30 research outputs found

    Integrated Mathematical and Experimental Study of Cell Migration and Shape

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    Cell migration plays an essential role in many of physiological and pathological processes, including morphogenesis, inflammation, wound healing, and tumor metastasis. It is a complex process that involves multi-scale interactions between the cell and the extracellular matrix (ECM). Cells migrate through stromal ECM with native and cell-derived curvature at micron-meter scale are context-specific. How does the curvature of ECM mechanically change cell morphology and motility? Can the diverse migration behaviors from genetically identical cells be predictively using cell migrating data? We address these questions using an integrated computational and experimental approach: we developed three-dimensional biomechanical cell model and measured and analyzed a large number of cell migration images over time. Our findings suggest that 1. substrate curvature determines cell shape through contact and regulating protrusion dynamics; 2. effective cell migration is characterized with long cellular persistence time, low speed variation, spatial-temporally coordinated protrusion and contraction; 3. the cell shape variation space is low dimensional; and 4. migration behavior can be determined by a single image projected in the low dimensional cell shape variation space

    Insights on the spatiotemporal organization of integrins and their ligands using quantitative biophysical tools

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    Cotutela Universitat Polit猫cnica de Catalunya i Universit脿 degli Studi di FirenzeThe migration of leukocytes from the blood stream to sites of injury and infection in the extravascular tissues is fundamental for the immune response. Two of the main receptors mediating this process are the integrins aL脽2 and a4脽1, both expressed on the leukocyte cell membrane, which bind to their respective ligands ICAM-1 and VCAM-1, expressed on endothelial cell (EC) membrane. The dynamic and lateral organization of integrins on the cell membrane has been shown to be crucial in the regulation of cell adhesion. Likewise, organization of ligands in small domains (clusters) would probably reinforce the bonds formed with the integrins, thus increasing leukocyte adhesion. However, how the spatiotemporal behavior of integrins and their ligands is affected by the influence of external factors, such as mechanical and/or biochemical stimuli, has not been extensively studied. In addition, the impact of such spatiotemporal changes in the process of leukocyte adhesion and migration has not been addressed yet. The main aim of this thesis has been to address these questions using a combination of state-of-art biophysical tools, including advanced cell imaging, single molecule dynamic approaches, cell mechanical stimulation, and custom-designed algorithms for data quantification. From the technical side, our general approach involved the use of single particle tracking (SPT) approaches to monitor the dynamics of individual molecules implicated in the process of the cell adhesion, in combination with different super-resolution microscopy techniques, such as STED and STORM, to visualize changes in their nanoscale organization upon the influence of biochemical and/or mechanical stimuli. To mechanically stimulate cells we developed two different approaches; namely, a mechanical stretching device and a parallel-plate flow chamber (PPFC). We integrated these devices into our single molecule set-up and succeeded in recording the diffusion of integrins on cells plated on the mechanical stretching device upon varying stress conditions. As part of this thesis, we also applied different custom-made data analysis algorithms existent in the Lab and developed novel algorithms aimed at tracking and quantifying changes in the migratory behavior of T-cells on ECs exposed to shear stress. Using this powerful palette of tools we discovered that, as a consequence of prolonged shear flow exposure, ICAM-1 undergoes a global reorganization on the EC membrane accompanied by the formation of ICAM-1 nanoclusters. These nanoclusters were found to co-localize with shear flow-induced actin-enriched patch-like structures. Moreover, we showed that T-cells migrate faster and interact for shorter period of times on ECs mechanically stimulated as compared to ECs not subjected to shear stimulation. Hence, from these results, we concluded that continuous shear flow regulates the spatial organization of cell adhesion receptors on ECs, which in turn modulates leukocyte migration. In addition, we showed that chemokine (CXCL12) stimulation leads to rapid and transient activation of the a4脽1 expressed on T cells. These changes in activation profile directly correlate with talin