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

Cotutela Universitat Politècnica de Catalunya i Università 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 aLß2 and a4ß1, 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 a4ß1 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 a4ß1 remains unaffected upon CXCL12 and/or VCAM-1 stimulation. Our data, thus, indicate that docking by talin of the chemokine-activated a4ß1 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 migración de leucocitos desde el torrente sanguíneo a sitios de lesión e infección es fundamental en la respuesta inmune. Dos de los principales receptores que median este proceso son las integrinas aLß2 y a4ß1, 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 células endoteliales (CE). Se ha demostrado que la dinámica y la organización lateral de las integrinas sobre la membrana celular son cruciales en la regulación de la adhesión celular. Asimismo, es muy probable que la organización de los ligandos en pequeños dominios refuerce los enlaces formados con las integrinas, fortaleciendo así la adhesión celular. Sin embargo, cómo el comportamiento espacio-temporal de las integrinas y sus ligandos es afectado por factores externos, tales como estímulos mecánicos y/o bioquímicos, no ha sido ampliamente estudiado. Además, el impacto de estos cambios espacio-temporales en el proceso de adhesión y migración leucocitaria aún no ha sido abordado. El objetivo principal de esta tesis ha sido abordar estas interrogantes usando una combinación de herramientas biofísicas de última generación que incluyen avanzadas técnicas de visualización, dinámica de moléculas individuales, estimulación mecánica de células y desarrollo de algoritmos para la cuantificación de datos. Con este fin hemos utilizado métodos de seguimiento de partículas individuales para monitorizar la dinámica de moléculas individuales, en combinación con técnicas de microscopía de super-resolución, como STED y STORM, para visualizar cambios en su organización a nano-escala tras la estimulación bioquímica y/o mecánica. Para estimular las células mecánicamente hemos desarrollado dos métodos: un dispositivo de estiramiento y una cámara de flujo de placa paralela. Ambos dispositivos fueron integrados a nuestro montaje experimental de detección de moléculas individuales y el primero fue exitosamente utilizado en la caracterización de la difusión de integrinas en células cultivadas en el dispositivo de estiramiento mecánico. Como parte de esta tesis también usamos diferentes algoritmos, existentes en nuestro grupo, para el análisis de datos y desarrollamos otros nuevos para el seguimiento y cuantificación del comportamiento migratorio de células T sometidas a flujo continuo. Utilizando estas herramientas descubrimos que, a causa de la exposición a flujo mecánico, ICAM-1 se reorganiza sobre la membrana de las CEs y forma nano-agregados. Se encontró además que estos agregados se colocalizan con regiones ricas en actina con estructura de parches. Además mostramos que las células T migran más rápido e interactúan por periodos más cortos de tiempo con CEs estimuladas mecánicamente respecto a CEs no estimuladas. De estos resultados concluimos que el flujo mecánico regula la organización espacial de los receptores de adhesión en las CEs, que a su vez modula la migración de leucocitos. También demostramos que la estimulación con quimioquinas (CXCL12) conduce a una rápida y transitoria activación de a4ß1. Estos cambios en la activación de a4ß1 se correlacionaron con el reclutamiento de talina, la inmovilización de la integrina y su reducida difusión lateral. Además, la coestimulación con CXCL12 y el ligando VCAM-1 potenció la inmovilización de a4ß1. Adicionalmente, imágenes de superresolución revelaron que la organización a nanométrica de a4ß1 no se ve afectada por la estimulación 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 inmovilización de a4ß1 y facilitando así, la interacción con su ligando y una mayor adhesión. En resumen, esta tesis indica que las células del sistema inmune responden a estímulos mecánicos y bioquímicos mediante el reajuste rápido de la organización espacio-temporal de integrinas y ligandos sobre la membrana celular, modulando así su adhesión y migraciónPostprint (published version

Influência das instruções aos autores e normas de publicação nas estratégias linguístico-discursivas utilizadas na produção do género textual caso clínico na era digital

