O Diâmetro Abdominal Sagital (DAS) é uma medida antropométrica relacionada com a gordura visceral e empregada para avaliar a obesidade abdominal, uma variável associada à síndrome metabólica. Sua utilização é indicada na prática clínica para avaliação de risco cardiometabólico em adolescentes obesos. OBJETIVO: Verificar a concordância entre o DAS e a circunferência abdominal (CA) na avaliação da obesidade central e sua associação com os critérios da Síndrome Metabólica e HOMA-IR em adolescentes obesos. CASUÍSTICA E MÉTODOS: Estudo de corte transversal constituído por 83 adolescentes obesos entre 14 e 18 anos, (46 do sexo feminino e 37 do sexo masculino) matriculados no Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, nos ambulatórios das Unidades de Endocrinologia Pediátrica e de Adolescentes. Foram submetidos a avaliações antropométricas (IMC, Escore Z do IMC, percentual de gordura corporal, circunferência abdominal, diâmetro abdominal sagital), laboratoriais (HDL-c, triglicérides (TG), glicemia (GLIS) e insulina para o cálculo do HOMA-IR) e de pressão arterial sistólica (PAS) e diastólica (PAD), utilizadas para classificação dos critérios da SM. RESULTADOS: Todos os adolescentes apresentaram valores elevados de IMC (36,9±6,7 kg/m2), Z-IMC (+3,27±0,94) e 91,6% da casuística tiveram valores alterados no percentual de gordura corporal (41,4% para o grupo feminino e 36% para o grupo masculino), confirmando a obesidade grave do grupo. Considerando o percentil >=90 do Center for Disease Control and Prevention (CDC), no National Health and Nutrition Examination Survey (NHANES 2011-2014), 85,5% dos adolescentes apresentaram valores elevados de CA (117,7 ± 14,7) e 89,2% valores alterados no DAS (26,9±3,7). Quanto às variáveis laboratoriais, 32,5% dos pacientes apresentaram diminuição de HDL-c e níveis aumentados de: TG (10,8%); GLIS (3,6%); PAS (32,5%,); PAD (21,7%) e HOMA-IR (79,5%), considerando toda a amostra. De acordo com os critérios utilizados pelo International Diabetes Federation, 27,7% da casuística apresentou SM. O DAS demonstrou estar significantemente correlacionado com as variáveis PAS (r=0,489 p < 0,001), PAD(r=0,277 p 0,011) e HOMA-IR (r=0,462 p < 0,001) nos grupos geral, feminino, masculino, com e sem SM. A correlação encontrada entre as medidas do DAS e CA no grupo geral e feminino foi de r = 0,91 (p 0,000) e, no grupo masculino, de r = 0,93 (p 0,000). A concordância entre a CA e o DAS é significante (Kappa k = 0.511; p < 0,001). Nos grupos geral, feminino e masculino com SM, a concordância é mais expressiva (Kappa k = 1,00; p < 0,001.). Esses resultados mostram que os adolescentes apresentavam risco cardiometabólico aumentado e expressiva obesidade central, identificada pelo DAS e CA, apesar de 73,5% deles estarem medicados. O DAS oferece vantagem metodológica na sua mensuração. CONCLUSÕES: Nas condições deste estudo, conclui-se que: as medidas antropométricas CA e DAS se equivalem para o grupo de adolescentes avaliados na classificação da SM; O DAS é preditor de PAS, PAD e HOMA-IR e forte indicador de risco cardiometabólico em adolescentes obesos / The sagittal abdominal diameter (SAD) is an anthropometric measure related to visceral fat and used to evaluate abdominal obesity, a variable associated with the metabolic syndrome. Studies have suggested its employment in the clinical practice for estimating cardiometabolic risk of obese adolescents. OBJECTIVE: To verify the concordance between SAD and abdominal circumference in the assessment of central obesity and its association with the Metabolic Syndrome cluster and with HOMA-IR in obese adolescents. CASUISTICS AND METHODS: In a cross-sectional study, 83 obese adolescents between 14 and 18 years (46 females and 37males) with body mass index (BMI) of 36.9 ± 6.7 kg/m2, followed at the Pediatric Endocrinology Unit and at the Adolescent Unit of the Children\'s Institute, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo were submitted to anthropometric (BMI, BMI Z Score, body fat percentage, abdominal circumference, sagittal abdominal diameter), laboratory (HDL-c, triglycerides, glycemia and insulin for calculating HOMA-IR) and systolic and diastolic blood pressure assessments aimed for classification of metabolic syndrome. Previous consentment was given by all patients and their families. RESULTS: All adolescents presented elevated BMI and Z-BMI, and high body fat percentage was displayed by 91.6% of the patients (41.4% for the female group and 36% for the male group), confirming severe obesity. Considering the >= 90 percentile cut-off values as provided by the Anthropometric Reference Data for Children and Adults: United States 2011-2014 for abdominal circumference and SAD, 85.5% of the patients presented high abdominal circumference values (117.7±14.7) and 89.2% presented elevated values of SAD (26.9±3.7). With regard to laboratory variables, 32.5% of the patients displayed decreased HDL-c and increased values of: triglycerides (10.8%); glycemia (3.6%); systolic blood pressure (32.5%); diastolic blood pressure (21.7%) and HOMA-IR (79.5%). According to the criteria of the International Diabetes Federation (IDF), 27.7% of patients presented metabolic syndrome. SAD was significantly correlated with systolic (r=0.489 p < 0.001) and diastolic (r=0.277 p 0.011) blood pressures and HOMA-IR (r=0.462 p < 0.001) in the general, female and male groups, with and without metabolic syndrome. The correlation between SAD and abdominal