recruitment, restricted lateral diffusion and integrin immobilization. Moreover, co-stimulation with CXCL12 and the ligand VCAM-1 potentiated integrin immobilization. In addition, superresolution imaging revealed that the nanoscale organization of a4脽1 remains unaffected upon CXCL12 and/or VCAM-1 stimulation. Our data, thus, indicate that docking by talin of the chemokine-activated a4脽1 to the actin cytoskeleton favors integrin immobilization, which likely facilitates ligand interaction and increased adhesiveness. The overall finding of this thesis indicates that cells of the immune system respond to mechanical and biochemical stimuli by rapidly readjusting the spatiotemporal behavior of integrins and ligands on the cell membrane modulating in turn cell adhesion and migration.La migraci贸n de leucocitos desde el torrente sangu铆neo a sitios de lesi贸n e infecci贸n es fundamental en la respuesta inmune. Dos de los principales receptores que median este proceso son las integrinas aL脽2 y a4脽1, que se expresan en la membrana celular de los leucocitos y se unen a sus respectivos ligandos ICAM-1 y VCAM-1, ambos localizados sobre la membrana de c茅lulas endoteliales (CE). Se ha demostrado que la din谩mica y la organizaci贸n lateral de las integrinas sobre la membrana celular son cruciales en la regulaci贸n de la adhesi贸n celular. Asimismo, es muy probable que la organizaci贸n de los ligandos en peque帽os dominios refuerce los enlaces formados con las integrinas, fortaleciendo as铆 la adhesi贸n celular. Sin embargo, c贸mo el comportamiento espacio-temporal de las integrinas y sus ligandos es afectado por factores externos, tales como est铆mulos mec谩nicos y/o bioqu铆micos, no ha sido ampliamente estudiado. Adem谩s, el impacto de estos cambios espacio-temporales en el proceso de adhesi贸n y migraci贸n leucocitaria a煤n no ha sido abordado. El objetivo principal de esta tesis ha sido abordar estas interrogantes usando una combinaci贸n de herramientas biof铆sicas de 煤ltima generaci贸n que incluyen avanzadas t茅cnicas de visualizaci贸n, din谩mica de mol茅culas individuales, estimulaci贸n mec谩nica de c茅lulas y desarrollo de algoritmos para la cuantificaci贸n de datos. Con este fin hemos utilizado m茅todos de seguimiento de part铆culas individuales para monitorizar la din谩mica de mol茅culas individuales, en combinaci贸n con t茅cnicas de microscop铆a de super-resoluci贸n, como STED y STORM, para visualizar cambios en su organizaci贸n a nano-escala tras la estimulaci贸n bioqu铆mica y/o mec谩nica. Para estimular las c茅lulas mec谩nicamente hemos desarrollado dos m茅todos: un dispositivo de estiramiento y una c谩mara de flujo de placa paralela. Ambos dispositivos fueron integrados a nuestro montaje experimental de detecci贸n de mol茅culas individuales y el primero fue exitosamente utilizado en la caracterizaci贸n de la difusi贸n de integrinas en c茅lulas cultivadas en el dispositivo de estiramiento mec谩nico. Como parte de esta tesis tambi茅n usamos diferentes algoritmos, existentes en nuestro grupo, para el an谩lisis de datos y desarrollamos otros nuevos para el seguimiento y cuantificaci贸n del comportamiento migratorio de c茅lulas T sometidas a flujo continuo. Utilizando estas herramientas descubrimos que, a causa de la exposici贸n a flujo mec谩nico, ICAM-1 se reorganiza sobre la membrana de las CEs y forma nano-agregados. Se encontr贸 adem谩s que estos agregados se colocalizan con regiones ricas en actina con estructura de parches. Adem谩s mostramos que las c茅lulas T migran m谩s r谩pido e interact煤an por periodos m谩s cortos de tiempo con CEs estimuladas mec谩nicamente respecto a CEs no estimuladas. De estos resultados concluimos que el flujo mec谩nico regula la organizaci贸n espacial de los receptores de adhesi贸n en las CEs, que a su vez modula la migraci贸n de leucocitos. Tambi茅n demostramos que la estimulaci贸n con quimioquinas (CXCL12) conduce a una r谩pida y transitoria activaci贸n de a4脽1. Estos cambios en la activaci贸n de a4脽1 se correlacionaron con el reclutamiento de talina, la inmovilizaci贸n de la integrina y su reducida difusi贸n lateral. Adem谩s, la coestimulaci贸n con CXCL12 y el ligando VCAM-1 potenci贸 la inmovilizaci贸n de a4脽1. Adicionalmente, im谩genes de superresoluci贸n revelaron que la organizaci贸n a nanom茅trica de a4脽1 no se ve afectada por la estimulaci贸n con CXCL12 y/o VCAM-1. Estos datos indican que las integrinas activadas por CXCL12 se unen al citoesqueleto por medio de talina, favoreciendo la inmovilizaci贸n de a4脽1 y facilitando as铆, la interacci贸n con su ligando y una mayor adhesi贸n. En resumen, esta tesis indica que las c茅lulas del sistema inmune responden a est铆mulos mec谩nicos y bioqu铆micos mediante el reajuste r谩pido de la organizaci贸n espacio-temporal de integrinas y ligandos sobre la membrana celular, modulando as铆 su adhesi贸n y migraci贸nPostprint (published version

    Ultrasound Imaging Innovations for Visualization and Quantification of Vascular Biomarkers

