2,861 research outputs found

    Converging organoids and extracellular matrix::New insights into liver cancer biology

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    Converging organoids and extracellular matrix::New insights into liver cancer biology

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    Primary liver cancer, consisting primarily of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), is a heterogeneous malignancy with a dismal prognosis, resulting in the third leading cause of cancer mortality worldwide [1, 2]. It is characterized by unique histological features, late-stage diagnosis, a highly variable mutational landscape, and high levels of heterogeneity in biology and etiology [3-5]. Treatment options are limited, with surgical intervention the main curative option, although not available for the majority of patients which are diagnosed in an advanced stage. Major contributing factors to the complexity and limited treatment options are the interactions between primary tumor cells, non-neoplastic stromal and immune cells, and the extracellular matrix (ECM). ECM dysregulation plays a prominent role in multiple facets of liver cancer, including initiation and progression [6, 7]. HCC often develops in already damaged environments containing large areas of inflammation and fibrosis, while CCA is commonly characterized by significant desmoplasia, extensive formation of connective tissue surrounding the tumor [8, 9]. Thus, to gain a better understanding of liver cancer biology, sophisticated in vitro tumor models need to incorporate comprehensively the various aspects that together dictate liver cancer progression. Therefore, the aim of this thesis is to create in vitro liver cancer models through organoid technology approaches, allowing for novel insights into liver cancer biology and, in turn, providing potential avenues for therapeutic testing. To model primary epithelial liver cancer cells, organoid technology is employed in part I. To study and characterize the role of ECM in liver cancer, decellularization of tumor tissue, adjacent liver tissue, and distant metastatic organs (i.e. lung and lymph node) is described, characterized, and combined with organoid technology to create improved tissue engineered models for liver cancer in part II of this thesis. Chapter 1 provides a brief introduction into the concepts of liver cancer, cellular heterogeneity, decellularization and organoid technology. It also explains the rationale behind the work presented in this thesis. In-depth analysis of organoid technology and contrasting it to different in vitro cell culture systems employed for liver cancer modeling is done in chapter 2. Reliable establishment of liver cancer organoids is crucial for advancing translational applications of organoids, such as personalized medicine. Therefore, as described in chapter 3, a multi-center analysis was performed on establishment of liver cancer organoids. This revealed a global establishment efficiency rate of 28.2% (19.3% for hepatocellular carcinoma organoids (HCCO) and 36% for cholangiocarcinoma organoids (CCAO)). Additionally, potential solutions and future perspectives for increasing establishment are provided. Liver cancer organoids consist of solely primary epithelial tumor cells. To engineer an in vitro tumor model with the possibility of immunotherapy testing, CCAO were combined with immune cells in chapter 4. Co-culture of CCAO with peripheral blood mononuclear cells and/or allogenic T cells revealed an effective anti-tumor immune response, with distinct interpatient heterogeneity. These cytotoxic effects were mediated by cell-cell contact and release of soluble factors, albeit indirect killing through soluble factors was only observed in one organoid line. Thus, this model provided a first step towards developing immunotherapy for CCA on an individual patient level. Personalized medicine success is dependent on an organoids ability to recapitulate patient tissue faithfully. Therefore, in chapter 5 a novel organoid system was created in which branching morphogenesis was induced in cholangiocyte and CCA organoids. Branching cholangiocyte organoids self-organized into tubular structures, with high similarity to primary cholangiocytes, based on single-cell sequencing and functionality. Similarly, branching CCAO obtain a different morphology in vitro more similar to primary tumors. Moreover, these branching CCAO have a higher correlation to the