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BRAMBILLA, MARCO. "Rewiring yeast nitrogen and mRNA metabolism for eliciting industrially relevant phenotypes. The Saccharomyces cerevisiae glutamate synthase (Glt1) and the poly(A) binding protein (Pab1) as case studies." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2018. http://hdl.handle.net/10281/198932.
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Le attuali produzioni di energia e materiali si basano soprattutto sullo sfruttamento di fonti fossili, ma la loro non rinnovabilità e la necessità di una maggiore sostenibilità ambientale han contribuito ad aumentare l’interesse verso risorse alternative. In questo contesto si collocano le bioraffinerie, che sono volte alla conversione di biomasse rinnovabili in prodotti commerciabili. Per implementare bioindustrie competitive, molte sfide devono essere superate, tra cui il miglioramento delle prestazioni dei microrganismi, cell factory, utilizzati nei processi produttivi. Tra tutte, il lievito Saccharomyces cerevisiae è la principale cell factory per la produzione di bioetanolo come biocarburante. L'obiettivo principale di questa tesi è stato di ingegnerizzare ceppi di S. cerevisiae per ottenere una cell factory con caratteristiche rilevanti a livello industriale, tra cui aumentate performances di crescita e un incremento della termotolleranza. In particolare, è stato studiato e quindi indotto un profondo rewiring cellulare, selezionando due proteine: la glutammato sintasi Glt1, un enzima del metabolismo centrale dell'azoto, CNM e la principale polyA binding protein Pab1, un fattore chiave nel metabolismo dell'mRNA. Gli effetti fisiologici della delezione e overespressione di GLT1 sono stati valutati coltivando S. cerevisiae con diverse fonti di azoto. I risultati mostrano che l'aggiunta di solfato d’ammonio, glutammato o glutammina, ma non la modulazione dell’espressione di GLT1, influenza notevolmente la crescita, il contenuto proteico, la vitalità e l'accumulo di specie reattive dell'ossigeno ROS. Questi dati evidenziano la plasticità del CNM di S. cerevisiae rispetto a variazioni ambientale e confermano la sua robustezza contro perturbazioni interne. Inoltre, sebbene la modulazione dell'espressione di GLT1 potrebbe non indurre una riprogrammare cellulare, la caratterizzazione fisiologica descritta puo essere utile per selezionare altri target pio promettenti per riprogrammazioni cellulari. L’induzione del rewiring cellulare è stato poi valutato selezionando Pab1 come target. A tal scopo, un ceppo recante la copia endogena di PAB1 è stato trasformato con una mutant library plasmidica di PAB1 e poi sottoposto a screening per isolare ceppi con maggior termotolleranza. I cloni isolati han mostrato, in drop test, una maggior crescita ad alte temperature e alte concentrazioni di etanolo. Tra tutte, la variante PAB1 S40.7 conferisce una maggiore termotolleranza tramite l’espressione dei soli primi 20 amminoacidi di Pab1. Tale fenotipo è stato anche confermato in bioreattore a 40°C. Una possibile ragione della maggior termotolleranza del ceppo S40.7 potrebbe essere collegata ad un minor accumulo di ROS rispetto al ceppo di controllo. Nel complesso, questi risultati han dimostrato che Pab1 è un promettente target per indurre fenotipi complessi con tratti migliorati, tra cui una maggiore termotolleranza. Infine, Pab1 è stata caratterizzata per determinare il ruolo dei suoi domini nel suo reclutamento all'interno di stress granules, SG, aggregati di mRNP non tradotti che si formano in condizioni stressanti. Questa caratterizzazione ha mostrato che l'associazione di Pab1 negli SG è principalmente dovuta agli RNA Recognition Motives RRM, il cui numero è importante per un efficiente reclutamento. Sebbene i domini P e C non partecipino direttamente all'associazione di Pab1 negli SG, la loro presenza rafforza o diminuisce, rispettivamente, la localizzazione di varianti sintetiche di Pab1 prive di almeno un RRM in questi aggregati. Questo lavoro suggerisce inoltre che i domini di Pab1 potrebbero essere sfruttati razionalmente per scopi di biologia sintetica. Nel complesso, i risultati di questa tesi evidenziano la difficoltà ma allo stesso tempo la capacità del rewiring cellulare nell’indurre fenotipi industrialmente rilevanti e, in particolare, Pab1 è emerso come target promettente per questo approccio.Nowadays, for the production of energy and materials, our society mainly relies on fossil sources, but many concerns arise from their utilization, like greenhouse gases emission and non-renewability within the time of their consumption. Hence, biorefineries, which convert renewable biomasses into products and energy, could be a promising alternative. Despite some biorefineries are now at commercial scale, many challenges must be overcome to implement competitive bio-based industries, such as improving the performances of microorganisms, named cell factory, used during the production processes. Above all, the yeast Saccharomyces cerevisiae is the most prominent cell factory for producing bioethanol as biofuel. The main objective of this thesis was to engineer S. cerevisiae strains with biotechnological interesting traits, among which improved growth performances and increased thermotolerance. For this purpose, we investigated and applied a cellular rewiring, by selecting two targets: the glutamate synthase Glt1 that is an enzyme of the central nitrogen metabolism, CNM, and the main polyA binding protein Pab1, a master regulator of mRNA metabolism. Regarding GLT1, the physiological effects of its deletion and over-expression were assessed by growing yeasts in the presence of different nitrogen sources. Results showed that the supplementation of ammonium sulfate, glutamate or glutamine considerably affects growth, protein content, viability and reactive oxygen species, ROS, accumulation. Conversely, GLT1 modulation does not significantly influence these parameters. Overall, these data highlight the plasticity of the S. cerevisiae CNM in respect to the environment and confirm its robustness against internal perturbation. Moreover, even though the sole modulation of GLT1 expression might not reprogram the entire cell, the physiological characterization of this study might be helpful to guide the selection of other more promising candidate for the application of the rewiring approaches. Then, the induction of cellular reprogramming was assessed selecting Pab1. To this purpose, a strain carrying the unaltered PAB1 chromosomal allele was transformed with a PAB1 plasmid mutant library and then screened for isolating strains with high thermotolerance. The isolated clones showed growth improvement at both high temperatures and ethanol concentration by drop tests. Among all, the PAB1 S40.7 variant was further characterized because, strikingly, it dominantly confers higher thermotolerance by expressing just the first 20 amino acidic residues of Pab1. This improved phenotype was also confirmed in bioreactor at 40°C. Remarkably, the S40.7 strain accumulates less ROS compared to the control strain, thus possibly explaining its increased thermotolerance. Overall, these results demonstrated that Pab1 is a powerful candidate to evoke complex phenotypes with improved traits, among which higher thermotolerance. Finally, Pab1 was characterized to uncover the role of its domains in the recruitment of the protein within stress granules, aggregates of untranslated mRNPs that form during stressful conditions. This characterization shows that Pab1 association into these aggregates relies mainly on RNA recognition motives, RRM, whose number is important for an efficient recruitment. Although the P and C domains do not directly participate in Pab1 association to stress granules, their presence strengthens or decreases, respectively, the distribution of synthetic Pab1 variants lacking at least one RRM into these aggregates. As additional outcome, this part of the work is suggesting that Pab1 domains might be rationally exploited for synthetic biology purposes. Overall, the results of this thesis highlight and confirm the difficulty but at the same time the power of cellular rewiring in evoking industrially relevant phenotype. Pab1 is undoubtedly emerging as a pivotal element that deserves more attention for future strain design and tailoring.
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Grenier, St-Sauveur Valérie. "Caractérisation et fonction de la protéine Nab2 chez Schizosaccharomyces pombe." Mémoire, Université de Sherbrooke, 2014. http://savoirs.usherbrooke.ca/handle/11143/105.
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L’étude qui vous est présentée dans ce mémoire est réalisée chez l’organisme Schizosaccharomyces pombe et elle porte sur la caractérisation de la protéine Nab2, une protéine liant les queues poly(A) ainsi que sur son implication dans la régulation génique. Tout d’abord, il faut savoir qu’il existe quatre modes de régulation (transcriptionnel, post-transcriptionnel, traductionnel et post-traductionnel) dans lesquels interviennent différents types de protéines, en particulier des protéines liant les queues poly(A) des ARN, aussi connues sous le nom de PABPs. Ces protéines reconnaissent l’ARN à l’aide de différents domaines de liaison et elles se subdivisent en deux catégories : les PABPs nucléaires ou cytoplasmiques, représentées respectivement par PABPN1 et PABPC1 chez les mammifères. Comprendre la fonction des PABPs revêt un intérêt particulier puisqu’elles sont impliquées à différents stades de la régulation génique. Des maladies ont aussi été associées à deux PABPs nucléaires humaines, PABPN1 et ZC3H14, mais aucune association entre leur fonction réciproque et la maladie n’a pu être établie. Une des façons de comprendre leur rôle est d’étudier celui de leurs orthologues respectifs. Chez la levure à fission, un orthologue de PABPN1 a été caractérisé et il s’agit de Pab2. S. pombe possède cependant une seconde PABP nucléaire, Nab2, qui est caractérisée dans ces travaux. Des méthodes in vivo et in vitro ont été utilisées afin de confirmer le statut de la protéine, à savoir qu’il s’agit bel et bien d’une PABP nucléaire et que celle-ci est non essentielle. L’identification de partenaires protéiques de Nab2 par spectrométrie de masse a aussi permis de relier Nab2 avec des processus de régulation génique tels que l’épissage et la dégradation. Puisque Pab2 est aussi associée à des fonctions en lien avec la dégradation, il est possible de faire un parallèle entre ces deux protéines et de supposer qu’elles interagissent ensemble. La deuxième partie de ces travaux porte donc sur l’étude de la relation fonctionnelle entre Nab2 et Pab2 et elle a permis de montrer un mécanisme de régulation opportuniste basé sur la liaison de la cible ARN par l’une ou l’autre de ces PABPs. En effet, l’étude de la régulation du gène modèle RPL30-2 indique que Nab2 et Pab2 ont des rôles opposés puisqu’ils sont respectivement des régulateurs positifs et négatifs de l’expression de son transcrit.
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Grenier, St-Sauveur Val??rie. "Caract??risation et fonction de la prot??ine Nab2 chez Schizosaccharomyces pombe." Mémoire, Universit?? de Sherbrooke, 2014. http://savoirs.usherbrooke.ca/handle/11143/105.
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L?????tude qui vous est pr??sent??e dans ce m??moire est r??alis??e chez l???organisme Schizosaccharomyces pombe et elle porte sur la caract??risation de la prot??ine Nab2, une prot??ine liant les queues poly(A) ainsi que sur son implication dans la r??gulation g??nique. Tout d???abord, il faut savoir qu???il existe quatre modes de r??gulation (transcriptionnel, post-transcriptionnel, traductionnel et post-traductionnel) dans lesquels interviennent diff??rents types de prot??ines, en particulier des prot??ines liant les queues poly(A) des ARN, aussi connues sous le nom de PABPs. Ces prot??ines reconnaissent l???ARN ?? l???aide de diff??rents domaines de liaison et elles se subdivisent en deux cat??gories : les PABPs nucl??aires ou cytoplasmiques, repr??sent??es respectivement par PABPN1 et PABPC1 chez les mammif??res. Comprendre la fonction des PABPs rev??t un int??r??t particulier puisqu???elles sont impliqu??es ?? diff??rents stades de la r??gulation g??nique. Des maladies ont aussi ??t?? associ??es ?? deux PABPs nucl??aires humaines, PABPN1 et ZC3H14, mais aucune association entre leur fonction r??ciproque et la maladie n???a pu ??tre ??tablie. Une des fa??ons de comprendre leur r??le est d?????tudier celui de leurs orthologues respectifs. Chez la levure ?? fission, un orthologue de PABPN1 a ??t?? caract??ris?? et il s???agit de Pab2. S. pombe poss??de cependant une seconde PABP nucl??aire, Nab2, qui est caract??ris??e dans ces travaux. Des m??thodes in vivo et in vitro ont ??t?? utilis??es afin de confirmer le statut de la prot??ine, ?? savoir qu???il s???agit bel et bien d???une PABP nucl??aire et que celle-ci est non essentielle. L???identification de partenaires prot??iques de Nab2 par spectrom??trie de masse a aussi permis de relier Nab2 avec des processus de r??gulation g??nique tels que l?????pissage et la d??gradation. Puisque Pab2 est aussi associ??e ?? des fonctions en lien avec la d??gradation, il est possible de faire un parall??le entre ces deux prot??ines et de supposer qu???elles interagissent ensemble. La deuxi??me partie de ces travaux porte donc sur l?????tude de la relation fonctionnelle entre Nab2 et Pab2 et elle a permis de montrer un m??canisme de r??gulation opportuniste bas?? sur la liaison de la cible ARN par l???une ou l???autre de ces PABPs. En effet, l?????tude de la r??gulation du g??ne mod??le RPL30-2 indique que Nab2 et Pab2 ont des r??les oppos??s puisqu???ils sont respectivement des r??gulateurs positifs et n??gatifs de l???expression de son transcrit.
