Integrating quantitative proteomics and metabolomics in a cellular model of diabetic retinopathy

Curs 2013-2014Diabetic retinopathy is the leading cause of visual loss in individuals under the age of 55. Most investigations into the pathogenesis of diabetic retinopathy have been concentrated on the neural retina since this is where clinical lesions are manifested. Recently, however, various abnormalities in the structural and secretory functions of retinal pigment epithelium that are essential for neuroretina survival, have been found in diabetic retinopathy. In this context, here we study the effect of hyperglycemic and hypoxic conditions on the metabolism of a human retinal pigment epithelial cell line (ARPE-19) by integrating quantitative proteomics using tandem mass tagging (TMT), untargeted metabolomics using MS and NMR, and 13C-glucose isotopic labeling for metabolic tracking. We observed a remarkable metabolic diversification under our simulated in vitro hyperglycemic conditions of diabetes, characterized increased flux through polyol pathways and inhibition of the Krebs cycle and oxidative phosphorylation. Importantly, under low oxygen supply RPE cells seem to consume rapidly glycogen storages and stimulate anaerobic glycolysis. Our results therefore pave the way to future scenarios involving new therapeutic strategies addressed to modulating RPE metabolic impairment, with the aim of regulating structural and secretory alterations of RPE. Finally, this study shows the importance of tackling biomedical problems by integrating metabolomic and proteomics results.Director/a: Oscar Yanes Torrado, Jordi Planas Cuch

From spectrometric data to metabolic networks: an integrated view of cell metabolism

La biologia molecular ha avançat considerablement gràcies a importants progressos com la seqüenciació del ADN o la seva modificació per CRISPR. Tot i això, per entendre el metabolisme requerim estudiar els perfils metabòlics i les seves reaccions metabòliques. L™objectiu d™aquesta tesi és contribuir en aquest estudi del metabolism, el qual unifica dels camps de la proteòmica i la metabolòmica. Tradicionalment, l™anàlisi de dades òmiques es basa en el tractament independent de les diferents variables encara que està profundament establert que els mecanismes moleculars són controlats per la interacció de diferents molècules, i per tant seria més correcte tractar les dades de la mateixa manera. Avui dia, s™han descrit una gran quantitat de vies metabòliques, incluint els enzims responsables de les transformacions dels metabòlits que les formen, aquesta informació s™ha recopilat en bases de dades, que a la vegada poden ser utilitzades per a construir xarxes metabòliques. En aquesta tesi, s™han utilitzat xarxes metabòliques per a desenvolupar un algoritme que prediu metabòlits desregulats basant-se en el perfil d™expressió d™enzims gràcies a proteòmica quantitativa. Per a validar tals prediccions, és possible mesurar l™abundància d™aquests metabòlits, o el seu flux, o sigui la velocitat a la que s™han transformat, utilitzant experiments de marcatge amb isòtops estables, mesures completades mitjançant metabolòmica. Aqui, mostrem els productes del desenvolupament de dos mètodes per a l™anàlisi de dades de metabolòmica per a experiments amb isòtops estables: el primer per a la quantificació dirigida del flux en metabòlits del metabolisme central; i un segon, per la detecció no-dirigida de metabòlits marcats amb isòtops en altres vies metabòliques. Aquests mètodes han sigut provats en diferents estudis on han aportat resultats remarcables, revelant nous mecanismes moleculars en una complicació de la diabetes o en relació al metabolisme del càncer.La biología molecular ha avanzado considerablemente gracias a progresos como la secuenciación de ADN o su modificación por CRISPR. Sin embargo, para entender el metabolismo es indispensable estudiar los perfiles metabólicos y sus reacciones metabólicas. El objetivo de esta tesis es contribuir en el estudio del metabolismo, el cual implica los campos de la proteómica y la metabolómica. Tradicionalmente, el análisis de datos ómicas se basa en el tratamiento independiente de las diferentes variables aunque está profundamente aceptado que los mecanismos moleculares son controlados por la interacción de diferentes moléculas, y por lo tanto sería más correcto tratar los datos de esa manera. Hoy día, se han descrito una gran cantidad de vías metabólicas, incluyendo las enzimas responsables de las transformaciones de los metabolitos que las forman, esta información se ha recopilado en bases de datos, que a su vez pueden ser utilizadas para construir redes metabólicas . En esta tesis, se han utilizado redes metabólicas para desarrollar un algoritmo que predice metabolitos desregulados basándose en el perfil de expresión de enzimas por proteómica cuantitativa. Para validar tales predicciones, es posible medir la abundancia de estos metabolitos, o su flujo, o sea la velocidad a la que se han transformado, utilizando experimentos de marcado con isótopos estables, estas medidas se obtienen por metabolómica. Aquí, mostramos los productos del desarrollo de dos métodos para el análisis de datos de metabolómica para experimentos con isótopos estables: el primero para la cuantificación dirigida del flujo en metabolitos del metabolismo central; y un segundo, para la detección no-dirigida de metabolitos marcados con isótopos en otras vías metabólicas. Estos métodos han sido probados en diferentes estudios donde han aportado resultados interesantes, revelando nuevos mecanismos moleculares en una complicación de la diabetes o en relación al metabolismo del cáncer.Understanding the molecular basis of life has been in the spotlight of biochemistry research for more than a century already. Molecular biology has taken medicine forward thanks to technological breakthroughs like DNA sequencing and CRISPR editing. However, in order to understand metabolism we must rely on the study of metabolite profiles and metabolic reactions. The purpose of this thesis to contribute to this area, which unites the fields of proteomics and metabolomics. Traditionally, omics data analysis treats variables independently even if it is strongly settled that molecular mechanisms involve the interaction of diverse pathways, therefore data should be analyzed correspondingly. A vast amount of metabolic pathways have been described, together with enzymes that are responsible for metabolite transformations, this information has been assembled in databases that, in turn, can be used to build metabolic networks. In here, we use metabolic networks to predict metabolite dysregulation based on quantitative proteomics profiles. To validate the predictions, it is possible to measure the abundance of metabolites or their flux, namely the rate at which they are transformed, using stable isotope labelling experiments, both measurements can be performed by metabolomics. In this thesis, two different metabolomics-based stable isotope labelling approaches have been developed, one for the study of central carbon metabolites and one for the unbiased detection of deregulated fluxes in other metabolic pathways. These approaches have been tested on different datasets and have proven valuable to obtain remarkable results, unraveling molecular mechanisms in diabetes complications or novel metabolic hallmarks of cancer

