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This article presents the theories related to bullying and, at the same time, a pedagogical strategy to promote personal relationships between students. This shows the importance of playful-cooperative games in the development of personal relationships, as well as the use of practices of good treatment and inclusion from diversity, framed in the context of school coexistence. This work is based on a qualitative paradigm, which goes with the nonparticipantaction research, comprising a sample of 38 students between the ages of 14 and 16. The results obtained show that playful-cooperative games contribute to the improvement and strengthening of the intra and interpersonal relationships, to achieve a school coexistence, denoting that physical education can contribute to personal relationships, promoting learning scenarios based on equal opportunities, and counteracting discrimination., Este artigo apresenta as teorias relacionadas ao assédio e uma estratégia pedagógica para promover as relações pessoais entre os alunos. Isso mostra a importância dos jogos lúdicos-cooperativos no desenvolvimentodas relações pessoais, bem como a utilização de práticas de bom trato e inclusão a partir da diversidade, enquadradas no contexto de convivência escolar. Este trabalho baseia-se em um paradigma qualitativo, que acompanha a pesquisa-ação não participante, composta por uma amostra de 38 alunos com idades entre 14 e 16 anos. Os resultados obtidos mostram que os jogos lúdicos cooperativos contribuem para a melhoria e fortalecimento das relações intra e interpessoais, para o alcance da convivência escolar, denotando que a educação física pode contribuir para as relações pessoais, promovendo cenários de aprendizagem baseados na igualdade de oportunidades e combatendo a discriminação., El artículo plantea las teorías relacionadas con el acoso escolar y, a la vez, sugiere una estrategia pedagógica para favorecer las relaciones personales entre estudiantes. Se evidencia la importancia de los juegos lúdicocooperativos en el desarrollo de las relaciones personales, además del uso de prácticas del buen trato y la inclusión desde la diversidad, enmarcadas en el contexto de la convivencia escolar. El trabajo se sustenta en un paradigma cualitativo, de la mano de la investigación acción no participante, con una muestra de 38 estudiantes de edades entre los 14 y los 16 años de edad. Los resultados obtenidos permiten apreciar que los juegos lúdicocooperativos contribuyen al mejoramiento de la relación intrapersonal e interpersonal, para lograr una convivencia escolar. La educación física puede contribuir en las relaciones personales, ya que promueve escenarios de aprendizaje basados en la igualdad de oportunidades y, contrarresta las discriminaciones.

This research explores Chinese queer women’s practices for using lesbian social and dating apps such as Rela and HER in Australia. It highlights how social and cultural contexts played instrumental roles in shaping the development trajectories and technological infrastructures of Rela and HER and the Australia-based Chinese queer women’s digital intimacy practices. Findings in this thesis enrich our understanding of queer diasporas and their digital media use in cross-cultural and transnational contexts.

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product—the alarmone nucleotide (p)ppGpp—through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product—the alarmone nucleotide (p)ppGpp—through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product—the alarmone nucleotide (p)ppGpp—through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product—the alarmone nucleotide (p)ppGpp—through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity., SCOPUS: ar.j, info:eu-repo/semantics/published

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product—the alarmone nucleotide (p)ppGpp—through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product—the alarmone nucleotide (p)ppGpp—through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.