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Penheiter, Kristi L. "Functional characterization of the Paf1 complex in Saccharomyces cerevisiae by identification of Paf1 target genes /." Connect to full text via ProQuest. IP filtered, 2005.
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Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005.Typescript. Includes bibliographical references (leaves 126-149). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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de, Carli da Costa Lima Tamara. "Caracterização funcional de homólogos à proteína de ligação a Cauda Poli-A (PABP) de Leishmania major." Universidade Federal de Pernambuco, 2007. https://repositorio.ufpe.br/handle/123456789/6362.
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Previous issue date: 2007A proteína de ligação à cauda poli-A (PABP) se liga a seqüência de poli adenosinas presente na extremidade 3 do mRNA e possui uma multiplicidade de funções dentro da célula. Dentre as funções atribuídas a PABP destaca-se sua participação em eventos da síntese de proteínas, tais como iniciação e terminação da tradução e reciclagem dos ribossomos, e seu envolvimento no transporte de alguns mRNAs do núcleo para o citoplasma. O objetivo deste trabalho é caracterizar homólogos de proteínas relevantes à iniciação da tradução em tripanosomatídeos. O estudo da PABP teve início com a busca em bancos de dados de L. major de possíveis homólogos, onde foi encontrado tanto o homólogo previamente descrito (LmPABP1) como mais dois homólogos (LmPABP2 e LmPABP3), sendo que o último não possui ortólogos em Trypanosoma. Os três genes foram amplificados, clonados, as proteínas expressas e utilizadas para produção de soro policlonal. Em seguida foi realizada a quantificação dos níveis intracelulares das três proteínas em extratos da forma promastigota de L. major sendo a LmPABP2 a mais abundante, a LmPABP3 em níveis intermediários e a LmPABP1 a menos abundante. No entanto, as LmPABP2-3 são detectadas com uma única forma, enquanto que a LmPABP1 está presente em duas isoformas, provavelmente uma delas devido a fosforilação. Análises da expressão durante o ciclo evolutivo do parasita mostrou que as três proteínas encontravam-se presentes em todas as formas evolutivas, porém a LmPABP3 mostrou um decréscimo na fase estacionária de crescimento e a LmPABP1 apresenta-se fosforilada apenas nas primeiras horas após o repique. Experimentos de localização subcelular indicam que a LmPABP1 está presente na fração citoplasmática, enquanto que LmPABP2-3 estão presentes tanto na fração citoplasmática quanto na nuclear, porém com predominância da fração nuclear. Estudos adicionais precisam ser feitos pra entender como estas proteínas diferem funcionalmente e quais seus papéis na síntese protéica
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Siddiqui, Nadeem. "Structure and function of the PABC domain." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=102727.
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The poly (A)-binding protein (PABP) is an essential protein found in all eukaryotes and functions in mRNA metabolic and translational processes. Structurally, PABP consists of two distinct regions. The N-terminal half contains four RNA recognition motifs that bind to the poly (A)-tail of mRNA, while the C-terminal segment contains a unique peptide binding module referred to as the PABC domain. The function of this domain in PABP is to recruit proteins containing a very specific 'PAM-2' motif to the mRNP complex. Unique to metazoans, a PABC domain is also found in the hyperplastic discs tumor suppressor (HYD), which is an E3 ubiquitin ligase.This thesis completes a structural investigation of PABC domains from various species by nuclear magnetic resonance spectroscopy. In particular, we report the solution structure of PABC from the parasite Trypanosoma cruzi and plant Triticum aevestium PABP. Both domains consist of five alpha-helices which fold into a structure highly comparable to the human PABC domain from PABP and HYD. All four PABCs interact with a similar PAM-2 sequence and show comparable peptide binding surfaces. The human PABC-PAM-2 complex structure displays a PAM-2 peptide interacting with specific residues within the domain. Sequence analyses and peptide surface mapping studies show that these residues are highly conserved, which indicates an analogous mechanism of peptide recognition throughout animal, parasite, and plant species. An exception to these observations was found in the PABC domain from Saccharomyces cerevisiae PABP. Yeast PABC recognizes a variation of the typical PAM-2 motif but mediates its interaction through a similar mechanism as human PABC.
The PAM-2 motif encloses a signature sequence which was used to successfully identify new interacting partners for the PABC domain via a bioinformatics screen. In mammalian systems, the identified proteins are implicated in either RNA metabolic, translational, or ubiquitin associated functions. This thesis concludes with a model illustrating a unique cross-talk between major gene expression pathways mediated by the PABC domain and its binding partners.
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Tsui, Hoyee. "The role of p21-activated kinase 1 (Pak1) in the heart." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/the-role-of-p21activated-kinase-1-pak1-in-the-heart(8c34d7bc-a2aa-4ae0-a197-91ed905212f5).html.
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Heart failure is associated with a high mortality rate and is one of the most prevalent diseases worldwide whereby susceptibility increases with age. The development of heart failure occurs over an extensive period of time in which arrhythmias and hypertrophy are both very prevalent manifestations throughout this progression. Arrhythmias are defined as an irregular rhythm originating from intracellular calcium dysregulation, which can be fatal. Cardiac hypertrophy is a compensatory condition induced by increased workload involving augmented cardiomyocyte growth accompanied by myocardial remodelling. However, under prolonged periods of increased stress this compensatory mechanism can lead to cardiac dysfunction. The current treatments for heart failure are mainly aimed at relieving symptoms or itself possess proarrhythmic ability. Therefore it is fundamental to elucidate the pathways involved in arrhythmias and hypertrophy for the development of more effective treatment. p21 activated protein kinase (Pak1) is a novel gene involved in the regulation of cardiac function, however, the mechanisms involved remain inconclusive. This study has demonstrated Pak1 to be both antiarrhythmic and antihypertrophic, emphasizing Pak1 as a credible therapeutic target for simultaneously treating both manifestations. The antiarrhythmic properties of Pak1 were demonstrated through cardiomyocyte-specific Pak1 knockout (Pak1cko) mouse model which underwent Isoproterenol (ISO) stimulation for 2 weeks. Compared with ISO treated control group, the Pak1cko group had increased calcium irregularities and particularly a prolongation in sarcoplasmic reticulum (SR) calcium refill. The absence of Pak1 abrogated the transcriptional up-regulation of sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) under stressed conditions. Further analysis in neonatal rat cardiomyocytes (NRCMs) revealed this regulation to be through activation of the transcription factor, SRF. The antihypertrophic effects of Pak1 were further illustrated through cardiomyocyte-specific overexpressed constitutively-active Pak1 (Pak1cTG) mice which were subjected to transverse aortic constriction (TAC) for 3 weeks. Compared to TAC control group, Pak1cTG mice had improved cardiac performance accompanied with diminished fibrosis. Further analysis led to the discovery of a novel antihypertrophic pathway of Pak1 involving positive regulation of the E3ligase, Fbxo32 through activation of Smad3. This pathway is vital in the prevention of calcineurin (PP2B) accretion. Berberine administration in TAC treated mice corroborated that Fbxo32 up-regulation is sufficient in the prevention of hypertrophy. In conclusion, my study has demonstrated that Pak1 conveys antiarrhythmic influence through the up-regulation of SERCA2a. In the prevention of pathological hypertrophy, Pak1 inhibits PP2B through positive regulation of Fbxo32. Overall, my thesis has advanced the knowledge about cardioprotective pathways initiated by Pak1 under stressed conditions, presenting Pak1 as a promising therapeutic target.
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Ganem, Carine. "Implication de Cak1 (CDK activating kinase) dans le processus de transcription par l'ARN polymérase II chez Saccharomyces cerevisiae." Paris 11, 2002. http://www.theses.fr/2002PA112180.
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Chez la levure S. Cerevisiae, les rôles physiologiques connus de la protéine essentielle Cak1 (kinase activatrice des kinases cycline-dépendantes (CDK)) sont de phosphoryler Cdc28, régulateur majeur du cycle cellulaire, ainsi que Kin28, kinase du domaine carboxy-terminal de la plus grosse sous-unité de l'ARN polymérase II. Des mutants conditionnels de cakI semblent affectés dans le contrôle du cycle cellulaire, mais aussi dans la formation de la paroi des spores, la différentiation pseudohyphale, ainsi que dans la réponse au stress osmotique. Dans ce travail, nous avons montré que Cak1 est impliquée dans le processus de transcription non seulement via la phosphorylation de Kin28, mais également via le complexe transcriptionnel multimérique Paf1 et via la protéine Ssu72. Une étude plus poussée de Ssu72 nous a permis de dévoiler sa nature de protéine phosphatase, et les liens étroits, tant génétiques que moléculaires, qu'elle tisse avec plusieurs autres protéines impliquées dans l'expression des gènes, telles que Sua7 (TFIIB), Kin28, Pafl, Ctr9 ou Fcp1. La multiplicité des effets des mutations dans le gène CAK1 pourrait ainsi s'expliquer par un rôle de modulateur de l'activité de transcriptionIn the budding yeast S. Cerevisae, the known physiological roles of the essential protein Cak1 (Cyclin-dependant kinases (CDKs) Activating Kinase) are to phosphorylate Cdc28, the main regulator of progression through cell cycle, and Kin28, the carboxy-terminal domain (CTD) kinase of the largest RNA polymerase II subunit. Conditionnal mutants of cakl appear affected in cell cycle control, but also in spore-wall morphogenesis, pseudohyphal differentiation, and response to high osmolarity. In this work, we show that Cak1 is implicated in transcription process not only through Kin28 phosphorylation, but also through the multimeric transcriptional complex Paf1 and the Ssu72 protein. A more extensive study of Ssu72 enabled us to reveal its phosphatase activity, as well as its genetic and molecular interactions with several other proteins involved in gene expression, such as Sua7 (TFIIB), Kin28, Paf1, Ctr9 or Fcp1. Thus, the multiple effects of cakl mutations could be explained by the role of Cak1 as a modulator of transcriptional activity
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Angathevar, Veluchamy Raaja Raajan. "Structure and activity of Pseudomonas aeruginosa PAO1 biofilms." Thesis, Montana State University, 2006. http://etd.lib.montana.edu/etd/2006/angathevarveluchamy/AngathevarVeluchamyR0506.pdf.
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Roy, Guylaine. "Characterization of PABP-interacting proteins 1 and 2." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84317.
