The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of 'mature' snRNPs, or the reclamation of unassembled snRNP components.

Genetic Engineering of Induced Pluripotent Stem Cells and Reprogramming to Motor Neurons to Elucidate Selective Motor Neuron Death in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease, fatal within 1 to 5 years after onset of symptoms. About 3 out of 100’000 persons are diagnosed with ALS and there is still no cure available [1, 2]. 95% of all cases occur sporadically and the aetiology remains largely unknown [XXXX]. However, up to now 16 genes were identified to play a role in the development of familial ALS. One of these genes is FUS that encodes for the protein fused in sarcoma/translocated in liposarcoma (FUS/TLS). Mutations in this gene are responsible for some cases of sporadic as well as of inherited ALS [3]. FUS belongs to the family of heterogeneous nuclear ribonucleoproteins and is predicted to be involved in several cellular functions like transcription regulation [4], RNA splicing [5, 6], mRNA transport in neurons [7] and microRNA processing [8]. Aberrant accumulation of mutated FUS has been found in the cytoplasm of motor neurons from ALS patients [9]. The mislocalization of FUS is based on a mutation in the nuclear localization signal of FUS [10]. However, it is still unclear if the cytoplasmic localization of FUS leads to a toxic gain of cytoplasmic function and/or a loss of nuclear function that might be crucial in the course of ALS. The goal of this project is to characterize the impact of ALS-associated FUS mutations on in vitro differentiated motor neurons. To this end, we edit the genome of induced pluripotent stem cells (iPSC) using transcription activator-like effector nucleases (TALENs) [11,12] to create three isogenic cell lines, each carrying an ALS-associated FUS mutation (G156E, R244C and P525L). These iPSC’s will then be differentiated to motor neurons according to a recently establishe protocol (Ref Wichterle) and serve to study alterations in the transcriptome, proteome and metabolome upon the expression of ALS-associated FUS. With this approach, we hope to unravel the molecular mechanism leading to FUS-associated ALS and to provide new insight into the emerging connection between misregulation of RNA metabolism and neurodegeneration, a connection that is currently implied in a variety of additional neurological diseases, including spinocerebellar ataxia 2 (SCA-2), spinal muscular atrophy (SMA), fragile X syndrome, and myotonic dystrophy.

Genetic Engineering of Induced Pluripotent Stem Cells and Reprogramming to Motor Neurons to Elucidate Selective Motor Neuron Death in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease, fatal within 1 to 5 years after onset of symptoms. About 3 out of 100’000 persons are diagnosed with ALS and there is still no cure available [1, 2]. 95% of all cases occur sporadically and the aetiology remains largely unknown [3]. However, up to now 16 genes were identified to play a role in the development of familial ALS. One of these genes is FUS that encodes for the protein fused in sarcoma (FUS). Mutations in this gene are responsible for some cases of sporadic as well as of inherited ALS [4]. FUS belongs to the family of heterogeneous nuclear ribonucleoproteins and is predicted to be involved in several cellular functions like transcription regulation, RNA splicing, mRNA transport in neurons and microRNA processing [5] Aberrant accumulation of mutated FUS has been found in the cytoplasm of motor neurons from ALS patients [6]. The mislocalization of FUS is based on a mutation in the nuclear localization signal of FUS [7]. However, it is still unclear if the cytoplasmic localization of FUS leads to a toxic gain of cytoplasmic function and/or a loss of nuclear function that might be crucial in the course of ALS. The goal of this project is to characterize the impact of ALS-associated FUS mutations on in vitro differentiated motor neurons. To this end, we edit the genome of induced pluripotent stem cells (iPSC) using transcription activator-like effector nucleases (TALENs) [8,9] to create three isogenic cell lines, each carrying an ALS-associated FUS mutation (G156E, R244C and P525L). These iPSC’s will then be differentiated to motor neurons according to a recently established protocol [10] and serve to study alterations in the transcriptome, proteome and metabolome upon the expression of ALS-associated FUS. With this approach, we hope to unravel the molecular mechanism leading to FUS-associated ALS and to provide new insight into the emerging connection between misregulation of RNA metabolism and neurodegeneration, a connection that is currently implied in a variety of additional neurological diseases, including spinocerebellar ataxia 2 (SCA-2), spinal muscular atrophy (SMA), fragile X syndrome, and myotonic dystrophy. [1] Cleveland, D.W. et al. (2001) Nat Rev Neurosci 2(11): 806-819 [2] Sathasivam, S. (2010) Singapore Med J 51(5): 367-372 [3] Schymick, J.C. et al. (2007) Hum Mol Genet Vol 16: 233-242 [4] Pratt, A.J. et al. (2012). Degener Neurol Neuromuscul Dis 2012(2): 1-14 [5] Lagier-Tourenne, C. Hum Mol Genet, 2010. 19(R1): p. R46-64 [6] Mochizuki, Y. et al. (2012) J Neurol Sci 323(1-2): 85-92 [7] Dormann, D. et al. (2010) EMBO J 29(16): 2841-2857 [8] Hockemeyer, D. et al. (2011) Nat Biotech 29(8): 731-734 [9] Joung, J.K. and J.D. Sander (2013) Nat Rev Mol Cell Biol 14(1): 49-55 [10]Amoroso, M.W. et al. (2013) J Neurosci 33(2): 574-586.

