Background: Beta blockers are recommended by the American College of Cardiology/American Heart Association Guidelines for high and intermediate-risk cardiac patients undergoing non-cardiac surgery. Beta blockers are a class of drugs that moderate the effects of increased catecholamine levels on the heart by selectively blocking beta receptors in the heart and blood vessels, resulting in a lower heart rate and blood pressure. Beta blocker use perioperatively has been shown to reduce the risk of ischemia and infarction. Purpose: The purpose of this project is to address beta blocker use in a group of anesthesia providers who routinely attend to high-risk and intermediate-risk cardiac patients undergoing non-cardiac surgery in a medium-sized private hospital in suburban South Florida. There are barriers to the implementation of the published guidelines for beta blocker administration, including lack of awareness of the best current practice and a lack of a formal beta blocker protocol at the institutional level. Methods: A simple and inexpensive beta blocker protocol was implemented and evaluated by various means. Beta blocker administration practices were examined and documented prior to and after protocol implementation. Beta blocker usage was examined prior to and after protocol implementation Findings/Implications: It was hypothesized that increased anesthesia provider awareness would lead to increased administration of perioperative beta blockers to high-risk and intermediate-risk cardiac patients undergoing non-cardiac procedures. Although there was a knowledge increase related to the new beta blocker protocol, no change in practice was observed.

Made available in DSpace on 2014-07-29T15:25:14Z (GMT). No. of bitstreams: 1 Tese de doutorado Luiza Cristina Lacerda Jacomini.pdf: 1113139 bytes, checksum: 6f7dbdd64a41cff3100ce05df4ddcf61 (MD5) Previous issue date: 2010-08-05 / Lacerda Jacomini, LC. Investigation on dysautonomia, epinephrine and norepinephrine-evoked pain, and effect of beta-blocker in fibromyalgia and systemic lupus erythematosus. 2010, 169 p. Doctoral thesis - Universidade Federal de Goiás, Goiânia. Dysautonomia is a condition in which an altered autonomic function affects the health in an adverse way. The present study aims: to search for the presence of epinephrine and norepinephrine-evoked pain; to evaluate the cardiovascular autonomic function and the effect of propranolol in women with fibromyalgia (FM), systemic lupus erythematosus (SLE) and controls (CTR). For each objective a separate research was developed, including a clinical trial. Epinephrine and norepinephrine-evoked pain were diagnosed when the subcutaneous injections containing these substances (10 micrograms/ 0.1 mL saline solution) induced greater pain than the saline solution did (n=7). Autonomic function was assessed through the standard Ewing tests battery, through heart rate responses to Valsalva maneuver, deep breathing, standing and blood pressure responses to ortostatism and to hand grip (n=7). Functional symptoms related to autonomic manifestations were checked. In a randomized, double-blind, placebo-controlled, crossover clinical trial with 6 women with FM, SLE and CTR, propranolol (80 mg/day po/4 weeks) was added to the usual schedule of prescribed medicines and its effect was examined regarding: pain, fatigue, tender points, blood pressure, heart rate, health related quality of life (SF-36) and fibromyalgia impact questionnaire. Epinephrine-evoked pain was diagnosed in SLE and FM groups and norepinephrine-evoked pain was diagnosed in FM group. Epinephrine and norepinephrine-evoked pain intensity has a trend to be greater in FM patients when compared to healthy CTR. FM and SLE patients had an elevated number of functional symptoms related to autonomic manifestations. Cardiovascular autonomic function was altered in FM and SLE groups. Parasympathetic cardiovascular autonomic function tests were mainly abnormal in SLE patients while in FM patients both, parasympathetic and sympathetic tests were abnormal. Propranolol reduced tender points count and the number of symptoms related to autonomic manifestations in FM group. Four in six patients presented significant improvement in health related quality of life evaluated by SF-36. These results suggest that FM belong to the group of sympathetically maintained pain syndromes. The study demonstrates that FM and SLE patients have cardiovascular autonomic function alterations which can be detected by simple, standardized, non-invasive and inexpensively methodology and that propranolol has a potential benefit in FM treatment. / Lacerda Jacomini, LC. Pesquisa de disautonomia, dor evocada por adrenalina e noradrenalina e efeito de beta-bloqueador na fibromialgia e no lupus eritematoso sistêmico. 