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The 3' poly(A) tail of eukaryotic mRNAs and the poly(A) binding protein (PABP) play key roles in the regulation of translation. Recently, our group identified two human PABP-interacting proteins (Paip), Paip1 and Paip2, which stimulate and repress translation, respectively. Paip2 also inhibits the binding of PABP to the poly(A) tail and competes with Paip1 for binding to PABP. My research project was divided into two parts to allow me to gain a greater understanding of the roles of these two PABP-interacting proteins. First, in order to study the mechanism of interaction of Paip1 and PABP, their binding sites were mapped by Far Western and GST pull-downs experiments. The Paip1-PABP interaction involves two distinct binding regions in each protein. The PABP interacting motif-1 (PAM1) of Paip1 is rich in acidic amino acids and is located in the C-terminus (a.a. 440--479). PAM1 interacts with the RNA recognition motifs (RRMs) 1 and 2 of PABP. PAM2 consists of a 15 amino acid stretch residing in the N-terminus of Paip1 (a.a. 123--137) and interacts with the C-terminal domain of PABP. In addition, the stoichiometry and the kinetic and thermodynamic constants for the Paip1-PABP interaction were determined using a Surface Plasmon Resonance (SPR)-biosensor. Paip1 interacts with PABP with an apparent KD of 1.9 nM and with a 1:1 stoichiometry. In the second part of my thesis research, the Drosophila Paip2 (dPaip2) was isolated and characterized in order to ascertain a biological role for Paip2. dPaip2 was found to be the bona fide homologue of human Paip2 since it interacts with the Drosophila PABP (dPABP) via two independent binding sites, interferes with the ability of dPABP to bind to poly(A), and represses translation. Ectopic overexpression of dPaip2 in wings resulted in a size reduction phenotype, which was due to a decrease in the cell number but not to a difference in cell size. Clones of cells overexpressing dPaip2 in wing discs also contai
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Fantus, Daniel. "The polyadenlyate binding protein (PABP) and translational control /." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80261.
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Translation is the process by which proteins are synthesized from messenger RNAs. This complex process is regulated by a multitude of factors (1). One such factor is the Polyadenylate Binding Protein (PABP), an abundant protein associated with messenger ribonucleoprotein particles (mRNPs) (18). As a result of its interaction with both poly(A) RNA and eukaryotic initiation factor 4G (eIF4G), a component of the tripartite eIF4F cap-binding complex, PABP elicits mRNA circularization and stimulates translation (2). Although the mechanism by which mRNA circularization facilitates translation is not clear, one model suggests that circularization promotes ribosome recycling. Recently, an interaction between PABP and the termination factor, eukaryotic Release Factor3 (eRF3/GSPT) was demonstrated in mammals (71). By interacting with both eRF3 and eIF4G, it was postulated that PABP physically bridges the stop codon to the 5' cap structure, enabling terminating ribosomes to reinitiate translation on the same mRNA. To explore this idea, several PABP mutants lacking the ability to bind eRF3 were generated and added to a PABP-depleted Krebs-2 Cell-Free Extract to study the effects on translation. The PABP mutants stimulated translation as effectively as wild-type PABP, suggesting that the eRF3-PABP interaction is dispensable for translation and ribosome recycling. RNA-binding protein localization may be regulated by methylation and protein-protein interactions (98). PABP was also recently identified as a Coactivator-Associated Arginine Methyltransferase I (CARMI) substrate (23). Here, we show that PABP methylation does not modulate nuclear shuttling or its interactions with 14-3-3, a novel PABP-interacting protein. Translation in "non-nuclear" rabbit reticulocyte lysate is robust and PABP-independent, suggesting a loss of inhibitory control by nuclear factors. We reveal for the first time that the general RNA-binding protein YB-1 renders translation in
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楊雋永 and Chun-wing Yeung. "PAK1, PAK2 and PAK4 in gestational trophoblastic disease." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B40721905.
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Price, Bianca Louise. "Characterisation of rhamnolipid biosynthesis in Pseudomonas aeruginosa PA01." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609565.
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El, Bestawy Walid. "Transcriptional regulation of the mouse PAC1 receptor gene." Thesis, University of Strathclyde, 2013. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=22708.
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The G-protein coupled receptor PAC1 has been implicated in playing a role in neural growth, development and stress-protection, cognition, homeostasis, immunomodulation and anti-inflammatory effects. Previously, an investigation of PAC1 gene (mADCYAP1r1) revealed a minimum promoter region -113bp to +206 relative to the transcriptional start site. Binding of the Zac1 transcription factor has been investigated but no detailed analysis has been carried out of the region downstream or upstream of -2598bp from exon 1 and a full characterisation of the Transcription Factors (TF) that govern expression of the receptor has not been performed. The aim of this study was to characterise the promoter of the mAdcyap1r1 gene and to identify transcription factor binding sites, explore their role in controlling expression. Since the mAdcyap1r1 gene is well conserved across vertebrate species, a cross-species DNA comparison was performed to detect evolutionary conserved regions. Mouse, rat, human and chimpanzee sequences were aligned, and several cross-species conserved regions rich in transcription factor binding sites have been identified and reported. 3 upstream regions AdU1, AdU2 and AdU3; 2 downstream AdD1 and AdD2; and a new minimum promoter region Ad1 -80 and including most of exon 1 to +353. 279 transcription factor binding sites were found in these regions. Luciferase reporter gene assays of these regions was performed in Neuro-2a cell lines, a-T3 neuroendocrine models and Cos-7 cells as a negative control that don't express PAC1. Results indicated a functional new basal promoter region, Ad1, that was expressed in Neuro-2a. Upstream and downstream regions showed Neuro-2a specificity but were 20 fold lower than expression from Ad1. Hydrogen peroxide treatment gave increased expression via the AdU1, Ad1 and AdD2 regions mainly in Neuro-2a cells. Electrophoretic Mobility Shift Assays indicated binding of nuclear extracts to most promoter regions. Promoter Expression data gave functional insight to the bioinformatic analysis, and transcription factor binding sites indicate further roles to be investigated in future studies.
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Nelson, Shona Margaret. "Studies on biofilm growth of Pseudomonas aeruginosa PA01." Thesis, Aston University, 1993. http://publications.aston.ac.uk/12581/.
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The development of in vitro techniques to model the surface-associated mode of growth is a prerequisite to understanding more fully the physiological changes involved in such a growth strategy. Key factors believed to influence bacterial persistence in chronic infections are those of the biofilm mode of growth and slow growth rate. Methods for controlling Pseudomonas aeruginosa biofilm population growth rates were investigated in this project. This microorganism was incompatible with the in vitro 47mm diameter membrane filter-based biofilm technique developed for the study of Escherichia coli and Staphylococcus epidermidis by Gilbert et al (Appl. Environ. Microbiol. 1989, 55, 1308-1311). Two alternative methods were designed. The first comprised a 25mm diameter cellulose acetate membrane filter supported in an integral holder. This was found to be limited to the study of low microbial population densities with low flow rates. The second, based on a cylindrical cellulose fibre depth filter, permitted rapid flow rates to be studied and allowed growth rate control of biofilm and eluted cells. Model biofilms released cells to the perfusing medium as they grew and divided. The viability of released cells was reduced during, and shortly after, inclusion of ciprofloxacin in the perfusate. Outer membrane profiles of biofilm populations exhibited at least two bands not apparent in planktonic cells grown in batch and chemostat culture, and LPS profiles of biofilm populations showed variation with growth rate. Cell surface hydrophobicity of resuspended biofilm cells varied little with growth rate, whilst it decreased markedly for cells released from the biofilms as growth rate increased. Cells released from the biofilm were more hydrophilic than their sessile counterparts. Differing growth rates, LPS profiles and hydrophobicity are proposed to have a bearing on the release of cells from the adherent population.
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Yeung, Chun-wing. "PAK1, PAK2 and PAK4 in gestational trophoblastic disease." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B40721905.
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Smith, Stephen. "The regulation and role of PAK1 in macrophages." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445920/.
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The p21-activated kinases (PAKs) are a family of serine/threonine kinases that are activated by the Rho GTPases, Rac1-3, Cdc42, Chp, TC10 and Wrch-1, as well as a number of lipids and PAK-interacting proteins. PAKs have been implicated in a variety of cellular processes including cell polarisation, migration, chemotaxis and gene transcription. The aim of this study was to determine the regulation and function of PAK1, using bone marrow-derived macrophages (BMMs) as a model system. CSF-1 stimulation of BMMs induced rapid phosphorylation and activation of PAK1, and CSF-1 and TNFa also promoted an increase in PAK1 protein levels after 2 to 5 hours. The rise in PAK1 protein was not due to changes in gene transcription, mRNA translation or reduced proteasomal degradation. Wildtype and PAK1"7" BMMs were compared to determine the roles of PAK1 in a number of macrophage functions. PAK1 was required for maximal ERK, p38 and JNK activation in response to CSF-1 although it did not appear to signal via c-Raf or MEK1. PAK1 phosphorylated Op18 and LIMK downstream of CSF-1, which regulate microtubule and actin reorganisation respectively. PAK1 also regulated MLC phosphorylation although this was not a CSF-1-induced response. PAK1_/" BMMs adhered more rapidly than WT BMMs and transiently spread to a greater area than WT BMMs. PAK1 promotion of ERK activity at the lamellipodial edge was required for the stability of lamellipodial extension during cell spreading with a greater number of smaller lamellipodia produced in PAK1"7" BMMs compared to WT BMMs. However, PAK2, active Cdc42, total ERK, £-PIX and Rac1 all localised normally at the cell periphery in spreading PAK1"7" BMMs. PAK1 was also required for membrane ruffling after CSF-1 stimulation but was dispensable for macrophage polarisation, migration and chemotaxis towards CSF-1. PAK1, therefore, contributes to CSF-1 and cell adhesion induced signalling in macrophages.
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St-André, Olivier. "La régulation des gènes méiotiques par la protéine PAB2." Mémoire, Université de Sherbrooke, 2011. http://hdl.handle.net/11143/5567.
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Les travaux décrits dans ce mémoire visent à élucider le rôle de la protéine Pab2 dans la régulation des gènes méiotiques. La méiose est un phénomène conservé à travers l'évolution chez les organismes eucaryotes. Chez les eucaryotes unicellulaires comme la levure, la méiose accomplie [i.e. accomplit] autant un rôle de reproduction sexuelle qu'un rôle de protection de l'organisme. Chez la levure à fission Schizosaccharomyces pombe , le processus de méiose résultant de la reproduction de deux individus génère des spores, des structures résistantes aux conditions environnementales détrimentales. Lorsque la cellule enclenche la méiose, plusieurs centaines de gènes sont temporellement induits pour exprimer des gènes spécifiques aux processus méiotiques. Afin de s'assurer que des gènes méiotiques ne soient pas exprimés pendant la mitose au détriment de la cellule, des mécanismes de surveillance doivent rigoureusement surveiller l'expression de ces gènes. Afin d'éclaircir le rôle de Pab2 dans cette surveillance, nous avons utilisé une approche génétique combinée à des essais biochimiques. Des expériences d'immunoprécipitation de la chromatine combinées à des essais de substitution de promoteur ont démontré que la régulation de Pab2 sur les gènes méiotiques est posttranscriptionnelle. De plus, des expériences d'immunoprécipitation de la protéine Pab2 suivies d'analyse d'ARN ont démontré que la protéine Pab2 forme un complexe in vivo avec les ARN qu'elle régule. Des analyses dans le laboratoire ont démontré que Pab2 possède une interaction fonctionnelle et physique avec des composantes de l'exosome. Afin de déterminer l'implication de l'exosome dans la régulation des gènes méiotiques, nous avons utilisé différentes souches de levure [i.e. levures] possédant des délétions dans les gènes des composantes de l'exosome ou des facteurs coopérant avec ce complexe, en présence ou absence de Pab2. Ces expériences ont démontré que l'exosome nucléaire, particulièrement la sous-unité Rrp6, était majoritairement responsable de la dégradation des transcrits méiotiques, et ce sans l'aide de complexes coopérateurs comme le TRAMP. Afin de déterminer le facteur de sélectivité de la régulation par Pab2 et par l'exosome, nous avons utilisé des souches thermosensibles pour la protéine Mmi 1, un autre agent posttranscriptionnel impliqué dans la surveillance de l'expression des gènes méiotiques. Ces expériences ont démontré que Pab2 coopère avec Mmi1 dans la dégradation des transcrits méiotiques. Notamment, l'augmentation de l'expression des gènes méiotiques en absence de Pab2 est suffisante pour contourner la voie dépendante de meiRNA, qui est un ARN non-codant essentiel à l'initiation de la méiose. Ces résultats constituent l'évidence que la protéine Pab2 liant les queues poly(A) participe au maintient du silence de l'expression des gènes méiotiques pendant la mitose.