The secretome of induced pluripotent stem cells reduces lung fibrosis in part by hepatocyte growth factor.

INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic lung disease, resulting in respiratory insufficiency and reduced survival. Pulmonary fibrosis is a result of repeated alveolar epithelial microinjuries, followed by abnormal regeneration and repair processes in the lung. Recently, stem cells and their secretome have been investigated as a novel therapeutic approach in pulmonary fibrosis. We evaluated the potential of induced pluripotent stem cells (iPSC) conditioned media (iPSC-cm) to regenerate and repair the alveolar epithelium in vitro and improve bleomycin induced lung injury in vivo. METHODS IPSC-cm was collected from cultured iPSC derived from human foreskin fibroblasts and its biological effects on alveolar epithelial wound repair was studied in an alveolar wound healing assay in vitro. Furthermore, iPSC-cm was intratracheally instilled 7 days after bleomycin induced injury in the rat lungs and histologically and biochemically assessed 7 days after instillation. RESULTS iPSC-cm increased alveolar epithelial wound repair in vitro compared with medium control. Intratracheal instillation of iPSC-cm in bleomycin-injured lungs reduced the collagen content and improved lung fibrosis in the rat lung in vivo. Profibrotic TGFbeta1 and alpha-smooth muscle actin (alpha-sma) expression were markedly reduced in the iPSC-cm treated group compared with control. Antifibrotic hepatocyte growth factor (HGF) was detected in iPSC-cm in biologically relevant levels, and specific inhibition of HGF in iPSC-cm attenuated the antifibrotic effect of iPSC-cm, indicating a central role of HGF in iPSC-cm. CONCLUSION iPSC-cm increased alveolar epithelial wound repair in vitro and attenuated bleomycin induced fibrosis in vivo, partially due to the presence of HGF and may represent a promising novel, cell free therapeutic option against lung injury and fibrosis.

Effect of Combined Systemic and Local Morpholino Treatment on the Spinal Muscular Atrophy Δ7 Mouse Model Phenotype

BACKGROUND: Spinal muscular atrophy (SMA) is a fatal motor neuron disease of childhood that is caused by mutations in the SMN1 gene. Currently, no effective treatment is available. One possible therapeutic approach is the use of antisense oligos (ASOs) to redirect the splicing of the paralogous gene SMN2, thus increasing functional SMN protein production. Various ASOs with different chemical properties are suitable for these applications, including a morpholino oligomer (MO) variant with a particularly excellent safety and efficacy profile. OBJECTIVE: We investigated a 25-nt MO sequence targeting the negative intronic splicing silencer (ISS-N1) 10 to 34 region. METHODS: We administered a 25-nt MO sequence against the ISS-N1 region of SMN2 (HSMN2Ex7D[-10-34]) in the SMAΔ7 mouse model and evaluated the effect and neuropathologic phenotype. We tested different concentrations (from 2 to 24 nM) and delivery protocols (intracerebroventricular injection, systemic injection, or both). We evaluated the treatment efficacy regarding SMN levels, survival, neuromuscular phenotype, and neuropathologic features. RESULTS: We found that a 25-nt MO sequence against the ISS-N1 region of SMN2 (HSMN2Ex7D[-10-34]) exhibited superior efficacy in transgenic SMAΔ7 mice compared with previously described sequences. In our experiments, the combination of local and systemic administration of MO (bare or conjugated to octaguanidine) was the most effective approach for increasing full-length SMN expression, leading to robust improvement in neuropathologic features and survival. Moreover, we found that several small nuclear RNAs were deregulated in SMA mice and that their levels were restored by MO treatment. CONCLUSION: These results indicate that MO-mediated SMA therapy is efficacious and can result in phenotypic rescue, providing important insights for further development of ASO-based therapeutic strategies in SMA patients.

