9,727 research outputs found
André Chambon Villes et développement éducatif local : les cas d’Évry, d’Amiens et de Calais
No full textBier Bernard. André Chambon Villes et développement éducatif local : les cas d’Évry, d’Amiens et de Calais . In: Agora débats/jeunesses, 38, 2004. Jeunes et violences. pp. 117-119
RNA-targeting CRISPR–Cas systems
No full textCRISPR–Cas is a widespread adaptive immune system in bacteria and archaea that protects against viral infection by targeting specific invading nucleic acid sequences. Whereas some CRISPR–Cas systems sense and cleave viral DNA, type III and type VI CRISPR–Cas systems sense RNA that results from viral transcription and perhaps invasion by RNA viruses. The sequence-specific detection of viral RNA evokes a cell-wide response that typically involves global damage to halt the infection. How can one make sense of an immune strategy that encompasses broad, collateral effects rather than specific, targeted destruction? In this Review, we summarize the current understanding of RNA-targeting CRISPR–Cas systems. We detail the composition and properties of type III and type VI systems, outline the cellular defence processes that are instigated upon viral RNA sensing and describe the biological rationale behind the broad RNA-activated immune responses as an effective strategy to combat viral infection.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BN/Stan Brouns LabScience Centre & Programmerin
CRISPR interference : a structural perspective
No full textThis article was made open access through BIS OA funding. The laboratory is funded by grants from the Biotechnology and Biological Sciences Research Council (BBSRC).CRISPR (cluster of regularly interspaced palindromic repeats) is a prokaryotic adaptive defence system, providing immunity against mobile genetic elements such as viruses. Genomically encoded crRNA (CRISPR RNA) is used by Cas (CRISPR-associated) proteins to target and subsequently degrade nucleic acids of invading entities in a sequence-dependent manner. The process is known as ‘interference’. In the present review we cover recent progress on the structural biology of the CRISPR/Cas system, focusing on the Cas proteins and complexes that catalyse crRNA biogenesis and interference. Structural studies have helped in the elucidation of key mechanisms, including the recognition and cleavage of crRNA by the Cas6 and Cas5 proteins, where remarkable diversity at the level of both substrate recognition and catalysis has become apparent. The RNA-binding RAMP (repeat-associated mysterious protein) domain is present in the Cas5, Cas6, Cas7 and Cmr3 protein families and RAMP-like domains are found in Cas2 and Cas10. Structural analysis has also revealed an evolutionary link between the small subunits of the type I and type III-B interference complexes. Future studies of the interference complexes and their constituent components will transform our understanding of the system.Peer reviewe
Structural studies of CRISPR-associated proteins
No full textClustered regularly interspaced short palindromic repeats (CRISPRs) act to prevent viral infection and horizontal gene transfer in prokaryotes. The genomic CRISPR array contains short sequences (“spacers”) that are derived from foreign genetic elements. The CRISPR array is transcribed and processed into CRISPR RNAs (crRNAs) used in the sequence-specific degradation of foreign nucleic acids. This process is called interference and is mediated by CRISPR-associated (Cas) proteins.
This thesis has focused on the structural and functional characterisation of four Cas proteins from the CRISPR/Cas system of Sulfolobus solfataricus. The crystal structure of Cmr7 (Sso1725), a Sulfolobales-specific subunit of the ssRNA-degrading CMR complex, allowed for the identification of a putative protein-binding site, though no specific function could be ascribed to the protein. Cas6 (Sso1437) is the enzyme responsible for crRNA maturation and the characterisation of this protein allowed for the molecular rationalisation of its atypical RNA cleavage mechanism. Csa5 and Cas8a2 are subunits of the aCascade complex that targets dsDNA. Csa5 (Sso1398) was shown to have a putative role in R-loop stabilisation during interference while the role of Cas8a2 (Sso1401) was not determined. The structures of these two proteins were used to define relationships between the subunits of interference complexes from various CRISPR/Cas systems.
