Immunoreceptor MerTK: A journey from the membrane into the nucleus of human dendritic cells

[eng] Discrimination between foreign and potentially harmful antigens, and the body’s own tissue is one of the most crucial first steps that lays at the basis of a proper immune response. Immunoreceptors are cell membrane embedded molecules that aid immune cells in identifying and interacting with its environment. Because of their key importance, they are therefore a frequent subject of research in immunology. It is becoming increasingly clear that the organization of immunoreceptors in space and time on the plasma membrane directly impacts on the way they function. Over the past two decades, novel microscopy techniques and biophysical tools have been developed and exploited to directly visualize molecular events in immune cells with unprecedented spatial and temporal resolution. These technical advances have led to the emergence of a new, active field of research: Nano-immunology. Biophysical tools and super-resolution imaging have been exploited in this thesis to unravel the spatiotemporal behaviour of different immunoreceptors, with a particular emphasis on the tyrosine kinase immunoreceptor MerTK. These studies have contributed to further our understanding of immune cell biology at the molecular level. In Part I of this thesis, I will discuss several advanced imaging techniques and microfabrication approaches that I used throughout my doctoral studies. For each technique, the fundamental principles as well as the quantitative analysis associated to them will be explained. Their specific advantages in the field of nano-immunology will be highlighted. In addition, an example of how each technique has been exploited to answer a specific biological question in the field will be given. All of the examples presented in part I correspond to publications that I co-authored during my PhD research. In Part II, I will address the subcellular organization of the immunoreceptor MerTK in human dendritic cells (DCs). By exploiting super-resolution STED nanoscopy, we discovered that MerTK organizes in small nanoclusters on the plasma membrane of tolerogenic DCs, where MerTK is highly expressed. Moreover, we will show that even though MerTK is a membrane receptor, it is also found at very high levels in the nucleus of DCs. To place this finding in the context of immunity, we established a direct correlation between DC differentiation and the amount of MerTK found in the nucleus. We enquired the route by which MerTK translocate to the nucleus, and dissected some of the main molecular factors involved in promoting this translocation. In a first attempt to identify its nuclear function, we additionally mapped the spatial relationship between MerTK and chromatin with nanometre accuracy using super-resolution STORM nanoscopy, in single intact DCs nuclei at different stages of their differentiation. We will finally place our findings in a broader perspective and suggest future lines of investigation that may further unravel the molecular mechanism of action of MerTK in particular, and the functional role of membrane receptors in the nucleus in general.[spa] Discriminar entre los antígenos ajenos y potencialmente peligrosos y los propios es uno de los pasos más cruciales en el inicio de una respuesta inmune. Los inmuno-receptores son moléculas inmersas en la membrana celular que ayudan a las células del sistema inmune a identificar y relacionarse con su entorno. Debido a su importancia, son sujeto intenso de estudio en el campo de la inmunología. Cada vez está más claro que la organización espaciotemporal de los inmuno-receptores en la membrana celular tiene un impacto directo en su función. En las dos últimas décadas, se han desarrollado un gran número de técnicas de microscopía y de biofísica innovadoras que están siendo utilizadas para visualizar de manera directa procesos a nivel molecular en células del sistema inmune con una resolución espacial y temporal sin precedentes. Estos avances técnicos han dado lugar a la aparición de un nuevo y activo campo de investigación: la nano-inmunología. En esta tesis se han utilizado técnicas de biofísica y microscopía de super-resolución para descifrar el comportamiento espaciotemporal de varios inmuno- receptores, con especial énfasis en el inmuno-receptor tirosina quinasa MerTK. Estos estudios han contribuido al mejor entendimiento de la biología celular del sistema inmune a nivel molecular. En la Parte I de esta tesis, analizaré varias técnicas de imagen y estrategias de micro-fabricación avanzadas utilizadas a lo largo de mi doctorado. En cada una de las técnicas se explicarán tanto los principios fundamentales como el análisis cuantitativo asociado. Se destacarán también sus ventajas específicas en el campo de la nano-inmunología. Además, se dará un ejemplo de cómo cada una de estas técnicas se ha aprovechado para dar respuesta a preguntas biológicas concretas. Todos los ejemplos expuestos en la Parte I de esta tesis se corresponden con publicaciones que he co-escrito durante mi doctorado. En la Parte II, abordaré el estudio de la organización sub-celular del inmuno-receptor MerTK expresado en células dendríticas humanas (DCs). Haciendo uso de la técnica de nanoscopía de superresolución STED, hemos descubierto que MerTK está organizado en pequeños nano-agregados en la membrana de células dendríticas tolerogénicas, donde MerTK está altamente expresado. Además, hemos descubierto que, aunque MerTK es un receptor de membrana, también se encuentra expresado a niveles muy altos en el núcleo de células dendríticas. Para posicionar este hallazgo en el contexto de la inmunología, hemos establecido una correlación directa entre la diferenciación de células dendríticas y la cantidad de MerTK en el núcleo. Adicionalmente, hemos investigado la ruta a través de la cual MerTK es translocado al núcleo y hemos analizado algunos de los principales factores moleculares involucrados en promover esta translocación. En un primer intento de identificar su función en el núcleo, hemos cartografiado además la relación espacial entre MerTK y cromatina con precisión nanométrica, utilizando la técnica de nanoscopía de superresolución STORM en células dendríticas intactas en varios estados de diferenciación. Finalmente, pondremos todos nuestros hallazgos en una perspectiva más amplia con la finalidad de sugerir líneas de investigación futuras que puedan descifrar con mas detalle el mecanismo molecular de acción de MerTK en particular, y la función de receptores de membrana en el núcleo en general

