Phosphorylation at Ser-181 of oncogenic KRAS is required for tumor growth

KRAS phosphorylation has been reported recently to modulate the activity of mutant KRAS protein in vitro. In this study, we defined S181 as a specific phosphorylation site required to license the oncogenic function of mutant KRAS in vivo. The phosphomutant S181A failed to induce tumors in mice, whereas the phosphomimetic mutant S181D exhibited an enhanced tumor formation capacity, compared with the wild-type KRAS protein. Reduced growth of tumors composed of cells expressing the nonphosphorylatable KRAS S181A mutant was correlated with increased apoptosis. Conversely, increased growth of tumors composed of cells expressing the phosphomimetic KRAS S181D mutant was correlated with increased activation of AKT and ERK, two major downstream effectors of KRAS. Pharmacologic treatment with PKC inhibitors impaired tumor growth associated with reduced levels of phosphorylated KRAS and reduced effector activation. In a panel of human tumor cell lines expressing various KRAS isoforms, we showed that KRAS phosphorylation was essential for survival and tumorigenic activity. Furthermore, we identified phosphorylated KRAS in a panel of primary human pancreatic tumors. Taken together, our findings establish that KRAS requires S181 phosphorylation to manifest its oncogenic properties, implying that its inhibition represents a relevant target to attack KRAS-driven tumors.

Novel Robot Solutions for Carrying out Field Joint Welding and Machining in the Assembly of the Vacuum Vessel of ITER

It is necessary to use highly specialized robots in ITER (International Thermonuclear Experimental Reactor) both in the manufacturing and maintenance of the reactor due to a demanding environment. The sectors of the ITER vacuum vessel (VV) require more stringent tolerances than normally expected for the size of the structure involved. VV consists of nine sectors that are to be welded together. The vacuum vessel has a toroidal chamber structure. The task of the designed robot is to carry the welding apparatus along a path with a stringent tolerance during the assembly operation. In addition to the initial vacuum vessel assembly, after a limited running period, sectors need to be replaced for repair. Mechanisms with closed-loop kinematic chains are used in the design of robots in this work. One version is a purely parallel manipulator and another is a hybrid manipulator where the parallel and serial structures are combined. Traditional industrial robots that generally have the links actuated in series are inherently not very rigid and have poor dynamic performance in high speed and high dynamic loading conditions. Compared with open chain manipulators, parallel manipulators have high stiffness, high accuracy and a high force/torque capacity in a reduced workspace. Parallel manipulators have a mechanical architecture where all of the links are connected to the base and to the end-effector of the robot. The purpose of this thesis is to develop special parallel robots for the assembly, machining and repairing of the VV of the ITER. The process of the assembly and machining of the vacuum vessel needs a special robot. By studying the structure of the vacuum vessel, two novel parallel robots were designed and built; they have six and ten degrees of freedom driven by hydraulic cylinders and electrical servo motors. Kinematic models for the proposed robots were defined and two prototypes built. Experiments for machine cutting and laser welding with the 6-DOF robot were carried out. It was demonstrated that the parallel robots are capable of holding all necessary machining tools and welding end-effectors in all positions accurately and stably inside the vacuum vessel sector. The kinematic models appeared to be complex especially in the case of the 10-DOF robot because of its redundant structure. Multibody dynamics simulations were carried out, ensuring sufficient stiffness during the robot motion. The entire design and testing processes of the robots appeared to be complex tasks due to the high specialization of the manufacturing technology needed in the ITER reactor, while the results demonstrate the applicability of the proposed solutions quite well. The results offer not only devices but also a methodology for the assembly and repair of ITER by means of parallel robots.