The phenomenon of isolated value in each blockchain system has become adistinct issue of the blockchain field. To address this problem, the demandof cross-chain intercommunication came up. In a narrow sense, cross-chainrefers to the process of asset interoperability between relatively independentblockchains. In this thesis, we mainly analyze the design principles, technicaldifficulties, and solutions of cross-chain intercommunication in this narrowsense. With the introduction of distributed ledger technology(DLT), we describethe interaction with other ledgers as the fundamental problem of currentblockchain technology.The implementation of cross-chain is mainly manifested as asset swap and assettransfer. So far, there are many existing application scenarios and projectsadopted from these manifestation. This paper will focus on these two implementations,illustrate their principles, locate the realization difficulties, andput forward corresponding possible solutions. Then we elaborated on eightpopular cross-chain projects underlying mechanism listed with three maincategories. A detailed comparison according to their interoperability level,consensus algorithm and application scenarios of the overall overview of 20cross-chain projects is presented as a table in the Appendix A.During the implementation process, we performed a simple atomic swap crosschainframework based on Hash Time Lock Contract between Bitshares andEthereum, then compare the performance with a wallet application presentby Ripple using Interledger Protocol. These two applications are representedthe two different use case of cross-chain realization.With limited number of projects to test out, our conclusion was reached aftera discussion with the relative merits of the two approaches. Interledger protocolhas a better solution from the aspects of the decentralization, scalability,and whether it supports traditional ledgers., Fenomenet isolerat värde i varje blockchain-system har blivit en distinkt frågaom blockchainfält. För att hantera detta problem kom kravet på interkommunikationmellan kedjor upp. I en smal mening hänvisar tvärkedjantill processen för interoperabilitet mellan tillgångar mellan relativt oberoendeblockchains. I denna avhandling analyserar vi huvudsakligen designprinciper,tekniska svårigheter och lösningar för interkommunikation mellan kedjor ien smal bemärkelse. Med introduktionen av distribuerad huvudboksteknologi(DLT) beskriver vi interaktionen med andra bokar som det grundläggandeproblemet med den nuvarande blockchain-tekniken.Tvärkedjans implementeringsform manifesteras huvudsakligen som tillgångsbyteoch överföring av tillgångar. Hittills finns det många befintliga applikationsscenarier och Pro projekt som antagits från dessa manifestation. Dettadokument kommer att fokusera på dessa två implementeringar, illustrera derasprinciper, lokalisera förståelsessvårigheterna och lägga fram motsvarandemöjliga lösningar. Sedan utarbetade vi åtta populära tvärkedjeprojekt underliggandemekanism listade med tre huvudkategorier. En detaljerad jämförelseberoende på deras driftskompatibilitetsnivå, konsensusalgoritm och tillämpningsscenarierav den övergripande översikten över 20 tvärkedjeprojekt presenterassom en tabell i Appendix A.Under genomförandeprocessen genomförde vi en enkel atomisk swap Cross-Chain ram baserad på hash Time lock kontrakt mellan Bitshares och Ethereum,sedan jämföra prestanda med en plånbok program som finns med Ripplemed Interledger Protocol. Dessa två applikationer föreställs de två olika användningsfallenav Cross-Chain genomförande.Med begränsade projekt att testa, nåddes vår slutsats efter en diskussion medrelativa fördelar med två metoder praktiskt taget. Interledger-protokollet haren bättre lösning med avseende på decentralisering, skalbarhet och huruvidadet stöder traditionella bokar.

The phenomenon of isolated value in each blockchain system has become adistinct issue of the blockchain field. To address this problem, the demandof cross-chain intercommunication came up. In a narrow sense, cross-chainrefers to the process of asset interoperability between relatively independentblockchains. In this thesis, we mainly analyze the design principles, technicaldifficulties, and solutions of cross-chain intercommunication in this narrowsense. With the introduction of distributed ledger technology(DLT), we describethe interaction with other ledgers as the fundamental problem of currentblockchain technology.The implementation of cross-chain is mainly manifested as asset swap and assettransfer. So far, there are many existing application scenarios and projectsadopted from these manifestation. This paper will focus on these two implementations,illustrate their principles, locate the realization difficulties, andput forward corresponding possible solutions. Then we elaborated on eightpopular cross-chain projects underlying mechanism listed with three maincategories. A detailed comparison according to their interoperability level,consensus algorithm and application scenarios of the overall overview of 20cross-chain projects is presented as a table in the Appendix A.During the implementation process, we performed a simple atomic swap crosschainframework based on Hash Time Lock Contract between Bitshares andEthereum, then compare the performance with a wallet application presentby Ripple using Interledger Protocol. These two applications are representedthe two different use case of cross-chain realization.With limited number of projects to test out, our conclusion was reached aftera discussion with the relative merits of the two approaches. Interledger protocolhas a better solution from the aspects of the decentralization, scalability,and whether it supports traditional ledgers., Fenomenet isolerat värde i varje blockchain-system har blivit en distinkt frågaom blockchainfält. För att hantera detta problem kom kravet på interkommunikationmellan kedjor upp. I en smal mening hänvisar tvärkedjantill processen för interoperabilitet mellan tillgångar mellan relativt oberoendeblockchains. I denna avhandling analyserar vi huvudsakligen designprinciper,tekniska svårigheter och lösningar för interkommunikation mellan kedjor ien smal bemärkelse. Med introduktionen av distribuerad huvudboksteknologi(DLT) beskriver vi interaktionen med andra bokar som det grundläggandeproblemet med den nuvarande blockchain-tekniken.Tvärkedjans implementeringsform manifesteras huvudsakligen som tillgångsbyteoch överföring av tillgångar. Hittills finns det många befintliga applikationsscenarier och Pro projekt som antagits från dessa manifestation. Dettadokument kommer att fokusera på dessa två implementeringar, illustrera derasprinciper, lokalisera förståelsessvårigheterna och lägga fram motsvarandemöjliga lösningar. Sedan utarbetade vi åtta populära tvärkedjeprojekt underliggandemekanism listade med tre huvudkategorier. En detaljerad jämförelseberoende på deras driftskompatibilitetsnivå, konsensusalgoritm och tillämpningsscenarierav den övergripande översikten över 20 tvärkedjeprojekt presenterassom en tabell i Appendix A.Under genomförandeprocessen genomförde vi en enkel atomisk swap Cross-Chain ram baserad på hash Time lock kontrakt mellan Bitshares och Ethereum,sedan jämföra prestanda med en plånbok program som finns med Ripplemed Interledger Protocol. Dessa två applikationer föreställs de två olika användningsfallenav Cross-Chain genomförande.Med begränsade projekt att testa, nåddes vår slutsats efter en diskussion medrelativa fördelar med två metoder praktiskt taget. Interledger-protokollet haren bättre lösning med avseende på decentralisering, skalbarhet och huruvidadet stöder traditionella bokar.

Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands - such as "starved" ribosomal complexes, deacylated tRNA and pppGpp - and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native ReIA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.

Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands - such as "starved" ribosomal complexes, deacylated tRNA and pppGpp - and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native ReIA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.

Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands - such as "starved" ribosomal complexes, deacylated tRNA and pppGpp - and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native ReIA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.

Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands - such as "starved" ribosomal complexes, deacylated tRNA and pppGpp - and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native ReIA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.

Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands - such as "starved" ribosomal complexes, deacylated tRNA and pppGpp - and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native ReIA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.

RelA is a ribosome associated multi-domain enzyme, which plays a crucial role in adaptation ofEscherichia coli to nutritional stress as such as amino acid deficiency. It detects the deficiency of aminoacids in the cell by monitoring whether a tRNA at the acceptor site (A-site) of the ribosome is chargedwith amino acid or not. When RelA detects uncharged, i.e. deacylated tRNA, it starts to producealarmone guanosine penta- or tetraphosphate, collectively referred to as (p)ppGpp. (p)ppGpp is aglobal metabolism regulator in bacteria. Increase in (p)ppGpp concentration alters crucial metabolicprocesses, such as DNA replication, gene expression, cell wall synthesis and translation. Thesechanges also include activation of different virulence factors and are proposed to drive formation of abacterial sub-population that is highly resilient to antibiotic treatment, the so-called persisters. For a long time the molecular mechanism of RelA’s activation by and interaction with the ribosomedeacylatedtRNA complex was unknown. Only recently several cryo-EM structures of RelA-ribosomecomplex have shed light on how C-terminal domains of RelA interact with ribosome-deacylated tRNAcomplex. Guided by these structures we investigated the role of RelA’s domains in this interaction byconstructing a set of RelA C-terminal truncates and subjecting these to biochemical and microbiologicalexperimentation. These experiments were complemented with mutations in ribosomal RNA atpositions that interact with RelA, namely A-site finger and sarcin-ricin loop. We have shown that only the full-length wild type RelA can be activated by ribosome-tRNA complex,whereas, the set of truncated proteins missing either one, two or three C-terminal domains do notrespond to the presence of uncharged tRNA in the A-site of the ribosome. However, these truncatedversions can still be activated by vacant 70S ribosome as well as pppGpp, suggesting that N-terminaldomain of RelA has an allosteric regulation site for (p)ppG

RelA is a ribosome associated multi-domain enzyme, which plays a crucial role in adaptation ofEscherichia coli to nutritional stress as such as amino acid deficiency. It detects the deficiency of aminoacids in the cell by monitoring whether a tRNA at the acceptor site (A-site) of the ribosome is chargedwith amino acid or not. When RelA detects uncharged, i.e. deacylated tRNA, it starts to producealarmone guanosine penta- or tetraphosphate, collectively referred to as (p)ppGpp. (p)ppGpp is aglobal metabolism regulator in bacteria. Increase in (p)ppGpp concentration alters crucial metabolicprocesses, such as DNA replication, gene expression, cell wall synthesis and translation. Thesechanges also include activation of different virulence factors and are proposed to drive formation of abacterial sub-population that is highly resilient to antibiotic treatment, the so-called persisters. For a long time the molecular mechanism of RelA’s activation by and interaction with the ribosomedeacylatedtRNA complex was unknown. Only recently several cryo-EM structures of RelA-ribosomecomplex have shed light on how C-terminal domains of RelA interact with ribosome-deacylated tRNAcomplex. Guided by these structures we investigated the role of RelA’s domains in this interaction byconstructing a set of RelA C-terminal truncates and subjecting these to biochemical and microbiologicalexperimentation. These experiments were complemented with mutations in ribosomal RNA atpositions that interact with RelA, namely A-site finger and sarcin-ricin loop. We have shown that only the full-length wild type RelA can be activated by ribosome-tRNA complex,whereas, the set of truncated proteins missing either one, two or three C-terminal domains do notrespond to the presence of uncharged tRNA in the A-site of the ribosome. However, these truncatedversions can still be activated by vacant 70S ribosome as well as pppGpp, suggesting that N-terminaldomain of RelA has an allosteric regulation site for (p)ppG