Desperate Affirmation: on the Aporetic Performativity of Memoria and Testimony, in the Light of W.G. Sebalds Story Max Ferber; with a Theological Response

Is ‘remembering’ an intentional activity, residing in the subject’s autonomy, or does it belong to the realm of receptivity, interrupting the subject? Or is it both at once? This jointly authored paper sets these questions in the context of a recently renewed interest in memoria in cultural theory and the humanities, as well as of an increasing pluralism in Western societies. The impossibility of sharing memories as a common good and a common truth is explored by putting the theme of historical responsibility, to which every gesture of memoria is tied, in a new light. The paper first demonstrates that the concept of performativity, as developed in particular by Jacques Derrida through a critical reading of Austin and Searle, can be a fruitful theoretical model in the analysis of memoria and of its double status: active and receptive at the same time. A reflection on the practice of testimony, again starting from Derrida, will further articulate this coherence between performativity and memoria. After this theoretical clarification, the value of performativity as a model for memoria will be tested through a detailed reading of the German writer W.G. Sebald’s (1944–2001) story ‘Max Ferber’, focussing on the delicate way this story stages an impossible testimonial drama. The authors will, finally, enquire as to the relevance of the performative model for a theological view of memoria and testimony

Online rules for container stacking

Container stacking rules are an important factor in container terminal efficiency. In this paper, we investigate two concepts to increase efficiency and compare them to several benchmark algorithms, using a discrete-event simulation tool. The first concept is to use knowledge about container departure times, in order to limit the number of reshuffles. We stack containers leaving shortly before each other on top of each other. The second concept is the trade-off between stacking further away in the terminal versus stacking close to the exit points and accepting more reshuffles. It is concluded that even the use of imperfect or imprecise departure time information leads to significant improvements in efficiency. Minimizing the difference in departure times proved to be important. It was also found that the trade-off between stacking further away in the terminal versus stacking close by the exit points and accepting more reshuffles leads to improvements over the benchmark

We\u27re Working On It: Transferring the Sloan Digital Sky Survey from Laboratory to Library

This article reports on the transfer of a massive scientific dataset from a national laboratory to a university library, and from one kind of workforce to another. We use the transfer of the Sloan Digital Sky Survey (SDSS) archive to examine the emergence of a new workforce for scientific research data management. Many individuals with diverse educational backgrounds and domain experience are involved in SDSS data management: domain scientists, computer scientists, software and systems engineers, programmers, and librarians. These types of positions have been described using terms such as research technologist, data scientist, e-science professional, data curator, and more. The findings reported here are based on semi-structured interviews, ethnographic participant observation, and archival studies from 2011-2013. The library staff conducting the data storage and archiving of the SDSS archive faced two performance problems. The preservation specialist and the system administrator worked together closely to discover and implement solutions to the slow data transfer and verification processes. The team overcame these slow-downs by problem solving, working in a team, and writing code. The library team lacked the astronomy domain knowledge necessary to meet some of their preservation and curation goals. The case study reveals the variety of expertise, experience, and individuals essential to the SDSS data management process. A variety of backgrounds and educational histories emerge in the data managers studied. Teamwork is necessary to bring disparate expertise together, especially between those with technical and domain education. The findings have implications for data management education, policy and relevant stakeholders. This article is part of continuing research on Knowledge Infrastructures