The Structural Basis for inorganic Pyrophosphatase Catalysis and Regulation

Inorganic pyrophosphatases (PPases) are essential enzymes for every living cell. PPases provide the necessary thermodynamic pull for many biosynthetic reactions by hydrolyzing pyrophosphate. There are two types of PPases: integral membrane-bound and soluble enzymes. The latter type is divided into two non-homologous protein families, I and II. Family I PPases are present in all kingdoms of life, whereas family II PPases are only found in prokaryotes, including archae. Family I PPases, particularly that from Saccharomyces cerevisiae, are among the most extensively characterized phosphoryl transfer enzymes. In the present study, we have solved the structures of wild-type and seven active site variants of S. cerevisiae PPase bound to its natural metal cofactor, magnesium ion. These structures have facilitated derivation of the complete enzyme reaction scheme for PPase, fulfilling structures of all the reaction intermediates. The main focus in this study was on a novel subfamily of family II PPases (CBSPPase) containing a large insert formed by two CBS domains and a DRTGG domain within the catalytic domain. The CBS domain (named after cystathionine beta-synthase in which it was initially identified) usually occurs as tandem pairs with two or four copies in many proteins in all kingdoms of life. The structure formed by a pair of CBS domains is also known as a Bateman domain. CBS domains function as regulatory units, with adenylate ligands as the main effectors. The DRTGG domain (designated based on its most conserved residues) occurs less frequently and only in prokaryotes. Often, the domain co-exists with CBS domains, but its function remains unknown. The key objective of the current study was to explore the structural rearrangements in the CBS domains induced by regulatory adenylate ligands and their functional consequences. Two CBS-PPases were investigated, one from Clostridium perfringens (cpCBS-PPase) containing both CBS and DRTGG domains in its regulatory region and the other from Moorella thermoacetica (mt CBS-PPase) lacking the DRTGG domain. We additionally constructed a separate regulatory region of cpCBS-PPase (cpCBS). Both full-length enzymes and cpCBS formed homodimers. Two structures of the regulatory region of cpCBS-PPase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate, were solved. The structures were significantly different, providing information on the structural pathway from bound adenylates to the interface between the regulatory and catalytic parts. To our knowledge, these are the first reported structures of a regulated CBS enzyme, which reveal large conformational changes upon regulator binding. The activator-bound structure was more open, consistent with the different thermostabilities of the activator- and inhibitor-bound forms of cpCBS-PPase. The results of the functional studies on wild-type and variant CBS-PPases provide support for inferences made on the basis of structural analyses. Moreover, these findings indicate that CBS-PPase activity is highly sensitive to adenine nucleotide distribution between AMP, ADP and ATP, and hence to the energy level of the cell. CBS-PPase activity is markedly inhibited at low energy levels, allowing PPi energy to be used for cell survival instead of being converted into heat.

Multiple forms of cotyledonary b-galactosidases from Vigna unguiculata quiescent seeds

Cotyledonary b-galactosidases were isolated and partially purified from Pitiúba cowpea (Vigna unguiculata (L.) Walp.) quiescent seeds. The purification steps consisted of precipitation of the crude extract with ammonium sulphate in the range of 20-60% saturation, acid precipitation, DEAE-Sephadex ion-exchange chromatography and Lactosyl-Sepharose affinity chromatography. This purification process gave rise to three b-galactosidases-rich fractions: b-gal I, b-gal II and b-gal III, which were purified about 5, 509, and 62 fold, respectively. They reached maximal enzyme activity at different pH ranges: 3.5-4.5 for b-gal I, 3.0-3.5 for b-gal II, and 3.0-4.0 for b-gal III. Their maximal activities were reached when the temperature of the assay medium was 60° C, and preincubation of the enzymes at different temperatures has shown that they were heat-stable up to 50° C. There were no significant differences among the partially purified enzymes as far as their response to the different effectors tested, except for Mn2+ and EDTA, which affected differently b-gal I, b-gal II, and b-gal III. They were slightly affected by Mg2+, Ca2+, Zn2+, Co2+, tartarate, molybdate, glucose, and lactose, strongly inhibited by Cu2+ and galactose, and inactivated by Hg2+. These chemical and physical properties are similar to the ones found for other plant b-galactosidases. Although through this process of purification three isoforms of this enzyme were obtained, isoelectric focusing in polyacrylamide slab gel of these enzyme-proteins suggest that cotyledons of Pitiúba cowpea quiescent seeds possess four isoforms of b-galactosidases.

