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Jacobsen, Theis. « Structure and assembly of bacterial type IV filaments unravelled by an integrative approach ». Electronic Thesis or Diss., Sorbonne université, 2022. http://www.theses.fr/2022SORUS146.
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La superfamille des filaments de type IV (TFF) est un groupe de machines moléculaires localisées dans la membrane des bactéries et des archées. Ces machines associent des polymères protéiques de manière non-covalente appelés des pili, qui s’étendent depuis la cellule pour réaliser plusieurs fonctions qui ont évolué spécifiquement pour s’adapter à des organismes hôtes différents. La superfamille TFF comprend le système de sécrétion de type II (T2SS) et les pili de type IVa (T4aP). Le T2SS induit la sécrétion de substrats chez les bactéries Gram-négatives. Ces substrats sont en général des enzymes qui dégradent les complexes carbohydrates, le peptidoglycane, les lipides, ce qui à terme entraîne la libération de nutriments. Les T4aP sont de longues fibres flexibles qui sont ancrées dans la membrane et permettent de nombreuses fonctions. Le mécanisme par lequel le T2SS et les T4aP remplissent ces différentes fonctions n’est toujours pas entièrement compris. Pour comprendre le mécanisme de sécrétion du T2SS, nous avons étudié par RMN la structure de la pseudopiline OutG, le composant majeur du pseudopilus chez Dickeya dadantii. Dans une seconde partie, nous avions pour objectif d’aborder la structure et l’assemblage des pilines mineures, des protéines qui composent le T4P d’Escherichia coli entérohémorragique. Nous avons optimisé la surexpression, la purification et le marquage de les pilines mineures pour leur étude par RMN. De plus, la modélisation des pilines et le cross-linking ont été réalisés sur les échantillons de pseudopili T4P et T2SS purifiés en tant que méthodologie pour déterminer la structure et les interactions des pilines et pseudopilines au sein du pilus natifThe type IV filament (TFF) superfamily is a group of molecular machineries located in the membrane of bacteria and archaea. These machineries assemble non-covalent protein polymers called pili extending away from the cell to perform multiple functions which have evolved specifically to adapt to different host organisms. The TFF superfamily includes the type II secretion system (T2SS) and the type IVa pili (T4aP). The T2SS promotes the secretion of substrates in Gram-negative bacteria. These substrates are in general enzymes degrading complex carbohydrates, peptidoglycan, and lipids, resulting in the release of nutrients. The T4aP are long flexible fibres anchored in the membrane and enable various functions such as twitching motility, DNA uptake and biofilm formation. The mechanism by which the T2SS and T4aP pilus fulfil their different functions is still not completely understood. To understand the mechanism of secretion by T2SS, we studied the structure of the pseudopilin OutG, the major component of the pseudopilus in Dickeya dadantii by Nuclear Magnetic Resonance (NMR). In a second part, we aimed to address the structure and the assembly of minor pilins, protein components of Enterohemorrhagic Escherichia coli T4aP. We optimised the overexpression, purification and labelling of the minor pilins for their structural study by NMR. Furthermore, molecular modelling of the minor pilins and crosslinking mass spectrometry were performed on whole T4aP and T2SS pseudopili purified samples as a methodology to determine the structure and the interactions of pilins and pseudopilins within the native pilus
2
Leh, David. « Optimisation du dimensionnement d'un réservoir composite type IV pour stockage très haute pression d'hydrogène ». Phd thesis, Université de Grenoble, 2013. http://tel.archives-ouvertes.fr/tel-00942731.
Texte intégralRésumé :
Ce travail a pour but de proposer une nouvelle approche du dimensionnement optimisé des réservoirs de stockage d'hydrogène de type IV visant à mieux répondre aux enjeux industriels. Les objectifs scientifiques et techniques consistent à disposer de modèles qualifiés pour la simulation du comportement de ces réservoirs, associés à des méthodologies de dimensionnement et d'optimisation fiables. La démarche s'appuie sur trois axes principaux :- proposer une démarche de conception prédictive en intégrant (i) un premier aspect lié à la ruine de la structure qui est la conséquence de mécanismes complexes et multiples d'endommagement s'initiant, s'accumulant et se développant dans un milieu anisotrope et (ii) des modèles de simulation de la structuration composite spécifique au procédé d'enroulement filamentaire, technologie employée largement dans la fabrication des réservoirs de stockage à haute pression. Leurs implémentations constituent une première avancée face à l'existant ;- choisir et évaluer les paramètres structuraux par une démarche d'optimisation où nous sommes amenés à utiliser (i) des méthodes de métamodélisation permettant de répondre aux contraintes de coûts, (ii) des méthodes spécifiques de tri et (iii) des méthodes à spectres larges qui recherchent des solutions sur une large population telles que des méthodes génétiques ;- qualifier la démarche dans sa globalité par une comparaison entre calculs et essais. Ainsi, la finalité de ce travail est de développer et valider des modèles et méthodes pour permettre de mieux concevoir, tester et fabriquer à moindre coût un réservoir avec une structure calculée optimisée.
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Roumagnac, Agnès. « Hyperlipoprotéinémie type IV ». Paris 5, 1988. http://www.theses.fr/1988PA05P031.
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Elliott, Jayne Louise. « Properties and interactions of type III intermediate filaments with CRYAB ». Thesis, Durham University, 2013. http://etheses.dur.ac.uk/7017/.
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Glial fibrillary acidic protein is a type three intermediate filament found within astrocytes in the central nervous system and mutations have been found as the cause of Alexander disease, resulting in protein aggregates of Rosenthal fibers with αBcrystallin. Here two glial fibrillary acidic protein mutants of R79C and R239H, located in the LNDR and rod 2A domain respectively, were assembled in vitro and their morphology, assembly competence and interactions with αB-crystallin were assessed. Both mutants were unable to form usual filament lengths but instead were similar to unit length filaments with R239H forming aggregates due to such high protein interactions and the R79C mutant having much lower assembly competent protein interactions. R239H had a much greater affinity for αB-crystallin, likely a reflection that it has one of the most severe phenotypes. An absence of divalent cations equally affected GFAP assembly resulting in compromised compaction and increased filament-filament interactions. The R120G αB-crystallin mutant results in cardiomyopathy and cataract due to aberrant interactions with desmin intermediate filaments, due to an increased oligomer size and therefore these interactions were studied and compared to wild-type interactions. Temperature and pH also alter the oligomer size of αB-crystallin and were therefore investigated with αB-crystallin-type III intermediate filament interactions. It was found that ischemic conditions and increased temperatures promote their association, demonstrated by increased co-pelleting under high speed sedimentations. There was a preference for binding to desmin filaments thus showing how desmin-wild-type αB-crystallin interactions are important for homeostasis in muscle. Passive microrheology was used to complement this and investigate how αB-crystallin may be modulating desmin filaments under equilibrium. From these experiments wild-type αB-crystallin reduced the frequency-dependent passive viscosity η(f) and the G’, whereas the cardiomyopathy- and cataract- causing R120G mutant increased the η(f).
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Brunmark, Charlott. « Type IV collagen and renal disease ». Lund : Dept. of Nephrology, University of Lund, 1994. http://books.google.com/books?id=owdrAAAAMAAJ.
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Louw, Cassandra Alexandrovna. « Characterisation of Trypanosomal Type III and Type IV Hsp40 proteins ». Thesis, Rhodes University, 2009. http://hdl.handle.net/10962/d1003985.
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The heat shock protein-70 (Hsp70) family of molecular chaperones are ubiquitous highly conserved proteins that are critical for the viability of cellular homeostasis. The ATPase activity of Hsp70 proteins is critical to their function as the affinity of a given Hsp70 for non-native substrate is modulated by ATP binding and hydrolysis. When bound to ATP, Hsp70s possess a low affinity for a given substrate protein, while the hydrolysis of ATP to ADP causes a conformational change that results in a high affinity for substrate proteins. The basal ATPase activity of Hsp70s is too low to facilitate their function in vivo, and co-chaperones are essential to modulate the efficient protein folding by Hsp70. Heat shock protein-40 (Hsp40) heat shock proteins are essential for the in vivo function of Hsp70s by stimulating the ATPase activity of these proteins and facilitating transfer of substrates. The Type III class of Hsp40 proteins have not been well characterised due to their poor levels of conservation at the primary sequence level. This is due to the fact that Type III Hsp40s only contain a J-domain and a poorly conserved C-terminal region. The newly identified Type IV class of Hsp40s, contain an abrogated HPD tripeptide motif in the J-domain and have also not been extensively studied. Trypanosoma brucei (T. brucei) is a unicellular flagellated protozoan parasite. It is the causative agent of Human African Trypansomiasis (HAT) which results in thousands of deaths and devastating agricultural losses in many parts of Africa. T. brucei undergoes a complex lifecycle that is characterised by the transition from an insect vector to a mammalian host in markedly different conditions of temperature, pH, nutrient availability and respiratory requirements. It has been proposed that molecular chaperones may enhance the survival of these parasites due to their cytoprotective effect in combating cellular stress. Due to the fact that T. brucei infection is invariably fatal if left untreated, and that no novel treatment regimens have been developed recently, the identification of potential novel drug targets among proteins essential to the parasite’s survival in the host organism is an attractive aspect of T. brucei research. Because Type III Hsp40s are poorly conserved with respect to Hsp40s found in the human host, the identification of any of these proteins found to be essential to T. brucei survival in humans could potentially make attractive novel drug targets. An in depth in silico investigation into the Type III Hsp40 complement as well as partner Hsp70 proteins in T.brucei was performed. T. brucei possesses 65 Hsp40 proteins, of which 47 were classed as Type III and 6 of which were identified as being putative Type IV Hsp40s. A small but significant number (5) of Type III TbHsp40s contained tetratricopeptide (TPR) domains in addition to the J-domain. The J-domains of the Type III TbHsp40 complement were found to be conserved with respect to those of canonical Hsp40 proteins, although the mutation of certain residues that play a key role in Hsp40-Hsp70 interaction was noted. Potential partnerships of these proteins in the parasite was also investigated. The coding regions of three previously uncharacterised TbHsp40s were successfully amplified from T. brucei TREU927 genomic DNA and cloned into an expression vector. Tbj1, a Tcj1 ortholog, was selected for further study and successfully expressed and biochemically characterised. Tbj1 expressed in E. coli was found to be insoluble, but large amounts were recovered with the aid of a denaturing purification followed by refolding elution strategies, and the bulk of the protein recovered was in compact monomeric form as determined by size-exclusion chromatography fast protein liquid chromatography (SEC-FPLC). The addition of Tbj1 to a thermally aggregated substrate resulted in increased levels of aggregation, although Tbj1 was able to assist two Hsp70 proteins in the suppression of aggregation. Tbj1 proved unable to stimulate the ATPase activity of these same Hsp70s, and could not rescue temperature sensitive cells when replacing E.coli DnaJ and CbpA. It was concluded that Tbj1 does not possess independent chaperone activity, but could display Hsp40 co-chaperone properties under certain circumstances. This could allude to a specialised function in the T. brucei parasite. The lack of human orthologues to Tbj1 could result in the attractiveness of this protein as a novel drug target.