transcriptomic profile of patient-paired tumor tissue and an increased drug resistance to gemcitabine and cisplatin, the standard chemotherapy regimen for CCA patients in the clinic. As discussed, CCAO represent the epithelial compartment of CCA. Proliferation, invasion, and metastasis of epithelial tumor cells is highly influenced by the interaction with their cellular and extracellular environment. The remodeling of various properties of the extracellular matrix (ECM), including stiffness, composition, alignment, and integrity, influences tumor progression. In chapter 6 the alterations of the ECM in solid tumors and the translational impact of our increased understanding of these alterations is discussed. The success of ECM-related cancer therapy development requires an intimate understanding of the malignancy-induced changes to the ECM. This principle was applied to liver cancer in chapter 7, whereby through a integrative molecular and mechanical approach the dysregulation of liver cancer ECM was characterized. An optimized agitation-based decellularization protocol was established for primary liver cancer (HCC and CCA) and paired adjacent tissue (HCC-ADJ and CCA-ADJ). Novel malignancy-related ECM protein signatures were found, which were previously overlooked in liver cancer transcriptomic data. Additionally, the mechanical characteristics were probed, which revealed divergent macro- and micro-scale mechanical properties and a higher alignment of collagen in CCA. This study provided a better understanding of ECM alterations during liver cancer as well as a potential scaffold for culture of organoids. This was applied to CCA in chapter 8 by combining decellularized CCA tumor ECM and tumor-free liver ECM with CCAO to study cell-matrix interactions. Culture of CCAO in tumor ECM resulted in a transcriptome closely resembling in vivo patient tumor tissue, and was accompanied by an increase in chemo resistance. In tumor-free liver ECM, devoid of desmoplasia, CCAO initiated a desmoplastic reaction through increased collagen production. If desmoplasia was already present, distinct ECM proteins were produced by the organoids. These were tumor-related proteins associated with poor patient survival. To extend this method of studying cell-matrix interactions to a metastatic setting, lung and lymph node tissue was decellularized and recellularized with CCAO in chapter 9, as these are common locations of metastasis in CCA. Decellularization resulted in removal of cells while preserving ECM structure and protein composition, linked to tissue-specific functioning hallmarks. Recellularization revealed that lung and lymph node ECM induced different gene expression profiles in the organoids, related to cancer stem cell phenotype, cell-ECM integrin binding, and epithelial-to-mesenchymal transition. Furthermore, the metabolic activity of CCAO in lung and lymph node was significantly influenced by the metastatic location, the original characteristics of the patient tumor, and the donor of the target organ. The previously described in vitro tumor models utilized decellularized scaffolds with native structure. Decellularized ECM can also be used for creation of tissue-specific hydrogels through digestion and gelation procedures. These hydrogels were created from both porcine and human livers in chapter 10. The liver ECM-based hydrogels were used to initiate and culture healthy cholangiocyte organoids, which maintained cholangiocyte marker expression, thus providing an alternative for initiation of organoids in BME. Building upon this, in chapter 11 human liver ECM-based extracts were used in combination with a one-step microfluidic encapsulation method to produce size standardized CCAO. The established system can facilitate the reduction of size variability conventionally seen in organoid culture by providing uniform scaffolding. Encapsulated CCAO retained their stem cell phenotype and were amendable to drug screening, showing the feasibility of scalable production of CCAO for throughput drug screening approaches. Lastly, Chapter 12 provides a global discussion and future outlook on tumor tissue engineering strategies for liver cancer, using organoid technology and decellularization. Combining multiple aspects of liver cancer, both cellular and extracellular, with tissue engineering strategies provides advanced tumor models that can delineate fundamental mechanistic insights as well as provide a platform for drug screening approaches.<br/

