Molecular cloning of the transcription factor TFIIB homolog from Sulfolobus shibatae.

The Archaea (archaebacteria) constitute a group of prokaryotes that are phylogenetically distinct from Eucarya (eukaryotes) and Bacteria (eubacteria). Although Archaea possess only one RNA polymerase, evidence suggests that their transcriptional apparatus is similar to that of Eucarya. For example, Archaea contain a homolog of the TATA-binding protein which interacts with the TATA-box like A-box sequence upstream of many archaeal genes. Here, we report the cloning of a Sulfolobus shibatae gene that encodes a protein (transcription factor TFB) with striking homology to the eukaryotic basal transcription factor TFIIB. We show by primer extension analysis that transcription of the S. shibatae TFB gene initiates 27 bp downstream from a consensus A-box element. Significantly, S. shibatae TFB contains an N-terminal putative metal-binding region and two imperfect direct repeats--structural features that are well conserved in eukaryotic TFIIBs. This suggests that TFB may perform analogous functions in Archaea and Eucarya. Consistent with this, we demonstrate that S. shibatae TFB promotes the binding of S. shibatae TBP to the A-box element of the Sulfolobus 16S/23S rRNA gene. Finally, we show that S. shibatae TFB is significantly more related to TFB of the archaeon Pyrococcus woesei than it is to eukaryotic TFIIBs. These data suggest that TFB arose in the common archaeal/eukaryotic ancestor and that the lineages leading to P. woesei and S. shibatae separated after the divergence of the archaeal and eukaryotic lines of descent.

In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus.

Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. Only one of the proteins encoded by the virus, here referred to as replication initiator protein (Rep protein), is indispensable for replication. We show that the Rep protein of tomato yellow leaf curl virus initiates viral-strand DNA synthesis by introducing a nick in the plus strand within the nonanucleotide 1TAATATT decreases 8AC, identical among all geminiviruses. After cleavage, the Rep protein remains bound to the 5' end of the cleaved strand. In addition, we show that the Rep protein has a joining activity, suggesting that it acts as a terminase, thus resolving the nascent viral single strand into genome-sized units.

Dual Regulation of the mitotic exit network (MEN) by PP2A-Cdc55 phosphatase.

Exit from mitosis in budding yeast is triggered by activation of the key mitotic phosphatase Cdc14. At anaphase onset, the protease separase and Zds1 promote the downregulation of PP2A(Cdc55) phosphatase, which facilitates Cdk1-dependent phosphorylation of Net1 and provides the first wave of Cdc14 activity. Once Cdk1 activity starts to decline, the mitotic exit network (MEN) is activated to achieve full Cdc14 activation. Here we describe how the PP2A(Cdc55) phosphatase could act as a functional link between FEAR and MEN due to its action on Bfa1 and Mob1. We demonstrate that PP2A(Cdc55) regulates MEN activation by facilitating Cdc5- and Cdk1-dependent phosphorylation of Bfa1 and Mob1, respectively. Downregulation of PP2A(Cdc55) initiates MEN activity up to Cdc15 by Bfa1 inactivation. Surprisingly, the premature Bfa1 inactivation observed does not entail premature MEN activation, since an additional Cdk1-Clb2 inhibitory signal acting towards Dbf2-Mob1 activity restrains MEN activity until anaphase. In conclusion, we propose a clear picture of how PP2A(Cdc55) functions affect the regulation of various MEN components, contributing to mitotic exit.

Um modelo multiescala concorrente para representar o processo de fissuração do concreto.