Stress Tensor Correlators in the Schwinger-Keldysh Formalism

We express stress tensor correlators using the Schwinger-Keldysh formalism. The absence of off-diagonal counterterms in this formalism ensures that the +- and -+ correlators are free of primitive divergences. We use dimensional regularization in position space to explicitly check this at one loop order for a massless scalar on a flat space background. We use the same procedure to show that the ++ correlator contains the divergences first computed by `t Hooft and Veltman for the scalar contribution to the graviton self-energy.Comment: 14 pages, LaTeX 2epsilon, no figures, revised for publicatio

Stochastic Spacetime and Brownian Motion of Test Particles

The operational meaning of spacetime fluctuations is discussed. Classical spacetime geometry can be viewed as encoding the relations between the motions of test particles in the geometry. By analogy, quantum fluctuations of spacetime geometry can be interpreted in terms of the fluctuations of these motions. Thus one can give meaning to spacetime fluctuations in terms of observables which describe the Brownian motion of test particles. We will first discuss some electromagnetic analogies, where quantum fluctuations of the electromagnetic field induce Brownian motion of test particles. We next discuss several explicit examples of Brownian motion caused by a fluctuating gravitational field. These examples include lightcone fluctuations, variations in the flight times of photons through the fluctuating geometry, and fluctuations in the expansion parameter given by a Langevin version of the Raychaudhuri equation. The fluctuations in this parameter lead to variations in the luminosity of sources. Other phenomena which can be linked to spacetime fluctuations are spectral line broadening and angular blurring of distant sources.Comment: 15 pages, 3 figures. Talk given at the 9th Peyresq workshop, June 200

Webometric analysis of departments of librarianship and information science: a follow-up study

This paper reports an analysis of the websites of UK departments of library and information science. Inlink counts of these websites revealed no statistically significant correlation with the quality of the research carried out by these departments, as quantified using departmental grades in the 2001 Research Assessment Exercise and citations in Google Scholar to publications submitted for that Exercise. Reasons for this lack of correlation include: difficulties in disambiguating departmental websites from larger institutional structures; the relatively small amount of research-related material in departmental websites; and limitations in the ways that current Web search engines process linkages to URLs. It is concluded that departmental-level webometric analyses do not at present provide an appropriate technique for evaluating academic research quality, and, more generally, that standards are needed for the formatting of URLs if inlinks are to become firmly established as a tool for website analysis

Immiscible fluid displacement in porous media with spatially correlated particle sizes

Immiscible fluid displacement in porous media is fundamental for many environmental processes, including infiltration of water in soils, groundwater remediation, enhanced recovery of hydrocarbons and CO2 geosequestration. Microstructural heterogeneity, in particular of particle sizes, can significantly impact immiscible displacement. For instance, it may lead to unstable flow and preferential displacement patterns. We present a systematic, quantitative pore-scale study of the impact of spatial correlations in particle sizes on the drainage of a partially-wetting fluid. We perform pore-network simulations with varying flow rates and different degrees of spatial correlation, complemented with microfluidic experiments. Simulated and experimental displacement patterns show that spatial correlation leads to more preferential invasion, with reduced trapping of the defending fluid, especially at low flow rates. Numerically, we find that increasing the correlation length reduces the fluid-fluid interfacial area and the trapping of the defending fluid, and increases the invasion pattern asymmetry and selectivity. Our experiments, conducted for low capillary numbers, support these findings. Our results delineate the significant effect of spatial correlations on fluid displacement in porous media, of relevance to a wide range of natural and engineered processes

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 μs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.Peer ReviewedPostprint (author's final draft