948 resultados para Two-domain architecture
Resumo:
A Monte Carlo simulation study of the vacancy-assisted domain growth in asymmetric binary alloys is presented. The system is modeled using a three-state ABV Hamiltonian which includes an asymmetry term. Our simulated system is a stoichiometric two-dimensional binary alloy with a single vacancy which evolves according to the vacancy-atom exchange mechanism. We obtain that, compared to the symmetric case, the ordering process slows down dramatically. Concerning the asymptotic behavior it is algebraic and characterized by the Allen-Cahn growth exponent x51/2. The late stages of the evolution are preceded by a transient regime strongly affected by both the temperature and the degree of asymmetry of the alloy. The results are discussed and compared to those obtained for the symmetric case.
Resumo:
Actualment un típic embedded system (ex. telèfon mòbil) requereix alta qualitat per portar a terme tasques com codificar/descodificar a temps real; han de consumir poc energia per funcionar hores o dies utilitzant bateries lleugeres; han de ser el suficientment flexibles per integrar múltiples aplicacions i estàndards en un sol aparell; han de ser dissenyats i verificats en un període de temps curt tot i l’augment de la complexitat. Els dissenyadors lluiten contra aquestes adversitats, que demanen noves innovacions en arquitectures i metodologies de disseny. Coarse-grained reconfigurable architectures (CGRAs) estan emergent com a candidats potencials per superar totes aquestes dificultats. Diferents tipus d’arquitectures han estat presentades en els últims anys. L’alta granularitat redueix molt el retard, l’àrea, el consum i el temps de configuració comparant amb les FPGAs. D’altra banda, en comparació amb els tradicionals processadors coarse-grained programables, els alts recursos computacionals els permet d’assolir un alt nivell de paral•lelisme i eficiència. No obstant, els CGRAs existents no estant sent aplicats principalment per les grans dificultats en la programació per arquitectures complexes. ADRES és una nova CGRA dissenyada per I’Interuniversity Micro-Electronics Center (IMEC). Combina un processador very-long instruction word (VLIW) i un coarse-grained array per tenir dues opcions diferents en un mateix dispositiu físic. Entre els seus avantatges destaquen l’alta qualitat, poca redundància en les comunicacions i la facilitat de programació. Finalment ADRES és un patró enlloc d’una arquitectura concreta. Amb l’ajuda del compilador DRESC (Dynamically Reconfigurable Embedded System Compile), és possible trobar millors arquitectures o arquitectures específiques segons l’aplicació. Aquest treball presenta la implementació d’un codificador MPEG-4 per l’ADRES. Mostra l’evolució del codi per obtenir una bona implementació per una arquitectura donada. També es presenten les característiques principals d’ADRES i el seu compilador (DRESC). Els objectius són de reduir al màxim el nombre de cicles (temps) per implementar el codificador de MPEG-4 i veure les diferents dificultats de treballar en l’entorn ADRES. Els resultats mostren que els cícles es redueixen en un 67% comparant el codi inicial i final en el mode VLIW i un 84% comparant el codi inicial en VLIW i el final en mode CGA.
Resumo:
The amount of installed wind power has been growing exponentially during the past ten years. As wind turbines have become a significant source of electrical energy, the interactions between the turbines and the electric power network need to be studied more thoroughly than before. Especially, the behavior of the turbines in fault situations is of prime importance; simply disconnecting all wind turbines from the network during a voltage drop is no longer acceptable, since this would contribute to a total network collapse. These requirements have been a contributor to the increased role of simulations in the study and design of the electric drive train of a wind turbine. When planning a wind power investment, the selection of the site and the turbine are crucial for the economic feasibility of the installation. Economic feasibility, on the other hand, is the factor that determines whether or not investment in wind power will continue, contributing to green electricity production and reduction of emissions. In the selection of the installation site and the turbine (siting and site matching), the properties of the electric drive train of the planned turbine have so far been generally not been taken into account. Additionally, although the loss minimization of some of the individual components of the drive train has been studied, the drive train as a whole has received less attention. Furthermore, as a wind turbine will typically operate at a power level lower than the nominal most of the time, efficiency analysis in the nominal operating point is not sufficient. This doctoral dissertation attempts to combine the two aforementioned areas of interest by studying the applicability of time domain simulations in the analysis of the economicfeasibility of a wind turbine. The utilization of a general-purpose time domain simulator, otherwise applied to the study of network interactions and control systems, in the economic analysis of the wind energy conversion system is studied. The main benefits of the simulation-based method over traditional methods based on analytic calculation of losses include the ability to reuse and recombine existing models, the ability to analyze interactions between the components and subsystems in the electric drive train (something which is impossible when considering different subsystems as independent blocks, as is commonly done in theanalytical calculation of efficiencies), the ability to analyze in a rather straightforward manner the effect of selections other than physical components, for example control algorithms, and the ability to verify assumptions of the effects of a particular design change on the efficiency of the whole system. Based on the work, it can be concluded that differences between two configurations can be seen in the economic performance with only minor modifications to the simulation models used in the network interaction and control method study. This eliminates the need ofdeveloping analytic expressions for losses and enables the study of the system as a whole instead of modeling it as series connection of independent blocks with no lossinterdependencies. Three example cases (site matching, component selection, control principle selection) are provided to illustrate the usage of the approach and analyze its performance.