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Chapman, Tajekesa Kudzaishe Pamacheche. "Regulation of PABP1 function by differential post-translational modification." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25875.
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Post-transcriptional control of gene expression is critical for normal cellular function and viability. Poly(A)-binding protein (PABP) 1 is the prototypical member of a family of RNA-binding proteins which are key post-transcriptional regulators. PABP1 is multifunctional, acting as a primary determinant of translation efficiency and mRNA stability, regulating the fate of specific mRNAs, and participating in microRNAmediated regulation and nonsense-mediated mRNA decay. As well as binding various mRNAs, PABP1 achieves its multifunctionality through protein-protein interactions with numerous PABP-interacting motif (PAM)-2 motif-containing protein partners. These have been identified to bind the same site within the C-terminal PABC domain, therefore it is unclear how different PABP1 functions are coordinated. Recently, PABP1 was found to exhibit extensive post-translational modification (PTM), including putative lysine acetylation/methylation switches, which were suggested as a potential mechanism by which interactions with different PAM2 motifcontaining proteins may be regulated. In particular, in silico molecular modelling of the acetylation or dimethylation of the position 606 lysine residue (Lys606) within the PABC domain, using available structures of PABC in complex with PAM2 peptides of eukaryotic release factor (eRF)-3a and PABP-interacting protein (Paip)-2, suggested that modification of this residue, which is critical in PABC-PAM2 interactions, may differentially affect these PABP1 interactions. To examine the role of the Lys606 modification as a molecular switch to dictate PABC-mediated protein-protein interactions, site-specifically acetylated recombinant PABC domain was generated using cutting–edge amber codon suppression recoding technology. Following sequential purification by affinity, ion exchange and size exclusion chromatography, recombinant PABC protein quality was analysed by biophysical approaches such as thermal denaturation assay (TDA), dynamic light scattering (DLS), circular dichroism (CD) and liquid chromatography mass spectrometry (LCMS). Biochemical and biophysical analysis of PABC-PAM2 interactions was subsequently undertaken using GST-pulldown analysis, with the well characterised Paip2 protein, and Surface Plasmon Resonance (SPR) using PAM2 peptides of eRF3, Paip2 and trinucleotide repeat-containing (Tnrc) 6C (or GW182) proteins. These revealed that PABC Lys606 acetylation significantly increased the affinity and increased the association rate for eRF3 peptide. In contrast, effects on Paip2 peptide binding were less suggestive. Furthermore, although approaches to decipher the biological relevance of Lys606 and its modifications within cells are in their infancy, they reflect the complexity of studying PTM function in vitro. Overall, these data provide support for the hypothesis that Lys606 modification status confers selectivity between PABP1 protein partners suggesting a potential mechanism for how its multi-functionality may be coordinated.
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SCELLES, VERONIQUE. "Inhibiteur de l'activateur du plasminogene (pai1) et maladies metaboliques." Aix-Marseille 2, 1993. http://www.theses.fr/1993AIX20837.
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Rasselet, Damien. "Etude de nanocomposites basés sur des alliages PLA/PA11." Thesis, IMT Mines Alès, 2019. http://www.theses.fr/2019EMAL0001/document.
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L’acide polylactique (PLA) est l’un des polymères biosourcés qui suscite le plus d’intérêt, mais ses propriétés thermomécaniques nécessitent d’être améliorées. Pour ce faire, les méthodes les plus utilisées et étudiées sont de le mélanger avec d’autres polymères ou bien d’y ajouter des charges minérales nanométriques (nanoparticules), afin de constituer un nanocomposite à matrice PLA. C’est dans la combinaison de ces deux approches que s’inscrivent ces travaux de thèse, consacrés à l’élaboration et à la caractérisation des propriétés de nanocomposites à base d’un alliage de PLA et de polyamide 11 (PA11) 80/20 m/m. L’objectif de cette thèse est l’obtention d’un matériau biosourcé aux propriétés thermiques, mécaniques et de réaction au feu améliorées par le contrôle de sa morphologie et l’ajout de nanoparticules et de retardateurs de flamme (RF). Pour y parvenir, deux techniques de compatibilisation, destinées à améliorer l’adhésion interfaciale entre le PLA et le PA11, ont été évaluées. La première consistait à incorporer des nanoparticules de silice. Il a été noté d’importantes modifications de la morphologie et des propriétés rhéologiques du mélange d’étude, selon leur localisation dans le mélange étudié fonction de la nature chimique de la surface de la silice. La deuxième consistait à introduire un copolymère époxyde multifonctionnel réactif, dénommé Joncryl. La réactivité de ce copolymère avec le PLA et le PA11 a permis de compatibiliser le mélange d’étude, conduisant à une morphologie plus fine et à l’obtention de propriétés mécaniques supérieures à celles du mélange d’étude, en particulier avec l’ajout de 3%m de Joncryl. Des échantillons basés sur les mélanges compatibilisés par cette méthode ont été préparés par le procédé de fabrication additive FDM. Une étude de l’impact de ce procédé sur la morphologie et les propriétés mécaniques obtenues a été entreprise. Enfin, une meilleure réaction au feu pour le mélange compatibilisé avec 3%m de Joncryl a pu être obtenue par l’ajout combiné de nanoparticules de phyllosilicates et de RFPolylactic acid (PLA) is one of the biobased polymers that generates the most interest, but its thermomechanical properties need to be improved. To do that, the most used and studied methods consist of blending PLA with other polymers or adding nanoscaled mineral fillers (nanoparticles) to get a PLA based nanocomposite. This PhD work is dedicated to the elaboration and properties characterization of nanocomposites based on a filled PLA and polyamide 11 80/20 wt/wt blend. The aim is to obtain a biobased material with improved thermal, mechanical and fire reaction properties by controlling its morphology through the addition of nanoparticles and flame retardants additives.To achieve that, two compatibilization techniques, aiming to improve PLA-PA11 interfacial adhesion, were evaluated. The first one consisted of adding silica nanoparticles. Important changes of the blend morphology and rheological properties were noticed, depending on the localization of the two different silica nanoparticles used into the polymer blend phases. The second one consisted of introducing a reactive multifunctional epoxy copolymer, named Joncryl. The reactivity of this copolymer with PLA and PA11 allowed to compatibilize the blend, leading to a fine morphology and higher mechanical properties compared to those of the pristine blend. Samples of compatibilized blends obtained through this method were processed using FDM additive manufacturing process. A study of the influence of this process on the morphology and mechanical properties obtained for these samples was performed. Finally, a better fire reaction of compatibilized polymer blend with 3%wt Joncryl was obtained by the combined addition of phyllosilicates nanoparticles and flame retardants
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Harb, Maya. "Effet de l'interaction de la protéine NSP3 du Rotavirus avec les protéines cellulaires, eIF4G et RoXaN, sur la localisation subcellulaire de PABP." Paris 11, 2008. http://www.theses.fr/2008PA114848.
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La protéine non structurale du Rotavirus, NSP3, interagit avec l’extrémité 3’ des ARNm viraux, avec le facteur d’initiation de la traduction eIF4G, ainsi qu’avec la protéine cellulaire RoXaN. NSP3, en se fixant à l’eIF4G décroche la poly(A) binding protein cytoplasmique (PABPC1) du complexe d’initiation de la traduction, et empêche ainsi l’initiation de la traduction des ARNm cellulaires. De cette façon, la traduction des ARNm viraux est favorisée. Ce travail avait pour objet l’étude du rôle de l’interaction de NSP3 avec RoXaN dont la fonction était jusqu’alors inconnue. Dans une première partie de ce travail, nous avons d’abord délimité la région minimale de NSP3 nécessaire à l’interaction avec RoXaN. Sur la base d’un modèle tridimentionel de cette région, nous avons identifié des mutants n’interagissant plus avec le domaine LD de RoXaN. Ces mutants ont d’abord été testés afin de voir si l’interaction NSP3/RoXaN était impliquée dans la régulation de la traduction des ARNm viraux. Nous n’avons vu aucun effet de RoXaN dans la traduction. Dans la deuxième partie de ce travail, nous avons montré que l’infection par le Rotavirus induisait l’accumulation nucléaire de la PABPC1. En utilisant les mutants de NSP3 n’interagissant plus avec RoXaN, nous avons montré que l’interaction NSP3/RoXaN était nécessaire, en plus de l’interaction de NSP3 avec l’eIF4G, pour la localisation nucléaire de PABPC1. De plus, nous avons montré que le domaine LD de RoXaN correspondait à un signal d’export nucléaire. A partir de nos résultats, nous faisons l’hypothèse que RoXaN joue un rôle dans l’export nucléaire de PABPC1 liée aux ARNm cellulaires polyadénylés. Ainsi, l’interaction RoXaN-NSP3 en bloquant cet export, renforcerait l’inhibition de la traduction des ARNm cellulaires et pourrait aussi bloquer l’export d’ARNm de gènes impliqués dans la réponse antiviraleRotavirus nonstructural protein NSP3 interacts specifically with the 3' end of viral mRNAs, with the eukaryotic translation initiation factor eIF4G, and with RoXaN, a cellular protein of yet-unknown function. By evicting cytoplasmic poly(A) binding protein (PABP-C1) from translation initiation complexes, NSP3 shuts off the translation of cellular polyadenylated mRNAs. We show here that PABP-C1 evicted from eIF4G by NSP3 accumulates in the nucleus of rotavirus-infected cells. Through modeling of the NSP3-RoXaN complex, we have identified mutations in NSP3 predicted to interrupt its interaction with RoXaN without disturbing the NSP3 interaction with eIF4G. Using these NSP3 mutants and a deletion mutant unable to associate with eIF4G, we show that the nuclear localization of PABP-C1 not only is dependent on the capacity of NSP3 to interact with eIF4G but also requires the interaction of NSP3 with a specific region in RoXaN, the leucine- and aspartic acid-rich (LD) domain. Furthermore, we show that the RoXaN LD domain functions as a nuclear export signal and that RoXaN tethers PABP-C1 with RNA. This work identifies RoXaN as a cellular partner of NSP3 involved in the nucleocytoplasmic localization of PABP-C1
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Langley, Roger Sean. "Metal binding by Pseudomonas aeruginosa PAO1 and isogenic lipopolysaccharide mutants." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ33244.pdf.
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Landreau, Emmanuel. "Matériaux issus de ressources renouvelables. Mélanges amidon plastifié/PA11 compatibilisés." Reims, 2008. http://theses.univ-reims.fr/exl-doc/GED00000801.pdf.
