Academic literature on the topic 'RSC complex'

The RSC complex remodels chromatin structure and regulates gene transcription. We used cryo–electron microscopy to determine the structure of yeast RSC bound to the nucleosome. RSC is delineated into the adenosine triphosphatase motor, the actin-related protein module, and the substrate recruitment module (SRM). RSC binds the nucleosome mainly through the motor, with the auxiliary subunit Sfh1 engaging the H2A-H2B acidic patch to enable nucleosome ejection. SRM is organized into three substrate-binding lobes poised to bind their respective nucleosomal epitopes. The relative orientations of the SRM and the motor on the nucleosome explain the directionality of DNA translocation and promoter nucleosome repositioning by RSC. Our findings shed light on RSC assembly and functionality, and they provide a framework to understand the mammalian homologs BAF/PBAF and the Sfh1 ortholog INI1/BAF47, which are frequently mutated in cancers.

ABSTRACT RSC is an essential chromatin remodeling complex in Saccharomyces cerevisiae that performs central roles in transcriptional regulation and cell cycle progression. Here we identify Htl1 as a novel factor that associates with the RSC complex both physically and functionally. We isolated HTL1 through a genetic screen for mutants that displayed additive growth defects with a conditional mutation in the protein kinase C gene (PKC1), which has been suggested through genetic connections to interact functionally with RSC. Several lines of evidence connect HTL1 to RSC function. First, an htl1Δ mutant displayed temperature-sensitive growth and a G2/M cell cycle arrest at restrictive temperatures, a phenotype similar to that of strains with conditional mutations in essential RSC components. Second, we isolated RSC3, which encodes a component of the RSC complex, as a dosage suppressor of the htl1Δ growth arrest. Third, an htl1Δ mutant displayed additive growth defects with conditional rsc3 alleles. Fourth, overexpression of HTL1 suppressed the growth defect of a strain with a conditional mutation in another RSC component, RSC8. Finally, we demonstrate that Htl1 is a nuclear protein that can associate in vivo with a fraction of the RSC complex. We propose that an RSC-Htl1 complex acts coordinately with protein kinase C to regulate the G2/M transition.

ABSTRACT The accurate segregation of chromosomes requires the kinetochore, a complex protein machine that assembles onto centromeric DNA to mediate attachment of replicated sister chromatids to the mitotic spindle apparatus. This study reveals an important role for the yeast RSC ATP-dependent chromatin-remodeling complex at the kinetochore in chromosome transmission. Mutations in genes encoding two core subunits of RSC, the ATPase Sth1p and the Snf5p homolog Sfh1p, interact genetically with mutations in genes encoding kinetochore proteins and with a mutation in centromeric DNA. RSC also interacts genetically and physically with the histone and histone variant components of centromeric chromatin. Importantly, RSC is localized to centromeric and centromere-proximal chromosomal regions, and its association with these loci is dependent on Sth1p. Both sth1 and sfh1 mutants exhibit altered centromeric and centromere-proximal chromatin structure and increased missegregation of authentic chromosomes. Finally, RSC is not required for centromeric deposition of the histone H3 variant Cse4p, suggesting that RSC plays a role in reconfiguring centromeric and flanking nucleosomes following Cse4p recruitment for proper chromosome transmission.

ABSTRACT RSC is an essential, multisubunit chromatin remodeling complex. We show here that the Rsc4 subunit of RSC interacted via its C terminus with Rpb5, a conserved subunit shared by all three nuclear RNA polymerases (Pol). Furthermore, the RSC complex coimmunoprecipitated with all three RNA polymerases. Mutations in the C terminus of Rsc4 conferred a thermosensitive phenotype and the loss of interaction with Rpb5. Certain thermosensitive rpb5 mutations were lethal in combination with an rsc4 mutation, supporting the physiological significance of the interaction. Pol II transcription of ca. 12% of the yeast genome was increased or decreased twofold or more in a rsc4 C-terminal mutant. The transcription of the Pol III-transcribed genes SNR6 and RPR1 was also reduced, in agreement with the observed localization of RSC near many class III genes. Rsc4 C-terminal mutations did not alter the stability or assembly of the RSC complex, suggesting an impact on Rsc4 function. Strikingly, a C-terminal mutation of Rsc4 did not impair RSC recruitment to the RSC-responsive genes DUT1 and SMX3 but rather changed the chromatin accessibility of DNases to their promoter regions, suggesting that the altered transcription of DUT1 and SMX3 was the consequence of altered chromatin remodeling.

ABSTRACT Repair of chromosome double-strand breaks (DSBs) is central to cell survival and genome integrity. Nonhomologous end joining (NHEJ) is the major cellular repair pathway that eliminates chromosome DSBs. Here we report our genetic screen that identified Rsc8 and Rsc30, subunits of the Saccharomyces cerevisiae chromatin remodeling complex RSC, as novel NHEJ factors. Deletion of RSC30 gene or the C-terminal truncation of RSC8 impairs NHEJ of a chromosome DSB created by HO endonuclease in vivo. rsc30Δ maintains a robust level of homologous recombination and the damage-induced cell cycle checkpoints. By chromatin immunoprecipitation, we show recruitment of RSC to a chromosome DSB with kinetics congruent with its involvement in NHEJ. Recruitment of RSC to a DSB depends on Mre11, Rsc30, and yKu70 proteins. Rsc1p and Rsc2p, two other RSC subunits, physically interact with yKu80p and Mre11p. The interaction of Rsc1p with Mre11p appears to be vital for survival from genotoxic stress. These results suggest that chromatin remodeling by RSC is important for NHEJ.

Ploidy is tightly regulated in eukaryotic cells and is critical for cell function and survival. Cells coordinate multiple pathways to ensure replicated DNA is segregated accurately to prevent abnormal changes in chromosome number. In this study, we characterize an unanticipated role for the Saccharomyces cerevisiae “remodels the structure of chromatin” (RSC) complex in ploidy maintenance. We show that deletion of any of six nonessential RSC genes causes a rapid transition from haploid to diploid DNA content because of nondisjunction events. Diploidization is accompanied by diagnostic changes in cell morphology and is stably maintained without further ploidy increases. We find that RSC promotes chromosome segregation by facilitating spindle pole body (SPB) duplication. More specifically, RSC plays a role in distributing two SPB insertion factors, Nbp1 and Ndc1, to the new SPB. Thus, we provide insight into a role for a SWI/SNF family complex in SPB duplication and ploidy maintenance.

Faithful chromosome segregation during mitosis is fundamental for cell viability and genome stability. For a correct division, all kinetochores must be attached to the mitotic spindle and cohesion must be timely removed. Anaphase is triggered by the Anaphase Promoting Complex bound to its regulatory subunit Cdc20 (APC-Cdc20) that polyubiquitylates securin (Pds1 in budding yeast), whose role is to maintain inactive the protease separase (Esp1 in budding yeast) until anaphase onset. Once active, separase cleaves cohesin, thus triggering sister chromatid separation. Separase also promotes cyclinB proteolysis and mitotic exit due to its involvement in the Cdc14-early anaphase release (FEAR) pathway that promotes a partial activation of the Cdc14 phophatase, which is in turn key for CDK inactivation and mitotic exit. Cdc14 is maintained inactive throughout most of the cell cycle bound to its inhibitor Net1/Cfi1 and trapped in the nucleolus. At the beginning of anaphase Cdc14 is released from the nucleolus into the nucleus by the FEAR pathway; subsequently, Cdc14 is released also in the cytoplasm by the MEN (Mitotic Exit Network) pathway. In this way Cdc14 is fully active and can trigger mitotic exit by cyclinB-CDK inactivation. The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism conserved in all eukaryotic organisms that ensures the correct segregation of the genetic material. In fact, it inhibits the metaphase to anaphase transition until all kinetochores are properly attached to the mitotic spindle by inactivating the APC-Cdc20 complex, thus providing the time for error correction. Cells do not arrest indefinitely upon SAC activation. After a variable period of time cells escape from the metaphase arrest also in the presence of a damaged mitotic spindle or faulty kinetochore attachments to spindle microtubules. This process is referred to as adaptation or mitotic slippage and is often involved in the resistance to chemotherapeutic compounds that target the mitotic spindle. In spite of its importance, the adaptation process is still little known. Within this context, the goals of my Ph.D. were: (1) to characterize the molecular mechanisms underlying SAC adaptation and (2) to search for factors involved in this process. For these purposes we used the yeast Saccharomyces cerevisiae as a model organism. (1) We characterized the adaptation process in either the presence or the absence of mitotic spindle perturbations. We depolymerized spindles by using two different drugs that alter microtubule dynamics, i.e. nocodazole and benomyl, whereas we induced SAC hyperactivation without spindle damage by overproducing Mad2 (GAL1-MAD2 cells), one of the key proteins for SAC signal generation and maintenance. We observed that in all the conditions cells are able to adapt, but with different kinetics. In particular, cells adapt faster in benomyl, while in nocodazole and with high levels of Mad2 cells need more time to slip out of mitosis. The few data available about SAC adaptation in higher eukaryotes indicate that SAC adaptation is accompanied by chromatid separation, a decrease in mitotic CDK activity and mitotic exit. Indeed, like in mammalian cells, yeast securin and cyclinB are degraded and sister chromatids are separated during adaptation. In addition, cyclinB stabilization, as well as Cdc20 and Cdc5 (polo kinase) inactivation, markedly delay adaptation, while the only yeast CKI (Sic1) is not involved in this process. Finally, when yeast cells adapt the SAC is likely to be turned off, as shown by the disassembly of the Mad1/Bub3 checkpoint complex. (2) To search for factors involved in SAC adaptation, we performed a genetic screen using GAL1-MAD2 cells. In particular, we screened for mutants that would remain arrested for prolonged times in mitosis upon MAD2 overexpression. We identified Rsc2, a non-essential component of the RSC chromatin remodelling complex, as a regulator of SAC adaptation in yeast. We demonstrated that RSCRsc2 is involved in fine tuning mitotic exit during the unperturbed cell cycle. Its activity becomes particularly important in conditions that would activate the SAC, as it contributes to cyclinB degradation. In the absence of Rsc2 Net1 phosphorylation and the early anaphase release of Cdc14 from the nucleolus are impaired, whereas expression of a dominant allele of CDC14 that loosens Net1 inhibition (CDC14TAB6-1) is sufficient to restore mitotic exit in conditions where Rsc2 becomes essential for this process. We further demonstrated that the ATPase activity of RSC is required for mitotic exit regulation, suggesting that its chromatin-remodelling activity is involved in this process. By studying possible genetic interactions between the RSC2 deletion and FEAR or MEN mutations, we found that RSC2 deletion confers synthetic lethality or sickness to MEN but not to FEAR mutants. Altogether, our data suggest that RSCRsc2 is a novel component of the FEAR pathway. Finally, we demonstrated that Rsc2 interacts in vivo and in vitro with the polo kinase Cdc5, which controls mitotic exit at different levels. Since RSC binds to acetylated histone tails, it is possible that histone transacetylases are also involved in SAC adaptation. We tested if the SAGA (Spt-Ada-Gcn5 Acetyltransferase) complex is involved in SAC adaptation by deleting ADA2 or GCN5 in yeast. Indeed, SAGA seems involved in adaptation, although the contribution of Ada2 and Gcn5 in the process differs depending on the conditions used to activate the SAC. Finally, since we found that upon treatment with benomyl (a microtubule destabilizer) cells adapt dividing nuclei, we wondered if SAC adaptation could be linked to the presence of cytoplasmic microtubules that are still partially detectable in these conditions. We therefore asked whether motor proteins and microtubule regulators are involved in mitotic slippage. Indeed, we found that in the absence of Kip2 and Bik1, which specifically bind to cytoplasmic microtubules, cells divide nuclei and exit mitosis slower than wild type cells, demonstrating that cytoplasmic microtubules and associated proteins could accelerate SAC adaptation. In conclusion, SAC adaptation is a very complex process whose timing probably depends on the interplay between different mechanisms. An important aim for a complete comprehension of this process, as well as for the development of new and more efficient cancer therapies, will be to identify novel factors implicated in adaptation and clarify how their function might be linked to one another.

