Endoglycan mediates malignant leukocyte rolling on e-selectin and signals through SYK and the MAPK pathway

Selectins play a key role regulating leukocyte migration into tissues by mediating leukocyte tethering (capture) and rolling on inflamed endothelium and/or on adherent leukocytes or platelets. During leukocyte rolling, endothelial E- or P-selectin bind to glycoprotein ligands carrying sialyl Lewis χ (sLex) determinant. P-selectin glycoprotein ligand-1 (PSGL-1) is a common ligand for L-, P- and E-selectin, which sequentially cooperates with CD44 and E- selectin ligand-1 (ESL-1) to roll on E-selectin. During rolling on endothelial selectins, PSGL-1 and CD44 signal through Src family kinases and Syk, leading to αι_β2 integrin partial activation and slow rolling on intercellular adhesion molecule-1 (ICAM-1). Leukocyte exposure to chemokines then leads to firm adhesion. Little information is available on ligands that mediate malignant leukocyte rolling on E- selectin. We defined these ligands on U937 monoblasts by immunoadsorbtion and immunoblotting using mAb raised against CD43, CD44, PSGL-1, sLex/CLA determinants and E-selectin/IgM chimera. Immunoblotting and blot rolling assays demonstrated that PSGL-1, CD43, CD44 and a -125 kDa sLex/CLA positive ligand contribute to support E-seiectin- dependent rolling. This -125 kDa ligand is endoglycan, a member of the CD34 family of sialomucins. Endoglycan was frequently detected by flow cytometry on primary leukemia, lymphoma and multiple myeloma ceils (in -50% of cases). Endoglycan, immunopurified from U937 cells, as well as endoglycan/IgG chimera efficiently supported E-selectin dependent rolling. Membrane fractionation on sucrose gradient demonstrated that endoglycan is expressed in lipid rafts. We tested the hypothesis that it signals, like PSGL-1 and CD44, through Src kinases and the MAPK pathway. Indeed, endoglycan engagement induced Syk and ERK phosphorylation in a iipid raft-dependent manner. Syk activation was dependent on Src kinase activity. Downstream of Syk, endoglycan activated PI3K and Akt as well as Bruton's tyrosine kinase and p38 MAPK. Thus, endoglycan is a ligand for endothelial selectins which may contribute to regulate leukemia, lymphoma and multiple myeloma cell trafficking and interactions with bone marrow microenvironment. - Les sélectines contrôlent la migration tissulaire des leucocytes en assurant leur capture et leur roulement sur l'endothélium vasculaire enflammé et/ou sur des plaquettes ou des leucocytes adhérant à la paroi vasculaire. Lors du roulement leucocytaire, les sélectines endothéliales (E- et P-sélectine) se lient à des ligands porteurs du saccharide sialyl Lewis χ (sLex). PSGL-1 est un ligand commun des sélectines qui coopère avec CD44 et ESL-1 pour permettre la capture et le roulement des neutrophiles. Lorsque PSGL-1 et CD44 se lient aux sélectines endothéliales, elles induisent la phosphorylation des kinases Src et de Syk conduisant à l'activation partielle de l'intégrine aLp2 et au ralentissement des leucocytes sur les sélectines et ICAM-1. Les chimiokines induisent ensuite l'adhésion ferme des leucocytes. Les ligands des sélectines qui assurent le roulement, sur la E-sélectine, des cellules issues d'hémopathies malignes sont peu connus. Nous avons caractérisé ces ligands en les purifiant avec des anticorps dirigés contre CD43, CD44, PSGL-1, sLex/CLA et en utilisant la chimère E-sélectine/IgM. Des tests d'adhésion ont montré que PSGL-1, CD43, CD44 et une glycoprotéine de ~125 kDa soutiennent les interactions cellulaires dépendant de la E- sélectine. Le ligand de -125 kDa a été identifié comme étant l'endoglycan. Il a été détecté, par cytométrie de flux, sur les cellules leucémiques, les cellules de lymphomes ou de myélome multiple, dans ~50% des cas analysés. Sa forme membranaire, immunopurifiée, ou recombinante (endoglycan/lgG) soutient les interactions cellulaires dépendant de la E- sélectine. Nous avons montré qu'il réside dans les rafts lipidiques membranaires puis avons testé l'hypothèse que l'endoglycan, comme PSGL-1 et CD44, induit une signalisation via les kinases de type Src et la voie des MAPK. Nous avons pu observer que son engagement induit la phosphorylation de Syk et de ERK pour autant que la structure des rafts soit préservée. En aval de Syk, l'endoglycan active la PI3K, Akt, Btk et la MAPK p38. Ces résultats montrent que l'endoglycan est un ligand des sélectines endothéliales qui pourrait participer au contrôle du trafic et des interactions des cellules leucémiques, de lymphomes ou de myélomes multiples avec leur microenvironnement. - Le sang est un élément clé du fonctionnement de notre corps. La circulation sanguine permet la communication et le transfert de molécules et cellules entre divers organes. Lors d'une inflammation aiguë due à une réaction allergique, une infection ou une blessure, on observe un oedème local accompagné de rougeur, de chaleur et souvent de douleurs. Au sein des tissus enflammés, on observe des globules blancs (leucocytes) et diverses molécules inflammatoires qui attirent les leucocytes dans les tissus lésés (chimiokines). Le sang est composé de globules rouges, de plaquettes et de leucocytes spécialisés dans les défenses immunes. Pour atteindre le site d'inflammation, les leucocytes doivent quitter la circulation sanguine. Ils utilisent pour cela des molécules d'adhésion présentes à leur surface qui se lient à d'autres molécules d'adhésion de la paroi sanguine. Leurs interactions permettent aux leucocytes de rouler à la surface du vaisseau sanguin. Lorsqu'ils roulent au voisinage d'un site d'inflammation, les leucocytes sont exposés à des chimiokines qui induisent leur arrêt et les dirigent dans les tissus enflammés. Ce processus physiologique est aussi impliqué dans des pathologies telles que l'infarctus, l'artériosclérose ou la thrombose. Il peut être détourné à des fins moins louables par des cellules cancéreuses pour permettre leur dissémination (métastatisation). Dans ce travail de thèse, nous avons caractérisé une molécule d'adhésion qui soutient l'adhésion des leucocytes aux sélectines endothéliales: l'endoglycan. Nous avons observé que cette molécule d'adhésion est fréquemment exprimée par les cellules malignes de nombreuses maladies du sang comme les leucémies, les lymphomes et le myélome multiple. Nous avons également pu montrer que l'endoglycan envoie des signaux à l'intérieur des cellules malignes lorsqu'elles se lient aux sélectines endothéliales. Ces signaux pourraient jouer un rôle déterminant dans la régulation des interactions des cellules malignes avec leur microenvironnement. Elles pourraient peut-être aussi favoriser leur survie et leur prolifération.

