L’hémoglobine est une protéine contenue dans les globules rouges dont la principale fonction est le transport de l’oxygène. Chaque molécule d’hémoglobine est un tétramère constitué de deux paires de globines identiques de type α et β. La β-thalassémie est une maladie génétique hématopoïétique provenant de mutations du gène encodant l'hémoglobine. Ce désordre se caractérise par une diminution ou une absence totale de la synthèse de la chaîne β-globine résultant principalement en une anémie hémolytique sévère ainsi que des complications multisystémiques, telles que la splénomégalie, des déformations osseuses et une dysfonction hépatique et rénale. Actuellement, les transfusions sanguines chroniques représentent le traitement standard des patients β-thalassémiques. Cette thérapie nécessite l’administration conjointe d’un traitement chélateur de fer puisqu’elle entraîne une accumulation pathologique du fer, considéré à ce jour comme la source principale des complications cardiovasculaires de la β-thalassémie. Néanmoins, malgré le traitement efficace de la surcharge de fer transfusionnelle, l’insuffisance cardiaque demeure encore la principale cause de mortalité chez les patients atteints de β-thalassémie. Cette observation indique possiblement la présence d’un mécanisme complémentaire dans le développement de la physiopathologie cardiaque β-thalassémique. L’objectif du présent projet consistait donc à étudier les altérations cardiovasculaires de la β-thalassémie indépendamment de la surcharge de fer transfusionnelle. En utilisant un modèle murin non-transfusé de la β-thalassémie majeure, nous avons d’abord évalué in vivo, par méthode d’imagerie novatrice échographique à haute fréquence, les propriétés hémodynamiques vasculaires. Nos résultats d’index de Pourcelot ainsi que de résistance vasculaire périphérique totale ont démontré une perturbation de l’écoulement microcirculatoire chez les souris β-thalassémiques non-transfusées. Subséquemment, nous avons étudié la fonction endothéliale de régulation du tonus vasculaire de vaisseaux mésentériques isolés. Nos résultats ont révélé un dysfonctionnement de la réponse vasodilatatrice dépendante de l’endothélium chez les souris β-thalassémiques malgré une augmentation de l’expression de l’enzyme de synthèse du monoxyde d’azote ainsi qu’un remodelage de la carotide commune caractérisé par un épaississement de la paroi vasculaire. Finalement, notre étude échocardiographique de la fonction et la morphologie cardiaque a montré, chez les souris β-thalassémiques, le développement d’une hypertrophie et une dysfonction ventriculaire gauche en l’absence de transfusions sanguines chroniques ou de dépôts directs de fer dans le myocarde. L’ensemble des résultats présentés dans le cadre de cette thèse indique la présence d’une pathologie cardiovasculaire chez les souris β-thalassémiques non-transfusés. Nos travaux permettent de proposer un mécanisme de la pathophysiologie cardiovasculaire β-thalassémique, indépendant de la charge de fer transfusionnelle, impliquant les effets compensatoires d’une anémie chronique combinés à une vasculopathie complexe initiée par les érythrocytes endommagés et l’hémolyse intravasculaire. / Hemoglobin is the major protein in red blood cells and is responsible of the oxygen transport. Each hemoglobin molecule is a tetramer consisting of two identical α- and β-globin subunits. β-thalassemia is a genetic hematopoietic disease caused by mutations in hemoglobin genes. This disorder is characterized by a decrease or absence of production of β-globin chain leading mainly to a severe hemolytic anemia and several systemic manifestations, including splenomegaly, skeletal deformities as well as hepatic and renal dysfunctions. Chronic blood transfusions remain the standard treatment for β-thalassemic patients. This therapy requires iron chelating management since it leads to pathological iron accumulation which is currently considered the main cause of cardiovascular complications of β-thalassemia. However, despite adequate control of transfusional iron loading, heart failure remains the leading cause of mortality in β-thalassemia. This issue is possibly indicative of additional pathogenic mechanisms underlying the development of the β-thalassemic cardiac pathology. The objective of the present research project was to study cardiovascular alterations of β-thalassemia independently of transfusional iron overloading. Using an untransfused murine model of β-thalassemia major, we have evaluated in vivo, by non-invasive high-frequency ultrasound imaging, vascular hemodynamic properties. Our results of Pourcelot indices and total peripheral vascular resistance have shown microcirculatory flow disturbances in untransfused β-thalassemic mice. Consequently, we have studied ex vivo the endothelial vasomotor function in isolated mesenteric arterioles. Our findings have pointed out endothelium-dependent vasodilator dysfunction in β-thalassemic mice despite increased expression of nitric oxide synthase, as well as remodeling of the common carotid artery wall. Lastly, our echocardiography studies of heart morphology and function in β-thalassemic mice have demonstrated the development of left ventricle hypertrophy and dysfunction in the absence of chronic blood transfusions or direct myocardial iron deposits. In conclusion, findings presented in this thesis have demonstrated for the first time development of severe cardiovascular complications in untransfused β-thalassemic mice. Based on our results, we have proposed a novel mechanism, independent of direct myocardial iron deposition, responsible for the cardiovascular complications in β-thalassemia. This model combines compensatory effects of chronic anemia with a complex vasculopathy initiated by abnormal erythrocytes and intravascular hemolysis.

