Distroglicanopatías: análisis en pacientes, generación del knockout condicional de Pomt1 en fotorreceptores, y estudios genéticos y funcionales de FKTN y de FKRP en células en cultivo

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 25 -11-2016Esta tesis tiene embargado el acceso al texto completo hasta el 25-05-2018Las distroglicanopatías son un grupo heterogéneo de distrofias musculares de herencia recesiva que también pueden presentar en diferentes grados afectación nerviosa y ocular. A nivel molecular se caracterizan por la pérdida de la glicosilación del alfa-­‐ distroglicano (α-­‐DG). El DG es una proteína de la matriz extracelular (MEC) formada por dos subunidades (α-­‐DG y β-­‐DG) que conecta proteínas de la MEC (p. ej. laminina, perlecano o pikachurina) con el citoesqueleto de actina, a través de su unión citoplasmática a la distrofina. La conexión con las proteínas de la MEC se realiza a través de los residuos glicosílicos, principalmente O-­‐manosilglicanos, presentes en la región mucina del α-­‐DG. Hasta la fecha, 18 genes se han relacionado con el proceso de O-­‐manosilación del α-­‐ DG. Entre ellos encontramos genes que codifican tanto glicosiltransferasas como proteínas que intervienen en la generación de compuestos intermedios de la glicosilación. La proteína O-­‐manosiltransferasa 1 (POMT1) es la primera enzima, que conjuntamente con su homóloga POMT2, introduce la manosa inicial. Este monosacárido es esencial para la generación de las diferentes estructuras glicosiladas del α-­‐DG. Por otra parte, las proteínas FKTN y FKRP son enzimas que actúan en pasos posteriores del proceso de glicosilación. Recientemente se ha descrito que ambas introducen residuos de ribitol 5-­‐fosfato en uno de los residuos del α-­‐DG. Mutaciones en POMT1, FKTN y FKRP generan desde distroglicanopatías muy graves, como pueden ser el síndrome de Walker-­‐Warburg (WWS) o la distrofia muscular congénita tipo Fukuyama (FCMD), a presentaciones clínicas más leves como las distrofias musculares de cintura tipo 2 (LGMD2). En este trabajo hemos analizado dos pacientes con sospecha de distroglicanopatía, los cuales finalmente han mostrado un déficit en la vía de autofagia con una alteración en la glicosilación del α-­‐DG. Este hecho plantea una posible relación entre la glicosilación del α-­‐ DG y esta ruta de degradación. Por otra parte, hemos generado un modelo knockout condicional de ratón para el gen Pomt1 en los fotorreceptores de la retina. Hemos demostrado cómo la mutación del gen Pomt1 está asociada a la pérdida de glicosilación del α-­‐DG, la cual genera una malformación de la sinapsis entre los fotorreceptores y la células bipolares (sinapsis ribbon) y una disfunción visual. Por último, hemos generado líneas celulares de mioblastos de ratón knockout para los genes Fktn y Fkrp. En estas células hemos realizado estudios comparativos de glicoproteómica y del perfil de expresión con el objetivo de aproximarnos a la función de estos genes de una manera indirecta. También hemos llevado a cabo estudios de la señal de retención en el aparato de Golgi de estas proteínas en las líneas silvestres y knockout.Dystroglycanopathies are a heterogenous group of recessive muscular dystrophies which can also present different grades of nervous and ocular affectation. At a molecular level, the absence of α-­‐dystroglycan (α-­‐DG) glycosylation is the main characteristic of these diseases. Dystroglycan is a matrix extracelular (ECM) protein conformed by two subunits (α-­‐DG and β-­‐DG), that conects other ECM proteins (such as laminin, perlecan or pikachurin) with the actin cytoskeleton through its cytoplasmatic connection with dystrophin. The interaction with ECM proteins is mediated by its glycan residues, mainly O-­‐ mannosylglycans, located in the mucine región of α-­‐DG. To date, 18 genes have been related with α-­‐DG O-­‐mannosylation process. These genes codified for glycosyltransferases or proteins that generate glycosylation intermediates. Protein O-­‐mannosyltransferase 1 (POMT1) is the first enzyme, together with its homologous POMT2, which introduces the inital mannose. This monosacharide is essential for the generation of different mannosylated cores. On the other hand, FKTN and FKRP are enzymes that act in posterior steps of the glycosylation process. It has recently been described that both of them introduces ribitol 5-­‐phosphate in a specific α-­‐DG core. Mutationzs in POMT1, FKTN and FKRP are able to cause from severe dystroglycanopathies, such as Walker-­‐Warburg syndrome (WWS) or Fukuyama congenital muscular dystrophy (FCMD), to less severe dystroglycanopathies like limb-­‐girdle muscular dystrophies type 2 (LGMD2). In this work, we have analyzed two patients with dystroglycanopathy suspicion. The genetic-­‐molecular characterization has finally revealed an imparment in the autophagy pathway but we have also detected α-­‐DG glyscosylation anomalies. The data seem to highlight a posible relation between autophagy and α-­‐DG. On the other hand, we have generated a conditional knockout mouse model for Pomt1 gene in the retinal photoreceptors. We have demonstrated how the mutation of POMT1 is associated with the loss of α-­‐DG glycosylation, which causes a destructing in the synapsis between both photoreceptor and bipolar cells (ribbon synapsis) and an associated visual imparment. Finally, we have generated mouse myoblast cell lines knockout for Fktn and Fkrp. In these cell lines we have performed comparative glycoproteomic and transcriptional profile analysis with the aim to approach, indirectly, to the function of these proteins. We have also analyzed the signal for Golgi retention in wild-­‐type and knockout cell lines