Resumo:
A small carbonatite dyke swarm has been identified at Naantali, southwest Finland. Several swarms of shoshonitic lamprophyres are also known along the Archean-Proterozoic boundary in eastern Finland and northwest Russia. These intrusions, along with the carbonatite intrusion at Halpanen, eastern Finland, represent a stage of widespread low-volume mantle-sourced alkaline magmatism in the Svecofennian Domain. Using trace element and isotope geochemistry coupled with precise geochronology from these rocks, a model is presented for the Proterozoic metasomatic evolution of the Fennoscandian subcontinental lithospheric mantle. At ~2.2-2.06 Ga, increased biological production in shallow seas linked to continental rifting, resulted in increased burial rates of organic carbon. Subduction between ~1.93-1.88 Ga returned organic carbon-enriched sediments of mixed Archean and Proterozoic provenance to the mantle. Dehydration reactions supplied water to the mantle wedge, driving arc volcanism, while mica, amphibole and carbonate were brought deeper into the mantle with the subducting slab. The cold subducted slab was heated conductively from the surrounding warm mantle, while pressures continued to gradually increase as a result of crustal thickening. The sediments began to melt in a two stage process, first producing a hydrous alkaline silicate melt, which infiltrated the mantle wedge and crystallised as metasomatic veins. At higher temperatures, carbonatite melt was produced, which preferentially infiltrated the pre-existing metasomatic vein network. At the onset of post-collisional extension, deep fault structures formed, providing conduits for mantle melts to reach the upper crust. Low-volume partial melting of the enriched mantle at depths of at least 110 km led to the formation of first carbonatitic magma and subsequently lamprophyric magma. Carbonatite was emplaced in the upper crust at Naantali at 1795.7 ± 6.8 Ma; lamprophyres along the Archean-Proterozoic boundary were emplaced between 1790.1 ± 3.3 Ma and 1781 ± 20 Ma.
