New insights into McArdie disease : characterization of the murine model and development of new diagnosis and therapeutic approaches /

La malaltia de McArdle (glicogenosis tipus V), és la glicogenosis muscular més freqüent i està causada per la manca de la isoforma muscular de la glicogen fosforilasa. Els pacients són incapaços d'obtenir l'energia de les seves reserves de glicogen al múscul, i com a conseqüència, presenten intolerància a l'exercici amb fatiga prematura i contractures, a vegades acompanyada per rabdomiolisis i mioglobinuria. Actualment, no hi ha cap teràpia capaç de restaurar l'activitat de l'enzim en pacients, però el recent desenvolupat model murí de la malaltia de McArdle obre les portes a l'estudi de les conseqüències fenotípiques de la indisponibilitat del glicogen muscular, així com, a l'estudi de noves aproximacions terapèutiques per a aquest trastorn. Aquesta tesi doctoral s'ha basat en la caracterització fenotípica del model murí de la malaltia de McArdle, centrant tots els esforços en l'estudi de la regulació del metabolisme del glicogen pel que fa a la manca de la glicogen fosforilasa i la subsequent acumulació de glicogen, així com en el desenvolupament d'un model in vitro de la malaltia que imita l'abscència de la isoforma muscular de la glicogen fosforilasa i l'acumulacio de glicogen observada en el múscul esquelètic dels pacients amb la malaltia de McArdle. Utilitzant aquest model, hem demostrat l'eficiència del valproat de sodi, un inhibidor de les histones desacetilases, com a possible teràpia per a la malatia de McArdle, ja que hem observat la reexpressió de la isoforma cerebral de la glicogen fosforilasa i una reducció dosi-depenent en l'acumulació de glicogen després del tractament amb aquest fàrmac. A més, hem desenvolupat un test no invasiu, funcional i complementari per al diagnòstic de la malaltia de McArdle mitjançant la citometria de flux, que permet la detecció de l'expressió de la isoforma muscular de la glicogen fosforilasa en limfòcits T, sense necessitat de dur a terme una biòpsia muscular.McArdle disease (glycogenosis type V), the most common muscle glycogenosis, is caused by inherited deficiency of the muscle isoform of glycogen phosphorylase. Patients are unable to obtain energy from their muscle glycogen, thus typically experience exercise intolerance with premature fatigue and contractures, sometimes accompanied by rhabdomyolysis and myoglobinuria. Currently, there is no therapy to restore the enzyme activity in patients, but the recently developed p.R50X/p.R50X knock-in McArdle mice opens the door to the study of the phenotypic consequences of muscle glycogen unavailability, as well as, to the evaluation and testing of new therapeutic approaches for this disorder. The main goal of this PhD thesis was to deepen into the phenotypic characterization of the murine model of McArdle disease, focusing our efforts on the study of the regulation of glycogen metabolism from a glycogen phosphorylase deficiency and the subsequent high glycogen content in the skeletal muscle cell, as well as, the development of an in vitro model of the disease that mimics the absence of the muscular isoform of glycogen phosphorylase, and subsequent glycogen accumulation observed in skeletal muscle from McArdle patients. Using the in vitro model, we demonstrated the efficacy of sodium valproate, an inhibitor of histone deacetylase, as a potential therapy for McArdle disease, thus we observed the re-expression of the brain isoform of glycogen phosphorylase and a dose-dependent reduction in glycogen accumulation. In addition, we developed a non-invasive, functional and complementary test for the diagnosis of McArdle disease by flow cytometry, which allows the detection of muscle isoform of glycogen phosphorylase expression in T lymphocytes, without the need to perform a muscular biopsy

Vascular Interstitial Cells in Retinal Arteriolar Annuli Are Altered During Hypertension

Hypertension; Interstitial cells; Arteriolar annuliHipertensió; Cèl·lules intersticials; Arteriolar annuliHipertensión; Células intersticiales; Arteriolar annuliPurpose: It has been suggested that arteriolar annuli localized in retinal arterioles regulate retinal blood flow acting as sphincters. Here, the morphology and protein expression profile of arteriolar annuli have been analyzed under physiologic conditions in the retina of wild-type, β-actin-Egfp, and Nestin-gfp transgenic mice. Additionally, to study the effect of hypertension, the KAP transgenic mouse has been used. Methods: Cellular architecture has been studied using digested whole mount retinas and transmission electron microscopy. The profile of protein expression has been analyzed on paraffin sections and whole mount retinas by immunofluorescence and histochemistry. Results: The ultrastructural analysis of arteriolar annuli showed a different cell population found between endothelial and muscle cells that matched most of the morphologic criteria established to define interstitial Cajal cells. The profile of protein expression of these vascular interstitial cells (VICs) was similar to that of interstitial Cajal cells and different from the endothelial and smooth muscle cells, because they expressed β-actin, nestin, and CD44, but they did not express CD31 and α-SMA or scarcely express F-actin. Furthermore, VICs share with pericytes the expression of NG2 and platelet-derived growth factor receptor beta (PDGFR-β). The high expression of Ano1 and high activity of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase observed in VICs was diminished during hypertensive retinopathy suggesting that these cells might play a role on the motility of arteriolar annuli and that this function is altered during hypertension. Conclusions: A novel type of VICs has been described in the arteriolar annuli of mouse retina. Remarkably, these cells undergo important molecular modifications during hypertensive retinopathy and might thus be a therapeutic target against this disease