RelA is a ribosome associated multi-domain enzyme, which plays a crucial role in adaptation ofEscherichia coli to nutritional stress as such as amino acid deficiency. It detects the deficiency of aminoacids in the cell by monitoring whether a tRNA at the acceptor site (A-site) of the ribosome is chargedwith amino acid or not. When RelA detects uncharged, i.e. deacylated tRNA, it starts to producealarmone guanosine penta- or tetraphosphate, collectively referred to as (p)ppGpp. (p)ppGpp is aglobal metabolism regulator in bacteria. Increase in (p)ppGpp concentration alters crucial metabolicprocesses, such as DNA replication, gene expression, cell wall synthesis and translation. Thesechanges also include activation of different virulence factors and are proposed to drive formation of abacterial sub-population that is highly resilient to antibiotic treatment, the so-called persisters. For a long time the molecular mechanism of RelA’s activation by and interaction with the ribosomedeacylatedtRNA complex was unknown. Only recently several cryo-EM structures of RelA-ribosomecomplex have shed light on how C-terminal domains of RelA interact with ribosome-deacylated tRNAcomplex. Guided by these structures we investigated the role of RelA’s domains in this interaction byconstructing a set of RelA C-terminal truncates and subjecting these to biochemical and microbiologicalexperimentation. These experiments were complemented with mutations in ribosomal RNA atpositions that interact with RelA, namely A-site finger and sarcin-ricin loop. We have shown that only the full-length wild type RelA can be activated by ribosome-tRNA complex,whereas, the set of truncated proteins missing either one, two or three C-terminal domains do notrespond to the presence of uncharged tRNA in the A-site of the ribosome. However, these truncatedversions can still be activated by vacant 70S ribosome as well as pppGpp, suggesting that N-terminaldomain of RelA has an allosteric regulation site for (p)ppG

RelA is a ribosome associated multi-domain enzyme, which plays a crucial role in adaptation ofEscherichia coli to nutritional stress as such as amino acid deficiency. It detects the deficiency of aminoacids in the cell by monitoring whether a tRNA at the acceptor site (A-site) of the ribosome is chargedwith amino acid or not. When RelA detects uncharged, i.e. deacylated tRNA, it starts to producealarmone guanosine penta- or tetraphosphate, collectively referred to as (p)ppGpp. (p)ppGpp is aglobal metabolism regulator in bacteria. Increase in (p)ppGpp concentration alters crucial metabolicprocesses, such as DNA replication, gene expression, cell wall synthesis and translation. Thesechanges also include activation of different virulence factors and are proposed to drive formation of abacterial sub-population that is highly resilient to antibiotic treatment, the so-called persisters. For a long time the molecular mechanism of RelA’s activation by and interaction with the ribosomedeacylatedtRNA complex was unknown. Only recently several cryo-EM structures of RelA-ribosomecomplex have shed light on how C-terminal domains of RelA interact with ribosome-deacylated tRNAcomplex. Guided by these structures we investigated the role of RelA’s domains in this interaction byconstructing a set of RelA C-terminal truncates and subjecting these to biochemical and microbiologicalexperimentation. These experiments were complemented with mutations in ribosomal RNA atpositions that interact with RelA, namely A-site finger and sarcin-ricin loop. We have shown that only the full-length wild type RelA can be activated by ribosome-tRNA complex,whereas, the set of truncated proteins missing either one, two or three C-terminal domains do notrespond to the presence of uncharged tRNA in the A-site of the ribosome. However, these truncatedversions can still be activated by vacant 70S ribosome as well as pppGpp, suggesting that N-terminaldomain of RelA has an allosteric regulation site for (p)ppG