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Pour développer un matériau issu de ressources renouvelables, l'amidon plastifié a donc été mélangé au polyamide 11, issu de l'huile de ricin, dans le but d'améliorer ses propriétés mécaniques et sa résistance à l'eau. Malgré la forte polarité des liaisons amide, le mélange a besoin d'être compatibilisé. Les différents compatibisants étudiés sont des polysaccharides anioniques susceptibles de développer des interactions avec le polyamide et l'amidon: alginate de sodium, carraghenane et carboxymethylcellulose. Des tests en mélangeur interne montrent que seule la carboxyméthylcellulose (CMC) permet une amélioration effective des propriétés mécaniques des mélanges. L'optimisation de la quantité de plastifiant, de compatibilisant et du procédé de mélange permet d'obtenir un matériau contenant majoritairement de l'amidon plastifié (70%) qui conserve un allongement et une contrainte à la rupture élevée (130%, 15MPa) avec seulement 1% de CMC. Ces matériaux ont été réalisés en extrudeuse bivis afin d'être étudié en détail. La morphologie étudiée par: MEB, extraction sélective, rhéologie et résistance électrique; montre que le PA 11 forme une phase continue jusqu'à plus de 80% d'amidon plastifié. L'amidon forme des nodules sphériques qui percolent à partir de 30% d'amidon plastifié pour former une phase partiellement co-continue. Il subsiste cependant une fraction dispersée jusqu'à 80% d'amidon. La CMC diminue la tension interfaciale et ainsi diminuer la taille des nodules et de prévenir leur coalescence. Des tests de compostage sur les mélanges ont montré que la minéralisation de l'amidon est importante quelque soit sa proportion, mais que le PA reste inchangéTo develop renewable resources based material, plasticized starch were blend with polyamide 11, a bio-based polymer from castor oil, to improve its mechanical properties and water resistance. Through the high polarity of the amide group, the blends need a compatibilizer to be efficient. The different molecules tested are polysaccharides with anionic groups known to interact with polyamide: sodium alginate, carraghenan and sodium carboxymethylcellulose. Tests runs in blender show that only sodium carboxymethylcellulose (CMC) can improve blend tensile properties. Optimization of the plasticizer, the compatibilizer level and the blend process lead to a mainly starch based material (70%) with a high tensile strength (15 MPa) and elongation at break (130%) with only 1% of CMC. These materials were blends in a twin screw extruder to be studied. SEM, solvent extraction, rheology and electrical resistance mesurment show a continuous PA phase up to 80% of starch. The polysaccharide has a mainly nodular morphology with a partial percolation around 30% starch. Isolated nodules co-exist with a co-continous structure up very high starch content. CMC reduce interfacial tension and nodule size preventing their coalescence. Compostability test on blends, show that starch mineralization is complete whatever its concentration is, but PA remain resistant to biodegradation
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Landreau, Emmanuel Tighzert Lan Bliard Christophe. "Matériaux issus de ressources renouvelables. Mélanges amidon plastifié/PA11 compatibilisés." Reims : S.C.D. de l'Université, 2008. http://scdurca.univ-reims.fr/exl-doc/GED00000801.pdf.
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Alsayegh, Maha. "DEF6 is associated with the translational initiation regulating protein synthesis of eIF4E, 4E-T and PABP and colocalises with eIF4E and PABP in the immunological synapse." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/51984/.
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DEF6 is an atypical guanine nucleotide exchange factor (GEF) for Rho GTPases Rac1 and Cdc42, which is highly expressed in mature T cells. DEF6 is recruited to the immunological synapse (IS) following phosphorylation by LCK in response to T cell receptor (TCR) stimulation during inflammation. In resting Jurkat T cells, DEF6 has been known to associate with polysomes and under cellular stress conditions to form cytoplasmic granules colocalising with mRNA decapping enzyme subunit 1 (DCP1), a marker of P-bodies. Hence DEF6 has been linked to mRNA regulation, which includes mRNA translation initiation and repression by P-body formation. To elucidate the structural requirements for P-body localisation of DEF6, N-terminal truncation mutants lacking either the C-terminal coiled coil domain or the pleckstrin homology (PH) domain in conjunction with the coiled coil domain were established and coexpressed with DCP1 in COS7. Both GFP-tagged mutant DEF6 proteins spontaneously colocalised with DCP1 indicating that the N-terminal end of DEF6 is sufficient for P- body association and that the coiled coil domain facilitates a confirmation that masks the N-terminal end of DEF6. Exchange of serine and/or threonine residues in the C- terminal end with either phosphomimic or phosphosilent amino acids resulted in formation of GFP-DEF6 aggregates or granules, respectively. While GFP-DEF6 granules partly overlapped with DCP1, GFP-DEF6 aggregates appeared to trap DCP1. In resting Jurkat cells endogenous DEF6 associated with nascent mRNA translation and colocalised with eukaryotic translation initiation factor 4E (eIF4E) as well as poly A binding protein (PABP). eIF4E and the eIF4E-binding protein 4E-T but not PABP were shown to interact with DEF6 in vitro. Moreover, siRNA-mediated knockdown and ectopic overexpression of DEF6 in Jurkat cells established a positive correlation of DEF6 expression and eIF4E, PABP and 4E-T protein expression. RT-PCR revealed that the alteration in expression of these proteins was not due to transcriptional regulation and inhibition of protein degradation pathways could not rescue downregulation of eIF4E. Colocalisation of DEF6 with eIF4E and PABP but not with 4E-T was also observed in the IS upon TCR-mediated signalling. Together these results strongly suggest that DEF6 is involved in active mRNA translation in resting and activated Jurkat T cells controlling the expression of components of the translational initiation complex as well as P-bodies.
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Abreu, Ieda Santos. "Influência do diabetes mellitus do tipo 2 na expressão dos receptores ativados por protease do tipo 1 (PAR1) e do tipo 2 (PAR2) em pacientes com periodontite crônica." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/23/23146/tde-18032015-161437/.
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O receptor ativado por protease do tipo 1 (PAR1) parece estar associado ao reparo periodontal, enquanto o tipo 2 (PAR2) com a inflamação periodontal. Esses receptores podem ser ativados pelas proteases gingipaina, uma protease secretada pela Porphyromonas gingivalis, um importante periodontopatógeno, e pela proteinase-3 de neutrófilos (P3), que é liberada por neutrófilos quando expostos a um estímulo inflamatório. Uma vez que o diabetes é reconhecido como um fator de risco importante para a doença periodontal, o objetivo deste estudo foi investigar a expressão de PAR1 e de PAR2 e de seus ativadores, gingipaina e P3 no fluido gengival (FG) de pacientes diabéticos com periodontite crônica, antes e após tratamento periodontal não cirúrgico. Amostras de FG e os parâmetros clínicos, como profundidade de sondagem (PS), nível clínico de inserção (NCI), sangramento à sondagem (SS) e índice de placa (IP) foram coletados de pacientes sistemicamente saudáveis e de pacientes com diabetes mellitus do tipo 2 (DMT2) com periodontite crônica , no baseline e após o tratamento periodontal não cirúrgico. As expressões gênicas de PAR1, PAR2, gingipaina e P3 no FG foram quantificadas por qPCR. Os parâmetros clínicos melhoraram significativamente após a terapia periodontal (p <0,01). O diabetes levou ao aumento da expressão de PAR1 no fluido gengival e na presença da periodontite crônica diminuiu significativamente a expressão de PAR1, PAR2 e P3 (p<0,05). Além disso, o tratamento periodontal não cirúrgico em diabéticos resultou no aumento da expressão de PAR1 e de PAR2 (p<0,05). Dentro dos limites do presente estudo, sugerimos que os PARs podem estar associados com a inflamação periodontal em diabéticos.Protease activated receptor type 1 (PAR1) seems to play a role in periodontal repair, while PAR2 is associated with periodontal inflammation. These receptors can be activated by gingipain, a protease released from Porphyromonas gingivalis, an important periodontal pathogen, and neutrophil proteinase-3 (P3), which is released by neutrophils when exposed to an inflammatory stimulus. Since diabetes is known risk factor to periodontal disease, the aim of this study was to investigate PAR1 and PAR2 mRNA expression at the gingival crevicular fluid (GCF) in diabetic patients with chronic periodontitis, before and after non-surgical periodontal treatment. GCF samples and clinical parameters consisting of measuring probing depth (PD), clinical attachment level (CAL), bleeding on probing (BOP) and plaque index (PI) were collected from systemically healthy patients and patients with type 2 diabetes mellitus with chronic periodontitis, at baseline and after nonsurgical periodontal treatment. PAR1 and PAR2, as well the expression of the activators gingipain and P3 at the GCF were quantified by qPCR. The clinical parameters improved significantly after periodontal therapy (p <0.01). Diabetes led to increased expression of PAR1 in the GCF, and the presence of chronic periodontitis significantly decreased the expression of PAR1, PAR2 and P3 (p <0.05). Moreover, non-surgical periodontal treatment in diabetics resulted in increased expression of PAR1 and PAR2 (p <0.05). Within the limits of this study, we suggest that PARs may be associated with periodontal inflammation in diabetics.
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Löfgren, Emma. "The role of PAR1 and PAR4 in platelet PAI-1 secretion." Thesis, Department of Physics, Chemistry and Biology, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-20813.
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In Sweden about 26000 people are affected by myocardial infarction (MI) every year. Coronary vascular diseases and cerebrovascular diseases with blood flow disruption caused by thrombi are actually the most common cause of death. A contributing mechanism may be if the fibrinolytic system is uncapable to dissolve clots and it might in turn be related to the high amount of plasminogen activator inhibitor 1 (PAI-1) released from platelets. This study investigated if there is any difference between the thrombin activation of the platelet protease activated receptors (PAR) PAR1 and PAR4 concerning PAI-1 secretion. An ELISA assay was modified in order to measure PAI-1 released from stimulated platelets. Concerning PAI-1 secretion this study concluded that PAR1 activation is slightly more sensitive to PAI-1 secretion than PAR4. Both thrombin receptors are really potent in this respect, activating only one of PAR1 or PAR4 is enough to get a strong response. This study could also confirm that platelet PAR1 undergoes desensitization when stimulated with a low dose of agonist. Despite platelets poor protein synthesis machinery it seems like they have the ability to synthesize large amounts of PAI-1.
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Yang, Zhe. "Functional Characterization of the Arginine Transaminase Pathway in Pseudomonas aeruginosa PAO1." Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/biology_diss/29.
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Arginine utilization in Pseudomonas aeruginosa with multiple catabolic pathways represents one of the best examples of metabolic versatility of this organism. To identify genes of this complex arginine network, we employed DNA microarray to analyze the transcriptional profiles of this organism in response to L-arginine. While most genes in arginine uptake, regulation and metabolism have been identified as members of the ArgR regulon in our previous study, eighteen putative transcriptional units of 38 genes including the two known genes of the arginine dehydrogenase (ADH) pathway, kauB and gbuA, were found inducible by exogenous L-arginine but independent of ArgR. The potential physiological functions of those candidate genes in L-arginine utilization were studied by growth phenotype analysis in knockout mutants. The insertion mutation of aruH encoding an L-arginine:pyruvate transaminase abolished the capability to grow on L-arginine of an aruF mutant devoid of a functional arginine succinyltransferase (AST) pathway, the major route of arginine utilization. The aruH gene was cloned and over-expressed in E. coli. Taking L-arginine and pyruvate as the substrates, the reaction products of recombinant enzyme were identified by MS and HPLC as 2-ketoarginine and L-alanine. Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of ping-pong kinetics mechanism, and the apparent Km and catalytic efficiency (Kcat/Km) were 1.6 ± 0.1 mM and 24.1 mM-1 s-1 for pyruvate and 13.9 ± 0.8 mM and 2.8 mM-1 s-1 for L-arginine. Recombinant AruH showed an optimal pH at 9.0 and substrate specificity with an order of preference being Arg > Lys > Met > Leu > Orn > Gln. These data led us to propose the arginine transaminase (ATA) pathway that removes the α-amino group of L-arginine via transamination instead of oxidative deamination by dehydrogenase or oxidase as originally proposed. In the same genetic locus, we also identified a two-component system, AruRS, for the regulation of arginine-responsive induction of the ATA pathway. Our latest DNA microarray experiments under D-arginine conditions also revealed PA3863 as the candidate gene encoding D-arginine dehydrogenase which might lead to the recognition of a wider network of arginine metabolism than we previously recognized.
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Wang, Xiaoqi. "Role of the PAT1 gene of S. cerevisiae in genome stability." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389018.
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Derbali, Habib. "La caractérisation de l'amidotransférase ARNt-dépendante (AdT) de Pseudomonas aeruginosa PA01." Thesis, Université Laval, 2008. http://www.theses.ulaval.ca/2008/25218/25218.pdf.
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Penney, Mary. "Pad1 : a novel subunit of the 26S proteasome in fission yeast." Thesis, University of Edinburgh, 1998. http://hdl.handle.net/1842/11250.
