Measuring the mechanical properties of plant cells by combining micro-indentation with osmotic treatments

A combination of osmotic treatments, micro-indentation with cellular force microscopy, and inverse finite-element modelling gives an estimate for both turgor pressure and cell wall elasticity in plant cell

Visualization and manipulation of repair and regeneration in biological systems using light

Tissue repair after an injury is a fundamental process in biomedicine. It can involve regeneration, which uses new growth to restore tissue function. The interest in repair and regeneration is motivated by the desire to treat injuries and diseases and has attracted researchers for centuries. In the last decades, it evolved in the field of regenerative medicine, which has the ultimate goal of providing strategies for regenerating human cells, tissues, or even organs, for instance, via engineering principles. Already since the first experiments on regeneration by Abraham Trembley, novel findings in biomedicine, repair, and regeneration have been enabled or accompanied by research in optics, for example, on the development of novel microscopy techniques. Nowadays, novel optical techniques are advancing, which allow to understand the role of single cells in tissue repair processes. Moreover, repair processes within cells can be visualized and manipulated. Ultimately, optics can provide enabling techniques for regenerative therapies. This habilitation thesis aims to present several of these advances. On a single cell level, femtosecond laser nanosurgery was used to target specific intracellular structures during concurrent imaging in vitro. The relation of femtosecond laser nanosurgery to the cell state and cellular staining was investigated. Manipulation of single Z-discs in cardiomyocytes using a femtosecond oscillator laser system was accomplished, which allows to better elucidate the role of a single Z-disc in cardiomyocyte function. In particular, measurements on cell survival, (calcium-) homeostasis, and morphology yielded only minor deviations from control cells after single Z-disc ablation. A reduction in force generation was elucidated via traction force microscopy and gene expression level changes, for instance, an upregulation of -actinin were examined. Additionally, light-based systems to influence single cells in their alignment or to trigger single cells, for example, to activate other cells via optogenetics were applied. On the tissue scale, imaging via confocal microscopy or multiphoton microscopy has been applied for various contexts of regenerative approaches. Furthermore, a fiber-based imaging approach, which could later be used for longitudinal imaging in vivo and builds upon a fluorescence microscope system and an imaging fiber bundle in combination with reconstruction via a neural network, was developed. As another imaging strategy, an abdominal imaging window served to image the mouse liver in vivo via multiphoton microscopy in successive imaging sessions. Manipulation in tissue was applied in colonoids, which resemble the structure of the colon on an in vitro scale, and revealed different cell dynamics dependent on the location of the damage. In particular, activation of the Wnt signaling pathway after crypt damage was observed. Cell ablation via a femtosecond laser amplifier system during concurrent two-photon microscopy was also established during in vivo liver imaging to study micro-regenerative processes. Furthermore, laser-based delivery processes with novel materials or in the context of genome editing using CRISPR/Cas9 technology were investigated as enabling technologies for regenerative medicine. In conclusion, this thesis addresses the question of how optics can help to illuminate future directions in research on tissue repair and regeneration, as well as, regenerative therapies by addressing (longitudinal) imaging in a complex environment, sophisticated cell-manipulation strategies, and the application of novel materials for laser-based delivery

