Estudio del tráfico intracelular del transportador neuronal de glicina GlyT2: modulación por lipid rafts, ubiquitinación e interacción con Na/K ATPasa

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura:13-04-201

Molecular basis of the dominant negative effect of a glycine transporter 2 mutation associated with hyperekplexia

"This research was originally published in Journal of Biological Chemistry. Esther Arribas-González, Jaime de Juan-Sanz, Carmen Aragón, and Beatriz López-Corcuera. Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia. Journal of Biological Chemistry. 2015; 290:2150-2165. © the American Society for Biochemistry and Molecular Biology."Hyperekplexia or startle disease is a rare clinical syndrome characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. This neurological disorder can have serious consequences in neonates, provoking brain damage and/or sudden death due to apnea episodes and cardiorespiratory failure. Hyperekplexia is caused by defective inhibitory glycinergic neurotransmission. Mutations in the human SLC6A5 gene encoding the neuronal GlyT2 glycine transporter are responsible for the presynaptic form of the disease. GlyT2 mediates synaptic glycine recycling, which constitutes the main source of releasable transmitter at glycinergic synapses. Although the majority of GlyT2 mutations detected so far are recessive, a dominant negative mutant that affects GlyT2 trafficking does exist. In this study, we explore the properties and structural alterations of the S512R mutation in GlyT2. We analyze its dominant negative effect that retains wild-type GlyT2 in the endoplasmic reticulum (ER), preventing surface expression. Weshow that the presence of an arginine rather than serine 512 provoked transporter misfolding, enhanced association to the ER-chaperone calnexin, altered association with the coat-protein complex II component Sec24D, and thereby impeded ER exit. The S512R mutant formed oligomers with wild-type GlyT2 causing its retention in the ER. Overexpression of calnexin rescued wild-type GlyT2 from the dominant negative effect of the mutant, increasing the amount of transporter that reached the plasma membrane and dampening the interaction between the wild-type and mutant GlyT2. The ability of chemical chaperones to overcome the dominant negative effect of the disease mutation on the wild-type transporter was demonstrated in heterologous cells and primary neuronsThis work was supported by Spanish “Ministerio de Economía y Competitividad” Grant SAF2011-28674, by the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), and by an institutional grant from the “Fundación Ramón Areces.

La caracterización del ambiente de aprendizaje en un laboratorio de química general mediante métodos de investigación social

Desde una crítica al paradigma proceso-producto, que asume la causalidad entre el diseño del proceso de aprendizaje y su desarrollo, consideramos la caracterización de los ambientes de aprendizaje un requisito para la evaluación de innovaciones educativas. En este trabajo han sido incorporados métodos de la investigación social como los inspirados en la Teoría Fundamentada, a la caracterización del ambiente de aprendizaje en un contexto experimental. Mediante dichos métodos se ha llevado a cabo un estudio de caso en el que han sido analizadas las preguntas formuladas en el laboratorio y las reflexiones reflejadas en sus cuadernos por los estudiantes, con objeto de proponer un marco conceptual para la evaluación de dichos ambientes de aprendizaje. Los resultados justifican el uso de tales métodos, al ser notablemente contradictorios con los procedentes de la aplicación de un cuestionario basado en el conocido Science Laboratory Environment Inventory.From a critical view point of process-product paradigm, which accepts a causal relationship between learning process design and its classroom development, the characterization of Learning Environments as requirement for educational innovations assessment is considered. In this paper, research methods from Social Sciences as those inspired in the Grounded Theory have been applied to experimental learning environments. Students generated questions during laboratory sessions and self-reports have been analyzed by means of a case study in order to propose a framework to assess these learning environments. The results justify the use of suggested methodology since they are significantly contradictory with the ones from a questionnaire based on the well known Science Laboratory Environment Inventory

“Delirium” en el paciente oncológico

Delirium is a functional disorder of the central nervous system, very common among oncological patients, consequence of the presence in the organism of “false neurotransmitters” that could be exogenous or endogenous and give place to alterations inside several aspects (cognitive, perceptive, behavior, attention). The restoration of the symptoms is acute and the course is fluctuating along the day. In an important percentage of the cases the situation is reversible. Nevertheless, in other occasions it is an indicator of a situation premortem of the patient (“terminal agitation”). Most of cases etiology is multifactorial and there can be implied both, the proper tumor and the treatments administered to fight it or to solve the symptoms that this one causes (chemotherapy, opioids, steroids). The managing of this entity implies several questions of psychological area (information to the family), of environmental hygiene (skills of reorientation) and pharmacologist (control the symptoms with neuroleptics, benzodiacepines). Nevertheless, the most important point is to eliminate the ethiologic factor, in case of this one exists

