Collagen-Based Mechanical Anisotropy of the Tectorial Membrane: Implications for Inter-Row Coupling of Outer Hair Cell Bundles

The tectorial membrane (TM) in the mammalian cochlea displays anisotropy, where mechanical or structural properties differ along varying directions. The anisotropy arises from the presence of collagen fibrils organized in fibers of approximately 1 microm diameter that run radially across the TM. Mechanical coupling between the TM and the sensory epithelia is required for normal hearing. However, the lack of a suitable technique to measure mechanical anisotropy at the microscale level has hindered understanding of the TM's precise role.Here we report values of the three elastic moduli that characterize the anisotropic mechanical properties of the TM. Our novel technique combined Atomic Force Microscopy (AFM), modeling, and optical tracking of microspheres to determine the elastic moduli. We found that the TM's large mechanical anisotropy results in a marked transmission of deformations along the direction that maximizes sensory cell excitation, whereas in the perpendicular direction the transmission is greatly reduced.Computational results, based on our values of elastic moduli, suggest that the TM facilitates the directional cooperativity of sensory cells in the cochlea, and that mechanical properties of the TM are tuned to guarantee that the magnitude of sound-induced tip-link stretching remains similar along the length of the cochlea. Furthermore, we anticipate our assay to be a starting point for other studies of biological tissues that require directional functionality

Variación normal de superficies

Treball Final de Grau en Matemàtica Computacional. Codi: MT1054. Curs: 2021/2022Este documento presenta el proyecto fin de grado de la asignatura MT1054-Trabajo Fin de Grado del grado en Matemática Computacional. Se estudiará la variación normal del área de superficies de forma teórica y computacional. Empezaremos con conceptos básicos sobre las superficies regulares, vistos durante los estudios de grado, para poder introducir la variación normal de una superficie parametrizada regular. Demostraremos que la variación normal es una familia uni-paramétrica de superficies parametrizadas regulares y calcularemos la primera y segunda derivada del área. Por otra parte, desarrollaremos un programa en Mathematica que nos permitirá calcular el área y representar como varían las superficies. Finalmente explicaremos el funcionamiento del código y presentaremos dos ejemplos de superficies y de su variación normal

Low-cost, open-source device for simultaneously subjecting rodents to different circadian cycles of light, food, and temperature

Exposure of experimental rodents to controlled cycles of light, food, and temperature is important when investigating alterations in circadian cycles that profoundly influence health and disease. However, applying such stimuli simultaneously is difficult in practice. We aimed to design, build, test, and open-source describe a simple device that subjects a conventional mouse cage to independent cycles of physiologically relevant environmental variables. The device is based on a box enclosing the rodent cage to modify the light, feeding, and temperature environments. The device provides temperature-controlled air conditioning (heating or cooling) by a Peltier module and includes programmable feeding and illumination. All functions are set by a user-friendly front panel for independent cycle programming. Bench testing with a model simulating the CO2 production of mice in the cage showed: a) suitable air renewal (by measuring actual ambient CO2), b) controlled realistic illumination at the mouse enclosure (measured by a photometer), c) stable temperature control, and d) correct cycling of light, feeding, and temperature. The cost of all the supplies (retail purchased by e-commerce) was <300 US$. Detailed technical information is open-source provided, allowing for any user to reliably reproduce or modify the device. This approach can considerably facilitate circadian research since using one of the described low-cost devices for any mouse group with a given light-food-temperature paradigm allows for all the experiments to be performed simultaneously, thereby requiring no changes in the light/temperature of a general-use laboratory

Lung Extracellular Matrix Hydrogels-Derived Vesicles Contribute to Epithelial Lung Repair

The use of physiomimetic decellularized extracellular matrix-derived hydrogels is attracting interest since they can modulate the therapeutic capacity of numerous cell types, including mesenchymal stromal cells (MSCs). Remarkably, extracellular vesicles (EVs) derived from MSCs display similar functions as their parental cells, mitigating tissue damage in lung diseases. However, recent data have shown that ECM-derived hydrogels could release other resident vesicles similar to EVs. Here, we aim to better understand the contribution of EVs and ECM-vesicles released from MSCs and/or lung-derived hydrogel (L-HG) in lung repair by using an in vitro lung injury model. L-HG derived-vesicles and MSCs EVs cultured either in L-HG or conventional plates were isolated and characterized. The therapeutic capacity of vesicles obtained from each experimental condition was tested by using an alveolar epithelial wound-healing assay. The number of ECM-vesicles released from acellular L-HG was 10-fold greater than EVs from conventional MSCs cell culture revealing that L-HG is an important source of bioactive vesicles. MSCs-derived EVs and L-HG vesicles have similar therapeutic capacity in lung repair. However, when wound closure rate was normalized by total proteins, the MSCs-derived EVs shows higher therapeutic potential to those released by L-HG. The EVs released from L-HG must be considered when HG is used as substrate for cell culture and EVs isolation