Hybrid bio-robotics: from the nanoscale to the macroscale

[eng] Hybrid bio-robotics is a discipline that aims at integrating biological entities with synthetic materials to incorporate features from biological systems that have been optimized through millions of years of evolution and are difficult to replicate in current robotic systems. We can find examples of this integration at the nanoscale, in the field of catalytic nano- and micromotors, which are particles able to self-propel due to catalytic reactions happening in their surface. By using enzymes, these nanomotors can achieve motion in a biocompatible manner, finding their main applications in active drug delivery. At the microscale, we can find single-cell bio-swimmers that use the motion capabilities of organisms like bacteria or spermatozoa to transport microparticles or microtubes for targeted therapeutics or bio-film removal. At the macroscale, cardiac or skeletal muscle tissue are used to power small robotic devices that can perform simple actions like crawling, swimming, or gripping, due to the contractions of the muscle cells. This dissertation covers several aspects of these kinds of devices from the nanoscale to the macro-scale, focusing on enzymatically propelled nano- and micromotors and skeletal muscle tissue bio-actuators and bio-robots. On the field of enzymatic nanomotors, there is a need for a better description of their dynamics that, consequently, might help understand their motion mechanisms. Here, we focus on several examples of nano- and micromotors that show complex dynamics and we propose different strategies to analyze their motion. We develop a theoretical framework for the particular case of enzymatic motors with exponentially decreasing speed, which break the assumptions of constant speed of current methods of analysis and need different strategies to characterize their motion. Finally, we consider the case of enzymatic nanomotors moving in complex biological matrices, such as hyaluronic acid, and we study their interactions and the effects of the catalytic reaction using dynamic light scattering, showing that nanomotors with negative surface charge and urease-powered motion present enhanced parameters of diffusion in hyaluronic acid. Moving towards muscle-based robotics, we investigate the application of 3D bioprinting for the bioengineering of skeletal muscle tissue. We demonstrate that this technique can yield well-aligned and functional muscle fibers that can be stimulated with electric pulses. Moreover, we develop and apply a novel co-axial approach to obtain thin and individual muscle fibers that resemble the bundle-like organization of native skeletal muscle tissue. We further exploit the versatility of this technique to print several types of materials in the same process and we fabricate bio-actuators based on skeletal muscle tissue with two soft posts. Due to the deflection of these cantilevers when the tissue contracts upon stimulation, we can measure the generated forces, therefore obtaining a force measurement platform that could be useful for muscle development studies or drug testing. With these applications in mind, we study the adaptability of muscle tissue after applying various exercise protocols based on different stimulation frequencies and different post stiffness, finding an increase of the force generation, especially at medium frequencies, that resembles the response of native tissue. Moreover, we adapt the force measurement platform to be used with human-derived myoblasts and we bioengineer two models of young and aged muscle tissue that could be used for drug testing purposes. As a proof of concept, we analyze the effects of a cosmetic peptide ingredient under development, focusing on the kinematics of high stimulation contractions. Finally, we present the fabrication of a muscle-based bio-robot able to swim by inertial strokes in a liquid interface and a nanocomposite-laden bio-robot that can crawl on a surface. The first bio-robot is thoroughly characterized through mechanical simulations, allowing us to optimize the skeleton, based on a serpentine or spring-like structure. Moreover, we compare the motion of symmetric and asymmetric designs, demonstrating that, although symmetric bio-robots can achieve some motion due to spontaneous symmetry breaking during its self-assembly, asymmetric bio-robots are faster and more consistent in their directionality. The nanocomposite-laden crawling bio-robot consisted of embedded piezoelectric boron nitride nanotubes that improved the differentiation of the muscle tissue due to a feedback loop of piezoelectric effect activated by the same spontaneous contractions of the tissue. We find that bio-robots with those nanocomposites achieve faster motion and stronger force outputs, demonstrating the beneficial effects in their differentiation. This research presented in this thesis contributes to the development of the field of bio-hybrid robotic devices. On enzymatically propelled nano- and micromotors, the novel theoretical framework and the results regarding the interaction of nanomotors with complex media might offer useful fundamental knowledge for future biomedical applications of these systems. The bioengineering approaches developed to fabricate murine- or human-based bio-actuators might find applications in drug screening or to model heterogeneous muscle diseases in biomedicine using the patient’s own cells. Finally, the fabrication of bio-hybrid swimmers and nanocomposite crawlers will help understand and improve the swimming motion of these devices, as well as pave the way towards the use of nanocomposite to enhance the performance of future actuators.[spa] La bio-robótica híbrida es una disciplina cuyo objetivo es la integración de entidades biológicas con materiales sintéticos para superar los desafíos existentes en el campo de la robótica blanda, incorporando características de los sistemas biológicos que han sido optimizadas durante millones de años de evolución natural y no son fáciles de reproducir artificialmente. Esta tesis cubre varios aspectos de este tipo de dispositivos desde la nanoescala a la macroescala, enfocándose en nano- y micromotores propulsados enzimáticamente y bio-actuadores y bio-robots basados en tejido muscular esquelético. En el campo de nanomotores enzimáticos, existe la necesidad de encontrar mejores modelos que puedan describir la dinámica de su movimiento para llegar a entender sus mecanismos de propulsión subyacentes. Aquí, nos enfocamos en diversos ejemplos de nano- y micromotores que muestran dinámicas de movimiento complejas y proponemos diferentes estrategias que se pueden utilizar para analizar y caracterizar este movimiento. Moviéndonos hacia robots basados en células musculares, investigamos la aplicación de la técnica de bioimpresión en 3D para la biofabricación de músculo esquelético. Demostramos que esta técnica puede producir fibras musculares funcionales y bien alineadas que puede ser estimuladas y contraerse con pulsos eléctricos. Investigamos la versatilidad de esta técnica para imprimir varios tipos de materiales en el mismo proceso y fabricamos bio-actuadores basados en músculo esquelético. Debido a los movimientos de unos postes gracias a las contracciones musculares, podemos obtener medidas de la fuerza ejercida, obteniendo una plataforma de medición de fuerzas que podría ser de utilidad para estudios sobre el desarrollo del músculo o para testeo de fármacos. Finalmente, presentamos la fabricación de un bio-robot basado en músculo esquelético capaz de nadar en la superficie de un líquido y un bio-robot con nanocompuestos incrustados que puede arrastrarse por una superficie sólida. El primer de ellos es minuciosamente caracterizado a través de simulaciones mecánicas, permitiéndonos optimizar su esqueleto, basado en una estructura tipo serpentina o muelle. El segundo bio-robot contiene nanotubos piezoeléctricos incrustados en su tejido, los cuales ayudan en la diferenciación del músculo debido a una retroalimentación basada en su efecto piezoeléctrico y activada por las contracciones espontáneas del tejido. Mostramos que estos bio-robots pueden generar un movimiento más rápido y una mayor generación de fuerza, demostrando los efectos beneficiales en la diferenciación del tejido

