Identification of residues and domains of Raf important for function in vivo and in vitro

Random mutagenesis and genetic screens for impaired Raf function in Caenorhabditis elegans were used to identify six loss-of-function alleles of lin-45 raf that result in a substitution of a single amino acid. The mutations were classified as weak, intermediate, and strong based on phenotypic severity. We engineered these mutations into the homologous residues of vertebrate Raf-1 and analyzed the mutant proteins for their underlying biochemical defects. Surprisingly, phenotype strength did not correlate with the catalytic activity of the mutant proteins. Amino acid substitutions Val-589 and Ser-619 severely compromised Raf kinase activity, yet these mutants displayed weak phenotypes in the genetic screen. Interestingly, this is because these mutant Raf proteins efficiently activate the MAPK (mitogen-activated protein kinase) cascade in living cells, a result that may inform the analysis of knockout mice. Equally intriguing was the observation that mutant proteins with non-functional Ras-binding domains, and thereby deficient in Ras-mediated membrane recruitment, displayed only intermediate strength phenotypes. This confirms that secondary mechanisms exist to couple Ras to Raf in vivo. The strongest phenotype in the genetic screens was displayed by a S508N mutation that again did not correlate with a significant loss of kinase activity or membrane recruitment by oncogenic Ras in biochemical assays. Ser-508 lies within the Raf-1 activation loop, and mutation of this residue in Raf-1 and the equivalent Ser-615 in B-Raf revealed that this residue regulates Raf binding to MEK. Further characterization revealed that in response to activation by epidermal growth factor, the Raf-S508N mutant protein displayed both reduced catalytic activity and aberrant activation kinetics: characteristics that may explain the C. elegans phenotype.

Core outcome domains for chronic pain clinical trials: IMMPACT recommendations

Objective. To provide recommendations for the core outcome domains that should be considered by investigators conducting clinical trials of the efficacy and effectiveness of treatments for chronic pain. Development of a core set of outcome domains would facilitate comparison and pooling of data, encourage more complete reporting of outcomes, simplify the preparation and review of research proposals and manuscripts, and allow clinicians to make informed decisions regarding the risks and benefits of treatment. Methods. Under the auspices of the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT), 27 specialists from academia. governmental agencies, and the pharmaceutical industry participated in a consensus meeting and identified core outcome domains that should be considered in clinical trials of treatments for chronic pain. Conclusions. There was a consensus that chronic pain clinical trials should assess outcomes representing six core domains: (1) pain, (2) physical functioning, (3) emotional functioning, (4) participant ratings of improvement and satisfaction with treatment, (5) symptoms and adverse events, (6) participant disposition (e.g. adherence to the treatment regimen and reasons for premature withdrawal from the trial). Although consideration should be given to the assessment of each of these domains, there may be exceptions to the general recommendation to include all of these domains in chronic pain trials. When this occurs, the rationale for not including domains should be provided. It is not the intention of these recommendations that assessment of the core domains should be considered a requirement for approval of product applications by regulatory agencies or that a treatment must demonstrate statistically significant effects for all of the relevant core domains to establish evidence of its efficacy. (C) 2003 International Association for the Study of Pain.

The trans-membrane protein p25 forms highly specialized domains that regulate membrane composition and dynamics

Trans-membrane proteins of the p24 family are abundant, oligomeric proteins predominantly found in cis-Golgi membranes. They are not easily studied in vivo and their functions are controversial. We found that p25 can be targeted to the plasma membrane after inactivation of its canonical KKXX motif (KK to SS, p25SS), and that p25SS causes the co-transport of other p24 proteins beyond the Golgi complex, indicating that wild-type p25 plays a crucial role in retaining p24 proteins in cis-Golgi membranes. We then made use of these observations to study the intrinsic properties of these proteins, when present in a different membrane context. At the cell surface, the p25SS mutant segregates away from both the transferrin receptor and markers of lipid rafts, which are enriched in cholesterol and glycosphingolipids. This suggests that p25SS localizes to, or contributes to form, specialized membrane domains, presumably corresponding to oligomers of p25SS and other p24 proteins. Once at the cell surface, p25SS is endocytosed, together with other p24 proteins, and eventually accumulates in late endosomes, where it remains confined to well-defined membrane regions visible by electron microscopy. We find that this p25SS accumulation causes a concomitant accumulation of cholesterol in late endosomes, and an inhibition of their motility - two processes that are functionally linked. Yet, the p25SS-rich regions themselves seem to-exclude not only Lamp1 but also accumulated cholesterol. One may envision that p25SS accumulation, by excluding cholesterol from oligomers, eventually overloads neighboring late endosomal membranes with cholesterol beyond their capacity (see Discussion). In any case, our data show that p25 and presumably other p24 proteins are endowed with the intrinsic capacity to form highly specialized domains that control membrane composition and dynamics. We propose that p25 and other p24 proteins control the fidelity of membrane transport by maintaining cholesterol-poor membranes in the Golgi complex.