New corrole–BODIPY conjugates have been synthesized in high yield under mild conditions. Upon excitation at the absorption maximum of the BODIPY antenna chromophore, the fluorescence intensity of the free base corrole–BODIPY conjugate increases by ca. 300%, and significant phosphorescence intensity is observed for the iridium(III) complex of the conjugate, while almost no phosphorescence is observed for the parent iridium(III) corrole, due to through-bond energy transfer from the BODIPY antenna-chromophore to the corrole core.
Original publication is available at http://dx.doi.org/10.1039/c5ra07250f
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Les protéines se liant à l’ARN de la famille IMP sont les protéines oncofoetales conservées, qui régulent le transport, la stabilité et la traduction de plusieurs ARNm cibles. Les IMPs sont impliqués dans la tumorigenèse et dans le développement embryonnaire par le contrôle de la prolifération cellulaire, la différenciation, la migration, la polarisation et d`autres processus cellulaires. IMP-3 est difficilement détectable dans des tissus adultes normaux, mais il est surexprimé dans les nombreux cancers, où il a été caractérisé comme un marqueur d’agressivité et de la croissance tumorale rapide, ainsi que d’un pronostic défavorable pour les patients. Dans notre étude, nous avons utilisé une lignée cellulaire RD de rhabdomyosarcome (RMS), où IMPs étaient initialement décrits comme des protéines régulatrices de l`ARNm de IGF-2. Nous avons essayé d'élucider le mécanisme par lequel IMP3 régule l’expression des cyclines D1 et D3, contribuant ainsi à la compréhension des processus oncogéniques dans les RMS et autres cancers.Nous avons montré que IMP3 régule l'expression des cyclines D1 et D3 d'une manière significative in vivo. Nous avons également démontré, qu'en absence de IMP3, les ARNm des cyclines sont exportés vers le cytoplasme et s’associent avec les polyribosomes, mais ne sont pas traduits. En outre, l'inhibition d`IMP3 n'a pas d'influence sur la stabilité des ARNm des cyclines. Nous démontrons que dans des cellules cancéreuses humaines, IMP3 interagit avec plusieurs protéines se liant à l'ARN, et que nombre de ces protéines a un effet sul l’expression des cyclines, ce que suggère l'existence d'un complexe régulateur multiprotéique sur les 3'UTR des cyclines D1 et D3. Nos résultat montrent que l'inhibition de deux protéines clés de RNA-induced silencing complex (RISC) (AGO2 et GW182/TNRC6), rétablit les niveaux d'expression des cyclines D1 et D3, qui ont été considérablement diminués en l’absence d’IMP3 ou de ses partenaires protéiques ILF3/NF90 et PTBP1. Nous concluons que les complexes d`IMP3 et RISC peuvent concourir pour la régulation des ARNm des cyclines. Nous avons également identifié les miARNs qui peuvent être impliqués dans ce processus, ainsi que les domaines fonctionnellement importants dans les 3 'UTR des cyclines, où se passe la competition entre les complexes d’IMP-3 et RISC. Nos résultats sont compatibles avec l'existence de IMP3 - contenant complexe multiprotéique, qui est associé à 3'UTRs des cyclines et régule leur traduction en les protégeant contre la répression traductionnelle par miRISC
RNA-binding proteins of the IMP family (IGF2 mRNA-binding proteins 1-3) are conserved oncofetal proteins, regulating transport, stability and decay of multiple mRNAs. IMPs are involved in embryonic developement and tumorigenesis by controlling cell proliferation, differentation, migration, polarization and many other important aspects of cell function. IMP-3 is hardly detectable in normal adult tissues, but is overexpressed in many cancers, where it has been reported as a marker of tumor aggressiveness, rapid growth, and bad prognosis for patients. In our research we utilized a rhabdomyosarcoma (RMS) cell line RD, where IMPs were first described as IGF-2 mRNA regulating proteins. We aimed to elucidate the mechanism by which IMP3 regulates the expression of cyclins D1 and D3, thereby contributing to the understanding of oncogenic processes in RMS.In this study, we show that IMP3 regulates the expression of cyclin D1 and D3 in a significant manner in vivo. We also demonstrate that in the absence of IMP3, the mRNAs of the cyclins are exported to the cytoplasm and associated with polyribosomes, but not translated. IMP3 inhibition does not influence the stability of cyclin mRNAs. We demonstrate that in human cancer cells, IMP3 interacts with multiple RNA-binding proteins, and that a number of these IMP-3 partners impacts on the expression of cyclins D1 and D3. These observations suggest the existence of a regulatory IMP-3 containing RNP complex on the 3’UTR of mRNAs of cyclin D1 and D3. Our results show that an inhibition of two key proteins of RNA-induced silencing complex (RISC) (AGO2 and GW182/TNRC6) rescues the expression of cyclin D1 and D3 proteins, which is significantly decreased in the absence of IMP3 or its protein partners ILF3/NF90 and PTBP1. Therefore, IMP3 and RISC complexes can compete for cyclin mRNAs translational repression/activation. We also identified a number of miRNAs that can be involved in this process, and characterized functionally important regions within 3’ UTRs of the cyclins, where the competition between IMP-3 and RISC complexes takes place. Our results are consistent with the existence of IMP3 - containing multiprotein complex, which is associated with 3’UTRs of the cyclins and regulates their translation by protecting them from miRISC-dependent translational repression

