Characterization of DnmA, the Dnmt2 homolog in Dictyostelium discoideum

Eukaryotic DNA m5C methyltransferases (MTases) play a major role in many epigenetic regulatory processes like genomic imprinting, X-chromosome inactivation, silencing of transposons and gene expression. Members of the two DNA m5C MTase families, Dnmt1 and Dnmt3, are relatively well studied and many details of their biological functions, biochemical properties as well as interaction partners are known. In contrast, the biological functions of the highly conserved Dnmt2 family, which appear to have non-canonical dual substrate specificity, remain enigmatic despite the efforts of many researchers. The genome of the social amoeba Dictyostelium encodes Dnmt2-homolog, the DnmA, as the only DNA m5C MTase which allowed us to study Dnmt2 function in this organism without interference by the other enzymes. The dnmA gene can be easily disrupted but the knock-out clones did not show obvious phenotypes under normal lab conditions, suggesting that the function of DnmA is not vital for the organism. It appears that the dnmA gene has a low expression profile during vegetative growth and is only 5-fold upregulated during development. Fluorescence microscopy indicated that DnmA-GFP fusions were distributed between both the nucleus and cytoplasm with some enrichment in nuclei. Interestingly, the experiments showed specific dynamics of DnmA-GFP distribution during the cell cycle. The proteins colocalized with DNA in the interphase and were mainly removed from nuclei during mitosis. DnmA functions as an active DNA m5C MTase in vivo and is responsible for weak but detectable DNA methylation of several regions in the Dictyostelium genome. Nevertheless, gel retardation assays showed only slightly higher affinity of the enzyme to dsDNA compared to ssDNA and no specificity towards various sequence contexts, although weak but detectable specificity towards AT-rich sequences was observed. This could be due to intrinsic curvature of such sequences. Furthermore, DnmA did not show denaturant-resistant covalent complexes with dsDNA in vitro, although it could form covalent adducts with ssDNA. Low binding and methyltransfer activity in vitro suggest the necessity of additional factor in DnmA function. Nevertheless, no candidates could be identified in affinity purification experiments with different tagged DnmA fusions. In this respect, it should be noted that tagged DnmA fusion preparations from Dictyostelium showed somewhat higher activity in both covalent adduct formation and methylation assays than DnmA expressed in E.coli. Thus, the presence of co-purified factors cannot be excluded. The low efficiency of complex formation by the recombinant enzyme and the failure to define interacting proteins that could be required for DNA methylation in vivo, brought up the assumption that post-translational modifications could influence target recognition and enzymatic activity. Indeed, sites of phosphorylation, methylation and acetylation were identified within the target recognition domain (TRD) of DnmA by mass spectrometry. For phosphorylation, the combination of MS data and bioinformatic analysis revealed that some of the sites could well be targets for specific kinases in vivo. Preliminary 3D modeling of DnmA protein based on homology with hDNMT2 allowed us to show that several identified phosphorylation sites located on the surface of the molecule, where they would be available for kinases. The presence of modifications almost solely within the TRD domain of DnmA could potentially modulate the mode of its interaction with the target nucleic acids. DnmA was able to form denaturant-resistant covalent intermediates with several Dictyostelium tRNAs, using as a target C38 in the anticodon loop. The formation of complexes not always correlated with the data from methylation assays, and seemed to be dependent on both sequence and structure of the tRNA substrate. The pattern, previously suggested by the Helm group for optimal methyltransferase activity of hDNMT2, appeared to contribute significantly in the formation of covalent adducts but was not the only feature of the substrate required for DnmA and hDNMT2 functions. Both enzymes required Mg2+ to form covalent complexes, which indicated that the specific structure of the target tRNA was indispensable. The dynamics of covalent adduct accumulation was different for DnmA and different tRNAs. Interestingly, the profiles of covalent adduct accumulation for different tRNAs were somewhat similar for DnmA and hDNMT2 enzymes. According to the proposed catalytic mechanism for DNA m5C MTases, the observed denaturant-resistant complexes corresponded to covalent enamine intermediates. The apparent discrepancies in the data from covalent complex formation and methylation assays may be interpreted by the possibility of alternative pathways of the catalytic mechanism, leading not to methylation but to exchange or demethylation reactions. The reversibility of enamine intermediate formation should also be considered. Curiously, native gel retardation assays showed no or little difference in binding affinities of DnmA to different RNA substrates and thus the absence of specificity in the initial enzyme binding. The meaning of the tRNA methylation as well as identification of novel RNA substrates in vivo should be the aim of further experiments.

Transformers 1

A 35 minute overview of Transformer construction and design elements by Prof Jan Sykulski of the University of Southampton.

Transformers 3

A 36 minute covering transformer testing, use in three-phase connections and power systems by Prof Jan Sykulski of the University of Southampton.

