in the 5.9 GHz band and about which types of radios should be used for vehicular safety. Although wireless technology is available for safety-critical communications, few applications have been developed to improve railroad crossing safety. To prevent collisions between trains and vehicles, we present a vehicle-to-train (V2T) communication system that uses DSRC radios to provide safety warnings to motorists. Although the term V2T is used, the emphasis is on communication from the train to vehicles. We present a high-level design, or architecture, of the warning system that includes goals for safety notification time and vi distance. We conducted measurements of radio channels near 5.9 GHz as well as measurements of 5.9 GHz DSRC radio link performance at the same locations (railroad crossings in open spaces, shadowed or obstructed environments, and rural and suburban environments). The measurements were performed to help decide whether the V2T warning system architecture would work. A DSRC system can provide good communication range; however, that range could be reduced if the DSRC system experiences interference from other radios or if the signal is partially blocked due to objects between the DSRC radios. The environmental factors are the most important influence on DSRC performance. By knowing the relationship between DSRC and environmental factors, manufacturers and operators can set up the radios to perform well in environments of interest. Although DSRC performance and radio channel characteristics were measured separately, they were measured in the same locations near railroad crossings. This made it possible to perform a statistical analysis of the relationship between DSRC performance and propagation channel characteristics. This analysis indicated that additional measurements will be required to collect enough data to develop robust statistical models that relate DSRC performance directly to measured channel characteristics. However, the results of the V2T measurements that we conducted near rural and suburban railroad crossings with varying numbers and types of obstacles to the radio signals provide a strong indication that DSRC can be used for to provide V2T safety warnings. The 5.9 GHz band has been sought after by several stakeholders, including traditional mobile operators and others who support use of the band for DSRC, unlicensed Wi-Fi, and CellularVehicle-to-Everything (C-V2X) communication. The FCC and National Highway Traffic Safety Administration (NHTSA), the two major organizations that are responsible for vii regulations related to vehicular communications, have not finalized the rules regarding this band. The relative merits of the above mentioned communication standards and coexistence issues between these standards are complex. There has been considerable research devoted to understanding the performance of these standards, but in some instances there are gaps in needed research. We have analyzed regulation scenarios that FCC and NHTSA are likely to consider and have identified the technical challenges associated with these potential regulatory scenarios. The technical challenges are presented and for each a survey of relevant technical literature is presented. In our opinion for the most challenging technical requirements that could result from new regulations are interoperability between DSRC and C-V2X and the ability to detect either adjacent channel or co-channel coexisting interference. We conducted initial measurements to evaluate the feasibility of adjacent channel coexistence between DSRC, Wi-Fi, and C-V2X, which is one of the possible regulatory scenarios. From the measurements, we observed almost no effect on DSRC performance when other types of radios used frequencies adjacent to the frequencies used by the DSRC radios. Based on our results, we concluded that adjacent channel coexistence between DSRC, C-V2X, and Wi-Fi is possible.