Study of the period function of a two-parameter family of centers

In this paper we study the period function of ẍ = (1 x) p − (1 x) q , with p, q ∈ R and p > q. We prove three independent results. The first one establishes some regions in the parameter space where the corresponding center has a monotonous period function. This result extends the previous ones by Miyamoto and Yagasaki for the case q = 1. The second one deals with the bifurcation of critical periodic orbits from the center. The third one is addressed to the critical periodic orbits that bifurcate from the period annulus of each one of the three isochronous centers in the family when perturbed by means of a one-parameter deformation. These three results, together with the ones that we obtained previously on the issue, leads us to propose a conjectural bifurcation diagram for the global behaviour of the period function of the family

The criticality of centers of potential systems at the outer boundary

The number of critical periodic orbits that bifurcate from the outer boundary of a potential center is studied. We call this number the criticality at the outer boundary. Our main results provide sufficient conditions in order to ensure that this number is exactly 0 and 1. We apply them to study the bifurcation diagram of the period function of X = −y∂ x ((x 1) p − (x 1) q )∂ y with q < p. This family was previously studied for q = 1 by Y. Miyamoto and K. Yagasaki

Analytic tools to bound the criticality at the outer boundary of the period annulus

In this paper we consider planar potential differential systems and we study the bifurcation of critical periodic orbits from the outer boundary of the period annulus of a center. In the literature the usual approach to tackle this problem is to obtain a uniform asymptotic expansion of the period function near the outer boundary. The novelty in the present paper is that we directly embed the derivative of the period function into a collection of functions that form a Chebyshev system near the outer boundary. We obtain in this way explicit sufficient conditions in order that at most n 0 critical periodic orbits bifurcate from the outer boundary. These theoretical results are then applied to study the bifurcation diagram of the period function of the family ẍ= xp − xq , p, q ∈ R with p > q

Enhancing Localized Pesticide Action through Plant Foliage by Silver-Cellulose Hybrid Patches

Efficacy and efficiency of pesticide application in the field through the foliage still face many challenges. There exists a mismatch between the hydrophobic character of the leaf and the active molecule, low dispersion of the pesticides on the leaves' surface, runoff loss, and rolling down of the active molecules to the field, decreasing their efficacy and increasing their accumulation to the soil. We produced bacterial cellulose-silver nanoparticles (BC-AgNPs) hybrid patches by in situ thermal reduction under microwave irradiation in a scalable manner and obtaining AgNPs strongly anchored to the BC. Those hybrids increase the interaction of the pesticide (AgNPs) with the foliage and avoids runoff loss and rolling down of the nanoparticles. The positive antibacterial and antifungal properties were assessed in vitro against the bacteria Escherichia coli and two agro-economically relevant pathogens: the bacterium Pseudomonas syringae and the fungus Botrytis cinerea. We showed in vivo inhibition of the infection in Nicotiana benthamiana and tomato leaves, as proven by the suppression of the expression of defense molecular markers and reactive oxygen species production. The hydrogel-like character of the bacterial cellulose matrix increases the adherence to the foliage of the patches

Redundant roles of the phosphatidate phosphatase family in triacylglycerol synthesis in human adipocytes.