2010, 169 p. Tese de doutorado - Universidade Federal de Goiás, Goiânia. Disautonomia é uma condição na qual a função autonômica alterada afeta a saúde de modo adverso. Os objetivos do presente estudo foram: pesquisar a presença de dor evocada por adrenalina (AD) e por noradrenalina (NA), avaliar a função autonômica cardiovascular e o efeito do propranolol, em mulheres com fibromialgia (FM), lupus eritematoso sistêmico (LES) e controles (CTR). Para cada objetivo foi desenvolvida uma etapa de estudo, sendo incluído um ensaio clínico. A dor evocada por AD ou NA foi diagnosticada quando estas substâncias produziram dor maior que soro fisiológico quando injetadas (10 microgramas/ 0,1 mL de soro fisiológico), via subcutânea (n=7). A função autonômica foi testada usando-se a bateria de testes de Ewing (respostas da frequência cardíaca à manobra de Valsalva, respiração profunda e ao ortostatismo e respostas da pressão arterial ao ortostatismo e à preensão sustentada) (n=7). Foram pesquisados sintomas funcionais relacionados às manifestações autonômicas. No ensaio-clínico randomizado, duplo-cego, placebo controlado e cruzado em grupos de 6 mulheres com LES, FM e CTR, o propranolol (80 mg/dia, via oral/4 semanas) foi adicionado ao esquema terapêutico das pacientes e seu efeito avaliado segundo as variáveis: dor, fadiga, tender points, pressão arterial, frequência cardíaca, qualidade de vida e questionário do impacto da FM. A dor evocada por AD ocorreu nos grupos FM e LES comparada ao CTR e, por NA ocorreu no grupo FM. Os escores de dor evocada por AD e por NA, no grupo FM, tiveram uma tendência a serem maiores do que os do grupo CTR. Pacientes com FM e com LES apresentaram elevado número de sintomas funcionais relacionados a manifestações autonômicas. A função autonômica cardiovascular estava alterada no LES e na FM. No grupo LES, os testes de função parassimpática tiveram frequência maior de respostas anormais e no grupo FM estavam alterados tanto os da função parassimpática como da simpática. O propranolol reduziu o número de tender points e de sintomas funcionais relacionados a manifestações autonômicas no grupo FM. Quatro em 6 pacientes do grupo FM apresentaram melhora significativa na qualidade de vida avaliada pelo SF-36. Os resultados sugerem que a FM faz parte do grupo de doenças com dor simpaticamente mantida. O estudo demonstrou que as pacientes com LES e FM têm alterações da função autonômica cardiovascular detectáveis por metodologia simples, padronizada, não invasiva e de baixo custo e que o propranolol tem um benefício potencial no tratamento da FM.

This dissertation consists of six essays in health economics.The first essay, “Economic evaluations in health care: Basic principles and special topics”, serves as an introduction to economic evaluations in health care, including estimations of costs, health effects, and the discount rate. Special topics of interest for the rest of the studies are also discussed, e.g. the role of modelling in cost-effectiveness analysis, and methods for dealing with incomplete observations in clinical trial data. The main theme of the second essay, “Consumption and production by age in Sweden: Basic facts and health economic implications”, is a fairly detailed compilation of consumption and production figures by age in Sweden. The purpose of this is to use the difference between consumption and production in each age group as a measure of the average costs of added years of life in the general population. In economic evaluations of health care interventions, only future costs for related ill­nesses have typically been included in the analysis. However, the health economist David Meltzer has argued that future costs for un­related illnesses and general consumption should also be in­cluded in eco­nomic evaluations. Otherwise, the analysis will not be consistent with expected utility maximiza­tion. The third essay is entitled “The possibility of predicting health care costs in the future from predicted changes in age structure and age specific mortality: The case of Sweden”. Changes in the age structure, especially the growing number of elderly people, have raised concerns about increasing costs for health and elderly care in the future. However, the number of elderly per se is not the main problem, since the growing number of elderly people is a result of better health and hence lower morta­lity. The main purpose of the study is to investigate if future health care costs can be predicted based on forecasts of future changes in age structure and mortality rates. It is shown here that at least in Sweden and in the U.S., there is a linear relationship between age-specific mortality and age-specific health care costs. When these relationships are applied retrospectively to old data, however, the predictions are underestimates of the actual costs. These results are in line with earlier studies, which show that the future age structure is not likely to have a great impact on the future health care costs. The fourth essay is called “Cost effectiveness of bisoprolol in the treatment of chronic congestive heart failure in Sweden: Analysis using data from the Cardiac Insufficiency Bisoprolol Study II” (with Niklas Zethraeus and Bengt Jönsson). Treatment of heart failure with beta blockers was introduced in Sweden already in the 1970s, but it was not until the 1990s that large-scale clinical trials established the efficacy of beta blockers in reducing heart failure mortality. The study consists of an economic evaluation of the beta blocker bisoprolol added to standard treatment of chronic heart failure, compared with placebo added to the same standard treatment. The study raises a number of methodological issues. At the forefront are the inclusion of costs of added years of life, and the question of how to model health effects that extend beyond the clinical trial on which the economic evaluation is based. The results indi­cate that treatment with bisoprolol is