Um dos géneros textuais mais antigos da literatura médica mundial é o caso clínico, com algumas das primeiras referências datadas de 1600 a.C. A sua evolução é percetível, embora não seja acentuada do ponto de vista diacrónico, tendo sido alvo, inclusive, de um período de diminuição da sua produção na segunda metade do século XX, aquando da instituição do método quantitativo defendido pela investigação biomédica. No entanto, o seu valor pedagógico na formação de novos médicos e na prática clínica fez com que a comunidade médica contestasse a sua não aceitação em revistas médicas e, a partir dos anos 90, o género foi retomado, tendo lugar lançamentos como o da secção Case Reports da revista científica Lancet

PLANT: A Method for Detecting Changes of Slope in Noisy Trajectories

Time traces obtained from a variety of biophysical experiments contain valuable information on underlying processes occurring at the molecular level. Accurate quantification of these data can help explain the details of the complex dynamics of biological systems. Here, we describe PLANT (Piecewise Linear Approximation of Noisy Trajectories), a segmentation algorithm that allows the reconstruction of time-trace data with constant noise as consecutive straight lines, from which changes of slopes and their respective durations can be extracted. We present a general description of the algorithm and perform extensive simulations to characterize its strengths and limitations, providing a rationale for the performance of the algorithm in the different conditions tested. We further apply the algorithm to experimental data obtained from tracking the centroid position of lymphocytes migrating under the effect of a laminar flow and from single myosin molecules interacting with actin in a dual-trap force-clamp configuration.The authors gratefully acknowledge financial support fromthe European Commission (FP7-ICT-2011-7, grant number 288263), Erasmus Mundus Doctorate Program Europhoton-ics (grant number 159224-1-2009-1-FR-ERA MUNDUS-EMJD), Spanish Ministry of Economy and Competi-tiveness (“Severo Ochoa” Programme for Centres of Excellence in Research & Development SEV-2015-0522,and FIS2014-56107-R grants), Generalitat de Catalunyathrough the CERCA program, Italian Ministry of Uni-versity and Research (FIRB “Futuro in Ricerca” 2013grant n. RBFR13V4M2 and Flagship Project NANOMAX),Fundaci ́o Privada CELLEX (Barcelona), Ente Cassa diRisparmio di Firenze, Human Frontier Science Program (GARGP0027/2012) and LaserLab Europe 4 (GA 654148). C.M.acknowledges funding from the Spanish Ministry of Econ-omy and Competitiveness (MINECO) and the EuropeanSocial Fund (ESF) through the Ram ́on y Cajal program 2015(RYC-2015-17896).Peer ReviewedPostprint (author's final draft

High sensitivity measurements of the CMB power spectrum with the extended Very Small Array

We present deep Ka-band ($\nu \approx 33$ GHz) observations of the CMB made with the extended Very Small Array (VSA). This configuration produces a naturally weighted synthesized FWHM beamwidth of $\sim 11$ arcmin which covers an $\ell$-range of 300 to 1500. On these scales, foreground extragalactic sources can be a major source of contamination to the CMB anisotropy. This problem has been alleviated by identifying sources at 15 GHz with the Ryle Telescope and then monitoring these sources at 33 GHz using a single baseline interferometer co-located with the VSA. Sources with flux densities \gtsim 20 mJy at 33 GHz are subtracted from the data. In addition, we calculate a statistical correction for the small residual contribution from weaker sources that are below the detection limit of the survey. The CMB power spectrum corrected for Galactic foregrounds and extragalactic point sources is presented. A total $\ell$-range of 150-1500 is achieved by combining the complete extended array data with earlier VSA data in a compact configuration. Our resolution of $\Delta \ell \approx 60$ allows the first 3 acoustic peaks to be clearly delineated. The is achieved by using mosaiced observations in 7 regions covering a total area of 82 sq. degrees. There is good agreement with WMAP data up to $\ell=700$ where WMAP data run out of resolution. For higher $\ell$-values out to $\ell = 1500$, the agreement in power spectrum amplitudes with other experiments is also very good despite differences in frequency and observing technique.Comment: 16 pages. Accepted in MNRAS (minor revisions