Deficit actual tool use in schizophrenia is linked to structural alterations in key regions of planning and executing of tool use and connecting fibers

Introduction: Schizophrenia patients frequently suffer from complex motor abnormalities including fine and gross motor disturbances, abnormal involuntary movements, neurological soft signs and parkinsonism. These symptoms occur early in the course of the disease, continue in chronic patients and may deteriorate with antipsychotic medication. Furthermore gesture performance is impaired in patients, including the pantomime of tool use. Whether schizophrenia patients would show difficulties of actual tool use has not yet been investigated. Human tool use is complex and relies on a network of distinct and distant brain areas. We therefore aim to test if schizophrenia patients had difficulties in tool use and to assess associations with structural brain imaging using voxel based morphometry (VBM) and tract based spatial statistics (TBSS). Methode: In total, 44 patients with schizophrenia (DSM-5 criteria; 59% men, mean age 38) underwent structural MR imaging and performed the Tool-Use test. The test examines the use of a scoop and a hammer in three conditions: pantomime (without the tool), demonstration (with the tool) and actual use (with a recipient object). T1-weighted images were processed using SPM8 and DTI-data using FSL TBSS routines. To assess structural alterations of impaired tool use we first compared gray matter (GM) volume in VBM and white matter (WM) integrity in TBSS data of patients with and without difficulties of actual tool use. Next we explored correlations of Tool use scores and VBM and TBSS data. Group comparisons were family wise error corrected for multiple tests. Correlations were uncorrected (p < 0.001) with a minimum cluster threshold of 17 voxels (equivalent to a map-wise false positive rate of alpha < 0.0001 using a Monte Carlo procedure). Results: Tool use was impaired in schizophrenia (43.2% pantomime, 11.6% demonstration, 11.6% use). Impairment was related to reduced GM volume and WM integrity. Whole brain analyses detected an effect in the SMA in group analysis. Correlations of tool use scores and brain structure revealed alterations in brain areas of the dorso-dorsal pathway (superior occipital gyrus, superior parietal lobule, and dorsal premotor area) and the ventro-dorsal pathways (middle occipital gyrus, inferior parietal lobule) the action network, as well as the insula and the left hippocampus. Furthermore, significant correlations within connecting fiber tracts - particularly alterations within the bilateral corona radiata superior and anterior as well as the corpus callosum -were associated with Tool use performance. Conclusions: Tool use performance was impaired in schizophrenia, which was associated with reduced GM volume in the action network. Our results are in line with reports of impaired tool use in patients with brain lesions particularly of the dorso-dorsal and ventro-dorsal stream of the action network. In addition an effect of tool use on WM integrity was shown within fiber tracts connecting regions important for planning and executing tool use. Furthermore, hippocampus is part of a brain system responsible for spatial memory and navigation.The results suggest that structural brain alterations in the common praxis network contribute to impaired tool use in schizophrenia.

A large animal model of spinal muscular atrophy and correction of phenotype.

OBJECTIVES Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein, which results in motoneuron loss. Therapeutic strategies to increase SMN levels including drug compounds, antisense oligonucleotides, and scAAV9 gene therapy have proved effective in mice. We wished to determine whether reduction of SMN in postnatal motoneurons resulted in SMA in a large animal model, whether SMA could be corrected after development of muscle weakness, and the response of clinically relevant biomarkers. METHODS Using intrathecal delivery of scAAV9 expressing an shRNA targeting pig SMN1, SMN was knocked down in motoneurons postnatally to SMA levels. This resulted in an SMA phenotype representing the first large animal model of SMA. Restoration of SMN was performed at different time points with scAAV9 expressing human SMN (scAAV9-SMN), and electrophysiology measurements and pathology were performed. RESULTS Knockdown of SMN in postnatal motoneurons results in overt proximal weakness, fibrillations on electromyography indicating active denervation, and reduced compound muscle action potential (CMAP) and motor unit number estimation (MUNE), as in human SMA. Neuropathology showed loss of motoneurons and motor axons. Presymptomatic delivery of scAAV9-SMN prevented SMA symptoms, indicating that all changes are SMN dependent. Delivery of scAAV9-SMN after symptom onset had a marked impact on phenotype, electrophysiological measures, and pathology. INTERPRETATION High SMN levels are critical in postnatal motoneurons, and reduction of SMN results in an SMA phenotype that is SMN dependent. Importantly, clinically relevant biomarkers including CMAP and MUNE are responsive to SMN restoration, and abrogation of phenotype can be achieved even after symptom onset.