Analysis of students' generated questions in laboratory learning environments

In order to attain a reliable laboratory work assessment, we argue taking the Learning Environment as a core concept and a research paradigm that considers the factors affecting the laboratory as a particularly complex educational context. With regard to Laboratory Learning Environments (LLEs), a well known approach is the SLEI (Science Laboratory Environment Inventory). The aim of this research is to design and apply an alternative and qualitative assessment tool to characterize Laboratory Learning Environments in an introductory course of organic chemistry. An alternative and qualitative assessment tool would be useful for providing feed-back for experimental learning improvement; serving as a complementary triangulation tool in educational research on LLEs; and generating meaningful categories in order to design quantitative research instruments. Toward this end, spontaneous questions by students have been chosen as a reliable source of information. To process these questions, a methodology based on the Grounded Theory has been developed to provide a framework for characterizing LLEs. This methodology has been applied in two case studies. The conclusions lead us to argue for using more holistic assessment tools in both everyday practice and research. Likewise, a greater attention should be paid to metacognition to achieve suitable self-perception concerning students¿ previous knowledge and manipulative skills.Llorens Molina, JA.; Llorens De Jaime, JM.; Sanz Berzosa, I. (2012). Analysis of students' generated questions in laboratory learning environments. Journal of Technology and Science Education. 2(1):46-55. doi:10.3926/jotse.36S46552

Regulación de la neurotransmisión glicinérgica en procesos de dolor inflamatorio: una nueva vía de acción de la prostaglandina E2 en médula espinal

Glycinergic inhibitory neurotransmission is terminated by reuptake through specific transporters, GlyT1 (glial isoform) and GlyT2 (neuronal isoform). In this work we describe that Prostaglandin E2 (PGE2, an important mediator of inflammatory pain) activates GlyT2-mediated recapture of glycine via interaction with the EP3 receptor. Moreover, in these conditions a diminished ubiquitination of GlyT2 is observed. Ubiquitination is an important modification for the correct trafficking of this transporter.  We propose that the reduction of ubiquitination leads to accumulate GlyT2 in the neuronal surface, which could explain the PGE2-mediated activation of GlyT2. Therefore, our results suggest that GlyT2 is an interesting therapeutic target and its inhibition could contribute to reduce inflammatory pain.La acción de la glicina como neurotransmisor inhibidor es finalizada por su recaptación del espacio sináptico a través de dos transportadores específicos, GlyT1 (isoforma glial) y GlyT2 (isoforma neuronal). En este trabajo describimos un mecanismo mediante el cual la unión de la prostaglandina E2 (un importante mediador del dolor inflamatorio) a sus receptores EP3 activa la recaptación de glicina llevada a cabo por GlyT2. Esta activación coincide con una disminución de la ubiquitinación del transportador, modificación post-traduccional necesaria para su correcto tráfico intracelular. Una menor ubiquitinación de GlyT2 produce una acumulación del transportador en la superficie neuronal, lo que explica la activación observada. Por tanto, los resultados de este trabajo sugieren que GlyT2 es una interesante diana terapéutica cuya inhibición podría contribuir a la reducción del dolor inflamatorio

The role of CCR5/CXCR3 expressing CD8+ cells in liver damage and viral control during persistent hepatitis C virus infection

20 p.Background/Aims:CXCR3 and CCR5 play a major role in recruiting cytotoxic T cells (Tc) and secreting secondary type 1 cytokines (Tc1) in the liver. HCV could impair their expression as a survival mechanism. The role of these chemokine receptors on CD8+ cells in chronic hepatitis C is analysed. Methods:Serum, chemokines, peripheral blood and intrahepatic lymphocytes from chronic hepatitis C patients were studied. CXCR3 / CCR5 expressing CD8+ cells were quantified by flow-cytometry. Serum chemokines concentration (CXCL10/CCL3) was measured by ELISA. Basal data were correlated with liver inflammation. Longitudinal data were obtained during treatment and correlated with virologic response. Results:CCR5/CXCR3 expressing CD8+ cells were enriched in the liver and correlated with inflammation. Chronic HCV patients presented the same frequency of CCR5high/CXCR3high expressing CD8+ cells in peripheral blood as in healthy controls but higher serum concentration of CXCL10/CCL3. Treatment with PEG-interferon a-2b plus ribavirin increased CCR5high/CXCR3high expressing CD8+ cells frequency in peripheral blood and decreased CXCL10/CCL3 serum concentration. Increase in CXCR3high expressing CD8+ cells after 24 weeks of treatment was correlated with SVR. Conclusions:In chronic hepatitis C, anti-viral treatment induces an increase in CD8+ cells expressing chemokine receptors associated with Tc1 response and a reduction in their ligands. Achievement of viral control is associated with an increase in CXCR3high expressing CD8+ cells during treatmentSchering-Plough-SpainJunta de Comunidades de Castilla-La Manch

