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Introduction: Bone marrow-derived mesenchymal stem cells (BM-MSC) are a potential therapeutic tool due to their ability for migrating and producing neuroprotector factors when transplanted. The aim of this study was to evaluate the short-time effects of a BM-MSC experimental therapy in the hyh mouse model with severe obstructive hydrocephalus. Methods: BM-MSC were characterized in vitro and then injected into the ventricles of hyh mice. Wild-type and saline-injected hyh mice were used as controls. Samples were studied by analyzing and comparing mRNA, protein and metabolites level expression in control and damaged tissue. Results: Undifferentiated BM-MSC were found to: i) spread into the periventricular astrocyte reaction region after four days post-injection, and, ii) be producing neuroprotector factors (GDNF and VEGF). Astrocytes located in periventricular edematous region increased their aquaporin-4 expression, as well as Slit2 expression (neuroprotective and anti-inflammatory molecule). There was also a significant reduction of osmolytes such as taurine and neuroexcytotoxic glutamate. Halved apoptotic cell death was detected in the periventricular walls. Conclusions: BM-MSC lead to recovery of the severe neurodegenerative conditions associated to congenital hydrocephalus mediated by reactive astrocytes. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Supported by Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech, and PI15/0619 (ISCIII/FEDER).

Introduction: In obstructive congenital hydrocephalus, cerebrospinal fluid accumulation is associated with high intracranial pressure (ICP), ischemia/hypoxia, metabolic impairment, neuronal damage and astrocytic reaction. The hyh mutant mice exhibit two different forms of hydrocephalus evolution: severe and moderate. A study was carried out in hyh mice to detect a metabolite profile that define the tissue response in each hydrocephalus form. Methods: Metabolites levels in brain cortex were analyzed with 1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance (1H HR-MAS) spectroscopy. The study was complemented with ICP recording and histopathological analysis. Results: Mice with severe hydrocephalus were found to have higher ICP and stronger astrocytic reaction. Several metabolites including glutamate and glutamine were found to correlate with the severity of hydrocephalus. The whole metabolite profile may be explained based in differential astrocyte reactions, neurodegenerative and ischemic conditions. The glutamate transporter EAAT2 and the metabolite taurine were found as key histopathological markers for the damaged parenchyma. Conclusions: Spectroscopy allowed the detection of a metabolite profile related to intracranial pressure and hydrocephalus severity, and therefore can be useful to monitor the efficacy of experimental therapies. Supported by Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech, and PI15/0619 (ISCIII/FEDER). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Supported by Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech, and PI15/0619 (ISCIII/FEDER).

In congenital hydrocephalus, cerebrospinal fluid accumulation is associated to ischemia/hypoxia, metabolic impairment, neuronal damage and astrocytic reaction, which cause significant mortality and life-long neurological complications. Currently, there are no effective therapies for congenital hydrocephalus. Bone marrow-derived mesenchymal stem cells (BM-MSC) are considered as a potential therapeutic tool for neurodegenerative diseases due to their ability for migrating and producing neuroprotector factors when they are transplanted. The aim of this research was to study the ability of BM-MSC to reach the degenerated regions and to detect their neuroprotector effects, using an animal model of congenital hydrocephalus, the hyh mouse. Fluorescent BM-MSC were analyzed by flow-cytometry and multilineage cell differentiation. BM-MSC were brain-ventricle injected into hyh mice. Wild-type and saline-injected hyh mice were used as controls. Inmunohistochemical, RT-PCR and High Resolution Magic Angle Spinning spectroscopy (HRMAS) analyses were carried out. After administration, integrated BM-MSC were identified inside the periventricular astrocyte reaction. They were detected producing glial-derived neuroprotector factor (GDNF), neural growth factor (NGF), and brain-derived neuroprotector factor (BDNF). Tissue recovery was detected with a reduction of apoptotic cells in the periventricular walls and of the levels of glutamate, glutamine, taurine, and creatine, all of them markers of tissue damage in hydrocephalus. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. ISCIII PI15/00619 y FEDER

