Proyectos

The quality of fetal and early post-natal environment influences lifelong health and predicts the risk for a range of non-communicable chronic diseases (NCDs). These observations form the basis of the "Developmental Origins of Health and Disease" (DOHaD hypothesis), which indicates that the intrauterine signals that compromise fetal growth also act to "program" tissue differentiation in a manner that predisposes later illnesses. Interestingly, the DOHaD hypothesis asserts that some aging-associated diseases that occur in adults are closely related to the development and conditions in the intrauterine environment. Thus, aging and aging-associated diseases can be viewed, at least in part, as the result of a developmental program activated early in embryogenesis and persists throughout the organism's lifespan. On the other hand, one of the main consequences of this programming is Fetal Growth Restriction (FGR) and which remains a leading cause of perinatal morbidity and mortality, affecting about 10% of pregnancies, but the incidence is reportedly sixfold greater in low-income countries depending on the region surveyed's nutrition and health access availability. FGR is clinically defined as a fetal weight below the 10th percentile of normal for gestational age, associated with some loss of fetal-placental blood flow diagnosed by ultrasound, and it is a condition in which the potential growth of the fetus is negatively influenced by environmental and maternal factor; the short-term consequences of FGR are low birth weight (LBW) and the corresponding phenotype, which is associated with increased perinatal morbidity and mortality. Besides, the long-term effects include a 2 to 3-fold increase in the risk of developing cerebrovascular disease in adulthood. Indeed, many neurodevelopmental dysfunctions originated in the antenatal period, but few studies have focused on how growth restriction interferes with normal brain development of the blood-brain barrier (BBB) in the FGR neonate. The BBB is a cellular network formed by a monolayer of neuro-endothelial and mural cells. The BBB regulates the transport of molecules into and out of the central nervous system (CNS) (selective permeability and integrity of the BBB). In cerebrovascular aging, BBB breakdown and dysfunction lead to leakages of components into the central nervous system (CNS), contributing to neurological deficits; growing evidence from genomic data shows that FGR vascular dysfunction is mediated by aging, with a series of prominent hallmarks, including genetic and epigenetic alterations. These aging-associated epigenetic changes include DNA methylation, histone modification, chromatin remodeling, and non-coding RNA (ncRNA) regulation; however, how this mechanism regulates the aging process and contributes to aging-related BBB dysfunction remains elusive. We hypothesize that the impaired fetal growth conditions associated with epigenetic programming of aging-related DNA methylation, chromatin remodeling, and miRNA-omic profile of complex junctional genes in the neuro-endothelium, which can alter BBB integrity and permeability, increasing cerebral damage which impacts the perinatal and adulthood neurocognitive function. This hypothesis will be addressed by the study of the effects of gestational chronic hypoxia on the aging epigenetic programming of gene expression of junctional complexes: Tight junction, adherens junction, and Gap junction family’s molecules, as important regulators of the permeability para and transcellular of the BBB. For this, we will use a well-established Guinea pig model of cerebrovascular programming (DOHaD model) to demonstrate DNA methylation shift, chromatin remodeling, miRNA-omic profile, and transcriptomic analyses in neuro-endothelial cells isolated from cortex and hippocampus from animals gestated under hypobaric hypoxia at two stages of life (juvenile period and adulthood). The methodology for this project is an in vivo assess locomotor, exploratory activity, and memory acquisition evaluation, and in vitro determinations of epigenetic regulation of aging in BBB from the cortex, hypothalamus, and neuro- endothelial cell culture primary at different stages of life in animals gestated under hypoxia. Our expected outcome is to improve the knowledge about neuro-endothelial epigenetic programming by aging induced by the FGR and enhance the characterization of those epigenomic patterns and mechanisms associated with BBB breakdown by intrauterine hypoxia. This project aims to demonstrate that the effect of gestational hypoxia can accelerate the permeability of the BBB by epigenetic mechanisms not yet studied and that these changes continue throughout life, producing further deterioration of brain function