AIMS/HYPOTHESIS: In mammals, the evolutionary conserved family of Mg(2+)-dependent phosphatidate phosphatases (PAP1), involved in phospholipid and triacylglycerol synthesis, consists of lipin-1, lipin-2 and lipin-3. While mutations in the murine Lpin1 gene cause lipodystrophy and its knockdown in mouse 3T3-L1 cells impairs adipogenesis, deleterious mutations of human LPIN1 do not affect adipose tissue distribution. However, reduced LPIN1 and PAP1 activity has been described in participants with type 2 diabetes. We aimed to characterise the roles of all lipin family members in human adipose tissue and adipogenesis. METHODS: The expression of the lipin family was analysed in adipose tissue in a cross-sectional study. Moreover, the effects of lipin small interfering RNA (siRNA)-mediated depletion on in vitro human adipogenesis were assessed. RESULTS: Adipose tissue gene expression of the lipin family is altered in type 2 diabetes. Depletion of every lipin family member in a human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocyte cell line, alters expression levels of adipogenic transcription factors and lipid biosynthesis genes in early stages of differentiation. Lipin-1 knockdown alone causes a 95% depletion of PAP1 activity. Despite the reduced PAP1 activity and alterations in early adipogenesis, lipin-silenced cells differentiate and accumulate neutral lipids. Even combinatorial knockdown of lipins shows mild effects on triacylglycerol accumulation in mature adipocytes. CONCLUSIONS/INTERPRETATION: Overall, our data support the hypothesis of alternative pathways for triacylglycerol synthesis in human adipocytes under conditions of repressed lipin expression. We propose that induction of alternative lipid phosphate phosphatases, along with the inhibition of lipid hydrolysis, contributes to the maintenance of triacylglycerol content to near normal levels.This study was supported by research grants from the ‘Instituto de Salud Carlos III’ (ISCIII, Spanish Ministry of Economy and Competitiveness) (PI10/00967 and CP11/0 0021 to MM); the R. Barri Private Foundation (PV12142S to MM); the Medical Research Council (G0701446 to SS); and National Institutes of Health Grant (GM028140 to GMC). CIBER de Diabetes y Enfermedades Metabólicas asociadas (CB07708/0012) is an initiative of the ISCIII. MM acknowledges support from the ‘Miguel Servet’ tenure track programme (CP11/00021), from the Fondo de Investigación Sanitaria (FIS) co-financed by the European Regional Development Fund (ERDF), and supported by a Salvador de Madariaga Mobility fellowship from the Spanish Ministry of Education (PR2011-0584). AT is the recipient of a FI-DGR fellowship (9015-97318/2012) from the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR)This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Springer

Role of the Transforming Growth Factor-β in regulating hepatocellular carcinoma oxidative metabolism.

Transforming Growth Factor beta (TGF-β) induces tumor cell migration and invasion. However, its role in inducing metabolic reprogramming is poorly understood. Here we analyzed the metabolic profle of hepatocellular carcinoma (HCC) cells that show diferences in TGF-β expression. Oxygen consumption rate (OCR), extracellular acidifcation rate (ECAR), metabolomics and transcriptomics were performed. Results indicated that the switch from an epithelial to a mesenchymal/migratory phenotype in HCC cells is characterized by reduced mitochondrial respiration, without signifcant diferences in glycolytic activity. Concomitantly, enhanced glutamine anaplerosis and biosynthetic use of TCA metabolites were proved through analysis of metabolite levels, as well as metabolic fuxes from U-13C6-Glucose and U-13C5-Glutamine. This correlated with increase in glutaminase 1 (GLS1) expression, whose inhibition reduced cell migration. Experiments where TGF-β function was activated with extracellular TGF-β1 or inhibited through TGF-β receptor I silencing showed that TGF-β induces a switch from oxidative metabolism, coincident with a decrease in OCR and the upregulation of glutamine transporter Solute Carrier Family 7 Member 5 (SLC7A5) and GLS1. TGF-β also regulated the expression of key genes involved in the fux of glycolytic intermediates and fatty acid metabolism. Together, these results indicate that autocrine activation of the TGF-β pathway regulates oxidative metabolism in HCC cells