cost-effective. A drawback of the analysis in the fourth study was that the expected survival after the end of follow-up was modelled deterministically. This makes it impossible to assess the uncertainty of the cost-effectiveness estimate in a realistic way. The fifth essay is entitled “Assessing uncertainty in cost-effectiveness analysis by combining resampling of clinical trial data with stochastic modelling: The economic evaluation of bisoprolol for heart failure revisited”. Here, the drawback with the fourth study that was mentioned above is addressed by using resamp­ling of the clinical trial data in combination with stochastic modelling of the expected survival after the end of follow-up in the clinical trial. The methodology is inspired by the bootstrap method, which is a simulation technique whereby various statistics, like the mean and variance, can be estimated through repeated resampling from the original sample. The difference from the traditional boot­strap method is that resampling of observations from the clinical trial data is combined with stochastic modelling of the expected remaining lifetime of the patients who were alive at the end of the clinical trial. Cost-effectiveness acceptability curves for treatment of heart failure with bisoprolol were obtained as a result of the analysis. The sixth essay, “Survival analysis techniques for estimating the costs attributable to head and neck cancer in Sweden”, concerns the estimation of average treatment cost attri­butable to a disease when the data contain censored, i.e. incomplete, observations. For various reasons, censored observations are common in medical and epidemiological studies. As a result, the length of the survival time or the size of the costs for those who are alive at the end of follow-up are not exactly known. This is of course problematic if we want to estimate the average survival time or the average cost for all patients, both survivors and non-survivors included. In this study, the Kaplan-Meier sample-average estimator is used for overcoming the problem with censored observations. It is a method that has been proposed specifically for handling censored cost data. / Diss. Stockholm: Handelshögsk., 2002

Diss. Stockholm: Handelshögsk., 2002.

No description available.

En France, le syndrome coronaire aigu (SCA) représente environ 100 000 hospitalisations par an. Il est recommandé, en prévention secondaire du SCA, un traitement combinant quatre classes médicamenteuses : bêtabloquants, antiagrégants plaquettaires, statines, et inhibiteurs de l’enzyme de conversion ou antagonistes des récepteurs à l’angiotensine II(combinaison BASI). L’objectif de ce travail était l’étude, en situation réelle de soin et en population générale, de l’utilisation et de l’effectivité de la combinaison thérapeutique recommandée en prévention secondaire du SCA. Nous avons d’abord exploré le potentiel représenté par les bases de données médicoadministratives françaises pour cette évaluation. Nous avons ensuite étudié l’utilisation de la combinaison BASI : 42 % des patients étaient traités par la combinaison BASI en post-SCA et 57 % d’entre eux étaient encore traités à deux ans ; la persistance au traitement était plus faible chez les patients âgés, chez ceux ayant d’autres co-morbidités et chez ceux ayant eu un SCA de nature autre qu’un infarctus du myocarde.Nous avons enfin étudié l’effectivité de la combinaison BASI : la combinaison BASI était la combinaison thérapeutique la plus effective à long terme après un SCA chez les patients avec antécédent d’insuffisance cardiaque ; chez les patients sans antécédent de ce type la combinaison sans bêtabloquants n’était pas associée à une augmentation du risque.Ces résultats permettent de reconsidérer l’intérêt à long terme de l’ensemble de la combinaison BASI en post-SCA chez tous les patients et mettent en avant la nécessité de renforcer les stratégies d’éducation thérapeutique. / Acute coronary syndrome (ACS) causes approximately 100,000 hospitalisations per year in France. In secondary prevention of ACS, guidelines advocate pharmacological treatment combining four drug classes: beta-blockers, antiplatelet agents, statins and angiotensin converting enzyme inhibitors or angiotensin receptor blockers (recommended combination).The aim of this work was to study, in real life and among the general population, the use and the effectiveness of the recommended combination for secondary prevention of ACS.Firstly, we explored the potential represented by the French claims databases in this context. Secondly, we studied the use of the recommended combination: 42% of patients were treated with the recommended combination in post-ACS and 57% of them were still treated two years after; persistence to combination was lower in older patients, in those with other comorbidities and those who had an ACS different of myocardial infarction. Thirdly, we studied the effectiveness of the recommended combination: the recommended combination was the most effective combination therapy at long-term in post-ACS patients with history of heart failure; in patients without such history the combination without betablockers was not associated with an increased risk. These results could help reconsidering the long-term interest of the full recommended combination in all ACS patients and highlight the need to strengthen patient education strategies.