The role of T cell subsets and cytokines in the regulation of intracellular bacterial infection

Cellular immune responses are a critical part of the host's defense against intracellular bacterial infections. Immunity to Brucella abortus crucially depends on antigen-specific T cell-mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. T lymphocytes that proliferate in response to B. abortus were characterized for phenotype and cytokine activity. Human, murine, and bovine T lymphocytes exhibited a type 1 cytokine profile, suggesting an analogous immune response in these different hosts. In vivo protection afforded by a particular cell type is dependent on the antigen presented and the mechanism of antigen presentation. Studies using MHC class I and class II knockout mice infected with B. abortus have demonstrated that protective immunity to brucellosis is especially dependent on CD8+ T cells. To target MHC class I presentation we transfected ex vivo a murine macrophage cell line with B. abortus genes and adoptively transferred them to BALB/c mice. These transgenic macrophage clones induced partial protection in mice against experimental brucellosis. Knowing the cells required for protection, vaccines can be designed to activate the protective T cell subset. Lastly, as a new strategy for priming a specific class I-restricted T cell response in vivo, we used genetic immunization by particle bombardment-mediated gene transfer

Cytokine production profile of heart-infiltrating T cells in Chagas' disease cardiomyopathy

The hallmark of chronic Chagas' disease cardiomyopathy (CCC) is the finding of a T cell-rich inflammatory mononuclear cell infiltrate in the presence of extremely few parasites in the heart lesions. The scarcity of parasites in affected heart tissue casts doubt on the direct participation of Trypanosoma cruzi in CCC heart tissue lesions, and suggests the possible involvement of autoimmunity. The cells in the infiltrate are presumably the ultimate effectors of tissue damage, and there is evidence that such cells recognize cardiac myosin in molecular mimicry with T. cruzi proteins rather than primary reactivity to T. cruzi antigens (Cunha-Neto et al. (1996) Journal of Clinical Investigation, 98: 1709-1712). Recently, we have studied heart-infiltrating T cells at the functional level. In this short review we summarize the studies about the role of cytokines in human and experimental T. cruzi infection, along with our data on heart-infiltrating T cells in human Chagas' cardiomyopathy. The bulk of evidence points to a significant production of IFN-g and TNF-a which may be linked to T. cruzi-induced IL-12 production

Mapping and signaling of neural pathways involved in the regulation of hydromineral homeostasis

Several forebrain and brainstem neurochemical circuitries interact with peripheral neural and humoral signals to collaboratively maintain both the volume and osmolality of extracellular fluids. Although much progress has been made over the past decades in the understanding of complex mechanisms underlying neuroendocrine control of hydromineral homeostasis, several issues still remain to be clarified. The use of techniques such as molecular biology, neuronal tracing, electrophysiology, immunohistochemistry, and microinfusions has significantly improved our ability to identify neuronal phenotypes and their signals, including those related to neuron-glia interactions. Accordingly, neurons have been shown to produce and release a large number of chemical mediators (neurotransmitters, neurohormones and neuromodulators) into the interstitial space, which include not only classic neurotransmitters, such as acetylcholine, amines (noradrenaline, serotonin) and amino acids (glutamate, GABA), but also gaseous (nitric oxide, carbon monoxide and hydrogen sulfide) and lipid-derived (endocannabinoids) mediators. This efferent response, initiated within the neuronal environment, recruits several peripheral effectors, such as hormones (glucocorticoids, angiotensin II, estrogen), which in turn modulate central nervous system responsiveness to systemic challenges. Therefore, in this review, we shall evaluate in an integrated manner the physiological control of body fluid homeostasis from the molecular aspects to the systemic and integrated responses.

Epidermal growth factor receptor (EGFR) mutations in lung cancer: preclinical and clinical data

Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing of the NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermal growth factor receptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by the mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activation of signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials of the covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC.

