Luis Marques

A Pax3/Dmrt2/Myf5 regulatory cascade functions at the onset of myogenesis.

All skeletal muscle progenitor cells in the body derive from the dermomyotome, the dorsal epithelial domain of developing somites. These multipotent stem cells express Pax3, and this expression is maintained in the myogenic lineage where Pax3 plays an important role. Identification of Pax3 targets is therefore important for understanding the mechanisms that underlie the onset of myogenesis. In a microarray screen of Pax3-GFP sorted cells, with analysis on Pax3 gain and loss of function genetic backgrounds, we identify Dmrt2, expressed in the dermomyotome, as a Pax3 target. In vitro gel shift analysis and chromatin immunoprecipitation with in vivo extracts show that Pax3 binds to a conserved 286 bp sequence, situated at −18 kb from Dmrt2. This sequence directs reporter transgene expression to the somite, and this is severely affected when the Pax3 site is mutated in the context of the locus. In Dmrt2 mutant embryos, somite maturation is perturbed and the skeletal muscle of the myotome is abnormal. We now report that the onset of myogenesis is also affected. This depends on activation, in the epaxial dermomyotome, of the myogenic determination gene, Myf5, through its early epaxial enhancer. This sequence contains sites that bind Dmrt2, which belongs to the DM class of DNA–binding proteins. Mutation of these sites compromises activity of the enhancer in transgenic embryos where the reporter transgene is under the control of the Myf5 epaxial enhancer. Transactivation of this site by Dmrt2 is demonstrated in vitro, and conditional overexpression of Dmrt2 in Pax3 expressing cells in the somite confirms the role of this factor in the activation of Myf5. These results reveal a novel genetic network, comprising a Pax3/Dmrt2/Myf5 regulatory cascade that operates in stem cells of the epaxial dermomyotome to initiate skeletal muscle formation.

Lightweight Dependable Adaptation for Wireless Sensor Networks

Achieving dependable and real-time operation in Wireless Sensor Networks (WSNs) is a hard and open problem. This can be an obstacle for many applications, namely in the automotive and medical domains, particularly if safety-critical control is envisaged. To overcome the communication uncertainties that are intrinsic to wireless and dynamic environments, a generic approach is to constantly adapt to environment conditions. This requires appropriate solutions to characterize such conditions. This paper contributes with a lightweight solution for a dependable characterization of network QoS metrics, which is appropriate to support dependable adaptation in WSNs. The proposed solution offers probabilistic guarantees, building on non-parametric stochastic analysis to achieve fast and effective results. The paper also provides an evaluation of the solution.

Dynamics of Akt activation during mouse embryo development: distinct subcellular patterns distinguish proliferating versus differentiating cells.

Akt is a highly conserved serine-threonine protein kinase which has been implicated in a wide variety of cellular functions, from the regulation of growth and metabolism, to activation of pro-survival pathways and cell proliferation, and promotion of differentiation in specific cell types. However, very little is known about the spatial and temporal pattern of Akt activity within cells and whether this pattern changes as cells enter and proceed in their differentiation programs. To address this issue we profiled Akt activation in E8.5-E13.5 mouse embryos and in C2C12 cells. We used a commercial antibody against Akt, phosphorylated on one of its activating residues, Thr-308, and performed high resolution confocal imaging of the immunofluorescence in labeled embryos. We observe strong Akt activity during mitosis in the dermomyotome, the neuroepithelium and some mesenchymal cells. This burst of activity fills the whole cell except for heterochromatin-positive areas in the nucleus. A surge in activity during mitosis is also observed in subconfluent C2C12 cells. Later on in the differentiation programs of skeletal muscle and neural cells, derivatives of the dermomyotome and neuroepithelium, respectively, we find robust, sustained Akt activity in the cytoplasm, but not in the nucleus. Concomitantly with skeletal muscle differentiation, Akt activity becomes concentrated in the sarcomeric Z-disks whereas developing neurons maintain a uniform cytoplasmic pattern of activated Akt. Our findings reveal unprecedented cellular and subcellular details of Akt activity during mouse embryo development, which is spatially and temporally consistent with proposed functions for Akt in mitosis and myogenic and neural differentiation and/or survival. Our results thus demonstrate a subcellular change in the pattern of Akt activation when skeletal muscle and neural progenitor cells cease dividing and progress in their differentiation programs.