Maneuver Optimization for Simultaneous Airspeed Calibration and Wind Estimation

[EN] The purpose of this work is to optimize systematically the maneuver required to identify the wind and calibrate the airspeed sensor of a subsonic aircraft using a GPS method. The optimization is based on sensitivity analyses that require a considerable number of flight simulations. To face this challenging computational effort, we adapted and parallelized a particle swarm optimization algorithm. We also introduced a new formulation of the sensor model in the Bernstein form. The results show stability using the selected formulation and bring out non-obvious aliasing and precision loss effects that depend on the maneuver configuration. The knowledge of these effects allowed us to fine-tune the maneuver in order to improve the estimation's precision. Finally, we validated the method using the JSBSim flight simulator under calm and light turbulence conditions.S

Large-eddy simulation analysis of the influence of the needle lift on the cavitation in diesel injector nozzles

The cavitation phenomenon has a strong influence on the internal flow and spray development in diesel injector nozzles. Despite its importance, there are many aspects which still remain unclear, especially for partial needle lifts when the injector is in the opening and closing phases. For that reason, the current paper is focused on the influence of the needle lift on the internal flow in a diesel nozzle. This study was carried out with three-dimensional simulations at a high injection pressure (160 MPa) using a homogeneous equilibrium model implemented in OpenFOAM to model the cavitation phenomenon. The nozzle was simulated with large-eddy simulation methods at six different needle lifts (10 mm, 30 mm, 50 mm, 75 mm, 100 mm and 250 mm), providing relevant information about the evolution of the internal flow, the turbulence development (the vorticity, the turbulence–cavitation interaction and the turbulent structures) and the flow characteristics in the nozzle outlet (the mass flow, the momentum flux and the effective velocity) with the needle position.Desantes Fernández, JM.; Salvador Rubio, FJ.; Carreres Talens, M.; Martínez López, J. (2015). Large-eddy simulation analysis of the influence of the needle lift on the cavitation in diesel injector nozzles. Proceedings of the Institution of Mechanical Engineers, Part D: Journal of Automobile Engineering. 229(4):407-423. doi:10.1177/0954407014542627S4074232294Faeth, G. ., Hsiang, L.-P., & Wu, P.-K. (1995). Structure and breakup properties of sprays. 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Computational study of the cavitation phenomenon and its interaction with the turbulence developed in diesel injector nozzles by Large Eddy Simulation (LES). Mathematical and Computer Modelling, 57(7-8), 1656-1662. doi:10.1016/j.mcm.2011.10.050Piomelli, U. (1999). Large-eddy simulation: achievements and challenges. Progress in Aerospace Sciences, 35(4), 335-362. doi:10.1016/s0376-0421(98)00014-1Launder, B. E., & Spalding, D. B. (1974). The numerical computation of turbulent flows. Computer Methods in Applied Mechanics and Engineering, 3(2), 269-289. doi:10.1016/0045-7825(74)90029-2Payri, F., Bermúdez, V., Payri, R., & Salvador, F. J. (2004). The influence of cavitation on the internal flow and the spray characteristics in diesel injection nozzles. Fuel, 83(4-5), 419-431. doi:10.1016/j.fuel.2003.09.010Payri, R., Salvador, F. J., Gimeno, J., & de la Morena, J. (2009). Study of cavitation phenomena based on a technique for visualizing bubbles in a liquid pressurized chamber. 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Experimental Characterization of the Thermodynamic Properties of Diesel Fuels Over a Wide Range of Pressures and Temperatures