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Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil
A candidíase é uma infecção oportunista provocada por diversas espécies de fungos do gênero Candida, frequentemente encontrados integrando a microbiota, da superfície cutânea, no trato gastrointestinal e cavidades mucosas do ser humano desde o seu nascimento. A incidência das infecções fúngicas sistêmicas têm aumentado consideravelmente nas últimas décadas em função do grande número de pacientes com SIDA, a grande quantidade de transplantes e condições crônicas como o câncer, a terapia prolongada com imunossupressores e o uso de agentes corticosteroides. Além disso, a exposição prolongada aos antifúngicos azólicos promove a seleção de patógenos resistentes. No presente estudo avaliou-se a atividade antifúngica do complexo Rutênio-pirocatecol (RPC) frente a um isolado clinico de Candida tropicalis resistente ao fluconazol. A metodologia empregada para os testes de susceptibilidade foi de acordo com o documento M27-A3 do National Committee for Clinical Laboratory Standards (NCCLS, 2008). Esplenócitos de camundongos Balb/c foram obtidos de forma asséptica para avaliar a citotoxicidade do composto para células de mamíferos. O estresse oxidativo promovido pelo composto foi avaliado através da reação ao ácido tiobarbitúrico (TBARS) e ensaios de fluorescência com a sonda diclorodihidrofluoresceína diacetato (DCFH2DA). O Calcofluor White foi empregado para avaliar a integridade da parede celular. A análise ultraestrutural foi realizada através da microscopia eletrônica de varredura e transmissão. Os resultados encontrados para os testes de atividade antifúngica foram analisados através do teste estatístico ANOVA e pós-teste Dunnett. Os resultados encontrados para os testes de atividade antifúngica do RPC mostraram uma Concentração Inibitória de 50% (IC50) de 20,3 μM, enquanto em esplesnócitos a concentração efetiva de 50% foi de 325 μM mostrando um índice de seletividade igual a 16. O referido composto também mostrou um elevado efeito pró-oxidante quando avaliamos os níveis de estresse oxidativo através da TBARS e por meio da sonda DCFH2DA. Quando as leveduras foram tratadas por 24 h com o referido composto, observamos na microscopia de varredura o desenvolvimento de pseudo-hifas com 9 μM, a formação de fissuras em sua parede e uma forte agregação das células com 18 μM, além disso, encontramos uma intensa redução na quantidade de células e muito debris celular com 38 μM. Na microscopia de transmissão observamos estruturas vesiculares no espaço periplasmático associado a grânulos eletrondensos, os quais também foram vistos associados a parede celular, quando tratadas por 3h com 40 μM. No tratamento por 24h com 60 μM observamos a referida estrutura granular eletrondensa no citoplasma envolta por membrana, uma grande quantidade destas estruturas no espaço citoplasmático e associado a parede da célula, além disso, também observamos trechos de membrana associado a estas estruturas no espaço extracelular. Em conclusão, a atividade antifúngica e o índice de seletividade do RPC contra uma cepa resistente é consideravelmente interessante devido as suas possibilidades de aplicações na descoberta de novos antifúngicos
Candidiasis is an opportunistic infection caused by several species of fungi of the genus Candida, often found is the microbiota, on the skin, gastrointestinal tract and mucous cavities of the human beings birth. The incidence of systemic fungal infections have increased considerably in recent decades due to the large number of AIDS patients, the large number of transplants and chronic conditions such as cancer, prolonged therapy promotes the selection of resistant pathogen with immunosuppressant and corticosteroid agents. Also prolonged exposure azole antifungals to make them strong candidates for patients resistance. In the present study we evaluated the antifungal activity of Ruthenium-pyrocatechol complex (RPC) against a clinical isolate of Candida tropicalis resistant to fluconazole. The methodology for susceptibility testing was in accordance with the M27-A3 document of there National Committee for Clinical Laboratory Standards (NCCLS, 2008). Splenocytes from Balb/c mice were obtained aseptically to evaluate the cytotoxicity of the compound to mammalian cells. Oxidative stress caused by the compound was assessed by reaction to thiobarbituric acid (TBARS) and fluorescence assays with the probe diclorodihidrofluoresceína diacetate (DCFH2DA). The Calcofluor White was used to evaluate the integrity of the cell wall. The ultrastructural analysis was performed by scanning and transmission electron microscopy. The results for the antifungal activity tests were analyzed using ANOVA and pos-test Dunnett test statistic. The results for the tests of antifungal activity of the RPC showed a 50% inhibitory concentration (IC50) of 20.3 μM while in splenocytes the 50% effective concentration was 325 μM showing a selectivity index of 16. The compound also showed that a high pro-oxidant effect when evaluated levels of oxidative stress by TBARS and through DCFH2DA staining. When yeast cells were treated for 24 h with this probe, in scanning microscopy we observed the development of pseudohyphae 9 μM, the formation of cracks on their fungal walls and in these cell aggregation with 18 μM furthermore found a remarkable reduction in the number of cells, and cell debris with 38 μM. In transmission microscopy vesicular structures observed in the periplasmic space associated with electrondense granules, which were also seen associated with the cell wall, when there cells were treated for 3 h with 40 μM. In the treatment for 24h with 60 μM observed that the grain structure in the clusters in periplasmic, a large amount of these structures in the cytoplasmic space and associated with the cell wall, moreover, we also observe membrane portions associated with these structures in the extracellular space. In conclusion, the antifungal activity and the selectivity index RPC against a resistant strain is pretty interesting because of its possible applications in the discovery of new antifungal agents.

Le complexe RSC est un des facteurs de remodelage de la chromatine capables de lever la barrière nucléosomale notamment lors de la transcription. Ce processus est effectué chez les eucaryotes par trois ARN polymérases (pol). Nous avons montré que le complexe RSC interagit avec les pol I et III. La protéine Rsc4 interagit par son domaine C-terminal avec la protéine ABC27, commune aux trois ARN polymérases. Nous avons isolé une mutation de la sous-unité Rsc4 qui abolit cette interaction. Les profils d'expression génomiques, établis par puces à ADN, ont permis de caractériser ses effets sur la transcription par la pol II. Curieusement, la majorité des gènes induits sont répartis sur le chromosome XII de manière non polaire. La présence de l'ADN ribosomique sur ce chromosome suggère un lien avec ce comportement particulier. Par ailleurs, la maturation de l'ARN 35S, transcrit par la pol I, est altérée, mais nous n'avons pas pu caractériser des défauts de transcription par les pol I et III
The RSC complex is one of the chromatin remodeling complexes that helps the transcripiton machinery to overcome the nucleosomal barrier. Eukaryotic transcription is carried out by three RNA polymerases. We have demonstrated that RSC complex interacts with pol I and III. The Rsc4 protein interacts by its C-teminal domain with the ABC27 protein, a subunit shared by the three eukaryotic RNA polymerases, We have isolated a mutation in the Rsc4 subunit that ablolish thi interaction. We performed genome profiling experiments using DNA microarrays to characterise pol II transcription defects. Surprisingly, the vast majority of the upregulated genes localised to the chromosome XII, spreading all along in a non-polar manner. We propose that the presence of the rDNA cluster on chromosome XII could be responsible for this peculiar transcriptional pattern. We have seen defects in the 35S RNA maturation but have been unable to clearly establish defects on pol I and pol III transcription

RFC plays an essential role in DNA replication by loading the sliding clamp PCNA onto DNA in order to tether DNA polymerase δ to DNA. RFC consists of five subunits, one large subunit and four small subunits. The large subunit of RFC contains an extended C-­terminal domain that is not present in the small subunits and whose function remains unknown. In addition to RFC, eukaryotic cells contain two more putative PCNA loaders known as RLCs. These other PCNA loaders have similar structures to RFC and contains the RFC small subunits, however the large subunit is replaced with a different protein, either Elg1 or Ctf18. The function of the three PCNA loaders is not clear. In this work the function of the Rfcl C-terminal domain (CTD) was examined. The analysis of an Rfcl CTD deletion mutant showed that the domain is essential for cell viability. rfcl-44, a temperature-sensitive mutant with a mutation in the C-terminal domain, displayed sensitivity to DNA damaging agents, abnormal chromosome structure and a synthetic lethal phenotype when combined with DNA replication mutants. rfc5 mutants were isolated as suppressors of rfcl-44 suggesting that the defect in rfcl-44 may be in the Rfcl-Rfc5 interaction. Ctf18, Dccl and Ctf8, components of Ctf18-RLC, were required for the viability of rfc1­-44 whilst Elg1 was not. Deletion of Elg1 restored the viability of rfc1-44 ctf18Δ double mutant cells, suggesting that Elg1 plays a negative role. The negative role of Elg1 was confirmed by over-expression of Elg1 in rfc1-44 cells showing a lethal phenotype at permissive temperature. These results suggest that RFC plays a key role in DNA replication and that Elg1-RLC and Ctf18-RLC can play negative and positive roles respectively when RFC function is impaired.

The work group in charge of editing the trauma disorders in the upcoming edition of the International Classification of Diseases (ICD-11) made several changes to the trauma criteria. Specifically, they simplified the criteria for posttraumatic stress disorder (PTSD) and added a new trauma disorder called complex PTSD (CPTSD). To assess the new and newly defined trauma disorders, Litvin, Kaminski and Riggs developed a self-report trauma measure called the Complex Trauma Inventory (CTI). Although the reliability and validity of the CTI has been supported, no empirically-derived cutoff scores exist. We determined the optimal CTI cutoff scores using receiver operating characteristic (ROC) analyses in a diverse sample of 82 participants who experienced trauma and were recruited from an inpatient trauma unit, student veteran organizations, and university classrooms. We used the Clinician-Administered Interview for Trauma Disorders (CAIT) to diagnose the presence of an ICD-11 trauma disorder, and we correlated the results of the CAIT with the Clinician-Administered PTSD Scale for the DSM-5 to establish the convergent validity of the CAIT, r = .945, p < .001. For the ROC analyses, the CTI was used as the index test and the CAIT was used as the criterion test. The area under the curve (AUC) analyses indicated good to excellent effect sizes, AUC = .879 to .904. We identified two sets of cutoff scores for the CTI: the first set prioritized the sensitivity of the CTI scores and ranged from .884 to .962; the second set prioritized the specificity of the CTI scores and the false-positive scores (1-specificity) ranged from .054 to .143. Our study enhanced the utility of the CTI and addressed another need in the trauma field by developing a structured clinical interview (CAIT) that can be used to diagnose the ICD-11 trauma disorders.