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Liou, Angela Yen-Chun. « Characterization of members of type IV and type IIC of human P-type ATPase ». Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/62488.
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P-type ATPases comprise a superfamily of proteins that play vital roles in the human body and can cause severe diseases if their functions are impaired. P₄-ATPases or type 4 of P-type ATPases are implicated in the ATP-dependent flipping of phospholipids across cell membranes. This generates and maintains transverse phospholipid asymmetry, a property important for biological processes including vesicle trafficking. ATP9A is a P₄-ATPase that remains poorly characterized despite its high expression in brain and testis. Interestingly, loss of Neo1p, the yeast ortholog of ATP9A, is lethal. The first part of this study investigates the functional properties and cellular localization of ATP9A. Human ATP9A was expressed in HEK293T cells and characterized using biochemical and cell-based approaches. ATP9A exhibited little if any phospholipid-dependent ATPase activity, but underwent hydroxylamine-sensitive phosphorylation, a characteristic feature of the P-type ATPase reaction cycle. A monoclonal antibody to ATP9A was generated for analysis of ATP9A in cells and brain tissues by western blotting and immunofluorescence microscopy. In transfected HEK293T cells ATP9A localized to perinuclear and peripheral punctate structures possibly related to the endocytic pathway. Our findings suggest that ATP9A undergoes autophosphorylation, but fails to dephosphorylate, possibly due to lack of an accessory protein or a specific substrate. Further studies on endogenous ATP9A should provide further insight into its physiological function and possible role in human disease.
On the other hand, Na⁺/K⁺-ATPase (NKA) belongs to type 2C of P-type ATPases and establishes Na⁺ and K⁺ gradients across cell membranes. NKA has been shown to interact with retinoschisin (RS1), an adhesion protein essential for normal retinal structure and function. Mutations in the gene encoding RS1 cause a macular degeneration disorder called X-linked retinoschisis (XLRS). RS1 is thought to be anchored to the membranes of photoreceptor and bipolar cells through interaction with the α3 and β2 isoforms of NKA. The second part aims to characterize the RS1-NKA complex by generating monoclonal antibodies specific for the components. Indeed, immunoaffinity purification of NKAβ2 from bovine retinal membranes co-immunoprecipitated the α3 subunit and RS1. Tandem affinity purification of the native protein complexes should enhance understanding of the molecular and cellular mechanisms underlying XLRS.Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
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Karuppiah, Vijaykumar. « Structural studies of type IV pilus biogenesis proteins ». Thesis, University of Manchester, 2010. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:101892.
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Berry, Jamie. « Structural characterization of type IV pilus biogenesis proteins ». Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/structural-characterization-of-type-iv-pilus-biogenesis-proteins(1e0d7119-58d5-4e5d-839d-daef8deb76ab).html.
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Type IV pili, or fimbriae, are long, thin proteinaceous appendages found on the surface of many well-known pathogens. They mediate a variety of important virulence functions for the organism, such as twitching motility, biofilm formation, uptake of genetic material and host cell recognition and adhesion. Pili are formed by the rapid polymerization and de-polymerization of the pilin subunit, and this is orchestrated by a complex macromolecular machine which spans the bacterial cell envelope, requiring a variety of gene products. The type IV pilus biogenesis system is closely related to the bacterial type II secretion system, one of six designated multi-protein cell envelope complexes which are dedicated to the specific secretion of exotoxins and virulence factors. Many of these secretion systems also produce fimbrial structures to facilitate the extrusion of their substrates or to communicate with the host. As they form crucial virulence factors, the secretion systems and the type IV pilus biogenesis system have become attractive potential antimicrobial targets and obtaining structural and functional information for the components of these systems is an important first step towards achieving this.Type IV pili appear on the surface of bacteria through an outer membrane pore, PilQ, which is a member of the secretin family. Secretins are also found in the type II and III secretion systems, but the way in which they are regulated remains unclear. PilQ forms a dodecameric chamber in the outer membrane with a large vestibule which reaches into the periplasm, composed of its N-terminal domains. In this project, N-terminal domains from PilQ were produced in recombinant form and their structures determined by NMR. One of these domains revealed an eight-stranded beta-sandwich structure which appears to be unique to type IV pilus secretins and has not been structurally characterized before. Another revealed an alpha/beta type fold which is common to secretins of other systems. In the second part of this project, the interaction formed between the N-terminal alpha/beta domains of PilQ and an essential inner membrane-anchored lipoprotein, PilP, was probed by NMR chemical shift perturbation. Based on changes to the 15N-HSQC spectra the binding site was mapped onto each protein to produce a computational model for the complex formed between the two. Using a recent cryo-EM structure for the Neisseria PilQ dodecamer determined by colleagues, it was possible to model the PilQ N-terminal domains in complex with PilP into the electron density map. This produced a model for the trans-periplasmic assembly formed by PilQ and PilP in the type IV pilus biogenesis system, and led to the conclusion that the PilQ dodecamer needs to disassemble considerably at the base to accommodate a pilus fibre. The novel beta-domains might therefore function to gate or open the secretin, and PilP may play a role in stabilizing the secretin during this and serve to connect the outer and inner membrane system components.
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Schröder, Gunnar. « TraG-like transporter proteins of type IV secretion systems ». [S.l. : s.n.], 2003. http://www.diss.fu-berlin.de/2003/179/index.html.
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Sukmana, Andreas Binar Aji. « Understanding PilB, The Type IV Pilus (T4P) Assembly ATPase ». Thesis, Virginia Tech, 2018. http://hdl.handle.net/10919/83819.
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The type IV pilus (T4P) is a dynamic long thin fiber found on the surface of many bacterial groups. T4P is a versatile nanomachine; it plays many important roles such as for surface attachment, virulence factor, and surface motility apparatus. This research focuses on understanding the kinetics of PilB, the T4P assembly ATPase. PilB crystal structure exhibits an elongated hexamer with 2-fold symmetry indicating a symmetric rotary mechanism model. Except for its structure, the symmetric rotary mechanism of PilB has not been demonstrated experimentally. Its conformation and relatively low activity constrained previous in vitro studies of PilB. This study identified PilB from thermophilic organism Chloracidobacterium thermophilum (Ct) to be a model for in vitro studies. An active CtPilB was successfully expressed and purified as a hexamer. Malachite green phosphate assay was used to examine CtPilB ATPase activity. The examination indicated that CtPilB is a robust ATPase with a complex kinetics profile. The profile has a stepwise incline in ATPase activity as a function of [ATP] that led to a decline in higher [ATP]. The decline was confirmed to be a substrate inhibition by the enzyme-coupled assay. As for the incline, the detailed mechanism is still less clear to explain the multiphasic profile. The overall incline did not conform with classical Michaelis-Menten kinetic but the first part of the incline was shown to conform with Michaelis-Menten kinetics. The complex kinetics profile of PilB is consistent with the symmetric rotary mechanism of catalysis.Master of Science
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Luna-Rico, Areli-Noemi. « Enterobacterial type IV pili : structure, assembly and molecular function ». Thesis, Sorbonne Paris Cité, 2018. https://theses.md.univ-paris-diderot.fr/LUNARICO_Areli_4_va_20180629.pdf.
