Most teleost fish species are adapted to use amino acids as preferred energy source over carbohydrates and thus require high levels of dietary amino acids. In commercial aquaculture this requirement is met with fish meal-based diet. This practice is now incompatible with sustainable aquaculture and marine resources have to be replaced by plant feedstuffs naturally rich in starch and with specific amino acid profiles. One major obstacle linked with such a substitution is that the plant products are naturally rich in starch and carbohydrates which are poorly used by teleost fish. When dietary starch level is increased, fish respond by prolonged hyperglycaemia accompanied by an absence of inhibition of hepatic gluconeogenic gene expression. A better understanding of the reasons of the poor nutritional utilisation of carbohydrates in rainbow trout is essential to develop diets in agreement with fish growth and metabolism, environmental and economic constraints. In mammals, excessive nutrients including amino acids, contribute to the development of insulin resistance through a negative feed back loop on the insulin signalling pathway. We suspect that high dietary amino acid intake by rainbow trout can thus have undesirable effects on insulin sensitivity, particularly on insulin-regulated gene expression. This could explain the absence of post-prandial down-regulation of the expression of insulin target genes such as gluconeogenic genes and the restoration of their inhibition by reducing dietary protein levels. Thus, according to mammalian studies, we put forward the hypothesis that interactions between insulin, amino acids and glucose play a key role in the poor efficiency of rainbow trout to use dietary carbohydrates. In order to test this hypothesis, trials will be undertaken in rainbow trout both in vivo and in vitro 1) to analyse the long term effect of a high carbohydrate/low protein diet on the expression of metabolism-related genes and 2) to identify the molecular signalling events controlling the expression of these genes. This study will be performed on two metabolically active tissues: the liver and the skeletal muscle. To address the long term effect of an increase of dietary carbohydrate on hepatic and skeletal muscle gene expression, fish will be fed on high carbohydrate/low protein or high protein/low carbohydrate during 9 weeks and expression of several candidate genes will be measured in fasted and re-fed fish. Then, to identify unexpected genes affected by these diet manipulations, transcriptome analysis will be performed in re-fed fish. Signalling studies will also be developed to determine the molecular mechanisms that govern differential gene expression. We will focus our attention on the activation of the IRS1/PI3 kinase/Akt, mTOR/S6K1, PKA/PP2A and AMPK, key transduction factors of the insulin, amino acids, glucose and energy signalling pathways, respectively. In order to evaluate the potential negative feed back of amino acids on insulin signalling, the serine phosphorylation of IRS1 will be investigated. Since this in vivo experiment will not allow us to differentiate the relative effect of insulin, amino acids and glucose on the regulation of expression of target genes, complementary experiments will be developed in vitro using primary cell cultures of hepatocytes and satellite cells, respectively. In order to specify how insulin, amino acids and glucose interact to regulate target gene expression, signalling and gene expression studies will be conducted in these cellular models. We will first characterize the effect of amino acids on the mTOR/S6K1 signalling pathway and compare signalling effect of different amino acids, particularly branched-chain amino acids as leucine, known as a potent activator of the mTOR/S6K1 pathway. Then interactions between insulin, amino acids and glucose will be analysed. Stimulations combining different concentrations of amino acids, glucose and insulin will be performed to mimic what may occur in vivo after dietary manipulations or to place the cells in condition of excessive nutrient supply. Then experiments based on the use of drugs, agonist or antagonist of transduction molecules, will help us to determine how amino acids, glucose and insulin interacts to regulate target gene expression and what are the essential intracellular mediators of these regulations. The new data that will emerge from this scientific project will provide knowledge about the role of amino acids and glucose in the control of insulin action in fish, especially on insulin regulation of gene expression. The expected results might also help us to better understand the role played by amino acids in the development of insulin resistance. Data from this project will help us to develop fish diets adapted to environmental and economical requirements.

The production of tree fruits in most countries is predominantly a small farm/orchard endeavor. However, tree fruits are significant economic drivers worldwide in particular in many low to mid-income countries. Tree fruits not only provide income but also represent a substantial portion of the normal dietary intake; they are high in nutritional value and important components of a healthy diet. Fruit tree plantations are ideally suited to small farm settings as well as urban plantings and marginalized lands. They provide protection from drought and localized desertification. Additionally, fruit trees are important parts of many forest ecosystems worldwide and as a tree species they are important for land preservation and carbon assimilation. As with other tree species, Prunus fruit tree breeding programs however are continually challenged to find genetic solutions for producing nutritionally improved varieties while facing dynamic problems of disease and pests and ever-changing environmental landscapes. The most critical challenge in stone fruit agriculture are the current uncontrolled spread of Plum Pox Virus (PPV, a devastating disease of all stone fruits), the potential loss of production associated with insufficient winter chilling due to global warming, and the necessity to rapidly couple genetic solutions to these problems with the goal of increased nutritional quality of the fruit product. Unfortunately, these important characters are quantitative in genetic nature and thus present significant challenges to rapid genetic manipulation for varietal improvement. Genomics resources (genes, markers etc.) offer the tools to substantially augment the traditional breeders toolbox to improve the speed and efficiency of the character selection process. However, in order to capitalize on the use of the Prunus genomics resources and translate them into breeding programs, the genetic resolution of trait containing intervals in the genome needs to be substantially improved. This is no simple task as the physical limitations (time, space) required for tree species genetics complicates studies to refine QTL intervals into marker selectable units. Thus, new approaches to QTL resolution (e.g. association studies and candidate gene approaches) are rapidly supplementing more traditional approaches (F1 or F2 mapping populations) with the promise of higher resolution of QTLs for marker assisted breeding purposes. In this proposal, we address these major challenges by capitalizing on the robust genomics/genetics data in key Prunus species, the rich germplasm resources in European and other partner countries, innovative genotyping by whole genome sequencing and marker assisted breeding strategies to explore the translation of markers to accelerate improvement of varieties: A) in the short term (during the proposal research period) for genes controlling resistance to PPV, and chilling requirement (CR) and bloom date (BD); B) in the long term (part of the continuing research, post proposed period) for genes significantly impacting nutritional value of Prunus fruits. To tackle these specific aims we have developed a partnership of laboratories with particular expertise and resources ideally suited to this project. We propose to establish a Chaire of Excellence in the French host institution, INRA UMR BFP, hosting as scientific coordinator the reference senior scientist in the field of Prunus genomics, Pr A.G. Abbott (a world renowned expert in fruit tree genomics and genetics). We will be assisted locally by a bio-informatic-specialized research unit, CBiB, allowing us to optimize the analysis of large data sets. We will be supported by both the French Apricot breeding institution (INRA UGAFL) and the laboratory of Forestry genomics (Biogeco, Cestas-Pierroton) in order to ensure optimal translation of our results to fruit and forest trees.

