The development of a first myocardial infarction is associated with a large number of contributing factors. Age, male sex, hypertension, smoking, diabetes, body mass index and hypercholesterolemia are considered as established risk factors. The primary aim of the present dissertation was to evaluate whether specific biomarkers could improve the prediction of subjects at risk for a first myocardial infarction when considered in addition to established cardiovascular risk factors. The biomarkers investigated include: tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), thrombomodulin (TM), von Willebrand factor (VWF), dehydroepiandrosterone sulfate (DHEAS), lipoprotein (a) (Lp(a)), leptin, apolipoproptein A1 (ApoA1), proinsulin, homocysteine and homozygosity for the 5,10- methylenetetrahydrofolate reductase (MTHFR) C>T genotype. A secondary objective was to determine whether a first myocardial infarction leads to increased plasma homocysteine concentrations and whether the association between homocysteine and myocardial infarction was greater at follow-up compared to baseline. The study population consisted of 36 405 subjects screened and included in the Västerbotten Intervention Program and the Northern Sweden MONICA cohorts between January 1, 1985 and September 30, 1994. A nested incident case-referent study design was used. Seventy eight cases with a first myocardial infarction were identified, and from the same cohort twice as many sex and age matched referents were randomly selected. Moreover, a follow-up health survey (average 8.5 years between surveys) was conducted with 50 cases and 56 matched referents. High plasma levels of tPA and PAI-1 mass concentration, VWF, proinsulin, leptin and Lp(a) and low plasma levels of ApoA1 were associated with subsequent development of a first myocardial infarction in univariate conditional logistic regression analysis. For PAI-1 and tPA, this relation was found in both men and women. For tPA, but not for PAI-1 and VWF, this association was independent of established risk factors. In women, high plasma concentrations of TM were associated with significant increases in risk of a first myocardial infarction. No predictive values of DHEAS, homocysteine or for the point mutation C677>T in the gene for MTHFR was found regarding the risk of a first myocardial infarction. The summarised importance of haemostatic and metabolic variables (proinsulin, lipids including Lp(a) and leptin) in predicting first myocardial infarction in men, as well as possible interactions among these variables, were studied. High tPA and Lp(a) and low ApoA1 remained significant risk markers in multivariate analysis independent of established risk factors. There were non-significant synergic interactions between high Lp(a) and leptin and tPA respectively, and between high Lp(a) and low ApoA1. In the follow-up study plasma homocysteine and plasma creatinine increased significantly, and plasma albumin decreased significantly over time. Conditional univariate logistic regression indicated that high homocysteine at follow-up but not at baseline was associated with first myocardial infarction but the relation disappeared in multivariate analyses including plasma creatinine and plasma albumin. High plasma creatinine remained associated with first myocardial infarction at both baseline and follow-up. In conclusion, the present results support the hypothesis that biomarkers, in addition to the traditional cardiovascular risk factors, carry predictive information on the risk of developing a first myocardial infarction.