Este trabalho propõe uma técnica de modelagem multiescala concorrente do concreto considerando duas escalas distintas: a mesoescala, onde o concreto é modelado como um material heterogêneo, e a macroescala, na qual o concreto é tratado como um material homogêneo. A heterogeneidade da estrutura mesoscópica do concreto é idealizada considerando três fases distintas, compostas pelos agregados graúdos e argamassa (matriz), estes considerados materiais homogêneos, e zona de transição interfacial (ZTI), tratada como a parte mais fraca entre as três fases. O agregado graúdo é gerado a partir de uma curva granulométrica e posicionado na matriz de forma aleatória. Seu comportamento mecânico é descrito por um modelo constitutivo elástico-linear, devido a sua maior resistência quando comparado com as outras duas fases do concreto. Elementos finitos contínuos com alta relação de aspecto em conjunto com um modelo constitutivo de dano são usados para representar o comportamento não linear do concreto, decorrente da iniciação de fissuras na ZTI e posterior propagação para a matriz, dando lugar à formação de macrofissuras. Os elementos finitos de interface com alta relação de aspecto são inseridos entre todos os elementos regulares da matriz e entre os da matriz e agregados, representando a ZTI, tornando-se potenciais caminhos de propagação de fissuras. No estado limite, quando a espessura do elemento de interface tende a zero (h ?0) e, consequentemente, a relação de aspecto tende a infinito, estes elementos apresentam a mesma cinemática da aproximação contínua de descontinuidades fortes (ACDF), sendo apropriados para representar a formação de descontinuidades associados a fissuras, similar aos modelos coesivos. Um modelo de dano à tração é proposto para representar o comportamento mecânico não linear das interfaces, associado à formação de fissuras, ou até mesmo ao eventual fechamento destas. A fim de contornar os problemas causados pela malha de elementos finitos de transição entre as malhas da macro e da mesoescala, que, em geral, apresentam diferenças expressivas 5 de refinamento, utiliza-se uma técnica recente de acoplamento de malhas não conformes. Esta técnica é baseada na definição de elementos finitos de acoplamento (EFAs), os quais são capazes de estabelecer a continuidade de deslocamento entre malhas geradas de forma completamente independentes, sem aumentar a quantidade total de graus de liberdade do problema, podendo ser utilizados tanto para acoplar malhas não sobrepostas quanto sobrepostas. Para tornar possível a análise em multiescala em casos nos quais a região de localização de deformações não pode ser definida a priori, propõe-se uma técnica multiescala adaptativa. Nesta abordagem, usa-se a distribuição de tensões da escala macroscópica como um indicador para alterar a modelagem das regiões críticas, substituindo-se a macroescala pela mesoescala durante a análise. Consequentemente, a malha macroscópica é automaticamente substituída por uma malha mesoscópica, onde o comportamento não linear está na iminência de ocorrer. Testes numéricos são desenvolvidos para mostrar a capacidade do modelo proposto de representar o processo de iniciação e propagação de fissuras na região tracionada do concreto. Os resultados numéricos são comparados com os resultados experimentais ou com aqueles obtidos através da simulação direta em mesoescala (SDM).

Cover to be unveiled : how meaning is revealed with sheer veils in paintings by Lucas Cranach The Elder

While scholars have questioned the meaning of Lucas Cranach the Elder’s sheer veils when associated with sensual nude figures, research about sheer veils adorning women in a religious context in his paintings has not yet been developed. Through a primarily iconographical approach, I explore who dons each type of veil, and when, to better understand why the same sheer veil is worn differently by various individuals and what that could mean relative to Cranach’s body of work. These veils exhibit artistic prowess, but analysis of their placement on individual figures also reveals how Cranach’s repeated use of sheer veils in his paintings trains the eye on underlying messages, unlocking meanings of these works for Cranach and his patrons and broader themes present in sixteenth century visual culture. My paper initiates this important discussion about how sheer veils – often overlooked in Cranach’s works – are used in both religious and secular contexts.

La communication hors du commun : aporétique de la communauté inachevée

En prenant pour appui initial le caractère équivoque de la communication, cette dissertation interroge les manières par lesquelles la vie en commun prend aussi effet comme œuvre de mort. S’inspirant du renouvellement de la recherche sur le thème de la communauté, l’interrogation se déploie en trois mouvements principaux. Chacun de ces mouvements ouvre et négocie trois grandes impasses : épistémologique, politique et éthique. La recherche propose de s’y frayer un chemin en s’appuyant principalement sur les travaux de Jean-Luc Nancy, Giorgio Agamben et Roberto Esposito. Le premier mouvement ouvre au voilement de l’idée de communication. L’idée de communication est voilée par une idéologie qui hérite elle-même d’une certaine conception humaniste de la communauté. Un examen de l’essai de Pic de la Mirandole Sur la dignité de l’homme permet d’exposer les valeurs associées à cette tradition qui recouvrent le caractère ambivalent de la communication. Ce premier mouvement mène au seuil de la situation politique contemporaine, marquée notamment par la nécessité de penser « notre » condition après la crise des valeurs humanistes. Le deuxième mouvement s’applique à l’examen de trois événements politiques contemporains. Chacun donne à comprendre comment s’exprime le péril associé à ce voilement : la fusillade au Collège Dawson de Montréal en 2006, un incident impliquant l’usage de gaz lacrymogènes lors de manifestations menées en 2013 à la Place Taksim à Istanbul, en Turquie, et une analyse de la crise de la dette publique grecque. L’aporie qui articule communication et incommunicabilité y est examinée à partir des thèmes de l’incommensurabilité des modes de vie en commun, de la biopolitique et du fascisme. Le fait que le péril qui menace de « nous » partager soit encore, malgré tout, ce que « nous » avons en partage invite à avancer là où aucune voie ne semble s’ouvrir. Le troisième mouvement présente les manières par lesquelles l’aporie de la communication peut être saisie en montrant qu’il est possible de penser par delà l’opposition de la communication et de la non-communication. Ce problème est abordé à l’horizon de la tradition philosophique concernant la question de l’être. Le saisissement du commun comme d’un propre — l’appropriation de l’inappropriable — ouvre à une conception de la communication « hors du commun ». Ces trois mouvements ne portent pas jusqu’à une conclusion. Ils ouvrent plutôt sur une autre conception de la communication. Celle-ci expose la possibilité sans cesse reconduite de l’événement fragile et intime dont « nous » sommes le nom.