Local migration quantification method for scratch assays

The scratch assay is an in vitro technique used to assess the contribution of molecular and cellular mechanisms to cell migration. The assay can also be used to evaluate therapeutic compounds before clinical use. Current quantification methods of scratch assays deal poorly with irregular cell-free areas and crooked leading edges which are features typically present in the experimental data. We introduce a new migration quantification method, called 'monolayer edge velocimetry', that permits analysis of low-quality experimental data and better statistical classification of migration rates than standard quantification methods. The new method relies on quantifying the horizontal component of the cell monolayer velocity across the leading edge. By performing a classification test on in silico data, we show that the method exhibits significantly lower statistical errors than standard methods. When applied to in vitro data, our method outperforms standard methods by detecting differences in the migration rates between different cell groups that the other methods could not detect. Application of this new method will enable quantification of migration rates from in vitro scratch assay data that cannot be analysed using existing methods

Acute effects of a session of electroconvulsive therapy on brain-derived neurotrophic factor plasma levels

Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are neurotrophins that play critical roles in brain neuronal function. Previous studies have established the association between BDNF and NGF signaling and severe mental disorders, but changes in BDNF plasma levels and electroconvulsive therapy (ECT) response are controversial. The aim of his study was to explore the acute effects of a single session of ECT on these neurotrophins signaling. Plasma levels of BDNF and NGF and their tyrosine kinase-type receptors expression in peripheral blood mononuclear cells (PBMCs) were determined before and two hours after a single ECT session in 30 subjects with a severe mental disorder. Two hours after an ECT session we found a statistically significant decrease of BDNF plasma levels (p=0.007). We did not find significant acute effects on NGF plasma levels or receptors expression in PBMCs. We found a significant inverse correlation between the time of convulsion and BDNF plasma levels decrease (r=-0.041, p=0.024). We have identified a decrease in BDNF plasma levels after 2h of a single ECT session. These results indicate the interest for future research in the role of neurotrophins in the response and safety of ECT

Isolation and characterization of exosome-enriched urinary extracellular vesicles from Dent's disease type 1 Spanish patients