circumference in the general and female groups was r = 0.91(p 0.000) and in the male group was r = 0.93 (p 0.000). The concordance between SAD and abdominal circumference was significant (Kappa coefficient k = 0.511; p < 0.001). In the general, male and female groups with metabolic syndrome, the concordance was more expressive (Kappa coefficient; k = 1.00 and p < 0.001). These results show that the adolescents presented increased cardiometabolic risk and significant central obesity identified by SAD and abdominal circumference although 73.5% of the studied patients were maintained under medication for clinical metabolic syndrome symptoms. SAD displayed methodological advantages concerning its measurement. CONCLUSIONS: Under the conditions of this study, it is concluded that: the anthropometric measurements of SAD and abdominal circumference are equivalent for metabolic syndrome classification of the studied adolescents; that SAD is a predictor of systolic and diastolic blood pressures and HOMA-IR and is a strong indicator of cardiometabolic risk in obese adolescents

O Diâmetro Abdominal Sagital (DAS) é uma medida antropométrica relacionada com a gordura visceral e empregada para avaliar a obesidade abdominal, uma variável associada à síndrome metabólica. Sua utilização é indicada na prática clínica para avaliação de risco cardiometabólico em adolescentes obesos. OBJETIVO: Verificar a concordância entre o DAS e a circunferência abdominal (CA) na avaliação da obesidade central e sua associação com os critérios da Síndrome Metabólica e HOMA-IR em adolescentes obesos. CASUÍSTICA E MÉTODOS: Estudo de corte transversal constituído por 83 adolescentes obesos entre 14 e 18 anos, (46 do sexo feminino e 37 do sexo masculino) matriculados no Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, nos ambulatórios das Unidades de Endocrinologia Pediátrica e de Adolescentes. Foram submetidos a avaliações antropométricas (IMC, Escore Z do IMC, percentual de gordura corporal, circunferência abdominal, diâmetro abdominal sagital), laboratoriais (HDL-c, triglicérides (TG), glicemia (GLIS) e insulina para o cálculo do HOMA-IR) e de pressão arterial sistólica (PAS) e diastólica (PAD), utilizadas para classificação dos critérios da SM. RESULTADOS: Todos os adolescentes apresentaram valores elevados de IMC (36,9±6,7 kg/m2), Z-IMC (+3,27±0,94) e 91,6% da casuística tiveram valores alterados no percentual de gordura corporal (41,4% para o grupo feminino e 36% para o grupo masculino), confirmando a obesidade grave do grupo. Considerando o percentil >=90 do Center for Disease Control and Prevention (CDC), no National Health and Nutrition Examination Survey (NHANES 2011-2014), 85,5% dos adolescentes apresentaram valores elevados de CA (117,7 ± 14,7) e 89,2% valores alterados no DAS (26,9±3,7). Quanto às variáveis laboratoriais, 32,5% dos pacientes apresentaram diminuição de HDL-c e níveis aumentados de: TG (10,8%); GLIS (3,6%); PAS (32,5%,); PAD (21,7%) e HOMA-IR (79,5%), considerando toda a amostra. De acordo com os critérios utilizados pelo International Diabetes Federation, 27,7% da casuística apresentou SM. O DAS demonstrou estar significantemente correlacionado com as variáveis PAS (r=0,489 p < 0,001), PAD(r=0,277 p 0,011) e HOMA-IR (r=0,462 p < 0,001) nos grupos geral, feminino, masculino, com e sem SM. A correlação encontrada entre as medidas do DAS e CA no grupo geral e feminino foi de r = 0,91 (p 0,000) e, no grupo masculino, de r = 0,93 (p 0,000). A concordância entre a CA e o DAS é significante (Kappa k = 0.511; p < 0,001). Nos grupos geral, feminino e masculino com SM, a concordância é mais expressiva (Kappa k = 1,00; p < 0,001.). Esses resultados mostram que os adolescentes apresentavam risco cardiometabólico aumentado e expressiva obesidade central, identificada pelo DAS e CA, apesar de 73,5% deles estarem medicados. O DAS oferece vantagem metodológica na sua mensuração. CONCLUSÕES: Nas condições deste estudo, conclui-se que: as medidas antropométricas CA e DAS se equivalem para o grupo de adolescentes avaliados na classificação da SM; O DAS é preditor de PAS, PAD e HOMA-IR e forte indicador de risco cardiometabólico em adolescentes obesos / The sagittal abdominal diameter (SAD) is an anthropometric measure related to visceral fat and used to evaluate abdominal obesity, a variable associated with the metabolic syndrome. Studies have suggested its employment in the clinical practice for estimating cardiometabolic risk of obese adolescents. OBJECTIVE: To verify the concordance between SAD and abdominal circumference in the assessment of central obesity and its association with the Metabolic Syndrome cluster and with HOMA-IR in obese adolescents. CASUISTICS AND METHODS: In a cross-sectional study, 83 obese adolescents between 14 and 18 years (46 females and 37males) with body mass index (BMI) of 36.9 ± 6.7 kg/m2, followed at the Pediatric Endocrinology Unit and at the Adolescent Unit of the Children\'s Institute, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo were submitted to anthropometric (BMI, BMI Z Score, body fat percentage, abdominal circumference, sagittal abdominal diameter), laboratory (HDL-c, triglycerides, glycemia and insulin for calculating HOMA-IR) and systolic and diastolic blood pressure assessments aimed for classification of metabolic syndrome. Previous consentment was given by all patients and their families. RESULTS: All adolescents presented elevated BMI and Z-BMI, and high body