Statusreproduktion und Mobilitätseffekte beruflicher Weiterbildung

Es wird für Westdeutschland untersucht, ob sowohl der intergenerationale Abstieg als auch der intergenerationale Statuserhalt Beweggründe für berufliche Weiterbildung sind. Des Weiteren wird untersucht, ob mit beruflicher Weiterbildung ein intergenerationaler Abstieg ausgeglichen oder das Risiko für intergenerationale Abwärtsmobilität minimiert werden kann. Mit Längsschnittdaten der Deutschen Lebensverlaufsstudie finden sich für westdeutsche Frauen und Männer in fünf aufeinander folgenden Geburtskohorten im Zeitraum von 1949 bis 1999 Indizien dafür, dass das Statuserhaltmotiv ein Weiterbildungsmotiv für Personen sein könnte, die einen intergenerationalen Statusverlust erfahren haben. Mit Weiterbildungsanstrengungen können im weiteren Berufsverlauf intergenerationale Abstiege ausgeglichen werden. Ferner haben statuskonsistente Teilnehmer geringere Risiken für einen intergenerationalen Abstieg als Nichtteilnehmer. Berufliche Weiterbildung hat statusreproduzierende Wirkungen im Berufsverlauf.

La communication hors du commun : aporétique de la communauté inachevée

En prenant pour appui initial le caractère équivoque de la communication, cette dissertation interroge les manières par lesquelles la vie en commun prend aussi effet comme œuvre de mort. S’inspirant du renouvellement de la recherche sur le thème de la communauté, l’interrogation se déploie en trois mouvements principaux. Chacun de ces mouvements ouvre et négocie trois grandes impasses : épistémologique, politique et éthique. La recherche propose de s’y frayer un chemin en s’appuyant principalement sur les travaux de Jean-Luc Nancy, Giorgio Agamben et Roberto Esposito. Le premier mouvement ouvre au voilement de l’idée de communication. L’idée de communication est voilée par une idéologie qui hérite elle-même d’une certaine conception humaniste de la communauté. Un examen de l’essai de Pic de la Mirandole Sur la dignité de l’homme permet d’exposer les valeurs associées à cette tradition qui recouvrent le caractère ambivalent de la communication. Ce premier mouvement mène au seuil de la situation politique contemporaine, marquée notamment par la nécessité de penser « notre » condition après la crise des valeurs humanistes. Le deuxième mouvement s’applique à l’examen de trois événements politiques contemporains. Chacun donne à comprendre comment s’exprime le péril associé à ce voilement : la fusillade au Collège Dawson de Montréal en 2006, un incident impliquant l’usage de gaz lacrymogènes lors de manifestations menées en 2013 à la Place Taksim à Istanbul, en Turquie, et une analyse de la crise de la dette publique grecque. L’aporie qui articule communication et incommunicabilité y est examinée à partir des thèmes de l’incommensurabilité des modes de vie en commun, de la biopolitique et du fascisme. Le fait que le péril qui menace de « nous » partager soit encore, malgré tout, ce que « nous » avons en partage invite à avancer là où aucune voie ne semble s’ouvrir. Le troisième mouvement présente les manières par lesquelles l’aporie de la communication peut être saisie en montrant qu’il est possible de penser par delà l’opposition de la communication et de la non-communication. Ce problème est abordé à l’horizon de la tradition philosophique concernant la question de l’être. Le saisissement du commun comme d’un propre — l’appropriation de l’inappropriable — ouvre à une conception de la communication « hors du commun ». Ces trois mouvements ne portent pas jusqu’à une conclusion. Ils ouvrent plutôt sur une autre conception de la communication. Celle-ci expose la possibilité sans cesse reconduite de l’événement fragile et intime dont « nous » sommes le nom.

Organizational aspects of e-referrals

Three different, well established systems for e-referral were examined. They ranged from a system in a single country handling a large number of cases (60,000 per year) to a global system covering many countries which handled fewer cases (150 per year). Nonetheless, there appeared to be a number of common features. Whether the purpose is e-transfer or e-consultation, the underlying model of the e-referral process is: the referrer initiates an e-request; the organization managing the process receives it, the organization allocates it for reply; the responder replies to the initiator. Various things can go wrong and the organization managing the e-referral process needs to be able to track requests through the system; this requires various performance metrics. E-referral can be conducted using email, or as messages passed either directly between computer systems or via a Web-link to a server. The experience of the three systems studied shows that significant changes in work practice are needed to launch an e-referral service successfully. The use of e-referral between primary and secondary care improves access to services and can be shown to be cost-effective.