Enfermedad de Dent; Exosomas; Vesículas extracelularesDent's disease; Exosomes; Extracelular vesiclesMalaltia de Dent; Exosomes; Vesícules extracel·lularsAntecedentes y objetivo La enfermedad de Dent tipo 1 (DD1) es una enfermedad hereditaria rara ligada al cromosoma X causada por mutaciones en el CLCN5 que se caracteriza principalmente por una disfunción del túbulo proximal, hipercalciuria, nefrolitiasis o nefrocalcinosis, enfermedad renal crónica progresiva y proteinuria de bajo peso molecular, rasgo distintivo de la enfermedad. En la actualidad no existe un tratamiento curativo específico, únicamente sintomático, y no previene la progresión de la enfermedad. En este estudio hemos aislado y caracterizado las vesículas extracelulares urinarias (uEV) enriquecidas en exosomas que nos permitirán identificar biomarcadores asociados a la progresión de DD1 y ayudarán a una mejor comprensión de las bases fisiopatológicas. Materiales y métodos A través de una convocatoria nacional de la Sociedad Española de Nefrología (SEN) y la Sociedad Española de Nefrología Pediátrica (AENP), se obtuvieron orinas de pacientes y controles de distintos hospitales españoles, las cuales se procesaron para obtener los uEV. Los datos de estos pacientes fueron proporcionados por los respectivos nefrólogos o extraídos del registro RENALTUBE. Los uEV se aislaron mediante ultracentrifugación, fueron caracterizados morfológicamente y se extrajo su contenido de proteína y micro-ARN. Resultados Se seleccionó a 25 pacientes y 10 controles, de los cuales se procesaron las orinas para aislar los uEV. Nuestros resultados mostraron que la concentración relativa de uEV/ml era menor en los pacientes que en los controles (0,26 × 106 vs. 1,19 × 106 uEV/ml; p < 0,01). Además, se vio que los uEV de los pacientes eran significativamente más grandes que los de los sujetos control (diámetro medio: 187,8 vs. 143,6 nm; p < 0,01). Por último, nuestros datos demostraron que se había extraído correctamente el ARN tanto de los exosomas de pacientes como de los controles. Conclusiones En este trabajo describimos el aislamiento y caracterización de uEV de pacientes con DD 1 y controles sanos, útiles para el posterior estudio de moléculas cargo diferencialmente expresadas en esta enfermedad.Background and objectives Dent's disease type 1 (DD1) is a rare X-linked hereditary pathology caused by CLCN5 mutations that is characterized mainly by proximal tubule dysfunction, hypercalciuria, nephrolithiasis/nephrocalcinosis, progressive chronic kidney disease, and low-weight proteinuria, the molecular hallmark of the disease. Currently, there is no specific curative treatment, only symptomatic and does not prevent the progression of the disease. In this study we have isolated and characterized urinary extracellular vesicles (uEVs) enriched in exosomes that will allow us to identify biomarkers associated with DD1 progression and a better understanding of the pathophysiological bases of the disease. Materials and methods Through a national call from the Spanish Society of Nephrology (SEN) and the Spanish Society of Pediatric Nephrology (AENP), urine samples were obtained from patients and controls from different Spanish hospitals, which were processed to obtain the uEVs. The data of these patients were provided by the respective nephrologists and/or extracted from the RENALTUBE registry. The uEVs were isolated by ultracentrifugation, morphologically characterized and their protein and microRNA content extracted. Results Twenty-five patients and 10 controls were recruited, from which the urine was processed to isolate the uEVs. Our results showed that the relative concentration of uEVs/ml is lower in patients compared to controls (0.26 × 106 vs. 1.19 × 106 uEVs/ml, P < 0.01). In addition, the uEVs of the patients were found to be significantly larger than those of the control subjects (mean diameter: 187.8 vs. 143.6 nm, P < 0.01). Finally, our data demonstrated that RNA had been correctly extracted from both patient and control exosomes. Conclusions In this work we describe the isolation and characterization of uEVs from patients with DD1 and healthy controls, that shall be useful for the subsequent study of differentially expressed cargo molecules in this pathology.Este trabajo ha sido financiado principalmente por la fundación SENEFRO (SEN2019 a AM), por ASDENT y por subvenciones del Ministerio de Ciencia e Innovación (SAF201789989 a AM) y de la Red de Investigación Renal REDinREN (12/0021/0013). El Grupo de Fisiopatología Renal tiene la Mención de Calidad de la Generalitat de Cataluña (2017 SGR)

Vascular interstitial cells in retinal arteriolar annuli are altered during hypertension

Research Areas: OphthalmologyABSTRACT - Purpose: It has been suggested that arteriolar annuli localized in retinal arterioles regulate retinal blood flow acting as sphincters. Here, the morphology and protein expression profile of arteriolar annuli have been analyzed under physiologic conditions in the retina of wild-type, β-actin–Egfp, and Nestin–gfp transgenic mice. Additionally, to study the effect of hypertension, the KAP transgenic mouse has been used.info:eu-repo/semantics/publishedVersio

Vascular Interstitial Cells in Retinal Arteriolar Annuli Are Altered During Hypertension.

Purpose: It has been suggested that arteriolar annuli localized in retinal arterioles regulate retinal blood flow acting as sphincters. Here, the morphology and protein expression profile of arteriolar annuli have been analyzed under physiologic conditions in the retina of wild-type, β-actin-Egfp, and Nestin-gfp transgenic mice. Additionally, to study the effect of hypertension, the KAP transgenic mouse has been used. Methods: Cellular architecture has been studied using digested whole mount retinas and transmission electron microscopy. The profile of protein expression has been analyzed on paraffin sections and whole mount retinas by immunofluorescence and histochemistry. Results: The ultrastructural analysis of arteriolar annuli showed a different cell population found between endothelial and muscle cells that matched most of the morphologic criteria established to define interstitial Cajal cells. The profile of protein expression of these vascular interstitial cells (VICs) was similar to that of interstitial Cajal cells and different from the endothelial and smooth muscle cells, because they expressed β-actin, nestin, and CD44, but they did not express CD31 and α-SMA or scarcely express F-actin. Furthermore, VICs share with pericytes the expression of NG2 and platelet-derived growth factor receptor beta (PDGFR-β). The high expression of Ano1 and high activity of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase observed in VICs was diminished during hypertensive retinopathy suggesting that these cells might play a role on the motility of arteriolar annuli and that this function is altered during hypertension. Conclusions: A novel type of VICs has been described in the arteriolar annuli of mouse retina. Remarkably, these cells undergo important molecular modifications during hypertensive retinopathy and might thus be a therapeutic target against this disease

Fascin-1 is released from proximal tubular cells in response to calcineurin inhibitors (CNIs) and correlates with isometric vacuolization in kidney transplanted patients