fat percentage was displayed by 91.6% of the patients (41.4% for the female group and 36% for the male group), confirming severe obesity. Considering the >= 90 percentile cut-off values as provided by the Anthropometric Reference Data for Children and Adults: United States 2011-2014 for abdominal circumference and SAD, 85.5% of the patients presented high abdominal circumference values (117.7±14.7) and 89.2% presented elevated values of SAD (26.9±3.7). With regard to laboratory variables, 32.5% of the patients displayed decreased HDL-c and increased values of: triglycerides (10.8%); glycemia (3.6%); systolic blood pressure (32.5%); diastolic blood pressure (21.7%) and HOMA-IR (79.5%). According to the criteria of the International Diabetes Federation (IDF), 27.7% of patients presented metabolic syndrome. SAD was significantly correlated with systolic (r=0.489 p < 0.001) and diastolic (r=0.277 p 0.011) blood pressures and HOMA-IR (r=0.462 p < 0.001) in the general, female and male groups, with and without metabolic syndrome. The correlation between SAD and abdominal circumference in the general and female groups was r = 0.91(p 0.000) and in the male group was r = 0.93 (p 0.000). The concordance between SAD and abdominal circumference was significant (Kappa coefficient k = 0.511; p < 0.001). In the general, male and female groups with metabolic syndrome, the concordance was more expressive (Kappa coefficient; k = 1.00 and p < 0.001). These results show that the adolescents presented increased cardiometabolic risk and significant central obesity identified by SAD and abdominal circumference although 73.5% of the studied patients were maintained under medication for clinical metabolic syndrome symptoms. SAD displayed methodological advantages concerning its measurement. CONCLUSIONS: Under the conditions of this study, it is concluded that: the anthropometric measurements of SAD and abdominal circumference are equivalent for metabolic syndrome classification of the studied adolescents; that SAD is a predictor of systolic and diastolic blood pressures and HOMA-IR and is a strong indicator of cardiometabolic risk in obese adolescents

[pt] O fenômeno de dessorção iônica a partir da incidência de pulsos de radiação laser ultravioleta sobre superfícies em vácuo foi estudada. A dessorção de três tipos diferentes de sólidos foi analisada: insulina, carbono (amorfo e grafite) e policristais de haletos alcalinos. Os processos fundamentais da interação da radiação laser com sólido e o vapor formado, assim como a evolução do plasma gerado, foram descritos satisfatoriamente através de um modelo térmico e da simulação de espectros de têmpo-de-vôo para os primeiros instante da expansão ao vácuo. Um novo método foi proposto para determinar as velocidades iniciais e o início da expansão livre em função da intensidade do laser para LDI (Laser Desorption Ionization) e MALDI (Matrix Assisted Laser Desorption Ionization). Embora o estudo da expansão do plasma gerado não tenha sido tratada dinamicamente pela sua complexidade, a análise utilizando varias combinações do sólido irradiado permitiram concluir que a dessorção induzida por laser pode ser caracterizada por dois processos fundamentais: i) a atomização seguida de recombinação dos constituintes do alvo formando aglomerados e ii) a emissão de aglomerados pré-formados do material. As estruturas geométricas mais estáveis das espécies detectadas foram caracterizadas utilizando a Teoria do Funcional da Densidade (DFT) e classificadas taxonomicamente em função de sua energia (método D-plot); determinou-se a influência da estabilidade dessas estruturas nas abundâncias relativas no espectro de massa. / [en] Ion desorption induced by ultraviolet laser radiation pulses was studied in surfaces in vacuum. The ion desorption from three different solids was analyzed: insulin, carbon (amorphous and graphite) and polycrystals of alkali halides. The main processes involved in the laser-solid and laser-vapor interactions, as well as in the plasma evolution, were well described by a thermal model and by the simulation of the time-of-flight spectra for the first moments of the plasma expansion to vacuum. A new method to determine the initial velocity and the beginning of the free expansion regime as a function of the laser intensity was proposed for LDI (Laser Desorption Ionization) and MALDI (Matrix Assisted Laser Desorption Ionization). Considering the complexity of the dynamical treatment of the expansion of the laser-generated plasma, an analysis by using several combinations of irradiated solids was performed. It was established that the desorption process is characterized by two main mechanisms: i) the atomization followed by recombination of the target elements in clusters and ii) the emission of preformed clusters of the target material. The most stable geometric structures of the measured species were characterized using Density Functional Theory (DFT) and classified taxonomically as a function of their internal total energy (D-plot method); the influence of the structure`s stability on the relative mass abundances was also determined. / [fr] Le phénomène de la désorption ionique induite par des pulses laser ultraviolets dans la surface de solides est étudié. Trois types différents de solides ont été analysés: insuline, carbone (amorphe, graphite et CO condensé) et halogénures alcalins polycristallins (CsI, KI, KBr). La dynamique des ions secondaires émis est analysée par