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein-Friesian (bMECCH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA). RESULTS The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

Central and peripheral defects in motor units of the diaphragm of spinal muscular atrophy mice.

Spinal muscular atrophy (SMA) is characterized by motoneuron loss and muscle weakness. However, the structural and functional deficits that lead to the impairment of the neuromuscular system remain poorly defined. By electron microscopy, we previously found that neuromuscular junctions (NMJs) and muscle fibres of the diaphragm are among the earliest affected structures in the severe mouse SMA model. Because of certain anatomical features, i.e. its thinness and its innervation from the cervical segments of the spinal cord, the diaphragm is particularly suitable to characterize both central and peripheral events. Here we show by immunohistochemistry that, at postnatal day 3, the cervical motoneurons of SMA mice receive less stimulatory synaptic inputs. Moreover, their mitochondria become less elongated which might represent an early stage of degeneration. The NMJs of the diaphragm of SMA mice show a loss of synaptic vesicles and active zones. Moreover, the partly innervated endplates lack S100 positive perisynaptic Schwann cells (PSCs). We also demonstrate the feasibility of comparing the proteomic composition between diaphragm regions enriched and poor in NMJs. By this approach we have identified two proteins that are significantly upregulated only in the NMJ-specific regions of SMA mice. These are apoptosis inducing factor 1 (AIFM1), a mitochondrial flavoprotein that initiates apoptosis in a caspase-independent pathway, and four and a half Lim domain protein 1 (FHL1), a regulator of skeletal muscle mass that has been implicated in several myopathies.

Neuropeptide Y restores non-receptor-mediated vasoconstrictive action in superior mesenteric arteries in portal hypertension

BACKGROUND & AIMS Vascular hyporeactivity to vasoconstrictors contributes to splanchnic arterial vasodilatation and hemodynamic dysregulation in portal hypertension. Neuropeptide Y (NPY), a sympathetic cotransmitter, has been shown to improve adrenergic vascular contractility in portal hypertensive rats and markedly attenuate hyperdynamic circulation. To further characterize the NPY-effects in portal hypertension, we investigated its role for non-receptor-mediated vasoconstriction in the superior mesenteric artery (SMA) of portal vein ligated (PVL) and sham-operated rats. METHODS Ex vivo SMA perfusion of PVL and sham rats was used to analyse the effects of NPY on pressure response to non-receptor-mediated vasoconstriction. Dose-response curves to KCl (30-300 mM) were used to bypass G protein-coupled receptor mechanisms. Potential involvement of the cyclooxygenase-pathway was tested by non-selective cyclooxygenase-inhibition using indomethacin. RESULTS KCl-induced vascular contractility but not vascular sensitivity was significantly attenuated in PVL rats as compared with sham rats. Administration of NPY resulted in an augmentation of KCl-evoked vascular sensitivity being not different between study groups. However, KCl-induced vascular contractility was markedly more enhanced in PVL rats, thus, vascular response was no more significantly different between PVL and sham rats after addition of NPY. Administration of indomethacin abolished the NPY-induced enhancement of vasoconstriction. CONCLUSIONS Receptor-independent vascular contractility is impaired in mesenteric arteries in portal hypertension. NPY improves non-receptor mediated mesenteric vasoconstriction more effective in portal hypertension than in healthy conditions correcting splanchnic vascular hyporesponsiveness. This beneficial vasoactive action of NPY adds to its well known more pronounced effects on adrenergic vasoconstriction in portal hypertension making it a promising therapeutic agent in portal hypertension.