La pharmacogénomique (PGx) étudie le concept selon lequel les déterminants génétiques peuvent aider à prédire la réponse clinique d’un patient aux médicaments. Les concentrations plasmatiques de ces derniers sont essentielles pour déterminer l’exposition, les profils pharmacocinétiques (PK), les effets cliniques et éventuellement les doses des médicaments, dont la plupart sont métabolisés par des enzymes hépatiques, les cytochromes P450 (CYPs). Néanmoins, la plupart des découvertes en matière de PGx concernant la prédiction des profils de concentrations des médicaments ont généralement recours à des plans d’études PK traditionnels avec une approche fonctionnelle. Bien qu’utile, cette méthodologie comporte des limites pour les études PGx, notamment le nombre restreint de sujets inclus, qui réduit la puissance statistique des associations PGx et limite l’identification de nouveaux variants génétiques moins fréquents. À l’inverse, les grandes cohortes observationnelles sont largement utilisées pour identifier des marqueurs génétiques physiopathologiques. Cette thèse de doctorat visait donc à 1) synthétiser les données publiées concernant les effets cliniques des polymorphismes génétiques de l’enzyme CYP2D6 sur le traitement au métoprolol, un agent β-bloquant. Les concentrations plasmatiques de métoprolol ont montré à plusieurs reprises qu’elles étaient fortement influencées par la PGx du CYP2D6; 2) développer une nouvelle méthode bioanalytique capable de quantifier les concentrations chirales de métoprolol des patients dans un contexte clinique; 3) mener une étude clinique en utilisant une grande cohorte observationnelle, ou biobanque, comme preuve de concept pour recréer l’association précédemment établie entre les phénotypes inférés des génotypes du CYP2D6 et les concentrations plasmatiques de métoprolol. Ces projets sont présentés en tant que chapitres de thèse et sous forme de manuscrits publiés. Le premier projet consistait en une revue systématique qui a permis d’extraire toutes les études relatives à la PGx du métoprolol-CYP2D6. La synthèse qualitative a suggéré que les métaboliseurs lents du CYP2D6, dépourvus de capacité enzymatique, avaient des valeurs plus élevées concernant les réductions de la fréquence cardiaque et de tension artérielle, ainsi que la survenue d’épisodes bradycardiques relativement aux autres phénotypes. Une méta-analyse ultérieure a confirmé la significativité de ces associations. Le deuxième projet a combiné des techniques bioanalytiques telles que la dérivation, l’extraction en phase solide et la chromatographie liquide avec spectrométrie de masse en tandem. Une méthode permettant de surmonter les limites analytiques antérieures a été validée avec succès pour mesurer les concentrations plasmatiques de (S)-métoprolol, l’énantiomère pharmacologiquement actif, et de son métabolite spécifique au CYP2D6. L’applicabilité d’une telle méthode a ensuite été démontrée grâce aux échantillons d’un groupe de patients issus de la Cohorte Hospitalière de l’Institut de Cardiologie de Montréal (ICM). Puis, le troisième projet présente la réalisation de l’étude LEVEL-PGx (LEVEraging Large observational cohort studies to identify pharmacogenetic determinants of drug dosing : A proof-of-concept study in the Montreal Heart Institute Hospital Cohort). L’étude portait sur un échantillon de >1000 patients sélectionnés dans la cohorte hospitalière de l’ICM, incluant leur génotypage pour CYP2D6 et la quantification du métoprolol racémique et de son métabolite spécifique au CYP2D6 dans des échantillons provenant de la Biobanque de l’ICM. Un seul échantillon unique et aléatoire par patient a été utilisé. Le recours à des modèles multivariables a validé le concept selon lequel de grandes cohortes transversales recueillant des échantillons biologiques pouvaient être utilisées afin d’identifier des associations PGx de concentrations de médicaments et ce, à des valeurs satisfaisant les seuils de significativité d’essais pangénomiques. D’autres analyses de cette cohorte ont indiqué que cette méthodologie parvenait à identifier des associations PGx qui influençaient la fréquence cardiaque au repos et la posologie du métoprolol à-travers les phénotypes du CYP2D6 et pour les déterminants génétiques uniques, même en présence de co-médications. Cependant, ces associations PGx avec les paramètres cliniques n’ont pas atteint une significativité applicable aux seuils pangénomiques. En résumé, par la reproduction d’une association PGx préalablement démontrée, l’ensemble des travaux présentés dans cette thèse suggère que l’identification et la découverte de nouveaux