XXI SPB CONGRESS BOOK

The University of Évora welcomes YOU at the XXI SPB National Congress of Biochemistry 2020 in 14-16 October 2021 either in person or online! Under challenging conditions, due to the COVID-19 pandemic, we have managed to organize the National Congress of Biochemistry in a hybrid format, where at least 2/3 of the participants will come to Évora in person. With the pandemic under control and we hope to carry out the Congress both successfully as well as safely. This is the main meeting point for Portuguese Biochemistry Academy, fostering the discussion and dissemination of high-quality research in Biochemistry, both fundamental and applied, taking place in Portugal. The Scientific Program covers a wide range of issues, from Health and Disease to Environment and Drugs development, where Biochemistry is either fundamental or instrumental in the study of complex and transdisciplinary problems in the society. The Congress is a moment of Science and Innovation in several Biochemistry domains, sharing experiences and fostering healthy confraternization. Despite the difficult context, over 160 confirmed registrations and >120 abstracts were submitted, involving the whole Portuguese Biochemical community. Moreover, this year for the first time the Congress has gone eco-friendly, with ePosters only where short poster presentations are encouraged. We hope the congress meets your expectations! The Organizing Committee, on behalf of the Portuguese Biochemical Society, looks forward meeting you, at Colégio do Espírito Santo, University of Évora!Sociedade Portuguesa de Bioquímica - SPB; Biochem; Instituto de Ciências da Terra - ICT; Fundação Eugénio de Almeida; Câmara Municipal de Évora; Universidade de Évora; Labor Spirit; Terrius; Nitrifresco; Evora Hotel

Life Sciences Program Tasks and Bibliography for FY 1997

This document includes information on all peer reviewed projects funded by the Office of Life and Microgravity Sciences and Applications, Life Sciences Division during fiscal year 1997. This document will be published annually and made available to scientists in the space life sciences field both as a hard copy and as an interactive internet web page

Role for the phytocalpain DEK1 in plant mechanosensing

The effect of mechanical stimulation in plants has been studied in depth for more than a century. This type of stress has been shown to trigger alterations in development such as stunting, thickened stems and differential cell wall deposition. These responses are very likely to be initiated at a subcellular level, but the molecular mechanisms transducing mechanical signals into intracellular responses still remain unknown in plants. In this thesis I test the hypothesis that the membrane anchored protein Defective Kernel 1 (DEK1) could act as a plant-specific mechanosensor in plants. Constitutive overexpression of the cytoplasmic CALPAIN domain DEK1 causes a phenotype in Arabidopsis, that that resembles that of mechanically stressed plants. The CALPAIN domain of DEK1 shows a very high homology with animal calpains; a class of calcium-dependent Cysteine proteases which undergo a calciumstimulated CALPAIN domain-releasing autolytic cleavage event during activation. A similar autolytic cleavage event has been observed in DEK1 which, together with the fact that the CALPAIN domain alone can rescue the embryo-lethality associated with loss of DEK1 function, has led to the suggestion that this domain represents an activated form of the protein. I show that like mechanically stressed plants, CALPAIN overexpressing plants show a modified call wall composition. Consistent with this, transcriptional analysis of these plants shows a deregulation of genes encoding cell wall modifying enzymes, amongst others. Other characteristics of mechanically stimulated plants which I have characterized in CALPAIN overexpressing lines include late flowering and thickened stems. Therefore, I proposed a model in which the CALPAIN domain of DEK1 acts as an effector which is normally activated by mechanical stimulation. In this model, the transmembrane domains of DEK1 would regulate activation (cleavage) of the CALPAIN domain, potentially in response to mechanical stress. In order to test this model further, CALPAIN overexpressing lines were generated in a dek1 mutant background. If the model is correct, these plants should not only behave as if responding constitutively to mechanical stimulation, but should also lack appropriate responses to applied mechanical stimuli due to lack of the mechanosensory integral membrane domain of DEK1. My results confirm that the absence of the transmembrane domains of DEK1 is indeed translated into a lack of some, but not all responses to mechanical stimulation compared to wild-type plants. Furthermore, the lack of the transmembrane domains of DEK1 correlates with the absence of a mechanically-triggered calcium flux in the plant. Thus my work suggests that the transmembrane domains of DEK1 are involved in sensing mechanical stimulation, via the regulation activity of a mechano-sensitive calcium flux at the plasma membrane. In summary, my proposal is that Defective Kernel 1 (DEK1) acts both as a key mechanosensory cellular component, and as the first effector of the signalling cascade in response to mechanical stimulation, via an autolytic activation in response to mechanical stress

