Protein complexes in base excision repair: Biochemical and kinetic analysis of mismatch uracil DNA glycosylase

Mismatch uracil DNA glycosylase (MUG) is an E. coli enzyme involved in the repair of ethenocytosine and uracil through the base excision repair pathway. MUG is known to bind the abasic site tightly. This may act to protect the abasic lesion, but the question then is how is the site handed over to the AP Endonuclease? Much has been made of the increase in turnover of some DNA glycosylases by AP endonucleases, but it is not clear whether this occurs via an active displacement mechanism or by passive diffusion. We are addressing these questions by studying the kinetics of MUG interactions with its product and Exonuclease III, the main AP Endonuclease in E. coli. We used fluorescence anisotropy and fluorescence resonance energy transfer assays to investigate MUG & DNA interactions. These revealed that MUG binds abasic DNA in a cooperative manner and that binding of two MUG‘s is needed for efficient DNA repair. Higher salt concentrations reduced cooperativity leading to a 1:1 binding and reduced MUG‘s activity. We also used these assays to investigate whether ExoIII displaces MUG from the DNA through an active or passive mechanism. MUG‘s role in the in vivo repair of etheno lesions has been explored by treating E. coli cells with urethane, a chemical, known to introduce etheno lesions in the DNA. A eukaryotic DNA glycosylase, hSMUG was shown to repair etheno lesions; hence we investigated hSMUG‘s ability to complement mug deficiency in E. coli cells. hSMUG was found to reduce cell viability, increase mutation rates and provided a boost in cell divisions in mug deficient cells whereas it didn‘t affect wild type cells. We then asked the question whether hAPE1, the AP endonuclease believed to increase the turnover rate of hSMUG, can reverse the effects of hSMUG expression in E. coli

Financiar o desenvolvimento internacional por meio da ajuda : um paradigma em redefinição

Trabalho de conclusão de curso (graduação)—Universidade de Brasília, Instituto de Relações Internacionais, 2016.Desde os anos 1950 o sistema de ajuda internacional para o desenvolvimento tem sido adotado como o principal mecanismo de financiamento do desenvolvimento internacional. A dificuldade em se alcançar melhorias nos indicadores sociais e econômicos dos países mais pobres após mais de meio século de fluxos de ajuda, contudo, tem levado estudiosos assim como a comunidade internacional a questionarem a continuidade do modelo de financiamento vigente. Desse modo, o presente trabalho volta-se ao estudo do percurso histórico do sistema de ajuda internacional, analisando as tendências atuais observadas na agenda do financiamento ao desenvolvimento mais precisamente a partir dos resultados da Terceira Conferência das Nações Unidas sobre Financiamento ao Desenvolvimento Internacional, realizada em Addis Abeba em julho de 2015.Since the 1950s, international aid has been adopted as the main financial mechanism to fund international development worldwide. The difficulty in achieving improvements in economic and social indicators in a huge number of recipient countries after more than half a century of aid flows, however, has led scholars as well as the international community to question the continuity of this model. Thus, the present study aims at analyzing the history of the international aid system while approaching new trends currently verified in the development financing agenda – in particular the outcomes of the Third UN Conference on Financing for Development, held in Addis Ababa in July 2015

Endommagement et microfissuration d'un composite à matrice céramique tissé 3D (approchemulti-échelle et évaluation ultrasonore)