Elucidating the role of mesenchymal stem cell participation in the tumor microenvironment

To meet the requirements for rapid tumor growth, a complex array of non-neoplastic vascular, fibroblastic, and immune cells are recruited to the tumor microenvironment. Understanding the origin, composition, and mechanism(s) for recruitment of these stromal components will help identify areas for therapeutic intervention. Previous findings have suggested that ex-vivo expanded bone marrow-derived MSC home to the sites of tumor development, responding to inflammatory signals and can serve as effective drug delivery vehicles. Therefore, we first sought to fully assess conditions under which MSC migrate to and incorporate into inflammatory microenvironments and the consequences of modulated inflammation. MSC delivered to animals bearing inflammatory insults were monitored by bioluminescence imaging and displayed specific tropism and selective incorporation into all tumor and wound sites. These findings were consistent across routes of tumor establishment, MSC administration, and immunocompetence. MSC were then used as drug delivery vehicles, transporting Interferon β to sites of pancreatic tumors. This therapy was effective at inhibiting pancreatic tumor growth under homeostatic conditions, but inhibition was lost when inflammation was decreased with CDDO-Me combination treatment. Next, to examine the endogenous tumor microenvironment, a series of tissue transplant experiments were carried out in which tissues were genetically labeled and engrafted in recipients prior to tumor establishment. Tumors were then analyzed for markers of tumor associated fibroblasts (TAF): α-smooth muscle actin (α-SMA), nerve glia antigen 2 (NG2), fibroblast activation protein (FAP), and fibroblast specific protein (FSP) as well as endothelial marker CD31 and macrophage marker F4/80. We determined the majority of α-SMA+, NG2+ and CD31+ cells were non-bone marrow derived, while most FAP+, FSP+, and F4/80+ cells were recruited from the bone marrow. In accord, transplants of prospectively isolated BM MSC prior to tumor development indicated that these cells were recruited to the tumor microenvironment and co-expressed FAP and FSP. In contrast, fat transplant experiments revealed recruited fat derived cells co-expressed α-SMA, NG2, and CD31. These results indicate TAF are a heterogeneous population composed of subpopulations with distinct tissues of origin. These models have provided a platform upon which further investigation into tumor microenvironment composition and tests for candidate drugs can be performed. ^

Evaluación de la profundidad de la fisuración superficial descendente en pavimentos asfálticos mediante técnicas de ultrasonidos. Validación teórico-práctica y modelos