Non-communicable diseases (NCDs) are responsible for 74% of worldwide human deaths, with cardiovascular causes in the first place (1). NCDs are determined by a combination of environmental, genetic and epigenetic factors. In fact, adverse intrauterine conditions, such as reduced oxygen availability (hypoxia) and oxidative stress, can increase the risk of developing diseases during life, a phenomenon known as Fetal Programming or Developmental Origins of Health and Disease (DOHaD). Intrauterine hypoxia (IUH) affects most of the pregnancies in high altitudes populations (> 2500m) (2-4) and 3-4% in lowlands, with uteroplacental and developmental complications (4,5). We, and a couple of others, have recently shown that IUH determines cardiovascular oxidative stress during lifespan affecting endothelial function and vasodilator capacity, similar to what is seen with aging. The hypoxia-induced responses during development are responsible for fetal survival, but also determine mechanisms that program postnatal cardiovascular function that may increase cardiovascular health risks and accelerate aging (6). This proposal aims to determine the mechanisms and trace the origins and outcomes of cardiovascular dysfunction resulting from intrauterine hypoxia and oxidative stress, and further identify the interrelated senescence mechanisms in the heart and blood vessels. To assess the aforementioned, we will study the effects of IUH on cardiovascular aging along lifespan, as important regulators of the function, structure and biomechanical properties of the cardiovascular system.

The brain is an energy intensive organ that requires a robust supply of nutrients and oxygen. The vasculature irrigating the brain is a huge and complex network of blood vessels fulfilling this requirement, while also protecting the neural tissue from blood-borne toxic substances. This regulated nutrient supply is accomplished by the formation of a highly selective molecular barrier, termed the blood-brain barrier (BBB). Dysfunction of the BBB or malformations of the vascular network are associated with pathological conditions that impair brain function, and can lead to death. Thus, appropriate morphogenesis and establishment of the brain vasculature is necessary for a healthy life. The brain vasculature forms during intrauterine development, matching brain growth in this same period. Anatomically, blood vessels grow first surrounding the brain primordium and then penetrate the parenchyma until they vascularize the periventricular zone. The molecular regulation of this patterned growth is not completely understood. Several signaling pathways are known to be involved in brain angiogenesis, including WNT, TGF-β, Hh, and NOTCH, which differentially regulate vascular growth. Recently, cholesterol has been shown to modulate angiogenic growth in other vascular beds by regulating the activity of the NOTCH pathway, suggesting that cholesterol levels could influence developmental angiogenesis in the brain. Interestingly, cholesterol is also required for signal transduction of the Hh pathway. In preliminary in vitro experiments, we have observed that brain endothelial cells activate an angiogenic program after cholesterol depletion. Here, we will extend those studies to in vivo models to determine the role of cholesterol in developmental brain angiogenesis. We propose that an increase in vascular cell cholesterol activates NOTCH and attenuates Hh signaling pathways, restricting sprouting angiogenesis and blood-brain barrier formation in mouse embryo brain vasculature. To test this hypothesis, we will study mouse embryos with altered cholesterol levels by dietary, pharmacological, and genetic manipulations. We expect these manipulations to induce a reduction or an increase in cholesterol levels in the brain vasculature during embryonic development, which we will evaluate by measuring cholesterol content in isolated vascular fragments. In all these models, we will (Specific aim 1) study vascularization in the brain during intrauterine development using immunofluorescence with specific antibodies against endothelium proteins. In addition, we will measure the levels of transcript and proteins of general key regulators of angiogenesis in isolated vascular fragments, using qPCR and Western blot. We will (Specific aim 2) also evaluate the state of the BBB in the brain vasculature of these models at a fetal stage when the barrier is already formed and functional. For this, we will use immunofluorescence to detect the presence of marker proteins of the BBB in vascular fragments, and we will measure their levels by Western blot. Further, we will test the functionality of the barrier by injecting a fluorescent tracer and evaluating its extravasation in the brain. Finally, we will (Specific aim 3) determine the activation of the NOTCH and Hh pathways in the brain vasculature of the models at the stage of maximal angiogenesis. We will use qPCR and Western blot to measure the levels of marker genes and proteins for these two pathways in vascular fragments, and Proximity Ligation In Situ Hybridization in tissue sections to evaluate the transcript levels of those markers in situ. We expect that the different models of dietary, pharmacological, and genetic interventions will increase or reduce cholesterol levels in the brain vasculature. These changes are expected to correlate with opposing effects on angiogenesis in the brain during development (i.e. low cholesterol will increase angiogenesis, while high cholesterol will inhibit it). In the same way, we expect that distinct cholesterol levels will have opposing effects on the integrity of the BBB. These changes in angiogenesis and BBB function are expected to be associated with concomitant disruption of the NOTCH and Hh pathways. In summary, in this proposal we aim to cover a knowledge gap regarding the role of cholesterol in the regulation of developmental angiogenesis in the brain. These experiments may uncover new mechanisms driving vascular growth and barrier establishment in the brain, which could lead to new strategies for the prevention and treatment of pathologies involving the brain vasculature.