AbstractChromatin architecture has a profound effect on the gene expression in eukaryotes. It is constantly modulated in the cells in response to different stress condition and during the normal physiological process in the cell. The chromatin is also modulated during the cell growth and division, where several proteins involved during the cell cycle are synthesized, and hence the gene expression profile and chromatin state of an actively dividing cell differ from that of a resting cell in G0 state. Candida albicans, which is a harmless commensal in human host and an opportunistic fungal pathogen also show dynamic chromatin architecture, and this is facilitated by the several epigenetic determinants, which modulate the chromatin architecture. In this context, RSC (Remodel the structure of chromatin) complex in C. albicans is previously shown to be crucial for cell viability and to carry out several DNA templated events, like kinetochore function and cohesion enrichment. To correlate the role of RSC in kinetochore function with the chromatin architecture at centromeric and non-centromeric region, here we have shown that the chromatin at non-CEN7 regions shows lesser occupancy of nucleosomes in absence of Sth1 protein (catalytic component of RSC complex), which is due to the reduced binding but not due to the reduced expression of the histones.

The maritime industry is transitioning toward zero emission. To ensure compliance with future emission reduction regulations, many different alternative fuels and technology options are being investigated and evaluated. However, as these are ongoing developments, this results in varying and changing data on the performance and requirements of options. On top of that, while regulatory ambitions are aiming for increasingly larger reductions of Green House Gases (GHG) and other harmful substances, the level and details of the future regulations are unknown and subject to ongoing scientific and societal discussions. The level of uncertainty regarding regulation and technology for the energy transition can be defined as being deeply uncertain, which means uncertainty cannot be ordered in terms of possibility or occurrence. Although uncertainty is not uncommon in ship design, ship owners and designers are faced with an unprecedented level of uncertainty and require new methods to deal with this. This paper therefore investigates and compares several methods that could be used to increase the feasibility of future energy carriers in the design process, while accounting for the uncertainty in regulation and technical details of alternative fuels. Three promising methods were identified in a literature research: Firstly, Dynamic Adaptive Policy Pathways (DAPP) evaluates alternative options and develops possible pathways to compliance. Secondly, Responsive Systems Comparison (RSC) determines performance of a design in established scenarios (epoch), also allowing evaluation including retrofit (changeability). Thirdly, Robust Decision making (RDM) explores the effect of uncertainties on a pre-specified design and analyses its vulnerability. Within this paper, a first comparison is carried out by applying each method to a general cargo ship case. The goal is to better understand the usability and potential of each method for the energy transition in shipping. Each of the researched methods was shown to allow for different insights in option performance in uncertain conditions during the early design stage. With DAPP providing a global, but clear overview of the possible future pathways toward emission reduction compliance of the design, RSC giving a more detailed insight of technology options in specific scenarios (including evaluation of changeability in a scenario) and RDM allowing a more in depth research of the alternative fuel’s parameters and the circumstances under which these might comply. With each method demonstrating its own strength, future research will develop more realistic and complex designs and processes to be applied to a combination of the beneficial aspects of two or more methods.

Abstract Aerial application of dispersants are an effective means of responding to oil spills in coastal waters and the deeper waters of the Outer Continental Shelf or Gulf of Mexico. To ensure the safety of responders and nearby wildlife, a buffer area is put in place around the spilled oil to be treated, within which spraying operations are conducted. In 2015, a research project was initiated to develop a prototype Decision Support Tool (DST) designed specifically for estimating the spray drift during the aerial application of dispersants on an oil spill. In 2019, an initiative was undertaken to further develop the DST and address known data gaps in the modeling used in the prototype, expand on the aircraft included in the tool, and include a contour plot output of dispersant deposition. The DST has been designed specifically for estimating the spray drift during the aerial application of dispersants on an oil spill through the use of complex Computational Fluid Dynamics (CFD) modeling. The DST program operational space was developed based on direct input from Oil Spill Response Operators (OSROs) for ten airframes currently used in the United States for aerial response operations, including both turbo propeller and turbo fan engine types. The DST employs a database of results generated using the latest in CFD modeling technology to examine flow structures and drift effects created by various operating conditions, coupled with specific configurations of different oil spill response aircraft and their spray systems (boom and nozzle configurations). The DST uses a Response Surface Curve (RSC) for each airframe to predict the drift extent of dispersant particles and mass deposition concentration, the RSC for each airframe was derived from a database of results generated using the latest CFD modeling technology. The studies conducted to generate data for the DST RSCs provided considerable insight into the relationships between the particle dispersant behavior for different airframe types. Trends were identified in particle dispersion behavior when airframes were flown with a heavy payload (full weight) compared to lighter payload (empty weight). These trends change depending on the airframe used and, more specifically, the location and arrangement of the boom used to release the droplets relative to the location of the main wing. Change in Particle Size Distribution (PSD) was also investigated for flight operations of one airframe and the impact on the drift extent reported. The DST will provide oil spill responders with information related to the extent of any areas potentially impacted by dispersant drift. This will assist the operational control personnel in establishing setback distances, information which becomes increasingly important as a spill escalates beyond a Tier 1 response where the size of the spill, and the resources committed, become significant. In addition, the DST generates a contour plot of mass deposition at ground level based on the operational and environmental parameters input to the program, providing the user with a graphical display of where the majority of the aerial dispersant is predicted to land. While the analysis and tool development are complete, a formal peer review has not been completed at the time of the paper publication.

Abstract Aerial application of dispersants are an effective means of responding to oil spills in coastal waters and the deeper waters of the Outer Continental Shelf or Gulf of Mexico. To ensure the safety of responders and nearby wildlife, a buffer area is put in place around the spilled oil to be treated, within which spraying operations are conducted. In 2015, a research project was initiated to develop a prototype Decision Support Tool (DST) designed specifically for estimating the spray drift during the aerial application of dispersants on an oil spill. In 2019, an initiative was undertaken to further develop the DST and address known data gaps in the modeling used in the prototype, expand on the aircraft included in the tool, and include a contour plot output of dispersant deposition. The DST has been designed specifically for estimating the spray drift during the aerial application of dispersants on an oil spill through the use of complex Computational Fluid Dynamics (CFD) modeling. The DST program operational space was developed based on direct input from Oil Spill Response Operators (OSROs) for ten airframes currently used in the United States for aerial response operations, including both turbo propeller and turbo fan engine types. The DST employs a database of results generated using the latest in CFD modeling technology to examine flow structures and drift effects created by various operating conditions, coupled with specific configurations of different oil spill response aircraft and their spray systems (boom and nozzle configurations). The DST uses a Response Surface Curve (RSC) for each airframe to predict the drift extent of dispersant particles and mass deposition concentration, the RSC for each airframe was derived from a database of results generated using the latest CFD modeling technology. The studies conducted to generate data for the DST RSCs provided considerable insight into the relationships between the particle dispersant behavior for different airframe types. Trends were identified in particle dispersion behavior when airframes were flown with a heavy payload (full weight) compared to lighter payload (empty weight). These trends change depending on the airframe used and, more specifically, the location and arrangement of the boom used to release the droplets relative to the location of the main wing. Change in Particle Size Distribution (PSD) was also investigated for flight operations of one airframe and the impact on the drift extent reported. The DST will provide oil spill responders with information related to the extent of any areas potentially impacted by dispersant drift. This will assist the operational control personnel in establishing setback distances, information which becomes increasingly important as a spill escalates beyond a Tier 1 response where the size of the spill, and the resources committed, become significant. In addition, the DST generates a contour plot of mass deposition at ground level based on the operational and environmental parameters input to the program, providing the user with a graphical display of where the majority of the aerial dispersant is predicted to land. While the analysis and tool development are complete, a formal peer review has not been completed at the time of the paper publication.

During the past 25 years EDF, AREVA and CEA developed a complete set of Codes and Standards for Nuclear Power Plants inside the French Association for Nuclear Codes and Standards (AFCEN). The list of these Codes are: RCC-M (Design of PWR Mechanical Components), RSE-M (Surveillance and Operation of PWR Mechanical Components), RCC-E (Electrical Components), RCC-G (Civil Engineering), RCC-I (Fire Protection), RCC-C (Design of PWR fuel) and in connection with CEA RCC-MR (Design of HTR Mechanical Components) and RCC-Mx (Design of Experimental Reactor Mechanical Components). This paper will present major Codes for Mechanical Components and the future developments of these Codes. For all these Codes, revisions are generally done every year and are associated to: - needs for new project, - recent R&D results, - regular user requests, - field experience. For all these different disciplines, modernization and State of the Art justification have to be justified. In parallel to that all these Codes have to be used in different international projects, and need some particular developments (as specific appendices) to assure usability under different national regulations and practices.

Abstract The BECM are proposed to classify spatial four-link mechanisms according to groups such as crank-rocker, double-rocker and double-crank (drag link). The BECM can determine the feasible regions and number of the extreme values exactly and solve them simply without complex derivation and calcultion. The paper concerns itself mainly with the RSSR linkage but it can be applied also to other types such as RSSP, RSCP, RSCR etc. The method is very simple and the geometric concept is very clear. Although the graphical methods is mainly introduced in this paper, certainly, it can also be coded in computer to solve out their accurate values.