Les interactions protéine-ligand interviennent dans de nombreux processus biochimiques et permettent notamment à certaines protéines sanguines d’assurer leur rôle de transport. Parmi ces protéines figurent notamment l’albumine, protéine la plus abondante du plasma, ou l’apolipoprotéine A1 (ApoA1), majoritaire au sein des lipoprotéines de haute densité (HDL). Dans un contexte diabétique, la glycation des protéines induit des modifications structurales affectant ainsi leur potentiel d’interaction.Le premier objectif de ce travail de thèse visait à déterminer l’impact de la glycation de l’albumine sur sa liaison au liraglutide, un médicament de plus en plus utilisé dans le traitement du diabète de type 2. Ensuite, la seconde partie de ce travail a consisté en la production d’une ApoA1 humaine recombinante fonctionnelle afin d’étudier ses propriétés d’interaction, sous forme libre ou associée aux phospholipides. La technique RMN (résonance magnétique nucléaire) a été utilisée sur les protéines préalablement marquées au fluor (pour le liraglutide) ou aux isotopes stables 13C/15N (pour l’ApoA1). La titration microcalorimétrique isotherme (ITC), méthode complémentaire à la RMN a été appliquée pour l’étude des interactions avec l’avantage de ne nécessiter aucun marquage. Différentes stratégies de clonage ont été explorées pour la surexpression de l’ApoA1 en bactérie Clearcoli.Les résultats obtenus démontrent une altération de l’affinité de l’albumine pour le liraglutide in vitro et in vivo, dépendante du degré de glycation. Ces résultats, enrichis d’une analyse lipidomique et peptidique, permettent d’expliquer les observations cliniques concernant la baisse de l’efficacité de médicaments liant l’albumine chez les patients ayant un diabète mal contrôlé. Concernant l’ApoA1, le choix de l’étiquette de fusion reste à optimiser, mais sa surexpression de manière soluble et abondante a été obtenue pour l’ApoA1 marquée et non marquée. / Protein-ligand interactions are involved in many biochemical processes. They are notably implicated in the role of transporter proteins in blood. Albumin, the most abundant plasma protein, and apolipoprotein A1 (ApoA1), which is the main component of high-density lipoprotein (HDL) belong to this class of proteins. In the context of diabetes, proteins are altered by glycation which leads to structural modifications and potentially affect their interactions.The first objective of this work was to determine the impact of albumin glycation on its binding to liraglutide, a drug increasingly used in the treatment of type 2 diabetes. Then, the second part of this work involved the production of recombinant functional human ApoA1 in order to study its interaction properties, in its lipid-free form or associated with phospholipids. The NMR (nuclear magnetic resonance) technique has been used on proteins previously labeled with fluorine (for liraglutide) or stable 13C/15N isotopes (for ApoA1). In addition, isothermal titration microcalorimetry (ITC), has been applied to the study of interactions with the advantage of not requiring any labeling. Various cloning strategies have been explored for the overexpression of ApoA1 in Clearcoli bacteria.The results demonstrate an alteration of the affinity of albumin for liraglutide in vitro and in vivo, depending on the degree of glycation. These results, supported by a lipidomic and peptide analysis, explain clinical observations concerning the decrease of efficacy of albumin-binding drugs in patients with poorly controlled diabetes. Regarding ApoA1, the choice of the fusion tag remains to be optimized, but both labeled and unlabeled ApoA1 were successfully overexpressed at high yields in a soluble form.

A corticoterapia tem sido utilizada frequentemente nas éguas suscetíveis. O uso de isuflupredona melhora a taxa de prenhez e altera o perfil proteico do líquido endometrial em relação a éguas não tratadas. A utilização de dexametasona diminui o acúmulo de líquido pós-cobertura, reduz o edema do útero, porém, desconhecem-se seus efeitos no perfil proteico do líquido endometrial. O objetivo do presente estudo foi analisar o efeito da dexametasona em éguas suscetíveis à endometrite persistente póscobertura sobre perfil proteico do líquido endometrial na presença ou ausência de infecção. Nove éguas suscetíveis foram utilizados, com idade entre 7 a 30 anos. Após a verificação dos sinais de estro as éguas foram submetidas a quatro tratamentos: (C) éguas não receberam nenhum tipo de tratamento e serviram como controle; (D) éguas receberam 40mg de dexametasona (IV), no momento da cobertura, com coleta da amostras após 6 horas, (I-6 e I-24) infusão intra-uterina de 1 x 109 de S. zooepidemicus/mL, com coleta da amostra após 6 e 24 horas; (I/D-6 e I/D-24) infusão intra-uterina de 1 x 109 S. zooepidemicus/mL e administração de 40mg de dexametasona (IV), com coleta da amostra após 6 e 24 horas. Todas as éguas foram submetidas a todos os tratamentos. As amostras foram