Plasticity of neutrophils reveals modulatory capacity

Neutrophils are widely known as proinflammatory cells associated with tissue damage and for their early arrival at sites of infection, where they exert their phagocytic activity, release their granule contents, and subsequently die. However, this view has been challenged by emerging evidence that neutrophils have other activities and are not so short-lived. Following activation, neutrophil effector functions include production and release of granule contents, reactive oxygen species (ROS), and neutrophil extracellular traps (NETs). Neutrophils have also been shown to produce a wide range of cytokines that have pro- or anti-inflammatory activity, adding a modulatory role for this cell, previously known as a suicide effector. The presence of cytokines almost always implies intercellular modulation, potentially unmasking interactions of neutrophils with other immune cells. In fact, neutrophils have been found to help B cells and to modulate dendritic cell (DC), macrophage, and T-cell activities. In this review, we describe some ways in which neutrophils influence the inflammatory environment in infection, cancer, and autoimmunity, regulating both innate and adaptive immune responses. These cells can switch phenotypes and exert functions beyond cytotoxicity against invading pathogens, extending the view of neutrophils beyond suicide effectors to include functions as regulatory and suppressor cells.

Development of a Control System for a Tele Operated Mobile Robot

Mobile robots are capable of performing spatial displacement motions in different environments. This motions can be calculated based on sensorial data (autonomous robot) or given by an operator (tele operated robot). This thesis is focused on the latter providing the control architecture which bridges the tele operator and the robot’s locomotion system and end effectors. Such a task might prove overwhelming in cases where the robot comprises a wide variety of sensors and actuators hence a relatively new option was selected: Robot Operating System (ROS). The control system of a new robot will be sketched and tested in a simulation model using ROS together with Gazebo in order to determine the viability of such a system. The simulated model will be based on the projected shape and main features of the real machine. A stability analysis will be performed first theoretically and afterwards using the developed model. This thesis concluded that both the physical properties and the control architecture are feasible and stable settling up the ground for further work with the same robot.

Formation of the Employer Brand Image

The purpose of this study was to explore the employer brand image and its formation of a Finnish (yet international) service company in B2B sector. In the research qualitative case study method was used to explore the most preferred attributes in employer attraction, the employer brand perceptions towards the case company, and the effectors behind these brand perceptions among the prospective group of employees. The research was conducted through in-depth semistructured interviews among 23-30 undergraduate or graduate business students in Finnish universities close to their graduation. After deriving the attributes of an attractive employer, and exploring their relation to the case company, the employer brand image of the case company was determined and its attractiveness level evaluated. Moreover, the formation of the employer brand image was investigated and its sources detected. The most important factors for the target group in employer attractiveness were found to be company reputation and company culture/work atmosphere. Also career opportunities and international opportunities were seen important. The case company was found to hold a positive, attractive image on company culture and international opportunities, but failing to hold a good reputation as a fair employer. The main and most impactful source of employer brand image was former and current employees - directly or indirectly. Company’s own actions played only small part in employer brand formation as credibility is a critical factor in brand image formation and corporate communication found not to be perceived genuine. Based on the findings, suggestions for further employer branding were made.

The acute effects of differential dietary fatty acids on PDHa activity in human skeletal muscle at the onset of exercise

Pyruvate dehydrogenase (PDH) is an important regulator of carbohydrate oxidation during exercise and its activity can be down-regulated by an increase in dietary fat. The purpose of this study was to determine the acute metabolic effects of differential dietary fatty acids on the activation of PDH in its active form (PDHa) at rest and at the onset of moderate-intensity exercise. University-aged male subjects (n=7) underwent 2 fat loading trials spaced at least 2 weeks apart. Subjects consumed saturated (SFA) or polyunsaturated (PUFA) fat over the course of 5 hours. Following this, participants cycled at 65% VO2 max for 15 min. Muscle biopsies were taken prior to and following fat loading and at 1 min exercise. Plasma free fatty acids increased from 0.15 ± 0.07 to 0.54 ± 0.19 mM over 5 hours with SFA and from 0.1 1 ± 0.04 to 0.35 ±0.13 mM with PUFA. PDHa activity was unchanged following fat loading, but increased at the onset of exercise in the SFA trial, from 1 .4 ± 0.4 to 2.2 ± 0.4 /xmol/min/kg wet wt. This effect was negated in the PUFA trial (1 .2 ± 0.3 to 1 .3 ± 0.3 pimol/min/kg wet wt.). PDH kinase (PDK) was unchanged in both trials, suggesting that the attenuation of PDHa activity with PUFA was a result of changes in the concentrations of intramitochondrial effectors, more specifically intramitochondrial NADH or Ca^*. Our findings suggest that attenuated PDHa activity participates in the preferential oxidation of PUFA during moderateintensity exercise.

