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étude structurale et fonctionnelle de la protéine a1 du bactériophage t5 : une dnase octamérique originale / structural and functional study of bacteriophage t5 a1 protein : an original octameric dnaseZangelmi, Léo 06 December 2018 (has links)
Les bactériophages neutralisent les systèmes de défense et détournent les fonctions vitales de leur hôte pour favoriser leur multiplication. Les gènes de phages qui gouvernent cette prise de contrôle de l’hôte restent mal connus, pourtant leur caractérisation présente un intérêt majeur pour mettre à jour des fonctions bactériennes spécifiquement ciblées par les phages et pour concevoir de nouveaux agents antibactériens.Le phage T5 injecte son ADN dans la bactérie Escherichia coli en deux étapes. Seuls les gènes précoces codés par 8% du génome entrent dans la cellule et le transfert s’arrête. Leur expression induit la dégradation du chromosome de l’hôte et l’inactivation de ses systèmes de restriction et de réparation de l’ADN. Après quelques minutes, le reste de la molécule d’ADN est injecté, ce qui permet la production de nouveaux phages. Deux gènes précoces A1 et A2 ont été identifiés comme essentiels pour la reprise du transfert de l’ADN et A1 est également nécessaire pour induire la dégradation de l’ADN de l’hôte. A1 et A2 sont les deux seuls gènes connus pour être impliqués dans la régulation de ce système original d’infection, mais leur fonction n’a jamais été identifiée.Ma thèse porte sur la caractérisation fonctionnelle et structurale des protéines A1 et A2. J’ai purifié A1 et démontré in vitro qu’elle avait une activité DNase dépendante du manganèse. Sa structure atomique a été résolue par cryomicroscopie électronique à 3.01 Å de résolution, montrant une organisation octamérique de symétrie D4 inédite pour une DNase. Chaque monomère (61kDa) contient un domaine exonuclease dont le site actif lie deux ions Mn2+ et qui s’apparente au site catalytique des domaines exonucléases de la DNA polymerase II et des DNAses associées aux systèmes de recombinaison homologue et de réparation de l’ADN comme Mre11. En construisant différents mutants de A1, j’ai identifié certains acides aminés essentiels pour l’activité catalytique et, par des expériences de complémentation fonctionnelle, j’ai montré que cette activité était indispensable pour l’infection. L’ensemble de ces résultats suggèrent que A1 est la DNase, jusqu’ici inconnue, responsable de la dégradation massive du génome de l’hôte au tout début de l’infection. Enfin, j’ai observé que la production de A1 pendant l’infection induit une forte activité recombinase. De nombreux autres bactériophages qui n’appartiennent pas à la famille des T5virus produisent également une protéine similaire à A1 dont la fonction n’a jamais été identifiée. Ce travail est un premier pas vers la compréhension de son rôle dans le mécanisme général d‘infection par les phages. Une deuxième partie de cette thèse porte sur la caractérisation structurale de A2. Des recherches de similarité indiquent la présence d’un domaine Helix-Turn-Helix typique des régulateurs transcriptionnels. J’ai purifié A2 et montré que cette protéine de 14 kDa est un dimère en solution. La caractérisation des propriétés biochimiques de A2 a permis de débuter l’étude de sa structure par RMN.Les résultats de ma thèse ont révélé la structure originale d’une DNase de bactériophage qui contrôle la dégradation du génome bactérien et la régulation du transport de l’ADN viral au début du cycle infectieux. Ces résultats soulèvent des questions intrigantes : comment l’ADN de T5 est-il protégé de l’activité DNase de A1 ? Comment A1 et A2 interagissent-elles lors des étapes de prise de contrôle de l’hôte ? / Bacteriophages defeat bacterial defences and hijack host cell machineries to establish a favourable environment for their multiplication. Early-expressed viral genes that govern host takeover are highly diverse from one phage to another and most of them have no assigned function. They thus represent a pool of novel genes whose products potentially subvert bacterial cell vital functions and could help in designing new antibacterial strategies.T5 phage uses a unique 2-step mechanism to deliver its DNA into its host Escherichia coli. At the onset of the infection, only 8 % of the genome enter the cell before the transfer temporarily stops. Expression of the genes encoded by this DNA portion leads to host chromosome degradation and inactivation of host restriction and DNA mending systems. After a few minutes, T5 DNA transfer resumes, allowing further phage multiplication. A1 and A2 are early genes required for DNA transfer completion and A1 is also necessary to trigger host DNA degradation. A1 and A2 are the only two genes known to