Background Bone marrow-derived mesenchymal stem cells (BM-MSC) are considered as a potential therapeutic tool for neurodegenerative diseases due to their ability for migrating into damaged tissue. Additionally, these cells have been proven to produce neuroprotective factors when they are transplanted into damaged tissue. In this research we uncover a neuroprotective role of BM-MSC on congenital hydrocephalus and we study the molecular mechanisms behind it. Materials and methods Fluorescent BM-MSC were analyzed by flow-cytometry and multilineage cell differentiation. They were brain-ventricle injected into hyh hydrocephalic mice. Wild-type and saline-injected hyh mice were used as controls. Inmunohistochemical analyses were performed in fixed brain sections. Inflammatory reaction and neuroprotective factors were studied using quantitative RT-PCR. Metabolites and osmolytes related to brain damage were studied by High Resolution Magic Angle Spinning spectroscopy (HRMAS). RT-PCR and HRMAS were performed in fresh tissue. Results Integrated BM-MSC were identified inside the periventricular astrocyte reaction. IL-1alpha/beta and IL6 levels indicated the absence of inflammatory response in the transplanted tissue after BM-MSC integration. BM-MSC were found producing neuroprotective factors, including GDNF, NGF, BDNF and VEGF. Reduction of osmolytes and neural/glial-function related metabolites levels were detected in hyh mice after BM-MSC injection. These levels mimicked the wild-type situation and indicate partial recovery. Conclusions The BM-MSC can play a neuroprotective effect on congenital hydrocephalus in the hyh mouse by the production of neuroprotective factors and the reduction of osmolytes and metabolites related to tissue damage. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Background Cerebrospinal fluid accumulation in hydrocephalus produces an elevation of intraventricular pressure with pathological consequences on the periventricular brain parenchyma including ischemia, oedema, oxidative stress, and accumulation of metabolic waste products. Here we studied in the hyh mouse, an animal model of congenital hydrocephalus, the role of reactive astrocytes in this clinical degenerative condition. Materials and Methods Wild type and hydrocephalic hyh mice at 30 days of postnatal age were used. Three metabolites related to the oxidative and neurotoxic conditions were analysed in ex vivo samples (glutathione, glutamine and taurine) using High Resolution Magic Angle Spinning (HR-MAS). Glutathione synthetase and peroxidase, glutamine synthetase, kidney-type glutaminase (KGA), and taurine/taurine transporter were immunolocated in brain sections. Results Levels of the metabolites were remarkably higher in hydrocephalic conditions. Glutathione peroxidase and synthetase were both detected in the periventricular reactive astrocytes and neurons. Taurine was mostly found free in the periventricular parenchyma and in the reactive astrocytes, and the taurine transporter was mainly present in the neurons located in such regions. Glutamine synthetase was found in reactive astrocytes. Glutaminase was also detected in the reactive astrocytes and in periventricular neurons. These results suggest a possible protective response of reactive astrocytes against oxidative stress and neurotoxic conditions. Conclusions Astrocyte reaction seems to trigger an anti-oxidative and anti-neurotoxic response in order to ameliorate pathological damage in periventricular areas of the hydrocephalic mice. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. PI15-00619 to AJJ.

Magnetic nanoparticles have been extensively investigated for in vivo nanomedical applications in the last decade. PEG coating improves the solubility and stability of the nanoparticles, and provides stealth properties by preventing opsonization and subsequent removal by the reticuloendothelial system. All these effects conferred by the PEG coating are dependent on its molecular weight. Therefore, the selection of the right MW of PEG is a crucial point in the design of new nanomaterials for in vivo applications. The aim of this work lies in the in vivo optimization of the circulation times of small iron oxide nanoparticles by coating them with PEG of different MWs. PEGylated small superparamagnetic iron oxide nanoparticles (PEG-SPIONs), using PEG MWs ranging from 600 to 8000, were synthesized following a ligand exchange methodology, resulting in highly stable and water-soluble nanoparticles. Semi-quantitative and quantitative MRI studies allowed us to track the pharmacokinetics and biodistribution of intravenously injected PEG-SPIONs (HD < 50 nm) in vivo up to one week. Results show that high MW PEGs (6000–8000) lead to nanoparticle aggregation and low MW PEGs (≤1500) are not able to stabilize the 6 nm iron oxide nanoparticles in physiological medium or confer stealth properties, thus leading to rapid recognition by the RES. In contrast, PEG3000-SPIONs show excellent in vivo behavior, they do not aggregate and they exhibit better stealth properties, giving rise to slower liver uptake and longer circulation times. In conclusion, 3000 Da turned out to be the optimal MW for the PEGylation of small nanoparticles (∼6 nm) designed for biomedical applications in which long circulation times together with moderate liver uptake are desirable.