la comparaison des résultats de modélisation avec leur distributions de vitesse mesurées. Un modèle thermique est proposé pour décrire l´interaction entre la radiation laser avec une pellicule de CsI polycristallin et aussi avec la plume émise dans ce processus. Dans le cadre de ce modèle, une nouvelle méthode est utilisée pour caractériser le régime de collision dans lê plasma, soit dans le cas du LDI (Laser Desorption Ionization), fait sur le CsI, soit dans le cas du MALDI (Matrix Assisted Laser Desorption Ionization), fait sur l´insuline dissoute dans une solution solide d´ ACHC. Il est rappelé que le ions CsI émis peuvent être reconstitués après une atomisation complète de la cible mais ceux des de l´insuline difficilement le peuvent. Pour mieux comprendre l´émission des agrégats ioniques Cn + et des halogénures alcalins, leurs structures moléculaires ont été étudiées théoriquement par DFT (Density Functional Theory). L´énergie totale de chaque isomère a été calculée et transformée dans une nouvelle quantité nommée déviation énergétique (D). Le graphique D-plot, oú D est présenté en fonction du nombre de monomères, n, s´est montré très utile pour classer les agrégats en familles et pour estimer leur stabilité, laquelle est liée vraisemblablement à ses abondances de désorption.

Les protéines 14-3-3 sont des protéines ubiquitaires conservées chez tous les eucaryotes. Elles interagissent avec de multiples partenaires, dont elles modulent la fonction et la localisation, et interviennent de ce fait dans de nombreux processus cellulaires. FTT-1 et FTT-2 sont les deux orthologues de 14-3-3 présents chez Caenorhabditis elegans. En utilisant l'approche de RNA interférence, nous avons montré que les protéines FTT sont impliquées dans la voie DAF-2/Insuline/IGF-1, régissant la longévité, la résistance au stress et le métabolisme chez C. elegans, et dont l'effecteur majeur est le facteur de transcription DAF-16, orthologue de Forkhead chez les mammifères. Ce travail de thèse a permis de caractériser les fonctions de chacune des protéines FTT dans la régulation des différents phénotypes engendrés par cette voie et de mettre en évidence le rôle prépondérant de FTT-2. D'autre part, nos données ont révélé la contribution supplémentaire de FTT-2 dans la régulation de la longévité et du métabolisme lipidique et de FTT-1 et FTT-2 dans la résistance au stress, à travers un mécanisme parallèle à la voie DAF-2/Ins/IGF-1 et ne mettant pas en jeu DAF-16. Les résultats de notre étude soulignent la complexité des fonctions des protéines FTT et démontrent leur participation à plusieurs processus importants pour la survie de C. elegans lors d'une exposition à un stress mais aussi lors du vieillissement physiologique du nématode.

Polycystic ovary syndrome (PCOS) is a complex clinical condition characterized by hyperandrogenism and chronic oligo/anovulation. Infrequent ovulation and metabolic alterations in women with PCOS are associated with subfertility and probably increased miscarriage rates compared with normal fertile women. The overall risk of developing type 2 diabetes and impaired glucose tolerance (IGT) is three- to sevenfold higher in PCOS women, and the onset of glucose intolerance seems to occur at an earlier age than in healthy controls. Women with PCOS also have several risk factors for cardiovascular disease, although it is unclear whether they actually experience more cardiovascular events than other women. Very few studies assessing the long-term reproductive and metabolic consequences in older women with previously confirmed PCOS have been conducted. In this long-term follow-up of women with PCOS, 84 women with a diagnosis of PCOS between 1987 and 1995 and age at the follow-up &gt; 35 years and an age-matched population-based group of control women participated. Data on reproductive outcome, ovarian reserve, endothelial function, insulin sensitivity and beta-cell function were collected. According to our results most women with PCOS had given birth and the rate of spontaneous pregnancies was relatively high. The rate of miscarriages was not increased in PCOS patients and the ultrasound findings together with increased levels of anti-müllerian hormone suggested that their ovarian reserve is superior to women of similar age. PCOS women displayed signs of endothelial dysfunction, but this was largely due to the increased prevalence of independent risk factors for cardiovascular disease such as increased BMI, triglycerides and blood pressures. IGT and type 2 diabetes occurred more often in PCOS women. Free androgen levels and beta-cell function decreased over time whereas insulin sensitivity remained unchanged. Obesity at young age and progressive weight-gain rendered them more prone to be insulin resistant at the follow-up. Beta-cell function was increased in PCOS women in comparison with control subjects but declined over time. Independent of PCOS phenotype at the index assessment and persistence of PCOS symptoms at the follow-up investigation, premenopausal women with PCOS had lower insulin sensitivity and increased beta cell function in comparison with control subjects. Conclusion: The long-term reproductive outcomes of PCOS are similar compared to women with normal ovaries. Although symptoms and androgen levels are normalized over time, women with PCOS continue to display reduced insulin sensitivity and increased beta-cell function and they also have an increased risk of IGT and type 2 diabetes.