Resumo:
Bakgrunden och inspirationen till föreliggande studie är tidigare forskning i tillämpningar på randidentifiering i metallindustrin. Effektiv randidentifiering möjliggör mindre säkerhetsmarginaler och längre serviceintervall för apparaturen i industriella högtemperaturprocesser, utan ökad risk för materielhaverier. I idealfallet vore en metod för randidentifiering baserad på uppföljning av någon indirekt variabel som kan mätas rutinmässigt eller till en ringa kostnad. En dylik variabel för smältugnar är temperaturen i olika positioner i väggen. Denna kan utnyttjas som insignal till en randidentifieringsmetod för att övervaka ugnens väggtjocklek. Vi ger en bakgrund och motivering till valet av den geometriskt endimensionella dynamiska modellen för randidentifiering, som diskuteras i arbetets senare del, framom en flerdimensionell geometrisk beskrivning. I de aktuella industriella tillämpningarna är dynamiken samt fördelarna med en enkel modellstruktur viktigare än exakt geometrisk beskrivning. Lösningsmetoder för den s.k. sidledes värmeledningsekvationen har många saker gemensamt med randidentifiering. Därför studerar vi egenskaper hos lösningarna till denna ekvation, inverkan av mätfel och något som brukar kallas förorening av mätbrus, regularisering och allmännare följder av icke-välställdheten hos sidledes värmeledningsekvationen. Vi studerar en uppsättning av tre olika metoder för randidentifiering, av vilka de två första är utvecklade från en strikt matematisk och den tredje från en mera tillämpad utgångspunkt. Metoderna har olika egenskaper med specifika fördelar och nackdelar. De rent matematiskt baserade metoderna karakteriseras av god noggrannhet och låg numerisk kostnad, dock till priset av låg flexibilitet i formuleringen av den modellbeskrivande partiella differentialekvationen. Den tredje, mera tillämpade, metoden kännetecknas av en sämre noggrannhet förorsakad av en högre grad av icke-välställdhet hos den mera flexibla modellen. För denna gjordes även en ansats till feluppskattning, som senare kunde observeras överensstämma med praktiska beräkningar med metoden. Studien kan anses vara en god startpunkt och matematisk bas för utveckling av industriella tillämpningar av randidentifiering, speciellt mot hantering av olinjära och diskontinuerliga materialegenskaper och plötsliga förändringar orsakade av “nedfallande” väggmaterial. Med de behandlade metoderna förefaller det möjligt att uppnå en robust, snabb och tillräckligt noggrann metod av begränsad komplexitet för randidentifiering.
Resumo:
Resonance energy transfer (RET) is a non-radiative transfer of the excitation energy from the initially excited luminescent donor to an acceptor. The requirements for the resonance energy transfer are: i) the spectral overlap between the donor emission spectrum and the acceptor absorption spectrum, ii) the close proximity of the donor and the acceptor, and iii) the suitable relative orientations of the donor emission and the acceptor absorption transition dipoles. As a result of the RET process the donor luminescence intensity and the donor lifetime are decreased. If the acceptor is luminescent, a sensitized acceptor emission appears. The rate of RET depends strongly on the donor–acceptor distance (r) and is inversely proportional to r6. The distance dependence of RET is utilized in binding assays. The proximity requirement and the selective detection of the RET-modified emission signal allow homogeneous separation free assays. The term lanthanide-based RET is used when luminescent lanthanide compounds are used as donors. The long luminescence lifetimes, the large Stokes’ shifts and the intense, sharply-spiked emission spectra of the lanthanide donors offer advantages over the conventional organic donor molecules. Both the organic lanthanide chelates and the inorganic up-converting phosphor (UCP) particles have been used as donor labels in the RET based binding assays. In the present work lanthanide luminescence and lanthanide-based resonance energy transfer phenomena were studied. Luminescence lifetime measurements had an essential role in the research. Modular frequency-domain and time-domain luminometers were assembled and used successfully in the lifetime measurements. The frequency-domain luminometer operated in the low frequency domain ( 100 kHz) and utilized a novel dual-phase lock-in detection of the luminescence. One of the studied phenomena was the recently discovered non-overlapping fluorescence resonance energy transfer (nFRET). The studied properties were the distance and temperature dependences of nFRET. The distance dependence was found to deviate from the Förster theory and a clear temperature dependence was observed whereas conventional RET was completely independent of the temperature. Based on the experimental results two thermally activated mechanisms were proposed for the nFRET process. The work with the UCP particles involved the measurement of the luminescence properties of the UCP particles synthesized in our laboratory. The goal of the UCP particle research is to develop UCP donor labels for binding assays. In the present work the effect of the dopant concentrations and the core–shell structure on the total up-conversion luminescence intensity, the red–green emission ratio, and the luminescence lifetime was studied. Also the non-radiative nature of the energy transfer from the UCP particle donors to organic acceptors was demonstrated for the first time in aqueous environment and with a controlled donor–acceptor distance.