Obesity is a major health problem in developed countries and a growing one in the developing world. Obesity-related diseases account for up to 8% of health costs in Europe. However, despite this, efficient anti-obesity treatments are currently lacking. Thus, because of the social and medical burden represented by obesity, intense research in recent years has been focusing on understanding the physiology of energy balance regulation, in order to identify possible therapeutic targets that will help halt obesity and its disastrous consequences. Such efforts have lately led to discover the role of several molecular pathways also known as “fuel sensing” mechanisms that, particularly within the hypothalamus, modulate feeding behavior and body weight, and whose activity can go awry in obesity. One of such cellular pathways is the mammalian Target of Rapamycin complex 1(mTORC1) signaling cascade. We have recently demonstrated that mTORC1 signaling, which is known to control protein synthesis and cell growth, integrates cellular fuel status with hormonal-related signaling in specific populations of hypothalamic neurons that use this information to regulate energy balance. Processes like neurogenesis and neuroinflammation in the adult hypothalamus have also been lately implicated in the regulation of energy balance. Recent evidence has shown that induction of neurogenesis in the adult hypothalamus is the mechanism underlying the ability of neurotrophic factors, like ciliary neurotrophic factor (CNTF), to maintain the body weight loss induced by the drug in diet-induced obese animals even beyond treatment cessation. Conversely, hypothalamic neuroinflammation has a causal role in the development of hyperphagia and obesity. Interestingly, neuroinflammation is known to modulate adult neurogenesis. Furthermore, we have shown that hypothalamic mTORC1 signaling critically mediates CNTF actions on food intake and body weight. Thus, in the present project we aim at determining the link among the mTORC1 pathway, the reciprocal modulation of hypothalamic adult neurogenesis and neuroinflammation and the consequent regulation of energy balance. In particular, we will study whether mTORC1 signaling has a critical role in mediating the long-term effects of CNTF treatment on body weight loss; we will establish whether such involvement is due to the modulation of either hypothalamic adult neurogenesis or neuroinflammation and we will finally clarify whether decreased hypothalamic neuroinflammation critically regulates energy balance by favoring neurogenesis through an mTORC1-dependent mechanism. To reach our goals we will combine genetic, pharmacological, behavioral, metabolic, neuroanatomical and molecular approaches. The use of these approaches will therefore allow us obtaining a very detailed characterization of the molecular and neuronal mechanisms that might underline the actual link among caloric intake, relative changes in brain neuroanatomy and molecular function and actual behavior. Taking into account the epidemic of obesity and the health threat that it represents, we expect that our studies will lead to a better understanding of the physiopathological mechanisms leading to this disease, thus helping find new therapeutic approaches to tackle and eventually prevent obesity and its associated consequences.

A first objective of this project is to validate molecular markers previously identified to identify oak trees to the species level and to source oak wood lots used by the barrel industry. A second one is to make these markers available to all interested end-users. Hence, it will become possible by the industry to control the wood to be sold or purchased. Close to the source, this involves forest managers that grow oak trees and sell their wood, and at the other extremity, winemakers that use oakwood to age their wines and alcohols. The French National Forest Office and the managers of private forests will be able to certify the conformity of the logs they sell, by using independent and incorruptible evidence provided by DNA testing. In addition, the molecular tools will allow oenologists to optimize wine and alcohols maturation, by better controlling the aromatic composition of the wood, which strongly depends on the oak species used and partly on its geographic origin. The work implies the validation, in an industrial context, of the results obtained during the last years in our lab using genetic characterization of DNA isolated from dry wood. A single test will be designed that combines markers useful for the diagnostic of species and of geographic origin. This test will be adapted to two circumstances, depending on whether the wood to be studied is obtained from oak logs (limited degradation of DNA) or from oak staves (highly degraded DNA, requiring a more sophisticated procedure). In the course of the project, the validated tools will be transferred to a company providing genotyping services to end-users.