A second aspect of this work has been the expression and purification of eukaryotic ion channels for structural studies. The acid sensing ion channel (ASIC) and FMRFamide-gated sodium channel (FaNaC) are gated ion channels with unknown mechanisms of channel activation. These ion channels must be expressed in eukaryotic systems and so human embryonic kidney (HEK) cells and baculovirus-insect cell expression systems were developed to express ASIC and FaNaC constructs. The expression and purification protocols have been optimised to allow for the preparation of soluble protein that will in future be used for crystallography and electron paramagnetic resonance (EPR) studies
Le Sylvicole inférieur au Méganticois : le cas du site Nepress (BiEr-21)
No full textLa découverte du site Nepress (BiEr-21) en 2004 et les saisons de fouilles subséquentes ont permis de découvrir de nombreux vestiges archéologiques. Ce mémoire a donc pour objectif de déterminer l’identité culturelle des occupants qui ont fréquenté le site, en prenant en considération les activités rituelles et la stratégie d’approvisionnement en matière lithique. Pour y parvenir, une analyse morpho-métrique de l’assemblage lithique a été effectuée. La distribution intra-site des artéfacts a également été prise en considération lors de l’analyse. Une séquence chronologique du Nord-Est américain remontant au Sylvicole inférieur est présentée dans ce mémoire. Une période d’occupation semble dominer sur le site Nepress, soit le Sylvicole inférieur. Cette manifestation est caractérisée par la présence d’artéfacts diagnostiques de la culture Meadowood. Ces objets sont un grattoir triangulaire bifacial Meadowood, ainsi qu’une imitation de pointe de type box-base.The discovery of the Nepress site (BiEr-21) in 2004 and the subsequent excavations have revealed many archaeological remains. This thesis has seeks to determine the cultural identity of the site’s occupants, taking into account their ritual activities and their lithic procurement strategy. To achieve this, a morpho-metric analysis of the lithic assemblage was undertaken. The intra-site artifact distribution was also taken into account. A chronological sequence for Northeastern North America going back to the Early Woodland is presented. The Early Woodland appears to dominate the occupation of the Nepress site. This is characterised by the presence of diagnostic artifacts of the Meadowood culture. These objects are a triangular bifacial Meadowood scraper, as well as an imitation of a projectile point
Le Sylvicole inférieur au Méganticois : le cas du site Nepress (BiEr-21)
No full textLa découverte du site Nepress (BiEr-21) en 2004 et les saisons de fouilles subséquentes ont permis de découvrir de nombreux vestiges archéologiques. Ce mémoire a donc pour objectif de déterminer l’identité culturelle des occupants qui ont fréquenté le site, en prenant en considération les activités rituelles et la stratégie d’approvisionnement en matière lithique. Pour y parvenir, une analyse morpho-métrique de l’assemblage lithique a été effectuée. La distribution intra-site des artéfacts a également été prise en considération lors de l’analyse. Une séquence chronologique du Nord-Est américain remontant au Sylvicole inférieur est présentée dans ce mémoire. Une période d’occupation semble dominer sur le site Nepress, soit le Sylvicole inférieur. Cette manifestation est caractérisée par la présence d’artéfacts diagnostiques de la culture Meadowood. Ces objets sont un grattoir triangulaire bifacial Meadowood, ainsi qu’une imitation de pointe de type box-base.The discovery of the Nepress site (BiEr-21) in 2004 and the subsequent excavations have revealed many archaeological remains. This thesis has seeks to determine the cultural identity of the site’s occupants, taking into account their ritual activities and their lithic procurement strategy. To achieve this, a morpho-metric analysis of the lithic assemblage was undertaken. The intra-site artifact distribution was also taken into account. A chronological sequence for Northeastern North America going back to the Early Woodland is presented. The Early Woodland appears to dominate the occupation of the Nepress site. This is characterised by the presence of diagnostic artifacts of the Meadowood culture. These objects are a triangular bifacial Meadowood scraper, as well as an imitation of a projectile point
Intra and Inter organisational determinants of electronic-based traceability adoption: evidences from the French agri-food industry
No full textTraceability, the ability to trace the origin of products throughout the supply chain, has become an instrument to assure food quality and safety in agri-food chains. This process is organized within both institutional and market constraints, yet it integrates also a technological sphere marked by the unprecedented development of information and communication technologies. This paper analyses the factors influencing firms’ behaviour, with regards to adopting electronic-based traceability, in the French agri-food industry. These factors (microeconomic determinants) related to firms’ internal characteristics and the factors related to their environment. We use data from the ICT and Electronic Commerce survey from 2002, carried out by the French National Institute of Statistics (INSEE). A Probit type model is used, which allow us to take into account the firm’s determinants for its organisational choice, differentiating from those adopting (or not) an electronic-based traceability tool. Our main results show that the choices of electronic-based traceability depend on and interact with their own organizational characteristics and those of their competitive, industrial and local environment. Traceability technologies evidence the complementarities between organisational and technological practices. Large industrial firms known for their established identity and a brand image seem distant from standard traceability practices, contrarily to agribusinesses, which are subjected to regulations and look forward to use traceability for both complying with their downstream contracts and add value to their regional specificities.Traceability, Technology adoption, Agri-food industry, Agribusiness, Food Consumption/Nutrition/Food Safety,