déterminants génétiques prédictifs des concentrations et des doses des médicaments pourrait s’effectuer par le biais de grandes cohortes observationnelles à l’échelle du génome. Ces approches permettraient de développer des modèles prédictifs plus précis de l’exposition et de la réponse aux médicaments, ce qui pourrait favoriser les découvertes PGx et, dans certains cas, éventuellement développer le potentiel translationnel d’une approche thérapeutique personnalisée selon le profil génétique des patients. / Pharmacogenomics (PGx) studies the concept that genetic determinants can help predict a patient’s clinical response to therapies. Drug concentrations are an essential component to determining the exposure, pharmacokinetic (PK) profiles, clinical effects, and potentially drug doses, most of which are metabolized through the cytochrome P450 (CYPs) liver enzymes. Nevertheless, most PGx discoveries regarding the prediction of drug concentration profiles have generally resorted to traditional PK study designs with a functional approach. Though useful, this methodology contains limitations for gene-drug interaction studies, most notably the restricted number of subjects included, which reduces the statistical power for PGx associations and limits the identification of new, less frequent genetic variants. On the opposite, large observational cohorts have long been utilized for identifying genetic markers of disease. This doctoral thesis therefore aimed to 1) synthesize published data regarding the clinical effects of CYP2D6 genetic polymorphism on metoprolol therapy. A β-blocker, metoprolol plasma concentrations have shown repeatedly to be heavily influenced by the PGx of the CYP2D6 enzyme; 2) develop a new bioanalytical method able to quantify patients’ chiral concentrations of metoprolol in a clinical setting; 3) conduct a clinical study using a large observational cohort, or biobank, as a proof of concept to recreate the previously established association between CYP2D6 genotype-inferred phenotypes and metoprolol plasma concentrations. Those projects are presented as thesis chapters in the form of published manuscripts. The first project was a systematic review that allowed us to find all studies pertaining to the PGx of metoprolol. The qualitative synthesis suggested that CYP2D6 poor metabolizers (PMs), without enzymatic capacity, had greater values regarding reductions in heart rate, blood pressures, and occurrences in bradycardia relative to non-PMs. A subsequent meta-analysis confirmed the significance of those associations. The second project combined bioanalytical techniques such as derivatization, solid phase extraction, and liquid chromatography-tandem mass spectrometry. A method overcoming previous analytical shortcomings was successfully validated to measure (S)-metoprolol plasma concentrations and its CYP2D6-specific metabolite. Its application was later demonstrated in a group of patients from the Montreal Heart Institute (MHI) Hospital Cohort. Then, the third project presents the conduct of the LEVEL-PGx study (LEVEraging Large observational cohort studies to identify pharmacogenetic determinants of drug dosing: A proof-of-concept study in the Montreal Heart Institute Hospital Cohort). The study implicated a sample of >1000 selected patients selected from the MHI Hospital Cohort, along with the genotyping of CYP2D6, and the quantification of racemic metoprolol and its CYP2D6-specific metabolite in samples from the MHI Biobank. A single, random sample per patient was used. Multivariable modeling validated the concept that large observational cohorts collecting biospecimens could be utilized to identify PGx associations of drug concentrations with genome-wide significance. Further analyses in our cohort indicated that the tested PGx associations influenced resting heart rate and metoprolol daily drug dosage across CYP2D6 phenotypes and for single genetic determinants, regardless of interfering comedications. However, such PGx associations with clinical parameters could not achieve genome-wide significance. In summary, the body of work presented in this thesis suggested that, using a previously validated PGx association, the identification of novel genetic determinants predictive of drug concentrations and dosage could be discovered and identified at the genome-wide level with large observational cohorts. These approaches would help develop more accurate predictive models of drug exposure and response, which could favor PGx discoveries and the translational potential of a personalized approach to treatments according to a patient’s genetic profile.

