Análisis transcriptómico y de microarrays para la identificación de genes biomarcadores de la utilización de los nutrientes de la dieta en músculo esquelético de dorada (Sparus aurata)

[spa] La acuicultura constituye una de las actividades productivas de mayor desarrollo en las últimas décadas. Entre los peces de origen marino, la dorada (Sparus aurata) es la especie de mayor producción en España y en Europa. Sin embargo, a pesar del interés en la producción, los recursos genómicos para estudiar el impacto de los nutrientes de la dieta en el metabolismo son limitados. Actualmente, el análisis de expresión génica a nivel de mRNA es una de las principales herramientas utilizadas en estudios de genómica funcional. En este sentido, el estudio del transcriptoma mediante tecnologías de pirosecuenciación de última generación y el desarrollo de microarrays, que determinan perfiles de expresión de decenas de miles de genes, representan un avance para los estudios de la funcionalidad génica. El objetivo de esta tesis doctoral fue realizar un análisis transcriptómico del músculo esquelético de dorada con aplicación para estudios nutricionales, e identificar mediante microarrays, genes biomarcadores de la utilización de nutrientes. Cinco grupos de doradas fueron alimentadas durante 23 días con dietas de diferente composición: HPLLLC, MPHLLC, LPLLHC, MPLLHC, LPHLLC (letras mayúsculas representan niveles de nutrientes [High, Medium and Low], letras en superíndice representan el tipo de nutriente [Protein, Lipid and Carbohydrate]). Adicionalmente, durante un periodo similar, un grupo de doradas se mantuvo en ayuno. Se llevaron a cabo dos diseños experimentales: el experimento 1 fue diseñado para estudiar el transcriptoma del músculo esquelético de dorada y el experimento 2 tuvo como finalidad estudiar el aprovechamiento de los nutrientes de la dieta y el efecto de la composición sobre la digestibilidad, ingesta, parámetros somáticos y metabolismo intermediario. El análisis transcriptómico realizado con RNA de músculo esquelético de doradas, permitió obtener 691433 secuencias de alta calidad, correspondientes a 275,9 megabases. 4799 anotaciones fueron asignadas a categorías de ontología génica (GO). El análisis GO mostró que los procesos biológicos más representados correspondieron a oxidación-reducción, ciclo celular y división celular. Las funciones moleculares más abundantes fueron: unión a proteínas, unión a nucleótidos y actividad transferasa. La información transcriptómica se utilizó para analizar, mediante RT-qPCR, el efecto del estado nutricional sobre la expresión de genes relacionados con la cadena de transporte electrónico, fosforilación oxidativa y translocación de ATP al espacio intermembrana. De los genes analizados, la expresión de citocromo C oxidasa subunidad 5B y la caja de unión a ATP subfamilia G mostró una fuerte dependencia de la composición de la dieta, incrementando la expresión de dichos genes en peces alimentados con la dieta de menor contenido proteico y mayor contenido de carbohidratos. La información transcriptómica se utilizó, asimismo, para estudiar el efecto del estado nutricional y la composición de la dieta sobre el patrón global de expresión en músculo mediante microarrays. El análisis GO indicó que el ayuno se asocia a expresión diferencial disminuida en genes con funciones moleculares de actividad antioxidante y de unión a proteínas. Al realizar la comparación entre peces alimentados con dietas de diferente composición, se observó que las dietas de alto/medio contenido en proteína y bajo contenido de carbohidratos promovieron un incremento en la expresión de genes implicados en funciones de crecimiento, regulación y organización del citoesqueleto. Adicionalmente, mediante RT-qPCR, se analizó la expresión de genes implicados en procesos como: regulación de la miogénesis, regulación del crecimiento y vía de señalización Akt/TOR. En esta tesis doctoral, podemos concluir que la expresión de citocromo C oxidasa subunidad 5B, caja de unión a ATP subfamilia G, GHRs y MyoD2 podrían resultar de utilidad en estudios biotecnológicos encaminados a optimizar la utilización de los nutrientes en las dietas de doradas en cultivo.[eng] The aquaculture production system is the worldwide fastest growing animal food-producing sector. Gilthead seabream (Sparus aurata) is the leading species for aquaculture production in Spain and, among the marine fish, in Europe. Despite the commercial interest in improving aquaculture production, at present the genomic resources to study the impact of dietary nutrients in fish metabolism and growth are limited.Currently, gene expression analysis at mRNA level represents one of the main tools for functional genomic studies. In this sense, the transcriptomic analysis through pyrosequencing reaction with the 454 sequencing platform and the study of changes in the global pattern of gene expression by means of microarrays, represent a huge progress to determine the control of gene expression and their functions.The aim of this PhD thesis was to perform a transcriptomic analysis in skeletal muscle from gilthead seabream fed submitted to different nutritional conditions in order to identify, using microarrays, potential biomarker genes of interest to improve the use of dietary nutrients. To this end, 5 groups of S. aurata were fed for 23 days the following diets: HPLLLC, MPHLLC, LPLLHC, MPLLHC and LPHLLC. The 5 diets differ in the nutrient composition; capital letters represent macronutrient levels (High, Medium or Low), and superscript letters represent macronutrient (Protein, Carbohydrate or Lipid). Additionally, during the same time period, a group of fish was deprived of food. Two experimental procedures were carried out: experiment 1 was designed to determine the transcriptome in skeletal muscle of gilthead seabream; and experiment 2 allowed us to analyze the metabolic effects of the diet composition on the digestibility, food intake, somatic parameters and expression of key genes related to growth and fish intermediary metabolism.Transcriptomic analysis performed with RNA isolated from S. aurata skeletal muscle, yielded 691433 sequences of high quality, that corresponded to 275.9 megabases. 4799 entries were assigned to different categories of Gene Ontology (GO). GO analysis showed that the most represented biological processes corresponded to oxidation-reduction, cell cycle and cell division processes. Among the most abundant molecular functions were found: protein binding, nucleotide binding and those involving transferase activity. Transcriptomic information was used to design specific primers to analyze, by RT-qPCR, the effects of nutritional status on the expression of genes related to the electron transport chain, oxidative phosphorylation and translocation of ATP to the mitochondrial intermembrane space. From the analyzed genes, the expression of cytochrome C oxidase subunit 5B and ATP binding cassette subfamily G showed a strong dependence on the diet composition; the expression of both genes is upregulated in the fish fed low protein/high carbohydrate diets.In addition, transcriptomic data was used to design oligonucleotide microarrays to allow analysis of changes in gene expression profiling in muscle of fish under different nutritional conditions. GO analysis indicated a decreased expression of genes associated with antioxidant activity and protein binding in muscle of starved fish. Feeding with diets of high/medium protein and low carbohydrate content increased the expression of genes involved in growth and the regulation and organization of the cytoskeleton. Furthemore, RT-qPCR was performed to determined the expressional changes of genes involved in regulation of myogenesis, growth and the Akt/TOR signaling pathway.The results presented in this thesis allowed us to conclude that changes in the expression of cytochrome C oxidase subunit 5B, ATP binding cassette subfamily G, growth hormone receptors and MyoD2 could be useful to develop biotechnological studies to optimize the use of dietary nutrients supplied to gilthead seabream in culture