No description available.

La phosphorylation est une modification post-traductionnelle modulant l’activité, la conformation ou la localisation d’une protéine et régulant divers processus. Les kinases et phosphatases sont responsables de la dynamique de phosphorylation et agissent de manière coordonnée. L’activation anormale ou la dérégulation de kinases peuvent conduire au développement de cancers ou de désordres métaboliques. Les récepteurs tyrosine kinase (RTKs) sont souvent impliqués dans des maladies et la compréhension des mécanismes régissant leur régulation permet de déterminer les effets anticipés sur leurs substrats. Dans ce contexte, le but de cette thèse est d’identifier les évènements de phosphorylation intervenant dans la voie de l’insuline chez la drosophile impliquant un RTK : le récepteur de l’insuline (InR). La cascade de phosphorylation déclenchée suite à l’activation du récepteur est conservée chez le mammifère. Afin d’étudier le phosphoprotéome de cellules S2 de drosophile, nous avons utilisé une étape d’enrichissement de phosphopeptides sur dioxyde de titane suivie de leur séparation par chromatographie liquide (LC) et mobilité ionique (FAIMS). Les phosphopeptides sont analysés par spectrométrie de masse en tandem à haute résolution. Nous avons d’abord démontré les bénéfices de l’utilisation du FAIMS comparativement à une étude conventionnelle en rapportant une augmentation de 50 % dans le nombre de phosphopeptides identifiés avec FAIMS. Cette technique permet de séparer des phosphoisomères difficilement distinguables par LC et l’acquisition de spectres MS/MS distincts où la localisation précise du phosphate est déterminée. Nous avons appliqué cette approche pour l’étude des phosphoprotéomes de cellules S2 contrôles ou traitées à l’insuline et avons identifié 32 phosphopeptides (sur 2 660 quantifiés) pour lesquels la phosphorylation est modulée. Étonnamment, 50 % des cibles régulées possèdent un site consensus pour la kinase CK2. Une stratégie d’inhibition par RNAi a été implémentée afin d’investiguer le rôle de CK2 dans la voie de l’insuline. Nous avons identifié 6 phosphoprotéines (CG30085, su(var)205, scny, protein CDV3 homolog, D1 et mu2) positivement régulées suite à l’insuline et négativement modulées après le traitement par RNAi CK2. Par essai kinase in vitro, nous avons identifié 29 cibles directes de CK2 dont 15 corrélaient avec les résultats obtenus par RNAi. Nous avons démontré que la phosphorylation de su(var)205 (S15) était modulée par l’insuline en plus d’être une cible directe de CK2 suite à l’expérience RNAi et à l’essai kinase. L’analyse des données phosphoprotéomiques a mis en évidence des phosphopeptides isomériques dont certains étaient séparables par FAIMS. Nous avons déterminé leur fréquence lors d’études à grande échelle grâce à deux algorithmes. Le script basé sur les différences de temps de rétention entre isomères a identifié 64 phosphoisomères séparés par LC chez la souris et le rat (moins de 1 % des peptides identifiés). Chez la drosophile, 117 ont été répertoriés en combinaison avec une approche ciblée impliquant des listes d’inclusion. Le second algorithme basé sur la présence d’ions caractéristiques suite à la fragmentation de formes qui co-éluent a rapporté 23 paires isomériques. L’importance de pouvoir distinguer des phosphoisomères est capitale dans le but d’associer une fonction biologique à un site de phosphorylation précis qui doit être identifié avec confiance. / Phosphorylation is a reversible post-translational modification that modulates protein activity, and can impart conformational changes and affect translocation of their protein substrates. Kinases and phosphatases are responsible for the dynamic of changes in protein phosphorylation and act in a coordinated manner. Abnormal activation or misregulation of kinase activity can lead to the development of cancers and metabolic disorders. Tyrosine kinase receptor (RTK) associated signaling pathways are often implicated in numerous diseases and the further understanding of mechanisms affecting their regulation is necessary to determine their activity and effects anticipated on their substrates. In this context, the primary objective of this thesis is to study the phosphorylation events arising from the activation of the insulin receptor (InR) following stimulation of drosophila S2 cells with insulin. The phosphorylation cascade triggered after InR activation is conserved in mammals. In order to study the phosphoproteome of drosophila S2 cells, we enriched phosphopeptides on titanium dioxide (TiO2) stationary phase prior to their separation by liquid chromatography (LC) and ion mobility (FAIMS) mass spectrometry (MS). Phosphopeptides were then analysed by tandem MS at high resolution. We first compared the benefits of FAIMS to conventional LC-MS, and observed a 50% increase in the number of identified phosphopeptides when using ion mobility. FAIMS enables the separation of phosphoisomers that are typically unresolved by LC, enabling high confidence assignment of modification sites via distinct MS/MS spectra. This approach was used to profile phosphorylation changes taking place between control and insulin-treated drosophila cells and enabled the identification of 32 phosphopeptides (out of 2 660 quantified) showing differential regulation. Interestingly, 50% of the regulated targets have a CK2 consensus site. These