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Nombreuses espèces bactériennes présentent des fibres à leur surface qui leur permettent d’interagir avec leur environnement. Les pili de type 4 (PT4) sont des fibres longues, fines et flexibles, impliquées dans des fonctions multiples telles que l'adhérence, la motilité, la sécrétion, l'import d'ADN et la formation des biofilms. Ils sont composés de milliers de copies de sous-unité majeure de piline et sont assemblés par un complexe protéique localisé dans l'enveloppe bactérienne. Dans cette étude, nous nous sommes intéressés aux PT4 chez Escherichia coli entérohémorragique (EHEC) de sérotype O157: H7. EHEC est un agent pathogène humain d'origine alimentaire qui provoque des épidémies de diarrhées hémorragiques et/ou des atteintes rénales sévères appelées syndrome hémolytique et urémique (SHU), qui peuvent être mortelles. Les PT4 chez EHEC sont composés de la sous-unité majeure PpdD et probablement des pilines mineures PpdA, PpdB, YgdB et PpdC en plus faible abondance.Dans cette étude notre objectif était de décrire la structure des PT4 chez EHEC, en tant que modèle de la famille des pili conservés chez les entérobactéries. La structure est indispensable pour décrire le mode d'action de ces organites. Dans cette étude, nous avons abordé la structure de l'EHEC T4P ainsi que les bases moléculaires de leur assemblage. En raison de la nature flexible et non covalente des fibres T4P, nous avons utilisé une approche intégrative incluant des données obtenues par cryo-microscopie électronique (cryo-ME), spectroscopie RMN, modélisation moléculaire et analyses biochimiques pour déterminer la structure des pili PpdD. Les interactions entre les sous-unités de pilines présentes dans la fibre ont été étudiées par mutagenèse dirigée et analyses fonctionnelles. Ces expériences nous ont permis d’identifier les résidus de PpdD essentiels pour l’assemblage des pili.Les T4aP ont été peu étudiés chez les entérobactéries. Bien que tous les gènes nécessaires soient présents chez E. coli, les conditions induisant leur expression restent inconnues. Cependant, les gènes codant pour les PT4 chez E. coli sont co-régulés avec des gènes codant pour la machinerie impliquée dans l’import d'ADN, ce qui suggère leur rôle dans la compétence naturelle. Afin de faciliter l'étude de PT4, nous avons réalisé une reconstitution fonctionnelle de l'EHEC T4PS chez E. coli K-12 non pathogène par l'expression contrôlée des gènes de PT4 clonés dans un opéron artificiel. Cette construction nous a permis de réaliser des analyses comparatives d'assemblage des pili par le système hétérologue (système de sécrétion de type 2 provenant d'une autre entérobactérie, Klebsiella oxytoca) et le système d'assemblage propre aux PT4 chez EHEC. Leur analyse par cryo-ME a montré que les pili assemblés sont identiques, indiquant ainsi que ce sont les pilines majeures qui déterminent la structure et la symétrie des fibres. Les résultats des études comparatives ont également apporté des informations importantes sur l'assemblage des pili. Nous avons identifié des composants de la machinerie d'assemblage qui interagissent avec la piline majeure PpdD. La formation des dimères de PpdD et ses interactions spécifiques avec les facteurs d’assemblage semblent importantes pour la stabilité de la piline avant l'assemblage en pilus. L’ensemble de nos résultats consitutent des bases pour de futures analyses structurales et fonctionnelles des pili chez les entérobactériesMany bacterial species display surface fibers to interact with the surrounding environment. Type 4 pili (T4P) are long and thin, flexible fibers involved in a variety of functions including adherence, motility, secretion, DNA uptake and biofilm formation. They are composed of thousands of copies of major pilin subunits and are assembled by a protein complex localized in the bacterial envelope. In this study we focused on the T4P from Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7. EHEC is a human foodborne pathogen that causes outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS), which can lead to a lethal outcome. EHEC T4P is composed of the major subunit PpdD and presumably in lower abundance by minor pilins PpdA, PpdB, YgdB and PpdC.This study has aimed to describe the structure of the T4P from EHEC, a representative of pili conserved in enterobacteria. Structural information provides insights that can relate to the mode of action of these organelles. In this project we addressed the structure of the EHEC T4P and the molecular basis of their assembly. Due to the flexible and non-covalent nature of T4P fibers we used an integrative approach to combine information obtained by cryo-electron microscopy, NMR spectroscopy, molecular modeling and biochemical analyses to obtain a high quality structure of the EHEC T4P. In addition, from functional, interaction and mutational analysis we gained insights into the molecular interactions between the pilin subunits present in the fiber. The T4P are poorly studied in enterobacterial systems; despite the presence of all the necessary genes, the conditions that trigger their expression in E. coli are unknown. In addition, in E. coli the T4P-related genes are coregulated with genes encoding the DNA uptake machinery, suggesting their role in natural competence. In order to facilitate the study of T4P, we achieved a functional reconstitution of the EHEC T4PS in the non-pathogenic E. coli K-12 through the controlled expression of the T4P genes cloned together as a single artificial operon. With this accomplishment we were able to perform comparative analyses between pili assembled by a heterologous system (the Type 2 secretion system from another enterobacterium, Klebsiella oxytoca) and by the cognate EHEC T4P assembly system. CryoEM analysis showed that these pili are identical, indicating that major pilins are the key determinants of fiber structure and symmetry. The results also led us to obtain important insights into pilus assembly and to characterize PpdD interactions with the assembly machinery. These interactions and PpdD dimerization were required for pilin stability prior to pilus assembly, highlighting important early steps involving targeting of subunits to the assembly machinery. Together, these results lay the foundations for future structural and functional studies of enterobacterial T4P
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Seow, Vui Yin. « DNA Transformation and Type IV Pili in Neisseria gonorrhoeae ». Electronic Thesis or Diss., Sorbonne université, 2024. http://www.theses.fr/2024SORUS049.
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La résistance aux antibiotiques, évidente chez des souches telles que Neisseria gonorrhoeae, est une crise sanitaire mondiale. Il est vital de comprendre son mécanisme, en particulier la transformation naturelle par les Pili de type IV de ces bactéries. Bien qu'elle ait été découverte en 1928, la transformation naturelle n'a toujours pas été élucidée. La dépendance de Neisseria gonorrhoeae à l'égard des Pili de type IV en fait un modèle idéal pour l'étude de ce processus.Cette thèse se lance dans une exploration intensive des processus complexes de transformation de l'ADN au sein de Neisseria gonorrhoeae, répondant ainsi à l'urgence de ce problème critique. Le rôle central de cette étude approfondie est attribué aux pili de type IV, un acteur multifonctionnel et essentiel dans l'orchestration de la transformation de l'ADN.Cette recherche pose méticuleusement les bases, en introduisant des techniques de biologie moléculaire essentielles pour le génie génétique chez N. gonorrhoeae. Nous explorons le développement d'outils, en particulier l'optimisation des milieux et les outils de microscopie adaptés à l'étude de l'absorption et de la transformation de l'ADN. En outre, nos recherches ont mis en évidence une corrélation intrigante entre l'amidon et les acides gras, qui a un impact significatif sur la croissance des gonocoques. Pour comprendre la dynamique des pili de type IV pendant l'absorption de l'ADN, nous avons mis au point des outils et un flux de travail rationalisé capables de visualiser et de quantifier à la fois les pili et les molécules d'ADN. En outre, nous avons automatisé l'analyse des micropiliers d'hydrogel afin d'étudier les propriétés mécaniques des rétractions des pili. En outre, des adaptations des revêtements des micropiliers nous ont permis d'étudier les rétractions des pili en interaction avec l'ADN.Cette étude approfondie comprend également l'examen du comportement des mutants ΔPilV, ΔPilC, et ΔPilD, dévoilant ainsi de profondes connaissances sur les mécanismes de régulation des pili de type IV et leur impact sur la dynamique de l'absorption de l'ADN. Il est particulièrement intéressant de noter que les mutations ΔPilV induisent des altérations dans la translocation de PilE, ce qui entraîne l'émergence de pili plus courts mais efficaces. Cette découverte souligne la nature adaptative de N. gonorrhoeae dans la manipulation de la diversité des pili de type IV pour optimiser les processus d'absorption de l'ADN, une révélation qui revêt une importance considérable dans la lutte contre la résistance aux antibiotiques.En outre, nos études sur d'autres pilines mineures mettent en lumière les altérations du phénotype sans entraver le mécanisme de rétraction des pili. Nos études sur les mutants PilV et PilD mettent en évidence l'influence des modifications post-traductionnelles sur PilE, accentuant ainsi la composition hétérogène des pili de type IV et leur fonctionnalité robuste en tant que polymère.Nous incluons également une courte étude examinant l'interaction entre les espèces de Neisseria commensales et pathogènes dans le contexte de l'absorption de l'ADN, ce qui élargit le champ des implications, invitant à une enquête plus approfondie et élargissant les horizons de ce domaine captivant.Bien que cette expédition laisse certaines questions sans réponse, sa profondeur et son ampleur permettent de mieux comprendre les mécanismes complexes de transformation de l'ADN et le rôle dynamique joué par les pili de type IV dans la remarquable adaptabilité de Neisseria gonorrhoeae. Non seulement cette thèse apporte des solutions aux questions existantes, mais elle ouvre également de nouvelles voies pour la recherche future, suscitant la curiosité et la fascination pour l'élucidation des complexités fonctionnelles des structures pili et leurs implications profondes dans la transformation de l'ADNAntibiotic resistance, evident in strains like Neisseria gonorrhoeae, is a global health crisis. Understanding its mechanism, particularly natural transformation through Type IV Pili in these bacteria, remains vital. Despite discovery in 1928, the specifics of natural transformation remain elusive. Neisseria gonorrhoeae's dependence on Type IV Pili makes it an ideal model for studying this process.This thesis embarks on an intensive exploration into the intricate processes of DNA transformation within Neisseria gonorrhoeae, responding to the urgency of this critical concern. Central to this comprehensive study is the pivotal role attributed to Type IV Pili, a multifunctional and essential player in orchestrating DNA transformation.This research meticulously lays the groundwork, introducing molecular biology techniques essential for genetic engineering within N. gonorrhoeae. We explores tool development, particularly in medium optimization and microscopy tools tailored to study DNA uptake and transformation. Moreover, our investigations uncovered an intriguing correlation between starch and fatty acid, significantly impacting gonococcal growth. To understand Type IV pili dynamics during DNA uptake, we engineered tools and a streamlined workflow capable of visualizing and quantifying both pili and DNA molecules. Additionally, we automated the analysis of hydrogel micropillars to delve into the mechanical properties of pili retractions. Furthermore, adaptations to the micropillar coatings enabled us to study pili retractions interacting with DNA.This in-depth investigation also involves scrutinizing the behaviour of ΔPilV, ΔPilC, and ΔPilD mutants, unveiling profound insights into the regulatory mechanisms of Type IV Pili and their consequential impact on the dynamics of DNA uptake. Particularly noteworthy is the revelation that ΔPilV mutations induce alterations in PilE translocation, resulting in the emergence of shorter yet efficient pili. This discovery underscores the adaptive nature of N. gonorrhoeae in manipulating the diversity of Type IV Pili to optimize DNA uptake processes, a revelation that holds immense significance in combating antibiotic resistance.Furthermore, our studies on other minor pilins sheds light on phenotype alterations without impeding the mechanics of pili retraction. Our studies on PilV and PilD mutants, highlight the influence of posttranslational modifications on PilE, thereby accentuating the heterogeneous composition of Type IV Pili and their robust functionality as a polymer.We also include a short study examining the interplay between commensal and pathogenic Neisseria species within the context of DNA uptake broadens the scope of implications, inviting further inquiry and expanding the horizons of this captivating field.While this expedition leaves certain questions unanswered, its depth and breadth offer extensive insights into the intricate mechanisms of DNA transformation and the dynamic role played by Type IV Pili in the remarkable adaptability of Neisseria gonorrhoeae. Not only does this thesis provide solutions to existing queries, but it also illuminates novel pathways for future research, sparking curiosity and fascination in unravelling the functional intricacies of pili structures and their profound implications in DNA transformation
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Coelho, Nuno Miranda. « Dynamic behavior of type IV collagen at cell-biomaterial interface ». Doctoral thesis, Universitat Politècnica de Catalunya, 2012. http://hdl.handle.net/10803/132248.