Resumo:
The aim of this dissertation is to bridge and synthesize the different streams of literature addressing ecosystem architecture through a multiple‐lens perspective. In addition, the structural properties of and processes to design and manage the architecture will be examined. With this approach, the oft‐neglected actor‐structure duality is addressed and both the position and structure, and action and process are under scrutiny. Further, the developed framework and empirical evidence offer valuable insights on how firms collectively create value and individually appropriate value. The dissertation is divided into two parts. The first part comprises a literature review, as well as the conclusions of the whole study, and the second part includes six research publications. The dissertation is based on three different reasoning logics: abduction, induction and deduction; related qualitative and quantitative methodologies are utilized in the empirical examination of the phenomenon in the information and communication technology industry. The results suggest firstly that there are endogenous and exogenous structural properties of the ecosystem architecture. Out of these, the former ones can be more easily influenced by a particular actor whereas the latter ones are taken more or less for granted. Secondly, the exogenous ecosystem design properties influence the value creation potential of the ecosystem whereas the endogenous ecosystem design properties influence the value appropriation potential of a particular actor in the ecosystem. Thirdly, the study suggests that there is a relationship between endogenous and exogenous structural properties in that the endogenous properties can be leveraged to create and reconfigure the exogenous properties whereas the exogenous properties prose opportunities and restrictions on the use of endogenous properties. In addition, the study suggests that there are different emergent and engineered processes to design and manage ecosystem architecture and to influence both the endogenous and exogenous structural properties of ecosystem architecture. This study makes three main contributions. First, on the conceptual level, it brings coherence and direction to the fast growing body of literature on novel inter‐organizational arrangements, such as ecosystems. It does this by bridging and synthetizing three different streams of literature, namely the boundary, design and orchestration conception. Secondly, it sets out a framework that enhances our understanding of the structural properties of ecosystem architecture; of the processes to design and manage ecosystem architecture; and of their influence on the value creation potential of the ecosystem and the value capture potential of a particular firm. Thirdly, it offers empirical evidence of the structural properties and processes.
Resumo:
Cyber security is one of the main topics that are discussed around the world today. The threat is real, and it is unlikely to diminish. People, business, governments, and even armed forces are networked in a way or another. Thus, the cyber threat is also facing military networking. On the other hand, the concept of Network Centric Warfare sets high requirements for military tactical data communications and security. A challenging networking environment and cyber threats force us to consider new approaches to build security on the military communication systems. The purpose of this thesis is to develop a cyber security architecture for military networks, and to evaluate the designed architecture. The architecture is described as a technical functionality. As a new approach, the thesis introduces Cognitive Networks (CN) which are a theoretical concept to build more intelligent, dynamic and even secure communication networks. The cognitive networks are capable of observe the networking environment, make decisions for optimal performance and adapt its system parameter according to the decisions. As a result, the thesis presents a five-layer cyber security architecture that consists of security elements controlled by a cognitive process. The proposed architecture includes the infrastructure, services and application layers that are managed and controlled by the cognitive and management layers. The architecture defines the tasks of the security elements at a functional level without introducing any new protocols or algorithms. For evaluating two separated method were used. The first method is based on the SABSA framework that uses a layered approach to analyze overall security of an organization. The second method was a scenario based method in which a risk severity level is calculated. The evaluation results show that the proposed architecture fulfills the security requirements at least at a high level. However, the evaluation of the proposed architecture proved to be very challenging. Thus, the evaluation results must be considered very critically. The thesis proves the cognitive networks are a promising approach, and they provide lots of benefits when designing a cyber security architecture for the tactical military networks. However, many implementation problems exist, and several details must be considered and studied during the future work.
Resumo:
By coupling the Boundary Element Method (BEM) and the Finite Element Method (FEM) an algorithm that combines the advantages of both numerical processes is developed. The main aim of the work concerns the time domain analysis of general three-dimensional wave propagation problems in elastic media. In addition, mathematical and numerical aspects of the related BE-, FE- and BE/FE-formulations are discussed. The coupling algorithm allows investigations of elastodynamic problems with a BE- and a FE-subdomain. In order to observe the performance of the coupling algorithm two problems are solved and their results compared to other numerical solutions.