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    The existence of plaque in the carotid arteries, which provide circulation to the brain, is a known risk for stroke and dementia. Alas, this risk factor is present in 25% of the adult population. Proper assessment of carotid plaque may play a significant role in preventing and managing stroke and dementia. However, current plaque assessment routines have known limitations in assessing individual risk for future cardiovascular events. There is a practical need to derive new vascular biomarkers that are indicative of cardiovascular risk based on hemodynamic information. Nonetheless, the derivation of these biomarkers is not a trivial technical task because none of the existing clinical imaging modalities have adequate time resolution to track the spatiotemporal dynamics of arterial blood flow that is pulsatile in nature. The goal of this dissertation is to devise a new ultrasound imaging framework to measure vascular biomarkers related to turbulent flow, intra-plaque microvasculature, and blood flow rate. Central to the proposed framework is the use of high frame rate ultrasound (HiFRUS) imaging principles to track hemodynamic events at fine temporal resolution (through using frame rates of greater than 1000 frames per second). The existence of turbulent flow and intra-plaque microvessels, as well as anomalous blood flow rate, are all closely related to the formation and progression of carotid plaque. Therefore, quantifying these biomarkers can improve the identification of individuals with carotid plaque who are at risk for future cardiovascular events. To facilitate the testing and the implementation of the proposed imaging algorithms, this dissertation has included the development of new experimental models (in the form of flow phantoms) and a new HiFRUS imaging platform with live scanning and on-demand playback functionalities. Pilot studies were also carried out on rats and human volunteers. Results generally demonstrated the real-time performance and the practical efficacy of the proposed algorithms. The proposed ultrasound imaging framework is expected to improve carotid plaque risk classification and, in turn, facilitate timely identification of at-risk individuals. It may also be used to derive new insights on carotid plaque formation and progression to aid disease management and the development of personalized treatment strategies

    Imaging Sensors and Applications

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    In past decades, various sensor technologies have been used in all areas of our lives, thus improving our quality of life. In particular, imaging sensors have been widely applied in the development of various imaging approaches such as optical imaging, ultrasound imaging, X-ray imaging, and nuclear imaging, and contributed to achieve high sensitivity, miniaturization, and real-time imaging. These advanced image sensing technologies play an important role not only in the medical field but also in the industrial field. This Special Issue covers broad topics on imaging sensors and applications. The scope range of imaging sensors can be extended to novel imaging sensors and diverse imaging systems, including hardware and software advancements. Additionally, biomedical and nondestructive sensing applications are welcome

    Quantitative Phenotyping of Brain Endothelial Cell-Cell Junctions for Physiological and Pathophysiological Applications

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    The integrity of endothelial cell-cell junctions is required for the maintenance of normal physiological processes. The expression of junctional proteins is particularly important in the endothelial cells of the blood-brain barrier (BBB), the cellular unit that protects the brain via regulated transport between the peripheral blood and the central nervous system. Dysfunction of the BBB is linked with decreased junctional protein localization and is implicated in several diseases including Alzheimer鈥檚 disease and multiple sclerosis. On the other hand, the tight junctions of the BBB impede the delivery of medications targeting the brain. Therefore, understanding the key players driving junction stability could hold significant promise for therapeutic discovery and drug delivery applications. Despite this, the mechanisms underlying junction disruption aren鈥檛 fully understood. While several studies have linked different junction protein patterns with altered barrier function, the quantification of this parameter remains limited due to the lack of efficient measurement techniques. Here, we aimed to investigate the influence of junction phenotype on brain endothelial barrier properties. To accomplish this, we developed the Junction Analyzer Program (JAnaP) to semi-automatically calculate edge-localization protein phenotypes. Application of the JAnaP to measure the junctional proteins VE-cadherin and ZO-1 in different physiological and pathophysiological conditions revealed that discontinuous junctions contribute more to barrier permeability compared to continuous, linear junctions. Continuous junctions were also increased in endothelial cells with decreased contractility, mediated biochemically or by lowered subendothelial matrix stiffness. Finally, breast cancer cell secreted factors increased immature adherens junctions, likely through VEGF signaling, but minimally affected tight junction presentation. Thus far, the development and application of the JAnaP has revealed insights into the effects of junction patterns on barrier function, the mechanobiology of endothelial cells, and the response of brain endothelial cells to biochemical cues involved in breast cancer metastasis. Understanding the conditions driving altered junction presentation, and the resultant effects on barrier integrity, could lead to the development of therapeutics capable of traversing the BBB for delivery to the brain or for diseases associated with BBB dysfunction. Future use of this program holds significant potential for physiological and pathophysiological study in various endothelial and epithelial cell systems

    Measurement of subtle blood-brain barrier disruption in cerebral small vessel disease using dynamic contrast-enhanced magnetic resonance imaging