Texte intégralRésumé :
The initial molecular events that take place at biomaterials interface mimic to a certain extent the natural interaction of cells with the extracellular matrix (ECM). In this thesis we describe the fate of adsorbed type IV collagen (Col IV) - the main structural component of the basement membrane (BM) - as an important target in vascular tissue engineering. We studied the adsorption kinetic of Col IV on different model surfaces varying in wettability, chemistry and charge, and followed how it alters the molecular organization of the adsorbed protein layer. We strived to learn how it will affect the subsequent cellular interaction. AFM studies revealed specific substratum– dependent adsorption pattern of Col IV ranging from single molecular deposition to fine
meshwork formation at high coating concentrations, which is characteristic for hydrophilic and NH2 functionalized substrates. Conversely, the formation of a complex networks consisting of molecular aggregates were found on hydrophobic and COOH modified surfaces. Complex structures were found also when a family of model substrates with tailored density of OH, CH3 and NH2 functions were used. Human umbilical endothelial cells (HUVEC) and fibroblasts were employed to study the biological response on these substrata. We found that fibroblast not only interact with adsorbed Col IV but also tend to reorganize it in fibril like pattern, which is strongly dependent on the materials surface properties. Following the trend of adsorption NH2>CH3>COOH>OH the reorganization pattern of Col IV improve with lowering the amount of protein. However, the cells interact better with hydrophilic and NH2 surfaces, thus acting independently on the amount of adsorbed Col IV. This trend was confirmed by the quantitative measurements of cell adhesion and spreading, as well as, the expression of p-FAK, α1 and α2 integrins – all reflecting the proper functioning of cell adhesion machinery. This is the first study that addresses the relationship between microscopic observation for remodeling of surface associated Col IV and it´s dynamic behavior in nano scale. We found that cells remodel Col IV in two ways: by mechanical reorganization and via proteolytic degradation. We identify the role of FN in the reorganization process and the involvement of MMP2 and MMP9 in the pericellular degradation activity of both HUVEC and fibroblasts. The later was further quantified via FITC labeled Col IV release and zymography. We found that in hydrophobic environment the degradation activity can override the Col IV organization process, which corroborates with the altered cell morphology, abrogated cell adhesion machinery and altered capability of HUVEC to form capillary-like structures. Taken together these results support our view that the ability of cells to remodel surface associated proteins affects strongly the biological performance of a biomaterial. They also show that the appropriate chemical functionalization (NH2, OH), combined with Col IV pre-adsorption, comprises a prospective biomimetic modification that might provide insights for the improved endothelization of cardiovascular implants.
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Tuchscherer, Robin G. « Investigation of the cracking problem of short type IV girders ». Full-text Adobe Acrobat (PDF) file, 2006. http://www.engr.utexas.edu/research/fsel/FSEL_reports/Thesis/Tuchscherer,%20Robin.pdf.
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Anderson, Emma S. « The Type IV Oligodendrocyte : experimental studies on chicken white matter / ». Linköping : Univ, 2002. http://www.bibl.liu.se/liupubl/disp/disp2002/med720s.pdf.
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Wheatcroft, Alison Clare. « Detection of type IV collagen degradation in inflammatory bowel disease ». Thesis, University of Sheffield, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310765.
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Connery, Sarah. « Reconstitution and localisation studies of a type IV secretion system ». Thesis, Birkbeck (University of London), 2014. http://bbktheses.da.ulcc.ac.uk/75/.
Texte intégralRésumé :
Bacterial conjugation is the transport of a DNA molecule from a donor cell to a recipient. Since bacteria do not reproduce sexually, conjugation is a major contributor to prokaryotic genome plasticity and the spread of antibiotic resistance genes. A Type IV Secretion System (T4SS) mediates the DNA transport during conjugation. T4SSs are large macromolecular assemblies embedded in the membrane of bacteria, and are associated with pathogenesis, bacterial conjugation and natural transformation. They are a versatile family of secretion systems, who transport a wide variety of substrates, such as virulence proteins, DNA––protein complexes as well as only DNA. Here, we investigate the minimal requirements for conjugation, and the T4SS’s localisation within the cell. We show that the conjugative T4SS of the plasmid pR388 requires a total of 14 genes to efficiently mobilise DNA from a donor cell to a recipient cell and is arranged around the cell circumfence in a helical array. Our study of two reconstituted and fully functional conjugative T4SSs opens doors for further structural and functional analysis.
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Walker, Nigel Stuart. « Type IV creep cavitation in low alloy ferritic steel weldments ». Thesis, University of Bristol, 1997. http://hdl.handle.net/1983/efa6973c-9a3d-4a95-8297-61f12cbde92d.
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Nikraftar, Sarah. « Localization of Type IV Pilin Polymerization Proteins in Clostridium perfringens ». Thesis, Virginia Tech, 2015. http://hdl.handle.net/10919/71742.
Texte intégralRésumé :
Clostridium perfringens is a spore-forming anaerobic Gram-positive rod which has gliding motility through type IV Pili (TFP). Since the discovery of TFP in Gram-positive bacteria is relatively new, more studies are required to understand the mechanism and interaction of the proteins of this machinery. Moreover, the similarities between TFP and type 2 secretion system (T2SS) suggest that C. perfringens has also a T2SS.
We studied the localization of TFP ATPases, PilB1, PilB2 and PilT in Bacillus subtilis to compare the localization in an organism other than C. perfringens and which lacks any known genes similar to TFP. Unlike the case in C. perfringens, PilB1 in B. subtilis localized to the poles in the absence of PilT, with some central foci at the future division sites. Colocalization of PilB1 was also studied with PilT and the results suggested that PilB1 needs PilT to migrate from the poles to the center. Localization of PilB2 in B. subtilis, was similar to the results in C. perfringens and to the localization of PilB1 in B. subtilis. We have not been able to co-express PilB2 with PilT yet. Succeeding in this study will help us better understand the interactions between PilB proteins and PilT.
In another project, we studied a von Willebrand factor Type A-Domain Containing protein (vWA) which is secreted from C. perfringens strain 13. We overexpressed and purified this protein and tested the effects on mammalian cells. We found that the vWA is probably not a toxin but since it seems to bind to macrophage membranes, we propose that the vWA could be part of a toxin complex, probably the subunit of the complex that binds to the host cells.Master of Science
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Hendrick, William Anthony. « Molecular Analysis of Type IV Pilus Assembly in Clostridium perfringens ». Diss., Virginia Tech, 2016. http://hdl.handle.net/10919/81696.
Texte intégralRésumé :
Clostridium perfringens is a Gram-positive anaerobe capable of causing disease in humans and many animals. C. perfringens is able to move across surfaces in a manner that is dependent on growth and type IV pili.
Type IV pili are filaments that can be extended away from the cell by rapid polymerization, and retracted by depolymerization. Furthering the understanding of the initial and final energetic states of the pilins will reveal insights into possible mechanisms of type IV pilus assembly. Toward that end, a pilin was purified from the Gram-negative pathogen Pseudomonas aeruginosa and incorporated into an artificial membrane. The pilin was probed by a solid state nuclear magnetic resonance (ssNMR) technique that can determine the angle and depth of insertion of a helical peptide, as well as fluorescent and electron microscopy.
All type IV pilus systems involve the action of an assembly ATPase to provide energy to polymerize the pilus. One proposed mechanism involves two primary proteins: an ATPase and an integral membrane core protein (IMCP). Other type IV pilus proteins are thought to play supportive roles in aiding the traversal of the cell envelope. In order to evaluate this model, the assembly ATPase PilB2 and IMCP PilC2 from C. perfringens were purified and examined for interactions. The evidence presented here suggest that PilB2 and PilC2 do not interact directly, and cannot function as a core assembly apparatus.
The carbonic anhydrase (Cpb) from C. perfringens strain 13 was characterized both biochemically and physiologically. Cpb belongs to the type I subclass of the β class and is the first β class enzyme investigated from a strictly anaerobic bacteria. Kinetic analyses revealed a two-step, pingpong, zinc-hydroxide mechanism of catalysis. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semi-defined medium and an atmosphere without CO₂. The grew well in nutrient-rich media with or without CO₂ in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO₂ fixation reactions by supplying bicarbonate to carboxylases.Ph. D.
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ARJOMAND, MOHAMAD-SHAHREYAR. « Le neuroblastome type iv-s : a propos de 2 cas ». Université Louis Pasteur (Strasbourg) (1971-2008), 1993. http://www.theses.fr/1993STR1M092.
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Lau, En-Lieng. « Molecular analysis of the autosomal dominant spastic paraplegia type IV (SPG4) ». [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=962848255.
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Kelly, John James. « Characteristics and properties of phosphodiesterase type IV in guinea-pig macrophages ». Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283687.
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Stratford, G. C. « Type IV cracking in 1¼Cr - ½Mo low alloy steel welds ». Thesis, Swansea University, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.639124.