Thrombin-induced contraction in alveolar epithelial cells probed by traction microscopy

Contractile tension of alveolar epithelial cells plays a major role in the force balance that regulates the structural integrity of the alveolar barrier. The aim of this work was to study thrombin-induced contractile forces of alveolar epithelial cells. A549 alveolar epithelial cells were challenged with thrombin, and time course of contractile forces was measured by traction microscopy. The cells exhibited basal contraction with total force magnitude 55.0 ± 12.0 nN (mean ± SE, n = 12). Traction forces were exerted predominantly at the cell periphery and pointed to the cell center. Thrombin (1 U/ml) induced a fast and sustained 2.5-fold increase in traction forces, which maintained peripheral and centripetal distribution. Actin fluorescent staining revealed F-actin polymerization and enhancement of peripheral actin rim. Disruption of actin cytoskeleton with cytochalasin D (5 µM, 30 min) and inhibition of myosin light chain kinase with ML-7 (10 µM, 30 min) and Rho kinase with Y-27632 (10 µM, 30 min) markedly depressed basal contractile tone and abolished thrombin-induced cell contraction. Therefore, the contractile response of alveolar epithelial cells to the inflammatory agonist thrombin was mediated by actin cytoskeleton remodeling and actomyosin activation through myosin light chain kinase and Rho kinase signaling pathways. Thrombin-induced contractile tension might further impair alveolar epithelial barrier integrity in the injured lung

A Fast and Efficient Decellularization Method for Tissue Slices

The study and use of decellularized extracellular matrix (dECM) in tissue engineering, regenerative medicine, and pathophysiology have become more prevalent in recent years. To obtain dECM, numerous decellularization procedures have been developed for the entire organ or tissue blocks, employing either perfusion of decellularizing agents through the tissue's vessels or submersion of large sections in decellularizing solutions. However, none of these protocols are suitable for thin tissue slices (less than 100 μm) or allow side-by-side analysis of native and dECM consecutive tissue slices. Here, we present a detailed protocol to decellularize tissue sections while maintaining the sample attached to a glass slide. This protocol consists of consecutive washes and incubations of simple decellularizing agents: ultrapure water, sodium deoxycholate (SD) 2%, and deoxyribonuclease I solution 0.3 mg/mL (DNase I). This novel method has been optimized for a faster decellularization time (2-3 h) and a better correlation between dECM properties and native tissue-specific biomarkers, and has been tested in different types of tissues and species, obtaining similar results. Furthermore, this method can be used for scarce and valuable samples such as clinical biopsies

Development of a physiomimetic model of acute respiratory distress syndrome by using ECM hydrogels and organ-on-a-chip devices

Acute Respiratory Distress Syndrome is one of the more common fatal complications in COVID-19, characterized by a highly aberrant inflammatory response. Pre-clinical models to study the effect of cell therapy and anti-inflammatory treatments have not comprehensively reproduced the disease due to its high complexity. This work presents a novel physiomimetic in vitro model for Acute Respiratory Distress Syndrome using lung extracellular matrix-derived hydrogels and organ-on-a-chip devices. Monolayres of primary alveolar epithelial cells were cultured on top of decellullarized lung hydrogels containing primary lung mesenchymal stromal cells. Then, cyclic stretch was applied to mimic breathing, and an inflammatory response was induced by using a bacteriotoxin hit. Having simulated the inflamed breathing lung environment, we assessed the effect of an anti-inflammatory drug (i.e., dexamethasone) by studying the secretion of the most relevant inflammatory cytokines. To better identify key players in our model, the impact of the individual factors (cyclic stretch, decellularized lung hydrogel scaffold, and the presence of mesenchymal stromal cells) was studied separately. Results showed that developed model presented a more reduced inflammatory response than traditional models, which is in line with what is expected from the response commonly observed in patients. Further, from the individual analysis of the different stimuli, it was observed that the use of extracellular matrix hydrogels obtained from decellularized lungs had the most significant impact on the change of the inflammatory response. The developed model then opens the door for further in vitro studies with a better-adjusted response to the inflammatory hit and more robust results in the test of different drugs or cell therapy