The LFA-1-associated molecule PTA-1 (CD226) on T cells forms a dynamic molecular complex with protein 4.1G and human discs large

Clustering of the T cell integrin, LFA-1, at specialized regions of intercellular contact initiates integrin-mediated adhesion and downstream signaling, events that are necessary for a successful immunological response. But how clustering is achieved and sustained is not known. Here we establish that an LFA-1-associated molecule, PTA-1, is localized to membrane rafts and binds the carboxyl-terminal domain of isoforms of the actin-binding protein 4.1G. Protein 4.1 is known to associate with the membrane-associated guanylate kinase homologue, human discs large. We show that the carboxyl-terminal peptide of PTA-1 also can bind human discs large and that the presence or absence of this peptide greatly influences binding between PTA-1 and different isoforms of 4.1G. T cell stimulation with phorbol ester or PTA-1 cross-linking induces PTA-1 and 4.1G to associate tightly with the cytoskeleton, and the PTA-1 from such activated cells now can bind to the amino-terminal region of 4.1G. We propose that these dynamic associations provide the structural basis for a regulated molecular adhesive complex that serves to cluster and transport LFA-1 and associated molecules.

Increased site 1 affinity improves biopotency of porcine growth hormone - Evidence against diffusion dependent receptor dimerization

Based on phage display optimization studies with human growth hormone (GH), it is thought that the biopotency of GH cannot be increased. This is proposed to be a result of the affinity of the first receptor for hormone far exceeding that which is required to trap the hormone long enough to allow diffusion of the second receptor to form the ternary complex, which initiates signaling. We report here that despite similar site 1 kinetics to the hGH/hGH receptor interaction, the potency of porcine GH for its receptor can be increased up to 5-fold by substituting hGH residues involved in site 1 binding into pGH. Based on extensive mutations and BIAcore studies, we show that the higher potency and site 1 affinity of hGH for the pGHR is primarily a result of a decreased off-rate associated with residues in the extended loop between helices 1 and 2 that interact with the two key tryptophans Trp(104) and Trp(169) in the receptor binding hot spot. Our mutagenic analysis has also identified a second determinant (Lys(165)), which in addition to His(169), restricts the ability of non-primate hormones to activate hGH receptor. The increased biopotency of GH that we observe can be explained by a model for GH receptor activation where subunit alignment is critical for effective signaling.

The role of the complement system in ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury is a common clinical event with the potential to seriously affect, and sometimes kill, the patient. Interruption of blood supply causes ischemia, which rapidly damages metabolically active tissues. Paradoxically, restoration of blood flow to the ischemic tissues initiates a cascade of pathology that leads to additional cell or tissue injury. I/R is a potent inducer of complement activation that results in the production of a number of inflammatory mediators. The use of specific inhibitors to block complement activation has been shown to prevent local tissue injury after I/R. Clinical and experimental studies in gut, kidney, limb, and liver have shown that I/R results in local activation of the complement system and leads to the production of the complement factors C3a, C5a, and the membrane attack complex. The novel inhibitors of complement products may find wide clinical application because there are no effective drug therapies currently available to treat I/R injuries.

Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K-m) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-Angstrom resolution (final R-free value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to A protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

Endosome-to-cytosol transport of viral nucleocapsids

During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra- endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid ( LBPA) and its putative effector Alix/ AIP1, and is regulated by phosphatidylinositol- 3-phosphate ( PtdIns( 3) P) signalling via the PtdIns( 3) P- binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back- fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns( 3) P and their effectors.

Family violence: Walking the tight rope between maternal alienation and child safety

Mothers are often alienated from their children when child abuse is suspected or confirmed, whether she is the primary abuser of the child or not. An abusive or violent partner often initiates the process of maternal alienation from children as a control mechanism. When the co-occurrence of maternal and child abuse is not recognised, nurses and health professionals risk further alienating a mother from her children, which can have detrimental effects in both the short and long term. Evidence shows that when mothers are supported and have the necessary resources there is a reduction in the violence and abuse she and her children experience; this occurs even in situations where the mother is the primary abuser of her children. The family-centred care philosophy, which is widely accepted as the best approach to nursing care for children and their families, creates tension for nurses caring for children who are the victims of abuse as this care generally occurs away from the context of the family. This fragmented approach to caring for abused children can inadvertently undermine the mother-child relationship and further contribute to maternal alienation. This paper discusses the complexity of family violence for nurses negotiating the 'tight rope' between the prime concern for the safety of children and further contributing to maternal alienation, within a New Zealand context. The premise that restoration of the mother-child relationship is paramount for the long-term wellbeing of both the children and the mother provides the basis for discussing implications for nursing practice.