Le comportement mécanique non linéaire des composites à matrice céramique SiC/SiC tissés 3D résultede la microfissuration de ses constituants fragiles. Cet endommagement induit une variation descomposantes du tenseur de rigidité. La caractérisation ultrasonore de ce tenseur a nécessité l utilisationd un algorithme d optimisation génétique robuste à un mélange prononcé des modes acoustiques. Le suivisous charge de ces propriétésmacroscopiques a conduit à identifier les mécanismes d endommagement etles réseaux de fissuration. Trois régions de microfissuration matricielle : inter-fils, intra-fils transversaux etintra-fils longitudinaux, ont été localisés par analyse micrographique. Les densités associées ont été corréléesaux cinétiques d endommagement mesurées par ultrasons, reliant la réponse macroscopique dumatériauà son endommagement microstructural. L introduction de ces réseaux et de leur cinétique d évolutiondans une modélisation multi-échelle du composite, a permis de confirmer les variations expérimentales desrigidités et d accéder aux longueurs de décohésion de chaque réseau, quantités difficilement mesurables.The non-linear mechanical behaviour of CMC involves the initiation and growth of micro-cracks. Ata macroscopic scale, mechanisms of damage can be associated with changes in the stiffness tensor components.By using an ultrasonic device coupled with a tensile machine, a spectro-interferometry methodallows the characterisation of materials during their damaging and thus, the measurement of the state ofmaterial cracking. Ultrasonic test results have yielded a typical behaviour of the 3D SiC/SiC composite. Twoarrays of multi-scale cracks are detected: i) crack perpendicular to the load, i.e., the transverse cracking, ii)debonding on interfaces yarn / matrix or fibre / matrix. The simultaneous use of ultrasonic characterisationresults and micrographic observations under load lead to make assumptions about the kinetics of crackingof these materials: i) transverse matrix cracking between yarns, ii) superimposed on the transverse crackingof transversal yarns, iii) when those both arrays of cracking saturate, transverse cracking reaches the longitudinalyarns. This microscopic cracking array is coupled to matrix/fibre debonding. The micrographicobservations made during a tensile test have been used to estimate the density of transverse cracking versusthe applied stress. The lengths of the decohesion are not measured but can be estimated by comparisonwith amulti-scale modelling.BORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

Specialization of an Exonuclease III family enzyme in the repair of 3′ DNA lesions during base excision repair in the human pathogen Neisseria meningitidis

We have previously demonstrated that the two Exonuclease III (Xth) family members present within the obligate human pathogen Neisseria meningitidis, NApe and NExo, are important for survival under conditions of oxidative stress. Of these, only NApe possesses AP endonuclease activity, while the primary function of NExo remained unclear. We now reveal further functional specialization at the level of 3′-PO4 processing for NExo. We demonstrate that the bi-functional meningococcal glycosylases Nth and MutM can perform strand incisions at abasic sites in addition to NApe. However, no such functional redundancy exists for the 3′-phosphatase activity of NExo, and the cytotoxicity of 3′-blocking lesions is reflected in the marked sensitivity of a mutant lacking NExo to oxidative stress, compared to strains deficient in other base excision repair enzymes. A histidine residue within NExo that is responsible for its lack of AP endonuclease activity is also important for its 3′-phosphatase activity, demonstrating an evolutionary trade off in enzyme function at the single amino acid level. This specialization of two Xth enzymes for the 3′-end processing and strand-incision reactions has not previously been observed and provides a new paradigm within the prokaryotic world for separation of these critical functions during base excision repair

Differential modes of DNA binding by mismatch uracil DNA glycosylase from Escherichia coli: implications for abasic lesion processing and enzyme communication in the base excision repair pathway

Mismatch uracil DNA glycosylase (Mug) from Escherichia coli is an initiating enzyme in the base-excision repair pathway. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. Since Mug is known to bind its product tightly, inhibiting enzyme turnover, understanding how Mug binds DNA is of significance when considering how Mug interacts with downstream enzymes in the base-excision repair pathway. We have demonstrated differential binding modes of Mug between its substrate and abasic DNA product using both band shift and fluorescence anisotropy assays. Mug binds its product cooperatively, and a stoichiometric analysis of DNA binding, catalytic activity and salt-dependence indicates that dimer formation is of functional significance in both catalytic activity and product binding. This is the first report of cooperativity in the uracil DNA glycosylase superfamily of enzymes, and forms the basis of product inhibition in Mug. It therefore provides a new perspective on abasic site protection and the findings are discussed in the context of downstream lesion processing and enzyme communication in the base excision repair pathway

Damage and microcraking of a 3D woven ceramic matrix composite : multi-scale approach and ultrasonic evaluation