2 nd Brazilian Consensus on Chagas Disease, 2015

Abstract Chagas disease is a neglected chronic condition with a high burden of morbidity and mortality. It has considerable psychological, social, and economic impacts. The disease represents a significant public health issue in Brazil, with different regional patterns. This document presents the evidence that resulted in the Brazilian Consensus on Chagas Disease. The objective was to review and standardize strategies for diagnosis, treatment, prevention, and control of Chagas disease in the country, based on the available scientific evidence. The consensus is based on the articulation and strategic contribution of renowned Brazilian experts with knowledge and experience on various aspects of the disease. It is the result of a close collaboration between the Brazilian Society of Tropical Medicine and the Ministry of Health. It is hoped that this document will strengthen the development of integrated actions against Chagas disease in the country, focusing on epidemiology, management, comprehensive care (including families and communities), communication, information, education, and research

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Measurement of the top quark forward-backward production asymmetry and the anomalous chromoelectric and chromomagnetic moments in pp collisions at √s = 13 TeV

Abstract The parton-level top quark (t) forward-backward asymmetry and the anomalous chromoelectric (d̂ t) and chromomagnetic (μ̂ t) moments have been measured using LHC pp collisions at a center-of-mass energy of 13 TeV, collected in the CMS detector in a data sample corresponding to an integrated luminosity of 35.9 fb−1. The linearized variable AFB(1) is used to approximate the asymmetry. Candidate t t ¯ events decaying to a muon or electron and jets in final states with low and high Lorentz boosts are selected and reconstructed using a fit of the kinematic distributions of the decay products to those expected for t t ¯ final states. The values found for the parameters are AFB(1)=0.048−0.087+0.095(stat)−0.029+0.020(syst),μ̂t=−0.024−0.009+0.013(stat)−0.011+0.016(syst), and a limit is placed on the magnitude of | d̂ t| < 0.03 at 95% confidence level. [Figure not available: see fulltext.

Measurement of the azimuthal anisotropy of Y(1S) and Y(2S) mesons in PbPb collisions at root s(NN)=5.02 TeV

The second-order Fourier coefficients (v(2)) characterizing the azimuthal distributions of Y(1S) and Y(2S) mesons produced in PbPb collisions at root s(NN) = 5.02 TeV are studied. The Y mesons are reconstructed in their dimuon decay channel, as measured by the CMS detector. The collected data set corresponds to an integrated luminosity of 1.7 nb(-1). The scalar product method is used to extract the v2 coefficients of the azimuthal distributions. Results are reported for the rapidity range vertical bar y vertical bar < 2.4, in the transverse momentum interval 0 < pT < 50 GeV/c, and in three centrality ranges of 10-30%, 30-50% and 50-90%. In contrast to the J/psi mesons, the measured v(2) values for the Y mesons are found to be consistent with zero. (C) 2021 The Author(s). Published by Elsevier B.V.Peer reviewe

Development and validation of HERWIG 7 tunes from CMS underlying-event measurements

This paper presents new sets of parameters (“tunes”) for the underlying-event model of the HERWIG7 event generator. These parameters control the description of multiple-parton interactions (MPI) and colour reconnection in HERWIG7, and are obtained from a fit to minimum-bias data collected by the CMS experiment at s=0.9, 7, and 13Te. The tunes are based on the NNPDF 3.1 next-to-next-to-leading-order parton distribution function (PDF) set for the parton shower, and either a leading-order or next-to-next-to-leading-order PDF set for the simulation of MPI and the beam remnants. Predictions utilizing the tunes are produced for event shape observables in electron-positron collisions, and for minimum-bias, inclusive jet, top quark pair, and Z and W boson events in proton-proton collisions, and are compared with data. Each of the new tunes describes the data at a reasonable level, and the tunes using a leading-order PDF for the simulation of MPI provide the best description of the dat