Les transfusions sanguines sont fréquemment employées pour corriger l’anémie secondaire à une arthroplastie de la hanche ou du genou. Il n’y a cependant pas consensus sur les indications de transfuser. La tendance actuelle est d’utiliser une stratégie transfusionnelle restrictive (soit un seuil de 75-80 g/L d’hémoglobine) mais les conséquences d’une telle pratique sur la récupération fonctionnelle et la qualité de vie des patients sont mal connues. Dans un premier temps, nous avons caractérisé la pratique transfusionnelle au Centre hospitalier de l’Université de Montréal (CHUM). Notre hypothèse était que, devant l’imprécision des recommandations, la pratique transfusionnelle serait variable. Une étude rétrospective de 701 dossiers de patients ayant subi une arthroplastie de la hanche ou du genou a été réalisée. Nous avons observé que les transfusions étaient utilisées de la même façon dans les trois hôpitaux et que les médecins basaient leur décision de transfuser principalement sur un seul chiffre, la concentration d’hémoglobine, adoptant une stratégie restrictive. Soixante-six pourcent des patients avaient une concentration d’hémoglobine inférieure à 100 g/L au départ de l’hôpital. Dans un deuxième temps, nous avons évalué l’impact de cette anémie postopératoire sur la récupération fonctionnelle et la qualité de vie des patients. Notre hypothèse était qu’il existe une concentration d’hémoglobine en dessous de laquelle celles-ci sont atteintes. Une étude de cohorte prospective et observationnelle a été menée chez 305 patients regroupés selon leur concentration d’hémoglobine postopératoire. Les groupes d’hémoglobine (≤ 80, 81-90, 91-100 et > 100 g/L) étaient similaires dans l’évolution de la distance de marche en six minutes, de l’évaluation de l’effort fourni, de la force de préhension et des scores de qualité de vie. L’anémie modérée n’est donc pas associée à une atteinte de la récupération fonctionnelle et de la qualité de vie à court terme. D’autres études devront déterminer les conséquences à long terme d’une stratégie transfusionnelle restrictive sur ces patients. / Red blood cell transfusions are frequently used to treat anemia after total hip or knee arthroplasties. The indications for transfusions remain unclear despite published guidelines. Clinicians have adopted a restrictive transfusion threshold (75-80 g/L) but the consequences of such a strategy on functional outcome and quality of life are not known. First, we characterized the transfusion practice inside the Centre hospitalier de l’Université de Montréal (CHUM). Our hypothesis was that transfusion practice varies inside the CHUM due to uncertainty. A retrospective study of 701 charts of patients operated for a hip or knee arthroplasty was conducted. We observed that there was no difference among hospitals regarding the way transfusions are used and that physicians mainly based their decision to transfuse on a single variable, the hemoglobin concentration, adopting a restrictive transfusion strategy. Sixty-six percent of patients had a hemoglobin concentration under 100 g/L after surgery. Second, we evaluated the impact of this postoperative anemia on functional outcome and quality of life. We hypothesized that a threshold hemoglobin concentration exists below which these become impaired. A prospective, observational cohort study was conducted in 305 patients categorized in groups according to their postoperative hemoglobin concentration. Hemoglobin groups (≤ 80, 81-90, 91-100 and > 100 g/L) were similar in the evolution of the distance walked in six minutes, perception of effort, maximal dominant hand strength and quality of life scores. Thus, moderate anemia is not associated with an impaired functional recovery or quality of life early after hip and knee arthroplasties. Further studies will be required to determine the long-term consequences of a restrictive transfusion strategy in these patients.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

O diabetes mellitus tipo 1 (DM1) está associado com condições pró-oxidantes, próinflamatórias e elevado risco cardiovascular, enquanto o exercício físico pode ser considerado um dos melhores instrumentos não farmacológicas para o tratamento do DM1. Nesse contexto, exercícios que propiciem um menor risco hipoglicêmico e diversos benefícios sobre a saúde devem ser estimulados. Um dos objetivos da tese foi verificar a influência da realização de exercícios de força (SE) antes ou depois do exercício intervalado de alta intensidade (HIIE) sobre o comportamento glicêmico durante e logo após uma sessão de esforço (estudo transversal) (manuscrito original 1). Entretanto, o principal objetivo desta tese foi comparar os efeitos do treinamento intervalado de alta intensidade (HIIT), do treinamento de força (ST) e da combinação destes (ST+HIIT), sobre marcadores sanguíneos inflamatórios, de estresse oxidativo (OS) e metabolismo glicêmico em pacientes com DM1 através de um ensaio clínico randomizado (ECR) (manuscrito original 2). Com relação ao estudo transversal (manuscrito 1), em três visitas, adultos fisicamente ativos