preliminary experiments were followed-up by RNAi mediated inhibition of CK2 and revealed that 6 phosphoproteins (CG30085, su(var)205, scny, protein CDV3 homolog, D1 and mu2) were positively modulated after insulin stimulation and negatively regulated after CK2 RNAi treatment. Using in vitro kinase assay, we identified 29 direct CK2 targets, of which 15 were correlated with results from the CK2 RNAi experiment. We demonstrated specifically that the su(var)205 (S15) is regulated by insulin and is a direct CK2 target based on RNAi and kinase assays. Our phosphoproteomics data also highlighted the presence of isomeric phosphopeptides, several of which could be distinguished using FAIMS. We developed two algorithms to determine the occurrence of phosphoisomers in large scale studies. The first algorithm based on differences in retention times between isomers identified 64 candidates in mouse and rat phosphoproteome datasets corresponding to less than 1% of all identified phosphopeptides. We also identified 117 isomer candidates in drosophila using a targeted LC-MS/MS approach with inclusion lists. The second algorithm is based on the presence of characteristic fragment ions present in MS/MS spectra of co-eluting or partially resolved species and allowed the identification of 23 isomeric pairs. The ability to distinguish phosphoisomers in large-scale phosphoproteome datasets is of significance to correlate phosphorylation events taking place on specific residues with biological activities.

L’insuline est une hormone essentielle qui induit des réponses complexes dans l’organisme pour maintenir l’homéostasie du glucose et des lipides. La résistance à son action est un phénomène pathologique observé dans un large éventail de situations, allant de l’obésité et du syndrome métabolique à la stéatose hépatique et au diabète de type 2, qui aboutissent au développement de l’athérosclérose et de la mortalité. Des avancées remarquables ont été réalisées dans notre compréhension des mécanismes moléculaires responsables du développement de la résistance à l’action de l’insuline. En particulier, l’induction d’un stress cellulaire par des taux élevés d’acides gras libres (AGL) et des cytokines, via l’activation des protéines Ser/Thr kinases, qui augmente la phosphorylation sur des résidus sérine, des molécules critiques impliquées dans la signalisation insulinique (p. ex. IR, IRS et p85) et conduit à la diminution de la réponse cellulaire à l’insuline. Cependant, la plupart des chercheurs ont limité leur travail dans l’investigation du rôle des protéines kinases susceptibles de modifier la réponse cellulaire à l’insuline. Donc, peu de données sont disponibles sur le rôle des Protéines Ser/Thr phosphatases (PS/TPs), même si il est bien établi que la phosphorylation de ces protéines est étroitement régulée par un équilibre entre les activités antagonistes des Ser/Thr kinases et des PS/TPs. Parmi les PS/TPS, PPM1A (également connu sous le nom PP2Cα) est une phosphatase particulièrement intéressante puisqu’il a été suggéré qu’elle pourrait jouer un rôle dans la régulation du métabolisme lipidique et du stress cellulaire. Ainsi, en se basant sur des résultats préliminaires de notre laboratoire et des données de la littérature, nous avons émis l’hypothèse selon laquelle PPM1A pourrait améliorer la sensibilité à l’insuline en diminuant l’activité des protéines kinases qui seraient activées par le stress cellulaire induit par l’augmentation des AGL. Ces effets pourraient finalement améliorer le métabolisme glucidique et lipidique dans l’hépatocyte. Ainsi, pour révéler le rôle physiologique de PPM1A à l’échelle d’un animal entier, nous avons généré un modèle animal qui la surexprime spécifiquement dans le foie. Nous décrivons ici notre travail afin de générer ce modèle animal ainsi que les premières analyses pour caractériser le phénotype de celui-ci. Tout d’abord, nous avons remarqué que la surexpression de PPM1A chez les souris C57BL/6J n’a pas d’effets sur le gain de poids sur une longue période. Deuxièmement, nous avons observé que PPM1A a peu d’effets sur l’homéostasie du glucose. Par contre, nous avons montré que sa surexpression a des effets significatifs sur l’homéostasie du glycogène et des triglycérides. En effet, nous avons observé que le foie des souris transgéniques contient moins de glycogène et de triglycérides que le foie de celles de type sauvage. De plus, nos résultats suggèrent que les effets de la surexpression de PPM1A pourraient refléter son impact sur la synthèse et la sécrétion des lipides hépatiques puisque nous avons observé que sa surexpression conduit à l’augmentation la triglycéridémie chez les souris transgéniques. En conclusion, nos résultats prouvent l’importance de PPM1A comme modulateur de l’homéostasie hépatique du glucose et des lipides. Des analyses supplémentaires restent cependant nécessaires pour confirmer ceux-ci et éclaircir l’impact moléculaire de PPM1A et surtout pour identifier ses substrats. / Insulin is a key hormone that elicits complex responses in the body to maintain glucose and lipid homeostasis. Impaired sensitivity to insulin is present throughout a spectrum of inter-related disorders ranging from obesity and metabolic syndrome to hepatic steatosis and type 2 diabetes, which promotes atherogenesis and mortality. Remarkable strides have been achieved in the molecular mechanisms responsible for the