Nuevas herramientas para el análisis de interacciones y adquisición automatizada inteligente en microscopia óptica

Tesis doctoral inédita, leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 08 de febrero de 2016La comunidad científica necesita de nuevas tecnologías y métodos que permitan profundizar en el entendimiento de los distintos procesos biológicos. En esta tesis presentamos nuevos abordajes en microscopía óptica que permiten resolver algunas de las limitaciones existentes en este campo. Por una parte, presentamos el desarrollo de un nuevo método denominado FRET que posibilita medir la interacción entre moléculas utilizando el fenómeno de transferencia de energía de la fluorescencia por resonancia, que permite resolver muchas de las limitaciones de los métodos existentes, haciendo este tipo de estudios más fiables y sensibles cuando se compara con algunos de los métodos más usados. Este nuevo método ha sido validado en estudios de interacción entre las moléculas CD44 y MT1-MMP, demostrando su utilidad en el campo. Aun así, el mayor inconveniente de dicho tipo de ensayos y en general de las adquisiciones de imagen complejas es el gran consumo de tiempo y recursos. Las opciones disponibles de automatización son aún muy básicas y poco eficientes. Por ejemplo, las técnicas de screening convencional (o adquisición aleatoria automatizada) necesitan de muestras homogéneas y con una localización estandarizada que muchas veces resulta imposible de conseguir en biología, ya sea por la baja frecuencia del evento de estudio como por su heterogeneidad. En este trabajo presentamos también el desarrollo y validación de una nueva técnica de “screening inteligente” denominado iMSRC (Intelligent Matrix Screening Remote Control) que posibilita realizar de forma fácil e intuitiva la adquisición de imágenes dirigida por análisis de imagen automatizada en microscopios de fluorescencia estándar y en microscopios confocales. Esta nueva herramienta permite la realización de estudios de microscopía óptica basados en la localización de eventos de baja frecuencia en una muestra y otros ensayos complejos, que antes eran prácticamente inviables.The scientific community needs new technologies and methods which may allow us to study biological processes in depth. In this Ph.D. Thesis we present new approaches in optical microscopy which that allow solving some of the current limitations in this field. On the one hand, we present here the development of a new method named FRET which allows the measurement of molecular interactions by using the fluorescence resonance energy transfer phenomenon. This novel method overcomes most of the limitations from previously existing methods. The measurements with this new method proved to be more reliable and sensitive than most common ones based in optical microscopy. Furthermore, we have validated this method in the study of the interaction between CD44 and MT1MMP. The main problem of this kind of assays and of complex image acquisition in microscopy in general is that they require long acquisition time and resources. The currently available options for automation are still very simple and poorly efficient. For example conventional screening approaches (random automated acquisition of images) require homogenous samples and the location of the events of interest must be predefined. This is often difficult to achieve in most common assays characterized by biological heterogeneity or when trying to quantify rare events. To overcome these limitations we have developed and validated the iMSRC (intelligent matrix screening remote control), a new platform for running intelligent screenings (automated acquisition driven by image analysis) in a user friendly manner, and compatible with conventional and confocal microscopes. This new approach allow performing microscopy studies based on the detection of low frequency events in a sample and other complex assays that were almost impossible before

The balance between gyrase and topoisomerase I activities determines levels of supercoiling, nucleoid compaction, and viability in bacteria