Texte intégralRésumé :
Service experience has shown the main form of medium to long term damage and failure in low alloy steel weldments to be "TYPE IV" creep cracking in the intercritically transformed region of the heat affected zone (HAZ). This research programme aimed to define the fabricational, loading and microstructural factors which lead to this form of damage. Research welds, which were an accurate model of the geometry and microstructure of steam pipe weldments, were fabricated in tubular testpieces using standard welding techniques. The welds were subjected to a post-weld heat treatment (PWHT) of 700oC or 750oC for two hours. Whilst the heat treatment reduced the hardness of the welds, significant changes in microstructure were only seen in the 750oC PWHT weld. Uniaxial creep tests were performed on base metal and cross-weld specimens. Post-weld heat treatment increased the creep deformation and reduced the failure life of base metal specimens. For cross-weld specimens in the as-welded condition, the susceptibility for low ductility TYPE IV failure in the HAZ was invariably found to be linked to the sub-surface development of creep cavities and cracks. All specimens tested in the as-welded condition failed in a low ductility TYPE IV mode. For all cross-weld specimens, the susceptibility to low ductility failure was linked to factors which affect the base metal ductility, such as PWHT and test temperature. Thus, specimens with a PWHT of 750oC or those with a PWHT of 700oC tested at a temperature greater than 580oC were found to fail in a high ductility manner in the base metal region. Tubular specimens were tested at elevated temperature under internal pressure and applied end-load conditions. Results showed that deformation and fracture were dependant on temperature and axial stress and that sub-surface TYPE IV damage had developed in the HAZ of 700oC PWHT welds.
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Jasim, Sarah. « Type IV secretion genes in cagE-negative Aggregatibacter actinomycetemcomitans serotype b ». Thesis, Umeå universitet, Tandläkarutbildning, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-156060.
Texte intégralRésumé :
Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium that plays an important role in the development of aggressive periodontitis. The cagE gene, which encodes a putative exotoxin, has been found to be present in highly leukotoxic serotype b strains. Both in the JP2 genotype, in which there is a 530-bp deletion in the leukotoxin operon’s promoter region, and in the non-JP2 genotype, and it was recently shown that the cagE gene could serve as a genetic marker for highly leukotoxic serotype b strains. It was also noticed that the cagE gene and the virB4 gene did not seem to be found in the same strain. The aim of this study was to determine whether the cagE negative strains of A. actinomycetemcomitans serotype B harboured the virB4 gene and vice versa. We hypothesize that the virB4 gene would be present in the serotype b samples that lacked the cagE gene and vice versa. In order to screen the samples for the presence of the virB4 gene conventional polymerase chain reaction (PCR) and quantitative PCR (qPCR) was used. Indeed, we found that the cagE gene and the virB4 gene seldom co-existed in the same strain of A. actinomycetemcomitans, i.e. it can function as a marker for non-cagE genotype strains. However, it could also be concluded that it was common for a strain to lack both of these genes.
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Liu, Hongyan. « ROLES OF TYPE IV SECRETION EFFECTOR ECH0825 IN EHRLICHIA CHAFFEENSIS INFECTION ». The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1376665876.
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Hockenberry, Alyson Marie, et Alyson Marie Hockenberry. « Dissection of the Type IV Pilus Retraction Motor in Neisseria Gonorrhoeae ». Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/622992.
Texte intégralRésumé :
Bacteria of the Neisseria are predominately commensal, though N. gonorrhoeae and N. meningitidis are capable of causing disease. Both of these species often asymptomatically colonize humans, a trait reminiscent of their commensal cousins. The factors that shift the balance between asymptomatic carriage and disease are unknown. Pathogenic Neisseria use retractile surface structures called Type IV pili to coordinate community behavior and to initiate and sustain infection. Previously, the contributions of pilus retraction have been studied by deleting the pilus retraction motor, PilT. Recent findings suggest the speed and force exerted by pilus retraction is responsive to environmental cues. By examining several PilT mutants that maintain the ability to retract pili, I show retraction, per se, is not required for N. gonorrhoeae social interactions with bacteria or with human cells. Furthermore, Type IV pilus retraction by the commensal N. elongata affects the host cell differently than retraction by N. gonorrhoeae. These observations collectively suggest pilus retraction properties shape the host cell response to Neisseria colonization and could tip the balance of asymptomatic colonization to symptomatic disease.
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Sutten, Eric Lynn. « The immunogenicity of the type IV secretion system in Anaplasma marginale ». Pullman, Wash. : Washington State University, 2009. http://www.dissertations.wsu.edu/Thesis/Summer2009/e_sutten_072409.pdf.
Texte intégralRésumé :
Thesis (M.S. in veterinary science)--Washington State University, August 2009.Title from PDF title page (viewed on Aug. 7, 2009). "Department of Veterinary Microbiology and Pathology." Includes bibliographical references.
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Couchman, Edward. « Investigating the Type IV pili of Clostridium difficile and Clostridium sordellii ». Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/48055.
Texte intégralRésumé :
Type IV pili (T4P) are the only type of bacterial pili known to be produced by both Gram-negative and Gram-positive organisms. Though the main pilus shaft consists primarily of only one protein (the major pilin), T4P are unusual in their complexity, requiring multiple (10 or more) different protein components for assembly. Like most types of pili, T4P often function as virulence factors. In particular, T4P frequently operate as adhesins, enabling bacteria on which they are present to stick to each other (to form a biofilm or suchlike) or to adhere directly to host cells. Many T4P systems are able to retract, in which case the T4P may mediate flagella-independent motility. Most research into T4P has historically been performed on Gram-negative organisms, with T4P-encoding genes only being identified in Gram-positive organisms more recently. In particular, all sequenced species of the genus Clostridium are known to encode T4P, but only minimal investigation of these systems has been performed to date. In this study, the T4P of Clostridium difficile were investigated. C. difficile is an important pathogen, being the leading cause of antibiotic-associated diarrhoea in the developed world and thus a considerable burden on Western healthcare systems. By investigating the T4P of this species it was hoped to further elucidate its mechanisms of pathogenicity. Data is presented demonstrating the control of T4P expression by cyclic-di-GMP, and identifying which genes are essential for T4P production in C. difficile. Additionally, a genomic analysis of the related pathogen Clostridium sordellii was performed, using the first high quality genome sequence produced for this species. Genes encoding T4P were identified, analysed and investigated. Furthermore, plasmids carrying the genes encoding the species’ key virulence factors (Lethal Toxin, TcsL, and in some cases haemorrhagic toxin, TcsH) were identified. These plasmids appear to be unstable, a fact with significant implications for diagnosis of C. sordellii disease.
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Moody, Jessica Ashley. « Epigenetic modifications and conserved, non-coding DNA play a role in regulation of type IV collagen gene expression ». [College Station, Tex. : Texas A&M University, 2008. http://hdl.handle.net/1969.1/ETD-TAMU-2839.
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Mohammed, Safiuddin Adil. « Impact of AASHTO LRFD bridge design specifications on the design of Type C and AASHTO Type IV girder bridges ». Texas A&M University, 2005. http://hdl.handle.net/1969.1/4841.
Texte intégralRésumé :
This research study is aimed at assisting the Texas Department of Transportation
(TxDOT) in making a transition from the use of the AASHTO Standard Specifications
for Highway Bridges to the AASHTO LRFD Bridge Design Specifications for the design
of prestressed concrete bridges. It was identified that Type C and AASHTO Type IV are
among the most common girder types used by TxDOT for prestressed concrete bridges.
This study is specific to these two types of bridges. Guidelines are provided to tailor
TxDOT's design practices to meet the requirements of the LRFD Specifications.
Detailed design examples for an AASHTO Type IV girder using both the
AASHTO Standard Specifications and AASHTO LRFD Specifications are developed
and compared. These examples will serve as a reference for TxDOT bridge design
engineers. A parametric study for AASHTO Type IV and Type C girders is conducted
using span length, girder spacing, and strand diameter as the major parameters that are
varied. Based on the results obtained from the parametric study, two critical areas are
identified where significant changes in design results are observed when comparing
Standard and LRFD designs. The critical areas are the transverse shear requirements and
interface shear requirements, and these are further investigated.
The interface shear reinforcement requirements are observed to increase
significantly when the LRFD Specifications are used for design. New provisions for
interface shear design that have been proposed to be included in the LRFD
Specifications in 2007 were evaluated. It was observed that the proposed interface shear
provisions will significantly reduce the difference between the interface shear
reinforcement requirements for corresponding Standard and LRFD designs.The transverse shear reinforcement requirements are found to be varying
marginally in some cases and significantly in most of the cases when comparing LRFD
designs to Standard designs. The variation in the transverse shear reinforcement
requirement is attributed to differences in the shear models used in the two
specifications. The LRFD Specifications use a variable truss analogy based on the
Modified Compression Field Theory (MCFT). The Standard Specifications use a
constant 45-degree truss analogy method for its shear design provisions. The two
methodologies are compared and major differences are noted.
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Kwapong, A. A. « Natural product inhibitors of bacterial type-IV secretion systems and efflux pumps ». Thesis, University College London (University of London), 2016. http://discovery.ucl.ac.uk/1521062/.
Texte intégralRésumé :
Multidrug-resistance is a global health concern and in bacteria it may be conferred by the acquisition of multiple-drug resistance genes and/or by the action of multidrug-efflux pumps. The current study targeted these processes as a means to combat the spread of multidrug resistance genes among bacteria, and reinstate the efficacy of antibiotics against efflux-mediated drug-resistant strains. Our aim was therefore to isolate and characterise natural products that function by either inhibiting bacterial conjugation and/or by potentiating antibiotic activity against efflux-related multidrug-resistant (MDR) strains. Selected medicinal plants, some of which have antibacterial properties (Amoracia rusticana, Borago officinalis, Brassica oleracea, Lepidium sativum, Myristica Iowiana, Sinapis alba, Uncaria tomentosa and Zingiber officinale) were extracted with solvents of increasing polarity. The extracts were then screened against Escherichia coli conjugation pairs of donors (pKM101, IncN; TP114, IncI2; pUB307, IncP; and R7K, IncW), and recipients (ER1793 and JM109), and Staphylococcus aureus strains expressing distinct efflux-related multidrug-resistance pumps; SA-1199B (NorA) and XU212 (TetK). The active extracts were further fractionated using various chromatographic techniques (Thin Layer Chromatography, Solid Phase Extraction, Vacuum Liquid Chromatography, Column Chromatography and High Performance Liquid Chromatography). The compounds, which were isolated from the bioactive fractions, were then characterized by the use of spectroscopic techniques (NMR, MS, IR and UV) and re-assessed for anti-conjugation and antibiotic potentiation activity. The isolated glucosinolates from the Brassica plants showed moderate activity (10 - 50% reduction) against the conjugal transfer of the tested plasmids while the isothiocyanates, which are degradation products of the glucosinolates, showed better broad-range anti-conjugal activity. An amide, isolated from M. lowiana, showed significant anti-conjugal inhibitory activity (16.7 ± 2.0%) against the R7K plasmid. Its anti-conjugal activity was plasmid specific and non-toxic to human dermal fibroblasts, adult cells. In addition, a gingerol compound isolated from Z. officinale, the isolated amide from M. lowiana, and benzyl isothiocyanate, significantly potentiated the activity of norfloxacin and tetracycline against SA-1199B (NorA) and XU212 (TetK), respectively. Their potentiation activity ranged from 2 to 512-fold. In conclusion, the study identified natural product inhibitors of the type-IV secretion-related processes and efflux pump systems. Compounds with such anti-conjugative and antibiotic potentiation activity could help decrease the spread of multidrug resistance genes via conjugation and prolonging the efficacy of existing antibiotics.