realizaram 30 min de SE antes de 30 min de HIIE ou realizaram a ordem inversa da sessão (HIIE+SE) ou permaneceram em repouso nesse período (REST). A glicemia capilar foi mensurada a cada 15 min durante e até 60 min da recuperação. Comparando-se com os valores basais, a condição HIIE+SE reduziu a glicemia em 30, 45 e 60 min, enquanto SE+HIIE adiou esta queda glicêmica para a partir de 60 min. HIIE+SE também acarretou uma maior glicemia em 105 min quando comparado a 60 min. A quantidade ingerida de carboidratos durante as sessões, bem como a dose insulínica no mesmo dia antes e depois dos protocolos, além dos episódios noturnos de hipoglicemia, foram similares entre as três condições. Conclui-se que pacientes com DM1 propensos a desenvolver hipoglicemia associada ao exercício devem realizar SE antes do HIIE na mesma sessão. Com relação ao estudo principal (ECR) (manuscrito original 2), após 4 semanas de um período controle, pacientes fisicamente inativos com DM1 foram randomizados para realização de 10 semanas de HIIT, ST ou ST+HIIT, praticados 3x/sem. As sessões de HIIT duraram 25 min, as de ST 40 min, e as de ST+HIIT ~65 min. Os desfechos foram analisados através do modelo de equações de estimativas generalizadas (GEE), com post hoc de Bonferroni. ST, HIIT e ST+HIIT melhoraram parâmetros glicêmicos e antioxidantes, mas não os marcadores plasmáticos de inflamação e de OS. Interessantemente, as intervenções reduziram as concentrações de receptores solúveis para produtos finais da glicação avançada. Entretanto, o conteúdo intracelular das proteínas de choque térmico de 70 kDa aumentou somente depois do HIIT. Enquanto a dose diária de insulina utilizada reduziu apenas no grupo ST+HIIT, todos os protocolos induziram benefícios antropométricos, cardiorrespiratórios e funcionais. Sob uma perspectiva prática, conclui-se que um maior volume (ST+HIIT) de treinamento é necessário para o benefício adicional da redução insulínica diária. Já o HIIT, por exemplo, é diretamente aplicável para pessoas que reclamam da falta de tempo, podendo ser recomendado devido a vantagem extra com relação a proteínas anti-inflamatórios em células imunológicas. / Type 1 diabetes mellitus (DM1) is associated with prooxidant and proinflammatory conditions, besides an increased cardiovascular risk, while exercise may be considered one of the best nonpharmacological tools for DM1 treatment. In this context, exercises linked with a lower hypoglycemic risk and several health benefits should be stimulated. One of the goals of this thesis was to verify the influence of performing strength exercises (SE) before or after highintensity interval exercise (HIIE) on glycaemia during and postexercise (cross-sectional study) (original manuscript 1). However, the main objective of this thesis was to compare the effects of high-intensity interval training (HIIT), strength training (ST) or their combination (ST+HIIT), on blood inflammatory, oxidative stress (OS) and glycemic markers in DM1 patients using a randomized clinical trial (ECR) (original manuscript 2). Regarding the crosssectional study (original manuscript 1), in three visits, physically active adults performed 30 min of SE before 30 min of HIIE or performed the reverse order (HIIE+SE) or rested for 30 min (REST). Capillary glycaemia was measured each 15 min during and 60 min postexercise recovery. HIIE+SE lowered glycaemia at 30, 45 and 60 min compared with baseline concentrations, while SE+HIIE postponed this glucose decayment to 60 min and thereafter. HIIE+SE increased glycaemia at 105 min compared with 60 min. Carbohydrates ingested during exercise, insulin dosage at same day before and after protocols, and nocturnal hypoglycemia episodes were similar among the three conditions. DM1 patients prone to develop exercise-associated hypoglycemia should perform SE before HIIE in a single session. Regarding the main study (ECR) (original manuscript 2), after 4-week control period, physically inactive patients with DM1 were randomly assigned to 10-week HIIT, ST or ST+HIIT protocol, performed 3 x/week. HIIT sessions lasted 25 min, ST lasted 40 min and ST+HIIT sessions lasted ~65 min. Blood biochemical, anthropometric, strength and cardiorespiratory fitness variables were assessed. Outcomes were analyzed via generalized estimating equations (GEE), with Bonferroni post hoc analysis. ST, HIIT and ST+HIIT improved glycemic and antioxidant parameters, but not plasma inflammatory or OS markers. Noteworthy, interventions reduced soluble receptors for advanced glycation end products levels. However, intracellular heat shock protein 70 content increased only after HIIT. While daily insulin dosage decreased only in the ST+HIIT group, all training models induced anthropometric and functional benefits. From a practical clinical perspective, a higher volume (SE+HIIT) of training is required for the additional benefit of daily insulin reduction. The HIIT, for example, is directly applicable for people who claim lack of time, and it may be 13 recommended due to extra advantage concerning anti-inflammatory proteins at immunological cells.