Nuevos blancos en la respuesta vegetal a la deficiencia de boro: N-glicosilación de proteínas y la regulación del desarrollo de la raíz)

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 22-01-2016Esta tesis tiene embargado el acceso al texto completo hasta el 22-07-201

Life Sciences Program Tasks and Bibliography for FY 1996

This document includes information on all peer reviewed projects funded by the Office of Life and Microgravity Sciences and Applications, Life Sciences Division during fiscal year 1996. This document will be published annually and made available to scientists in the space life sciences field both as a hard copy and as an interactive Internet web page

Studies on NET4B and associated proteins

The cytoskeleton is an essential component of the eukaryotic cell, determining both the cellular architecture and function. In plant cells, the cytoskeleton is composed of two distinct networks of filamentous proteins; microtubules and actin microfilaments, as well as a plethora of associated proteins that organise or regulate the network. Metazoans use a range of adaptor proteins, such as spectrin, α-actinin, filamin, and the FERM-domain proteins, that link actin to membrane structures. However these protein families are absent in plants, despite the predominance of the actin cytoskeleton in organelle and endomembrane trafficking. Recently a novel plant-specific superfamily of actin-binding proteins has been identified, termed the Networked (NET) family. The NET family is composed of thirteen members in Arabidopsis thaliana, divided into four phylogenetic clades, with members of each subclade associating with specific membrane compartments (Deeks et al. 2012; Wang et al. 2014). The NET4s are the only NET subfamily that can be found universally throughout the genomes of the Tracheophytes, and in A. thaliana NET4A associates with actin surrounding the vacuole. NET4B remains a relatively uncharacterised member of the NET family and was thus the focus of this project. Through live cell imaging and an actin cosedimentation assay, NET4B was shown to bind actin filaments in vivo and in vitro. The expression pattern of NET4B in plants was investigated using NET4Bpromoter::GUS lines, demonstrating a high expression in roots and guard cells. Immunogold labelling of plant roots with a NET4B specific antibody revealed its preferential localisation to the tonoplast. The NET4s therefore represent novel actin-vacuole adaptors in plants, and this project investigates the role of these proteins in plant cell growth and identifies key interacting partners that implicate the NET4s in signalling events

Preclinincal development of Avaren-Fc: a novel lectin-Fc fusion protein targeting cancer-associated high-mannose glycans.

This dissertation explores the anticancer activity of Avaren-Fc (AvFc), a novel lectin-Fc fusion protein or “lectibody” targeting cancer and virus-associated high-mannose glycans. Previously, we have shown that AvFc recognizes a broad selection of established cancer cell lines from a wide array of tissue types, can potently induce antibody-dependent cell-mediated cytotoxicity (ADCC) against them, and exhibits anti-cancer activity in vivo. However, the exact mechanism of action remains elusive. We hypothesized that the primary mechanism of action is through Fc-mediated effector functions, and the purpose of this dissertation is to explore this question through the use of Fc variants that either increase or decrease ADCC activity relative to the WT molecule using the B16F10 murine melanoma model. Chapters 1 and 2 give a comprehensive overview of glycosylation and its role in cancer and disease, the molecule AvFc, the mechanism of action of the various Fc-mediated effector functions, and the current status of plant-made cancer biologics. Chapter 4 discusses the efficacy of AvFc in a human liver chimeric mouse model of HCV infection, which helped not only to establish AvFc’s activity in vivo but also demonstrated its safety and feasibility as a drug candidate. The bulk of the data obtained regarding the anticancer activity of AvFc are contained in Chapter 5, which establishes that Fc-mediated functions are the primary mechanisms of action and that AvFc administration is associated with the recruitment of FcγR-bearing cells to the tumor microenvironment. Interestingly, these studies also indicated that the presence of pre-existing immunity in the presence of anti-drug antibodies to AvFc did not obviate its activity in vivo. Further exploration of the anticancer activity of AvFc is detailed in Chapter 6, which discusses the use of AvFc as a therapeutic for ovarian cancer (OVCA) and details its in vitro and in vivo activities. The results presented herein provide evidence to suggest that cancer-associated high-mannose glycans may be a viable pharmacological target and that AvFc is a unique and potent first-in-class agent with significant anticancer capabilities through recognition of this glycobiomarker, warranting its further development as a therapeutic against cancers with limited therapeutic options such as OVCA