RelA is a ribosome associated multi-domain enzyme, which plays a crucial role in adaptation ofEscherichia coli to nutritional stress as such as amino acid deficiency. It detects the deficiency of aminoacids in the cell by monitoring whether a tRNA at the acceptor site (A-site) of the ribosome is chargedwith amino acid or not. When RelA detects uncharged, i.e. deacylated tRNA, it starts to producealarmone guanosine penta- or tetraphosphate, collectively referred to as (p)ppGpp. (p)ppGpp is aglobal metabolism regulator in bacteria. Increase in (p)ppGpp concentration alters crucial metabolicprocesses, such as DNA replication, gene expression, cell wall synthesis and translation. Thesechanges also include activation of different virulence factors and are proposed to drive formation of abacterial sub-population that is highly resilient to antibiotic treatment, the so-called persisters. For a long time the molecular mechanism of RelA’s activation by and interaction with the ribosomedeacylatedtRNA complex was unknown. Only recently several cryo-EM structures of RelA-ribosomecomplex have shed light on how C-terminal domains of RelA interact with ribosome-deacylated tRNAcomplex. Guided by these structures we investigated the role of RelA’s domains in this interaction byconstructing a set of RelA C-terminal truncates and subjecting these to biochemical and microbiologicalexperimentation. These experiments were complemented with mutations in ribosomal RNA atpositions that interact with RelA, namely A-site finger and sarcin-ricin loop. We have shown that only the full-length wild type RelA can be activated by ribosome-tRNA complex,whereas, the set of truncated proteins missing either one, two or three C-terminal domains do notrespond to the presence of uncharged tRNA in the A-site of the ribosome. However, these truncatedversions can still be activated by vacant 70S ribosome as well as pppGpp, suggesting that N-terminaldomain of RelA has an allosteric regulation site for (p)ppG

The nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p) ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p) ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p) ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p) ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to beta-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p) ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.

The nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p) ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p) ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p) ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p) ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to beta-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p) ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.

The nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p) ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p) ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p) ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p) ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to beta-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p) ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.

The nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p) ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p) ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p) ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p) ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to beta-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p) ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.

The nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p) ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p) ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p) ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p) ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to beta-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p) ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.

The nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p) ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p) ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p) ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p) ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to beta-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p) ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.

The biochemical mechanisms of transcription specificity in the p53 family and NFkappaB family of transcription factors in the context of DNA sequence recognition are not well-defined. Given the loose consensus of the DNA binding sites recognized by the p53 and NFkappaB family, we investigated the structural and biochemical contributions to DNA sequence recognition. In chapter 2, we show the effects of perturbing a loop feature in the DNA-binding domain of p73 on cooperative binding that is selective for different DNA sequences using x-ray crystallography and fluorescence polarization. This is the first structural evidence of a L1 loop-dependent mechanism of sequence recognition that implicates the role of L1 loop acetylation to selective activation of apoptotic genes. In chapter 3, we continued to investigate the structural mechanism of recognition by crystallizing wild-type p73 DNA-binding domain with a native p53 DNA response element, which showed conserved features of DNA recognition. In the final chapter 4, the effects of mutating a native NFkappaB DNA response element were investigated with RelA using mammalian luciferase assay, electrophoretic mobility shift assay, and in vitro chromatin transcription assay to corroborate the “ruler” mechanism of recognition observed in crystal structures. The RelA transactivation domain was shown to contribute to sequence specificity that has not been shown before. Together, these studies show structural features that are important in sequence recognition and further refine mechanisms of transcription specificity based on cooperative, multi-domain interactions.

The biochemical mechanisms of transcription specificity in the p53 family and NFkappaB family of transcription factors in the context of DNA sequence recognition are not well-defined. Given the loose consensus of the DNA binding sites recognized by the p53 and NFkappaB family, we investigated the structural and biochemical contributions to DNA sequence recognition. In chapter 2, we show the effects of perturbing a loop feature in the DNA-binding domain of p73 on cooperative binding that is selective for different DNA sequences using x-ray crystallography and fluorescence polarization. This is the first structural evidence of a L1 loop-dependent mechanism of sequence recognition that implicates the role of L1 loop acetylation to selective activation of apoptotic genes. In chapter 3, we continued to investigate the structural mechanism of recognition by crystallizing wild-type p73 DNA-binding domain with a native p53 DNA response element, which showed conserved features of DNA recognition. In the final chapter 4, the effects of mutating a native NFkappaB DNA response element were investigated with RelA using mammalian luciferase assay, electrophoretic mobility shift assay, and in vitro chromatin transcription assay to corroborate the “ruler” mechanism of recognition observed in crystal structures. The RelA transactivation domain was shown to contribute to sequence specificity that has not been shown before. Together, these studies show structural features that are important in sequence recognition and further refine mechanisms of transcription specificity based on cooperative, multi-domain interactions.