O diabetes mellitus tipo 1 (DM1) está associado com condições pró-oxidantes, próinflamatórias e elevado risco cardiovascular, enquanto o exercício físico pode ser considerado um dos melhores instrumentos não farmacológicas para o tratamento do DM1. Nesse contexto, exercícios que propiciem um menor risco hipoglicêmico e diversos benefícios sobre a saúde devem ser estimulados. Um dos objetivos da tese foi verificar a influência da realização de exercícios de força (SE) antes ou depois do exercício intervalado de alta intensidade (HIIE) sobre o comportamento glicêmico durante e logo após uma sessão de esforço (estudo transversal) (manuscrito original 1). Entretanto, o principal objetivo desta tese foi comparar os efeitos do treinamento intervalado de alta intensidade (HIIT), do treinamento de força (ST) e da combinação destes (ST+HIIT), sobre marcadores sanguíneos inflamatórios, de estresse oxidativo (OS) e metabolismo glicêmico em pacientes com DM1 através de um ensaio clínico randomizado (ECR) (manuscrito original 2). Com relação ao estudo transversal (manuscrito 1), em três visitas, adultos fisicamente ativos realizaram 30 min de SE antes de 30 min de HIIE ou realizaram a ordem inversa da sessão (HIIE+SE) ou permaneceram em repouso nesse período (REST). A glicemia capilar foi mensurada a cada 15 min durante e até 60 min da recuperação. Comparando-se com os valores basais, a condição HIIE+SE reduziu a glicemia em 30, 45 e 60 min, enquanto SE+HIIE adiou esta queda glicêmica para a partir de 60 min. HIIE+SE também acarretou uma maior glicemia em 105 min quando comparado a 60 min. A quantidade ingerida de carboidratos durante as sessões, bem como a dose insulínica no mesmo dia antes e depois dos protocolos, além dos episódios noturnos de hipoglicemia, foram similares entre as três condições. Conclui-se que pacientes com DM1 propensos a desenvolver hipoglicemia associada ao exercício devem realizar SE antes do HIIE na mesma sessão. Com relação ao estudo principal (ECR) (manuscrito original 2), após 4 semanas de um período controle, pacientes fisicamente inativos com DM1 foram randomizados para realização de 10 semanas de HIIT, ST ou ST+HIIT, praticados 3x/sem. As sessões de HIIT duraram 25 min, as de ST 40 min, e as de ST+HIIT ~65 min. Os desfechos foram analisados através do modelo de equações de estimativas generalizadas (GEE), com post hoc de Bonferroni. ST, HIIT e ST+HIIT melhoraram parâmetros glicêmicos e antioxidantes, mas não os marcadores plasmáticos de inflamação e de OS. Interessantemente, as intervenções reduziram as concentrações de receptores solúveis para produtos finais da glicação avançada. Entretanto, o conteúdo intracelular das proteínas de choque térmico de 70 kDa aumentou somente depois do HIIT. Enquanto a dose diária de insulina utilizada reduziu apenas no grupo ST+HIIT, todos os protocolos induziram benefícios antropométricos, cardiorrespiratórios e funcionais. Sob uma perspectiva prática, conclui-se que um maior volume (ST+HIIT) de treinamento é necessário para o benefício adicional da redução insulínica diária. Já o HIIT, por exemplo, é diretamente aplicável para pessoas que reclamam da falta de tempo, podendo ser recomendado devido a vantagem extra com relação a proteínas anti-inflamatórios em células imunológicas. / Type 1 diabetes mellitus (DM1) is associated with prooxidant and proinflammatory conditions, besides an increased cardiovascular risk, while exercise may be considered one of the best nonpharmacological tools for DM1 treatment. In this context, exercises linked with a lower hypoglycemic risk and several health benefits should be stimulated. One of the goals of this thesis was to verify the influence of performing strength exercises (SE) before or after highintensity interval exercise (HIIE) on glycaemia during and postexercise (cross-sectional study) (original manuscript 1). However, the main objective of this thesis was to compare the effects of high-intensity interval training (HIIT), strength training (ST) or their combination (ST+HIIT), on blood inflammatory, oxidative stress (OS) and glycemic markers in DM1 patients using a randomized clinical trial (ECR) (original manuscript 2). Regarding the crosssectional study (original manuscript 1), in three visits, physically active adults performed 30 min of SE before 30 min of HIIE or performed the reverse order (HIIE+SE) or rested for 30 min (REST). Capillary glycaemia was measured each 15 min during and 60 min postexercise recovery. HIIE+SE lowered glycaemia at 30, 45 and 60 min compared with baseline concentrations, while SE+HIIE postponed this glucose decayment to 60 min and thereafter. HIIE+SE increased glycaemia at 105 min compared with 60 min. Carbohydrates ingested during exercise, insulin dosage at same day before and after protocols, and nocturnal hypoglycemia episodes were similar among the three conditions. DM1 patients prone to develop exercise-associated hypoglycemia should perform SE before HIIE in a single session. Regarding the main study (ECR) (original manuscript 2), after 4-week control period, physically inactive patients with DM1 were randomly assigned to 10-week HIIT, ST or ST+HIIT protocol, performed 3 x/week. HIIT sessions lasted 25 min, ST lasted 40 min and ST+HIIT sessions lasted ~65 min. Blood biochemical, anthropometric, strength and cardiorespiratory fitness variables were assessed. Outcomes were analyzed via generalized estimating equations (GEE), with Bonferroni post hoc analysis. ST, HIIT and ST+HIIT improved glycemic and antioxidant parameters, but not plasma inflammatory or OS markers. Noteworthy, interventions reduced soluble receptors for advanced glycation end products levels. However, intracellular heat shock protein 70 content increased only after HIIT. While daily insulin dosage decreased only in the ST+HIIT group, all training models induced anthropometric and functional benefits. From a practical clinical perspective, a higher volume (SE+HIIT) of training is required for the additional benefit of daily insulin reduction. The HIIT, for example, is directly applicable for people who claim lack of time, and it may be 13 recommended due to extra advantage concerning anti-inflammatory proteins at immunological cells.