Resumo:
The capabilities and thus, design complexity of VLSI-based embedded systems have increased tremendously in recent years, riding the wave of Moore’s law. The time-to-market requirements are also shrinking, imposing challenges to the designers, which in turn, seek to adopt new design methods to increase their productivity. As an answer to these new pressures, modern day systems have moved towards on-chip multiprocessing technologies. New architectures have emerged in on-chip multiprocessing in order to utilize the tremendous advances of fabrication technology. Platform-based design is a possible solution in addressing these challenges. The principle behind the approach is to separate the functionality of an application from the organization and communication architecture of hardware platform at several levels of abstraction. The existing design methodologies pertaining to platform-based design approach don’t provide full automation at every level of the design processes, and sometimes, the co-design of platform-based systems lead to sub-optimal systems. In addition, the design productivity gap in multiprocessor systems remain a key challenge due to existing design methodologies. This thesis addresses the aforementioned challenges and discusses the creation of a development framework for a platform-based system design, in the context of the SegBus platform - a distributed communication architecture. This research aims to provide automated procedures for platform design and application mapping. Structural verification support is also featured thus ensuring correct-by-design platforms. The solution is based on a model-based process. Both the platform and the application are modeled using the Unified Modeling Language. This thesis develops a Domain Specific Language to support platform modeling based on a corresponding UML profile. Object Constraint Language constraints are used to support structurally correct platform construction. An emulator is thus introduced to allow as much as possible accurate performance estimation of the solution, at high abstraction levels. VHDL code is automatically generated, in the form of “snippets” to be employed in the arbiter modules of the platform, as required by the application. The resulting framework is applied in building an actual design solution for an MP3 stereo audio decoder application.
Resumo:
This thesis aims to find an effective way of conducting a target audience analysis (TAA) in cyber domain. There are two main focal points that are addressed; the nature of the cyber domain and the method of the TAA. Of the cyber domain the object is to find the opportunities, restrictions and caveats that result from its digital and temporal nature. This is the environment in which the TAA method is examined in this study. As the TAA is an important step of any psychological operation and critical to its success, the method used must cover all the main aspects affecting the choice of a proper target audience. The first part of the research was done by sending an open-ended questionnaire to operators in the field of information warfare both in Finland and abroad. As the results were inconclusive, the research was completed by assessing the applicability of United States Army Joint Publication FM 3-05.301 in the cyber domain via a theory-based content analysis. FM 3- 05.301 was chosen because it presents a complete method of the TAA process. The findings were tested against the results of the questionnaire and new scientific research in the field of psychology. The cyber domain was found to be “fast and vast”, volatile and uncontrollable. Although governed by laws to some extent, the cyber domain is unpredictable by nature and not controllable to reasonable amount. The anonymity and lack of verification often present in the digital channels mean that anyone can have an opinion, and any message sent may change or even be counterproductive to the original purpose. The TAA method of the FM 3-05.301 is applicable in the cyber domain, although some parts of the method are outdated and thus suggested to be updated if used in that environment. The target audience categories of step two of the process were replaced by new groups that exist in the digital environment. The accessibility assessment (step eight) was also redefined, as in the digital media the mere existence of a written text is typically not enough to convey the intended message to the target audience. The scientific studies made in computer sciences and both in psychology and sociology about the behavior of people in social media (and overall in cyber domain) call for a more extensive remake of the TAA process. This falls, however, out of the scope of this work. It is thus suggested that further research should be carried out in search of computer-assisted methods and a more thorough TAA process, utilizing the latest discoveries of human behavior. ---------------------------------------------------------------------------------------------------------------------------------- Tämän opinnäytetyön tavoitteena on löytää