    Spatiotemporal Control of Chemical Reaction Networks using Droplet Microfluidics

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    A number of cellular organisms, such as yeast, bacteria and slime moulds, exhibit dynamic behaviour, in particular switching and rhythms that are controlled by feedback mechanisms in enzyme-catalysed reactions. The mechanisms of these processes are well understood, and recently there has been a focus on generating similar reactions in synthetic biocatalytic systems to establish bioinspired analogues for applications in materials and medicine. In this context, compartmentalisation of biochemical reactions within synthetic cell models such as micelles, vesicles, and W/O/W-based double emulsions is attracting growing attention for applications in the field of therapeutics. In this respect, it is necessary to adopt easier-to-use stimuli-responsive (react to pH, temperature or light) biochemical reactions, to apply artificial cell models to the biomedical context, and regulate artificial cell communication in a spatiotemporal controlled way. As a first step, it is crucial to control the output of a chemical reaction that maybe exploited for applications in the field of programmable materials and biomedicine. Droplet emulsion and synthetic vesicle systems have been widely employed as bioinspired micro- or nanoreactors for production of materials such as hydrogels and ceramic particles. They also provide test platform for biomimetic cell like behaviour. To address this, we have developed and fine-tuned a platform with synthetic bottom-up chemistry that has enabled us to systematically and thoroughly investigate the effects of entrapment on a feedback-driven enzymatic reaction. As a result of this process, we have revealed a system that is more intricate than originally thought. Firstly, taking advantage from pressure driven droplet microfluidics, we developed a system of enzyme-encapsulated (urea-urease) double emulsion (W/O/W) droplets to obtain a localised pH pulse, with a controllable induction time to program material properties. The urease-catalysed hydrolysis of urea (urea-urea reaction), has a feedback through the production of the base (NH3). This leads to a change from an acidic to a basic pH after an induction time (Tind), resulting in an environment with auto-changing pH conditions. Reaction was initiated by addition of urea and a pulse in base (ammonia) was observed in the droplets after a time lag of the order of minutes. The pH-time profile can be manipulated by the diffusion timescale of urea and ammonia through the oil layer, resulting in localised pH changes not accessible in bulk solutions. Secondly, we performed a computational investigation of the nonlinear reaction chemistry (urea-urease) within the designed platform of the W/O/W-based reactor. A radially distributed reaction diffusion model is presented for a layered sphere mimicking a double emulsion. Here we have combined the experiments with simulations (shell-core model) to demonstrate the influence of urea transport triggered by the shell, the core and the external solution surrounding the cell model (µ-reactor) on the induction time/period (Tind) of urea-urease reaction. Third, inspired from natural cellular systems (e.g. bacterial quorum sensing), we focus on the use of urea-urease reaction confined to double emulsions to investigate chemical communications. We observed a system that resulted in a system of microreactors acting as individual units with distinct induction periods (Tind) for the first time. We show that in contrast to other systems, the release of ammonia can accelerate the reaction in all the droplets but there is no evident synchronisation of activity characterised by a wide distribution of induction times across the population of micro-reactors. However, the investigation of behaviour of population/group of µ-reactors as a function of substrate urea concentration and the density of µ-reactors highlights the possibility of transitions to collective behaviours. Finally, we aimed to use the double emulsion template for potential biomedical and therapeutic applications using the autocatalytic urea-urease reaction. We used the platform to produce thiol-acrylate gels in the form of double emulsion loaded gel films and spherical microcapsules for potential drug delivery applications. In addition, we employed the encapsulated double emulsion platform of the enzyme urease to study the inhibition of the enzyme itself; which is important in the development of anti-microbials for ureolytic bacteria. By building this platform, we have not only learned how to control the kinetic output of the reaction (urea-urease), but have also demonstrated its potential in future applications

    Functional Nanomaterials and Polymer Nanocomposites: Current Uses and Potential Applications

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    This book covers a broad range of subjects, from smart nanoparticles and polymer nanocomposite synthesis and the study of their fundamental properties to the fabrication and characterization of devices and emerging technologies with smart nanoparticles and polymer integration

    Mechanical characterization, constitutive modeling and applications of ultra-soft magnetorheological elastomers

