TRANSFER OF ANALYTICAL METHODS FOR THE CHARACTERISATION OF NATURAL PRODUCTS TO R&D PROCESSES OF IDOKI

IDOKI SCF Technologies S.L. is a technology-based company, set up on September 2006 in Derio (Biscay) with the main scope of developing extraction and purification processes based on the use of supercritical fluid extraction technology (SFE) in food processing, extraction of natural products and the production of personal care products. IDOKI¿s researchers have been working on many different R&D projects so far, most of them using this technology. However, the optimization of a SFE method for the different matrices cannot be performed unless we have an analytical method for the characterisation of the extracts obtained in each experiment. The analytical methods are also essential for the quality control of the raw materials that are going to be used and also for the final product. This PhD thesis was born to tackle this problem and therefore, it is based on the development of different analytical methods for the characterisation of the extracts and products. The projects that we could include in this thesis were the following: the extraction propolis, the recovery of agroindustrial residues (soy and wine) and the dealcoholisation of wine.On the one hand, for the extraction of propolis, several UV-Vis spectroscopic methods were used in order to measure the antioxidant capacity and the total polyphenol and flavonoid content of the extracts. A SFC method was also developed in order to measure more specific phenolic compounds. On the other hand, for the recovery of agroindustrial residues UV-Vis spectroscopy was used to determine the total polyphenol content and two SFC methods were developed to analyse different phenolic compounds. Extraction methods such as MAE, FUSE and rotary agitation were also evaluated for the characterisation of the raw materials.Finally, for the dealcoholisation of wine, the development of a SBSE-TD-GC-MS and DHS-TD-GC-MS methods for the analysis of aromas and a NIR spectroscopic method for the determination of ethanol content with the help of chemometrics was necessary. Most of these methods are typically used in IDOKI¿s lab as routine analyses apart from others not included in this PhD thesis.

The Interplay between Natural Selection and Susceptibility to Melanoma on Allele 374F of SLC45A2 Gene in a South European Population

We aimed to study the selective pressures interacting on SLC45A2 to investigate the interplay between selection and susceptibility to disease. Thus, we enrolled 500 volunteers from a geographically limited population (Basques from the North of Spain) and by resequencing the whole coding region and intron 5 of the 34 most and the 34 least pigmented individuals according to the reflectance distribution, we observed that the polymorphism Leu374Phe (L374F, rs16891982) was statistically associated with skin color variability within this sample. In particular, allele 374F was significantly more frequent among the individuals with lighter skin. Further genotyping an independent set of 558 individuals of a geographically wider population with known ancestry in the Spanish population also revealed that the frequency of L374F was significantly correlated with the incident UV radiation intensity. Selection tests suggest that allele 374F is being positively selected in South Europeans, thus indicating that depigmentation is an adaptive process. Interestingly, by genotyping 119 melanoma samples, we show that this variant is also associated with an increased susceptibility to melanoma in our populations. The ultimate driving force for this adaptation is unknown, but it is compatible with the vitamin D hypothesis. This shows that molecular evolution analysis can be used as a useful technology to predict phenotypic and biomedical consequences in humans.

Planificación estratégica de una asociación orientada a la gestión de actividades y servicios recreativo-deportivos en el medio natural

La idea del proyecto consiste en crear una asociación orientada a la gestión de actividades recreativo-deportivas en el medio natural. En un enclave puramente rural, la asociación tendrá su sede en Jaurrieta, un pueblo del Pirineo Navarro con menos de 200 habitantes. La intención es que el pueblo de Jaurrieta y el propio Valle de Salazar, formen parte de esta asociación, involucrando a un gran número de personas. Se considera que la unión y la heterogeneidad de cada una de estas personas, potenciarán positivamente la propuesta. Con el objetivo de ofrecer actividades recreativo-deportivas y servicios complementarios, se aprovecharán los recursos naturales del entorno para proporcionar al visitante un mayor conocimiento de la zona y su estilo de vida rural. La oferta de actividades recreativo-deportivas en el medio natural, estará dirigida a aquellas personas que decidan pasar su tiempo de ocio en el Pirineo Navarro. Por esto, surge la necesidad de elaborar un “Plan Estratégico” que facilite la creación y desarrollo de esta asociación. Como misión o fin último de la propuesta, se pretende contribuir en el desarrollo rural del Valle de Salazar; desde una perspectiva de progreso económico y social.

Natural Variation In Recovery From A Short Period Of Anoxia Due To A cGMP-Dependent Protein Kinase in Drosophila melanogaster

[EN] Protein Kinase G (PKG) or cGMP-dependent protein kinases (PKG) have been shown to play an important role in resistance to abiotic stressors such as high temperatures or oxygen deprivation in Drosophila melanogaster. In Drosophila, the foraging gene encodes a PKG; natural variants for this gene exist, which differ in the level of expression of PKG: rovers (forR allele) which express high PKG levels, and sitters (forS allele) which express lower PKG levels. This project explores the differences in recovery from short periods of anoxia between natural variants (focusing on forS2, flies with a sitter gene in a rover background), as well as mutants with insertions in the foraging gene and RNAi recombinants that show a reduced PKG expression. The parameters measured were time to recovery and level of activity after anoxia. The results showed lower activity after anoxia in sitters than in rovers, reflecting a worse recovery from the anoxic coma in flies with lower PKG levels.

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of alpha-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg(-1), 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle-regulated by both diet and CB1 receptor activity-through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB1-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.

18S rRNA V9 metabarcoding for diet characterization: a critical evaluation with two sympatric zooplanktivorous fish species

The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired-end (PE; 2 9 150 bp) technology. To critically evaluate the method's performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual-based diet analysis methods. The high sensitivity and semi-quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR-based results. This molecular approach provides an alternative cost and time effective tool for food-web analysis.