AtTRB1, a telomeric DNA-binding protein from Arabidopsis, is concentrated in the nucleolus and shows highly dynamic association with chromatin

AtTRB1, 2 and 3 are members of the SMH (single Myb histone) protein family, which comprises double-stranded DNA-binding proteins that are specific to higher plants. They are structurally conserved, containing a Myb domain at the N-terminus, a central H1/H5-like domain and a C-terminally located coiled-coil domain. AtTRB1, 2 and 3 interact through their Myb domain specifically with telomeric double-stranded DNA in vitro, while the central H1/H5-like domain interacts non-specifically with DNA sequences and mediates protein–protein interactions. Here we show that AtTRB1, 2 and 3 preferentially localize to the nucleus and nucleolus during interphase. Both the central H1/H5-like domain and the Myb domain from AtTRB1 can direct a GFP fusion protein to the nucleus and nucleolus. AtTRB1–GFP localization is cell cycle-regulated, as the level of nuclear-associated GFP diminishes during mitotic entry and GFP progressively re-associates with chromatin during anaphase/telophase. Using fluorescence recovery after photobleaching and fluorescence loss in photobleaching, we determined the dynamics of AtTRB1 interactions in vivo. The results reveal that AtTRB1 interaction with chromatin is regulated at two levels at least, one of which is coupled with cell-cycle progression, with the other involving rapid exchange.

Design, implementation and characterisation of a contactless power transfer system for rotating applications

This paper describes the design, implementation and characterisation of a contactless power transfer system for rotating applications. The power transfer system is based upon a zero-voltage-switched, full-bridge, DC-DC converter, but utilises a non-standard transformer. This transformer allows power transfer between its primary and secondary windings while also allowing free rotation between these windings. The aim of this research is to develop a solution that could replace mechanical slip-rings in certain applications where a non-contacting system would be advantageous. Based upon the design method presented in this paper, a 2 kW prototype system is constructed. Results obtained from testing the 2 kW prototype are presented and discussed. This discussion considers how the performance of the transformer varies with rotation and also the overall efficiency of the system

Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell

Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.

Localization to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division

The subcellular localization of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) (group I and group II coronaviruses, respectively) nucleoproteins (N proteins) were examined by confocal microscopy. The proteins were shown to localize either to the cytoplasm alone or to the cytoplasm and a structure in the nucleus. This feature was confirmed to be the nucleolus by using specific antibodies to nucleolin, a major component of the nucleolus, and by confocal microscopy to image sections through a cell expressing N protein. These findings are consistent with our previous report for infectious bronchitis virus (group III coronavirus) (J. A. Hiscox et al., J. Virol. 75:506-512, 2001), indicating that nucleolar localization of the N protein is a common feature of the coronavirus family and is possibly of functional significance. Nucleolar localization signals were identified in the domain III region of the N protein from all three coronavirus groups, and this suggested that transport of N protein to the nucleus might be an active process. In addition, our results suggest that the N protein might function to disrupt cell division. Thus, we observed that approximately 30% of cells transfected with the N protein appeared to be undergoing cell division. The most likely explanation for this is that the N protein induced a cell cycle delay or arrest, most likely in the G2/M phase. In a fraction of transfected cells expressing coronavirus N proteins, we observed multinucleate cells and dividing cells with nucleoli (which are only present during interphase). These findings are consistent with the possible inhibition of cytokinesis in these cells.

A comparison between bond graphs switching modelling techniques implemented on a boost dc-dc converter

A Bond Graph is a graphical modelling technique that allows the representation of energy flow between the components of a system. When used to model power electronic systems, it is necessary to incorporate bond graph elements to represent a switch. In this paper, three different methods of modelling switching devices are compared and contrasted: the Modulated Transformer with a binary modulation ratio (MTF), the ideal switch element, and the Switched Power Junction (SPJ) method. These three methods are used to model a dc-dc Boost converter and then run simulations in MATLAB/SIMULINK. To provide a reference to compare results, the converter is also simulated using PSPICE. Both quantitative and qualitative comparisons are made to determine the suitability of each of the three Bond Graph switch models in specific power electronics applications

Analytical calculation of resonant inductance for zero voltage switching in phase-shifted full-bridge converters

The phase shift full bridge (PSFB) converter allows high efficiency power conversion at high frequencies through zero voltage switching (ZVS); the parasitic drain-to-source capacitance of the MOSFET is discharged by a resonant inductance before the switch is gated resulting in near zero turn-on switching losses. Typically, an extra inductance is added to the leakage inductance of a transformer to form the resonant inductance necessary to charge and discharge the parasitic capacitances of the PSFB converter. However, many PSFB models do not consider the effects of the magnetizing inductance or dead-time in selecting the resonant inductance required to achieve ZVS. The choice of resonant inductance is crucial to the ZVS operation of the PSFB converter. Incorrectly sized resonant inductance will not achieve ZVS or will limit the load regulation ability of the converter. This paper presents a unique and accurate equation for calculating the resonant inductance required to achieve ZVS over a wide load range incorporating the effects of the magnetizing inductance and dead-time. The derived equations are validated against PSPICE simulations of a PSFB converter and extensive hardware experimentations.