Amino acid starvation has a profound effect on gene expression in E. coli, and activate the stringent response. Stress response in E. coli is primarily regulated by the stringent response, which is initiated by the protein RelA. Based on preliminary results from our group, a model has been proposed in which E. coli degrades up to 80% of its total tRNA during amino acid starvation, in order to mitigate misincorperations of amino acids independently of RelA. This model proposes that the RNA chaperone Hfq and the novel group of small regulatory RNAs known as tRNA linked repeats (TLR) are involved in this process. Two different experimental approaches are pursued in this project, in which the translational fidelity of wild type, ΔHfq and ΔTLR22 strains in balanced growth and arginine starvation are investigated by a dual luciferase based mistranslation reporter assay and 2-dimensional gel electrophoresis. The findings do not show a decrease in translational fidelity during arginine starvation for the strains investigated.

Amino acid starvation has a profound effect on gene expression in E. coli, and activate the stringent response. Stress response in E. coli is primarily regulated by the stringent response, which is initiated by the protein RelA. Based on preliminary results from our group, a model has been proposed in which E. coli degrades up to 80% of its total tRNA during amino acid starvation, in order to mitigate misincorperations of amino acids independently of RelA. This model proposes that the RNA chaperone Hfq and the novel group of small regulatory RNAs known as tRNA linked repeats (TLR) are involved in this process. Two different experimental approaches are pursued in this project, in which the translational fidelity of wild type, ΔHfq and ΔTLR22 strains in balanced growth and arginine starvation are investigated by a dual luciferase based mistranslation reporter assay and 2-dimensional gel electrophoresis. The findings do not show a decrease in translational fidelity during arginine starvation for the strains investigated.

The electrical grid contains protection functions to ensure a secure and efficient disconnection of faulty plant parts in accordance with Swedish law, applicable standards and industry practice. The aim with this bachelor thesis has been to develop a way of testing relay protection equipment to ensure the applicable standards are followed and at the same time show the result in a way that is easy to understand and makes it possible to use the result in E.ONs risk and vulnerable analysis. A risk and vulnerable analysis is required under regulation EIFS 2013:3. The work has consists of four stages. The first stage has been to examine the existing situation and see how the work is performed. The second stage has been to study the literature about the subject and the demands that E.ON places on the testing procedure. The third stage has been to develop a report template. The fourth stage has been to test the proposed method in reality and to guarantee the desired function of the protection. It has been discovered that the most appropriate way of reporting testing is a template where relay parameters and the testing protocol are collected into one file where an automatized assessment is done of the protection equipment. This approach also makes it possible to use the results directly in E.ONs risk and vulnerability analysis. The report contains a theoretical background of the protection equipment for electrical plants and grids. The report also shows the development of the testing plan together with the proposed reporting templates.The work shows that a periodical testing of electrical protections should consist of the following parts:• Visual inspection• Verification of tripping, blocking and signalling circuits• Verification of relay protection equipment• Measuring of operating valuesThe way of testing has been a bit simplified. This has been done by excluding those parts from the testing that are not necessary in modern protection equipment. A saving in the data handl, Validerat; 20150608 (global_studentproject_submitter)

Syftet med detta examensarbete är att pressentera ett antal tekniska lösningar för att förbättra säkerheten och kapaciteten i ställverk 59. Ställverk 59 är den vanligaste ställverkstypen på Trafikverkets järnvägsnät. Grundkonstruktionen är utvecklad på 1950-talet, men ställverkstypen nybyggs fortfarande trots konstruktionens ålder. Ända sedan järnvägen infördes har det funnits behov av att styra trafiken. Att spåren är fria från hinder och att tåget inte kör för fort i växelkurvor är två grundläggande krav för trafikstyrning. Dessa problem har man genom tiderna löst på olika sätt. I Trafikverkets modernare anläggningar används spårledningar för hinderfrihetskontroll och en kombination av optisk signalering och hyttsignalering för att ange tillåten hastighet. För att kunna ge körsignal till ett tåg krävs att alla växlar bortom signalen är låsta i rätt läge så att tåget leds till avsett spår. Dessutom krävs att signaler på anslutande spår visar stopp, så att två tåg inte riskerar att krocka. Ett vanligt problem är att en tungkontrollkontakt går sönder. I ställverk 59 innebär det att tåg måste passera signaler i stopp vilket får konsekvenser för såväl tidtabell som säkerhet. Genom att separera växelkontrollen så att växelns driv och tungkontrollkontakter kontrolleras separat kan man tillåta att körsignal med hastighetsbegränsning på 40 km/h ges trots att en tungkontrollkontakt är trasig. Passagekontrollen i ställverk 59 uppfyller inte Trafikverkets krav för att låsa upp tågvägar. Den befintliga kopplingen motsvarar endast en förenklad variant av bakändespassage. Därför pressenteras en ny krets som uppfyller kraven. Kopplingen innebär att såväl framändespassage som bakändespassage måste ha registrerats för att få låsa upp en tågväg automatiskt. Om denna koppling införs kan även kretsen för tågvägsupplåsning på tid (stoppanmälan) förbättras så att tiden börjar mätas så snart en framändespassage in på huvudspåret har registreras, istället för vid en bakändespassage. Funkt