Two enzymes are responsible for maintaining supercoiling in the human pathogen Streptococcus pneumoniae, gyrase (GyrA2GyrB2) and topoisomerase I. To attain diverse levels of topoisomerase I (TopoI, encoded by topA), two isogenic strains derived from wild-type strain R6 were constructed: PZn topA, carrying an ectopic topA copy under the control of the ZnSO4-regulated PZn promoter and its derivative ΔtopAPZn topA, which carries a topA deletion at its native chromosomal location. We estimated the number of TopoI and GyrA molecules per cell by using Western-blot and CFUs counting, and correlated these values with supercoiling levels. Supercoiling was estimated in two ways. We used classical 2D-agarose gel electrophoresis of plasmid topoisomers to determine supercoiling density (σ) and we measured compaction of nucleoids using for the first time super-resolution confocal microscopy. Notably, we observed a good correlation between both supercoiling calculations. In R6, with σ = -0.057, the average number of GyrA molecules per cell (2,184) was higher than that of TopoI (1,432), being the GyrA:TopoI proportion of 1:0.65. In ΔtopAPZn topA, the number of TopoI molecules depended, as expected, on ZnSO4 concentration in the culture media, being the proportions of GyrA:TopoI molecules in 75, 150, and 300 μM ZnSO4 of 1:0.43, 1:0.47, and 1:0.63, respectively, which allowed normal supercoiling and growth. However, in the absence of ZnSO4, a higher GyrA:TopoI ratio (1:0.09) caused hyper-supercoiling (σ = -0.086) and lethality. Likewise, growth of ΔtopAPZn topA in the absence of ZnSO4 was restored when gyrase was inhibited with novobiocin, coincidentally with the resolution of hyper-supercoiling (σ change from -0.080 to -0.068). Given that TopoI is a monomer and two molecules of GyrA are present in the gyrase heterotetramer, the gyrase:TopoI enzymes proportion would be 1:1.30 (wild type R6) or of 1:1.26-0.86 (ΔtopAPZn topA under viable conditions). Higher proportions, such as 1:0.18 observed in ΔtopAPZn topA in the absence of ZnSO4 yielded to hyper-supercoiling and lethality. These results support a role of the equilibrium between gyrase and TopoI activities in supercoiling maintenance, nucleoid compaction, and viability. Our results shed new light on the mechanism of action of topoisomerase-targeting antibiotics, paving the way for the use of combination therapies.This work was supported by project PID2021-124738OB-100 to AGC, financed by MCIN/AEI/10.13039/501100011033/FEDER, UE.S

Podoplanin associates with CD44 to promote directional cell migration

This article is under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.-- et al.Podoplanin is a transmembrane glycoprotein up-regulated in different human tumors, especially those derived from squamous stratified epithelia (SCCs). Its expression in tumor cells is linked to increased cell migration and invasiveness; however, the mechanisms underlying this process remain poorly understood. Here we report that CD44, the major hyaluronan (HA) receptor, is a novel partner for podoplanin. Expression of the CD44 standard isoform (CD44s) is coordinately up-regulated together with that of podoplanin during progression to highly aggressive SCCs in a mouse skin model of carcinogenesis, and during epithelial-mesenchymal transition (EMT). In carcinoma cells, CD44 and podoplanin colocalize at cell surface protrusions. Moreover, CD44 recruitment promoted by HA-coated beads or cross-linking with a specific CD44 antibody induced corecruitment of podoplanin. Podoplanin-CD44s interaction was demonstrated both by coimmunoprecipitation experiments and, in vivo, by fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM), the later confirming its association on the plasma membrane of cells with a migratory phenotype. Importantly, we also show that podoplanin promotes directional persistence of motility in epithelial cells, a feature that requires CD44, and that both molecules cooperate to promote directional migration in SCC cells. Our results support a role for CD44-podoplanin interaction in driving tumor cell migration during malignancy.This work was supported by grant SAF2007-63821 from the Spanish Ministry of Science and Innovation (to M.Q.), the Royal Society University Research Fellowship (to M.P.), Medical Research Council (to G.E.J.) EU FP7 T3Net Consortium (GEJ), and Cancer Research UK (to G.E.J. and E.M.V.).Peer reviewe

Physiologic and Transcriptomic Effects Triggered by Overexpression of Wild Type and Mutant DNA Topoisomerase I in Streptococcus pneumoniae

Topoisomerase I (TopoI) in Streptococcus pneumoniae, encoded by topA, is a suitable target for drug development. Seconeolitsine (SCN) is a new antibiotic that specifically blocks this enzyme. We obtained the topARA mutant, which encodes an enzyme less active than the wild type (topAWT) and more resistant to SCN inhibition. Likely due to the essentiality of TopoI, we were unable to replace the topAWT allele by the mutant topARA version. We compared the in vivo activity of TopoIRA and TopoIWT using regulated overexpression strains, whose genes were either under the control of a moderately (PZn) or a highly active promoter (PMal). Overproduction of TopoIRA impaired growth, increased SCN resistance and, in the presence of the gyrase inhibitor novobiocin (NOV), caused lower relaxation than TopoIWT. Differential transcriptomes were observed when the topAWT and topARA expression levels were increased about 5-fold. However, higher increases (10-15 times), produced a similar transcriptome, affecting about 52% of the genome, and correlating with a high DNA relaxation level with most responsive genes locating in topological domains. These results confirmed that TopoI is indeed the target of SCN in S. pneumoniae and show the important role of TopoI in global transcription, supporting its suitability as an antibiotic target.This research and the APC were funded by project PID2021-124738OB-100 to AGC, financed by MCIN/AEI/10.13039/501100011033/FEDER, UE. M.G.-L. is the beholder of a PhD Contract from Instituto de Salud Carlos III.S