development of insulin resistance that has been associated with a chronic inflammatory state and an activation of cellular stress responses. In particular, the activation of cellular stress by elevated levels of free fatty acids (FFA) and cytokines, via upstream protein Ser/Thr kinases, increase the serine phosphorylation of critical molecules involved in insulin signaling pathway (e.g. IR, IRS and p85) and leads to decreased insulin response. However, most of the investigators have limited their works to stress-activated kinases capable of altering the cellular insulin responsiveness. Conversely, limited data are available on upstream Protein Ser/Thr phosphatases (PS/TPs), even if it is well established that the activity of stress-activated kinases is tightly regulated by a delicate balance between the opposing activities of both Ser/Thr kinases and PS/TPs. Among the PS/TPs associated with insulin resistance conditions, PPM1A (also known as PP2Cα) is of particular interest in the regulation of lipid metabolism and cellular stress. Based on our recent findings and preliminary data, we postulate that PPM1A plays a significant role in insulin resistance via dephosphorylation and lessening of FFA-activated stress kinases, mainly in the liver, an important organ in glucose and lipid metabolism. More specifically, we hypothesize that increasing PPM1A activity might improve the insulin responsiveness by down regulating the activity of stress-activated kinases and by improving lipid metabolism in the hepatocyte. Thus, to reveal the physiological role of PPM1A in whole animal, we generated an animal model that overexpresses PPM1A specifically in the liver. In the present research report, we describe our work to generate this animal model as well as the initial analyses to characterize the phenotype of these mice. Accordingly, we first noticed that overexpression of PPM1A in C57BL/6J mice has no effects on weight gain over a long period. Secondly, we observed that PPM1A has subtle effects on glucose homeostasis. However and more importantly, we showed that overexpression of PPM1A has a significant effect on both glycogen and triglycerides homeostasis. Indeed, we observed that the liver of PPM1A transgenic mice had less glycogen and triglycerides than their littermates’ wild type mice. Our results suggest that these effects might reflect the impact of PPM1A on lipids synthesis and secretion since we observed that overexpression of PPM1A leads to increase the triglyceridemia in the transgenic mice. En conclusion, our results pinpoint PPM1A as an important modulator of hepatic glucose and lipid metabolism. However, further analyses are needed to confirm these results, to decipher the molecular impact of PPM1A and particularity to identify its substrates.

Introduction De nombreuses études indiquent que la prématurité, qui représente 8 % des naissances, est associée à des indices précoces de dysfonction vasculaire, d’élévation de la pression sanguine et de survenue de diabète de type 2. Les enfants nés prématurément sont plus sujets aux blessures oxydatives de par l’immaturité de leurs défenses antioxydantes et de leur exposition à des situations pro-oxydantes (exposition à l’air ambiant, à un supplément d’oxygène, ou à une exposition aux infections). Cependant, les conséquences à long terme des blessures oxydatives induites par une exposition à l’oxygène en période périnatale restent méconnues. Le but de ce doctorat a été de mettre en évidence certains mécanismes pouvant relier les dommages de la prématurité induits par l’oxygène, et le risque à long terme de développer des maladies cardiovasculaires et métaboliques dans le concept global d’une programmation développementale de l’hypertension et des pathologies reliées au syndrome métabolique. Matériels et méthodes Des ratons Sprague-Dawley (SD) ont été exposés à 80 % O2 (O2) vs air ambiant (AA) du 3ème au 10ème jour de vie. Concernant les paramètres cardiovasculaires, nous avons mesuré au cours de la croissance, la pression sanguine à la queue (de la 4ème semaine à la 15ème semaine) et à l’âge adulte : la réactivité vasculaire à l’angiotensine II (AngII) et au carbachol (ex vivo, carotides) avec ou sans le tempol; la production d’oxyde nitrique (NO) en présence ou non L-arginine et de L-sépiaptérine (aorte, immunohistochimie) ainsi que l’expression de la nitric oxyde synthase endothéliale (eNOS) (aorte, immunohistochimie et western blot); le stress oxydant vasculaire (aorte, chemiluminescence) par la mesure de la production d’anions superoxide en présence ou non des inhibiteurs de la nicotinamide-adenine-dinucleotide-phosphate (NADPH oxydase) et de la nitric oxyde synthase endotheliale (eNOS), l’apocynine, et N-nitro-L-arginine methyl ester (L-NAME) respectivement, ainsi que le stress oxydant circulant par la mesure des niveaux plasmatiques de malondialdéhyde (MDA, HPLC); la densité microvasculaire a été évaluée au niveau du muscle tibial antérieur, immunohistochimie); la vitesse d’onde pulsée (VOP) (entre la valve aortique et juste avant la bifurcation ilio-fémorale) a été mesurée par ultrason; le nombre de néphrons a été compté par digestion acide. L’ontogenèse de la plupart de ces mécanismes a été regardée à l’âge de 4 semaines. Concernant les paramètres métaboliques, le poids a été mesuré au cours de la croissance. À l’âge adulte, la composition corporelle et la tolérance au glucose