NF-B proteins belong to a family of ubiquitous transcription factors involved in a number of cellular responses. While the pathways of NF-B activation and input into the regulation of gene activity have been comprehensively investigated, its cytoplasmic functions are poorly understood. In this study we addressed effects of the compartmentalisation of NF-B proteins RelA/p65 and p50 in relation to the inhibitor IB-, using fibronectin (FN) and epidermal growth factor (EGF) for environmental stimulation of epidermoid carcinoma A431 cells. We thus assessed the presence of NF-B family proteins in the cytosol, membrane, nuclear and cytoskeletal fractions with a special attention to the cytoskeletal fraction to define whether NF-B was active or not. Sub-cellular fractionation demonstrated that the proportion of RelA/p65 differed in diverse sub-cellular fractions, and that the cytoskeleton harboured about 7% thereof. Neither the nuclear nor the cytoskeleton fraction did contain IB-. The cytoskeleton binding of RelA/p65 and p50 was further confirmed by co-localisation and electron microscopy data. During 30-min EGF stimulation similar dynamics were found for RelA/p65 and IB- in the cytosol, RelA/p65 and p50 in the nucleus and p50 and IB- in the membrane. Furthermore, EGF stimulation for 30min resulted in a threefold accumulation of RelA/p65 in cytoskeletal fraction. Our results suggest that nuclear-, membrane- and cytoskeleton-associated NF-B are dynamic and comprise active pools, whereas the cytoplasmic is more constant and likely non-active due to the presence of IB-. Moreover, we discovered the existence of a dynamic, IB--free pool of RelA/p65 associated with cytoskeletal fraction, what argues for a special regulatory role of the cytoskeleton in NF-B stimulation., Funding Agencies|Swedish Institute|879/2009|Swedish Research Council|2010-3045|European Science Foundation||postdoctoral fellowship at the Faculty of Health Science||Linkoping University||Russian Foundation for Basic Research|10-04-00174a

HTLV-1 is more pathogenic than HTLV-2 despite having a similar genome and closely related transactivating oncoproteins. Both Tax-1 protein from HTLV-1 and Tax-2 from HTLV-2 activate the NF-κB pathway. The mechanisms involved in Tax-1 deregulation of this signalling pathway have been thoroughly investigated, but little is known about regulation by Tax-2. We have compared the interaction of Tax-1 and Tax-2 with two key NF-κB signalling factors: TAK1-binding protein 2 (TAB2), an adaptor involved in the activation of TAK1 kinase, and RelA, the active subunit of the canonical RelA/p50 NF-κB transcription factor. Tax-2 formed stable complexes with both RelA and TAB2. These two NF-κB factors colocalized with Tax proteins in dotted cytoplasmic structures targeted by calreticulin, a multi-process calcium-buffering chaperone. Co-expression of RelA and/or TAB2 markedly increased Tax-mediated NF-κB activation. These findings provide new insights into the role of RelA, TAB2 and Tax in the deregulation of the NF-κB pathway. © 2010 Elsevier Inc., SCOPUS: ar.j, info:eu-repo/semantics/published