coletadas e submetidas à eletroforese bidimensional para separação proteica e espectrometria de massa para a identificação das bandas proteicas relevantes. A corticoterapia provocou alteração na proteômica do líquido endometrial de éguas suscetíveis, caracterizada pelo aumento (TTR) e/ou diminuição (ApoA1) na densidade óptica de proteínas da fase aguda da inflamação. Conclui-se que a utilização da dexametasona em éguas com e sem presença de infecção altera a proteômica do fluido endometrial de éguas suscetíveis. Sugere-se que a dosagem ou a frequência de aplicação da dexametasona deva ser aumentada. / Corticotherapy has often been used in susceptible mares. The use of isuflupredona improves pregnancy rate and alters the protein profile of the endometrial fluid in relation to untreated mares. The use of dexamethasone decreases the post breeding fluid accumulation, reduces the uterine edema, however is unaware of its effects on the protein profile of endometrial fluid. The aim of the present study was analyze the effect of dexamethasone in mares susceptible to post-breeding persistent endometritis on the protein profile of endometrial fluid in the presence or absence of infection. Nine susceptible mares were used, aged 7-30 years old. After checking the signs of estrus, mares were subjected to four treatments: (C) mares received no treatment and served as controls; (D) mares received 40 mg of dexamethasone at breeding time, with collection of samples after 6 hours; (I-6 and I-24) intrauterine infusion of 1 x 109 S. zooepidemicus/ml and the sample was collected after 6 and 24 hours; (I/D-6 and I/D-24) intrauterine infusion of 1 x 109 S. zooepidemicus/ml and 40 mg of dexamethasone administration, collecting the sample after 6 and 24 hours. All of the mares were subjected to all treatments. Samples were collected and subjected to two-dimensional electrophoresis for protein separation and mass spectrometry for the identification of relevant protein bands. Corticotherapy resulted in alteration of the protein profile of the endometrial fluid of susceptible mares, characterized by an increase (TTR) and/or decrease (ApoA1) in optical density of the acute phase of proteins of inflammation. We conclude that the use of dexamethasone in mares with and without the presence of infection alters the protein profile of endometrial fluid of susceptible mares. It is suggested that the dosage or frequency of application of dexamethasone should be increased.

A corticoterapia tem sido utilizada frequentemente nas éguas suscetíveis. O uso de isuflupredona melhora a taxa de prenhez e altera o perfil proteico do líquido endometrial em relação a éguas não tratadas. A utilização de dexametasona diminui o acúmulo de líquido pós-cobertura, reduz o edema do útero, porém, desconhecem-se seus efeitos no perfil proteico do líquido endometrial. O objetivo do presente estudo foi analisar o efeito da dexametasona em éguas suscetíveis à endometrite persistente póscobertura sobre perfil proteico do líquido endometrial na presença ou ausência de infecção. Nove éguas suscetíveis foram utilizados, com idade entre 7 a 30 anos. Após a verificação dos sinais de estro as éguas foram submetidas a quatro tratamentos: (C) éguas não receberam nenhum tipo de tratamento e serviram como controle; (D) éguas receberam 40mg de dexametasona (IV), no momento da cobertura, com coleta da amostras após 6 horas, (I-6 e I-24) infusão intra-uterina de 1 x 109 de S. zooepidemicus/mL, com coleta da amostra após 6 e 24 horas; (I/D-6 e I/D-24) infusão intra-uterina de 1 x 109 S. zooepidemicus/mL e administração de 40mg de dexametasona (IV), com coleta da amostra após 6 e 24 horas. Todas as éguas foram submetidas a todos os tratamentos. As amostras foram coletadas e submetidas à eletroforese bidimensional para separação proteica e espectrometria de massa para a identificação das bandas proteicas relevantes. A corticoterapia provocou alteração na proteômica do líquido endometrial de éguas suscetíveis, caracterizada pelo aumento (TTR) e/ou diminuição (ApoA1) na densidade óptica de proteínas da fase aguda da inflamação. Conclui-se que a utilização da dexametasona em éguas com e sem presença de infecção altera a proteômica do fluido endometrial de éguas suscetíveis. Sugere-se que a dosagem ou a frequência de aplicação da dexametasona deva ser aumentada. / Corticotherapy has often been used in susceptible mares. The use of isuflupredona improves pregnancy rate and alters the protein profile of the endometrial fluid in relation to untreated mares. The use of dexamethasone decreases the post breeding fluid accumulation, reduces the uterine edema, however is unaware of its effects on the protein profile of endometrial fluid. The aim of the present study was analyze the effect of dexamethasone in mares susceptible to post-breeding persistent endometritis on the protein profile of endometrial fluid in the presence or absence of infection. Nine susceptible mares were used, aged 