Inside the French Association for Design, Construction and Surveillance Rules of Nuclear Power Plant Components (AFCEN) a set of 7 Codes has been developed and are periodically updated. This paper will review the major improvements in the past 3 years of the mechanical component codes: RCC-M: Rules for Design and Construction of Mechanical Components for PWR Nuclear Islands RCC-MRx: Rules for Design and Construction of Mechanical Components for High Temperature, Experimental and fusion reactors RSE-M: Rules for surveillance and ISI of Mechanical Components for PWR Nuclear Islands After a general overview of the way of developments of these Codes, the paper will present the major improvements of these Codes since 2007, many of them have a complete new edition issued in 2010 or 2012. The conclusion of the paper will present the major objectives and topics of development of these Codes for new international project under the pressure of Code Harmonization by Regulators (MDEP) Standards Development Organization Board (SDO) and Industry organization (CORDEL).

Review question / Objective: This study is to assess the efficacy and safety of ketamine in treating refractory and super-refractory status epilepticus in pediatric and adult populations. Rationale: Refractory status epilepticus (RSE) is either generalized or complex partial status epilepticus (SE) that fails to respond to first and second-line therapies. Super refractory status epilepticus (SRSE) is SE that remains unresponsive despite 24 hours of therapy with general anesthesia [1, 2]. Both RSE and SRSE pose significant challenges for the managing intensivist. There exists a race against time for control of epileptic activity in the RSE/SRSE patient to preserve cortical function and reduce morbidity/mortality. However, despite the best intentions, and not uncommonly, standard frontline antiepileptic drugs (AEDs) fail to control or reduce seizure activity once seizures approach the 30-minute mark. The following review provides an analysis of ketamine in treating RSE/SRSE, focusing on the potential target population, dosing, concerns, and the role of early administration.

This deliverable focuses on the proling activities developed in the project with the partner's applications. To perform this proling activities, a couple of benchmarks were dened in collaboration with WP5. The rst benchmark is an embarrassingly parallel benchmark that performs a read and then multiple writes of the same object, with the objective of stressing the memory and storage systems and evaluate the overhead when these reads and writes are performed in parallel. A second benchmark is dened based on the Continuation Multi Level Monte Carlo (C-MLMC) algorithm. While this algorithm is normally executed using multiple levels, for the proling and performance analysis objectives, the execution of a single level was enough since the forthcoming levels have similar performance characteristics. Additionally, while the simulation tasks can be executed as parallel (multi-threaded tasks), in the benchmark, single threaded tasks were executed to increase the number of simulations to be scheduled and stress the scheduling engines. A set of experiments based on these two benchmarks have been executed in the MareNostrum 4 supercomputer and using PyCOMPSs as underlying programming model and dynamic scheduler of the tasks involved in the executions. While the rst benchmark was executed several times in a single iteration, the second benchmark was executed in an iterative manner, with cycles of 1) Execution and trace generation; 2) Performance analysis; 3) Improvements. This had enabled to perform several improvements in the benchmark and in the scheduler of PyCOMPSs. The initial iterations focused on the C-MLMC structure itself, performing re-factors of the code to remove ne grain and sequential tasks and merging them in larger granularity tasks. The next iterations focused on improving the PyCOMPSs scheduler, removing existent bottlenecks and increasing its performance by making the scheduler a multithreaded engine. While the results can still be improved, we are satised with the results since the granularity of the simulations run in this evaluation step are much ner than the one that will be used for the real scenarios. The deliverable nishes with some recommendations that should be followed along the project in order to obtain good performance in the execution of the project codes.

In this project, we studied the photosynthetic apparatus during dehydration and rehydration of the homoiochlorophyllous resurrection plant Craterostigmapumilum (retains most of the photosynthetic components during desiccation). Resurrection plants have the remarkable capability to withstand desiccation, being able to revive after prolonged severe water deficit in a few days upon rehydration. Homoiochlorophyllous resurrection plants are very efficient in protecting the photosynthetic machinery against damage by reactive oxygen production under drought. The main purpose of this BARD project was to unravel these largely unknown protection strategies for C. pumilum. In detail, the specific objectives were: (1) To determine the distribution and local organization of photosynthetic protein complexes and formation of inverted hexagonal phases within the thylakoid membranes at different dehydration/rehydration states. (2) To determine the 3D structure and characterize the geometry, topology, and mechanics of the thylakoid network at the different states. (3) Generation of molecular models for thylakoids at the different states and study the implications for diffusion within the thylakoid lumen. (4) Characterization of inter-system electron transport, quantum efficiencies, photosystem antenna sizes and distribution, NPQ, and photoinhibition at different hydration states. (5) Measuring the partition of photosynthetic reducing equivalents between the Calvin cycle, photorespiration, and the water-water cycle. At the beginning of the project, we decided to use C. pumilum instead of C. wilmsii because the former species was available from our collaborator Dr. Farrant. In addition to the original two dehydration states (40 relative water content=RWC and 5% RWC), we characterized a third state (15-20%) because some interesting changes occurs at this RWC. Furthermore, it was not possible to detect D1 protein levels by Western blot analysis because antibodies against other higher plants failed to detect D1 in C. pumilum. We developed growth conditions that allow reproducible generation of different dehydration and rehydration states for C. pumilum. Furthermore, advanced spectroscopy and microscopy for C. pumilum were established to obtain a detailed picture of structural and functional changes of the photosynthetic apparatus in different hydrated states. Main findings of our study are: 1. Anthocyan accumulation during desiccation alleviates the light pressure within the leaves (Fig. 1). 2. During desiccation, stomatal closure leads to drastic reductions in CO2 fixation and photorespiration. We could not identify alternative electron sinks as a solution to reduce ROS production. 3. On the supramolecular level, semicrystalline protein arrays were identified in thylakoid membranes in the desiccated state (see Fig. 3). On the electron transport level, a specific series of shut downs occur (summarized in Fig. 2). The main events include: Early shutdown of the ATPase activity, cessation of electron transport between cyt. bf complex and PSI (can reduce ROS formation at PSI); at higher dehydration levels uncoupling of LHCII from PSII and cessation of electron flow from PSII accompanied by crystal formation. The later could severe as a swift PSII reservoir during rehydration. The specific order of events in the course of dehydration and rehydration discovered in this project is indicative for regulated structural transitions specifically realized in resurrection plants. This detailed knowledge can serve as an interesting starting point for rationale genetic engineering of drought-tolerant crops.

Une grande partie des enfants non scolarisés dans le monde vivent dans des zones de conflit. Un des principaux défis dans l’élimination des obstacles à l’instruction des filles marginalisées dans ces contextes réside dans la compréhension des formes multiples et croisées de la marginalisation et des changements dans leurs dynamiques au cours des conflits violents. Les recherches menées en République démocratique du Congo (RDC) dans le cadre du projet d’éducation REALISE identifient des éléments clés à prendre en compte dans les programmes d’éducation destinés aux filles marginalisées dans les zones de conflit, tels que l’éducation inclusive pour les filles et les garçons, les liens entre l’éducation et la consolidation de la paix, et les activités périscolaires favorisant les liens sociaux.

Comprehensive park management to fulfill the NPS mission requires an accurate inventory of the geologic features of a park unit, but Comprehensive park management to fulfill the NPS mission requires an accurate inventory of the geologic features of a park unit, but park managers may not have the needed information, geologic expertise, or means to complete such an undertaking; therefore, the Geologic Resources Inventory (GRI) provides information and resources to help park managers make decisions for visitor safety, planning and protection of infrastructure, and preservation of natural and cultural resources. Information in the GRI report may also be useful for interpretation. park managers may not have the needed information, geologic expertise, or means to complete such an undertaking; therefore, the Geologic Resources Inventory (GRI) provides information and resources to help park managers make decisions for visitor safety, planning and protection of infrastructure, and preservation of natural and cultural resources. Information in the GRI report may also be useful for interpretation. This report synthesizes discussions from a scoping meeting for Redwood National and State Parks (referred to as the “parks” throughout this report) held in 2004 and a follow-up conference call in 2019. Two GRI–compiled GIS data sets of the geology and geohazards of the parks are the principal deliverables of the GRI. The GRI GIS data are available on the GRI publications website http://go.nps.gov/gripubs and through the NPS Integrated Resource Management Applications (IRMA) portal https://irma.nps.gov/App/Portal/Home. Enter “GRI” as the search text and select a park from the unit list. Writing of this report was based on those data and the interpretations of the source map authors (see “GRI Products” and “Acknowledgements”). A geologic map poster illustrates the geology GRI GIS data set and serves as a primary figure for this GRI report. No poster was prepared for the geohazards GRI GIS data set. Additionally, figure 7 of this report illustrates the locations of the major geologic features in the parks. Unlike the poster, which is divided into a northern and southern portion to show detail while accommodating the parks’ length, figure 7 is a single-page, simplified map. The features labeled on figure 7 are discussed in the “Geologic History, Features, and Processes” chapter. To provide a context of geologic time, this report includes a geologic time scale (see "Geologic History, Features, and Processes"). The parks’ geologic story encompasses 200 million years, starting in the Jurassic Period. Following geologic practice, the time scale is set up like a stratigraphic column, with the oldest units at the bottom and the youngest units at the top. Organized in this manner, the geologic time scale table shows the relative ages of the rock units that underlie the parks and the unconsolidated deposits that lie at the surface. Reading the “Geologic Event” column in the table, from bottom to top, will provide a chronologic order of the parks’ geologic history. The time scale includes only the map units within the parks that also appear on the geologic map poster; that is, map units of the geohazards data are not included. Geology is a complex science with many specialized terms. This report provides definitions of geologic terms at first mention, typically in parentheses following the term. Geologic units in the GRI GIS data are referenced in this report using map unit symbols; for example, map unit KJfrc stands for the Cretaceous (K) and Jurassic (J) Franciscan Complex (f), Redwood Creek schist (rc), which underlies a portion of the Redwood Creek watershed (see “GRI Products”).