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Georgiadou, Michaella. « Functional analysis of the type IV pilus assembly machinery in Neisseria meningitidis ». Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/23787.
Texte intégralRésumé :
Type IV pili (Tfp) are one of the most widespread virulence factors in prokaryotes. Their inherent capacity to mediate an astonishing array of functions differentiates them from other pili and contributes to the pathogenesis of many important human pathogens. Previous intensive efforts by our group in Neisseria meningitidis identified 23 proteins dedicated to Tfp biology, 15 of which are essential for pilus biogenesis. Though these proteins are widely accepted to exert their functions within a large multiprotein complex, the mechanisms governing the biogenesis and functionality of these organelles remained poorly defined. Consequently, the first objective of my project was to perform a large-scale analysis to identify fundamental interactions between 11 Pil proteins from N. meningitidis. To achieve this, we employed the bacterial adenylate cyclase two-hybrid system, which uncovered 20 different binary interactions, many of which are novel and represents the most complex interaction network between Pil proteins reported to date. Significantly, this study revealed that PilE, PilM, PilN and PilO involved in pilus assembly, indeed interact and provided us with a strong foundation to proceed to our main objective, which was to perform a detailed functional analysis of this poorly characterized subcomplex. Using a battery of assays we determined the membrane topology of PilN and PilO, mapped the interaction domains between PilE, PilM, PilN and PilO, and showed that a widely conserved N-terminal motif in PilN is essential for both PilM-PilN interactions and pilus assembly. Furthermore, we established by stability and co-immunoprecipitation studies that PilP (another protein involved in pilus assembly) forms a complex with PilM, PilN and PilO. Finally, we attempted to reconstitute a minimal Tfp assembly machinery in E. coli, however these efforts necessitate further improvements. Taken together, this study has shed light on the molecular mechanisms of Tfp biology and provides a useful blueprint for future studies.
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Sun, Ben Shuang. « Type IV crack characterisation and modelling of high chromium ferritic steel weldments ». Thesis, Loughborough University, 2005. https://dspace.lboro.ac.uk/2134/22734.
Texte intégralRésumé :
In this thesis, the heat affected zone (HAZ) of Gleeble simulated welds, the weldments and the creep specimens for several types of 9%-12% Cr ferritic steels were studied by focusing on the Type IV cracking in the fine grained zone (FZ). The field emission gun transmission electron microscopy (FEGTEM) and scanning electron microscopy (SEM) were used to measure the phosphorus segregation on the grain boundary (GB) and the creep fracture morphologies respectively. Meanwhile the well-developed grain boundary segregation and precipitation (GBSP) model was applied to simulate the experimental results. The experimental results have showed that the HAZ zone was characterised by softening and Type IV cracking. All the high Cr ferritic steel welds gave a microstructure of mainly tempered martensite and M23C6 precipitates after the post weld heat treatment (PWHT). There was no δ-ferrite observed in the HAZ. The Type IV cracking exhibited a mixed cracking mechanism in which the intergranular grain boundary separation is dominant due to the crack initiation by voids and the faster M23C6 growth with the service time. A new model on the mechanism of the Type IV cracking is established. The FEGTEM research has also showed obvious non-equilibrium phosphorus segregation at the grain boundaries, which is affected significantly by the quenching temperature. The phosphorus GB segregation deteriorates the weak grain boundaries. The experimental results were well in agreement with the GBSP modelling.
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Netto, Diogo dos Santos. « Development of a database for classification and analysis of type IV secretion systems ». Laboratório Nacional de Computação Científica, 2008. http://www.lncc.br/tdmc/tde_busca/arquivo.php?codArquivo=198.
Texte intégralRésumé :
The type IV secretion system can be classified as a large family of macromolecule transporters divided in three recognized sub-families involved in different bacterial functions. The major sub-family of T4SS is the conjugation system, which allows transfer of genetic material as a nucleoprotein via cell contact among bacteria. Analogously to bacterial conjugation, the T4SS can transfer genetic material from bacteria to eukaryotic cells; such is the case of T-DNA transfer of Agrobacterium tumefaciens to host plant cells. The system of effector proteins transport constitutes the second sub-family, being indispensable for infection processes of several mammalian and plants pathogens. The third sub-family corresponds to the DNA uptake/release system involved in genetic transformation competence, independently of cell contact, as it was described to the systems VirB/D4 from Campylobacter jejuni and ComB form Helicobacter pylori.
Several essential features of T4SS are well known, but the knowledge in support of an uncomplicated classification or proper protein annotation of system subunits remains confusing, which in same cases can avoid making inferences about evolution of the system in bacterial species.
The purpose of this work was to organize, classify and integrate the knowledge about T4SS through building a database devoted to this bacterial secretion system.
The T4SS database was created using the SGBD MySQL and Perl programming language and with a web interface (HTML/CGI) that gives access to the database. Currently, this database hold genomic data from 43 bacteria and 10 plasmids acquired from the GenBank NCBI, these organisms comprise groups from Actionobacteria to Gram-negative Proteobacteria including symbiotic and pathogenic bacteria.
By applying Bidirectional Best-Hits method was possible to get a core set of 75 clusters with 974 proteins involved in the T4SS. Also, during this procedure BlastP, Muscle e ClustalW algorithms were applied. The database was manually annotated supported by cross references built-in the T4SS annotation pages, such as the UniProtKB/Swiss-Prot, COG, InterPro and TCDB as well as by the methods for signal peptide and transmembrane regions prediction.
All T4SS protein records scattered into 75 ortholog clusters were organized into five different classes of type IV secretion system proteins: (i) Type IVA Mpf/T4CP; (ii) Type IVA Dtr; (iii) F-type plasmid; (iv) IncP-1-type plasmid; (v) Type IVB Icm/Dot. All 974 proteins were annotated into 68 well-known families, which can be involved in conjugation, effector translocator, DNA uptake/release or even can be bifunctional proteins.
Also, by using the Maximum Likelihood method were built 70 unrooted phylogenetic trees that represents just 70 clusters instead of 75, this is due to five clusters had only two protein sequences, five unrooted phylogenetic trees were built for each group of first hierarchical classification, one unrooted phylogenetic trees including proteins from archetype systems of all groups, one unrooted phylogenetic trees from 16S sequence of each organism and one rooted tree including a sequence from a Gram-positive bacteria as an external group.
The phylogenetic analyses show that some proteins of T4SS are more divergent than others, which indicate that for a particular function few sequence mutations were needed, but other proteins required many sequence mutations to get another functions. Thus, these results proved that proteins belong to the same cluster show different functions: conjugation, DNA uptake/release or effector translocator. Consequently, it was possible verify that similar functions were grouped together within phylogenetic tree, which allowed to annotate a probable function of some uncharacterized proteins, that is possibly due to the sequence similarity may reveal a similar evolution to get the same function. Thus, the phylogenetic trees allowed confirming the protein annotation as well as inferring whether uncharacterized proteins would encompass a known function. The T4SS database will be an open access, given to the users searching and submission sequence tools, which will permit to get insights about classification and phylogeny of T4SS sequence of interest.