7-30 years old. After checking the signs of estrus, mares were subjected to four treatments: (C) mares received no treatment and served as controls; (D) mares received 40 mg of dexamethasone at breeding time, with collection of samples after 6 hours; (I-6 and I-24) intrauterine infusion of 1 x 109 S. zooepidemicus/ml and the sample was collected after 6 and 24 hours; (I/D-6 and I/D-24) intrauterine infusion of 1 x 109 S. zooepidemicus/ml and 40 mg of dexamethasone administration, collecting the sample after 6 and 24 hours. All of the mares were subjected to all treatments. Samples were collected and subjected to two-dimensional electrophoresis for protein separation and mass spectrometry for the identification of relevant protein bands. Corticotherapy resulted in alteration of the protein profile of the endometrial fluid of susceptible mares, characterized by an increase (TTR) and/or decrease (ApoA1) in optical density of the acute phase of proteins of inflammation. We conclude that the use of dexamethasone in mares with and without the presence of infection alters the protein profile of endometrial fluid of susceptible mares. It is suggested that the dosage or frequency of application of dexamethasone should be increased.

A corticoterapia tem sido utilizada frequentemente nas éguas suscetíveis. O uso de isuflupredona melhora a taxa de prenhez e altera o perfil proteico do líquido endometrial em relação a éguas não tratadas. A utilização de dexametasona diminui o acúmulo de líquido pós-cobertura, reduz o edema do útero, porém, desconhecem-se seus efeitos no perfil proteico do líquido endometrial. O objetivo do presente estudo foi analisar o efeito da dexametasona em éguas suscetíveis à endometrite persistente póscobertura sobre perfil proteico do líquido endometrial na presença ou ausência de infecção. Nove éguas suscetíveis foram utilizados, com idade entre 7 a 30 anos. Após a verificação dos sinais de estro as éguas foram submetidas a quatro tratamentos: (C) éguas não receberam nenhum tipo de tratamento e serviram como controle; (D) éguas receberam 40mg de dexametasona (IV), no momento da cobertura, com coleta da amostras após 6 horas, (I-6 e I-24) infusão intra-uterina de 1 x 109 de S. zooepidemicus/mL, com coleta da amostra após 6 e 24 horas; (I/D-6 e I/D-24) infusão intra-uterina de 1 x 109 S. zooepidemicus/mL e administração de 40mg de dexametasona (IV), com coleta da amostra após 6 e 24 horas. Todas as éguas foram submetidas a todos os tratamentos. As amostras foram coletadas e submetidas à eletroforese bidimensional para separação proteica e espectrometria de massa para a identificação das bandas proteicas relevantes. A corticoterapia provocou alteração na proteômica do líquido endometrial de éguas suscetíveis, caracterizada pelo aumento (TTR) e/ou diminuição (ApoA1) na densidade óptica de proteínas da fase aguda da inflamação. Conclui-se que a utilização da dexametasona em éguas com e sem presença de infecção altera a proteômica do fluido endometrial de éguas suscetíveis. Sugere-se que a dosagem ou a frequência de aplicação da dexametasona deva ser aumentada. / Corticotherapy has often been used in susceptible mares. The use of isuflupredona improves pregnancy rate and alters the protein profile of the endometrial fluid in relation to untreated mares. The use of dexamethasone decreases the post breeding fluid accumulation, reduces the uterine edema, however is unaware of its effects on the protein profile of endometrial fluid. The aim of the present study was analyze the effect of dexamethasone in mares susceptible to post-breeding persistent endometritis on the protein profile of endometrial fluid in the presence or absence of infection. Nine susceptible mares were used, aged 7-30 years old. After checking the signs of estrus, mares were subjected to four treatments: (C) mares received no treatment and served as controls; (D) mares received 40 mg of dexamethasone at breeding time, with collection of samples after 6 hours; (I-6 and I-24) intrauterine infusion of 1 x 109 S. zooepidemicus/ml and the sample was collected after 6 and 24 hours; (I/D-6 and I/D-24) intrauterine infusion of 1 x 109 S. zooepidemicus/ml and 40 mg of dexamethasone administration, collecting the sample after 6 and 24 hours. All of the mares were subjected to all treatments. Samples were collected and subjected to two-dimensional electrophoresis for protein separation and mass spectrometry for the identification of relevant protein bands. Corticotherapy resulted in alteration of the protein profile of the endometrial fluid of susceptible mares, characterized by an increase (TTR) and/or decrease (ApoA1) in optical density of the acute phase of proteins of inflammation. We conclude that the use of dexamethasone in mares with and without the presence of infection alters the protein profile of endometrial fluid of susceptible mares. It is suggested that the dosage or frequency of application of dexamethasone should be increased.

