La obesidad es uno de los problemas de salud más frecuentes en nuestro entorno y afecta a todos los segmentos de edad de nuestra población, con un incremento muy importante en las primeras décadas de vida. Así, durante los últimos años, la prevalencia de obesidad infanto-juvenil se ha incrementado hasta doblarse en algunos países desarrollados. La importancia de la obesidad radica en la comorbilidad asociada y en el riesgo cardiovascular añadido. En la actualidad se conoce poco sobre la epidemiología y la fisiopatología del síndrome metabólico en niños, debido a la falta de consenso en su definición y a la controversia de su existencia en la edad pediátrica. Considerando el aumento de la prevalencia de obesidad infantil y el hecho de que los niños están relativamente libres de comorbilidades y habitualmente ausentes de tratamientos farmacológicos, constituyen una población interesante para estudiar la secuencia de aparición de eventos asociados al aumento de peso y su fisiopatología. Particularmente, nos ha interesado la investigación de los posibles mediadores de la inflamación de bajo grado presente en la obesidad y su comportamiento después de tratar a los pacientes con dieta y cambios en el estilo de vida. Se ha escogido una población homogénea en cuanto a edad y estadío puberal para evitar factores de confusión. El proyecto de tesis consta de dos partes diferenciadas. La primera es el estudio transversal del grupo de niños obesos prepuberales en comparación con un grupo de niños sanos controles, apareados por edad y sexo donde se analiza la prevalencia de rasgos del síndrome metabólico según 5 definiciones y el impacto de la obesidad sobre estos rasgos. También se compara las características de la dieta y del estilo de vida entre ambos grupos. La segunda parte, es el estudio longitudinal que está centrado en evaluar las concentraciones plasmáticas de retinol binding protein 4 (RBP-4), lipocalina-2, (adipocyte fatty acid-binding protein) A-FABP y brain derived neurotrophic factor (BDNF) en situación basal y a los dos años de una intervención dietética y en el estilo de vida, en función de la pérdida de peso obtenida y su correlación con los rasgos del síndrome metabólico. Uno de los puntos fuertes de esta tesis es el planteamiento experimental preciso de un estudio longitudinal de dos años de duración. Otro punto fuerte del trabajo es el poder disponer de dos grupos de pacientes obesos, los que han perdido peso y los que no, con suficiente número de pacientes en cada grupo, para poderlos comparar. Los puntos débiles de la tesis, podrían ser el hecho de utilizar medidas indirectas tanto para clasificar la obesidad (IMC) como para valorar la resistencia a la insulina (índice HOMA-IR). Esta tesis tiene tanto aplicabilidad en el estudio de la fisiopatología de la obesidad infantil como en la práctica clínica. En la fisiopatología, ha permitido comprender mejor la relación entre las lipocalinas y los parámetros de obesidad en niños prepuberales. En la práctica clínica, esta tesis corrobora que hay que insistir en las recomendaciones aceptadas para la ingesta de macronutrientes en niños prepuberales y que el estilo de vida es importante para la evolución de la obesidad. Esta tesis permite abrir nuevas líneas de investigación dentro del campo de la obesidad infantil. Habría que repetir el estudio en diferentes etnias para poder generalizar los resultados obtenidos. También se podría realizar el análisis de las lipocalines y el BDNF en los diferentes puntos del TTOG para permitir estudiar la regulación de estas proteínas en relación a los picos de glucemia e insulinemia. También sería necesario un mejor conocimiento de la biología del tejido adiposo y de la acción de estas lipocalines para poder generar dianas terapéuticas más específicas para combatir la obesidad en fases tempranas. / Obesity is one of the most common health problems in our environment and affects all segments of our population ages, with a very important increase in the first decades of life. Thus, in recent years, the prevalence of infant-juvenile obesity has doubled in some developed countries. The importance of obesity lies in comorbidity and associated cardiovascular risk added. At present little is known about the epidemiology and pathophysiology of metabolic syndrome in children due to lack of consensus on its definition and the controversy of its existence in childhood. Considering the increasing prevalence of childhood obesity and the fact that children are relatively free of comorbidity and usually absent from drug treatments, they are an interesting population to study the sequence of occurrence of events associated with increase in weight and its pathophysiology. Particularly we are interested in investigating potential mediators of low grade inflammation present in obesity and its behavior after treating patients with diet and lifestyle changes. We have chosen