The influence of pressure and temperature on some of the important thermodynamic properties of diesel fuels has been assessed for a set of fuels. The study focuses on the experimental determination of the speed of sound, density and compressibility (via the bulk modulus) of these fuels by means of a method that is thoroughly described in this paper. The setup makes use of a common-rail injection system in order to transmit a pressure wave through a high-pressure line and measure the time it takes for the wave to travel a given distance. Measurements have been performed in a wide range of pressures (from atmospheric pressure up to 200 MPa) and temperatures (from 303 to 353 K), in order to generate a fuel properties database for modelers on the field of injection systems for diesel engines to incorporate to their simulations.This work was partly sponsored by "Ministerio de Economia y Competitividad" (Spanish Ministry of Economy) in the frame of the project "Comprension de la influencia de combustibles no convencionales en el proceso de inyeccion y combustion tipo diesel", reference TRA2012-36932. The equipment used in this work hasDesantes Fernández, JM.; Salvador Rubio, FJ.; Carreres Talens, M.; Jaramillo-Císcar, D. (2015). Experimental Characterization of the Thermodynamic Properties of Diesel Fuels Over a Wide Range of Pressures and Temperatures. SAE International Journal of Fuel and Lubricants. 8(1):190-199. https://doi.org/10.4271/2015-01-0951S1901998

Insights into the room temperature magnetism of ZnO/Co3O4 mixtures

The origin of room temperature (RT) ferromagneticlike behavior in ZnO-based diluted magnetic semiconductors is still an unclear topic. The present work concentrates on the appearance of RT magnetic moments in just mixed ZnO/Co3O4 mixtures without thermal treatment. In this study, it is shown that the magnetism seems to be related to surface reduction of the Co3O4 nanoparticles, in which, an antiferromagnetic Co3O4 nanoparticle (core) is surrounded by a CoO-like shell. This singular superficial magnetism has also been found in other mixtures with semiconductors such as TiO2 and insulators such as Al2O3

Molecular Recognition, Transient Chirality and Sulfur Hydrogen Bonding in the Benzyl Mercaptan Dimer

The homodimers of transiently chiral molecules offer physical insight into the process of molecular recognition, the preference for homo or heterochiral aggregation and the nature of the non-covalent interactions stabilizing the adducts. We report the observation of the benzyl mercaptan dimer in the isolation conditions of a supersonic jet expansion, using broadband (chirped-pulse) microwave spectroscopy. A single homochiral isomer was observed for the dimer, stabilized by a cooperative sequence of S-H···S and S-H···π hydrogen bonds. The structural data, stabilization energies and energy decomposition describe these non-covalent interactions as weak and dispersion-controlled. A comparison is also provided with the benzyl alcohol dimer.This research was funded by the Spanish Ministerio de Ciencia e Innovación MICINN-FEDER, grants numbers PGC2018-098561-B-C21 and PGC2018-098561-B-C22. The APC were funded by PGC2018-098561-B-C22

Light-driven motion of charged domain walls in isolated ferroelectrics

©2022 American Physical SocietyLight-induced ferroelectric domain wall motion turns out to be a promising phenomenon to de- velop new photo-controlled devices. However, the physical origin of this ligh-matter coupling when material is irradiated with visible light remains unclear. Here, a phenomenological model predicting the motion of charged domain walls (CDWs) is developed. The photo-induced electronic reconstruc- tion mechanism is proposed as the primary absorption mechanism, leading to a linear dependence for the polarization perturbation with the light intensity. Domain walls motion is then driven by the energetic difference between domains in a CDW array, such that the macroscopic polarization can be easily tuned.Postprint (author's final draft

Increased YKL-40 but Not C-Reactive Protein Levels in Patients with Alzheimer’s Disease