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    Mención Internacional en el título de doctorSmart materials are bringing sweeping changes in the way humans interact with engineering devices. A myriad of state-of-the-art applications are based on novel ways to actuate on structures that respond under different types of stimuli. Among them, materials that respond to magnetic fields allow to remotely modify their mechanical properties and macroscopic shape. Ultra-soft magnetorheological elastomers (MREs) are composed of a highly stretchable soft elastomeric matrix in the order of 1 kPa and magnetic particles embedded in it. This combination allows large deformations with small external actuations. The type of the magnetic particles plays a crucial role as it defines the reversibility or remanence of the material magnetization. According to the fillers used, MREs are referred to as soft-magnetic magnetorheological elastomers (sMREs) and hard-magnetic magnetorheological elastomers (hMREs). sMREs exhibit strong changes in their mechanical properties when an external magnetic field is applied, whereas hMREs allow sustained magnetic effects along time and complex shape-morphing capabilities. In this regard, end-of-pipe applications of MREs in the literature are based on two major characteristics: the modification of their mechanical properties and macrostructural shape changes. For instance, smart actuators, sensors and soft robots for bioengineering applications are remotely actuated to perform functional deformations and autonomous locomotion. In addition, hMREs have been used for industrial applications, such as damping systems and electrical machines. From the analysis of the current state of the art, we identified some impediments to advance in certain research fields that may be overcome with new solutions based on ultrasoft MREs. On the mechanobiology area, we found no available experimental methodologies to transmit complex and dynamic heterogeneous strain patterns to biological systems in a reversible manner. To remedy this shortcoming, this doctoral research proposes a new mechanobiology experimental setup based on responsive ultra-soft MRE biological substrates. Such an endeavor requires deeper insights into the magneto-viscoelastic and microstructural mechanisms of ultra-soft MREs. In addition, there is still a lack of guidance for the selection of the magnetic fillers to be used for MREs and the final properties provided to the structure. Eventually, the great advances on both sMREs and hMREs to date pose a timely question on whether the combination of both types of particles in a hybrid MRE may optimize the multifunctional response of these active structures. To overcome these roadblocks, this thesis provides an extensive and comprehensive experimental characterization of ultra-soft sMREs, hMREs and hybrid MREs. The experimental methodology uncovers magneto-mechanical rate dependences under numerous loading and manufacturing conditions. Then, a set of modeling frameworks allows to delve into such mechanisms and develop three ground-breaking applications. Therefore, the thesis has lead to three main contributions. First and motivated on mechanobiology research, a computational framework guides a sMRE substrate to transmit complex strain patterns in vitro to biological systems. Second, we demonstrate the ability of remanent magnetic fields in hMREs to arrest cracks propagations and improve fracture toughness. Finally, the combination of soft- and hard-magnetic particles is proved to enhance the magnetorheological and magnetostrictive effects, providing promising results for soft robotics.Los materiales inteligentes están generando cambios radicales en la forma que los humanos interactúan con dispositivos ingenieriles. Distintas aplicaciones punteras se basan en formas novedosas de actuar sobre materiales que responden a diferentes estímulos. Entre ellos, las estructuras que responden a campos magnéticos permiten la modificación de manera remota tanto de sus propiedades mecánicas como de su forma. Los elastómeros magnetorreológicos (MREs) ultra blandos están compuestos por una matriz elastomérica con gran ductilidad y una rigidez en torno a 1 kPa, reforzada con partículas magnéticas. Esta combinación permite inducir grandes deformaciones en el material mediante la aplicación de campos magnéticos pequeños. La naturaleza de las partículas magnéticas define la reversibilidad o remanencia de la magnetización del material compuesto. De esta manera, según el tipo de partículas que contengan, los MREs pueden presentar magnetización débil (sMRE) o magnetización fuerte (hMRE). Los sMREs experimentan grandes cambios en sus propiedades mecánicas al aplicar un campo magnético externo, mientras que los hMREs permiten efectos magneto-mecánicos sostenidos a lo largo del tiempo, así como programar cambios de forma complejos. En este sentido, las aplicaciones de los MREs se basan en dos características principales: la modificación de sus propiedades mecánicas y los cambios de forma macroestructurales. Por ejemplo, los campos magnéticos pueden emplearse para inducir deformaciones funcionales en actuadores y sensores inteligentes, o en robótica blanda para bioingeniería. Los hMREs también se han aplicado en el ámbito industrial en sistemas de amortiguación y máquinas eléctricas. A partir del análisis del estado del arte, se identifican algunas limitaciones que impiden el avance en ciertos campos de investigación y que podrían resolverse con nuevas soluciones basadas en MREs ultra blandos. En el área de la mecanobiología, no existen metodologías experimentales para transmitir patrones de deformación complejos y dinámicos a sistemas biológicos de manera reversible. En esta investigación doctoral se propone una configuración experimental novedosa basada en sustratos biológicos fabricados con MREs ultra blandos. Dicha solución requiere la identificación de los mecanismos magneto-viscoelásticos y microestructurales de estos materiales, según el tipo de partículas magnéticas, y las consiguientes propiedades macroscópicas del material. Además, investigaciones recientes en sMREs y hMREs plantean la pregunta sobre si la combinación de distintos tipos de partículas magnéticas en un MRE híbrido puede optimizar su respuesta multifuncional. Para superar estos obstáculos, la presente tesis proporciona una caracterización experimental completa de sMREs, hMREs y MREs híbridos ultra blandos. Estos resultados muestran las dependencias del comportamiento multifuncional del material con la velocidad de aplicación de cargas magneto-mecánicas. El desarrollo de un conjunto de modelos teórico-computacionales permite profundizar en dichos mecanismos y desarrollar aplicaciones innovadoras. De este modo, la tesis doctoral ha dado lugar a tres bloques de aportaciones principales. En primer lugar, este trabajo proporciona un marco computacional para guiar el diseño de sustratos basados en sMREs para transmitir patrones de deformación complejos in vitro a sistemas biológicos. En segundo lugar, se demuestra la capacidad de los campos magnéticos remanentes en los hMRE para detener la propagación de grietas y mejorar la tenacidad a la fractura. Finalmente, se establece que la combinación de partículas magnéticas de magnetización débil y fuerte mejora el efecto magnetorreológico y magnetoestrictivo, abriendo nuevas posibilidades para el diseño de robots blandos.I want to acknowledge the support from the Ministerio de Ciencia, Innovación y Universidades, Spain (FPU19/03874), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 947723, project: 4D-BIOMAP).Programa de Doctorado en Ingeniería Mecánica y de Organización Industrial por la Universidad Carlos III de MadridPresidente: Ramón Eulalio Zaera Polo.- Secretario: Abdón Pena Francesch.- Vocal: Laura de Lorenzi