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Mutations in the fission yeast genes mts5-1 and mbc]-1 were isolated in a screen for Schizosaccharomyces pombe (S.pombe) mutants that are both resistant to the microtubule destabilising drug methybenzylcarbamylate (MBCR) and temperature sensitive (t.s.) for growth. This screen has so far been specific for mutations in genes encoding subunits of the 26S proteasome (Gordon et al., 1993). This study shows that these strains contain mutations in the pad1 and crm] genes respectively. Crm] and pad] have previously been shown to be positive and negative regulators respectively of the AP-1 transcription factor Papi (Toda et al., 1992, Shimanuki et al., 1995), the S. pombe homologue of the mammalian AP-1 transcription factors fos and fun, which is involved in the transcription of multidrug resistance genes (Shimanuki et al., 1995). The mts5-1 (pad]-]) strain has a metaphase arrest phenotype and an increased level of high molecular weight ubiquitinated proteins when incubated at the restrictive temperature. This is identical to the mts2-1 (Gordon et al., 1993) and mts3-1 (Gordon et al., 1996) mutants isolated in the same screen and which have been shown to encode subunits of the 26S proteasome. This study reclassifies pad]' as a subunit of the 26S proteasome, 'and data is provided which shows genetic interactions between Padi and three other subunits of the 26S proteasome, Mts3 (Gordon et al., 1996), Mts4 (Wilkinson et al., 1997) and Pus (C. Wilkinson pers. comm.). A putative function for the 19S cap subunit Padi as an isopeptidase is also investigated. Crml has been implicated in MDR through Papi, since Papi is responsible for the transcription of genes involved in resistance to a wide variety of drugs. 26S proteasome mutants are also shown to be resistant to the same range of drugs as the mbcl-] (cnn]-I) mutant, but to a lower level, and that papli\ cells are sensitive to MBC (MBCS). 26S proteasome mutants are shown to have elevated levels of Papi when incubated at the permissive temperature indicating that this protein is not being degraded as efficiently as in wild type cells. A c.s. cnn] mutant has been shown to over-express a non-essential 25KDa protein that has been shown to be a downstream target of Papi (Adachi and Yanagida, 1989). This protein is also shown to be over expressed in S.pombe proteasome mutants and cnn]-1. paplA 26S proteasome double mutants are t.s. and MBCS. This is consistent with the 26S proteasome being involved in the degradation of Papi and hence involved in pleiotropic multi-drug resistance.
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Briand, Stéphanie. "APP, PAT1, SET et mort neuronale : implications dans la Maladie d'Alzheimer." Paris 6, 2011. http://www.theses.fr/2011PA066012.
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La Maladie d’Alzheimer est une affection à neurodégénérescence importante qui comporte des plaques amyloïdes et des dégénérescences neurofibrillaires (DNFs). Les plaques sont majoritairement composées de peptide Aβ issu de la protéine APP. Les DNFs sont une agrégation de la protéine Tau hyperphosphorylée en filaments insolubles. Dans les neurones, l’accumulation du domaine cytoplasmique de l’APP allant de la membrane au site caspase (Jcasp) induit une apoptose in vitro et in vivo. Dans ce modèle comme en pathologie la protéine SET, impliquée dans la phosphorylation de Tau, se délocalise du noyau vers le cytoplasme. Le but du travail suivant a été de préciser la séquence des événements précoces conduisant au processus délétère en aval à l’aide du modèle Jcasp. Plusieurs données ont été identifiées : la protéine Egr-1 est potentiellement impliquée dans l’apoptose induite par le Jcasp car dans ce modèle son gène est précocement activé et elle est très vite phosphorylée. D’un autre côté, le signal de mort se traduit par un excès d’APP/APLP2 à la surface de la cellule dû à la plus forte affinité de PAT1 pour Jcasp qu’APP/APLP2. Cet excès induit la sortie de SET dans le cytoplasme, d’où elle induit une hyperphosphorylation de Tau. SET agirait par inhibition de la PP2A, principale protéine phosphatase capable de déphosphoryler Tau. Peut-être existe-t-il une relation entre l’APP clivée au site caspase et l’hyperphosphorylation de Tau via la délocalisation de SET du noyau vers le cytoplasme ? En contribuant à une meilleure compréhension des mécanismes impliqués dans la maladie d’Alzheimer, ces travaux pourraient participer à l’élaboration de nouvelles stratégies thérapeutique
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Derbali, Habib. "La caractérisation de l'amidotransférase ARNt-dépendante (AdT) de Pseudomonas aeruginosa PAO1." Master's thesis, Université Laval, 2008. http://hdl.handle.net/20.500.11794/19806.
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Eyraud, Vanessa. "Etude d’un insecticide naturel nommé PA1b : Mécanisme d’action et expression hétérologue." Thesis, Lyon, INSA, 2014. http://www.theses.fr/2014ISAL0020/document.
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Dans un contexte où l’utilisation de substance chimique en agriculture est de plus en plus décriée, il est nécessaire de trouver de nouveaux moyens de protections des cultures, tout en maintenant une agriculture économiquement performante. Ainsi, un peptide extrait de graines de pois nommée PA1b (Pea Albumin 1 sous-unité b), présentant une forte activité insecticide a été découvert au laboratoire. PA1b provoque chez l’insecte modèle du laboratoire, le charançon des céréales Sitophilus sp., 100% de mortalité. L’action de PA1b passe par la liaison à un récepteur présent chez les charançons sensibles, et cette liaison est absente chez les charançons résistants ; ce récepteur est une pompe à protons nommée V-ATPase pour Vacuolar ATPase. Elle est composée de 14 sous-unités organisées en deux complexes protéiques nommés V1 (intracellulaire) et V0 (membranaire). PA1b agissant à l’extérieur des cellules seul le complexe V0 composé des sous - unités a, c, d et e pouvait être le récepteur de notre toxine. Mon premier objectif de thèse a été de comprendre le mode d’action de PA1b, en identifiant d’abord la ou les sous-unités réceptrices de PA1b, puis en recherchant par quel mécanisme la liaison de PA1b induit la mort de l’insecte. Nous avons cloné chez le charançon tous les gènes du complexe Vo, puis j’ai complémenté des levures déficientes pour ces gènes. Ce travail, mais également celui réalisé en collaboration avec d’autres équipes, nous a permis de proposer un modèle de perception de PA1b qui implique les sous-unités c et e de la V-ATPase, et permet également de proposer des hypothèses pour les différentes résistances au peptide. Par des méthodes d’immunohistologie et de biochimie, j’ai ensuite montré de manière concordante que la liaison de PA1b à la V-ATPase déclenche un phénomène d’apoptose qui conduit à la mort cellulaire, puis à la mort de l’insecte. Le second objectif de ma thèse était la mise en place d’un système de production hétérologue de PA1b. Grâce à l’expression hétérologue par infiltration de feuille de tabac (Nicotiana benthamiana) nous avons mis en place une technique de production efficace de la protéine PA1b. Après avoir déterminé les parties du gène codant PA1b nécessaires à la production de la protéine fonctionnelle, le système de production a ensuite été simplifié par la construction d’une cassette d’expression. Ainsi six isoformes de PA1b présents chez le pois, dont l’activité individuelle restait inconnue, ont été produits et testés, permettant de montrer que le caractère amphiphile de PA1b était primordial pour son activité. Par cette technique nous avons mis en place un système de production rapide et efficace permettant de tester la toxicité de nombreux isoformes de PA1b. Ce travail sera une aide précieuse pour le projet, dont l’un des objectifs majeurs est l’optimisation de PA1b, c’est-à-dire de déterminer la séquence peptidique la plus toxiqueIn a context where chemical pesticides are increasingly criticized, new crops protection strategies that do not affect agriculture efficiency and productivity, must be found. A new peptide extracted from pea (Pisum sativum) seeds, named PA1b (Pea albumin 1 subunit b), and showing an important insecticide activity, was discovered in our laboratory. PA1b induces 100% mortality in our insect model, the cereal weevil, Sitophilus sp. PA1b acts by interacting with a receptor, this interaction is present in sensitive weevil, but not present in resistant weevil. The PA1b receptor is the vacuolar H+ -ATPase (V-ATPase), a multi-subunit proton pump. The V-ATPase is composed of two functional protein complexes named V1 (in the cytoplasm) and V0 (in the membrane). As PA1b is known to act only in the extracellular space, thus only the V0 complex, composed on the subunits a, c, d and e, can be the toxin receptor. The first aim of this thesis is to understand the PA1b mode of action: (i) identifying the subunit(s) acting as the receptor(s), (ii) understanding how the binding mechanism of PA1b on the receptor lead to the insect death. The weevil V0 complex genes were cloned and we used them for a functional complementation tests in yeasts strains deleted for these genes. Our data, together with those obtained through collaboration, lead to the proposal of model for the PA1b perception signaling which would involve subunits c and e of the V-ATPase. The identification of the PA1b receptor allows us to propose a hypothetical model explaining resistance mechanism to the peptide. Using immunohistology and biochemistry methods, we showed that PA1b-receptor interaction induced cells death triggered by apoptosis thus leading to insect death. The second aim of this thesis was the development of a PA1b heterologous production system. Through Agrobacterium tumefaciens mediated transient transformation by infiltration in tobacco leaves (Nicotiana benthamiana) an efficient system for PA1b production was developed. After identification of the essential parts of the complex PA1 gene necessary for efficient PA1b production, we created an expression cassette to simplify our heterologous production system. We used the system to produce six pea PA1b-isoformes with unknown individual toxic activity. The isoforms toxicity was experimentally determined, and our data showed that the amphiphilic properties of PA1b are essential for the maintenance of its toxic activity. For the first time, we implemented a quick and efficient production system, which allows to produce and to test many naturals or synthetics PA1b isoforms. This work will be useful to achieve one of the most important objectives of the research on this molecule, that is the identification
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El, Atmani Asmaa. "Implication de PAR1 dans la progression du cancer de la prostate." Thesis, Paris Est, 2009. http://www.theses.fr/2009PEST0047.
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Le phénomène métastatique est important à comprendre puisque de manière irrémédiable, une fois engagé, il conduit le plus souvent au décès des patients. Le cancer de la prostate représente un bon modèle car sa progression du stade hormono-dépendant vers le stade d'hormono-échappement s’accompagne par l’apparition de métastases. Les Protease Activated Receptors (PAR1-4) sont des récepteurs qui jouent un rôle important dans l'hémostase et l'inflammation et dont l’implication dans la prolifération et l'invasion des cellules tumorales a été décrite dans plusieurs tissus. L’étude comparative de l'expression in vitro de PAR1 a confirmé son rôle dans la prolifération et l'invasion des lignées prostatiques normales et tumorales hormono-sensibles comme hormono-indépendantes. Son expression in vivo dans des tissus prostatiques à différents stades pathologiques a montré une surexpression de PAR1 chez les patients ayant atteint le stade d'hormono-échappement, associée à un mauvais pronostic. Son absence s’avère par contre de bon pronostic chez les patients hormonodépendants. L'ensemble des résultats obtenus nous permet de proposer PAR1 comme un nouveau marqueur pronostique pour le cancer de la prostate. L’activation des PARs, comme celle de plusieurs récepteurs de chémokines, apparaît comme un élément fondateur de la transition vers l’état métastatique. Le décryptage de cette combinatoire permettra de mieux comprendre les phénomènes impliqués dans cette transition et permettra de développer des thérapies ciblées pour prévenir l’apparition délétère de métastasesMetastasis is nowadays an important field of research as, once engaged, patients will generally die from their metastatic cancer. Prostate cancer represents an interesting model as its progression from hormone-naïve to hormone-independent status lead to metastatic disease. Protease Activated Receptors (PAR1-4) are G-protein-coupled receptors that play crucial roles in blood coagulation and inflammation but that are likely to play fundamental role in tumor cells proliferation and invasion. In vitro analysis of PAR1 expression in prostate cancer cell lines has confirmed the role of PAR1 in prostate cancer proliferation and invasion. Its expression in vivo in prostate cancer tissues have shown a constant surexpression in hormonerefractory ones, associated with a worse prognosis. However, its absence in hormone-naïve tissues is associated with a good prognosis. These results prompted us to recommend PAR1 as a new prognostic marker associated with prostate cancer progression. PAR activation, as well as several chemokine receptors, seems to be a founder feature of cancer transition to metastasis. Deciphering the pattern of receptor activation will allow a better understanding of the events that drive transition to metastasis and thus the development of new specific targeted therapeutics aimed at stopping deleterious metastatic evolution
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Lévêque, Maxime. "Évaluation du potentiel d’un polymère bio-sourcé, PA11, pour applications piézoélectriques." Thesis, Lille 1, 2014. http://www.theses.fr/2014LIL10194.