La restauration rapide du flux sanguin est essentielle pour limiter l’étendue de l’infarctus myocardique mais elle est à l’origine de lésions irréversibles. Le préconditionnement ischémique à distance (RIPC) qui désigne l’application non invasive de brèves séquences d’ischémie/reperfusion au niveau d’un organe à distance du cœur peut prévenir la survenue de ces lésions de reperfusion. De nombreuses études suggèrent une implication de facteurs de transcription et de facteurs humoraux dans la cardioprotection induite par le RIPC, mais leurs identités restent inconnues. Dans la première partie du travail, nous avons donc étudié le rôle potentiel du facteur de transcription induit par l’hypoxie (HIF-1α) dans la phase précoce du RIPC. Nous avons ainsi démontré, en utilisant deux modèles expérimentaux : les souris transgéniques déficientes en HIF-1α et les rats traités par un inhibiteur pharmacologique de HIF-1α, que HIF-1α n’est pas indispensable pour cette phase du RIPC. Dans la deuxième partie de ce travail, nous avons essayé d’identifier, de façon directe cette fois, le ou les facteurs humoraux responsables de l’effet protecteur du RIPC. En se basant sur les résultats des études protéomiques démontrant une augmentation des concentrations plasmatiques d’apolipoprotéine A1 (Apo A1) suite au RIPC, nous avons alors cherché à mettre en évidence si cette protéine pourrait être un facteur circulant du RIPC. L’Apo A1, injectée directement avant la réalisation de l’infarctus chez le rat, était capable de reproduire l’effet cardioprotecteur et d’activer les mêmes voies de signalisation du RIPC. L’Apo A1 pourrait donc être un facteur humoral du RIPC. / Although early restoration of blood flow to the ischemic heart is essential to reduce the extent of myocardial infarction, reperfusion per se may cause irreversible tissue injury. Remote ischemic preconditioning (RIPC), the phenomenon whereby brief episodes of I/R are applied in distant tissues or organs, can protect the myocardium against reperfusion injuries. Several studies suggest that transcription factors and humoral mediators may be involved in RIPC mechanisms ; however the actual identity of these factors remains unknown. Therefore, in the first part of the study, we aimed to identify the role of hypoxia inducible factor (HIF-1α) in the acute phase of RIPC. We have thus demonstrated, using two animal models: partially HIF-1α-deficient mice and rats pretreated with a pharmacological inhibitor of HIF-1α, that HIF-1α is not crucial for this phase of RIPC. In the second part of the study, we have used a more direct approach and attempted to identify one or more humoral mediators of RIPC. Based on the results of proteomic studies showing an increase in plasmatic apolipoprotein A1 (Apo A1) levels following RIPC, we thus sought to determine if Apo A1 may constitute a blood-borne factor involved in RIPC’s protective effects. Our findings indicated that Apo A1 injected before myocardial infarction in rats acutely protected the heart, recapitulating RIPC-induced cardioprotection and that Apo A1 share some common signaling pathways with RIPC. Apo A1 may then be a humoral mediator of RIPC.