    Leveraging elasticity theory to calculate cell forces: From analytical insights to machine learning

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    Living cells possess capabilities to detect and respond to mechanical features of their surroundings. In traction force microscopy, the traction of cells on an elastic substrate is made visible by observing substrate deformation as measured by the movement of embedded marker beads. Describing the substrates by means of elasticity theory, we can calculate the adhesive forces, improving our understanding of cellular function and behavior. In this dissertation, I combine analytical solutions with numerical methods and machine learning techniques to improve traction prediction in a range of experimental applications. I describe how to include the normal traction component in regularization-based Fourier approaches, which I apply to experimental data. I compare the dominant strategies for traction reconstruction, the direct method and inverse, regularization-based approaches and find, that the latter are more precise while the former is more stress resilient to noise. I find that a point-force based reconstruction can be used to study the force balance evolution in response to microneedle pulling showing a transition from a dipolar into a monopolar force arrangement. Finally, I show how a conditional invertible neural network not only reconstructs adhesive areas more localized, but also reveals spatial correlations and variations in reliability of traction reconstructions

    Modern meat: the next generation of meat from cells

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    Modern Meat is the first textbook on cultivated meat, with contributions from over 100 experts within the cultivated meat community. The Sections of Modern Meat comprise 5 broad categories of cultivated meat: Context, Impact, Science, Society, and World. The 19 chapters of Modern Meat, spread across these 5 sections, provide detailed entries on cultivated meat. They extensively tour a range of topics including the impact of cultivated meat on humans and animals, the bioprocess of cultivated meat production, how cultivated meat may become a food option in Space and on Mars, and how cultivated meat may impact the economy, culture, and tradition of Asia

    Ovarian hormones shape brain structure, function, and chemistry: A neuropsychiatric framework for female brain health