P-selectin glycoprotein ligand-1 decameric repeats regulate selectin-dependent rolling under flow conditions.

P-selectin glycoprotein ligand-1 (PSGL-1) interacts with selectins to support leukocyte rolling along vascular wall. L- and P-selectin bind to N-terminal tyrosine sulfate residues and to core-2 O-glycans attached to Thr-57, whereas tyrosine sulfation is not required for E-selectin binding. PSGL-1 extracellular domain contains decameric repeats, which extend L- and P-selectin binding sites far above the plasma membrane. We hypothesized that decamers may play a role in regulating PSGL-1 interactions with selectins. Chinese hamster ovary cells expressing wild-type PSGL-1 or PSGL-1 molecules exhibiting deletion or substitution of decamers with the tandem repeats of platelet glycoprotein Ibalpha were compared in their ability to roll on selectins and to bind soluble L- or P-selectin. Deletion of decamers abrogated soluble L-selectin binding and cell rolling on L-selectin, whereas their substitution partially reversed these diminutions. P-selectin-dependent interactions with PSGL-1 were less affected by decamer deletion. Videomicroscopy analysis showed that decamers are required to stabilize L-selectin-dependent rolling. Importantly, adhesion assays performed on recombinant decamers demonstrated that they directly bind to E-selectin and promote slow rolling. Our results indicate that the role of decamers is to extend PSGL-1 N terminus far above the cell surface to support and stabilize leukocyte rolling on L- or P-selectin. In addition, they function as a cell adhesion receptor, which supports approximately 80% of E-selectin-dependent rolling.