be involved in the regulation of this original infection system, but their function yet remains to be characterized.The objectives of this work were to characterize the function and structure of A1 and A2 proteins. I have purified the A1 protein and shown that it has a manganese-dependent DNase activity in vitro. Cryo Electron Microscopy at 3.01 Å resolution unravelled its structure, showing an octameric organization with a D4 symmetry, which is unprecedented for a DNase. Each monomer (61 kDa) carries an exonuclease domain harbouring an active site with two Mn2+ ions. This site is similar to those from the exonuclease domain of the DNA polymerase II and from DNases involved in DNA mending and recombination events like Mre11. I identified essential catalytic residues for the DNase activity and demonstrated that this activity is crucial for infection by engineering A1 mutant proteins and by doing functional complementation assays. Taken together, my results suggest that A1 could then be the elusive DNase responsible for the massive host genome degradation observed during T5 phage infection. Eventually, I uncovered a recombinase activity associated to A1 production during infection. Similar proteins to A1 with unknown functions are produced in several other bacteriophages outside of the T5virus family. This work is a first step towards understanding the role of this protein in the general mechanism of infection by bacteriophages. In a second part, I worked on the structural characterisation of A2 protein. Similarity searches revealed a helix-turn-helix domain typically found in transcriptional regulators. I purified and demonstrated the dimeric organisation of this 14-kDa protein in solution. This initial characterization of A2 has opened avenues for further NMR studies.During my Ph.D., I uncovered the structure of an original bacteriophage DNase that controls bacterial genome degradation and that regulates viral DNA transport at the beginning of the infectious cycle. These results open the intriguing question about the mechanism for T5 DNA protection from A1 DNase activity as well as about the interplay between A1 and A2 during the host takeover.
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Transferência gênica em células espermáticas de Mus musculus e Ramdia quelen / Genic transfer in sperm cells in Mus musculus and Ramdia quelemAmaral, Marta Gonçalves 28 December 2009 (has links)
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Previous issue date: 2009-12-28 / Transgenic animals have been used as biological models in studies of the genes functions and their mechanisms of action, as well as to improve animal production. Researchers are trying to produce transgenic animals that will be organs donors in xenotransplants. Another use of the transgenic animal is in the production of recombinant proteins for pharmaceutical interest, starting from several tissues and corporal fluids of different animal species. Using TMGT (Testis Mediated Gene Transfer), the efficiency of pEGFP transgene transmission in mice using non surgical TMGT was evaluated, without epididymis electroporation; using transfectants as DMSO, liposomes, and for the first time the DMA. To evaluate the efficiency of non surgical TMGT in F0 the EGFP expression was evaluated in vivo and detection in genome was conducted by PCR analysis. Moreover, we evaluated which transfectants were more efficient in transgene transmission and if it induce histological damage in testis, by histological analysis. EGFP expression was not detected in F0 through the ultraviolet light.The result of the PCR analysis shows that liposomes and DMSO were the best transfectants for pEGFP in F0. The histological analysis shows that injections of DMSO with exogenous DNA could affect the development of the germ cell of seminal tubules. The purpose about SMGT was to evaluate the interaction of the spermatozoa of silver catfish with pEGFP
vector. It was observed that the semen after three washes in isosmotic solution and at 1000 x g centrifugation could eliminate seminal plasma proteins and preserve cellular motility. The time of action of DNase in the seminal plasma was 30 minutes, the temperature of action of DNase ranged between 33-53°C and its inhibition was detected at 70°C. In the presence of EDTA 30mM the activity of DNase was inhibited. Through PCR it was detected that in the DNA of the silver catfish s spermatozoids, the amplicon of EGFP at different concentrations of pEGFP vector (5-100 ng/106 spermatozoa). We demonstrate that spermatozoa of the silver catfish need to be washed to remove seminal plasma before contact with exogenous DNA, after several washes exogenous DNA was internalized in spermatozoa. / Os animais transgênicos vêm sendo empregados como modelos