This deliverable presents the activities performed on the ExaQUte project task 4.5 Development of interface to fast local storage. The activities have been focused in two aspects: reduction of the storage space used by applications and design and implementation of an interface that optimizes the use of fast local storage by MPI simulations involved in the project applications. In the rst case, for one of the environments involved in the project (PyCOMPSs) the default behavior is to keep all intermediate les until the end of the execution, in case these les are reused later by any additional task. In the case of the other environment (HyperLoom), all les are deleted by default. To unify these two behaviours, the calls \delete object" and \detele le"have been added to the API and a ag \keep" that can be set to true to keep the les and objects that maybe needed later on. We are reporting results on the optimization of the storage needed by a small case of the project application that reduces the storage needed from 25GB to 350MB. The second focus has been on the de nition of an interface that enables the optimization of the use of local storage disk. This optimization focuses on MPI simulations that may be executed across multiple nodes. The added annotation enables to de ne access patters of the processes in the MPI simulations, with the objective of giving hints to the runtime of where to allocate the di erent MPI processes and reduce the data transfers, as well as the storage usage.

Objetivo: analisar o processo de implementação e conformação dos modos de produção do cuidado da política de Rede de Atenção às Urgências e Emergências (RUE), em duas regiões de saúde: Campinas (SP) e Passo Fundo (RS). Procura, ainda, identificar analisadores da produção micropolítica presentes no processo de pactuação e implementação da RUE nessas regiões; analisar as possíveis mudanças no processo de gestão a partir da implementação das RUE; e caracterizar as transformações ocorridas nos modos de produção do cuidado em saúde a partir da implementação das RUE. Metodologia: a pesquisa tem caráter qualitativo, com abordagem micropolítica, e caracteriza-se como estudo de caso e foi desenvolvida através de revisão de literatura, análise de documentos oficiais, coleta de depoimentos de gestores municipais e estaduais, entrevistas narrativas com usuários, e entrevistas em profundidade com gerentes de serviços. No total, foram entrevistados 61 sujeitos. A análise teve como referência a ‘Abordagem do Ciclo de Políticas’. A pesquisa foi desenvolvida por pesquisadores dos programas de Pós-Graduação em Saúde Coletiva da Escola Paulista de Medicina – Universidade Federal de São Paulo (Unifesp-EPM) e da Imed-Faculdade Meridional de Passo Fundo (RS), com apoio de gestores regionais e municipais de saúde e dos Conselhos de Secretários Municipais de Saúde (COSEMS) dos estados de São Paulo e do Rio Grande do Sul. Resultados: Os principais resultados apontam para uma política pública de caráter plural e multifacetado, formulada a partir de diversas influências sociais, econômicas, políticas e teóricas, que expressa como intencionalidade a ampliação do acesso e o cuidado integral em situações de urgência e emergência em saúde. No contexto da prática, apesar da ênfase aos aspectos organizativos e ao financiamento, é observada a política ‘em cena’ onde podem ser identificadas ações de gestão e de produção de cuidado induzidas pela política, mantidas apesar da política e produzidas para além da política da RUE. A relação entre a política oficial e a ação micropolítica dos gestores, tornou-a uma produção singular no campo da governança regional; a necessidade de autonomia dos usuários e a dimensão do cuidado familiar apontam para caminhos na construção da integralidade; há evidências de produções vivas induzidas pela política que qualificam o cuidado, embora iniquidades sejam mantidas ou produzidas; e, a necessidade de articulação entre os componentes em rede, embora evocada, traduz-se em conexões frágeis e não regulares. Considerações Finais: A compreensão dos complexos processos que envolvem as políticas públicas de saúde, os interesses e poderes que as atravessam, tem potencial para fortalecer os atores implicados com a luta pela promoção da equidade em saúde e pela justiça social.

High Grain Protein Content (GPC) is a desirable trait in breadmaking and pasta wheat varieties because of its positive effects on quality and nutritional value. However, selection for GPC is limited by our poor understanding of the genes involved in the accumulation of protein in the grain. The long-term goal of this project is to provide a better understanding of the genes controlling GPC in wheat. The specific objectives of this project were: a) to develop a high-density genetic map of the GPC gene in tetraploid wheat, b) to construct a T. turgidum Bacterial Artificial Chromosome (BAC) library, c) to construct a physical map of the GPC gene and identify a candidate for the GPC gene. A gene with a large effect on GPC was detected in Triticum turgidum var. dicoccoides and was previously mapped in the short arm of chromosome 6B. To define better the position of the Gpc-B1 locus we developed homozygous recombinant lines with recombination events within the QTL region. Except for the 30-cM region of the QTL these RSLs were isogenic for the rest of the genome minimizing the genetic variability. To minimize the environmental variability the RSLs were characterized using 10 replications in field experiments organized in a Randomized Complete Block Design, which were repeated three times. Using this strategy, we were able to map this QTL as a single Mendelian locus (Gpc-B1) on a 2.6-cM region flanked by RFLP markers Xcdo365 and Xucw67. All three experiments showed that the lines carrying the DIC allele had an average absolute increase in GPC of 14 g/kg. Using the RFLP flanking markers, we established the microcolinearity between a 2.l-cM region including the Gpc-B1 gene in wheat chromosome 6BS and a 350-kb region on rice chromosome 2. Rice genes from this region were used to screen the Triticeae EST collection, and these ESTs were used to saturate the Gpc-B1 region with molecular markers. With these new markers we were able to map the Gpc-B1 locus within a 0.3-cM region flanked by PCR markers Xucw83 and Xucw71. These flanking markers defined a 36-kb colinear region with rice, including one gene that is a potential candidate for the Gpc-B1 gene. To develop a physical map of the Gpc-B1 region in wheat we first constructed a BAC library of tetraploid wheat, from RSL#65 including the high Gpc-B1 allele. We generated half- million clones with an average size of l3l-kb (5.1 X genome equivalents for each of the two genomes). This coverage provides a 99.4% probability of recovering any gene from durum wheat. We used the Gpc-BI flanking markers to screen this BAC library and then completed the physical map by chromosome walking. The physical map included two overlapping BACs covering a region of approximately 250-kb, including two flanking markers and the Gpc-B1 gene. Efforts are underway to sequence these two BACs to determine if additional wheat genes are present in this region. Weare also developing new RSLs to further dissect this region. We developed PCR markers for flanking loci Xucw79andXucw71 to facilitate the introgression of this gene in commercial varieties by marker assisted selection (httQ://maswheat.ucdavis.edu/ orotocols/HGPC/index.hlm). Using these markers we introgressed the Gpc-B1 gene in numerous pasta and common wheat breeding lines.