Gene markers of dietary macronutrient composition and growth in the skeletal muscle of gilthead sea bream (Sparus aurata)

To increase our current knowledge on the nutritional regulation of growth and gene expression pattern in fish skeletal muscle, the effect of dietary macronutrient composition was assessed on digestibility, nutrient retention, growth performance, and the mRNA levels of key genes involved in functionality, growth and development of the skeletal muscle in gilthead sea bream (Sparus aurata). Long-term starvation decreased the expression of myogenic regulatory factors such as Myod2, Myf5, myogenin (Myog) and Myf6 in the skeletal muscle of S. aurata. The supply of high or medium protein, low carbohydrate diets enhanced growth parameters, feed efficiency ratio, feed conversion ratio and significantly upregulated myod2. However, the supply of low protein, high carbohydrate diets restricted growth and stimulated the mRNA levels of myostatin, while downregulated follistatin (fst), igf1, mtor and rps6. Microarray analysis revealed igfals, tnni2, and gadd45a as gene markers upregulated by diets enriched with protein, lipids and carbohydrates, respectively. The results of the present study show that in addition to myod2, fst, igf1, mtor and rps6, the expression levels of igfals, tnni2 and remarkably gadd45a in the skeletal muscle can be used as markers to evaluate the effect of dietary macronutrient changes on fish growth and muscle development in S. aurata.This work was supported by the Ministerio de Economía, Industria y Competitividad, Spain (grant no. AGL2016-78124-R; cofunded by the European Regional Development Fund, European Commission) and the Agencia Nacional de Investigacion ´ y Desarrollo, Chile (Becas Chile/ 2011–72111506). The authors thank Piscicultura Marina Mediterranea (Burriana, Castellon, ´ Spain) for providing S. aurata juveniles.Peer ReviewedPostprint (published version

Gene markers of dietary macronutrient composition and growth in the skeletal muscle of gilthead sea bream (Sparus aurata)