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Glutathione (γ-L-glutamyl-L-cysteinylglycine) is the major non-protein thiol body and is involved in cellular antioxidant defense. The free glutathione is present mainly in its reduced form (GSH) and can be converted to the oxidized form (GSSG) in the presence of reactive oxygen species (ROS). The GSH/GSSG ratio high is very important for the cellular redox state and a reduction of this ratio is often used as an indicator of oxidative stress. The enzyme glutathione reductase (GR) catalyzes the reduction of GSSG to GSH using NADPH. Because of the great importance of the antioxidant glutathione and considering that it is present in almost all organisms, numerous studies involving several attempts to detect GSH, GSSG and GR in biological systems has been performed. The objective of this study was to validate an automated analytical method, based on a spectrophotometric method proposed by Mannervick and Carlberg in 1985 to measurement the GR activity which is the oxidation of NADPH, which is monitored spectrophotometrically at a wavelength of 340 nm using the automated analyzer Cobas Mira®, determine its limits of reference for a healthy population and further evaluate the pre-analytical interference that influence the analytical phase of hemoglobin, bilirubin and lipemia. The automated method for measuring of the GR activity was validated as recommended by the EMEA and ANVISA. Since it was linear (r2 = 0.990), precise, with a coefficient of variation (CV) in precision intraassay of 5.7% (50 U/L) and 3.4% (100 U/L) and precision interassay CV of 9.5% (50 U/L) and 9.9% (100 U/L). In addition, we observed a recovery of 114.1% with this method considered accurate. The reference limits were evaluated as recommended by the International Federation of Clinical Chemistry (IFCC), and were 21.7 U / L to 60.3 U / L, for a healthy population. In the simulation of hemolysis, lipemia and jaundice in plasma samples, we evaluated the pre-analytical interference in the activity of the GR. All concentrations of Intralipid® (0.67, 1.25, 2.5, 5 and 10 mg / dL), hemoglobin standard (0.0625, 0.125, 0.25, 0.5 and 1 g / dL), and bilirubin (0.9, 1.9, 3.8, 7.5, 15 and 30 mg / dL) resulted in a difference from the original value of GR, and verified a percentage greater than 5%, and this percentage considered for enzymes, analytical interference. Thus, it was concluded that the automated method developed was linear, precise, accurate, simple and inexpensive, and can be adapted to the Cobas Mira® analyzer. The reference limits for a healthy population were established. Furthermore, it was demonstrated that hemoglobin, lipemia and bilirubin interfere in the measurement of the GR activity. / A glutationa (L-γ-glutamil-L-cysteinylglycine) é o principal tiol não proteico do organismo e está envolvida na defesa celular antioxidante. A glutationa livre está presente principalmente na sua forma reduzida (GSH) e pode ser convertida para a forma oxidada (GSSG) na presença de espécies reativas de oxigênio (EROs). A razão GSH/GSSG elevada é muito importante para o estado redox celular e uma redução desta razão é frequentemente utilizada como um indicador do estresse oxidativo. A enzima glutationa redutase (GR) catalisa a redução de GSSG a GSH utilizando NADPH. Devido a grande importância antioxidante da glutationa e considerando que a mesma está presente em quase todos os organismos, numerosas pesquisas envolvendo as mais diversas tentativas de detecção de GSH, GSSG e GR em sistemas biológicos tem sido realizadas. Assim, o objetivo deste estudo foi validar um método analítico automatizado, baseado em um método espectrofotométrico proposto por Mannervick e Carlberg em 1985 para a mensuração da atividade da GR que consiste na oxidação do NADPH, o qual é monitorado espectrofotometricamente no comprimento de onda de 340 nm utilizando o analisador automatizado Cobas Mira®, determinar seus limites de referência para uma população saudável e ainda avaliar a interferência pré-analítica que influenciam na fase analítica da hemoglobina, lipemia e bilirrubina. O método automatizado para a mensuração da atividade da enzima GR foi validado seguindo recomendações da ANVISA e EMEA. Sendo que o mesmo foi linear (r2=0,990), preciso, apresentando um coeficiente de variação (CV) na precisão intraensaio de 5,7% (50 U/L) e 3,4% (100 U/L) e na precisão interensaio um CV de 9,5% (50U/L) e 9,9% (100 U/L). Além disso, foi observada uma recuperação de 114,1%, sendo este método considerado exato. Os limites de referência foram avaliados seguindo recomendações da International Federation of Clinical Chemistry (IFCC), sendo que foram de 21,7 U/L a 60,3 U/L, para uma população saudável. Na simulação da hemólise, lipemia e icterícia em amostras de plasma, avaliou-se a interferência pré-analítica na atividade da GR. Todas as concentrações utilizadas de Intralipid® (0,67; 1,25; 2,5; 5 e 10 mg/dL), de padrão de hemoglobina (0,0625; 0,125; 0,25; 0,5 e 1 g/dL) e de bilirrubina (0,9; 1,9; 3,8; 7,5; 15 e 30 mg/dL) resultaram em uma diferença do valor original de GR, sendo verificada uma porcentagem maior que 5%, sendo essa porcentagem considerada, para enzimas, interferência analítica. Dessa forma, foi possível concluir que o método automatizado desenvolvido foi linear, preciso, exato, simples e de baixo custo, podendo ser adaptado ao analisador Cobas Mira®. Os limites de referência para uma população saudável também foram estabelecidos. Além disso, foi demonstrado que a hemoglobina, a lipemia e a bilirrubina interferem na mensuração da atividade da GR.