The proposal focuses on understanding the neuro-vascular aging mechanisms associated with alterations in fetal growth by intrauterine hypoxia using molecular biology and physiology as an area. The aim of the study is to demonstrate that impaired fetal growth conditions are associated with epigenetic programming of aging-related DNA methylation, chromatin remodeling, and miRNA-omic profile of junctional complex genes in the neuroendothelium, which can alter BBB integrity and permeability, increasing cerebral damage which impacts the juvenile and adulthood neurocognitive function.

Analizar los procesos de enseñanza, integración curricular, evaluación y acreditación en carreras del área de salud en grado y posgrado y su relación con la trayectoria académica de los estudiantes y la actividad docente en Argentina y Chile.

Frailty is increasingly becoming an important public health challenge worldwide because it is associated with older age, and with adverse outcomes such as reduced quality of life, increased mortality rates, hospitalizations, falls, depression, and dementia. Frailty is defined as dynamic state affecting an individual who experiences losses in one or more domains of human functioning (physical, psychological, social) that are caused by the influence of a range of variables, and which increases the risk of adverse outcomes. This more integral conceptual definition promotes the collaboration of scientists, social and behavioral professionals as well as clinicians from diverse specialties. In this proposal an interdisciplinary group (Biochemistry, Geriatric, Occupational Therapist, Kinesiologist, social worker, bioengineer, statistician among others) aims to evaluate frailty in Chile with a biopsychosocial approach with the final purpose to identify and manage frailty while taking into consideration all the dimensions. Additionally, we aim to design a multidomain personalized person-base intervention for a healthy aging that can uncover a circulating microRNA biomarker panel that can allow an early-detection of frailty, leading to a new multidimensional geriatric assessment. We propose the following hypothesis: A personalized multidimensional training program reduces the frailty prevalence, increasing adherence and participation in the program among community-living older adults. This intervention will be paralleled by a distinctive miRNA profile reflecting the multiple domains of frailty, as well as improvements in diverse psychosocial traits.

Durante las últimas décadas, la expectativa de vida promedio ha aumentado dramáticamente, mientras que la salud no ha aumentado proporcionalmente. Para Chile se espera que para el 2050 el número de adultos mayores superará el 30% de la población con un incremento del 109% respecto al 2015, superando el 75% proyectado para la población mundial. Entender el proceso de envejecimeinto y encontrar marcadores biológicos con capacidad diagnóstica y pronóstica, permitirá promover estrategias para aumentar el número de años de vida saludable, disminuyendo los gastos en salud asociado al envejecimiento. Varios estudios han permitido avanzar y entender los mecanismos moleculares de pérdidad de función muscular durante el evenjecimiento, más conocida como sarcopenia. Sin embargo, la complejidad del proceso y el insuficiente conocimiento de los mecanismos subjacentes dificultan el diseño de estrategias terapéuticas eficaces. Hasta el momento, la actividad física y el ejercicio siguen siendo la estrategia más eficaz para previnir y tratar la sarcopenia. Por lo cual, identificar nuevos biomarcadores musculares en combinación con marcadores clínicos bien establecidos de parámetros físicos y funcionales, fortalecería la actual evaluación geriátrica, al utilizar un enfoque interdiciplinario. Los microRNA (miRNA, 18–25 nt de largo) han ganado interés debido a que son moléculas altamente conservadas en todas las especies, y actúan como reguladores positivos y/o negativos de la expresión génica. Entender la relación reguladora directa entre los miARN y el ARN mensajero (mRNA) desempeña en el músculo esquelética es fundamentaltanto para el proceso de transcripción del ARNm y traducción de proteínas como para entender el deterioro de la función muscular. Sin embargo, la regulación de la red post-transcripcional miARN-ARNm y los mecanismos genéticos involucrados en el proceso de envejecimiento del músculo esquelético humano están lejos de ser elucidados. En el presente estudio, nuestro objetivo es explorar e integrar coperfiles emparejados de miARN y ARNm durante la pérdida de función muscular producidad durante el envejecimeinto en personas mayores. Este análisis integral permitirá la identificación de nuevos blancos de miARN y estrategias reguladoras que controlan la expresión génica en la pérdidad de función del músculo esquelético y cómo éstas son modificadas por el entrenamiento de fuerza. Se hipotetiza que existen redes de regulación post-transcripcional miRNA/mRNA que se expresan de manera diferencial en el músculo esquelético de personas jóvenes y personas mayores, que estarían asociadas con cambios en la función muscular durante el envejecimiento. Además, el entrenamiento de fuerza modifica estas redes de regulación, lo que podría contribuir a la mejora de la función muscular y la prevención o atenuación de la sarcopenia. Objetivos generales - Identificar las redes de regulación post-transcripcional miRNA/mRNA involucradas en la pérdida de función musclular en personas mayores que son moduladas por un entrenamiento de fuerza, para proponer nuevos biomarcadores de función muscular durante el envejecimeinto que se correlacionan con parámetros clínicos/funcionales. Objetivos específicos 1. Determinar las redes de regulación post-transcripcional miRNA/mRNA que se expresan diferencialmente en el músculo esquelético de personas jóvenes y personas mayores. 2. Identificar las redes de regulación post-transcripcional miRNA/mRNA que se modifican después de 12 semanas de entrenamiento de fuerza en personas mayores 3. Correlacionar las redes miRNA/mRNA identificadas con parámetros bioquímicos, físicos y funcionales de función muscular, antes y después de 12 semanas de entrenamiento de fuerza en personas mayores Este proyecto busca Identificar nuevos biomarcadores de función muscular durante el envejecimiento, podrían ser utilizados en futuros estudios y en la práctica clínica para evaluar la salud muscular en personas mayores. Además, de contribuir a una mejor comprensión de los mecanismos moleculares que subyacen a la pérdida de masa muscular en el envejecimiento y cómo la intervención del entrenamiento de fuerza puede modificar esos mecanismos. Finalmente, el uso de tecnologías avanzadas de secuenciación (mARN y miARN) y bioinformática permitirá impulsar el desarrollo y la mejora de técnicas de secuenciación y análisis de datos en el campo de la genómica y la transcriptómica en el área del envejecimiento, como también, promover la colaboración a nivel nacional e internacional a través de las bases de datos y recursos bioinformáticos generados que estarán a disposición de investigadores del área.