Cette étude porte sur une des innovations contemporaines liées à l’économie de la publication scientifique : les accords dits transformants, un objet relativement circonscrit au sein des relations entre consortiums de bibliothèques et éditeurs scientifiques, et temporellement situé entre 2015 et 2020. Ce type d’accords a pour objectif affiché d’organiser la transition du modèle traditionnel de l’abonnement à des revues (souvent proposées par regroupements thématiques ou collections) vers celui de l’accès ouvert en opérant une réaffectation des budgets qui y sont consacrés. Notre travail d’analyse sociologique constitue une première étude systématique de cet objet, fondée sur la recension de 197 accords. Le corpus ainsi constitué inclut des accords caractérisés par la coprésence d’une composante d’abonnement et d’une composante de publication en accès ouvert, même minimale (« jetons » de publication offerts, réduction sur les APC...). En conséquence, ont été exclus de l’analyse les accords portant uniquement sur du financement centralisé de publication en accès ouvert, que ce soit avec des éditeurs ne proposant que des revues avec paiement par l’auteur (PLOS, Frontiers, MDPI...) ou des éditeurs dont une partie du catalogue est constitué de revues en accès ouvert. L’accord le plus ancien de notre corpus a été signé en 2010, les plus récents en 2020 – les accords ne commençant qu’en 2021, même annoncés au cours de l’étude, n’ont pas été retenus. Plusieurs résultats se dégagent de notre analyse. Tout d’abord, on note une grande diversité des acteurs impliqués avec 22 pays et 39 éditeurs, même si certains consortiums (Pays-Bas, Suède, Autriche, Allemagne) et éditeurs (CUP, Elsevier, RSC, Springer) en ont signé beaucoup plus que d’autres. Ensuite, la durée des accords, comprise entre une et six années, révèle une distribution très inégalitaire, avec plus de la moitié des accords (103) signés pour 3 ans, ainsi qu’une faible proportion pour 4 ans ou plus (22 accords). Enfin, en dépit d’appels répétés à la transparence, moins de la moitié des accords (96) ont un texte accessible au moment de cette étude, sans qu’on puisse observer une tendance récente à une plus grande disponibilité. L’analyse montre également des degrés d’ouverture très variables, allant d’une simple information sur le répertoire ESAC en passant par la mise à disposition d’un format annotable jusqu’à l’attribution d’un DOI et d’une licence de réutilisation (CC-BY), en incluant le détail des sommes monétaires. Parmi les 96 accords disponibles, dont 47 signés en 2020, 62 ont fait l’objet d’une analyse en profondeur. C’est à notre connaissance la première analyse à cette échelle, sur un type de matériel non seulement inédit, mais qui était auparavant soumis à des clauses de confidentialité. Fondée sur une lecture minutieuse, l’étude décrit de manière fine leurs propriétés, depuis la matérialité du document jusqu’aux formules financières, en passant par leur morphologie et l’ensemble des droits et devoirs des parties. Les contenus des accords sont donc analysés comme une collection dont nous cherchons à déterminer les points communs et les variations, à travers des codages explicites sur certaines de leurs caractéristiques. L’étude pointe également des incertitudes, et notamment leur caractère « transitionnel », qui demeure fortement discuté. D’un point de vue morphologique, les accords montrent une grande diversité en matière de taille (de 7 à 488 pages) et de structure. Néanmoins, par définition, ils articulent tous deux objets essentiels : d’une part, les conditions de réalisation d’une lecture d’articles de revues, sous forme d’abonnement, mêlant des préoccupations d’accès et de sécurité ; d’autre part, les modalités de publication en accès ouvert, articulant la gestion d’un nouveau type de workflow à toute une série d’options possibles. Parmi ces options, mentionnons notamment le périmètre des revues considérées (hybrides et/ou accès ouvert), les licences disponibles, le degré d’obligation de cette publication, les auteurs éligibles ou le volume d’articles publiables. L’un des résultats les plus importants de cette analyse approfondie est la mise au jour d’un découplage presque complet, au sein même des accords, entre l’objet abonnement et l’objet publication. Bien entendu, l’abonnement est systématiquement configuré dans un monde fermé, soumis à paiement qui déclenche des séries d’identification des circulations légitimes tant du contenu informationnel que des usagers. Il insiste notamment sur les interdictions de réutilisation ou même de copie des articles scientifiques. À l’opposé, la publication en accès ouvert est attachée à un monde régi par l’accès gratuit au contenu, ce qui induit des préoccupations de gestion du workflow et des modalités d’accessibilité. De plus, les différents éléments constitutifs de ces objets contractuels ne sont pas couplés : d’un côté, les lecteurs sont constitués de l’ensemble des membres des institutions abonnées, de l’autre, seuls les auteurs correspondants (« corresponding authors ») sont concernés ; les listes de revues accessibles à la lecture et celles réservées à la publication en accès ouvert sont le plus souvent distinctes ; les workflows ont des objectifs et des organisations matérielles totalement différentes, etc. L’articulation entre les deux objets contractuels relève uniquement d’une formule de distribution financière qui, outre des combinaisons particulières entre l’un et l’autre, permet d’attribuer des étiquettes distinctes aux accords (offset agreement, publish & read, read & publish, read & free articles, read & discount). Au-delà de cette distribution, l’étude des arrangements financiers montre une gamme de dispositions allant d’une prévisibilité budgétaire totale, donc identique aux accords d’abonnement antérieurs, à une incertitude sur le volume de publication ou sur le montant définitif des sommes échangées. Les modalités concrètes de calcul des montants associés à la publication en accès ouvert sont relativement variées. S’il existe effectivement des formules récurrentes (volume d’articles multiplié par un prix individuel, reprise de la moyenne des sommes totales d’APC des années précédentes...), le calcul des sommes en jeu est toujours le résultat d’une négociation singulière entre un consortium et un éditeur scientifique, et aboutit parfois à des formules originales et complexes. À ce titre, l’espace des possibles en matière de formules financières n’est jamais totalement clos. Par ailleurs, la volonté des consortiums d’opérer une « transformation » de leurs accords vers la publication à coût constant renvoie à des définitions diversifiées du « coût » (inclusion ou non des dépenses d’APC préexistantes) et de la constance (admission ou pas d’une « inflation » à 2 ou 3%). De plus, nous n’avons observé aucune disposition contractuelle permettant d’anticiper les sommes en jeu au-delà de l’horizon temporel de l’accord courant. La grande diversité des accords provient d’une part des conditions initiales des relations entre consortiums et éditeurs scientifiques – les sommes dépensées en abonnement étant le point de départ des nouveaux accords –, d’autre part des objectifs de chaque partie. Même si cette étude excluait volontairement les négociations, les accords portent des traces de ces objectifs. Ainsi, de nombreux accords sont de nature explicitement expérimentale, quand certains visent un contrôle budgétaire strict, ou d’autres ambitionnent, dans la période plus récente, la publication du plus grand nombre possible d’articles en accès ouvert. C’est dans ce dernier cas qu’on touche à l’ambiguïté des attentes générales sur les accords transformants. En effet, pour les consortiums, la dimension « transformante » consiste essentiellement à transférer les sommes traditionnellement allouées à l’abonnement vers la publication en accès ouvert. Mais l’objectif n’est jamais de transformer le modèle économique des revues, c'est-à-dire de faire basculer des revues sous abonnement ou hybrides en revues entièrement en accès ouvert. D’ailleurs, aucune clause ne vise une telle fin – à l’exception du modèle d’accord proposé par l’éditeur ACM. Du côté des éditeurs, et notamment de Springer, le caractère cumulatif des accords nationaux passés vise à projeter un monde de la publication où l’accès ouvert devient de fait quantitativement très dominant, sans pour autant modifier de manière pérenne le modèle économique de leurs revues. Notre étude montre que les accords transformants actuels ne permettent pas d’assurer de manière durable une transition de l’économie de la publication vers l’accès ouvert, dans la mesure où ils n’offrent pas de garantie sur le contrôle des dépenses ni sur la pérennité de l’ouverture des contenus. L’avenir des relations entre consortium et éditeur demeure largement indéterminé.Cette étude porte sur une des innovations contemporaines liées à l’économie de la publication scientifique : les accords dits transformants, un objet relativement circonscrit au sein des relations entre consortiums de bibliothèques et éditeurs scientifiques, et temporellement situé entre 2015 et 2020. Ce type d’accords a pour objectif affiché d’organiser la transition du modèle traditionnel de l’abonnement à des revues (souvent proposées par regroupements thématiques ou collections) vers celui de l’accès ouvert en opérant une réaffectation des budgets qui y sont consacrés. Notre travail d’analyse sociologique constitue une première étude systématique de cet objet, fondée sur la recension de 197 accords. Le corpus ainsi constitué inclut des accords caractérisés par la coprésence d’une composante d’abonnement et d’une composante de publication en accès ouvert, même minimale (« jetons » de publication offerts, réduction sur les APC...). En conséquence, ont été exclus de l’analyse les accords portant uniquement sur du financement centralisé de publication en accès ouvert, que ce soit avec des éditeurs ne proposant que des revues avec paiement par l’auteur (PLOS, Frontiers, MDPI...) ou des éditeurs dont une partie du catalogue est constitué de revues en accès ouvert. L’accord le plus ancien de notre corpus a été signé en 2010, les plus récents en 2020 – les accords ne commençant qu’en 2021, même annoncés au cours de l’étude, n’ont pas été retenus. Plusieurs résultats se dégagent de notre analyse. Tout d’abord, on note une grande diversité des acteurs impliqués avec 22 pays et 39 éditeurs, même si certains consortiums (Pays-Bas, Suède, Autriche, Allemagne) et éditeurs (CUP, Elsevier, RSC, Springer) en ont signé beaucoup plus que d’autres. Ensuite, la durée des accords, comprise entre une et six années, révèle une distribution très inégalitaire, avec plus de la moitié des accords (103) signés pour 3 ans, ainsi qu’une faible proportion pour 4 ans ou plus (22 accords). Enfin, en dépit d’appels répétés à la transparence, moins de la moitié des accords (96) ont un texte accessible au moment de cette étude, sans qu’on puisse observer une tendance récente à une plus grande disponibilité. L’analyse montre également des degrés d’ouverture très variables, allant d’une simple information sur le répertoire ESAC en passant par la mise à disposition d’un format annotable jusqu’à l’attribution d’un DOI et d’une licence de réutilisation (CC-BY), en incluant le détail des sommes monétaires. Parmi les 96 accords disponibles, dont 47 signés en 2020, 62 ont fait l’objet d’une analyse en profondeur. C’est à notre connaissance la première analyse à cette échelle, sur un type de matériel non seulement inédit, mais qui était auparavant soumis à des clauses de confidentialité. Fondée sur une lecture minutieuse, l’étude décrit de manière fine leurs propriétés, depuis la matérialité du document jusqu’aux formules financières, en passant par leur morphologie et l’ensemble des droits et devoirs des parties. Les contenus des accords sont donc analysés comme une collection dont nous cherchons à déterminer les points communs et les variations, à travers des codages explicites sur certaines de leurs caractéristiques. L’étude pointe également des incertitudes, et notamment leur caractère « transitionnel », qui demeure fortement discuté. D’un point de vue morphologique, les accords montrent une grande diversité en matière de taille (de 7 à 488 pages) et de structure. Néanmoins, par définition, ils articulent tous deux objets essentiels : d’une part, les conditions de réalisation d’une lecture d’articles de revues, sous forme d’abonnement, mêlant des préoccupations d’accès et de sécurité ; d’autre part, les modalités de publication en accès ouvert, articulant la gestion d’un nouveau type de workflow à toute une série d’options possibles. Parmi ces options, mentionnons notamment le périmètre des revues considérées (hybrides et/ou accès ouvert), les licences disponibles, le degré d’obligation de cette publication, les auteurs éligibles ou le volume d’articles publiables. L’un des résultats les plus importants de cette analyse approfondie est la mise au jour d’un découplage presque complet, au sein même des accords, entre l’objet abonnement et l’objet publication. Bien entendu, l’abonnement est systématiquement configuré dans un monde fermé, soumis à paiement qui déclenche des séries d’identification des circulations légitimes tant du contenu informationnel que des usagers. Il insiste notamment sur les interdictions de réutilisation ou même de copie des articles scientifiques. À l’opposé, la publication en accès ouvert est attachée à un monde régi par l’accès gratuit au contenu, ce qui induit des préoccupations de gestion du workflow et des modalités d’accessibilité. De plus, les différents éléments constitutifs de ces objets contractuels ne sont pas couplés : d’un côté, les lecteurs sont constitués de l’ensemble des membres des institutions abonnées, de l’autre, seuls les auteurs correspondants (« corresponding authors ») sont concernés ; les listes de revues accessibles à la lecture et celles réservées à la publication en accès ouvert sont le plus souvent distinctes ; les workflows ont des objectifs et des organisations matérielles totalement différentes, etc. L’articulation entre les deux objets contractuels relève uniquement d’une formule de distribution financière qui, outre des combinaisons particulières entre l’un et l’autre, permet d’attribuer des étiquettes distinctes aux accords (offset agreement, publish & read, read & publish, read & free articles, read & discount). Au-delà de cette distribution, l’étude des arrangements financiers montre une gamme de dispositions allant d’une prévisibilité budgétaire totale, donc identique aux accords d’abonnement antérieurs, à une incertitude sur le volume de publication ou sur le montant définitif des sommes échangées. Les modalités concrètes de calcul des montants associés à la publication en accès ouvert sont relativement variées. S’il existe effectivement des formules récurrentes (volume d’articles multiplié par un prix individuel, reprise de la moyenne des sommes totales d’APC des années précédentes...), le calcul des sommes en jeu est toujours le résultat d’une négociation singulière entre un consortium et un éditeur scientifique, et aboutit parfois à des formules originales et complexes. À ce titre, l’espace des possibles en matière de formules financières n’est jamais totalement clos. Par ailleurs, la volonté des consortiums d’opérer une « transformation » de leurs accords vers la publication à coût constant renvoie à des définitions diversifiées du « coût » (inclusion ou non des dépenses d’APC préexistantes) et de la constance (admission ou pas d’une « inflation » à 2 ou 3%). De plus, nous n’avons observé aucune disposition contractuelle permettant d’anticiper les sommes en jeu au-delà de l’horizon temporel de l’accord courant. La grande diversité des accords provient d’une part des conditions initiales des relations entre consortiums et éditeurs scientifiques – les sommes dépensées en abonnement étant le point de départ des nouveaux accords –, d’autre part des objectifs de chaque partie. Même si cette étude excluait volontairement les négociations, les accords portent des traces de ces objectifs. Ainsi, de nombreux accords sont de nature explicitement expérimentale, quand certains visent un contrôle budgétaire strict, ou d’autres ambitionnent, dans la période plus récente, la publication du plus grand nombre possible d’articles en accès ouvert. C’est dans ce dernier cas qu’on touche à l’ambiguïté des attentes générales sur les accords transformants. En effet, pour les consortiums, la dimension « transformante » consiste essentiellement à transférer les sommes traditionnellement allouées à l’abonnement vers la publication en accès ouvert. Mais l’objectif n’est jamais de transformer le modèle économique des revues, c'est-à-dire de faire basculer des revues sous abonnement ou hybrides en revues entièrement en accès ouvert. D’ailleurs, aucune clause ne vise une telle fin – à l’exception du modèle d’accord proposé par l’éditeur ACM. Du côté des éditeurs, et notamment de Springer, le caractère cumulatif des accords nationaux passés vise à projeter un monde de la publication où l’accès ouvert devient de fait quantitativement très dominant, sans pour autant modifier de manière pérenne le modèle économique de leurs revues. Notre étude montre que les accords transformants actuels ne permettent pas d’assurer de manière durable une transition de l’économie de la publication vers l’accès ouvert, dans la mesure où ils n’offrent pas de garantie sur le contrôle des dépenses ni sur la pérennité de l’ouverture des contenus. L’avenir des relations entre consortium et éditeur demeure largement indéterminé.