L’analyse protéomique par spectrométrie de masse s’est imposée comme une méthode incontournable pour la caractérisation des protéines. Grâce aux progrès de l’instrumentation et de la bioinformatique, l’interprétation automatisée des spectres MS/MS permet aujourd’hui d’identifier des milliers de protéines dans un type cellulaire. Cependant, cette méthodologie s’applique encore difficilement aux organismes dont les génomes n’ont pas été séquencés, et donc pour lesquels il n’existe pas de banques de séquences peptidiques de référence. Notre travail a porté sur le développement et l’application d’une méthodologie d’interprétation des données MS/MS pour les organismes à génomes non séquencés. Cette méthodologie est basée sur le séquençage de novo suivi de recherche MS-BLAST. Ainsi nous avons pu : Identifier les différents partenaires de complexes protéiques tels que les protéines des complexes TgGAP50, TgAlba, TgSORTLR impliqués dans la motilité, la virulence ou le trafic intracellulaire des protéines du parasite Toxoplasma gondii, Identifier et caractériser des variants d’hémoglobine humaine, Identifier les protéines différentiellement exprimées lors des interactions vigne et champignons à génomes non séquencés dans la maladie de l’esca, Caractériser finement la N-glycosylation de l’invertase vacuolaire du raisin. Nous avons pu réaliser nos études sur des échantillons d’origines très différentes : homme, plantes, champignons, parasites et nous avons apporté des éléments de réponses moléculaires aux questions biologiques. / The proteomic analysis by mass spectrometry is now an essential method for the characterization of proteins. Thanks to advances in instrumentation and bioinformatics, automated interpretation of MS/MS spectra can now identify thousands of proteins in a cell type. However, this methodology remains poorly applied to the organisms that genomes are not sequenced and therefore where there is no database of reference for peptides sequences. Our work has focused on the development and application of a methodology for the interpretation of MS/MS data for the organisms that genomes are not sequenced. This methodology is based on the de novo sequencing followed by MS-BLAST search. Thus we have: Identify different partners of protein complexes such as proteins TgGAP50, TgAlba and TgSORTLR complex, involved in motility, virulence or intracellular protein trafficking of Toxoplasma gondii, Identify and characterize human hemoglobin variants, Identify the proteins differentially expressed during interaction of vines and fungi that genomes are not sequenced in esca disease, Finely characterize the N-glycosylation of the grape vacuolar invertase. We have achieved our studies on samples of very different origins: human, plants, fungi, parasites, and we provided evidence of molecular responses to biological questions.

O número de pacientes com diagnóstico de diabetes aumenta a cada dia. Infarto agudo do miocárdio (IAM) e acidente vascular cerebral (AVC) constituem as principais causas de óbito neste grupo. Ruptura da placa aterosclerótica coronariana é o mecanismo fisiopatológico para (IAM) em 2 a cada 3 casos e as características destas placas mais vulneráveis e propensas a ruptura como:remodelamento positivo do segmento vascular afetado e placa não calcificada com baixa atenuação podem ser avaliadas pela Angiotomografia. Objetivo: Avaliar a frequência de doença arterial coronariana e as principais características de vulnerabilidade dessas placas ateroscleróticas em diabéticos assintomáticos considerando o grau de controle glicêmico através da Angiotomografia Computadorizada Multislice. Desenho do estudo e Métodos: 90 pacientes diabéticos tipo 2 assintomáticos, avaliados,entre junho de 2011 a setembro de 2012, entre 40 e 65 anos de idade, tempo de duração do diabetes inferior a 10 anos, submetidos a avaliação clínica, laboratorial e Angiotomografia Computadorizada de artérias coronárias com 320 colunas de detectores. Resultados: Dos 90 pacientes, 42,2% (n=38) apresentaram doença arterial coronariana a Angiotomo sendo n=11 no grupo A1c < 7% e n=27 no grupo A1c >=7% com diferença estatística (p=0,0006). 14 indivíduos apresentaram doença arterial coronariana significativa (obstrução do lúmen superior a 50%), n=3 no grupo A1c<7% e n=11 no A1c>=7% (p=0,02). O tipo de placa não calcificada predominou no grupo A1c>=7% (p=0,005) e 29% dos diabéticos com doença coronária apresentaram lesões ateroscleróticas classificadas como mais vulneráveis que predominaram no grupo A1c>=7% (p=0,04). Conclusão: O paciente diabético assintomático apresenta além de elevada frequência de doença arterial coronariana possui grande número de placas classificadas como vulneráveis pela Angiotomo e portanto predispostas a ruptura e evento coronariano agudo, principalmente no grupo A1c > =7% / The number of diabetic patients increase every day. Acute myocardial infarction (AMI) and Stroke are the leading causes of death in this group. Coronary atherosclerotic plaque rupture is the pathophysiologic mechanism for (AMI) in 2 every 3 cases and the characteristics of these plaques more vulnerable and prone to rupture as positive remodeling of the vascular segment affected and non-calcified plaque with low attenuation can be evaluated by Angiotomography. Objective: To evaluate the frequency of coronary artery disease and the main characteristics of these vulnerable atherosclerotic plaques in asymptomatic diabetic considering the degree of glycemic control by Multislice Computed Angiotomography. Study Design and Methods: 90 asymptomatic type 2 diabetic patients, evaluated between June 2011 and September 2012, between 40 and 65 years of age, duration of diabetes less than 10 years, underwent clinical, laboratory and Angiotomography Computed coronary arteries with 320 columns of detectors. Results: Of 90 patients, 42.2% (n = 38) had coronary artery disease being the Angiotomo n = 11 in group A1c <7% and n = 27 in group A1c> = 7% with statistical difference (p = 0.0006 ). 14 individuals showed significant coronary artery disease (obstruction of the lumen than 50%), n = 3 in the A1c <7% and n = 11 in A1c> = 7% (p = 0.02). The type of noncalcified plaque predominated in A1c> = 7% (p = 0.005) and 29% of diabetics with coronary disease showed atherosclerotic lesions classified as most vulnerable group that predominated in A1c> = 7% (p = 0.04) . Conclusion: Diabetic patients asymptomatic features besides high frequency of coronary artery disease has a large number of plaques classified as vulnerable by Angiotomo and therefore prone to rupture and acute coronary event, especially in the group A1c> = 7%

Diabetes mellitus (DM) é uma das principais causas de doença renal crônica terminal. Na doença renal diabética (DRD) observa-se um curso bifásico no padrão glicêmico, na fase inicial o aumento da resistência insulínica induz a hiperglicemia e, com perda progressiva da taxa de filtração glomerular, há redução na depuração dos medicamentos anti-hiperglicemiantes e insulina, aumentando o risco de hipoglicemias. Portanto, diante da perda da função renal, a reavaliação da terapia hipoglicemiante e ajustes constantes nas doses de insulina são necessários, com intuito de otimizar o controle glicêmico e minimizar seus efeitos colaterais. A revisão da literatura mostra diversos pontos sem resposta, principalmente relacionados à dose, ajuste da terapia insulínica, seguimento e monitoração do controle glicêmico em portadores de DM e DRC. O objetivo deste ensaio randomizado, cruzado, controlado foi comparar o controle glicêmico do tratamento com insulina glargina à insulina NPH em portadores de DM2 e DRD estágios 3 e 4. Pacientes e métodos: Trinta e quatro pacientes foram randomizados para receber insulina glargina uma vez ao dia ou insulina NPH em três aplicações diárias. Insulina lispro foi prescrita três vezes ao dia, em aplicações pré-prandiais nos dois grupos. Após 24 semanas de terapia, os pacientes tiveram seu esquema de insulina trocado para terapia insulínica oposta. Testes laboratoriais foram realizados após 12, 24, 36 e 48 semanas de estudo. O sistema de monitorização continua de glicose (CGMS) foi instalado ao término de cada terapia. Resultados: Dos 34 pacientes incluídos, 29 completaram as 48 semanas propostas no estudo, 2 pacientes perderam seguimento por má adesão e 3 pacientes não completaram o estudo em decorrência a eventos adversos (1 óbito, 1 ingresso em hemodiálise e 1 evento cardiovascular, todos em uso de insulina NPH). Após 24 semanas de tratamento com insulina glargina houve uma redução estatisticamente significante da média da HbA1c de 8,86 ± 1,4% para 7,95 ± 1,1% (p=0,0285), esta diferença não foi observada com a insulina NPH (8,21 ± 1,29% para 8,44 ± 1,32%). Durante o uso de insulina glargina o número de eventos noturnos de hipoglicemia foi menor comparado a insulina NPH (p=0,046); além disso, hipoglicemia grave ocorreu apenas na terapêutica com NPH. Conclusão: O tratamento com insulina glargina foi associado a melhor controle glicêmico e a redução do risco de hipoglicemia noturna quando comparada à insulina NPH,em pacientes portadores de DM e DRC estágios 3 e 4 / Diabetes mellitus is the leading cause of chronic kidney disease (CKD). Kidney disease diagnosis and its progression require re-evaluation of hypoglycemic therapy and constant dosing adjustments, to optimize glycemic control and minimize its side effects. Long acting insulin analogs and its pharmacokinetics have not been studied in different stages of kidney disease, nor is there consensus defining appropriate dose adjustment in patients with type 2 diabetes (T2DM) and CKD. The aim of this randomized, cross-over, open-label controlled clinical trial is to compare the glycemic response to intensive insulin treatment with NPH insulin or insulin glargine in T2DM patients and CKD stages 3 and 4. The primary efficacy end point was change in A1C from baseline. Thirty-four patients were randomized to receive insulin glargine once a day or NPH insulin, three times a day. Insulin lispro was prescribed as prandial insulin to both