Neuroinflammation is a common feature in Alzheimer's (AD) and Parkinson's (PD) disease. In the last few decades, a testable hypothesis was proposed that protein-unfolding events might occur due to neuroinflammatory cascades involving alterations in the crosstalk between glial cells and neurons. Here, we tried to clarify the pattern of two of the most promising biomarkers of neuroinflammation in cerebrospinal fluid (CSF) in AD and PD. This study included cognitively unimpaired elderly patients, patients with mild cognitive impairment, patients with AD dementia, and patients with PD. CSF samples were analyzed for YKL-40 and C-reactive protein (CRP). We found that CSF YKL-40 levels were significantly increased only in dementia stages of AD. Additionally, increased YKL-40 levels were found in the cerebral orbitofrontal cortex from AD patients in agreement with augmented astrogliosis. Our study confirms that these biomarkers of neuroinflammation are differently detected in CSF from AD and PD patients

R-RAS2 overexpression in tumors of the human central nervous system

Malignant tumors of the central nervous system (CNS) are the 10th most frequent cause of cancer mortality. Despite the strong malignancy of some such tumors, oncogenic mutations are rarely found in classic members of the RAS family of small GTPases. This raises the question as to whether other RAS family members may be affected in CNS tumors, excessively activating RAS pathways. The RAS-related subfamily of GTPases is that which is most closely related to classical Ras and it currently contains 3 members: RRAS, RRAS2 and RRAS3. While R-RAS and R-RAS2 are expressed ubiquitously, R-RAS3 expression is restricted to the CNS. Significantly, both wild type and mutated RRAS2 (also known as TC21) are overexpressed in human carcinomas of the oral cavity, esophagus, stomach, skin and breast, as well as in lymphomas. Hence, we analyzed the expression of R-RAS2 mRNA and protein in a wide variety of human CNS tumors and we found the R-RAS2 protein to be overexpressed in all of the 90 CNS cancer samples studied, including glioblastomas, astrocytomas and oligodendrogliomas. However, R-Ras2 was more strongly expressed in low grade (World Health Organization grades I-II) rather than high grade (grades III-IV) tumors, suggesting that R-RAS2 is overexpressed in the early stages of malignancy. Indeed, R-RAS2 overexpression was evident in pre-malignant hyperplasias, both at the mRNA and protein levels. Nevertheless, such dramatic changes in expression were not evident for the other two subfamily members, which implies that RRAS2 is the main factor triggering neural transformation.This work was supported by grants SAF2012-31279 from the ‘Comisión Interministerial de Ciencia y Tecnología’ and the ‘Ramón y Cajal’ program (RYC-2010-06251, to B.C.). We also thank the Fundación Ramón Areces for its institutional support of the ‘Centro de Biología Molecular Severo Ochoa’

Is a neutral expression also a neutral stimulus?: a study with functional magnetic resonance

Although neutral faces do not initially convey an explicit emotional message, it has been found that individuals tend to assign them an affective content. Moreover, previous research has shown that affective judgments are mediated by the task they have to perform. Using functional magnetic resonance imaging in 21 healthy participants, we focus this study on the cerebral activity patterns triggered by neutral and emotional faces in two different tasks (social or gender judgments). Results obtained, using conjunction analyses, indicated that viewing both emotional and neutral faces evokes activity in several similar brain areas indicating a common neural substrate. Moreover, neutral faces specifically elicit activation of cerebellum, frontal and temporal areas, while emotional faces involve the cuneus, anterior cingulated gyrus, medial orbitofrontal cortex, posterior superior temporal gyrus, precentral/postcentral gyrus and insula. The task selected was also found to influence brain activity, in that the social task recruited frontal areas while the gender task involved the posterior cingulated, inferior parietal lobule and middle temporal gyrus to a greater extent. Specifically, in the social task viewing neutral faces was associated with longer reaction times and increased activity of left dorsolateral frontal cortex compared with viewing facial expressions of emotions. In contrast, in the same task emotional expressions distinctively activated the left amygdale. The results are discussed taking into consideration the fact that, like other facial expressions, neutral expressions are usually assigned some emotional significance. However, neutral faces evoke a greater activation of circuits probably involved in more elaborate cognitive processing.This research was supported by a grant from Ministerio de Ciencia y Tecnología, Spain (MICINN-PSI-2009-09067)