Fascin-1; Nephrotoxicity; TransplantFascina-1; Nefrotoxicitat; TrasplantamentFascina-1; Nefrotoxicidad; TrasplanteImmunosuppression based on calcineurin inhibitors (CNIs) has greatly improved organ transplantation, although subsequent nephrotoxicity significantly hinders treatment success. There are no currently available specific soluble biomarkers for CNI-induced nephrotoxicity and diagnosis relies on renal biopsy, which is costly, invasive and may cause complications. Accordingly, identification of non-invasive biomarkers distinguishing CNI-induced kidney tubular damage from that of other etiologies would greatly improve diagnosis and enable more precise dosage adjustment. For this purpose, HK-2 cells, widely used to model human proximal tubule, were treated with CNIs cyclosporine-A and FK506, or staurosporine as a calcineurin-independent toxic compound, and secretomes of each treatment were analyzed by proteomic means. Among the differentially secreted proteins identified, only fascin-1 was specifically released by both CNIs but not by staurosporine. To validate fascin-1 as a biomarker of CNI-induced tubular toxicity, fascin-1 levels were analyzed in serum and urine from kidney-transplanted patients under CNIs treatment presenting or not isometric vacuolization (IV), which nowadays represents the main histological hallmark of CNI-induced tubular damage. Patients with chronic kidney disease (CKD) and healthy volunteers were used as controls. Our results show that urinary fascin-1 was only significantly elevated in the subset of CNI-treated patients presenting IV. Moreover, fascin-1 anticipated the rise of sCr levels in serially collected urine samples from CNI-treated pulmonary-transplanted patients, where a decline in kidney function and serum creatinine (sCr) elevation was mainly attributed to CNIs treatment. In conclusion, our results point towards fascin-1 as a putative soluble biomarker of CNI-induced damage in the kidney tubular compartment

Cognitive reserve assessment scale in health (CRASH): its validity and reliability

Background: the cognitive reserve (CR) concept has not been precisely defined in severe mental disorders and has been estimated using heterogeneous methods. This study aims to investigate and develop the psychometric properties of the Cognitive Reserve Assessment Scale in Health (CRASH), an instrument designed to measure CR in people with severe mental illness; (2) Methods: 100 patients with severe mental illness (non-affective psychoses and affective disorders) and 66 healthy controls were included. The internal consistency and convergent validity of CRASH were assessed. Spearman's correlations coefficients were also performed to examine the relationship between CRASH and neuropsychological tests, psychosocial functioning, and clinical course; (3) Results: the internal consistency was high (Cronbach's alpha coefficient = 0.903). The CRASH global score had a large positive correlation with the Cognitive reserve questionnaire total score (r = 0.838, p < 0.001), demonstrating good convergent validity. The correlation coefficients between the CRASH total scores and clinical, functional, and neuropsychological performance were different between groups. In order to provide clinical interpretation, severity classification based on diagnosis (non-affective psychotic disorders, affective disorders, and healthy controls) have been created; (4) Conclusions: CRASH is the first CR measure developed specifically for patients with severe mental illness, facilitating reliable and valid measurement of this construct. The scale may aid in the stratification of patients and the implementation of personalized interventions

Monografies del Montseny - 37

Antoni Pladevall Font va fer possible el naixement de la col.lecció completa de les Monografies del Montseny, n’ha estat el director durant molts anys, ha publicat més de seixanta treballs d’investigació històrica, i ha estat el mestre que ha mostrat el camí a seguir en la tasca d’aprofundiment de la història del Montseny i el seu entorn més proper. En el pròleg del volum 37 de les Monografies del Montseny es glosa la figura d’Antoni Pladevall Font, qui l’any 1966 inicià la col·laboració en el Servei de Conservació i Catalogació de Monuments Històrics a la Diputació de Barcelona; dos anys més tard, passà a exercir com a professor d’història medieval a la Facultat de Teologia de Barcelona. Obtingué el títol d’Arxiver Municipal de Vic l’any 1979, mentre treballa a la Diputació, i al 1981començà a treballar en el Departament de Cultura de la Generalitat fins a la seva jubilació l’any 1999. L’aleshores conseller de Cultura de la Generalitat Max Cahner li va encarregar d’organitzar el Servei de Conservació i Restauració de Monuments Arquitectònics on se li va assignar la secció d’inventari i la de cap de servei en funcions. Dos anys després hi va exercir com a director general del Patrimoni Arquitectònic. En aquesta etapa va col·laborar amb la Fundació i Editorial de la Gran Enciclopèdia Catalana, en la qual va exercir la direcció d’obres tan importants com la “Catalunya Romànica” i “L’Art Gòtic de Catalunya”, i va assessorar la “Gran Geografia Comarcal de Catalunya”.Objectius de Desenvolupament Sostenible::11 - Ciutats i Comunitats SosteniblesObjectius de Desenvolupament Sostenible::4 - Educació de QualitatPostprint (published version