T4SS Database is accessible at the URL http://www.t4ss.lncc.br.O T4SS pode ser classificado como uma família de transportadores de macromoléculas envolvidos em diferentes funções bacterianas. A maior subfamília do T4SS é a do sistema de conjugação, o qual permite a transferência de material genético entre bactérias. Analogamente à conjugação, o sistema pode transferir material genético entre bactérias e eucariotos, tal como a transferência de T-DNA de Agrobacterium tumefaciens. O sistema de transporte de proteínas efetoras constitui uma segunda subfamília do T4SS, sendo indispensável nos processos de infecção de vários patógenos de mamíferos e plantas. A última subfamília corresponde ao sistema DNA-uptake/release" que funciona independente de contato com uma célula alvo, representado pelos sistemas VirB/D4 de Campylobacter jejuni e ComB de Helicobacter pylori. Muitas características básicas do T4SS são bem conhecidas, entretanto o conhecimento para a classificação simples e intuitiva ou a anotação apropriada das proteínas ainda não está claro, impedindo em alguns casos estabelecer correlações evolutivas deste sistema em bactérias. O objetivo deste trabalho foi o de organizar, classificar e integrar o conhecimento do T4SS através da construção de um banco de dados especializado para este sistema secretório bacteriano. O banco de dados T4SS foi criado utilizando o SGBD MySQL e a linguagem de programação Perl e com uma interface web (HTML/CGI) que fornece acesso ao banco. Este banco consta atualmente com 43 genomas bacterianos e 10 plasmídeos obtidos do GenBank NCBI, estes organismos vão desde Actinobactérias até Proteobactérias Gram-negativas, incluindo simbiontes e patogênicos. Foi utilizada a metodologia do Bidirectional Best-Hits", com a qual foi possível obter um conjunto mínimo de 75 clusters" com 974 proteínas envolvidas no T4SS. Também, durante este procedimento foram utilizados os algoritmos BlastP, Muscle e ClustalW. O banco foi anotado manualmente utilizando referências cruzadas incluídas nas páginas de anotação do T4SS, tais como UniProtKB/Swiss-Prot, COG, InterPro e TCDB e métodos para predição de regiões de peptídeos sinal e transmembrana. As análises do banco T4SS permitiram criar uma classificação hierárquica e funcional para as proteínas do T4SS, consistindo em cinco grupos: (i) Type IVA Mpf/T4CP; (ii) Type IVA Dtr; (iii) F-type plasmid; (iv) IncP-1-type plasmid; (v) Type IVB Icm/Dot). As 974 proteínas foram anotadas em 68 famílias conhecidas, as quais podem estar envolvidas em conjugação, transferência de T-DNA, transferência de proteínas efetoras, DNA-uptake/release" ou bem serem proteínas bifuncionais. Também, através do método de máxima verossimilhança foram geradas 70 árvores filogenéticas não enraizadas (NR) representando apenas 70 clusters, já que cinco clusters apresentaram apenas duas seqüências de proteínas, cinco árvores filogenéticas NR foram criadas para cada grupo da primeira categoria hierárquica, uma árvore NR com representantes de todos os grupos, uma árvore NR gerada a partir das seqüências 16S de cada organismo e uma árvore de um cluster incluindo uma seqüência de bactéria Gram-positiva como grupo externo. As análises filogenéticas mostram que determinadas proteínas do sistema são mais divergentes que outras, indicando que para uma determinada função poucas mutações de seqüências foram necessárias, já outras proteínas precisaram de maiores mutações para adquirir outras funções. Por isso, verifica-se que proteínas de um mesmo cluster apresentam diferentes funções: conjugação, DNA-uptake/release", traslocadores de proteínas efetoras. Conseqüentemente, foi possível verificar que funções semelhantes se agruparam juntas nas árvores filogenéticas, permitindo anotar uma função provável das proteínas ainda não caracterizadas (unknown"), isto possivelmente devido a que em virtude de sua semelhança de seqüências, possivelmente evoluíram para realizar a mesma função. Assim, as arvores possuíram a finalidade de confirmar a anotação e contribuíram permitindo inferir se os unknown" ou probable" podem ser de uma determinada classificação funcional. O banco T4SS será de uso público, oferecendo ao usuário ferramentas de buscas e submissão de seqüências, as quais permitirão inferir respostas sobre a classificação e filogenia da seqüência T4SS de interesse. O banco de dados T4SS pode ser acessado na URL: http://www.t4ss.lncc.br.
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Gurung, Ishwori. « Deciphering type IV pilus biology in the Gram-positive opportunistic pathogen Streptococcus sanguinis ». Thesis, Imperial College London, 2017. http://hdl.handle.net/10044/1/55879.
Texte intégralRésumé :
Type IV pili (Tfp) are the paradigm of a large group of diverse and functionally versatile nanomachines, intensively studied in Gram-negative bacteria. However, details regarding the molecular mechanisms of Tfp biogenesis and/or mediated functions are still unclear. Thus, owing to the inherent lack of outer cell wall in Gram-positive bacteria, my PhD has focused on molecular characterisation of Tfp in a simpler such bacterium Streptococcus sanguinis. My work has shown that the naturally competent S. sanguinis produces bona fide retractable Tfp enabling twitching motility, but dispensable for competence. Unlike Gram-negative Tfp, we show that S. sanguinis Tfp are unusual since they are composed of two pilin proteins, a feature likely to be shared by other Gram-positive Tfp-expressing species. All the genes involved in Tfp biology in S. sanguinis are found within a pil locus encoding 21 proteins. A systematic genetic study highlighted that 10 proteins only are required for Tfp biogenesis, whilst another four modulate twitching motility. To enhance genetic manipulation of S. sanguinis, a markerless mutagenesis strategy was devised enabling us to make various mutations in situ, which helped us characterise some of these proteins further. Via this methodology, the last six genes of the pil locus were found to be completely dispensable for Tfp biology. To get an overall structural picture of Tfp in S. sanguinis, the structure of one of the major pilins (PilE1) was determined by NMR. Moreover, three pilin-like proteins within the pil locus were found to be minor Tfp components. Collectively, my work has established S. sanguinis as a robust Gram-positive model organism for studying Tfp, which paves way for interesting future studies.
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Ramont, Laurent. « Contrôle de l'invasion tumorale par le collagène de type IV et TGF-β1 ». Reims, 2003. http://www.theses.fr/2003REIMM202.
Texte intégralRésumé :
@Au cours de l'invasion tumorale, les cellules cancéreuses traversent les membranes basales des vaisseaux sanguins en dégradant un de leurs constituants majeurs, le collagène de type IV. Cette dégradation implique la sécrétion et l'activation de protéinases, dont les métalloprotéinases matricielles (MMPs) et le système activateur du plasminogène (uPA, tPA, PAI-1). Nous avons comparé les effets de TGF-b1 et du peptide a3(IV) 185-203 du domaine NC1 de la chaîne a3(IV) du collagène de type IV dans un modèle de mélanome de souris in vivo et in vitro. Le TGF-b1 et le peptide a3(IV) 185-203 inhibent la croissance tumorale in vivo. In vitro, ils diminuent la migration des cellules de mélanome à travers des membranes recouvertes d'un gel de Matrigel. Le peptide a3(IV) 185-203 induit une diminution de la prolifération cellulaire et diminue la synthèse de la pro-MMP2. Le TGF-b1 et le peptide a3(IV) 185-203 induisent tous deux une forte diminution de l'activité uPA et tPA, ainsi que de l'activité génératrice de plasmine. L'ensemble de ces résultats indiquent que TGF-b1 et le peptide a3(IV) 185-203 contrôlent les propriétés invasives des cellules de mélanome par des mécanismes différents. Ils pourraient donc jouer un rôle protecteur complémentaire au cours de l'invasion tumorale@Tumor invasion is characterized by a sequential proteolytic degradation of extracellular matrix and basement membranes. The degradation of basement membranes by tumor cells involves secretion and activation of proteinases, such as matrix metalloproteinases (MMPs) and the plasminogen activation system (u-PA, t-PA, PAI-1). We studied the effect of TGF-b1 and the peptide 185-203 of the NC1 domain a3(IV) chain. TGF-b1 inhibited in vivo tumor growth after subcutaneous injection of B16F1 cells in syngenic mice. TGF-b1 did not alter B16F1 cell proliferation, but strongly decreased their migration though Matrigel-coated membranes. TGF-b1 triggered a large decrease of u-PA and t-PA and a strong increase of PAI-1 synthesis. The [NC1 a3(IV) 185-203] peptide inhibited in vivo tumor growth after subcutaneous injection of B16F1 cells in syngenic mice. The peptide inhibited cell proliferation and the migration of B16F1 though Matrigel-coated membranes. The [NC1 a3(IV) 185-203] also inhibed the expression of pro-MMP-2. These results suggest that TGF-b1 may inhibit melanoma tumor growth by decreasing plasmin activity. The peptide of the [NC1 a3(IV) 185-203] may inhibit melanoma tumor growth by decreasing MMP activity
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Borchiellini, Carole. « Rôle du collagène de type IV au cours du développement de Drosophila melanogaster ». Aix-Marseille 2, 1995. http://www.theses.fr/1995AIX22073.
Texte intégralRésumé :
L'objectif de notre etude s'inscrit dans l'ensemble des travaux devolus a l'analyse de la fonction des membranes basales, tant chez les vertebres que chez les invertebres, et presente donc un interet d'ordre general. Ces etudes sont justifiees par l'extraordinaire variete de fonctions attribuees aux membranes basales au cours du developpement. La presence de molecules identiques (ou similaires) dans les membranes basales, chez des especes eloignees phylogenetiquement suggere que les processus impliques dans le developpement reposent sur des mecanismes qui ont ete conserves au cours de l'evolution. Parmi ces molecules, le collagene est un composant constant de tous les organismes, des eponges jusqu'a l'homme. L'etude in vivo de la fonction du collagene de type iv, chez la drosophile, par le biais de mutants dominants negatifs, a permis de demontrer son role structural et informatif majeur dans le controle des migrations cellulaires necessaires a certains mouvements morphogenetiques essentiels tels que la retraction de la bande germinative et la fermeture de l'epiderme dorsal. Nous avons egalement mis en evidence son implication durant les processus de neurogenese et de myogenese, notamment dans l'etablissement des jonctions muscle-epiderme. Cette etude a permis, egalement, d'envisager un role du collagene dans l'etablissement de l'asymetrie droite-gauche. Il ressort de cette etude que l'integrite fonctionnelle de ce reseau collagenique est necessaire pour permettre les echanges de signaux entre la membrane basale et les recepteurs cellulaires (integrines et autres recepteurs des macromolecules matricielles). Nous avons etabli que le collagene de type iv peut participer directement a ces echanges d'informations avec les cellules adjacentes et/ou contribuer au positionnement adequat d'autres macromolecules de la matrice extracellulaire vis a vis de ces cellules. En outre, compte tenu de la grande conservation de la structure des membranes basales, en general et du collagene de type iv, en particulier, a travers toute la phylogenese, nos resultats peuvent egalement participer a une meilleure comprehension des mecanismes du developpement chez les vertebres
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Murray, Samantha Rose. « Characterization of Type IV Pilus System Genes and Their Regulation in Clostridium perfringens ». Thesis, Virginia Tech, 2017. http://hdl.handle.net/10919/86173.
Texte intégralRésumé :
Clostridium perfringens is a Gram-positive (Gr+) anaerobic pathogen that was found to contain Type IV pilus (T4P) system genes within the genomes of all its sequenced strains. T4P are widely used in Gram-negative organisms for aggregation, biofilm formation, adherence, and DNA uptake. Because few examples of T4P-utilizing Gram-positive bacteria are studied to date, we wanted to characterize the T4P system in this Gr+ bacterium.
To understand the regulation of T4P genes and therefore better understand their expression, we employed the highly powerful next-generation sequencing tool RNA-seq in a variety of conditions. RNA-seq uncovered previously unknown regulatory mechanisms surrounding T4P genes as well as provided transcriptional information for most of the genes in the C. perfringens strain 13 genome. We also utilized reporter gene assays to look at post-transcriptional regulation of T4P promoters.