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Les développements récents des applications d’électronique portable demande de plus en plus l’utilisation de systèmes de récupération d’énergie à partir de sources environnantes de basse énergie. La conversion piézoélectrique à partir de sources vibratoire pourrait être un moyen intéressant pour l’alimentation électrique de petits systèmes. Dans ce travail, nous investiguons les potentialités de films de polymère bio-sourcé à base de polyamide 11 (PA11). Dans un premier temps, les effets de traitements physiques ou chimiques sur le développement des phases cristallines du PA11 ont été longuement étudiés. Dans un deuxième temps, des nano-argiles ont été utilisées comme modificateurs de structure dans la matrice PA11. Des nanocomposites de PA11/argile ont été réalisés en utilisant des argiles en feuillets et en nanotubes par extrusion. L’effet de ces argile n’est pas significatif sur la qualité structurale des composites obtenus. L’ajout d’argile en feuillets (cloisite 20A, cloisite 10A et cloisite Na+)conduit à une amélioration des caractéristiques mécaniques alors que l’ajout de silicates tubulaires (nanotubes d’halloysite) n’a pas d’effet significatif. Parmi tous les nanocomposites préparés, le nanocomposite chargé de Cloisite Na+ a montré la meilleure propriétés piézoélectriques. Il semble que la polarité plus élevée de la Cloisite Na+ puisse être à l’origine de la meilleure réponse à la polarisation de ces composites. Une méthode spécifique de quantification de la récupération d’énergie vibratoire a été développée pour ces composites polymères/argile. Les capacités de récupération d’énergie par vibration ont été étudiées sur l’argile PA11 chargée de Cloisite Na+In the last few years it has been an increasing demand for the elaboration of flexible energy conversion sources due to the rapid increase in the usage of portable electronic devices. The piezoelectric conversion from vibration sources could be an interesting way to charge small systems. In this work, we investigate the potential of bio-based polyamide 11 (PA11) polymer films. At first, the effect of physical and chemical treatments on the development of crystalline phases in polyamide 11 has been thoroughly investigated. In a second step, nanoclays have been used as structural modifiers in PA 11 matrix. Nanocomposites were prepared using layered and tubular clays by melt-blending process and it was found that addition of layered silicates (cloisite 20A, cloisite 10A and cloisite Na+) results in an increase in mechanical properties, while the addition of tubular silicates (halloysite nanotube) has no significant effect. Addition of clay is not significant on the structural quality of the obtained nanocomposites. PA11 nanocomposite loaded with Cloisite Na+ have shown the best piezoelectric properties. It appears that the higher value of the polarity of Cloisite Na + may be responsible of the best answer to the polarization of the composites. A specific method for the quantification of energy vibration recovery has been developed for these nanocomposites. The capabilities of vibrational energy recovery were studied on PA11 loaded with Cloisite Na+
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Bruneau, Joannie. "Gata4 et Cdx2 sont des régulateurs transcriptionnels intestinaux du gène encodant la protéine sécrétoire de type lectine Pap1." Mémoire, Université de Sherbrooke, 2011. http://hdl.handle.net/11143/5542.
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Gata4 est un facteur de transcription exprimé par les entérocytes. Nos études ont démontré que Gata4 pouvait réguler l'expression de Pap1 chez le rat. Ce gène encode pour une protéine sécrétoire de type lectine impliquée dans le contrôle de la prolifération bactérienne. Puisqu'il a été démontré que des lectines antibactériennes de la famille Pap sont sécrétées dans la lumière intestinale en réponse à des stimuli inflammatoires, le but de cette étude était de définir l'implication transcriptionnelle de Gata4 dans la réponse inflammatoire des cellules épithéliales intestinales. Afin de caractériser l'effet de Gata4 sur la régulation transcriptionnelle de Pap1 dans les cellules Caco-2/15, nous avons utilisé des essais luciférase et généré différents mutants de la protéine Gata4 et du promoteur Pap1 . Nous avons également utilisé des immunobuvardages et des analyses de gel de rétention afin de mesurer la quantité et l'affinité de Gata4 pour l'ADN dans les cellules IEC-6/Cdx2. Les essais luciférase ont démontré que Gata4, en combinaison avec Cdx2, amène un effet synergique important sur l'activité du promoteur de Pap1 de l'ordre d'environ 8 fois. Différents mutants de la protéine Gata4 ont montré une abolition du potentiel transcriptionnel, démontrant que l'effet observé est spécifique. Cependant, la cotransfection d'un mutant du domaine en doigt de zinc (Zn) localisé en N-terminal, en combinaison avec Cdx2, augmente radicalement l'activation du promoteur de 18 fois. Des résultats préliminaires ont également démontré que la surexpression de ce mutant dans les cellules IEC-6/Cdx2 augmente fortement l'expression endogène du gène Pap1 . Cet effet pourrait être médié par des interactions avec les cofacteurs Fog. En effet, la cotransfection de Fog1 réprime l'effet synergique observé avec Gata4 de type sauvage mais non avec le mutant du domaine en doigt de Zn en N-terminal. Les mutants générés du promoteur Pap1 ont permis d'identifier le site Cdx2 et le site Gata le plus proximal du site d'initiation de la transcription comme nécessaire à l'effet transcriptionnel de Gata4 et Cdx2. En utilisant comme modèle les cellules IEC-6/Cdx2, nous avons montré qu'une induction avec des LPS n'a pas d'effet significatif sur la quantité totale de la protéine Gata4 mais des résultats préliminaires montrent une modulation de la phosphorylation de Gata4 sur la sérine 105. Par gel de rétention, nous avons montré que GATA4 a une affinité pour plusieurs sites sur le promoteur du gène Pap1 et qu'elle est augmentée en condition de stress cellulaire induit par les LPS. Cette étude nous permet de mieux comprendre l'implication de Gata4 dans la réponse inflammatoire de la cellule épithéliale intestinale.
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Kwok, Yuen-yuen. "Cloning and characterization of PAC1 receptor splice variants in goldfish (Carassius auratus)." Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B30708072.
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Haglund, Linda. "Thrombin receptor signalling in platelets: PAR1, but not PAR4, is rapidly desensitized." Thesis, Linköping University, Department of Physics, Chemistry and Biology, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-18455.
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Platelets play a key role in primary haemostasis but are also related to the pathogenesis of arterial thrombosis. Thrombin is the most effective agonist inducing platelet activation. Human platelets express two G-protein coupled thrombin receptors (GPCRs), called protease activated receptor (PAR)1 and PAR4. The aim of this study was to clarify differences in the activities of PAR1 and PAR4, especially focusing on their resistance towards the platelet inhibitor nitric oxide (NO) and their ability to undergo desensitization. For this, PAR1- and PAR4- activating peptides (APs) (SFLLRN and AYPGKF, respectively) were used. Different aspects of platelet activities were studied: aggregation and the rise in intracellular Ca2+ concentrations ([Ca2+]i). Aggregation was analyzed with lumiaggregometry, and [Ca2+]i were studied using the fura-2 method. PKC substrate phosphorylation and the expression of PAR1 surface receptors were also analyzed, using Western blot and flow cytometry, respectively. The results from this study showed that NO exerted similar inhibitory effects on the two thrombin receptors. However, PAR1 and PAR4 differed in their ability to undergo desensitization. In cumulative dose-response studies, a low concentration of PAR1-AP induced desensitization of platelets towards higher PAR1-AP concentrations. This was not the case when studying PAR4-AP. The mechanism behind the desensitization of PAR1 to some part involved PKC, at least when studying the mobilization of intracellular Ca2+. PAR1 desensitization did not seem to involve receptor internalization and neither did it affect the activity of PAR4. This thus suggests that PAR4 might be a more suitable therapeutic target in the future management of thrombosis.
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Chou, Han Ting. "L-Lysine Decarboxylase and Cadaverine Gamma-Glutamylation Pathways in Pseudomonas Aeruginosa PAO1." Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/biology_diss/103.
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In comparison to other Pseudomonas, P. aeruginosa grows poorly in L-lysine as a sole source of nutrient while fast growth mutants can be obtained. The proposed catabolic pathway involves lysine decarboxylation to cadaverine and its subsequent degradation through g-glutamylation pathway to d-aminovalerate and glutarate. The lysine decarboxylase A (ldcA) gene, previously identified as a member of the ArgR regulon of L-arginine metabolism, was found essential for L-lysine catabolism. The ldcA gene encodes a decarboxylase which takes L-lysine but not L-arginine as substrate. Contrarily, the ldcA expression was inducible by L-arginine but not by L-lysine. This peculiar arginine control on lysine utilization was also noted from uptake experiments. The lack of lysine-responsive control on lysine catabolism and its tight connection to arginine regulatory network provided an explanation of lysine as poor nutrient for P. aeruginosa. Catabolism of cadaverine, a product from lysine decarboxylation, was investigated and compared to that of putrescine, another diamine of similar biochemical properties that is derived from arginine and ornithine. While the g-glutamylation pathway was first reported in E. coli for putrescine utilization, an expanded version of this pathway was found in P. aeruginosa with redundant enzymes for polyamine degradation. The PauR protein was identified as a transcriptional repressor of genes for the catabolism of putrescine and cadaverine, as well as their corresponding downstream metabolites, g-aminobutyrate (GABA) and d-aminovalerate (AMV). PauR shows distinct dimer configuration after glutaraldehyde crosslinkage, and possible conformational changes could be triggered by the presence of putrescine and cadaverine, but not GABA. A newly identified ABC transport system, encoded by the agtABCD operon, was found important for the uptake of GABA and AMV; and expression of which is controlled by the AgtSR two-component system. The CbrAB two-component system was proposed to regulate the catabolite repression control protein Crc through a small RNA CrcZ. A consensus CbrB recognition sequence was proposed based on the conserved palindromic nucleotide sequence in the upstream activating sequence of the crcZ promoter. Genetic studies indicated utilization of arginine, lysine and diamines (but not histidine, GABA and AMV) might be under CbrAB regulation through the CbrAB/CrcZ/Crc system in P. aeruginosa.
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Taverner, Claire Jeanne. "Regulation of proliferation and cell survival by thrombin through the PAR1 receptor." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620458.
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Kwok, Yuen-yuen, and 郭圓圓. "Cloning and characterization of PAC1 receptor splice variants in goldfish (Carassius auratus)." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B30708072.
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Vretenbrant, Öberg Karin. "The role of platelet thrombin receptors PAR1 and PAR4 in platelet activation." Doctoral thesis, Linköpings universitet, Klinisk kemi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-51935.
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Platelets play a pivotal role in coagulation and haemostasis. Their most prominent task is to seal damaged blood vessels by the formation of a platelet plug at the damaged area. Once the injury is covered, platelets retract the coagulum to close the wound and allow the blood to flow freely in the vessel. Platelets are strongly activated by the essential enzyme thrombin, formed in the coagulation cascade. Activation of the platelet thrombin receptors PAR1 and PAR4 leads to shape change, secretion of granule content, and aggregation, all of which can be accomplished by each receptor individually. However more and more findings indicate that there are differences between the receptors and that they have different physiological functions. This thesis presents studies performed to elucidate the relative role of PAR1 and PAR4 in platelet activation and coagulation. We have studied the effects on platelet activation and coagulation, and revealed a possible physiological role for PAR4 in the stabilisation of the coagulum. We also investigated the relative role of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine with and without inhibition of COX-1. We demonstrated that PAR4 interacts with adrenergic receptors and causes an aggregation of platelets dependent on released ATP and its receptor P2X1, thereby circumventing the inhibition by aspirin. Not only is this an interesting specific role for PAR4, but it may also be of clinical importance considering that COX-1 inhibition is the most common treatment for patients with cardiovascular disease to prevent thrombosis. We show that the number of PAR1 receptors varied between donors and that this variation was correlated to the response on receptor activation. The number of PAR1 receptors on the platelet surface was decreased after PAR1 stimulation but increased after stimulation of other receptors. In a final attempt to elucidate the nature of PAR1 and PAR4 we used mathematics to evaluate the effect of co-stimulation of the receptors. We found a strong synergistic effect for both platelet activation and aggregation. This indicates that PAR1 and PAR4 interact in a yet unknown way to regulate or amplify the effect of each other rather than merely transmitting the incoming signal the same way.
