Biotechnological applications at GBMI: industry-driven "bio-research"

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Proposta de Mòdul Professional en mecanitzat convencional i CNC per alumnes que formen part del Programa de Formació i Inserció (PFI)

Aquest treball final de Màster, és una proposta on es pretén donar cobertura a un petit buit que hi ha dins els programes de formació i inserció (PFI). No hi ha cap PFI d'auxiliar en mecanitzat, per tant l'autor plantejarà tres mòduls d'aprenentatge en mecanitzat. Dins d'un curs sencer PFI hi ha tres blocs de formació. Els blocs de formació general i tutoria, tant el temari com les hores, ja venen determinats pel currículum establert per la Generalitat a la RESOLUCIÓ ENS/2250/2014, de 6 d'octubre, de l'estructura dels programes de formació i inserció. Però en canvi, les hores destinades al bloc de formació professional, poden ser entre 360h a 410h. L'autor vol fer la proposta de creació de tres mòduls professionals (MP) sencers i bàsics, i les corresponents Unitats Formatives (UF) necessàries i bàsiques, per tal d'assegurar una formació adequada. L'estructura serà la següent: del bloc destinat a la formació professional es plantegen tres MP, un destinat a la introducció de treballs amb eines manuals i trepants (llima, serra, broques, mascles de roscar, ...). Aquest mòdul es documenta amb més detall i servirà d'exemple per els altres dos. Un altre MP destinat a l'aprenentatge de mecanitzat en màquines convencionals (torn i fresadora) i un darrer MP a la introducció de mecanitzat en màquines CNC. En aquest TFM l'autor també a fet un breu recull d'història i del perquè es van crear els PFI, dels seus antecedents, estructures i el seu àmbit legislatiu

Ligand binding mechanisms in human cone visual pigments

Vertebrate vision starts with light absorption by visual pigments in rod and cone photoreceptor cells of the retina. Rhodopsin, in rod cells, responds to dim light, whereas three types of cone opsins (red, green, and blue) function under bright light and mediate color vision. Cone opsins regenerate with retinal much faster than rhodopsin, but the molecular mechanism of regeneration is still unclear. Recent advances in the area pinpoint transient intermediate opsin conformations, and a possible secondary retinal-binding site, as determinant factors for regeneration. In this Review, we compile previous and recent findings to discuss possible mechanisms of ligand entry in cone opsins, involving a secondary binding site, which may have relevant functional and evolutionary implicationsPeer ReviewedPostprint (author's final draft

Novel ligands for the treatment of retinal degenerative diseases

Programa de doctorat: Doctorat en Polímers i Biopolímer

The zinc binding receptor GPR39 interacts with 5-HT1A and GalR(1) to form dynamic heteroreceptor complexes with signaling diversity

GPR39 is a class A G protein-coupled receptor involved in zinc binding and glucose homeostasis regulation, among other physiological processes. GPR39 was originally thought to be the receptor for obestatin peptide but this view has been challenged. However, activation of this receptor by zinc has been clearly established. Recent studies suggest that low GPR39 expression, due to deficient zinc levels, is involved in major depressive disorder. We have previously reported that zinc can alter receptor-receptor interactions and favor specific receptor interactions. In order to unravel the effect of zinc on specific G protein-coupled receptor association processes, we have performed FRET and co-immunopurification studies with GPR39 and 5-HT1A and GalR(1) which have been shown to dimerize. Our results suggest that zinc can modulate the formation of specific 5-HT1A-GPR39 and GalR(1)-5-HT1A-GPR39 heteroreceptor complexes under our experimental conditions.; We have analyzed the differences in signaling between the mono-homomeric receptors 5-HT1A, GalR(1) and GPR39 and the heteroreceptor complexes between them Our results show that the GPR39-5-HT1A heterocomplex has additive functionalities when compared to the monomeric-homomeric receptors upon receptor activation. In addition, the heterocomplex including also GalR(1) shows a different behavior, upon exposure to the same agonists. Furthermore, these processes appear to be regulated by zinc. These findings provide a rationale for the antidepressive effect widely described for zinc because pro-depressive heterocomplexes are predominant at low zinc concentration levels. (C) 2015 Elsevier B.V. All rights reserved.Peer ReviewedPostprint (author's final draft

Effect of trace metal Ions on the conformational stability of the visual photoreceptor rhodopsin