tehokas tapa kohdeyleisöanalyysin tekemiseksi kybertoimintaympäristössä. Työssä keskitytään kahteen ilmiöön: kybertoimintaympäristön luonteeseen ja kohdeyleisöanalyysin metodiin. Kybertoimintaympäristön osalta tavoitteena on löytää sen digitaalisesta ja ajallisesta luonteesta juontuvat mahdollisuudet, rajoitteet ja sudenkuopat. Tämä on se ympäristö jossa kohdeyleisöanalyysiä tarkastellaan tässä työssä. Koska kohdeyleisöanalyysi kuuluu olennaisena osana jokaiseen psykologiseen operaatioon ja on onnistumisen kannalta kriittinen tekijä, käytettävän metodin tulee pitää sisällään kaikki oikean kohdeyleisön valinnan kannalta merkittävät osa-alueet. Tutkimuksen ensimmäisessä vaiheessa lähetettiin avoin kysely informaatiosodankäynnin ammattilaisille Suomessa ja ulkomailla. Koska kyselyn tulokset eivät olleet riittäviä johtopäätösten tekemiseksi, tutkimusta jatkettiin tarkastelemalla Yhdysvaltojen armeijan kenttäohjesäännön FM 3-05.301 soveltuvuutta kybertoimintaympäristössä käytettäväksi teorialähtöisen sisällönanalyysin avulla. FM 3-05.301 valittiin koska se sisältää kokonaisvaltaisen kohdeyleisöanalyysiprosessin. Havaintoja verrattiin kyselytutkimuksen tuloksiin ja psykologian uusiin tutkimuksiin. Kybertoimintaympäristö on tulosten perusteella nopea ja valtava, jatkuvasti muuttuva ja kontrolloimaton. Vaikkakin lait hallitsevat kybertoimintaympäristöä jossakin määrin, on se silti luonteeltaan ennakoimaton eikä sitä voida luotettavasti hallita. Digitaalisilla kanavilla usein läsnäoleva nimettömyys ja tiedon tarkastamisen mahdottomuus tarkoittavat että kenellä tahansa voi olla mielipide asioista, ja mikä tahansa viesti voi muuttua, jopa alkuperäiseen tarkoitukseen nähden vastakkaiseksi. FM 3-05.301:n metodi toimii kybertoimintaympäristössä, vaikkakin jotkin osa-alueet ovat vanhentuneita ja siksi ne esitetään päivitettäväksi mikäli metodia käytetään kyseisessä ympäristössä. Kohdan kaksi kohdeyleisökategoriat korvattiin uusilla, digitaalisessa ympäristössä esiintyvillä ryhmillä. Lähestyttävyyden arviointi (kohta 8) muotoiltiin myös uudestaan, koska digitaalisessa mediassa pelkkä tekstin läsnäolo ei sellaisenaan tyypillisesti vielä riitä halutun viestin välittämiseen kohdeyleisölle. Tietotekniikan edistyminen ja psykologian sekä sosiologian aloilla tehty tieteellinen tutkimus ihmisten käyttäytymisestä sosiaalisessa mediassa (ja yleensä kybertoimintaympäristössä) mahdollistavat koko kohdeyleisöanalyysiprosessin uudelleenrakentamisen. Tässä työssä sitä kuitenkaan ei voida tehdä. Siksi esitetäänkin että lisätutkimusta tulisi tehdä sekä tietokoneavusteisten prosessien että vielä syvällisempien kohdeyleisöanalyysien osalta, käyttäen hyväksi viimeisimpiä ihmisen käyttäytymiseen liittyviä tutkimustuloksia.
Resumo:
Two adjacent tracts of tropical secondary forest, situated in Itambé do Mato Dentro, south-eastern Brazil, which had been regenerating for 15 and 40 years after clearing, were compared with the purpose of detecting differences in species diversity and composition, species guild composition (regeneration, stratification and dispersion), and stand structure. Four and three 1,125 m² plots laid on the 15- and 40-year-old stands, respectively, sampled 2,430 trees with diameter at the base of the stem > 5 cm. The number of species (S = 199) was high for this forest type and significantly higher for the older stand. Tree density was significantly higher in the younger stand, particularly for smaller trees, whereas the two stands did not differ in both basal area and volume per hectare. Trees of shade-tolerant and understory species were significantly more abundant in the older stand. Though sharing a large proportion of species (49%), the two stands differed significantly in the abundance of many species. Live stumps probably contributed to the relatively quick restoration of some forest characteristics, particularly species diversity, basal area and volume.
Resumo:
Temperate and temperate x tropical germplasms were introduced into the maize breeding of the Department of Genetics (ESALQ-USP). Twenty populations designated as P-1 to P-20 and a check population were evaluated in a randomized complete block design with two replications at Piracicaba, SP, Brazil. Plots were 2.0 m long, spaced 1.0 m apart, with 10 plants per plot after thinning. The following traits were evaluated: PH, plant height; EH, ear height; TB, tassel branch number; KW, 100 kernels weight; EL, ear length; ED, ear diameter; KR, kernels per row, and RN, number of rows of kernels. The means of the exotic populations showed a variation in performance for all traits. The population means were greater than check means for KW, EL, and KR. Populations P-9, P-10, P-12, and P-13 showed low relative ear placement values (EH/PH index), indicating that these materials could contribute to lower ear placement in local populations. P-1 and P-9 showed a high potential to reduce TB. The phenotypic correlation coefficients among populations were positive for most combinations, except for RN with PH, EH, TB, and KW. Predictions of 19 composite means were obtained for all traits.