Dynamics of the pedestal transport during edge localized mode cycles at ASDEX Upgrade

The dynamic behaviour of the ion and electron energy, particle and momentum transport measured during type-I edge localized mode (ELM) cycles at ASDEX Upgrade is presented. Fast measurements of the ion and electron temperature profiles revelead that the ion and electron energy transport recover on different timescales, with the electrons recovering on a slower timescale (Cavedon et al 2017 Plasma Phys. Control. Fusion 59 105007). The dominant mechanism for the additional energy transport in the electron channel that could cause the delay in the electron temperature gradient (VTe) recovery is attributed to the depletion of energy caused by the ELM. The local sources and sinks for the electron channel in the steep gradient region are much smaller compared to the energy flux arriving from the pedestal top, indicating that the core plasma may dictate the local dynamics of the VTe recovery during the ELM cycle. A model for the edge momentum transport based on toroidal torque balance that takes into account the existence of poloidal impurity asymmetries has been developed. The analysis of the profile evolution during the ELM cycle shows that the model captures the dynamics of the rotation both before the ELM crash and during the recovery phase.European Commission (Euratom) Grant agreement No. 633053H2020 Marie-Sklodowska Curie programme (grant agreement No. 708257)European Union’s Horizon 2020 (grant agreement No. 805162

Disturbed circadian rhythm and retinal degeneration in a mouse model of Alzheimer’s disease

The circadian clock is synchronized to the 24 h day by environmental light which is transmitted from the retina to the suprachiasmatic nucleus (SCN) primarily via the retinohypothalamic tract (RHT). Circadian rhythm abnormalities have been reported in neurodegenerative disorders such as Alzheimer's disease (AD). Whether these AD-related changes are a result of the altered clock gene expression, retina degeneration, including the dysfunction in RHT transmission, loss of retinal ganglion cells and its electrophysiological capabilities, or a combination of all of these pathological mechanisms, is not known. Here, we evaluated transgenic APP/PS1 mouse model of AD and wild-type mice at 6- and 12-month-old, as early and late pathological stage, respectively. We noticed the alteration of circadian clock gene expression not only in the hypothalamus but also in two extra-hypothalamic brain regions, cerebral cortex and hippocampus, in APP/PS1 mice. These alterations were observed in 6-month-old transgenic mice and were exacerbated at 12 months of age. This could be explained by the reduced RHT projections in the SCN of APP/PS1 mice, correlating with downregulation of hypothalamic GABAergic response in APP/PS1 mice in advanced stage of pathology. Importantly, we also report retinal degeneration in APP/PS1 mice, including Aβ deposits and reduced choline acetyltransferase levels, loss of melanopsin retinal ganglion cells and functional integrity mainly of inner retina layers. Our findings support the theory that retinal degeneration constitutes an early pathological event that directly affects the control of circadian rhythm in AD

Empleo de herramientas virtuales para la creación de material didáctico de acceso libre

[SPA]Potenciar el aprendizaje autónomo del alumno siguiendo un determinado itinerario es uno de los postulados del denominado Plan Bolonia; en este contexto, además de otras funciones, el profesor ha de actuar como guía, enseñando y ayudando a alcanzar las habilidades y competencias marcadas como objetivos en cada materia. En algunos casos, esto significa la introducción paulatina de cambios en la metodología docente y la introducción de nuevas herramientas para facilitar el trabajo autónomo del alumno. Como profesores responsables de la experiencia piloto de adaptación al EEES de la materia Citología e Histología Vegetal y Animal iniciada en el año 2007 en la Universidad de Vigo, consideramos oportuno crear una herramienta propia para nuestro ámbito de conocimiento: un atlas histológico virtual. Asimismo, creímos que dicho atlas debía cumplir una serie de requisitos, tales como: abarcar la mayor parte de los aspectos relacionados con la Citología e Histología, disponer de gran cantidad de ilustraciones originales, ser fácil de usar, intuitivo y ameno, ser interactivo y potenciar el autoaprendizaje, diseñarse mediante programas de software libre, y ser de acceso y uso libre. [ENG]Promoting the autonomous learning of the students following a specific itinerary is one of the postulates of the so called Bologna Process; in this context, besides other functions, the professor should act as a guide, teaching and helping 2078 students to achieve the skills and competences marked like objectives in each subject. In some cases, this means a gradual introduction of changes in the teaching methodology and the introduction of new tools to facilitate the autonomous work of the students. As the responsible professors for the pilot experience of adaptation to the EEES of the matter Cytology and Animal and Plant Histology started in 2007 at the University of Vigo, we considered opportune to create our own tool for cell biology teaching: a virtual histological atlas. Likewise, we believed that the atlas should meet a series of requirements, such as: to cover most aspects of Cytology and Histology, to include a high number of original illustrations, to be easy to use, intuitive and mild (fun) pleasant, to be interactive and encourage self-learning, to be designed by free software, and to be of free access and use.Campus Mare Nostrum, Universidad Politécnica de Cartagena, Universidad de Murcia, Región de Murci