groups. After six months, patients switched to the other insulin therapy group. Laboratory tests were performed at baseline at 12, 24, 36 and 48 weeks. A continuous glucose monitoring system was implemented after 24 weeks and at the end of protocol. Results: Total of 29 subjects have completed the two branches of study, 2 patients dropped out due to low compliance and other 3 patients as a result of adverse events (1 death, 1 ingress on dialysis program, 1 cardiovascular event; all of them were on NPH therapy). After 24 weeks, average of A1c decreased on glargine group compared to baseline 8,86 ± 1,4% to 7,95 ± 1,1% (p=0,0285), but this difference was not observed on NPH group. There were no differences of insulin doses between both groups. Glargine group showed a tendency of lower risk of nocturnal hypoglycemia compared to NPH group (p=0,046). Conclusion: Insulin glargine improved glycemic control by reducing HbA1c without gain weight and with reduced tendency toward nocturnal hypoglycemic events compared with NPH insulin

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2015-10-15T19:58:50Z No. of bitstreams: 2 Tese - NELSON RASSI - 2014.pdf: 2531801 bytes, checksum: 4a281a91c2ca563e8fead55207f00d61 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Cláudia Bueno (claudiamoura18@gmail.com) on 2015-10-15T20:31:04Z (GMT) No. of bitstreams: 2 Tese - NELSON RASSI - 2014.pdf: 2531801 bytes, checksum: 4a281a91c2ca563e8fead55207f00d61 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-15T20:31:04Z (GMT). No. of bitstreams: 2 Tese - NELSON RASSI - 2014.pdf: 2531801 bytes, checksum: 4a281a91c2ca563e8fead55207f00d61 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-09-13 / Background: The number of patients with Type 2 Diabetes Mellitus (T2DM) in Brazil has, in recent decades, increased substantially and insulin therapy is often necessary in a large portion of this population in order to achieve appropriate glycemic control. Objective: To evaluate glycemic control achieved with recombinant human insulin NPH - Gansulin and compares it with human NPH insulin - Humulin N® in patients with Type 2 Diabetes Mellitus. Subjects and methods: A prospective, double-blind, randomized, parallel, single center with 37 individuals with type 2 diabetes using insulin NPH insulin. For statistical analyzes were used: the multiple comparison test of Tukey-Kramer test, Wilcoxon paired comparison test and Chi- Square. It was regarded level of significance value lower than 5% (p<0.05). Results: Insulins NPH and Humulin Gansulin showed similar reductions in HbA1c at the end of the study compared to baseline. Initial HbA1c 7.91% in the Humulin group was reduced to 6.56% (p<0.001) at the end of the study whereas in the Gansulin the glycated hemoglobin was reduced from 8.18% to 6.65% (p<0.001). At the end of the study there was no significant difference between the glycated hemoglobin levels (p=0.2410), fasting blood glucose (p=0.9257) and glucose at bedtime (p=0.3906) between the two types of insulin. Regarding the number of hypoglycemic events, there was no significant difference between the two insulins and no severe hypoglycemic episodes were recorded. Conclusion: The NPH Gansulin (Insuneo N®) presented glycemic control similar to that presented by human insulin Humulin N® in patients with DM2. It was considered level of significance value less than 5%. / Fundamento: O número de pacientes com Diabetes Mellitus Tipo 2 (DM2) no Brasil tem, nas últimas décadas, aumentado substancialmente e a terapia insulínica é necessária em uma grande parcela desta população com a finalidade de adquirir controle glicêmico adequado. Objetivo: Avaliar o controle glicêmico obtido com a insulina humana recombinante NPH – Gansulin e compará-la com o da insulina humana NPH – Humulin N® em pacientes com Diabetes Mellitus Tipo 2 (DM2). Sujeitos e métodos: Estudo prospectivo, duplo cego, randomizado, paralelo e monocêntrico com 37 indivíduos portadores de diabetes tipo 2, em uso de insulina NPH. Para as análises estatísticas foram utilizados: o teste de comparações múltiplas de Tukey-Kramer, o teste de comparação pareada de Wilcoxon e o teste Chi-Square. Foi considerado como nível de significância o valor inferior a 5% (p<0,05). Resultados: As insulinas NPH Humulin e Gansulin apresentaram reduções semelhantes da HbA1c ao final do estudo, quando comparadas aos valores iniciais. A HbA1c inicial de 7,91% do grupo Humulin foi reduzida para 6,56% (p<0,001), enquanto que na do Gansulin, a redução foi de 8,18% para 6,65% (p<0,001). Ao final do estudo não houve diferença significativa entre os valores de hemoglobina glicada (p=0,2410), glicemia jejum (p=0,9257) e glicemia ao deitar (p=0,3906) entre os dois tipos de insulina. Em relação ao número de eventos hipoglicêmicos, não se observou diferença significativa entre as duas insulinas e não foram registrados episódios hipoglicêmicos graves. Conclusão: A insulina NPH Gansulin apresentou controle glicêmico semelhante ao apresentado pela insulina humana Humulin N® em pacientes com DM2.