Resumo:
Abnormal production of interferon alpha (IFN-a) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-a. IFN-a can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-a inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-a receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-aB on Daudi cells, but did not block IFN-aB activity at higher concentrations (>6.25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-aB on Hep 2/c cells challenged with encephalomyocarditis virus.
Resumo:
JNK1 is a MAP-kinase that has proven a significant player in the central nervous system. It regulates brain development and the maintenance of dendrites and axons. Several novel phosphorylation targets of JNK1 were identified in a screen performed in the Coffey lab. These proteins were mainly involved in the regulation of neuronal cytoskeleton, influencing the dynamics and stability of microtubules and actin. These structural proteins form the dynamic backbone for the elaborate architecture of the dendritic tree of a neuron. The initiation and branching of the dendrites requires a dynamic interplay between the cytoskeletal building blocks. Both microtubules and actin are decorated by associated proteins which regulate their dynamics. The dendrite-specific, high molecular weight microtubule associated protein 2 (MAP2) is an abundant protein in the brain, the binding of which stabilizes microtubules and influences their bundling. Its expression in non-neuronal cells induces the formation of neurite-like processes from the cell body, and its function is highly regulated by phosphorylation. JNK1 was shown to phosphorylate the proline-rich domain of MAP2 in vivo in a previous study performed in the group. Here we verify three threonine residues (T1619, T1622 and T1625) as JNK1 targets, the phosphorylation of which increases the binding of MAP2 to microtubules. This binding stabilizes the microtubules and increases process formation in non-neuronal cells. Phosphorylation-site mutants were engineered in the lab. The non-phosphorylatable mutant of MAP2 (MAP2- T1619A, T1622A, T1625A) in these residues fails to bind microtubules, while the pseudo-phosphorylated form, MAP2- T1619D, T1622D, Thr1625D, efficiently binds and induces process formation even without the presence of active JNK1. Ectopic expression of the MAP2- T1619D, T1622D, Thr1625D in vivo in mouse brain led to a striking increase in the branching of cortical layer 2/3 (L2/3) pyramidal neurons, compared to MAP2-WT. The dendritic complexity defines the receptive field of a neuron and dictates the output to the postsynaptic cells. Previous studies in the group indicated altered dendrite architecture of the pyramidal neurons in the Jnk1-/- mouse motor cortex. Here, we used Lucifer Yellow loading and Sholl analysis of neurons in order to study the dendritic branching in more detail. We report a striking, opposing effect in the absence of Jnk1 in the cortical layers 2/3 and 5 of the primary motor cortex. The basal dendrites of pyramidal neurons close to the pial surface at L2/3 show a reduced complexity. In contrast, the L5 neurons, which receive massive input from the L2/3 neurons, show greatly increased branching. Another novel substrate identified for JNK1 was MARCKSL1, a protein that regulates actin dynamics. It is highly expressed in neurons, but also in various cancer tissues. Three phosphorylation target residues for JNK1 were identified, and it was demonstrated that their phosphorylation reduces actin turnover and retards migration of these cells. Actin is the main cytoskeletal component in dendritic spines, the site of most excitatory synapses in pyramidal neurons. The density and gross morphology of the Lucifer Yellow filled dendrites were characterized and we show reduced density and altered morphology of spines in the motor cortex and in the hippocampal area CA3. The dynamic dendritic spines are widely considered to function as the cellular correlate during learning. We used a Morris water maze to test spatial memory. Here, the wild-type mice outperformed the knock-out mice during the acquisition phase of the experiment indicating impaired special memory. The L5 pyramidal neurons of the motor cortex project to the spinal cord and regulate the movement of distinct muscle groups. Thus the altered dendrite morphology in the motor cortex was expected to have an effect on the input-output balance in the signaling from the cortex to the lower motor circuits. A battery of behavioral tests were conducted for the wild-type and Jnk1-/- mice, and the knock-outs performed poorly compared to wild-type mice in tests assessing balance and fine motor movements. This study expands our knowledge of JNK1 as an important regulator of the dendritic fields of neurons and their manifestations in behavior.