Les récepteurs couplés aux protéines G (RCPG) constituent la plus grande famille de protéines membranaires du génome humain. Ils transmettent les signaux extracellulaires provenant de plusieurs stimuli comme les odeurs, les ions, les hormones et les neurotransmetteurs, à l'intérieur des cellules. En se liant aux RCPGs, ces molécules contribuent à la stabilisation des changements conformationnels activateurs qui se propagent jusqu'au domaine intracellulaire des récepteurs. Ces derniers engagent ensuite un ou plusieurs effecteurs, comme les protéines G hétérotrimériques et les β-arrestines (βarrs), qui activent une cascade d'événements moléculaires menant à la réponse cellulaire.Récemment, la publication de structures cristallines de RCPGs liant des ligands diffusibles a offert une opportunité de raffiner à une résolution atomique les modèles des mécanismes de transduction des signaux. Dans la première partie de cette thèse, nous avons donc exploré les déterminants de la signalisation du récepteur prototypique β2-adrénergique (β2AR), induite par les β-bloqueurs. En ne tenant compte que de leur efficacités sur le β2AR dans les voies de l'adénylate cyclase (AC) et des protéines kinases activées par les facteurs mitogéniques (MAPK), les β-bloqueurs peuvent être classés en 3 groupes distincts (agoniste inverse AC / agoniste MAPK, antagoniste neutre AC / agoniste MAPK et agoniste inverse AC / agoniste inverse MAPK). Afin de déterminer le lien entre leur efficacité et leur mode de liaison, nous avons réalisé des expériences d'arrimages moléculaires in silico entre des β-bloqueurs de chacun des groupes et la structure cristalline du β2AR liée au carazolol. De manière intéressante, les ligands à l'intérieur d'un groupe partagent un mode de liaison, alors que ceux des ligands entre les groupes divergent, suggérant que le mode de liaison des β-bloqueurs pourrait être utilisé pour prédire leur l'efficacité. En accord avec cette hypothèse, nous avons prédit et confirmé l'efficacité agoniste MAPK du carazolol, un inverse agoniste AC du β2AR se liant au récepteur de manière similaire au groupe inverse agoniste AC / agoniste MAPK. De manière intéressante, le groupement aryl des ligands agonistes inverses agonistes AC / agoniste MAPK, le seul groupement chimique variable de ce groupe, est prédite pour lier la région des 3e et 5e hélices transmembranaires (TM3 et TM5). Nous avons donc émis l'hypothèse que cette région pourrait être un déterminant de l'efficacité de ces ligands. En accord avec cette dernière, la mutation de 2 résidus (T118I, S203A) localisés proches du site de liaison des groupements aryls des β-bloqueurs, prévient l'efficacité agoniste inverse de l'ICI-118551 sur la voie de l'AC sans affecter l'efficacité d'un agoniste, indiquant que cette région est importante pour la transmission de l'effet agoniste inverse, du moins sur la voie de l'AC. Les βarrs sont des protéines d'échafaudage qui coordonnent la formation de complexes avec plusieurs dizaines d'effecteurs de signalisation. Originalement identifiées pour leur rôle dans la désensibilisation et l'internalisation des RCPGs, elles sont aussi d'importants effecteurs de la signalisation des RCPGs indépendante des protéines G hétérotrimériques. Cependant, contrairement aux protéines G hétérotrimériques, il n'existe que peu d'outils pour les étudier. Ainsi, la deuxième partie de la thèse est dédiée au développement d'outils pour l'étude des βarrs. À cette fin, nous avons d'abord tenté de transposer une méthode de mesure de l'interaction entre 2 protéines par la technologie de transfert d'énergie de bioluminescence par résonance (BRET) en microscopie et chez des souris transgéniques afin de mesurer de manière subcellulaire et dans un contexte natif l'engagement de la βarr à des RCPGs. Ainsi, nous avons établi les preuves de principe que le BRET peut être utilisé pour localiser l'interaction entre la βarr et le récepteur de la vasopressine de type 2 (V2R) sur une cellule au microscope et pour détecter l'interaction entre la βarr et le β2AR sur des tissus de souris transgéniques exprimant ces protéines fusionnées avec des partenaires BRET. Finalement, il n'existe aucun inhibiteur pharmacologique ciblant les βarrs. Ainsi, grâce à la combinaison d'approches de criblage virtuel sur un modèle de la structure des βarrs et d'essais de validation cellulaire, nous avons développé un inhibiteur pharmacologique des βarrs. À l'aide de cet outil, nous avons confirmé l'implication des βarrs dans l'activation des MAPK par le V2R, mais aussi montré un nouveau rôle des βarrs dans le recyclage du β2AR. Les connaissances et outils développés dans cette thèse permettront de mieux comprendre les déterminants moléculaires de la signalisation des RCPGs et entre autres, grâce à des nouvelles approches pour étudier le rôle cellulaire et physiologique des βarrs. / G Protein-Coupled Receptors (GPCR) are members of the largest family of membrane protein in the human genome. They transduce the signal from a variety of stimuli like odors, ions, hormones and neurotransmitters, inside the cells. By binding directly to the receptors, these molecules stabilize activating conformational changes that are allosterically propagated through transmembrane to intracellular domains. Effectors like heterotrimeric G protein and β-arrestins (βarrs) are then engaged by activated receptors and trigger a cascade of signalling events leading to a cellular response. Recently, the resolution of the crystal structure of GPCR that bind to freely diffusible ligands