biológicos em estudos das funções dos genes e dos seus mecanismos de ação, bem como para melhorar a produção animal. Pesquisadores vêm tentando produzir animais transgênicos que serão doadores de órgãos em xenotransplantes. Outra utilização da transgênese animal é a produção de proteínas recombinantes de interesse farmacêutico a partir de diversos tecidos e fluidos corporais de diferentes espécies de animais. Em relação à TMGT (Testis Mediated Gene Transfer) foi avaliada a eficiência da transmissão do transgene EGFP em camundongos, utilizando a TMGT não cirúrgica, sem o uso de eletroporação no epidídimo; utilizando transfectantes como o DMSO, lipossomos, e pela primeira vez o DMA. A detecção da expressão do EGFP foi avaliada in vivo na F0 e por PCR, para comprovar a eficiência da TMGT não cirúrgica. Também foi analisado qual dos transfectantes propiciou a maior taxa de transmissão e se eles causaram danos histológicos aos testículos, através de análise histológica. Não foi detectada a expressão de EGFP, através da luz ultravioeta na F0. Os resultados da análise de PCR demonstraram que o lipossomo e o DMSO foram os melhores transfectantes do pEGFP na F0. A análise histológica demonstrou que a injeção de DMSO com o DNA exógeno,
pode comprometer o desenvolvimento das células germinativas do túbulo seminal. O objetivo em relação à SMGT (Sperm-mediated gene transfer) foi avaliar a interação dos espermatozóides de silver catfish (Rhandia quelen) com o vetor pEGFP. Foi observado que o sêmen após três lavagens em solução isosmótica e centrifugadas à 1000 x g, eliminaram as proteínas do plasma seminal e preservaram a motilidade celular. O tempo de atividade da DNase no plasma seminal foi de 30 minutos, a temperatura de atividade da DNase variou entre 33-53°C e sua inativação ocorreu aos 70°C. Na presença de EDTA 30mM a atividade da DNase foi inibida. Através da PCR foi detectada a presença do pEGFP no DNA dos espermatozoides do silver catfish, que incorporaram o vetor em diferentes concentrações (5-100 ng/106 espermatozoides). Concluímos que os espermatozoides do silver catfish precisam ser lavados para retirada do da DNase do plasma seminal antes de entrar em contato com o DNA exógeno, e que após as lavagens ocorreu a internalização do DNA exógeno no espermatozoide.
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The Role of XRCC1 in the Repair of DNA Strand Breaks in Skeletal Muscle DifferentiationBurns, Leanne E. 22 September 2011 (has links)
Caspase-3 has demonstrated a non-apoptotic function in several developmental programs including skeletal muscle differentiation, yet the mechanism of action has not been fully elucidated. Under apoptotic conditions Caspase-3 induces DNA fragmentation through activation of CAD. Recent observations have demonstrated CAD activity and the resulting DNA strand breaks are also vital for skeletal muscle differentiation. These breaks are transient in nature, suggesting an active DNA repair program to maintain genomic integrity. The aim of this study was to delineate the DNA repair mechanism coordinated with caspase/CAD mediated DNA damage. It was found that XRCC1 formed punctate nuclear foci early in myoblast differentiation concurrent to the induction of DNA damage. Caspase-3 inhibition caused attenuation of the formation of DNA lesions and XRCC1 foci in differentiating myoblasts. Targeted reduction in XRCC1 expression impaired myoblast differentiation. These results suggest that XRCC1 may play a role in repairing the DNA damage associated with myoblast differentiation.
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Effects of three deafness-causing gamma-actin mutations on actin structure and functionKruth, Karina Annette 01 December 2013 (has links)
Hearing requires proper function of the auditory hair cell, which is critically dependent upon its actin-based cytoskeletal structure. Eleven point mutations in gamma (γ) nonmuscle actin have been identified as causing progressive autosomal dominant nonsyndromic hearing loss (DFNA20/26); however, exactly why these mutations lead to deafness is unclear. Organization, stability, and repair of the hair cell cytoskeleton are highly regulated by actin binding proteins (ABPs), and two of the mutations, K118M and K118N, are located near an area of the actin monomer believed to be important in actin-ABP interactions. A third mutation, D51N, is located in a region of the actin monomer believed to be important for polymerization dynamics and stability in filamentous actin. I therefore hypothesized that the K118M/N mutations cause hearing loss due to impaired regulation of the actin cytoskeleton within the hair cell, whereas the D51N mutation likely interferes with polymerization dynamics and actin filament stability or flexibility.