Lung Extracellular Matrix Hydrogels Enhance Preservation of Type II Phenotype in Primary Alveolar Epithelial Cells

One of the main limitations of in vitro studies on lung diseases is the difficulty of maintaining the type II phenotype of alveolar epithelial cells in culture. This fact has previously been related to the translocation of the mechanosensing Yes-associated protein (YAP) to the nuclei and Rho signaling pathway. In this work, we aimed to culture and subculture primary alveolar type II cells on extracellular matrix lung-derived hydrogels to assess their suitability for phenotype maintenance. Cells cultured on lung hydrogels formed monolayers and maintained type II phenotype for a longer time as compared with those conventionally cultured. Interestingly, cells successfully grew when they were subsequently cultured on a dish. Moreover, cells cultured on a plate showed the active form of the YAP protein and the formation of stress fibers and focal adhesions. The results of chemically inhibiting the Rho pathway strongly suggest that this is one of the mechanisms by which the hydrogel promotes type II phenotype maintenance. These results regarding protein expression strongly suggest that the chemical and biophysical properties of the hydrogel have a considerable impact on the transition from ATII to ATI phenotypes. In conclusion, culturing primary alveolar epithelial cells on lung ECM-derived hydrogels may facilitate the prolonged culturing of these cells, and thus help in the research on lung diseases

Col·leccions singulars a les biblioteques de la Universitat Autònoma de Barcelona

Les biblioteques de la Universitat Autònoma de Barcelona són, com la mateixa institució, entitats amb una història curta; però, malgrat que van començar la seva activitat fa menys de cinquanta anys, els seus fons han assolit una importància considerable i superen en quantitat els de moltes universitats centenàries del nostre context. Les col·leccions de fons antics de les biblioteques de la UAB són, per aquesta mateixa raó, limitades, si bé se n'han anat creant algunes d'especialitzades d'importància i valor singulars. Aquest llibre pretén donar-les a conèixer al món universitari, però també al públic en general. Moltes són col·leccions úniques, fruit del treball persistent del personal bibliotecari, del professorat i de la generositat de moltes persones particulars, que han donat o llegat a la nostra universitat els seus arxius, les seves biblioteques personals o les seves col·leccions especialitzades. Aquestes col·leccions comprenen molts àmbits de les ciències i de les humanitats i, molt sovint, es tracta de col·leccions úniques al nostre país. Hi trobareu també un ampli ventall de tipologies documentals en llengües diverses, des dels mapes fins als audiovisuals, des de les revistes i diaris fins als cartells, des dels fons antics fins als més actuals, arxius personals i fons institucionals. Les biblioteques de la Universitat, a més d'inventariar i catalogar aquests fons documentals, també porten a terme una tasca constant de preservació i difusió, que sovint inclou la digitalització dels documents, que després es posen a l'abast del públic general mitjançant el dipòsit digital institucional (ddd.uab.cat)

Contractile response of alveolar epithelial cells to biochemical or mechanical stimulation probed by traction microscopy