a homogeneous population in terms of age and pubertal stage to avoid confounding factors. The thesis project consists of two distinct parts. The first is the cross-sectional study group of obese prepubertal children compared with a group of young healthy controls matched for age and gender, in which we analyze the prevalence of metabolic syndrome according to 5 definitions and the impact of obesity on these traits. It also compares the characteristics of diet and lifestyle differences between groups. The second part is the longitudinal study that focuses on evaluating the plasma concentrations of retinol binding protein 4 (RBP-4), lipocalina-2, adipocyte fatty acid¬binding protein (A-FABP) and BDNF on baseline and after two years of a dietary intervention and lifestyle depending on the weight loss and its correlation with the features of the metabolic syndrome. One of the strengths of this thesis is the precise experimental approach of a longitudinal study lasting two years. Another strong point of the work is to have two groups of obese patients, those who have lost weight and those not, with sufficient numbers of patients in each group in order to compare them. Also the fact that the group of obese patients have been compared with a group of healthy patients of similar age and sex, is a strong point of the work. The limitations of the thesis are the use of indirect measures for classifying obesity (BMI) and to assess insulin resistance by indirect index (HOMA IR). This thesis has applicability in both the study of the pathophysiology of childhood obesity and in clinical practice. In the pathophysiology has allowed a better understanding of the relationship between lipocalins and parameters of obesity in prepubertal children. In clinical practice, this thesis confirms that we must insist on the recommendations accepted by the macronutrient intake in prepubertal children and that lifestyle is important for the evolution of obesity. This thesis can open new lines of research within the field of childhood obesity. Repeating the study in different ethnic groups in order to generalize the results should be made. One could also perform the analysis of lipocalines and BDNF in the various points to allow TTOG study the regulation of these proteins in relation to blood glucose and insulin peaks. It also would require a better understanding of the biology of adipose tissue and action of these lipocalines to generate more specific therapeutic targets to combat obesity in early stages.

Basic research into the histology, endocrine control, and local regulation of prepuberal ruminant mammogenesis was conducted to provide a better understanding of this important developmental period. Histologic features of prepuberal ruminant mammary parenchymal morphogenesis were examined in tissue samples taken from ewe lambs at 2 (n = 5), 3 (n = 15), 6 (n = 26), 9 (n = 7), 12 (n = 5), and 13 wk (n = 20), and from Holstein heifers at 4 (n = 1) and 6 mo (n = 2). Examination of approximately 8000 histologic sections revealed that mammary parenchymal morphogenesis in sheep and cattle occurs through the proliferation of highly arborescent ductal structures embedded in a dense stroma. These observations contrast strongly with models of mammogenesis based on murine mammary development. The formation of luminal spaces and the expansion of ducts also differed from murine mammogenesis models. Luminal spaces were shown to develop through a progressive separation of opposing sides in initially solid ductal structures. Likewise, our investigation of prepubertal ovine mammogenesis revealed that parenchymal weight, 3H-thymidine labeling, stromal weight, and parenchymal DNA were all unaffected by ovariectomy (P > 0.05), in marked contrast to the dramatic reduction in mammary development following ovariectomy in rats, mice, and heifers. Responsiveness to exogenous estrogen (0.1 mg/kg) was demonstrated by increased 3H-thymidine labeling (P < 0.05) in both intact and ovariectomized lambs. Three dimensional collagen gel cultures of bovine mammary organoids from the peripheral (OUTER) and medial (INNER) parenchymal zones were used to characterize the proliferative and morphogenetic responses to local-acting growth factors. The proliferation of OUTER cells was 2 to 3 times greater than INNER cells (P < 0.0001) in response to IGF-I stimulation. Dramatic differences in the morphology of INNER and OUTER organoids were also observed. INNER cells grew into smooth-edged colonies when treated with heifer serum but stellate colonies when treated with other mitogens. OUTER cells grew into stellate colonies regardless of mitogen treatment. These investigations highlight the fact that a great deal more research into the basic physiology of prepuberal ruminant mammogenesis is required and that dogma developed in murine model systems may not be applicable to ruminant mammary physiology. / Ph. D.