Le comportement mécanique non linéaire des composites à matrice céramique SiC/SiC tissés 3D résultede la microfissuration de ses constituants fragiles. Cet endommagement induit une variation descomposantes du tenseur de rigidité. La caractérisation ultrasonore de ce tenseur a nécessité l’utilisationd’un algorithme d’optimisation génétique robuste à un mélange prononcé des modes acoustiques. Le suivisous charge de ces propriétésmacroscopiques a conduit à identifier les mécanismes d’endommagement etles réseaux de fissuration. Trois régions de microfissuration matricielle : inter-fils, intra-fils transversaux etintra-fils longitudinaux, ont été localisés par analyse micrographique. Les densités associées ont été corréléesaux cinétiques d’endommagement mesurées par ultrasons, reliant la réponse macroscopique dumatériauà son endommagement microstructural. L’introduction de ces réseaux et de leur cinétique d’évolutiondans une modélisation multi-échelle du composite, a permis de confirmer les variations expérimentales desrigidités et d’accéder aux longueurs de décohésion de chaque réseau, quantités difficilement mesurables.The non-linear mechanical behaviour of CMC involves the initiation and growth of micro-cracks. Ata macroscopic scale, mechanisms of damage can be associated with changes in the stiffness tensor components.By using an ultrasonic device coupled with a tensile machine, a spectro-interferometry methodallows the characterisation of materials during their damaging and thus, the measurement of the state ofmaterial cracking. Ultrasonic test results have yielded a typical behaviour of the 3D SiC/SiC composite. Twoarrays of multi-scale cracks are detected: i) crack perpendicular to the load, i.e., the transverse cracking, ii)debonding on interfaces yarn / matrix or fibre / matrix. The simultaneous use of ultrasonic characterisationresults and micrographic observations under load lead to make assumptions about the kinetics of crackingof these materials: i) transverse matrix cracking between yarns, ii) superimposed on the transverse crackingof transversal yarns, iii) when those both arrays of cracking saturate, transverse cracking reaches the longitudinalyarns. This microscopic cracking array is coupled to matrix/fibre debonding. The micrographicobservations made during a tensile test have been used to estimate the density of transverse cracking versusthe applied stress. The lengths of the decohesion are not measured but can be estimated by comparisonwith amulti-scale modelling

Deux ans de formations de formateurs à l’Association des archivistes français

Grippon Alice. Deux ans de formations de formateurs à l’Association des archivistes français. In: La Gazette des archives, n°218, 2010-2. La formation professionnelle : enjeux d’hier et d’aujourd’hui. pp. 75-82

25 ans d’activité du centre de formation de l’Association des archivistes français : évolution chiffrée

Grippon Alice. 25 ans d’activité du centre de formation de l’Association des archivistes français : évolution chiffrée. In: La Gazette des archives, n°218, 2010-2. La formation professionnelle : enjeux d’hier et d’aujourd’hui. pp. 51-73

Endommagement et microfissuration d’un composite à matrice céramique tissé 3D : approchemulti-échelle et évaluation ultrasonore

The non-linear mechanical behaviour of CMC involves the initiation and growth of micro-cracks. Ata macroscopic scale, mechanisms of damage can be associated with changes in the stiffness tensor components.By using an ultrasonic device coupled with a tensile machine, a spectro-interferometry methodallows the characterisation of materials during their damaging and thus, the measurement of the state ofmaterial cracking. Ultrasonic test results have yielded a typical behaviour of the 3D SiC/SiC composite. Twoarrays of multi-scale cracks are detected: i) crack perpendicular to the load, i.e., the transverse cracking, ii)debonding on interfaces yarn / matrix or fibre / matrix. The simultaneous use of ultrasonic characterisationresults and micrographic observations under load lead to make assumptions about the kinetics of crackingof these materials: i) transverse matrix cracking between yarns, ii) superimposed on the transverse crackingof transversal yarns, iii) when those both arrays of cracking saturate, transverse cracking reaches the longitudinalyarns. This microscopic cracking array is coupled to matrix/fibre debonding. The micrographicobservations made during a tensile test have been used to estimate the density of transverse cracking versusthe applied stress. The lengths of the decohesion are not measured but can be estimated by comparisonwith amulti-scale modelling.Le comportement mécanique non linéaire des composites à matrice céramique SiC/SiC tissés 3D résultede la microfissuration de ses constituants fragiles. Cet endommagement induit une variation descomposantes du tenseur de rigidité. La caractérisation ultrasonore de ce tenseur a nécessité l’utilisationd’un algorithme d’optimisation génétique robuste à un mélange prononcé des modes acoustiques. Le suivisous charge de ces propriétésmacroscopiques a conduit à identifier les mécanismes d’endommagement etles réseaux de fissuration. Trois régions de microfissuration matricielle : inter-fils, intra-fils transversaux etintra-fils longitudinaux, ont été localisés par analyse micrographique. Les densités associées ont été corréléesaux cinétiques d’endommagement mesurées par ultrasons, reliant la réponse macroscopique dumatériauà son endommagement microstructural. L’introduction de ces réseaux et de leur cinétique d’évolutiondans une modélisation multi-échelle du composite, a permis de confirmer les variations expérimentales desrigidités et d’accéder aux longueurs de décohésion de chaque réseau, quantités difficilement mesurables