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Marnef, Aline. "Investigating the roles of Pat1 proteins in the control of gene expression." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610150.
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Davis, Hannah E. "The role of the pak1 protein kinase in fission yeast cell polarity." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/13592.
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p21-activated kinase (paklp) is essential in fission yeast and plays roles in cell polarity and mating. The pakl-34 mutant has a specific mutation that does not affect essential functions but causes highly penetrant defects in cell polarity and morphology. The pakl-34 strain has a specific defect in bipolar growth and the potential to help dissect the role of paklp in mediating cell polarity. Tagging experiments demonstrated that both wild type and pakl-34 proteins localize to cell tips and septa. I hypothesised that pak-34p may be deficient in kinase activity. I adapted a two-dimensional gel electophoresis approach, called Difference Gel Electrophoresis (DIGE), to screen for paklp substrates. Wild type and pak1 mutant strains were compared in this manner and differential proteins identified by mass spectrometry. General results showed very few differences between wild type and pakl-34 strains and huge differences between wild type and pak1 kinase-dead strains, indicating that the kinase-dead strain may not be suitable for dissecting the paklp mechanism. Specific results identified hxklp as a potential substrate but it was generally concluded that the DIGE approach may not have sufficient sensitivity and/or scope for the screening of paklp substrates. In parallel to the hypothesis-driven DIGE approach I attempted to find paklp substrates by a candidate approach, investigating the phosphorylation state of a paklp regulator, ral3p. I found differences in ral3p phosphorylation, between wild type and pak1mutants, by SDS-polyacrylamide gel electrophoresis and lambda phosphatase treatment. I also looked for differences in the sizes of paklp and pakl-34p complexes using sucrose gradients. This study (1) describes a new way to screen for novel protein kinase substrates in fission yeast and (2) suggests that hxklp and ral3p are substrates for paklp.
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Rabelo, Jacilane de Holanda. "Pabc-pattern: uma estratégia para codificar lições aprendidas em organizações de software." Universidade Federal do Amazonas, 2013. http://tede.ufam.edu.br/handle/tede/2929.
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Previous issue date: 2013-02-19CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
The knowledge acquired by the members of a software organization is one of its most important assets. Therefore, the proper management of this knowledge has become a real need in the software development industry. The capture of lessons learned has become a practice developed by some of these organizations in order to manage and transfer knowledge. One of the most common approaches to encourage knowledge transfer is codification. Knowledge codification can facilitate the storage of lessons learned for a future usage. This MA research aims at elaborating a strategy to facilitate the codification of lessons learned in software organizations, consequently promoting this practice. The strategy was denominated PABC-Pattern, and three studies were developed: the first compared the approach initially called ABC-Pattern with another visual approach the Mind Map; the second compared the ABC-Pattern with the codification structure supported by the ACKNOWLEDGE tool. Results from the first study indicated that the participants had a preference in using the ABC-Pattern approach due to its usage and utility. For this preference, it follows some of the reasons identified: a) more codification details; b) a more detailed description of the knowledge s scenario which is more clarifying; c) the possibility of acquiring more information with less effort; and d) the possibility of describing both the problem and solution. The main contribution for the second study were the improvement suggestions for the ABC-Pattern, in which enabled an evolution of the strategy. The second version of the strategy was denominated PABC-Pattern and was experimentally evaluated by a third study, in an observational study, with the help of the Technology Information professionals. The results showed that PABC-Pattern found good acceptance as an approach to codify and share knowledge, and also that industry professionals would recommend its usage where they work
O conhecimento adquirido pelos membros de uma organização de software é um dos seus ativos mais importantes. A gestão adequada deste conhecimento tornou-se uma necessidade real na indústria de desenvolvimento de software. A coleta de lições aprendidas é uma das práticas utilizadas por parte dessas organizações para gerenciar e transferir o conhecimento. Uma das abordagens para incentivar a transferência de conhecimento é a codificação. A codificação do conhecimento pode facilitar o registro das lições aprendidas para sua consulta posterior. O objetivo desta pesquisa de mestrado é elaborar uma estratégia para facilitar a codificação das lições aprendidas em organizações de software, de modo a incentivar esta prática. A estratégia foi nomeada PABC-Pattern. Foram realizados três estudos: o primeiro comparou a abordagem inicialmente chamada ABC-Pattern com outra abordagem visual, o Mapa Mental; o segundo comparou a abordagem ABC-Pattern com a estrutura de codificação apoiada pela ferramenta ACKNOWLEDGE. Os resultados do primeiro estudo indicaram que os participantes tinham uma preferência em utilizar a abordagem ABC-Pattern, devido à sua facilidade de uso e utilidade. Foram identificadas algumas das razões para essa preferência: a) mais detalhes de codificação; b) uma descrição mais detalhada do cenário do conhecimento, que é mais esclarecedora, c) a possibilidade de adquirir mais informações com menos esforço, e d) a possibilidade de descrever tanto o problema e a solução. A principal contribuição do segundo estudo foram as sugestões de melhoria para ABC-Pattern que possibilitaram uma evolução da estratégia. A segunda versão da estratégia foi chamada de PABC-Pattern e foi avaliada experimentalmente através de um terceiro estudo, dessa vez de observação, com profissionais da área de Tecnologia da Informação. Os resultados possibilitaram apontar que PABC-Pattern teve uma boa aceitação como abordagem para codificar e compartilhar o conhecimento e que os profissionais da indústria recomendariam sua utilização na empresa em que trabalham.
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Abdel-Malek, Suzanne Mohamed Amin. "Studies on the mechanism of zinc pyrithione resistance in Pseudomonas aeruginosa PAO1." Thesis, Abertay University, 2002. https://rke.abertay.ac.uk/en/studentTheses/9dedb4be-5764-4460-993b-67553e8075fb.
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Previous workers have suggested that it is possible to passage Pseudomonas aeruginosa towards resistance against selected antimicrobial agents (Isothiazolone biocides). These passages in sub-minimal inhibitory concentrations (sub-MIC) of antimicrobial agents also exhibited corresponding shifts in outer membrane protein profiles. The aim of this project was to elucidate whether or not such adaptive resistance was evidenced with consecutive passages in sub-MIC of the Pyrithione group of antimicrobial agents and whether or not such passages yielded comparable outer membrane protein shifts. Pseudomonas aeruginosa, an opportunistic Gram-negative bacteria known for its resistance to a wide range of antimicrobials was found to develop an adaptive resistanceto the biocides Zinc Pyrithione (ZnPT) and Sodium Pyrithione (NaPT) and Cetrimide. Investigation of the mechanism of resistance involved SDS-PAGE analysis of outer membranes of biocide-sensitive and biocide-resistant cells, SDS-PAGE LPS analysis,changes in cell surface hydrophobicity, cross resistance, multiple antibiotic resistance, efflux systems and stress response. Results indicate that the ZnPT-resistant phenotype exhibited an increase in cell surface hydrophobicity and LPS changes. When outer membrane protein profiles of ZnPT-resistant cells and ZnPT-sensitive cells were compared, a 26.30 kDa protein was observed to be missing from the outer membranes of the former but was expressed inthe latter. Furthermore, this protein was observed to reappear when the ZnPT-resistant cells were grown in a ZnPT-free media. The ZnPT-resistant phenotype exhibited resistance towards NaPT and Cetrimide but did not show resistance towards a selection of antibiotics, which indicates that ZnPT is not an inducer of the mar regulon in P.aeruginosa. Experiments using efflux pump inhibitors suggested the possible involvement of an efflux mechanism in the resistance developed towards ZnPT. SDS-PAGE of cell lysates of ZnPT-resistant and ZnPT-sensitive cells has shown that resistant cells expresses cytoplasmic proteins that were not expressed in sensitive cells. Using western blot analysis these proteins were found to be dissimilar to P.aeruginosa heat shock proteins, suggesting that they could be ZnPT-specific stress proteins or molecular chaperones that assist the cells in recovering from ZnPT-induced cellular damage.
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Seo, Young Hoon. "Purification and characterization of pyocin S2 : bacteriocin produced by Pseudomonas aeruginosa PA01 /." The Ohio State University, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487688973686079.
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NASCIMENTO, JUNIOR Agrinaldo Jacinto do. "A Influência de Cátions na Membrana de Lipopolissacarídeos de Pseudomonas aeruginosa PAO1." Universidade Federal de Pernambuco, 2013. https://repositorio.ufpe.br/handle/123456789/12203.
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license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5)Made available in DSpace on 2015-03-12T16:57:48Z (GMT). No. of bitstreams: 2 TESE_Agrinaldo Jacinto do Nascimento Júnior.pdf: 6927683 bytes, checksum: bf123d71858e87a718a988da950251f3 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-12-19
FACEPE CAPES INAMI
A membrana externa de bactérias Gram- negativas é constituida majoritariamente de lipopolissacárideo (LPS) e fosfolipídeo. Cada unidade de LPS é constituida por até três partes, o lipídeo A, o Core e outra nem sempre presente chamada antígeno-O. O lípideo A tem um maior carater apolar, embora possua grupos fosfatos. Já o core é a região mais carregada do LPS e possui não apenas açucares fosforilados, mas também carboxilados. Desta maneira, a superfície da membrana de LPS é negativa e a interação com os cátions necessária para neutralizar a carga da membrana. Por isso, estas membranas possuem alta capacidade em adsorver cátions e assim são candidatas a serem utilizadas como agente biorremediadores, na captura por exemplo de radionuclídeos. Numa perspectiva de saúde, o LPS atua como um antigeno para o sistema imunológico de mamíferos e a sua ação pode auxiliar a causar não apenas febre, mas até levar a morte individuos imunocomprometidos. A bactéria Gram-negativa Pseudomonas aeruginosa é um destes patógenos nosocomiais oportunistas e tem sido apontada como uma das principais responsáveis por provocar a morte de pacientes portadores de fibroce cística. A estrutura supramolecular das membranas de LPS afetam não apenas a sua permeabilidade, mas ainda a ativação do sistema imunológico do hospedeiro no momento da infecção. Para uma descrição a nível molecular da influência dos cátions, Na+ , K+, Ca2+, Mg2+ , Zn2+ e Ba2+ na membrana de LPS utilizamos uma abordagem teórica baseada na dinâmica molecular clássica atomística. O modelo da membrana foi uma bicamada constituida de 72 unidades de LPS de Pseudomonas aeruginosa do quimiotipo PAO1 ancorados em 180 moléculas de 1,2-dipalmitoil-3-fosfatidil-etanolamina (DPPE) considerando o pH = 7 e temperatura de 300K. Os resultados das análises das simulações indicaram que as membranas de LPS suportam um nível de hidratação maior do que as bicamadas de fosfolipídeos e além disso os cátions tendem a provocar a ligação cruzada entre as unidades de LPS. Ainda, o aumento do raio de hidratação dos cátions, bem como a diminuição da ligação cruzada entre as unidades de LPS tendem a promover a transição da membrana de um estado lamelar para não lamelar. Deste modo, os resultados sugerem que a escolha combinada da valência do cátion e sua capacidade relativa de coordenar os grupos fosfatos e moléculas de água podem servir como regra para modular propriedades das membranas de LPS como estrutura supramolecular, fluidez e hidratação.