Specific Targeting of Human Inflamed Endothelium and In Situ Vascular Tissue Transfection by the Use of Ultrasound Contrast Agents

ObjectivesWe used human umbilical cord segments as an ex vivo model to investigate the possible clinical diagnostic and therapeutic applications of microbubbles (MBs).BackgroundMicrobubbles are commonly used in clinical practice as ultrasound contrast agents. Several studies have addressed the in vivo applications of MBs for specific targeting of vascular dysfunction or sonoporation in animal models, but to date no human tissue model has been established.MethodsPrimary venular endothelial cell monolayers were targeted with MBs conjugated to an antibody against a highly expressed endothelial marker (tetraspanin CD9), and binding was assessed under increasing flow rates (0.5 to 5 dynes/cm2). Furthermore, CD9-coupled MB endothelial targeting was measured under flow conditions by contrast-enhanced ultrasound analysis in an ex vivo human macrovascular model (umbilical cord vein), and the same tissue model was used for the detection of inflamed vasculature with anti-intercellular adhesion molecule (ICAM)-1–coated MBs. Finally, plasmids encoding fluorescent proteins were sonoporated into umbilical cord vessels.ResultsSpecific endothelial targeting in the in vitro and ex vivo models described previously was achieved by the use of MBs covered with an anti-CD9. Furthermore, we managed to induce inflammation in umbilical cord veins and detect it with real-time echography imaging using anti–ICAM-1–coupled MBs. Moreover, expression and correct localization of green fluorescent protein and green fluorescent protein-tagged ICAM-1 were assessed in this human ex vivo model without causing vascular damage.ConclusionsIn the absence of clinical trials to test the benefits and possible applications of ultrasound contrast agents for molecular imaging and therapy, we have developed a novel ex vivo human model using umbilical cords that is valid for the detection of inflammation and for exogenous expression of proteins by sonoporation

Disturbed circadian rhythm and retinal degeneration in a mouse model of Alzheimer's disease

The circadian clock is synchronized to the 24 h day by environmental light which is transmitted from the retina to the suprachiasmatic nucleus (SCN) primarily via the retinohypothalamic tract (RHT). Circadian rhythm abnormalities have been reported in neurodegenerative disorders such as Alzheimer's disease (AD). Whether these AD-related changes are a result of the altered clock gene expression, retina degeneration, including the dysfunction in RHT transmission, loss of retinal ganglion cells and its electrophysiological capabilities, or a combination of all of these pathological mechanisms, is not known. Here, we evaluated transgenic APP/PS1 mouse model of AD and wild-type mice at 6- and 12-month-old, as early and late pathological stage, respectively. We noticed the alteration of circadian clock gene expression not only in the hypothalamus but also in two extra-hypothalamic brain regions, cerebral cortex and hippocampus, in APP/PS1 mice. These alterations were observed in 6-month-old transgenic mice and were exacerbated at 12 months of age. This could be explained by the reduced RHT projections in the SCN of APP/PS1 mice, correlating with downregulation of hypothalamic GABAergic response in APP/PS1 mice in advanced stage of pathology. Importantly, we also report retinal degeneration in APP/PS1 mice, including Aβ deposits and reduced choline acetyltransferase levels, loss of melanopsin retinal ganglion cells and functional integrity mainly of inner retina layers. Our findings support the theory that retinal degeneration constitutes an early pathological event that directly affects the control of circadian rhythm in AD.This study was supported by grants from Instituto de Salud Carlos III (PI2021/00679; PI22CIII/00042), Hospital Universitario 12 de Octubre Research Institute (2022/0068), FEDER, and CIBERNED (CB07/502, PI2021/03).S

Experimental study of the impact of ion orbit losses on the edge radial electric field at the ASDEX Upgrade tokamak

Spanish Ministry of Science, Innovation and Universities (grant FPU17/06273)EUROfusion Consortium 63305