The role of microRNAs and retinoid signaling during spinal cord regeneration in the adult newt.

The molecular events after spinal cord injury that lead to the establishment of a permissive environment and epimorphic regeneration remain unclear. Two molecular pathway regulators that may converge to create a spinal cord regeneration-permissive environment in the urodele are retinoic acid (RA) and microRNAs (miRNAs). Recent evidence suggests that RARβ-mediated signaling is necessary for tail and caudal spinal cord regeneration in the adult newt. MicroRNAs are attractive candidates as mediators of retinoid signaling during regeneration, as their pleiotropic effects are vital in situations where global changes in gene expression are required. Thus, the overall aim of this thesis was to determine if miRNAs are involved in tail and caudal spinal cord regeneration in the adult newt, and if they act as regulators and/or effectors of retinoid signaling during this process. I have demonstrated here, for the first time, that multiple miRNAs are dysregulated in response to spinal cord injury in the adult newt, as well as in response to inhibition of retinoid signaling. Two of these miRNAs, miR-133a and miR-1, appear to target RARβ2 transcripts both in vivo and in vitro. Inhibition of RA signaling via RARβ with a selective antagonist, LE135, alters the pattern of expression of these miRNAs, which leads to an inhibition of tail regeneration. These data are indicative of a negative feed back loop, albeit potentially an indirect one. I also aimed to examine which miRNAs are affected by inhibiting RA synthesis during regeneration, and provided a long list of miRNAs that are dysregulated. These data provide the foundation for future studies on the putative roles of these miRNAs, as well as their function in retinoid signaling. Overall, these studies provide the first evidence for a role for miRNAs as mediators of retinoid signaling during caudal spinal cord regeneration in any system.

Régulation de la proprotéine convertase subtilisine / kexine type 9 (PCSK9) dans les cellules intestinales Caco-2/15

La proprotéine convertase subtilisine/kexine type 9 (PCSK9) favorise la dégradation post-transcriptionnelle du récepteur des lipoprotéines de faible densité (LDLr) dans les hépatocytes et augmente le LDL-cholestérol dans le plasma. Cependant, il n’est pas clair si la PCSK9 joue un rôle dans l’intestin. Dans cette étude, nous caractérisons les variations de la PCSK9 et du LDLr dans les cellules Caco-2/15 différentiées en fonction d’une variété d’effecteurs potentiels. Le cholestérol (100 µM) lié à l’albumine ou présenté en micelles a réduit de façon significative l’expression génique (30%, p<0,05) et l’expression protéique (50%, p<0,05) de la PCSK9. Étonnamment, une diminution similaire dans le LDLr protéique a été enregistrée (45%, p<0,05). Les cellules traitées avec le 25-hydroxycholestérol (50 µM) présentent également des réductions significatives dans l’ARNm (37%, p<0,01) et la protéine (75%, p<0,001) de la PCSK9. Une baisse des expressions génique (30%, p<0,05) et protéique (57%, p<0,01) a également été constatée dans le LDLr. Des diminutions ont aussi été observées pour la HMG CoA réductase et la protéine liant l’élément de réponse aux stérols SREBP-2. Il a été démontré que le SREBP-2 peut activer transcriptionnellement la PCSK9 par le biais de la liaison de SREBP-2 à son élément de réponse aux stérols situé dans la région proximale du promoteur de la PCSK9. Inversement, la déplétion du contenu cellulaire en cholestérol par l’hydroxypropyl-β-cyclodextrine a augmenté l’expression génique de la PCSK9 (20%, p<0,05) et son contenu protéique (540%, p<0,001), en parallèle avec les niveaux protéiques de SREBP-2. L’ajout des acides biliaires taurocholate et déoxycholate dans le milieu apical des cellules intestinales Caco-2/15 a provoqué une baisse d’expression génique (30%, p<0,01) et une hausse d’expression protéique (43%, p<0,01) de la PCSK9 respectivement, probablement via la modulation du FXR (farnesoid X receptor). Ces données combinées semblent donc indiquer que la PCSK9 fonctionne comme un senseur de stérols dans le petit intestin.