Frailty is increasingly becoming an important public health challenge worldwide because it is associated with older age, and with adverse outcomes such as reduced quality of life, increased mortality rates, hospitalizations, falls, depression, and dementia. Frailty is defined as dynamic state affecting an individual who experiences losses in one or more domains of human functioning (physical, psychological, social) that are caused by the influence of a range of variables, and which increases the risk of adverse outcomes. This more integral conceptual definition promotes the collaboration of scientists, social and behavioral professionals as well as clinicians from diverse specialties. In this proposal an interdisciplinary group (Biochemistry, Geriatric, Occupational Therapist, Kinesiologist, social worker, bioengineer, statistician among others) aims to evaluate frailty in Chile with a biopsychosocial approach with the final purpose to identify and manage frailty while taking into consideration all the dimensions. Additionally, we aim to design a multidomain personalized person-base intervention for a healthy aging that can uncover a circulating microRNA biomarker panel that can allow an early-detection of frailty, leading to a new multidimensional geriatric assessment. We propose the following hypothesis: A personalized multidimensional training program reduces the frailty prevalence, increasing adherence and participation in the program among community-living older adults. This intervention will be paralleled by a distinctive miRNA profile reflecting the multiple domains of frailty, as well as improvements in diverse psychosocial traits.

Analizar los procesos de enseñanza, integración curricular, evaluación y acreditación en carreras del área de salud en grado y posgrado y su relación con la trayectoria académica de los estudiantes y la actividad docente en Argentina y Chile.

