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

Isolation of Pyrenochaeta terrestris from Onion Roots

A comparison was made of various techniques for isolating Pyrenochaeta terrestris (Hansen) Gorenz et al. from growing onion roots. Water agar was as suitable as potato dextrose agar (0.5% dextrose) and is recommended to avoid contamination. Texas L 303, Iowa Yellow Globe 51, and Trapp\u27s Downing Yellow Globe used as trap crops were equally efficient. Maximum infection of the early planting occurred earlier than a later planting

The Effects of Stress Tensor Fluctuations upon Focusing

We treat the gravitational effects of quantum stress tensor fluctuations. An operational approach is adopted in which these fluctuations produce fluctuations in the focusing of a bundle of geodesics. This can be calculated explicitly using the Raychaudhuri equation as a Langevin equation. The physical manifestation of these fluctuations are angular blurring and luminosity fluctuations of the images of distant sources. We give explicit results for the case of a scalar field on a flat background in a thermal state.Comment: 26 pages, 1 figure, new material added in Sect. III and in Appendices B and

Searching Data: A Review of Observational Data Retrieval Practices in Selected Disciplines

A cross-disciplinary examination of the user behaviours involved in seeking and evaluating data is surprisingly absent from the research data discussion. This review explores the data retrieval literature to identify commonalities in how users search for and evaluate observational research data. Two analytical frameworks rooted in information retrieval and science technology studies are used to identify key similarities in practices as a first step toward developing a model describing data retrieval

Appointment scheduling with unscheduled arrivals and reprioritization

Inspired by the real life problem of a radiology department in a Dutch hospital, we study the problem of scheduling appointments, taking into account unscheduled arrivals and reprioritization. The radiology department offers CT diagnostics to both scheduled and unscheduled patients. Of these unscheduled patients, some must be seen immediately, while others may wait for some time. Herein a trade-off is sought between acceptable waiting times for appointment patients and unscheduled patients’ lateness. In this paper we use a discrete event simulation model to determine the performance of a given appointment schedule in terms of waiting time and lateness. Also we propose a constructive and local search heuristic that embeds this model and optimizes the schedule. For smaller instances, we verify the simulation model as well as compare our search heuristics’ performance with optimal schedules obtained using a Markov reward process. In addition we present computational results from the case study in the Dutch hospital. These results show that a considerable decrease of waiting time is possible for scheduled patients, while still treating unscheduled patients on time

The rotating molecular core and precessing outflow of the young stellar object Barnard 1c

We investigate the structure of the core surrounding the recently identified deeply embedded young stellar object Barnard 1c which has an unusual polarization pattern as traced in submillimeter dust emission. Barnard 1c lies within the Perseus molecular cloud at a distance of 250 pc. It is a deeply embedded core of 2.4 solar masses (Kirk et al.) and a luminosity of 4 +/- 2 solar luminosities. Observations of CO, 13CO, C18O, HCO+ and N2H+ were obtained with the BIMA array, together with the continuum at 3.3 mm and 2.7 mm. Single-dish measurements of N2H+ and HCO+ with FCRAO reveal the larger scale emission in these lines, The CO and HCO+ emission traces the outflow, which coincides in detail with the S-shaped jet recently found in Spitzer IRAC imaging. The N2H+ emission, which anticorrelates spatially with the C18O emission, originates from a rotating envelope with effective radius ~ 2400 AU and mass 2.1 - 2.9 solar masses. N2H+ emission is absent from a 600 AU diameter region around the young star. The remaining N2H+ emission may lie in a coherent torus of dense material. With its outflow and rotating envelope, B1c closely resembles the previously studied object L483-mm, and we conclude that it is a protostar in an early stage of evolution. We hypothesize that heating by the outflow and star has desorbed CO from grains which has destroyed N2H+ in the inner region and surmise that the presence of grains without ice mantles in this warm inner region can explain the unusual polarization signature from B1c.Comment: 17 pages, 17 figures (9 colour). Accepted to The Astrophysical Journal. For higher resolution images, see http://astrowww.phys.uvic.ca/~brenda/preprints.htm

Spatially Explicit Data: Stewardship and Ethical Challenges in Science

Scholarly communication is at an unprecedented turning point created in part by the increasing saliency of data stewardship and data sharing. Formal data management plans represent a new emphasis in research, enabling access to data at higher volumes and more quickly, and the potential for replication and augmentation of existing research. Data sharing has recently transformed the practice, scope, content, and applicability of research in several disciplines, in particular in relation to spatially specific data. This lends exciting potentiality, but the most effective ways in which to implement such changes, particularly for disciplines involving human subjects and other sensitive information, demand consideration. Data management plans, stewardship, and sharing, impart distinctive technical, sociological, and ethical challenges that remain to be adequately identified and remedied. Here, we consider these and propose potential solutions for their amelioration