ont été évaluées. Résultats À l’âge de 4 semaines, aucune différence n’a été observée dans la pression sanguine, la réactivité vasculaire et le stress oxydant, mais chez les rats O2 vs AA, la densité microvasculaire est moindre, et des changements histologiques suggèrent la présence d’une rigidité artérielle augmentée. À l’âge adulte chez les rats O2 vs AA (n = 6-8 /groupe) : i) les pressions sanguines systoliques et diastoliques sont augmentées; ii) la réactivité vasculaire à l’AngII est augmentée et celle au carbachol est diminuée, le tempol prévient ces dysfonctions; iii) la production de NO est plus faible au niveau basal et après stimulation par le carbachol, mais est restaurée après la pré-incubation avec L-arginine et L-sépiaptérine; iv) l’expression d’eNOS est diminuée par immunohistochimie et augmentée par western blot; v) les niveaux d’anions superoxide, au niveau basal et en réponse à l’AngII, sont augmentés et sont induits par la NADPH oxydase et le non-couplage d’eNOS; vi) les niveaux plasmatiques de MDA sont augmentés; vii) La densité microvasculaire est moindre; viii) la VOP est augmentée; ix) le nombre de néphrons par rein est réduit; x) le poids est plus faible au cours de la croissance et un catch up est observé à l’âge adulte; la composition corporelle n’est pas différente entre les groupes; xi) la tolérance au glucose est diminuée. Conclusion Ces résultats supportent l’hypothèse d’une programmation développementale des maladies cardiovasculaires et métaboliques à l’âge adulte à la suite d’un stress hyperoxique néonatal. / Introduction Many studies showed that prematurity, which represents 8 % of birth, is associated with early indices of vascular dysfunction, increased blood pressure and Type 2 diabetes. Prematurity babies are more susceptible to oxidative injury, consequence of the immaturity of their antioxidant defences, and exposure to pro-oxidant situations (oxygen supplementation, infection). However, the long-term consequences of oxidative injury induced by oxygen exposure in the neonatal period are unknown. The aim of these PhD studies was to unravel some mechanisms that might underlie the damage induced by oxygen and the long-term risk of developing vascular and metabolic diseases in the overall concept of developmental programming of hypertension and metabolic syndrome-related diseases. Materials and methods Sprague-Dawley pups were kept with their mother in 80 % O2 (O2) or room air (RA) from day 3 to 10 of life. Cardiovascular parameters, tail blood pressure was measured between 4 and 15 weeks of life. In adulthood : vascular reactivity (ex vivo carotid rings) to angiotensine II (AngII) and carbachol with and without tempol was studied; studies of nitric oxide (NO) production with and without L-arginine and L-sépiaptérine (aorta, immunohistochemistry) and endothelial nitric oxide synthase expression (eNOS; aorta, immunohistochemistry, western blot) were performed; vascular oxidative stress (aorta, using chemiluminescence) by measuring superoxide anion production with and without inhibitors of nicotinamide-adenine-dinucleotide-phosphate (NADPH oxydase) and nitric oxyde synthase endotheliale (eNOS), apocynin and N-nitro-L-arginine methyl ester (L-NAME) respectively, and circulating oxidative stress by measuring the plasma levels of malondialdéhyde (MDA, HPLC) were evaluated; microvascular density was assessed on tibialis anterior muscle sections; pulse wave velocity (PWV) was measured by ultrasound, between aortic valve and ilio-femoral bifurcation; nephrons were counted after hydrochloric acid digestion. The main observations were also evaluated at 4 weeks of age. Metabolic parameters: body weight has been measured during the growth. In adulthood, body composition, glucose tolerance were evaluated. Results A 4 weeks of age, no difference was observed regarding blood pressure, vascular reactivity, and oxidative stress indices, but in rats O2 vs. RA (n = 6-8 /group), microvascular rarefaction and histological changes suggesting enhanced vascular stiffness were present. To adulthood, rats O2 vs. RA (n = 6-8/group) : i) systolic and diastolic blood pressures are increased; ii) vascular reactivity to Ang II is increased and to carbachol is decreased, these dysfunction were totally abolished by co-incubation of the vessel rings with tempol; iii) NO-production is decreased in basal condition and after carbachol stimulation, but is restored after pre-incubation of aorta sections with L- arginine and L-sépiaptérine; iv) eNOS expression is decreased by immunohistochemistry but increased by western blot; v) vascular superoxide anion levels are increased in basal condition, after AngII stimulation and this is mediated by NADPH oxydase and eNOS uncoupling; vi) the plasma levels of MDA are increased; vii) microvascular density is decreased; viii) PWV is increased; ix) nephron count per kidney is decreased; x) body weight is less during growth, but a catch up is observed in adulthood, body composition is similar; xi) the glucose tolerance is decreased in adults. Conclusion These results support the hypothesis of developmental programming of vascular and metabolic diseases in adulthood, after exposure to hyperoxic stress in the neonatal period. / Thèse réalisée dans le cadre d'une cotutelle entre l'Université de Montréal et l'Université d'Auvergne en France

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