Adipose tissue expansion associated with obesity drives to a chronic low-grade inflammation state that alters metabolically relevant tissues, predisposing to developing metabolic diseases. This sustained inflammation causes alteration in SkM glucose uptake, impairs protein metabolism and causes mitochondrial dysfunction. Skeletal muscle (SkM) is a relevant organ responsible for whole-body metabolism and homeostasis. It has been recently proposed that obesity causes a phenomenon known as "anabolic resistance," where Skm cannot respond to anabolic stimuli induced by a nutritional challenge and muscle contraction and where inflammation could be a relevant mediator. The inflammatory response is regulated by the priming and activation of the NLRP3 inflammasome. However, the response to the NLRP3 inflammasome in human skeletal muscle (Hskm) has not been described, nor their possible relation with protein metabolism. Nowadays, the ketone body β-hydroxybutyrate (βHB) produced on liver mitochondria under ketogenic diets, caloric restriction or ingested exogenously as a supplement, has emerged as a possible muscle-sparing molecule. Still, the mechanism by which βHB could benefit protein metabolism or mitochondrial function is not understood. Interestingly, it has been shown that βHB by itself is a selective inhibitor of the NLRP3 inflammasome on human monocytes, suggesting an important role in controlling the inflammation response. Nevertheless, the anti-inflammatory role of βHB has not been tested on Hskm, neither the modulation of NLRP3 inflammasome nor their possible role in restoring anabolic resistance. To evaluate the effects of β- hydroxybutyrate supplementation on the anabolic response, NRLP3 inflammasome activation and mitochondrial dysfunction in skeletal muscle from obese individuals. Aim 1.- To determine the acute response of βHB supplementation on skeletal muscle inflammation, protein synthesis, and mitochondrial function in obese individuals. 15 lean and 15 obese men and women will be submitted to an acute protein intake (Whey protein 25g) with or without the previous ingestion of a Ketone Monoester (470 mg/kg)(Kme, ΔG®) in a cross-over protocol. Basal and postprandial (MPS) will be assessed in Vastus Lateralis (VL) muscle biopsy obtained at basal (0), 2, and after 4 hours of each condition respectively and determined afterward by LC-MS/MS. Plasma inflammation markers will be determined by ELISA while protein levels of mTOR signaling (pmTOR, 4EB-BP1, p70S6K and NLRP3 components) by immunoblot. Also, we will determine Hskm mitochondrial oxygen consumption and hydrogen peroxide (H2O2) as markers of mitochondrial function. Expected result: We expect that βHB supplementation causes a sensitizing effect on protein synthesis on obese Skm samples, a decrease in plasma and Skm inflammation markers, decrease or inactivation of the NLRP3 inflammasome along with restoration of mitochondrial function. Aim 2.- To analyze the chronic effects of βHB intake on the anabolic resistance response and its association with NRLP3 inactivation and skeletal muscle function in obese individuals. To determine the chronic effects of βHB supplementation anabolic resistance, individuals will be submitted to a three times per day βHB intake (357mg/kg)(Kme, ΔG®) or placebo for 2-weeks. Continuous Glucose Monitors will be placed on each participant's arm to determine day-to-day glucose control and three times a day determination of capillary βHB. At the end of the second week, obese individuals will be submitted to an acute protein intake and VL muscle biopsy at basal (0), 2, and after 4 hours of each condition to evaluate MPS. Physical fitness will be determined by total strength, power and maximal oxygen consumption. Skm and plasma inflammation will be determined by immunoblot (mTOR, pmTOR, 4EB-BP1, p70S6K, NLRP3, Casp-1, IL-1β, IL-18) and ELISA, respectively. In contrast, Skm mitochondrial oxygen consumption and H2O2 will be assessed to determine mitochondrial function. Expected result: we expected that obese individuals that are supplemented chronically with βHB will have an increased response to a whey protein leucine-enriched challenge reflected on the increase of MPS related to NLRP3 attenuation mitochondrial function restoration. Aim 3.- To determine the connection between the NLRP3 inactivation, mitochondrial function, and the attenuation of skeletal muscle anabolic resistance induced by βHB treatment in vitro. Primary human skeletal muscle (Hskm) from obese and lean donors obtained in biopsies from aim 1 will be stimulated with ATP (5mM), LPS (1μg/ml), ATP+LPS within the presence or absence of different concentration of βHB (0,5-5mM) and submitted to a leucine challenge (1.5mM), oxygen consumption and H2O2 determination. To test cause-effect Hskm will be transfected with a control or an NLRP3-pooled specific siRNA and submitted to leucine challenge. Here, p70S6K, p70S6K p-4EBP1, 4EBP1, mTOR, p-mTOR, NLRP3, Casp-1, IL-1β, IL-18 and Puromycin incorporation will be measured by immunoblot to test anabolic resistance. Expected result: We expected that βHB decreases NLRP3 inflammasome while increasing protein synthesis on Hskm of obese individuals, associated with Hskm myotube diameter. Thus, we propose a novel exogenous βHB intake approach to improve muscle function in obese populations by reducing systemic and Hskm inflammation, restoring skeletal muscle protein metabolism mediated by NLRP3 attenuation and improving mitochondrial function