Nuclear Factor κB (NF-κB) proteins make up a large family of eukaryotic transcription factors that regulate many important cellular functions, including cell signaling, cell growth, development, cell death by apoptosis, and immune and inflammatory responses, and has been shown to be involved in multiple human diseases. NF-κB proteins are subject to careful regulation in the cell, and are regulated at multiple levels. A family of inhibitory proteins exist (called IκB proteins) which bind to NF-κB and cause it to be localized in the cytoplasm. There are multiple signal transduction pathways that can lead to the activation of NF-κB. These signal transduction pathways are induced by contact with a wide variety of molecules, and therefore a huge number of molecules can act as NF-κB activators. One such molecule is lipopolysaccharide (LPS), a bacterial cell wall component, which activates NF-κB via Toll-Like Receptor 4 (TLR4). Following TLR4 activation, IkB is degraded, releasing NF-κB. NF-κB then triggers the transcription of IkB, thus down-regulating its own activity. These complex feedbacks create oscillations that have previously been observed in electrophoretic mobility shift assays (EMSA) and live-cell imaging studies. In order to better understand and characterize NF-κB oscillations, we created macrophage cell lines stably expressing a fluorescently labeled monomer of the NF-κB dimer, RelA-GFP, and conducted time-lapse fluorescence imaging studies of these cells. We examined nuclear NF-κB oscillations in both wild-type and transfected cell lines upon activation by bacterial LPS. LPS chemotypes from various organisms were tested for their effects on oscillatory patterns. Our data show that all LPS were able to elucidate oscillations, a novel finding for RAW 264.7 macrophage cells. The data also show that while the responses to the different LPS chemotypes do lead to different oscillatory dynamics, they do not show any easily discernable patterns. This suggests that

The NF-?B/RelA family of transcription factors regulates inducible transcription of a large number of genes in response to diverse stimuli. Little is known, however, about the location of NF-?B in the cytoplasm and the transport mechanism to the nucleus. We found that NF-?B is associated with the actin-binding protein a-actinin-4. NF-?B and a-actinin-4 co-localized along actin stress fibers and in membrane lamellae in A431 cells. After a 30-min stimulation with EGF or TNF-a, a-actinin-4 and p65 were found in the nucleus. Disruption of cytoskeleton by cytochalasin D prior to treatment with TNF-a led to increase of p65 nuclear translocation. Antibodies to p65 subunit of NF-?B co-immunoprecipitated a-actinin-4 from A431 cell lysates and nuclear extracts, but a-actinin-1 and ß-actin were not found in the precipitates. Affinity chromatography experiments displayed that p65 and p50 subunits of NF-?B can bind to matrix-bound chicken gizzard a-actinin. We suggest that the a-actinin-4 is important for the NF-?B nuclear translocation and its functions inside the nucleus. © 2007 Elsevier Inc. All rights reserved.

The activation of the transcription factor NF-kB is central to the control of the cellular response triggered by many stimuli. Once released from the inhibitory molecule IkB, NF-kB is translocated to the nucleus, and it has to be phosphorylated to activate transcription. In protein kinase C (PKC)-deficient cells, NF-kB is transcriptionally inactive and the phosphorylation of the RelA subunit in response to tumor necrosis factor (TNF-alpha) is severely impaired. In vitro assays showed that PKC directly phosphorylates RelA. Here we demonstrate that Ser311 accounts for PKC phosphorylation of RelA and that this site is phosphorylated in vivo in response to TNF-alpha. Also, an inactivating mutation of that residue severely impairs RelA transcriptional activity, blocks its anti-apoptotic function and abrogates the interaction of RelA with the co-activator CBP as well as its recruitment, and that of RNA polymerase II (Pol II) with the interleukin-6 (IL-6) promoter. The interaction of endogenous CBP with endogenous RelA is inhibited in PKC-/- cells, as well as the binding of Pol II to the IL-6 promoter. These results demonstrate the mechanism whereby PKC regulates NF-kB activation in vivo

Compared to conventional techniques in computational fluid dynamics, the lattice Boltzmann method (LBM) seems to be a completely different approach to solve the incompressible Navier-Stokes equations. The aim of this article is to correct this impression by showing the close relation of LBM to two standard methods: relaxation schemes and explicit finite difference discretizations. As a side effect, new starting points for a discretization of the incompressible Navier-Stokes equations are obtained.

Compared to conventional techniques in computational fluid dynamics, the lattice Boltzmann method (LBM) seems to be a completely different approach to solve the incompressible Navier-Stokes equations. The aim of this article is to correct this impression by showing the close relation of LBM to two standard methods: relaxation schemes and explicit finite difference discretizations. As a side effect, new starting points for a discretization of the incompressible Navier-Stokes equations are obtained.

Compared to conventional techniques in computational fluid dynamics, the lattice Boltzmann method (LBM) seems to be a completely different approach to solve the incompressible Navier-Stokes equations. The aim of this article is to correct this impression by showing the close relation of LBM to two standard methods: relaxation schemes and explicit finite difference discretizations. As a side effect, new starting points for a discretization of the incompressible Navier-Stokes equations are obtained.