Le récepteur V2 (V2R) de la vasopressine est un récepteur couplé aux protéines G (RCPG), jouant un rôle fondamental dans le maintien de l’homéostasie hydrosodique. À l’instar de nombreux RCPGs, il est capable d’interagir avec plusieurs types de protéines G hétérotrimériques et possède des voies de signalisation peu explorées aux mécanismes mal compris. Ces voies non canoniques font l’objet des travaux exposés dans ce mémoire. Il s’agit d’explorer les caractéristiques et mécanismes de la signalisation de V2R via G12, et de la voie d’activation d’ERK 1/2 par transactivation du récepteur de l’insulin-like growth factor 1, IGF1R. Par des études de transfert d’énergie de résonance de bioluminescence (BRET), nous exposons la capacité de V2R à interagir avec la sous-unité Gα12 ainsi que la modulation de la conformation de l’hétérotrimère G12 par l’agoniste de V2R, l’arginine-vasopressine. Ces travaux dévoilent également la modulation de l’interaction entre Gα12 et son effecteur classique RhoA, suggérant un engagement de RhoA, ainsi que la potentialisation via Gα12 de la production d’AMP cyclique. À l’aide de diverses méthodes d’inhibition sélective, nos résultats précisent les mécanismes de la transactivation. Ils supportent notamment le rôle initiateur de l’activation de Src par V2R et l’absence d’implication des ligands connus d’IGF1R dans la transactivation. La métalloprotéase MMP 3 apparaît par ailleurs comme un bon candidat pour réguler la transactivation. Ce projet met en lumière des modes de signalisation peu explorés de V2R, dont l’implication physiologique et physiopathologique pourrait s’avérer significative, au-delà d’un apport fondamental dans la compréhension de la signalisation des RCPGs. / Vasopressin V2 receptor is a G protein coupled receptor (GPCR) responsible for the homeostatic regulation of water and sodium recapture from the urine to the bloodstream. Akin to numerous GPCRs, this receptor can interact with more than one heterotrimeric G protein subtype, and is still associated with some poorly explored signaling pathways with indefinite mechanisms. These non-canonical pathways are the focus of this project. This work aims at unveiling the characteristics and mechanisms underlying G12 mediated signaling by V2R and ERK 1/2 activation through the transactivation of the tyrosine kinase Insulin-like growth factor 1 receptor (IGF1R). Using bioluminescence resonance energy transfer (BRET) experiments, we reveal V2R’s ability to interact with the Gα12 subunit, as well as the modulation of G12 heterotrimer’s conformation in response to V2R agonist arginine vasopressin (AVP). AVP-induced modulation of Gα12’s interaction with its classical effector RhoA upon stimulation with AVP suggests the engagement of RhoA, and our data also reveals that Gα12 potentiates AVP-induced cAMP production. Using diverse selective inhibition strategies, our results further define the mechanism of transactivation. Our data support a starter position of AVP-induced Src activation and discard IGF1R known agonists as the potential autocrine/paracrine factor responsible for IGF1R activation. Furthermore, our results suggest that the metalloproteinase MMP 3 is a good candidate for IGF1R transactivation. This project sheds light on lesser known signaling pathways involving V2R, which could reveal important on a physiological and pathophysiological scale, besides bringing a better understanding of the principles of GPCR signaling.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Advances in computing and wireless communication technologies have increased interest in smart vehicles, vehicles equipped with significant computing, communication and sensing capabilities to provide services to travelers. Smart vehicles can be exploited to improve driving safety and comfort as well as optimize surface transportation systems. Wireless communications among vehicles and between vehicles and roadside infrastructures represent an important class of vehicle communications. One can envision creating an integrated radio network leveraging various wireless technologies that work together in a seamless fashion. Based on cost-performance tradeoffs, different network configurations may be appropriate for different environments. An understanding of the properties of different vehicular network architectures is absolutely necessary before services can be successfully deployed. Based on this understanding, efficient data services (e.g., data dissemination services) can be designed to accommodate application requirements. This thesis examines several research topics concerning both the evaluation and design of vehicular networks. We explore the properties of vehicle-to-vehicle (v2v) communications. We study the spatial propagation of information along the road using v2v communications. Our analysis identifies the vehicle traffic characteristics that significantly affect information propagation. We also evaluate the feasibility of propagating information along a highway. Several design alternatives exist to build infrastructure-based vehicular networks. Their characteristics have been evaluated in a realistic vehicular environment. Based on these evaluations, we have developed some insights into the design of future broadband vehicular networks capable of adapting to varying vehicle traffic conditions. Based on the above analysis, opportunistic forwarding that exploit vehicle mobility to overcome vehicular network partitioning appears to be a viable approach for data dissemination using v2v communications for applications that can tolerate some data loss and delay. We introduce a methodology to design enhanced opportunistic forwarding algorithms. Practical algorithms derived from this methodology have exhibited different performance/overhead tradeoffs. An in-depth understanding of wireless communication performance in a vehicular environment is necessary to provide the groundwork for realizing reliable mobile communication services. We have conducted an extensive set of field experiments to uncover the performance of short-range communications between vehicles and between vehicles and roadside stations in a specific highway scenario.