The goal of my thesis was to investigate the effects of these three mutations, K118M, K118N, and D51N, on actin dynamics and regulation. I show in Chapter 2 that the K118M/N mutations differentially affect regulation of actin by the Arp2/3 complex, but also, surprisingly, that the K118N mutation accelerates polymerization dynamics. Chapter 3 details a continued investigation of the K118M/N mutations to ascertain their effects on actin structure and dynamics, particularly with regard to how they may affect polymerization. Chapter 4 provides both an in vivo and in vitro characterization of the D51N mutation, which revealed that not only does the mutation significantly accelerate actin polymerization, it also causes significant effects on yeast mitochondrial morphology and cytoskeletal regulation.
The work detailed within this thesis provides new insight into how the K118M/N and D51N mutations affect actin structure and dynamics and how these effects could lead to deafness. More importantly, this work provides a strong foundation for many future studies, ranging from structural investigation of the K118N and D51N actins as F-actin mimics, to the potential role of mitochondria in actin-based disease.
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Genome-wide Analysis of Chromatin Structure across Diverse Human Cell TypesWinter, Deborah R. January 2013 (has links)
<p>Chromatin structure plays an important role in gene regulation, especially in differentiating the diverse cell types in humans. In this dissertation, we analyze the nucleosome positioning and open chromatin profiles genome-wide and investigate the relationship with transcription initiation, the activity of regulatory elements, and expression levels. We mainly focus on the results of DNase-seq experiments, but also employ annotations from MNase-seq, FAIRE-seq, ChIP-seq, CAGE, and RNA microarrays. Our methods are based on computational approaches including managing large data sets, statistical analysis, and machine learning. We find that different transcription initiation patterns lead to distinct chromatin structures, suggesting diverse regulatory strategies. Moreover, we present a tool for comparing genome-wide annotation tracks and evaluate DNase-seq against a unique assay for detecting open chromatin. We also demonstrate how DNase-seq can be used to successfully predict rotationally stable nucleosomes that are conserved across cell types. We conclude that DNase-seq can be used to study genome-wide chromatin structure in an effort to better understand how it regulates gene expression.</p> / Dissertation
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The Role of XRCC1 in the Repair of DNA Strand Breaks in Skeletal Muscle DifferentiationBurns, Leanne E. 22 September 2011 (has links)
Caspase-3 has demonstrated a non-apoptotic function in several developmental programs including skeletal muscle differentiation, yet the mechanism of action has not been fully elucidated. Under apoptotic conditions Caspase-3 induces DNA fragmentation through activation of CAD. Recent observations have demonstrated CAD activity and the resulting DNA strand breaks are also vital for skeletal muscle differentiation. These breaks are transient in nature, suggesting an active DNA repair program to maintain genomic integrity. The aim of this study was to delineate the DNA repair mechanism coordinated with caspase/CAD mediated DNA damage. It was found that XRCC1 formed punctate nuclear foci early in myoblast differentiation concurrent to the induction of DNA damage. Caspase-3 inhibition caused attenuation of the formation of DNA lesions and XRCC1 foci in differentiating myoblasts. Targeted reduction in XRCC1 expression impaired myoblast differentiation. These results suggest that XRCC1 may play a role in repairing the DNA damage associated with myoblast differentiation.
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Role of Caspase 3/Caspase Activated DNase induced DNA Strand Breaks during Skeletal Muscle Differentiation.Larsen, Brian D. 21 February 2012 (has links)
Cell fate decisions incorporate distinct and overlapping mechanisms. The activity of caspase 3 was initially understood to be a cell death restricted event, however numerous studies have implicated this enzyme in the regulation of both differentiation and proliferation. How the activity of caspase 3 promotes a non-death cell fate remains unclear. Here we examine the role caspase 3 activity plays during skeletal muscle differentiation; in particular we explore the hypothesis that the mechanism of inducing DNA strand breaks during cell death is also a key feature of differentiation, albeit with a distinctly different outcome. We delineate the transient formation of Caspase 3/Caspase activated DNase (CAD) dependent DNA strand breaks during differentiation. The formation of these breaks is essential for differentiation and the regulation of specific genes. In particular expression of the cell cycle inhibitor p21 is related to the formation of a DNA strand break within the gene’s promoter element. Further, we explored the genome wide association of CAD using Chromatin Immunoprecipitation coupled to high through put sequencing (ChIP-seq). This approach identified a potential role for Caspase3/CAD in regulating the expression of Pax7. Here, a CAD directed DNA strand break in the Pax7 gene is correlated with decreased Pax7 expression, an outcome that has been shown to be critical for progress of the myogenic differentiation program. The regulation of Pax7 expression through a CAD induced DNA strand break raises an intriguing connection between this regulation and oncogenic transformation observed in alveolar rhabdomyosarcoma. The putative site of CAD induced DNA strand breaks that promote decreased Pax7 expression during differentiation corresponds to site of chromosomal translocations responsible for Pax7 fusion events in alveolar rhabdomyosarcoma.