Trace metals are essential elements that play key roles in a number of biochemical processes governing human visual physiology in health and disease. Several trace metals, such as zinc, have been shown to play important roles in the visual phototransduction process. In spite of this, there has been little research conducted on the direct effect of trace metal elements on the visual photoreceptor rhodopsin. In the current study, we have determined the effect of several metal ions, such as iron, copper, chromium, manganese, and nickel, on the conformational stability of rhodopsin. To this aim, we analyzed, by means of UV-visible and fluorescence spectroscopic methods, the effects of these trace elements on the thermal stability of dark rhodopsin, the stability of its active Metarhodopsin II conformation, and its chromophore regeneration. Our results show that copper prevented rhodopsin regeneration and slowed down the retinal release process after illumination. In turn, Fe3+, but not Fe2+, increased the thermal stability of the dark inactive conformation of rhodopsin, whereas copper ions markedly decreased it. These findings stress the important role of trace metals in retinal physiology at the photoreceptor level and may be useful for the development of novel therapeutic strategies to treat retinal disease.This research was funded by grants from Ministry of Science and Innovation, Spain (PID2019-104817GB-I00), and from the Government of Catalonia to Research Consolidated Groups (2021 SGR 00342) to P.G. F.W. is the recipient of a predoctoral grant from the Secretariat for Universities and Research of the Ministry of Business and Knowledge of the Government of Catalonia and the European Social Fund (2021 FI_B 00228).Peer ReviewedPostprint (published version

Effect of sodium valproate on the conformational stability of the visual G Protein-coupled receptor rhodopsin

Rhodopsin is the G protein-coupled receptor of rod photoreceptor cells that mediates vertebrate vision at low light intensities. Mutations in rhodopsin cause inherited retinal degenerative diseases such as retinitis pigmentosa. Several therapeutic strategies have attempted to address and counteract the deleterious effect of rhodopsin mutations on the conformation and function of this photoreceptor protein, but none has been successful in efficiently preventing retinal degeneration in humans. These approaches include, among others, the use of small molecules, known as pharmacological chaperones, that bind to the receptor stabilizing its proper folded conformation. Valproic acid, in its sodium valproate form, has been used as an anticonvulsant in epileptic patients and in the treatment of several psychiatric disorders. More recently, this compound has been tested as a potential therapeutic agent for the treatment of retinal degeneration associated with retinitis pigmentosa caused by rhodopsin mutations. We now report on the effect of sodium valproate on the conformational stability of heterologously expressed wild-type rhodopsin and a rhodopsin mutant, I307N, which has been shown to be an appropriate model for studying retinal degeneration in mice. We found no sign of enhanced stability for the dark inactive conformation of the I307N mutant. Furthermore, the photoactivated conformation of the mutant appears to be destabilized by sodium valproate as indicated by a faster decay of its active conformation. Therefore, our results support a destabilizing effect of sodium valproate on rhodopsin I307N mutant associated with retinal degeneration. These findings, at the molecular level, agree with recent clinical studies reporting negative effects of sodium valproate on the visual function of retinitis pigmentosa patients.This research was supported by grant PID2019-104817GB-I00 from Ministerio de Ciencia e Innovación (MICINN).Peer ReviewedPostprint (published version

Human blue cone opsin regeneration involves secondary retinal binding with analog specificity

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigmentsPeer ReviewedPostprint (author's final draft

Flavonoid allosteric modulation of mutated visual rhodopsin associated with retinitis pigmentosa

Dietary flavonoids exhibit many biologically-relevant functions and can potentially have beneficial effects in the treatment of pathological conditions. In spite of its well known antioxidant properties, scarce structural information is available on the interaction of flavonoids with membrane receptors. Advances in the structural biology of a specific class of membrane receptors, the G protein-coupled receptors, have significantly increased our understanding of drug action and paved the way for developing improved therapeutic approaches. We have analyzed the effect of the flavonoid quercetin on the conformation, stability and function of the G protein-coupled receptor rhodopsin, and the G90V mutant associated with the retinal degenerative disease retinitis pigmentosa. By using a combination of experimental and computational methods, we suggest that quercetin can act as an allosteric modulator of opsin regenerated with 9-cis-retinal and more importantly, that this binding has a positive effect on the stability and conformational properties of the G90V mutant associated with retinitis pigmentosa. These results open new possibilities to use quercetin and other flavonoids, in combination with specific retinoids like 9-cis-retinal, for the treatment of retinal degeneration associated with retinitis pigmentosa. Moreover, the use of flavonoids as allosteric modulators may also be applicable to other members of the G protein-coupled receptors superfamily.Peer ReviewedPostprint (author's final draft

Zinc is involved in depression by modulating G-protein-coupled receptor heterodimerization

The final publication is available at Springer via http://dx.doi.org/10.1007/s12035-015-9153-y5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G protein-coupled receptors superfamily, and they have been described to heterodimerize triggering an anomalous physiological state that would underlie depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody and employed ligand-assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with Förster resonance energy transfer (FRET) measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modeling suggests that there is a coincidence between zinc-binding sites and heterodimerization interfaces for the serotonin receptor. Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease.Peer ReviewedPostprint (author’s final draft