A new method to measure rolling resistance in treadmill cycling.

The purpose of this study was to evaluate a new method of measuring rolling resistance in treadmill cycling and to establish its sensitivity and reproducibility. One participant was asked to keep a bicycle in equilibrium on a treadmill without pedalling at a constant speed of 5.56 m x s(-1), which was held in place in the front by a dynamometer. For each condition, the method consisted of 11 measurements of the force required to hold the cycle at different treadmill slopes (0-10%, increment 1%). The coefficient of rolling resistance was calculated based on the forces applied to the bicycle in equilibrium. To test the sensitivity of the method, the bicycle was successively equipped with three tyre types (700 x 28, 700 x 23, 700 x 22) and inflation pressure was set at 150, 300, 600, 900, and 1100 kPa. To test the reproducibility of the method, a second experimenter repeated all measurements done with the 700 x 23 tyres. The method was sensitive enough to detect an effect of both tyre type and inflation pressure (P < 0.001: two-way ANOVA). The measurement of the coefficient of rolling resistance by two separate experimenters resulted in a small bias of 0.00029 (95% CI, -0.00011 to 0.00068). In conclusion, the new method is sensitive and reliable, as well as being simple and affordable.

Molecular basis of leukocyte rolling on PSGL-1. Predominant role of core-2 O-glycans and of tyrosine sulfate residue 51.

Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at N-terminal Thr or Tyr residues. Simultaneous expression of core-2 beta1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and P-selectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptor-counterreceptor pair.

Under the Stones, the beach. Vínculos generacionales y pulsiones subversivas en el cine a través de los Rolling Stones

Este trabajo busca investigar el fenómeno de lo generacional en el cine a través de su relación con la música. Para ello, utiliza como punto de partida la filmografía de los Rolling Stones y aplica elconcepto de aura de Walter Benjamin. Así, explora las tensiones generacionales entre jóvenes y adultos, que se reflejan en los ámbitos del creador, la audiencia y la industria como tensiones entre invención y comercialización, entre libertad y represión o entre rebeldía y conservadurismo. De ese modo, procura identificar en la puesta en escena la capacidad contracultural del cine que apela a lo juvenil y oponerla a los simulacros oportunistas que utiliza el sistema de la industria cultural.

Program hosting in communications environment with Reactor and thread pool approach

In this thesis concurrent communication event handling is implemented using thread pool approach. Concurrent events are handled with a Reactor design pattern and multithreading is implemented using a Leader/Followers design pattern. Main focus is to evaluate behaviour of implemented model by different numbers of concurrent connections and amount of used threads. Furthermore, model feasibility in a PeerHood middleware is evaluated. Implemented model is evaluated with created test environment which enables concurrent message sending from multiple connections to the system under test. Messages round trip times are measured in the tester application. In the evaluation processing delay into system is simulated and influence of delay to the average round trip time is analysed.

Thread Scheduling Mechanisms for Multiple-Context Parallel Processors

Scheduling tasks to efficiently use the available processor resources is crucial to minimizing the runtime of applications on shared-memory parallel processors. One factor that contributes to poor processor utilization is the idle time caused by long latency operations, such as remote memory references or processor synchronization operations. One way of tolerating this latency is to use a processor with multiple hardware contexts that can rapidly switch to executing another thread of computation whenever a long latency operation occurs, thus increasing processor utilization by overlapping computation with communication. Although multiple contexts are effective for tolerating latency, this effectiveness can be limited by memory and network bandwidth, by cache interference effects among the multiple contexts, and by critical tasks sharing processor resources with less critical tasks. This thesis presents techniques that increase the effectiveness of multiple contexts by intelligently scheduling threads to make more efficient use of processor pipeline, bandwidth, and cache resources. This thesis proposes thread prioritization as a fundamental mechanism for directing the thread schedule on a multiple-context processor. A priority is assigned to each thread either statically or dynamically and is used by the thread scheduler to decide which threads to load in the contexts, and to decide which context to switch to on a context switch. We develop a multiple-context model that integrates both cache and network effects, and shows how thread prioritization can both maintain high processor utilization, and limit increases in critical path runtime caused by multithreading. The model also shows that in order to be effective in bandwidth limited applications, thread prioritization must be extended to prioritize memory requests. We show how simple hardware can prioritize the running of threads in the multiple contexts, and the issuing of requests to both the local memory and the network. Simulation experiments show how thread prioritization is used in a variety of applications. Thread prioritization can improve the performance of synchronization primitives by minimizing the number of processor cycles wasted in spinning and devoting more cycles to critical threads. Thread prioritization can be used in combination with other techniques to improve cache performance and minimize cache interference between different working sets in the cache. For applications that are critical path limited, thread prioritization can improve performance by allowing processor resources to be devoted preferentially to critical threads. These experimental results show that thread prioritization is a mechanism that can be used to implement a wide range of scheduling policies.