An enhanced model for small-signal analysis of the phase-shifted full-bridge converter

This paper presents an in-depth critical discussion and derivation of a detailed small-signal analysis of the Phase-Shifted Full-Bridge (PSFB) converter. Circuit parasitics, resonant inductance and transformer turns ratio have all been taken into account in the evaluation of this topology’s open-loop control-to-output, line-to-output and load-to-output transfer functions. Accordingly, the significant impact of losses and resonant inductance on the converter’s transfer functions is highlighted. The enhanced dynamic model proposed in this paper enables the correct design of the converter compensator, including the effect of parasitics on the dynamic behavior of the PSFB converter. Detailed experimental results for a real-life 36V-to-14V/10A PSFB industrial application show excellent agreement with the predictions from the model proposed herein.1

Bond graph models of DC-DC converters operating in both CCM and DCM

In this paper, Bond Graphs are employed to develop a novel mathematical model of conventional switched-mode DC-DC converters valid for both continuous and discontinuous conduction modes. A unique causality bond graph model of hybrid models is suggested with the operation of the switch and the diode to be represented by a Modulated Transformer with a binary input and a resistor with fixed conductance causality. The operation of the diode is controlled using an if-then function within the model. The extracted hybrid model is implemented on a Boost and Buck converter with their operations to change from CCM to DCM and to return to CCM. The vector fields of the models show validity in a wide operation area and comparison with the simulation of the converters using PSPICE reveals high accuracy of the proposed model, with the Normalised Root Means Square Error and the Maximum Absolute Error remaining adequately low. The model is also experimentally tested on a Buck topology.

Magnetic Susceptibility Measurements at Ultra-low Temperatures

We report the design and operation of a device for ac magnetic susceptibility measurements that can operate down to 1 mK. The device, a modification of the standard mutual inductance bridge, is designed with detailed consideration of the thermalization and optimization of each element. First, in order to reduce local heating, the primary coil is made with superconducting wire. Second, a low-temperature transformer which is thermally anchored to the mixing chamber of a dilution refrigerator, is used to match the output of the secondary coil to a high-sensitivity bridge detector. The careful thermal anchoring of the secondary coil and the matching transformer is required to reduce the overall noise temperature and maximize sensitivity. The sample is immersed in liquid (3)He to minimize the Kapitza thermal resistance. The magnetic susceptibility of several magnetic compounds, such as the well-known spin gap compound NiCl(2)-4SC(NH(2))(2) and other powdered samples, have been successfully measured to temperatures well below 10 mK.

As relações de trabalho nas organizações de economia solidária : um paralelo Brasil-França