THESIS SUMMARY:GENERAL AIMThe general aim of this thesis was to study the generation of contractile force by human alveolar epithelial cells in culture in response to biochemical or mechanical stimuli using traction microscopy.SPECIFIC AIMS1. To implement a traction microscopy setup to measure the contractile force generated by human alveolar epithelial cells in culture.1.1. To implement and validate a software to determine the deformation field induced by adhered cells on the elastic substrate, following previously described algorithms.1.2. To implement and validate a software to determine the traction field induced by adhered cells and other contractility parameters, following previously described algorithms.1.3. To implement a software to determine the contour of an adhered cell from a brightfield or phase contrast image of the cell.2. To study the contractile response of human alveolar epithelial cells in response to thrombin.2.1. To determine the gel substrate conditions and gel fabrication procedure which enable suitable cell culture and optimal detection of traction forces exerted by human alveolar epithelial cells. These gel conditions include: concentration of polyacrilamide gel components to provide optimal gels stiffness; concentration of fluorescent beads to optimally compute gel deformation; and suitable gel coating to enable cell attachment.2.2. To determine the gel elastic properties (Young's modulus) by atomic force microscopy.2.3. To measure the time-course of the contractile response to thrombin challenge.2.4. To study the distribution of contractile forces exerted by adhered cells before and after thrombin stimulation.2.5. To measure actin polymerization and reorganization induced by thrombin challenge.2.6. To study the role of the actin cytoskeleton in the contractile response to thrombin by pre-treatments with cytochalasin D.2.7. To study the role of pathways signalling MLC phosphorilation in the contractile response to thrombin by pre-treatments with ML7 and Y-27632.3. To study the contractile response of human alveolar epithelial cells subjected to stretch.3.1. To determine a suitable gel substrate that firmly attaches to a flexible membrane, allowing biaxial stretch application (max ~15%) and cell culture.3.2. To determine the gel elastic properties (Young's modulus) of the gel at different strain levels by atomic force microscopy.3.3. To implement and validate a stretching device to apply controlled biaxial and uniform strains to cultured cells and simultaneously measure contractile forces by deforming the gel substrate to which they are adhered.3.4. To adapt the existing traction microscopy algorithms and software to allow computation of large bead displacements (~20 &#956;m) and corresponding stretch fields.3.5. To measure contractile forces exerted by human alveolar epithelial cells before, during and after being subjected to a stepwise deformation of up to 11.5% linear strain.3.6. To assess the role of actin polymerization in the contractile response to stretch.3.7. To assess the role of actomyosin crossbridges attachment or detachment in the contractile response to stretch.3.8. To assess temporal changes in cell contractility after stretch release."Estudi de la contracció de cèl·lules epitelials alveolars en resposta a estímuls inflamatoris i de deformació mitjançant microscopia de tracció"TEXT:L'epiteli alveolar forma una barrera cel·lular semipermeable entre l'espai alveolar i l'interstici del pulmó, permetent l'intercanvi gasós a la vegada que restringeix el pas de líquid, macromolècules i cèl·lules cap a l'alvèol. El trencament de la monocapa, degut a la formació de forats entre cèl·lules adjacents, pot donar lloc a l'augment de la permeabilitat i l'entrada de líquid a l'alvèol, característics del dany pulmonar agut. La integritat de la monocapa epitelial es regeix per un equilibri dinàmic de forces als punts d'unió cèl·lula-cèl·lula, i cèl·lula-matriu. Les forces en joc es divideixen en una component de tensió centrípeta i una component d'adhesió centrífuga. La component centrípeta es deguda a les forces de contracció generades activament per la maquinària contràctil cel·lular i el retrocés passiu degut a la deformació cíclica a la que es troben sotmeses les cèl·lules alveolars durant la respiració. Per tal de garantir la integritat de la monocapa, les forces d'adhesió han de ser capaces de contrarestar la tensió centrípeta. L'equilibri de forces als punts d'unió pot veure's compromès degut a estímuls inflamatoris o bé mecànics.El projecte de tesi es centra en el paper de les forces actives de contracció sobre la integritat de la monocapa alveolar en resposta a estímuls característics del dany pulmonar agut. Per tal d'estudiar aquesta component contràctil, el present projecte ha utilitzat la microscopia de tracció. Aquesta tècnica permet mesurar la força que cèl·lules adherents aïllades realitzen sobre el seu substrat, així com la seva distribució espaial i evolución temporal. La tècnica consisteix en cultivar cèl·lules adherents sobre substrats elàstics que contenen microesferes fluorescents. Comparant la posició de les microesferes quan la cèl·lula es troba adherida al substrat i un cop aquesta ha estat desenganxada amb tripsina, podem calcular la força que la cèl·lulaadherent realitzava sobre el substrat.El projecte de tesi inclou dos estudis concrets, dedicats a dos estímuls característics de dany pulmonar agut als qual poden trobar-se sotmeses les cèl·lules epitelials alveolars. El primer estudi (secció 3) es centra en l'efecte que el mediador inflamatori trombina provoca sobre la realització de forces contràctils per part de cèl·lules alveolars epitelials. El segon estudi (secció 4) es centra en la resposta contràctil de cèl·lules alveolars epitelials a l'aplicar deformacions externes, simulant les condicions de respiració mecànica que requereixen molts pacients amb dany pulmonar agut. </i