ADAM: A Decentralized Parallel Computer Architecture Featuring Fast Thread and Data Migration and a Uniform Hardware Abstraction

The furious pace of Moore's Law is driving computer architecture into a realm where the the speed of light is the dominant factor in system latencies. The number of clock cycles to span a chip are increasing, while the number of bits that can be accessed within a clock cycle is decreasing. Hence, it is becoming more difficult to hide latency. One alternative solution is to reduce latency by migrating threads and data, but the overhead of existing implementations has previously made migration an unserviceable solution so far. I present an architecture, implementation, and mechanisms that reduces the overhead of migration to the point where migration is a viable supplement to other latency hiding mechanisms, such as multithreading. The architecture is abstract, and presents programmers with a simple, uniform fine-grained multithreaded parallel programming model with implicit memory management. In other words, the spatial nature and implementation details (such as the number of processors) of a parallel machine are entirely hidden from the programmer. Compiler writers are encouraged to devise programming languages for the machine that guide a programmer to express their ideas in terms of objects, since objects exhibit an inherent physical locality of data and code. The machine implementation can then leverage this locality to automatically distribute data and threads across the physical machine by using a set of high performance migration mechanisms. An implementation of this architecture could migrate a null thread in 66 cycles -- over a factor of 1000 improvement over previous work. Performance also scales well; the time required to move a typical thread is only 4 to 5 times that of a null thread. Data migration performance is similar, and scales linearly with data block size. Since the performance of the migration mechanism is on par with that of an L2 cache, the implementation simulated in my work has no data caches and relies instead on multithreading and the migration mechanism to hide and reduce access latencies.

Learning Commonsense Categorical Knowledge in a Thread Memory System

If we are to understand how we can build machines capable of broad purpose learning and reasoning, we must first aim to build systems that can represent, acquire, and reason about the kinds of commonsense knowledge that we humans have about the world. This endeavor suggests steps such as identifying the kinds of knowledge people commonly have about the world, constructing suitable knowledge representations, and exploring the mechanisms that people use to make judgments about the everyday world. In this work, I contribute to these goals by proposing an architecture for a system that can learn commonsense knowledge about the properties and behavior of objects in the world. The architecture described here augments previous machine learning systems in four ways: (1) it relies on a seven dimensional notion of context, built from information recently given to the system, to learn and reason about objects' properties; (2) it has multiple methods that it can use to reason about objects, so that when one method fails, it can fall back on others; (3) it illustrates the usefulness of reasoning about objects by thinking about their similarity to other, better known objects, and by inferring properties of objects from the categories that they belong to; and (4) it represents an attempt to build an autonomous learner and reasoner, that sets its own goals for learning about the world and deduces new facts by reflecting on its acquired knowledge. This thesis describes this architecture, as well as a first implementation, that can learn from sentences such as ``A blue bird flew to the tree'' and ``The small bird flew to the cage'' that birds can fly. One of the main contributions of this work lies in suggesting a further set of salient ideas about how we can build broader purpose commonsense artificial learners and reasoners.

Rolling.

Forma parte de una colección de no ficción, que tiene como objetivo ayudar a los jóvenes lectores a conocer y comprender el mundo que les rodea. Como introducción a los principios científicos que constituyen la acción de rodar. Ilustrados con fotografías en color, los experimentos hacen hincapié en la necesidad del niño de investigar para favorecer el aprendizaje por descubrimiento. Hay glosario para desarrollar las habilidades ligüísticas.