La fisuración iniciada en la superficie de los pavimentos asfálticos constituye uno de los más frecuentes e importantes modos de deterioro que tienen lugar en los firmes bituminosos, como han demostrado los estudios teóricos y experimentales llevados a cabo en la última década. Sin embargo, este mecanismo de fallo no ha sido considerado por los métodos tradicionales de diseño de estos firmes. El concepto de firmes de larga duración se fundamenta en un adecuado seguimiento del proceso de avance en profundidad de estos deterioros y la intervención en el momento más apropiado para conseguir mantenerlos confinados como fisuras de profundidad parcial en la capa superficial más fácilmente accesible y reparable, de manera que pueda prolongarse la durabilidad y funcionalidad del firme y reducir los costes generalizados de su ciclo de vida. Por lo tanto, para la selección de la estrategia óptima de conservación de los firmes resulta esencial disponer de metodologías que posibiliten la identificación precisa in situ de la fisuración descendente, su seguimiento y control, y que además permitan una determinación fiable y con alto rendimiento de su profundidad y extensión. En esta Tesis Doctoral se presentan los resultados obtenidos mediante la investigación sistemática de laboratorio e in situ llevada a cabo para la obtención de datos sobre fisuración descendente en firmes asfálticos y para el estudio de procedimientos de evaluación de la profundidad de este tipo de fisuras empleando técnicas de ultrasonidos. Dichos resultados han permitido comprobar que la metodología no destructiva propuesta, de rápida ejecución, bajo coste y sencilla implementación (principalmente empleada hasta el momento en estructuras metálicas y de hormigón, debido a las dificultades que introduce la naturaleza viscoelástica de los materiales bituminosos) puede ser aplicada con suficiente fiabilidad y repetibilidad sobre firmes asfálticos. Las medidas resultan asimismo independientes del espesor total del firme. Además, permite resolver algunos de los inconvenientes frecuentes que presentan otros métodos de diagnóstico de las fisuras de pavimentos, tales como la extracción de testigos (sistema destructivo, de alto coste y prolongados tiempos de interrupción del tráfico) o algunas otras técnicas no destructivas como las basadas en medidas de deflexiones o el georradar, las cuales no resultan suficientemente precisas para la investigación de fisuras superficiales. Para ello se han realizado varias campañas de ensayos sobre probetas de laboratorio en las que se han estudiado diferentes condiciones empíricas como, por ejemplo, distintos tipos de mezclas bituminosas en caliente (AC, SMA y PA), espesores de firme y adherencias entre capas, temperaturas, texturas superficiales, materiales de relleno y agua en el interior de las grietas, posición de los sensores y un amplio rango de posibles profundidades de fisura. Los métodos empleados se basan en la realización de varias medidas de velocidad o de tiempo de transmisión del pulso ultrasónico sobre una única cara o superficie accesible del material, de manera que resulte posible obtener un coeficiente de transmisión de la señal (mediciones relativas o autocompensadas). Las mediciones se han realizado a bajas frecuencias de excitación mediante dos equipos de ultrasonidos diferentes dotados, en un caso, de transductores de contacto puntual seco (DPC) y siendo en el otro instrumento de contacto plano a través de un material especialmente seleccionado para el acoplamiento (CPC). Ello ha permitido superar algunos de los tradicionales inconvenientes que presenta el uso de los transductores convencionales y no precisar preparación previa de las superficies. La técnica de autocalibración empleada elimina los errores sistemáticos y la necesidad de una calibración local previa, demostrando el potencial de esta tecnología. Los resultados experimentales han sido comparados con modelos teóricos simplificados que simulan la propagación de las ondas ultrasónicas en estos materiales bituminosos fisurados, los cuales han sido deducidos previamente mediante un planteamiento analítico y han permitido la correcta interpretación de dichos datos empíricos. Posteriormente, estos modelos se han calibrado mediante los resultados de laboratorio, proporcionándose sus expresiones matemáticas generalizadas y gráficas para su uso rutinario en las aplicaciones prácticas. Mediante los ensayos con ultrasonidos efectuados en campañas llevadas a cabo in situ, acompañados de la extracción de testigos del firme, se han podido evaluar los modelos propuestos. El máximo error relativo promedio en la estimación de la profundidad de las fisuras al aplicar dichos modelos no ha superado el 13%, con un nivel de confianza del 95%, en el conjunto de todos los ensayos realizados. La comprobación in situ de los modelos ha permitido establecer los criterios y las necesarias recomendaciones para su utilización sobre firmes en servicio. La experiencia obtenida posibilita la integración de esta metodología entre las técnicas de auscultación para la gestión de su conservación. Abstract Surface-initiated cracking of asphalt pavements constitutes one of the most frequent and important types of distress that occur in flexible bituminous pavements, as clearly has been demonstrated in the technical and experimental studies done over the past decade. However, this failure mechanism has not been taken into consideration for traditional methods of flexible pavement design. The concept of long-lasting pavements is based on adequate monitoring of the depth and extent of these deteriorations and on intervention at the most appropriate moment so as to contain them in the surface layer in the form of easily-accessible and repairable partial-depth topdown cracks, thereby prolonging the durability and serviceability of the pavement and reducing the overall cost of its life cycle. Therefore, to select the optimal maintenance strategy for perpetual pavements, it becomes essential to have access to methodologies that enable precise on-site identification, monitoring and control of top-down propagated cracks and that also permit a reliable, high-performance determination of the extent and depth of cracking. This PhD Thesis presents the results of systematic laboratory and in situ research carried out to obtain information about top-down cracking in asphalt pavements and to study methods of depth evaluation of this type of cracking using ultrasonic techniques. These results have demonstrated that the proposed non-destructive methodology –cost-effective, fast and easy-to-implement– (mainly used to date for concrete and metal structures, due to the difficulties caused by the viscoelastic nature of bituminous materials) can be applied with sufficient reliability and repeatability to asphalt pavements. Measurements are also independent of the asphalt thickness. Furthermore, it resolves some of the common inconveniences presented by other methods used to evaluate pavement cracking, such as core extraction (a destructive and expensive procedure that requires prolonged traffic interruptions) and other non-destructive techniques, such as those based on deflection measurements or ground-penetrating radar, which are not sufficiently precise to measure surface cracks. To obtain these results, extensive tests were performed on laboratory specimens. Different empirical conditions were studied, such as various types of hot bituminous mixtures (AC, SMA and PA), differing thicknesses of asphalt and adhesions between layers, varied temperatures, surface textures, filling materials and water within the crack, different sensor positions, as well as an ample range of possible crack depths. The methods employed in the study are based on a series of measurements of ultrasonic pulse velocities or transmission times over a single accessible side or surface of the material that make it possible to obtain a signal transmission coefficient (relative or auto-calibrated readings). Measurements were taken at low frequencies by two short-pulse ultrasonic devices: one equipped with dry point contact transducers (DPC) and the other with flat contact transducers that require a specially-selected coupling material (CPC). In this way, some of the traditional inconveniences presented by the use of conventional transducers were overcome and a prior preparation of the surfaces was not required. The auto-compensating technique eliminated systematic errors and the need for previous local calibration, demonstrating the potential for this technology. The experimental results have been compared with simplified theoretical models that simulate ultrasonic wave propagation in cracked bituminous materials, which had been previously deduced using an analytical approach and have permitted the correct interpretation of the aforementioned empirical results. These models were subsequently calibrated using the laboratory results, providing generalized mathematical expressions and graphics for routine use in practical applications. Through a series of on-site ultrasound test campaigns, accompanied by asphalt core extraction, it was possible to evaluate the proposed models, with differences between predicted crack depths and those measured in situ lower than 13% (with a confidence level of 95%). Thereby, the criteria and the necessary recommendations for their implementation on in-service asphalt pavements have been established. The experience obtained through this study makes it possible to integrate this methodology into the evaluation techniques for pavement management systems.