O objetivo desta pesquisa é o estudo das relações que se estabelecem entre o trabalhador e seu trabalho em organizações não convencionais, isto é, que não se identificam com o modo capitalista de produção e que reivindicam, pelo contrário, a criatividade e a originalidade de um trabalho mais autônomo, mais justo e mais responsável: a economia solidária. O que significa, para o trabalhador, atuar nestas organizações? É a pergunta que orienta este estudo. Dito de outro modo: a experiência vivida no trabalho cotidiano pelo trabalhador da economia solidária manifesta-se mediante práticas profissionais e práticas sociais reveladoras de novas formas de inserção no trabalho e na sociedade? Seria essa experiência fruto de uma transformação que ocorre em alguns segmentos do mundo do trabalho e observável em outros contextos econômicos ou um reflexo da situação particularmente fragilizada dos trabalhadores brasileiros? Tratar-se-á então de entender a singularidade da participação dos trabalhadores a este projeto, seu impacto sobre o desenvolvimento de seu trabalho e das relações estabelecidas com a organização e de estudar as transformações que podem ocorrer nas relações sociais a partir do trabalho. Este será analisado sob uma dimensão subjetiva, como experiência de construção identitária, e sob uma dimensão institucional, como socialização para e pela solidariedade. O conceito de “trabalho solidário”, que reúne essas dimensões, será analisado apoiando-se, em grande parte, na obra de François Dubet e sua teoria do ator, da estrutura social e da socialização. A pesquisa se inscreve numa perspectiva de contribuição à Sociologia do Trabalho e das relações de trabalho sem a pretensão de realizar uma sociologia da economia solidária, nas suas múltiplas relações com a sociedade. O que está em foco é a transformação das relações entre o trabalhador e seu trabalho. O argumento defendido pela pesquisa é que as relações que nascem de uma experiência cotidiana do trabalho nas organizações da economia solidária são peculiares e diversificadas, mas interpelam e desafiam o conjunto das relações sociais. Portanto, o trabalho realizado nessas organizações talvez seria, apesar de suas ambigüidades, suscetível de estimular novas formas de relações sociais por meio de uma socialização assentada na solidariedade. Três tipos de organizações “referências” (tipos ideais) são construídos a partir do tipo de produção dominante e dos valores e objetivos que motivam a ação: organizações de produção, associações culturais e organizações humanitárias. A comparação Brasil – França, através das organizações investigadas em Porto Alegre e Paris, procura homologias, isto é: correspondências na construção da ação apesar de contextos diferentes, assim como a reconstrução de processos e procura de especificidades que possam enriquecer as interações. No decorrer da investigação, o que se encontrou, tanto no Brasil quanto na França, foi de um lado, um discurso “oficial” (mentores, militantes da economia solidária e pesquisadores) que descreve a tarefa que se atribui a economia solidária: a responsabilização de todos para transformar a sociedade. Por outro lado, encontrou-se, através do discurso dos trabalhadores, o relato da realidade quotidiana que aparece como um mundo de tensões e contradições. Para entender essa aparente incompatibilidade, foi preciso recriar os mecanismos de construção do trabalho solidário: em que medida pode-se falar de experiência social e em que medida a socialização para e pela solidariedade é bem sucedida. Para tanto, foi necessário recompor o processo de construção das relações sociais dentro e fora do trabalho, manifestado mediante estratégias dos atores que precisam se posicionar frente às lógicas de ação desenvolvidas pelas organizações. A seguir, analisou-se a possibilidade de encontrar semelhanças e diferenças entre o Brasil e a França na construção deste trabalho solidário. Enfim, procurou-se responder à pergunta que originou esta pesquisa: seria mesmo o trabalho solidário gerador de novas relações sociais no trabalho e no âmbito mais amplo da sociedade?

Héritage et transmission des savoirs dans une communauté de pêcheurs portugaise

La pêche est une activité complexe qui fait appel aux sens, à la perception et à l'analyse, aboutissant à un savoir-faire qui est l’ensemble des comportements maîtrisés. Ce savoir s'acquiert par transmission, et par l'expérience que seule la pratique confère. On ne peut donc dissocier dans l'analyse l'apprentissage didactique de l'acquisition empirique. Longtemps les générations de pêcheurs se sont succédées les unes aux autres et rares étaient ceux dont le chemin s'écartait du modèle traditionnel. Depuis quelques années, et en raison de la défection de la jeunesse pour le métier, les aînés n'exercent plus leur activité de formateur. Pour ne pas interrompre la transmission de ce 'capital cognitif' qui permet à un groupe de se perpétuer comme de se transformer, et pour pallier le manque de formateurs, dû à la disparition des anciens, d'autres voies sont empruntées.

A Fogueira do conhecimento: religação de saberes e formação

Les récits sur les expériences de vie se constituent en apprentissages signifiants dans le processus d´ auto-formation des éducateurs. La formation est un phenomène qui extrapôle l ambiance scolaire, incluant les expériences qui servent de matrice pour la construction de la connaissance au long de la vie. Dans cette perspective, «la connaissance de soi», telle quelle proposée par SOUZA, NOVOA et JOSSO, est la notion centrale au tour de laquelle se développe cette dissertation. La recherche a comme idée principale transposer pour la realité des professeurs l´exercice reflexif de son enseignement, médiant la redécouverte de ses expériences de vie, a travers des histoires de soi que, potentialisés, peuvent transformer ses pratiques dans la classe scolaire. Je prends comme point de départ mes propres expériences comme éducatrice assumant la conviction de la indissociation entre sujet et objet de la connaissance, comme propose Édgar Morin pour parler de la science de la complexité. Je fais aussi l´usage des récits de six professeurs du réseau publique de l enseigment, révélateurs de la construction de la connaissance appuyée dans la cohérence de la praxis pédagogique avec son mode de comprendre et sentir le monde. Les oeuvres «Mes Démons» d Édgar Morin, «O Tempo e Eu» de Luis da Câmara Cascudo et «O Banquete dos Deuses» de Daniel Munduruku, ont élargit le champs des récits d expériences que se constituent en matrices des processus de formation. Le travail avec les récits de formation démontrent qu´à partir de la réflection du sujet sur sa propre expérience, il est possible de se projéter des nouvelles configurations de la connaissance tenant comme base, la reliaison entre vie, idées, et pratiques pédagogiques. À partir de la métaphore du bûcher il est possible de se comprendre la force de combustion des expériences de vie dans la formation des enseignants