To increase our current knowledge on the nutritional regulation of growth and gene expression pattern in fish skeletal muscle, the effect of dietary macronutrient composition was assessed on digestibility, nutrient retention, growth performance, and the mRNA levels of key genes involved in functionality, growth and development of the skeletal muscle in gilthead sea bream (Sparus aurata). Long-term starvation decreased the expression of myogenic regulatory factors such as Myod2, Myf5, myogenin (Myog) and Myf6 in the skeletal muscle of S. aurata. The supply of high or medium protein, low carbohydrate diets enhanced growth parameters, feed efficiency ratio, feed conversion ratio and significantly upregulated myod2. However, the supply of low protein, high carbohydrate diets restricted growth and stimulated the mRNA levels of myostatin, while downregulated follistatin (fst), igf1, mtor and rps6. Microarray analysis revealed igfals, tnni2, and gadd45a as gene markers upregulated by diets enriched with protein, lipids and carbohydrates, respectively. The results of the present study show that in addition to myod2, fst, igf1, mtor and rps6, the expression levels of igfals, tnni2 and remarkably gadd45a in the skeletal muscle can be used as markers to evaluate the effect of dietary macronutrient changes on fish growth and muscle development in S. aurata

A transcriptomic approach to study the effect of long-term starvation and diet composition on the expression of mitochondrial oxidative phosphorylation genes in gilthead sea bream (Sparus aurata)

Background: The impact of nutritional status and diet composition on mitochondrial oxidative phosphorylation (OXPHOS) in fish remains largely unknown. To identify biomarkers of interest in nutritional studies, herein we obtained a deep-coverage transcriptome by 454 pyrosequencing of liver and skeletal muscle cDNA normalised libraries from long-term starved gilthead sea bream (Sparus aurata) and fish fed different diets. Results: After clean-up of high-throughput deep sequencing reads, 699,991 and 555,031 high-quality reads allowed de novo assembly of liver and skeletal muscle sequences, respectively (average length: 374 and 441 bp; total megabases: 262 and 245 Mbp). An additional incremental assembly was completed by integrating data from both tissues (hybrid assembly). Assembly of hybrid, liver and skeletal muscle transcriptomes yielded, respectively, 19,530, 11,545 and 10,599 isotigs (average length: 1330, 1208 and 1390 bp, respectively) that were grouped into 15,954, 10,033 and 9189 isogroups. Following annotation, hybrid transcriptomic data were used to construct an oligonucleotide microarray to analyse nutritional regulation of the expression of 129 genes involved in OXPHOS in S. aurata. Starvation upregulated cytochrome c oxidase components and other key OXPHOS genes in the liver, which exhibited higher sensitive to food deprivation than the skeletal muscle. However, diet composition affected OXPHOS in the skeletal muscle to a greater extent than in the liver: most of genes upregulated under starvation presented higher expression among fish fed a high carbohydrate/low protein diet. Conclusions: Our findings indicate that the expression of coenzyme Q-binding protein (COQ10), cytochrome c oxidase subunit 6A2 (COX6A2) and ADP/ATP translocase 3 (SLC25A6) in the liver, and cytochrome c oxidase subunit 5B isoform 1 (COX5B1) in the liver and the skeletal muscle, are sensitive markers of the nutritional condition that may be relevant to assess the effect of changes in the feeding regime and diet composition on fish farming

A transcriptomic approach to study the effect of long-term starvation and diet composition on the expression of mitochondrial oxidative phosphorylation genes in gilthead sea bream (Sparus aurata)

Background: The impact of nutritional status and diet composition on mitochondrial oxidative phosphorylation (OXPHOS) in fish remains largely unknown. To identify biomarkers of interest in nutritional studies, herein we obtained a deep-coverage transcriptome by 454 pyrosequencing of liver and skeletal muscle cDNA normalised libraries from long-term starved gilthead sea bream (Sparus aurata) and fish fed different diets. Results: After clean-up of high-throughput deep sequencing reads, 699,991 and 555,031 high-quality reads allowed de novo assembly of liver and skeletal muscle sequences, respectively (average length: 374 and 441 bp; total megabases: 262 and 245 Mbp). An additional incremental assembly was completed by integrating data from both tissues (hybrid assembly). Assembly of hybrid, liver and skeletal muscle transcriptomes yielded, respectively, 19,530, 11,545 and 10,599 isotigs (average length: 1330, 1208 and 1390 bp, respectively) that were grouped into 15,954, 10,033 and 9189 isogroups. Following annotation, hybrid transcriptomic data were used to construct an oligonucleotide microarray to analyse nutritional regulation of the expression of 129 genes involved in OXPHOS in S. aurata. Starvation upregulated cytochrome c oxidase components and other key OXPHOS genes in the liver, which exhibited higher sensitive to food deprivation than the skeletal muscle. However, diet composition affected OXPHOS in the skeletal muscle to a greater extent than in the liver: most of genes upregulated under starvation presented higher expression among fish fed a high carbohydrate/low protein diet. Conclusions: Our findings indicate that the expression of coenzyme Q-binding protein (COQ10), cytochrome c oxidase subunit 6A2 (COX6A2) and ADP/ATP translocase 3 (SLC25A6) in the liver, and cytochrome c oxidase subunit 5B isoform 1 (COX5B1) in the liver and the skeletal muscle, are sensitive markers of the nutritional condition that may be relevant to assess the effect of changes in the feeding regime and diet composition on fish farming

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