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    Cerebral small vessel disease (SVD) is a common cause of strokes and dementia. The pathogenesis of SVD is poorly understood, but imaging and biochemical investigations suggest that subtle blood-brain barrier (BBB) leakage may contribute to tissue damage. The most widely-used imaging method for assessing BBB integrity and other microvascular properties is dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). DCE-MRI has primarily been applied in situations where contrast uptake in tissue is typically large and rapid (e.g. neuro-oncology); the optimal approach for quantifying BBB integrity in diseases where the BBB remains largely intact and the reliability of resulting measurements is unclear. The main purpose of this thesis was to assess and improve the reliability of quantitative assessment of subtle BBB disruption, in order to illuminate its potential role in cerebral SVD. Firstly, a systematic literature review was performed in order to provide an overview of DCE-MRI methods in the brain. This review found large variations in MRI procedures and data analysis methods, resulting in widely varying estimates of tracer kinetic parameters. Secondly, this thesis focused on the analysis of DCE-MRI data acquired in an on-site clinical study of mild stroke patients. After performing basic DCE-MRI processing (e.g. selection of a vascular input function), this work aimed to determine the tracer kinetic modelling approach most suitable for assessing subtle BBB disruption in this cohort. Using data-driven model selection and computer simulations, the Patlak model was found to provide accurate estimates of blood plasma volume and low-level BBB leakage. Thirdly, this thesis aimed to investigate two potential pitfalls in the quantification of subtle BBB disruption. Contrast-free measurements in healthy volunteers revealed that a signal drift of approximately 0.1 %/min occurs during the DCE-MRI acquisition; computer simulations showed that this drift introduces significant systematic errors when estimating low-level tracer kinetic parameters. Furthermore, tracer kinetic analysis was performed in an external patient cohort in order to investigate the inter-study comparability of DCE-MRI measurements. Due to the nature of the acquisition protocol it proved difficult to obtain reliable estimates of BBB leakage, highlighting the importance of study design. Lastly, this thesis examined the relationship between quantitative MRI parameters and clinical measurements in cerebral SVD, with a focus on the estimates of blood volume and BBB leakage obtained in the internal SVD patient cohort. This work did not provide evidence that BBB leakage in normal-appearing tissue increases with SVD burden or predicts disease progression; however, increased BBB leakage was found in white matter hyperintensities. Furthermore, this work raises the possibility of a role for blood plasma volume and dietary salt intake in cerebral SVD. The work described in this thesis has demonstrated that it is possible to estimate subtle BBB disruption using DCE-MRI, provided that the measurement and data analysis strategies are carefully optimised. However, absolute values of tracer kinetic parameters should be interpreted with caution, particularly when making comparisons between studies, and sources of error and their influence should be estimated where possible. The exact roles of BBB breakdown and other microvascular changes in SVD pathology remain to be defined; however, the work presented in this thesis contributes further insights and, together with technical advances, will facilitate improved study design in the future

    Modelling and Identification of Immune Cell Migration during the Inflammatory Response

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    Neutrophils are the white blood cells that play a crucial role in the response of the innate immune system to tissue injuries or infectious threats. Their rapid arrival to the damaged area and timely removal from it define the success of the inflammatory process. Therefore, understanding neutrophil migratory behaviour is essential for the therapeutic regulation of multiple inflammation-mediated diseases. Recent years saw rapid development of various in vivo models of inflammation that provide a remarkable insight into the neutrophil function. The main drawback of the \textit{in vivo} microscopy is that it usually focuses on the moving cells and obscures the external environment that drives their migration. To evaluate the effect of a particular treatment strategy on neutrophil behaviour, it is necessary to recover the information about the cell responsiveness and the complex extracellular environment from the limited experimental data. This thesis addresses the presented inference problem by developing a dynamical modelling and estimation framework that quantifies the relationship between an individual migrating cell and the global environment. \par The first part of the thesis is concerned with the estimation of the hidden chemical environment that modulates the observed cell migration during the inflammatory response in the injured tail fin of zebrafish larvae. First, a dynamical model of the neutrophil responding to the chemoattractant concentration is developed based on the potential field paradigm of object-environment interaction. This representation serves as a foundation for a hybrid model that is proposed to account for heterogeneous behaviour of an individual cell throughout the migration process. An approximate maximum likelihood estimation framework is derived to estimate the hidden environment and the states of multiple hybrid systems simultaneously. The developed framework is then used to analyse the neutrophil tracking data observed in vivo under the assumption that each neutrophil at each time can be in one of three migratory modes: responding to the environment, randomly moving, and stationary. The second part of the thesis examines the process of neutrophil migration at the subcellular scale, focusing on the subcellular mechanism that translates the local environment sensing into the cell shape change. A state space model is formulated based on the hypothesis that links the local protrusions of the cell membrane and the concentration of the intracellular pro-inflammatory signalling protein. The developed model is tested against the local concentration data extracted from the in vivo time-lapse images via the classical expectation-maximisation algorithm
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