Background: Changes in the profile of metabolites in brain and/or fluids can be useful to evaluate the severity and evolution of hydrocephalus, and consequently to help in treatment decisions. Magnetic Resonance Spectroscopy can be used in ex vivo samples for such purposes. This study was designed to evaluate the levels of metabolites in the hyh mouse brain with congenital hydrocephalus by High Resolution Magic Angle Spinning (HR-MAS) Magnetic Resonance Spectroscopy (MRS). Materials and Methods: Wild type and hydrocephalic hyh mice at 30 days of postnatal age were sacrificed (n = 10-15 mice/disease condition), and hippocampus and neocortex were quickly dissected out, frozen in dry ice, and stored at -80ºC, before analysis by HR-MAS. Results: Similar levels of choline (Cho) were detected in the hippocampus and neorcortex of hydrocephalic mice compared to control samples, however phosphocholine (PCh) and glycerophosphorylcholine (GPC) displayed lower levels. These molecules are implied in the Kennedy pathway of phosphatidylcholine metabolism. The antioxidant tripeptide glutathione (GSH) was detected in higher quantities in the hydrocephalic mice, probably revealing a response to an oxidative metabolism. Other metabolites displayed remarkably higher levels in samples from hydrocephalic mice, such as creatine (Cr), taurine (Tau) and glutamine (Gln). Conclusions: HR-MAS was found as a reliable technique to detect differential levels of metabolites in small tissue biopsies samples from mice models with hydrocephalus. This technique represents a valuable tool for monitoring the degree of severity and/or the evolution of the disease in such models. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Supported by the grants PI12/0631 to AJJ; SERAM to MIML

El presente trabajo pretende iniciar una serie de estudios sobre el metabolismo nitrogenado en árboles adultos de la conífera Pinus pinaster (Aiton) en condiciones naturales. Durante un año completo se ha caracterizado el metabolismo de las acículas de árboles de P. pinaster que crecen en el paraje natural de Los Reales de Sierra Bermeja, Estepona (Málaga). Para ello se han seguido dos aproximaciones diferentes. En primer lugar se ha analizado la expresión génica mediante el empleo de un microarray para genes de P. pinaster desarrollado en nuestro laboratorio. Por otra parte se ha observado la acumulación de los distintos metabolitos en las acículas a lo largo del año mediante H1-NMR. Para la determinación de los metabolitos presentes en las muestras se ha generado una base de datos de espectros H1-NMR de 90 metabolitos. Para el análisis de los datos se han empleado herramientas estadísticas de clustering. Una de las principales técnicas que se han empleado para el análisis de las muestras ha sido el análisis de las redes de co-expresión de genes (WGCNA). Los resultados de los análisis de los datos sugieren que las acículas de P. pinaster presentan diferentes niveles de actividad a lo largo del año pudiéndose dividir en dos períodos principales, uno ligado al otoño y al invierno y el otro a la primavera y al verano.

Centro de ocio y cultura Castillo de Alicante. Convocatoria Abril. Plan 1996. Proyecto fin de carrera. Universidad Politécnica de Madrid. Escuela Técnica Superior de Arquitectura

Objective. To investigate if histogram analysis and visually assessed heterogeneity of diffusion-weighted imaging (DWI) with apparent diffusion coefficient (ADC) mapping can predict molecular subtypes of invasive breast cancers. Materials and Methods. In this retrospective study, 91 patients with invasive breast carcinoma who underwent preoperative magnetic resonance imaging (MRI) with DWI at our institution were included. Two radiologists delineated a 2-D region of interest (ROI) on ADC maps in consensus. Tumors were also independently classified into low and high heterogeneity based on visual assessment of DWI. First-order statistics extracted through histogram analysis within the ROI of the ADC maps (mean, 10th percentile, 50th percentile, 90th percentile, standard deviation, kurtosis, and skewness) and visually assessed heterogeneity were evaluated for associations with tumor receptor status (ER, PR, and HER2 status) as well as molecular subtype. Results. HER2-positive lesions demonstrated significantly higher mean (p=0.034), Perc50 (p=0.046), and Perc90 (p=0.040), with AUCs of 0.605, 0.592, and 0.652, respectively, than HER2-negative lesions. No significant differences were found in the histogram values for ER and PR statuses. Neither quantitative histogram analysis based on ADC maps nor qualitative visual heterogeneity assessment of DWI images was able to significantly differentiate between molecular subtypes, i.e., luminal A versus all other subtypes (luminal B, HER2-enriched, and triple negative) combined, luminal A and B combined versus HER2-enriched and triple negative combined, and triple negative versus all other types combined. Conclusion. Histogram analysis and visual heterogeneity assessment cannot be used to differentiate molecular subtypes of invasive breast cancer.

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina. Departamento de Bioquímica. Fecha de lectura: 20 de Diciembre de 2006.