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    There are robust sex differences in brain anatomy, function, as well as neuropsychiatric and neurodegenerative disease risk (1-6), with women approximately twice as likely to suffer from a depressive illness as well as Alzheimer’s Disease. Disruptions in ovarian hormones likely play a role in such disproportionate disease prevalence, given that ovarian hormones serve as key regulators of brain functional and structural plasticity and undergo major fluctuations across the female lifespan (7-9). From a clinical perspective, there is a wellreported increase in depression susceptibility and initial evidence for cognitive impairment or decline during hormonal transition states, such as the postpartum period and perimenopause (9-14). What remains unknown, however, is the underlying mechanism of how fluctuations in ovarian hormones interact with other biological factors to influence brain structure, function, and chemistry. While this line of research has translational relevance for over half the population, neuroscience is notably guilty of female participant exclusion in research studies, with the male brain implicitly treated as the default model and only a minority of basic and clinical neuroscience studies including a female sample (15-18). Female underrepresentation in neuroscience directly limits opportunities for basic scientific discovery; and without basic knowledge of the biological underpinnings of sex differences, we cannot address critical sexdriven differences in pathology. Thus, my doctoral thesis aims to deliberately investigate the influence of sex and ovarian hormones on brain states in health as well as in vulnerability to depression and cognitive impairment:Table of Contents List of Abbreviations ..................................................................................................................... i List of Figures .............................................................................................................................. ii Acknowledgements .....................................................................................................................iii 1 INTRODUCTION .....................................................................................................................1 1.1 Lifespan approach: Sex, hormones, and metabolic risk factors for cognitive health .......3 1.2 Reproductive years: Healthy models of ovarian hormones, serotonin, and the brain ......4 1.2.1 Ovarian hormones and brain structure across the menstrual cycle ........................4 1.2.2 Serotonergic modulation and brain function in oral contraceptive users .................6 1.3 Neuropsychiatric risk models: Reproductive subtypes of depression ...............................8 1.3.1 Hormonal transition states and brain chemistry measured by PET imaging ...........8 1.3.2 Serotonin transporter binding across the menstrual cycle in PMDD patients .......10 2 PUBLICATIONS ....................................................................................................................12 2.1 Publication 1: Association of estradiol and visceral fat with structural brain networks and memory performance in adults .................................................................................13 2.2 Publication 2: Longitudinal 7T MRI reveals volumetric changes in subregions of human medial temporal lobe to sex hormone fluctuations ..............................................28 2.3 Publication 3: One-week escitalopram intake alters the excitation-inhibition balance in the healthy female brain ...............................................................................................51 2.4 Publication 4: Using positron emission tomography to investigate hormone-mediated neurochemical changes across the female lifespan: implications for depression ..........65 2.5 Publication 5: Increase in serotonin transporter binding across the menstrual cycle in patients with premenstrual dysphoric disorder: a case-control longitudinal neuro- receptor ligand PET imaging study ..................................................................................82 3 SUMMARY ...........................................................................................................................100 References ..............................................................................................................................107 Supplementary Publications ...................................................................................................114 Author Contributions to Publication 1 .....................................................................................184 Author Contributions to Publication 2 .....................................................................................186 Author Contributions to Publication 3 .....................................................................................188 Author Contributions to Publication 4 .....................................................................................190 Author Contributions to Publication 5 .....................................................................................191 Declaration of Authenticity ......................................................................................................193 Curriculum Vitae ......................................................................................................................194 List of Publications ................................................................................................................195 List of Talks and Posters ......................................................................................................19

    (b2023 to 2014) The UNBELIEVABLE similarities between the ideas of some people (2006-2016) and my ideas (2002-2008) in physics (quantum mechanics, cosmology), cognitive neuroscience, philosophy of mind, and philosophy (this manuscript would require a REVOLUTION in international academy environment!)

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    (b2023 to 2014) The UNBELIEVABLE similarities between the ideas of some people (2006-2016) and my ideas (2002-2008) in physics (quantum mechanics, cosmology), cognitive neuroscience, philosophy of mind, and philosophy (this manuscript would require a REVOLUTION in international academy environment!
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