Co-responses of bacterial and fungal communities to fire management treatments in Mediterranean pyrophytic ecosystems

Producción CientíficaCistus scrublands are pyrophytic ecosystems and occur widely across Mediterranean regions. Management of these scrublands is critical to prevent major disturbances, such as recurring wildfires. This is because management appears to compromise the synergies necessary for forest health and the provision of ecosystem services. Furthermore, it supports high microbial diversity, opening questions of how forest management practices impact belowground associated diversity as research related to this issue is scarce. This study aims to investigate the effects of different fire prevention treatments and site history on bacterial and fungi co-response and co-occurrence patterns over a fire-risky scrubland ecosystem. Two different site histories were studied by applying three different fire prevention treatments and samples were analyzed by amplification and sequencing of ITS2 and 16S rDNA for fungi and bacteria, respectively. The data revealed that site history, especially regarding fire occurrence, strongly influenced the microbial community. Young burnt areas tended to have a more homogeneous and lower microbial diversity, suggesting environmental filtering to a heat-resistant community. In comparison, young clearing history also showed a significant impact on the fungal community but not on the bacteria. Some bacteria genera were efficient predictors of fungal diversity and richness. For instance, Ktedonobacter and Desertibacter were a predictor of the presence of the edible mycorrhizal bolete Boletus edulis. These results demonstrate fungal and bacterial community co-response to fire prevention treatments and provide new tools for forecasting forest management impacts on microbial communities.Junta de Castilla y León (VA050P17

Risk analysis for UAV safe operations: a rationalization for an agricultural environment

The road to the automation of the agricultural processes passes through the safe operation of the autonomous vehicles. This requirement is a fact in ground mobile units, but it still has not well defined for the aerial robots (UAVs) mainly because the normative and legislation are quite diffuse or even inexistent. Therefore, to define a common and global policy is the challenge to tackle. This characterization has to be addressed from the field experience. Accordingly, this paper presents the work done in this direction, based on the analysis of the most common sources of hazards when using UAV's for agricultural tasks. The work, based on the ISO 31000 normative, has been carried out by applying a three-step structure that integrates the identification, assessment and reduction procedures. The present paper exposes how this method has been applied to analyze previous accidents and malfunctions during UAV operations in order to obtain real failure causes. It has allowed highlighting common risks and hazardous sources and proposing specific guards and safety measures for the agricultural context

Post-transplant cyclophosphamide and sirolimus based graft-versus-host disease prophylaxis after allogeneic stem cell transplantation for acute myeloid leukemia

Post-transplant cyclophosphamide (PTCy) has emerged as a promising graft-versus-host disease (GvHD) prophylaxis in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, no studies have reported the efficacy of a GvHD prophylaxis based on PTCy with sirolimus (Sir-PTCy) in patients with acute myeloid leukemia (AML). In this retrospective study, we analyze the use of sirolimus in combination with PTCy, with or without mycophenolate mofetil (MMF), on 242 consecutive adult patients with AML undergoing a myeloablative first allo-HSCT from different donor types, in three European centers between January 2017 and December 2020. Seventy-seven (32%) patients received allo-HSCT from HLA-matched sibling donor, 101 (42%) from HLA-matched and mismatched unrelated donor, and 64 (26%) from haploidentical donor. Except for neutrophil and platelet engraftment, which was slower in the haploidentical cohort, no significant differences were observed in major transplant outcomes according to donor type in univariate and multivariate analysis. GvHD prophylaxis with Sir-PTCy, with or without MMF, is safe and effective in patients with AML undergoing myeloablative allo-HSCT, resulting in low rates of transplant-related mortality, relapse/progression, and acute and chronic GvHD in all donor settings