This report is one of a series of reports documenting the methods and findings of a multi-year, multi-disciplinary project coordinated by the Pacific Earthquake Engineering Research Center (PEER) and funded by the California Earthquake Authority (CEA). The overall project is titled “Quantifying the Performance of Retrofit of Cripple Walls and Sill Anchorage in Single-Family Wood-Frame Buildings,” henceforth referred to as the “PEER–CEA Project.” The overall objective of the PEER–CEA Project is to provide scientifically based information (e.g., testing, analysis, and resulting loss models) that measure and assess the effectiveness of seismic retrofit to reduce the risk of damage and associated losses (repair costs) of wood-frame houses with cripple wall and sill anchorage deficiencies as well as retrofitted conditions that address those deficiencies. Tasks that support and inform the loss-modeling effort are: (1) collecting and summarizing existing information and results of previous research on the performance of wood-frame houses; (2) identifying construction features to characterize alternative variants of wood-frame houses; (3) characterizing earthquake hazard and ground motions at representative sites in California; (4) developing cyclic loading protocols and conducting laboratory tests of cripple wall panels, wood-frame wall subassemblies, and sill anchorages to measure and document their response (strength and stiffness) under cyclic loading; and (5) the computer modeling, simulations, and the development of loss models as informed by a workshop with claims adjustors. This report is a product of Working Group 3 (WG3), Task 3.1: Selecting and Scaling Ground-motion records. The objective of Task 3.1 is to provide suites of ground motions to be used by other working groups (WGs), especially Working Group 5: Analytical Modeling (WG5) for Simulation Studies. The ground motions used in the numerical simulations are intended to represent seismic hazard at the building site. The seismic hazard is dependent on the location of the site relative to seismic sources, the characteristics of the seismic sources in the region and the local soil conditions at the site. To achieve a proper representation of hazard across the State of California, ten sites were selected, and a site-specific probabilistic seismic hazard analysis (PSHA) was performed at each of these sites for both a soft soil (Vs30 = 270 m/sec) and a stiff soil (Vs30=760 m/sec). The PSHA used the UCERF3 seismic source model, which represents the latest seismic source model adopted by the USGS [2013] and NGA-West2 ground-motion models. The PSHA was carried out for structural periods ranging from 0.01 to 10 sec. At each site and soil class, the results from the PSHA—hazard curves, hazard deaggregation, and uniform-hazard spectra (UHS)—were extracted for a series of ten return periods, prescribed by WG5 and WG6, ranging from 15.5–2500 years. For each case (site, soil class, and return period), the UHS was used as the target spectrum for selection and modification of a suite of ground motions. Additionally, another set of target spectra based on “Conditional Spectra” (CS), which are more realistic than UHS, was developed [Baker and Lee 2018]. The Conditional Spectra are defined by the median (Conditional Mean Spectrum) and a period-dependent variance. A suite of at least 40 record pairs (horizontal) were selected and modified for each return period and target-spectrum type. Thus, for each ground-motion suite, 40 or more record pairs were selected using the deaggregation of the hazard, resulting in more than 200 record pairs per target-spectrum type at each site. The suites contained more than 40 records in case some were rejected by the modelers due to secondary characteristics; however, none were rejected, and the complete set was used. For the case of UHS as the target spectrum, the selected motions were modified (scaled) such that the average of the median spectrum (RotD50) [Boore 2010] of the ground-motion pairs follow the target spectrum closely within the period range of interest to the analysts. In communications with WG5 researchers, for ground-motion (time histories, or time series) selection and modification, a period range between 0.01–2.0 sec was selected for this specific application for the project. The duration metrics and pulse characteristics of the records were also used in the final selection of ground motions. The damping ratio for the PSHA and ground-motion target spectra was set to 5%, which is standard practice in engineering applications. For the cases where the CS was used as the target spectrum, the ground-motion suites were selected and scaled using a modified version of the conditional spectrum ground-motion selection tool (CS-GMS tool) developed by Baker and Lee [2018]. This tool selects and scales a suite of ground motions to meet both the median and the user-defined variability. This variability is defined by the relationship developed by Baker and Jayaram [2008]. The computation of CS requires a structural period for the conditional model. In collaboration with WG5 researchers, a conditioning period of 0.25 sec was selected as a representative of the fundamental mode of vibration of the buildings of interest in this study. Working Group 5 carried out a sensitivity analysis of using other conditioning periods, and the results and discussion of selection of conditioning period are reported in Section 4 of the WG5 PEER report entitled Technical Background Report for Structural Analysis and Performance Assessment. The WG3.1 report presents a summary of the selected sites, the seismic-source characterization model, and the ground-motion characterization model used in the PSHA, followed by selection and modification of suites of ground motions. The Record Sequence Number (RSN) and the associated scale factors are tabulated in the Appendices of this report, and the actual time-series files can be downloaded from the PEER Ground-motion database Portal (https://ngawest2.berkeley.edu/)(link is external).