The flexibility matrix o timber composite beams with a discrete connection system

This work presents a method for the analysis of timber composite beams which considers the slip in the connection system, based on assembling the flexibility matrix of the whole structure. This method is based on one proposed by Tommola and Jutila (2001). This paper extends the method to the case of a gap between two pieces with an arbitrary location at the first connector, which notably broadens its practical application. The addition of the gap makes it possible to model a cracked zone in concrete topping, as well as the case in which forming produces the gap. The consideration of induced stresses due to changes in temperature and moisture content is also described, while the concept of equivalent eccentricity is generalized. This method has important advantages in connection with the current European Standard EN 1995-1-1: 2004, as it is able to deal with any type of load, variable section, discrete and non-regular connection systems, a gap between the two pieces, and variations in temperature and moisture content. Although it could be applied to any structural system, it is specially suited for the case of simple supported and continuous beams. Working examples are presented at the end, showing that the arrangement of the connection notably modifies shear force distribution. A first interpretation of the results is made on the basis of the strut and tie theory. The examples prove that the use of EC-5 is unsafe when, as a rule of thumb, the strut or compression field between the support and the first connector is at an angle with the axis of the beam of less than 60º.

Integration versus segregation in functional brain networks

We propose a new methodology to evaluate the balance between segregation and integration in functional brain networks by using singular value decomposition techniques. By means of magnetoencephalography, we obtain the brain activity of a control group of 19 individuals during a memory task. Next, we project the node-to-node correlations into a complex network that is analyzed from the perspective of its modular structure encoded in the contribution matrix. In this way, we are able to study the role that nodes play I/O its community and to identify connector and local hubs. At the mesoscale level, the analysis of the contribution matrix allows us to measure the degree of overlapping between communities and quantify how far the functional networks are from the configuration that better balances the integrated and segregated activity

Bending continuous structures with SMAs: a novel robotic fish design

In this paper, we describe our research on bio-inspired locomotion systems using deformable structures and smart materials, concretely shape memory alloys (SMAs). These types of materials allow us to explore the possibility of building motor-less and gear-less robots. A swimming underwater fish-like robot has been developed whose movements are generated using SMAs. These actuators are suitable for bending the continuous backbone of the fish, which in turn causes a change in the curvature of the body. This type of structural arrangement is inspired by fish red muscles, which are mainly recruited during steady swimming for the bending of a flexible but nearly incompressible structure such as the fishbone. This paper reviews the design process of these bio-inspired structures, from the motivations and physiological inspiration to the mechatronics design, control and simulations, leading to actual experimental trials and results. The focus of this work is to present the mechanisms by which standard swimming patterns can be reproduced with the proposed design. Moreover, the performance of the SMA-based actuators’ control in terms of actuation speed and position accuracy is also addressed.