provided the opportunity to refine at an atomic level the models describing the mecanisms of receptor signal transduction. In the first section of this thesis, we have explored the determinants of the prototypical β2-adrenergic receptor (β2AR) signalling induced by β-blockers. Given their efficacy on Adenylate Cyclase (AC) and Mitogen-Activated Protein Kinase (MAPK) pathways, β-blockers can be classified within 3 signalling groups (AC inverse agonist / MAPK agonist, AC neutral antagonist / MAPK agonist and inverse agonist for AC and MAPK). In order to gain insight on the relation between their efficacy and binding mode, we performed in silico binding experiments between β-blockers from each group and the β2AR crystal structure bound to carazolol. Interestingly, ligands within a group share similar binding mode in contrast to those of different groups, suggesting that β-blockers binding mode could be used to predict their efficacy. In accordance to this hypothesis, we have predicted and confirmed that carazolol, an AC inverse agonist that bind to β2AR in a similar way than the AC inverse agonist / MAPK agonist group, is indeed an agonist for MAPK pathway. Moreover, aryl chemical function from AC inverse agonist / MAPK agonist ligands, barely the only variable structure feature of this group, was predicted to bind β2AR nearby the transmembrane helices 3 and 5 (TM3 and TM5). We thus have predicted that this region would be a determinant of the AC inverse agonist / MAPK agonist ligand efficacy. Accordingly, we found that mutation of 2 residues (T118I, S203A) close to the aryl moiety binding site prevents inverse agonist efficacy of ICI-118551 on AC pathway, without affecting agonist efficacy, indicating that this receptor region is important for the efficacy of these group of β-blockers, at least on AC inverse agonism.βarrs are scaffolding proteins that coordinate protein complex formation with dozen of signalling effectors. First identified for their role on GPCR desensitization and internalization, βarrs are also an important heterotrimeric G protein independent GPCR signalling effectors. However, in contrast to heterotrimeric G protein, only a few tools are available for their study. Thus, the second section of this thesis aim at developing tools for the study of βarrs. For this purpose, we had attempted to transpose a method to measure protein-protein interaction that use Bioluminescence Resonance Energy Transfer (BRET) technology, in microscopy and in transgenic mice, in order to detect subcellular localization and in a native context the engagement of βarr to RCPGs. Thus, we have established a proof of principle that BRET can be combined with microscopy to locate an interaction between βarr and the type 2 vasopressin receptor (V2R) within a cell. Moreover, we have established a second proof of principle that we can detect βarrs recruitment to β2AR on cells extracted from tissues of transgenic mice expressing these proteins fused to BRET partner. Finally, there is no pharmacological inhibitor of βarrs. Thus, using a combination of virtual screening and cellular validation approches, we have developed the first pharmacological βarrs inhibitor. With this novel tool, we have confirmed the implication of βarrs in V2R-mediated MAPK activation, but also showed a new role of βarrs in β2AR recycling.The finding and the tools presented in this thesis should allow to better understand the molecular determinants of GPCR signalling, and among other things, by proposing new tools to study βarrs cellular and physiological roles.

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Les substances pharmaceutiques font partie du groupe des contaminants émergents du fait de leur intérêt récent dans les études environnementales comparativement à des polluants étudiés depuis plus longtemps tels que les pesticides. Elles correspondent aux principes actifs des médicaments et, à ce titre, sont responsables des propriétés pharmacologiques des médicaments. Ce sont donc des molécules biologiquement actives qui peuvent agir sur les organismes vivants présents dans les écosystèmes impactés. L’origine des substances pharmaceutiques dans l’environnement est variable mais les principales sources sont liées à leur utilisation en médecine humaine ou vétérinaire. Une fois consommées, les substances pharmaceutiques sont excrétées dans les urines ou les fèces et se retrouvent dans les eaux usées (consommation humaine) ou dans les déchets d’élevage (consommation vétérinaire). Dans le premier cas, elles peuvent être rejetées directement dans le milieu, ou indirectement, avec les eaux usées traitées ou les boues résiduaires, après traitement dans les stations d’épuration (STEP). Dans le deuxième cas, elles atteignent directement le milieu lorsque les animaux sont élevés en prairie ou indirectement lorsque les déchets d’élevage sont épandus sur les sols agricoles pour les fertiliser. Ces travaux de thèse se sont attachés à étudier l’origine et le devenir de ces substances dans ces 2 cas de figure. Ainsi en se basant sur des critères de consommation, de présence dans l’environnement par rapport à des études antérieures, de toxicité et d’écotoxicité, d’originalité et de disponibilité des composés standards de référence, 32 puis 78 molécules appartenant aux classes thérapeutiques des antibiotiques, des anticancéreux, des béta-bloquants, des anti-VIH et des inhibiteurs de phosphodiestérase de type 5 (PDE 5) ont été étudiées dans 2 continuums : i) effluents hospitaliers - eaux usées brutes