Design and development of a proof-of-concept platooning application using the HIDENETS architecture

This paper describes the design and development of a proof-of-concept platooning application, which operates in a mobile and dynamic environment and makes use of architectural and middleware solutions that were proposed in the scope of the HIDENETS project. With this application it is possible to demonstrate the practical feasibility of a hybrid system architecture, with realms of operation with distinct synchrony properties, and the benefits of adopting such architecture. In particular, we show that it is possible to improve the performance and behavior of the platooning application, which operates over an intrinsically uncertain environment (due to mobility and wireless communication), and still secure fundamental safety-critical requirements.

Fighting Uncertainty in Highly Dynamic Wireless Sensor Networks with Probabilistic Models

Real-time operation in Wireless Sensor Networks (WSNs) is conditioned not only by the current technological level (e.g., limited computing power) but also inherently by the target problem itself: WSNs are required to operate in very open and uncertain environments, subject to external radio interferences, highly dynamic network load, etc. Current WSN solutions either provide only best-effort real-time guarantees or make (generally implicit) assumptions on the dynamics of the open environment. These assumptions, in turn, are either very relaxed (i.e., compatible only with undemanding real-time requirements) or very hard to justify. When dealing with WSNs supporting highly dynamic applications and operating environments (e.g., media streaming, robot control, vehicle coordination, etc.) this problem cannot be ignored. Accordingly, we argue for, and show the efficacy of, using probabilistic models to characterize dynamic WSN QoS, which is the first step to tackle the problem head on. Using our network monitoring technique, we demonstrate that it is possible to meet probabilistic real-time objectives.

Middleware Support for Adaptive Real-Time Applications in Wireless Sensor Networks

This position paper describes initial efforts and ideas for the development of a middleware framework to support the operation of adaptive Wireless Sensor Networks applications with real-time and dependability requirements. We identify a set of underlying services that need to be implemented as part of this framework, explaining why they are needed and what they provide. In order to illustrate how this middleware can be used and its potential benefits, we consider the well-known LQER routing protocol to show how it must be changed to incorporate probabilistic real-time requirements and meet them in a dependable way.

Data Validity and Dependable Perception in Networked Sensor-Based Systems

Although the technology and applications of wireless sensor networks have greatly increased over the last years, ensuring a dependable real-time operation despite faults and temporal uncertainties is still an on-going research topic. The problems are particularly significant when considering that future applications will interact with their environment not only for supervision or monitoring, but also to directly control physical (real-time) entities, sometimes with safety-critical requirements. We believe that reasoning in terms of data validity might be a good way to approach the problem. The ability to know if sensor data flowing in the system is valid – data validity awareness –, is a first step to achieve a dependable operation. But more than that, it should be possible to ensure, given requirements for data validity throughout the operation, a dependable perception of the environment. In this paper we essentially discuss the problem, analyzing some of the issues that need to be addressed to achieve these goals. Particularly, we introduce fundamental concepts and relevant definitions, we elaborate on the main impediments to achieve data validity awareness and describe relevant means to deal with these impediments. Finally, we address the issue of ensuring a dependable perception and present some research ideas in this direction.

Towards dependable and stable perception in smart environments with timing and value faults

Future physical environments are expected to be pervasively enriched with sensors, which mobile embedded applications can use to safely interact in and with that environment. Unfortunately, due to the open and uncertain nature of the environment and the wireless communication, it is not possible to provide strict a priori guarantees with regard to the quality and timeliness with which such environments can be perceived. In this paper we take a look at the threats to a reliable perception of the environment, considering both timing and value faults. We discuss how such threats can be mitigated and we explore possible paths towards an integrated architecture to efficiently achieve a dependable and stable perception of smart environments in the presence of timing and value faults.

A simple way to teach the physiological role of ethylene at school laboratories

Ethylene plays an important role in plant physiology, being responsible for the maturation of fruits. In specialised laboratories this gaseous plant regulator is quantified through complex and expensive gaschromatography equipment; this technology is not available in schools, so ethylene is neglected by teachersperforming an experimental study about the control of plant growth and development. To provide an indirect but reliable indication of ethylene production by fruits, we developed a simple bioassay based on the titrationof malic acid.