Static-priority scheduling over wireless networks with multiple broadcast domains

We propose a wireless medium access control (MAC) protocol that provides static-priority scheduling of messages in a guaranteed collision-free manner. Our protocol supports multiple broadcast domains, resolves the wireless hidden terminal problem and allows for parallel transmissions across a mesh network. Arbitration of messages is achieved without the notion of a master coordinating node, global clock synchronization or out-of-band signaling. The protocol relies on bit-dominance similar to what is used in the CAN bus except that in order to operate on a wireless physical layer, nodes are not required to receive incoming bits while transmitting. The use of bit-dominance efficiently allows for a much larger number of priorities than would be possible using existing wireless solutions. A MAC protocol with these properties enables schedulability analysis of sporadic message streams in wireless multihop networks.

Factorization by invariant embedding of elliptic problems: circular and star-shaped domains

Dissertação apresentada para obtenção do grau de Doutor em Matemática na especialidade de Equações Diferenciais, pela Universidade Nova de Lisboa,Faculdade de Ciências e Tecnologia

Functional domains of Bacillus subtilis transcription factor AraR and identification of aminoacids important for nucleoprotein complex assembly and effector-binding.

Journal of Bacteriology (Apr 2006) 3024-3036

Hox genes control the specification of global vertebral domains

Dissertação apresentada para obtenção do grau de doutor em Biologia pelo Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa

Feature Extraction in Densely Sensed Environments: Extensions to Multiple Broadcast Domains

The vision of the Internet of Things (IoT) includes large and dense deployment of interconnected smart sensing and monitoring devices. This vast deployment necessitates collection and processing of large volume of measurement data. However, collecting all the measured data from individual devices on such a scale may be impractical and time consuming. Moreover, processing these measurements requires complex algorithms to extract useful information. Thus, it becomes imperative to devise distributed information processing mechanisms that identify application-specific features in a timely manner and with a low overhead. In this article, we present a feature extraction mechanism for dense networks that takes advantage of dominance-based medium access control (MAC) protocols to (i) efficiently obtain global extrema of the sensed quantities, (ii) extract local extrema, and (iii) detect the boundaries of events, by using simple transforms that nodes employ on their local data. We extend our results for a large dense network with multiple broadcast domains (MBD). We discuss and compare two approaches for addressing the challenges with MBD and we show through extensive evaluations that our proposed distributed MBD approach is fast and efficient at retrieving the most valuable measurements, independent of the number sensor nodes in the network.

Integration of crowdsourced information with traditional crises and disaster management information using linked data

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

A Web based geospatial application for disaster preparedness in Uganda

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

The role of temporary accommodation buildings for post-disaster housing reconstruction

The number of houses damaged or destroyed after disasters is frequently large, and re-housing of homeless people is one of the most important tasks of reconstruction programmes. Reconstruction works often last long and during that time, it is essential to provide victims with the minimum conditions to live with dignity, privacy, and protection. This research intends to demonstrate the crucial role of temporary accommodation buildings to provide spaces where people can live and gradually resume their life until they have a permanent house. The study also aims to identify the main problems of temporary accommodation strategies and to discuss some principles and guidelines in order to reach better design solutions. It is found that temporary accommodation is an issue that goes beyond the simple provision of buildings, since the whole space for temporary settlement is important. Likewise, temporary accommodation is a process that should start before a disaster occurs, as a preventive pre-planning. In spite of being temporary constructions, these housing buildings are one of the most important elements to provide in emergency scenarios, contributing for better recovery and reconstruction actions.

Robust learning in safety-related domains : machine learning methods for solving safety-related application problems

Magdeburg, Univ., Fak. für Informatik, Diss., 2009

Assessing the role of Piccolo and Bassoon zinc finger domains on synapse vesicle recycling and maintenance

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2013