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Identifying gene regulatory interactions using functional genomics dataJohansson, Annelie January 2014 (has links)
Previously studies used correlation of DNase I hypersensitivity sites sequencing (DNase-seq) experiments to predict interactions between enhancers and its target promoter gene. We investigate the correlation methods Pearson’s correlation and Mutual Information, using DNase-seq data for 100 cell-types in regions on chromosome one. To assess the performances, we compared our results of correlation scores to Hi-C data from Jin et al. 2013. We showed that the performances are low when comparing it to the Hi-C data, and there is a need of improved correlation metrics. We also demonstrate that the use of Hi-C data as a gold standard is limited, because of its low resolution, and we suggest using another gold standard in further studies.
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Role of Caspase 3/Caspase Activated DNase induced DNA Strand Breaks during Skeletal Muscle Differentiation.Larsen, Brian D. January 2012 (has links)
Cell fate decisions incorporate distinct and overlapping mechanisms. The activity of caspase 3 was initially understood to be a cell death restricted event, however numerous studies have implicated this enzyme in the regulation of both differentiation and proliferation. How the activity of caspase 3 promotes a non-death cell fate remains unclear. Here we examine the role caspase 3 activity plays during skeletal muscle differentiation; in particular we explore the hypothesis that the mechanism of inducing DNA strand breaks during cell death is also a key feature of differentiation, albeit with a distinctly different outcome. We delineate the transient formation of Caspase 3/Caspase activated DNase (CAD) dependent DNA strand breaks during differentiation. The formation of these breaks is essential for differentiation and the regulation of specific genes. In particular expression of the cell cycle inhibitor p21 is related to the formation of a DNA strand break within the gene’s promoter element. Further, we explored the genome wide association of CAD using Chromatin Immunoprecipitation coupled to high through put sequencing (ChIP-seq). This approach identified a potential role for Caspase3/CAD in regulating the expression of Pax7. Here, a CAD directed DNA strand break in the Pax7 gene is correlated with decreased Pax7 expression, an outcome that has been shown to be critical for progress of the myogenic differentiation program. The regulation of Pax7 expression through a CAD induced DNA strand break raises an intriguing connection between this regulation and oncogenic transformation observed in alveolar rhabdomyosarcoma. The putative site of CAD induced DNA strand breaks that promote decreased Pax7 expression during differentiation corresponds to site of chromosomal translocations responsible for Pax7 fusion events in alveolar rhabdomyosarcoma.
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The Role of XRCC1 in the Repair of DNA Strand Breaks in Skeletal Muscle DifferentiationBurns, Leanne E. January 2011 (has links)
Caspase-3 has demonstrated a non-apoptotic function in several developmental programs including skeletal muscle differentiation, yet the mechanism of action has not been fully elucidated. Under apoptotic conditions Caspase-3 induces DNA fragmentation through activation of CAD. Recent observations have demonstrated CAD activity and the resulting DNA strand breaks are also vital for skeletal muscle differentiation. These breaks are transient in nature, suggesting an active DNA repair program to maintain genomic integrity. The aim of this study was to delineate the DNA repair mechanism coordinated with caspase/CAD mediated DNA damage. It was found that XRCC1 formed punctate nuclear foci early in myoblast differentiation concurrent to the induction of DNA damage. Caspase-3 inhibition caused attenuation of the formation of DNA lesions and XRCC1 foci in differentiating myoblasts. Targeted reduction in XRCC1 expression impaired myoblast differentiation. These results suggest that XRCC1 may play a role in repairing the DNA damage associated with myoblast differentiation.
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