Influence of Endodontic Treatment and Retreatment on the Fatigue Failure Load, Numbers of Cycles for Failure, and Survival Rates of Human Canine Teeth
No full textIntroduction: This study evaluated the effects of endodontic treatment and retreatment on the fatigue failure load, numbers of cycles for failure, and survival rates of canine teeth. Methods: Sixty extracted canine teeth, each with a single root canal, were selected and randomly divided into 4 groups (n = 15): untreated, teeth without endodontic intervention; prepared, teeth subjected only to rotary instrumentation; filled, teeth receiving complete endodontic treatment; and retreated, teeth retreated endodontically. After the different endodontic interventions, the specimens were subjected to fatigue testing by the stepwise method: 200 N (x 5000 load pulses), 300 N, 400 N, 500.N, 600 N, 800 N, and 900 N at a maximum of 30,000 load pulses each or the occurrence of fracture. Data from load to failure and numbers of cycles for fracture were recorded and subjected to Kaplan-Meier and Log Rank tests (P < .05), in addition to Weibull analysis. The fractures of the specimens were classified as repairable or catastrophic. Results: The retreated, filled, and untreated groups presented statistically significantly higher fatigue failure loads and numbers of cycles for failure than did the prepared group. Weibull analysis showed no statistically significant difference among the treatments for characteristic load to failure and characteristic number of cycles for failure, although, for number of cycles, a higher Weibull modulus was observed in filled and retreated conditions. The predominant mode of failure was catastrophic. Conclusion: Teeth subjected to complete endodontic treatment and retreatment behaved similarly in terms of fatigue failure load and number of cycles to failure when compared with untreated teeth
The gRAMP CRISPR-Cas effector is an RNA endonuclease complexed with a caspase-like peptidase
No full textType III CRISPR-Cas immunity is widespread in prokaryotes and is generally mediated by multisubunit effector complexes. These complexes recognize complementary viral transcripts and can activate ancillary immune proteins. Here, we describe a type III-E effector from Candidatus “Scalindua brodae” (Sb-gRAMP), which is natively encoded by a single gene with several type III domains fused together. This effector uses CRISPR RNA to guide target RNA recognition and cleaves single-stranded RNA at two defined positions six nucleotides apart. Sb-gRAMP physically combines with the caspase-like TPR-CHAT peptidase to form the CRISPR-guided caspase (Craspase) complex, suggesting a potential mechanism of target RNA-induced protease activity to gain viral immunity.Accepted Author ManuscriptBN/Stan Brouns LabBN/Technici en AnalistenBT/Biocatalysi
PAM-repeat associations and spacer selection preferences in single and co-occurring CRISPR-Cas systems
No full textBackground: The adaptive CRISPR-Cas immune system stores sequences from past invaders as spacers in CRISPR arrays and thereby provides direct evidence that links invaders to hosts. Mapping CRISPR spacers has revealed many aspects of CRISPR-Cas biology, including target requirements such as the protospacer adjacent motif (PAM). However, studies have so far been limited by a low number of mapped spacers in the database. Results: By using vast metagenomic sequence databases, we map approximately one-third of more than 200,000 unique CRISPR spacers from a variety of microbes and derive a catalog of more than two hundred unique PAM sequences associated with specific CRISPR-Cas subtypes. These PAMs are further used to correctly assign the orientation of CRISPR arrays, revealing conserved patterns between the last nucleotides of the CRISPR repeat and PAM. We could also deduce CRISPR-Cas subtype-specific preferences for targeting either template or coding strand of open reading frames. While some DNA-targeting systems (type I-E and type II systems) prefer the template strand and avoid mRNA, other DNA- and RNA-targeting systems (types I-A and I-B and type III systems) prefer the coding strand and mRNA. In addition, we find large-scale evidence that both CRISPR-Cas adaptation machinery and CRISPR arrays are shared between different CRISPR-Cas systems. This could lead to simultaneous DNA and RNA targeting of invaders, which may be effective at combating mobile genetic invaders. Conclusions: This study has broad implications for our understanding of how CRISPR-Cas systems work in a wide range of organisms for which only the genome sequence is known.BN/Stan Brouns La
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