Assemblage oligomérique des récepteurs couplés aux protéines G avec les RAMPs

Les récepteurs couplés aux protéines G (RCPGs) constituent la plus grande classe de récepteurs membranaires impliqués dans la transmission des signaux extracellulaires. Traditionnellement, la transmission de la signalisation par les RCPGs implique l’activation d’une protéine G hétéro-trimérique qui pourra à son tour moduler l’activité de divers effecteurs intracellulaires. Ce schéma classique de signalisation s’est complexifié au fils des années et l’on sait maintenant qu’en plus d’interagir avec les protéines G, les RCPGs s’associent avec une panoplie d’autres protéines afin de transmettre adéquatement les signaux extracellulaires. En particulier, la découverte d’une famille de protéines transmembranaires modulant la fonction des RCPGs, baptisées protéines modifiant l’activité des récepteurs (« receptor activity-modifying proteins » ; RAMPs), a changé la façon de concevoir la signalisation par certains RCPGs. Dans le cas du récepteur similaire au récepteur de la calcitonine (« calcitonin-like receptor » ; CLR), l’association avec les RAMPs permet l’acheminement à la surface cellulaire du récepteur tout en modulant ses propriétés pharmacologiques. Lorsqu’il est associé avec RAMP1, le CLR fonctionne comme un récepteur du peptide relié au gène de la calcitonine (« calcitonin gene-related peptide » ; CGRP), alors qu’il devient un récepteur de l’adrénomedulline lorsqu’il interagit avec RAMP2 ou RAMP3. D’autre part, en plus d’interagir avec des protéines accessoires transmembranaires telles les RAMPs, les RCPGs peuvent aussi s’associer entre eux pour former des oligomères de récepteurs. Dans cette thèse, nous nous sommes penchés sur les interactions entre les RCPGs et les RAMPs, et plus particulièrement sur l’interrelation entre ce type d’association RCPG/RAMP et l’assemblage en oligomères de récepteurs, en utilisant le récepteur du CGRP comme modèle d’étude. Une première étude nous a tout d’abord permis de confirmer l’interaction entre le récepteur CLR et RAMP1, dans un contexte de cellules vivantes. Nous avons démontré que ce complexe CLR/RAMP1 active la protéine G et recrute la protéine de signalisation -arrestine suite à une stimulation par le CGRP. Ensuite, nous avons déterminé que même s’il doit obligatoirement former un hétéro-oligomère avec les RAMPs pour être actif, le CLR conserve malgré tout sa capacité à interagir avec d’autres RCPGs. En plus d’observer la présence d’homo-oligomère de CLR, nous avons constaté que tout comme les RCPGs, les RAMPs peuvent eux-aussi s’associer entre eux pour former des complexes oligomériques pouvant comprendre différents sous-types (RAMP1/RAMP2 et RAMP1/RAMP3). Cette observation de la présence d’homo-oligomères de CLR et de RAMP1, nous a amené à nous questionner sur la stœchiométrie d’interaction du complexe CLR/RAMP1. Dans une deuxième étude ayant pour but d’établir la composition moléculaire du récepteur CGRP1 in vivo, nous avons développé une nouvelle approche permettant l’étude de l’interaction entre trois protéines dans un contexte de cellules vivantes. Cette technique baptisée BRET/BiFC, est basée sur le transfert d’énergie de résonance de bioluminescence entre un donneur luminescent, la Renilla luciférase, et un accepteur fluorescent, la protéine fluorescente jaune (YFP), reconstituée suite au ré-assemblage de ces deux fragments. En utilisant cette approche, nous avons pu déterminer que le récepteur CGRP1 est constitué d’un homo-oligomère de CLR interagissant avec un monomère de RAMP1. En démontrant un assemblage oligomérique asymétrique pour le récepteur CGRP1 à partir d’une nouvelle approche biophysique, nous croyons que les travaux présentés dans cette thèse ont contribué à élargir nos connaissances sur le fonctionnement de la grande famille des RCPGs, et seront utile à la poursuite des recherches sur les complexes protéiques impliqués dans la signalisation.