The wealth of RNA-seq data acted as a jumping-off point for many smaller projects involving transcriptional regulators that may influence T4P expression. We investigated a novel small RNA in close proximity to the major T4P operon, as well as two little-characterized transcriptional regulators that function in the same conditions as T4P genes. RNA-seq also provided data to develop a method for protein purification from C. perfringens without induction.Master of Science
41
Kobayashi, Kaori. « Expression of 17 β-hydroxysteroid dehydrogenase type IV in chick retinal pigment epithelium ». Kyoto University, 1997. http://hdl.handle.net/2433/202174.
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Lavallée, Annet. « Identification et caractérisation d'un locus de fimbriae de type IV chez Actinobacillus pleuropneumoniae / ». Thèse, Trois-Rivières : Université du Québec à Trois-Rivières, 2007. http://www.uqtr.ca/biblio/notice/resume/30024847R.pdf.
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Lavallée, Annet. « Identification et caractérisation d'un locus de fimbriae de type IV chez Actinobacillus pleuropneumoniae ». Thèse, Université du Québec à Trois-Rivières, 2007. http://depot-e.uqtr.ca/1506/1/030024847.pdf.
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Perrin, Ian James. « Computer-based type IV creep CDM design of low alloy ferritic steel weldments ». Thesis, University of Manchester, 1995. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.617095.
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Raboisson, Pierre. « Purification de la phosphodiesterase de type-iv a l'aid'e molecules a structure adenine ». Strasbourg 1, 1995. http://www.theses.fr/1995STR15073.
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Hartman, Andrea H. « Use of an Inducible Promoter to Characterize Type IV Pili Homologues in Clostridium perfringens ». Thesis, Virginia Tech, 2012. http://hdl.handle.net/10919/76874.
Texte intégralRésumé :
Researchers of Clostridium perfringens, a Gram-positive anaerobic pathogen, were lacking a tightlyregulated, inducible promoter system in their genetic toolbox. We constructed a lactose-inducible plasmid-based system utilizing the transcriptional regulator, BgaR. Using the E. coli reporter GusA, we characterized its induction in three different strains of C. perfringens. We then used a newly-developed mutation system to create in-frame deletion mutants in three genes with homology to Type IV pilins, and we used the promoter system described above to complement the mutants. We analyzed each pilin for localization and expression, as well as tested each of the mutants for various phenotypes frequently associated with type IV pili (TFP) and type II secretion systems. PilA2, PilA3, and PilA4 localized to the poles of the cells. PilA2 was expressed in the wildtype when C. perfringens was grown on agar plates, and the PilA3 mutant lacked a von Willebrand factor A domain-containing protein in its secretome. We used our promoter system to express GFP-tagged versions of the TFP ATPase homologues and view them in cells growing on surfaces. We saw that PilB1 and PilB2 co-localized nearly all of the time, while a portion of PilT was independent of the PilB proteins. PilT appeared necessary for the localization of PilB, and it localized independently of TFP proteins in Bacillus subtilis. PilT's typical localization in Bacillus subtilis was disrupted when the GTPase and polymerization activity of cell division protein FtsZ was blocked, suggesting that PilT associates with cell division proteins.Master of Science
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Fisher, Patrick William. « Type-5 Phosphodiesterase Inhibition in the Prevention of Doxorubicin Cardiomyopathy ». VCU Scholars Compass, 2005. http://scholarscompass.vcu.edu/etd/1162.
Texte intégralRésumé :
Prior studies have demonstrated the effect of diazoxide in protecting against apoptosis via mitochondrial KATP channel opening in vitro. The current investigations are designed to determine if sildenafil, a phosphodiesterase-5 inhibitor and known mitochondrial KATP channel opener, would protect against chronic doxorubicin cardiomyopathy both in vivo and in vitro.Male ICR mice were randomized to 1 of 4 treatments: saline, sildenafil (0.7 mg/kg IP), doxorubicin (5 mg/kg IP), and sildenafil (0.7 mg/kg IP)+doxorubicin. Apoptosis was determined using the terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling and in situ oligo ligation methods. Desmin distribution was determined via immunofluorescence. Bcl-2 was analyzed by Western blot. Left ventricular function was measured in Langendorff mode. Electrocardiographical analysis measured changes indicative of doxorubicin cardiotoxicity (ST-prolongation). In vitro studies using adult ventricular cardiomyocytes were exposed to doxorubicin (1 μM), sildenafil (1 μM) with or without NG-nitro-L-arginine methyl ester (L-NAME; 100 μM), or 5-hydroxydecanoate (5-HD; 100 μM) 1 hour before doxorubicin and incubated for 18 hours. Doxorubicin-treated mice demonstrated increased apoptosis and desmin disruption, which was attenuated in the sildenafil+doxorubicin group. Bcl-2 decreased in the doxorubicin group but was maintained at basal levels in the sildenafil+doxorubicin group. Left ventricular developed pressure and rate pressure product were significantly depressed in the doxorubicin group but attenuated in the sildenafil+doxorubicin group. ST-interval significantly increased in the doxorubicin group over 8 weeks. In the sildenafil+doxorubicin group, ST-interval remained unchanged from baseline. Doxorubicin significantly increased apoptosis, caspase-3 activation, and disruption of mitochondrial membrane potential in vitro,. In contrast, sildenafil significantly protected against doxorubicin cardiotoxicity; however, protection was abolished by both L-NAME and 5-HD. Cell viability studies using spectrophotometer and flow cytometric techniques demonstrated that sildenafil did not affect the antitumor efficacy of doxorubicin in PC-3 cells in vitro. In fact, flow cytometry data indicate that sildenafil, when combined with doxorubicin, was synergistic in the antineoplastic action of doxorubicin. Prophylactic treatment with sildenafil prevented apoptosis and left ventricular dysfunction in a chronic model of doxorubicin-induced cardiomyopathy. Moreover, these studies provide relevant clinical data on the safety and efficacy of sildenafil, leading the way for clinical trials in humans receiving doxorubicin chemotherapy.
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Deak, Noémi. « Ligands soufrés (IV)/(VI) de type pince pour la stabilisation des métallylènes : synthèse, caractérisation et applications ». Thesis, Toulouse 3, 2017. http://www.theses.fr/2017TOU30393/document.
Texte intégralRésumé :
Ce travail de thèse présente la synthèse, la caractérisation et la réactivité de métallylènes stabilisés par différents ligands pinces fonctionnalisés par des substituants soufrés à différents états d'oxydation. Les métallylènes, analogues lourds des carbènes, présentent dans leur état fondamental singulet une paire d'électrons et une orbitale p vacante. Ces caractéristiques donnent à ces espèces un comportement et une réactivité particulière. La littérature décrit de nombreux exemples de métallylène stabilisés par différents types de ligands, parmi lesquels les ligands de type pince gagnent actuellement en importance. Dans la chimie des complexes de métaux de transition, il a déjà été démontré que les ligands de type pince constituent un type de plateforme efficace grâce aux possibilités de modulation des propriétés des complexes liées à la modulation du squelette du ligand. Au cours des dernières décennies, ces ligands se sont révélés efficaces pour la stabilisation de métallylènes stables. Cependant, un seul exemple de métallylène stabilisé par un ligand de type pince contenant du soufre a été reporté dans la littérature. Au cours de cette étude, des ligands pinces O,C,O chélatants contenant des groupement sulfonyles et sulfinyles ont été conçus, synthétisés et complètement caractérise par les méthodes physico-chimiques et computationnelle et leurs effets sur la stabilisation des métallylènes ont été étudiés. Dans un premier temps, un ligand pince de type bis-sulfone a été obtenu et étudié pour la synthèse de nouveaux métallylènes. A partir de ce ligand, un germylène et un stannylène ont été caractérisés, le germylène étant le premier exemple dans la littérature d'une espèce de germanium divalente stabilisée par un ligand pince de donneur d'oxygène. La réactivité des métallylènes a été testée pour obtenir des produits de cycloaddition avec l'ortho-benzoquinone et des complexes des métaux de transition (fer et tungstène). Il a été démontré que la bis-sulfone se comporte comme un ligand ajustable de type pince O,C,O-chélatant, la coordination pouvant être possible par l'un ou l'autre des atomes d'oxygène des groupements sulfonyles. [...]This work presents the synthesis, characterization and reactivity of metallylenes stabilized by different functionalized pincer ligands with substituents containing sulfur atoms in different oxidation states. Metallylenes, the heavier analogues of carbenes, have in their singlet ground state a lone pair of electron and a vacant p orbital. These features give the particular behavior and reactivity of these species. The literature describes many examples of metallylenes stabilized by different types of ligands, among which lately the pincer type ligands are gaining importance. In the chemistry of transitional metal complexes it was shown for a long time that the pincer-type ligands are a versatile ligand platform thanks to the possibilities of modulating the properties of the complexes through small changes of the ligand backbone. In the last few decades these ligands proved to be effective for obtaining stable metallylenes. However, in the literature there is one sole example of metallylene stabilized through sulfur containing pincer-type ligands. For this study, para substituted sulfonyl and sulfinyl containing O,C,O-chelating pincer ligands were designed, synthesized and completely characterized by the physico-chemical and computational methods and their effects on the stabilization of metallylenes was investigated. First, a bis-sulfone pincer ligand was obtained and studied for the synthesis of novel metallylenes. With this ligand a germylene and stannylene were obtained and characterized, the germylene being the first example in the literature of a divalent germanium species stabilized by an oxygen donor pincer ligand. The reactivity of the metallylenes was tested for obtaining cycloadducts with ortho-benzoquinone and transition metal complexes (iron and tungsten). It was shown that the bis-sulfone behaves as an adjustable O,C,O-chelating pincer type ligand, the coordination could be possible with either one of the oxygen atoms of the sulfonyl groups. [...]
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Engledow, Amanda Suzanne. « Role of type IV secretion systems in trafficking of virulence determinants of Burkholderia cenocepacia ». Thesis, [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1841.
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Soininen, Raija. « Structure of the gene for the [alpha] 1 chain of human type IV collagen ». Oulu, Finland : University of Oulu, 1989. http://catalog.hathitrust.org/api/volumes/oclc/20482376.html.
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