et traitées – eaux de surface, et ii) eaux usées brutes et traitées - eaux de surface - eaux de captage souterraines. En s’appuyant sur les mêmes critères de sélection, le devenir de 7 antibiotiques a été étudié dans des lisiers porcins dans des filières simples de traitement du lisier (fosse de stockage), dans des filières complexes de traitement du lisier (système de traitement ressemblant à des mini STEP) et dans des mésocosmes en conditions contrôlées. Pour pouvoir réaliser l’ensemble de ces études, des protocoles analytiques mettant en œuvre une étape d’extraction par SPE (Solide Phase Extraction) ou d’extraction ASE (Extraction Accélérée par Solvant) puis de purification par SPE et d’analyse par LC/MS/MS (Chromatographie en phase liquide couplée à la spectrométrie de masse en tandem) ont été développés. Ces protocoles, en remplissant des critères de qualité tels que des limites de détection et de quantification compatibles avec des analyses environnementales (de l’ordre du ng/l à la dizaine de ng/l), une bonne linéarité, précision, justesse et performance, ont permis d’analyser la phase dissoute des échantillons d’eaux et la phase dissoute et solide des échantillons de lisiers. Il ressort des analyses des échantillons aqueux que : i) les béta-bloquants, les anti-VIH et les antibiotiques appartenant aux familles des macrolides, des fluoroquinolones et des sulfonamides, sont les molécules les plus représentatifs de la contamination du milieu naturel parmi les classes étudiées ; ii) les rejets de STEP sont une source majeure de la contamination des systèmes aquatiques ; iii) les eaux usées sont davantage contaminées en hiver qu’en été ; et iv) les eaux de surface sont davantage contaminées en été qu’en hiver. / Pharmaceutical substances belong to the group of emerging contaminants due to their recent interest in environmental studies in comparison with pollutants who have been studied for a longer time like pesticides. They correspond to the active ingredient of drugs and by this mean are responsible for their pharmacological properties. Consequently they are biologically active molecules that can act on living organisms present in impacted ecosystems. The origin of pharmaceuticals in the environment is variable but the main sources are related to their use in human and veterinary medicine. Once consumed, pharmaceutical substances are excreted in urine or feces and are found in wastewater (human consumption) or animal manure (veterinary consumption). In the first case, they can be discharged directly in the environment, or indirectly, with treated wastewater or sludge from sewage treatment plants (SWTP). In the second case, they directly reach the environment when animals are bred on grassland or indirectly when livestock wastes are spread on agricultural soils as fertilizer. This PhD work has been focused on the study of the origin and fate of pharmaceutical substances in these 2 cases. Thus according to consumption data, occurrence in the environment reported in previous studies, toxicity and ecotoxicity data, originality and availability of reference standard compounds, 32 then 78 molecules belonging to 5 different therapeutic classes (antibiotics, antineoplastics, beta-blockers, anti-HIV, phosphodiesterase type 5 inhibitors (PDE 5 inhibitors)) were studied in 2 continuums : i) hospital wastewater effluents – raw and treated wastewater – surface water, and ii) raw and treated wastewater – surface water – ground water. Based on the same selection criteria, the fate of 7 antibiotics was studied in pig manure in simple manure storage facilities (storage tank), in aerobic manure treatment facilities (treatment system like in small SWTP) and in mesocosms under controlled conditions. In order to achieve all these studies, analytical protocols implementing an extraction step by SPE (Solid Phase Extraction) or an ASE extraction (Accelerated Solvent Extraction) followed by a SPE purification and an analytical step by LC / MS / MS (liquid chromatography tandem mass spectrometry) have been developed. These protocols, by filling out quality criteria such as limits of detection and quantification compatible with environmental analysis (ng/l to dozen of ng/l), good linearity, precision, accuracy and performance, were used to analyze the dissolved phase of water samples and dissolved and solid phases of pig manure samples. The water samples analysis shows : i) beta-blockers, anti-HIV and antibiotic belonging to the families of macrolides, fluoroquinolones and sulfonamides are the most representative molecules of the environmental contamination from the classes studied; ii) SWTP releases are a major source of aquatic systems’ contamination; iii) wastewaters are more contaminated in winter than in summer; and iv) surface water are more contaminated in summer than in winter. The pig manure samples analysis shows : i) the levels of contamination of manure by antibiotics are high, from a few µg/l to mg/l; ii) the manure level of contamination is not related to the physiological stage of pigs; iii) the interest to store manure before spreading in order to reduce the antibiotics contamination is not highlighted; iv) oxytetracycline, tetracycline, tylosin and marbofloxacin are mainly present in the solid phase whereas sulfadiazine, lincomycin and monensin are mainly present in the liquid phase of manure; v) the separation of solid and liquid phases reduce manure contamination in aerobic treatment facilities; and vi) antibiotics degradation is mainly aerobic.Key words: ,